Antiretroviral Therapy Restores Diversity in the T-Cell Receptor Vβ Repertoire of CD4 T-Cell Subpopulations among Human Immunodeficiency Virus Type 1-Infected Children and Adolescents

Human immunodeficiency virus (HIV) type 1 infection perturbs the T-cell receptor (TCR) Vβ repertoire. The TCR CDR3 length diversity of individual Vβ families was examined within CD45RA and CD45RO CD4 T cells to assess the impact of the virus on clonality throughout CD4 T-cell activation and differentiation. A cross-sectional and longitudinal cohort study of 13 HIV-infected and 8 age-matched healthy children and adolescents examined the Vβ CDR3 length profiles within CD4 T-cell subsets by the use of spectratyping. HIV-infected subjects demonstrated higher numbers of perturbations in CD4 CD45RA T cells (5.8 ± 4.9 Vβ families) than healthy individuals (1.6 ± 1.8 Vβ families) (P = 0.04). Surprisingly, CD4 CD45RO central memory T cells from infected subjects showed no increased perturbations compared to the perturbations for the same cells from healthy subjects (2.9 ± 3.1 and 1.1 ± 1.8 Vβ families, respectively; P = 0.11). CD4 CD45RA TCR perturbations were higher among infected subjects with >25% CD4 cells than healthy subjects (mean number of perturbed Vβ families, 6.6 ± 5.4; P = 0.04). No correlations between perturbations in CD4 subsets and pretherapy age or viral load were evident. In contrast to CD8 T cells, HIV induces TCR disruptions within CD45RA but not CD45RO CD4 T cells. Therapy-induced viral suppression resulted in increases in thymic output and the normalization of the diversity of TCR within CD45RA CD4 T cells after 2 months of treatment. Perturbations occur prior to CD4 T-cell attrition and normalize with effective antiretroviral therapy. The impact of HIV on the diversity of TCR within naïve, central memory, and effector memory CD4 T cells is distinctly different from that in CD8 T cells.Human immunodeficiency virus type 1 (HIV-1) infection alters T-cell homeostasis by both impairing thymic output and inducing chronic T-cell activation. These disruptions are manifest by the increased level of expression of T-cell activation markers and decreased numbers of naïve T cells from the thymus (10, 12, 51). Oligoclonal T-cell expansion results in perturbations of the T-cell receptor (TCR) Vβ repertoire within both CD4 and CD8 T cells, with CD8 T cells being affected to a greater extent than CD4 T cells (7, 12, 16, 29, 50). Many of these abnormalities occur prior to CD4 T-cell attrition and are not fully reconstituted when viral replication is controlled by antiretroviral therapy (6, 17, 30). Multiple mechanisms have been postulated to contribute to this processes of aberrant T-cell activation and clonal expansion, including microbial translocation across the gastrointestinal tract as a result of virus-induced intestinal fibrosis (4, 5, 40) and the loss of immune regulation due to chronic HIV-induced antigenemia (8, 22).CD4 and CD8 T cells are heterogeneous populations that differ functionally and in their expression of activation and differentiation markers, forming the basis of their classification as naïve, central memory (CM), or effector memory (EM) T cells (42). Isoforms of CD45 (CD45RA and CD45RO) are frequently used to subdivide CD4 and CD8 T cells into functional subsets (1, 13, 25, 44, 45). Oligoclonal expansions and deletions within T-cell subpopulations can be measured by analysis of the hypervariable CDR3 region of the TCR (37). CDR3 length variation reflects changes within the TCR Vβ repertoire during antigen-induced T-cell activation (24, 25, 34). Differences in CDR3 length diversity within the CD4 or the CD8 CD45RA or CD45RO subset enable assessments of disruptions of the TCR repertoire and the detection of oligoclonal expansion that would have been missed if the analysis were limited to unfractionated T cells (25, 26). While optimal control of viral replication by antiretroviral therapy (ART) corrects many T-cell abnormalities and slows the progression to AIDS, it is not clear if therapy completely restores the TCR repertoire or fully diminishes T-cell activation (7, 14, 27, 51). In the present study, we examined the relationship of TCR diversity, thymic output, and the expression of T-cell activation markers within the CD45RA and CD45RO subpopulations of CD4 and CD8 T cells before and after the initiation of ART to determine the extent to which the control of viral replication restores the TCR repertoire.     

Staphylococcus aureus Induces Microglial Inflammation via a Glycogen Synthase Kinase 3β-Regulated Pathway

A proinflammatory role for glycogen synthase kinase 3β (GSK-3β) has been demonstrated. Here, we addressed its roles on heat-inactivated Staphylococcus aureus-induced microglial inflammation. Heat-inactivated S. aureus induced tumor necrosis factor alpha (TNF-α) and nitric oxide (NO) production, at least in part, via a Toll-like receptor 2-regulated pathway. Neutralization of TNF-α largely blocked heat-inactivated S. aureus-induced NO. Heat-inactivated S. aureus activated GSK-3β, and inhibiting GSK-3β reduced TNF-α production as well as inducible NO synthase (iNOS)/NO biosynthesis. While activation of NF-κB was essential for heat-inactivated S. aureus-induced TNF-α and NO, inhibiting GSK-3β blocked heat-inactivated S. aureus-induced NF-κB p65 nuclear translocation. Additionally, inhibiting GSK-3β enhanced heat-inactivated S. aureus-induced interleukin-10 (IL-10) production (IL-10 is an anti-inflammatory cytokine which inhibits TNF-α production). Neutralization of IL-10 reduced TNF-α downregulation caused by GSK-3β inhibition. These results suggest that GSK-3β regulates heat-inactivated S. aureus-induced TNF-α and NO production in microglia mainly by activating NF-κB and probably by inhibiting IL-10.Staphylococcus aureus, a gram-positive bacterium, causes a variety of diseases, such as bacteremia, peritonitis, subcutaneous and brain abscess, and life-threatening staphylococcal septic shock (15). The mechanisms that lead to staphylococcal septic shock are multifactorial but involve especially immunogenic and toxic injuries (10, 40). Cell wall components and secreted virulence factors, including enterotoxins and exotoxins, have been shown to be inflammatory and cytotoxic to the host. Pathogen-associated molecular patterns are recognized by the innate immune system through a family of pattern recognition receptors, such as Toll-like receptors (TLRs) (2, 6, 26). Microglia, the resident macrophages in the brain, express TLR2 to recognize S. aureus peptidoglycan and play a critical role in neuroinflammation (7, 35, 37). Induction of neuroinflammation by S. aureus is partially mediated by TLR2- and nuclear factor-κB (NF-κB)-regulated pathways (23, 26, 36, 51).Infection of S. aureus causes the deregulated production of inflammatory cytokines, including tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), and IL-10, and chemokines, including monocyte chemoattractant protein 1 (MCP-1) and RANTES (regulated on activation, normal T cell expressed and secreted protein) (24, 25, 32, 45). TNF-α, a potent proinflammatory cytokine, causes severe inflammatory responses, including cytokine and chemokine production and inducible nitric oxide (NO) synthase (iNOS)/NO biosynthesis in S. aureus infection (49). The deregulated generation of NO contributes to S. aureus-induced circulatory failure and liver injury (34). IL-10, a potent anti-inflammatory cytokine, inhibits the synthesis of the proinflammatory cytokines (TNF-α, IL-1, IL-6, IL-12, IL-18, and IL-10 itself), chemokines (IL-8, MCP-1, and RANTES), and iNOS/NO (4, 30, 43). IL-10 knockout mice display high mortality and are more susceptible to S. aureus-induced brain abscess (48). Exogenous IL-10 inhibits lethal sepsis, hepatic injury, and TNF-α production induced by staphylococcal enterotoxin B in mice (46, 48).Inhibiting glycogen synthase kinase 3β (GSK-3β) downregulates TLR-mediated inflammatory responses but increases IL-10 production (41, 53). Since NF-κB is important for inflammatory activation, GSK-3β is also involved in activating NF-κB in response to inflammatory stimuli (17-21, 29, 44, 50, 52). Therefore, GSK-3β inhibitors have been used to confer anti-inflammation against TNF-α administration, endotoxemia, experimental colitis, type II collagen-induced arthritis, ovalbumin-induced asthma, and experimental autoimmune encephalomyelitis (5, 12-14, 18, 20, 31, 41, 50, 52). Notably, current studies also show the effects of GSK-3β inhibition in reducing gram-negative coccobacillus Francisella-induced inflammation (55). GSK-3β inhibitors have also been widely used to reduce microglial inflammation and neurotoxicity (31, 54). In search of strategies against S. aureus-induced microglial inflammation, we investigated the possible effects of GSK-3β inhibition. In the present study, we report that inhibiting GSK-3β blocks NF-κB activation, TNF-α production, and iNOS/NO biosynthesis, but increases IL-10 production in heat-inactivated S. aureus-stimulated microglia.     

