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T regulatory cells (Tregs) control immune homeostasis by preventing inappropriate responses to self and nonharmful foreign antigens. Tregs use multiple mechanisms to control immune responses, all of which require these cells to be near their targets of suppression; however, it is not known how Treg-to-target proximity is controlled. Here, we found that Tregs attract [CD4.sup.+] and [CD8.sup.+] T cells by producing chemokines. Specifically, Tregs produced both CCL3 and CCL4 in response to stimulation, and production of these chemokines was critical for migration of target T cells, as Tregs from [Ccl3.sup.-/-] mice, which are also deficient for CCL4 production, did not promote migration. Moreover, CCR5 expression by target T cells was required for migration of these cells to supernatants conditioned by Tregs. Tregs deficient for expression of CCL3 and CCL4 were impaired in their ability to suppress experimental autoimmune encephalomyelitis or islet allograft rejection in murine models. Moreover, Tregs from subjects with established type 1 diabetes were impaired in their ability to produce CCL3 and CCL4. Together, these results demonstrate a previously unappreciated facet of Treg function and suggest that chemokine secretion by Tregs is a fundamental aspect of their therapeutic effect in autoimmunity and transplantation., Introduction T regulatory cells (Tregs) are essential for preventing immunity to self antigens (Ags) and nonharmful foreign Ags; they also play an active role in suppressing immunity to tumors and [...]

Type 1 diabetes is characterized by destruction of insulin-producing β cells in the pancreatic islets by effector T cells. Tregs, defined by the markers CD4 and FoxP3, regulate immune responses by suppressing effector T cells and are recruited to sites of action by the chemokine CCL22. Here, we demonstrate that production of CCL22 in islets after intrapancreatic duct injection of double-stranded adeno-associated virus encoding CCL22 recruits endogenous Tregs to the islets and confers long-term protection from autoimmune diabetes in NOD mice. In addition, adenoviral expression of CCL22 in syngeneic islet transplants in diabetic NOD recipients prevented β cell destruction by autoreactive T cells and thereby delayed recurrence of diabetes. CCL22 expression increased the frequency of Tregs, produced higher levels of TGF-β in the [CD4.sup.+] T cell population near islets, and decreased the frequency of circulating autoreactive [CD8.sup.+] T cells and [CD8.sup.+] IFN-γ-producing T cells. The protective effect of CCL22 was abrogated by depletion of Tregs with a CD25-specific antibody. Our results indicate that islet expression of CCL22 recruits Tregs and attenuates autoimmune destruction of β cells. CCL22-mediated recruitment of Tregs to islets may be a novel therapeutic strategy for type 1 diabetes., Introduction The complex pathogenesis of autoimmune diabetes in NOD mice and humans involves multiple immune components. The autoimmune response recurs when βcells are transplanted into type 1 diabetic patients or [...]

Fas and TNF-R1 are cysteine-rich cell surface receptors related to the low-affinity nerve growth factor receptor family. Engagement of these receptors by their respective ligands, FasL and tumor necrosis factor, leads to apoptosis that is signaled through a conserved intracellular portion of the receptor termed the 'death domain.' We have cloned a new member of this family, lymphocyte-associated receptor of death (LARD), which leads to spontaneous apoptosis when expressed in 293T cells. The expression of LARD is more tightly regulated than that of either Fas or TNF-R1 as it is found predominantly on lymphocytes (T and B cells) but not on macrophages or a number of transformed lymphocyte cell lines. Alternative pre-mRNA splicing generates at least 11 distinct isoforms of LARD. The full-length isoform, LARD-1, extends to include the transmembrane and death domains, whereas the other isoforms encode potentially secreted molecules. Naive B and T cells express very little LARD-1 but express combinations of the other isoforms. Upon T cell activation, a programmed change in alternative splicing occurs so that the full-length, membrane-bound LARD-1 predominates. This may have implications for the control of lymphocyte proliferation following activation.

