Infection of human T-cell lymphotropic virus type 1 is most frequent in individuals of African origin.

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.

HTLV-I/II associated disease in England and Wales, 1993-7: retrospective review of serology requests.

Apart from HIV two exogenous retroviruses (human T cell leukaemia viruses type I (HTLV-I) and type II (HTLV-II)) infect humans. HTLV-I infection is endemic in Japan, the Caribbean, Africa, and Melanesia and is found among immigrants from these regions in Europe. HTLV-I infection is associated with a 1-5% lifetime risk of adult T cell leukaemia/lymphoma, 1 a 0.25% lifetime risk of HTLV-I associated myelopathy, 2 and other inflammatory conditions (uveitis, alveolitis, and arthritis).1 HTLV-II infection is endemic in some native American and African peoples and among injecting drug users and has been associated with neurological disease.1 Between 1986 and 1992, 100 cases of HTLV-I associated myelopathy and 44 cases of adult T cell leukaemia/lymphoma were diagnosed in the United Kingdom.3 Adult T cell leukaemia/lymphoma was first described in 1977 and patients with it have a mean life expectancy of only six months, so most of the 44 cases were probably incident cases. …

Lymph node T cell responses predict the efficacy of live attenuated SIV vaccines

Live attenuated simian immunodeficiency virus (SIV) vaccines (LAVs) remain the most efficacious of all vaccines in nonhuman primate models of HIV and AIDS, yet the basis of their robust protection remains poorly understood. Here we show that the degree of LAV-mediated protection against intravenous wild-type SIVmac239 challenge strongly correlates with the magnitude and function of SIV-specific, effector-differentiated T cells in the lymph node but not with the responses of such T cells in the blood or with other cellular, humoral and innate immune parameters. We found that maintenance of protective T cell responses is associated with persistent LAV replication in the lymph node, which occurs almost exclusively in follicular helper T cells. Thus, effective LAVs maintain lymphoid tissue-based, effector-differentiated, SIV-specific T cells that intercept and suppress early wild-type SIV amplification and, if present in sufficient frequencies, can completely control and perhaps clear infection, an observation that provides a rationale for the development of safe, persistent vectors that can elicit and maintain such responses.

The role of the immune response during SIVagm infection of the African green monkey natural host.

The African green monkey (AGM) is one of many African species endemically infected with simian immunodeficiency virus (SIV). Like the other natural hosts, AGMs do not succumb to AIDS and understanding the basis for this resistance to disease progression would be of enormous theoretical and practical importance. Early efforts by our group that concentrated on identifying immune mechanisms presumed to keep the virus under control failed to find any obvious candidates. The presumption of virus control was invalidated by the finding that SIVagm replicates in AGMs with the same vigor as HIV-1 does in humans. Focus therefore shifted to identifying possible immunopathologic features present in disease susceptible hosts but absent in the AGM natural host. The apparent immunologic tolerance of AGMs to the SIVagm core protein led to the development of a hypothesis implicating anti-Gag antibodies in the formation of immune complexes, virus trapping in the lymph nodes and immune dysfunction. The idea proved difficult to test in vivo and present work focuses on the possibility that Gag tolerance at the T-cell level plays an important role in preventing the catastrophic demise of the immune system characteristic of immunodeficiency virus infection of the heterologous primate host.

Autoimmunity due to molecular mimicry as a cause of neurological disease

One hypothesis that couples infection with autoimmune disease is molecular mimicry. Molecular mimicry is characterized by an immune response to an environmental agent that cross-reacts with a host antigen, resulting in disease. This hypothesis has been implicated in the pathogenesis of diabetes, lupus and multiple sclerosis (MS). There is limited direct evidence linking causative agents with pathogenic immune reactions in these diseases. Our study establishes a clear link between viral infection, autoimmunity and neurological disease in humans. As a model for molecular mimicry, we studied patients with human T-lymphotropic virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP), a disease that can be indistinguishable from MS (refs. 5,6,7). HAM/TSP patients develop antibodies to neurons. We hypothesized these antibodies would identify a central nervous system (CNS) autoantigen. Immunoglobulin G isolated from HAM/TSP patients identified heterogeneous nuclear ribonuclear protein-A1 (hnRNP-A1) as the autoantigen. Antibodies to hnRNP-A1 cross-reacted with HTLV-1-tax, the immune response to which is associated with HAM/TSP (refs. 5,9). Immunoglobulin G specifically stained human Betz cells, whose axons are preferentially damaged. Infusion of autoantibodies in brain sections inhibited neuronal firing, indicative of their pathogenic nature. These data demonstrate the importance of molecular mimicry between an infecting agent and hnRNP-A1 in autoimmune disease of the CNS.

Isolation from African Sykes' monkeys (Cercopithecus mitis) of a lentivirus related to human and simian immunodeficiency viruses.

Analysis of serum samples from 100 wild-caught or colony-born Sykes' monkeys (Cercopithecus mitis) in Kenya revealed that 59 animals had antibodies cross-reactive to human immunodeficiency virus type 2 (HIV-2) and to simian immunodeficiency viruses (SIVs). A lentivirus, designated SIVsyk, was isolated from five of six seropositive asymptomatic Sykes' monkeys, but in four cases isolation was possible only after depletion of CD8+ lymphocytes and cocultivation of the CD4(+)-enriched cell population with peripheral blood mononuclear cells from seronegative Sykes' monkeys. SIVsyk resembled other SIVs and HIVs morphologically, had an Mg2(+)-dependent reverse transcriptase enzyme, and replicated in and was cytopathic for CEMx174 and Sup-T1 cells. SIVsyk differred substantially from other SIVs, however, in that it failed to replicate in normal human, mangabey, and macaque peripheral blood mononuclear cells and serum from seropositive Sykes' monkeys immunoprecipitated env antigens from HIV-1 as well as from HIV-2, SIVsmm, and SIVagm. These data demonstrate a high prevalence of natural infection in Sykes' monkeys in Kenya with a lentivirus that appears to be unique with respect to its host range and antigenic cross-reactivity.

Kaposi's sarcoma-associated herpesvirus/human herpesvirus type 8-positive solid lymphomas: a tissue-based variant of primary effusion lymphoma.

Kaposi's sarcoma-associated herpesvirus (KSHV), also termed human herpesvirus type 8, is consistently identified in Kaposi's sarcoma, primary effusion lymphoma (PEL), and multicentric Castleman's disease. Here we report four cases of KSHV-bearing solid lymphomas that occurred in AIDS patients (cases 1 to 3) and in a human immunodeficiency virus (HIV)-seronegative person (case 4). The patients presented extranodal masses in the abdomen (cases 1, 3, and 4) or skin (case 2), and nodal involvement, together with Kaposi's sarcoma (case 3). The gastrointestinal tract was involved in two patients (cases 1 and 3). The patients did not develop a lymphomatous effusion. KSHV was detected in the tumor cells of all cases by immunohistochemistry and by polymerase chain reaction. Epstein-Barr virus was detected in two of the HIV-related cases. All KSHV-positive solid lymphomas exhibited PEL-like cell morphology. To investigate the relationship of these disorders to PEL and to other AIDS-associated diffuse large cell lymphomas, KSHV-positive solid lymphomas were tested for the expression of a set of genes that were previously shown by gene profiling analysis to define PEL tumor cells. The results showed that expression of this set of genes in KSHV-positive lymphomas is similar to that of PEL but distinct from KSHV-negative AIDS-associated diffuse large cell lymphomas. Because pathobiological features of KSHV-positive solid lymphomas closely mimic those of PEL, our results suggest that KSHV-positive solid lymphomas should be considered as a tissue-based variant of classical PEL, irrespective of HIV status.

Antibody-Based HIV-1 Vaccines: Recent Developments and Future Directions

The Global HIV Vaccine Enterprise convened a two-day workshop in May of 2007 to discuss humoral immune responses to HIV and approaches to design vaccines that induce viral neutralizing and other potentially protective antibody responses. The goals of this workshop were to identify key scientific issues, gaps, and opportunities that have emerged since the Enterprise Strategic Plan was first published in 2005 [1], and to make recommendations that Enterprise stakeholders can use to plan new activities. Most effective viral vaccines work, at least in part, by generating antibodies that inactivate or neutralize the invading virus, and the existing data strongly suggest that an optimally effective HIV-1 vaccine should elicit potent antiviral neutralizing antibodies. However, unlike acute viral pathogens, HIV-1 chronically replicates in the host and evades the antibody response. This immune evasion, along with the large genetic variation among HIV-1 strains worldwide, has posed major obstacles to vaccine development. Current HIV vaccine candidates do not elicit neutralizing antibodies against most circulating virus strains, and thus the induction of a protective antibody response remains a major priority for HIV-1 vaccine development. For an antibody-based HIV-1 vaccine, progress in vaccine design is generally gauged by in vitro assays that measure the ability of vaccine-induced antibodies to neutralize a broad spectrum of viral isolates representing the major genetic subtypes (clades) of HIV-1 [2]. Although it is not known what magnitude and breadth of neutralization will predict protection in vaccine recipients, it is clear that current vaccine immunogens elicit antibodies that neutralize only a minority of circulating isolates. Thus, much progress needs to be made in this area. Also, though virus neutralization is considered a critical benchmark for a vaccine, this may not be the only benchmark for predicting success with antibody-based HIV-1 vaccine immunogens. The main targets for neutralizing antibodies to HIV-1 are the surface gp120 and trans-membrane gp41 envelope glycoproteins (Env) that mediate receptor and coreceptor binding and the subsequent membrane fusion events that allow the virus to gain entry into cells [3]. Antibodies neutralize the virus by binding these viral spikes and blocking virus entry into susceptible cells, such as CD4+ T cells [4,5]. In order to chronically replicate in the host, the virus exploits several mechanisms to shield itself against antibody recognition, including a dense outer coating of sugar molecules (N-linked glycans) and the strategic positioning of cysteine–cysteine loop structures on the gp120 molecule [6–8]. These shielding mechanisms, although highly effective, have vulnerabilities imposed by fitness constraints. Information on the precise location and molecular structure of these vulnerable regions could be valuable for the rational design of improved vaccine immunogens. Participants in the workshop identified four areas that, if given proper attention, could provide key information that would bring the field closer to an effective antibody-based HIV-1 vaccine: (1) structure-assisted immunogen design, (2) role of Fc receptors and complement, (3) assay standardization and validation, and (4) immunoregulation of B cell responses.

“Rapid-Impact Interventions”: How a Policy of Integrated Control for Africa's Neglected Tropical Diseases Could Benefit the Poor

Over the past two decades there have been significant achievements in the control of a handful of important human tropical infections [1]. These achievements include the substantive reductions in the prevalence and incidence of the so-called neglected diseases such as lymphatic filariasis, onchocerciasis, guinea worm, leprosy, and trachoma (Box 1) [2]. Each of these neglected diseases is a poverty-promoting and often stigmatizing condition occurring primarily in rural areas of low-income countries (Box 2) [3]. They are ancient afflictions, described in the Bible and other ancient texts, which have burdened humanity for millennia [3]. But now, as a result of aggressive regional vertical interventions, there is a possibility that some neglected tropical infections could be eventually controlled to the point of elimination in some areas of endemicity [2–8]. In the case of guinea worm infection, disease eradication might also soon be possible [9]. Box 2. Common Features of the Neglected Tropical Diseases          Ancient afflictions that have burdened humanity for centuries      Poverty-promoting conditions      Associated with stigma      Rural areas of low-income countries and fragile states      No commercial markets for products that target these diseases      Interventions, when applied, have a history of success

Chronic lymphocytic leukemia with t(14;19)(q32;q13) is characterized by atypical morphologic and immunophenotypic features and distinctive genetic features.

The t(14;19)(q32;q13) involving the IGH@ and BCL3 loci is an infrequent cytogenetic abnormality detected in B-cell malignancies. We describe the clinicopathologic, cytogenetic, and molecular genetic characteristics of 14 cases of chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) with t(14;19)(q32;q13). All patients (10 men and 4 women) had lymphocytosis; 10 had lymphadenopathy. Blood and bone marrow lymphocytes were predominantly small, but cytologically and immunophenotypically atypical. In all cases, t(14;19) was found in the neoplastic stem line; it was the sole abnormality in 4. Ten cases showed additional cytogenetic abnormalities, including trisomy 12 in 9 and complex karyotypes in 7. Fluorescence in situ hybridization demonstrated IGH@/BCL3 fusion gene in all cases. In all cases, the IGHV genes were unmutated, but only 7 expressed ZAP70. Seven cases preferentially used IGHV4-39. Our results indicate that t(14;19)(q32;q13) identifies a subset of CLL/SLL with distinctive clinicopathologic and genetic features. Furthermore, t(14;19) may represent an early, possibly primary, genetic event.

CD8+ T cells are activated in an antigen-independent manner in HIV-infected individuals.

Hyperactivation of T cells, particularly of CD8(+) T cells, is a hallmark of chronic HIV 1 (HIV-1) infection. Little is known about the antigenic specificities and the mechanisms by which HIV-1 causes activation of CD8(+) T cells during chronic infection. We report that CD8(+) T cells were activated during in vivo HIV-1 replication irrespective of their Ag specificity. Cytokines present during untreated HIV-1 infection, most prominently IL-15, triggered proliferation and expression of activation markers in CD8(+) T cells, but not CD4(+) T cells, in the absence of TCR stimulation. Moreover, LPS or HIV-1-activated dendritic cells (DCs) stimulated CD8(+) T cells in an IL-15-dependent but Ag-independent manner, and IL-15 expression was highly increased in DCs isolated from viremic HIV-1 patients, suggesting that CD8(+) T cells are activated by inflammatory cytokines in untreated HIV-1 patients independent of Ag specificity. This finding contrasts with CD4(+) T cells whose in vivo activation seems biased toward specificities for persistent Ags. These observations explain the higher abundance of activated CD8(+) T cells compared with CD4(+) T cells in untreated HIV-1 infection.

Seroepidemiologic survey of captive Old-World primates for antibodies to human and simian retroviruses, and isolation of a lentivirus from sooty mangabeys (Cercocebus atys).

Sera from 526 Old-World monkeys and apes, representing 50 species and 20 genera and living in US zoos and vivaria, were screened for antibodies to HTLV-I, HTLV-III/LAV, and simian-AIDS retrovirus, type I (SRV-I). Sera were screened initially by ELISA, and ELISA-positive sera, as well as ELISA-negative sera from cage contacts, were further tested by Western blotting. A large number of false-positive and a small number of false-negative ELISA sera were identified. Although most true positive reactions were directed to a single retrovirus, a number of individuals from 4 species were positive for more than one retrovirus. Specific seroreactivity to HTLV-I was found in 39/526 (7%) animals of 15 species. True positive reactions to SRV-I were found in 21/516 (4%) animals, including talapoins and 2 species of macaques. Specific serologic reactions to HTLV-III/LAV were detected in 23/526 (4%) monkeys. Many of the HTLV-III/LAV seropositive animals were from one mixed-species zoo exhibit, containing sooty mangabeys, mandrills, Kolb's guenons, and talapoins. A type D virus was isolated from the blood of 3/10 SRV-I antibody-positive Tonkeana macaques, but from none of 11 seropositive talapoins. A lentivirus was isolated from the blood of 4/7 HTLV-III/LAV seropositive sooty mangabeys, but not from seropositive talapoins in the same exhibit or from 2 seropositive colobus from another zoo. The sooty mangabey lentivirus produced generalized lymphadenopathy, leukopenia, and decreased levels of T4 lymphocytes in 2 experimentally infected rhesus macaques.