CD28 Exerts Protective and Detrimental Effects in a Pulmonary Model of Paracoccidioidomycosis

T-cell immunity has been claimed as the main immunoprotective mechanism against Paracoccidioides brasiliensis infection, the most important fungal infection in Latin America. As the initial events that control T-cell activation in paracoccidioidomycosis (PCM) are not well established, we decided to investigate the role of CD28, an important costimulatory molecule for the activation of effector and regulatory T cells, in the immunity against this pulmonary pathogen. Using CD28-deficient (CD28^−/−) and normal wild-type (WT) C57BL/6 mice, we were able to demonstrate that CD28 costimulation determines in pulmonary paracoccidioidomycosis an early immunoprotection but a late deleterious effect associated with impaired immunity and uncontrolled fungal growth. Up to week 10 postinfection, CD28^−/− mice presented increased pulmonary and hepatic fungal loads allied with diminished production of antibodies and pro- and anti-inflammatory cytokines besides impaired activation and migration of effector and regulatory T (Treg) cells to the lungs. Unexpectedly, CD28-sufficient mice progressively lost the control of fungal growth, resulting in an increased mortality associated with persistent presence of Treg cells, deactivation of inflammatory macrophages and T cells, prevalent presence of anti-inflammatory cytokines, elevated fungal burdens, and extensive hepatic lesions. As a whole, our findings suggest that CD28 is required for the early protective T-cell responses to P. brasiliensis infection, but it also induces the expansion of regulatory circuits that lately impair adaptive immunity, allowing uncontrolled fungal growth and overwhelming infection, which leads to precocious mortality of mice.It has long been appreciated that cellular immunity is the most important resistance mechanism against fungal infections (14, 36, 64). CD4⁺ and CD8⁺ T-cell subpopulations have been described to have a fundamental role in the control of fungal growth, and disease severity is also controlled by regulatory T (Treg) cells, which prevent tissue pathology by controlling excessive inflammatory reactions (25, 45, 46, 65). Similar to other deep mycoses, the severity of paracoccidioidomycosis (PCM), the most severe pulmonary mycosis in Latin America, is controlled by cellular immunity and cytokine-activated phagocytes that are able to kill Paracoccidioides brasiliensis, the etiological agent of this infection (10, 20, 30, 60, 61). In humans and in murine models of PCM, resistance to the disease is associated with the secretion of gamma interferon (IFN-γ) and other type 1 cytokines, whereas impaired Th1 immunity and the prevalent secretion of Th2 cytokines correlate with a systemic and progressive disease (2, 6, 39, 59, 76). Studies with CD4⁺ and CD8⁺ T-cell-deficient mice revealed that both T-cell subsets are involved in the protective immunity against P. brasiliensis infection and indicated the prominent role of CD8⁺ T cells (3, 21, 25). Besides the prevalent Th2 immunity, recent investigations have described alternative mechanisms underlying T-cell dysfunction in humans and experimental PCM. Increased apoptosis and overexpression of Fas and FasL in T cells suggest that activation-induced cell death (AICD) is a mechanism that controls T-cell expansion during the active disease (13, 19). In addition, the increased expression of CTLA-4 and the expansion of Treg cells were associated with severe patterns of the disease (24, 45, 46, 56). Thus, in addition to cytokine imbalance, other regulatory mechanisms can actively participate in the unresponsiveness of T cells in P. brasiliensis-infected hosts.Optimal activation, proliferation, and cytokine production by antigen-specific T cells require two distinct signals from dendritic cells or other antigen-presenting cells (APCs). After T-cell receptor (TCR) occupancy by the antigen epitope/major histocompatibility (MHC) complex (first signal), a second signal is mediated by costimulatory molecules (43, 63), such as CD28 on T cells and their counter-receptors CD80 (B7-1) and CD86 (B7-2) expressed by APCs (1, 34). Soluble molecules, such as cytokines and chemokines, also participate in the activation process, which drives and controls T-cell numbers and fates (1). CD28 enhances the TCR-triggered activation of naïve T cells, promotes interleukin-2 (IL-2) secretion and prevents T-cell anergy (1, 37). Alternatively, CD28-independent T-cell activation can occur if a strong and sustained antigen-specific signal is available (40, 81). Like CD28, two other molecules, cytotoxic T-lymphocyte antigen-4 (CTLA-4) and mouse inducible costimulatory molecule (ICOS), are selectively expressed by T cells, but the expression of these molecules depends on previous cell activation (50, 71). More recently, evidence has emerged that CD28 family members are also crucial regulators of natural and induced regulatory (CD4⁺CD25⁺Foxp3⁺) T cells (9). These cells are induced in the thymus and in the periphery, respectively, and control self-tolerance and the activation of several components of innate and adaptive immunity (68). Treg cells can suppress immune responses through the production of immunosuppressive cytokines (mainly IL-10 and transforming growth factor β [TGF-β]), through the induction of the apoptosis of effector T cells and through the modification of the functional properties of antigen-presenting cells (70, 78).Immunoprotection against microorganisms has been shown to be either CD28 dependent or independent. CD28-deficient (CD28^−/−) mice are highly susceptible to infection with Salmonella enterica serovar Typhimurium due to the poor ability of these mice to secrete IFN-γ (51). During some viral and parasitic infections, CD28 was shown to be required to mediate CD8⁺ T-cell immunoprotection (8, 53). In contrast, CD28^−/− mice infected with Mycobacterium bovis or Listeria monocytogenes control the bacterial burden and develop cell-mediated immunity (35, 52). In primary and opportunistic fungal infections, CD28 costimulation controls protective immunity, the expansion and function of regulatory T cells, and the intensity of inflammatory reactions (5, 54, 55, 66, 84).Because CD28 is critical for T-cell activation in fungal infections, we investigated its role in a murine model of P. brasiliensis infection. We show that CD28 costimulation exerts contrasting roles in pulmonary PCM. Early in infection, CD28 expression results in efficient adaptive immunity that is able to control fungal growth. Late in infection, however, this costimulatory molecule induces significant expansion of regulatory T cells, diminished immunity, and uncontrolled fungal growth that eventually leads to the death of the mice. In contrast, the absence of CD28 costimulation results in impaired T-cell immunity, which appeared to be compensated by the absence of Treg cell expansion. This weak but persistent immunity was able to partially control fungal growth, organize granulomatous lesions, and guarantee the enhanced survival of the mice, suggesting the relative protection conferred by CD28-independent mechanisms.     

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.     

α-Galactosylceramide Promotes Killing of Listeria monocytogenes within the Macrophage Phagosome through Invariant NKT-Cell Activation

