Fatal autoimmunity in mice reconstituted with human hematopoietic stem cells encoding defective FOXP3.

Mice reconstituted with a human immune system provide a tractable in vivo model to assess human immune cell function. To date, reconstitution of murine strains with human hematopoietic stem cells (HSCs) from patients with monogenic immune disorders have not been reported. One obstacle precluding the development of immune-disease specific "humanized" mice is that optimal adaptive immune responses in current strains have required implantation of autologous human thymic tissue. To address this issue, we developed a mouse strain that lacks murine major histocompatibility complex class II (MHC II) and instead expresses human leukocyte antigen DR1 (HLA-DR1). These mice displayed improved adaptive immune responses when reconstituted with human HSCs including enhanced T-cell reconstitution, delayed-type hypersensitivity responses, and class-switch recombination. Following immune reconstitution of this novel strain with HSCs from a patient with immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome, associated with aberrant FOXP3 function, mice developed a lethal inflammatory disorder with multiorgan involvement and autoantibody production mimicking the pathology seen in affected humans. This humanized mouse model permits in vivo evaluation of immune responses associated with genetically altered HSCs, including primary immunodeficiencies, and should facilitate the study of human immune pathobiology and the development of targeted therapeutics.

Human anti-CAIX antibodies mediate immune cell inhibition of renal cell carcinoma in vitro and in a humanized mouse model in vivo.

BACKGROUND: Carbonic anhydrase (CA) IX is a surface-expressed protein that is upregulated by the hypoxia inducible factor (HIF) and represents a prototypic tumor-associated antigen that is overexpressed on renal cell carcinoma (RCC). Therapeutic approaches targeting CAIX have focused on the development of CAIX inhibitors and specific immunotherapies including monoclonal antibodies (mAbs). However, current in vivo mouse models used to characterize the anti-tumor properties of fully human anti-CAIX mAbs have significant limitations since the role of human effector cells in tumor cell killing in vivo is not directly evaluated. METHODS: The role of human anti-CAIX mAbs on CAIX(+) RCC tumor cell killing by immunocytes or complement was tested in vitro by antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) and antibody-dependent cellular phagocytosis (ADCP) as well as on CAIX(+) RCC cellular motility, wound healing, migration and proliferation. The in vivo therapeutic activity mediated by anti-CAIX mAbs was determined by using a novel orthotopic RCC xenograft humanized animal model and analyzed by histology and FACS staining. RESULTS: Our studies demonstrate the capacity of human anti-CAIX mAbs that inhibit CA enzymatic activity to result in immune-mediated killing of RCC, including nature killer (NK) cell-mediated ADCC, CDC, and macrophage-mediated ADCP. The killing activity correlated positively with the level of CAIX expression on RCC tumor cell lines. In addition, Fc engineering of anti-CAIX mAbs was shown to enhance the ADCC activity against RCC. We also demonstrate that these anti-CAIX mAbs inhibit migration of RCC cells in vitro. Finally, through the implementation of a novel orthotopic RCC model utilizing allogeneic human peripheral blood mononuclear cells in NOD/SCID/IL2Rγ(-/-) mice, we show that anti-CAIX mAbs are capable of mediating human immune response in vivo including tumor infiltration of NK cells and activation of T cells, resulting in inhibition of CAIX(+) tumor growth. CONCLUSIONS: Our findings demonstrate that these novel human anti-CAIX mAbs have therapeutic potential in the unmet medical need of targeted killing of HIF-driven CAIX(+)RCC. The orthotopic tumor xenografted humanized mouse provides an improved model to evaluate the in vivo anti-tumor capabilities of fully human mAbs for RCC therapy.

Cathepsin L activity controls adipogenesis and glucose tolerance.

