The Candida Expert

Introduction

Many of the doctors who have consulted with me have asked for more information and references to better educate themselves, their patients and fellow doctors. To assist them, I gathered together a few of the references that we are including in our online Candida Library. In this article, you’ll find over 75 research references that provide information on how Candida goes from a harmless normal constituent of the gastrointestinal tract to a pathogenic systemic problem that can affect anyone and everyone. I’m providing this information here for you to become better educated about Candida, like the many doctors with whom I’ve consulted. Pass it along to your family, friends, and doctors, if you feel that it can also assist them in learning and understanding more about a problem that affects virtually everyone. Please click here to download this Candida Fact Sheet as a PDF so you can email or print it out.

Candida Facts

The human digestive tract is said to contain some 100 trillion cells compared to about only 10 trillion human cells in the body. This particular arrangement has led to man being classified as a “super-organism,” whose health is directly related to the function of the thousands of species of micro-organisms that make up the 100 trillion cells in the intestinal tract. For years, research suggested that there were 400-500 species that made up this microbial population. Recent advances in research have now put that number at anywhere from 3,300 to 5,700 or more, (9) to upwards of 30,000 species. The intestinal tract houses what has been called “the densest ecosystem on the planet,” and is approximately 25-28 ft long. The surface area of the intestinal tract measures approximately 200 square meters, roughly the size of a tennis court.

Modern medicine states that systemic Candida exists only in immunocompromised individuals, as a result of AIDS, immunosuppressive therapy, such as in organ transplants, or chemotherapy. Science states otherwise, and extends that list to include: diabetes, premature infants, surgical patients; (7)(10)(66) hematological malignancies; (8) hospitalized patients, especially in Intensive Care Units, or having major injuries;(10) burn victims; (54) nutritional deficiencies; (22) as well as aging. (22)(35)(36)(37) alcoholism, cirrhosis, tuberculosis, cancer, corticosteroids, marrow hyperplasia;

Researchers continuously broaden the scope of those being affected. Valdimarsson et al. state that there are no common immunological denominators. (1)may appear following even a slight modification of the host. (55) Berg et al. on behalf of Biocodex Pharmaceuticals states that Candida spreads in immunocompetent individuals. (68) Senet states that the pathogenic behavior of Candida

The widespread use of antibiotics, which induce neutropenia, an abnormally low number of neutrophils (white blood cells), and immune system suppression is commonly attributed by science to be the most consistent cause of systemic Candida.(3)(9)(12)(13)(14)(16)(17)(18)(19)(20)(21)(22)(55)(56)(57)(64)(67)(68)(69)(76)(77) Corticosteroids suppress immune system function. (11)(17)(68) Intestinal homeostasis is critical for human health. (6)(7)(55)(57)(68)(71)

Stress can cause accumulation of iron at the luminal surface of intestinal cells (75) and iron overload leads to impaired neutrophil function. (14) Stress can lead to immunosuppression facilitating the spread of Candida. (55) Sanchez et al. discuss the affect of starches vs. sugars on the immune system response to Candida. (29)

Macrophages, which are widely distributed immune system cells that play an indispensable role in homeostasis and defense, and are cells that function as a first line of defense against invading microorganisms, are historically ineffective against Candida albicans. (40)(41)

While evidence suggests that intestinal Dendritic Cells are critical for regulation of immunity in the gut, (50) Dendritic Cells are poor in both intracellular killing and damaging of C. albicans hyphae, (48) and only kill as effectively as macrophages. (51) Ingestion of hyphae by Dendritic Cells inhibits Th1 immune responses. (52)

Candida Albicans’ Secreted Aspartyl Proteases (SAPs) are a highly specific family of enzymes that assists in its ability to cause disease in the body. SAPs are believed to play a role in Candida’s ability to induce inflammation, invade and breakdown tissue barriers, digest proteins for nutrients, destroy and evade immune defenses, and spread throughout the body. (25)(33)(34)(58)(59)(60)(61)(62)(63)(65) Research has shown that the destructive effects of protease enzymes are associated with diabetes, hypertension, and immune system suppression. (25)(31)(34)(62)

Conclusion

As impressive as I find the above research to be, it is just a small representation of the research on Candida albicans and its effects in humans. With over 26,000 studies on Candida albicans since the introduction of antibiotics in the late 1940s, there is much more to be analyzed and reported. What is readily apparent from this data is the fact that systemic fungal Candida infections are a common occurrence in most individuals as a result of antibiotic use and other contributing factors.

– Dr. Jeffrey McCombs, DC

References

1. Immunological phenomena associated with chronic mucocutaneous candidiasis have recently been intensively studied by many workers (reviewed by Kirkpatrick, Rich & Bennett, 1971). The results have shown that there is no common immunological denominator in this disease. The most common finding, however, is defective cellular immunity, which may or may not be accompanied by failure of in vitro lymphocyte transformation.

Immunological Feautures in a Case of Chronic Granulomatous Candidiasis and its Treatment with Transfer Factor

2. The ability of Candida albicans to establish an infection involves multiple components of this fungal pathogen, but its ability to persist in host tissue may involve primarily the immunosuppressive property of a major cell wall glycoprotein, mannan. Mannan and oligosaccharide fragments of mannan are potent inhibitors of cell-mediated immunity and appear to reproduce the immune deficit of patients with the mucocutaneous form of candidiasis. However, neither the exact structures of these inhibitory species nor their mechanisms of action have yet been clearly defined. Different investigators have proposed that mannan or mannan catabolites act upon monocytes or suppressor T lymphocytes, but research from unrelated areas has provided still other possibilities for consideration. These include interference with cytokine activities, lymphocyte-monocyte interactions, and leukocyte homing. To stimulate further research of the immunosuppressive property of C. albicans mannan, we have reviewed (i) the relationship of mannan to other antigens and virulence factors of the fungus; (ii) the chemistry of mannan, together with methods for preparation of mannan and mannan fragments; and (iii) the historical evidence for immunosuppression by Candida mannan and the mechanisms currently proposed for this property; and (iv) we have speculated upon still other mechanisms by which mannan might influence host defense functions. It is possible that understanding the immunosuppressive effects of mannan will provide clues to novel therapies for candidiasis that will enhance the efficacy of both available and future anti-Candida agents. Immunosuppressive properties observed for isolated Candida mannan and its catabolites in vivo and in vitro provide additional evidence that fungal mannan is responsible for patient immune dysfunction.

References (Continued)

3. Phagocytic cells of the innate immune system, such as macrophagesand neutrophils, are a primary line of defense against microbialinfections. Patients with defects in innate immunity, such asthose with chronic granulomatous disease or neutropenia, areextremely sensitive to a variety of infections. When a phagocyterecognizes the presence of an invading cell, it engulfs themicrobe with its membrane to form the phagosome, an intracellularcompartment containing the microbe. This compartment maturesby fusion with lysosomes to create the phagolysosome, an organellereplete with antimicrobial compounds and an acidic pH. Internalizationcreates a hostile environment for the microorganism, which,of course, is the intent. The phagolysosome is a precarious neighborhood even before theonslaught of antimicrobial compounds. Engulfment by the macrophagethrusts the microorganism into an alien milieu, one devoid ofkey nutrients necessary for metabolism and division. Survivingthe antimicrobial assault in the phagolysosome depends on themicrobe’s ability to synthesize the proteins and other cellularcomponents necessary to counteract these stresses. Thus, a pathogenmust find the requisite nutrients to provide the building blocksfor these complex macromolecules and the energy with which tosynthesize them.

In this article we consider the initial responses of severalmicrobes to nutrient deprivation inside the macrophage. Thefirst of these, Mycobacterium tuberculosis, the bacterium thatcauses tuberculosis, resides for prolonged periods within themacrophage, in which it can proliferate and subsequently spreadthroughout the body. The second, the yeast Saccharomyces cerevisiae,is killed efficiently by the macrophage. The third, the opportunisticfungal pathogen Candida albicans, survives ingestion by changingrapidly from a yeast to a filamentous morphology, lysing themacrophage from the inside out. Once free, C. albicans cellsare able to disseminate through the body. The interaction ofC. albicans with the macrophage is transient, as opposed tothe long-term persistence of M. tuberculosis. Although the outcomesof this macrophage capture are quite different among the threemicrobes, the initial responses of all three to the internalenvironment are remarkably similar: induction of the glyoxylatecycle, a pathway that permits the utilization of compounds withtwo carbons (C2 compounds), such as acetate, to satisfy cellularcarbon requirements.

Systemic fungal infections have increased dramatically in prevalenceand severity over the last few decades, in concert with thenumber of patients living for extended periods with significantimmune dysfunction. AIDS, cancer chemotherapy, and organ transplantationhave all contributed to this rise, as has the widespread useof antibiotics. The most common systemic fungal infection iscandidiasis, which accounts for well over half of these invasivemycoses (3). A single species, C. albicans, causes the majorityof these infections. C. albicans, which also causes oropharyngealthrush and vaginitis, is normally a commensal of the mammaliangastrointestinal tract, in which it lives without adverse effectson the host. Both C. albicans and S. cerevisiae are readily phagocytosedby cultured macrophages in the presence of serum. While themacrophages efficiently kill S. cerevisiae, engulfment inducesC. albicans cells to grow in a filamentous morphology. Thesehyphal filaments can penetrate through the membrane of the phagocyticcell, releasing the fungal cell back into the extracellularmedium while killing the macrophage in the process. The differentoutcomes are not surprising; C. albicans is a common pathogenwhile S. cerevisiae is rarely found in human hosts.

The primary function of the glyoxylate cycle is to permit growthwhen C2 compounds, such as ethanol and acetate, are the onlysources of carbon. The glyoxylate pathway (also dubbed the glyoxylate shunt, forclear reasons) bypasses these decarboxylations, allowing C2compounds to serve as carbon sources in gluconeogenesis andto be incorporated into glucose and, from there, into aminoacids, DNA, and RNA. Glucose, as the preferred carbon sourcein most organisms, can be both converted into five-carbon sugars(such as ribose and deoxyribose) via the pentose phosphate pathwayand catabolized to acetyl-CoA via glycolysis. In microorganisms, however, glucose is frequently not available,and simple carbon compounds provide the only accessible carbon.

With the population of immunocompromisedpeople on the rise, the frequency of invasive fungal infectionscontinues to increase, making the need for effective treatmentsmore imperative.

Life and Death in a Macrophage: Role of the Glyoxylate Cycle in Virulence

4. Fungal pathogens can be recognized by the immune system via their beta-glucan, a potent proinflammatory molecule that is present at high levels but is predominantly buried beneath a mannoprotein coat and invisible to the host. To investigate the nature and significance of “masking” this molecule, we characterized the mechanism of masking and consequences of unmasking for immune recognition. We found that the underlying beta-glucan in the cell wall of Candida albicans is unmasked by subinhibitory doses of the antifungal drug caspofungin, causing the exposed fungi to elicit a stronger immune response. Using a library of bakers’ yeast (Saccharomyces cerevisiae) mutants, we uncovered a conserved genetic network that is required for concealing beta-glucan from the immune system and limiting the host response. Perturbation of parts of this network in the pathogen C. albicans caused unmasking of its beta-glucan, leading to increased beta-glucan receptor-dependent elicitation of key proinflammatory cytokines from primary mouse macrophages. By creating an anti-inflammatory barrier to mask beta-glucan, opportunistic fungi may promote commensal colonization and have an increased propensity for causing disease.

5. The interaction of Candida albicans with macrophages is considered a crucial step in the development of an adequate immune response in systemic candidiasis. An in vitro model of phagocytosis that includes a differential staining procedure to discriminate between internalized and non-internalized yeast was developed. Upon optimization of a protocol to obtain an enriched population of ingested yeasts, a thorough genomics and proteomics analysis was carried out on these cells. Both proteins and mRNA were obtained from the same sample and analyzed in parallel. The combination of two-dimensional PAGE with MS revealed a total of 132 differentially expressed yeast protein species upon macrophage interaction. Among these species, 67 unique proteins were identified. This is the first time that a proteomics approach has been used to study C. albicans-macrophage interaction. We provide evidence of a rapid protein response of the fungus to adapt to the new environment inside the phagosome by changing the expression of proteins belonging to different pathways. The clear down-regulation of the carbon-compound metabolism, plus the up-regulation of lipid, fatty acid, glyoxylate, and tricarboxylic acid cycles, indicates that yeast shifts to a starvation mode. There is an important activation of the degradation and detoxification protein machinery. The complementary genomics approach led to the detection of specific pathways related to the virulence of Candida. Network analyses allowed us to generate a hypothetical model of Candida cell death after macrophage interaction, highlighting the interconnection between actin cytoskeleton, mitochondria, and autophagy in the regulation of apoptosis. In conclusion, the combination of genomics, proteomics, and network analyses is a powerful strategy to better understand the complex host-pathogen interactions.

6. Intestinal homeostasis is critical for efficient energy extraction from food and protection from pathogens. Its disruption can lead to an array of severe illnesses with major impacts on public health, such as inflammatory bowel disease characterized by self-destructive intestinal immunity. However, the mechanisms regulating the equilibrium between the large bacterial flora and the immune system remain unclear. Intestinal lymphoid tissues generate flora-reactive IgA-producing B cells, and include Peyer’s patches and mesenteric lymph nodes, as well as numerous isolated lymphoid follicles (ILFs). Here we show that peptidoglycan from Gram-negative bacteria is necessary and sufficient to induce the genesis of ILFs in mice through recognition by the NOD1 (nucleotide-binding oligomerization domain containing 1) innate receptor in epithelial cells, and -defensin 3- and CCL20-mediated signalling through the chemokine receptor CCR6. Maturation of ILFs into large B-cell clusters requires subsequent detection of bacteria by toll-like receptors. In the absence of ILFs, the composition of the intestinal bacterial community is profoundly altered. Our results demonstrate that intestinal bacterial commensals and the immune system communicate through an innate detection system to generate adaptive lymphoid tissues and maintain intestinal homeostasis.

