The percentage of lymphocytic subsets in the blood of cases with neuralgic amyotrophy (NA), and the proliferative response of blood lymphocytes cultured with different nerve extracts, obtained from normal subjects at postmortem, were examined in 6 patients with NA and in 18 age-matched controls with shoulder pain not related to NA. Most (5/6) NA patients had decreased CD3 values and increased CD4/CD8 ratios due to a decreased of the CD8 subset. Lymphocytes of NA patients increased their blastogenic activity in cultures with nerve extracts from different brachial plexus nerves and its branches, but not in cultures with extracts of sacral plexus nerves. Cultures did not respond to nerve extracts in any of the control cases, although mitogenic activity was similarly elicited in cultured lymphocytes stimulated with phytohemagglutinin in both control cases and NA patients. These results suggest that NA is probably an immune mediated disease.
In the current study we evaluated the effects of immunoproteasome inhibition using ONX 0914 (formerly PR-957) to ameliorate graft-versus-host disease (GVHD). ONX 0914, an LMP7-selective epoxyketone inhibitor of the immunoproteasome, has been shown to reduce cytokine production in activated monocytes and T cells and attenuate disease progression in mouse models of rheumatoid arthritis, colitis, systemic lupus erythematosus, and, more recently, encephalomyelitis. Inhibition of LMP7 with ONX 0914 in the B10.BR→CBA MHC-matched/minor histocompatibility antigen (miHA)-disparate murine blood and marrow transplant (BMT) model caused a modest but significant improvement in the survival of mice experiencing GVHD. Concomitant with these results, in vitro mixed lymphocyte cultures revealed that stimulator splenocytes, but not responder T cells, treated with ONX 0914 resulted in decreased IFN-γ production by allogeneic T cells in both MHC-disparate (B10.BR anti-B6) and miHA-mismatched (B10.BR anti-CBA) settings. In addition, a reduction in the expression of the MHC class I-restricted SIINFEKL peptide was observed in splenocytes from transgenic C57BL/6-Tg(CAG-OVA)916Jen/J mice exposed to ONX 0914. Taken together, these data support that LMP7 inhibition in the context of BMT modulates allogeneic responses by decreasing endogenous miHA presentation and that the consequential reduction in allogeneic stimulation and cytokine production reduces GVHD development.
Graft-versus-host disease (GVHD) is the primary complication of blood and marrow transplantion used to treat hematological diseases. Epithelial cells are apoptotic targets during GVHD. Gut epithelium is a key factor in the progression from localized to systemic GVHD, as damage to the intestines allows endotoxin transfer into general circulation, triggering cytokine release, organ damage and systemic inflammation. However, cellular mechanisms that regulate tissue damage in GVHD are poorly understood. Using the established MHC-matched, miHA-mismatched B6→BALB.B irradiation model of GVHD, we generated a novel primary cell culture system for in vitro study of gut epithelial cell injury by extracting intestinal epithelial cells from BALB.B mice for co-culture with B6 T cells. Our cultures are representative in their heterogeneity, containing both cytokeratin 15+ epithelial cells and smooth muscle cells that are easily differentiated from one another for analysis, with no contaminating dendritic cells or fibroblasts. In the skin, CD4+ and CD8+ T cells target cytokeratin 15+ stem cells of the basal membrane. Therefore, our in vitro gut epithelial cells may be targeted, and apoptosis induced, as in vivo. With this apoptotic co-culture model system in place, we will determine which of the perforin/granzyme, TRAIL, or Fas/FasL apoptotic pathways is critical at the early stages of intestinal GVHD, providing insight into potential therapeutic targets for prevention or treatment of GVHD.
Gastrointestinal GVHD is a major complication of allogeneic blood and marrow transplantation. In vitro models for screening potential donor T cell reactivity to host intestinal epithelial cells (IEC) as a portent of GVHD have been hampered by difficulty in maintaining primary IEC cultures. To approach this problem, conditional reprogramming (CR) technology was combined with tissue-engineering scaffolds composed of biocompatible polycaprolactone (PCL)/collagen nanofibers to enable a long-term preservation of primary murine IECs. Conditioned medium (CM) used for CR contains the Rho-associated kinase inhibitor Y-27632, an anti-apoptotic agent that can render CRIEC unsuitable for cell damage assays indicative of GVHD. Thus, we sought to create an enabling biomimetic extracellular matrix (ECM) platform that could support viable and functional CR murine-IEC (m-CRIEC) in the absence of Y-27632. Methods: Small intestine-derived IEC were plated on fibronectin-coated coverslips with CM+Y-27632. The m-CRIEC were removed from this media and seeded onto nanofibrous matrices in the presence of RPMI-1640 media. IEC normally reside on the thin fibrous basement membrane (BaMe) consisting of intermingled networks of laminins and type IV collagen, which provides cell anchoring and barrier functions. The BaMe interacts with cells through integrin receptors and other plasma membrane molecules, influencing cell phenotype and survival. Using electrospinning, BaMelike fibrous meshes were prepared. Slow degradable PCL was used as the fiber matrix phase in which Type I collagen (representing ECM molecules) was dispersed. Results: Flow cytometric analysis and microscopic inspection of m-CRIEC growing 7 days under CM+Y-27632depleted conditions presented with comparable viability and phenotypic display to that of m-CRIEC growing in CM+Y-27632; however only cells placed on nanofibers maintained cobblestone morphology and integrity (Figure 1). Conclusions: We have demonstrated that nanofibrous meshes can provide a physiologically relevant ECM-like microenvironment for the ex vivo maintenance of IEC. This biomimetic approach should prove particularly beneficial for the assessment of intestinal GVHD potential elicited by donor T cells. Figure 1. Morphology of m-CRIECs cultured in CRC medium, RPMI, and RPMI on nanofibrous mesh for 7 days to evaluate morphology and viability. Loss of cobble stone morphology (black arrow) was observed in cultures without nanofibrous mesh.Abstracts / Biol Blood Marrow Transplant 21 (2015) S322eS354 S354
observed in 3 patients (25%). There was no response in 2 patients (16%). The overall response rate at 4 weeks was 83%. Infectious complications were common, including bacteremia (41%), adenovirus viremia (50%), and CMV viremia (33%). Relapse of acute GVHD was observed in 42% of complete responders. Fifty percent of patients are currently surviving at a median follow up time of 775 days following first alemtuzumab course. We conclude that alemtuzumab is an effective treatment for steroid refractory GVHD in pediatric patients with a tolerable spectrum of complications. The dose, timing, and length of treatment should be optimized in a prospective study.
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