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Using standard techniques during mechanical thrombectomy, the Blood and Clot Thrombectomy Registry and Collaboration (BACTRAC) protocol (NCT03153683) isolates intracranial arterial blood distal to the thrombus and proximal systemic blood in the carotid artery. We augmented the current protocol to study leukocyte subpopulations both distal and proximal to the thrombus during human stroke (n = 16 patients), and from patients with cerebrovascular disease (CVD) undergoing angiography for unrelated conditions (e.g. carotid artery stenosis; n = 12 patients). We isolated leukocytes for flow cytometry from small volume (+ T cells and classical dendritic cells were significantly lower than CVD controls and correlated to within-patient edema volume and last known normal. This novel protocol successfully isolates leukocytes from small volume intracranial blood samples of stroke patients at time of mechanical thrombectomy and can be used to confirm preclinical results, as well as identify novel targets for immunotherapies.

BACKGROUND. Prolyl hydroxylase inhibitors (PHI) promote stabilization of hypoxia-inducible factor-1 alpha and affect signaling cascades of inflammation and cell death. Their beneficial use in experimental models of ulcerative colitis and lung allograft rejection led us to test the effect of the PHI dimethyl oxalyl glycine (DMOG) in the pathophysiology of graft versus host disease (GVHD). METHODS. Acute GVHD was induced in lethally irradiated BALB/c mice. DMOG was administered intraperitoneally on alternate days for the first 2-weeks posttransplant, and then twice a week till day +50, while controls received vehicle only. Animals were monitored for clinical GVHD and analyzed at day +7 and at day +50. RESULTS. DMOG treatment of allogeneic recipients improved survival by day +50, which was associated with decreased early gut injury and serum tumor necrosis factor-α compared with allogeneic controls. DMOG treatment of allogeneic recipients resulted in increased hypoxia-inducible factor-1 alpha expression and reduced apoptosis in the terminal ileum via Fas-associated protein with death domain protein repression along with decreased T-cell infiltration. Reduced pathology in colon after DMOG treatment associates with intestinal epithelium integrity and reduced damage caused by diminished recruitment of neutrophils. CONCLUSIONS. Taken together, we show protective effects of DMOG on early gut GVHD and improved survival in a model of allogeneic hematopoietic cell transplantation, providing the rationale for further evaluation of PHIs, in the prevention and treatment of acute GVHD.

BACKGROUND. Gastrointestinal acute graft-versus-host disease (GVHD) occurring after allogeneic hematopoietic cell transplant is an allo-reactive T cell and inflammatory cytokine driven organ injury with epithelial apoptosis as 1 of its hallmark findings and is associated with significant mortality. Tumor necrosis factor (TNF)-alpha-induced protein 8 (TNFAIP8 or TIPE) acts as a negative mediator of apoptosis via inhibition of caspase-3 activation, promotes cell proliferation and Tipe(−/−) deficiency is associated with increased inflammation. METHODS. To evaluate the role of TIPE in acute GVHD, naive C57BL/6 and Tipe(−/−) C57BL/6 mice were conditioned with 1000 cGy single dose total body irradiation, followed by transplantation of 10 million bone marrow cells and 20 million splenocytes from either syngeneic C57BL/6 or allogeneic BALB/c donors. RESULTS. Allo TIPE-deficient mice developed exacerbated gut GVHD compared with allo controls and had significantly decreased survival (6 wk overall survival: 85% versus 37%; P < 0.05), higher clinical GVHD scores, more profound weight loss, increased serum proinflammatory cytokines (interleukin-17A, TNF, interleukin-6, and interferon-γ). T-cell infiltration into the ileum was increased; epithelial proliferation was decreased along with significantly higher levels of chemokines KC and monokine induced by gamma interferon. Using bone marrow chimeric experiments, TIPE was found to have a role in both hematopoietic and nonhematopoietic cells. CONCLUSIONS. Absence of TIPE results in excessive inflammation and tissue injury after allo-HCT, supporting that TIPE confers immune homeostasis and has tissue-protective function during the development of gut GVHD and may be a potential future target to prevent or treat this complication after allogeneic HCT.

