Anthrax spores can be disposed of easily after they are dispersed.

Secondary aerosolization of viable Bacillus anthracis spores in a contaminated US Senate Office.

CONTEXT Bioterrorist attacks involving letters and mail-handling systems in Washington, DC, resulted in Bacillus anthracis (anthrax) spore contamination in the Hart Senate Office Building and other facilities in the US Capitol's vicinity. OBJECTIVE To provide information about the nature and extent of indoor secondary aerosolization of B anthracis spores. DESIGN Stationary and personal air samples, surface dust, and swab samples were collected under semiquiescent (minimal activities) and then simulated active office conditions to estimate secondary aerosolization of B anthracis spores. Nominal size characteristics, airborne concentrations, and surface contamination of B anthracis particles (colony-forming units) were evaluated. RESULTS Viable B anthracis spores reaerosolized under semiquiescent conditions, with a marked increase in reaerosolization during simulated active office conditions. Increases were observed for B anthracis collected on open sheep blood agar plates (P<.001) and personal air monitors (P =.01) during active office conditions. More than 80% of the B anthracis particles collected on stationary monitors were within an alveolar respirable size range of 0.95 to 3.5 micro m.   CONCLUSIONS Bacillus anthracis spores used in a recent terrorist incident reaerosolized under common office activities. These findings have important implications for appropriate respiratory protection, remediation, and reoccupancy of contaminated office environments.

A plasma membrane component able to bind and alter virions of poliovirus type 1: studies on cell-free alteration using a simplified assay.

Abstract Intact HeLa cells can bind virions of poliovirus type 1 and can subsequently convert them to altered particles by incubation at 37°. Altered particles sediment more slowly than virions, have lost VP-4, and are disrupted by sodium dodecyl sulfate, but their RNA is intact and ribonuclease insensitive. These characteristics allow assay of altered particles and of particles that have released their RNA, using nuclease digestion with or without sodium dodecyl sulfate treatment. With this simple assay procedure, quantitative parameters of binding and alteration can be measured. The binding-altering activity can be localized in plasma membranes, and pure membranes can be shown to carry out alteration. The membrane-bound activity is abolished by proteases and by nonionic detergents. Only altered particles are formed by membranes; release of RNA is not caused by membranes but is caused by intact cells. Binding and alteration are processes that are closely associated; it is possible that alteration is a consequence of binding by multiple receptors in a fluid membrane.

Nanotoxicology: An Emerging Discipline Evolving from Studies of Ultrafine Particles

Although humans have been exposed to airborne nanosized particles (NSPs; < 100 nm) throughout their evolutionary stages, such exposure has increased dramatically over the last century due to anthropogenic sources. The rapidly developing field of nanotechnology is likely to become yet another source through inhalation, ingestion, skin uptake, and injection of engineered nanomaterials. Information about safety and potential hazards is urgently needed. Results of older biokinetic studies with NSPs and newer epidemiologic and toxicologic studies with airborne ultrafine particles can be viewed as the basis for the expanding field of nanotoxicology, which can be defined as safety evaluation of engineered nanostructures and nanodevices. Collectively, some emerging concepts of nanotoxicology can be identified from the results of these studies. When inhaled, specific sizes of NSPs are efficiently deposited by diffusional mechanisms in all regions of the respiratory tract. The small size facilitates uptake into cells and transcytosis across epithelial and endothelial cells into the blood and lymph circulation to reach potentially sensitive target sites such as bone marrow, lymph nodes, spleen, and heart. Access to the central nervous system and ganglia via translocation along axons and dendrites of neurons has also been observed. NSPs penetrating the skin distribute via uptake into lymphatic channels. Endocytosis and biokinetics are largely dependent on NSP surface chemistry (coating) and in vivo surface modifications. The greater surface area per mass compared with larger-sized particles of the same chemistry renders NSPs more active biologically. This activity includes a potential for inflammatory and pro-oxidant, but also antioxidant, activity, which can explain early findings showing mixed results in terms of toxicity of NSPs to environmentally relevant species. Evidence of mitochondrial distribution and oxidative stress response after NSP endocytosis points to a need for basic research on their interactions with subcellular structures. Additional considerations for assessing safety of engineered NSPs include careful selections of appropriate and relevant doses/concentrations, the likelihood of increased effects in a compromised organism, and also the benefits of possible desirable effects. An interdisciplinary team approach (e.g., toxicology, materials science, medicine, molecular biology, and bioinformatics, to name a few) is mandatory for nanotoxicology research to arrive at an appropriate risk assessment.

ACAM2000™: The new smallpox vaccine for United States Strategic National Stockpile

Smallpox was eradicated more than 30 years ago, but heightened concerns over bioterrorism have brought smallpox and smallpox vaccination back to the forefront. The previously licensed smallpox vaccine in the United States, Dryvax (Wyeth Laboratories, Inc.), was highly effective, but the supply was insufficient to vaccinate the entire current US population. Additionally, Dryvax had a questionable safety profile since it consisted of a pool of vaccinia virus strains with varying degrees of virulence, and was grown on the skin of calves, an outdated technique that poses an unnecessary risk of contamination. The US government has therefore recently supported development of an improved live vaccinia virus smallpox vaccine. This initiative has resulted in the development of ACAM2000 (Acambis, Inc.), a single plaque-purified vaccinia virus derivative of Dryvax, aseptically propagated in cell culture. Preclinical and clinical trials reported in 2008 demonstrated that ACAM2000 has comparable immunogenicity to that of Dryvax, and causes a similar frequency of adverse events. Furthermore, like Dryvax, ACAM2000 vaccination has been shown by careful cardiac screening to result in an unexpectedly high rate of myocarditis and pericarditis. ACAM2000 received US Food and Drug Administration (FDA) approval in August 2007, and replaced Dryvax for all smallpox vaccinations in February 2008. Currently, over 200 million doses of ACAM2000 have been produced for the US Strategic National Stockpile. This review of ACAM2000 addresses the production, characterization, clinical trials, and adverse events associated with this new smallpox vaccine.

Identification and transmission of Piper yellow mottle virus and Cucumber mosaic virus infecting black pepper (Piper nigrum) in Sri Lanka

Sri Lankan black pepper with symptoms of yellow mottle disease contained a mixture of viruses: Piper yellow mottle virus (PYMV) particles (30 × 130 nm), Cucumber mosaic virus (CMV, 30 nm diameter isometric particles), and unidentified, isometric virus-like particles (30 nm diameter). An effective purification procedure is described for PYMV. Immunosorbent and conventional electron microscopy successfully detected badnavirus particles only when at least partially purified extracts were used. PYMV was confirmed as the cause of the disease, with the other two viruses apparently playing no part in producing symptoms. PYMV was transmitted by grafting, by the insect vectors citrus mealy bug (Planococcus citri) and black pepper lace bug (Diconocoris distanti), but not by mechanical inoculation or through seeds. The CMV isolate was transmitted to indicator plants by mechanical inoculation and by the vector Aphis gossypii, but not by Myzus persicae; but neither mechanical nor insect transmission of CMV to black pepper was successful. A sensitive polymerase chain reaction assay was developed to detect PYMV in black pepper.

Propagation and recovery of intact, infectious Epstein-Barr virus from prokaryotic to human cells.

With current techniques, genetic alterations of herpesviruses are difficult to perform, mostly because of the large size of their genomes. To solve this problem, we have designed a system that allows the cloning of any gamma-herpesvirus in Escherichia coli onto an F factor-derived plasmid. Immortalized B cell lines were readily established with recombinant Epstein-Barr virus (EBV), demonstrating that the F factor-cloned EBV genome has all the characteristics of wild-type EBV. Because any genetic modification is possible in E. coli, this experimental approach opens the way to the genetic analysis of all EBV functions. Moreover, it is now feasible to generate attenuated EBV strains in vitro such that vaccine strains can be designed. Because we incorporated the genes for hygromycin resistance and green fluorescent protein onto the E. coli cloned EBV genome, the still open question of the EBV target cells other than B lymphocytes will be addressed.

Classification, functions, and clinical relevance of extracellular vesicles.

Both eukaryotic and prokaryotic cells release small, phospholipid-enclosed vesicles into their environment. Why do cells release vesicles? Initial studies showed that eukaryotic vesicles are used to remove obsolete cellular molecules. Although this release of vesicles is beneficial to the cell, the vesicles can also be a danger to their environment, for instance in blood, where vesicles can provide a surface supporting coagulation. Evidence is accumulating that vesicles are cargo containers used by eukaryotic cells to exchange biomolecules as transmembrane receptors and genetic information. Because also bacteria communicate to each other via extracellular vesicles, the intercellular communication via extracellular cargo carriers seems to be conserved throughout evolution, and therefore vesicles are likely to be a highly efficient, robust, and economic manner of exchanging information between cells. Furthermore, vesicles protect cells from accumulation of waste or drugs, they contribute to physiology and pathology, and they have a myriad of potential clinical applications, ranging from biomarkers to anticancer therapy. Because vesicles may pass the blood-brain barrier, they can perhaps even be considered naturally occurring liposomes. Unfortunately, pathways of vesicle release and vesicles themselves are also being used by tumors and infectious diseases to facilitate spreading, and to escape from immune surveillance. In this review, the different types, nomenclature, functions, and clinical relevance of vesicles will be discussed.

Cancer cell–autonomous contribution of type I interferon signaling to the efficacy of chemotherapy

Some of the anti-neoplastic effects of anthracyclines in mice originate from the induction of innate and T cell–mediated anticancer immune responses. Here we demonstrate that anthracyclines stimulate the rapid production of type I interferons (IFNs) by malignant cells after activation of the endosomal pattern recognition receptor Toll-like receptor 3 (TLR3). By binding to IFN-α and IFN-β receptors (IFNARs) on neoplastic cells, type I IFNs trigger autocrine and paracrine circuitries that result in the release of chemokine (C-X-C motif) ligand 10 (CXCL10). Tumors lacking Tlr3 or Ifnar failed to respond to chemotherapy unless type I IFN or Cxcl10, respectively, was artificially supplied. Moreover, a type I IFN–related signature predicted clinical responses to anthracycline-based chemotherapy in several independent cohorts of patients with breast carcinoma characterized by poor prognosis. Our data suggest that anthracycline-mediated immune responses mimic those induced by viral pathogens. We surmise that such 'viral mimicry' constitutes a hallmark of successful chemotherapy.

Human and other faeces as breeding media of the trachoma vector Musca sorbens.

The fly Musca sorbens Wiedemann (Diptera: Muscidae) apparently transmits Chlamydia trachomatis, causing human trachoma. The literature indicates that M. sorbens breeds predominantly in isolated human faeces on the soil surface, but not in covered pit latrines. We sought to identify breeding media of M. sorbens in a rural Gambian village endemic for trachoma. Test breeding media were presented for oviposition on soil-filled buckets and monitored for adult emergence. Musca sorbens emerged from human (6/9 trials), calf (3/9), cow (3/9), dog (2/9) and goat (1/9) faeces, but not from horse faeces, composting kitchen scraps or a soil control (0/9 of each). After adjusting for mass of medium, the greatest number of flies emerged from human faeces (1426 flies/kg). Median time for emergence was 9 (inter quartile range = 8-9.75) days post-oviposition. Of all flies emerging from faeces 81% were M. sorbens. Male and female flies emerging from human faeces were significantly larger than those from other media, suggesting that they would be more fecund and live longer than smaller flies from other sources. Female flies caught from children's eyes were of a similar size to those from human faeces, but significantly larger than those from other media. We consider that human faeces are the best larval medium for M. sorbens, although some breeding also occurs in animal faeces. Removal of human faeces from the environment, through the provision of basic sanitation, is likely to greatly reduce fly density, eye contact and hence trachoma transmission, but if faeces of other animals are present M. sorbens will persist.

From Cell Differentiation to Cell Collectives: Bacillus subtilis Uses Division of Labor to Migrate

The organization of cells, emerging from cell-cell interactions, can give rise to collective properties. These properties are adaptive when together cells can face environmental challenges that they separately cannot. One particular challenge that is important for microorganisms is migration. In this study, we show how flagellum-independent migration is driven by the division of labor of two cell types that appear during Bacillus subtilis sliding motility. Cell collectives organize themselves into bundles (called "van Gogh bundles") of tightly aligned cell chains that form filamentous loops at the colony edge. We show, by time-course microscopy, that these loops migrate by pushing themselves away from the colony. The formation of van Gogh bundles depends critically on the synergistic interaction of surfactin-producing and matrix-producing cells. We propose that surfactin-producing cells reduce the friction between cells and their substrate, thereby facilitating matrix-producing cells to form bundles. The folding properties of these bundles determine the rate of colony expansion. Our study illustrates how the simple organization of cells within a community can yield a strong ecological advantage. This is a key factor underlying the diverse origins of multicellularity.

Cytomegalovirus US2 destroys two components of the MHC class II pathway, preventing recognition by CD4+ T cells

Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus that causes life-threatening disease in patients who are immunosuppressed for bone marrow or tissue transplantation or who have AIDS (ref. 1). HCMV establishes lifelong latent infections and, after periodic reactivation from latency, uses a panel of immune evasion proteins to survive and replicate in the face of robust, fully primed host immunity. Monocyte/macrophages are important host cells for HCMV, serving as a latent reservoir and as a means of dissemination throughout the body. Macrophages and other HCMV-permissive cells, such as endothelial and glial cells, can express MHC class II proteins and present antigens to CD4+ T lymphocytes. Here, we show that the HCMV protein US2 causes degradation of two essential proteins in the MHC class II antigen presentation pathway: HLA-DR-α and DM-α. This was unexpected, as US2 has been shown to cause degradation of MHC class I (refs. 5,6), which has only limited homology with class II proteins. Expression of US2 in cells reduced or abolished their ability to present antigen to CD4+ T lymphocytes. Thus, US2 may allow HCMV-infected macrophages to remain relatively 'invisible' to CD4+ T cells, a property that would be important after virus reactivation.

Infection-induced NETosis is a dynamic process involving neutrophil multitasking in vivo

Neutrophil extracellular traps (NETs) are released as neutrophils die in vitro in a process requiring hours, leaving a temporal gap that invasive microbes may exploit. Neutrophils capable of migration and phagocytosis while undergoing NETosis have not been documented. During Gram-positive skin infections, we directly visualized live polymorphonuclear cells (PMNs) in vivo rapidly releasing NETs, which prevented systemic bacterial dissemination. NETosis occurred during crawling, thereby casting large areas of NETs. NET-releasing PMNs developed diffuse decondensed nuclei, ultimately becoming devoid of DNA. Cells with abnormal nuclei showed unusual crawling behavior highlighted by erratic pseudopods and hyperpolarization consistent with the nucleus being a fulcrum for crawling. A requirement for both Toll-like receptor 2 and complement-mediated opsonization tightly regulated NET release. Additionally, live human PMNs injected into mouse skin developed decondensed nuclei and formed NETS in vivo, and intact anuclear neutrophils were abundant in Gram-positive human abscesses. Therefore early in infection NETosis involves neutrophils that do not undergo lysis and retain the ability to multitask.

