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To fulfill the bioenergetic and biosynthetic demand of proliferation, T cells reprogram their metabolic pathways from fatty acid β-oxidation and pyruvate oxidation via the TCA cycle to the glycolytic, pentose-phosphate, and glutaminolytic pathways. Two of the top-ranked candidate transcription factors potentially responsible for the activation-induced T cell metabolic transcriptome, HIF1α and Myc, were induced upon T cell activation, but only the acute deletion of Myc markedly inhibited activation-induced glycolysis and glutaminolysis in T cells. Glutamine deprivation compromised activation-induced T cell growth and proliferation, and this was partially replaced by nucleotides and polyamines, implicating glutamine as an important source for biosynthetic precursors in active T cells. Metabolic tracer analysis revealed a Myc-dependent metabolic pathway linking glutaminolysis to the biosynthesis of polyamines. Therefore, a Myc-dependent global metabolic transcriptome drives metabolic reprogramming in activated, primary T lymphocytes. This may represent a general mechanism for metabolic reprogramming under patho-physiological conditions., (Copyright © 2011 Elsevier Inc. All rights reserved.)

During apoptosis, the BCL-2 protein family controls mitochondrial outer membrane permeabilization (MOMP), but the dynamics of this regulation remain controversial. We employed chimeric proteins composed of exogenous BH3 domains inserted into a tBID backbone that can activate the proapoptotic effectors BAX and BAK to permeabilize membranes without being universally sequestered by all antiapoptotic BCL-2 proteins. We thus identified two "modes" whereby prosurvival BCL-2 proteins can block MOMP, by sequestering direct-activator BH3-only proteins ("MODE 1") or by binding active BAX and BAK ("MODE 2"). Notably, we found that MODE 1 sequestration is less efficient and more easily derepressed to promote MOMP than MODE 2. Further, MODE 2 sequestration prevents mitochondrial fusion. We provide a unified model of BCL-2 family function that helps to explain otherwise paradoxical observations relating to MOMP, apoptosis, and mitochondrial dynamics., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Caspase-8 has two opposing biological functions--it promotes cell death by triggering the extrinsic pathway of apoptosis, but also has a survival activity, as it is required for embryonic development, T-lymphocyte activation, and resistance to necrosis induced by tumour necrosis factor-α (TNF-α) and related family ligands. Here we show that development of caspase-8-deficient mice is completely rescued by ablation of receptor interacting protein kinase-3 (RIPK3). Adult animals lacking both caspase-8 and RIPK3 display a progressive lymphoaccumulative disease resembling that seen with defects in CD95 or CD95-ligand (also known as FAS and FASLG, respectively), and resist the lethal effects of CD95 ligation in vivo. We have found that caspase-8 prevents RIPK3-dependent necrosis without inducing apoptosis by functioning in a proteolytically active complex with FLICE-like inhibitory protein long (FLIP(L), also known as CFLAR), and this complex is required for the protective function.

We have selectively targeted monocyte/macrophages overexpressing an immunomodulatory molecule, latency-associated peptide (LAP), a naturally occurring antagonist for transforming growth factor-beta1, to murine skin utilizing UV light to produce a cutaneous influx of transduced monocyte/macrophages. Bone marrow (BM) cells from BALB/c mice were transduced in vitro with a retroviral construct containing green fluorescent protein (GFP) for detection and human LAP (hLAP) as a test molecule. The transduced BM cells were then cultured in vitro with granulocyte-macrophage colony-stimulating factor (GM-CSF) to produce differentiation to monocyte/macrophages. More than 80% of transduced BM cells were CD11b-positive and MOMA-positive by fluorescence-activated cell-sorter analysis and secreted LAP by ELISA after 10 days of culture in granulocyte-macrophage colony-stimulating factor (GM-CSF). Transduced monocyte/macrophages containing either GFP or hLAP-GFP were then injected intravenously into BALB/c mice. One-half of recipients in each group were exposed to UVB (72 mJ) to induce monocyte/macrophage infiltration into skin. Recipients were sacrificed 60 h after UV irradiation. We found transduced cutaneous macrophages expressing GFP by examining with fluorescence microscopy frozen skin sections of recipient mice immunostained with antibodies to GFP and to macrophage marker F4/80. We identified hLAP sequences by polymerase chain reaction (PCR) of total DNA in recipient blood and UV-irradiated skin but not in unirradiated skin. LAP sequences were also detected at much lower levels in other organs (lung, spleen, and liver) by PCR. Therefore, we have shown that genetically altered monocytic cells can be injected intravenously and targeted to mouse skin by UV exposure. This macrophage-based gene-transfer method may be a potentially useful immunotherapeutic approach for delivering monocyte/macrophage-derived products to skin.