7. Candida albicans is a component of the normal flora of the alimentary tract and also is found on the mucocutaneous membranes of the healthy host. However, when immune defenses are compromised or the normal microflora balance is disrupted, Candida transforms itself into an opportunistic pathogenic killer. Candida is the leading cause of invasive fungal disease in premature infants, diabetics, and surgical patients, and of oropharyngeal disease in AIDS patients. As the induction of cell-mediated immunity to Candida is of critical importance in host defense, we sought to determine whether human dendritic cells (DC) could phagocytose and degrade Candida and subsequently present Candida antigens to T cells. Immature DC obtained by culture of human monocytes in the presence of granulocyte-macrophage colony-stimulating factor and interleukin-4 phagocytosed unopsonized Candida in a time-dependent manner, and phagocytosis was not enhanced by opsonization of CandidaCandida by the mannose-fucose receptor. Upon ingestion, DC killed Candida as efficiently as human M, and fungicidal activity was not enhanced by the presence of fresh serum. Although phagocytosis of Candida by DC stimulated the production of superoxide anion, inhibitors of the respiratory burst (or NO production) did not inhibit killing of Candida, even when phagocytosis was blocked by preincubation of DC with cytochalasin D. Further, although apparently only modest phagolysosomal fusion occurred upon DC phagocytosis of Candida, killing of Candida under anaerobic conditions was almost equivalent to killing under aerobic conditions. Finally, DC stimulated Candida-specific lymphocyte proliferation in a concentration-dependent manner after phagocytosis of both viable and heat-killed Candida cells. These data suggest that, in vivo, such interactions between DC and C. albicans may facilitate the induction of cell-mediated immunity. in serum. Like macrophages (M), DC recognized

Despite appropriate therapy, mortality from systemic Candida infections in immunocompromised individuals is nearly 30%. In human immunodeficiency virus-infected individuals who have not yet developed advanced immunodeficiency, the prevalence of oropharyngeal Candida is from 7 to 48% of patients. As the immunodeficiency in AIDS patients progresses, the prevalence of oral candidiasis increases to 43 to 93%. Furthermore, the development of oral candidiasis in the early stages of human immunodeficiency virus infection is highly predictive of worsening immunodeficiency.

8. Systemic candidiasis remains a major cause of disease and death, particularly among patients suffering from hematological malignancies. In an attempt to contribute to the discovery of useful biomarkers for its diagnosis and therapeutic monitoring, we embarked on a mapping of Candida albicans immunogenic proteins specifically recognized by antibodies produced during the natural course of systemic Candida infection in this high-risk population. About 85 immunoreactive protein species were detected with systemic candidiasis patients’ serum specimens by using immunoproteomics (i.e., two-dimensional electrophoresis followed by Western blotting), and identified through a combination of peptide mass fingerprinting by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS), de novo peptide sequencing using nano-electrospray ionization-ion trap (ESI-IT) MS, and genomic database searches. This proteomic approach has led to the characterization of 42 different housekeeping enzymes as C. albicans antigens. Their biological significance is also discussed. Furthermore, this study is the first to report that 26 of them exhibit antigenic properties in C. albicans, and 35 of them become targets of the human antibody response to systemic candidiasis. Our findings suggest that the production of antibodies to C. albicans phosphoglycerate kinase and alcohol dehydrogenase during systemic candidiasis could be associated with a differentiation of the human immune response. We also highlight the relationship between changes in maintenance of circulating levels of specific anti-Candida antibodies and patients’ outcome. Some of these variations, especially the rise of high anti-enolase antibody concentrations, appear to be related to recovery from systemic candidiasis in these patients, which might serve as markers for predicting their outcome.

9. Humans rely on their native microbiota for nutrition and resistance to colonization by pathogens; furthermore, recent discoveries have shown that symbiotic microbes make essential contributions to the development, metabolism, and immune response of the host. Co-evolved, beneficial, human–microbe interactions can be altered by many aspects of a modern lifestyle, including urbanization, global travel, and dietary changes, but in particular by antibiotics. The acute effects of antibiotic treatment on the native gut microbiota range from self-limiting “functional” diarrhea to life-threatening pseudomembranous colitis. The long-term consequences of such perturbations for the human–microbial symbiosis are more difficult to discern, but chronic conditions such as asthma and atopic disease have been associated with childhood antibiotic use and an altered intestinal microbiota. Because many chemical transformations in the gut are mediated by specific microbial populations, with implications for cancer and obesity, among other conditions, changes in the composition of the gut microbiota could have important but undiscovered health effects.

The Pervasive Effects of an Antibiotic on the Human Gut Microbiota, as Revealed by Deep 16S rRNA Sequencing

10. A new study has uncovered the genetic wiring diagram underlying the infectiousness of Candida albicans, a fungus that causes thrush in babies, vaginal infections in women, and life-threatening infections in chemotherapy and AIDS patients. The study, led by Dr. Gerald R. Fink, Director of the Whitehead Institute for Biomedical Research, reveals that one key to Candida’s infectiousness lies in its ability to switch from a rounded form to filamentous forms. Fungal infections in hospitalized patients have almost doubled throughout the 1980s, often with life-threatening results in individuals with weakened immune systems. Candida, in particular, poses a serious threat and is associated with high mortality rates in patients undergoing chemotherapy. Candida is also a major cause of infection in hospitalized patients, especially those in Intensive Care Units, patients after major injuries or surgery, patients with burns, and premature babies.

In this study, Dr. Fink and his colleagues used molecular biology techniques to identify the components of the filamentation circuit in yeast. With the recently completed yeast genome to guide them, the scientists began to knock out suspicious genes and, by a process of elimination, discovered the culprits that are responsible for filamentation. Once scientists identified the key yeast filamentation genes, they simply plucked out the analogous genes in Candida. “Candida albicans is three hundred million years apart evolutionarily from yeast-as far away in evolution as humans are from turtles-and yet, the basic logic circuit was conserved,” says Dr. Fink.

Discovery Of Genetic Pathways May Provide New Ways To Combat Candida Infections

11. The mechanisms by which corticosteroids (CCs) improve the outcome of AIDS patients with severe Pneumocystis carinii pneumonia (PCP) are unclear. We studied IL-1b and TNFα release from alveolar macrophages (AMs) of patients receiving CCs for the treatment of PCP and also the effect of in vitro hydrocortisone on this release. Cytokine release from AMs of AIDS patients with pulmonary complications not receiving CCs (group 1) was compared with that from AM of those receiving CCs for PCP (group 2). The AMs of HIV-negative normal subjects (group 3) served as controls. AU participants were nonsmokers or exsmokers. We found that lipopolysaccharide-stimulated AM from group 2 released significantly less interleukin-1 beta (IL-1b) and tumor necrosis factor alpha (TNFα) than AM from group 1 and was similar to that from group 3

Effect of corticosteroids on IL 1β and TNFα releae by alveolar macrophages from patients with AIDS and Pneumocystis carinii pneumonia

12. A transporter in the colon called SLC5A8 plays an important role in enabling the colon to get the last bit of good out of food before the unusable is flushed away, according to research currently published online as an accelerated communication in the Journal of Biological Chemistry. The finding that SLC5A8 is the transporter helps clarify why fruits and vegetables are good for you and why antibiotics, which wipe out good bacteria along with bad, should only be taken when absolutely necessary, upset the model and colonic cells get sick and may even become cancerous. “We do not make the enzyme to digest cellulose; bacteria make the enzyme in the colon,” says Dr. Ganapathy. “Therefore, you need to eat dietary fiber to provide the food for bacteria. Otherwise, they are not going to su'”/>rvive there. Antibiotics can wipe out good bacteria as well, leaving a void where disease-causing bacteria can grow.” “The gut is a huge immune organ; there are more immune cells in our gut than there are in the rest of the body put together,” says Dr. Robert G. Martindale, MCG gastrointestinal surgeon and nutritionist with a special interest in probiotics giving patients good bacteria to restore a healthy flora. “The work that Dr. Ganapathy is doing is showing very nicely that if, in fact, we keep this short-chain fatty acid transporter healthy, we then can keep the whole immune system healthy.” Immune cells also have a specific receptor for short-chain fatty acids on the cell surface, and Drs. Ganapathy and Martindale are pursuing the idea that the SLC5A8 transporter is delivering these fatty acids to immune cells to interact with the receptors and keep the immune cells vigilant as well.

13. Although Candida albicansis present in many mammals including humans, normal bacterial flora and various immune factors usually restrict the growth of C. albicansin the alimentary tracts of immune competent hosts. Infection of the alimentary tract mucosae, including the mucosae of the oropharynx, esophagus, and gastrointestinal tract, with C. albicans is occurring with greater frequency, presumably because of the increased population of immune compromised individuals. Recent evidence suggests that cell-mediated immunity, and more specifically, CD41 T lymphocytes, play an important role in resistance to mucosal candidiasis. Patient populations with AIDS or other defects in cellular immune function show an increased incidence of mucocutaneous, but not necessarily disseminated, candidiasis, whereas patients with phagocytic cell defects, such as those that occur in patients with neutropenic or chronic granulomatous disease states, show a higher incidence of disseminated candidiasis. A combination of defective cell-mediated immunity and phagocytic cell defects in athymic beige (bg/bg nu/nu) mice was found to predispose them to severe mucosal candidiasis with subsequent Candidadissemination. Existing mouse models of mucosal candidiasis use combinations of chemically induced immune suppression, elimination or alteration of the host microflora by administration of antibiotics, high inocula, trauma, infant animals, or animals with congenital, functional, physiological, immunological, or metabolic defects to facilitate colonization of the gastrointestinal tract by C. albicans.

New Model Of Oropharyngeal and Gastrointestinal Colonization by Candida albicans in CD41 T-Cell-Deficient Mice for Evaluation of Antifungal Agents

14. Underlying acquired immunity to the fungus Candida albicans is usually present in adult immunocompetent individuals and is presumed to prevent mucosal colonization progressing to symptomatic infection. Exploration of immunological events leading to Candida resistance or susceptibility has indicated the central role of the innate and adaptive immune systems, the relative contribution of which may vary depending on the site of the primary infection. Nevertheless, acquired resistance to infection results from the development of Th1 responses. Cytokines produced by Th1 cells activate phagocytic cells to a Candidacidal state. In contrast, cytokines produced by Th2 cells inhibit Th1 development and deactivate phagocytic effector cells. Because reciprocal influences have been recognized between innate and adaptive Th immunity, it appears that an integrated immune response determines the life-long commensalism of the fungus at the mucosal level, as well as the transition from mucosal saprophyte to pathogen. However, if the ability of C. albicans to establish a disseminated infection involves neutropenia as a major predisposing factor, its ability to persist in infected tissues or to behave as a commensal may involve primarily downregulation of host cell-mediated adaptive immunity. As a commensal, C. albicans may be endowed with the ability to elude the host’s immunological surveillance, thus allowing its persistence on mucosal surfaces. Th1 and Th2 CD41 T-cells develop from a common, naïve CD41 T-cell precursor, and several parameters have been shown to influence the pathway of differentiation of CD41 T-cell precursors. Among these, cytokines appear to play a major role, acting not only as modulators of antifungal effector functions but also as key regulators in the development of the different Th subsets from precursor Th cells. Studies in mice have shown that development of protective AntiCandidal Th1 responses requires the concerted actions of several cytokines, such as interferon (IFN)-g, transforming growth factor (TGF)-b, interleukin (IL)-6 [31], tumor necrosis factor (TNF)-a, and IL-12, in the relative absence of inhibitory Th2 cytokines, such as IL-4 and IL-10, which inhibit development of Th1 responses. Early in infection, neutralization of Th1 cytokines (IFN-g and IL-12) leads to the onset of Th2 rather than Th1 responses, while neutralization of Th2 cytokines (IL-4 and IL-10) allows development of Th1- rather than Th2-cell responses. TNF/lymphotoxin (LT)-a and IL-6 deficiencies render mice highly susceptible to C. albicans infections. Studies in humans have reinforced this concept, by showing that acquired immunity to C. albicans correlates with the expression of local or peripheral Th1 reactivity, whereas susceptibility to the infection seen in thermally injured patients, in patients with human immunodeficiency virus (HIV) infection, or in patients with chronic mucocutaneous or hepatosplenic candidiasis correlates with a biased Th2 response to the fungus. Altogether these data demonstrate that susceptibility to primary and secondary C. albicans infections in cytokine-deficient mice correlates with the failure to develop antiCandidal, protective Th1 responses and with the occurrence of unprotective IL-4- and IL-10-producing Th2 cells. However, an important immunoregulatory role has been attributed to neutrophils recently. Neutrophils, more than macrophages, were endowed with the ability to produce directive cytokines such as IL-10 and IL-12. Most importantly, IL-12 appeared to be released in response to a low-virulence Candida strain that initiates Th1 development in vivo, but IL-10 was released in response to a virulent strain. Human neutrophils also produced bioactive IL-12 in response to a mannoprotein fraction of C. albicans, capable of inducing Th1 cytokine expression in peripheral blood mononuclear cells. By producing directive cytokines such as IL-10 and IL-12, neutrophils influenced antifungal Th-cell development, as evidenced by the inability of neutropenic mice to mount protective antiCandidal Th1 responses. Production of IL-12 by neutrophils occurred independently of TNF-a and IFN-g. It was impaired upon iron overload but increased upon in vitro priming with IL-4 through upregulation of IL-4 receptor expression. Human studies confirm the multiple and complex role neutrophils have in candidiasis. First, risk factors for invasive fungal infections are not the same in all neutropenic patients. Secondly, chronic systemic candidiasis initiated by neutropenia may persist in spite of normal neutrophil counts and adequate antifungal therapy. Third, some patients, particularly transplant recipients who have adequate or even normal neutrophil counts, may be at high risk for invasive mycoses.

Innate and adaptive immunity in Candida albicans infections and saprophytism

15. Candida albicans is a common commensal organism that occasionally causes opportunistic infections. As shown by theincreased number of fungal infections in AIDS, the frequency ofcandidiasis has rapidly increased during the last 2 decades. In addition to AIDS, immunosuppression is inducedby treatments of solid malignant tumors, lymphoproliferative disorders,and organ transplantation. In immunocompromised patients, Candidacells easily invade the host’s organs and multiply, causing lethaldamage to the lungs, kidneys, liver, andintestines.