Exploitative and Hierarchical Antagonism in a Cooperative Bacterium

Social organisms that cooperate with some members of their own species, such as close relatives, may fail to cooperate with other genotypes of the same species. Such noncooperation may take the form of outright antagonism or social exploitation. Myxococcus xanthus is a highly social prokaryote that cooperatively develops into spore-bearing, multicellular fruiting bodies in response to starvation. Here we have characterized the nature of social interactions among nine developmentally proficient strains of M. xanthus isolated from spatially distant locations. Strains were competed against one another in all possible pairwise combinations during starvation-induced development. In most pairings, at least one competitor exhibited strong antagonism toward its partner and a majority of mixes showed bidirectional antagonism that decreased total spore production, even to the point of driving whole populations to extinction. Differential response to mixing was the primary determinant of competitive superiority rather than the sporulation efficiencies of unmixed populations. In some competitive pairings, the dominant partner sporulated more efficiently in mixed populations than in clonal isolation. This finding represents a novel form of exploitation in bacteria carried out by socially competent genotypes and is the first documentation of social exploitation among natural bacterial isolates. Patterns of antagonistic superiority among these strains form a highly linear dominance hierarchy. At least some competition pairs construct chimeric, rather than segregated, fruiting bodies. The cooperative prokaryote M. xanthus has diverged into a large number of distinct social types that cooperate with clone-mates but exhibit intense antagonism toward distinct social types of the same species. Most lengthy migration events in nature may thus result in strong antagonism between migratory and resident populations, and this antagonism may have large effects on local population sizes and dynamics. Intense mutual antagonism appears to be more prevalent in this prokaryotic social species than has been observed in the eukaryotic social slime mold Dictyostelium discoideum, which also exhibits multicellular development. The finding of several cases of facultative social exploitation among these natural isolates suggests that such exploitation may occur frequently in nature in many prokaryotes with cooperative traits.

Viral Paratransgenesis in the Malaria Vector Anopheles gambiae

Paratransgenesis, the genetic manipulation of insect symbiotic microorganisms, is being considered as a potential method to control vector-borne diseases such as malaria. The feasibility of paratransgenic malaria control has been hampered by the lack of candidate symbiotic microorganisms for the major vector Anopheles gambiae. In other systems, densonucleosis viruses (DNVs) are attractive agents for viral paratransgenesis because they infect important vector insects, can be genetically manipulated and are transmitted to subsequent generations. However, An. gambiae has been shown to be refractory to DNV dissemination. We discovered, cloned and characterized the first known DNV (AgDNV) capable of infection and dissemination in An. gambiae. We developed a flexible AgDNV-based expression vector to express any gene of interest in An. gambiae using a two-plasmid helper-transducer system. To demonstrate proof-of-concept of the viral paratransgenesis strategy, we used this system to transduce expression of an exogenous gene (enhanced green fluorescent protein; EGFP) in An. gambiae mosquitoes. Wild-type and EGFP-transducing AgDNV virions were highly infectious to An. gambiae larvae, disseminated to and expressed EGFP in epidemiologically relevant adult tissues such as midgut, fat body and ovaries and were transmitted to subsequent mosquito generations. These proof-of-principle data suggest that AgDNV could be used as part of a paratransgenic malaria control strategy by transduction of anti-Plasmodium peptides or insect-specific toxins in Anopheles mosquitoes. AgDNV will also be extremely valuable as an effective and easy-to-use laboratory tool for transient gene expression or RNAi in An. gambiae.

Rapid method for the isolation of lipoproteins from human serum by precipitation with polyanions.

Procedures are described for the isolation of lipoproteins from human serum by precipitation with polyanions and divalent cations. A mixture of low and very low density lipoproteins can be prepared without ultracentrifugation by precipitation with heparin and either MnCl(2) alone or MgCl(2) plus sucrose. In both cases the precipitation is reversible, selective, and complete. The highly concentrated isolated lipoproteins are free of other plasma proteins as judged by immunological and electrophoretic methods. The low density and very low density lipoproteins can then be separated from each other by ultracentrifugation. The advantage of the method is that large amounts of lipoproteins can be prepared with only a single preparative ultracentrifugation. Polyanions other than heparin may also be used; when the precipitation of the low and very low density lipoproteins is achieved with dextran sulfate and MnCl(2), or sodium phosphotungstate and MgCl(2), the high density lipoproteins can subsequently be precipitated by increasing the concentrations of the reagents. These lipoproteins, containing small amounts of protein contaminants, are further purified by ultracentrifugation at d 1.22. With a single preparative ultracentrifugation, immunologically pure high density lipoproteins can be isolated from large volumes of serum.

Getting started with yeast.

Publisher Summary The yeast Saccharomyces cerevisiae is now recognized as a model system representing a simple eukaryote whose genome can be easily manipulated. Yeast has only a slightly greater genetic complexity than bacteria and shares many of the technical advantages that permitted rapid progress in the molecular genetics of prokaryotes and their viruses. Some of the properties that make yeast particularly suitable for biological studies include rapid growth, dispersed cells, the ease of replica plating and mutant isolation, a well-defined genetic system, and most important, a highly versatile DNA transformation system. Being nonpathogenic, yeast can be handled with little precautions. Large quantities of normal baker's yeast are commercially available and can provide a cheap source for biochemical studies. The development of DNA transformation has made yeast particularly accessible to gene cloning and genetic engineering techniques. Structural genes corresponding to virtually any genetic trait can be identified by complementation from plasmid libraries. Plasmids can be introduced into yeast cells either as replicating molecules or by integration into the genome. In contrast to most other organisms, integrative recombination of transforming DNA in yeast proceeds exclusively via homologous recombination. Cloned yeast sequences, accompanied by foreign sequences on plasmids, can therefore be directed at will to specific locations in the genome.

Advantages of larval control for African malaria vectors: Low mobility and behavioural responsiveness of immature mosquito stages allow high effective coverage

Based on sensitivity analysis of the MacDonald-Ross model, it has long been argued that the best way to reduce malaria transmission is to target adult female mosquitoes with insecticides that can reduce the longevity and human-feeding frequency of vectors. However, these analyses have ignored a fundamental biological difference between mosquito adults and the immature stages that precede them: adults are highly mobile flying insects that can readily detect and avoid many intervention measures whereas mosquito eggs, larvae and pupae are confined within relatively small aquatic habitats and cannot readily escape control measures. We hypothesize that the control of adult but not immature mosquitoes is compromised by their ability to avoid interventions such as excito-repellant insecticides. We apply a simple model of intervention avoidance by mosquitoes and demonstrate that this can substantially reduce effective coverage, in terms of the proportion of the vector population that is covered, and overall impact on malaria transmission. We review historical evidence that larval control of African malaria vectors can be effective and conclude that the only limitations to the effective coverage of larval control are practical rather than fundamental. Larval control strategies against the vectors of malaria in sub-Saharan Africa could be highly effective, complementary to adult control interventions, and should be prioritized for further development, evaluation and implementation as an integral part of Rolling Back Malaria.

Separation of mother and daughter cells.

Publisher Summary The budding yeast Saccharomyces cerevisiae ( S. cerevisiae ) divides asymmetrically. In vegetative growth, yeast cells reproduce by budding, and the position where the bud forms ultimately determines the plane of cell division. This chapter describes the detailed procedures for the separation and isolation of mothers and daughters. These protocols have been used by investigators studying aging, bud site selection, and other aspects of asymmetric cell division. The chapter describes the procedures for performing life span analysis by micromanipulation and the steps for the large-scale collection of old cells. At the beginning and the end of a life span, it can be difficult to distinguish mothers from daughters. At most points in the life span, daughter cells are smaller than the mothers that produced them. In addition, mother cells will generally bud a second time before their daughter cells form their first bud. One method for effective isolation of virgin daughter cells from mother cells, but not for recovery of old mothers, is called a “baby machine. ” Mother cells are attached to a membrane and allowed to divide. Daughter cells from these attached cells are eluted continuously by washing the membrane.

Unifying Viral Genetics and Human Transportation Data to Predict the Global Transmission Dynamics of Human Influenza H3N2

Information on global human movement patterns is central to spatial epidemiological models used to predict the behavior of influenza and other infectious diseases. Yet it remains difficult to test which modes of dispersal drive pathogen spread at various geographic scales using standard epidemiological data alone. Evolutionary analyses of pathogen genome sequences increasingly provide insights into the spatial dynamics of influenza viruses, but to date they have largely neglected the wealth of information on human mobility, mainly because no statistical framework exists within which viral gene sequences and empirical data on host movement can be combined. Here, we address this problem by applying a phylogeographic approach to elucidate the global spread of human influenza subtype H3N2 and assess its ability to predict the spatial spread of human influenza A viruses worldwide. Using a framework that estimates the migration history of human influenza while simultaneously testing and quantifying a range of potential predictive variables of spatial spread, we show that the global dynamics of influenza H3N2 are driven by air passenger flows, whereas at more local scales spread is also determined by processes that correlate with geographic distance. Our analyses further confirm a central role for mainland China and Southeast Asia in maintaining a source population for global influenza diversity. By comparing model output with the known pandemic expansion of H1N1 during 2009, we demonstrate that predictions of influenza spatial spread are most accurate when data on human mobility and viral evolution are integrated. In conclusion, the global dynamics of influenza viruses are best explained by combining human mobility data with the spatial information inherent in sampled viral genomes. The integrated approach introduced here offers great potential for epidemiological surveillance through phylogeographic reconstructions and for improving predictive models of disease control.

Large intestine-targeted nanoparticle-releasing oral vaccine to control genitorectal viral infection

Both rectal and vaginal mucosal surfaces serve as transmission routes for pathogenic microorganisms. Vaccination through large intestinal mucosa, previously proven protective for both of these mucosal sites in animal studies, can be achieved successfully by direct intracolorectal (i.c.r.) administration, but this route is clinically impractical. Oral vaccine delivery seems preferable but runs the risk of the vaccine's destruction in the upper gastrointestinal tract. Therefore, we designed a large intestine-targeted oral delivery with pH-dependent microparticles containing vaccine nanoparticles, which induced colorectal immunity in mice comparably to colorectal vaccination and protected against rectal and vaginal viral challenge. Conversely, vaccine targeted to the small intestine induced only small intestinal immunity and provided no rectal or vaginal protection, demonstrating functional compartmentalization within the gut mucosal immune system. Therefore, using this oral vaccine delivery system to target the large intestine, but not the small intestine, may represent a feasible new strategy for immune protection of rectal and vaginal mucosa.

Inducible caspase-9 suicide gene controls adverse effects from alloreplete T cells after haploidentical stem cell transplantation.

To test the feasibility of a single T-cell manipulation to eliminate alloreactivity while sparing antiviral and antitumor T cells, we infused 12 haploidentical hematopoietic stem cell transplant patients with increasing numbers of alloreplete haploidentical T cells expressing the inducible caspase 9 suicide gene (iC9-T cells). We determined whether the iC9-T cells produced immune reconstitution and if any resultant graft-versus-host disease (GVHD) could be controlled by administration of a chemical inducer of dimerization (CID; AP1903/Rimiducid). All patients receiving >10(4) alloreplete iC9-T lymphocytes per kilogram achieved rapid reconstitution of immune responses toward 5 major pathogenic viruses and concomitant control of active infections. Four patients received a single AP1903 dose. CID infusion eliminated 85% to 95% of circulating CD3(+)CD19(+) T cells within 30 minutes, with no recurrence of GVHD within 90 days. In one patient, symptoms and signs of GVHD-associated cytokine release syndrome (CRS-hyperpyrexia, high levels of proinflammatory cytokines, and rash) resolved within 2 hours of AP1903 infusion. One patient with varicella zoster virus meningitis and acute GVHD had iC9-T cells present in the cerebrospinal fluid, which were reduced by >90% after CID. Notably, virus-specific T cells recovered even after AP1903 administration and continued to protect against infection. Hence, alloreplete iC9-T cells can reconstitute immunity posttransplant and administration of CID can eliminate them from both peripheral blood and the central nervous system (CNS), leading to rapid resolution of GVHD and CRS. The approach may therefore be useful for the rapid and effective treatment of toxicities associated with infusion of engineered T lymphocytes. This trial was registered at www.clinicaltrials.gov as #NCT01494103.

Ecology, economics and political will: the vicissitudes of malaria strategies in Asia.

The documented history of malaria in parts of Asia goes back more than 2,000 years, during which the disease has been a major player on the socioeconomic stage in many nation states as they waxed and waned in power and prosperity. On a much shorter time scale, the last half century has seen in microcosm a history of large fluctuations in endemicity and impact of malaria across the spectrum of rice fields and rain forests, mountains and plains that reflect the vast ecological diversity inhabited by this majority aggregation of mankind. That period has seen some of the most dramatic changes in social and economic structure, in population size, density and mobility, and in political structure in history: all have played a part in the changing face of malaria in this extensive region of the world. While the majority of global malaria cases currently reside in Africa, greater numbers inhabited Asia earlier this century before malaria programs savored significant success, and now Asia harbors a global threat in the form of the epicenter of multidrug resistant Plasmodium falciparum which is gradually encompassing the tropical world. The latter reflects directly the vicissitudes of economic change over recent decades, particularly the mobility of populations in search of commerce, trade and personal fortunes, or caught in the misfortunes of physical conflicts. The period from the 1950s to the 1990s has witnessed near "eradication" followed by resurgence of malaria in Sri Lanka, control and resurgence in India, the influence of war and postwar instability on drug resistance in Cambodia, increase in severe and cerebral malaria in Myanmar during prolonged political turmoil, the essential disappearance of the disease from all but forested border areas of Thailand where it remains for the moment intractable, the basic elimination of vivax malaria from many provinces of central China. Both positive and negative experiences have lessons to teach in the debate between eradication and control as alternative strategies. China has for years held high the goal of "basic elimination", eradication by another name, in sensible semi-defiance of WHO dictates. The Chinese experience makes it clear that, given community organization, exhaustive attention to case detection, management and focus elimination, plus the political will at all levels of society, it is possible both to eliminate malaria from large areas of an expansive nation and to implement surveillance necessary to maintain something approaching eradication status in those areas. But China has not succeeded in the international border regions of the tropical south where unfettered population movement confounds the program. Thailand, Malaysia and to an extent Vietnam have also reached essential elimination in their rice field plains by vigorous vertical programs but fall short at their forested borders. Economics is central to the history of the rise and fall of nations, and to the history of disease in the people who constitute nations. The current love affair with free market economics as the main driving force for advance of national wealth puts severe limitations on the essential involvement of communities in malaria management. The task of malaria control or elimination needs to be clearly related to the basic macroeconomic process that preoccupies governments, not cloistered away in the health sector Historically malaria has had a severe, measurable, negative impact on the productivity of nations. Economic models need rehoning with political aplomb and integrating with technical and demographic strategies. Recent decades in Chinese malaria history carry some lessons that may be relevant in this context.

How absolute is zero? An evaluation of historical and current definitions of malaria elimination

Decisions to eliminate malaria from all or part of a country involve a complex set of factors, and this complexity is compounded by ambiguity surrounding some of the key terminology, most notably "control" and "elimination. " It is impossible to forecast resource and operational requirements accurately if endpoints have not been defined clearly, yet even during the Global Malaria Eradication Program, debate raged over the precise definition of "eradication. " Analogous deliberations regarding the meaning of "elimination" and "control" are basically nonexistent today despite these terms' core importance to programme planning. To advance the contemporary debate about these issues, this paper presents a historical review of commonly used terms, including control, elimination, and eradication, to help contextualize current understanding of these concepts. The review has been supported by analysis of the underlying mathematical concepts on which these definitions are based through simple branching process models that describe the proliferation of malaria cases following importation. Through this analysis, the importance of pragmatic definitions that are useful for providing malaria control and elimination programmes with a practical set of strategic milestones is emphasized, and it is argued that current conceptions of elimination in particular fail to achieve these requirements. To provide all countries with precise targets, new conceptual definitions are suggested to more precisely describe the old goals of "control" - here more exactly named "controlled low-endemic malaria" - and "elimination. " Additionally, it is argued that a third state, called "controlled non-endemic malaria," is required to describe the epidemiological condition in which endemic transmission has been interrupted, but malaria resulting from onwards transmission from imported infections continues to occur at a sufficiently high level that elimination has not been achieved. Finally, guidelines are discussed for deriving the separate operational definitions and metrics that will be required to make these concepts relevant, measurable, and achievable for a particular environment.

Intermittent prophylaxis with oral truvada protects macaques from rectal SHIV infection.