Cysteine proteases play an important part in human pathobiology. This report shows the participation of cathepsin L (CatL) in adipogenesis and glucose intolerance. In vitro studies demonstrate the role of CatL in the degradation of the matrix protein fibronectin, insulin receptor (IR) and insulin-like growth factor-1 receptor (IGF-1R), essential molecules for adipogenesis and glucose metabolism. CatL inhibition leads to the reduction of human and murine pre-adipocyte adipogenesis or lipid accumulation, protection of fibronectin from degradation, accumulation of IR and IGF-1R beta-subunits, and an increase in glucose uptake. CatL-deficient mice are lean and have reduced levels of serum glucose and insulin but increased levels of muscle IR beta-subunits, fibronectin and glucose transporter (Glut)-4, although food/water intake and energy expenditure of these mice are no less than their wild-type littermates. Importantly, the pharmacological inhibition of CatL also demonstrates reduced body weight gain and serum insulin levels, and increased glucose tolerance, probably due to increased levels of muscle IR beta-subunits, fibronectin and Glut-4 in both diet-induced obese mice and ob/ob mice. Increased levels of CatL in obese and diabetic patients suggest that this protease is a novel target for these metabolic disorders.

Mast cells promote atherosclerosis by releasing proinflammatory cytokines.

Mast cells contribute importantly to allergic and innate immune responses by releasing various preformed and newly synthesized mediators. Previous studies have shown mast cell accumulation in human atherosclerotic lesions. This report establishes the direct participation of mast cells in atherogenesis in low-density lipoprotein receptor-deficient (Ldlr(-/-)) mice. Atheromata from compound mutant Ldlr(-/-) Kit(W-sh)(/W-sh) mice showed decreased lesion size, lipid deposition, T-cell and macrophage numbers, cell proliferation and apoptosis, but increased collagen content and fibrous cap development. In vivo, adoptive transfer of syngeneic wild-type or tumor necrosis factor (TNF)-alpha-deficient mast cells restored atherogenesis to Ldlr(-/-)Kit(W-sh/W-sh) mice. Notably, neither interleukin (IL)-6- nor interferon (IFN)-gamma-deficient mast cells did so, indicating that the inhibition of atherogenesis in Ldlr(-/-)Kit(W-sh/W-sh) mice resulted from the absence of mast cells and mast cell-derived IL-6 and IFN-gamma. Compared with wild-type or TNF-alpha-deficient mast cells, those lacking IL-6 or IFN-gamma did not induce expression of proatherogenic cysteine proteinase cathepsins from vascular cells in vitro or affect cathepsin and matrix metalloproteinase activities in atherosclerotic lesions, implying that mast cell-derived IL-6 and IFN-gamma promote atherogenesis by augmenting the expression of matrix-degrading proteases. These observations establish direct participation of mast cells and mast cell-derived IL-6 and IFN-gamma in mouse atherogenesis and provide new mechanistic insight into the pathogenesis of this common disease.

Anti-CD99 Antibody Therapy Triggers Macrophage-Dependent Ewing Cell Death In Vitro and Myeloid Cell Recruitment In Vivo.

BACKGROUND: Ewing sarcoma is a rare tumor of the bone or soft tissues characterized by diffuse membranous staining for CD99. As this tumor remains incurable in the metastatic, relapsed, and refractory settings, we explored the downstream immune implications of targeting CD99. METHODS: We discovered a human anti-CD99 antibody (NOA2) by phagemid panning and investigated NOA2 immune cell-mediated cytotoxicity in vitro and in vivo focusing on the myeloid cell compartment, given that M2 macrophages are present in human tumors and associated with a poor prognosis. RESULTS: NOA2 is capable of inducing immune effector cell-mediated Ewing death in vitro via engagement of macrophages. Mice with metastatic Ewing tumors, treated with NOA2, experience tumor growth arrest and an associated increase in intratumoral macrophages. Further, incubation of macrophages and Ewing cells with NOA2, in conjunction with anti-PILRα antibody blockade in vitro, results in the reactivation of previously dormant macrophages possibly due to interrupted binding of Ewing CD99 to macrophage PILRα. CONCLUSIONS: These studies are the first to demonstrate the role of human immune effector cells in anti-CD99-mediated Ewing tumor death. We propose that the engagement of CD99 by NOA2 results in the recruitment of intratumoral macrophages. In addition, interruption of the CD99:PILRα checkpoint axis may be a relevant therapeutic approach to activate tumor-associated macrophages.

Immune-restoring CAR-T cells display antitumor activity and reverse immunosuppressive TME in a humanized ccRCC mouse model.