α-Galactosylceramide (α-GalCer) has been exploited for the treatment of microbial infections. Although amelioration of infection by α-GalCer involves invariant natural killer T (iNKT)-cell activation, it remains to be determined whether macrophages (Mφ) participate in the control of microbial pathogens. In the present study, we examined the participation of Mφ in immune intervention in infection by α-GalCer using a murine model of listeriosis. Phagocytic and bactericidal activities of peritoneal Mφ from C57BL/6 mice, but not iNKT cell-deficient mice, were enhanced after intraperitoneal injection of α-GalCer despite the absence of iNKT cells in the peritoneal cavity. High levels of gamma interferon (IFN-γ) and nitric oxide (NO) were detected in the peritoneal cavities of mice treated with α-GalCer and in culture supernatants of peritoneal Mφ from mice treated with α-GalCer, respectively. Although enhanced bactericidal activity of peritoneal Mφ by α-GalCer was abrogated by endogenous IFN-γ neutralization, this was only marginally affected by NO inhibition. Similar results were obtained by using a listeriolysin O-deficient strain of Listeria monocytogenes. Moreover, respiratory burst in Mφ was increased after α-GalCer treatment. Our results suggest that amelioration of listeriosis by α-GalCer is, in part, caused by enhanced killing of L. monocytogenes within phagosomes of Mφ activated by IFN-γ from iNKT cells residing in an organ(s) other than the peritoneal cavity.Listeria monocytogenes, a Gram-positive facultative intracellular bacterium, is the causative agent of listeriosis, with an overall mortality rate of 30% (76). A major virulence factor of L. monocytogenes is listeriolysin O (LLO), a 58-kDa protein encoded by the hly gene (26, 42, 65). LLO promotes intracellular survival of L. monocytogenes in professional phagocytes such as macrophages (Mφ) by promoting listerial escape from the phagosome into the cytosol (10, 22, 26, 42, 62, 65). Cells of the innate immune system play a pivotal role as a first line of defense against L. monocytogenes infection and among these, mononuclear phagocytes are critical (56, 61). Activation of Mφ by gamma interferon (IFN-γ) is mandatory for elimination of L. monocytogenes (31, 35). Nitric oxide (NO) synthesized by inducible NO synthase, which is localized in the cytosol of professional phagocytes, participates in killing of L. monocytogenes (48, 52, 69, 71). Similarly, reactive oxygen intermediates (ROI) play a role in killing of L. monocytogenes within the phagosome (52, 53, 59).Natural killer T (NKT) cells represent a unique T-lymphocyte population expressing NKR-P1B/C (NK1.1; CD161), which is a type 2 membrane glycoprotein of the C-type lectin superfamily (6). In the mouse, the majority of NKT cells express an invariant T-cell receptor (TCR) α chain encoded by Vα14/Jα18 gene segments and a TCRVβ highly biased toward Vβ8.2, Vβ7, and Vβ2 (invariant NKT [iNKT] cells) (6). In contrast to conventional T cells, which recognize antigenic peptides presented by polymorphic major histocompatibility complex class I or class II molecules, iNKT cells recognize glycolipid antigens, including α-galactosylceramide (α-GalCer), a synthetic glycolipid originally isolated from a marine sponge, presented by the nonpolymorphic antigen presentation molecule CD1d (6, 40). iNKT cells are highly versatile and promptly produce both type 1 and type 2 cytokines, such as IFN-γ and interleukin-4 (IL-4), respectively, upon activation through their TCRs (1, 15-17, 79). IL-15 is an essential growth factor of both iNKT cells and NK cells and, hence, both cell populations are absent in IL-15-deficient (IL-15^−/−) mice (58). The numbers of iNKT cells are also markedly reduced in SJL mice because of a large deletion in their TCRVβ genetic region (5, 78).In vivo administration of α-GalCer causes prompt release of various cytokines by iNKT cells, which are involved in the control of various diseases, e.g., tumor rejection and prevention of autoimmune diseases (33, 41, 67, 70). Although α-GalCer has been reported to enhance host resistance to some microbial pathogens (27-29, 37, 39, 44, 55, 64), its potential role in protection against intracellular bacterial infections remains enigmatic.We have recently described that α-GalCer ameliorates murine listeriosis, which is, in part, caused by accelerated infiltration of inflammatory cells into the liver (18), although iNKT cells themselves exacerbate disease (19). Because Mφ play a central role in the elimination of L. monocytogenes, we considered the possibility that Mφ participate in enhanced resistance to L. monocytogenes infection caused by α-GalCer treatment. In the present study, we examined the influence of α-GalCer on listericidal activities of Mφ using a virulent and an avirulent strain of L. monocytogenes.     

Involvement of SOCS3 in Regulation of CD11c+ Dendritic Cell-Derived Osteoclastogenesis and Severe Alveolar Bone Loss

To investigate the role of suppressor of cytokine signaling (SOCS) molecules in periodontal immunity and RANKL-mediated dendritic cell (DC)-associated osteoclastogenesis, we analyzed SOCS expression profiles in CD4⁺ T cells and the effect of SOCS3 expression in CD11c⁺ DCs during periodontal inflammation-induced osteoclastogenesis and bone loss in nonobese diabetic (NOD) versus humanized NOD/SCID mice. Our results of ex vivo and in vitro analyses showed that (i) there is significantly higher SOCS3 expression associated with RANKL⁺ T-cell-mediated bone loss in correlation with increased CD11c⁺ DC-mediated osteoclastogenesis; (ii) the transfection of CD11c⁺ DC using an adenoviral vector carrying a dominant negative SOCS3 gene significantly abrogates TRAP and bone-resorptive activity; and (iii) inflammation-induced TRAP expression, bone resorption, and SOCS3 activity are not associated with any detectable change in the expression levels of TRAF6 and mitogen-activated protein kinase signaling adaptors (i.e., Erk, Jnk, p38, and Akt) in RANKL⁺ T cells. We conclude that SOCS3 plays a critical role in modulating cytokine signaling involved in RANKL-mediated DC-derived osteoclastogenesis during immune interactions with T cells and diabetes-associated severe inflammation-induced alveolar bone loss. Therefore, the development of SOCS3 inhibitors may have therapeutic potential as the target to halt inflammation-induced bone loss under pathological conditions in vivo.Inflammatory bone diseases affect a large portion of the skeletal system, particularly those portions underlying mucosal surfaces, where inflammation-induced bone loss is closely associated with elevated osteoclast (OC) activity (15, 48). Inflammation-induced bone loss is readily manifested in periodontal disease (i.e., periodontitis), resulting in attachments loss, including periodontal connective tissues and supporting alveolar bone, as a consequence of the interplays between the specific subgingival biofilm and the host''s immune/inflammatory responses (36, 37). The tumor necrosis factor (TNF) family member receptor activator of NF-κB ligand (RANKL), its receptor, RANK, and the natural antagonist, osteoprotegrin (OPG), have been shown to be the key regulators of the differentiation, activation, and survival of OCs and OC. precursors (5, 16-18, 23-25, 44, 55, 58). In addition, it is now clear that the host responses, especially T-cell immunity, play a pivotal role in regulating osteoclastogenesis and homeostasis of the bone tissues (termed osteoimmunology [5]). Activated CD4⁺ T cells express RANKL, which can directly trigger osteoclastogenesis and bone loss, and they can also negatively regulate RANKL activity via OPG production. For instance, it has been shown that OPG treatment results in significantly reduced bone loss in arthritis, osteoporosis, and cancer-related bone metastasis (16-18, 24, 45, 48, 55, 58). In murine models of periodontitis, OPG injections yield a robust inhibition of alveolar bone loss of up to 80 to 100%, suggesting that the RANKL-RANK/OPG axis is the key pathway controlling periodontal osteoclastogenesis (6, 27, 46, 49, 53). In addition to its importance in regulating bone remodelling, RANKL-RANK signaling is also critical for lymph node formation and organogenesis and is involved with dendritic cell (DC) survival and DC-T-cell interactions (23, 55, 59) in which modulation of a complex cytokine network in osteoclastogenesis in vivo has been suggested and shown (45, 52, 53).DCs are efficient antigen-presenting cells (APCs) necessary for regulating T-cell immunity (9). They are detected in diseased tissues of periodontitis and arthritic joints, where they form aggregates with T-cell infiltrates in the inflammatory foci (9, 40, 53) and are believed to contribute indirectly to inflammation-induced bone loss. Recent studies showed that Flt3⁺ monocyte/macrophage (Mo/MQ) precursors may differentiate to OCs, microglia, or DCs, thereby suggesting that these cell types may share common progenitors (2, 9, 26, 32). We recently reported the development of functional OCs from a CD11c⁺ DC subset(s) capable of inducing bone resorption in vitro and post-adoptive transfer in vivo (called DC-derived OCs [DDOC]) (1-4). Moreover, under arthritic conditions, human Mo-derived DCs have been shown to trans-differentiate into OCs in vitro, collectively suggesting that DCs may contribute directly to pathological bone loss (1, 2, 38). Thus, further understanding of the molecular mechanisms during DC-T-cell interactions driving disease pathogenesis and the resulting bone loss is imperative to facilitate the development of novel therapeutics for future treatments.Suppressors of cytokine signaling (SOCS) family and cytokine-inducible SH2 domain-containing protein (CIS) are cytoplasmic adaptor proteins that regulate various cytokine responses in leukocytes via negative feedback loops to inhibit inflammatory stimuli (62). There are eight members, including SOCS 1 to 7 and CIS, and each contains an N terminus, a central SH2 domain, and a C-terminal SOCS box. SOCS inhibits cytokine signaling by binding to phosphorylated JAKs and cytokine receptors and interacting with E3 ubiquitin ligases to polyubiquitylate JAKs for degradation (62). Several studies have investigated the roles of SOCS family members in regulating the development and function of immune cells. For instance, SOCS1 and -3 contain a kinase inhibitory region (KIR) in the N terminus, which inhibits JAK tyrosine kinase activity (35, 60, 61). As cytokine signaling activates JAK/STAT pathways, yielding activated SOCS molecules, SOCS1 is known to bind to JAKs with inhibitory catalytic activity and SOCS3 binds to its proximal sites on cytokine receptors, thereby directly inhibiting JAK signaling (62). These studies established the involvement of SOCS1 and SOCS3 molecules in regulating APCs (i.e., DCs) and T-cell functions during both innate and adaptive immunity (61, 62). Meanwhile, researchers have just begun to elucidate the effects of SOCS expression on the development of OCs and OC precursors, whereby specific SOCS molecules regulating osteoclastogenesis via cytokine signaling have been shown (35, 62). To date, however, the role(s) of SOCS family molecules and their impact on modulating RANKL expression and alveolar bone destruction in periodontal lesions remain unclear. Of particular interest to us is SOCS3, as it has been previously shown to be involved during inflammation-induced osteoclastogenesis and its activity is closely associated with key osteotropic cytokines, such as interleukin-1 (IL-1), TNF-α, transforming growth factor β, IL-6, IL-17, etc. (8, 10, 11, 35, 43, 60).We and others have established the NOD and humanized NOD/SCID mouse models to study periodontal inflammation and immunity and discovered that diabetic NOD mice manifest enhanced alveolar bone loss associated with increases in (i) T-cell proliferation and RANKL expression postinfection by anaerobic Aggregatibactor actinomycetemcomitans compared to prediabetic and nondiabetic NOD mice, and (ii) the Th1-inducing capability of the CD11c⁺ DC subset (14, 27-29). The enhanced alveolar bone loss in diabetic NOD and the physiological relevance of humanized NOD/SCID to periodontitis (14, 27, 49, 63) provide robust systems to study disease pathogenesis during the host-microbe interactions and alveolar bone loss. Herein, we employed these established animal models to assess the role of key SOCS molecules during periodontal immunity and RANKL-mediated alveolar bone loss by studying SOCS expression profiles in pathogen-reactive CD4⁺ T cells and the impact of halting functional SOCS3 on the development and activity of CD11c⁺ DDOC.     