HIV continues to spread globally, mainly through sexual contact. Despite advances in treatment and care, preventing transmission with vaccines or microbicides has proven difficult. A promising strategy to avoid transmission is prophylactic treatment with antiretroviral drugs before exposure to HIV. Clinical trials evaluating the efficacy of daily treatment with the reverse transcriptase inhibitors tenofovir disoproxil fumarate (TDF) or Truvada (TDF plus emtricitabine) are under way. We hypothesized that intermittent prophylactic treatment with long-acting antiviral drugs would be as effective as daily dosing in blocking the earliest stages of viral replication and preventing mucosal transmission. We tested this hypothesis by intermittently giving prophylactic Truvada to macaque monkeys and then exposing them rectally to simian-human immunodeficiency virus (SHIV) once a week for 14 weeks. A simple regimen with an oral dose of Truvada given 1, 3, or 7 days before exposure followed by a second dose 2 hours after exposure was as protective as daily drug administration, possibly because of the long intracellular persistence of the drugs. In addition, a two-dose regimen initiated 2 hours before or after virus exposure was effective, and full protection was obtained by doubling the Truvada concentration in both doses. We saw no protection if the first dose was delayed until 24 hours after exposure, underscoring the importance of blocking initial replication in the mucosa. Our results show that intermittent prophylactic treatment with an antiviral drug can be highly effective in preventing SHIV infection, with a wide window of protection. They strengthen the possibility of developing feasible, cost-effective strategies to prevent HIV transmission in humans.

Prions Adhere to Soil Minerals and Remain Infectious

An unidentified environmental reservoir of infectivity contributes to the natural transmission of prion diseases (transmissible spongiform encephalopathies [TSEs]) in sheep, deer, and elk. Prion infectivity may enter soil environments via shedding from diseased animals and decomposition of infected carcasses. Burial of TSE-infected cattle, sheep, and deer as a means of disposal has resulted in unintentional introduction of prions into subsurface environments. We examined the potential for soil to serve as a TSE reservoir by studying the interaction of the disease-associated prion protein (PrPSc) with common soil minerals. In this study, we demonstrated substantial PrPSc adsorption to two clay minerals, quartz, and four whole soil samples. We quantified the PrPSc-binding capacities of each mineral. Furthermore, we observed that PrPSc desorbed from montmorillonite clay was cleaved at an N-terminal site and the interaction between PrPSc and Mte was strong, making desorption of the protein difficult. Despite cleavage and avid binding, PrPSc bound to Mte remained infectious. Results from our study suggest that PrPSc released into soil environments may be preserved in a bioavailable form, perpetuating prion disease epizootics and exposing other species to the infectious agent.

Comparison of agar dilution, microdilution, and disk elution methods for measuring the synergy of cefotaxime and its metabolite against anaerobes.

The activities of cefotaxime (CTX) and desacetyl cefotaxime (des-CTX) were tested both singly and in combination against 173 anaerobic clinical isolates. The MIC of CTX for 50% of 60 Bacteroides fragilis isolates was 22.4 micrograms/ml in broth, compared with 47.4 micrograms/ml in agar. This reduced efficacy in agar was seen with all species tested and is in apparent conflict with reported clinical efficacy of the drug. Synergy between CTX and des-CTX was observed with 70 to 100% of the isolates, including 60% of all Bacteroides spp. tested. The susceptibility results in a synergy system correlated well with those noted in a broth-disk elution method incorporating 32 micrograms of CTX and 8 micrograms of des-CTX per ml. The correlation was poorer when the broth-disk method contained 16 micrograms of CTX and 8 micrograms of des-CTX per ml.

A search for the presence of the enteroviral capsid protein VP1 in pancreases of patients with Type 1 (insulin-dependent) diabetes and pancreases and hearts of infants who died of coxsackieviral myocarditis

Using an antiserum raised to a recombinant coxsackie virus B3 capsid protein, VP1, an immunocytochemical technique was developed which was capable of detecting the presence of all coxsackie B viruses in formalin fixed paraffin embedded infected tissue culture cells. This technique was tested on autopsy heart and pancreas from 21 patients who were thought to have died of acute coxsackievirus B myocarditis. Cardiac myocytes were positive for the VP1 protein in 12 of 20 cases where the heart was available for study. Insulitis was present in the pancreas in seven of these cases and in all seven islet endocrine cells containing VP1 were found. VP1 was only rarely found in exocrine pancreas. In heart and pancreas, cells shown to contain VP1 usually showed signs of necrosis. Autopsy pancreases from 88 patients who had died at clinical presentation of Type 1 (insulin-dependent) diabetes mellitus showed no evidence of the presence of VP1. The continuing destruction of insulin-secreting B cells seen at the time of death in the diabetic pancreas is unlikely to be due to a direct cytopathic effect of a coxsackie B virus. However, this study does not exclude the possibility that a persistent infection of B cells by a defective enterovirus may result in their destruction by an autoimmune mechanism.

Population dynamics of a pathogen: the conundrum of vivax malaria

Building a mathematical model of population dynamics of pathogens within their host involves considerations of factors similar to those in ecology, as pathogens can prey on cells in the host. But within the multicellular host, attacked cell types are integrated with other cellular systems, which in turn intervene in the infection. For example, immune responses attempt to sense and then eliminate or contain pathogens, and homeostatic mechanisms try to compensate for cell loss. This review focuses on modeling applied to malarias, diseases caused by single-cell eukaryote parasites that infect red blood cells, with special concern given to vivax malaria, a disease often thought to be benign (if sometimes incapacitating) because the parasite only attacks a small proportion of red blood cells, the very youngest ones. However, I will use mathematical modeling to argue that depletion of this pool of red blood cells can be disastrous to the host if growth of the parasite is not vigorously check by host immune responses. Also, modeling can elucidate aspects of new field observations that indicate that vivax malaria is more dangerous than previously thought. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s12551-010-0034-3) contains supplementary material, which is available to authorized users.

Using Routine Surveillance Data to Estimate the Epidemic Potential of Emerging Zoonoses: Application to the Emergence of US Swine Origin Influenza A H3N2v Virus

BACKGROUND Prior to emergence in human populations, zoonoses such as SARS cause occasional infections in human populations exposed to reservoir species. The risk of widespread epidemics in humans can be assessed by monitoring the reproduction number R (average number of persons infected by a human case). However, until now, estimating R required detailed outbreak investigations of human clusters, for which resources and expertise are not always available. Additionally, existing methods do not correct for important selection and under-ascertainment biases. Here, we present simple estimation methods that overcome many of these limitations. METHODS AND FINDINGS Our approach is based on a parsimonious mathematical model of disease transmission and only requires data collected through routine surveillance and standard case investigations. We apply it to assess the transmissibility of swine-origin influenza A H3N2v-M virus in the US, Nipah virus in Malaysia and Bangladesh, and also present a non-zoonotic example (cholera in the Dominican Republic). Estimation is based on two simple summary statistics, the proportion infected by the natural reservoir among detected cases (G) and among the subset of the first detected cases in each cluster (F). If detection of a case does not affect detection of other cases from the same cluster, we find that R can be estimated by 1-G; otherwise R can be estimated by 1-F when the case detection rate is low. In more general cases, bounds on R can still be derived. CONCLUSIONS We have developed a simple approach with limited data requirements that enables robust assessment of the risks posed by emerging zoonoses. We illustrate this by deriving transmissibility estimates for the H3N2v-M virus, an important step in evaluating the possible pandemic threat posed by this virus. Please see later in the article for the Editors' Summary.

Evolution of novel cooperative swarming in the bacterium Myxococcus xanthus

Cooperation among individuals is necessary for evolutionary transitions to higher levels of biological organization. In such transitions, groups of individuals at one level (such as single cells) cooperate to form selective units at a higher level (such as multicellular organisms). Though the evolution of cooperation is difficult to observe directly in higher eukaryotes, microorganisms do offer such an opportunity. Here we report the evolution of novel cooperative behaviour in experimental lineages of the bacterium Myxococcus xanthus. Wild-type strains of M. xanthus exhibit socially dependent swarming across soft surfaces by a mechanism known as ‘S-motility’ that requires the presence of extracellular type IV pili. In lineages of M. xanthus unable to make pili, a new mechanistic basis for cooperative swarming evolved. Evolved swarming is mediated, at least in part, by enhanced production of an extracellular fibril matrix that binds cells—and their evolutionary interests—together. Though costly to individuals, fibril production greatly enhanced population expansion in groups of interconnected cells. These results show that fundamental transitions to primitive cooperation can readily occur in bacteria.

How nascent phagosomes mature to become phagolysosomes.

Phagocytosis mediates the clearance of apoptotic bodies and also the elimination of microbial pathogens. The nascent phagocytic vacuole formed upon particle engulfment lacks microbicidal and degradative activity. These capabilities are acquired as the phagosome undergoes maturation; a progressive remodeling of its membrane and contents that culminates in the formation of phagolysosomes. Maturation entails orderly sequential fusion of the phagosomal vacuole with specialized endocytic and secretory compartments. Concomitantly, the phagosomal membrane undergoes both inward and outward vesiculation and tubulation followed by fission, thereby recycling components and maintaining its overall size. Here, we summarize what is known about the molecular machinery that governs this complex metamorphosis of phagosome maturation.

Calreticulin exposure dictates the immunogenicity of cancer cell death

Anthracyclin-treated tumor cells are particularly effective in eliciting an anticancer immune response, whereas other DNA-damaging agents such as etoposide and mitomycin C do not induce immunogenic cell death. Here we show that anthracyclins induce the rapid, preapoptotic translocation of calreticulin (CRT) to the cell surface. Blockade or knockdown of CRT suppressed the phagocytosis of anthracyclin-treated tumor cells by dendritic cells and abolished their immunogenicity in mice. The anthracyclin-induced CRT translocation was mimicked by inhibition of the protein phosphatase 1/GADD34 complex. Administration of recombinant CRT or inhibitors of protein phosphatase 1/GADD34 restored the immunogenicity of cell death elicited by etoposide and mitomycin C, and enhanced their antitumor effects in vivo. These data identify CRT as a key feature determining anticancer immune responses and delineate a possible strategy for immunogenic chemotherapy.

A culture-independent sequence-based metagenomics approach to the investigation of an outbreak of Shiga-toxigenic Escherichia coli O104:H4.

IMPORTANCE Identification of the bacterium responsible for an outbreak can aid in disease management. However, traditional culture-based diagnosis can be difficult, particularly if no specific diagnostic test is available for an outbreak strain. OBJECTIVE To explore the potential of metagenomics, which is the direct sequencing of DNA extracted from microbiologically complex samples, as an open-ended clinical discovery platform capable of identifying and characterizing bacterial strains from an outbreak without laboratory culture. DESIGN, SETTING, AND PATIENTS In a retrospective investigation, 45 samples were selected from fecal specimens obtained from patients with diarrhea during the 2011 outbreak of Shiga-toxigenic Escherichia coli (STEC) O104:H4 in Germany. Samples were subjected to high-throughput sequencing (August-September 2012), followed by a 3-phase analysis (November 2012-February 2013). In phase 1, a de novo assembly approach was developed to obtain a draft genome of the outbreak strain. In phase 2, the depth of coverage of the outbreak strain genome was determined in each sample. In phase 3, sequences from each sample were compared with sequences from known bacteria to identify pathogens other than the outbreak strain. MAIN OUTCOMES AND MEASURES The recovery of genome sequence data for the purposes of identification and characterization of the outbreak strain and other pathogens from fecal samples. RESULTS During phase 1, a draft genome of the STEC outbreak strain was obtained. During phase 2, the outbreak strain genome was recovered from 10 samples at greater than 10-fold coverage and from 26 samples at greater than 1-fold coverage. Sequences from the Shiga-toxin genes were detected in 27 of 40 STEC-positive samples (67%). In phase 3, sequences from Clostridium difficile, Campylobacter jejuni, Campylobacter concisus, and Salmonella enterica were recovered. CONCLUSIONS AND RELEVANCE These results suggest the potential of metagenomics as a culture-independent approach for the identification of bacterial pathogens during an outbreak of diarrheal disease. Challenges include improving diagnostic sensitivity, speeding up and simplifying workflows, and reducing costs.

Dynamic capsule restructuring by the main pneumococcal autolysin LytA in response to the epithelium

Bacterial pathogens produce complex carbohydrate capsules to protect against bactericidal immune molecules. Paradoxically, the pneumococcal capsule sensitizes the bacterium to antimicrobial peptides found on epithelial surfaces. Here we show that upon interaction with antimicrobial peptides, encapsulated pneumococci survive by removing capsule from the cell surface within minutes in a process dependent on the suicidal amidase autolysin LytA. In contrast to classical bacterial autolysis, during capsule shedding, LytA promotes bacterial survival and is dispersed circumferentially around the cell. However, both autolysis and capsule shedding depend on the cell wall hydrolytic activity of LytA. Capsule shedding drastically increases invasion of epithelial cells and is the main pathway by which pneumococci reduce surface bound capsule during early acute lung infection of mice. The previously unrecognized role of LytA in removing capsule to combat antimicrobial peptides may explain why nearly all clinical isolates of pneumococci conserve this enzyme despite the lethal selective pressure of antibiotics.

Human Cytomegalovirus-Encoded Interleukin-10 Homolog Inhibits Maturation of Dendritic Cells and Alters Their Functionality

Interleukin-10 (IL-10) suppresses the maturation and cytokine production of dendritic cells (DCs), key regulators of adaptive immunity, and prevents the activation and polarization of naïve T cells towards protective gamma interferon-producing effectors. We hypothesized that human cytomegalovirus (HCMV) utilizes its viral IL-10 homolog (cmvIL-10) to attenuate DC functionality, thereby subverting the efficient induction of antiviral immune responses. RNA and protein analyses demonstrated that the cmvIL-10 gene was expressed with late gene kinetics. Treatment of immature DCs (iDCs) with supernatant from HCMV-infected cultures inhibited both the lipopolysaccharide-induced DC maturation and proinflammatory cytokine production. These inhibitory effects were specifically mediated through the IL-10 receptor and were not observed when DCs were treated with supernatant of cells infected with a cmvIL-10-knockout mutant. Incubation of iDCs with recombinant cmvIL-10 recapitulated the inhibition of maturation. Furthermore, cmvIL-10 had pronounced long-term effects on those DCs that could overcome this inhibition of maturation. It enhanced the migration of mature DCs (mDCs) towards the lymph node homing chemokine but greatly reduced their cytokine production. The inability of mDCs to secrete IL-12 was maintained, even when they were restimulated by the activated T-cell signal CD40 ligand in the absence of cmvIL-10. Importantly, cmvIL-10 potentiates these anti-inflammatory effects, at least partially, by inducing endogenous cellular IL-10 expression in DCs. Collectively, we show that cmvIL-10 causes long-term functional alterations at all stages of DC activation.

Therapeutic Clearance of Amyloid by Antibodies to Serum Amyloid P Component.

BACKGROUND The amyloid fibril deposits that cause systemic amyloidosis always contain the nonfibrillar normal plasma protein, serum amyloid P component (SAP). The drug (R)-1-[6-[(R)-2-carboxy-pyrrolidin-1-yl]-6-oxo-hexanoyl]pyrrolidine-2-carboxylic acid (CPHPC) efficiently depletes SAP from the plasma but leaves some SAP in amyloid deposits that can be specifically targeted by therapeutic IgG anti-SAP antibodies. In murine amyloid A type amyloidosis, the binding of these antibodies to the residual SAP in amyloid deposits activates complement and triggers the rapid clearance of amyloid by macrophage-derived multinucleated giant cells. METHODS We conducted an open-label, single-dose-escalation, phase 1 trial involving 15 patients with systemic amyloidosis. After first using CPHPC to deplete circulating SAP, we infused a fully humanized monoclonal IgG1 anti-SAP antibody. Patients with clinical evidence of cardiac involvement were not included for safety reasons. Organ function, inflammatory markers, and amyloid load were monitored. RESULTS There were no serious adverse events. Infusion reactions occurred in some of the initial recipients of larger doses of antibody; reactions were reduced by slowing the infusion rate for later patients. At 6 weeks, patients who had received a sufficient dose of antibody in relation to their amyloid load had decreased liver stiffness, as measured with the use of transient elastography. These patients also had improvements in liver function in association with a substantial reduction in hepatic amyloid load, as shown by means of SAP scintigraphy and measurement of extracellular volume by magnetic resonance imaging. A reduction in kidney amyloid load and shrinkage of an amyloid-laden lymph node were also observed. CONCLUSIONS Treatment with CPHPC followed by an anti-SAP antibody safely triggered clearance of amyloid deposits from the liver and some other tissues. (Funded by GlaxoSmithKline; ClinicalTrials.gov number, NCT01777243.).