Diversification in the HIV-1 Envelope Hyper-variable Domains V2, V4, and V5 and Higher Probability of Transmitted/Founder Envelope Glycosylation Favor the Development of Heterologous Neutralization Breadth

A recent study of plasma neutralization breadth in HIV-1 infected individuals at nine International AIDS Vaccine Initiative (IAVI) sites reported that viral load, HLA-A*03 genotype, and subtype C infection were strongly associated with the development of neutralization breadth. Here, we refine the findings of that study by analyzing the impact of the transmitted/founder (T/F) envelope (Env), early Env diversification, and autologous neutralization on the development of plasma neutralization breadth in 21 participants identified during recent infection at two of those sites: Kigali, Rwanda (n = 9) and Lusaka, Zambia (n = 12). Single-genome analysis of full-length T/F Env sequences revealed that all 21 individuals were infected with a highly homogeneous population of viral variants, which were categorized as subtype C (n = 12), A1 (n = 7), or recombinant AC (n = 2). An extensive amino acid sequence-based analysis of variable loop lengths and glycosylation patterns in the T/F Envs revealed that a lower ratio of NXS to NXT-encoded glycan motifs correlated with neutralization breadth. Further analysis comparing amino acid sequence changes, insertions/deletions, and glycan motif alterations between the T/F Env and autologous early Env variants revealed that extensive diversification focused in the V2, V4, and V5 regions of gp120, accompanied by contemporaneous viral escape, significantly favored the development of breadth. These results suggest that more efficient glycosylation of subtype A and C T/F Envs through fewer NXS-encoded glycan sites is more likely to elicit antibodies that can transition from autologous to heterologous neutralizing activity following exposure to gp120 diversification. This initiates an Env-antibody co-evolution cycle that increases neutralization breadth, and is further augmented over time by additional viral and host factors. These findings suggest that understanding how variation in the efficiency of site-specific glycosylation influences neutralizing antibody elicitation and targeting could advance the design of immunogens aimed at inducing antibodies that can transition from autologous to heterologous neutralizing activity.

The Global Spread of Hepatitis C Virus 1a and 1b: A Phylodynamic and Phylogeographic Analysis

BACKGROUND Hepatitis C virus (HCV) is estimated to affect 130-180 million people worldwide. Although its origin is unknown, patterns of viral diversity suggest that HCV genotype 1 probably originated from West Africa. Previous attempts to estimate the spatiotemporal parameters of the virus, both globally and regionally, have suggested that epidemic HCV transmission began in 1900 and grew steadily until the late 1980s. However, epidemiological data suggest that the expansion of HCV may have occurred after the Second World War. The aim of our study was to elucidate the timescale and route of the global spread of HCV. METHODS AND FINDINGS We show that the rarely sequenced HCV region (E2P7NS2) is more informative for molecular epidemiology studies than the more commonly used NS5B region. We applied phylodynamic methods to a substantial set of new E2P7NS2 and NS5B sequences, together with all available global HCV sequences with information in both of these genomic regions, in order to estimate the timescale and nature of the global expansion of the most prevalent HCV subtypes, 1a and 1b. We showed that transmission of subtypes 1a and 1b "exploded" between 1940 and 1980, with the spread of 1b preceding that of 1a by at least 16 y (95% confidence interval 15-17). Phylogeographic analysis of all available NS5B sequences suggests that HCV subtypes 1a and 1b disseminated from the developed world to the developing countries. CONCLUSIONS The evolutionary rate of HCV appears faster than previously suggested. The global spread of HCV coincided with the widespread use of transfused blood and blood products and with the expansion of intravenous drug use but slowed prior to the wide implementation of anti-HCV screening. Differences in the transmission routes associated with subtypes 1a and 1b provide an explanation of the relatively earlier expansion of 1b. Our data show that the most plausible route of the HCV dispersal was from developed countries to the developing world. Please see later in the article for the Editors' Summary.

HIV-1 regulation of latency in the monocyte-macrophage lineage and in CD4+ T lymphocytes.

The introduction in 1996 of the HAART raised hopes for the eradication of HIV-1. Unfortunately, the discovery of latent HIV-1 reservoirs in CD4+ T cells and in the monocyte-macrophage lineage proved the optimism to be premature. The long-lived HIV-1 reservoirs constitute a major obstacle to the eradication of HIV-1. In this review, we focus on the establishment and maintenance of HIV-1 latency in the two major targets for HIV-1: the CD4+ T cells and the monocyte-macrophage lineage. Understanding the cell-type molecular mechanisms of establishment, maintenance, and reactivation of HIV-1 latency in these reservoirs is crucial for efficient therapeutic intervention. A complete viral eradication, the holy graal for clinicians, might be achieved by strategic interventions targeting latently and productively infected cells. We suggest that new approaches, such as the combination of different kinds of proviral activators, may help to reduce dramatically the size of latent HIV-1 reservoirs in patients on HAART.

Virus-specific cytotoxic T-lymphocyte responses select for amino-acid variation in simian immunodeficiency virus Env and Nef

Cytotoxic T-lymphocyte (CTL) responses to human immunodeficiency virus arise early after infection, but ultimately fail to prevent progression to AIDS. Human immunodeficiency virus may evade the CTL response by accumulating amino-acid replacements within CTL epitopes. We studied 10 CTL epitopes during the course of simian immunodeficiency virus disease progression in three related macaques. All 10 of these CTL epitopes accumulated amino-acid replacements and showed evidence of positive selection by the time the macaques died. Many of the amino-acid replacements in these epitopes reduced or eliminated major histocompatibility complex class I binding and/or CTL recognition. These findings strongly support the CTL 'escape' hypothesis.

Characteristics of the Earliest Cross-Neutralizing Antibody Response to HIV-1

Recent cross-sectional analyses of HIV-1+ plasmas have indicated that broadly cross-reactive neutralizing antibody responses are developed by 10%-30% of HIV-1+ subjects. The timing of the initial development of such anti-viral responses is unknown. It is also unknown whether the emergence of these responses coincides with the appearance of antibody specificities to a single or multiple regions of the viral envelope glycoprotein (Env). Here we analyzed the cross-neutralizing antibody responses in longitudinal plasmas collected soon after and up to seven years after HIV-1 infection. We find that anti-HIV-1 cross-neutralizing antibody responses first become evident on average at 2.5 years and, in rare cases, as early as 1 year following infection. If cross-neutralizing antibody responses do not develop during the first 2-3 years of infection, they most likely will not do so subsequently. Our results indicate a potential link between the development of cross-neutralizing antibody responses and specific activation markers on T cells, and with plasma viremia levels. The earliest cross-neutralizing antibody response targets a limited number of Env regions, primarily the CD4-binding site and epitopes that are not present on monomeric Env, but on the virion-associated trimeric Env form. In contrast, the neutralizing activities of plasmas from subjects that did not develop cross-neutralizing antibody responses target epitopes on monomeric gp120 other than the CD4-BS. Our study provides information that is not only relevant to better understanding the interaction of the human immune system with HIV but may guide the development of effective immunization protocols. Since antibodies to complex epitopes that are present on the virion-associated envelope spike appear to be key components of earliest cross-neutralizing activities of HIV-1+ plasmas, then emphasis should be made to elicit similar antibodies by vaccination.

Analysis of the interaction of primate retroviruses with the human RNA interference machinery.

The question of whether retroviruses, including human immunodeficiency virus type 1 (HIV-1), interact with the cellular RNA interference machinery has been controversial. Here, we present data showing that neither HIV-1 nor human T-cell leukemia virus type 1 (HTLV-1) expresses significant levels of either small interfering RNAs or microRNAs in persistently infected T cells. We also demonstrate that the retroviral nuclear transcription factors HIV-1 Tat and HTLV-1 Tax, as well as the Tas transactivator encoded by primate foamy virus, fail to inhibit RNA interference in human cells. Moreover, the stable expression of physiological levels of HIV-1 Tat did not globally inhibit microRNA production or expression in infected human cells. Together, these data argue that HIV-1 and HTLV-1 neither induce the production of viral small interfering RNAs or microRNAs nor repress the cellular RNA interference machinery in infected cells.

Viral Infection of Engrafted Human Islets Leads to Diabetes

Type 1 diabetes (T1D) is characterized by the destruction of the insulin-producing β-cells of pancreatic islets. Genetic and environmental factors both contribute to T1D development. Viral infection with enteroviruses is a suspected trigger for T1D, but a causal role remains unproven and controversial. Studies in animals are problematic because of species-specific differences in host cell susceptibility and immune responses to candidate viral pathogens such as coxsackievirus B (CVB). In order to resolve the controversial role of viruses in human T1D, we developed a viral infection model in immunodeficient mice bearing human islet grafts. Hyperglycemia was induced in mice by specific ablation of native β-cells. Human islets, which are naturally susceptible to CVB infection, were transplanted to restore normoglycemia. Transplanted mice were infected with CVB4 and monitored for hyperglycemia. Forty-seven percent of CVB4-infected mice developed hyperglycemia. Human islet grafts from infected mice contained viral RNA, expressed viral protein, and had reduced insulin levels compared with grafts from uninfected mice. Human-specific gene expression profiles in grafts from infected mice revealed the induction of multiple interferon-stimulated genes. Thus, human islets can become severely dysfunctional with diminished insulin production after CVB infection of β-cells, resulting in diabetes.

High levels of viral replication contrast with only transient changes in CD4(+) and CD8(+) cell numbers during the early phase of experimental infection with simian immunodeficiency virus SIVmnd-1 in Mandrillus sphinx.

Early events during human immunodeficiency virus infections are considered to reflect the capacity of the host to control infection. We have studied early virus and host parameters during the early phase of simian immunodeficiency virus SIVmnd-1 nonpathogenic infection in its natural host, Mandrillus sphinx. Four mandrills were experimentally infected with a primary SIVmnd-1 strain derived from a naturally infected mandrill. Two noninfected control animals were monitored in parallel. Blood and lymph nodes were collected at three time points before infection, twice a week during the first month, and at days 60, 180, and 360 postinfection (p.i.). Anti-SIVmnd-1 antibodies were detected starting from days 28 to 32 p.i. Neither elevated temperature nor increased lymph node size were observed. The viral load in plasma peaked between days 7 to 10 p.i. (2 x 10(6) to 2 x 10(8) RNA equivalents/ml). Viremia then decreased 10- to 1,000-fold, reaching the viral set point between days 30 to 60 p.i. The levels during the chronic phase of infection were similar to that in the naturally infected donor mandrill (2 x 10(5) RNA equivalents/ml). The CD4(+) cell numbers and percentages in blood and lymph nodes decreased slightly (<10%) during primary infection, and CD8(+) cell numbers increased transiently. All values returned to preinfection infection levels by day 30 p.i. CD8(+) cell numbers or percentages, in peripheral blood and lymph nodes, did not increase during the 1 year of follow-up. In conclusion, SIVmnd-1 has the capacity for rapid and extensive replication in mandrills. Despite high levels of viremia, CD4(+) and CD8(+) cell numbers remained stable in the post-acute phase of infection, raising questions regarding the susceptibility of mandrill T cells to activation and/or cell death in response to SIVmnd-1 infection in vivo.

Prevalence of frequent headache in a population sample.

PURPOSE Patients with daily or near-daily headaches are commonly seen in neurology practices and in headache subspecialty centers, but there is little information on the prevalence of this condition in the general population. We present the first US-based study describing the prevalence and characteristics of frequent headache in the general population. METHODS In Baltimore County, Maryland, 13 343 individuals 18 to 65 years of age were selected by random-digit dialing and interviewed by telephone about their headaches. Subjects reporting 180 or more headaches per year were classified as having frequent headache. Three mutually exclusive subtypes of frequent headache were identified: frequent headache with migrainous features, chronic tension-type headache, and unclassified frequent headache. RESULTS The overall prevalence of frequent headache was 4.1% (5.0% female, 2.8% male; 1.8:1 female to male ratio). Frequent headache was 33% more common in Caucasians (4.4%) than in African Americans (3.3%). In both males and females, prevalence was highest in the lowest educational category. Among frequent headache sufferers, more than half (52% female, 56% male) met criteria for chronic tension-type headache, almost one third (33% female, 25% male) met criteria for frequent headache with migrainous features, and the remainder (15% female, 19% male) were unclassified. Overall, 30% of female and 25% of male frequent headache sufferers met International Headache Society (IHS) criteria for migraine (with or without aura). CONCLUSIONS Frequent headache is common in the general population and is more prevalent in Caucasians and in those with less than a high school education. Chronic tension-type headache is more common than frequent headache with migrainous features, though the latter is more disabling. Although more common in females than males, the female preponderance of frequent headache is less marked than in migraine. The sex ratio varies by frequent headache subtype.

Paediatric HIV infection.

By the year 2000 there will be six million pregnant women and five to ten million children infected with HIV-1. Intervention strategies have been planned and in some instances already started. A timely and cost-effective strategy needs to take into account that most HIV-1 infected individuals reside in developing countries. Further studies are needed on immunological and virological factors affecting HIV-1 transmission from mother to child, on differential disease progression in affected children, and on transient infection.

Nonproductive human immunodeficiency virus type 1 infection in nucleoside-treated G0 lymphocytes.

Productive infection by human immunodeficiency virus type 1 (HIV-1) requires the activation of target cells. Infection of quiescent peripheral CD4 lymphocytes by HIV-1 results in incomplete, labile, reverse transcripts. We have previously identified G1b as the cell cycle stage required for the optimal completion of the reverse transcription process in T lymphocytes. However, the mechanism(s) involved in the blockage of reverse transcription remains undefined. In this study we investigated whether nucleotide levels influence viral reverse transcription in G0 cells. For this purpose the role of the enzyme ribonucleotide reductase was bypassed, by adding exogenous deoxyribonucleosides to highly purified T cells in the G0 or the G1a phase of the cell cycle. Our data showed a significant increase in the efficiency of the reverse transcription process following the addition of the deoxyribonucleosides. To define the stability and functionality of these full reverse transcripts, we used an HIV-1 reporter virus that expresses the murine heat-stable antigen on the surfaces of infected cells. Following activation of infected quiescent cells treated with exogenous nucleosides, no increased rescue of productive infection was seen. Thus, in addition to failure to complete reverse transcription, there was an additional nonreversible blockage of productive infection in quiescent T cells. These experiments have important relevance in the gene therapy arena, in terms of improving the ability of lentivirus vectors to enter metabolically inactive cells, such as hematopoietic stem cells.