Staphylococcus aureus Superantigens Elicit Redundant and Extensive Human Vβ Patterns

Staphylococcus aureus can produce a wide variety of exotoxins, including toxic shock syndrome toxin 1 (TSST-1), staphylococcal enterotoxins, and staphylococcal enterotoxin-like toxins. These toxins share superantigenic activity. To investigate the β chain (Vβ) specificities of each of these toxins, TSST-1 and all known S. aureus enterotoxins and enterotoxin-like toxins were produced as recombinant proteins and tested for their ability to induce the selective in vitro expansion of human T cells bearing particular Vβ T-cell receptors (TCR). Although redundancies were observed between the toxins and the Vβ populations, each toxin induced the expansion of distinct Vβ subsets, including enterotoxin H and enterotoxin-like toxin J. Surprisingly, the Vβ signatures were not associated with a specific phylogenic group of toxins. Interestingly, each human Vβ analyzed in this study was stimulated by at least one staphylococcal superantigen, suggesting that the bacterium derives a selective advantage from targeting the entire human TCR Vβ panel.Staphylococcus aureus produces a broad range of exoproteins, including staphylococcal enterotoxins (SEs) and toxic shock syndrome toxin 1 (TSST-1) (9). These toxins were initially implicated in staphylococcal food poisoning (SEs) and TSS (TSST-1) (39). Since the first characterization of SEA and SEB in 1959 to 1960 by Casman and Bergdoll, 18 different SEs have been described; they are designated SEA to SEV, in the chronological order of their discovery (2, 5, 41). Some were renamed SE-like toxins (SEl), because either no emetic properties were detected or because they were not tested in primate models (21, 41).SEs, SEls, and TSST-1 share certain structural and biological properties. They have similar sizes (23 to 29 kDa), and their crystal structures, established for SEA, SEB, SEC, SED, SEH, SElI, SElK, and TSST-1, reveal significant homology in their secondary and tertiary conformations (26). However SEs, SEls, and TSST-1 can be divided into four phylogenic groups based on their primary amino acid sequences (41).SEs, SEls, and TSST-1 share superantigenic activity (24). Superantigens (SAgs), unlike conventional antigens, do not need to be processed by antigen-presenting cells (APC) before being presented to T cells. They can directly stimulate T cells by cross-linking major histocompatibility complex class II molecules on APC with the variable portion of the T-cell antigen receptor β chain (TCR Vβ) or the T-cell antigen receptor α chain for SEH (TCR Vα), thereby inducing polyclonal cell proliferation (19, 36, 37). SAg binding sites lie outside the peptide-binding groove and therefore do not depend on T-cell antigenic specificity but rather on the Vβ and/or Vα region of the TCR (8, 19, 37). It was assumed that each SAg elicited a specific pattern of Vβ and/or Vα activation (24). As SAgs are active at very low concentrations (less than 1 pg/ml) (44), which are barely detectable in vivo, SAg-related diseases might theoretically be identified by determining TCR Vβ specificities in vitro. For example, an expansion of Vβ2 T cells on the one hand and of Vβ3, -14, and -17 T cells on the other hand, which correspond to TSST-1 and SEB superantigenic activities, respectively, has been detected in patients with TSS (6, 10, 25, 29). Such an approach would be particularly useful for investigating suspected SAg-related diseases, including some inflammatory disorders, Kawasaki disease, and atopy (11). However, the list of staphylococcal SAg (SSAg) Vβ specificities is not exhaustive, and different activation profiles have been obtained with different methods (41).To determine the Vβ specificities of S. aureus SAgs, we produced all known SSAgs as recombinant proteins and investigated their Vβ TCR specificities in vitro, using commercial antibodies.     

Mobilization of CD34+ Progenitor Cells in Association with Decreased Proliferation in the Bone Marrow of Macaques after Administration of the Fms-Like Tyrosine Kinase 3 Ligand

Fms-like tyrosine kinase 3 ligand (FLT3-L) is critical for the differentiation and self-renewal of CD34⁺ progenitor cells in primates and has been used therapeutically to mobilize progenitor and dendritic cells in vivo. However, little is known regarding the expansion of progenitor cells outside of peripheral blood, particularly in bone marrow (BM), where progenitor cells primarily reside. Evaluation of FLT3-L-mediated cell mobilization during lentivirus infections, where the numbers of CD34⁺ progenitor cells are reduced, is limited. We enumerated frequencies and absolute numbers of CD34⁺ progenitor cells in blood and BM of naive and SIV- or SHIV-infected macaques during and after the administration of FLT3-L. Flow cytometric analyses revealed that, while CD34⁺ cells increased in the circulation, no expansion was observed in BM. Furthermore, in the BM intracellular Ki67, a marker of cell proliferation, was downregulated in CD34⁺ progenitor cells but was upregulated significantly in the bulk cell population. Although the exact mechanism(s) remains unclear, these data suggest that CD34⁺ cell mobilization in blood was the result of cellular emigration from BM and not the proliferation of CD34⁺ cells already in the periphery. It is possible that the decreased progenitor cell proliferation observed in BM is evidence of a negative regulatory mechanism preventing hyperproliferation and development of neoplastic cells.The cytokine receptor Fms-like tyrosine kinase 3 (FLT3) is expressed at high levels on both primitive and early lymphoid/myeloid CD34⁺ progenitor cells (3, 21). Interaction with its cognate ligand (FLT3-L), found in both soluble and membrane-bound isoforms, contributes to the regulation of self-renewal and differentiation potential of these cells (43, 44). However, dysregulation of FLT3/FLT3-L signaling can result in the development of various leukemias (1, 6, 22, 29), and increased serum levels of FLT3-L are often indicative of other hematologic and autoimmune abnormalities (17, 25, 39). Nonetheless, after both murine and human FLT3-L were cloned in the early 1990s (24), this hematopoietic cytokine was used effectively in vitro to expand and maintain CD34⁺ progenitor cells (26, 32, 33) and, in combination with other growth factors, was used to induce differentiation of myeloid lineage cells (4), dendritic cells (2, 15), natural killer (NK) cells (42), erythroid precursors (12), and even endothelial cells (41). In addition, FLT3-L was shown to specifically suppress apoptosis of CD34⁺ progenitor cells (27).Early in vivo studies in mice demonstrated that FLT3-L administration not only mobilized and expanded murine CD34⁺ progenitor cells but also promoted expansion of human CD34⁺ cells transferred into SCID mice (8, 9, 24). In nonhuman primates FLT3-L was used to expand dendritic cell subsets (7, 30, 35), to treat radiation-induced myelosuppression (13, 14, 19), and as an adjuvant for various vaccines (23, 40). Although CD34⁺ cells primarily reside in the bone marrow (BM), examination of mobilization of these cells in vivo in nonhuman primates has been limited and typically restricted to analyses of blood (5, 18, 28). CD34⁺ cell mobilization and hematopoiesis is of particular interest in macaque models of lentivirus infections because, during both HIV and SIV infections, BM damage and reduced hematopoiesis is evident early after infection and is associated with decreased numbers and clonogenic potential of CD34⁺ progenitors, despite low levels of infection and virus replication in these cells (10, 16, 20, 34, 36, 37). Therefore, in the present study we quantified and characterized mobilization of CD34⁺ progenitor cells in BM in relation to that observed in peripheral blood by examining BM aspirates taken at various times during and after FLT3-L administration to naive and SIV- or SHIV-infected macaques.     