Vps22/EAP30 in ESCRT-II mediates endosomal sorting of growth factor and chemokine receptors destined for lysosomal degradation.

The ubiquitin-binding protein Hrs and endosomal sorting complex required for transport (ESCRT)-I and ESCRT-III are involved in sorting endocytosed and ubiquitinated receptors to lysosomes for degradation and efficient termination of signaling. In this study, we have investigated the role of the ESCRT-II subunit Vps22/EAP30 in degradative protein sorting of ubiquitinated receptors. Vps22 transiently expressed in HeLa cells was detected in endosomes containing endocytosed epidermal growth factor receptors (EGFRs) as well as Hrs and ESCRT-I and ESCRT-III. Depletion of Vps22 by small interfering RNA, which was accompanied by decreased levels of other ESCRT-II subunits, greatly reduced degradation of EGFR and its ligand EGF as well as the chemokine receptor CXCR4. EGFR accumulated on the limiting membranes of early endosomes and aberrantly small multivesicular bodies in Vps22-depleted cells. Phosphorylation and nuclear translocation of extracellular-signal-regulated kinase1/2 downstream of the EGF-activated receptor were sustained by depletion of Hrs or the ESCRT-I subunit Tsg101. In contrast, this was not the case when Vps22 was depleted. These results indicate an important role for Vps22 in ligand-induced EGFR and CXCR4 turnover and suggest that termination of EGF signaling occurs prior to ESCRT-II engagement.

Inter-viral conflicts that exploit host CRISPR immune systems of Sulfolobus.

Infection of Sulfolobus islandicus REY15A with mixtures of different Sulfolobus viruses, including STSV2, did not induce spacer acquisition by the host CRISPR immune system. However, coinfection with the tailed fusiform viruses SMV1 and STSV2 generated hyperactive spacer acquisition in both CRISPR loci, exclusively from STSV2, with the resultant loss of STSV2 but not SMV1. SMV1 was shown to activate adaptation while itself being resistant to CRISPR-mediated adaptation and DNA interference. Exceptionally, a single clone S-1 isolated from an SMV1 + STSV2-infected culture, that carried STSV2-specific spacers and had lost STSV2 but not SMV1, acquired spacers from SMV1. This effect was also reproducible on reinfecting wild-type host cells with a variant SMV1 isolated from the S-1 culture. The SMV1 variant lacked a virion protein ORF114 that was shown to bind DNA. This study also provided evidence for: (i) limits on the maximum sizes of CRISPR loci; (ii) spacer uptake strongly retarding growth of infected cultures; (iii) protospacer selection being essentially random and non-directional, and (iv) the reversible uptake of spacers from STSV2 and SMV1. A hypothesis is presented to explain the interactive conflicts between SMV1 and the host CRISPR immune system.

Cytokine production patterns and lymphoproliferative responses in volunteers orally immunized with attenuated vaccine strains of Salmonella typhi.

New recombinant strains of attenuated Salmonella typhi used as live oral vaccines elicit potent immune responses. This study examined the patterns of cytokine production and proliferation to specific S. typhi antigens in subjects orally immunized with attenuated S. typhi vaccines CVD 906, CVD 908, and CVD 908 expressing the circumsporozoite protein of Plasmodium falciparum. After immunization, sensitized lymphocytes were found in subjects' blood that exhibited significantly increased proliferative responses and interferon-gamma production to purified S. typhi flagella when compared with preimmunization levels. Significant negative correlations were observed between interleukin-4 production and both interferon-gamma production and proliferation to S. typhi flagella. These results demonstrate that oral immunization with attenuated S. typhi strains alone or with those carrying a foreign gene elicits strong systemic cell-mediated immunity to purified S. typhi antigens, including the production of cytokines compatible with T1-type responses.

Downregulation of TAP1 in B lymphocytes by cellular and Epstein-Barr virus-encoded interleukin-10.

Virally infected cells degrade intracellular viral proteins proteolytically and present the resulting peptides in association with major histocompatibility complex (MHC) class I molecules to CD8+ cytotoxic T lymphocytes (CTLs). These cells are normally prone to CTL-mediated elimination. However, several viruses have evolved strategies to avoid detection by the immune system that interfere with the pathway of antigen presentation. Epstein-Barr virus (EBV) expresses a predominantly late protein, the BCRF1 gene product vIL-10, that is similar in sequence to the human interleukin-10 (hIL-10). We show here that vIL-10 affects the expression of one of the two transporter proteins (TAPs) associated with antigen presentation. Similarly, hIL-10 showed the same activity. Expression of the LMP2 and TAP1 genes but not expression of TAP2 or LMP7 is efficiently downregulated, indicating a specific IL-10 effect on the two divergently transcribed TAP1 and LMP2 genes. Downregulation of TAP1 by IL-10 hampers the transport of peptide antigens into the endoplasmatic reticulum, as shown in the TAP-specific peptide transporter assay, their loading onto empty MHC I molecules, and the subsequent translocation to the cell surface. As a consequence, IL-10 causes a general reduction of surface MHC I molecules on B lymphocytes that might also affect the recognition of EBV-infected cells by cytotoxic T cells.

Breaching the basement membrane: who, when and how?

The basement membrane (BM), a specialized network of extracellular matrix macromolecules, surrounds epithelial, endothelial, muscle, fat and nerve cells. During development, immune surveillance and disease states ranging from cancer to fibrosis, host cells penetrate the BM by engaging tissue-invasive programs, the identity of which remain largely undefined. Although it is commonly assumed that all cells employ similar mechanisms to cross BM barriers, accumulating evidence indicates that cells might selectively mobilize protease-dependent or -independent invasion programs. New data indicate that protease-dependent transmigration is largely reliant on a group of membrane-anchored metalloenzymes, termed the membrane-type matrix metalloproteinases, which irreversibly remodel BM structure. By contrast, mechanisms that enable protease-independent transmigration remain undefined and potentially involve the reversible disassembly of the BM network. Further characterization of the molecular mechanisms underlying BM transmigration should provide important insights into pathophysiologic tissue remodeling events and also enable the development of novel therapeutics.

Lessons from the past: managing insecticide resistance in malaria control and eradication programmes.

The distribution of insecticide-treated bednets to help combat the burden of malaria in sub-Saharan Africa has accelerated in the past 5 years. Additionally, many countries are also considering, or have already begun, indoor residual spraying campaigns. These are positive developments, since vector control has repeatedly proven to be an effective means of reducing malaria transmission. However, the sustainability of these insecticide-based interventions relies on the continuing susceptibility of the anopheles vectors to the limited number of available insecticides. Continual monitoring for early signs of insecticide resistance and the adoption of carefully considered resistance management strategies are therefore required. Regrettably, this essential monitoring component is frequently given a low priority in the push to meet ambitious coverage targets. We outline the key requirements for establishing an insecticide resistance surveillance system and urge all those involved in malaria vector control, either directly or as facilitators, to ensure that these measures are incorporated into control programmes. Failure to act now will inevitably lead to a future breakdown in disease control and jeopardise hopes of eradicating this major public-health problem.

Novel proteinaceous infectious particles cause scrapie.

After infection and a prolonged incubation period, the scrapie agent causes a degenerative disease of the central nervous system in sheep and goats. Six lines of evidence including sensitivity to proteases demonstrate that this agent contains a protein that is required for infectivity. Although the scrapie agent is irreversibly inactivated by alkali, five procedures with more specificity for modifying nucleic acids failed to cause inactivation. The agent shows heterogeneity with respect to size, apparently a result of its hydrophobicity; the smallest form may have a molecular weight of 50,000 or less. Because the novel properties of the scrapie agent distinguish it from viruses, plasmids, and viroids, a new term "prion" is proposed to denote a small proteinaceous infectious particle which is resistant to inactivation by most procedures that modify nucleic acids. Knowledge of the scrapie agent structure may have significance for understanding the causes of several degenerative diseases.

Activation of liver X receptors and retinoid X receptors prevents bacterial-induced macrophage apoptosis.

Microbe-macrophage interactions play a central role in the pathogenesis of many infections. The ability of some bacterial pathogens to induce macrophage apoptosis has been suggested to contribute to their ability to elude innate immune responses and successfully colonize the host. Here, we provide evidence that activation of liver X receptors (LXRs) and retinoid X receptors (RXRs) inhibits apoptotic responses of macrophages to macrophage colony-stimulating factor (M-CSF) withdrawal and several inducers of apoptosis. In addition, combined activation of LXR and RXR protected macrophages from apoptosis caused by infection with Bacillus anthracis, Escherichia coli, and Salmonella typhimurium. Expression-profiling studies demonstrated that LXR and RXR agonists induced the expression of antiapoptotic regulators, including AIM/CT2, Bcl-X(L), and Birc1a. Conversely, LXR and RXR agonists inhibited expression of proapoptotic regulators and effectors, including caspases 1, 4/11, 7, and 12; Fas ligand; and Dnase1l3. The combination of LXR and RXR agonists was more effective than either agonist alone at inhibiting apoptosis in response to various inducers of apoptosis, and it acted synergistically to induce expression of AIM/CT2. Inhibition of AIM/CT2 expression in response to LXR/RXR agonists partially reversed their antiapoptotic effects. These findings reveal unexpected roles of LXRs and RXRs in the control of macrophage survival and raise the possibility that LXR/RXR agonists may be exploited to enhance innate immunity to bacterial pathogens that induce apoptotic programs as a strategy for evading host responses.

Combining indoor residual spraying and insecticide-treated nets for malaria control in Africa: a review of possible outcomes and an outline of suggestions for the future

Insecticide-treated nets (ITNs) and indoor residual spraying (IRS) are currently the preferred methods of malaria vector control. In many cases, these methods are used together in the same households, especially to suppress transmission in holoendemic and hyperendemic scenarios. Though widespread, there has been limited evidence suggesting that such co-application confers greater protective benefits than either ITNs or IRS when used alone. Since both methods are insecticide-based and intradomicilliary, this article hypothesises that outcomes of their combination would depend on effects of the candidate active ingredients on mosquitoes that enter or those that attempt to enter houses. It is suggested here that enhanced household level protection can be achieved if the ITNs and IRS have divergent yet complementary properties, e.g. highly deterrent IRS compounds coupled with highly toxic ITNs. To ensure that the problem of insecticide resistance is avoided, the ITNs and IRS products should preferably be of different insecticide classes, e.g. pyrethroid-based nets combined with organophosphate or carbamate based IRS. The overall community benefits would however depend also on other factors such as proportion of people covered by the interventions and the behaviour of vector species. This article concludes by emphasizing the need for basic and operational research, including mathematical modelling to evaluate IRS/ITN combinations in comparison to IRS alone or ITNs alone.

[Pharmacokinetics and biotransformation of the antimycotic drug ciclopiroxolamine in animals and man after topical and systemic administration].

1. Following the dermal application of the carbon-14 labelled broad spectrum antimycotic 6-cyclohexyl-1-hydroxy-4-methyl-2(1H)-pyridone, 2-aminoethanol salt (ciclopiroxolamine, Hoe 296, Batrafen) in the form of a 1% aqueous cream to healthy human dorsal skin (penetration time: 6 h; occlusive dressing for 5 h), percutaneous absorption accounted on average for 1.3% of the dose applied. Excretion occurred via the kidney, with biological half-lives of 1.7 h. As can be seen from penetration studies of cadaverous skin, the horny layer contained the highest concentrations, with values of 2300-4500 microgram/cm3. The levels determined in the corium were still above the minimum inhibitory concentrations. These concentrations were already obtained at the first test stage (1.5 h after application) and did not change virtually at all over the longer penetration period. According to studies using histoautoradiography, ciclopirox can penetrate the skin via the epidermis and the hair follicles. When ciclopirox-14C-olamine aqueous cream was spread on the surface of fingernails, the radioactive-labelled compound penetrated right through the nail. The percutaneous absorption in dogs was higher, at 5-15% of the dose, than it was in humans. 2. After vaginal application (1 mg/kg) of ciclopirox-14C-olamine in the form of a 1% aqueous cream to bitches, between 42 and 97% of the dose (depending on the animal) was recovered in the urine and faeces, the remainder having penetrated into the tampon used to close the vagina. 3. Ciclopirox is excreted by dogs and man in the urine, primarily as a glucuronide. In humans another glucuronide with properties similar to those of the original substance was detected. Two conjugated, relatively non-polar metabolites were also present in small amounts. The metabolite patterns after oral and dermal application were similar. The binding of ciclopirox to serum proteins in humans was 96 +/- 2% in a concentration range of 0.01-11.0 microgram/ml. 4. Placental transfer was low in the rats studied. Though there was good absorption by the mother animal, the radioactivity in the foetal tissues was always lower than that of the maternal blood.

Cell death and immune privilege.

The host response to pathogens involves complex inflammatory responses and immune reactions. While these are central to host defense and vital to clearing infections, they are often accompanied by injury to surrounding tissue. Most organ systems can tolerate these responses without permanent consequences. However, there are sites that limit the spread of inflammation because it can threaten organ function. The most prominent examples of these are the eye, brain, and reproductive organs (testis, ovary), where even minor bouts of inflammation can have long-term consequences for the survival of the organism. In these organs immune responses either do not proceed, or proceed in a manner different from other areas; thus, they are called "immunologically privileged. " Here a functioning immune response can be the culprit that leads to disease.

Grass plants bind, retain, uptake, and transport infectious prions.

Prions are the protein-based infectious agents responsible for prion diseases. Environmental prion contamination has been implicated in disease transmission. Here, we analyzed the binding and retention of infectious prion protein (PrP(Sc)) to plants. Small quantities of PrP(Sc) contained in diluted brain homogenate or in excretory materials (urine and feces) can bind to wheat grass roots and leaves. Wild-type hamsters were efficiently infected by ingestion of prion-contaminated plants. The prion-plant interaction occurs with prions from diverse origins, including chronic wasting disease. Furthermore, leaves contaminated by spraying with a prion-containing preparation retained PrP(Sc) for several weeks in the living plant. Finally, plants can uptake prions from contaminated soil and transport them to aerial parts of the plant (stem and leaves). These findings demonstrate that plants can efficiently bind infectious prions and act as carriers of infectivity, suggesting a possible role of environmental prion contamination in the horizontal transmission of the disease.

Neutrophils Suppress Intraluminal NK Cell-Mediated Tumor Cell Clearance and Enhance Extravasation of Disseminated Carcinoma Cells.

UNLABELLED Immune cells promote the initial metastatic dissemination of carcinoma cells from primary tumors. In contrast to their well-studied functions in the initial stages of metastasis, the specific roles of immunocytes in facilitating progression through the critical later steps of the invasion-metastasis cascade remain poorly understood. Here, we define novel functions of neutrophils in promoting intraluminal survival and extravasation at sites of metastatic dissemination. We show that CD11b(+)/Ly6G(+) neutrophils enhance metastasis formation via two distinct mechanisms. First, neutrophils inhibit natural killer cell function, which leads to a significant increase in the intraluminal survival time of tumor cells. Thereafter, neutrophils operate to facilitate extravasation of tumor cells through the secretion of IL1β and matrix metalloproteinases. These results identify neutrophils as key regulators of intraluminal survival and extravasation through their cross-talk with host cells and disseminating carcinoma cells. SIGNIFICANCE This study provides important insights into the systemic contributions of neutrophils to cancer metastasis by identifying how neutrophils facilitate intermediate steps of the invasion-metastasis cascade. We demonstrate that neutrophils suppress natural killer cell activity and increase extravasation of tumor cells. Cancer Discov; 6(6); 630-49. ©2016 AACR.This article is highlighted in the In This Issue feature, p. 561.