Murine sclerodermatous graft-versus-host disease (Scl GVHD), produced by transplanting B10.D2 bone marrow and spleen cells to lethally irradiated BALB/cJ mice, is a model for human scleroderma. Mice with Scl GVHD have skin thickening, lung fibrosis, cutaneous mononuclear cell infiltration, and upregulation of cutaneous transforming growth factor beta1 (TGF-beta1) and type I collagen mRNAs by day 21 after bone marrow transplantation. Elevated TGF-beta1 appears to be the critical cytokine driving fibrosis in Scl GVHD, which can be prevented with antibodies to TGF-beta administered early in disease. Here we demonstrate that we can also prevent skin thickening in mice with Scl GVHD with a naturally occurring antagonist to TGF-beta1, human latency-associated peptide (LAP). By quantitative real-time PCR analysis and immunostaining, LAP treatment also abrogates the upregulation of cutaneous TGF-beta1 and connective tissue growth factor mRNAs and type I collagen synthesis in Scl GVHD. In contrast to anti-TGF-beta antibodies, LAP at 4 ng total per mouse has no significant suppressive effect on cutaneous influx of T cells and monocytes or immune cell activation. LAP may be a potential new therapy in scleroderma and other TGF-beta-driven fibrosing disease that targets TGF-beta more specifically, without affecting systemic critical roles of TGF-beta on immune cell function.

Murine sclerodermatous graft-vs-host disease (Scl GVHD) models human scleroderma, with prominent skin thickening, lung fibrosis, and up-regulation of cutaneous collagen mRNA. Fibrosis in Scl GVHD may be driven by infiltrating TGF-beta1-producing mononuclear cells. Here we characterize the origin and types of those cutaneous effector cells, the cytokine and chemokine environments, and the effects of anti-TGF-beta Ab on skin fibrosis, immune cell activation markers, and collagen and cytokine synthesis. Donor cells infiltrating skin in Scl GVHD increase significantly at early time points post-transplantation and are detectable by PCR analysis of Y-chromosome sequences when female mice are transplanted with male cells. Cutaneous monocyte/macrophages and T cells are the most numerous cells in Scl GVHD compared with syngeneic controls. These immune cells up-regulate activation markers (MHC class II I-A(d) molecules and class A scavenger receptors), suggesting Ag presentation by cutaneous macrophages in early fibrosing disease. Early elevated cutaneous mRNA expression of TGF-beta1, but not TGF-beta2 or TGF-beta3, and elevated C-C chemokines macrophage chemoattractant protein-1, macrophage inflammatory protein-1alpha, and RANTES precede subsequent skin and lung fibrosis. Therefore, TGF-beta1-producing donor mononuclear cells may be critical effector cells, and C-C chemokines may play important roles in the initiation of Scl GVHD. Abs to TGF-beta prevent Scl GVHD by effectively blocking the influx of monocyte/macrophages and T cells into skin and by abrogating up-regulation of TGF-beta1, thereby preventing new collagen synthesis. The Scl GVHD model is valuable for testing new interventions in early fibrosing diseases, and chemokines may be new potential targets in scleroderma.

Scleroderma, a debilitating acquired connective tissue disease, is characterized by fibrosis, particularly of the skin and lungs. Monocyte-produced TGF-beta1, a potent stimulus for collagen synthesis, is thought to drive the fibrosis. Here, we thoroughly characterize a murine sclerodermatous graft-vs-host disease (Scl GVHD) model for scleroderma that reproduces important features of scleroderma including skin thickening, lung fibrosis, and up-regulation of cutaneous collagen mRNA, which is preceded by monocyte infiltration and the up-regulation of cutaneous TGF-beta1 mRNA. Most importantly, we can prevent fibrosis in both the skin and lungs of mice with Scl GVHD by inhibiting TGF-beta with neutralizing Abs. The murine Scl GVHD model provides the unique opportunity to study basic immunologic mechanisms that drive fibrosing diseases and GVHD itself and will be useful for testing new therapies for these diseases.