Biologic and Immunologic Effects of Knockout of Human Cytomegalovirus pp65 Nuclear Localization Signal

The human cytomegalovirus (CMV) pp65 protein contains two bipartite nuclear localization signals (NLSs) at amino acids (aa) 415 to 438 and aa 537 to 561 near the carboxy terminus of CMV pp65 and a phosphate binding site related to kinase activity at lysine-436. A mutation of pp65 with K436N (CMV pp65mII) and further deletion of aa 537 to 561 resulted in a novel protein (pp65mIINLSKO, where NLSKO indicate NLS knockout) that is kinaseless and that has markedly reduced nuclear localization. The purpose of this study was to biologically characterize this protein and its immunogenicity compared to that of native pp65. Unlike the native CMV pp65, following either DNA- or recombinant adeno-associated virus-based transduction of CMV pp65mIINLSKO into cells in vitro, the first observation of pp65mIINLSKO expression was in the cytoplasm and pp65mIINLSKO was expressed at higher levels than the native protein. The CMV pp65mIINLSKO mRNA was more abundant earlier than CMV pp65 mRNA (at 4 h and 8 h, respectively), but the half-lives of the proteins were the same. This modification altered the antigenic processing of CMV pp65 in vitro, as measured by the improved efficiency of cytotoxic killing in a pp65mIINLSKO-transduced human HLA A*0201 target cell line. In HHDII mice expressing HLA A*0201, pp65mIINLSKO was as immunogenic as CMV pp65. By RNA microarray analysis, expression of the CMV pp65mIINLSKO had less of an effect on cell cycle pathways than the native CMV pp65 did and a greater effect on cell surface signaling pathways involving immune activity. It is concluded that the removal of the primary NLS motif from pp65 does not impair its immunogenicity and should be considered in the design of a vaccine.A major immunodominant protein of human cytomegalovirus (CMV) is the tegument protein CMV pp65 (UL83) (15, 16, 23, 36). The biologic function of CMV pp65 is unclear, but as a nucleotropic protein which enters the nucleus immediately after infection (10, 28, 31, 33), CMV pp65 binds to polo-like kinase 1 (PLK-1), an enzyme important in mitosis (11), and it is likely that the protein has specific effects on cell cycle events (14, 22, 30). Despite this potential for cell toxicity, CMV pp65 has been proposed to be a critical antigen in any anti-CMV vaccine (17, 32). CMV pp65 has been shown to have protein kinase activity (8, 39), and a mutation at a critical phosphate binding site (CMV pp65mII) removed the kinase activity without altering the antigenicity (39). UL83 is considered an early-late gene, with synthesis beginning between 12 and 24 h after infection, during which time the protein product accumulates in the nucleus. However, at late times after infection, this CMV pp65 is exported back to the cytoplasm by means of the exportin system (31). CMV pp65 contains elements of the prototypic nuclear localization signal (NLS) in which arginine and lysine predominate within a bipartite motif in which short regions of basic amino acids are separated by 10 or more nonconserved amino acids (13, 24, 35). The nuclear localization signals of CMV pp65 consist of at least two such motifs located in the carboxy-terminal region of the polypeptide (33). One of these (termed the A-B motif by Schmolke et al. [33] but simplified to region A in this paper) is a classic bipartite signal located at amino acids (aa) 415 to 438, in which two arginine- and lysine-rich motifs are separated by 18 aa. When this region A is deleted, however, there is very little change in nuclear localization, indicating that there are other components to the NLS. A second NLS of CMV pp65 consists of a basic region of amino acids between aa 537 and 561; this region was termed the C-D motif by Schmolke et al. (33) and is termed region B in this paper. When this region was deleted by Schmolke et al. (33), there was a more dramatic reduction in the nuclear localization of CMV pp65, suggesting that this is the dominant NLS. However, the combined deletion of both the A and the B regions leads to the more complete inhibition of nuclear localization (33).Although CMV pp65 has been the prototypic antigen for the demonstration of CMV-specific T-cell immunity (2, 17, 34), it is likely that other proteins of CMV will be necessary for the development of a vaccine that generates humoral and cellular protection. CMV pp65-specific T-cell responses have been used for the development of other immunotherapeutic approaches to the control of CMV infection (4, 19, 20). Because of the importance of CMV pp65 in a vaccine strategy, we have explored the effects of mutating elements of the protein in ways that preserve the class I-restricted cytotoxic T-cell epitopes while removing biologic signals that could have effects on normal cells. For example, we have demonstrated the immunogenicity of a kinaseless CMV pp65 in which the phosphate binding lysine-436 is mutated (8). By use of this approach, a phase I trial of an anti-CMV vaccine by using a mutation at this same active site has been completed (38). However, these mutations of CMVpp65 remain nucleotropic and, thus, potentially toxic to normal cellular processes.This current study shows that the removal of the nucleotropic properties of CMV pp65 resulted in earlier RNA and protein synthesis posttransfection and abundant accumulation in the cytoplasm, without alteration of the immunogenicity of the protein.     

Multiple T-Cell Responses to Human Immunodeficiency Virus Type 1 Are Enhanced by Dendritic Cells