The ascendancy of Amblyomma americanum as a vector of pathogens affecting humans in the United States.

Until the 1990s, Amblyomma americanum was regarded primarily as a nuisance species, but a tick of minor importance as a vector of zoonotic pathogens affecting humans. With the recent discoveries of Ehrlichia chaffeensis, Ehrlichia ewingii, and "Borrelia lonestari," the public health relevance of lone star ticks is no longer in question. During the next 25 years, the number of cases of human disease caused by A. americanum-associated pathogens will probably increase. Based on current trajectories and historic precedents, the increase will be primarily driven by biological and environmental factors that alter the geographic distribution and intensity of transmission of zoonotic pathogens. Sociologic and demographic changes that influence the likelihood of highly susceptible humans coming into contact with infected lone star ticks, in addition to advances in diagnostic capabilities and national surveillance efforts, will also contribute to the anticipated increase in the number of recognized cases of disease.

Ranking of elimination feasibility between malaria-endemic countries

Experience gained from the Global Malaria Eradication Program (1955-72) identified a set of shared technical and operational factors that enabled some countries to successfully eliminate malaria. Spatial data for these factors were assembled for all malaria-endemic countries and combined to provide an objective, relative ranking of countries by technical, operational, and combined elimination feasibility. The analysis was done separately for Plasmodium falciparum and Plasmodium vivax, and the limitations of the approach were discussed. The relative rankings suggested that malaria elimination would be most feasible in countries in the Americas and Asia, and least feasible in countries in central and west Africa. The results differed when feasibility was measured by technical or operational factors, highlighting the different types of challenge faced by each country. The results are not intended to be prescriptive, predictive, or to provide absolute assessments of feasibility, but they do show that spatial information is available to facilitate evidence-based assessments of the relative feasibility of malaria elimination by country that can be rapidly updated.

Rational design of a flavivirus vaccine by abolishing viral RNA 2'-O methylation.

Viruses that replicate in the cytoplasm cannot access the host nuclear capping machinery. These viruses have evolved viral methyltransferase(s) to methylate N-7 and 2'-O cap of their RNA; alternatively, they "snatch" host mRNA cap to form the 5' end of viral RNA. The function of 2'-O methylation of viral RNA cap is to mimic cellular mRNA and to evade host innate immune restriction. A cytoplasmic virus defective in 2'-O methylation is replicative, but its viral RNA lacks 2'-O methylation and is recognized and eliminated by the host immune response. Such a mutant virus could be rationally designed as a live attenuated vaccine. Here, we use Japanese encephalitis virus (JEV), an important mosquito-borne flavivirus, to prove this novel vaccine concept. We show that JEV methyltransferase is responsible for both N-7 and 2'-O cap methylations as well as evasion of host innate immune response. Recombinant virus completely defective in 2'-O methylation was stable in cell culture after being passaged for >30 days. The mutant virus was attenuated in mice, elicited robust humoral and cellular immune responses, and retained the engineered mutation in vivo. A single dose of immunization induced full protection against lethal challenge with JEV strains in mice. Mechanistically, the attenuation phenotype was attributed to the enhanced sensitivity of the mutant virus to the antiviral effects of interferon and IFIT proteins. Collectively, the results demonstrate the feasibility of using 2'-O methylation-defective virus as a vaccine approach; this vaccine approach should be applicable to other flaviviruses and nonflaviviruses that encode their own viral 2'-O methyltransferases.

Lethal complications of typhoid-cholera-vaccination. (Case report and review of the literature).

Simultaneous parenteral vaccination against typhoid and cholera lead to death through either anaphylactic shock or endotoxic shock in a 36-year-old male. At autopsy the charactertic features of shock as well as chronic interstitial myocarditis were noted. Moreover, fresh histiocytic and lymphocytic nodules were found in the liver, heart and meninges. A review of the literature dealing with lethal complications following parenteral tyhoid vaccinations shows an increased risk in debilitated persons (emaciation, stress, cold). Most of the fatalities occurred in persons who had previous disturbances of the cardiovascular system, as in the case reviewed here. Cardiac failure, Landry's paralysis, renal failure and disturbances of skin, joints and intestines may also follow typhoid vaccinations. However, these latter complications are usually not lethal. The patients presented here had many of the conditions which are known to aggravate the situation and to lead to a lethal culmination. The review of this case and the disucussion following it shows that only healthy persons should receive the parenteral typhoid vaccination. Hopefully, the presentation of this material will help prevent fatalities of this type in the future.

In vitro characterization of the anti-PD-1 antibody nivolumab, BMS-936558, and in vivo toxicology in non-human primates.

The programmed death-1 (PD-1) receptor serves as an immunologic checkpoint, limiting bystander tissue damage and preventing the development of autoimmunity during inflammatory responses. PD-1 is expressed by activated T cells and downmodulates T-cell effector functions upon binding to its ligands, PD-L1 and PD-L2, on antigen-presenting cells. In patients with cancer, the expression of PD-1 on tumor-infiltrating lymphocytes and its interaction with the ligands on tumor and immune cells in the tumor microenvironment undermine antitumor immunity and support its rationale for PD-1 blockade in cancer immunotherapy. This report details the development and characterization of nivolumab, a fully human IgG4 (S228P) anti-PD-1 receptor-blocking monoclonal antibody. Nivolumab binds to PD-1 with high affinity and specificity, and effectively inhibits the interaction between PD-1 and its ligands. In vitro assays demonstrated the ability of nivolumab to potently enhance T-cell responses and cytokine production in the mixed lymphocyte reaction and superantigen or cytomegalovirus stimulation assays. No in vitro antibody-dependent cell-mediated or complement-dependent cytotoxicity was observed with the use of nivolumab and activated T cells as targets. Nivolumab treatment did not induce adverse immune-related events when given to cynomolgus macaques at high concentrations, independent of circulating anti-nivolumab antibodies where observed. These data provide a comprehensive preclinical characterization of nivolumab, for which antitumor activity and safety have been demonstrated in human clinical trials in various solid tumors.

CRISPR Interference Limits Horizontal Gene Transfer in Staphylococci by Targeting DNA

Horizontal gene transfer (HGT) in bacteria and archaea occurs through phage transduction, transformation, or conjugation, and the latter is particularly important for the spread of antibiotic resistance. Clustered, regularly interspaced, short palindromic repeat (CRISPR) loci confer sequence-directed immunity against phages. A clinical isolate of Staphylococcus epidermidis harbors a CRISPR spacer that matches the nickase gene present in nearly all staphylococcal conjugative plasmids. Here we show that CRISPR interference prevents conjugation and plasmid transformation in S. epidermidis. Insertion of a self-splicing intron into nickase blocks interference despite the reconstitution of the target sequence in the spliced mRNA, which indicates that the interference machinery targets DNA directly. We conclude that CRISPR loci counteract multiple routes of HGT and can limit the spread of antibiotic resistance in pathogenic bacteria.

Natural Killer Cells for Immunotherapy – Advantages of the NK-92 Cell Line over Blood NK Cells

Natural killer (NK) cells are potent cytotoxic effector cells for cancer therapy and potentially for severe viral infections. However, there are technical challenges to obtain sufficient numbers of functionally active NK cells from a patient's blood since they represent only 10% of the lymphocytes and are often dysfunctional. The alternative is to obtain cells from a healthy donor, which requires depletion of the allogeneic T cells to prevent graft-versus-host reactions. Cytotoxic cell lines have been established from patients with clonal NK-cell lymphoma. Those cells can be expanded in culture in the presence of IL-2. Except for the NK-92 cell line, though, none of the other six known NK cell lines has consistently and reproducibly shown high antitumor cytotoxicity. Only NK-92 cells can easily be genetically manipulated to recognize specific tumor antigens or to augment monoclonal antibody activity through antibody-dependent cellular cytotoxicity. NK-92 is also the only cell line product that has been infused into patients with advanced cancer with clinical benefit and minimal side effects.

Key questions about the checkpoint blockade-are microRNAs an answer?

The introduction of immune-checkpoint blockade in the cancer therapy led to a paradigm change of the management of late stage cancers. There are already multiple FDA approved checkpoint inhibitors and many other agents are undergoing phase 2 and early phase 3 clinical trials. The therapeutic indication of immune checkpoint inhibitors expanded in the last years, but still remains unclear who can benefit. MicroRNAs are small RNAs with no coding potential. By complementary pairing to the 3' untranslated region of messenger RNA, microRNAs exert posttranscriptional control of protein expression. A network of microRNAs directly and indirectly controls the expression of checkpoint receptors and several microRNAs can target multiple checkpoint molecules, mimicking the therapeutic effect of a combined immune checkpoint blockade. In this review, we will describe the microRNAs that control the expression of immune checkpoints and we will present four specific issues of the immune checkpoint therapy in cancer: (1) imprecise therapeutic indication, (2) difficult response evaluation, (3) numerous immunologic adverse-events, and (4) the absence of response to immune therapy. Finally, we propose microRNAs as possible solutions for these pitfalls. We consider that in the near future microRNAs could become important therapeutic partners of the immune checkpoint therapy.

The association between sterilizing activity and drug distribution into tuberculosis lesions

Finding new treatment-shortening antibiotics to improve cure rates and curb the alarming emergence of drug resistance is the major objective of tuberculosis (TB) drug development. Using a matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging suite in a biosafety containment facility, we show that the key sterilizing drugs rifampicin and pyrazinamide efficiently penetrate the sites of TB infection in lung lesions. Rifampicin even accumulates in necrotic caseum, a critical lesion site where persisting tubercle bacilli reside. In contrast, moxifloxacin, which is active in vitro against a subpopulation of Mycobacterium tuberculosis that persists in specific niches under drug pressure and has achieved treatment shortening in mice, does not diffuse well in caseum, concordant with its failure to shorten therapy in recent clinical trials. We suggest that such differential spatial distribution and kinetics of accumulation in lesions may create temporal and spatial windows of monotherapy in specific niches, allowing the gradual development of multidrug-resistant TB. We propose an alternative working model to prioritize new antibiotic regimens based on quantitative and spatial distribution of TB drugs in the major lesion types found in human lungs. The finding that lesion penetration may contribute to treatment outcome has wide implications for TB.

Costs and financial feasibility of malaria elimination

The marginal costs and benefits of converting malaria programmes from a control to an elimination goal are central to strategic decisions, but empirical evidence is scarce. We present a conceptual framework to assess the economics of elimination and analyse a central component of that framework-potential short-term to medium-term financial savings. After a review that showed a dearth of existing evidence, the net present value of elimination in five sites was calculated and compared with effective control. The probability that elimination would be cost-saving over 50 years ranged from 0% to 42%, with only one site achieving cost-savings in the base case. These findings show that financial savings should not be a primary rationale for elimination, but that elimination might still be a worthy investment if total benefits are sufficient to outweigh marginal costs. Robust research into these elimination benefits is urgently needed.

Determination of Malaria Epidemiological Status in Iran’s Malarious Areas as Baseline Information for Implementation of Malaria Elimination Program in Iran

BACKGROUND According to willingness of the Ministry of Health, Iran and presence of appropriate conditions for disease elimination, national malaria control program decided to conduct a research to clarify malaria status in 2007 and to provide required information to perform the elimination program. This review is comprised of the basis of national malaria elimination program in vision of 2025, which was started in 2010. METHODS In this descriptive study, data were analyzed by applications of different variables at district level. All districts in the three south eastern provinces, in which malaria has local transmission, were considered. Malaria cases has been determined and studied based on the national malaria surveillance system. RESULTS Since vivax malaria is predominant in Sistan & Baluchestan Province, number of vivax cases is equal to malaria positive cases approximately. The important point is that Nikshahr contains the maximum number of local vivax cases in this province and the maximum number of falciparum cases is reported from Sarbaz district. Among all districts of Hormozgan Province, no case of autochthonous falciparum was detected except in Bandar Jask and one case in Minab. There was no case of autochthonous falciparum in Kerman Province, except in Kahnoj and Ghale Ganj that each of them had one case in 2007. CONCLUSION It appears that the report of locally transmitted cases in Iran is increasing over the past few years, before starting malaria elimination plan. Since the Afghan refugees started to return to their own country so the main source of reporting of imported malaria cases reduced and local cases would be demonstrated more clearly.

Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophage colony-stimulating factor

Antigen-presenting, major histocompatibility complex (MHC) class II-rich dendritic cells are known to arise from bone marrow. However, marrow lacks mature dendritic cells, and substantial numbers of proliferating less-mature cells have yet to be identified. The methodology for inducing dendritic cell growth that was recently described for mouse blood now has been modified to MHC class II-negative precursors in marrow. A key step is to remove the majority of nonadherent, newly formed granulocytes by gentle washes during the first 2-4 d of culture. This leaves behind proliferating clusters that are loosely attached to a more firmly adherent "stroma. " At days 4-6 the clusters can be dislodged, isolated by 1-g sedimentation, and upon reculture, large numbers of dendritic cells are released. The latter are readily identified on the basis of their distinct cell shape, ultrastructure, and repertoire of antigens, as detected with a panel of monoclonal antibodies. The dendritic cells express high levels of MHC class II products and act as powerful accessory cells for initiating the mixed leukocyte reaction. Neither the clusters nor mature dendritic cells are generated if macrophage colony-stimulating factor rather than granulocyte/macrophage colony-stimulating factor (GM-CSF) is applied. Therefore, GM-CSF generates all three lineages of myeloid cells (granulocytes, macrophages, and dendritic cells). Since > 5 x 10(6) dendritic cells develop in 1 wk from precursors within the large hind limb bones of a single animal, marrow progenitors can act as a major source of dendritic cells. This feature should prove useful for future molecular and clinical studies of this otherwise trace cell type.

Disaster Management and Community Planning, and Public Participation: How to Achieve Sustainable Hazard Mitigation

The paper offers first a brief historical overview of disaster management planning. Second, it reviews Australian and American research findings and show that they urge the field of disaster management to shift its focus from response and recovery to sustainable hazard mitigation. It is argued that in order for this shift to occur, it is necessary to integrate disaster management and community planning. Current practice seldom reflects such a synthesis, and this is one of the reasons why hazard awareness is absent from local decision-making processes. Third, it is asserted that if mitigative strategies are to be successfully implemented, then the disaster management process must incorporate public participation at the local decision-making level. The paper concludes with a case study of California's Portola Valley, which demonstrates that when public participation is integrated into disaster management planning and community planning, the result is sustainable hazard mitigation.

An adenovirus mutant that replicates selectively in p53-deficient human tumor cells.

The human adenovirus E1B gene encodes a 55-kilodalton protein that inactivates the cellular tumor suppressor protein p53. Here it is shown that a mutant adenovirus that does not express this viral protein can replicate in and lyse p53-deficient human tumor cells but not cells with functional p53. Ectopic expression of the 55-kilodalton EIB protein in the latter cells rendered them sensitive to infection with the mutant virus. Injection of the mutant virus into p53-deficient human cervical carcinomas grown in nude mice caused a significant reduction in tumor size and caused complete regression of 60 percent of the tumors. These data raise the possibility that mutant adenoviruses can be used to treat certain human tumors.

Ubiquitinylation of the cytosolic domain of a type I membrane protein is not required to initiate its dislocation from the endoplasmic reticulum.