The prevention and treatment of Candidal infection have therefore become important for immunocompromised patients. Althoughthe host’s defense system against Candida cells has not yet beencompletely clarified, it has been reported that both humoral andcellular immunities contribute to protection against Candida cells. In the former, antibodies to Candida cell antigensenhance phagocytosis of neutrophils and macrophages.Salivary proteins, such as secretory immunoglobulin A, secretorycomponents, histatins, lysozyme, lactoferrin, transferrin, lactoperoxidase,mucins, and defensins have also been nominated as the humoralagents that prevent Candida cell adhesion and growth in the oropharyngealcavity,whereas cellular agents, such as neutrophils, macrophages, andT and NK cells, play important roles in the front line againstCandida cells, exhibiting phagocytosis and killing.For sufficient phagocytosis, opsonization of Candida cells isrequired. However, macrophages can trap nonopsonizedblastoconidia by using their mannose receptors. To killthe trapped blastoconidia sufficiently, neutrophils and macrophagesgenerate reactive oxygen intermediates (ROI) and nitric oxide(NO). The generation of ROI and NO is regulatedby multiple cytokines. Among them, granulocyte-macrophagecolony-stimulating factor (GM-CSF), interferons, and prostaglandinsstrongly induce NO synthase (NOS) and activateother enzymes associated with ROI generation.However, the virulence of blastoconidia is correlated with theirresistance to phagocytes. It has been reported that Candidacells with high levels of hyphal wall protein 1 (HWP1) and C.albicans drug resistance proteins 1 and 2 (CDR1 and -2) were resistantnot only to antifungal drugs, but also to phagocytes.

Clinically, there are two types of candidiasis: body surface candidiasis, including mucocutaneous candidiasis, and deep (organ)candidiasis. Surface Candidal infection is relatively easily cured,but deep candidiasis is highly resistant to antifungal drug therapy. Along withthese approaches, we synthesized a short lactoferrin peptide,FKCRRWQWRM, and examined its influences on blastoconidia and phagocytes.We found that the peptide possessed superior activities in bothkinds of cells, suggesting its usefulness for the treatment ofcandidiasis.

16. Candida albicans infections often occur during or shortly after antibacterial treatment. Phagocytosis by polymorphonuclear neutrophil granulocytes (PMN) is the most important primarily defence mechanism against C. albicans. Certain antibiotics such as some fluoroquinolones (FQ) are known to influence phagocyte functions. Thus, we investigated the influence of older and newer FQ on the phagocytosis and killing of C. albicans by human PMN paying special attention to CD11b expression of these cells as an indicator of the degree of their activation. In order to obtain comprehensive and comparable results we tested 13 FQ over a wide range of concentrations and in a time dependent manner in a standardized approach. When used at therapeutic concentrations, the FQ tested did not influence to a clinically significant degree the phagocytosis or the killing of C. albicans by human PMN and also not their activation. However, at high concentrations those FQ with cyclopropyl-moiety at position N1 showed increase in CD11b expression and diminished phagocytosis and oxidative burst.

Influence of fluoroquinolones on phagocytosis and killing of Candida albicans by human polymorphonuclear neutrophils

17. We studied the effects of eight antibiotics, cyclosporin and corticosteroids on the in vitro secretion of GM-CSF and G-CSF by monocytes, T lymphocytes and endothelial cells. The aim was to evaluate a possible mechanism for these drugs in the delay of haemopoietic recovery after high-dose chemotherapy or bone marrow transplantation. Corticosteroids were prominent inhibitors of GM-CSF secretion by monocytes and T lymphocytes, but not by endothelial cells. In contrast, G-CSF secretion by monocytes was unchanged whereas that of endothelial cells was enhanced in the presence of corticosteroids. Cyclosporin efficiently down-regulated GM-CSF secretion by T lymphocytes and had also a minor effect on CSF secretion by endothelial cells, whereas monocyte secretion was unaffected. Stimulated T lymphocytes derived from patients under treatment with cyclosporin had impaired capacity to secrete GM-CSF compared to controls. Among the antibiotics, cephalosporins inhibited GM-CSF secretion by T lymphocytes, and GM- and G-CSF secretion by endothelial cells. Ciprofloxacin and sulphmethoxazole had minor effects on GM-CSF secretion by T lymphocytes and endothelial cells. No antibiotic significantly influenced GM-CSF secretion by monocytes.

Effects of immunosuppressive drugs and antibiotics on GM-CSF and G-CSF secretion in vitro by monocytes, T lymphocytes and endothelial cells

18. Some antimicrobial agents have been reported to modify the host immune andinflammatory responses both in vivo and in vitro. Fosfomycin (FOM) andclarithromycin (CAM) have immunomodulatory activity on human lymphocytefunction. In the present study, we examined the effects of FOM and CAM oncytokine synthesis by lipopolysaccharide (LPS)- stimulated human monocytesin comparison with that of dexamethasone in vitro. The three drugsdemonstrated positive or negative effects on the synthesis of variouscytokines by LPS-primed monocytes. They suppressed the synthesis of tumornecrosis factor alpha, interleukin 1 alpha (IL-1 alpha), IL-1 beta, theIL-1 receptor antagonist, and granulocyte- macrophage colony-stimulatingfactor in a concentration-dependent manner at concentrations between 1.6and 40 micrograms/ml. On the contrary, the drugs showed different actionson the synthesis of IL-6 and IL-10. Namely, FOM enhanced both IL-6 andIL-10 synthesis, CAM enhanced only IL-10 synthesis, but dexamethasonedeeply suppressed the synthesis of both cytokines. These data indicate thatantibacterial agents may modify acute-phase inflammatory responses throughtheir effects on cytokine synthesis by monocytes.

Modulatory effect of antibiotics on cytokine production by human monocytes in vitro

19. Experimental subcutaneous Candida albicans infections in mice were used to examine the manner in which this pathogen is cleared in animals recovering from cyclophosphamide-induced leucopenia. In this system, infections at the inoculation sites progressed rapidly during a 6 day period of leucopenia to form arrays of parallel filamentous organisms that effectively isolated those in the interior from contact by neutrophils, even when the leucopenia had resolved. Dense collections of organisms also developed at sites of metastatic infection in the kidneys. A majority of the organisms were found to be viable when they were retrieved from the infected subcutaneous sites of animals that had recovered from leucopenia and whose abscesses had begun to drain spontaneously. Removal of the protective arrays of fungal cells appeared to be accomplished by drainage of abscess contents through the surface of the skin or into the collecting system of the kidney. Drainage of the subcutaneous abscesses did not occur in the cyclophosphamide-treated animals until after the neutrophilic infiltrates had developed, suggesting that this drainage process was mediated by neutrophils rather than by the organisms themselves. In summary, the above findings demonstrate that C. albicans infections in leucopenic hosts may progress to the extent that they would be very difficult to clear solely through the microbicidal processes of returning neutrophils. However, neutrophils also appear to promote the removal of masses of viable fungal cells to the exterior of the body.

Arrays of Candida albicans pseudohyphae that protect the organisms from neutrophil fungicidal mechanisms in experimental infections of mice

20. Phagocytes are an essential defence against infection. Since drugs which affect their function may alter the outcome of infections, we have studied the effect of nine antibiotics on phagocyte function in vitro. The effects of antibiotics on the respiratory burst function of phagocytes from healthy adult donors were investigated using lucigenin-enhanced chemiluminescence in response to serum-opsonised zymosan. Aminoglycosides showed dose-dependent suppression of polymorphonuclear leucocyte chemiluminescence, except streptomycin which caused enhancement. Erythromycin caused profound suppression of chemiluminescence from both polymorphonuclear leucocytes and monocytes. Benzylpenicillin and the cephalosporins caused variable suppression of phagocyte chemiluminescence: cefotaxime increased monocyte chemiluminescence in some experiments. None of the drugs produced suppression at clinically relevant plasma concentrations, but erythromycin and some other drugs are preferentially concentrated in phagocytes to levels which suppress their oxidative metabolism in vitro. It is therefore possible that some antibiotics alter phagocyte function: ex vivo studies of phagocyte function in patients taking antibiotics would be valuable.

21. Drug-induced neutropenia is one of the most common causes of neutropenia. Drugs can decrease neutrophil production through toxic, idiosyncratic, or hypersensitivity mechanisms or increase peripheral neutrophil destruction through immune mechanisms. It may result from aminopyrine, propylthiouracil , penicillin, or other antibiotics. Severe dose-related neutropenia occurs predictably after cytotoxic cancer drugs or radiation therapy suppresses bone marrow production. Neutropenia due to ineffective marrow production can occur in megaloblastic anemias caused by vitamin B12 or folate deficiency. Usually, macrocytic anemia and sometimes mild thrombocytopenia develop simultaneously. Infections can cause neutropenia by impairing neutrophil production or by inducing immune destruction or rapid use of neutrophils.

22. With the increasing number of immune compromised patients, fungi have emerged as major causes of human disease. Risk factors for systemic candidiasis include presence of intravascular catheters, receipt of broad-spectrum antibiotics, injury to the gastrointestinal mucosa and neutropenia. Within a species, the fungal morphotype (e.g. yeast, pseudohyphae and hyphae of Candida albicans) may be an important determinant of the host response. Whereas yeasts and spores are often effectively phagocytosed, the larger size of hyphae precludes effective ingestion.

Differentiation of CD4+ T cells along a T-helper (Th) cell type 1 (Th1) or type 2 (Th2) pathway and development of specific Th responses, is an essential determinant of the host’s susceptibility or resistance to invasive fungal infections. Development of Th1 responses is influenced by the concerted action of cytokines, such as interferon (INF)-c, interleukin (IL)-6, tumour necrosis factor (TNF)-a, and IL-12, in the relative absence of Th2 cytokines, such as IL-4 and IL-10 (Romani, 2002).

Oropharyngeal candidiasis (OPC) is among the most common mycotic infections of immunocompromised patients. Development of infection depends upon both systemic and local determinants. Risk factors for oral candidiasis include extremes in age, diabetes mellitus, particularly when glycemic control is poor, nutritional deficiencies, use of broad spectrum antibiotics and immunosuppression (especially of cell-mediated immunity) (Klein et al., 1984; Guggenheimer et al., 2000). Local factors that promote infection include dentures, salivary abnormalities, treatment with inhaled steroids, and destruction of mucosal barriers with radiotherapy for head and neck cancers or cytotoxic chemotherapy. Human immunodeficiency virus (HIV) is one of the most important predisposing conditions worldwide. AIDS patients have a particularly high incidence of mucosal candidiasis, which is often recurrent and, when it involves the esophagus, can be disabling (Sangeorzan et al., 1994). Local defence mechanisms against mucosal infection include salivary proteins, such as lactoferrin, beta-defensins, histatins, lysozyme, transferrin, lactoperoxidase, mucins, and secretory immunoglobulin A. These impair adhesion and growth of Candida in the oropharyngeal cavity. Development of OPC has been associated with a salivary Th2-type cytokine profile (Leigh et al., 1998).

Cell-mediated immunity plays the dominant role in prevention of candidiasis at the gastrointestinal surfaces. In AIDS, development of oropharyngeal and oesophageal candidiasis correlates with declining CD4+ lymphocyte counts. OPC is also associated with T cell immunosuppression from corticosteroid therapy, organ transplantation, cancer chemotherapy and chronic mucocutaneous candidiasis (CMC). Candida species have emerged as an important cause of bloodstream and deep tissue infections. Risk factors for Candidaemia include breakdown of mucosal barriers due to cytotoxic chemotherapy and surgical procedures, neutropenia, changes in the gut flora due to antibiotics, and invasive interventions that breach the skin, such as intravenous lines and drains (Wey et al., 1989). Common sites of dissemination include the bloodstream, kidney, liver, spleen, and endovascular structures. Quantitative and qualitative abnormalities of neutrophils and monocytes are associated with systemic candidiasis. Patients with lymphoma, leukaemia, chronic granulomatous disease, and recipients of intensive cancer chemotherapy with resultant neutropenia are at increased risk for disseminated infection. Similar to the situation with Aspergillus hyphae, the large size of Candida hyphae and pseudohyphae may preclude phagocytosis. Achieving a balance between Th1 and Th2 cytokines may be important for optimal antifungal protection while minimizing immune-mediated damage. In vivo models indicate that T regulatory cells attenuate Th1 antifungal responses, induce tolerance to the fungus and participate in the development of long lasting protective immunity after yeast priming (Montagnoli et al., 2002; Romani, 2004).

Dendritic cells play an important role in linking innate with adaptive immunity. Dendritic cells that ingest the yeast form induce differentiation of CD4+ T cells toward a Th1 pathway. In contrast, hyphae induce Th2 responses (d’Ostiani et al., 2000). Neutrophils, macrophages and natural killer (NK) cells also modulate adaptive responses to the fungus. Neutrophils differentially induce Th1 and Th2 responses depending on whether the exposure is to yeast or hyphae.

The syndrome of chronic disseminated candidiasis (CDC, also known as hepatosplenic candidiasis) predominantly affects patients with haematological malignancies upon recovery from neutropenia. CDC is characterized by increased serum levels of IL-10 and local production of Th2-inducing cytokines by hepatocytes and by infected mononuclear cells (Roilides et al., 1998b; Letterio et al., 2001). Thus, although neutropenia is a major predisposing factor, the propensity for persistence of the fungus in infected tissues may be a consequence of cell-mediated immune dysregulation with suppression of Th1 and overexpression of Th2 responses.

23. Neutrophil-mediated inflammation is terminated through the programmed cell death or apoptosis of the neutrophil, a process that can be inhibited by soluble mediators released during an inflammatory response. It has been reported, however, that the phagocytosis of intact bacteria can accelerate apoptosis. We evaluated the effects of the phagocytosis of a common nosocomial pathogen, Candida albicans, on the expression of apoptosis. Phagocytosis of killed Candida induced a dose-dependent increase in the apoptosis of normal neutrophils after 18 h of in vitro culture, from 40.7 +/- 9.1% to 81.7 +/- 4.5%, while supernatants from neutrophil: Candida co-cultures actually inhibited apoptosis. Induction of apoptosis was not dependent on phagocytosis, since opsonization of yeast with serum failed to increase apoptosis, while inhibition of phagocytosis with latrunculin B resulted in a slightly increased apoptotic rate. Increased apoptosis induced by Candida was associated with increased activity of the membrane-associated apoptotic enzyme, caspase 8, and with increased expression of the active form of the key executioner caspase, caspase 3. Increased apoptosis was associated with depletion of intracellular glutathione (GSH), and could be inhibited by the addition of exogenous GSH. These data demonstrate an important physiologic role for host-pathogen interactions in the resolution of inflammation and suggest that the response to an invading pathogen is an important stimulus to the restoration of normal immunologic homeostasis.