Human immunodeficiency virus type 1 (HIV-1)-specific T-cell reactivity has been related to protection from disease progression. Optimal T-cell reactivity to HIV-1 presumably requires antigen processing and presentation by professional antigen-presenting cells, particularly dendritic cells (DC). Here we examined whether multiple HIV-1-specific T-cell functions are enhanced by stimulation with HIV-1 peptide-loaded DC derived from HIV-1-infected subjects on antiretroviral therapy. We first found that mature DC increased the number of gamma interferon (IFN-γ)-producing T cells detected by enzyme-linked immunospot assay to overlapping 15-mer peptides of HIV-1 Gag and Nef, compared to stimulation with peptide-loaded, immature DC or to peptides without DC. IFN-γ production was lower in response to large pools of the Gag and Nef peptides, regardless of presentation by DC. We further observed that HIV-1 peptide-loaded, mature DC stimulated greater CD8⁺ and CD4⁺ T-cell proliferation than did the peptides without DC and that T-cell proliferation was lower in response to larger pools of the peptides. The lower T-cell IFN-γ and proliferation responses to the larger peptide pools were related to lower T-cell viability. Finally, the number of polyfunctional CD8⁺ and CD4⁺ T cells stimulated by HIV-1 peptide-loaded, mature DC, defined as positive by intracellular staining for more than one immune mediator (IFN-γ, interleukin 2, tumor necrosis factor alpha, macrophage inhibitory protein 1β, or CD107a), was greater than that stimulated by the peptides alone. These results indicate that DC can enhance multiple types of HIV-1-specific T-cell functions.Considerable evidence supports the idea that T-cell immunity to human immunodeficiency virus type 1 (HIV-1) is important in control of HIV-1 infection (10). Specific correlates of T-cell immunity that are associated with protection against or progression of HIV-1 infection have nonetheless been difficult to determine. Such immune correlates could be useful in defining the efficacy of prophylactic and therapeutic vaccines for HIV-1 infection. Many studies of T-cell immunity in HIV-1 infection have shown that the number of T cells exhibiting gamma interferon (IFN-γ) production in the enzyme-linked immunospot (ELISPOT) assay is decreased in association with progressive infection (4, 51). Proliferation of T cells in response to HIV-1 antigens as measured by uptake of the succinimidyl ester of carboxyfluorescein diacetate (CFSE) has also been related to less progressive HIV-1 infection (19, 33, 53). Recently, the quality of the CD8⁺ T-cell functional response to HIV-1 peptides as defined by intracellular cytokine staining (ICS) for more than one immune mediator, i.e., IFN-γ, interleukin 2 (IL-2), tumor necrosis factor alpha (TNF-α), macrophage inhibitory protein 1β (MIP-1β), and/or cytotoxic degranulation molecule CD107a (11, 44), has been associated with slow progression and better control of HIV-1 infection (5).Although these are all valid measures of anti-HIV-1 T-cell immunity, they usually do not account for a role of professional antigen-presenting cells (APC), particularly dendritic cells (DC), which are necessary for optimal processing and presentation of antigens to T cells (2). Indeed, it is likely that during HIV-1 infection, DC are required to take up, process, and present HIV-1 antigens via their major histocompatibility complex (MHC) class I and II molecules for priming and boosting of anti-HIV-1 CD8⁺ and CD4⁺ T-cell responses (40). We have previously shown that IFN-γ production by CD8⁺ T cells from HIV-1-infected persons is enhanced by stimulation with DC loaded with HIV-1 antigens and matured with CD40L or a cocktail of various proinflammatory cytokines and a Toll-like receptor 3 ligand (15, 20, 21). Myeloid DC loaded with peptides representing dominant epitopes of HIV-1 proteins stimulated significantly more epitope-specific, IFN-γ-producing CD8⁺ T cells than did peptides added directly to peripheral blood mononuclear cells (PBMC). There is little information, however, as to whether these professional APC can similarly enhance other T-cell functions that could be critical to control of HIV-1 infection, particularly their proliferative capacity and ability to produce multiple immune mediators. Moreover, many current approaches for measuring the magnitude and breadth of T-cell responses use pools of various numbers of synthetic peptides, usually 15 or 20 amino acids (aa) in length, which overlap by 10 to 11 aa (1, 3, 7, 9, 13, 14, 17, 24, 25, 27, 32, 37, 45, 48, 49), developed by Kern et al. (26) and Maecker et al. (31). Such studies have not accounted for a role of APC in processing that is required to reduce these peptides to their optimal, 8- to 10-mer length for presentation by MHC class I molecules to CD8⁺ T cells (43), or to 13- to 17-mers for presentation by MHC class II to CD4⁺ T cells (46). These are important considerations in determining correlates of T-cell immunity in HIV-1 infection and in response to HIV-1 vaccines.We have analyzed the magnitude of several types of T-cell responses during HIV-1 infection stimulated by autologous DC loaded with different-size pools of overlapping HIV-1 peptides. We assessed T-cell responses in HIV-1- infected persons for single-cell IFN-γ production by using a conventional ELISPOT assay; for CD8⁺ and CD4⁺ T-cell proliferation by using uptake of CFSE dye; and for production of IFN-γ, IL-2, TNF-α, MIP-1β, and CD107a by CD8⁺ and CD4⁺ T cells by using ICS. We found that, in addition to enhancing IFN-γ production detected by ELISPOT assay, DC loaded with HIV-1 peptide singlets or smaller pools of HIV-1 peptides enhanced HIV-1-specific T-cell proliferation and polyfunctional CD8⁺ and CD4⁺ T-cell responses.     

Lipoprotein Lipase and Hydrofluoric Acid Deactivate Both Bacterial Lipoproteins and Lipoteichoic Acids,but Platelet-Activating Factor-Acetylhydrolase Degrades Only Lipoteichoic Acids

To identify the Toll-like receptor 2 ligand critically involved in infections with gram-positive bacteria, lipoprotein lipase (LPL) or hydrogen peroxide (H₂O₂) is often used to selectively inactivate lipoproteins, and hydrofluoric acid (HF) or platelet-activating factor-acetylhydrolase (PAF-AH) is used to selectively inactivate lipoteichoic acid (LTA). However, the specificities of these chemical reactions are unknown. We investigated the reaction specificities by using two synthetic lipoproteins (Pam₃CSK₄ and FSL-1) and LTAs from pneumococci and staphylococci. Changes in the structures of the two synthetic proteins and the LTAs were monitored by mass spectrometry, and biological activity changes were evaluated by measuring tumor necrosis factor alpha production by mouse macrophage cells (RAW 264.7) following stimulation. PAF-AH inactivated LTA without reducing the biological activities of Pam₃CSK₄ and FSL-1. Mass spectroscopy confirmed that PAF-AH monodeacylated pneumococcal LTA but did not alter the structure of either Pam₃CSK₄ or FSL-1. As expected, HF treatment reduced the biological activity of LTA by more than 80% and degraded LTA. HF treatment not only deacylated Pam₃CSK₄ and FSL-1 but also reduced the activities of the lipoproteins by more than 60%. Treatment with LPL decreased the biological activities by more than 80%. LPL also removed an acyl chain from the LTA and reduced its activity. Our results indicate that treatment with 1% H₂O₂ for 6 h at 37°C inactivates Pam₃CSK₄, FSL-1, and LTA by more than 80%. Although HF, LPL, and H₂O₂ treatments degrade and inactivate both lipopeptides and LTA, PAF-AH selectively inactivated LTA with no effect on the biological and structural properties of the two lipopeptides. Also, the ability of PAF-AH to reduce the inflammatory activities of cell wall extracts from gram-positive bacteria suggests LTA to be essential in inflammatory responses to gram-positive bacteria.Bacterial sepsis is a leading cause of death within intensive care units (43). Although bacterial sepsis was traditionally associated with gram-negative (Gr−) bacteria, recently, the prevalence of sepsis caused by gram-positive (Gr+) bacteria has rapidly increased (2, 3, 38). In fact, in 2000, Gr+ bacteria accounted for 52% of sepsis cases whereas Gr− bacteria accounted for only 37.6% (7, 31, 38). In bacterial sepsis, the innate immune system provides both the initial immune responses and the early inflammatory responses (1, 8, 12). Early responses to infections with Gr+ and Gr− bacteria have been shown in previous studies to involve different cytokine profiles (9, 16, 25, 51, 54). Other studies have found that infections with Gr− bacteria activate Toll-like receptor 4 (TLR4) primarily with lipopolysaccharide (LPS), a membrane component of Gr− bacteria (26, 27, 44, 53). In contrast, infections with Gr+ bacteria involve TLR2, but the nature of the key TLR2 ligand is still controversial (34, 52, 56).Two components of the cell walls of Gr+ bacteria have been proposed to be TLR2 ligands. One group of studies suggests that lipoteichoic acid (LTA) is the key ligand (10, 46, 49, 57). LTA is a polyphosphate attached to the cell membrane via a diacyl glycolipid and is an abundant component of the envelopes of Gr+ bacteria (47). Highly purified LTA, as well as its synthetic analogs, has been shown to trigger TLR2-mediated inflammatory responses (10, 15, 20, 35). However, the biological role of the LTA is unclear because it is difficult to purify natural LTA without introducing contaminants or damaging the structure of the LTA (41). Another group proposes bacterial lipoproteins as the critical ligand (22). Lipoproteins are a functionally diverse class of bacterial membrane proteins characterized by an N-terminal lipid moiety (4) and are TLR2 ligands (22-24). Although synthetic analogs of lipoproteins were found to be potent TLR2 ligands (5, 6, 42), natural lipoproteins are difficult to purify, and their properties are poorly understood.To avoid the technical difficulties involved in purification, a different investigational approach was developed. This approach uses methods to selectively inactivate either LTA or lipoproteins in bacterial culture supernatants or crude bacterial cell wall extracts (22-24, 49). LTA inactivation is usually performed with hydrofluoric acid (HF) or platelet-activating factor-acetylhydrolase (PAF-AH) (23, 48, 49), which, respectively, hydrolyzes the phosphodiester bonds in the LTA or deacylates one of its acyl chains (17, 28, 36, 55). Lipoprotein inactivation is commonly achieved by deacylation with a lipoprotein lipase (LPL) or by oxidation with hydrogen peroxide (H₂O₂) (22, 24, 62). Despite their wide use, the reaction selectivities of these methods have not been evaluated. Thus, we investigated the reaction specificities of these methods by studying the impacts of these four reactions on the biological properties as well as the chemical structures of LTA and lipoprotein analogs.     

