Variation in Susceptibility of Bloodstream Isolates of Candida glabrata to Fluconazole According to Patient Age and Geographic Location in the United States in 2001 to 2007

We examined the susceptibilities to fluconazole of 642 bloodstream infection (BSI) isolates of Candida glabrata and grouped the isolates by patient age and geographic location within the United States. Susceptibility of C. glabrata to fluconazole was lowest in the northeast region (46%) and was highest in the west (76%). The frequencies of isolation and of fluconazole resistance among C. glabrata BSI isolates were higher in the present study (years 2001 to 2007) than in a previous study conducted from 1992 to 2001. Whereas the frequency of C. glabrata increased with patient age, the rate of fluconazole resistance declined. The oldest age group (≥80 years) had the highest proportion of BSI isolates that were C. glabrata (32%) and the lowest rate of fluconazole resistance (5%).Candidemia is without question the most important of the invasive mycoses (6, 33, 35, 61, 65, 68, 78, 86, 88). Treatment of candidemia over the past 20 years has been enhanced considerably by the introduction of fluconazole in 1990 (7, 10, 15, 28, 29, 31, 40, 56-58, 61, 86, 90). Because of its widespread usage, concern about the development of fluconazole resistance among Candida spp. abounds (2, 6, 14, 32, 47, 53, 55, 56, 59, 60, 62, 80, 86). Despite these concerns, fluconazole resistance is relatively uncommon among most species of Candida causing bloodstream infections (BSI) (5, 6, 22, 24, 33, 42, 54, 56, 65, 68, 71, 86). The exception to this statement is Candida glabrata, of which more than 10% of BSI isolates may be highly resistant (MIC ≥ 64 μg/ml) to fluconazole (6, 9, 15, 23, 30, 32, 36, 63-65, 71, 87, 91). Suboptimal fluconazole dosing practices (low dose [<400 mg/day] and poor indications) may lead to an increased frequency of isolation of C. glabrata as an etiological agent of candidemia in hospitalized patients (6, 17, 29, 32, 35, 41, 47, 55, 60, 68, 85) and to increased fluconazole (and other azole) resistance secondary to induction of CDR efflux pumps (2, 11, 13, 16, 43, 47, 50, 55, 69, 77, 83, 84) and may adversely affect the survival of treated patients (7, 10, 29, 40, 59, 90). Among the various Candida species, C. glabrata alone has increased as a cause of BSI in U.S. intensive care units since 1993 (89). Within the United States, the proportion of fungemias due to C. glabrata has been shown to vary from 11% to 37% across the different regions (west, midwest, northeast, and south) of the country (63, 65) and from <10% to >30% within single institutions over the course of several years (9, 48). It has been shown that the prevalence of C. glabrata as a cause of BSI is potentially related to many disparate factors in addition to fluconazole exposure, including geographic characteristics (3, 6, 63-65, 71, 88), patient age (5, 6, 25, 35, 41, 42, 48, 63, 82, 92), and other characteristics of the patient population studied (1, 32, 35, 51). Because C. glabrata is relatively resistant to fluconazole, the frequency with which it causes BSI has important implications for therapy (21, 29, 32, 40, 41, 45, 56, 57, 59, 80, 81, 86, 90).Previously, we examined the susceptibilities to fluconazole of 559 BSI isolates of C. glabrata and grouped the isolates by patient age and geographic location within the United States over the time period from 1992 to 2001 (63). In the present study we build upon this experience and report the fluconazole susceptibilities of 642 BSI isolates of C. glabrata collected from sentinel surveillance sites throughout the United States for the time period from 2001 through 2007 and stratify the results by geographic region and patient age. The activities of voriconazole and the echinocandins against this contemporary collection of C. glabrata isolates are also reported.     

Shiga Toxin 2 Targets the Murine Renal Collecting Duct Epithelium

Hemolytic-uremic syndrome (HUS) caused by Shiga toxin-producing Escherichia coli infection is a leading cause of pediatric acute renal failure. Bacterial toxins produced in the gut enter the circulation and cause a systemic toxemia and targeted cell damage. It had been previously shown that injection of Shiga toxin 2 (Stx2) and lipopolysaccharide (LPS) caused signs and symptoms of HUS in mice, but the mechanism leading to renal failure remained uncharacterized. The current study elucidated that murine cells of the glomerular filtration barrier were unresponsive to Stx2 because they lacked the receptor glycosphingolipid globotriaosylceramide (Gb₃) in vitro and in vivo. In contrast to the analogous human cells, Stx2 did not alter inflammatory kinase activity, cytokine release, or cell viability of the murine glomerular cells. However, murine renal cortical and medullary tubular cells expressed Gb₃ and responded to Stx2 by undergoing apoptosis. Stx2-induced loss of functioning collecting ducts in vivo caused production of increased dilute urine, resulted in dehydration, and contributed to renal failure. Stx2-mediated renal dysfunction was ameliorated by administration of the nonselective caspase inhibitor Q-VD-OPH in vivo. Stx2 therefore targets the murine collecting duct, and this Stx2-induced injury can be blocked by inhibitors of apoptosis in vivo.Shiga toxin-producing Escherichia coli (STEC) is the principal etiologic agent of diarrhea-associated hemolytic-uremic syndrome (HUS) (42, 60, 66). Renal disease is thought to be due to the combined action of Shiga toxins (Shiga toxin 1 [Stx1] and Stx2), the primary virulence factors of STEC, and bacterial lipopolysaccharide (LPS) on the renal glomeruli and tubules (6, 42, 60, 66). Of these, Stx2 is most frequently associated with the development of HUS (45). Shiga toxin enters susceptible cell types after binding to the cell surface receptor glycosphingolipid globotriaosylceramide (Gb₃) and specifically depurinates the 28S rRNA, thereby inhibiting protein synthesis (42, 60, 66). The damage initiates a ribotoxic stress response consisting of mitogen-activated protein (MAP) kinase activation, and this response can be associated with cytokine release and cell death (21, 22, 25-27, 61, 69, 73). This cell death is often caspase-dependent apoptosis (18, 61). Gb₃ is expressed by human glomerular endothelial cells, podocytes, and multiple tubular epithelial cell types, and damage markers for these cells can be detected in urine samples from HUS patients (10-12, 15, 49, 73). Shiga toxin binds to these cells in renal sections from HUS patients, and along with the typical fibrin-rich glomerular microangiopathy, biopsy sections demonstrate apoptosis of both glomerular and tubular cell types (9, 29, 31).Concomitant development of the most prominent features of HUS: anemia, thrombocytopenia, and renal failure, requires both Shiga toxin and LPS in the murine model (30, 33). Nevertheless, our previous work demonstrated that renal failure is mediated exclusively by Stx2 (33). While it is established that Gb₃ is the unique Shiga toxin receptor (46), the current literature regarding the mechanism by which Shiga toxin causes renal dysfunction in mice is inconsistent. Even though Gb₃ has been localized to some murine renal tubules and tubular damage has been observed (19, 23, 46, 53, 65, 68, 72, 74), the specific types of tubules affected have been incompletely characterized. Although multiple groups have been unable to locate the Shiga toxin receptor Gb₃ in glomeruli in murine renal sections (19, 53), one group has reported that murine glomerular podocytes possess Gb₃ and respond to Stx2 in vitro (40), and another group has reported that renal tubular capillaries express the Gb₃ receptor (46). Furthermore, murine glomerular abnormalities, including platelet and fibrin deposition, occur in some murine HUS models (28, 30, 33, 46, 59, 63). We demonstrate here that murine glomerular endothelial cells and podocytes are unresponsive to Stx2 because they do not produce the glycosphingolipid receptor Gb₃ in vitro or in vivo. Further, murine renal tubules, including collecting ducts, express Gb₃ and undergo Stx2-induced apoptosis, resulting in dysfunctional urine production and dehydration.     

Cytomegalovirus Infection- and Age-Dependent Changes in Human CD8+ T-Cell Cytokine Expression Patterns

T cells are strongly affected by immune aging, a phenomenon that leads to increased susceptibility to infections and decreased vaccination efficacy in elderly individuals. Cytomegalovirus (CMV) infection induces vigorous T-cell immune responses in humans and is thought to be a driving force of immune aging. In the present study we analyzed CMV-induced quantitative and qualitative differences in the cytokine-expressing T-cell repertoire from individuals of different age groups after in vitro stimulation. The CMV pp65 peptide pool and the superantigen Staphylococcus enterotoxin B (SEB) induced higher proportions of CD8⁺ effector T cells expressing gamma interferon (IFN-γ), tumor necrosis factor alpha (TNF-α), and granulocyte-macrophage colony-stimulating factor in the oldest study group, while only SEB induced increased responses in the middle-aged study group. Notably, CMV-specific multiple cytokine expression patterns revealed higher proportions of IFN-γ- and TNF-α-coexpressing CD8⁺ T cells exclusively in the oldest study group. These qualitative differences were absent in SEB-induced CD8⁺ effector T cells, although quantitative differences were detected. We report age-dependent qualitative changes in CMV-specific CD8⁺ T-cell cytokine patterns which are biocandidate markers of immune exhaustion in elderly individuals.The term immune aging describes a conglomerate of features that affect immune functions and phenotype at late stages of human life (reviewed in reference 24). Clinically, immune aging is associated with higher susceptibility to diseases caused by “new” respiratory infections with bacteria (e.g., pneumococci) and viruses (e.g., influenza virus) (6, 7). The phenotype of immune aging includes several features and affects both innate and adaptive immunity. In particular, marked changes in T-cell immunity have been well documented (18, 20). These differences include changes on the single-cell level (e.g., T-cell receptor [TCR]- and coreceptor-dependent signal transduction [reviewed in reference 21]), as well as changes on the T-cell population level, which show (i) a decline in naïve T-cell numbers, (ii) a reduction in TCR repertoire diversity, and (iii) increases in memory and effector T-cell proportions (reviewed in reference 24). Longitudinal studies in human populations indicate that a subset of immune aging features can be used as biomarkers of mortality (25, 33). These studies identified cytomegalovirus (CMV) as a biomarker in the so-called immune risk profile (IRP).Human CMV infection is common in all parts of the world, with infection rates between 60% in developed countries and up to 100% in developing countries (11). The CMV infection rate steadily increases during an individual''s life time, with the highest prevalence in the very old (9). CMV infection is usually asymptomatic and leads to life-long persistence of the pathogen. Reactivation and development of clinical symptoms are rare and largely restricted to immunocompromised and immunosuppressed patients (10, 29). CMV infection rapidly induces immunodominant T-cell responses, mainly against two proteins, the immediate-early protein 1 (IE-1) and the matrix 65-kDa phosphoprotein (pp65) (14, 19, 34). This T-cell response is like a double-edged sword, because studies in humans and animal models suggest a protective role against CMV reactivation on the one hand (1, 12, 31) and, on the other hand, the T-cell response leads to vigorous and persistent clonal expansion of CD8⁺ T cells (reviewed in reference 22). These T-cell clones reach high numbers and thus dominate the peripheral blood T-cell repertoire (8, 28). In contrast to other chronic infection- or vaccine-induced T-cell responses, CMV-specific T-cell numbers are stable or even increase over time and lead to extremely high proportions of clonally expanded T cells in very old individuals (26). CMV-specific CD8⁺ and CD4⁺ T cells show some phenotypic peculiarities and continuously become dysfunctional or exhausted (5, 26). It has been suggested that CMV-induced changes in T-cell repertoire and function, although crucial for protection against CMV reactivation, challenge the maintenance of a balanced T-cell repertoire and in this way compromise host immunity against infections and efficacies of new vaccines in elderly individuals (reviewed in reference 24). Furthermore, it has been hypothesized that CMV infection itself is the driving force of immune aging (reviewed in reference 16).Quantitative assessment of T-cell responses (e.g., gamma interferon [IFN-γ] and tumor necrosis factor alpha [TNF-α] expression) is a common tool used to predict disease susceptibility and vaccination efficacy, but for some diseases (e.g., tuberculosis) these parameters are not sufficient for use as biomarkers of protection (13). Recent studies have strengthened the crucial role of qualitative differences in the T-cell response (i.e., T-cell-expressed cytokine patterns) for the prediction of disease susceptibility (3, 17, 36). Our own previous studies suggest a role of granulocyte-macrophage colony-stimulating factor (GM-CSF) as a T-cell biomarker in childhood tuberculosis (23).Here we analyzed proportions of cytokine-expressing T-cell subpopulations (i.e., for IFN-γ, TNF-α, GM-CSF, and interleukin-2 [IL-2]) and determined the patterns of cytokine coexpression after in vitro stimulation with CMV pp65 and the superantigen staphylococcus enterotoxin B (SEB) in individuals from different age groups. In this way we characterized concomitantly age-dependent, CMV-specific and nonspecific, quantitative as well as qualitative differences in the T-cell response.     