Bacterial polysaccharides (PS) are T-independent type 2 Ags that elicit restricted Ab responses of IgM and IgG3 in mice and IgM and predominantly IgG2 in humans. Immunodeficiency in the dominant IgG subclass made to PS is associated with chronic sinus and pulmonary infections with PS-encapsulated bacteria. To elucidate the biologic role of the dominant IgG subclass in the immune response to PS and to make an animal model of human IgG subclass deficiency, we generated mice with a targeted disruption of the exon encoding the CH1 domain of the gamma 3 heavy-chain constant region gene. Homozygotes had no detectable serum IgG3, and their splenocytes did not produce IgG3 after LPS stimulation. IgG3(-/-) mice immunized with PS from Pseudomonas aeruginosa LPS O-side chain or Streptococcus pneumoniae type 19F capsule did not produce any IgG3 anti-PS Abs, in contrast to wild-type mice in which IgG3 was the major IgG subclass. Immunizing both wild-type and IgG3(-/-) mice with 19F PS-protein conjugate elicited IgG1 Abs. We conclude that IgG3(-/-) mice have a selective deficiency in the dominant murine IgG subclass made to T-independent type 2 Ags and may be a useful animal model of IgG subclass deficiency. In addition, we show that the anti-PS Ab class switching to IgG1 that occurs when mice are immunized with a PS-protein conjugate vaccine does not require sequential Ig expression or an intact, upstream gamma 3 heavy-chain gene.

Unmethylated bacterial DNA containing a high frequency of the CpG motif, is mitogenic and induces T-cell independent, murine B-cell proliferation. These stimulatory effects are also induced by synthetic oligonucleotides that contain one or more unmethylated CpG dinucleotides (CpG oligo). Such mitogenicity is not seen with highly methylated vertebrate DNA, which has a lower prevalence of the CpG motif than bacterial DNA. Due to their stimulatory effects, CpG oligo have been proposed for use as vaccine adjuvants. In order to determine if a synthetic CpG oligo that was stimulatory for B-cell proliferation could augment the murine antibody response to protective bacterial polysaccharide epitopes (Pseudomonas aeruginosa LPS-O polysaccharide side chain; high-molecular-weight polysaccharide or high-MW PS), BALB/c mice were injected with mitogenic doses of CpG oligo simultaneously with high-MW PS, and antibody titers were measured by ELISA weekly for 4 weeks. Controls received PBS, a nonstimulatory control oligo plus PS, CpG alone, or PS alone. Despite evidence of B-cell mitogenicity and an increase in total IgM in CpG oligo-treated mice, CpG oligo treatment plus PS significantly decreased the high-MW PS antibody response compared to PS alone. The blunting of the anti-PS antibody response could be eliminated by vaccinating the animals with PS prior to CpG oligo. We conclude that despite in vitro and in vivo evidence of B-cell proliferation, this CpG oligo reduces PS-specific antibody responses in an animal model when given simultaneously with a bacterial polysaccharide. Based on results in this model, oligonucleotides containing stimulatory unmethylated CpG dinucleotides may not be useful adjuvants when given simultaneously with bacterial PS vaccines.

Inflammation and infection associated with bacterial pathogens, primarily Pseudomonas aeruginosa (Pa), are the primary causes of morbidity and mortality for cystic fibrosis (CF) patients. CF patients may be predisposed to these bacterial infections by a defect in phagocytosis due to "opsonin-receptor mismatch," in which a complement receptor (CR1) and an important opsonin (iC3b) are destroyed by proteolytic enzymes. We show that opsonin-receptor mismatch can be mitigated in vitro using a bispecific Ab (bsAb) to cross-link neutrophils via the beta-chain of leukocyte integrins (CD18) to bacterial epitopes or C3d on opsonized Pa. Two chemically cross-linked bsAb were constructed with mAb specific for C3d (or the O-specific side chain of Fisher Devlin Immunotype 1 Pa) and CD18. Using an in vitro model of elastase-mediated opsonin-receptor mismatch, these bsAb specifically enhanced Pa phagocytosis and killing, with the anti-C3d-containing bsAb restoring the levels of phagocytosis to approximately those for the non-elastase-treated opsonic control. These results encourage the further investigation of bsAb as therapeutic agents for bacterial infection in the lungs of CF patients.

