Misconduct: lower ranks take most of the blame.
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Age Factors↗
Biomedical subjects
Shawn G Clouthier
Publications and source records attributed to Shawn G Clouthier.
Intact-protein-based high-resolution three-dimensional quantitative analysis system for proteome profiling of biological fluids.
The substantial complexity and vast dynamic range of protein abundance in biological fluids, notably serum and plasma, present a formidable challenge for comprehensive protein analysis. Integration of multiple technologies is required to achieve high-resolution and high-sensitivity proteomics analysis of biological fluids. We have implemented an orthogonal three-dimensional intact-protein analysis system (IPAS), coupled with protein tagging and immunodepletion of abundant proteins, to quantitatively profile the human plasma proteome. Following immunodepletion, plasma proteins in each of paired samples are concentrated and labeled with a different Cy dye, before mixing. Proteins are subsequently separated in three dimensions according to their charge, hydrophobicity, and molecular mass. Differences in the abundance of resolved proteins are determined based on Cy dye ratios. We have applied this strategy to profile the plasma proteome for changes that occur with acute graft-versus-host disease (GVHD), following allogeneic bone marrow transplantation (BMT). Using capillary HPLC ESI Q-TOF MS, we identified 75 proteins in the micromolar to femtomolar range that exhibited quantitative differences between the pre- and post-GVHD samples. These proteins included serum amyloid A, apolipoproteins A-I/A-IV, and complement C3 that are well-known acute-phase reactants likely reflecting the post-BMT inflammatory state. In addition, we identified some potentially interesting immunologically relevant molecules including vitamin D-binding protein, fetuin, vitronectin, proline-rich protein 3 and 4, integrin-alpha, and leukocyte antigen CD97. IPAS provides a combination of comprehensive profiling and quantitative analysis, with a substantial dynamic range, for disease-related applications.
Adult↗
Lacrimal gland involvement in graft-versus-host disease: a murine model.
PURPOSE: To describe lacrimal gland involvement in a murine model of acute graft-versus-host disease (GVHD). METHODS: Histopathologic examination was performed on lacrimal glands of mice affected by GVHD at 1, 2, 4, and 6 weeks after allogeneic bone marrow transplantation (BMT). Histopathologic scoring, based on characteristic GVHD findings in human disease involved evaluation of periductal inflammation, apoptosis, ductal stasis, ductal debris, and fibrosis. CD3, CD4, CD8, CD20, and CD68 antibodies were used to stain leukocyte subsets in GVHD lacrimal gland infiltrates. Lacrimal glands from syngeneic BMT mice were used in control experiments. RESULTS: Patchy periductal inflammation and focal fibrosis were significantly elevated as early as 2 weeks after allogeneic BMT. Histopathologic scoring of lacrimal glands after allogeneic BMT was significantly different at 4 (P = 0.005) and 6 (P < 0.0001) weeks when compared with scores in syngeneic control mice. The leukocytes in lacrimal gland GVHD infiltrates were predominately CD3+ T lymphocytes, most of which were CD8+, with fewer CD4+ cells present. CONCLUSIONS: This study describes the first murine model of lacrimal gland GVHD with features that closely mimic those described in human disease and indicates that lacrimal involvement occurs in acute GVHD.
Acute Disease↗
Rare incorporation of bone marrow-derived cells into kidney after folic acid-induced injury.
Results obtained in recent experiments suggest that bone marrow-derived cells (BMDCs) engraft into tissues and differentiate into various somatic cell types. However, it is unclear whether injury is required for the phenomenon to occur at appreciable frequencies. In this study we tested whether BMDCs engraft into kidneys and differentiate into renal cells in the absence or presence of toxic injury. Renal damage was induced by delivery of folic acid (FA) to bone marrow (BM)-recipient mice 1 or 9 months after bone marrow transplant, and kidneys were examined for donor-derived cells 2,4, or 8 weeks after injury. Donor-derived cells were abundant in the renal interstitium of injured kidneys and were detected in glomeruli of vehicle- and FA-treated mice. Most of these cells expressed the common leukocyte antigen CD45 and display morphological characteristics of white blood cells. No donor-derived renal tubule cells (RTCs) were detected in kidney sections of BM-recipient mice. However, in cell culture, a cluster of seven donor-derived cells of 4 x 10(6) RTCs examined (approximately 0.0002%) displayed morphological characteristics of RTCs. CD45+ cells of donor origin were also detected in glomeruli and glomerular outgrowths. Nested polymerase chain reaction analysis for the male-specific Sry gene in cultured RTCs and glomerular outgrowths confirmed the presence of donor-derived cells. These results suggest that BMDCs may incorporate into glomeruli as specialized glomerular mesangial cells; however, BMDCs rarely contribute to the repair of renal tubules in uninjured or FA-treated mouse kidneys.
Acute Kidney Injury↗
Donor T-cell production of RANTES significantly contributes to the development of idiopathic pneumonia syndrome after allogeneic stem cell transplantation.