Acanthamoeba culbertsoni Elicits Soluble Factors That Exert Anti-Microglial Cell Activity

Acanthamoeba culbertsoni is an opportunistic pathogen that causes granulomatous amoebic encephalitis (GAE), a chronic and often fatal disease of the central nervous system (CNS). A hallmark of GAE is the formation of granulomas around the amoebae. These cellular aggregates consist of microglia, macrophages, lymphocytes, and neutrophils, which produce a myriad of proinflammatory soluble factors. In the present study, it is demonstrated that A. culbertsoni secretes serine peptidases that degrade chemokines and cytokines produced by a mouse microglial cell line (BV-2 cells). Furthermore, soluble factors present in cocultures of A. culbertsoni and BV-2 cells, as well as in cocultures of A. culbertsoni and primary neonatal rat cerebral cortex microglia, induced apoptosis of these macrophage-like cells. Collectively, the results indicate that A. culbertsoni can apply a multiplicity of cell contact-independent modes to target macrophage-like cells that exert antiamoeba activities in the CNS.Acanthamoeba culbertsoni belongs to a group of free-living amoebae, such as Balamuthia mandrillaris, Naegleria fowleri, and Sappinia pedata, that can cause disease in humans (46, 56). Acanthamoeba spp. are found worldwide and have been isolated from a variety of environmental sources, including air, soil, dust, tap water, freshwater, seawater, swimming pools, air conditioning units, and contaminated contact lenses (30). Trophozoites feed on bacteria and algae and represent the infective form (47, 56). However, under unfavorable environmental conditions, such as extreme changes in temperature or pH, trophozoites transform into a double-walled, round cyst (22, 45).Acanthamoeba spp. cause an infection of the eye known as amoebic keratitis (AK), an infection of the skin referred to as cutaneous acanthamoebiasis, and a chronic and slowly progressing disease of the central nervous system (CNS) known as granulomatous amoebic encephalitis (GAE) (22, 23, 30, 56). GAE is most prevalent in humans who are immunocompromised (30, 33, 40) and has been reported to occur among individuals infected with the human immunodeficiency virus (HIV) (28). It has been proposed that Acanthamoeba trophozoites access the CNS by passage through the olfactory neuroepithelium (32) or by hematogenous spread from a primary nonneuronal site of infection (23, 24, 33, 53).In immune-competent individuals, GAE is characterized by the formation of granulomas. These cellular aggregates consist of microglia, macrophages, polymorphonuclear cells, T lymphocytes, and B lymphocytes (24, 30). The concerted action of these immune cells results in sequestration of amoebae and is instrumental in slowing the progression of GAE. This outcome is consistent with the observation that granulomas are rarely observed in immunocompromised individuals (34) and in mice with experimentally induced immune suppression following treatment with the cannabinoid delta-9-tetrahydrocannabinol (Δ⁹-THC) (8).Microglia are a resident population of macrophages in the CNS. These cells, along with CNS-invading peripheral macrophages, appear to play a critical early effector role in the control of Acanthamoeba spread during GAE (4, 5, 29, 31). In vitro, microglia have been shown to produce an array of chemokines and cytokines in response to Acanthamoeba (31, 51). However, these factors appear not to have a deleterious effect on these amoebae (29).Acanthamoeba spp. produce serine peptidases, cysteine peptidases, and metallopeptidases (1, 2, 9, 10, 14, 16, 18, 19, 21, 25, 26, 37, 38, 41, 42, 52). In the present study, it is demonstrated that serine peptidases secreted by A. culbertsoni degrade chemokines and cytokines that are produced by immortalized mouse BV-2 microglia-like cells. In addition, soluble factors present in cocultures of A. culbertsoni and BV-2 cells induced apoptosis of the BV-2 cells. Collectively, these results suggest a mode through which A. culbertsoni can evade immune responsiveness in the CNS.     

Borrelia burgdorferi BmpA Is a Laminin-Binding Protein

The Borrelia burgdorferi BmpA outer surface protein plays a significant role in mammalian infection by the Lyme disease spirochete and is an important antigen for the serodiagnosis of human infection. B. burgdorferi adheres to host extracellular matrix components, including laminin. The results of our studies indicate that BmpA and its three paralogous proteins, BmpB, BmpC, and BmpD, all bind to mammalian laminin. BmpA did not bind mammalian type I or type IV collagens or fibronectin. BmpA-directed antibodies significantly inhibited the adherence of live B. burgdorferi to laminin. The laminin-binding domain of BmpA was mapped to the carboxy-terminal 80 amino acids. Solubilized collagen inhibited BmpA-laminin binding, suggesting interactions through the collagen-binding domains of laminin. These results, together with previous data, indicate that BmpA and its paralogs are targets for the development of preventative and curative therapies for Lyme disease.Early during the course of Lyme disease, humans frequently produce antibodies directed against a Borrelia burgdorferi antigen originally described as “P39” (66). Antibodies recognizing P39 are considered to be specific and diagnostic for Lyme disease spirochete infection (5, 18, 30, 62, 64). The antigenic protein was subsequently identified as BmpA (Borrelia membrane protein A) (65). The bmpA gene is located on the main borrelial chromosome, adjacent to three paralogous genes named bmpB, bmpC, and bmpD, which together form a complex operon (3, 4, 28, 32, 55, 56, 65). These other Bmp proteins are also often antigenic in infected humans (14). In addition to the serological data described above, examination of B. burgdorferi within skin and joint tissues confirmed the production of BmpA protein during mammalian infection (21, 49). BmpA is located in the borrelial outer membrane (46), where it is exposed to the external environment and can be a target of bactericidal antibodies (49, 63; F. Cabello, personal communication). BmpA and its paralogs have been implicated as playing roles in some symptoms of Lyme disease (49, 72). B. burgdorferi mutants in which bmpA or bmpB is specifically deleted are unable to persist in mouse joint tissues (49), indicating an important role for these proteins in the maintenance of mammalian infection. Despite the extensive research conducted on these important antigens, functions for the Bmp proteins had not been determined previously.B. burgdorferi is an extracellular organism, frequently found associated with its hosts'' connective tissues (6-9, 16, 17, 24, 26, 31, 36, 39, 48). In the laboratory, B. burgdorferi shows affinity for various host extracellular matrix (ECM) components, such as type I collagen, fibronectin, and decorin (16, 33, 34, 50, 74). We recently determined that B. burgdorferi also adheres to mammalian laminin, an important component of many mammalian ECMs (13). Ligand affinity blot analyses of a B. burgdorferi cell fraction enriched for outer membrane components revealed that the type strain, B31, can produce several distinct laminin-binding proteins, one of which we previously identified as being the surface-exposed outer membrane lipoprotein ErpX (11, 13, 69). We now present data indicating that BmpA and its paralogs are also laminin-binding proteins.     