24. The surface of the pathogenic yeast Candida albicans is coated with phospholipomannan (PLM), a phylogenetically unique glycolipid composed of _-1,2-oligomannosides and phytoceramide. This study compared the specific contribution of PLM to the modulation of signaling pathways linked to the survival of C. albicans in macrophages in contrast to Saccharomyces cerevisiae. The addition of PLM to macrophages incubated with S. cerevisiae mimicked each of the disregulation steps observed with C. albicans and promoted the survival of S. cerevisiae. Externalization of membranous phosphatidylserine, loss of mitochondrial integrity, and DNA fragmentation induced by PLM showed that this molecule promoted yeast survival by inducing host cell death. These findings suggest strongly that PLM is a virulence attribute of C. albicans and that elucidation of the relationship between structure and apoptotic activity is an innovative field of research.

The yeast C. albicanshas been reported to inhibit tumor necrosis factor-_-induced DNA fragmentation in macrophages (33) and to induce apoptosis of macrophages (24) and neutrophils. Macrophages undergo apoptotic cell death after infection with C. albicans strains capable of hyphal formation (24), and activation of caspase 3 has been observed after endocytosis of C. albicans by neutrophils. Extensive literature exists on the effects of surface glycolipids from pathogens on the control of host cell apoptosis.

25. Interactions of microorganisms with integrins are central tothe host defense mechanisms. The leukocyte integrin CD11b/CD18is the principal adhesion receptor on leukocytes for Candida albicans,a major opportunistic pathogen. In this study we have investigatedthe roles of three regions within the receptor, the inserted(I) and lectin-like domains within the CD11b subunit, and the CD18subunit, in CD11b/CD18-C. albicans interactions. We report fourmajor findings. 1) A mutation in CD18 exerts a dominant negativeeffect on the function of the CD11b/CD18 complex. This interpretationis based on the observation that in the absence of CD18, theCD11b subunit alone binds C. albicans well, but a single pointmutation at Ser138 of CD18 abolishes CD11b/CD18 binding of thefungus. 2) The lectin-like domain is not sufficient for CD11b/CD18-C.albicans interactions. Rather, the lectin-like domain appearsto influence CD11b/CD18 binding activity by modulating the functionof the I domain. 3) The I domain is the primary binding sitefor C. albicans in the receptor and is sufficient to supportan efficient interaction. 4) We have identified specific aminoacid sequences within the I domain that engage the microorganism.Compared with other ligands of CD11b/CD18, C. albicans has someunique as well as common contact sites within the I domain ofthe receptor. Such unique contact sites may underlie the abilityof C. albicans to modulate CD11b/CD18 function and raise thepossibility for selective interference of the microorganism-hostleukocyte interactions.

Interaction of the Fungal Pathogen Candida albicans with Integrin CD11b/CD18: Recognition by the I Domain Is Modulated by the Lectin-Like Domain and the CD18 Subunit

26. Studies of host-parasite relationships at the cellular level, using Candida albicans and rabbit alveolar macrophages or guinea pig neutrophils are presented. Guinea pig neutrophils killed the intracellular Candida cells presumed by myeloperoxidase-halide-hydrogen peroxide system. In contrast, rabbit alveolar macrophages did not kill the intracellular Candida cells although their phagocytic rate was almost comparable to that of neutrophils. Phagocytizing macrophages were eventually destroyed by the intracellular proliferation of Candida cells and formation of germ tubes and pseudomycelia. No significant improvement of Candidacidal activity was observed with macrophages from normal and immunized rabbits in immune serum. The mode of phagocytosis by macrophages and neutrophils were also studied under the scanning electron microscope.

27. We examined the in vitro effect of Candida albicans on NO production by macrophages. Candida albicans suppressed not only NO production but also expression of inducible NO synthase (iNOS) mRNA by murine IFN-γ and bacterial LPS-stimulated peritoneal macrophages. The suppression was not associated with inhibition but rather stimulation of IL-1β production. This effect was observed when more than 1 × 103/ml of Candida albicanswere added to macrophage cultures (1 × 106 cells/ml) and reached a maximal level at 1 × 106/ml. The NO inhibitory effect of Candida albicans was mediated predominantly by as yet unidentified soluble factor(s) and to a lesser extent by direct contact. In addition, heat- or paraformaldehyde-killed Candida albicans did not show this inhibitory activity. Culture supernatant of Candida albicans also inhibited NO production by activated macrophages in a dose-dependent manner, and increased IL-1β production. Finally, the inhibitory effect was not mediated by IL-10 and transforming growth factor- Candida albicans beta (TGF-β), since neutralizing antibodies to these cytokines did not influence -induced reduction in macrophage NO production. Our results suggest that Candida albicans may evade host defence mechanism(s) through a soluble factor-mediated suppression of NO production by stimulated macrophages, and that the effect is independent of production of immunosuppressive cytokines such as IL-10 and TGF-β. Candida albicans, an ubiquitous fungal microorganism, forms part of the normal microflora in the gastrointestinal tract and vagina even in individuals who do not have an apparent immunological dysfunction, suggesting the presence of certain mechanisms that evade the host defence system against this pathogen. It has been well documented that the host defence mechanism against mucosal infection with Candida albicansis mediated mainly by cellular immunity. In experimental models, protection against candidiasis is closely associated with the synthesis of IL-12 and induction of Th1 cells. Neutralization of endogenously synthesized IFN-γ and IL-12 by specific antibodies prevents the development of protective Th1 responses and exacerbates infections with Candida albicans. A number of macrophage-derived cytokines, including IL-10 and TGF-β, exert anti-inflammatory effects by inhibiting the production of proinflammatory cytokines, such as IL-1, IL-6, and tumour necrosis factor-alpha (TNF-α). In addition, these cytokines also suppress the production of NO by IFN-γ-stimulated macrophages.

Several investigators have demonstrated alterations by Candida albicans of the fungicidal activity of phagocytic cells. For example, Hilger & Danley and Danley et al. indicated that live Candida albicans suppressed the release of H2O2 by neutrophils, while dead organisms did not. On the other hand, Smail et al. demonstrated that Candida albicans produced a crude hyphal inhibitory product which inhibited superoxide anion production and release of azurophilic and specific granule components by activated neutrophils. Furthermore, Diamond et al.Candida albicans demonstrated that released small peptides which inhibited adhesion of the fungus and neutrophils. Other investigations also described the suppressive effects of Candida albicans on both cellular and humoral immunity. Considered together, these observations demonstrate the presence of several mechanisms by which Candida albicans evades the host defence systems and multiplies in host tissue. The present results may describe a new mechanism that allows Candida albicans to resist macrophage fungicidal activity.

28. Host resistance against infections caused by the yeast Candida albicans is mediated predominantly by polymorphonuclear leukocytes and macrophages. Antigens of Candida stimulate lymphocyte proliferation and cytokine synthesis, and in both humans and mice, these cytokines enhance the Candidacidal functions of the phagocytic cells. In systemic candidiasis in mice, cytokine production has been found to be a function of the CD4+ T helper (Th) cells. The Th1 subset of these cells, characterized by the production of gamma interferon and interleukin-2, is associated with macrophage activation and enhanced resistance against reinfection, whereas the Th2 subset, which produces interleukins-4, -6, and -10, is linked to the development of chronic disease. However, other models have generated divergent data. Mucosal infection generally elicits Th1-type cytokine responses and protection from systemic challenge, and identification of cytokine mRNA present in infected tissues of mice that develop mild or severe lesions does not show pure Th1- or Th2-type responses. Furthermore, antigens of C. albicans, mannan in particular, can induce suppressor cells that modulate both specific and nonspecific cellular and humoral immune responses, and there is an emerging body of evidence that molecular mimicry may affect the efficiency of anti-Candida responses within defined genetic contexts.

Production and function of cytokines in natural and acquired immunity to Candida albicans infection.

29. This study was designed to test a) whether carbohydrates otherthan glucose decreased the phagocytic capacity of neutrophilsin normal human subjects, b) the duration of this effect, andc) the effect of fasting on neutrophilic phagocytosis. Venousblood was drawn from the arm after an overnight fast and at0.5, 1, 2, 3, or 5 hr postprandial and this was incubated witha suspension of Staphylococcus epidermidis. The phagocytic index(mean number of bacteria viewed within each neutrophil) wasdetermined by microscopic examination of slides prepared withWright’s stain. Oral 100-g portions of carbohydrate from glucose,fructose, sucrose, honey, or orange juice all significantlydecreased the capacity of neutrophils to engulf bacteria asmeasured by the slide technique. Starch ingestion did not havethis effect. The decrease in phagocytic index was rapid followingthe ingestion of simple carbohydrates. The greatest effectsoccurred between 1 and 2 hr postprandial, but the values werestill significantly below the fasting control values 5 hr afterfeeding (P < 0.001). The decreased phagocytic index was notsignificantly associated with the number of neutrophils. Thesedata suggest that the function and not the number of phagocyteswas altered by ingestion of sugars. This implicates glucoseand other simple carbohydrates in the control of phagocytosisand shows that the effects last for at least 5 hr. On the otherhand, a fast of 36 or 60 hr significantly increased (P <0.001) the phagocytic index.

30. The innate immune response was once considered to be a limited set of responses that aimed to contain an infection by primitive ‘ingest and kill’ mechanisms, giving the host time to mount a specific humoral and cellular immune response. In the mid-1990s, however, the discovery of Toll-like receptors heralded a revolution in our understanding of how microorganisms are recognized by the innate immune system, and how this system is activated. Several major classes of pathogen-recognition receptors have now been described, each with specific abilities to recognize conserved bacterial structures.

The first receptor on the surface of macrophages to be described as a mannan receptor was the C‑type-lectin mannose receptor (MR). By contrast, recognition of the shorter linear structures of O‑bound mannan is performed by TLR4, and results in cytokine production. Interestingly, TLR4 stimulation is lost during the germination of yeast into hyphae, which leads to a loss of interferon-γ (IFNγ) production capacity. The lectin domain mediates recognition of both the yeast and hyphal forms of C. albicans, as well as several other fungi. Recognition by CR3 does not trigger protective host responses, such as the respiratory burst, and can repress pro-inflammatory signals. Dectin 1 is a myeloid-expressed transmembrane receptor and possesses a single extracellular, nonclassical C-type-lectin-like domain that specifically recognizes β-(1,3)-glucans. Dectin 1 can recognize several fungi, including C. albicansyeast, although it does not appear to recognize C. albicanshyphae. In addition to mannoproteins, mannans and β-glucans, other structures of C. albicanscan also be recognized as fungal PAMPs. Recently, it has been demonstrated that chitin induces recruitment of immune cells that mainly release IL-4 and IL-13.

More is known about the receptors that are involved in the induction of cytokine production by C. albicans. At least four TLRs (TLR2, TLR4, TLR6 and TLR9) are involved in triggering these responses. Moreover, TLR2 ligands fail to induce

the release of IL-12 and TH1-type IFNγ, thus promoting conditions that are favourable for TH2- or regulatory T cell (TReg)-type responses. C. albicansinduces immunosuppression through TLR2-mediated IL-10 release, and this leads to the generation of CD4+CD25+ TReg cells with immunosuppressive potential. In addition to inducing direct anti-inflammatory effects, C. albicanshas developed strategies to either block or avoid recognition by stimulatory PRRs.

The challenge ahead is to understand the level of complexity that underlies the response that is triggered by pathogen recognition. In this Review, we use the fungal pathogen Candida albicans as a model for the complex interaction that exists between the host pattern-recognition systems and invading microbial pathogens.

An integrated model of the recognition of Candida albicans by the innate immune system

31. In a paper published June 30 in the online version of Hypertension, Frank DeLano, a research scientist at UC San Diego, and Geert Schmid-Schönbein, a professor of bioengineering, describe how they successfully reversed the SHR animals’ symptoms of high blood pressure, a pre-diabetes condition called insulin resistance, and immune suppression. H. Glenn Bohlen, a professor in the Department of Cellular and Integrative Physiology at Indiana University Medical School, wrote in an accompanying editorial in Hypertension that the new study will likely be important to people suffering from obesity as well as hypertension. “With the national and international emphasis on obesity and its attendant cardiovascular problems, there is a tendency to forget that essential hypertension affects about the same percentage of humans as does serious obesity and an even higher percentage of the population than does type 2 diabetes mellitus,” wrote Bohlen. “The elegant study by Delano and Schmid-Schönbein points to a potentially very important overlap of an insulin resistance mechanism with hypertension in the spontaneously hypertensive rat (SHR).”The researchers showed that the SHR animals have protease activity in their circulation that cleaves more than just insulin receptors. In these animals, proteases also cleave significant numbers of CD18, an important binding receptor on the surface of infection-fighting leukocytes. CD18 gives these cells the ability to adhere to the walls of blood vessels as a way to home in on infections. With the loss of CD18 receptors, leukocytes of the SHR animals are unable to bind to the wall of blood vessels, resulting in a compromised immune system. “These results point to a single mechanism that explains multiple and diverse cell dysfunctions encountered in hypertensive rats, and they also suggest that a similar mechanism may be operating in humans suffering simultaneously from hypertension, diabetes, and other metabolic conditions,” said Schmid-Schönbein The team went on to test whether administration of a protease-blocking drug could reverse the multiple metabolic complications in the rat strain. They administered doxycycline, a seemingly unlikely Candidate to have such a beneficial effect. Infectious disease specialists often prescribe doxycycline, an antibiotic, to counter bacterial infections. However, in laboratory tests doxycycline also blocks the activity of certain proteases in the SHR strain of rat. The researchers found that protein receptors on the surface of SHR cells become clipped off as the animals develop hypertension. They used a novel visualization technique to show that after several weeks of ingesting doxycycline in their drinking water, the SHR rats developed cells that again bristled with normal CD18 and insulin receptors. The animals’ metabolic conditions simultaneously improved; blood pressure normalized and symptoms of immune suppression disappeared. Hypertension, insulin resistance and immune suppression links

Frank DeLano, a research scientist at UC San Diego, and Geert Schmid-Schönbein

32. Chronic inflammation rather than invasion is characteristic of some forms of superficial candidiasis such as denture stomatitis. We hypothesized that Candida albicans may play a critical role in the pathogenesis of inflammatory lesions observed in chronic candidiasis by activating the proinflammatory cytokine interleukin-1β (IL-1β) from epithelial stores of the precursor. The aim of this study was therefore to demonstrate the proteolytic cleavage and activation of the inactive precursor of IL-1β (pro-IL-1β) by C. albicans. After incubation of either blastospores or hyphae with the inactive precursor, proteolytic cleavage was monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis Western immunoblotting analysis, and the biological activity of the cleavage products was tested in a bioassay. We report here that late-stationary-growth-phase blastospores as well as hyphae of C. albicans, but not exponentially growing cells, can efficiently cleave pro-IL-1β to yield fragments of molecular masses compatible with mature biologically active IL-1β (17 to 19 kDa). Assays conducted in the presence of selected proteinase inhibitors suggest that the cleavage of pro-IL-1β involves the participation of one or more aspartyl proteinases. Cleavage products showed a dose-dependent IL-1β-like activity in a thymocyte proliferation bioassay, which was inhibited by anti-IL-1β neutralizing antibodies. The present data thus suggest a role for C. albicans proteinases in the activation and maintenance of the inflammatory response at epithelial surfaces.