Idiopathic pneumonia syndrome (IPS) is a major cause of mortality following allogeneic stem cell transplantation (allo-SCT). Clinical and experimental data support a role for conditioning-induced inflammation and alloreactive T-cell responses in IPS pathophysiology, but the mechanisms by which donor leukocytes are ultimately recruited to the lung are not fully understood. RANTES is a chemokine ligand that is up-regulated during inflammation and promotes the recruitment of T cells and macrophages to sites of tissue damage. Using a lethally irradiated murine SCT model (B6 --> B6D2F1), we evaluated the role of donor leukocyte-derived RANTES in the development of IPS. Pulmonary mRNA and protein levels of RANTES were significantly elevated in allo-SCT recipients compared to syngeneic controls and were associated with enhanced mRNA expression of CCR5 and CCR1 and with inflammatory cell infiltration into the lung. Allo-SCT with RANTES-/- donor cells significantly decreased IPS and improved survival. Combinations of allogeneic wild-type or RANTES-/- bone marrow with wild-type or RANTES-/- T cells demonstrated that the expression of RANTES by donor T cells was critical to the development of lung injury after SCT. These data reveal that donor T cells can help regulate leukocyte recruitment to the lung after allo-SCT and provide a possible mechanism through which inflammation engendered by SCT conditioning regimens is linked to allo-specific T-cell responses during the development of IPS.
Animals↗
Both perforin and Fas ligand are required for the regulation of alloreactive CD8+ T cells during acute graft-versus-host disease.
Fas ligand (FasL) and perforin pathways not only are the major mechanisms of T cell-mediated cytotoxicity but also are involved in homeostatic regulation of these T cells. In the present study, we tested whether CD8+ donor T cells that are deficient in both perforin and FasL (cytotoxic double deficient [cdd]) could induce graft-versus-host disease (GVHD) in a major histocompatibility complex class I-mismatched lethally irradiated murine model. Interestingly, recipients of cdd CD8+ T cells demonstrated significantly greater serum levels of interferon gamma and tumor necrosis factor alpha and histopathologic damage from GVHD than wild-type (wt) T cells on day 30 after allogeneic bone marrow transplantation (P<.05). Wt and either perforin-deficient or FasL-deficient CD8+ T cells expanded early after transplantation followed by a contraction phase in which the majority of expanded CD8+ T cells were eliminated. In contrast, cdd CD8+ T cells exhibited prolonged expansion and reduced apoptosis to alloantigen stimulation in vivo and in vitro. Together these results suggest that donor cdd CD8+ T cells expand continuously and cause lethal GVHD, and that both perforin and FasL are required for the contraction of allo-reactive CD8+ T cells.
Acute Disease↗
Donor-derived TNF-alpha regulates pulmonary chemokine expression and the development of idiopathic pneumonia syndrome after allogeneic bone marrow transplantation.
Idiopathic pneumonia syndrome (IPS) is a significant cause of mortality after allogeneic bone marrow transplantation (allo-BMT), and tumor necrosis factor-alpha (TNF-alpha) is a significant effector molecule in this process. However, the relative contribution of donor-versus host-derived TNF-alpha to the development of IPS has not been elucidated. Using a lethally irradiated parent --> F1 mouse IPS model, we showed that 5 weeks after transplantation allo-BMT recipients developed significant lung injury compared with syngeneic controls, which was associated with increased bronchoalveolar lavage (BAL) fluid levels of TNF-alpha, elevated numbers of donor-derived TNF-alpha-secreting T cells, and increased pulmonary macrophage production of TNF-alpha to lipopolysaccharide (LPS) stimulation. Allo-BMT with TNF-alpha(-/-) donor cells resulted in significantly reduced IPS severity, whereas utilization of TNF-alpha-deficient mice as BMT recipients had no effect on IPS. We next determined that TNF-alpha secretion from both donor accessory cells (monocytes/macrophages) and T cells significantly contributed to the development of IPS. Importantly, the absence of donor T-cell-derived TNF-alpha resulted in a significant decrease in inflammatory chemokine production in the lung and near complete abrogation of IPS. Collectively, these data demonstrate that donor TNF-alpha is critical to the development of IPS and reveal a heretofore unknown mechanism for T-cell-derived TNF-alpha in the evolution of this process.
Animals↗
Plagiarism run amuck: why the cat ought not guard the canary.
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Authorship↗
A critical role for CCR2/MCP-1 interactions in the development of idiopathic pneumonia syndrome after allogeneic bone marrow transplantation.