Human cytomegalovirus US2 and US11 target newly synthesized class I major histocompatibility complex (MHC) heavy chains for rapid degradation by the proteasome through a process termed dislocation. The presence of US2 induces the formation of class I MHC heavy chain conjugates of increased molecular weight that are recognized by a conformation-specific monoclonal antibody, W6/32, suggesting that these class I MHC molecules retain their proper tertiary structure. These conjugates are properly folded glycosylated heavy chains modified by attachment of an estimated one, two, and three ubiquitin molecules. The folded ubiquitinated class I MHC heavy chains are not observed in control cells or in cells transfected with US11, suggesting that US2 targets class I MHC heavy chains for dislocation in a manner distinct from that used by US11. This is further supported by the fact that US2 and US11 show different requirements in terms of the conformation of the heavy chain molecule. Although ubiquitin conjugation may occur on the cytosolic tail of the class I MHC molecule, replacement of lysines in the cytosolic tail of heavy chains with arginine does not prevent their degradation by US2. In an in vitro system that recapitulates US2-mediated dislocation, heavy chains that lack these lysines still occur in an ubiquitin-modified form, but in the soluble (cytoplasmic) fraction. Such ubiquitin conjugation can only occur on the class I MHC lumenal domain and is likely to take place once class I MHC heavy chains have been discharged from the endoplasmic reticulum. We conclude that ubiquitinylation of class I MHC heavy chain is not required during the initial step of the US2-mediated dislocation reaction.

Beyond pattern recognition: five immune checkpoints for scaling the microbial threat

Pattern recognition by the innate immune system enables the detection of microorganisms, but how the level of microbial threat is evaluated — a process that is crucial for eliciting measured antimicrobial responses with minimal inflammatory tissue damage — is less well understood. New evidence has shown that features of microbial viability can be detected by the immune system and thereby induce robust responses that are not warranted for dead microorganisms. Here, we propose five immune checkpoints that, as defined here, collectively determine the gravity of microbial encounters.

Molecular mechanisms underlying tumor dormancy.

Evidence suggests that dormant, microscopic tumors are not only common, but are highly prevalent in otherwise healthy individuals. Due to their small size and non-invasive nature, these dormant tumors remain asymptomatic and, in most cases, undetected. With advances in diagnostic imaging and molecular biology, it is now becoming clear that such neoplasms can remain in an asymptomatic, dormant stage for considerable periods of time without expanding in size. Although a number of processes may play a role in thwarting the expansion of microscopic tumors, one critical mechanism behind tumor dormancy is the ability of the tumor population to induce angiogenesis. Although cancer can arise through multiple pathways, it is assumed that essentially most tumors begin as microscopic, non-angiogenic neoplasms which cannot expand in size until vasculature is established. It is now becoming clear that cancer does not progress through a continuous exponential growth and mass expansion. Clinical cancer is usually manifested only in late, unavoidably symptomatic stages of the disease when tumors are sufficiently large to be readily detected. While dormancy in primary tumors is best defined as the time between the carcinogenic transformation event and the onset of inexorable progressive growth, it can also occur as minimal residual or occult disease from treated tumors or as micro-metastases. The existence of dormant tumors has important implications for the early detection and treatment of cancer. Elucidating the regulatory machinery of these processes will be instrumental in identifying novel early cancer biomarkers and could provide a rationale for the development of dormancy-promoting tumor therapies. Despite the high prevalence of microscopic, dormant tumors in humans and the significant clinical implications of their early detection, this area in cancer research has, to date, been under-investigated. In this mini review observations, models and experimental approaches to study tumor dormancy are summarized. Additionally, analogies and distinctions between the concepts of "tumor dormancy" and that of the "cellular dormancy" of tumor cells, as well as between the "exit from tumor dormancy" and the "onset of the angiogenic switch" are discussed.

In vivo electroporation in the embryonic mouse central nervous system

This protocol describes a basic method for in vivo electroporation in the nervous system of embryonic mice. Delivery of electric pulses following microinjection of DNA into the brain ventricle or the spinal cord central canal enables efficient transfection of genes into the nervous system. Transfection is facilitated by forceps-type electrodes, which hold the uterus and/or the yolk sac containing the embryo. More than ten embryos in a single pregnant mouse can be operated on within 30 min. More than 90% of operated embryos survive and more than 90% of these survivors express the transfected genes appropriately. Gene expression in neurons persists for a long time, even at postnatal stages, after electroporation. Thus, this method could be used to analyze roles of genes not only in embryonic development but also in higher order function of the nervous system, such as learning.

Th2-mediated host protective immunity to intestinal nematode infections.

Despite many years of study, relatively little is known about the effector mechanisms that operate against intestine-dwelling nematodes. Most of the current understanding comes from studies of laboratory model systems in rodents. It is clear that when an intestinal helminth infection takes place the immune system generates a strong Th2-mediated response, which regulates a variety of responses characteristic of helminth infections such as eosinophilia, intestinal mastocytosis and elevated IgE production. The ability to modulate the host's immune response in vivo with cytokine-specific monoclonal antibodies and recombinant cytokines, together with the use of animals with disruption of key genes involved in the immune response, have provided powerful tools with which to dissect the potential effector mechanisms operating. In the absence of a T-cell compartment the host is unable to expel the parasite. If a Th1-dominated response is generated, protective immunity is almost universally compromised. Thus, it it would appear that some aspect of a Th2-mediated response controls effector mechanisms. Although it is clear that for some infections the mast cell appears to be involved in protection, probably through the generation of a non-specific inflammatory response, how these cells become activated remains unclear. Data from infections in transgenic animals suggest that activation is not through the high-affinity receptor for IgE. Such studies also call into doubt the importance of conventional interactions between effector leucocytes and antibody. There is little evidence to support a protective role for eosinophilia in any system. New data also imply that, although interleukin 4 (IL-4) is generally important (and can exert effects independent of an adaptive immune response), it is not always sufficient to mediate protection; other Th2 cytokines (e.g. IL-13) may warrant closer investigation. It is apparent that a number of potential Th2-controlled effector mechanisms (some of which may be particularly important at mucosal surfaces) remain to be explored. Overall, it is likely that worm expulsion is the result of a combination of multiple mechanisms, some of which are more critical to some species of parasite than to others.

The Growth and Sporulation of the Benign and Malignant Tertian Malarial Parasites in the Culture Tube and in the Human Host

Researches on the cultivation of the parasites of malaria in Liverpool were commenced some time ago at my suggestion by Dr. Sinton, and then, with better success, by Drs. J. G. Thomson and McLellan, and by Dr. D. Thomson. We are greatly obliged to Sir Edwin Durning-Lawrence, Bart., for giving us the services of Dr. J. G. Thomson for this important enquiry.— Ronald Ross, 21st May, 1913.

Role of the Parasite-Derived Prostaglandin D2 in the Inhibition of Epidermal Langerhans Cell Migration during Schistosomiasis Infection

Epidermal Langerhans cells (LCs) play a key role in immune defense mechanisms and in numerous immunological disorders. In this report, we show that percutaneous infection of C57BL/6 mice with the helminth parasite Schistosoma mansoni leads to the activation of LCs but, surprisingly, to their retention in the epidermis. Moreover, using an experimental model of LC migration induced by tumor necrosis factor (TNF)-α, we show that parasites transiently impair the departure of LCs from the epidermis and their subsequent accumulation as dendritic cells in the draining lymph nodes. The inhibitory effect is mediated by soluble lipophilic factors released by the parasites and not by host-derived antiinflammatory cytokines, such as interleukin-10. We find that prostaglandin (PG)D2, but not the other major eicosanoids produced by the parasites, specifically impedes the TNF-α–triggered migration of LCs through the adenylate cyclase–coupled PGD2 receptor (DP receptor). Moreover, the potent DP receptor antagonist BW A868C restores LC migration in infected mice. Finally, in a model of contact allergen-induced LC migration, we show that activation of the DP receptor not only inhibits LC emigration but also dramatically reduces the contact hypersensitivity responses after challenge. Taken together, we propose that the inhibition of LC migration could represent an additional stratagem for the schistosomes to escape the host immune system and that PGD2 may play a key role in the control of cutaneous immune responses.

Activation of the NLRP3 inflammasome in dendritic cells induces IL-1β–dependent adaptive immunity against tumors

The therapeutic efficacy of anticancer chemotherapies may depend on dendritic cells (DCs), which present antigens from dying cancer cells to prime tumor-specific interferon-γ (IFN-γ)–producing T lymphocytes. Here we show that dying tumor cells release ATP, which then acts on P2X7 purinergic receptors from DCs and triggers the NOD-like receptor family, pyrin domain containing-3 protein (NLRP3)-dependent caspase-1 activation complex ('inflammasome'), allowing for the secretion of interleukin-1β (IL-1β). The priming of IFN-γ–producing CD8+ T cells by dying tumor cells fails in the absence of a functional IL-1 receptor 1 and in Nlpr3-deficient (Nlrp3−/−) or caspase-1–deficient (Casp-1−/−) mice unless exogenous IL-1β is provided. Accordingly, anticancer chemotherapy turned out to be inefficient against tumors established in purinergic receptor P2rx7−/− or Nlrp3−/− or Casp1−/− hosts. Anthracycline-treated individuals with breast cancer carrying a loss-of-function allele of P2RX7 developed metastatic disease more rapidly than individuals bearing the normal allele. These results indicate that the NLRP3 inflammasome links the innate and adaptive immune responses against dying tumor cells.

Epstein-Barr virus and virus human protein interaction maps.

A comprehensive mapping of interactions among Epstein-Barr virus (EBV) proteins and interactions of EBV proteins with human proteins should provide specific hypotheses and a broad perspective on EBV strategies for replication and persistence. Interactions of EBV proteins with each other and with human proteins were assessed by using a stringent high-throughput yeast two-hybrid system. Overall, 43 interactions between EBV proteins and 173 interactions between EBV and human proteins were identified. EBV-EBV and EBV-human protein interaction, or "interactome" maps provided a framework for hypotheses of protein function. For example, LF2, an EBV protein of unknown function interacted with the EBV immediate early R transactivator (Rta) and was found to inhibit Rta transactivation. From a broader perspective, EBV genes can be divided into two evolutionary classes, "core" genes, which are conserved across all herpesviruses and subfamily specific, or "noncore" genes. Our EBV-EBV interactome map is enriched for interactions among proteins in the same evolutionary class. Furthermore, human proteins targeted by EBV proteins were enriched for highly connected or "hub" proteins and for proteins with relatively short paths to all other proteins in the human interactome network. Targeting of hubs might be an efficient mechanism for EBV reorganization of cellular processes.

Interaction between broad-spectrum antibiotics and the combined oral contraceptive pill. A literature review.

There is considerable variation in opinion about the importance of drug interactions between the combined oral contraceptive pill (COCP) and broad-spectrum antibiotics. Clinical practice varies widely, especially between doctors in Europe and those in the US. Rifampicin and griseofulvin induce hepatic enzymes and do appear to have a genuine interaction with the COCP, leading to reduced efficacy. The situation with the broad-spectrum antibiotics is less clear. There are relatively few prospective studies of the pharmacokinetics of concurrent COCP and antibiotic use and few, if any, demonstrate a convincing basis for any reduced contraceptive efficacy. There is evidence, however, that variable contraceptive steroid handling could make some women, at some times, more susceptible to COCP failure. Given the serious consequences of unwanted pregnancy, the cautious approach of using additional or alternative contraception during short courses of broad-spectrum antibiotics and the initial weeks of long-term antibiotic administration may be justified to safeguard the few unidentifiable women who may be at risk. Conflicting opinion and advice is potentially confusing to both professionals and patients, and instructions for additional precautions during and after concurrent COCP and antibiotic use are complicated. Many women are ignorant of, or confused about, the circumstances that can cause OC to fail. Health professionals who prescribe the COCP must continue to strive to educate women about the mode of action and about the times when there is the greatest danger of failure. Professionals who feel that concurrent antibiotic use represents a real threat to contraceptive efficacy of the COCP should be prepared to present the advice for additional contraceptive precautions in a simple and consistent way, backed up with written information and reinforced at regular intervals.

MHC class I antigen presentation: learning from viral evasion strategies

The cell surface display of peptides by MHC class I molecules to lymphocytes provides the host with an important surveillance mechanism to protect against invading pathogens. However, in turn, viruses have evolved elegant strategies to inhibit various stages of the MHC class I antigen presentation pathway and prevent the display of viral peptides. This Review highlights how the elucidation of mechanisms of viral immune evasion is important for advancing our understanding of virus–host interactions and can further our knowledge of the MHC class I presentation pathway as well as other cellular pathways.

THE USE OF LEAD CITRATE AT HIGH pH AS AN ELECTRON-OPAQUE STAIN IN ELECTRON MICROSCOPY

Aqueous solutions of lead salts (1, 2) and saturated solutions of lead hydroxide (1) have been used as stains to enhance the electron-scattering properties of components of biological materials examined in the electron microscope. Saturated solutions of lead hydroxide (1), while staining more intensely than either lead acetate or monobasic lead acetate (l , 2), form insoluble lead carbonate upon exposure to air. The avoidance of such precipitates which contaminate surfaces of sections during staining has been the stimulus for the development of elaborate procedures for exclusion of air or carbon dioxide (3, 4). Several modifications of Watson's lead hydroxide stain (1) have recently appeared (5-7). All utilize relatively high pH (approximately 12) and one contains small amounts of tartrate (6), a relatively weak complexing agent (8), in addition to lead. These modified lead stains are less liable to contaminate the surface of the section with precipitated stain products. The stain reported here differs from previous alkaline lead stains in that the chelating agent, citrate, is in sufficient excess to sequester all lead present. Lead citrate, soluble in high concentrations in basic solutions, is a chelate compound with an apparent association constant (log Ka) between ligand and lead ion of 6.5 (9). Tissue binding sites, presumably organophosphates, and other anionic species present in biological components following fixation, dehydration, and plastic embedding apparently have a greater affinity for this cation than lead citrate inasmuch as cellular and extracellular structures in the section sequester lead from the staining solution. Alkaline lead citrate solutions are less likely to contaminate sections, as no precipitates form when droplets of fresh staining solution are exposed to air for periods of up to 30 minutes. The resultant staining of the sections is of high intensity in sections of Aralditeor Epon-embedded material. Cytoplasmic membranes, ribosomes, glycogen, and nuclear material are stained (Figs. 1 to 3). STAIN SOLUTION: Lead citrate is prepared by

Basics of PD-1 in self-tolerance, infection, and cancer immunity

Successful cancer treatment requires understanding host immune response against tumor cells. PD-1 belongs to the CD28 superfamily of receptors that work as “checkpoints” of immune activation. PD-1 maintains immune self-tolerance to prevent autoimmunity and controls T-cell reaction during infection to prevent excessive tissue damage. Tumor cells that arise from normal tissue acquire mutations that can be targeted by lymphocytes. Accumulating lines of evidence suggest that tumor cells evade host immune attack by expressing physiological PD-1 ligands and stimulating PD-1 on the lymphocytes. Based on this idea, researchers have successfully demonstrated that systemic administration of monoclonal antibodies that inhibit the binding of PD-1 to the ligands reactivated T cells and augmented the anti-cancer immune response. In this review, I summarize the basics of T-cell biology and its regulation by PD-1 and discuss the current understanding and questions about this multifaceted molecule.