Protection of Sheep against Rift Valley Fever Virus and Sheep Poxvirus with a Recombinant Capripoxvirus Vaccine

Rift Valley fever (RVF) is an epizootic viral disease of sheep that can be transmitted from sheep to humans, particularly by contact with aborted fetuses. A capripoxvirus (CPV) recombinant virus (rKS1/RVFV) was developed, which expressed the Rift Valley fever virus (RVFV) Gn and Gc glycoproteins. These expressed glycoproteins had the correct size and reacted with monoclonal antibodies (MAb) to native glycoproteins. Mice vaccinated with rKS1/RVFV were protected against RVFV challenge. Sheep vaccinated with rKS1/RVFV twice developed neutralizing antibodies and were significantly protected against RVFV and sheep poxvirus challenge. These findings further document the value of CPV recombinants as ruminant vaccine vectors and support the inclusion of RVFV genes encoding glycoproteins in multivalent recombinant vaccines to be used where RVF occurs.Rift Valley fever (RFV) virus (RVFV) is a mosquito-borne member of the genus Phlebovirus, family Bunyaviridae. It is widely distributed in Africa, causing endemic and epidemic disease in both humans and livestock, including sheep, cattle, and goats. RVF was first described in Kenya and was shown to be caused by a filterable virus transmissible via blood (9). Acute RVF in lambs is characterized by fever and death within 24 to 48 h of being detected (43). Signs in adult sheep include fever, mucopurulent nasal discharge, hemorrhagic diarrhea, and abortion in pregnant ewes (43). RVFV can be transmitted from infected sheep to humans, particularly when humans are exposed to aborted sheep fetuses and blood.Attenuated live RVFV vaccines are available for use in livestock. A mutagen-attenuated RVFV vaccine induces protective immune responses in lambs and appears to be safe (25); however, other studies documented teratogenic effects on lambs from vaccinated pregnant ewes similar to those caused by the attenuated RVFV strain Smithburn (18). An inactivated RVFV vaccine induces neutralizing antibody responses in humans (33), and its use in sheep would not induce teratogenic effects or abortions. However, the inactivated vaccine requires 3 doses (33) and is expensive to produce. Efforts to make RVFV vaccines without these disadvantages include an attenuated RVFV developed by reverse genetics and lacking the NSs and NSm genes (4) and other new-generation RVFV vaccines (reviewed in reference 19) that protect mice against virus challenge (7, 16, 24, 27).The middle (M) RNA segment of the RVFV genome encodes the viral glycoproteins Gn and Gc (8, 20), and recombinant vaccinia virus expressing these glycoproteins induces neutralizing antibody and protective immunity to RVFV in mice (7). Vaccinia virus is safe for animals, but there is some risk to humans, as it was reported previously to spread from human vaccinees to contacts (28, 55) and to cause serious clinical disease in human immunodeficiency virus-infected patients (36). Although modified vaccinia virus Ankara is a safer alternative for humans (6, 57), there are animal poxviruses with naturally restricted host ranges for vaccine vectors in animals (1, 13, 30, 31, 40, 46, 47, 52, 53).For ruminants, the genus Capripoxvirus (CPV) of the family Poxviridae has been an effective recombinant vector to induce protective immunity against several other viruses (3, 17, 29, 32, 40, 41, 51). This genus has three closely related species causing sheep pox, goat pox, and lumpy skin disease (LSD) of cattle. A recombinant LSD vaccine expressing the Gn and Gc glycoproteins of RVFV induced protection against RVFV challenge in mice (52, 53) and sheep (52). The three species of CPV have 96 to 97% nucleotide identity (49) and are restricted to ruminants, with no evidence of human infections (10, 11). Furthermore, attenuated CPV vaccines are in use in Africa and the Middle East to control ruminant poxvirus disease (11, 21). The use of a CPV vector to deliver virus vaccines to ruminants also induces immunity to the CPV vector, thus increasing the valence of the vaccine (3, 17, 39, 40). We report here the construction of a recombinant CPV that expresses the RVFV Gn and Gc glycoproteins and induces protective immunity against RVFV and sheep poxvirus (SPV) challenge in sheep.     

Hag Mediates Adherence of Moraxella catarrhalis to Ciliated Human Airway Cells

Moraxella catarrhalis is a human pathogen causing otitis media in infants and respiratory infections in adults, particularly patients with chronic obstructive pulmonary disease. The surface protein Hag (also designated MID) has previously been shown to be a key adherence factor for several epithelial cell lines relevant to pathogenesis by M. catarrhalis, including NCIH292 lung cells, middle ear cells, and A549 type II pneumocytes. In this study, we demonstrate that Hag mediates adherence to air-liquid interface cultures of normal human bronchial epithelium (NHBE) exhibiting mucociliary activity. Immunofluorescent staining and laser scanning confocal microscopy experiments demonstrated that the M. catarrhalis wild-type isolates O35E, O12E, TTA37, V1171, and McGHS1 bind principally to ciliated NHBE cells and that their corresponding hag mutant strains no longer associate with cilia. The hag gene product of M. catarrhalis isolate O35E was expressed in the heterologous genetic background of a nonadherent Haemophilus influenzae strain, and quantitative assays revealed that the adherence of these recombinant bacteria to NHBE cultures was increased 27-fold. These experiments conclusively demonstrate that the hag gene product is responsible for the previously unidentified tropism of M. catarrhalis for ciliated NHBE cells.Moraxella catarrhalis is a gram-negative pathogen of the middle ear and lower respiratory tract (29, 40, 51, 52, 69, 78). The organism is responsible for ∼15% of bacterial otitis media cases in children and up to 10% of infectious exacerbations in patients with chronic obstructive pulmonary disease (COPD). The cost of treating these ailments places a large financial burden on the health care system, adding up to well over $10 billion per annum in the United States alone (29, 40, 52, 95, 97). In recent years, M. catarrhalis has also been increasingly associated with infections such as bronchitis, conjunctivitis, sinusitis, bacteremia, pneumonia, meningitis, pericarditis, and endocarditis (3, 12, 13, 17-19, 24, 25, 27, 51, 67, 70, 72, 92, 99, 102-104). Therefore, the organism is emerging as an important health problem.M. catarrhalis infections are a matter of concern due to high carriage rates in children, the lack of a preventative vaccine, and the rapid emergence of antibiotic resistance in clinical isolates. Virtually all M. catarrhalis strains are resistant to β-lactams (34, 47, 48, 50, 53, 65, 81, 84). The genes specifying this resistance appear to be gram positive in origin (14, 15), suggesting that the organism could acquire genes conferring resistance to other antibiotics via horizontal transfer. Carriage rates as high as 81.6% have been reported for children (39, 104). In one study, Faden and colleagues analyzed the nasopharynx of 120 children over a 2-year period and showed that 77.5% of these patients became colonized by M. catarrhalis (35). These investigators also observed a direct relationship between the development of otitis media and the frequency of colonization. This high carriage rate, coupled with the emergence of antibiotic resistance, suggests that M. catarrhalis infections may become more prevalent and difficult to treat. This emphasizes the need to study pathogenesis by this bacterium in order to identify vaccine candidates and new targets for therapeutic approaches.One key aspect of pathogenesis by most infectious agents is adherence to mucosal surfaces, because it leads to colonization of the host (11, 16, 83, 93). Crucial to this process are surface proteins termed adhesins, which mediate the binding of microorganisms to human cells and are potential targets for vaccine development. M. catarrhalis has been shown to express several adhesins, namely UspA1 (20, 21, 59, 60, 77, 98), UspA2H (59, 75), Hag (also designated MID) (22, 23, 37, 42, 66), OMPCD (4, 41), McaP (61, 100), and a type 4 pilus (63, 64), as well as the filamentous hemagglutinin-like proteins MhaB1, MhaB2, MchA1, and MchA2 (7, 79). Each of these adhesins was characterized by demonstrating a decrease in the adherence of mutant strains to a variety of human-derived epithelial cell lines, including A549 type II pneumocytes and Chang conjunctival, NCIH292 lung mucoepidermoid, HEp2 laryngeal, and 16HBE14o-polarized bronchial cells. Although all of these cell types are relevant to the diseases caused by M. catarrhalis, they lack important aspects of the pathogen-targeted mucosa, such as the features of cilia and mucociliary activity. The ciliated cells of the respiratory tract and other mucosal membranes keep secretions moving out of the body so as to assist in preventing colonization by invading microbial pathogens (10, 26, 71, 91). Given this critical role in host defense, it is interesting to note that a few bacterial pathogens target ciliated cells for adherence, including Actinobacillus pleuropneumoniae (32), Pseudomonas aeruginosa (38, 108), Mycoplasma pneumoniae (58), Mycoplasma hyopneumoniae (44, 45), and Bordetella species (5, 62, 85, 101).In the present study, M. catarrhalis is shown to specifically bind to ciliated cells of a normal human bronchial epithelium (NHBE) culture exhibiting mucociliary activity. This tropism was found to be conserved among isolates, and analysis of mutants revealed a direct role for the adhesin Hag in binding to ciliated airway cells.     

The Purified and Recombinant Legionella pneumophila Chaperonin Alters Mitochondrial Trafficking and Microfilament Organization