Proteolytic Activation of the Interleukin-1β Precursor by Candida albicans

33. This study examined CD11/CD18-mediated adhesion in neutrophil emigrationduring acute and recurrent Pseudomonas aeruginosa-induced pneumonia.Neutrophil emigration during acute pneumonia was studied in anti-CD18antibody or murine-IgG-pretreated rabbits 4 hours after intrabronchialinstillation of P. aeruginosa. To examine emigration in recurrentpneumonias, rabbits given P. aeruginosa on day 0 received anti-CD18antibody or IgG on day 7. A second instillate was placed either at theinitial site or in a separate lobe, and emigration into alveolar spaces wasquantitated morphometrically after 4 hours. The results show that CD11/CD18was required for neutrophil emigration in acute pneumonias and in recurrentpneumonias that occurred at a site distant from the initial infection.However, when the recurrent pneumonia occurred in the previously inflamedsite, CD11/CD18 was not required. When the same number of organisms wereinstilled on days 0 and 7, emigration was reduced to 15 to 20 percent ofthe number that migrated initially and only CD18-independent adhesionpathways were used. Increasing the concentration of organisms threefoldincreased emigration through both CD18-dependent and CD18-independentpathways. These data indicate that P. aeruginosa inducesCD11/CD18-dependent emigration during acute pneumonia and recurrentpneumonia at previously uninflamed sites. However, adhesion pathways arealtered in regions of chronic inflammation, and a greater proportion ofneutrophil emigration occurs through CD11/CD18-independent pathways.

Role of CD 11/CD 18 in neutrophil emigration during acute and recurrent Pseudomonas aeruginosa-induced pneumonia in rabbits

34. Some secreted proteins do not remain cell associated but forage into the extracellular environment. If such proteins are hydrolytic enzymes, they have capacity to hydrolyze large or complex substrates into small units that can be transported into the cell as a source of nutrition. If the degradation of host targets facilitates colonization or invasion, then such enzymes also function as virulence factors. In the last few years, several studies have examined clinical isolates for one or more phospholipase, proteinase, or hemolytic activities. These studies demonstrate the production of these activities by some but not all isolates. There are differences based on the site of isolation or the presence of type 2 diabetes mellitus. One of the characteristics of most of the hydrolytic activities is that there are multiple enzymes that may be expressed under different conditions.

35. The study indicates that host age is a determining factor in yeast carriage. From the neonatal period, humans go through several dentition periods, and the emergence and substitution of teeth and changes in living habits greatly change the environment of the oral cavity, and therefore influence colonization by oral commensal organisms, certainly

including Candida spp. Russell and Lay showed that the frequency of oral yeast carriage at birth was low, doubled by the time that infants were discharged from the hospital at about seven-day old, and increased sharply after one month old. Kleinegger et al. demonstrated that the frequency of oral yeast carriage was 44% of the examined individuals in a group aged from 0.5 to 1.5, 24% in a 5-7-year-old group, 40% in a 15- to 18-year-old group.

In a review by Odd in 1988, the highest reported frequencies were 71% of school children in the United Kingdom, and 56% of children in Israel. Different sampling and identification methods for Candida spp. would also certainly influence the results. Because of the uneven distribution of C. albicans throughout the oral cavity, swab samples can yield false-negative culture more often than oral rinse samples or imprint culture. The number of false-negative cultures was reduced by the swabbing of two sitesrather than one. In conclusion, our observations indicate that there is an increased risk of dental caries with C. albicans carriage rates in preschool and school children.

Oral Candida albicans carriage in healthy preschool and school children

36. The prevalence of opportunistic fungal infections has increaseddramatically among the aged population in recent years. Thiswork investigated the effect of ageing on murine defences againstCandida albicans. Aged C57BL/6 mice that were experimentallyinfected intravenously had a significantly impaired survivaland a higher tissue fungal burden compared with young mice.In vitro production of tumour necrosis factor (TNF)- by macrophagesfrom aged mice in response to yeast cells and hyphae of C. albicanswas significantly lower than production by macrophages fromyoung mice. In vitro production of cytokines, such as TNF- andgamma interferon (IFN-), by antigen-stimulated splenocytes frommice intravenously infected with C. albicans cells was alsodiminished in old mice. This decrease in production of T helper1 cytokines in old mice correlated with a diminished frequencyof IFN–producing CD4+ T lymphocytes, although the ability todevelop an acquired resistance upon vaccination (primary sublethalinfection) of mice with the low-virulence PCA2 strain was notaffected in aged mice. The diversity of antigens recognizedby C. albicans-specific antibodies in sera from infected agedmice was clearly diminished when compared with that from infectedyoung mice. Taken together, these data show that aged mice developan altered innate and adaptive immune response to C. albicansand are more susceptible to systemic primary candidiasis.

37. Previous work by our group showed that aged C57BL/6 mice develop an altered innate and adaptive immune response to Candida albicans and are more susceptible to systemic primary candidiasis. In this work, we used young (2-3 months old) and aged (18-20 months old) C57BL/6 mice to study in vitro the influence of aging on (1) the fungicidal activity of neutrophils and macrophages, (2) the production of cytokines by resident peritoneal macrophages in response to C. albicans, and (3) cell surface Toll-like receptor (TLR) 2 expression on resident peritoneal macrophages. Our results indicate that murine phagocytes have a fungicidal activity well preserved with aging. In vitro production of proinflammatory cytokines (IL-6, IL-1beta, and tumor necrosis factor-alpha and chemokines (MIP-2) by purified (CD11b(+)) peritoneal macrophages in response to yeasts and hyphae of C. albicans was significantly lower in aged mice as compared with young mice. However, the production of IL-10 by macrophages, in response to C. albicans, was similar in both young and aged animals. Moreover, baseline TLR2 surface expression level was lower on aged macrophages than on control macrophages. Taken together, these data indicate that the increased susceptibility to C. albicans disseminated infections in aged mice is correlated with defects in TLR2 expression and in cytokine production, but not with an impaired fungicidal activity.

Influence of aging on murine neutrophil and macrophage function against Candida albicans.

38. The pathogenic yeast Candida albicans and its derived moleculesstimulate a wide range of macrophage secretory functions and mayadapt to escape being killed by this phagocyte. In this study, phagocytosisof C. albicans and of the nonpathogenic yeast Saccharomycescerevisiae was shown to be associated with phosphorylation ofthe mitogen-activated protein kinase (MAPK)/extracellularlyregulated kinase (ERK) pathway in the absence of significantactivation of either p38MAPK or stress-activated protein kinase/c-JunN-terminal kinase. However, although 80% of endocytosed C. albicanssurvived after 1 h, 80% of S. cerevisiae cells were killed.Considerable quantitative differences were observed betweenthe two species in the sequential phosphorylation of MAPK/ERKkinase (MEK), extracellularly regulated kinase-1, and 90-kDa-ribosomalS6 kinases. A lower level of activation of the pathway by C.albicans was associated with a species-specific overexpressionof the MEK phosphatase MAPK phosphatase (MKP)-1. Killing ofboth C. albicans and S. cerevisiae could be reduced using PD98059,which mimics MKP-1 and inhibits MEK phosphorylation, suggestingthat specific MKP-1 activation by C. albicans could contributeto its ability to escape the yeast lytic potential of macrophages.

Role of extracellular signal-regulated protein kinase cascade in macrophage killing of Candida albicans

39. Candida albicans is the most common opportunistic fungal pathogen of humans, causing systemic disease in immunocompromised patients. Host resistance to C. albicans infections is mediated predominantly by neutrophils and monocytes/macrophages. We have previously shown that exposure of a human epithelial cell line (HEp2) to C. albicans or to a culture filtrate of C. albicans caused actin rearrangement in the HEp2 cells. Since shifting of actin from the filamentous to the globular form may be crucial to the activity of phagocytes, we assessed in the present study the effect of the C. albicans metabolite (lyophilized culture filtrate) on the cytoskeleton of murine peritoneal macrophages and on their phagocytic activity. Our results showed a significant decrease in phagocytosis of C. albicans, ranging from 53-63% and a 25% reduction for C. glabrata cells. Using confocal laser scanning microscopy an actin rearrangement in the macrophages could be demonstrated that may be associated with the decrease of phagocytosis. We also tested the effect of mannan and of the secreted aspartic proteinase (Sap) inhibitor–pepstatin, on the activity of the metabolite in order to define the putative component and found no influence. In conclusion, our data indicate that a C. albicans metabolite affects phagocytic activity of macrophages, probably by alterations in their cytoskeleton.

40. Macrophages are widely distributed immune system cells that play an indispensable role in homeostasis and defense. They can be phenotypically polarized by the microenvironment to mount specific functional programs. Polarized macrophages can be broadly classified in two main groups: classically activated macrophages (or M1), whose prototypical activating stimuli are IFNgamma and LPS, and alternatively activated macrophages (or M2), further subdivided in M2a (after exposure to IL-4 or IL-13), M2b (immune complexes in combination with IL-1beta or LPS) and M2c (IL-10, TGFbeta or glucocorticoids). M1 exhibit potent microbicidal properties and promote strong IL-12-mediated Th1 responses, whilst M2 support Th2-associated effector functions. Beyond infection M2 polarized macrophages play a role in resolution of inflammation through high endocytic clearance capacities and trophic factor synthesis, accompanied by reduced pro-inflammatory cytokine secretion. Similar functions are also exerted by tumor-associated macrophages (TAM), which also display an alternative-like activation phenotype and play a detrimental pro-tumoral role. Here we review the main functions of polarized macrophages and discuss the perspectives of this field.

41. Macrophages are cells that function as a first line of defence against invading microorganisms. One of the hallmarks of macrophages is their ability to become activated in response to exogenous ‘danger signals’. Most microbes have molecular patterns (PAMPS) that are recognized by macrophages and trigger this activation response. There are many aspects of the activation response to PAMPS that are recapitulated when macrophages encounter endogenous danger signals. In response to damaged or stressed self, macrophages undergo physiological changes that include the initiation of signal transduction cascades from germline-encoded receptors, resulting in the elaboration of chemokines, cytokines and toxic mediators. This response to endogenous mediators can enhance inflammation, and thereby contribute to autoimmune pathologies. Often the overall inflammatory response is the result of cooperative activation signals from both exogenous and endogenous signals. Macrophage activation plays a critical role, not only in the initiation of the inflammatory response but also in the resolution of this response. The clearance of granulocytes and the elaboration of anti-inflammatory mediators by macrophages contribute to the dissolution of the inflammatory response. Thus, macrophages are a key player in the initiation, propagation and resolution of inflammation. This review summarizes our understanding of the role of macrophages in inflammation. We pay particular attention to the endogenous danger signals that macrophages may encounter and the responses that these signals induce. The molecular mechanisms responsible for these responses and the diseases that result from inappropriately controlled macrophage activation are also examined.

42. Phagocytosis is the first step of defense against infections from the innate immune system, as it is the process of internalization of pathogens by cells with phagocytic activity, such as macrophages, which is followed by pathogen killing and destruction. Thus, phagocytosis assays are used as assays for one function of the innate immune system. As fungal infections are of increasing relevance and phagocytic mechanisms are dependent on the pathogenic organism and its viability, we established a microtiter plate phagocytosis assay based on viable, fluorescence-labeled Candida albicans. The distinction between internalized yeast cells and cells attached to macrophages was done via quenching of FITC—fluorescence by trypan blue, and the remaining fluorescence was quantified and used as indicator of the phagocytosis efficiency. As a proof of principle we showed that compounds acting on the dynamics of the actin cytoskeleton of the macrophages reduced the phagocytosis efficiency in a concentration dependent manner.

Phagocytosis Assay Based on Living Candida albicans for the Detection of Effects of Chemicals on Macrophage Function

43. Production of reactive oxygen species (ROS) is an important aspect of phagocyte-mediated host responses. Since phagocytes play a crucial role in the host response to Candida albicans, we examined the ability of Candida to modulate phagocyte ROS production. ROS production was measured in the murine macrophage cell line J774 and in primary phagocytes using luminol-enhanced chemiluminescence. J774 cells, murine polymorphonuclear leukocytes (PMN), human monocytes, and human PMN treated with live C. albicans produced significantly less ROS than phagocytes treated with heat-killed C. albicans. Live C. albicans also suppressed ROS production in murine bone marrow-derived macrophages from C57BL/6 mice, but not from BALB/c mice. Live C. albicans also suppressed ROS in response to external stimuli. C. albicans and Candida glabrata suppressed ROS production by phagocytes, whereas Saccharomyces cerevisiae stimulated ROS production. The cell wall is the initial point of contact between Candida and phagocytes, but isolated cell walls from both heat-killed and live C. albicans stimulated ROS production. Heat-killed C. albicans has increased surface exposure of 1,3-beta-glucan, a cell wall component that can stimulate phagocytes. To determine whether surface 1,3-beta-glucan exposure accounted for the difference in ROS production, live C. albicans cells were treated with a sublethal dose of caspofungin to increase surface 1,3-beta-glucan exposure. Caspofungin-treated C. albicans was fully able to suppress ROS production, indicating that suppression of ROS overrides stimulatory signals from 1,3-beta-glucan. These studies indicate that live C. albicans actively suppresses ROS production in phagocytes in vitro, which may represent an important immune evasion mechanism.

Live Candida albicans suppresses production of reactive oxygen species in phagocytes.