The extracellular signal-related kinase 1 and 2 (ERK1/2) pathway is a highly conserved signaling cascade with numerous essential functions in development. The scaffold protein Shoc2 amplifies the activity of the ERK1/2 pathway and is an essential modulator of a variety of signaling inputs. Germline mutations in Shoc2 are associated with the human developmental disease known as the Noonan-like syndrome with loose anagen hair. Clinical manifestations of this disease include congenital heart defects, developmental delays, distinctive facial abnormalities, reduced growth and cognitive deficits along with hair anomalies. The many molecular details of pathogenesis of the Noonan-like syndrome and related developmental disorders, cumulatively called RASopathies, remain poorly understood. Mouse knockouts for Shoc2 are embryonic lethal, emphasizing the need for additional animal models to study the role of Shoc2 in embryonic development. Here, we characterize a zebrafish shoc2 mutant, and show that Shoc2 is essential for development, and that its loss is detrimental for the development of the neural crest and for hematopoiesis. The zebrafish model of the Noonan-like syndrome described here provides a novel system for the study of structure–function analyses and for genetic screens in a tractable vertebrate system.

Acute undifferentiated leukemia (AUL) is rare and defined by the absence of bona fide myeloid and lymphoid markers. Little is known about its incidence, survival and optimal management in the recent time period. Based on a case observed in our clinic, we queried the Surveillance, Epidemiology, and End Results database between 2000 and 2016. A total of 1,888 cases of AUL were diagnosed (1.34 per million person-years). The incidence of AUL has significantly decreased over time. Compared to other acute leukemias, patients with AUL have the highest median age (74 years); in contrast to acute myeloid leukemia (AML, 65) and acute lymphoblastic leukemia (ALL, 12). Excluding patients with preexisting malignancies, 1,444 patients with AUL were analyzed for survival. Only 35% of AUL patients had received chemotherapy. Comparatively, 94% of ALL and 71% of AML cases received chemotherapy. Among AUL patients who received chemotherapy, the median survival was 12 months as opposed to 1 month in the group who did not receive chemotherapy (or unknown status). Among adults, AUL patients had the worst prognosis, with a median overall survival (OS) of 9 months, compared to 27 months in ALL and 13 months in AML. Among children, the median OS was superior for all three groups of leukemias, the OS of AUL patients being better than in AML and very similar to ALL. On multivariate analysis, older age and time period were associated with worse outcome. We describe here the largest series of cases with AUL published to date.

Allogeneic hematopoietic stem cell transplant (allo-HSCT) is often used to treat acute leukemia or defects of hematopoiesis. Its widespread use is hampered by graft-versus-host disease (GVHD), which has high morbidity and mortality in both acute and chronic subtypes. Chronic GVHD (cGVHD) occurs most frequently in skin and often is characterized by pathogenic fibrosis. Mast cells (MCs) are known to be involved in the pathogenesis of other fibrotic diseases. In a murine model of cGVHD after allo-HSCT, C57BL/6J recipients of allogeneic LP/J donor cells develop sclerodermatous dermal cGVHD which is significantly decreased in mast cell-deficient B6.Cg-KitW-sh/HNihrJaeBsmGlliJ recipients. MCs survive conditioning and are associated with fibrosis, chemokine production, and recruitment of GVHD effector cells to the skin. Chemokine production by MCs is blocked by drugs used to treat cGVHD. The importance of MCs in skin cGVHD is mirrored by increased MCs in the skin of patients with dermal cGVHD. We show for the first time a role for MCs in skin cGVHD that may be targetable for preventive and therapeutic intervention in this disease.

Key Clinical Message IgG4‐RD can also present in the skeletal muscle, mimicking several other diseases. It is unusual for this relatively new classification of diseases to present in the muscles and can be mistakenly diagnosed as other autoimmune diseases rendering a delay in the appropriate management and progression of the disease.

Introduction Graft versus host disease (GVHD) is a major complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT). The pathophysiology of acute GVHD involves donor T cell- and inflammatory cytokine-mediated injury to the patient's tissues and causes significant morbidity and mortality. Defibrotide is a mixture of single stranded nucleotides that has a number of pharmacological effects that contribute to its endothelial protective properties. We hypothesized that preventive treatment with defibrotide will reduce the incidence and severity of GVHD due to its endothelial protective and anti-inflammatory properties and potentially does not alter graft versus leukemia (GVL) responses. Methods Recipient B10.BR mice received Cyclophosphamide 120 mg/kg per day on day-3 and -2, and received lethal total body irradiation (750cGy) on day 0, followed by the infusion of donor C57BL/6 T-cell depleted bone marrow cells. To induce GVHD, respective groups were co-infused with splenocytes, while non-GVHD controls received BM cells only. Mice were treated with defibrotide at the dose of 800mg/kg per body weight or vehicle control for the first week daily and then three times/week thereafter. For GVL experiments, C57BL/6 recipients were lethally irradiated (12Gy) with TBI and transplanted with C3H.SW donor TCD bone marrow alone or in combination with 3 million T-cells along with C1498-luc and treated with defibrotide or vehicle controls. Results Allogeneic defibrotide treated recipients demonstrated significantly better survival when compared to control group (83.34% vs 54.55% on day +28). Clinical GVHD scores were significantly worse after day +14 in control group when compared to defibrotide treated recipients. Both at day +7 and on day +28 allogeneic defibrotide treated recipients showed significantly less organ GVHD in gut, lung and liver (p Conclusion Defibrotide treatment through its anti-inflammatory and endothelium-protective effects reduces the incidence and severity of acute GVHD, while not impairing GVL activity.