One of the major barriers that have restricted successful use of chimeric antigen receptor (CAR) T cells in the treatment of solid tumors is an unfavorable tumor microenvironment (TME). We engineered CAR-T cells targeting carbonic anhydrase IX (CAIX) to secrete anti-PD-L1 monoclonal antibody (mAb), termed immune-restoring (IR) CAR G36-PDL1. We tested CAR-T cells in a humanized clear cell renal cell carcinoma (ccRCC) orthotopic mouse model with reconstituted human leukocyte antigen (HLA) partially matched human leukocytes derived from fetal CD34+ hematopoietic stem cells (HSCs) and bearing human ccRCC skrc-59 cells under the kidney capsule. G36-PDL1 CAR-T cells, haploidentical to the tumor cells, had a potent antitumor effect compared to those without immune-restoring effect. Analysis of the TME revealed that G36-PDL1 CAR-T cells restored active antitumor immunity by promoting tumor-killing cytotoxicity, reducing immunosuppressive cell components such as M2 macrophages and exhausted CD8+ T cells, and enhancing T follicular helper (Tfh)-B cell crosstalk.

Mast cell tryptase deficiency attenuates mouse abdominal aortic aneurysm formation.

RATIONALE: Mast cells (MCs) contribute to the formation of abdominal aortic aneurysms (AAAs) by producing biologically active mediators. Tryptase is the most abundant MC granule protein and participates in MC activation, protease maturation, leukocyte recruitment, and angiogenesis-all processes critical to AAA pathogenesis. OBJECTIVE: To test the hypothesis that tryptase participates directly in AAA formation. METHODS AND RESULTS: Immunohistochemistry demonstrated enhanced tryptase staining in media and adventitia of human and mouse AAA lesions. Serum tryptase levels correlated significantly with the annual expansion rate of AAA before (r = 0.30, P = 0.003) and after (r = 0.29, P = 0.005) adjustment for common AAA risk factors in a patient follow-up study, and associated with risks for later surgical repair or overall mortality before (P = 0.009, P = 0.065) and after (P = 0.004, P = 0.001) the adjustment. Using MC protease-6-deficient mice (Mcpt6(-/-)) and aortic elastase perfusion-induced experimental AAAs, we proved a direct role of this tryptase in AAA pathogenesis. Whereas all wild-type (WT) mice developed AAA at 14 or 56 days postperfusion, Mcpt6(-/-) mice were fully protected. AAA lesions from Mcpt6(-/-) mice had fewer inflammatory and apoptotic cells, and lower chemokine levels, than did those from WT mice. MC from WT mice restored reduced AAA lesions and lesion inflammatory cell content in MC-deficient Kit(W-sh/W-sh) mice, but those prepared from Mcpt6(-/-) mice did not. Mechanistic studies demonstrated that tryptase deficiency affected endothelial cell (EC) chemokine and cytokine expression, monocyte transmigration, smooth-muscle cell apoptosis, and MC and AAA lesion cysteinyl cathepsin expression and activities. CONCLUSIONS: This study establishes the direct participation of MC tryptase in the pathogenesis of experimental AAAs, and suggests that levels of this protease can serve as a novel biomarker for abdominal aortic expansion.

Cathepsin K deficiency reduces elastase perfusion-induced abdominal aortic aneurysms in mice.

OBJECTIVE: Cathepsin K (CatK) is one of the most potent mammalian elastases. We have previously shown increased expression of CatK in human abdominal aortic aneurysm (AAA) lesions. Whether this protease participates directly in AAA formation, however, remains unknown. METHODS AND RESULTS: Mouse experimental AAA was induced with aortic perfusion of a porcine pancreatic elastase. Using this experimental model, we demonstrated that absence of CatK prevented AAA formation in mice 14 days postperfusion. CatK deficiency significantly reduced lesion CD4(+) T-cell content, total lesion and medial cell proliferation and apoptosis, medial smooth muscle cell (SMC) loss, elastinolytic CatL and CatS expression, and elastin fragmentation, but it did not affect AAA lesion Mac-3(+) macrophage accumulation or CD31(+) microvessel numbers. In vitro studies revealed that CatK contributed importantly to CD4(+) T-cell proliferation, SMC apoptosis, and other cysteinyl cathepsin and matrix metalloproteinase expression and activities in SMCs and endothelial cells but played negligible roles in microvessel growth and monocyte migration. AAA lesions from CatK-deficient mice showed reduced elastinolytic cathepsin activities compared with those from wild-type control mice. CONCLUSIONS: This study demonstrates that CatK plays an essential role in AAA formation by promoting T-cell proliferation, vascular SMC apoptosis, and elastin degradation and by affecting vascular cell protease expression and activities.