The vaccine that keeps on giving: Bacille Calmette-Guérin (BCG) is a tuberculosis vaccine that is frequently given to infants in countries with a high incidence of tuberculosis infection. However, emerging evidence suggests that this vaccine may have additional beneficial effects for the infants. Following on previous observations of reduced neonatal mortality in BCG-vaccinated infants, Brook et al. performed a systematic study of mouse models and human infants in multiple countries. The authors demonstrated that BCG vaccine given during the neonatal period protected the infants from death due to sepsis and showed that this could be attributed to a rapid increase in neutrophils driven by the vaccine. Death from sepsis in the neonatal period remains a serious threat for millions. Within 3 days of administration, bacille Calmette-Guérin (BCG) vaccination can reduce mortality from neonatal sepsis in human newborns, but the underlying mechanism for this rapid protection is unknown. We found that BCG was also protective in a mouse model of neonatal polymicrobial sepsis, where it induced granulocyte colony-stimulating factor (G-CSF) within hours of administration. This was necessary and sufficient to drive emergency granulopoiesis (EG), resulting in a marked increase in neutrophils. This increase in neutrophils was directly and quantitatively responsible for protection from sepsis. Rapid induction of EG after BCG administration also occurred in three independent cohorts of human neonates. [ABSTRACT FROM AUTHOR]

Autoimmune diabetes in nonobese diabetic (NOD) mice can be prevented by a single injection of complete Freund's adjuvant (CFA), but the mechanisms mediating protection remains unclear. We previously showed that NOD mice immunized with CFA have a markedly reduced incidence of diabetes that is associated with a significant decrease in the number of β-cell-specific, autoreactive cytotoxic T lymphocytes and, furthermore, that the effect of CFA is mediated by natural killer (NK) cells. In this study, we report one mechanism by which NK cells regulate the onset of diabetes. Administration of CFA produced a rapid increase in NK cell frequency and function, including cytotoxicity and IFN-γ secretion. By co-transferring NK cells from IFN-γ-deficient (or wild-type) NOD mice and spleen cells from diabetic NOD mice to NOD/SCID recipients, we show that IFN-γ secretion by NK cells significantly influences the effect of CFA protection. In contrast, NK cytotoxicity does not appear to participate in CFA-mediated protection from diabetes. Our findings demonstrate that NK cells mediate the protective effects of CFA through secretion of IFN-γ. [ABSTRACT FROM AUTHOR]

Background. Neisseria meningitidis continues to be an important cause of invasive bacterial disease among children and young adults worldwide. In Canada, N. meningitidis strains that bear serogroups B and C polysaccharide capsules predominate. We report the first documented case of invasive meningococcal disease in an immunocompetent host caused by an acapsular strain of N. meningitidis containing the capsule null locus (cnl). Methods. Analysis of the isolate was performed with use of serological and molecular methods, including multilocus sequence typing and cnl gene identification. Analysis of 16S ribosomal RNA (rRNA) and porA genes was also performed to confirm the identity of the bacterium. Results. The patient was a healthy, immunocompetent 13-year-old child, and N. meningitidis was recovered from a sample of her cerebrospinal fluid before death. The isolate was nontypeable by both conventional antisera and indirect whole-cell enzyme-linked immuosorbent assay methods using antibodies to serogroups B, C, Y, and W135. The isolate was further identified as a cnl strain, serotype 15 (ST-198). N. meningitidis-specific DNA was identified in the isolate and in the pre- and postmortem specimens by 16S rRNA and porA gene analysis. Conclusions. This is the first reported case of fatal meningococcal disease caused by an acapsular cnl strain of N. meningitidis that was isolated from an immunocompetent host. Routine molecular diagnostic methods targeted at the cnl locus failed to detect this organism, indicating a need to determine the incidence of infection with cnl strains among patients with culture-negative invasive disease. [ABSTRACT FROM AUTHOR]