Introduction Graft versus host disease (GVHD) leads to significant post-transplant morbidity and mortality after allogeneic hematopoietic cell transplant (allo-HCT). Vedolizumab (VDZ) is a monoclonal antibody targeting the homing of T cells to the intestine through binding of integrin α4β7 of their surface. We hypothesized that interfering with T cell homing to the GI tract results in decreased GVHD severity and improved outcome in an established humanized mouse model of allo HCT. Methods Sublethally irradiated RAG2-/- γc-/- mice were transplanted with 30 million human PBMCs (huPBMCs). Vedolizumab or control IgG was given intraperitoneally at a dose of 4mg/kg body weight on day -1 and day +15 (preventive approach) or day +8 and day +22 (therapeutic approach). Time points were chosen in order to start α4β7 blockade prior to clinical onset of GVHD (preventive approach) and when animals started to get significantly suffer from GVHD (therapeutic approach). As non-GVHD controls, animals received sublethal irradiation without huPBMCs administration and received VDZ or control IgG to assess for direct conditioning and VDZ toxicity. Results HuPBMC CD45 splenocyte analysis on day +28 after infusion demonstrated strong human leukocyte engraftment with more than 80% of spleen cells being huCD45 positive. Both preventive and therapeutic administration of Vedolizumab resulted in significantly improved survival in HuPBMC treated recipients when compared to IgG treated GVHD controls at week 8 (62.6% Vs 12.5% and 50% Vs 12.5% respectively). All recipients, which did not receive huPBMCs survived, indicating VDZ safety in this model. Clinical GVHD scores after day 21 after transplant were significantly lower in VDZ huPBMCs treated animals until end of analysis compared to control-treated recipients using both the preventive and therapeutic approaches, along with significant decreases in GI tract pathology scores on day +28. No differences in pathology were seen in liver or lung at this time point. Serum cytokine analysis revealed decreases in TNF (p Conclusion Vedolizumab reduces the severity of intestinal GVHD by targeting donor T cell migration into the intestinal tract both when given early prior to onset of clinical disease and given at later time point, and improves overall survival both in the preventive and therapeutic setting. Given these promising results, targeting T cell homing with VDZ potentially presents a novel option for the management of intestinal GVHD.

7050 Background: Allogeneic hematopoietic stem cell transplantation (allo-HCT) is a potentially curative treatment for patients with malignant neoplasms or inborn defects of hematopoiesis. Benefits of allo-HSCT are hampered by graft-versus-host-disease (GVHD), which can be debilitating and potentially lethal. In chronic GVHD (cGVHD), inflammation and aberrant wound healing lead to pathological fibrosis across multiple organs, most frequently in the skin, yet the exact pathophysiology is not well-understood. Mast cells (MCs) are primarily known for their role in atopic disease. However, recent studies have demonstrated new roles for MCs, showing that they can be involved in wound healing and in the pathogenesis of fibrotic disease. Given these new paradigms and the MC tropism to skin, alongside their reported role in other fibrotic diseases, we investigated whether MCs may play a role in the pathogenesis of dermal cGVHD. Methods: Cells: MCs were grown ex vivo from murine bone marrow. Transplant: 8 Gy radiation, followed by injection of LP/J marrow and splenocytes into C57BL/6J (WT) or B6.Cg-KitW-sh MC-deficient recipients. Results: Ex vivo, we show that MCs survive and are functional after lethal radiation, such as that used in conditioning prior to HCT. In a murine model of cGVHD WT mice had significantly more cGVHD symptoms than MC-deficient mice as measured by clinical scoring. This scoring correlated with a significant increase in skin pathology, collagen deposition, and expression of pro-fibrotic genes in WT as compared to B6.Cg-KitW-sh mice. Dermal MC numbers were increased in WT mice, but were nearly undetectable in B6.Cg-KitW-sh mice, implying that the MCs that are present were recipient-derived and had survived conditioning. Skin from WT but not B6.Cg-KitW-sh mice was enriched in cGVHD effector cells and in inflammatory cytokines and chemokines. Murine MCs, upon stimulation were sources of many of these factors, production of which was blocked when treated with ibrutinib and ruxolitinib, drugs used in cGVHD treatment. Conclusions: In summary, we show here a previously unknown role for MCs in the pathogenesis of dermal cGVHD, suggesting that MCs may be targetable to prevent and treat this devastating complication of allo HCT.