Cathepsin L activity is essential to elastase perfusion-induced abdominal aortic aneurysms in mice.

OBJECTIVE: The development of abdominal aortic aneurysms (AAA) requires extensive aortic wall matrix degradation. Human AAA lesions express high levels of cathepsin L (CatL), one of the most potent mammalian elastases. Whether this protease participates directly in AAA pathogenesis, however, is unknown. METHODS AND RESULTS: We generated experimental AAA with aortic elastase perfusion in mice and established an essential role of CatL in AAA formation. After 14 days postperfusion, most wild-type (Ctsl(+/+)) mice developed AAA, but none of the CatL-deficient (Ctsl(-/-)) mice did. AAA lesion macrophage contents, CD4(+) T cell numbers, CD31(+) and laminin-5 angiogenic fragment γ2(+) microvessel numbers, and elastin fragmentation were all significantly lower in Ctsl(-/-) mice than in Ctsl(+/+) mice. While lesions from Ctsl(-/-) mice contained fewer Ki67(+) proliferating cells than did Ctsl(+/+) mice, the absence of CatL did not affect lesion apoptotic cell contents or medial smooth-muscle cell loss significantly. Mechanistic studies indicated that the absence of CatL reduced lesion chemokine monocyte chemotactic protein-1 content, macrophage and T-cell in vitro transmigration, and angiogenesis, and altered the expression and activities of matrix metalloproteinases and other cysteinyl cathepsins in inflammatory cells, vascular cells, and AAA lesions. CONCLUSION: CatL contributes to AAA formation by promoting lesion inflammatory cell accumulation, angiogenesis, and protease expression.

Deficiency of antigen-presenting cell invariant chain reduces atherosclerosis in mice.

BACKGROUND: Adaptive immunity and innate immunity play important roles in atherogenesis. Invariant chain (CD74) mediates antigen-presenting cell antigen presentation and T-cell activation. This study tested the hypothesis that CD74-deficient mice have reduced numbers of active T cells and resist atherogenesis. METHODS AND RESULTS: In low-density lipoprotein receptor-deficient (Ldlr(-/-)) mice, CD74 deficiency (Ldlr(-/-)Cd74(-/-)) significantly reduced atherosclerosis and CD25(+)-activated T cells in the atheromata. Although Ldlr(-/-)Cd74(-/-) mice had decreased levels of plasma immunoglobulin (Ig) G1, IgG2b, and IgG2c against malondialdehyde-modified LDL (MDA-LDL), presumably as a result of impaired antigen-presenting cell function, Ldlr(-/-)Cd74(-/-) mice showed higher levels of anti-MDA-LDL IgM and IgG3. After immunization with MDA-LDL, Ldlr(-/-)Cd74(-/-) mice had lower levels of all anti-MDA-LDL Ig isotypes compared with Ldlr(-/-) mice. As anticipated, only Ldlr(-/-) splenocytes responded to in vitro stimulation with MDA-LDL, producing Th1/Th2 cytokines. Heat shock protein-65 immunization enhanced atherogenesis in Ldlr(-/-) mice, but Ldlr(-/-) Cd74(-/-) mice remained protected. Compared with Ldlr(-/-) mice, Ldlr(-/-)Cd74(-/-) mice had higher anti-MDA-LDL autoantibody titers, fewer lesion CD25(+)-activated T cells, impaired release of Th1/Th2 cytokines from antigen-presenting cells after heat shock protein-65 stimulation, and reduced levels of all plasma anti-heat shock protein-65 Ig isotypes. Cytofluorimetry of splenocytes and peritoneal cavity cells of MDA-LDL- or heat shock protein-65-immunized mice showed increased percentages of autoantibody-producing marginal zone B and B-1 cells in Ldlr(-/-)Cd74(-/-) mice compared with Ldlr(-/-) mice. CONCLUSIONS: Invariant chain deficiency in Ldlr(-/-) mice reduced atherosclerosis. This finding was associated with an impaired adaptive immune response to disease-specific antigens. Concomitantly, an unexpected increase in the number of innate-like peripheral B-1 cell populations occurred, resulting in increased IgM/IgG3 titers to the oxidation-specific epitopes.