2B4 is a SLAM-related receptor expressed on natural killer (NK) cells and cytotoxic T cells. It can regulate killing and gamma interferon secretion by NK cells, as well as T-cell-mediated cytotoxicity. There are conflicting data regarding the mechanism of action of 2B4. In these studies, we attempted to understand better the nature and basis of 2B4 signaling. Our studies showed that engagement of 2B4 on NK cells triggered a tyrosine phosphorylation signal implicating 2B4, Vav-1, and, to a lesser extent, SHIP-1 and c-Cbl. Structure-function analyses demonstrated that this response was defined by a series of tyrosine-based motifs in the cytoplasmic region of 2B4 and was not influenced by the extracellular or transmembrane segment of 2B4. In addition, the 2B4-induced signal was absolutely dependent on coexpression of SAP, a Src homology 2 (SH2) domaincontaining adaptor associating with SLAM-related receptors and mutated in X-linked lymphoproliferative disease. It was also observed that 2B4 was detectably associated with the Src-related protein tyrosine kinase FynT in an immortalized NK cell line. Mutation of arginine 78 of SAP, a residue critical for binding of SAP to FynT, eliminated 2B4-mediated protein tyrosine phosphorylation, implying that SAP promotes 2B4 signaling most probably by recruiting FynT. Finally, despite the similarities in the signaling modalities of 2B4 and its relative SLAM, the natures of the tyrosine phosphorylation signals induced by these two receptors were found to be different. These differences were not caused by variations in the extent of binding to SAP but rather were dictated by the tyrosine-based sequences in the cytoplasmic domain of the receptors. Taken together, these data lead to a better understanding of 2B4 signaling. Furthermore, they provide firm evidence that the signals transduced by the various SLAM-related receptors are unique and that the specificity of these signals is defined by the distinctive arrays of intracytoplasmic tyrosines in the receptors. [ABSTRACT FROM AUTHOR]

Type 1 diabetes is an autoimmune disease in which pancreatic beta-cells are destroyed by cytotoxic T-cells that recognize peptide epitopes presented by HLA class I molecules. The identification of human beta-cell epitopes may significantly improve the prospects for immunodiagnosis and immunotherapy in type 1 diabetes. Using algorithms to predict nonameric beta-cell peptides that would bind to the common HLA allele, HLA-A*0201, we identified a potential epitope from the leader sequence of islet amyloid polypeptide (human islet amyloid polypeptide [IAPP] precursor protein [preproIAPP] 5-13: KLQVFLIVL). Peripheral blood mononuclear cells (PBMCs) were isolated from 18 HLA-A*0201 patients with type 1 diabetes (9 with recent-onset [<180 days; range, 1-120 days] and 9 with long-standing diabetes [>180 days; range, 183-3,273 days]) and 9 healthy, nondiabetic control subjects. PBMCs were screened for peptide recognition using interferon-gamma enzyme-linked immunospot (ELISpot) assays. Of the nine patients with recent-onset type 1 diabetes, six had ELISpot responses to preproIAPP 5-13 that were >3 SDs above the mean of the nondiabetic control subjects (P = 0.002). In contrast, no patients with type 1 diabetes for >180 days had a response above this threshold. In summary, preproIAPP 5-13 is a novel HLA class I epitope recognized by a significant proportion of cytotoxic T-cells from HLA-A*0201 patients with recent-onset type 1 diabetes and may prove to be a useful tool for the prediction and/or prevention of this disease. [ABSTRACT FROM AUTHOR]

Vigorous HIV-1-specific CD8+ cytotoxic T lymphocyte (CTL) responses play an important role in the control of HIV-1 replication and the induction of a strong, broadly cross-reactive CTL response remains an important goal of HIV vaccine development. It is known that the display of high levels of class I MHC–viral peptide complexes at the cell surface of target cells is necessary to elicit a strong CTL response. We now report two strategies to enhance the presentation of defined HIV-1 epitope-specific CTL target structures, by incorporating subdominant HIV-1 reverse transcriptase (RT) CTL epitope sequences into the human class I MHC molecule HLA-A2. We show that either incorporation of HIV-1 CTL epitopes into the signal sequence of HLA or tethering of epitopes to the HLA-A2 heavy chain provide simple ways to create effective CTL target structures that can be recognized and lysed by human HLA-A2-restricted RT-specific CD8+ CTL. Moreover, cells expressing these epitope-containing HLA-A2 constructs stimulated the generation of primary epitope-specific CTL in vitro. These strategies offer new options in the design of plasmid DNA-based vaccines or immunotherapeutics for the induction of CTL responses against subdominant HIV-1 epitopes. [ABSTRACT FROM PUBLISHER]

Suspected colonies of methicillin-resistant Staphylococcus aureus (MRSA) on chromogenic, MRSA Select (BioRad) medium were confirmed using routine microbiological methods, and a multiplex real-time PCR ( n = 108). Although the specificity of MRSA Select assessed at 24 h of incubation was much higher than that of 48 h (91.4 vs. 60 %), extending the incubation time to 48 h, along with PCR confirmation, increased the total number of true positive samples by 27.8 %. These results provide a cost effective method for sensitive and specific detection of MRSA. [ABSTRACT FROM AUTHOR]