A Viral microRNA Cluster Strongly Potentiates the Transforming Properties of a Human Herpesvirus

Epstein-Barr virus (EBV), an oncogenic human herpesvirus, induces cell proliferation after infection of resting B lymphocytes, its reservoir in vivo. The viral latent proteins are necessary for permanent B cell growth, but it is unknown whether they are sufficient. EBV was recently found to encode microRNAs (miRNAs) that are expressed in infected B cells and in some EBV-associated lymphomas. EBV miRNAs are grouped into two clusters located either adjacent to the BHRF1 gene or in introns contained within the viral BART transcripts. To understand the role of the BHRF1 miRNA cluster, we have constructed a virus mutant that lacks all its three members (Δ123) and a revertant virus. Here we show that the B cell transforming capacity of the Δ123 EBV mutant is reduced by more than 20-fold, relative to wild type or revertant viruses. B cells exposed to the knock-out virus displayed slower growth, and exhibited a two-fold reduction in the percentage of cells entering the cell cycle S phase. Furthermore, they displayed higher latent gene expression levels and latent protein production than their wild type counterparts. Therefore, the BHRF1 miRNAs accelerate B cell expansion at lower latent gene expression levels. Thus, this miRNA cluster simultaneously enhances expansion of the virus reservoir and reduces the viral antigenic load, two features that have the potential to facilitate persistence of the virus in the infected host. Thus, the EBV BHRF1 miRNAs may represent new therapeutic targets for the treatment of some EBV-associated lymphomas.

Measurement and clinical monitoring of human lymphocyte clonality by massively parallel VDJ pyrosequencing.

The complex repertoire of immune receptors generated by B and T cells enables recognition of diverse threats to the host organism. In this work, we show that massively parallel DNA sequencing of rearranged immune receptor loci can provide direct detection and tracking of immune diversity and expanded clonal lymphocyte populations in physiological and pathological contexts. DNA was isolated from blood and tissue samples, a series of redundant primers was used to amplify diverse DNA rearrangements, and the resulting mixtures of barcoded amplicons were sequenced using long-read ultra deep sequencing. Individual DNA molecules were then characterized on the basis of DNA segments that had been joined to make a functional (or nonfunctional) immune effector. Current experimental designs can accommodate up to 150 samples in a single sequence run, with the depth of sequencing sufficient to identify stable and dynamic aspects of the immune repertoire in both normal and diseased circumstances. These data provide a high-resolution picture of immune spectra in normal individuals and in patients with hematological malignancies, illuminating, in the latter case, both the initial behavior of clonal tumor populations and the later suppression or re-emergence of such populations after treatment.

Human Macrophages Infected with a High Burden of ESAT-6-Expressing M. tuberculosis Undergo Caspase-1- and Cathepsin B-Independent Necrosis

Mycobacterium tuberculosis (Mtb) infects lung macrophages, which instead of killing the pathogen can be manipulated by the bacilli, creating an environment suitable for intracellular replication and spread to adjacent cells. The role of host cell death during Mtb infection is debated because the bacilli have been shown to be both anti-apoptotic, keeping the host cell alive to avoid the antimicrobial effects of apoptosis, and pro-necrotic, killing the host macrophage to allow infection of neighboring cells. Since mycobacteria activate the NLRP3 inflammasome in macrophages, we investigated whether Mtb could induce one of the recently described inflammasome-linked cell death modes pyroptosis and pyronecrosis. These are mediated through caspase-1 and cathepsin-B, respectively. Human monocyte-derived macrophages were infected with virulent (H37Rv) Mtb at a multiplicity of infection (MOI) of 1 or 10. The higher MOI resulted in strongly enhanced release of IL-1β, while a low MOI gave no IL-1β response. The infected macrophages were collected and cell viability in terms of the integrity of DNA, mitochondria and the plasma membrane was determined. We found that infection with H37Rv at MOI 10, but not MOI 1, over two days led to extensive DNA fragmentation, loss of mitochondrial membrane potential, loss of plasma membrane integrity, and HMGB1 release. Although we observed plasma membrane permeabilization and IL-1β release from infected cells, the cell death induced by Mtb was not dependent on caspase-1 or cathepsin B. It was, however, dependent on mycobacterial expression of ESAT-6. We conclude that as virulent Mtb reaches a threshold number of bacilli inside the human macrophage, ESAT-6-dependent necrosis occurs, activating caspase-1 in the process.

Screening for inhibition of Vibrio cholerae VipA-VipB interaction identifies small-molecule compounds active against type VI secretion.

The type VI secretion system (T6SS) is the most prevalent bacterial secretion system and an important virulence mechanism utilized by Gram-negative bacteria, either to target eukaryotic cells or to combat other microbes. The components show much variability, but some appear essential for the function, and two homologues, denoted VipA and VipB in Vibrio cholerae, have been identified in all T6SSs described so far. Secretion is dependent on binding of an α-helical region of VipA to VipB, and in the absence of this binding, both components are degraded within minutes and secretion is ceased. The aim of the study was to investigate if this interaction could be blocked, and we hypothesized that such inhibition would lead to abrogation of T6S. A library of 9,600 small-molecule compounds was screened for their ability to block the binding of VipA-VipB in a bacterial two-hybrid system (B2H). After excluding compounds that showed cytotoxicity toward eukaryotic cells, that inhibited growth of Vibrio, or that inhibited an unrelated B2H interaction, 34 compounds were further investigated for effects on the T6SS-dependent secretion of hemolysin-coregulated protein (Hcp) or of phospholipase A1 activity. Two compounds, KS100 and KS200, showed intermediate or strong effects in both assays. Analogues were obtained, and compounds with potent inhibitory effects in the assays and desirable physicochemical properties as predicted by in silico analysis were identified. Since the compounds specifically target a virulence mechanism without affecting bacterial replication, they have the potential to mitigate the virulence with minimal risk for development of resistance.

Preventing bacterial DNA release and absent in melanoma 2 inflammasome activation by a Legionella effector functioning in membrane trafficking.

Legionella pneumophila, the causative agent of Legionnaires' pneumonia, resides in a distinct vacuole structure called Legionella-containing vacuole (LCV). The LCV resists fusion with the lysosome and permits efficient bacterial replication in host macrophages, which requires a Dot/Icm type IVB secretion system. Dot/Icm-translocated effector SdhA is critical for L. pneumophila intracellular growth and functions to prevent host cell death. Here, we show that the absence of SdhA resulted in elevated caspase-1 activation and IL-1β secretion as well as macrophage pyroptosis during Legionella infection. These inflammasome activation phenotypes were independent of the established flagellin-NAIP5-NLRC4 axis, but relied on the DNA-sensing AIM2 inflammasome. We further demonstrate that Legionella DNA was released into macrophage cytosol, and this effect was significantly exaggerated by the absence of SdhA. SdhA bears a functional Golgi-targeting GRIP domain that is required for preventing AIM2 inflammasome activation. Ectopically expressed SdhA formed a unique ring-shape membrane structure, further indicating a role in membrane trafficking and maintaining LCV membrane integrity. Our data together suggest a possible link, mediated by the function of SdhA, between LCV trafficking/maturation and suppression of host innate immune detection.

Subcellular steroid/nuclear receptor dynamics.

Steroid hormones, thyroid hormones, retinoic acids, and vitamin D bind to their receptors, which are now called steroid/nuclear receptors, and liganded receptors translocate either intracellularly or intranuclearly and form large protein complexes with cofactors to induce or repress gene transcription. Therefore, steroid/nuclear receptors are ligand-dependent transcription factors. With the advent of green fluorescent protein (GFP) and its color variants, the subcellular distribution of many steroid/nuclear receptors has been found to be much more dynamic than previously thought, with some of the receptors shuttling between the cytoplasm and nucleus. Steroid/nuclear receptors can be divided into three categories based on their unliganded distribution: those that are primarily in the nucleus, those in the cytoplasm, and those with mixed cytoplasmic and nuclear distributions. However, in all cases, the addition of a ligand leads to almost complete nuclear translocation of the receptors. Hormonal stimulation induces intranuclear receptor distribution from a homogeneous pattern to a heterogeneous dot-like image. Ligand binding to steroid/nuclear receptors leads to the recruitment of many proteins including cofactors to provoke the redistribution of receptor complexes in the nucleus. This focal organization could involve more complex events than simple DNA binding sites for transcription. Protein activities and interactions of steroid/nuclear receptors can be imaged and localized in a single cell.

Nrf2 protects human alveolar epithelial cells against injury induced by influenza A virus

BACKGROUND Influenza A virus (IAV) infection primarily targets respiratory epithelial cells and produces clinical outcomes ranging from mild upper respiratory infection to severe pneumonia. Recent studies have shown the importance of lung antioxidant defense systems against injury by IAV. Nuclear factor-erythroid 2 related factor 2 (Nrf2) activates the majority of antioxidant genes. METHODS Alveolar type II (ATII) cells and alveolar macrophages (AM) were isolated from human lungs not suitable for transplantation and donated for medical research. In some studies ATII cells were transdifferentiated to alveolar type I-like (ATI-like) cells. Alveolar epithelial cells were infected with A/PR/8/34 (PR8) virus. We analyzed PR8 virus production, influenza A nucleoprotein levels, ROS generation and expression of antiviral genes. Immunocytofluorescence was used to determine Nrf2 translocation and western blotting to detect Nrf2, HO-1 and caspase 1 and 3 cleavage. We also analyzed ingestion of PR8 virus infected apoptotic ATII cells by AM, cytokine levels by ELISA, glutathione levels, necrosis and apoptosis by TUNEL assay. Moreover, we determined the critical importance of Nrf2 using adenovirus Nrf2 (AdNrf2) or Nrf2 siRNA to overexpress or knockdown Nrf2, respectively. RESULTS We found that IAV induced oxidative stress, cytotoxicity and apoptosis in ATI-like and ATII cells. We also found that AM can ingest PR8 virus-induced apoptotic ATII cells (efferocytosis) but not viable cells, whereas ATII cells did not ingest these apoptotic cells. PR8 virus increased ROS production, Nrf2, HO-1, Mx1 and OAS1 expression and Nrf2 translocation to the nucleus. Nrf2 knockdown with siRNA sensitized ATI-like cells and ATII cells to injury induced by IAV and overexpression of Nrf2 with AdNrf2 protected these cells. Furthermore, Nrf2 overexpression followed by infection with PR8 virus decreased virus replication, influenza A nucleoprotein expression, antiviral response and oxidative stress. However, AdNrf2 did not increase IFN-λ1 (IL-29) levels. CONCLUSIONS Our results indicate that IAV induces alveolar epithelial injury and that Nrf2 protects these cells from the cytopathic effects of IAV likely by increasing the expression of antioxidant genes. Identifying the pathways involved in protecting cells from injury during influenza infection may be particularly important for developing new therapeutic strategies.

Prevention of malaria in long-term travelers.

CONTEXT Long-term travelers, defined here as those traveling for periods of 6 months or longer, face particular challenges regarding malaria prevention. Current guidelines for malaria prevention primarily address prevention of Plasmodium falciparum infections in short-term travelers. OBJECTIVES To examine the risk of malaria in long-term travelers, recent developments in personal protective measures, and the safety and tolerability of malaria chemoprophylaxis during long-term use and to consider prevention strategies including continuous chemoprophylaxis, stand-by emergency self-treatment, seasonal prophylaxis, and strategies to prevent primary infection and relapses from P vivax malaria. EVIDENCE ACQUISITION Comprehensive search of scientific publications including MEDLINE via both OVID and PubMED for relevant studies and articles with a cutoff date of July 2006, using the search terms long-term travel and malaria prevention, long-term malaria chemoprophylaxis, and insect repellent and malaria. Additional references were obtained from searching the bibliographies of the selected articles, from dissertations, and from the proceedings of relevant conferences on travel medicine. There were no language restrictions. EVIDENCE SYNTHESIS Long-term travelers have a higher risk of malaria than short-term travelers. Long-term travelers underuse personal protective measures and adhere poorly to continuous chemoprophylaxis regimens. A number of strategies are used during long-term stays: discontinuation of chemoprophylaxis after the initial period, sequential regimens with different medications for chemoprophylaxis, stand-by emergency self-treatment, and seasonal chemoprophylaxis targeting high-incidence periods or locations. All strategies have advantages and drawbacks. Counterfeit drugs sold in countries endemic for malaria pose serious concern for long-term travelers who purchase their medications overseas. Vivax malaria causes significant illness in travelers, but relapses of vivax malaria are not prevented with the current first-line chemoprophylaxis regimens. Consensus guidelines are needed for prevention of malaria in long-term travelers. CONCLUSIONS Prevention of malaria in long-term travelers is a complex issue and requires expert advice from travel medicine specialists. Recommendations for prevention of malaria in long-term travelers must be individualized.

Inhibition of the dipeptidyl peptidase DPP4 (CD26) reveals IL-33-dependent eosinophil-mediated control of tumor growth

Post-translational modification of chemokines mediated by the dipeptidyl peptidase DPP4 (CD26) has been shown to negatively regulate lymphocyte trafficking, and its inhibition enhances T cell migration and tumor immunity by preserving functional chemokine CXCL10. By extending those initial findings to pre-clinical models of hepatocellular carcinoma and breast cancer, we discovered a distinct mechanism by which inhibition of DPP4 improves anti-tumor responses. Administration of the DPP4 inhibitor sitagliptin resulted in higher concentrations of the chemokine CCL11 and increased migration of eosinophils into solid tumors. Enhanced tumor control was preserved in mice lacking lymphocytes and was ablated after depletion of eosinophils or treatment with degranulation inhibitors. We further demonstrated that tumor-cell expression of the alarmin IL-33 was necessary and sufficient for eosinophil-mediated anti-tumor responses and that this mechanism contributed to the efficacy of checkpoint-inhibitor therapy. These findings provide insight into IL-33- and eosinophil-mediated tumor control, revealed when endogenous mechanisms of DPP4 immunoregulation are inhibited. Eosinophils have been described mainly in allergy settings but are increasingly appreciated as being involved in other aspects of immunity. Albert and colleagues use a clinically approved inhibitor of the dipeptidyl peptidase DPP4 to facilitate the recruitment of eosinophils to mouse tumors, where they are essential in tumor destruction.

Isolation of intact nuclei of high purity from mouse liver.

Current methods of nuclear isolation from liver disrupt the plasmalemmae via homogenization and separation of the nuclei by high centrifugal force (HCF) through gradients of sucrose or other substances for up to 80 min. The use of HCF for such a long time increases the potential for nuclear damage and degradation by endogenous proteases. We compared four combinations of alterations to classical nuclear isolation methods as follows. Mouse liver was gently crushed through a fine mesh with and without in vivo perfusion with collagenase. The cell suspension was centrifuged at 600 g to remove gross debris and then at moderate centrifugal force (MCF, 16,000 g) or high centrifugal force (HCF, 70,000 g) through sucrose gradients for 30 min. The purity of the isolated nuclei was assessed biologically and morphologically, including analyses of representative marker proteins for nuclei and cytoplasm. The results indicate that MCF and no collagenase provided the highest nuclear integrity and purity, whereas MCF with collagenase is a viable option if priority is given to yield. The method is especially suited for small samples and so should facilitate studies with human liver biopsies and livers from mice, the most widely used species for gene targeting.

Fusion proteins for versatile antigen targeting to cell surface receptors reveal differential capacity to prime immune responses.

Targeting of proteins to APCs is an attractive strategy for eliciting adaptive immune responses. However, the relationship between the choice of the targeted receptor and the quality and quantity of responses remains poorly understood. We describe a strategy for expression of Ags including hydrophobic proteins as soluble fusion proteins that are optimized for proteasome-dependent MHC class I-restricted cross-presentation and form stable complexes with a wide variety of targeting Abs. Upon s.c. immunization, these complexes were initially taken up by CD169+ lymph node subcapsular sinus macrophages. In the OVA model system, receptor-targeted antigenic complexes primed specific T and B cell responses in vitro and in vivo at least 100-fold more efficiently than Ag alone. Comparison of 10 targeting receptors allowed us to establish a ranking with respect to priming of CD8+ T cell responses and demonstrated striking differences with respect to the relative efficacy of CD8+ and CD4+ T cell subset and B cell priming. The described fusion proteins should help in developing optimized strategies for targeted delivery of protein Ags in the context of tolerization or vaccination.