Idiopathic pneumonia syndrome (IPS) is a major complication after allogeneic bone marrow transplantation (allo-BMT) and involves the infiltration of donor leukocytes and the secretion of inflammatory cytokines. We hypothesized that leukocyte recruitment during IPS is dependent in part upon interactions between chemokine receptor 2 (CCR2) and its primary ligand monocyte chemoattractant protein-1 (MCP-1). To test this hypothesis, IPS was induced in a lethally irradiated parent --> F1 mouse BMT model. Compared with syngeneic controls, pulmonary expression of MCP-1 and CCR2 mRNA was significantly increased after allo-BMT. Transplantation of CCR2-deficient (CCR2-/-) donor cells resulted in a significant reduction in IPS severity compared with transplantation of wild-type (CCR2+/+) cells and in reduced bronchoalveolar lavage (BAL) fluid cellularity and BAL fluid levels of tumor necrosis factor-alpha (TNF-alpha) and soluble p55 TNF receptor (sTNFRI). In addition, neutralization of MCP-1 resulted in significantly decreased lung injury compared with control-treated allogeneic recipients. Experimental data correlated with preliminary clinical findings; patients with IPS have elevated levels of MCP-1 in the BAL fluid at the time of diagnosis. Collectively, these data demonstrate that CCR2/MCP-1 interactions significantly contribute to the development of experimental IPS and suggest that interventions blocking these receptor-ligand interactions may represent novel strategies to prevent or treat this lethal complication after allo-BMT.
Animals↗
Plagiarism erodes scientific integrity.
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Education, Graduate↗
Role of CXCR3-induced donor T-cell migration in acute GVHD.
OBJECTIVE: The chemokine receptor CXCR3 has an important role in the migration of effector T cells. To investigate the role of CXCR3 on donor cells in acute graft vs host disease (GVHD) we used a well-defined experimental bone marrow transplantation (BMT) model where acute GVHD is mediated by donor CD8(+) T cells against minor histocompatibility antigens. METHODS; Lethally irradiated C3H.SW recipients were transplanted from either wild-type B6 or CXCR3(-/-) B6 donors. Donor T-cell expansion was analyzed in the spleen and small intestine of recipients by FACS. Donor T-cell function was analyzed by cytokine secretion. The severity of acute GVHD was assessed by histopathological analysis of intestine and liver, GVHD clinical scores, and survival after BMT. RESULTS: Significantly higher numbers of donor CD8(+) CXCR3(-/-) T cells were found in the spleen on days +7 and +14 compared to donor wild-type T cells. By contrast, the number of CD8(+) T cells in the small bowel of BMT recipients from CXCR3(-/-) donors was sevenfold lower than from wild-type donors. Systemic concentrations of INF-gamma and TNF-alpha were equivalent between groups. Animals that received CXCR3(-/-) donor T cells demonstrated diminished GI tract and liver damage and showed improved survival after BMT compared to recipients of wild-type donor cells (43% vs 0%, p<0.001). CONCLUSION: The migration of donor CD8(+) T cells to GVHD target organs such as the intestine depends on the expression of CXCR3 and contributes significantly to GVHD damage and overall mortality.
Acute Disease↗
Repifermin (keratinocyte growth factor-2) reduces the severity of graft-versus-host disease while preserving a graft-versus-leukemia effect.
Graft-versus-host disease (GVHD) is the principal complication after allogeneic bone marrow transplantation (BMT). Reductions in systemic GVHD are frequently associated with a corresponding diminishment of the graft-versus-leukemia (GVL) response. In this study, we tested the effects of a novel recombinant human keratinocyte growth factor, repifermin (keratinocyte growth factor-2), on the induction of GVHD in a well-defined murine BMT model (B6 --> B6D2F1). Administration of repifermin (5 mg/kg/d) to allogeneic BMT recipients resulted in a significant decrease in both systemic GVHD and target organ histopathology. Repifermin treatment also reduced serum levels of tumor necrosis factor alpha and lipopolysaccharide compared with control mice. In contrast, repifermin did not affect T-cell proliferation, cytokine production, or cytotoxic responses to host antigens. When 2000 host-derived P815 (H-2(d)) leukemia cells were added to the bone marrow inoculum, repifermin preserved GVL effects and resulted in significantly delayed mortality compared with control-treated allogeneic BMT recipients. Collectively, these data suggest that repifermin administration may represent a novel strategy to separate the toxicity of GVHD from the beneficial GVL effects after allogeneic BMT.
Animals↗
Graft-versus-host disease in the absence of the spleen after allogeneic bone marrow transplantation.
BACKGROUND: The spleen is considered to be an important secondary lymphoid organ where acute graft-versus-host disease (GVHD) is initiated by donor T cells that recognize host alloantigens after allogeneic bone-marrow transplantation (BMT). The influence of splenectomy on the development of GVHD prior to BMT has yet to be determined. METHODS: The mortality and severity of murine GVHD of unsplenectomized, splenectomized, and sham-operated recipients of allogeneic BMT were compared in a blinded fashion. Serum levels of interferon (IFN)-gamma were measured 7 days after BMT, as an index of systemic donor T-cell responses. RESULTS: Mortality and morbidity of acute GVHD were not significantly affected by splenectomy in a major histocompatibility complex (MHC)-mismatched, CD4-driven murine GVHD model and a minor histocompatibility antigen (MiHA)-mismatched, CD8-driven GVHD model. Serum levels of IFN-gamma also were not different between the groups. CONCLUSION: GVHD can readily develop after allogeneic BMT, even in the absence of the spleen, in these mouse models.
Animals↗