A portion of the total cellular pool of the Legionella pneumophila chaperonin, HtpB, is found on the bacterial cell surface, where it can mediate invasion of nonphagocytic cells. HtpB continues to be abundantly produced and released by internalized L. pneumophila and may thus have postinvasion functions. We used here two functional models (protein-coated beads and expression of recombinant proteins in CHO cells) to investigate the competence of HtpB in mimicking early intracellular trafficking events of L. pneumophila, including the recruitment of mitochondria, cytoskeletal alterations, the inhibition of phagosome-lysosome fusion, and association with the endoplasmic reticulum. Microscopy and flow cytometry studies indicated that HtpB-coated beads recruited mitochondria in CHO cells and U937-derived macrophages and induced transient changes in the organization of actin microfilaments in CHO cells. Ectopic expression of HtpB in the cytoplasm of transfected CHO cells also led to modifications in actin microfilaments similar to those produced by HtpB-coated beads but did not change the distribution of mitochondria. Association of phagosomes containing HtpB-coated beads with the endoplasmic reticulum was not consistently detected by either fluorescence or electron microscopy studies, and only a modest delay in the fusion of TrOv-labeled lysosomes with phagosomes containing HtpB-coated beads was observed. HtpB is the first Legionella protein and the first chaperonin shown to, by means of our functional models, induce mitochondrial recruitment and microfilament rearrangements, two postinternalization events that typify the early trafficking of virulent L. pneumophila.The gram-negative bacterium Legionella pneumophila is an intracellular parasite of amoebae (67) that has emerged as an accidental human pathogen capable of replicating in mononuclear phagocytes (38), primarily alveolar macrophages. Lung infection by L. pneumophila usually begins after the inhalation of contaminated water aerosol and manifests as an atypical pneumonia known as Legionnaires'' disease (86).The early events of L. pneumophila infection are well described at the cellular level. The first steps of infection are bacterial attachment to host cell receptors and subsequent internalization by conventional phagocytosis (27, 61, 75), coiling phagocytosis (12, 35), or macropinocytosis (84). Once internalized, L. pneumophila remains contained within a membrane-bound compartment, which is transformed into a specialized vacuole referred to as the Legionella-containing vacuole (LCV). The major cellular events of LCV conditioning include recruitment of vesicles and mitochondria (26, 33, 60), avoidance of both acidification and fusion with lysosomes (34, 37), and association with the endoplasmic reticulum (ER) (33, 42, 79, 80). Less known (or predictable) are the early changes in F-actin organization induced by L. pneumophila, which seem to be unrelated to the F-actin rearrangements required for its internalization (16, 44, 60, 78).At the molecular level, at least five bacterial gene products, RtxA, EnhC (13, 14), LpnE (58), LvhB2 (65), and HtpB (25) are involved in cell entry, confirming L. pneumophila''s flexibility in the use of alternate entry pathways. Conditioning of the LCV requires the Dot/Icm type IV secretion system of L. pneumophila, suggesting that the translocation of type IV-secreted effectors into host cells is essential for the recruitment of organelles and avoidance of acidification and fusion with lysosomes (3, 15, 53). Although a number of specific Dot/Icm effectors have been identified that mediate the sequestration of ER-derived vesicles to the LCV (19, 40, 48, 50, 63), no specific gene products have been linked to the recruitment of mitochondria or the inhibition of LCV-lysosome fusion.It has been hypothesized that the factors that mediate the internalization of L. pneumophila are preformed and may also participate in the early conditioning of the LCV (see, for example, reference 41). This hypothesis is supported by the following observations: (i) conditioning of the LCV begins within minutes after L. pneumophila internalization (18, 42, 49, 68), and (ii) antibiotic-treated legionellae (incapable of de novo protein synthesis) are not affected in their ability to attach to or enter host cells (26, 39) and resume intracellular growth immediately after removal of the antibiotic (39). It seems that L. pneumophila is prearmed to deploy a sequence of coordinated events (which follow a precise timing) immediately after making contact with a host cell, a notion also suggested by detailed microscopy studies conducted in the genetically tractable amoeba Dictyostelium discoideum (49). Moreover, some of the infection steps clearly have a short duration (49), suggesting that L. pneumophila may transiently alter a number of cellular processes. The transient nature of such effects sometimes depends on the host cell being infected. For instance, in human macrophages avoidance of both LCV acidification and fusion with lysosomes persists throughout the intracellular growth cycle (70, 85), whereas in murine macrophages, LCVs acidify and fuse with lysosomes toward the end of the L. pneumophila replicative phase (76). In contrast, other LCV conditioning processes, such as the association with mitochondria and ER, are structurally maintained (regardless of the cellular host being infected) until the replicative phase of the intracellular growth cycle has been completed (26, 33, 60).L. pneumophila HtpB is a member of the group 1 chaperonins, which includes the evolutionarily conserved and essential chaperonins of bacteria, mitochondria and plastids, with well-characterized roles in protein folding (28). The function of bacterial chaperonins, however, is not limited to protein folding. Chaperonins of bacteria can mediate adherence to mammalian cells (22), stabilize membrane lipids (81), paralyze insects (87), and activate eukaryotic signaling cascades (51, 88). The expression of HtpB is upregulated in the presence of L929 murine cells or human monocytes, and high levels of expression are maintained during the course of intracellular infection (21), leading to its accumulation in the lumen of the LCV, as shown in infected HeLa cells (24). The increased production of HtpB in L929 cells and monocytes correlates with virulence because spontaneous salt-tolerant, avirulent mutants of L. pneumophila are unable to upregulate the expression of HtpB upon contact with these host cells (21). In addition, HtpB is found in association with the L. pneumophila cytoplasmic membrane (5, 23), as well as on the bacterial cell surface (24), where it can mediate invasion of HeLa cells (25). As a L. pneumophila factor that mediates cell entry (25), and one that continues to be abundantly produced and released into the LCV after L. pneumophila internalization (24, 32), HtpB may participate in the early intracellular establishment of L. pneumophila. In the present study, we show that microbeads coated with purified HtpB (but not uncoated beads or beads coated with control proteins) are sufficient to attract mitochondria and transiently modify the organization of actin microfilaments in mammalian cells, two postinternalization events that typify the early trafficking of virulent L. pneumophila.     

Characterization of a Campylobacter jejuni VirK Protein Homolog as a Novel Virulence Determinant

Campylobacter jejuni is a leading cause of food-borne illness in the United States. Despite significant recent advances, its mechanisms of pathogenesis are poorly understood. A unique feature of this pathogen is that, with some exceptions, it lacks homologs of known virulence factors from other pathogens. Through a genetic screen, we have identified a C. jejuni homolog of the VirK family of virulence factors, which is essential for antimicrobial peptide resistance and mouse virulence.Campylobacter jejuni is a leading cause of infectious diarrhea in industrialized and developing countries (2, 67). Although most often self-limiting, C. jejuni infections can also lead to severe disease and harmful sequelae, such as Guillain-Barré syndrome (4, 55). Despite the significant progress made during the past few years, the mechanisms of C. jejuni pathogenesis remain poorly understood. A number of potential virulence factors have been identified, and in some cases, their role in virulence and/or colonization has been demonstrated in animal models of infection. For example, motility has been shown to be crucial in order for C. jejuni to colonize or cause disease in several animal models of infection (1, 15, 30, 54). A variety of surface structures, such as adhesins (34, 40, 64) and polysaccharides (5, 6), and glycosylation systems (38, 74), which presumably modify some of these surface structures, have also been shown to be important for infection. Additional studies have revealed the importance of specific metabolic pathways in C. jejuni growth both in vitro and within animals (16, 25, 31, 60, 76). The ability of C. jejuni to invade and survive within nonphagocytic cells has also been proposed to be an important virulence determinant (21, 41, 57, 58, 68, 75, 80).The available genome sequences of several C. jejuni strains have provided significant insight into C. jejuni physiology and metabolism (22, 32, 62, 63, 65). Remarkably, however, analysis of these C. jejuni genome sequences has revealed very few homologs of common virulence factors from other pathogens. A notable exception is the toxin CDT (cytolethal distending toxin), which is also encoded by several other important bacterial pathogens (36, 44, 45). In this paper we describe the identification of a transposon insertion mutant in C. jejuni 81-176, which results in increased susceptibility to antimicrobial peptides and a significant defect in the ability of the organism to cause disease in an animal model of infection. The insertion mutant was mapped to the CJJ81176_1087 open reading frame (Cj1069 in the C. jejuni NCT 11168 reference strain), which encodes a protein with very significant amino acid sequence similarity to the VirK (DUF535) family of virulence factors (13, 20, 56).     

Association of Single Nucleotide Polymorphisms in Cytotoxic T-Lymphocyte Antigen 4 and Susceptibility to Autoimmune Type 1 Diabetes in Tunisians

In addition to HLA and insulin genes, the costimulatory molecule CTLA-4 gene is a confirmed type 1 diabetes (T1D) susceptibility gene. Previous studies investigated the association of CTLA-4 genetic variants with the risk of T1D, but with inconclusive findings. Here, we tested the contributions of common CTLA-4 gene variants to T1D susceptibility in Tunisian patients and control subjects. The study subjects comprised 228 T1D patients (47.8% females) and 193 unrelated healthy controls (45.6% females). Genotyping for CTLA-4 CT60A/G (rs3087243), +49A/G (rs231775), and −318C/T (rs5742909) was performed by PCR-restriction fragment length polymorphism (RFLP) analysis. The minor-allele frequencies (MAF) for the three CTLA-4 variants were significantly higher in T1D patients, and significantly higher frequencies of homozygous +49G/G and homozygous CT60G/G genotypes were seen in patients, which was confirmed by univariate regression analysis (taking the homozygous wild type as a reference). Of the eight possible three-locus CTLA-4 haplotypes (+49A/G, −318C/T, and CT60A/G) identified, multivariate regression analysis confirmed the positive association of ACG (odds ratio [OR], 1.93; 95% confidence interval [CI], 1.26 to 2.94), GCG (OR, 2.40; 95% CI, 1.11 to 5.21), and GTA (OR, 4.67; 95% CI, 1.52 to 14.39) haplotypes with T1D, after confounding variables were adjusted for. Our results indicate that CTLA-4 gene variants are associated with increased T1D susceptibility in Tunisian patients, further supporting a central role for altered T-cell costimulation in T1D pathogenesis.Type 1 (insulin-dependent) diabetes (T1D) is the most prevalent form of diabetes in children and young adults and results from autoimmune CD4⁺ and CD8⁺ T-cell-directed destruction of insulin-producing pancreatic β islet cells in genetically susceptible individuals (3, 12), leading to irreversible hyperglycemia and related complications (13). There is a strong genetic component to T1D pathogenesis, evidenced by its clustering in families and by the contributions of a number of susceptibility gene variants to its pathogenesis (10, 12, 29). They include the human leukocyte antigen (HLA) locus, in particular the class II region (DR and DQ), which accounts for 40 to 50% of T1D familial clustering (1, 12, 18), and non-HLA susceptibility loci, several of which were mapped by genome-scanning (11, 29) and/or candidate gene (7, 18, 31) approaches. They include insulin promoter gene variants, which reportedly may modulate immunological tolerance by controlling the expansion of the autoreactive cell pool (26), and the T-cell costimulator cytotoxic T-lymphocyte antigen 4 (CTLA-4) transmembrane glycoprotein, which plays a key role in the fine tuning of T-cell immunity (9, 32, 33).CTLA-4 is a 40-kDa transmembrane glycoprotein expressed on resting and activated T cells and nonlymphoid cells (33), and along with the related CD28 costimulatory molecule, it regulates T-cell activation (and is itself primarily mediated by engagement of the T-cell receptor [TCR]) but does recognize major histocompatibility complex (MHC)-bound antigenic peptides (9, 33). CTLA-4 negatively regulates T-cell activation and effector function, in part by inhibiting Th1 (interleukin 2 [IL-2] and gamma interferon [IFN-γ]) cytokine production and IL-2 receptor α-chain (p55; Tac) expression by engaging antigen-presenting cell (APC)-bound B7.1 (CD80) and B7.2 (CD86) ligands (9, 33). Functionally, CTLA-4 attenuates T-cell signaling by interference with intracellular signal transduction events, including TCR signaling, and reduced CTLA-4 expression and/or activity results in uncontrolled T-cell-associated autoimmunity and lymphoproliferative disease (9, 21). In this regard, it was shown that CTLA-4 polymorphisms significantly influence the risk of autoimmune diseases, including Graves'' disease, systemic lupus erythematosus, autoimmune hypothyroidism, celiac disease, and type 1 diabetes (15, 21, 32).First observed in Italian subjects (25), and confirmed subsequently by case control and family studies, CTLA-4 polymorphic variants were linked with T1D pathogenesis (14, 20, 31, 32). While this association was detected in different ethnic groups (14, 23, 30), it appears more likely to be Caucasian selective (10, 29, 33) and absent from non-Caucasians (5, 6, 8, 19, 22). A recent report from the Type I Diabetes Genetics Consortium bearing on 2,300 affected sib pair families demonstrated that among the 24 single nucleotide polymorphisms (SNPs) genotyped in the CTLA-4 region, only the +49A/G and CT60 SNPs were replicated in the nine combined collections (27). In the present study, we investigated the association of three common CTLA-4 SNPs (−318C/T; +49A/G, and CT60A/G) and the corresponding haplotypes with T1D in Tunisian Arab patients.     