44. The two-stage neuroinflammatory process, containment and progression, proposed to underlie neurodegeneration may predicate on systemic inflammation arising from the gastrointestinal tract. Helicobacter infection has been described as one switch in the pathogenic-circuitry of idiopathic parkinsonism (IP): eradication modifies disease progression and marked deterioration accompanies eradication-failure. Moreover, serum Helicobacter-antibody-profile predicts presence, severity and progression of IP. Slow gastrointestinal-transit precedes IP-diagnosis and becomes increasingly-apparent after, predisposing to small-intestinal bacterial-overgrowth (SIBO). Although IP is well-described as a systemic illness with a long prodrome, there has been no comprehensive overview of the blood profile. Here, it is examined in relation to Helicobacter status and lactulose-hydrogen-breath-testing for SIBO. A robust finding of reduced lymphocyte count in 126 IP-probands and 79 spouses (without clinically-definite IP), compared with that in 381 controls (p<0.001 in each case), was not explained by Helicobacter-status or breath-hydrogen. This complements a previous report that spouses were ‘down-the-pathway’ to ‘clinically-definite’ disease. In 205 other controls without clinically-definite IP, there were strong associations between sporadic cardinal features and immunoglobulin class concentration, not explained by Helicobacter-status. Premonitory states for idiopathic parkinsonism associated with relative lymphopenia, higher serum immunoglobulin concentrations and evidence of enteric-nervous-system damage may prove viral in origin. Although only 8% of the above 79 spouses were urea-breath-test-positive for Helicobacter, all 8 spouses with clinically-definite IP were (P<0.0001). Transmission of a ‘primer’ to a Helicobacter-colonised recipient might result in progression to the diagnostic threshold. Twenty-five percent of the 126 probands were seropositive for anti-nuclear autoantibody. In 20 probands, monitored before and serially after anti-Helicobacter therapy, seropositivity marked a severe hypokinetic response (p=0.03). It may alert to continuing infection, even at low-density. Hyperhomocysteinemia is a risk factor for dementia and depression. Serum homocysteine exceeded the target in 43% of the 126 IP-probands. It was partially explained by serum B12 (12% variance, p<0.001), but not by Helicobacter-status (gastric-atrophy uncommon in IP) or levodopa treatment. Immune-inflammatory activation increases homocysteine production. Since an estimated 60% of probands are hydrogen-breath-test positive, SIBO, with its increased bacterial utilisation of B12, is a likely cause. Thus, two prognostic indicators in established IP fit with involvement of Helicobacter and SIBO.

Blood profile holds clues to role of infection in a premonitory state for idiopathic parkinsonism and of gastrointestinal infection in established disease.

45. Chronic inflammatory diseases like rheumatoid arthritis, inflammatory bowel disease, psoriasis, and liver disease cause “sickness behaviors,” including fatigue, malaise, and loss of social interest. However, it has been unclear how inflammation in other organs in the body can impact the brain and behavior. The researchers found that in mice with inflamed livers, white blood cells called monocytes infiltrated the brain. These findings support previous research demonstrating the presence of immune cells in the brain following organ inflammation, challenging the long-held belief that the blood-brain barrier prevents immune cells from accessing the brain.

Liver inflammation also stimulated cells in the blood to make an immune chemical (TNFα). When the researchers blocked the signaling of this immune chemical, microglia produced less CCL2, and monocytes stayed out of the brain. In the mice with inflamed livers, preventing the entry of monocytes into the brain reduced sickness behaviors; mice showed more mobility and social interaction. These findings suggest that people with chronic inflammatory diseases may benefit from treatments that limit monocyte access to the brain. “Sickness behavior significantly impacts quality of life. Our findings further our understanding and may generate potential new avenues for treatment of these often crippling symptoms,” said Swain. “The brain is the master coordinator of many of our bodies’ defense responses, so it must be able to sense injury and inflammation in distant body organs. This study starts to explain the peripheral communication signals that activate the brain,” said Nancy Rothwell, PhD, DSc, at the University of Manchester, an expert on brain inflammation who is unaffiliated with the study.

46. Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases that are a subfamily of metzincins. Matrix metalloproteinases are responsible for much of the turnover of extracellular matrix components and are key to a wide range of processes including tissue remodeling and release of biological factors. Imbalance between the MMPs and endogenous tissue inhibitors of metalloproteinases (TIMPs) can result in dysregulation of many biologic processes and lead to the development of malignancy, cardiovascular disease, and autoimmune and inflammatory disorders. MMP production by monocyte/macrophages is dependent on the cell type, state of differentiation, and/or level of activation and whether they are infected, e.g., by HIV-1. MMP expression by

HIV-1 infected monocytes and macrophages may alter cellular trafficking and contribute to HIVassociated pathology such as HIV-associated dementia (HAD). This review will provide a classification of the MMP super-family with particular reference to those produced by monocyte/macrophages, describe their regulation and function within the immune system, and indicate their possible roles in the pathogenesis of disease, including

HIV-associated dementia.

Dysregulation in the levels and control of MMPs can lead to pathological processes (including tumor growth and migration, arthritis, cirrhosis, aortic aneurysms, and fibrosis) and diseases (such as glaucoma, lupus, scleroderma, multiple sclerosis, and HIV-1 associated dementia). MMPs play an important role in immunological functions including ECM for leukocyte migration, modulating chemokine, and cytokine activity through both their activation and inactivation and defensins activation.

MMPs regulate numerous biologic processes through their proteolytic function, in both normal and pathological states. They alter the cellular milieu and cell behavior through proteolytic turnover of matrix components, by releasing molecules expressed on the surface of cells and by cleaving cell surface receptors or cell-cell adhesion proteins.

Cells of the monocyte/macrophage lineage, including blood monocytes, dendritic cells, and tissue macrophages such as microglia secrete diverse MMPs in large quantities.

Induction of MMPs can modulate DC functions including DC migration through endothelial barriers. Cell migration is critical for dendritic cells (DCs) in the initiation of the immune response.

HIV-infected patients frequently report gingival inflammation and may develop progressive periodontal tissue breakdown or require dental extraction due to weakened attachment. HIV-related periodontal disease, including gingivitis; periodontitis; and bacterial, viral and fungal infections has been associated with increased levels of MMP-1, -3, and -8 in saliva from HIV-infected individuals, and these MMPs may play a role in the development of HIV-associated periodontitis.

Matrix metalloproteinases, their production by monocytes and macrophages and their potential role in HIV-related diseases

47. A variety of morphological changes in the basement membrane (BM) are known to occur in inflammatory diseases. Modifications of the BM can be associated with significant changes in protein content. Candida albicans (C. albicans) is normally a commensal organism and is a member of the natural flora of a large number of healthy individuals. However, under certain conditions, C. albicans can invade host tissues, causing inflammation and tissue damage. The aim of this study was to investigate the effect of C. albicans on the expression and production of structural (laminin-5 and type IV collagen) and inflammatory [matrix metalloproteinases (MMPs) and their inhibitors] proteins by human oral epithelial cells. Using engineered normal human oral mucosa infected with 10(5) C. albicans/cm2 for different periods of time, we were able to demonstrate that this yeast promotes significant laminin-5 and type IV collagen gene activation and protein secretion. These effects were accompanied by MMP-2 and MMP-9 gene activation. Interestingly, only the levels of active MMP-9 rose. The increase in MMP levels was paralleled by a decrease in the secretion of type 2 matrix metalloproteinase tissue inhibitors (TIMP-2). Our results demonstrated that C. albicans has a significant effect on tissue structure through BM protein and MMP modulation. This might help C. albicans overcome the mechanical and biological defenses of the tissue and allow it to disseminate, causing severe infections. If C. albicans uses MMPs (mainly MMP-9) to disseminate, inhibition of this protease could be of interest in treating a variety of inflammatory disorders, including oral candidiasis.

48. Dendritic cells (DC) function as professional phagocytes to kill Candida albicans and subsequently present it to the adaptive immune system. Monocytes, macrophages and DC were generated from five individual donors and their Candida-killing capacity and cytokine release were assessed. Compared to monocytes and macrophages, DC from healthy volunteers were significantly less effective in C. albicans-stimulated cytokine release, killing of C. albicans blastoconidia and damaging of C. albicans hyphae. In conclusion, while important as antigen-presenting cells and initiators of the adaptive immune system, DC are poor in both intracellular killing and damaging of C. albicans hyphae. Effective handling of large numbers of C. albicans is the prime task of the innate immune system consisting of large numbers of neutrophils and monocytes.

Human dendritic cells are less potent at killing Candida albicans than both monocytes and macrophages

49. Morphological plasticity of Candida albicans is a major virulence factor. Using pH-dependent dimorphism we show, that human dendritic cells (DC) recognize filamentous forms and blastoconidia of a virulent C. albicans isolate (strain SC5314). Heat inactivated and viable blastoconidia are rapidly phagocytosed by human DC. However, viable yeast cells start to filament inside the DC at later stages of infection, leading to penetration and loss of cellular integrity. The cytokine burst of human DC induced upon contact with Candida is dominated by the granulocyte-activating, chemotactic factor IL-8 and the proinflammatory mediator TNF-alpha. Blastoconidia induce markedly lower cytokine levels than filamentous forms. Whereas IL-8 secretion is mainly cell mass dependent, release of TNF-alpha, a major proinflammatory cytokine, is clearly dependent on the morphology of Candida.

Polymorphism of Candida albicans is a major factor in the interaction with human dendritic cells.

50. Dendritic cells (DC) are antigen presenting cells that act as sentinels, acquiring antigen and transporting it to lymphoid tissue where they have the unique ability to activate naïve T cells. From this pivotal position at the intersection of innate and adaptive immunity, DC shape many aspects of the developing immune response. They can determine whether non-responsiveness (tolerance) or an active immune response occurs, whether a type 1 or type 2 response predominates, and they may control tissue specific homing of antigen specific effector cells. Microbial products play a central role in modulating DC function and influencing these different immune outcomes. Using molecules including toll-like receptors, DC recognise and respond to microbe specific molecular structures. DC can distinguish between and initiate different responses to even closely related organisms. Emerging evidence suggests that intestinal DC are critical for regulation of immunity in the gut. They are likely to be pivotal in the balance between tolerance and active immunity to commensal microorganisms that is fundamental to inflammatory conditions, including Crohn’s disease and ulcerative colitis. Here, we describe how interactions between DC and microbial products contribute to regulation of immune responses and explore the concept that interactions between DC and commensal organisms may be responsible for maintaining intestinal immune homeostasis.

The dendritic cell: its role in intestinal inflammation and relationship with gut bacteria

51. Candida albicans is a component of the normal flora of the alimentary tract and also is found on the mucocutaneous membranesof the healthy host. Candida is the leading cause of invasivefungal disease in premature infants, diabetics, and surgical patients,and of oropharyngeal disease in AIDS patients. As the inductionof cell-mediated immunity to Candida is of critical importancein host defense, we sought to determine whether human dendriticcells (DC) could phagocytose and degrade Candida and subsequentlypresent Candida antigens to T cells. Immature DC obtained by cultureof human monocytes in the presence of granulocyte-macrophage colony-stimulatingfactor and interleukin-4 phagocytosed unopsonized Candida in atime-dependent manner, and phagocytosis was not enhanced by opsonizationof Candida in serum. Like macrophages (M), DC recognized Candidaby the mannose-fucose receptor. Upon ingestion, DC killed Candidaas efficiently as human M, and fungicidal activity was not enhancedby the presence of fresh serum. Although phagocytosis of Candidaby DC stimulated the production of superoxide anion, inhibitorsof the respiratory burst (or NO production) did not inhibit killingof Candida, even when phagocytosis was blocked by preincubationof DC with cytochalasin D. Further, although apparently only modestphagolysosomal fusion occurred upon DC phagocytosis of Candida,killing of Candida under anaerobic conditions was almost equivalentto killing under aerobic conditions. Finally, DC stimulated Candida-specificlymphocyte proliferation in a concentration-dependent manner afterphagocytosis of both viable and heat-killed Candida cells. Thesedata suggest that, in vivo, such interactions between DC and C.albicans may facilitate the induction of cell-mediatedimmunity.

52. The fungus Candida albicans behaves as a commensal as well asa true pathogen of areas highly enriched in dendritic cells,such as skin and mucosal surfaces. The ability of the fungusto reversibly switch between unicellular yeast to filamentousforms is thought to be important for virulence. However, whetherit is the yeast or the hyphal form that is responsible for pathogenicityis still a matter of debate. Here we show the interaction, andconsequences, of different forms of C. albicans with dendriticcells. Immature myeloid dendritic cells rapidly and efficientlyphagocytosed both yeasts and hyphae of the fungus. Phagocytosisoccurred through different phagocytic morphologies and receptors,resulting in phagosome formation. However, hyphae escaped thephagosome and were found lying free in the cytoplasm of thecells. In vitro, ingestion of yeasts activated dendritic cellsfor interleukin (IL)-12 production and priming of T helper type1 (Th1) cells, whereas ingestion of hyphae inhibited IL-12 andTh1 priming, and induced IL-4 production. In vivo, generationof antifungal protective immunity was induced upon injectionof dendritic cells ex vivo pulsed with Candida yeasts but nothyphae. The immunization capacity of yeast-pulsed dendriticcells was lost in the absence of IL-12, whereas that of hypha-pulseddendritic cells was gained in the absence of IL-4. These resultsindicate that dendritic cells fulfill the requirement of a celluniquely capable of sensing the two forms of C. albicans interms of type of immune responses elicited. By the discriminativeproduction of IL-12 and IL-4 in response to the nonvirulentand virulent forms of the fungus, dendritic cells appear tomeet the challenge of Th priming and education in C. albicanssaprophytism and infections.