Cystatin C deficiency promotes inflammation in angiotensin II-induced abdominal aortic aneurisms in atherosclerotic mice.

An imbalance between cysteinyl cathepsins and their principal endogenous inhibitor cystatin C (CystC) may favor proteolysis in the pathogenesis of human abdominal aortic aneurysms (AAA), yet a direct role of CystC in AAA remains unproven. This study used CystC and apolipoprotein E (ApoE) compound mutant (CystC(-/-)ApoE(-/-)) mice to examine directly the role of cysteine protease/protease inhibitor imbalance in AAA formation in angiotensin II-induced AAA. CystC-deficiency increased lumenal diameter and lesion size compared with control mice. CystC(-/-) ApoE(-/-) lesions also demonstrated enhanced inflammatory cell accumulation, more severe elastin fragmentation, and fewer smooth muscle cells in the tunica media. Macrophage content, measured as percent positive area (23.2 +/- 1.4% versus 11.2 +/- 1.4%; P = 0.0003) and number of the CD4(+) T cells (ninefold; P = 0.048), increased significantly in CystC(-/-)ApoE(-/-) lesions. CystC deficiency increased cathepsin activity (5.5 fold; P = 0.001) in AAA, yielding greater elastin degradation and proangiogenic laminin-5 gamma2 peptide production, which may account for increased microvascularization in CystC(-/-)ApoE(-/-) compared with ApoE(-/-) lesions. Increased leukocyte adhesion molecule VCAM-1 expression and leukocyte proliferation might also promote inflammation in CystC-deficient AAA. These data indicate that CystC contributes to experimental AAA pathogenesis and that enhanced cysteine protease activity, due to the lack of CystC, favors inflammation in AAA lesions induced in atherosclerotic mice by promoting microvascularization and smooth muscle cell apoptosis as well as leukocytes adhesion and proliferation.

Critical role of mast cell chymase in mouse abdominal aortic aneurysm formation.

BACKGROUND: Mast cell chymase may participate in the pathogenesis of human abdominal aortic aneurysm (AAA), yet a direct contribution of this serine protease to AAA formation remains unknown. METHODS AND RESULTS: Human AAA lesions had high numbers of chymase-immunoreactive mast cells. Serum chymase level correlated with AAA growth rate (P=0.009) in a prospective clinical study. In experimental AAA produced by aortic elastase perfusion in wild-type (WT) mice or those deficient in the chymase ortholog mouse mast cell protease-4 (mMCP-4) or deficient in mMCP-5 (Mcpt4(-/-), Mcpt5(-/-)), Mcpt4(-/-) but not Mcpt5(-/-) had reduced AAA formation 14 days after elastase perfusion. Even 8 weeks after perfusion, aortic expansion in Mcpt4(-/-) mice fell by 50% compared with that of the WT mice (P=0.0003). AAA lesions in Mcpt4(-/-) mice had fewer inflammatory cells and less apoptosis, angiogenesis, and elastin fragmentation than those of WT mice. Although Kit(W-sh/W-sh) mice had protection from AAA formation, reconstitution with mast cells from WT mice, but not those from Mcpt4(-/-) mice, partially restored the AAA phenotype. Mechanistic studies suggested that mMCP-4 regulates expression and activation of cysteine protease cathepsins, elastin degradation, angiogenesis, and vascular cell apoptosis. CONCLUSIONS: High chymase-positive mast cell content in human AAA lesions, greatly reduced AAA formation in Mcpt4(-/-) mice, and significant correlation of serum chymase levels with human AAA expansion rate suggests participation of mast cell chymase in the progression of human and mouse AAA.

Mast cells modulate the pathogenesis of elastase-induced abdominal aortic aneurysms in mice.

