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We previously demonstrated that the non-calcemic pregnacalciferol (pD) analog 17,20S (OH)2pD suppressed TGF-β1-induced type I collagen production in cultured normal human dermal fibroblasts. In the present studies, we examined fibroblasts cultured from the lesional skin of patients with systemic sclerosis (scleroderma (SSc)) and assessed the effects of 17,20S(OH)2pD on fibrosis-related mediators. Dermal fibroblast lines were established from skin biopsies from patients with SSc and healthy controls. Fibroblasts were cultured with either 17,20S(OH)2pD or 1,25(OH)2D3 (positive control) with/without TGF-β1 stimulation and extracted for protein and/or mRNA for collagen synthesis and mediators of fibrosis (MMP-1, TIMP-1, PAI-1, BMP-7, PGES, GLI1, and GLI2). 1 7,20S(OH)2pD (similar to 1,25(OH)2D3) significantly suppressed net total collagen production in TGF-β1-stimulated normal donor fibroblast cultures and in cultures of SSc dermal fibroblasts. 17,20S(OH)2pD (similar to 1,25(OH)2D3) also increased MMP-1, BMP-7, and PGES and decreased TIMP-1 and PAI1 expression in SSc fibroblasts. Although 17,20S(OH)2pD had no effect on Gli1 or Gli2 in SSc fibroblasts, it increased Gli2 expression when cultured with TGF-β1 in normal fibroblasts. These studies demonstrated that 17,20S(OH)2pD modulates mediators of fibrosis to favor the reduction of fibrosis and may offer new noncalcemic secosteroidal therapeutic approaches for treating SSc and fibrosis.

We previously demonstrated that the non-calcemic pregnacalciferol (pD) analog 17,20S (OH)2pD suppressed TGF-β1-induced type I collagen production in cultured normal human dermal fibroblasts. In the present studies, we examined fibroblasts cultured from the lesional skin of patients with systemic sclerosis (scleroderma (SSc)) and assessed the effects of 17,20S(OH)2pD on fibrosis-related mediators. Dermal fibroblast lines were established from skin biopsies from patients with SSc and healthy controls. Fibroblasts were cultured with either 17,20S(OH)2pD or 1,25(OH)2D3 (positive control) with/without TGF-β1 stimulation and extracted for protein and/or mRNA for collagen synthesis and mediators of fibrosis (MMP-1, TIMP-1, PAI-1, BMP-7, PGES, GLI1, and GLI2). 1 7,20S(OH)2pD (similar to 1,25(OH)2D3) significantly suppressed net total collagen production in TGF-β1-stimulated normal donor fibroblast cultures and in cultures of SSc dermal fibroblasts. 17,20S(OH)2pD (similar to 1,25(OH)2D3) also increased MMP-1, BMP-7, and PGES and decreased TIMP-1 and PAI1 expression in SSc fibroblasts. Although 17,20S(OH)2pD had no effect on Gli1 or Gli2 in SSc fibroblasts, it increased Gli2 expression when cultured with TGF-β1 in normal fibroblasts. These studies demonstrated that 17,20S(OH)2pD modulates mediators of fibrosis to favor the reduction of fibrosis and may offer new noncalcemic secosteroidal therapeutic approaches for treating SSc and fibrosis.

Systemic sclerosis (SSc, scleroderma) is a chronic fibrotic disease involving TGF-β1. Low serum vitamin D (vit D) correlates with the degree of fibrosis and expression of TGF-β1. This study was designed to determine whether the noncalcemic vit D analog, 17,20S(OH)2pD, suppresses fibrosis and mediators of the TGF-β1 pathway in the bleomycin (BLM) model of fibrosis. Fibrosis was induced into the skin of female C57BL/6 mice by repeated injections of BLM (50 μg/100 μL) subcutaneously. Mice received daily oral gavage with either vehicle (propylene glycol) or 17,20S(OH)2pD using 5, 15, or 30 μg/kg for 21 days. The injected skin was biopsied, analyzed histologically, examined for total collagen by Sircol, and examined for mRNA expression of MMP-13, BMP-7, MCP-1, Gli1, and Gli2 by TR-PCR. Spleen was analyzed for lymphocytes using flow cytometry. Serum was analyzed for cytokines using a multiplexed ELISA. Results showed that all three doses of 17,20S(OH)2pD suppressed net total collagen production, dermal thickness, and total collagen content in the BLM fibrosis model. 17,20S(OH)2pD also increased MMP-13 expression, decreased MCP-1 and Gli-2 expression in vivo, and suppressed serum levels of IL-13, TNF-α, IL-6, IL-10, IL-17, and IL-12p70. In summary, 17,20S(OH)2pD modulates the mediators of fibrosis in vivo and suppresses total collagen production and dermal thickness. This antifibrotic property of 17,20S(OH)2pD offers new therapeutic approaches for fibrotic disorders.