Dendritic Cells Discriminate between Yeasts and Hyphae of the Fungus Candida albicans: Implications for Initiation of T Helper Cell Immunity in Vitro and in Vivo

53. The ability of Candida albicans to convert from the yeast (Y) form to mycelial forms through germ tube (GT) formation is considered a key feature of the transition of the organism from commensalism to virulence. We show here that human monocytes cultured with granulocyte-macrophage colony-stimulating factor and interleukin-4 (IL-4) after phagocytosis of Y forms did not differentiate into dendritic cells (DCs); they retained CD14, did not acquire CD1a, and were unable to express the maturation markers CD83 and CCR7. Moreover, they did not produce IL-12p70 but secreted IL-10. In addition, they spontaneously expressed high levels of tumor necrosis factor alpha (TNF-α), IL-6, and IL-8 mRNA transcripts and were able to induce proliferation of alloreactive memory but not naïve T lymphocytes. Conversely, monocytes that had phagocytosed GT forms differentiated into mature CD83+ and CCR7+ DCs; however, there was no up-regulation of CD40, CD80, and major histocompatibility complex class II, irrespective of lipopolysaccharide (LPS) treatment. In addition, these cells were unable to produce IL-12 even after LPS stimulation, but they were not functionally exhausted, as shown by their capacity to express TNF-α and IL-8 mRNA transcripts. These cells were able to prime naïve T cells but not to induce their functional polarization into effector cells. These data indicate that phagocytosis of Y and GT forms has profound and distinct effects on the differentiation pathway of monocytes. Thus, the differentiation of human monocytes into DCs appears to be tunable and exploitable by C. albicans to elude immune surveillance.

54. A new study has uncovered the genetic wiring diagram underlying the infectiousness of Candida albicans, a fungus that causes thrush in babies, vaginal infections in women, and life-threatening infections in chemotherapy and AIDS patients. The study, led by Dr. Gerald R. Fink, Director of the Whitehead Institute for Biomedical Research, reveals that one key to Candida’s infectiousness lies in its ability to switch from a rounded form to filamentous forms. Fungal infections in hospitalized patients have almost doubled throughout the 1980s, often with life-threatening results in individuals with weakened immune systems. Candida, in particular, poses a serious threat and is associated with high mortality rates in patients undergoing chemotherapy. Candida is also a major cause of infection in hospitalized patients, especially those in Intensive Care Units, patients after major injuries or surgery, patients with burns, and premature babies.

In this study, Dr. Fink and his colleagues used molecular biology techniques to identify the components of the filamentation circuit in yeast. With the recently completed yeast genome to guide them, the scientists began to knock out suspicious genes and, by a process of elimination, discovered the culprits that are responsible for filamentation. Once scientists identified the key yeast filamentation genes, they simply plucked out the analogous genes in Candida. “Candida albicans is three hundred million years apart evolutionarily from yeast-as far away in evolution as humans are from turtles-and yet, the basic logic circuit was conserved,” says Dr. Fink.

Discovery Of Genetic Pathways May Provide New Ways To Combat Candida Infections

55. Clinical studies show that any modification of the host immune status can facilitate the proliferation of endogenous Candida which, according to the importance of the immune deficiency, can provoke diseases ranging from benign localized mucocutaneous candidosis to sometimes lethal systemic invasions. The pathogenic behavior of Candida cells is mainly due to a very high phenotypic biodiversity. Following even very slight environmental modifications, it may change its behavior through the appearance of new or amplified properties such as tube formation, adherence, protease secretion, etc. Together with the impairment of host defenses, these new invasive properties lead to the so-called opportunistic pathogenicity of Candida cells. From a host point of view, after the integrity of surface teguments, the mucosal protection is ensured by the Th1 “cellular” immune response which, through pro-inflammatory cytokine production, boosts the efficacy of the phagocytes (Polymorphonuclear cells and macrophages). Neutrophils are of particular importance as deep seated Candida proliferation is mostly associated with neutropenia. Whatever the pathogenic process, it is mostly due to modifications provoked by increasing medical awareness which makes patients more susceptible to illness. A better knowledge of the precise mechanisms involved and would lead to improved strategies for prevention.

Stress is an often forgotten cause of temporary immunodeficiency. Neuroendocrine regulation and chronobiological effects may notably modulate the immune system and provide the opportunity of fungal proliferation.

As Wilson (1962) put it: “C. albicansis a better clinician and can discover abnormalities in persons much earlier in the course of the development of such abnormalities than we can with our chemical tests”.

The pathogenic behaviour of Candidamay appear following even a slight modification of the host.

56. Substantial clinical evidence has been accumulated, showing that candidiasis is a medical problem of increasing magnitude. That patients on antibiotics experience proliferation of Candida albicans in the alimentary canal is no longer a point for dispute. That the increased incidence of severe fungus infections is associated with antibiotic therapy, particularly in patients with subnormal defense mechanisms, is being increasingly accepted. A recent report has presented an analysis of published experimental and clinical data which indicate that the two clinically disparate conditions-the ostensibly benign proliferation of C. albicans, and the invasion and spread of the organism in debilitated patients-are interrelated. This report presents evidence that the antibiotics enhance the invasiveness of the C. albicans, not only by a direct effect on the intestinal flora, and on the Candida itself, but also by depressing the host defense mechanisms.

Mechanisms by Which Antibiotics Increase the Incidence and Severity of Candidiasis and Alter the Immunological Defenses

57. Mammals are superorganisms, being a composite of mammalian and microbial cells existing in symbiosis. Although the microbiota is not essential for life, commensal and intestinal epithelial cell interactions are critical for the maturation of the immune system. Antibiotic treatment alters this delicate balance by causing compositional changes in the intestinal microbiota, and may lead to a homeostatic imbalance through alterations in expression of IEC tight junction proteins, mucin, antimicrobial peptides, and cytokines. Dysregulation of the homeostasis between mammals and their intestinal symbionts has been shown to predispose the host to enteric infection, and may lead to development of inflammatory bowel diseases. The composition of the microbiota is significantly affected by the use of antibiotics, which are often used extensively, and can lead to antibiotic-associated diarrhea and development of secondary infections such as urinary tract infections. The alteration in microbiota composition is believed to reduce carbohydrate fermentation and impair metabolism of bile acids, as well as creating niches for pathogens to proliferate. A recent investigation by Sekirov et al. has shown that antibiotic-mediated disturbance in the composition, but not total numbers of the intestinal microbiota predisposes mice to higher colonization by Salmonella typhimurium and more severe pathology. This shows that altering the microbiota composition, without creating vacant niches in the microbial community, predisposes the host to enteric infection. The intestinal epithelium and its protective mucus layer cover are the primary defenses against pathogen permeation and commensal leakage into the underlying LP. Colonization of the gut by probiotics results in protection of the epithelial barrier by maintaining tight junction protein expression and preventing apoptosis upon chemically induced colitis. Therefore, changing the composition of the microbiota, through antibiotic administration, could change the strength of the IEC barrier through alterations in tight junction protein expression. Decreased expression of tight junction proteins would increase the permeability of the IEC barrier allowing commensal leakage into the underlying LP, leading to inflammation that is indicative of IBD. Current findings suggest that probiotic strains may protect the host from intestinal inflammation by induction of mucus-associated genes, which strengthens the mucus barrier and protects against colonization by enteric pathogens. Impaired antimicrobial defense results in enhanced bacterial penetration into the LP resulting in an inflammatory response and tissue damage. Antimicrobial proteins secreted by IECs (enterocytes and paneth cells) include defensins, cathelicidins, and C-type lectins (RegIIIβ and RegIIIγ). They function by disrupting bacterial surface structures and contribute to the maintenance of microbiota composition. A recent study showed that administration of a combination of the broad-spectrum antibiotics metronidazole, neomycin, and vancomycin led to significant depletion of the microbiota and decreased expression of RegIIIγ by IECs. RegIIIγ expression has been shown to rely on IEC stimulation by microbes and their products. This decrease in both microbiota and RegIIIγ resulted in increased intestinal colonization by vancomycin-resistant Enterococcus.

59. Secreted aspartyl proteinases (Saps) are important virulencefactors of Candida albicans during mucosal and disseminatedinfections and may also contribute to the induction of an inflammatoryhost immune response. These data show that the potential of specific Saps to causetissue damage correlates with an epithelium-induced proinflammatorycytokine response, which may be crucial in controlling and managingC. albicans infections at the vaginal mucosa in vivo. A characteristic feature of vaginal candidiasis is a chronicinflammation of the mucosa. Candida albicans, normally a commensalhabitant of mucosal surfaces, is the most frequent cause ofthis type of infection.

60. Potent drugs for the treatmentof C. albicans infections are available; however, problems withthe toxicity of amphotericin B and the development of resistanceto the other drugs have stimulated the search for new pharmaceuticalswith different drug targets. Secretedaspartic proteases (Saps) are known virulence factors of C.albicans, and many investigations performed in the last decadeshave revealed that these enzymes contribute to the pathogenicityof C. albicans in different ways. The Saps can provide nutrientsby degrading host proteins but also support adherence to hostsurfaces and invasion of tissue barriers. Doxycycline-induced expressionof most of the SAP genes allowed the growth of the sap2 mutants.Dense growth of transformants expressing SAP1, SAP2, SAP3, andSAP8 was reached after 48 h at pH 4.0 and 30°C, whereasgrowth of strains expressing SAP4, SAP5, and SAP6 was strongestafter 90 to 120 h at pH 5.0 and 37°C.

61. A well-known virulence attribute of the human-pathogenic yeastCandida albicans is the secretion of aspartic proteases (Saps),which may contribute to colonization and infection of differenthost niches by degrading tissue barriers, destroying host defencemolecules, or digesting proteins for nutrient supply. The roleof individual Sap isoenzymes, which are encoded by a large genefamily, for the pathogenicity of C. albicans has been investigatedby assessing the virulence of mutants lacking specific SAP genesand by studying the expression pattern of the SAP genes in variousmodels of superficial and systemic infections. Even mutants lacking all of the SAP1–SAP3 orthe SAP4–SAP6 genes displayed the same capacity to invadeand damage both oral and vaginal RHE as their wild-type parentalstrain, in contrast to a nonfilamentous efg1 mutant that wasavirulent under these conditions. We therefore conclude fromthese results that the secreted aspartic proteases Sap1p–Sap6pare not required for invasion of reconstituted human epithelia RHE by C. albicans.

Secreted aspartic proteases are not required for invasion of reconstituted human epithelia by Candida albicans

62. Candida albicans is a facultative pathogenic micro-organism that has developed several virulence traits enabling invasion of host tissues and avoidance of host defence mechanisms. Virulence factors that contribute to this process are the hydrolytic enzymes. Most of them are extracellularly secreted by the fungus. The most discussed hydrolytic enzymes produced by C. albicans are secreted aspartic proteinases (Saps). The role of these Saps for C. albicans infections was carefully evaluated in numerous studies, whereas only little is known about the physiological role of the secreted phospholipases (PL) and almost nothing about the involvement of lipases (Lip) in virulence. Saps as the best-studied member of this group of hydrolytic enzymes contribute to host tissue invasion by digesting or destroying cell membranes and by degrading host surface molecules. There is also some evidence that hydrolytic enzymes are able to attack cells and molecules of the host immune system to avoid or resist antimicrobial activity. High hydrolytic activity with broad substrate specificity has been found in several Candida species, most notably in C. albicans. This activity is attributed to multigene families with at least 10 members for Saps and Lips and several members for PLB.

63. Vaginal infections caused by the opportunistic yeast Candidaalbicans are a significant problem in women of child-bearingage. Several factors are recognized as playing a crucial rolein the pathogenesis of superficial candidiasis; these factorsinclude hyphal formation, phenotypic switching, and the expressionof virulence factors, including a 10-member family of secretedaspartic proteinases. Candida albicans is a common commensal fungal organism thatis part of the normal microflora of the genital and gastrointestinaltract of healthy individuals. Under appropriate conditions,C. albicans can become an opportunistic pathogen that has debilitatingand sometimes deadly consequences for its host. One commonmanifestation of Candida infection is superficial candidiasis,which is a significant problem encountered in clinics worldwide.The most common superficial infections are vaginal candidiasisand mucosal candidiasis of the oropharyngeal cavity and theesophagus. Several factors of C. albicans were previously identified asvirulence determinants; these factors include hyphal formation, phenotypic switching, and secretion of hydrolyticenzymes. Although hyphal formation is probablythe best-documented virulence determinant of C. albicans, theproduction of hydrolytic enzymes, specifically the secretedaspartic proteinases (Saps), as key virulence determinants hasbeen comprehensively studied. Ten SAP isogenes encode theSap proteins. The purported functions of Saps duringinfection include the digestion of host proteins for nutrientsupply, evasion of host defenses by degradation of immunoglobulinsand complement proteins, adherence, and degradation of hostbarriers during invasion. Due to theirwide substrate specificity and broad pH range, it is acceptedthat Saps contribute to the development of active Candida infections.

64. Candida albicans is the primary cause of candidemia in hospitalized patients, and the intestinal tract is considered the source of most systemic infections. C. glabrata has emerged as the second or third most frequent cause of candidemia, but little is known about its epidemiology and pathogenesis. Our goal was to compare the intestinal colonization and extra-intestinal dissemination of C. glabrata and C. albicans (wild type and filamentation-defective mutant). Mice were pretreated with antibacterial agents to alter their resident microflora, and then orally inoculated with C. glabrata and/or C. albicans. Elimination of detectable cecal bacteria facilitated colonization with both Candida species. Selective elimination of aerobic/facultative gram-negative bacilli did not noticeably affect Candida colonization, but Escherichia coli overgrowth inhibited colonization. In all situations, C. glabrata colonized the cecum equally well or better than C. albicans, and the ability of C. albicans to form filaments did not facilitate colonization. In vitro generation times had little relevance to the resulting cecal population levels of C. glabrata and C. albicans, and neither species readily disseminated to mesenteric lymph nodes. Thus, like C. albicans, the intestinal tract may be an epidemiological reservoir for C. glabrata and antibiotic-induced alterations in intestinal bacteria may facilitate colonization.

Comparative abilities of Candida glabrata and Candida albicans to colonize and translocate from the intestinal tract of antibiotic-treated mice

65. Candida albicans, when cultivated in a medium containing insoluble bovine achilles tendon as a nitrogen source, was able to produce a collagen degrading proteinase. The degradation of achilles tendon collagen by the proteinase was verified by morphological change and the release of hydroxyproline. The proteinase activity was inhibited by pepstatin.

66. Although healthy subjects may host fungal diseases, various predisposing factors that depress the immune system have been implicated in most patients developing fungal infections or fungal arthritis, or both. Alcoholism, cirrhosis, diabetes, tuberculosis, cancer, prematurity, treatment with corticosteroids, cytotoxic drugs, prolonged use of intravenous antibiotics, intravenous drug abuse, granulocytopenia, and marrow hyperplasia are among the predisposing factors. Neonates are the first group of patients in whom haematogenously originated Candida arthritis can occur. The illness is a hospital acquired disease of sick children with underlying diseases such as the respiratory distress syndrome, and gastrointestinal defects. C albicans, which is responsible for more than 80% of the reported cases, and C tropicalis are the species responsible for this disease. Arthritis is usually present with accompanying metaphysial osteomyelitis. Bone infection might originate from the infected synovium or via the metaphysical vessels. Polyarthritis occurs in most patients and the knee is the joint most often affected.