Abdominal aortic aneurysm (AAA), an inflammatory disease, involves leukocyte recruitment, immune responses, inflammatory cytokine production, vascular remodeling, neovascularization, and vascular cell apoptosis, all of which contribute to aortic dilatation. This study demonstrates that mast cells, key participants in human allergic immunity, participate in AAA pathogenesis in mice. Mast cells were found to accumulate in murine AAA lesions. Mast cell-deficient KitW-sh/KitW-sh mice failed to develop AAA elicited by elastase perfusion or periaortic chemical injury. KitW-sh/KitW-sh mice had reduced aortic expansion and internal elastic lamina degradation; decreased numbers of macrophages, CD3+ T lymphocytes, SMCs, apoptotic cells, and CD31+ microvessels; and decreased levels of aortic tissue IL-6 and IFN-gamma. Activation of mast cells in WT mice via C48/80 injection resulted in enhanced AAA growth while mast cell stabilization with disodium cromoglycate diminished AAA formation. Mechanistic studies demonstrated that mast cells participated in angiogenesis, aortic SMC apoptosis, and matrix-degrading protease expression. Reconstitution of KitW-sh/KitW-sh mice with bone marrow-derived mast cells from WT or TNF-alpha-/- mice, but not from IL-6-/- or IFN-gamma-/- mice, caused susceptibility to AAA formation to be regained. These results demonstrate that mast cells participate in AAA pathogenesis in mice by releasing proinflammatory cytokines IL-6 and IFN-gamma, which may induce aortic SMC apoptosis, matrix-degrading protease expression, and vascular wall remodeling, important hallmarks of arterial aneurysms.

Deficiency and inhibition of cathepsin K reduce body weight gain and increase glucose metabolism in mice.

OBJECTIVE: Previous studies demonstrated increased levels of cysteine proteases cathepsins in serum and adipose tissues from obese patients. We now provide evidence from a mouse model of obesity to suggest a direct participation of cathepsin K (CatK) in mouse body weight gain and glucose metabolism. METHODS AND RESULTS: Using real-time polymerase chain reaction, we detected 12-fold increase in CatK transcripts after adipogenesis of human preadipocytes. Using an immunohistology analysis, we consistently observed high levels of CatK expression in adipose tissues from obese humans and mice. Selective inhibition of CatK activity blocked the lipid accumulation in human and mouse preadipocytes. In mice, CatK deficiency reduced significantly diet-induced body weight gain and serum glucose and insulin levels. Similar results were obtained in diet-induced and genetically created (ob/ob) obese mice after animals were treated with a CatK-selective inhibitor. Mechanistic study demonstrated a role for CatK in degrading fibronectin, a matrix protein that controls adipogenesis. Deficiency or inhibition of CatK leads to fibronectin accumulation in muscle and adipose tissues. CONCLUSIONS: This study demonstrates an essential role of CatK in adipogenesis and mouse body weight gain, possibly via degradation of fibronectin, thus suggesting a novel therapeutic strategy for the control of obesity by regulating CatK activity.

Alcohol consumption increases susceptibility to pneumococcal pneumonia in a humanized murine HIV model mediated by intestinal dysbiosis.

Alcohol use in persons living with HIV (PLWH) worsens the severity of bacterial pneumonia. However, the exact mechanism(s) by which this occurs remain ill-defined. We hypothesized that alcohol in the setting of HIV infection decreases Streptococcus pneumoniae clearance from the lung through mechanisms mediated by the gut microbiota. Humanized BLT (bone marrow, liver, thymus) mice were infected with 1 × 104 TCID50 of HIV (BAL and JRCSF strains) via intraperitoneal (i.p.) injection. One week post-HIV infection, animals were switched to a Lieber-DeCarli 5% ethanol diet or an isocaloric control diet for 10 days. Alcohol-fed animals were also given two binges of 2 g/kg ethanol on days 5 and 10. Feces were also collected, banked, and the community structures were analyzed. Mice were then infected with 1 × 105 CFU (colony-forming units) of S. pneumoniae and were sacrificed 48 h later. HIV-infected mice had viral loads of ∼2 × 104 copies/mL of blood 1 week post-infection, and exhibited an ∼57% decrease in the number of circulating CD4+ T cells at the time of sacrifice. Fecal microbial community structure was significantly different in each of the feeding groups, as well as with HIV infection. Alcohol-fed mice had a significantly higher burden of S. pneumoniae 48 h post-infection, regardless of HIV status. In follow-up experiments, female C57BL/6 mice were treated with a cocktail of antibiotics daily for 2 weeks and recolonized by gavage with intestinal microbiota from HIV+ ethanol-fed, HIV+ pair-fed, HIV- ethanol-fed, or HIV- pair-fed mice. Recolonized mice were then infected with S. pneumoniae and were sacrificed 48 h later. The intestinal microbiota from alcohol-fed mice (regardless of HIV status) significantly impaired clearance of S. pneumoniae. Collectively, these data indicate that alcohol feeding, as well as alcohol-associated intestinal dysbiosis, compromise pulmonary host defenses against pneumococcal pneumonia. Determining whether HIV infection acts synergistically with alcohol use in impairing pulmonary host defenses will require additional study.