The epidermis increases pigmentation and epidermal thickness in response to ultraviolet exposure to protect against UV-associated carcinogenesis; however, the contribution of epidermal thickness has been debated. In a humanized skin mouse model that maintains interfollicular epidermal melanocytes, we found that forskolin, a small molecule that directly activates adenylyl cyclase and promotes cAMP generation, up-regulated epidermal eumelanin accumulation in fair-skinned melanocortin-1-receptor (Mc1r)-defective animals. Forskolin-induced pigmentation was associated with a reproducible expansion of epidermal thickness irrespective of melanization or the presence of epidermal melanocytes. Rather, forskolin-enhanced epidermal thickening was mediated through increased keratinocyte proliferation, indirectly through secreted factor(s) from cutaneous fibroblasts. We identified keratinocyte growth factor (Kgf) as a forskolin-induced fibroblast-derived cytokine that promoted keratinocyte proliferation, as forskolin induced Kgf expression both in the skin and in primary fibroblasts. Lastly, we found that even in the absence of pigmentation, forskolin-induced epidermal thickening significantly diminished the amount of UV-A and UV-B that passed through whole skin and reduced the amount of UV-B-associated epidermal sunburn cells. These findings suggest the possibility of pharmacologic-induced epidermal thickening as a novel UV-protective therapeutic intervention, particularly for individuals with defects in pigmentation and adaptive melanization.

A case of lymphoma of T-cell derivation with aberrant expression of three B-cell lineage markers (CD19, CD20, and CD79a), which was diagnosed on a left axillary excision, is described. Immunohistochemical studies and flow cytometry analysis demonstrated neoplastic cells expressing CD3, CD19, CD20, and CD79a with absence of CD4, CD8, CD10, CD30, CD34, CD56, CD68, TDT, MPO, PAX-5, and surface immunoglobulin. Gene rearrangement studies performed on paraffin blocks demonstrated monoclonal T-cell receptor gamma chain rearrangement with no evidence of clonal heavy chain rearrangement. The neoplastic cells were negative for Epstein-Barr virus (EBV) or Human Herpes Virus 8 (HHV-8). At the time of diagnosis, the PET scan demonstrated hypermetabolic neoplastic cells involving the left axilla, bilateral internal jugular areas, mediastinum, right hilum, bilateral lungs, and spleen. However, bone marrow biopsy performed for hemolytic anemia revealed normocellular bone marrow with trilineage maturation. The patient had no evidence of immunodeficiency or infection with EBV or HHV-8. This is the first reported case of a mature T-cell lymphoma with aberrant expression of three B-cell lineage markers. The current report also highlights the need for molecular gene rearrangement studies to determine the precise lineage of ambiguous neoplastic clones.

Feminist Audre Lorde states, "The master's tools will never dismantle the master's house." In this paper, however, we encourage women to seize computer toolsfor their own educational purposes in order to counteract the "null environment" they experience in the traditional classroom. We argue for a proactive feminist pedagogy that enhances women's computer learning by demystifying the computer and promoting technological equity in the classroom. To accomplish these goals, we introduce a quantitative and a qualitative software program which help to create a proactive environment that enhances women's computerized learning when used in conjunction with feminist pedagogical techniques. We also discuss the implications of introducing feminist proactive computer learning into the sociology curriculum and, more specifically, into the women's studies classroom.

The federal government is the nation’s largest employer, with nearly 3 million civilian employees. Almost 81 percent of these workers are in white-collar jobs; of these, women accounted for 47.6 percent of the federal white-collar workers in 1986. Over one and a half million women work for the federal government in white-collar jobs. The question arises: How well does the government, as a major employer, meet its affirmative action obligations?