The history of SIVS and AIDS: epidemiology, phylogeny and biology of isolates from naturally SIV infected non-human primates (NHP) in Africa.

Simian immunodeficiency virus (SIV) naturally infects non-human primates in Africa. To date, 40 SIVs have been described both in natural hosts and in heterologous species. These viruses are highly diverse and the majority cluster in 6 relatively equidistant phylogenetic lineages. At least 8 SIVs are currently considered as recombinant viruses, based on different clustering patterns in different genomic regions. Only three types of genomes are known, based on the number of accessory genes: vpr-containing genomes, vpr-vpx containing genomes and vpr-vpu-containing genomes. vpx resulted by a duplication of the vpr gene following non-homologous recombination and is characteristic of SIVs infecting the Papionini tribe of monkeys and HIV-2 in humans. vpu is characteristic of SIVcpz and HIV-1 and may have originated from a recombination involving SIVs from cercopitecini monkeys. SIV seems to be non-pathogenic in the vast majority of natural hosts in spite of a high levels of viral replication. This is probably a consequence of virus-host adaptation, in which the incubation period of the disease generally exceeds the life span of the African primate host. SIVs also have a high propensity for cross-species transmission. In the new host, the outcome may vary from inapparent infection to highly pathogenic, the former being reported for African monkeys, whereas the latter being observed in macaques and humans. The high diversity of SIVs was generated by a high mutation rate due to a low fidelity of the reverse-transcriptase and active viral and host cell turnover, host-dependent evolution and recombination. Cross-species transmission is not rare, however preferential host switching may drive the majority of cross-species transmissions. Numerous SIVs tested so far are able to grow in vitro on human PBMC, therefore it has been postulated that SIV represents a threat for infection of humans in Central Africa and that AIDS is a zoonosis. However, although the simian origin of the two HIV types is broadly acknowledged, there are no data that AIDS is acquired like a zoonosis. SIV may undergo adaptation in the new human host in order to emerge in the general population. The study of SIV in their natural hosts should provide important clues to the real threat to human populations and also elucidate the mechanisms associated with a long-term persistent viral infection without clinical consequences for the host.

Enterovirus-induced gene expression profile is critical for human pancreatic islet destruction

Virally induced inflammatory responses, beta cell destruction and release of beta cell autoantigens may lead to autoimmune reactions culminating in type 1 diabetes. Therefore, viral capability to induce beta cell death and the nature of virus-induced immune responses are among key determinants of diabetogenic viruses. We hypothesised that enterovirus infection induces a specific gene expression pattern that results in islet destruction and that such a host response pattern is not shared among all enterovirus infections but varies between virus strains. The changes in global gene expression and secreted cytokine profiles induced by lytic or benign enterovirus infections were studied in primary human pancreatic islet using DNA microarrays and viral strains either isolated at the clinical onset of type 1 diabetes or capable of causing a diabetes-like condition in mice. The expression of pro-inflammatory cytokine genes (IL-1-α, IL-1-β and TNF-α) that also mediate cytokine-induced beta cell dysfunction correlated with the lytic potential of a virus. Temporally increasing gene expression levels of double-stranded RNA recognition receptors, antiviral molecules, cytokines and chemokines were detected for all studied virus strains. Lytic coxsackievirus B5 (CBV-5)-DS infection also downregulated genes involved in glycolysis and insulin secretion. The results suggest a distinct, virus-strain-specific, gene expression pattern leading to pancreatic islet destruction and pro-inflammatory effects after enterovirus infection. However, neither viral replication nor cytotoxic cytokine production alone are sufficient to induce necrotic cell death. More likely the combined effect of these and possibly cellular energy depletion lie behind the enterovirus-induced necrosis of islets.

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.

Germinal Center T Follicular Helper Cells Are Highly Permissive to HIV-1 and Alter Their Phenotype during Virus Replication.

HIV-1 replication is concentrated within CD4(+) T cells in B cell follicles of secondary lymphoid tissues during asymptomatic disease. Limited data suggest that a subset of T follicular helper cells (TFH) within germinal centers (GC) is highly permissive to HIV-1. Whether GC TFH are the major HIV-1 virus-producing cells in vivo has not been established. In this study, we investigated TFH permissivity to HIV-1 ex vivo by spinoculating and culturing tonsil cells with HIV-1 GFP reporter viruses. Using flow cytometry, higher percentages of GC TFH (CXCR5(high)PD-1(high)) and CXCR5(+)programmed cell death-1 (PD-1)(low) cells were GFP(+) than non-GC TFH (CXCR5(+)PD-1(intermediate)) or extrafollicular (EF) (CXCR5(-)) cells. When sorted prior to spinoculation, however, GC TFH were substantially more permissive than CXCR5(+)PD-1(low) or EF cells, suggesting that many GC TFH transition to a CXCR5(+)PD-1(low) phenotype during productive infection. In situ hybridization on inguinal lymph node sections from untreated HIV-1-infected individuals without AIDS revealed higher frequencies of HIV-1 RNA(+) cells in GC than non-GC regions of follicle or EF regions. Superinfection of HIV-1-infected individuals' lymph node cells with GFP reporter virus confirmed the permissivity of follicular cells ex vivo. Lymph node immunostaining revealed 96% of CXCR5(+)CD4(+) cells were located in follicles. Within sorted lymph node cells from four HIV-infected individuals, CXCR5(+) subsets harbored 11-66-fold more HIV-1 RNA than CXCR5(-) subsets, as determined by RT PCR. Thus, GC TFH are highly permissive to HIV-1, but downregulate PD-1 and, to a lesser extent, CXCR5 during HIV-1 replication. These data further implicate GC TFH as the major HIV-1-producing cells in chronic asymptomatic HIV-1 infection.

LINE-1 Retrotransposition Activity in Human Genomes

Highly active (i.e., "hot") long interspersed element-1 (LINE-1 or L1) sequences comprise the bulk of retrotransposition activity in the human genome; however, the abundance of hot L1s in the human population remains largely unexplored. Here, we used a fosmid-based, paired-end DNA sequencing strategy to identify 68 full-length L1s that are differentially present among individuals but are absent from the human genome reference sequence. The majority of these L1s were highly active in a cultured cell retrotransposition assay. Genotyping 26 elements revealed that two L1s are only found in Africa and that two more are absent from the H952 subset of the Human Genome Diversity Panel. Therefore, these results suggest that hot L1s are more abundant in the human population than previously appreciated, and that ongoing L1 retrotransposition continues to be a major source of interindividual genetic variation.

HIV–1 Infects Multipotent Progenitor Cells Causing Cell Death and Establishing Latent Cellular Reservoirs

HIV causes a chronic infection characterized by depletion of CD4(+) T lymphocytes and the development of opportunistic infections. Despite drugs that inhibit viral spread, HIV infection has been difficult to cure because of uncharacterized reservoirs of infected cells that are resistant to highly active antiretroviral therapy (HAART) and the immune response. Here we used CD34(+) cells from infected people as well as in vitro studies of wild-type HIV to show infection and killing of CD34(+) multipotent hematopoietic progenitor cells (HPCs). In some HPCs, we detected latent infection that stably persisted in cell culture until viral gene expression was activated by differentiation factors. A unique reporter HIV that directly detects latently infected cells in vitro confirmed the presence of distinct populations of active and latently infected HPCs. These findings have major implications for understanding HIV bone marrow pathology and the mechanisms by which HIV causes persistent infection.

Simian immunodeficiency viruses of diverse origin can use CXCR4 as a coreceptor for entry into human cells.

Primary simian immunodeficiency virus (SIV) isolated from sooty mangabey (SIVsm [n = 6]), stumptail (SIVstm [n = 1]), mandrill (SIVmnd [n = 1]), and African green (SIVagm [n = 1]) primates were examined for their ability to infect human cells and for their coreceptor requirements. All isolates infected human peripheral blood mononuclear cells (PBMCs) from a CCR5(+/+) donor, and seven of eight isolates tested also infected CCR5(-/-) PBMCs. Analysis of coreceptor utilization using GHOST and U87 cell lines revealed that all of the isolates tested used CCR5 and the orphan receptors STRL33 and GPR15. Coreceptors such as CCR2b, CCR3, CCR8, and CX3CR1 were also utilized by some primary SIV isolates. More importantly, we found that CXCR4 was used as a coreceptor by the SIVstm, the SIVagm, and four of the SIVsm isolates in GHOST and U87 cells. These data suggest that primary SIV isolates from diverse primate species can utilize CXCR4 for viral entry, similar to what has been described for human immunodeficiency viruses.

Clinical and laboratory features of adult T-cell leukaemia lymphoma in Barbados.

We describe the clinical and pathological features of 23 Afro-Caribbean patients with adult T-cell leukaemia/lymphoma admitted to the Queen Elizabeth Hospital, Barbados over a 5 year period. There were 9 males and 14 females, with a median age of 38 years (range 14-84). Twelve had acute leukaemia, 10 lymphoma (including 4 with solitary extra nodal lesions) and 1 smouldering subtype. Two patients had a past history of tropical spastic paraparesis/HTLV I associated myelopathy (TSP/HAM). The prognosis was poor, with only 3 complete responses to chemotherapy (CHOP) lasting from 9 to 36 months. We conclude that ATLL in Barbados is similar to the disease in the other Caribbean islands and Japan, except that in Barbados the age of onset is over a decade younger than in Japan.

Neutralizing antibody responses in acute human immunodeficiency virus type 1 subtype C infection.

The study of the evolution and specificities of neutralizing antibodies during the course of human immunodeficiency virus type 1 (HIV-1) infection may be important in the discovery of possible targets for vaccine design. In this study, we assessed the autologous and heterologous neutralization responses of 14 HIV-1 subtype C-infected individuals, using envelope clones obtained within the first 2 months postinfection. Our data show that potent but relatively strain-specific neutralizing antibodies develop within 3 to 12 months of HIV-1 infection. The magnitude of this response was associated with shorter V1-to-V5 envelope lengths and fewer glycosylation sites, particularly in the V1-V2 region. Anti-MPER antibodies were detected in 4 of 14 individuals within a year of infection, while antibodies to CD4-induced (CD4i) epitopes developed to high titers in 12 participants, in most cases before the development of autologous neutralizing antibodies. However, neither anti-MPER nor anti-CD4i antibody specificity conferred neutralization breadth. These data provide insights into the kinetics, potency, breadth, and epitope specificity of neutralizing antibody responses in acute HIV-1 subtype C infection.

HIV seropositivity and tuberculosis in a large general hospital in Malawi.

The incidence of the acquired immunodeficiency syndrome (AIDS) in Malawi is one of the highest in Central Africa. Since tuberculosis is an important initial manifestations of the disease, consecutive patients admitted to the tuberculosis (TB) wards of Zomba General Hospital, Malawi, were asked for permission to undergo a human immunodeficiency virus (HIV)-antibodies test. In addition, two other studies were done: from September 1986 all medical in-patients, clinically suspected for immune deficiency and from April 1988 all blood donors were tested for HIV seropositivity. Seventy-five percent of the TB patients volunteered; 32 out of 125 (26%) were seropositive. In the high-risk age groups (20-40 years) this percentage rose to 32. Among the medical in-patients suspected of immune deficiency the seropositivity rose sharply from April 1987 to October 1988. Among the blood donors tested, 20% were seropositive.

A point mutation in KINDLIN3 ablates activation of three integrin subfamilies in humans

Monogenic deficiency diseases provide unique opportunities to define the contributions of individual molecules to human physiology and to identify pathologies arising from their dysfunction. Here we describe a deficiency disease in two human siblings that presented with severe bleeding, frequent infections and osteopetrosis at an early age. These symptoms are consistent with but more severe than those reported for people with leukocyte adhesion deficiency III (LAD-III). Mechanistically, these symptoms arose from an inability to activate the integrins expressed on hematopoietic cells, including platelets and leukocytes. Immortalized lymphocyte cell lines isolated from the two individuals showed integrin activation defects. Several proteins previously implicated in integrin activation, including Ras-associated protein-1 (RAP1) and calcium and diacylglycerol-regulated guanine nucleotide exchange factor-1 (CALDAG-GEF1), were present and functional in these cell lines. The genetic basis for this disease was traced to a point mutation in the coding region of the KINDLIN3 (official gene symbol FERMT3) gene. When wild-type KINDLIN-3 was expressed in the immortalized lymphocytes, their integrins became responsive to activation signals. These results identify a genetic disease that severely compromises the health of the affected individuals and establish an essential role of KINDLIN-3 in integrin activation in humans. Furthermore, allogeneic bone marrow transplantation was shown to alleviate the symptoms of the disease.

Beta interferon controls West Nile virus infection and pathogenesis in mice.

Studies with mice lacking the common plasma membrane receptor for type I interferon (IFN-αβR(-)(/)(-)) have revealed that IFN signaling restricts tropism, dissemination, and lethality after infection with West Nile virus (WNV) or several other pathogenic viruses. However, the specific functions of individual IFN subtypes remain uncertain. Here, using IFN-β(-)(/)(-) mice, we defined the antiviral and immunomodulatory function of this IFN subtype in restricting viral infection. IFN-β(-)(/)(-) mice were more vulnerable to WNV infection than wild-type mice, succumbing more quickly and with greater overall mortality, although the phenotype was less severe than that of IFN-αβR(-)(/)(-) mice. The increased susceptibility of IFN-β(-)(/)(-) mice was accompanied by enhanced viral replication in different tissues. Consistent with a direct role for IFN-β in control of WNV replication, viral titers in ex vivo cultures of macrophages, dendritic cells, fibroblasts, and cerebellar granule cell neurons, but not cortical neurons, from IFN-β(-)(/)(-) mice were greater than in wild-type cells. Although detailed immunological analysis revealed no major deficits in the quality or quantity of WNV-specific antibodies or CD8(+) T cells, we observed an altered CD4(+) CD25(+) FoxP3(+) regulatory T cell response, with greater numbers after infection. Collectively, these results suggest that IFN-β controls WNV pathogenesis by restricting infection in key cell types and by modulating T cell regulatory networks.

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.