Arthritis originated by haematogenous dissemination beyond the neonatal period is usually a complication of disseminated candidiasis in patients with serious underlying disorders or intravenous drug abusers. C albicans is again the causative organism in about 80% of cases, and C tropicalis is responsible for most of the remaining cases. Two distinct clinical presentations can be observed: (a) acute onset of constitutional and synovial symptoms (about two thirds ofpatients), with the aetiological diagnosis established within the first week, and (b) indolent presentation, with mild systemic and arthritic symptoms, and delay in the diagnosis for months or years.

67. A quantitative assay of Candidacidal activity based on differential staining of nonviable Candida albicans by methylene blue was developed and applied to studies of leukocytes from normal individuals and patients with fungal and other infections.

Serum factors were necessary for optimal phagocytosis of C. albicans but lacked direct Candidacidal activity. Normal human neutrophils (38 studies) killed 29.0 ± 7.4% of ingested C. albicans in 1 hr. Eosinophils and monocytes killed a smaller percentage. Neutrophil Candidacidal activity did not require protein or ribonucleic acid synthesis by the leukocyte but was inhibited by anaerobic conditions, potassium cyanide, and colchicine. Leukocytes of a patient with hereditary myeloperoxidase deficiency and of three children with chronic granulomatous disease phagocytized

C. albicans normally, yet failed to kill them. Our data suggest that the neutrophil can play an important role in resistance to Candida infection and that the lysosomal enzyme myeloperoxidase and its oxidant substrate hydrogen peroxide are the major participants in neutrophil Candidacidal activity. Candidacidal activity was virtually absent under anaerobic conditions and was considerably diminished by 10-3 M cyanide or 10-4 M colchicine.

Several workers have shown that serum will induce yeast-phase C. albicans to form pseudo-germ tubes and grow in the mycelial phase. This change occurs in a few hours, is specific for C. albicans, and has been proposed as a rapid means of differentiating C. albicans from other Candida species. In our studies, considerable difference was found between the Candidacidal abilities of normal human neutrophils and monocytes. Whereas neutrophils killed 29.0 ± 7.4% of the ingested C. albicans in 1 hr, monocytes killed only 4 ± 2%. Anaerobic conditions inhibit neutrophil Candidacidal and bactericidal activity, perhaps by curtailing the generation of hydrogen peroxide, the oxidant substrate of MPO. In one case, that of a child with the Swiss type of familial lymphocytopenic agammaglobulinemia, disseminated candidiasis followed antibiotic therapy for bacterial infection. Although the patient’s immunological deficiency cannot be discounted as a predisposing factor, antibiotic therapy has been reported to precede disseminated candidiasis in children. Substantial clinical and experimental evidence indicates that the neutrophil is a major participant in the host response to systemic Candida infection. In experimental Candida infections, the disappearance of organisms from the tissue lesions parallels the appearance of neutrophils. Leukopenia is a common predisposing factor in disseminated candidiasis in man. Finally, the present studies establish that normal neutrophils can kill ingested C. albicans, although serum itself lacks Candidacidal activity.

68. We previously reported that the primary defense mechanisms inhibiting translocation from the GI tract are an ecologically balanced GI microflora (preventing intestinal microbial overgrowth or colonization by exogenous microorganisms, or both), the host immune defense system, and the physical barrier of an intact intestinal mucosa [2]. Oral antibiotics are especially effective in disrupting the GI ecologic equilibrium, leading to intestinal overgrowth by certain normal flora bacteria and their subsequent translocation from the GI tract.

All mice were given streptomycin and penicillin ad libitum for 4 days (0-4) in drinking water to reduce their indigenous GI microflora.

Also, placing fresh C albicans in the drinking water each day was as effective a method of colonizing mice with the organism as was daily intragastric inoculation of C albicans.

Viable C albicans translocated across the GI mucosal barrier to the MLN in immunocompetent, antibiotic-decontaminated mice colonized with C albicans, but the translocating C albicans did not spread systemically from the MLN to other organs. S. boulardii treatment reduced the incidence of MLN positive for C albicans in these immunocompetent mice but did not reduce the mean numbers of C albicans per gram of MLN. The ileal and cecal populations of C albicans were not decreased whether S. boulardii was given prior to or following C albicans challenge.

Disruption of the intestinal microecology by oral antibiotics to cause intestinal microbial overgrowth and increased microbial translocation is clinically relevant since antibiotic therapy also predisposes humans to intestinal microbial overgrowth and infection by various bacteria and yeasts, such as C albicans.

With a half-life of 8-20 hours, PMNs are short-lived cells. In the absence of inflammatory stimuli, PMNs undergo genetically programmed cell death, or apoptosis, characterized by cytoplasmic shrinkage, nuclear condensation, membrane blebbing, DNA fragmentation, and formation of apoptotic bodies.[2] Early in the apoptotic process, phosphatidylserine is exposed on the cell surface by flipping from the inner to the outer leaflets of the cytoplasmic membrane. This event is thought to be important for macrophage recognition of cells undergoing apoptosis, thus allowing the cells to be removed after their death with minimal inflammation.[3] In addition, apoptosis of human PMNs is thought to be critical for control of the inflammatory process and maintenance of homeostasis, although its regulatory mechanisms are not completely understood. Numerous pathologic conditions (certain cancers, autoimmune and neurodegenerative disorders, sepsis), microbial pathogens (human immunodeficiency virus, herpes viruses, Mycobacterium tuberculosis), chemicals (N-formyl-methionyl-leucyl-phenylalanine), and drug therapies (cyclosporine, macrolides, cancer chemotherapy, glucocorticoids) have been shown to alter apoptosis, in part through fibroblast-associated (Fas, CD95), caspase-dependent, and cytokine-signaling mechanisms.[4-6]

Several of these cytokines are known to initiate and/or regulate apoptosis.[6-10] Certain protein and DNA inhibitors, such as erythromycin, clindamycin, and fluoroquinolones, have been shown to decrease the production of proinflammatory cytokines (TNF-a, IL-1, IL-6) by stimulated phagocytes while increasing production of the antiinflammatory cytokine IL-10.[20-23] Whereas TNF-a begins the caspase cascade leading to cellular apoptosis, IL-1 is a suppressor.[24] In addition, TNF-a is a potent inducer of nuclear factor-kB, a transcription factor contributing to the antiapoptotic pathway.[25,26] It is not clear how the same TNF stimulus can activate both apoptotic and antiapoptotic mechanisms or what factors can shift the balance.[6,24]

To our knowledge, the only other published study assessing the influence of antibiotics on PMN apoptosis found that erythromycin shortened neutrophil survival in a dose-dependent fashion.[31]

70. Two chemically mutagenized agerminative variants of Candidaalbicans were used to immunize mice against challenge with highlyvirulent cells of the parent strain. Bulk culturesof purified CD4+ lymphocytes from mice infected with eithermutant were compared for their ability to produce gamma interferon(IFN-gamma), interleukin-2 (IL-2), IL-4, and IL-6 in vitro.After stimulation with specific antigen, CD4+ cells from Vir-3-immunized mice released large amounts of the Th1-specificcytokines, IFN-gamma and IL-2, at a time when CD4+ cells fromVir- 13-infected mice predominantly secreted the characteristicTh2 cytokines, IL-4 and IL-6. These results were confirmed byquantitative analysis of cytokine-producing Th1 and Th2 cells.In addition, only mice infected with Vir- 3 displayed a highfrequency of CD8+ cells with the potential for in vitro lysisof yeast-primed bone marrow macrophages. Purified CD4+ cellsfrom Vir- 3-infected mice, but not a mixture of these cellswith CD4+ lymphocytes from mice infected with Vir- 13, couldadoptively transfer delayed-type hypersensitivity reactivityonto naive mice. Taken together, these data suggest that bothTh1 and Th2 CD4+ lymphocytes may be activated during experimentalC. albicans infection in mice.

71. The human colon contains the most abundant and diverse assemblage of bacteria in the body. Symbiotic interactions with and within this complex community are now recognized as important predictors of human health. Aberrant community structures are associated with complex diseases like obesity, irritable bowel syndrome, and immune dysfunction. Antibiotic administration can disrupt the colonic ecosystem, which, in turn, leaves patients vulnerable to gastrointestinal disease.

One measure of ecosystem stability, in terms of maintaining function, is the ability to resist invasion and subsequent dominance by immigrating organisms. For the gut ecosystem, antibiotic therapy represents a strong perturbation that shifts the relative proportion of community members, allowing opportunists to establish. Antibiotic therapies exclude members of the community by eradicating them directly or indirectly by breaking necessary mutualistic interactions. During such events in murine models, the community structure was disrupted and enteric pathogens reached high numbers. Similar observations underlie the proposed colonization resistance or barrier function, provided to the host by the gut microbiota, preventing the ingress of pathogens into the gut ecosystem.

During a case-control study of subjects taking capsules of 150 mg clindamycin (orally), each individual was sampled prior to antibiotic treatment and at set time points throughout the following 2-year posttreatment. The overall diversity of this division decreased upon antibiotic treatment and remained reduced during the entire 2 years of the study. The authors also show that the dominant community members changed markedly in relative abundance during the first 3 weeks of the posttreatment, suggesting that these effects were not exclusive to the rest of the microbiota.

Pseudomembranous colitis in the distal colon and rectum is fatal in 6%–30% of cases. Disease onset occurs several days to several weeks after initial antibiotic treatment and certain drugs, such as clindamycin, cephalosporins, fluoroquinolones, and -lactams, are associated with greater risk of CDAD.

Emerging Insights into Antibiotic-Associated Diarrhea and Clostridium difficile Infection through the Lens of Microbial Ecology

72. We have previously shown that whole cell preparations of Candida albicans are capable of inducing immunosuppressive B-cell activity both in vivo and in vitro. In an effort to characterize the components of the yeast which manifest this immunomodulatory activity, we have successfully generated a soluble extract with dithiothreitol which exerts immunosuppressive activity. This extract is capable of inducing antigen-nonspecific suppressor cells which inhibit the antibody response of normal cells in coculture. Both primary and secondary antibody responses are suppressed by these cells. Our results also show that the suppressor cell population is a member of the L3T4+ Ly-1+ Lyt-2- T-cell lineage. These results provide evidence that Candida extracts may possess clinically significant immunomodulatory activities.

The induction of T-suppressor cells with a soluble extract of Candida albicans

73. The surface of the pathogenic yeast Candida albicans is coated with phospholipomannan (PLM), a phylogenetically unique glycolipid composed of _-1,2-oligomannosides and phytoceramide. Externalization of membranous phosphatidylserine, loss of mitochondrial integrity, and DNA fragmentation induced by PLM showed that this molecule promoted yeast survival by inducing host cell death. These findings suggest strongly that PLM is a virulence attribute of C. albicans and that elucidation of the relationship between structure and apoptotic activity is an innovative field of research. In host tissues, C. albicans may be both intra- and extracellular. Macrophages undergo apoptotic cell death after infection with C. albicans strains capable of hyphal formation, and activation of caspase 3 has been observed after endocytosis of C. albicans by neutrophils.

74. The pathogenic yeast Candida albicans and its derived moleculesstimulate a wide range of macrophage secretory functions and mayadapt to escape being killed by this phagocyte. In this study, phagocytosisof C. albicans and of the nonpathogenic yeast Saccharomycescerevisiae was shown to be associated with phosphorylation ofthe mitogen-activated protein kinase (MAPK)/extracellularlyregulated kinase (ERK) pathway in the absence of significantactivation of either p38MAPK or stress-activated protein kinase/c-JunN-terminal kinase. However, although 80% of endocytosed C. albicanssurvived after 1 h, 80% of S. cerevisiae cells were killed.Considerable quantitative differences were observed betweenthe two species in the sequential phosphorylation of MAPK/ERKkinase (MEK), extracellularly regulated kinase-1, and 90-kDa-ribosomalS6 kinases. A lower level of activation of the pathway by C.albicans was associated with a species-specific overexpressionof the MEK phosphatase MAPK phosphatase (MKP)-1. Killing ofboth C. albicans and S. cerevisiae could be reduced using PD98059,which mimics MKP-1 and inhibits MEK phosphorylation, suggestingthat specific MKP-1 activation by C. albicans could contributeto its ability to escape the yeast lytic potential of macrophages.

Role of extracellular signal-regulated protein kinase cascade in macrophage killing of Candida albicans

75. Psychological stress (PS) is recognized as an important pathogenic factor which leads to metabolism disorder in many diseases. Previous studies have shown that systemic iron homeostasis in mammalians was changed under specific stress conditions. Our study showed that iron apparent absorption decreased and iron significantly accumulated in the apical poles of villous enterocytes in 3 d and 7 d PS groups. The expression of intestinal FPN1 in 3 d and 7 d PS groups was lower than that of control, while the change of intestinal ferritin was opposite. However, the expression of DMT1 did not change. These results demonstrate that PS can decrease iron absorption in rats, which might be related to regulation expression of iron transporters.

77. This paper reports the sensitivity, specificity, and predictive values of symptoms in the diagnosis of antibiotic-induced candidal vaginitis (AICV) among 74 women recruited from three primary care practices. All subjects, who were examined both pre- and post-antibiotic treatment for acute respiratory, urinary tract, or skin infections, were initially free of vaginitis. Twenty-four subjects developed candidal vaginitis (CV), indicated by vaginitis symptoms or signs and a positive candidal culture or KOH preparation; there were no mixed infections. Fifty women did not develop AICV and, of this group, four developed a nonyeast vaginitis. Aggregate symptoms (pruritus and/or discharge) had 87.5% sensitivity, 95.8% specificity, and positive and negative predictive values of 91.3% and 93.9%, respectively. These values are much higher than those reported in studies of CV that excluded women on antibiotics. We conclude that women who develop vaginitis symptoms while on short courses of antibiotics may be treated as AICV without confirmatory examination.

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[…] immunosuppression is not necessary for the spread of candida. For more research on this, view the Candida Facts Sheet article. Tests can only serve as indicators, not absolute measures of function in the body. Targeting […]