The K5 Capsule of Escherichia coli Strain Nissle 1917 Is Important in Mediating Interactions with Intestinal Epithelial Cells and Chemokine Induction

Escherichia coli strain Nissle 1917 has been widely used as a probiotic for the treatment of inflammatory bowel disorders and shown to have immunomodulatory effects. Nissle 1917 expresses a K5 capsule, the expression of which often is associated with extraintestinal and urinary tract isolates of E. coli. In this paper, we investigate the role of the K5 capsule in mediating interactions between Nissle 1917 and intestinal epithelial cells. We show that the loss of capsule significantly reduced the level of monocyte chemoattractant protein 1 (MCP-1), RANTES, macrophage inflammatory protein 2α (MIP-2α), MIP-2β, interleukin-8, and gamma interferon-inducible protein 10 induction by Nissle 1917 in both Caco-2 cells and MCP-1 induction in ex vivo mouse small intestine. The complementation of the capsule-minus mutation confirmed that the effects on chemokine induction were capsule specific. The addition of purified K5, but not K1, capsular polysaccharide to the capsule-minus Nissle 1917 at least in part restored chemokine induction to wild-type levels. The purified K5 capsular polysaccharide alone was unable to stimulate chemokine production, indicating that the K5 polysaccharide was acting to mediate interactions between Nissle 1917 and intestinal epithelial cells. The induction of chemokine by Nissle 1917 was generated predominantly by interaction with the basolateral surface of Caco-2 cells, suggesting that Nissle 1917 will be most effective in inducing chemokine expression where the epithelial barrier is disrupted.A probiotic has been defined as “live microorganisms which when administered in adequate amounts confer a health benefit on the host” (20). These benefits include the balancing and restoration of the intestinal microflora, repair of intestinal barrier functions (54), expression of bacteriocins (36), immunomodulatory effects (18, 43, 47, 53), and antagonizing epithelial colonization and invasion by pathogens (2). Escherichia coli strain Nissle 1917 was isolated from the feces of a soldier who did not develop diarrhea during a severe outbreak of shigellosis (38). Despite exhibiting a serotype (O6:K5:H1) that is characteristic of E. coli strains associated with urinary tract infections, Nissle 1917 apparently is nonpathogenic (25, 53) and has been used widely in preventing infectious diarrheal diseases (7, 14, 27, 37, 52, 53), the treatment of inflammatory bowel diseases such as ulcerative colitis and Crohn''s disease (7, 23, 32, 33), and to prevent the colonization of the digestive tract of neonates by pathogens (35). Recently, there has been a growing interest in investigating the immunomodulatory effect of Nissle 1917. Previous studies showed that colonization by Nissle 1917 may lead to an alteration of the hosts'' cytokine repertoire (13, 49), increased immunoglobulin A secretion (14), lymphocyte or macrophage activation (13), the modulation of CD4⁺ clonal expansion (47), the stimulation of antimicrobial peptide production by intestinal epithelial cells (39, 52, 54), and alterations of the pro- and anti-inflammatory balance of local cytokines (49). Recently it has been shown that Nissle 1917 activates γδT cells, stimulating CXCL8 and interleukin-6 (IL-6) release but inhibiting tumor necrosis factor alpha (TNF-α) secretion (26). Following activation, Nissle 1917 induced apoptosis in activated γδT cells, indicating a key role for Nissle 1917 in interacting with the subset of T cells that operate at the interface between the adaptive and innate immune responses (26). Nissle 1917 also has been shown to express a direct anti-inflammatory activity on epithelial cells by blocking TNF-α-induced IL-8 secretion through a NF-κB-independent mechanism (28). Although the immunomodulatory effects of Nissle 1917 are well documented, the contribution of individual microbial components in mediating such effects is less well understood. So far, only a role for flagellin in mediating the induction of human β-defensin expression by Nissle 1917 has been established (44). Nissle 1917 expresses a K5 capsule on its cell surface, and a number of roles for polysaccharide capsules in the virulence of E. coli have been proposed, including resistance to phagocytosis and complement-mediated killing and the increased colonization of the host (42). In contrast, in the case of other encapsulated pathogens, it has been shown that the expression of a polysaccharide capsule can affect the induction of chemokines following attachment to host cells (6, 17, 22, 24, 40, 41, 45, 50). The aim of the present study was to investigate the role of the K5 capsule in mediating the immunomodulatory activity of Nissle 1917.     

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).     

Human Brucellosis Is Characterized by an Intense Th1 Profile Associated with a Defective Monocyte Function

In animal models, a defective Th1 response appears to be critical in the pathogenesis of brucellosis, but the Th1 response in human brucellosis patients remains partially undefined. Peripheral blood from 24 brucellosis patients was studied before and 45 days after antibiotherapy. Twenty-four sex- and age-matched healthy donors were analyzed in parallel. Significantly increased levels of interleukin 1β (IL-1β), IL-2, IL-4, IL-6, IL-12p40, gamma interferon (IFN-γ), and tumor necrosis factor alpha (TNF-α), but not of IL-10, in serum and/or significantly increased percentages of samples with detectable levels of these cytokines, measured by enzyme-linked immunosorbent assays (ELISA), were found for untreated brucellosis patients, but these levels were reduced and/or normalized after treatment. Flow cytometry studies showed that the intracytoplasmic expression of IFN-γ, IL-2, and TNF-α, but not that of IL-4, by phorbol myristate-activated CD4⁺ CD3⁺ and CD8⁺ CD3⁺ T lymphocytes was significantly increased in untreated brucellosis patients and was also partially normalized after antibiotherapy. The percentage of phagocytic cells, the mean phagocytic activity per cell, and the phagocytic indices for monocytes at baseline were defective and had only partially reverted at follow-up. T lymphocytes from untreated brucellosis patients are activated in vivo and show Th1 cytokine production polarization, with strikingly high serum IFN-γ levels. In spite of this Th1 environment, we found deficient effector phagocytic activity in peripheral blood monocytes.Brucellosis is a zoonotic disease of worldwide distribution. Despite its control in many countries, it remains endemic in the Mediterranean and Middle Eastern regions (20, 28, 41, 42). Brucella melitensis is the most frequent cause of human brucellosis in these geographical areas (19). In Spain, it has been reported that the majority (more than 97.5%) of isolates were identified as Brucella melitensis (13, 44, 45).Brucella organisms are facultatively intracellular Gram-negative coccobacilli that reside and replicate in a vacuolar compartment within myelomonocytic cells of the infected host (14, 15, 47). The response to Brucella involves the whole gamut of the immune system, from innate to adaptive immunity (21). In murine models, passive transfer of immune cells resulted in an effective anti-Brucella defensive response mediated by CD4⁺ and CD8⁺ T lymphocytes (5, 6, 32, 37, 51, 52). Furthermore, the pattern of T-lymphocyte cytokine secretion is considered to be critical for the effectiveness of the protective anti-Brucella immune response (3, 7). It has been postulated that Th1 cytokines confer resistance, while Th2 cytokines facilitate the development of brucellosis (2, 3, 24, 25, 40, 43, 52). In animal models, gamma interferon (IFN-γ) induces macrophage activation and control of Brucella infection (16, 18, 43). In Brucella-infected mice, administration of recombinant IFN-γ enhances host resistance, resulting in a deep decrease in the number of viable bacteria (51). Moreover, host IFN-γ depletion results in an increase in the number of viable bacteria (17, 37, 52). Several abnormalities in the immune system have been found in human brucellosis (27, 46, 49). It has been found that T and NK lymphocytes show defective functions in brucellosis patients (46, 49). Since mice are naturally resistant to Brucella infections, it is possible to suggest that the immune response elicited by Brucella in humans might have different characteristics. Thus, susceptibility to, or protection from, human brucellosis conferred by T-lymphocyte cytokines has not been established.In this work, we have further investigated the pattern of T-lymphocyte and monocyte responses to human Brucella infection. We have prospectively studied (i) the levels of Th1, Th2, and regulatory cytokines in serum, (ii) the distribution, activation stage, and pattern of Th1/Th2 cytokine production by T lymphocytes, and (iii) the phagocytic activity of monocytes in a group of brucellosis patients before and after antimicrobial treatment.     