Natural history of adult T-cell leukemia/lymphoma and approaches to therapy

After cell-to-cell transmission, HTLV-I increases its viral genome by de novo infection and proliferation of infected cells. Proliferation of infected cells is clonal and persistent in vivo. During the carrier state, infected cells are selected in vivo by the host's immune system, the genetic and epigenetic environment of proviral integration sites, and other factors. In leukemic cells, tax gene expression is frequently impaired by genetic and epigenetic mechanisms. Such loss of Tax expression enables ATL cells to escape the host immune system. On the other hand, ATL cells acquire the ability to proliferate without Tax by intracellular genetic and epigenetic changes. Despite advances in support and the development of novel treatment agents, the prognosis for ATLL remains poor. A number of therapies, however, do appear to improve prognosis compared to CHOP (VEPA). These include interferon-α plus zidovudine (probably after 1–2 cycles of CHOP), intensive chemotherapy as in LSG-15 with G-CSF support and Allo-SCT (which includes the potential for cure). Emerging novel approaches include HDAC inhibitors, monoclonal antibodies, and proteasome inhibitors. Comparison between different therapeutic approaches is complicated by the range of natural history of ATLL, different recruitments of naïve-to-therapy, refractory or relapsed patients, and variations in the reporting of outcome that frequently excludes difficult-to-evaluate patients. Moreover, results from relatively small proof-of-principle studies have not been extended with randomized, controlled trials. As a result, currently, there is no clear evidence to support the value of any particular treatment approach over others. To avoid further unnecessary patient suffering and to identify optimal therapy as rapidly as possible, large randomized, controlled trials encompassing multicenter, international collaborations will be necessary.

Autosomal dominant lateral temporal epilepsy: two families with novel mutations in the LGI1 gene.

PURPOSE Mutations in the leucine rich, glioma inactivated gene (LGI1) were recently described in a small number of families with autosomal dominant lateral temporal epilepsy (ADLTE). ADLTE is characterized by partial seizures with symptoms suggestive of a lateral temporal onset, including frequent auditory aura. Here we report the results of clinical and genetic analyses of two newly identified families with ADTLE. METHODS We identified two families whose seizure semiology was suggestive of ADLTE. Evaluation included a detailed history and neurologic examination, as well as collection of DNA. The coding sequence of the LGI1 gene from affected subjects from both families was analyzed for evidence of mutation. RESULTS Each patient had a history of partial seizures, often with secondary generalization earlier in the course. Auditory aura was reported by approximately two thirds of affected patients in each pedigree. Novel mutations in LGI1 were detected in both families. A heterozygous single-nucleotide deletion at position 329 (del 329C) was detected in affected individuals from one family, whereas patients from the second family had a nonsynonymous variation, corresponding to C435G. CONCLUSIONS We identified two novel mutations in the LGI1 gene. The phenotype of these two families was similar to that of other kindreds with ADLTE, as auditory aura was absent in one third of affected individuals. Our results further support that LGI1 mutations should be considered in patients with a history of partial seizures if the semiology of seizures is consistent with the onset in the lateral temporal lobe.

Persistent LCMV infection is controlled by blockade of type I interferon signaling.

During persistent viral infections, chronic immune activation, negative immune regulator expression, an elevated interferon signature, and lymphoid tissue destruction correlate with disease progression. We demonstrated that blockade of type I interferon (IFN-I) signaling using an IFN-I receptor neutralizing antibody reduced immune system activation, decreased expression of negative immune regulatory molecules, and restored lymphoid architecture in mice persistently infected with lymphocytic choriomeningitis virus. IFN-I blockade before and after establishment of persistent virus infection resulted in enhanced virus clearance and was CD4 T cell-dependent. Hence, we demonstrate a direct causal link between IFN-I signaling, immune activation, negative immune regulator expression, lymphoid tissue disorganization, and virus persistence. Our results suggest that therapies targeting IFN-I may help control persistent virus infections.

Human T Cell Leukemia Virus Reactivation with Progression of Adult T-Cell Leukemia-Lymphoma

BACKGROUND Human T-cell leukemia virus-associated adult T-cell leukemia-lymphoma (ATLL) has a very poor prognosis, despite trials of a variety of different treatment regimens. Virus expression has been reported to be limited or absent when ATLL is diagnosed, and this has suggested that secondary genetic or epigenetic changes are important in disease pathogenesis. METHODS AND FINDINGS We prospectively investigated combination chemotherapy followed by antiretroviral therapy for this disorder. Nineteen patients were prospectively enrolled between 2002 and 2006 at five medical centers in a phase II clinical trial of infusional chemotherapy with etoposide, doxorubicin, and vincristine, daily prednisone, and bolus cyclophosphamide (EPOCH) given for two to six cycles until maximal clinical response, and followed by antiviral therapy with daily zidovudine, lamivudine, and alpha interferon-2a for up to one year. Seven patients were on study for less than one month due to progressive disease or chemotherapy toxicity. Eleven patients achieved an objective response with median duration of response of thirteen months, and two complete remissions. During chemotherapy induction, viral RNA expression increased (median 190-fold), and virus replication occurred, coincident with development of disease progression. CONCLUSIONS EPOCH chemotherapy followed by antiretroviral therapy is an active therapeutic regimen for adult T-cell leukemia-lymphoma, but viral reactivation during induction chemotherapy may contribute to treatment failure. Alternative therapies are sorely needed in this disease that simultaneously prevent virus expression, and are cytocidal for malignant cells.

Establishment of a cell line (NPC/HK1) from a differentiated squamous carcinoma of the nasopharynx.

A long-term cell culture epithelioid cell line was established from a recurrent squamous carcinoma of the nasopharynx of a Chinese male 17 1/2 years after radiation therapy. The cell line, designated NPC/HK1, has been passed 72 times over a period 1 year. The cells have been shown by light and electron microscopies to be of the squamous epithelial type. When they were transplanted subcutaneously into the back of athymic nude BALB/c (nu/nu) mice, tumors developed at the sites of inoculation, which on histological examination were shown to be well-differentiated squamous carcinomas, similar in morphology to the recurrent human tumor from which they were derived. Karyotypic analysis of cells from the cell line demonstrates an aneuploid human type with a modal chromosome number of 74 with both numerical and structural aberrations. Viral particles or Epstein-Barr viral nuclear antigen (EBNA) has not been demonstrated in the cells from the primary culture or several of the subcultures tested. The presence of EBNA in touch smears prepared from the biopsy tissue was inconclusive. Infection of the subcultured cells with EBV from P3HR1 and B95-8 cells was unsuccessful.

A talin-dependent LFA-1 focal zone is formed by rapidly migrating T lymphocytes

Cells such as fibroblasts and endothelial cells migrate through the coordinated responses of discrete integrin-containing focal adhesions and complexes. In contrast, little is known about the organization of integrins on the highly motile T lymphocyte. We have investigated the distribution, activity, and cytoskeletal linkage of the integrin lymphocyte function associated antigen-1 (LFA-1) on human T lymphocytes migrating on endothelial cells and on ligand intercellular adhesion molecule-1 (ICAM-1). The pattern of total LFA-1 varies from low expression in the lamellipodia to high expression in the uropod. However, high affinity, clustered LFA-1 is restricted to a mid-cell zone that remains stable over time and over a range of ICAM-1 densities. Talin is essential for the stability and formation of the LFA-1 zone. Disruption of the talin–integrin link leads to loss of zone integrity and a substantial decrease in speed of migration on ICAM-1. This adhesive structure, which differs from the previously described integrin-containing attachments displayed by many other cell types, we have termed the “focal zone. ”

Human Cytomegalovirus Latency-Associated Proteins Elicit Immune-Suppressive IL-10 Producing CD4+ T Cells

Human cytomegalovirus (HCMV) is a widely prevalent human herpesvirus, which, after primary infection, persists in the host for life. In healthy individuals, the virus is well controlled by the HCMV-specific T cell response. A key feature of this persistence, in the face of a normally robust host immune response, is the establishment of viral latency. In contrast to lytic infection, which is characterised by extensive viral gene expression and virus production, long-term latency in cells of the myeloid lineage is characterised by highly restricted expression of viral genes, including UL138 and LUNA. Here we report that both UL138 and LUNA-specific T cells were detectable directly ex vivo in healthy HCMV seropositive subjects and that this response is principally CD4⁺ T cell mediated. These UL138-specific CD4⁺ T cells are able to mediate MHC class II restricted cytotoxicity and, importantly, show IFNγ effector function in the context of both lytic and latent infection. Furthermore, in contrast to CDCD4⁺ T cells specific to antigens expressed solely during lytic infection, both the UL138 and LUNA-specific CD4⁺ T cell responses included CD4⁺ T cells that secreted the immunosuppressive cytokine cIL-10. We also show that cIL-10 expressing CD4⁺ T-cells are directed against latently expressed US28 and UL111A. Taken together, our data show that latency-associated gene products of HCMV generate CD4⁺ T cell responses in vivo, which are able to elicit effector function in response to both lytic and latently infected cells. Importantly and in contrast to CD4⁺ T cell populations, which recognise antigens solely expressed during lytic infection, include a subset of cells that secrete the immunosuppressive cytokine cIL-10. This suggests that HCMV skews the T cell responses to latency-associated antigens to one that is overall suppressive in order to sustain latent carriage in vivo.

Massive activation of archaeal defense genes during viral infection.

Archaeal viruses display unusually high genetic and morphological diversity. Studies of these viruses proved to be instrumental for the expansion of knowledge on viral diversity and evolution. The Sulfolobus islandicus rod-shaped virus 2 (SIRV2) is a model to study virus-host interactions in Archaea. It is a lytic virus that exploits a unique egress mechanism based on the formation of remarkable pyramidal structures on the host cell envelope. Using whole-transcriptome sequencing, we present here a global map defining host and viral gene expression during the infection cycle of SIRV2 in its hyperthermophilic host S. islandicus LAL14/1. This information was used, in combination with a yeast two-hybrid analysis of SIRV2 protein interactions, to advance current understanding of viral gene functions. As a consequence of SIRV2 infection, transcription of more than one-third of S. islandicus genes was differentially regulated. While expression of genes involved in cell division decreased, those genes playing a role in antiviral defense were activated on a large scale. Expression of genes belonging to toxin-antitoxin and clustered regularly interspaced short palindromic repeat (CRISPR)-Cas systems was specifically pronounced. The observed different degree of activation of various CRISPR-Cas systems highlights the specialized functions they perform. The information on individual gene expression and activation of antiviral defense systems is expected to aid future studies aimed at detailed understanding of the functions and interplay of these systems in vivo.

The descent of memory T-cell subsets

The immune system has evolved by continuously increasing its complexity to provide the host with an advantage over infectious agents. The development of immunological memory engenders long-lasting protection and lengthens the lifespan of the host. The generation of subsets of memory T cells with distinct homing and functional properties increases our defensive capabilities. However, the developmental relationship of memory T-cell subsets is a matter of debate. In this Opinion article, in light of recent developments, we suggest that it is probable that two distinct lineages comprise the memory CD8+ T-cell population generated in response to infection.

Simian immunodeficiency viruses replication dynamics in African non-human primate hosts: common patterns and species-specific differences.

METHODS To define potential common features of simian immunodeficiency virus (SIV) infections in different naturally infected host species, we compared the dynamics of viral replication in 31 African green monkeys (10 sabeus, 15 vervets and seven Caribbean AGMs), 14 mandrills and three sooty mangabeys (SMs) that were experimentally infected with their species-specific viruses. RESULTS After infection, these SIVs replicated rapidly reaching viral loads (VLs) of 10(5)-10(9) copies/ml of plasma between days 9-14 post-infection (p.i). Set point viremia was established between days 42 and 60 p.i., with levels of approximately 10(5)-10(6) copies/ml in SM and mandrills, and lower levels (10(3)-10(5) copies/ml) in AGMs. VL during the chronic phase did not correlate with viral genome structure: SIVmnd-2 (a vpx-containing virus) and SIVmnd-1 (which does not contain vpu or vpx) replicated to similar levels in mandrills. VL was dependent on virus strain: vervets infected with three different viral strains showed different patterns of viral replication. The pattern of viral replication of SIVagm.sab, which uses both CCR5 and CXCR4 co-receptors was similar to those of the other viruses. CONCLUSIONS Our results show a common pattern of SIV replication in naturally and experimentally infected hosts. This is similar overall to that observed in pathogenic SIV infection of macaques. This result indicates that differences in clinical outcome between pathogenic and non-pathogenic infections rely on host responses rather than the characteristics of the virus itself.

TLR activation triggers the rapid differentiation of monocytes into macrophages and dendritic cells

Leprosy enables investigation of mechanisms by which the innate immune system contributes to host defense against infection, because in one form, the disease progresses, and in the other, the infection is limited. We report that Toll-like receptor (TLR) activation of human monocytes induces rapid differentiation into two distinct subsets: DC-SIGN+ CD16+ macrophages and CD1b+ DC-SIGN− dendritic cells. DC-SIGN+ phagocytic macrophages were expanded by TLR-mediated upregulation of interleukin (IL)-15 and IL-15 receptor. CD1b+ dendritic cells were expanded by TLR-mediated upregulation of granulocyte-macrophage colony-stimulating factor (GM-CSF) and its receptor, promoted T cell activation and secreted proinflammatory cytokines. Whereas DC-SIGN+ macrophages were detected in lesions and after TLR activation in all leprosy patients, CD1b+ dendritic cells were not detected in lesions or after TLR activation of peripheral monocytes in individuals with the progressive lepromatous form, except during reversal reactions in which bacilli were cleared by T helper type 1 (TH1) responses. In tuberculoid lepromatous lesions, DC-SIGN+ cells were positive for macrophage markers, but negative for dendritic cell markers. Thus, TLR-induced differentiation of monocytes into either macrophages or dendritic cells seems to crucially influence effective host defenses in human infectious disease.

Adult T-cell leukemia/lymphoma in London: clinical experience of 21 cases.

Adult T-cell leukemia/lymphoma (ATLL) is uncommon in the United Kingdom and has so far been restricted to people of Afro-Caribbean extraction. Between 1981 and 1995, 21 cases presented to 2 inner London teaching hospitals where 17% of the population are of Afro-Caribbean origin. Clinical presentations were similar to those of the disease in HTLV-I-endemic areas. Major responses (CR + PR) were obtained in 10/16 assessable patients (63%) treated with combination chemotherapy. However, median survival was only 5.5 months. Disease progression and opportunistic infection were the major causes of treatment failure and death. Three patients (14%) relapsed in the central nervous system (CNS). Our cases confirm the profound immunosuppression in ATLL. The poor prognosis of acute and lymphoma types of ATLL highlight the need for new approaches to treatment such as zidovudine and alpha-interferon, incorporating prophylaxis against CNS disease and opportunistic infections.

Maternal alloantigens promote the development of tolerogenic fetal regulatory T cells in utero.

As the immune system develops, T cells are selected or regulated to become tolerant of self antigens and reactive against foreign antigens. In mice, the induction of such tolerance is thought to be attributable to the deletion of self-reactive cells. Here, we show that the human fetal immune system takes advantage of an additional mechanism: the generation of regulatory T cells (Tregs) that suppress fetal immune responses. We find that substantial numbers of maternal cells cross the placenta to reside in fetal lymph nodes, inducing the development of CD4+CD25highFoxP3+ Tregs that suppress fetal antimaternal immunity and persist at least until early adulthood. These findings reveal a form of antigen-specific tolerance in humans, induced in utero and probably active in regulating immune responses after birth.