Objective: The study examined the level of community integration achieved by patients discharged from the state hospital into the community in compliance with a regionalization policy in Vermont that sought to reduce the need for central hospitalization through expansion of community capacity., Methods: The population in residence at the state hospital on August 30, 1989, was tracked longitudinally as patients were discharged into one of Vermont's ten catchment areas. Structured interviews about the current status of the discharged individuals were conducted four years later with case managers, nursing home personnel, and community care home operators. Service utilization and hospitalization data were obtained from the Vermont Department of Mental Health database., Results: Of 122 patients in residence at the state hospital on the given date, 58 were discharged into the community, of whom 46 consented to participate in the study. At follow-up, about half lived in structured residential settings. Of the 46 followed, 87 percent were rehospitalized during the study period for periods ranging from three months to one year. Although participants had adequate levels of support both from within and outside the mental health system, their integration into the community was low in terms of their use of community resources, stigma-related problems, and difficulties gaining access to services., Conclusions: The regionalization policy accomplished some of its goals, especially those related to downsizing the state hospital, placing clients in community residential settings, and enhancing the range of community services. The more pervasive and insidious problems of community integration faced by consumers were not effectively mitigated by the policy.

Objective: In 1988 Vermont implemented a policy designed to reduce the state hospital census and expand community-based services. This qualitative study assessed perceptions of the policy's impact among mental health consumers, family members, and providers., Methods: Eleven focus groups were convened, which included 94 participants from across the state. Separate groups were held for consumers, family members, and providers. Trained facilitators guided discussion of the policy's effect on quality of life, housing and vocational status, community integration, and social networks. Audiotapes of the discussion were transcribed, and content was analyzed., Results: Several universal themes were noted. All participants reported that stigma was still a substantial barrier to integration and that community education to reduce stigma had not been effective. Tension between families and providers was a problem; family members felt that although providers depended on their supporting the consumer, they were not included in treatment planning. All participants noted that urban areas were better served by the policy's service packages. A lack of coordination of community services was reported to be a continuing problem across the state. In contrast to findings of previous studies, consumers in this study preferred not to live alone, which led to feelings of isolation., Conclusions: Service delivery in rural areas and system coordination throughout the state must be improved. Families' conflicting feelings of burden and isolation must be addressed. Further research should determine more clearly the range of housing preferences among consumers.

Human service workers devote a great deal of time in preparing to serve clients. The majority of this preparation is focused in one direction, with exposure to a limited type of client. The growing population of individuals with dual disorders of mental illness and substance abuse challenges this tradition in training, in that these clients pose unique, multiple, and overlapping characteristics, symptomatology, and behaviors requiring a synthesis of training approaches. Based on survey data collected in a county in New York State, this study discusses how providers from various agencies view individuals with dual disorders and proposes coordination and training efforts that can be designed to respond to providers' treatment concerns.

Nitric oxide (NO) is produced by murine macrophages in response to cytokines and/or gram-negative bacterial lipopolysaccharide. NO induction by gram-positive bacteria such as group B streptococci (GBS), the major etiologic agents of neonatal pneumonia and meningitis, has received little study. GBS as well as two other gram-positive bacterial species, Staphylococcus aureus and Staphylococcus epidermidis, were found to stimulate NO production in thioglycolate-elicited murine macrophages and in the mouse macrophage cell line J774A.1 in the presence of gamma interferon. Serotype Ia and III GBS were both stimulatory, as were asialo- and type antigen-deficient mutant strains of type III GBS. NO production was dose dependent, inhibitable by L-arginine analogs, and unaffected by polymyxin B. Since phagocytosis by murine and human phagocytes of GBS is dependent on complement receptor type 3 (CR3), the role of CR3 in the NO response to GBS was tested in the CR3-deficient myelomonocytic cell line WEHI-3. GBS did not induce NO, whereas S. aureus or lipopolysaccharide did induce NO in WEHI-3 cells. S. epidermidis, whose nonopsonic phagocytosis is also CR3 dependent, failed to induce NO in WEHI-3 cells. Monoclonal anti-CR3 (anti-CD11b or anti-CD18) in the presence of interferon also induced NO production in thioglycolate-elicited macrophages and in J774A.1 cells but not in WEHI-3 cells. This evidence suggests that ligated CR3 and gamma interferon act synergistically to induce NO production and that CR3 mediates the GBS-induced signal for NO production in interferon-treated macrophages.