Clinical and mutation profile of multicentric osteolysis nodulosis and arthropathy

​Multicentric osteolysis nodulosis and arthropathy (MONA) is an infrequently described autosomal recessive skeletal dysplasia characterized by progressive osteolysis and arthropathy. Inactivating mutations in MMP2, encoding matrix metalloproteinase‐2, are known to cause this disorder. Fifteen families with mutations in MMP2 have been reported in literature. In this study we screened thirteen individuals from eleven families for MMP2 mutations and identified eight mutations (five novel and three known variants). We characterize the clinical, radiographic and molecular findings in all individuals with molecularly proven MONA from the present cohort and previous reports, and provide a comprehensive review of the MMP2 related disorders. © 2015 Wiley Periodicals, Inc.

     

Assessing Documentation of Critical Imaging Result Follow-up Recommendations in Emergency Department Discharge Instructions

To facilitate follow-up of critical test results across transitions in patient care settings, we implemented an electronic discharge module that enabled care providers to include follow-up recommendations in the discharge instructions. We assessed the impact of this module on documentation of follow-up recommendations for critical imaging findings in Emergency Department (ED) discharge instructions. We studied 240 patients with critical imaging findings discharged from the ED before (n?=?80) and after (n?=?160) implementation of the module. We manually reviewed hand-written forms and electronic discharge instructions to determine if follow-up recommendations were documented. Follow-up recommendations in ED discharge instructions increased from 60.0% (48/80) to 73.8% (118/160) post-module implementation (p?=?0.03), a relative increase of 23%. There was no significant change in the rate of documented critical imaging findings in the discharge instructions (77.5% [62/80] before the intervention and 76.9% [123/160] after the intervention; p?=?0.91). Implementation of a discharge module was associated with increased documentation of critical imaging finding follow-up recommendations in ED discharge instructions. However, one in four patients still did not receive adequate follow-up recommendations, suggesting further opportunities for performance improvement exist.     

Immune Profiling of T Cells Infiltrating Vitreous Humor in Tubercular Uveitis

Purpose: To assess cellular composition and local cytokine response in vitreous humor of tubercular uveitis.

Methods: Cells were collected from vitreous cassettes and peripheral blood of 8 tubercular uveitis and 5 control subjects, undergoing vitrectomy and analyzed by flow cytometry for cellular composition, activation status, proinflammatory cytokine expression, and uptake of TLR9 ligand, CpG ODN 2216.

Results: CD3 + T cells with equal proportion of CD4+ and CD8 + T cells formed major fraction of infiltrating cells. The vitreous humor showed higher expression of recent activation marker, CD69, and proinflammatory cytokines, IFN-γ and IL-17A, in CD4 + T cells as compared to peripheral blood. Lastly, intraocular CD4 + T cells showed reduced uptake of ODN 2216 than peripheral blood.

Conclusions: Our results indicate that local antigenic stimuli trigger T cell infiltration and activation of CD4 + T cells that are hyporesponsive to TLR9 stimulation. These infiltrating T cells might be responsible in further aggravating ocular inflammation.     

Role of granulosa cell mitogen-activated protein kinase 3/1 in gonadotropin-mediated meiotic resumption from diplotene arrest of mammalian oocytes

In mammals, preovulatory oocytes are encircled by several layers of granulosa cells (GCs) in follicular microenvironment. These follicular oocytes are arrested at diplotene arrest due to high level of cyclic nucleotides from encircling GCs. Pituitary gonadotropin acts at the level of encircling GCs and increases adenosine 3′,5′-cyclic monophosphate (cAMP) and guanosine 3′,5′-cyclic monophosphate (cGMP) and activates mitogen-activated protein kinase 3/1 (MAPK3/1) signaling pathway. The MAPK3/1 disrupts the gap junctions between encircling GCs and oocyte. The disruption of gap junctions interrupts the transfer of cyclic nucleotides to the oocyte that results a drop in intraoocyte cAMP level. A transient decrease in oocyte cAMP level triggers maturation promoting factor (MPF) destabilization. The destabilized MPF finally triggers meiotic resumption from diplotene arrest in follicular oocyte. Thus, MAPK3/1 from GCs origin plays important role in gonadotropin-mediated meiotic resumption from diplotene arrest in follicular oocyte of mammals.     

Are alterations of tight junctions at molecular and ultrastructural level different in duodenal biopsies of patients with celiac disease and Crohn's disease?