Integration of antiretroviral therapy with tuberculosis treatment.

BACKGROUND We previously reported that integrating antiretroviral therapy (ART) with tuberculosis treatment reduces mortality. However, the timing for the initiation of ART during tuberculosis treatment remains unresolved. METHODS We conducted a three-group, open-label, randomized, controlled trial in South Africa involving 642 ambulatory patients, all with tuberculosis (confirmed by a positive sputum smear for acid-fast bacilli), human immunodeficiency virus infection, and a CD4+ T-cell count of less than 500 per cubic millimeter. Findings in the earlier-ART group (ART initiated within 4 weeks after the start of tuberculosis treatment, 214 patients) and later-ART group (ART initiated during the first 4 weeks of the continuation phase of tuberculosis treatment, 215 patients) are presented here. RESULTS At baseline, the median CD4+ T-cell count was 150 per cubic millimeter, and the median viral load was 161,000 copies per milliliter, with no significant differences between the two groups. The incidence rate of the acquired immunodeficiency syndrome (AIDS) or death was 6.9 cases per 100 person-years in the earlier-ART group (18 cases) as compared with 7.8 per 100 person-years in the later-ART group (19 cases) (incidence-rate ratio, 0.89; 95% confidence interval [CI], 0.44 to 1.79; P=0.73). However, among patients with CD4+ T-cell counts of less than 50 per cubic millimeter, the incidence rates of AIDS or death were 8.5 and 26.3 cases per 100 person-years, respectively (incidence-rate ratio, 0.32; 95% CI, 0.07 to 1.13; P=0.06). The incidence rates of the immune reconstitution inflammatory syndrome (IRIS) were 20.1 and 7.7 cases per 100 person-years, respectively (incidence-rate ratio, 2.62; 95% CI, 1.48 to 4.82; P<0.001). Adverse events requiring a switching of antiretroviral drugs occurred in 10 patients in the earlier-ART group and 1 patient in the later-ART group (P=0.006). CONCLUSIONS Early initiation of ART in patients with CD4+ T-cell counts of less than 50 per cubic millimeter increased AIDS-free survival. Deferral of the initiation of ART to the first 4 weeks of the continuation phase of tuberculosis therapy in those with higher CD4+ T-cell counts reduced the risks of IRIS and other adverse events related to ART without increasing the risk of AIDS or death. (Funded by the U.S. President's Emergency Plan for AIDS Relief and others; SAPIT ClinicalTrials.gov number, NCT00398996.).

Siglec-1 Is a Novel Dendritic Cell Receptor That Mediates HIV-1 Trans-Infection Through Recognition of Viral Membrane Gangliosides

Dendritic cells (DCs) are essential antigen-presenting cells for the induction of immunity against pathogens. However, HIV-1 spread is strongly enhanced in clusters of DCs and CD4(+) T cells. Uninfected DCs capture HIV-1 and mediate viral transfer to bystander CD4(+) T cells through a process termed trans-infection. Initial studies identified the C-type lectin DC-SIGN as the HIV-1 binding factor on DCs, which interacts with the viral envelope glycoproteins. Upon DC maturation, however, DC-SIGN is down-regulated, while HIV-1 capture and trans-infection is strongly enhanced via a glycoprotein-independent capture pathway that recognizes sialyllactose-containing membrane gangliosides. Here we show that the sialic acid-binding Ig-like lectin 1 (Siglec-1, CD169), which is highly expressed on mature DCs, specifically binds HIV-1 and vesicles carrying sialyllactose. Furthermore, Siglec-1 is essential for trans-infection by mature DCs. These findings identify Siglec-1 as a key factor for HIV-1 spread via infectious DC/T-cell synapses, highlighting a novel mechanism that mediates HIV-1 dissemination in activated tissues.

Axicabtagene Ciloleucel CAR T‐Cell Therapy in Refractory Large B‐Cell Lymphoma

Background In a phase 1 trial, axicabtagene ciloleucel (axi‐cel), an autologous anti‐CD19 chimeric antigen receptor (CAR) T‐cell therapy, showed efficacy in patients with refractory large B‐cell lymphoma after the failure of conventional therapy. Methods In this multicenter, phase 2 trial, we enrolled 111 patients with diffuse large B‐cell lymphoma, primary mediastinal B‐cell lymphoma, or transformed follicular lymphoma who had refractory disease despite undergoing recommended prior therapy. Patients received a target dose of 2×106 anti‐CD19 CAR T cells per kilogram of body weight after receiving a conditioning regimen of low‐dose cyclophosphamide and fludarabine. The primary end point was the rate of objective response (calculated as the combined rates of complete response and partial response). Secondary end points included overall survival, safety, and biomarker assessments. Results Among the 111 patients who were enrolled, axi‐cel was successfully manufactured for 110 (99%) and administered to 101 (91%). The objective response rate was 82%, and the complete response rate was 54%.With a median follow‐up of 15.4 months, 42% of the patients continued to have a response, with 40% continuing to have a complete response. The overall rate of survival at 18 months was 52%. The most common adverse events of grade 3 or higher during treatment were neutropenia (in 78% of the patients), anemia (in 43%), and thrombocytopenia (in 38%). Grade 3 or higher cytokine release syndrome and neurologic events occurred in 13% and 28% of the patients, respectively. Three of the patients died during treatment. Higher CAR T‐cell levels in blood were associated with response. Conclusions In this multicenter study, patients with refractory large B‐cell lymphoma who received CAR T‐cell therapy with axi‐cel had high levels of durable response, with a safety profile that included myelosuppression, the cytokine release syndrome, and neurologic events. (Funded by Kite Pharma and the Leukemia and Lymphoma Society Therapy Acceleration Program; ZUMA‐1 ClinicalTrials.gov number, NCT02348216.)

Distinct memory CD4+ T cells with commitment to T follicular helper- and T helper 1-cell lineages are generated after acute viral infection.

CD4(+) T follicular helper (Tfh) cells provide the required signals to B cells for germinal center reactions that are necessary for long-lived antibody responses. However, it remains unclear whether there are CD4(+) memory T cells committed to the Tfh cell lineage after antigen clearance. By using adoptive transfer of antigen-specific memory CD4(+) T cell subpopulations in the lymphocytic choriomeningitis virus infection model, we found that there are distinct memory CD4(+) T cell populations with commitment to either Tfh- or Th1-cell lineages. Our conclusions are based on gene expression profiles, epigenetic studies, and phenotypic and functional analyses. Our findings indicate that CD4(+) memory T cells "remember" their previous effector lineage after antigen clearance, being poised to reacquire their lineage-specific effector functions upon antigen reencounter. These findings have important implications for rational vaccine design, where improving the generation and engagement of memory Tfh cells could be used to enhance vaccine-induced protective immunity.

Apoptosis occurs predominantly in bystander cells and not in productively infected cells of HIV- and SIV-infected lymph nodes

Although 13 years have passed since identification of human immunodeficiency virus-1 (HIV-1) as the cause of AIDS, we do not yet know how HIV kills its primary target, the T cell that carries the CD4 antigen. We and others have shown an increase in the percentage of apoptotic cells among circulating CD4+ (and CD8+) T cells of HIV-seropositive individuals and an increase in frequency of apoptosis with disease progression. However, it is not known if this apoptosis occurs in infected or uninfected T cells. We show here, using in situ labelling of lymph nodes from HIV-infected children and SIV-infected macaques, that apoptosis occurs predominantly in bystander cells and not in the productively infected cells themselves. These data have implications for pathogenesis and therapy, namely, arguing that rational drug therapy may involve combination agents targeting viral replication in infected cells and apoptosis of uninfected cells.

Dengue is still an imported disease in China: a case study in Guangzhou.

Dengue virus and its four serotypes (DENV 1-4) infect approximately 390 million people worldwide each year, with most cases in tropical and subtropical regions. Because of repeated introduction of DENV from epidemic regions and suitable weather conditions, many regions have shifted from hypo-endemicity to hyper-endemicity over recent decades. Since the first dengue outbreak in 1978, it is crucial to understand the current situation in China over nearly 40 years. The purpose of the study was to examine whether dengue in China was endemic or not, which is essential for relevant dengue control and prevention strategy implementation in China. The study, combining epidemiological characteristics of dengue from the disease notification system, phylogenetic and phylogeographic analyses, showed that all four serotypes had been detected in Guangzhou, China, which was dominated by DENV 1-2. The Maximum Likelihood tree analytic results showed that the virus detected in Guangzhou localized in different clades, except of virus of 2002 and 2003 clustered together. There existed the mutual introductions between Guangzhou and Southeast Asia. Most of the viruses were imported from Southeast Asia and the sources of outbreaks in Guangzhou mainly originated from Thailand, Indonesia, and the Philippines. The study indicates that dengue in China still remains as an imported disease, with the possibility of localization.

Human tuberculous granulomas induce peripheral lymphoid follicle-like structures to orchestrate local host defence in the lung.

The human tuberculous granuloma provides the morphological basis for local immune processes central to the outcome of tuberculosis. Because of the scarcity of information in human patients, the aim of the present study was to gain insights into the functional and structural properties of infiltrated tissue. To this end, the mycobacterial load in lesions and dissemination to different tissue locations were investigated, as well as distribution, biological functions, and interactions of host immune cells. Analysis of early granuloma formation in formerly healthy lung tissue revealed a spatio-temporal sequence of cellular infiltration to sites of mycobacterial infection. A general structure of the developing granuloma was identified, comprising an inner cell layer with few CD8(+) cells surrounding the necrotic centre and an outer area of lymphocyte infiltration harbouring mycobacteria-containing antigen-presenting cells as well as CD4(+), CD8(+), and B cells in active follicle-like centres resembling secondary lymphoid organs. It is concluded that the follicular structures in the peripheral rim of granulomas serve as a morphological substrate for the orchestration of the enduring host response in pulmonary tuberculosis.

Nef induces multiple genes involved in cholesterol synthesis and uptake in human immunodeficiency virus type 1-infected T cells.

Several recent reports indicate that cholesterol might play an important role in human immunodeficiency virus type 1 (HIV-1) replication. We investigated the effects of HIV-1 infection on cholesterol biosynthesis and uptake using microarrays. HIV-1 increased gene expression of cholesterol genes in both transformed T-cell lines and primary CD4(+) T cells. Consistent with our microarray data, (14)C-labeled mevalonate and acetate incorporation was increased in HIV-1-infected cells. Our data also demonstrate that changes in cholesterol biosynthesis and uptake are only observed in the presence of functional Nef, suggesting that increased cholesterol synthesis may contribute to Nef-mediated enhancement of virion infectivity and viral replication.

Immune Activation with HIV Vaccines

The development of a safe and effective HIV vaccine is perhaps the most important and challenging goal remaining in HIV-AIDS research. Recent progress using a poxvirus vector prime and envelope protein boost strategy demonstrated a modest but statistically significant level of efficacy and established the concept that a vaccine could prevent HIV infection (1), and approaches to boost durability and efficacy are currently in the planning stages (2). But the results of two vaccine concepts based on recombinant adenovirus serotype-5 (rAd5) (3–5) pointed to a potential major problem—that such vaccines might increase susceptibility to HIV infection. This also raised the question of whether the problem extends to some or all of the other recombinant adenovirus vectors currently in development or to other vector-based vaccines.

Human herpesvirus 8 infection and Kaposi's sarcoma among human immunodeficiency virus-infected and -uninfected women.

Little is known about the epidemiology of human herpesvirus 8 (HHV-8) infections among women. A cross-sectional study was conducted of HHV-8 infection among human immunodeficiency virus (HIV)-infected and high-risk HIV-uninfected women. Serological tests with noninduced (latent) and induced (lytic) HHV-8 antigens were used to detect infection among 2483 participants of a multisite cohort. Reactivity to latent antigen was present in 4.1% and to induced antigens in 12.0% of women. Seven of 8 women who reported Kaposi's sarcoma had HHV-8 antibodies. Among HIV-positive women, HHV-8 infection was associated with use of crack, cocaine, or heroin (76% vs. 65%; P<.001), past syphilis (29% vs. 20%; P<.001), an injection drug-using male sex partner (61% vs. 53%; P=.014), black race (P=.010), and enrollment site (P=.015). In multivariate analysis, HIV infection, older age, past syphilis, black race, and enrollment site were independently associated with HHV-8 infection. In this cohort of North American women, HHV-8 infection was associated with HIV infection, drug use, and risky sexual behavior.

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.

Role of CD28-B7 interactions in generation and maintenance of CD8 T cell memory.

Although the role of CD28-B7 interaction in the activation of naive T cells is well established, its importance in the generation and maintenance of T cell memory is not well understood. In this study, we examined the requirement for CD28-B7 interactions in primary T cell activation and immune memory. Ag-specific CD8 T cell responses were compared between wild-type (+/+) and CD28-deficient (CD28(-/-)) mice following an acute infection with lymphocytic choriomeningitis virus (LCMV). During the primary response, there was a substantial activation and expansion of LCMV-specific CD8 T cells in both +/+ and CD28(-/-) mice. However, the magnitude of the primary CD8 T cell response to both dominant and subdominant LCMV CTL epitopes was approximately 2- to 3-fold lower in CD28(-/-) mice compared with +/+ mice; the lack of CD28-mediated costimulation did not lead to preferential suppression of CD8 T cell responses to the weaker subdominant epitopes. As seen in CD28(-/-) mice, blockade of B7-mediated costimulation by CTLA4-Ig treatment of +/+ mice also resulted in a 2-fold reduction in the anti-LCMV CD8 T cell responses. Loss of CD28/B7 interactions did not significantly affect the generation and maintenance of CD8 T cell memory; the magnitude of CD8 T cell memory was approximately 2-fold lower in CD28(-/-) mice as compared with +/+ mice. Further, in CD28(-/-) mice, LCMV-specific memory CD8 T cells showed normal homeostatic proliferation in vivo and also conferred protective immunity. Therefore, CD28 signaling is not necessary for the proliferative renewal and maintenance of memory CD8 T cells.

Chronic stress, glucocorticoid receptor resistance, inflammation, and disease risk.

We propose a model wherein chronic stress results in glucocorticoid receptor resistance (GCR) that, in turn, results in failure to down-regulate inflammatory response. Here we test the model in two viral-challenge studies. In study 1, we assessed stressful life events, GCR, and control variables including baseline antibody to the challenge virus, age, body mass index (BMI), season, race, sex, education, and virus type in 276 healthy adult volunteers. The volunteers were subsequently quarantined, exposed to one of two rhinoviruses, and followed for 5 d with nasal washes for viral isolation and assessment of signs/symptoms of a common cold. In study 2, we assessed the same control variables and GCR in 79 subjects who were subsequently exposed to a rhinovirus and monitored at baseline and for 5 d after viral challenge for the production of local (in nasal secretions) proinflammatory cytokines (IL-1β, TNF-α, and IL-6). Study 1: After covarying the control variables, those with recent exposure to a long-term threatening stressful experience demonstrated GCR; and those with GCR were at higher risk of subsequently developing a cold. Study 2: With the same controls used in study 1, greater GCR predicted the production of more local proinflammatory cytokines among infected subjects. These data provide support for a model suggesting that prolonged stressors result in GCR, which, in turn, interferes with appropriate regulation of inflammation. Because inflammation plays an important role in the onset and progression of a wide range of diseases, this model may have broad implications for understanding the role of stress in health.