Extracellular Vesicles from Cryptococcus neoformans Modulate Macrophage Functions

Cryptococcus neoformans and distantly related fungal species release extracellular vesicles that traverse the cell wall and contain a varied assortment of components, some of which have been associated with virulence. Previous studies have suggested that these extracellular vesicles are produced in vitro and during animal infection, but the role of vesicular secretion during the interaction of fungi with host cells remains unknown. In this report, we demonstrate by fluorescence microscopy that mammalian macrophages can incorporate extracellular vesicles produced by C. neoformans. Incubation of cryptococcal vesicles with murine macrophages resulted in increased levels of extracellular tumor necrosis factor alpha (TNF-α), interleukin-10 (IL-10), and transforming growth factor β (TGF-β). Vesicle preparations also resulted in a dose-dependent stimulation of nitric oxide production by phagocytes, suggesting that vesicle components stimulate macrophages to produce antimicrobial compounds. Treated macrophages were more effective at killing C. neoformans yeast. Our results indicate that the extracellular vesicles of C. neoformans can stimulate macrophage function, apparently activating these phagocytic cells to enhance their antimicrobial activity. These results establish that cryptococcal vesicles are biologically active.Cryptococcus neoformans is an encapsulated yeast that causes disease in diverse species, including humans. Infection is most commonly acquired by inhalation of environmental propagules. C. neoformans rarely causes disease in immunocompetent individuals, but patients with immunological disorders can develop disseminated and neural cryptococcosis (63).Extracellular microbial products have been amply demonstrated to modulate the interaction between host cells and pathogens. Many virulence factors and immunogens are released in their soluble forms by fungal cells to the extracellular space (4, 9, 16, 19, 37, 49, 53, 60, 62, 65, 67). C. neoformans, for instance, constitutively secretes large amounts of its capsular polysaccharide glucuronoxylomannan (GXM) (61). Disease progress is associated with detection of GXM, which is a potent modulator of the immune response (reviewed in reference 81). Other secreted virulence-related factors include galactoxylomannan (GalXM) (14), phospholipases (16), and urease (12, 62). In addition to acting as virulence factors, culture supernatant components are immunogenic, conferring protection against C. neoformans infection (51, 53).Phagocytes are particularly important effector cells in the control of systemic mycoses (54). The interaction of C. neoformans with phagocytes, including macrophages, monocytes, dendritic cells, and neutrophils, has been widely studied (23, 32, 43, 46, 50, 59, 68, 77). Cryptococcal GXM is antiphagocytic (34) and a powerful immunomodulator (45, 79). C. neoformans capsule size directly correlates with the efficacy of phagocytosis in vitro (6, 15, 82). Phagocytosis of C. neoformans can result in either fungal killing (24, 30) or survival (2, 3, 39-41, 71, 80). Killing of C. neoformans apparently involves the production of oxidative species (24), while the mechanisms of fungal escape include phagosome extrusion, cell-to-cell spread, and phagosomal permeabilization (2, 3, 40, 41, 71). Capsular polysaccharides and melanin are known to modulate the interaction of C. neoformans with phagocytes in favor of the fungus (27, 39, 47, 48, 71, 72, 74, 76), but the role of other structures in the outcome of yeast phagocytosis is virtually unknown.A number of recent studies have shown that GXM, GalXM, pigments, proteins, and lipids are trafficked in vesicles that traverse the cell wall (7, 14, 20, 56, 57, 62, 64, 65). Extracellular vesicles are also produced by the pathogens Candida albicans, C. parapsilosis, Sporothrix schenckii, and Histoplasma capsulatum, as well as by the model yeast Saccharomyces cerevisiae (1), suggesting that extracellular vesicle secretion is a general property of fungal cells. Secreted vesicles are heterogeneous. For instance, vesicles secreted by C. neoformans were classified into four different groups based on morphology and electron density (64). Additionally, vesicle diameter ranges from 30 to 400 nm, with the majority having dimensions of 100 to 150 nm (20, 64, 65). The combined use of serology, biochemistry, proteomics, and lipidomics led to the identification of 2 polysaccharides, phospholipids, 4 neutral lipids, and 76 proteins as extracellular vesicle components secreted by C. neoformans, which means that at least 81 different molecules are released to the extracellular milieu by vesicular secretion (14, 57, 64). It is likely that this number is an underestimate resulting from the difficulty of proteomic studies in vesicles from highly encapsulated cryptococcal cells, since a higher number of vesicular proteins were characterized in other fungi. For example, in H. capsulatum, proteomics and lipidomics of extracellular vesicles revealed an even more complex composition, including 283 proteins and 17 different phospholipids (1).In this study, we evaluated the influence of extracellular vesicles on the fate of C. neoformans after phagocytosis by mouse macrophages. Our results show that fungal vesicles are biologically active and stimulate macrophages. Moreover, our results demonstrate that vesicles from an acapsular mutant strain were more effective in eliciting macrophage activation and augmenting fungal killing than vesicles from encapsulated strains. Taken together, our findings suggest that fungal secretory vesicles have the potential to influence the interaction of C. neoformans with host cells.     

Meningococcal Porin PorB Prevents Cellular Apoptosis in a Toll-Like Receptor 2- and NF-κB-Independent Manner

Meningococcal porin PorB is an inhibitor of apoptosis induced via the intrinsic pathway in various cell types. This effect is attributed to prevention of mitochondrial depolarization and of subsequent release of proapoptotic mitochondrial factors. To determine whether apoptosis is globally inhibited by PorB, we compared the intrinsic and extrinsic pathways in HeLa cells. Interestingly, PorB does not prevent extrinsic apoptosis induced by tumor necrosis factor alpha plus cycloheximide, suggesting a unique mitochondrial pathway specificity. Several intracellular factors regulated by NF-κB, including members of the Bcl-2 family and of the inhibitor of apoptosis (IAP) family, play major roles in controlling apoptosis, and some of them are thought to contribute to the antiapoptotic effect of the gonococcal porin, PIB. However, most of the members of the Bcl-2 family and the IAP family are not induced by meningococcal PorB in HeLa cells, with the exception of Bfl-1/A1. Interestingly, PorB does not induce NF-κB activation in HeLa cells, likely due to a lack of Toll-like receptor 2 (TLR2) expression in these cells. Bfl-1/A1 expression is also regulated by CBF1, a nuclear component of the Notch signaling pathway, independent of NF-κB activation. Since HeLa cells are protected by PorB from intrinsic apoptosis events, regardless of TLR2 and NF-κB expression, the possibility of a contribution of alternative signaling pathways to this effect cannot be excluded. In this paper, we describe an initial dissection of the cascade of cellular events involved in the antiapoptotic effect of PorB in the absence of TLR2.Apoptosis, or programmed cell death, is characterized by morphological events, including membrane blebbing and nuclear and chromatin condensation, and by intracellular events, such as activation of cytosolic proteins and DNA degradation (26). A variety of different intracellular stress signals can trigger apoptosis, including bacterial infections, excessive calcium, chemical substances, DNA-damaging agents (intrinsic or mitochondrial pathway), and cell surface death receptor activation (extrinsic pathway). Both pathways are divided into three basic phases: (i) initiation, (ii) commitment, and (iii) execution, ending with cell death (71).Several intracellular protein families, such as the Bcl-2 family (40), caspases (8), and the inhibitors of apoptosis (IAPs) (16), play important roles in controlling apoptosis. Bcl-2 proteins have a dual role; they trigger apoptosis (Bax, Bak, and Bid [1]) or block it (Bcl-2, Bcl-xL, Bfl-1, and Mcl-1 [39]). Proapoptotic Bcl-2 proteins can induce release of mitochondrial factors, including cytochrome c (36), apoptosis-inducing factor (AIF) (70), and Smac/DIABLO (18), in both a mitochondrial membrane potential-dependent manner and a mitochondrial membrane potential-independent manner (2, 25, 74). These events lead to activation of caspase 9 and 6 (intrinsic pathway) and subsequent DNA degradation. Alternatively, proapoptotic Bcl-2 proteins can also directly activate caspase 8 (extrinsic pathway) (1, 68), but the two pathways converge at a downstream event, caspase 3 activation (24). Antiapoptotic Bcl-2 proteins act mostly by modulating mitochondrial functions directly by interacting with mitochondrial components of the permeability transition pore or indirectly by neutralizing proapoptotic Bcl-2 proteins (7, 69, 72). IAPs are a family of proteins that directly inhibit caspase activation (16, 32) and, similar to Bcl-2 proteins, are also regulated by NF-κB (9).Modulation of apoptosis by several intracellular and extracellular bacteria, mostly to avoid normal host defense responses, has been described previously. Many bacteria induce and/or prevent apoptosis, depending on the host cell type, growth conditions, or bacterial life cycle. Some examples of bacteria that inhibit apoptosis are Chlamydia (20, 22, 79), Shigella flexneri (11), Brucella (28), Porphyromonas gingivalis (56, 58), Neisseria meningitidis, and Neisseria gonorrhoeae (4, 23, 31, 42, 50, 55, 62, 65, 73). Our group and other workers have reported that live N. meningitidis and purified meningococcal porin inhibit apoptosis (15, 49, 50, 62, 75), potentially via multiple mechanisms. While meningococcal infection induces NF-κB-mediated upregulation of antiapoptotic genes, purified PorB and PorB from live bacteria directly interact with mitochondria and modulate their membrane potential, preventing release of cytochrome c.N. gonorrhoeae and purified gonococcal porin PIB induce NF-κB-mediated upregulation of antiapoptotic genes (4, 5, 23, 33, 55, 65), which could also contribute to prevention of apoptosis.A correlation between the antiapoptotic effect of PorB and activation of NF-κB has not been shown so far, although our group has demonstrated that PorB activates NF-κB in a Toll-like receptor 2 (TLR2)-dependent manner (43, 46, 48, 52). Interestingly, various human and murine cell types are protected from apoptosis by PorB (23. 49, 50, 51) regardless of TLR2 expression. To clarify the role of this receptor in the antiapoptotic effect of PorB, this work focused in particular on naturally TLR2-deficient HeLa cells (78) and aimed at dissecting the potential cascade of cellular events elicited by PorB leading to protection from apoptosis.     