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.     

Optimal CD8 T-Cell Response against Encephalitozoon cuniculi Is Mediated by Toll-Like Receptor 4 Upregulation by Dendritic Cells

CD8⁺ T-cell immunity has been shown to play an important role in the protective immune response against Encephalitozoon cuniculi. Although earlier studies suggest that dendritic cells (DC) are important for the induction of this response, the factors responsible for initiation of the dendritic cell response against this pathogen have not been evaluated. In the current study, we demonstrate that E. cuniculi infection causes strong Toll-like receptor 4 (TLR4)-dependent dendritic cell activation and a blockade of this molecule reduces the ability of DC to prime an antigen-specific CD8⁺ T-cell response. Pretreatment of DC with anti-TLR4 antibody causes a defect in both in vitro and in vivo CD8⁺ T-cell priming. These findings, for the first time, emphasize the contribution of TLR4 in the induction of CD8⁺ T-cell immunity against E. cuniculi infection.Microsporidia are small obligate intracellular parasites that, until recently, were thought to be protozoans; however, evidence now suggests that they are related to fungi (15, 17). Microsporidia can infect a vast number of species of vertebrates and invertebrates; of the 150 genera of microsporidia, however, only 7 have been found to infect humans (13). Severe infections have been reported predominantly for immunocompromised patients, such as patients with HIV and organ transplant recipients (2, 7, 23, 37). Acute infections have also been reported in travelers and the elderly (26, 27), and there is evidence of colonization of healthy, nonsymptomatic patients (34).Due to the prevalence of opportunistic microsporidian infections associated with the HIV-AIDS pandemic, recent research has focused on the host''s immune response to these pathogens. Early animal studies showed that cellular immunity was necessary to protect SCID mice from a lethal Encephalitozoon cuniculi challenge. Moreover, depletion of CD8⁺ T cells caused mice to succumb to intraperitoneal (i.p.) E. cuniculi infection (21), and previous studies in our laboratory have shown that cytotoxic lymphocytes play a major role in protection against this effect (20, 21).Recent reports from our laboratory have demonstrated that dendritic cells (DC) play an important role in the priming of the immune response against E. cuniculi (31, 32). T cells incubated with E. cuniculi-pulsed DC exhibited antigen-specific characteristics, specifically gamma interferon (IFN-γ) production, cytotoxicity, and proliferation (31, 32). In order to mount an immune response against a foreign pathogen, DC must first recognize the pathogen to initiate an appropriate response. One key method of recognition is through Toll-like receptors (TLRs), which were first discovered in Drosophila in response to infection with fungal pathogens (24). However, specific TLR molecules involved in DC activation during E. cuniculi infection have not been identified previously. We evaluated the upregulation of specific molecules involved in activation of the DC response after E. cuniculi infection. Different TLR molecules were tested, and TLR4 expression was found to be essential for induction of the optimal CD8⁺ T-cell response by these cells.     

Clonal Analysis of Colonizing Group B Streptococcus,Serotype IV,an Emerging Pathogen in the United States

Colonizing group B Streptococcus (GBS) capsular polysaccharide (CPS) type IV isolates were recovered from vaginal and rectal samples obtained from 97 (8.4%) nonpregnant women of 1,160 women enrolled in a U.S. multicenter GBS vaccine study from 2004 to 2008. Since this rate was much higher than the rate of prevalence of 0.4 to 0.6% that we found in previous studies, the isolates were analyzed by using surface protein profile identification, pulsed-field gel electrophoresis (PFGE), and multilocus sequence typing (MLST) to characterize them and identify trends in DNA clonality and divergence. Of the 101 type IV isolates studied, 53 expressed α and group B protective surface (BPS) proteins, 27 expressed BPS only, 20 expressed α only, and 1 had no detectable surface proteins. The isolates spanned three PFGE macrorestriction profile groups, groups 37, 38, and 39, of which group 37 was predominant. The isolates in group 37 expressed the α and BPS proteins, while those in groups 38 and 39 expressed the α protein only, with two exceptions. MLST studies of selective isolates from the four protein profile groups showed that isolates expressing α,BPS or BPS only were of a new sequence type, sequence type 452, while those expressing α only or no proteins were mainly of a new sequence type, sequence type 459. Overall, our study revealed a limited diversity in surface proteins, MLST types, and DNA macrorestriction profiles for type IV GBS. There appeared to be an association between the MLST types and protein expression profiles. The increased prevalence of type IV GBS colonization suggested the possibility that this serotype may emerge as a GBS pathogen.Group B Streptococcus (GBS) (Streptococcus agalactiae) is a leading cause of neonatal infection in the United States, with maternal vaginal or rectal colonization often resulting in the transmission of GBS to the infant during the perinatal period (8, 23). GBS isolates are classified according to nine capsular polysaccharide (CPS) types: types Ia, Ib, and II to VIII and the recently proposed type IX (9, 15, 21, 23, 46, 52). Isolates that do not express any of the known CPS types are designated nontypeable (NT) (2, 6, 21, 40). In addition to CPS, GBS may express one or more surface-localized proteins, including the α and β components of the c protein (24); the alpha-like R proteins, specifically R1, R4(Rib), and R1,R4 (also known as Alp3) (14, 17, 19, 30, 40); and the group B protective surface (BPS) protein (12). Certain protein profiles are associated with each capsular polysaccharide CPS type (2), for example, the c(α only) protein with types Ia and II, c(α + β) with type Ib, and R4(Rib) with type III (2, 14). BPS, expressed by fewer than 3% of colonizing isolates, can be found alone or with another protein in type Ia, II, and V isolates (12, 14).In the United States, the predominant serotypes over the past 2 decades, constituting 70 to 75% of all GBS isolates, have been type Ia, type III, and the more recently emerged type V (14, 15, 20, 52). The remaining isolates consisted primarily of types Ib and II, with types IV, VI, VII, and VIII making up a small fraction of the isolates. We found type IV to represent between 0.4 and 0.6% of colonizing GBS isolates (14, 15), but only rare type IV isolates were found in invasive GBS disease during that same time period (14, 43, 52).In contrast to the previously low percentage of type IV isolates reported for the United States, recent studies in the United Arab Emirates, Turkey, and Zimbabwe showed large proportions of type IV isolates among their GBS isolates. In the United Arab Emirates, type IV was the predominant serotype among colonized pregnant women, representing 26.3% of the GBS isolates (1). In eastern Turkey, it was the second most common serotype, at 8.3%, among colonizing isolates (10), and in Zimbabwe, it was the fourth most common serotype, comprising 5.1% of GBS isolates from colonized pregnant women and 4.0% of all GBS isolates from various sites, including blood and cerebrospinal fluid (CSF), from hospitalized patients (36).Immunization studies of humans (3, 28) and protection studies with mice (37) have shown the potential of vaccines against the common GBS serotypes to prevent invasive neonatal GBS disease through the vaccination of pregnant women (3, 28). The GBS strains described here are from a phase II randomized, double-blinded clinical trial of a GBS serotype III-tetanus toxoid (CPS III-TT) vaccine to prevent the vaginal acquisition of GBS type III in nonpregnant women in three areas of the United States: Pittsburgh (PA), Georgia, and Texas (S. Hillier, unpublished data). Because we found type IV isolates for almost 10% of these patients, we examined the type IV isolates for surface proteins and clonality.Pulsed-field gel electrophoresis (PFGE) was used in this analysis because it is a widely used method that can further characterize GBS isolates within particular CPS type and/or protein profile groups (2, 4, 6, 48). Multilocus sequence typing (MLST) was performed in order to assess the general relatedness of strains within and across laboratories (25, 50). Together, the discriminatory power of PFGE and the objectivity of MLST gave insight into the GBS type IV population genetic structure and the identification of emerging clones (2, 5, 13, 18, 19).