Characterization of Programmed Death-1 Homologue-1 (PD-1H) Expression and Function in Normal and HIV Infected Individuals

Chronic immune activation that persists despite anti-retroviral therapy (ART) is the strongest predictor of disease progression in HIV infection. Monocyte/macrophages in HIV-infected individuals are known to spontaneously secrete cytokines, although neither the mechanism nor the molecules involved are known. Here we show that overexpression of the newly described co-stimulatory molecule, PD1 homologue (PD-1H) in human monocyte/macrophages is sufficient to induce spontaneous secretion of multiple cytokines. The process requires signaling via PD-1H as cytokine secretion could be abrogated by deletion of the cytoplasmic domain. Such overexpression of PD-1H, associated with spontaneous cytokine expression is seen in monocytes from chronically HIV-infected individuals and this correlates with immune activation and CD4 depletion, but not viral load. Moreover, antigen presentation by PD-1H-overexpressing monocytes results in enhanced cytokine secretion by HIV-specific T cells. These results suggest that PD-1H might play a crucial role in modulating immune activation and immune response in HIV infection.

CD28 and ITK signals regulate autoreactive T cell trafficking

Activation of self-reactive T cells and their trafficking to target tissues leads to autoimmune organ destruction. Mice lacking the co-inhibitory receptor cytotoxic T lymphocyte antigen-4 (CTLA-4) develop fatal autoimmunity characterized by lymphocytic infiltration into nonlymphoid tissues. Here, we demonstrate that the CD28 co-stimulatory pathway regulates the trafficking of self-reactive Ctla4(-/-) T cells to tissues. Concurrent ablation of the CD28-activated Tec family kinase ITK does not block spontaneous T cell activation but instead causes self-reactive Ctla4(-/-) T cells to accumulate in secondary lymphoid organs. Despite excessive spontaneous T cell activation and proliferation in lymphoid organs, Itk(-/-); Ctla4(-/-) mice are otherwise healthy, mount antiviral immune responses and exhibit a long lifespan. We propose that ITK specifically licenses autoreactive T cells to enter tissues to mount destructive immune responses. Notably, ITK inhibitors mimic the null mutant phenotype and also prevent pancreatic islet infiltration by diabetogenic T cells in mouse models of type 1 diabetes, highlighting their potential utility for the treatment of human autoimmune disorders.

Bromodomain Proteins Contribute to Maintenance of Bloodstream Form Stage Identity in the African Trypanosome

Trypanosoma brucei, the causative agent of African sleeping sickness, is transmitted to its mammalian host by the tsetse. In the fly, the parasite's surface is covered with invariant procyclin, while in the mammal it resides extracellularly in its bloodstream form (BF) and is densely covered with highly immunogenic Variant Surface Glycoprotein (VSG). In the BF, the parasite varies this highly immunogenic surface VSG using a repertoire of ~2500 distinct VSG genes. Recent reports in mammalian systems point to a role for histone acetyl-lysine recognizing bromodomain proteins in the maintenance of stem cell fate, leading us to hypothesize that bromodomain proteins may maintain the BF cell fate in trypanosomes. Using small-molecule inhibitors and genetic mutants for individual bromodomain proteins, we performed RNA-seq experiments that revealed changes in the transcriptome similar to those seen in cells differentiating from the BF to the insect stage. This was recapitulated at the protein level by the appearance of insect-stage proteins on the cell surface. Furthermore, bromodomain inhibition disrupts two major BF-specific immune evasion mechanisms that trypanosomes harness to evade mammalian host antibody responses. First, monoallelic expression of the antigenically varied VSG is disrupted. Second, rapid internalization of antibodies bound to VSG on the surface of the trypanosome is blocked. Thus, our studies reveal a role for trypanosome bromodomain proteins in maintaining bloodstream stage identity and immune evasion. Importantly, bromodomain inhibition leads to a decrease in virulence in a mouse model of infection, establishing these proteins as potential therapeutic drug targets for trypanosomiasis. Our 1.25Å resolution crystal structure of a trypanosome bromodomain in complex with I-BET151 reveals a novel binding mode of the inhibitor, which serves as a promising starting point for rational drug design.

CD64 expression distinguishes monocyte-derived and conventional dendritic cells and reveals their distinct role during intramuscular immunization.

Although most vaccines are administered i.m., little is known about the dendritic cells (DCs) that are present within skeletal muscles. In this article, we show that expression of CD64, the high-affinity IgG receptor FcγRI, distinguishes conventional DCs from monocyte-derived DCs (Mo-DCs). By using such a discriminatory marker, we defined the distinct DC subsets that reside in skeletal muscles and identified their migratory counterparts in draining lymph nodes (LNs). We further used this capability to analyze the functional specialization that exists among muscle DCs. After i.m. administration of Ag adsorbed to alum, we showed that alum-injected muscles contained large numbers of conventional DCs that belong to the CD8α(+)- and CD11b(+)-type DCs. Both conventional DC types were capable of capturing Ag and of migrating to draining LNs, where they efficiently activated naive T cells. In alum-injected muscles, Mo-DCs were as numerous as conventional DCs, but only a small fraction migrated to draining LNs. Therefore, alum by itself poorly induces Mo-DCs to migrate to draining LNs. We showed that addition of small amounts of LPS to alum enhanced Mo-DC migration. Considering that migratory Mo-DCs had, on a per cell basis, a higher capacity to induce IFN-γ-producing T cells than conventional DCs, the addition of LPS to alum enhanced the overall immunogenicity of Ags presented by muscle-derived DCs. Therefore, a full understanding of the role of adjuvants during i.m. vaccination needs to take into account the heterogeneous migratory and functional behavior of muscle DCs and Mo-DCs revealed in this study.

Persistence of HIV-1 Receptor-Positive Cells after HSV-2 Reactivation: A Potential Mechanism for Increased HIV-1 Acquisition

To explore the mechanism by which herpes simplex virus (HSV)-2 infection is related to HIV-1 acquisition, we conducted in situ analysis of the cellular infiltrate from sequential biopsies of HSV-2 lesions from patients on and off antiviral therapy. CD4(+) and CD8(+) T cells and a mixed population of plasmacytoid and myeloid dendritic cells (DCs), including cells expressing the C-type lectin receptor DC-SIGN, persisted at sites of HSV-2 reactivation for months after healing, even with daily antiviral therapy. The CD4(+) T cells that persisted reacted to HSV-2 antigen, were enriched for expression of the chemokine receptor CCR5, and were contiguous to DCs expressing the interleukin-3 receptor CD123 or DC-SIGN. Ex vivo infection with a CCR5-tropic strain of HIV-1 revealed greater concentrations of integrated HIV-1 DNA in cells derived from healed genital lesion biopsies than in cells from control skin biopsies. The persistence and enrichment of HIV receptor-positive inflammatory cells in the genitalia help explain the inability of anti-HSV-2 therapy to reduce HIV acquisition.

Development of leukemia in donor cells after allogeneic stem cell transplantation--a survey of the European Group for Blood and Marrow Transplantation (EBMT).

Leukemia in donor cells (donor cell leukemia; DCL) has been reported as a rare but severe complication of allogeneic stem cell transplantation (SCT). However, the incidence, potential pathogenetic factors, therapeutic options and outcome of patients suffering from DCL and the leukemia risk of their donors are not well defined. A questionnaire survey was carried out within European Blood and Marrow Transplantation Group (EBMT) centers. Ninety-one EBMT centers participated in this survey, covering 10489 allogeneic SCT between 12/1982 and 09/2003. Fourteen cases of DCL, most with a myeloid phenotype (7 cases of acute myeloid leukemia, 3 each of acute lymphocytic leukemia and 1 case of chronic myeloid leukemia) were identified. Demonstration of donor cell origin included molecular analysis of chimerism in most cases. DCL type and cytogenetic alterations were independent from the original disease. The median time between transplantation and diagnosis of DCL was 17 months (4-164). No type of conditioning, donor, graft manipulation, graft-versus-host disease prophylaxis or subsequent complications were identified as risk factors for DCL. Chemotherapy induced remissions in DCL and 2 of 5 patients remain alive in remission after a second transplant. None of the stem cell donors developed hematologic malignancies (median follow-up period of 9 years; range 6-30 years). DCL is an extremely rare complication of allogeneic SCT in which treatment attempts with chemotherapy and a second SCT are justified. Donors are not at an increased risk of developing hematologic malignancies.

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.

Central role of Th2/Tc2 lymphocytes in pattern II multiple sclerosis lesions

OBJECTIVE Multiple sclerosis (MS) is a disease of the central nervous system with marked heterogeneity in several aspects including pathological processes. Based on infiltrating immune cells, deposition of humoral factors and loss of oligodendrocytes and/or myelin proteins, four lesion patterns have been described. Pattern II is characterized by antibody and complement deposition in addition to T-cell infiltration. MS is considered a T-cell-mediated disease, but until now the study of pathogenic T cells has encountered major challenges, most importantly the limited access of brain-infiltrating T cells. Our objective was to identify, isolate, and characterize brain-infiltrating clonally expanded T cells in pattern II MS lesions. METHODS We used next-generation sequencing to identify clonally expanded T cells in demyelinating pattern II brain autopsy lesions, subsequently isolated these as T-cell clones from autologous cerebrospinal fluid and functionally characterized them. RESULTS We identified clonally expanded CD8(+) but also CD4(+) T cells in demyelinating pattern II lesions and for the first time were able to isolate these as live T-cell clones. The functional characterization shows that T cells releasing Th2 cytokines and able to provide B cell help dominate the T-cell infiltrate in pattern II brain lesions. INTERPRETATION Our data provide the first functional evidence for a putative role of Th2/Tc2 cells in pattern II MS supporting the existence of this pathogenic phenotype and questioning the protective role that is generally ascribed to Th2 cells. Our observations are important to consider for future treatments of pattern II MS patients.

Phenotypic deficits in the HIV-1 envelope are associated with the maturation of a V2-directed broadly neutralizing antibody lineage

Broadly neutralizing antibodies (bnAbs) to HIV-1 can evolve after years of an iterative process of virus escape and antibody adaptation that HIV-1 vaccine design seeks to mimic. To enable this, properties that render HIV-1 envelopes (Env) capable of eliciting bnAb responses need to be defined. Here, we followed the evolution of the V2 apex directed bnAb lineage VRC26 in the HIV-1 subtype C superinfected donor CAP256 to investigate the phenotypic changes of the virus populations circulating before and during the early phases of bnAb induction. Longitudinal viruses that evolved from the VRC26-resistant primary infecting (PI) virus, the VRC26-sensitive superinfecting (SU) virus and ensuing PI-SU recombinants revealed substantial phenotypic changes in Env, with a switch in Env properties coinciding with early resistance to VRC26. Decreased sensitivity of SU-like viruses to VRC26 was linked with reduced infectivity, altered entry kinetics and lower sensitivity to neutralization after CD4 attachment. VRC26 maintained neutralization activity against cell-associated CAP256 virus, indicating that escape through the cell-cell transmission route is not a dominant escape pathway. Reduced fitness of the early escape variants and sustained sensitivity in cell-cell transmission are both features that limit virus replication, thereby impeding rapid escape. This supports a scenario where VRC26 allowed only partial viral escape for a prolonged period, possibly increasing the time window for bnAb maturation. Collectively, our data highlight the phenotypic plasticity of the HIV-1 Env in evading bnAb pressure and the need to consider phenotypic traits when selecting and designing Env immunogens. Combinations of Env variants with differential phenotypic patterns and bnAb sensitivity, as we describe here for CAP256, may maximize the potential for inducing bnAb responses by vaccination.

A C-Type Lectin Collaborates with a CD45 Phosphatase Homolog to Facilitate West Nile Virus Infection of Mosquitoes

West Nile virus (WNV) is the most common arthropod-borne flavivirus in the United States; however, the vector ligand(s) that participate in infection are not known. We now show that an Aedes aegypti C-type lectin, mosGCTL-1, is induced by WNV, interacts with WNV in a calcium-dependent manner, and facilitates infection in vivo and in vitro. A mosquito homolog of human CD45 in A. aegypti, designated mosPTP-1, recruits mosGCTL-1 to enable viral attachment to cells and to enhance viral entry. In vivo experiments show that mosGCTL-1 and mosPTP-1 function as part of the same pathway and are critical for WNV infection of mosquitoes. A similar phenomenon was also observed in Culex quinquefasciatus, a natural vector of WNV, further demonstrating that these genes participate in WNV infection. During the mosquito blood-feeding process, WNV infection was blocked in vivo with mosGCTL-1 antibodies. A molecular understanding of flaviviral-arthropod interactions may lead to strategies to control viral dissemination in nature.

Antibodies specific for Epstein-Barr virus nuclear antigen-1 cross-react with human heterogeneous nuclear ribonucleoprotein L.

Epstein-Barr virus (EBV) is associated with multiple sclerosis (MS), and antibodies to the EBV nuclear antigen-1 (EBNA-1) are consistently increased in MS patients. The hypothesis of this study is that anti-EBNA-1 antibodies cross-react with a self antigen in MS patients. We affinity purified anti-EBNA-1 antibodies from human plasma, used the anti-EBNA-1 to immunoprecipitate antigens from human brain, and identified bound antigens with mass spectrometry. Anti-EBNA-1 consistently bound heterogeneous nuclear ribonucleoprotein L (HNRNPL). We expressed both the long and short isoforms of this protein, and verified with Western blots and ELISA that the long isoform cross-reacts with EBNA-1. Immunohistochemistry demonstrated that anti-EBNA-1 bound to an antigen in the nucleus of cultured rat central nervous system cells. ELISA demonstrated the presence of antibodies to HNRNPL in the plasma of both healthy controls and MS patients, but anti-HNRNPL was not increased in MS patients. We conclude that HNRNPL is an autoantigen which cross-reacts with EBNA-1. The relevance of this autoantigen to MS and other autoimmune diseases remains to be investigated.

Expression of a human cytomegalovirus latency-associated homolog of interleukin-10 during the productive phase of infection.