Cathepsin L deficiency reduces diet-induced atherosclerosis in low-density lipoprotein receptor-knockout mice.

BACKGROUND: Remodeling of the arterial extracellular matrix participates importantly in atherogenesis and plaque complication. Increased expression of the elastinolytic and collagenolytic enzyme cathepsin L (Cat L) in human atherosclerotic lesions suggests its participation in these processes, a hypothesis tested here in mice. METHODS AND RESULTS: We generated Cat L and low-density lipoprotein receptor (LDLr) double-deficient (LDLr-/- Cat L-/-) mice by crossbreeding Cat L-null (Cat L-/-) and LDLr-deficient (LDLr-/-) mice. After 12 and 26 weeks of a Western diet, LDLr-/- Cat L-/- mice had significantly smaller atherosclerotic lesions and lipid cores compared with littermate control LDLr-/- Cat L+/- and LDLr-/- Cat L+/+ mice. In addition, lesions from the compound mutant mice showed significantly reduced levels of collagen, medial elastin degradation, CD4+ T cells, macrophages, and smooth muscle cells. Mechanistic studies showed that Cat L contributes to the degradation of extracellular matrix elastin and collagen by aortic smooth muscle cells. Smooth muscle cells from LDLr-/- Cat L-/- mice or those treated with a Cat L-selective inhibitor demonstrated significantly less degradation of elastin and collagen and delayed transmigration through elastin in vitro. Cat L deficiency also significantly impaired monocyte and T-lymphocyte transmigration through a collagen matrix in vitro, suggesting that blood-borne leukocyte penetration through the arterial basement membrane requires Cat L. Cysteine protease active site labeling demonstrated that Cat L deficiency did not affect the activity of other atherosclerosis-associated cathepsins in aortic smooth muscle cells and monocytes. CONCLUSIONS: Cat L directly participates in atherosclerosis by degrading elastin and collagen and regulates blood-borne leukocyte transmigration and lesion progression.

Structural characterization and transcriptional regulation of the gene encoding diapause hormone and pheromone biosynthesis activating neuropeptide in the cotton bollworm, Helicoverpa armigera.

We have cloned the gene encoding the diapause hormone and the pheromone biosynthesis activating neuropeptide in Helicoverpa armigera (Har-DH-PBAN). The Har-DH-PBAN gene contains six exons and five introns that fall in the same positions as in the Bombyx mori DH-PBAN gene (Bom-DH-PBAN). The transcription initiation site lays 29 bp upstream of the translation initiation site. Southern blot analysis suggests that a single copy of this gene is present per haploid genome. A structural comparison of DH-PBAN promoters between H. armigera and B. mori show similarities in the TATA box and in a potential binding site for a POU family transcription factor, POU-M2. However, testing of these DNA regions for factor binding in vitro and transcription assays in cell culture highlight significant differences in their regulation particularly in reference to the POU-M2 sites. Our results uncover common and different regulatory mechanisms at work in the control of DH-PBAN gene expression in H. armigera and B. mori.

Increased serum cathepsin S in patients with atherosclerosis and diabetes.