Comparative Evaluation of a Commercially Available Automated System for Extraction of Viral DNA from Whole Blood: Application to Monitoring of Epstein-Barr Virus and Cytomegalovirus Load

The NucliSENS easyMAG automated system was compared to the column-based Qiagen method for Epstein-Barr virus (EBV) or cytomegalovirus (CMV) DNA extraction from whole blood before viral load determination using the corresponding R-gene amplification kits. Both extraction techniques exhibited a total agreement of 81.3% for EBV and 87.2% for CMV.Epstein-Barr virus (EBV) and cytomegalovirus (CMV) infections represent a significant clinical threat for immunocompromised patients. The frequent determination of EBV and CMV viral load permits the early diagnosis of infection, start of preemptive or curative therapy, and monitoring of treatment efficiency (5, 17, 20). By comparison to serum, plasma, or white-blood-cell fractions, whole-blood samples are now recognized as the most suitable sample for the determination of viral loads for EBV and CMV (3, 4, 7, 9, 10, 12, 13, 18, 19).Although the methods relying on silica columns are time-consuming and need trained experimenters, these methods are considered the gold standard for the extraction of nucleic acids from whole-blood samples. Due to the large amount of genetic material in such samples, new extraction methods must be carefully evaluated, including those relying on automated devices (1, 7, 10, 14, 15). The fully automated NucliSENS easyMAG instrument (bioMérieux) using magnetic silica particles (2) allows the simultaneous process of up to 24 extractions. The use of magnetic particles eliminates the several centrifugation steps that could be a source of cross-contamination, and manual steps are limited to the loading of samples, reagents, and disposables. The performance of this method in the extraction of DNA from whole-blood samples prior to viral quantification has not been yet evaluated. The present study was undertaken to answer this question in the clinical context of EBV or CMV infection.The whole-blood specimens selected for this study included 80 samples for EBV analysis and 94 samples for CMV analysis, taken from patients hospitalized at the University Hospital of Saint-Etienne, Saint-Etienne, France, from December 2007 to September 2008. The samples were kept frozen at −20°C. After the samples were thawed, whole-blood aliquots were tested and kept at 4°C for up to 24 h for potential retest. Two hundred microliters of each selected sample was extracted by two different technicians either by the reference manual method, i.e., QIAamp column DNA blood extraction kit according to the manufacturer''s recommendations (Qiagen), or by the new specific B protocol on the NucliSENS easyMAG instrument. The latter protocol consists of the treatment of 200 μl of whole blood in 2 ml of lysis buffer and the capture of nucleic acids by 140 μl of magnetic silica. After incubation and washing procedures, nucleic acids were recovered in 50 μl of elution buffer. EBV and CMV loads were quantified by using the respective R-gene amplification kit (Argene Biosoft) according to the manufacturer''s recommendations. Both amplification kits have been previously validated for quantification of EBV and CMV load in whole blood (8, 11). DNA extracts from both methods were amplified in the same run using an ABI 7500 instrument (Applied Biosystems).     

Glyceraldehyde-3-Phosphate Dehydrogenase Is a Surface-Associated,Fibronectin-Binding Protein of Trichomonas vaginalis

Trichomonas vaginalis colonizes the urogenital tract of humans and causes trichomonosis, the most prevalent nonviral sexually transmitted disease. We have shown an association of T. vaginalis with basement membrane extracellular matrix components, a property which we hypothesize is important for colonization and persistence. In this study, we identify a fibronectin (FN)-binding protein of T. vaginalis. A monoclonal antibody (MAb) from a library of hybridomas that inhibited the binding of T. vaginalis organisms to immobilized FN was identified. The MAb (called ws1) recognized a 39-kDa protein and was used to screen a cDNA expression library of T. vaginalis. A 1,086-bp reactive cDNA clone that encoded a protein of 362 amino acids with identity to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was obtained. The gapdh gene was cloned, and recombinant GAPDH (rGAPDH) was expressed in Escherichia coli cells. Natural GAPDH and rGAPDH bound to immobilized FN and to plasminogen and collagen but not to laminin. MAb ws1 inhibited binding to FN. GAPDH was detected on the surface of trichomonads and was upregulated in synthesis and surface expression by iron. Higher levels of binding to FN were seen for organisms grown in iron-replete medium than for organisms grown in iron-depleted medium. In addition, decreased synthesis of GAPDH by antisense transfection of T. vaginalis gave lower levels of organisms bound to FN and had no adverse effect on growth kinetics. Finally, GAPDH did not associate with immortalized vaginal epithelial cells (VECs), and neither GAPDH nor MAb ws1 inhibited the adherence of trichomonads to VECs. These results indicate that GAPDH is a surface-associated protein of T. vaginalis with alternative functions.Trichomonas vaginalis, an extracellular protozoan parasite, is the cause of trichomonosis, the most prevalent nonviral sexually transmitted disease (47). In women, vaginitis due to T. vaginalis clinically manifests with symptoms of vaginal itching, odor, and discharge. Adverse health outcomes for women with this sexually transmitted disease include cervical cancer (46) and preterm delivery and low-birth-weight infants (25). There is a relationship between seropositivity to T. vaginalis and prostate cancer (43). This disease is significant due to its association with human immunodeficiency virus (33, 45). More recently, persistent, undetected T. vaginalis infections associated with asymptomatic carriage were found among women (40).T. vaginalis penetration of the mucous layer (28), followed by adherence to vaginal epithelial cells (VECs), is preparatory for colonization (9, 10). VEC adherence by parasites is mediated by numerous distinct trichomonad surface adhesins (5, 10, 18). Brief contact of T. vaginalis with VECs and fibronectin (FN) elicited dramatic changes in parasite morphology, suggesting a host-specific signaling of parasites (8, 9). Importantly, iron and cell contact by parasites each upregulated the expression of adhesins in a coordinated fashion via distinct mechanisms (2, 4, 6, 21, 29). Genetic approaches using antisense (AS) inhibition of synthesis (36, 37) and heterologous expression in Tritrichomonas foetus (26, 36) have reaffirmed the role of these T. vaginalis proteins as adhesins. T. vaginalis organisms secrete or release numerous metabolic enzymes, including adhesin AP65 (decarboxylating malic enzyme), α-enolase, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) during growth and multiplication (27). AP65 and α-enolase were found to reassociate with the parasite surface for the expression of adhesin function (19) and binding to plasminogen (35), respectively.There is an increased awareness of the existence of metabolic enzymes on the surfaces of bacterial pathogens, yeast, and parasites (12, 24, 35). These surface-associated enzymes appear to be novel virulence factors (17, 22, 38, 39). The anchorless glycolytic enzymes GAPDH (13, 31, 38) and α-enolase (39) are present on the surface of group A streptococcus. The surface-associated GAPDH of Candida albicans binds with strong affinity to FN and laminin (22). In enterohemorrhagic Escherichia coli and enteropathogenic E. coli, GAPDH is an extracellular protein that binds human plasminogen and fibrinogen and also interacts with intestinal epithelial cells (17).We demonstrate that GAPDH is another enzyme on the surface of T. vaginalis. A monoclonal antibody (MAb) that inhibited parasite associations with FN was immunoreactive with GAPDH. Importantly, iron was found to regulate gene expression and synthesis and surface placement of GAPDH. Both low-iron-grown trichomonads and AS-transfected parasites with decreased amounts of GAPDH had smaller amounts of surface GAPDH and corresponding lower levels of binding to FN. GAPDH was not involved in adherence of trichomonads to immortalized VECs. Interestingly, as with other microbial pathogens, T. vaginalis GAPDH also bound plasminogen and collagen but not laminin (17, 22).     

Role of Dendritic Cells and Alveolar Macrophages in Regulating Early Host Defense against Pulmonary Infection with Cryptococcus neoformans

Successful pulmonary clearance of the encapsulated yeast Cryptococcus neoformans requires a T1 adaptive immune response. This response takes up to 3 weeks to fully develop. The role of the initial, innate immune response against the organism is uncertain. In this study, an established model of diphtheria toxin-mediated depletion of resident pulmonary dendritic cells (DC) and alveolar macrophages (AM) was used to assess the contribution of these cells to the initial host response against cryptococcal infection. The results demonstrate that depletion of DC and AM one day prior to infection results in rapid clinical deterioration and death of mice within 6 days postinfection; this effect was not observed in infected groups of control mice not depleted of DC and AM. Depletion did not alter the microbial burden or total leukocyte recruitment in the lung. Mortality (in mice depleted of DC and AM) was associated with increased neutrophil and B-cell accumulation accompanied by histopathologic evidence of suppurative neutrophilic bronchopneumonia, cyst formation, and alveolar damage. Collectively, these data define an important role for DC and AM in regulating the initial innate immune response following pulmonary infection with C. neoformans. These findings provide important insight into the cellular mechanisms which coordinate early host defense against an invasive fungal pathogen in the lung.Cryptococcus neoformans, an opportunistic fungal pathogen acquired through inhalation, causes significant morbidity and mortality primarily in patients with impairments in host defense, including those with AIDS, those with lymphoid or hematological malignancies, or those receiving immunosuppressive therapy secondary to autoimmune disease or organ transplantation (31, 33, 60). The development of a T1 antigen-specific immune response characterized by gamma interferon production and classical activation of macrophages is required to eradicate the organism (4, 8, 21, 23, 24, 28). This adaptive immune response takes 2 to 3 weeks to develop and coincides with the CCR2-mediated recruitment of additional pulmonary dendritic cells (DC) and T cells to the lung (51, 66, 67). The role of initial, innate immune responses against the organism (prior to the development of adaptive immunity) is not well understood.Resident lung phagocytic cells, primarily DC and alveolar macrophages (AM), are likely the first immune cells exposed to C. neoformans upon inhalation of the organism into the lung. Both DC and AM express lectin receptors, including macrophage mannose receptor and DC-specific non-ICAM3 grabbing nonintergrin (DC-SIGN) (14, 15), which bind C. neoformans glycoantigens, including mannoproteins (42, 55). DC and AM phagocytose the organism in vitro and in vivo (29, 34, 63, 77, 78), and phagocytosis (and/or exposure to soluble glycoantigens or cryptococcal DNA) is associated with cytokine and chemokine production (5, 29, 40, 48, 49, 55, 61) and yeast lysis (77). It is unclear whether phagocytosis by resident DC and AM contributes to early clearance and/or the later development of adaptive immunity.DC represent an important interface between innate and adaptive immunity (reviewed in references 25, 53, 59, and 62). DC-cryptococcal interactions alter DC antigen-presenting functions and modulate resultant T-cell responses in vitro (10, 19, 55, 71). Following cryptococcal infection in vivo, DC migrate to thoracic lymph nodes (4, 52, 67). Thereafter, newly recruited DC colocalize with T cells within bronchovascular infiltrates in the lung (51). This is associated with interleukin-12 and gamma interferon production, yet direct evidence that DC-T-cell interactions modulate anticryptococcal responses in vivo remains sparse.A well-described murine model of in vivo DC depletion has been used to evaluate the contribution of DC to the development of dynamic, antigen-specific immune responses against a variety of antigens and microbial pathogens (16, 26, 27, 37, 56, 57, 69). Depletion results from the administration of diphtheria toxin (DT) to transgenic (Tg) mice in which the DT receptor (DTR) has been linked to the CD11c promoter. DT administration transiently depletes tissue DC (which express CD11c) for up to 96 h. This model has helped in assessment of the role of pulmonary DC in mediating adaptive immune responses against inhaled antigen (ovalbumin) and influenza virus (16, 69). In these studies, DT administration was associated with transient depletion of AM (which also express CD11c). The effect of CD11c cell depletion on innate immune responses has not been assessed (or reported) in these studies.In the current study, this established model of DT-mediated depletion of resident DC and AM was used to assess the in vivo contribution of these cells to the initial host response against cryptococcal infection. This objective is clinically relevant, as studies modulating DC numbers are in development for the treatment of patients with asthma (30, 32), autoimmunity (65, 68, 74), organ transplantation (12, 39, 43), and cancer (3, 11, 73). It is unknown whether manipulating the number of tissue DC will alter the innate or adaptive antifungal host defense in these patients. Our results demonstrate that DC and AM are critical regulators of the initial immune response against C. neoformans within the lung. Early mortality in DC- and AM-depleted mice precluded our ability to assess the role of these cells in the development of adaptive immunity.     