The human cytomegalovirus UL111A region is active during both productive and latent phases of infection. During productive infection, the virus expresses ORF79, a protein with oncogenic properties, and cmvIL-10, a functional homolog of human IL-10. During latent infection of myeloid progenitor cells, an alternately spliced variant of cmvIL-10, termed latency-associated (LA) cmvIL-10 has previously been identified. To determine whether LAcmvIL-10 transcription occurs during productive infection, we performed 5' and 3' RACE to map UL111A-region transcripts in productively infected human foreskin fibroblasts (HFFs). This analysis revealed the presence of a singly spliced UL111A-region transcript predicted to encode LAcmvIL-10. This transcript was expressed in HFFs with early (beta) kinetics, a temporal class that differs from that of ORF79 (alpha kinetics) and cmvIL-10 (gamma kinetics). These data identify and map a transcript encoding a latency-associated homolog of IL-10 which is expressed by the virus during the productive phase of infection.

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.

Gastrointestinal tract as a major site of CD4+ T cell depletion and viral replication in SIV infection.

Human and simian immunodeficiency virus (HIV and SIV) replicate optimally in activated memory CD4(+) T cells, a cell type that is abundant in the intestine. SIV infection of rhesus monkeys resulted in profound and selective depletion of CD4+ T cells in the intestine within days of infection, before any such changes in peripheral lymphoid tissues. The loss of CD4+ T cells in the intestine occurred coincident with productive infection of large numbers of mononuclear cells at this site. The intestine appears to be a major target for SIV replication and the major site of CD4+ T cell loss in early SIV infection.

The novel immunosuppressive enzyme IL4I1 is expressed by neoplastic cells of several B-cell lymphomas and by tumor-associated macrophages

We previously reported a strong IL4I1 gene expression in primary mediastinal B-cell lymphoma (PMBL) and recently identified the protein as a secreted L-phenylalanine oxidase, physiologically expressed by myeloid cells, which inhibits T-cell proliferation in vitro. Here, we analyzed the pattern of IL4I1 protein expression in 315 human lymphoid and non-lymphoid malignancies. Besides PMBL, IL4I1 expression in tumors was very frequent. IL4I1 was detected in tumor-associated macrophages from most of the tumors and in neoplastic cells from follicular lymphoma, classic and nodular lymphocyte predominant Hodgkin lymphomas and small lymphocytic lymphoma, three of which are germinal center derived. IL4I1-positive tumor cells were also detected in rare cases of solid cancers, mainly mesothelioma. The enzymatic activity paralleled protein expression, suggesting that IL4I1 is functional in vivo. Depending on the tumor type, IL4I1 may impact on different infiltrating lymphocyte populations with consequences on tumor evolution. In the particular case of follicular lymphoma cells, which are susceptible to antitumor cytotoxic T cells killing but depend on interactions with local T helper cells for survival, a high level of IL4I1 expression seems associated with the absence of bone marrow involvement and a better outcome. These findings plead for an evaluation of IL4I1 as a prognosis factor.

Human oocytes reprogram adult somatic nuclei of a type 1 diabetic to diploid pluripotent stem cells

The transfer of somatic cell nuclei into oocytes can give rise to pluripotent stem cells that are consistently equivalent to embryonic stem cells, holding promise for autologous cell replacement therapy. Although methods to induce pluripotent stem cells from somatic cells by transcription factors are widely used in basic research, numerous differences between induced pluripotent stem cells and embryonic stem cells have been reported, potentially affecting their clinical use. Because of the therapeutic potential of diploid embryonic stem-cell lines derived from adult cells of diseased human subjects, we have systematically investigated the parameters affecting efficiency of blastocyst development and stem-cell derivation. Here we show that improvements to the oocyte activation protocol, including the use of both kinase and translation inhibitors, and cell culture in the presence of histone deacetylase inhibitors, promote development to the blastocyst stage. Developmental efficiency varied between oocyte donors, and was inversely related to the number of days of hormonal stimulation required for oocyte maturation, whereas the daily dose of gonadotropin or the total number of metaphase II oocytes retrieved did not affect developmental outcome. Because the use of concentrated Sendai virus for cell fusion induced an increase in intracellular calcium concentration, causing premature oocyte activation, we used diluted Sendai virus in calcium-free medium. Using this modified nuclear transfer protocol, we derived diploid pluripotent stem-cell lines from somatic cells of a newborn and, for the first time, an adult, a female with type 1 diabetes.

14q deletions are associated with trisomy 12, NOTCH1 mutations and unmutated IGHV genes in chronic lymphocytic leukemia and small lymphocytic lymphoma.

Deletions of the long arm of chromosome 14 [del(14q)] are rare but recurrently observed in mature B-cell neoplasms, particularly in chronic lymphocytic leukemia (CLL). To further characterize this aberration, we studied 81 cases with del(14q): 54 of CLL and 27 of small lymphocytic lymphoma (SLL), the largest reported series to date. Using karyotype and fluorescence in situ hybridization (FISH), the most frequent additional abnormality was trisomy 12 (tri12), observed in 28/79 (35%) cases, followed by del13q14 (12/79, 15%), delTP53 (11/80, 14%) delATM (5/79, 6%), and del6q21 (3/76, 4%). IGHV genes were unmutated in 41/53 (77%) patients, with a high frequency of IGHV1-69 (21/52, 40%). NOTCH1 gene was mutated in 14/45 (31%) patients. There was no significant difference in cytogenetic and molecular abnormalities between CLL and SLL. Investigations using FISH and SNP-array demonstrated the heterogeneous size of the 14q deletions. However, a group with the same del(14)(q24.1q32.33) was identified in 48% of cases. In this group, tri12 (P = 0.004) and NOTCH1 mutations (P = 0.02) were significantly more frequent than in the other patients. In CLL patients with del(14q), median treatment-free survival (TFS) was 27 months. In conclusion, del(14q) is associated with tri12 and with pejorative prognostic factors: unmutated IGHV genes (with over-representation of the IGHV1-69 repertoire), NOTCH1 mutations, and a short TFS.

Reversing the tide — diagnosis and prevention of T2DM in populations of African descent

Abstract | Populations of African descent are at the forefront of the worldwide epidemic of type 2 diabetes mellitus (T2DM). The burden of T2DM is amplified by diagnosis after preventable complications of the disease have occurred. Earlier detection would result in a reduction in undiagnosed T2DM, more accurate statistics, more informed resource allocation and better health. An underappreciated factor contributing to undiagnosed T2DM in populations of African descent is that screening tests for hyperglycaemia, specifically, fasting plasma glucose and HbA1c, perform sub‐optimally in these populations. To offset this problem, combining tests or adding glycated albumin (a nonfasting marker of glycaemia), might be the way forward. However, differences in diet, exercise, BMI, environment, gene‐environment interactions and the prevalence of sickle cell trait mean that neither diagnostic tests nor interventions will be uniformly effective in individuals of African, Caribbean or African‐American descent. Among these three populations of African descent, intensive lifestyle interventions have been reported in only the African‐American population, in which they have been found to provide effective primary prevention of T2DM but not secondary prevention. Owing to a lack of health literacy and poor glycaemic control in Africa and the Caribbean, customized lifestyle interventions might achieve both secondary and primary prevention. Overall, diagnosis and prevention of T2DM requires innovative strategies that are sensitive to the diversity that exists within populations of African descent.

The epidemiology of sexually transmitted co-infections in HIV-positive and HIV-negative African-Caribbean women in Toronto

BACKGROUND HIV disproportionately affects African-Caribbean women in Canada but the frequency and distribution of sexually transmitted infections in this community have not been previously studied. METHODS We recruited women based on HIV status through a Toronto community health centre. Participants completed a socio-behavioural questionnaire using Audio Computer Assisted Self-Interview (ACASI) and provided blood for syphilis, HIV, hepatitis B and C, herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), and human cytomegalovirus (CMV) serology, urine for chlamydia and gonorrhea molecular testing and vaginal secretions for bacterial vaginosis (BV) and human papillomavirus (HPV). Differences in prevalence were assessed for statistical significance using chi-square. RESULTS We recruited 126 HIV-positive and 291 HIV-negative women, with a median age of 40 and 31 years, respectively (p < 0.001). Active HBV infection and lifetime exposure to HBV infection were more common in HIV-positive women (4.8% vs. 0.34%, p = 0.004; and 47.6% vs. 21.2%, p < 0.0001), as was a self-reported history of HBV vaccination (66.1% vs. 44.0%, p = 0.0001). Classical STIs were rare in both groups; BV prevalence was low and did not vary by HIV status. HSV-2 infection was markedly more frequent in HIV-positive (86.3%) than HIV-negative (46.6%) women (p < 0.0001). Vaginal HPV infection was also more common in HIV-positive than in HIV-negative women (50.8% vs. 22.6%, p < 0.0001) as was infection with high-risk oncogenic HPV types (48.4% vs. 17.3%, p < 0.0001). CONCLUSIONS Classical STIs were infrequent in this clinic-based population of African-Caribbean women in Toronto. However, HSV-2 prevalence was higher than that reported in previous studies in the general Canadian population and was strongly associated with HIV infection, as was infection with hepatitis B and HPV.

The Ratio of Monocytes to Lymphocytes in Peripheral Blood Correlates with Increased Susceptibility to Clinical Malaria in Kenyan Children

BACKGROUND Plasmodium falciparum malaria remains a major cause of illness and death in sub-Saharan Africa. Young children bear the brunt of the disease and though older children and adults suffer relatively fewer clinical attacks, they remain susceptible to asymptomatic P. falciparum infection. A better understanding of the host factors associated with immunity to clinical malaria and the ability to sustain asymptomatic P. falciparum infection will aid the development of improved strategies for disease prevention. METHODS AND FINDINGS Here we investigate whether full differential blood counts can predict susceptibility to clinical malaria among Kenyan children sampled at five annual cross-sectional surveys. We find that the ratio of monocytes to lymphocytes, measured in peripheral blood at the time of survey, directly correlates with risk of clinical malaria during follow-up. This association is evident among children with asymptomatic P. falciparum infection at the time the cell counts are measured (Hazard ratio (HR)  =  2.7 (95% CI 1.42, 5.01, P  =  0.002) but not in those without detectable parasitaemia (HR  =  1.0 (95% CI 0.74, 1.42, P  =  0.9). CONCLUSIONS We propose that the monocyte to lymphocyte ratio, which is easily derived from routine full differential blood counts, reflects an individual's capacity to mount an effective immune response to P. falciparum infection.

Central memory and effector memory T cell subsets: function, generation, and maintenance.

The memory T cell pool functions as a dynamic repository of antigen-experienced T lymphocytes that accumulate over the lifetime of the individual. Recent studies indicate that memory T lymphocytes contain distinct populations of central memory (TCM) and effector memory (TEM) cells characterized by distinct homing capacity and effector function. This review addresses the heterogeneity of TCM and TEM, their differentiation stages, and the current models for their generation and maintenance in humans and mice.

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.

Somatic Mutations of the Immunoglobulin Framework Are Generally Required for Broad and Potent HIV-1 Neutralization

Broadly neutralizing antibodies (bNAbs) to HIV-1 can prevent infection and are therefore of great importance for HIV-1 vaccine design. Notably, bNAbs are highly somatically mutated and generated by a fraction of HIV-1-infected individuals several years after infection. Antibodies typically accumulate mutations in the complementarity determining region (CDR) loops, which usually contact the antigen. The CDR loops are scaffolded by canonical framework regions (FWRs) that are both resistant to and less tolerant of mutations. Here, we report that in contrast to most antibodies, including those with limited HIV-1 neutralizing activity, most bNAbs require somatic mutations in their FWRs. Structural and functional analyses reveal that somatic mutations in FWR residues enhance breadth and potency by providing increased flexibility and/or direct antigen contact. Thus, in bNAbs, FWRs play an essential role beyond scaffolding the CDR loops and their unusual contribution to potency and breadth should be considered in HIV-1 vaccine design.

Novel syndromes associated with JC virus infection of neurons and meningeal cells: no longer a gray area.

PURPOSE OF REVIEW The availability of a growing number of immunomodulatory medications over the past few years has been associated with various JC virus (JCV)-associated brain syndromes in patients with autoimmune diseases, including multiple sclerosis, Crohn's disease, and psoriasis that had not been previously recognized as predisposing factors for progressive multifocal leukoencephalopathy. This review covers the three novel syndromes discovered in the last decade that are caused by JCV infection of neurons and meningeal cells. RECENT FINDINGS For more than 30 years, JCV was thought to exclusively infect oligodendrocytes and astrocytes in the white matter of the brain of immunosuppressed individuals. We now recognize that JCV-infected glial cells are frequently located at the gray-white matter junction or exclusively within the gray matter causing demyelination in the cortex. Mutations in JCV can trigger a change in tropism leading to involvement of other cell types, such as neurons and meningeal cells, causing clinically distinct entities. These new features of JCV infection provide challenges for clinicians taking care of affected patients and investigators studying the biology of this polyomavirus, its pathogenesis, and tropism. SUMMARY We hope that increasing awareness of these syndromes will lead to early diagnosis, and pave the way for new avenues of research to better understand all aspects of JCV pathogenesis and develop efficient therapies for our patients. However, we need to remain vigilant and open to the possibility that additional JC variants or yet unknown polyomaviruses may also be associated with neurological diseases.

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.

Evidence of a pluripotent human embryonic stem cell line derived from a cloned blastocyst.

Somatic cell nuclear transfer (SCNT) technology has recently been used to generate animals with a common genetic composition. In this study, we report the derivation of a pluripotent embryonic stem (ES) cell line (SCNT-hES-1) from a cloned human blastocyst. The SCNT-hES-1 cells displayed typical ES cell morphology and cell surface markers and were capable of differentiating into embryoid bodies in vitro and of forming teratomas in vivo containing cell derivatives from all three embryonic germ layers in severe combined immunodeficient mice. After continuous proliferation for more than 70 passages, SCNT-hES-1 cells maintained normal karyotypes and were genetically identical to the somatic nuclear donor cells. Although we cannot completely exclude the possibility that the cells had a parthenogenetic origin, imprinting analyses support a SCNT origin of the derived human ES cells.