Atherosclerosis and diabetes are closely associated and both involve extensive degradation of the aortic elastin. Increased elastase activity has been detected in diabetic animal aortae. We have demonstrated enhanced elastolytic cathepsin S in human atherosclerotic lesions but insufficient amounts of its endogenous inhibitor cystatin C, suggesting alterations of serum cathepsin S and/or cystatin C in patients with atherosclerosis or diabetes. In this study, we measured levels of both cathepsin S and cystatin C in sera from 240 patients by ELISA. Among these patients, 107 had a diagnosis of atherosclerotic stenosis, 103 were diabetic, and 30 had neither condition. Multiple linear regression analysis demonstrated that significantly higher serum levels of cathepsin S in patients with either atherosclerotic stenosis (p<0.04) or diabetes (p=0.0005) persisted after adjustment for cystatin C level, renal function, smoking, and serum glucose levels (p=0.008, p=0.0005). Furthermore, patients with acute (p=0.009) or previous myocardial infarction (p<0.02) or unstable angina pectoris (p<0.05) had elevated levels of cathepsin S after adjustment for smoking, creatinine, cystatin C, and serum glucose. In contrast, serum cystatin C levels were higher in diabetic patients (p=0.00001), but not in atherosclerotic subjects (p=0.14), than in the non-involved population after adjustment for age, smoking, and renal function. Although the pathophysiology of cathepsin S or cystatin C in atherosclerosis and diabetes requires further investigation, increased serum cathepsin S may serve as a biomarker for both diseases.

Cathepsin L expression and regulation in human abdominal aortic aneurysm, atherosclerosis, and vascular cells.

The cysteine protease cathepsin L is one of the most potent mammalian elastases and collagenases, widely expressed at basal levels in most tested tissues and cell types, and regulated by pro-inflammatory stimuli. The inflammatory arterial diseases abdominal aortic aneurysm (AAA) and atherosclerosis involve extensive vascular remodeling that requires elastolysis and collagenolysis. This study examined the hypothesis that cathepsin L is over-expressed in human AAA and atherosclerotic lesions and its expression in vascular cell types found in these lesions is regulated by pro-inflammatory cytokines. Immunohistochemical and tissue extract immunoblot analysis demonstrated increased expression of cathepsin L in human AAA and atheromata and localized its expression to lesional smooth muscle cells (SMC), endothelial cells (EC), and macrophages. In primary cultured human SMC, EC, and monocyte-derived macrophages, pro-inflammatory cytokines or growth factors induced the expression of cathepsin L and its activity against extracellular collagen and elastin. Patients with coronary artery stenosis (n=65) had higher serum cathepsin L levels than those without lesions detectable by quantitative coronary angiography (n=30) (1.47+/-0.33 ng/ml versus 0.60+/-0.06 ng/ml, p<0.02). A strong correlation between the percent of stenosis of left anterior descending coronary artery and serum cathepsin L levels in patients with stenosis (R=0.542, p<0.0001), also suggests involvement of cathepsin L in these vascular diseases.

Pancreatic cancer cell-derived IGFBP-3 contributes to muscle wasting.

BACKGROUND: Progressive loss of skeletal muscle, termed muscle wasting, is a hallmark of cancer cachexia and contributes to weakness, reduced quality of life, as well as poor response to therapy. Previous studies have indicated that systemic host inflammatory response regarding tumor development results in muscle wasting. However, how tumor directly regulates muscle wasting via tumor-derived secreted proteins is still largely unknown. METHODS: In this study, we performed bioinformatics analysis in two datasets of pancreatic ductal adenocarcinoma, which causes cancer cachexia and muscle wasting with the highest prevalence, and uncovered that IGFBP3, which encodes IGF-binding protein-3 (IGFBP-3), is dramatically up-regulated in pancreatic tumor samples. We also verified the wasting effect of IGFBP-3 on C2C12 muscle cells with biochemical and genetic assays. RESULTS: IGFBP-3 potently leads to impaired myogenesis and enhanced muscle protein degradation, the major features of muscle wasting, via IGF signaling inhibition. Moreover, conditioned medium from Capan-1 pancreatic cancer cells, which contains abundant IGFBP-3, significantly induces muscle cell wasting. This wasting effect is potently alleviated by IGFBP3 knockdown in Capan-1 cells or IGFBP-3 antibody neutralization. Strikingly, compared to muscle cells, IGF signaling and proliferation rate of Capan-1 cells were rarely affected by IGFBP-3 treatment. CONCLUSIONS: Our results demonstrated that pancreatic cancer cells induce muscle wasting via IGFBP-3 production.