Abnormalities of transmembrane and cytoplasmic proteins of tight junctions (TJ) have been implicated in pathogenesis of both celiac (CeD) and Crohn’s diseases (CD). Since disease pathogenesis in CeD and CD are different, we planned to study if there is any differential expression pattern of TJ marker proteins and ultrastructural changes, respectively, in duodenal villi vs crypts. Endoscopic duodenal biopsies from treatment naïve patients with CeD (n?=?24), active CD (n?=?28), and functional dyspepsia (as controls, n?=?15), both at baseline and 6 months after treatment, were subjected to light microscopic analysis (modified Marsh grading); immune-histochemical staining and Western blot analysis to see the expression of key TJ proteins [trans-membrane proteins (claudin-2, claudin-3, claudin-4, occludin, and JAM) and cytoplasmic protein (ZO-1)]. Transmission electron microscopy and image analysis of the TJs were also performed. There was significant overexpression of claudin-2 (pore-forming) and occludin (protein maintaining cell polarity) with under-expression of claudin-3 and claudin-4 (pore-sealing proteins) in treatment naïve CeD and active CD with simultaneous alteration in ultrastructure of TJs such as loss of penta-laminar structure and TJ dilatation. Normalization of some of these TJ proteins was noted 6 months after treatment. These changes were not disease specific and were not different in duodenal villi and crypts. Overexpression of pore-forming and under-expression of pore-sealing TJ proteins lead to dilatation of TJ. These changes are neither disease specific nor site specific and the end result of mucosal inflammation.     

Characterization of a robust serine protease from Bacillus subtilis K‐1

Process apt microbial proteases due to their wide range industrial applications have become the focus of intense scientific research during recent years. Considering the hostile process milieu, the proteases intended for application must be robust enough to withstand the extremes of temperature and pH, and presence of organic solvents and other potential enzyme inhibitors. Current study presents the characterization of a robust protease from a previously isolated bacterium Bacillus subtilis K‐1 (BSK‐1). Purification of BSK‐1 protease (5.21‐fold) was achieved to homogeneity by salt (ammonium sulfate) precipitation, and ion‐exchange (diethyl‐aminoethyl‐sephadex) and size exclusion chromatography (Sephadex G‐100). Molecular weight of BSK‐1 protease was determined by SDS‐PAGE analysis (42 kDa). Though the optimum temperature and pH for BSK‐1 protease activity was 50 °C and 10, respectively, but, the protease exhibited remarkable activity and stability over elevated temperatures (60–80 °C) and a broad pH range (pH 7–11). Protease showed resistance towards several organic solvents/other potential enzyme inhibitors. Drastic activity loss in presence of phenylmethylsulfonyl fluoride indicated that the enzyme is a serine protease. Kinetic parameters (K_m and V_max) for BSK‐1 protease were found to be 0.14 mg ml^?1 and 1176 mg min^?1, respectively. Putative amino acid sequence of BSK‐1 protease (derived from nucleotide sequence of protease gene) suggested that the enzyme belonged to peptidases S8/S53 super family with multidomain of S8. BSK‐1 protease being stable under harsh conditions may serve a model system for understanding the molecular basis of stability, and may help designing novel proteases that are suitable for industrial applications.     

Outcomes of Unrelated Donor Stem Cell Transplantion with Post-Transplant Cyclophosphamide for Graft-versus-Host Disease Prophylaxis in Patients with Severe Sickle Cell Disease

Unrelated donor (URD) hematopoietic cell transplantation (HCT) in children with sickle cell disease (SCD) is associated with a high incidence of rejection and graft-versus-host disease (GVHD). We report on the first 4 patients with severe SCD who underwent URD HCT using a novel myeloablative and immunosuppressive regimen composed of busulfan, fludarabine, and antithymocyte globulin with a single dose of post-transplant cyclophosphamide along with tacrolimus and mycophenolate mofetil for GVHD prophylaxis. Three patients engrafted and remain disease-free after a median follow-up period of 2.5 years. One patient had primary graft failure attributed to low stem cell content of the graft. Of interest, none of the engrafted patients developed acute or chronic GVHD. This preparative regimen along with the use of post-transplant cyclophosphamide offers a promising approach for unrelated donor transplants in patients with SCD and needs further corroboration in larger number of patients.     