Purpose Cryopreservation of sperm from human semen has been available since the 1950s. The actual utilization of available cryopreservation technology has been infrequently reported. We set out to examine the utilization and outcomes of cryopreserved sperm cells based on the indication for storage. Materials and methods A dataset was developed from retrospective review. The purposes for cryopreservation, eventual utilization, and outcomes of use for insemination were recorded. The types of utilization were accumulated as proportions for different purposes. The timing for use of samples for insemination procedures was evaluated using survival statistics. The frequency of patients arranging to destroy samples was also reported. Results From September 1988 through March 2015, 1442 samples were cryopreserved. Samples were cryopreserved for four primary purposes: infertility treatments focused on intrauterine insemination (IUI) or in vitro fertilization/intracellular injection (IVF/ICSI), for fertility preservation related to cancer treatment, or prior to military deployment. Total utilization rates were 19.3%. Samples cryopreserved for IUI were more likely to be used (64.3%), while samples cryopreserved as backup for IVF/ICSI were more likely to be destroyed (29.8%). Pregnancy rates varied based on the indication and ART used. Pregnancies per cycle were 35% for IVF/ICSI and were 10% for IUI. Conclusions Cryopreservation of sperm is a valuable and underutilized resource, particularly amongst male cancer patients. This technology can facilitate infertility treatments based on a variety of indications, including deployment-a patient cohort unique to our dataset.

Semen from asymptomatic men who are being evaluated as male partners in interfile couples have been reported to contain a variety of bacteria. Longitudinal studies of the variation of these bacteria over time and their resistance patterns have not been commonly reported. At our institution, residues from semen samples are routinely evaluated for bacteria, including antibiotic sensitivity profiles. We set out to profile the changes in semen bacteria and antibiotic resistance at our institution over time. A total of 72 semen isolates were examined for type of bacteria and sensitivity to a panel of antibiotics. The results were divided into two separate 5-year intervals (the first beginning in 2006, the second in 2011) and compared. The majority of bacteria were skin flora, with Streptococcus and Staphylococcus being the most prevalent. The resistance data for these two pathogens showed minimal statistically significant difference between the two time periods, although the Staphylococcus species did show a trend toward increasing resistance, suggesting that antibiotics currently used in sperm cell preparations may need to be varied.

Objective To determine whether an intraarticular injection of the neutrophil chemorepellent dipeptidyl peptidase IV (DPPIV; CD26) can attenuate inflammation and decrease the severity of arthritis in a murine model. Methods DBA/1 mice were immunized with type II collagen/Freund's complete adjuvant to produce collagen-induced arthritis (CIA). On day 25 postimmunization, recombinant human DPPIV (rhDPPIV) or phosphate buffered saline was injected intraarticularly, and arthritis severity scores were recorded 3 times per week. The hind legs of mice in both groups were fixed, decalcified, paraffin embedded, and sectioned. Pathologic scores for inflammation and neutrophil infiltration were recorded on a scale of 1–8, and the number of neutrophils was determined by morphometric cell counts. In addition, Mac-2–positive macrophages and articular damage were assessed using anti–Mac-2 antibodies and histologic staining, respectively. Results Injection of rhDPPIV reduced the mean score of arthritis severity in mice with CIA. DPPIV treatment reduced the overall extent of inflammation and articular damage around the arthritic joint and periarticular tissue, and also decreased neutrophil and macrophage infiltration. Conclusion A localized injection of the neutrophil chemorepellent DPPIV reduces inflammation and the severity of the disease in a murine model of arthritis.

A 14-year-old healthy male adolescent presented to the emergency department with a 1-month history of vomiting and was found to have severe hypertension secondary to renal failure. After a prolonged hospitalization and extensive workup his ultimate diagnosis was revealed. This case highlights the etiology and workup of hypertension as well as differential of hypertension and renal failure in the adolescent population.