Multiplexed Luminex xMAP Assay for Detection and Identification of Five Adenovirus Serotypes Associated with Epidemics of Respiratory Disease in Adults

Several serotypes of human adenovirus (HAdV) cause acute respiratory disease (ARD) among healthy adults, sometimes generating broad outbreaks with high attack rates and occasional fatalities. Timely serotype identification provides valuable epidemiological information and significantly contributes to prevention (vaccination) strategies. The prevalence of specific serotypes causing ARD varies geographically. HAdV-3, HAdV-4, HAdV-7, HAdV-14, and HAdV-21 are the serotypes most commonly found in adult populations in the Western Hemisphere. Unfortunately, conventional serotype identification is a tedious process which can take a week or longer. For this reason, new molecular methods for serotype identification are needed. Commercially available rapid antigen and PCR assays for the detection of HAdV are universal but do not distinguish between the different serotypes. We describe the development of a sensitive and specific multiplex assay capable of identifying serotypes 3, 4, 7, 14, and 21. Two sets of primers were used for nonspecific (universal) PCR amplification, and serotype-specific probes coupled to Luminex tags were used for target-specific extension (TSE). PCR and TSE primers were designed using known hexon gene sequences of HAdV. The TSE products of HAdV-3, HAdV-4, HAdV-7, HAdV-14, and HAdV-21 were correctly identified using the Luminex xMAP fluid microsphere-based array system. No cross-reactivity with other respiratory pathogens or other HAdV serotypes was observed. This multiplexed assay can be expanded to include more serotypes and will allow broad and rapid detection and identification of adenoviral serotypes in a high-throughput environment.Human adenoviruses (HAdVs) cause a wide range of diseases in humans, including upper and lower respiratory illness, urinary tract infections, conjunctivitis, and gastroenteritis. There are 51 different serotypes based on type-specific serum neutralization, and these are classified into six species (A, B, C, D, E, and F) on the basis of hemagglutination, oncogenic, and phylogenetic properties (13, 24, 28, 31).The most common serotypes known to cause respiratory illness in the adult population are 3, 4, 7, 11, 14, and 21 (21, 24, 25, 37). All of these can cause locally severe outbreaks with high attack rates. These types of outbreaks are rarely reported in civilian populations but are essentially continuous at military training camps, particularly with serotype 4 (HAdV-4) and, to a lesser extent, HAdV-7 (10). Some recent studies have suggested that specific serotypes cause more severe disease, especially in immunocompromised patients (8, 13, 14, 24, 28, 32). The military previously established universal vaccination of new recruits for HAdV-4 and -7, which reduced adenovirus-induced acute respiratory disease (ARD) by 95 to 99% (20). Production of these vaccines ceased in 1996, but similar replacement vaccines are currently in the final stages of licensure. In 2006 and thereafter, HAdV-14 and HAdV-21 produced extensive outbreaks of ARD in military recruits (20).It is vital to be able to identify the serotype and changes in the serotype over time in order to evaluate viral virulence, vaccine efficacy, and the potential impact of antiviral use. Identification of serotypes was traditionally done by virus isolation in cell culture, followed by neutralization tests, antibody studies, and/or antigen detection by immunofluorescence (3, 16, 19, 23). These techniques are time-consuming and labor-intensive (11, 34). Another technique that has been used is whole-genome restriction endonuclease analysis, which relies on large-scale viral culture to generate the full genomic substrate (2, 4, 5).More recently, PCR-based detection and discrimination methods have been developed (1, 21, 35, 36). These techniques are faster and can also detect coinfections when used in a multiplex assay (20, 21, 33, 35, 36), thus reducing cost, labor, and sample volume needed for analysis. Current PCR assays identify the six subgenera (A to F) or up to three serotypes per reaction mixture (1, 21, 33, 35, 36). Real-time PCR also exists for generic detection (but not discrimination) of all 51 serotypes (6, 7, 12, 21, 33), and sequence analysis of the genomic region coding for the seven hypervariable loops of the hexon (the primary antigenic determinant) can identify and discriminate all 51 serotypes with a single assay (27), albeit a relatively time-consuming and complex one.Luminex has designed an xMAP system that in theory can detect up to 100 pathogens in a single sample by coupling bioassays with digital signal processing in real time. The platform is a suspension array where specific capture moieties are covalently coupled to the surfaces of internally dyed microspheres (22). The diversity of these microspheres increases the number of targets that can be identified in a single sample approximately 20-fold over traditional real-time PCR.In this study we designed and tested a Luminex-based assay capable of detecting and identifying HAdV-3, HAdV-4, HAdV-7, HAdV-14, and HAdV-21 in a single reaction. The assay was tested, and the results indicate its potential as a diagnostic tool.     

Serum Bactericidal Antibody Response 1 Year after Meningococcal Polysaccharide Vaccination of Patients with Common Variable Immunodeficiency

Some patients with common variable immunodeficiency (CVID) can generate an antibody response following vaccination with Neisseria meningitidis polysaccharide, but the duration of this protection is unknown. In this study, serum bactericidal antibody (SBA) responses to serogroup C N. meningitidis of 23 patients with CVID and 23 sex- and age-matched controls were measured 1 year after vaccination with the plain A/C meningococcal polysaccharide vaccine. The fold rise in serum bactericidal antibody geometric mean titers of the control group from prevaccination to 1 year postvaccination was significantly higher than that of the patient group (5.41- versus 2.96-fold, P = 0.009). Of 23 CVID patients, 8 had a poor response to vaccine (<4-fold rise) 3 weeks after vaccination, and low titers remained when measured 1 year later. Of the 15 CVID patients who had a normal response to vaccine (≥4-fold rise) 3 weeks after vaccination, 6 cases failed to maintain protective SBA titers, whereas the remaining 9 had protective titers 1 year after vaccination. Only one of the 23 controls, who developed protective SBA titers after 3 weeks, lost the protective titers after 1 year. Among the patients, the presence of bronchiectasis and/or splenomegaly at enrollment was associated with poor SBA response to vaccine at 3 weeks and/or failure to maintain protective levels at 1 year. The results of this study demonstrate that a number of CVID patients can produce protective antibody titers that can persist for 1 year after vaccination, which lends strong support to the inclusion of polysaccharide vaccine in the immunization program for CVID patients.Common variable immunodeficiency (CVID) is the commonest symptomatic primary immunodeficiency disease and is a heterogeneous group of disorders, characterized by severe reduction of serum levels of IgG and IgA, with normal or low numbers of B cells in the absence of any recognized genetic abnormality (2, 11, 16, 30). Patients with CVID usually experience recurrent bacterial infections (1, 14) and carry an increased risk of autoimmunity (12, 28) and malignancies (4, 24). Various defects of B cells, T cells, and dendritic cells have been reported for CVID (26, 29, 34-36); however, the exact pathophysiology of the disease is still unclear (3, 15).Deployment of polysaccharide and protein vaccines in CVID is a subject of active debate. Although it is intuitive that CVID patients should have poor antibody responses to vaccine, it is apparent that some patients can produce normal antibody titers (5, 18, 21, 32, 33). We have reported that a protective antibody response was achieved 3 weeks following vaccination with polysaccharide meningococcal vaccine of a group of CVID patients (32, 33). In the current study, we measured serum bactericidal antibody (SBA) titers (7) of the same cohort of patients 1 year after the initial vaccination.     

Infection with Anaplasma phagocytophilum Induces Multilineage Alterations in Hematopoietic Progenitor Cells and Peripheral Blood Cells