Advance Directive Utilization Is Associated with Less Aggressive End-of-Life Care in Patients Undergoing Allogeneic Hematopoietic Cell Transplantation

Allogeneic hematopoietic cell transplantation (HCT) is associated with significant morbidity and mortality, making advance care planning (ACP) and management especially important in this patient population. A paucity of data exists on the utilization of ACP among allogeneic HCT recipients and the relationship between ACP and intensity of healthcare utilization in these patients. We performed a retrospective review of patients receiving allogeneic HCT at our institution from 2008 to 2015 who had subsequently died after HCT. Documentation and timing of advance directive (AD) completion were abstracted from the electronic medical record. Outcomes of interest included use of intensive care unit (ICU) level of care at any time point after HCT, within 30 days of death, and within 14 days of death; use of mechanical ventilation at any time after HCT; and location of death. Univariate logistic regression was performed to explore associations between AD completion and each outcome. Of the 1031 patients who received allogeneic HCT during the study period, 422 decedents (41%) were included in the analysis. Forty-four percent had AD documentation prior to death. Most patients (69%) indicated that if terminally ill, they did not wish to be subjected to life-prolonging treatment attempts. Race/ethnicity was significantly associated with AD documentation, with non-Hispanic white patients documenting ADs more frequently (51%) compared with Hispanic (22%) or Asian patients (35%; P?=?.0007). Patients with ADs were less likely to use the ICU during the transplant course (41% for patients with ADs versus 52% of patients without ADs; P?=?.03) and also were less likely to receive mechanical ventilation at any point after transplantation (21% versus 37%, P?<?.001). AD documentation was also associated with decreased ICU use at the end of life; relative to patients without ADs, patients with ADs were more likely to die at home or in hospital as opposed to in the ICU (odds ratio, .44; 95% confidence interval, .27 to .72). ACP remains underused in allogeneic HCT. Adoption of a systematic practice to standardize AD documentation as part of allogeneic HCT planning has the potential to significantly reduce ICU use and mechanical ventilation while improving quality of care at end of life in HCT recipients.     

Argininosuccinate Synthetase-1 (ASS1) Loss in High-Grade Neuroendocrine Carcinomas of the Urinary Bladder: Implications for Targeted Therapy with ADI-PEG 20

High-grade neuroendocrine carcinomas (HGNECs) of the urinary bladder encompass small cell (SCNEC) and large cell neuroendocrine carcinomas (LCNEC). Currently, recommended initial management is with systemic chemotherapy, followed by consolidative therapy with either radical cystectomy or radiotherapy in patients with localized disease. Nevertheless, survival in this setting remains poor. We therefore evaluated the potential to modify arginine metabolism as an alternative, targeted therapy approach in these carcinomas. In humans, arginine is a semi-essential amino acid and its synthesis enzyme argininosuccinate synthetase (ASS1) represents the rate-limiting step in arginine biosynthesis. Neoplasms that show low to absent ASS1 expression require extracellular arginine for cancer cell survival, and thus can be targeted using arginine-degrading enzymes such as pegylated arginine deiminase (ADI-PEG 20). An initial study by our group of 19 patients demonstrated that a high percentage of SCNEC lack ASS1 expression. Herein, we evaluated an expanded cohort of 74 radical cystectomy patients with HGNEC, including 63 SCNEC, 5 LCNEC, and 6 mixed morphology HGNEC patients. ASS1 expression was assessed through immunohistochemistry. Fifty-eight (of 74, 78%) patients with HGNEC showed absent ASS1 expression, including all patients with LCNEC and mixed morphology (11 of 11, 100%). Ten-year survival from disease-specific death was not statistically significant between ASS1-expressing and ASS1-deficient cases (p?=?0.75). Our results show that HGNEC of the bladder may be candidates for arginine deprivation therapy using drugs such as ADI-PEG 20. Further studies are needed to validate these findings and to determine the therapeutic efficacy of such agents.     

Influence of the stiffness of three-dimensional alginate/collagen-I interpenetrating networks on fibroblast biology

Wound dressing biomaterials are increasingly being designed to incorporate bioactive molecules to promote healing, but the impact of matrix mechanical properties on the biology of resident cells orchestrating skin repair and regeneration remains to be fully understood. This study investigated whether tuning the stiffness of a model wound dressing biomaterial could control the behavior of dermal fibroblasts. Fully interpenetrating networks (IPNs) of collagen-I and alginate were fabricated to enable gel stiffness to be tuned independently of gel architecture, polymer concentration or adhesion ligand density. Three-dimensional cultures of dermal fibroblasts encapsulated within matrices of different stiffness were shown to promote dramatically different cell morphologies, and enhanced stiffness resulted in upregulation of key-mediators of inflammation such as IL-10 and COX-2. These findings suggest that simply modulating the matrix mechanical properties of a given wound dressing biomaterial deposited at the wound site could regulate the progression of wound healing.