A 9-year-old African American boy presented with chronic urticaria and progressive spondyloarthritis. Laboratory tests were abnormal for persistently positive antinuclear antibodies and undetectable total hemolytic complement (CH50) despite normal levels of complement C2, C3, and C4. Ultimately, further testing revealed an underlying deficiency in the immune system that may be associated with autoimmune disease and thus have a bearing on the patient's urticaria and spondyloarthritis. This is a unique presentation of a rare disease and underscores the importance of evaluating for systemic disease in the workup of chronic urticaria.

Diffuse alveolar hemorrhage in a pediatric patient requires urgent and aggressive therapy. Here we report a young child with Wiskott-Aldrich syndrome and antiplatelet antibody manifesting as recurrent pulmonary hemorrhage due to pauci-immune capillaritis that was successfully treated with rituximab.

To determine whether an intraarticular injection of the neutrophil chemorepellent dipeptidyl peptidase IV (DPPIV; CD26) can attenuate inflammation and decrease the severity of arthritis in a murine model.DBA/1 mice were immunized with type II collagen/Freund's complete adjuvant to produce collagen-induced arthritis (CIA). On day 25 postimmunization, recombinant human DPPIV (rhDPPIV) or phosphate buffered saline was injected intraarticularly, and arthritis severity scores were recorded 3 times per week. The hind legs of mice in both groups were fixed, decalcified, paraffin embedded, and sectioned. Pathologic scores for inflammation and neutrophil infiltration were recorded on a scale of 1-8, and the number of neutrophils was determined by morphometric cell counts. In addition, Mac-2-positive macrophages and articular damage were assessed using anti-Mac-2 antibodies and histologic staining, respectively.Injection of rhDPPIV reduced the mean score of arthritis severity in mice with CIA. DPPIV treatment reduced the overall extent of inflammation and articular damage around the arthritic joint and periarticular tissue, and also decreased neutrophil and macrophage infiltration.A localized injection of the neutrophil chemorepellent DPPIV reduces inflammation and the severity of the disease in a murine model of arthritis.

Systemic sclerosis (SSc) is an acquired multiorgan connective tissue disease with variable mortality and morbidity dictated by clinical subset type. The etiology of the basic disease and pathogenesis of the systemic autoimmunity, fibrosis, and fibroproliferative vasculopathy are unknown and debated. In this review, the spectrum of vascular abnormalities and the options currently available to treat the vascular manifestations of SSc are discussed. Also discussed is how the hallmark pathologies (ie, how autoimmunity, vasculopathy, and fibrosis of the disease) might be effected and interconnected with modulatory input from lysophospholipids, sphingosine 1-phosphate, and lysophosphatidic acid.

Introduction We used DR1 transgenic mice and covalently linked DR1 multimers to characterize analog-specific inhibitory T cells in collagen-induced arthritis (CIA). Because of the low numbers of antigen-specific T cells in wild-type mice, functional T-cell studies in autoimmune arthritis have been challenging. The use of T-cell receptor (TCR) transgenic mice has provided useful information, but such T cells may not represent the heterogeneous T-cell response that occurs in natural settings. Our focus was to develop tools to identify and characterize the population of immunoregulatory T cells induced in wild-type mice by an analog peptide of CII259-273, which contains amino acid substitutions at positions 263 (N) and 266 (D) (analog peptide A12). Methods DR1 multimers, developed by loading empty class II molecules with exogenous peptide, provide a method for visualizing antigen-specific T cells with flow cytometry. However, the low binding avidity of A12 for the major histocompatibility complex (MHC) made this strategy untenable. To overcome this problem, we generated DR1 multimers in which the analog peptide A12 was covalently linked, hoping that the low-avidity analog would occupy enough binding clefts to allow detection of the responsive T cells. Results Staining with the tetramer revealed that A12-specific T cells were readily detectable at 10 days after immunization. These CD4(+) T cells are a highly selective subset of the TCR repertoire and have a limited clonality. Analysis of cytokine expression showed that cells detected by tetramer (A12) expressed primarily suppressive cytokines (interleukin-4 (IL-4) and IL-10) in response to collagen, compared with control cells. Although they did not express Fox-p3, they were extremely effective in preventing and suppressing inflammatory arthritis. Conclusions In summary, our studies showed that the use of covalently linked multimers allows characterization of analog-specific T cells that are otherwise difficult to detect. The suppressive character of the analog-specific T-cell response suggests that these cells attenuate autoimmunity and differ significantly in phenotype from the inflammatory T cells predominantly found in arthritic joints. Such reagents will become powerful tools to study T-cell responses in RA patients in upcoming clinical trials.