CD8 Cells of Patients with Diffuse Cutaneous Leishmaniasis Display Functional Exhaustion: The Latter Is Reversed, In Vitro, by TLR2 Agonists

Leishmania mexicana (Lm) causes localized (LCL) and diffuse (DCL) cutaneous leishmaniasis. DCL patients have a poor cellular immune response leading to chronicity. It has been proposed that CD8 T lymphocytes (CD8) play a crucial role in infection clearance, although the role of CD8 cytotoxicity in disease control has not been elucidated. Lesions of DCL patients have been shown to harbor low numbers of CD8, as compared to patients with LCL, and leishmanicidal treatment restores CD8 numbers. The marked response of CD8 towards Leishmania parasites led us to analyze possible functional differences between CD8 from patients with LCL and DCL. We compared IFNγ production, antigen-specific proliferation, and cytotoxicity of CD8 purified from PBMC against autologous macrophages (MO) infected with Leishmania mexicana (MOi). Additionally, we analyzed tissue biopsies from both groups of patients for evidence of cytotoxicity associated with apoptotic cells in the lesions. We found that CD8 cell of DCL patients exhibited low cytotoxicity, low antigen-specific proliferation and low IFNγ production when stimulated with MOi, as compared to LCL patients. Additionally, DCL patients had significantly less TUNEL+ cells in their lesions. These characteristics are similar to cellular "exhaustion" described in chronic infections. We intended to restore the functional capacity of CD8 cells of DCL patients by preincubating them with TLR2 agonists: Lm lipophosphoglycan (LPG) or Pam3Cys. Cytotoxicity against MOi, antigen-specific proliferation and IFNγ production were restored with both stimuli, whereas PD-1 (a molecule associated with cellular exhaustion) expression, was reduced. Our work suggests that CD8 response is associated with control of Lm infection in LCL patients and that chronic infection in DCL patients leads to a state of CD8 functional exhaustion, which could facilitate disease spread. This is the first report that shows the presence of functionally exhausted CD8 T lymphocytes in DCL patients and, additionally, that pre-stimulation with TLR2 ligands can restore the effector mechanisms of CD8 T lymphocytes from DCL patients against Leishmania mexicana-infected macrophages.

A chikungunya fever vaccine utilizing an insect-specific virus platform

Traditionally, vaccine development involves tradeoffs between immunogenicity and safety. Live-attenuated vaccines typically offer rapid and durable immunity but have reduced safety when compared to inactivated vaccines. In contrast, the inability of inactivated vaccines to replicate enhances safety at the expense of immunogenicity, often necessitating multiple doses and boosters. To overcome these tradeoffs, we developed the insect-specific alphavirus, Eilat virus (EILV), as a vaccine platform. To address the chikungunya fever (CHIKF) pandemic, we used an EILV cDNA clone to design a chimeric virus containing the chikungunya virus (CHIKV) structural proteins. The recombinant EILV/CHIKV was structurally identical at 10 Å to wild-type CHIKV, as determined by single-particle cryo-electron microscopy, and it mimicked the early stages of CHIKV replication in vertebrate cells from attachment and entry to viral RNA delivery. Yet the recombinant virus remained completely defective for productive replication, providing a high degree of safety. A single dose of EILV/CHIKV produced in mosquito cells elicited rapid (within 4 d) and long-lasting (>290 d) neutralizing antibodies that provided complete protection in two different mouse models. In nonhuman primates, EILV/CHIKV elicited rapid and robust immunity that protected against viremia and telemetrically monitored fever. Our EILV platform represents the first structurally native application of an insect-specific virus in preclinical vaccine development and highlights the potential application of such viruses in vaccinology.

Transcriptome analysis reveals human cytomegalovirus reprograms monocyte differentiation toward an M1 macrophage.

Monocytes are primary targets for human CMV (HCMV) infection and are proposed to be responsible for hematogenous dissemination of the virus. Monocytes acquire different functional traits during polarization to the classical proinflammatory M1 macrophage or the alternative antiinflammatory M2 macrophage. We hypothesized that HCMV induced a proinflammatory M1 macrophage following infection to promote viral dissemination because, biologically, a proinflammatory state provides the tools to drive infected monocytes from the blood into the tissue. To test this hypothesis of monocyte conversion from a normal quiescent phenotype to an inflammatory phenotype, we used Affymetrix Microarray to acquire a transcriptional profile of infected monocytes at a time point our data emphasized is a key temporal regulatory point following infection. We found that HCMV significantly up-regulated 583 (5.2%) of the total genes and down-regulated 621 (5.5%) of the total genes>or=1.5-fold at 4 h postinfection. Further ontology analysis revealed that genes implicated in classical M1 macrophage activation were stimulated by HCMV infection. We found that 65% of genes strictly associated with M1 polarization were up-regulated, while only 4% of genes solely associated with M2 polarization were up-regulated. Analysis of the monocyte chemokinome at the transcriptional level showed that 44% of M1 and 33% of M2 macrophage chemokines were up-regulated. Proteomic analysis using chemokine Ab arrays confirmed the secretion of these chemotactic proteins from HCMV-infected monocytes. Overall, the results identify that the HCMV-infected monocyte transcriptome displayed a unique M1/M2 polarization signature that was skewed toward the classical M1 activation phenotype.

Migratory dendritic cells transfer antigen to a lymph node-resident dendritic cell population for efficient CTL priming.

Skin dendritic cells (DCs) are thought to act as key initiators of local T cell immunity. Here we show that after skin infection with herpes simplex virus (HSV), cytotoxic T lymphocyte (CTL) activation required MHC class I-restricted presentation by nonmigratory CD8(+) DCs rather than skin-derived DCs. Despite a lack of direct presentation by migratory DCs, blocking their egress from infected skin substantially inhibited class I-restricted presentation and HSV-specific CTL responses. These results support the argument for initial transport of antigen by migrating DCs, followed by its transfer to the lymphoid-resident DCs for presentation and CTL priming. Given that relatively robust CTL responses were seen with small numbers of skin-emigrant DCs, we propose that this inter-DC antigen transfer functions to amplify presentation across a larger network of lymphoid-resident DCs for efficient T cell activation.

Modulation of cellular and viral promoters by mutant human p53 proteins found in tumor cells.

Wild-type p53 has recently been shown to repress transcription from several cellular and viral promoters. Since p53 mutations are the most frequently reported genetic defects in human cancers, it becomes important to study the effects of mutations of p53 on promoter functions. We, therefore, have studied the effects of wild-type and mutant human p53 on the human proliferating-cell nuclear antigen (PCNA) promoter and on several viral promoters, including the herpes simplex virus type 1 UL9 promoter, the human cytomegalovirus major immediate-early promoter-enhancer, and the long terminal repeat promoters of Rous sarcoma virus and human T-cell lymphotropic virus type I. HeLa cells were cotransfected with a wild-type or mutant p53 expression vector and a plasmid containing a chloramphenicol acetyltransferase reporter gene under viral (or cellular) promoter control. As expected, expression of the wild-type p53 inhibited promoter function. Expression of a p53 with a mutation at any one of the four amino acid positions 175, 248, 273, or 281, however, correlated with a significant increase of the PCNA promoter activity (2- to 11-fold). The viral promoters were also activated, although to a somewhat lesser extent. We also showed that activation by a mutant p53 requires a minimal promoter containing a lone TATA box. A more significant increase (25-fold) in activation occurs when the promoter contains a binding site for the activating transcription factor or cyclic AMP response element-binding protein. Using Saos-2 cells that do not express p53, we showed that activation by a mutant p53 was a direct enhancement. The mutant forms of p53 used in this study are found in various cancer cells. The activation of PCNA by mutant p53s may indicate a way to increase cell proliferation by the mutant p53s. Thus, our data indicate a possible functional role for the mutants of p53 found in cancer cells in activating several important loci, including PCNA.

Transcriptional Signature and Memory Retention of Human-Induced Pluripotent Stem Cells

Genetic reprogramming of somatic cells to a pluripotent state (induced pluripotent stem cells or iPSCs) by over-expression of specific genes has been accomplished using mouse and human cells. However, it is still unclear how similar human iPSCs are to human Embryonic Stem Cells (hESCs). Here, we describe the transcriptional profile of human iPSCs generated without viral vectors or genomic insertions, revealing that these cells are in general similar to hESCs but with significant differences. For the generation of human iPSCs without viral vectors or genomic insertions, pluripotent factors Oct4 and Nanog were cloned in episomal vectors and transfected into human fetal neural progenitor cells. The transient expression of these two factors, or from Oct4 alone, resulted in efficient generation of human iPSCs. The reprogramming strategy described here revealed a potential transcriptional signature for human iPSCs yet retaining the gene expression of donor cells in human reprogrammed cells free of viral and transgene interference. Moreover, the episomal reprogramming strategy represents a safe way to generate human iPSCs for clinical purposes and basic research.

Human cytomegalovirus encodes an MHC class I-like molecule (UL142) that functions to inhibit NK cell lysis.

Clinical and low passage strains of human CMV (HCMV) encode an additional MHC class I-related molecule UL142, in addition to the previously described UL18. The UL142 open reading frame is encoded within the ULb' region which is missing from a number of common high passage laboratory strains. Cells expressing UL142 following transfection, and fibroblasts infected with a recombinant adenovirus-expressing UL142, were used to screen both polyclonal NK cells and NK cell clones, in a completely autologous system. Analysis of 100 NK cell clones derived from five donors, revealed 23 clones that were inhibited by fibroblasts expressing UL142 alone. Small-interfering RNA-mediated knockdown of UL142 mRNA expression in HCMV-infected cells resulted in increased sensitivity to lysis. From these data we conclude that UL142 is a novel HCMV-encoded MHC class I-related molecule which inhibits NK cell killing in a clonally dependent manner.

Natural variation in the parameters of innate immune cells is preferentially driven by genetic factors

The quantification and characterization of circulating immune cells provide key indicators of human health and disease. To identify the relative effects of environmental and genetic factors on variation in the parameters of innate and adaptive immune cells in homeostatic conditions, we combined standardized flow cytometry of blood leukocytes and genome-wide DNA genotyping of 1,000 healthy, unrelated people of Western European ancestry. We found that smoking, together with age, sex and latent infection with cytomegalovirus, were the main non-genetic factors that affected variation in parameters of human immune cells. Genome-wide association studies of 166 immunophenotypes identified 15 loci that showed enrichment for disease-associated variants. Finally, we demonstrated that the parameters of innate cells were more strongly controlled by genetic variation than were those of adaptive cells, which were driven by mainly environmental exposure. Our data establish a resource that will generate new hypotheses in immunology and highlight the role of innate immunity in susceptibility to common autoimmune diseases. Both environmental factors and genetic factors influence human immunity. Albert and colleagues leverage data from the Milieu Intérieur Consortium to comprehensively describe the effects of lifestyle, environment and genetics on human innate and adaptive immunity.

Antigen availability determines CD8⁺ T cell-dendritic cell interaction kinetics and memory fate decisions.

T cells are activated by antigen (Ag)-bearing dendritic cells (DCs) in lymph nodes in three phases. The duration of the initial phase of transient, serial DC-T cell interactions is inversely correlated with Ag dose. The second phase, characterized by stable DC-T cell contacts, is believed to be necessary for full-fledged T cell activation. Here we have shown that this is not the case. CD8⁺ T cells interacting with DCs presenting low-dose, short-lived Ag did not transition to phase 2, whereas higher Ag dose yielded phase 2 transition. Both antigenic constellations promoted T cell proliferation and effector differentiation but yielded different transcriptome signatures at 12 hr and 24 hr. T cells that experienced phase 2 developed long-lived memory, whereas conditions without stable contacts yielded immunological amnesia. Thus, T cells make fate decisions within hours after Ag exposure, resulting in long-term memory or abortive effector responses, correlating with T cell-DCs interaction kinetics.

Imaging the expression of transfected genes in vivo.

Imaging the expression of successful gene transduction has been demonstrated in vivo for the first time by using an appropriate combination of "marker gene" and "marker substrate" in an experimental animal model. The herpes simplex virus 1 thymidine kinase (HSV1-tk) gene was selected as an example of a marker gene, and the recombinant STK retrovirus containing HSV1-tk was used to transduce RG2 glioma cells in vitro and in vivo. RG2TK+ cell lines expressing the HSV1-tk gene and three potential marker substrates for the HSV1-TK enzyme were evaluated. Radiolabeled 5-iodo-2'-fluoro-2'deoxy-1-beta-D-arabinofuranosyluracil (FIAU) was shown to be a substantially better marker substrate for the HSV1-TK enzyme than 5-iodo-2'-deoxyuridine or ganciclovir. The magnitude of FIAU accumulation in different RG2TK+ clones corresponded to their sensitivity to ganciclovir and to the level of HSV1-tk mRNA expression. Imaging the expression of HSV1-tk in transduced RG2 tumor cells was demonstrated in animals using quantitative autoradiography; 2-[14C]FIAU accumulation was shown to be high in RG2TK+ brain tumors growing in one hemisphere and very low in nontransduced RG2 tumors in the contralateral hemisphere. Transduction of RG2 tumor cells with the HSV-tk gene in vivo resulted in tumors which accumulated FIAU to high levels and produced clearly defined images. Given the level of FIAU accumulation in the transduced tumors, it is likely that a clinically applicable method for imaging HSV1-tk gene expression can be implemented using existing clinical imaging techniques.

Biological properties of herpes simplex virus 2 replication-defective mutant strains in a murine nasal infection model.

We used a mouse nasal model of herpes simplex virus 2 (HSV-2) infection to examine the biological properties of HSV-2 wild-type (wt), TK-negative, and replication-defective strains in vivo. Nasal septa tissue is the major site of wt viral replication post intranasal (i.n.) inoculation. The HSV-2 strain 186 syn(+)-1 wt virus caused lethal encephalitis at doses of 10(4) PFU and above per nostril, and at lower doses no neurons in the trigeminal ganglia were positive for the latency-associated transcript, indicating a lack of latent infection. The 186DeltaKpn TK-negative mutant virus replicated in nasal septa tissue but showed low-level replication in trigeminal ganglia at only one timepoint. In situ hybridization of trigeminal ganglia showed that the number of LAT-positive neurons was proportional to the inoculum dose from 10(3) to 10(6) PFU per nare. The replication-defective mutant virus 5BlacZ showed no replication in nasal septa tissue and no persistence of viral DNA at the inoculation site or the trigeminal ganglia. Nevertheless, inoculation of 5BlacZ or the double-mutant dl5-29 at distal sites reduced acute replication and latent infection of 186DeltaKpn following intranasal challenge. This infection model provides a biological system to test the properties of HSV-2 strains and shows that replication-defective mutant strains do not persist at sites of inoculation or in sensory ganglia but can induce immune protection that reduces the latent viral load of a challenge virus.

High-throughput sequencing reveals extensive variation in human-specific L1 content in individual human genomes.

Using high-throughput sequencing, we devised a technique to determine the insertion sites of virtually all members of the human-specific L1 retrotransposon family in any human genome. Using diagnostic nucleotides, we were able to locate the approximately 800 L1Hs copies corresponding specifically to the pre-Ta, Ta-0, and Ta-1 L1Hs subfamilies, with over 90% of sequenced reads corresponding to human-specific elements. We find that any two individual genomes differ at an average of 285 sites with respect to L1 insertion presence or absence. In total, we assayed 25 individuals, 15 of which are unrelated, at 1139 sites, including 772 shared with the reference genome and 367 nonreference L1 insertions. We show that L1Hs profiles recapitulate genetic ancestry, and determine the chromosomal distribution of these elements. Using these data, we estimate that the rate of L1 retrotransposition in humans is between 1/95 and 1/270 births, and the number of dimorphic L1 elements in the human population with gene frequencies greater than 0.05 is between 3000 and 10,000.