Elimination of HIV in South Africa through Expanded Access to Antiretroviral Therapy: A Model Comparison Study

BACKGROUND Expanded access to antiretroviral therapy (ART) using universal test and treat (UTT) has been suggested as a strategy to eliminate HIV in South Africa within 7 y based on an influential mathematical modeling study. However, the underlying deterministic model was criticized widely, and other modeling studies did not always confirm the study's finding. The objective of our study is to better understand the implications of different model structures and assumptions, so as to arrive at the best possible predictions of the long-term impact of UTT and the possibility of elimination of HIV. METHODS AND FINDINGS We developed nine structurally different mathematical models of the South African HIV epidemic in a stepwise approach of increasing complexity and realism. The simplest model resembles the initial deterministic model, while the most comprehensive model is the stochastic microsimulation model STDSIM, which includes sexual networks and HIV stages with different degrees of infectiousness. We defined UTT as annual screening and immediate ART for all HIV-infected adults, starting at 13% in January 2012 and scaled up to 90% coverage by January 2019. All models predict elimination, yet those that capture more processes underlying the HIV transmission dynamics predict elimination at a later point in time, after 20 to 25 y. Importantly, the most comprehensive model predicts that the current strategy of ART at CD4 count ≤350 cells/µl will also lead to elimination, albeit 10 y later compared to UTT. Still, UTT remains cost-effective, as many additional life-years would be saved. The study's major limitations are that elimination was defined as incidence below 1/1,000 person-years rather than 0% prevalence, and drug resistance was not modeled. CONCLUSIONS Our results confirm previous predictions that the HIV epidemic in South Africa can be eliminated through universal testing and immediate treatment at 90% coverage. However, more realistic models show that elimination is likely to occur at a much later point in time than the initial model suggested. Also, UTT is a cost-effective intervention, but less cost-effective than previously predicted because the current South African ART treatment policy alone could already drive HIV into elimination. Please see later in the article for the Editors' Summary.

Staphylococcus aureus infection dynamics

Staphylococcus aureus is a human commensal that can also cause systemic infections. This transition requires evasion of the immune response and the ability to exploit different niches within the host. However, the disease mechanisms and the dominant immune mediators against infection are poorly understood. Previously it has been shown that the infecting S. aureus population goes through a population bottleneck, from which very few bacteria escape to establish the abscesses that are characteristic of many infections. Here we examine the host factors underlying the population bottleneck and subsequent clonal expansion in S. aureus infection models, to identify underpinning principles of infection. The bottleneck is a common feature between models and is independent of S. aureus strain. Interestingly, the high doses of S. aureus required for the widely used "survival" model results in a reduced population bottleneck, suggesting that host defences have been simply overloaded. This brings into question the applicability of the survival model. Depletion of immune mediators revealed key breakpoints and the dynamics of systemic infection. Loss of macrophages, including the liver Kupffer cells, led to increased sensitivity to infection as expected but also loss of the population bottleneck and the spread to other organs still occurred. Conversely, neutrophil depletion led to greater susceptibility to disease but with a concomitant maintenance of the bottleneck and lack of systemic spread. We also used a novel microscopy approach to examine abscess architecture and distribution within organs. From these observations we developed a conceptual model for S. aureus disease from initial infection to mature abscess. This work highlights the need to understand the complexities of the infectious process to be able to assign functions for host and bacterial components, and why S. aureus disease requires a seemingly high infectious dose and how interventions such as a vaccine may be more rationally developed.

Using the yeast gene deletion collection to customize gene expression.

Substitute Teacher Despite the relative ease of genome manipulation in S. cerevisiae, researchers are always looking to learn still more convenient and rapid methods for substituting yeast promoters. Replacing a gene's native promoter with a heterolo-gous promoter of choice allows regulated expression and simplifies the task of discerning functional relevance. Although a host of clever chromosomal insertion strategies have been described over the years, the advent of the S. cerevisiae Genome Deletion Project provides an incredible resource for a further streamlined workflow. The strategy, explained by Liko et al. on p. 728 is appealingly simple. The genome deletion project resulted in a collection of strains in which a single ORF is replaced with a kanamycin resistance module. Although the purpose of the collection is to have a comprehensive resource of essentially all possible knockouts, the authors point out that for almost any given yeast promoter of interest there will be a strain in which the ORF imm...

Preexisting helminth infection induces inhibition of innate pulmonary anti-tuberculosis defense by engaging the IL-4 receptor pathway

Tuberculosis and helminthic infections coexist in many parts of the world, yet the impact of helminth-elicited Th2 responses on the ability of the host to control Mycobacterium tuberculosis (Mtb) infection has not been fully explored. We show that mice infected with the intestinal helminth Nippostrongylus brasiliensis (Nb) exhibit a transitory impairment of resistance to airborne Mtb infection. Furthermore, a second dose of Nb infection substantially increases the bacterial burden in the lungs of co-infected mice. Interestingly, the Th2 response in the co-infected animals did not impair the onset and development of the protective Mtb-specific Th1 cellular immune responses. However, the helminth-induced Th2 environment resulted in the accumulation of alternatively activated macrophages (AAMs) in the lung. Co-infected mice lacking interleukin (IL) 4Rα exhibited improved ability to control Mtb infection, which was accompanied by significantly reduced accumulation of AAMs. Moreover, IL-4Rα(-/-) mice adoptively transferred with wild-type macrophages had a significantly higher Mtb load in their lungs compared with those that received IL-4Rα(-/-) macrophages, suggesting a direct contribution for the IL-4R pathway to the heightened susceptibility of co-infected animals. The Th2 response can thus enhance the intracellular persistence of Mtb, in part by mediating the alternative activation of macrophages via the IL-4Rα signaling pathway.

Chimeric live, attenuated vaccine against Japanese encephalitis (ChimeriVax-JE): phase 2 clinical trials for safety and immunogenicity, effect of vaccine dose and schedule, and memory response to challenge with inactivated Japanese encephalitis antigen.

ChimeriVax-JE is a live, attenuated vaccine against Japanese encephalitis, using yellow fever (YF) 17D vaccine as a vector. In a double-blind phase 2 trial, 99 adults received vaccine, placebo, or YF 17D vaccine (YF-VAX). ChimeriVax-JE was well tolerated, with no differences in adverse events between treatment groups. Viremias resulting from administration of ChimeriVax-JE and YF-VAX were of short duration and low titer; 82 (94%) of 87 subjects administered graded doses (1.8-5.8 log(10)) of ChimeriVax-JE developed neutralizing antibodies. A second dose, administered 30 days later, had no booster effect. Previous inoculation with YF did not interfere with ChimeriVax-JE, but there was a suggestion (not statistically significant) that ChimeriVax-JE interfered with YF-VAX administered 30 days later. A separate study explored immunological memory both in subjects who had received ChimeriVax-JE 9 months before and in ChimeriVax-JE-naive subjects challenged with inactivated mouse-brain vaccine (JE-VAX). Anamnestic responses were observed in preimmune individuals. ChimeriVax-JE appears to be a safe vaccine that provides protective levels of neutralizing antibody after a single dose.

Immune complex relay by subcapsular sinus macrophages and non-cognate B cells drives antibody affinity maturation

Subcapsular sinus (SCS) macrophages capture antigens from lymph and present them intact for B cell encounter and follicular delivery. However, the properties of SCS macrophages are poorly defined. Here we show SCS macrophage development depended on lymphotoxin-alpha1beta2, and the cells had low lysosomal enzyme expression and retained opsonized antigens on their surface. Intravital imaging revealed immune complexes moving along macrophage processes into the follicle. Moreover, noncognate B cells relayed antigen opsonized by newly produced antibodies from the subcapsular region to the germinal center, and affinity maturation was impaired when this transport process was disrupted. Thus, we characterize SCS macrophages as specialized antigen-presenting cells functioning at the apex of an antigen transport chain that promotes humoral immunity.

A local macrophage chemokine network sustains protective tissue-resident memory CD4 T cells

CD8 tissue-resident memory T (TRM) cells provide efficient local control of viral infection, but the role of CD4 TRM is less clear. Here, by using parabiotic mice, we show that a preexisting pool of CD4 TRM cells in the genital mucosa was required for full protection from a lethal herpes simplex virus 2 (HSV-2) infection. Chemokines secreted by a local network of macrophages maintained vaginal CD4 TRM in memory lymphocyte clusters (MLCs), independently of circulating memory T cells. CD4 TRM cells within the MLCs were enriched in clones that expanded in response to HSV-2. Our results highlight the need for vaccine strategies that enable establishment of TRM cells for protection from a sexually transmitted virus and provide insights as to how such a pool might be established.

Kinetics of early T cell receptor signaling regulate the pathway of lytic granule delivery to the secretory domain.

Cytolytic granules mediate killing of virus-infected cells by cytotoxic T lymphocytes. We show here that the granules can take long or short paths to the secretory domain. Both paths utilized the same intracellular molecular events, which have different spatial and temporal arrangements and are regulated by the kinetics of Ca(2+)-mediated signaling. Rapid signaling caused swift granule concentration near the microtubule-organizing center (MTOC) and subsequent delivery by the polarized MTOC directly to the secretory domain-the shortest path. Indolent signaling led to late recruitment of granules that moved along microtubules to the periphery of the synapse and then moved tangentially to fuse at the outer edge of the secretory domain-a longer path. The short pathway is associated with faster granule release and more efficient killing than the long pathway. Thus, the kinetics of early signaling regulates the quality of the T cell cytolytic response.

Effects of repeated social stress on leukocyte distribution in bone marrow, peripheral blood and spleen

Leukocyte trafficking between the various body compartments has an important surveillance function that ensures the detection of antigen and enables the immune system to initiate a rapid and effective response. Repeated social defeat of group-housed male mice induced by daily, acute encounters with an aggressive conspecific substantially altered leukocyte trafficking and led to a gradual redistribution of immune cells in bone marrow, peripheral blood and spleen. Recurrent exposure to the stressor over a period of 2, 4 or 6 consecutive days was associated with cell mobilization and increased myelopoiesis in the bone marrow that was paralleled by an accumulation of neutrophils and monocytes in circulation and spleen. Substantial depletion of B cells in bone marrow and blood was associated with an increase in splenic B cells indicating a redirection of this cell type to the spleen. In contrast, T cells were markedly reduced in these immune compartments. The recruitment of CD11b+ leukocytes (i.e., monocytes/macrophages and neutrophils) from the bone marrow to the spleen might play a critical role in the development of functional glucocorticoid resistance in the murine spleen that was reported in context with repeated social defeat.

Treatment with cyclooxygenase-2 inhibitors enables repeated administration of vaccinia virus for control of ovarian cancer.

Metastatic ovarian cancer is the leading cause of death among women with gynecologic malignancies in the United States. The lack of effective treatment for patients with advanced ovarian cancer warrants development of innovative therapies. Cancer therapy using oncolytic viruses represents a promising new approach for controlling tumors. Vaccinia virus has been shown to preferentially infect tumor cells but not normal tissue. However, oncolytic therapy using recombinant viruses faces the limitation of viral clearance due to generation of neutralizing antibodies. In the current study, we found that cyclooxygenase-2 (Cox-2) inhibitors circumvented this limitation, enabling repeated administration of vaccinia virus without losing infectivity. We quantified the antivaccinia antibody response using enzyme-linked immunosorbent assay (ELISA) and neutralization assays to show that treatment of Cox-2 inhibitors inhibited the generation of neutralizing antibodies. Furthermore, we showed that combination treatment of Cox-2 inhibitors with vaccinia virus was more effective that either treatment alone in treating MOSEC/luc tumor-bearing mice. Thus, the combination of Cox-2 inhibitors and vaccinia virus represents a potential innovative approach to controlling ovarian tumors.

A novel viral transcript with homology to human interleukin-10 is expressed during latent human cytomegalovirus infection.

Human cytomegalovirus (CMV) establishes latent infections in hematopoietic cells such as granulocyte-macrophage progenitors (GM-Ps). During latency the virus is sequestered in a nonreplicating state, although limited transcriptional activity has been previously reported. In this study we sought to further examine viral gene expression during the latent phase of infection. Using an experimental model of latency, primary human GM-Ps were latently infected with CMV strain Toledo and extracted RNA subjected to reverse transcription-PCR by using CMV gene-specific primers. Using this approach, we detected transcription from the UL111.5A region of the viral genome. This transcription was also detected in GM-Ps latently infected with AD169 and Towne strains, indicating that expression was CMV strain independent. Significantly, we detected UL111.5A-region transcripts in mononuclear cells from healthy bone marrow and mobilized peripheral blood allograft donors, demonstrating expression during natural latent infection. Mapping experiments with RNA extracted from latently infected GM-Ps revealed the expression of a novel UL111.5A region transcript with a splicing pattern that differed from that reported during productive infection of permissive cells. This UL111.5A region transcript expressed during latent infection is predicted to encode a 139-amino-acid protein with homology to the potent immunosuppressor interleukin-10 (IL-10) and to the viral IL-10 homolog that is expressed during productive CMV infection. Expression of a latency-associated cmvIL-10 may confer upon the virus an ability to avoid immune recognition and clearance during the latent phase of infection.

Preparing the outbreak assistance laboratory network in the Netherlands for the detection of the influenza virus A(H1N1) variant.

BACKGROUND Late April 2009, human infection with variant influenza virus A(H1N1)v emerged in the Northern Americas posing a threat that this virus may become the next pandemic influenza virus. OBJECTIVES To prepare laboratories for surge capacity for molecular diagnosis of patients suspected for A(H1N1)v infection in the Netherlands. STUDY DESIGN A panel of 10 blinded specimens containing seasonal A(H1N1) or A(H3N2), or A/Netherlands/602/2009(H1N1)v influenza virus, or negative control was distributed to the outbreak assistance laboratories (OAL) together with influenza virus A (M-gene), swine influenza virus A (NP-gene) and influenza virus A(H1N1)v (H1v-gene) specific primers and probes and protocol (CDC Atlanta, USA). Laboratories were asked to implement and test this protocol. RESULTS All OAL were able to detect A(H1N1)v using the CDC M-gene reagents, the majority with similar sensitivity as the in-house M-gene based assays. RT-PCRs used in routine diagnostic setting in the OAL specifically designed to detect H1, H3, or NS1 from seasonal influenza A viruses, did not or at very low level cross-react with A(H1N1)v. The CDC swine NP-gene and H1v-gene RT-PCRs showed somewhat reduced sensitivity compared to the CDC and in-house M-gene RT-PCRs. In contrast, in-house developed A(H1N1)v specific H1v-gene and N1v-gene RT-PCRs showed equal sensitivity to CDC and in-house M-gene RT-PCRs. CONCLUSIONS The Dutch OAL are prepared for detection and specific identification of A(H1N1)v, although some level of cross-reactivity was observed with seasonal influenza viruses. Additionally, M-gene based generic influenza A virus detection is recommended to be able to detect emerging influenza A viruses in routine settings.

Isolation and partial characterization of an unusual human immunodeficiency retrovirus from two persons of west-central African origin.

An unusual human retrovirus was isolated from two patients with persistent generalized lymphadenopathy who originate from West-Central Africa and are currently residing in Belgium. Although the virus shared a number of the same biological and morphological properties as human immunodeficiency retrovirus type 1 (HIV-1) and HIV-2, significant antigenic differences could be demonstrated. Several of the viral proteins also differed in molecular weight from the corresponding HIV-1 and HIV-2 proteins. Partial chemical cleavage of the most highly conserved viral proteins resulted in patterns which differed from those of HIV-1 and HIV-2. Furthermore, nucleic acid hybridization experiments were capable of discriminating between the virus types. Sequence analysis of the viral U3 region revealed a unique enhancer organization not found in other immunodeficiency viruses. The data indicated that the new isolate is more closely related to HIV-1 than to HIV-2 but clearly differs in a number of important respects.