Infection with Anaplasma phagocytophilum, a gram-negative, lipopolysaccharide (LPS)-negative, obligate intracellular bacterium, results in multiple peripheral blood cytopenias. We hypothesized that infection with this organism would result in decreased bone marrow (BM) function and shifts in hematopoietic progenitor cells (HPCs) and lineage-committed cells in a well-established murine model of infection. HPCs and lineage-committed progenitors were enumerated in the BM and spleen during acute infection. BM cytokine production and BM CXCL12 expression were determined. Infection resulted in peripheral blood bicytopenia, marked decreases in the number of lineage-committed HPCs in the BM along with concurrent increases in the number of lineage-committed HPCs in the spleen, and a mixed, predominantly myelosuppressive BM cytokine environment. There was significant downregulation of CXCL12 in BM cells that may have been partially responsible for changes in HPC trafficking observed. Changes occurred in the absence of direct pathogen infection of BM cells. Hematopoietic lineage assessment demonstrated that there was loss of erythrocytes and B lymphocytes from the BM along with increased granulopoiesis. These changes were accompanied by splenomegaly due to lymphoid hyperplasia and increased hematopoiesis, most notably erythropoiesis. These changes largely mimic well-described inflammation and endotoxin-mediated effects on the BM and spleen; however, the numbers of peripheral blood neutrophils appear to be independently modulated as granulocytic hyperplasia does not result in neutrophilia. Our findings highlight a well-conserved series of events that we demonstrate can be instigated by an LPS-negative pathogen in the absence of an endotoxin-mediated acute proinflammatory response.Hematologic complications result from a wide variety of infectious diseases and may significantly complicate the prognosis and outcome of these diseases. Infection and inflammation elicit alterations in all hematopoietic subsets. For example, endotoxin mediates a proinflammatory cascade of events including the release of systemic, proinflammatory cytokines, granulocyte efflux from the bone marrow (BM), the release of colony-stimulating factors, and a compensatory granulocytic hyperplasia (49, 54, 61, 65). The expansion of the granulocytic compartment fuels a reactive neutrophilia that is balanced by reciprocal depletion of the BM lymphoid compartment by either mobilization or apoptosis of lymphocytes (58, 59). Inflammation, infection, and injury also profoundly alter erythropoiesis and generally result in anemia. The pathogenesis behind alterations in erythropoiesis involves multiple factors, including cytokine-mediated erythroid apoptosis, a blunted BM response to erythropoietin, reduced iron availability, suppression of erythroid progenitor cell proliferation, and BM egress of erythroid progenitor cells (34, 40, 50, 51). Infection and inflammation also modulate the numbers of platelets. Acute endotoxemia and acute viral infections are most frequently associated with thrombocytopenia. There are multiple mechanisms of thrombocytopenia, and they include increased platelet sequestration and destruction and inhibition of megakaryopoiesis (37). Platelet kinetics can also be modulated by inflammation-induced production of interleukin-6 (IL-6), which promotes a reactive thrombocytosis (37).Anaplasma phagocytophilum, the agent of granulocytic anaplasmosis (GA) (formerly granulocytic ehrlichiosis), is a gram-negative, lipopolysaccharide (LPS)-negative (38), obligate intracellular bacterium that resides primarily within circulating granulocytes (20, 25). GA is an emerging, tick-borne infectious disease (26). Unlike endotoxin-mediated events, infection with A. phagocytophilum typically results in multiple cytopenias in natural disease in both humans (5) and animals (7, 43, 48), as well as in animal models of infection (10, 12, 33). The cytopenias typically include mild nonregenerative anemia, mild to moderate leukopenia, and moderate to marked thrombocytopenia. The mechanism(s) underlying the cytopenias are not fully understood. Immune-mediated destruction or splenic sequestration of cells is unlikely as cytopenias occur within the first 2 to 5 days of infection, before a significant acquired immune response is mounted, SCID mice (lacking functional B and T cells) become cytopenic (10, 12, 33), and splenectomized mice become thrombocytopenic (12).Multiple cytopenias are frequently associated with alterations in BM production of cells. Primary BM progenitor cells are susceptible to A. phagocytophilum infection (36), and A. phagocytophilum DNA is present and may persist in BM during natural and experimental infections (8, 32). Nonetheless, pathological changes associated with infection are largely thought to be independent of direct pathogen infection (17, 53). Although routine histopathologic evaluation of BM from GA patients can reveal normo- to hypercellular marrow (39), pathogens can have profound effects on hematopoiesis with limited morphological alterations of BM. Such effects include nonvisible effects on stromal cells, increased apoptosis of proliferating cells, and a lack of differentiation and proliferation of immature hematopoietic precursors (ineffective hematopoiesis) (22, 24, 44). Hematopoietic cell trafficking and mobilization can be initiated by disruption of the CXCL12/CXCR4 axis in BM (15, 41, 59). The CXCL12/CXCR4 axis is a key regulator of stem cell and lineage-committed progenitor cell trafficking.The objective of this study was to characterize the hematopoietic response to infection with A. phagocytophilum, a pathogen that elicits multiple peripheral blood cytopenias. We hypothesized that infection would result in altered BM function. We found that infection with A. phagocytophilum resulted in rapid and profound multilineage deficits in hematopoietic progenitor proliferation or differentiation. This quantitative defect in hematopoiesis was accompanied by induction of myelosuppressive chemokines within the BM, shifts in BM hematopoietic subsets, including B-lymphocyte depletion, erythroid depletion, and granulocytic hyperplasia, and significant downregulation of CXCL12 in BM cells. Changes were independent of the pathogen burden or the route of pathogen inoculation. Our data, combined with data for characteristic multiple-lineage cytopenias, suggest that kinetic alterations in hematopoietic cell subsets may contribute to infection-induced cytopenias.     

New Approach for Diagnosis of Candidemia Based on Detection of a 65-Kilodalton Antigen

Nosocomial candidiasis is a major concern in tertiary care hospitals worldwide. This infection generally occurs in patients with degenerative and neoplastic diseases and is considered the fourth most frequent cause of bloodstream infections. Diagnosis of candidemia or hematogenous candidiasis has been problematic because clinical signs and symptoms are nonspecific, leading to delays in diagnosis and, consequently, delays in appropriate antifungal therapy. We developed an inhibition enzyme-linked immunosorbent assay (ELISA) for detection of a 65-kDa antigen in an experimental model of candidemia and for diagnosis of patients in intensive care units (ICUs) with suspected candidemia. An anti-65-kDa monoclonal antibody was tested for detection of the 65-kDa antigen produced by Candida albicans, Candida tropicalis, and Candida parapsilosis in murine candidemia models. The 65-kDa antigen was detected in sera at concentrations ranging from 0.012 to 3.25 μg/ml. A total of 20 human patients with candidemia were then evaluated with the inhibition ELISA using sequential sera. Sixteen (80%) patients had the 65-kDa antigen in concentrations ranging from 0.07 to 5.0 μg/ml. Sequential sera from patients with candidemia presented three different patterns of antigenemia of the 65-kDa molecule: (i) total clearance of antigenemia, (ii) initial clearance and relapse of antigenemia, and (iii) partial clearance of antigenemia. Our results indicate detection of the 65-kDa protein may be a valuable tool for the diagnosis of candidemia by C. albicans, C. tropicalis, and C. parapsilosis.Nosocomial candidiasis is a major concern in tertiary care hospitals worldwide. This infection generally occurs in patients with degenerative and neoplastic diseases exposed to broad-spectrum antibiotics, immunosuppressive drugs, and invasive medical procedures (10, 11, 32, 40, 48). The incidence of invasive candidiasis has increased over the past two decades, and candidemia is now considered the fourth most frequent cause of bloodstream infections (50, 53). A recent nationwide surveillance study, conducted in public general tertiary care hospitals in Brazil, found an incidence of 2.69 episodes/1,000 admissions, a rate 2 to 8 times higher than that observed in medical centers from Northern Hemisphere countries (10). In South America, most candidemic episodes are related to Candida albicans, Candida tropicalis, and Candida parapsilosis strains; Candida glabrata has been scarcely reported (9-12, 15, 21, 25, 42, 55). This is in contrast to United States and European medical centers, where C. glabrata is considered a major pathogen.Diagnosis of candidemia or hematogenous candidiasis has been problematic. The clinical signs and symptoms are nonspecific; therefore, the diagnosis and, consequently, appropriate antifungal therapy are delayed. Even in patients with autopsy-proven systemic candidiasis, positive diagnoses from blood cultures ranged from 40 to 60% (51-53).Antigen detection for the serodiagnosis of invasive Candida infections has been reported (5, 6, 13, 14, 18-20). Matthews and Burnie developed an immunobinding method for detection of a 47-kDa cytoplasmatic protein antigen in patients with systemic candidiasis (34). An immunoassay detecting a 48-kDa antigen of Candida, subsequently recognized as enolase (13, 33), is available for the diagnosis of invasive candidiasis (54). Latex agglutination tests are based on the detection of mannan, a cell wall component that is the most widely studied antigen in patients with candidiasis (5, 20). Commercial tests (Pastorex Candida assay and Cand-Tec assay) have been used to detect this molecule in sera. Colorimetric assays (Fungitec G and Fungitec G MT) detect β-d-glucan, a major structural component of the fungal cell wall, in serum and have been used for diagnosis of fungal infections. Studies show the concentration of β-d-glucan is increased in experimental models of fungal infections (35-38), as well as in the plasma of patients with mycosis (20, 36).Various tests have been developed based on detection of antibodies, antigens, and metabolites, although, they are all time-consuming and lack either specificity or sensitivity (50). In C. albicans, a 65-kDa mannoprotein (Mp65) is a structural and secreted component of the fungus. This mannoprotein is particularly observed in extracellular fractions of hyphal cells (1, 8, 17, 44-46). Mp65 is also present in both the structural and secretory mannoprotein material and is recognized by peripheral blood T cells of practically all healthy individuals (quasiuniversal antigen) (1, 44, 45), making it a potential target for immunodiagnosis of patients with suspected candidemia. Arancia et al. (1) used real-time PCR to detect and quantify C. albicans in sera from patients with invasive candidiasis. This assay was specific for a DNA fragment containing the gene for the 65-kDa mannoprotein of C. albicans (CaMP65). The assay was shown to be sensitive and specific for C. albicans, allowing quantitative detection of this fungus in clinical samples.The inhibition enzyme-linked immunosorbent assay (inh-ELISA) is an enzyme immunoassay (EIA) to detect circulating antigens in sera of patients with invasive fungal infections. This test uses a species-specific murine monoclonal antibody (MAb) with high sensitivity and specificity and is useful in diagnosis and follow-up of paracoccidioidomycosis patients. It is also useful for antigen detection in the cerebrospinal (28) and bronchoalveolar (27, 29-31) fluids of these patients.In this study, our intention was to demonstrate that inh-ELISA is a sensitive detection method able to detect nanograms of antigen in serum samples from patients with candidemia and not to compare it or show its superiority to other assays. Na and Song previously compared three serological methods of detecting C. albicans secreted aspartyl proteinase antigen (39); they found inh-ELISA had 93.9% sensitivity and 96.0% specificity and detected concentrations ranging from 6.3 to 19.0 ng/ml. The sensitivity and specificity for standard ELISA were 69.7 and 76.0%, respectively; while for capture ELISA, the sensitivity and specificity were 93.9 and 92.0%, respectively. The results of Na and Song showed inh-ELISA with MAb CAP1 effectively detected circulating secreted aspartyl proteinase antigen and suggest it may be useful for the diagnosis and treatment monitoring of invasive candidiasis.The aim of this study was to standardize an alternative inh-ELISA for detection of a 65-kDa antigen, present in C. albicans, C. tropicalis, and C. parapsilosis, using a MAb specific for the 65-kDa Candida protein. The assay could be used for diagnosis and follow-up of patients with candidemia. The present study involved five different stages: (i) identification of an immunodominant 65-kDa antigen of C. albicans that is common to C. tropicalis and C. parapsilosis, (ii) production of an anti-C. albicans 65-kDa-molecule MAb for detection of the immunodominant antigen mentioned above, (iii) application of the MAb to the inh-ELISA, (iv) characterization of antigenemia in an animal model, and (v) evaluation of the developed inh-ELISA with sera from patients with candidemia.