Suman Setty

IFN-γ activation of mesenchymal stem cells for treatment and prevention of graft versus host disease

Graft versus host disease (GVHD), mediated by donor T cells, is a significant source of morbidity and mortality following allogeneic stem cell transplantation. Mesenchymal stem cells (MSC) can successfully treat ongoing graft versus host disease, presumably due to their ability to suppress donor T cell proliferation. Little is known about the potential of MSC to prevent GVHD. Here we show that bone marrow‐isolated MSC can suppress the development of GVHD if given after donor T cell recognition of antigen. IFN‐γ was required to initiate MSC efficacy. Recipients of IFN‐γ–/– T cells did not respond to MSC treatment and succumbed to GVHD. MSC, pre‐treated with IFN‐γ, became immediately active and could suppress GVHD more efficiently than a fivefold‐greater number of MSC that were not activated. When given at the time of bone marrow transplantation, activated MSC could prevent GVHD mortality (100% survival, p=0.006). MSC activation was dependent on the magnitude of IFN‐γ exposure, with increased IFN‐γ exposure leading to increased MSC suppression of GVHD. Activated MSC present a new strategy for preventing GVHD using fewer MSC.

Chromatin Organization Measured by AluI Restriction Enzyme Changes with Malignancy and Is Regulated by the Extracellular Matrix and the Cytoskeleton

Given that expression of many genes changes when cells become malignant or are placed in different microenvironments, we asked whether these changes were accompanied by global reorganization of chromatin. We reasoned that sequestration or exposure of chromatin-sensitive sites to restriction enzymes could be used to detect this reorganization. We found that AluI-sensitive sites of nonmalignant cells were relatively more exposed compared to their malignant counterparts in cultured cells and human tumor samples. Changes in exposure and sequestration of AluI-sensitive sites in normal fibroblasts versus fibrosarcoma or those transfected with oncogenes, nonmalignant breast cells versus carcinomas and poorly metastatic versus highly invasive melanoma were shown to be independent of the cell cycle and may be influenced by proteins rich in disulfide bonds. Remarkably, regardless of degree of malignancy, AluI-sensitive sites became profoundly sequestered when cells were incubated with laminin, Matrigel, or a circular RGD peptide (RGD-C), but became exposed when cells were placed on collagen I or in serum-containing medium. Disruption of the actin cytoskeleton led to exposure, whereas disruption of microtubules or intermediate filaments exerted a sequestering effect. Thus, AluI-sensitive sites are more sequestered with increasing malignant behavior, but the sequestration and exposure of these sites is exquisitely sensitive to information conferred to the cell by molecules and biomechanical forces that regulate cellular and tissue architecture.

Distinguishing Fibrovascular Septa From Vasculogenic Mimicry Patterns

CONTEXT Molecular analyses indicate that periodic acid-Schiff (PAS)-positive (laminin-rich) patterns in melanomas are generated by invasive tumor cells by vasculogenic mimicry. Some observers, however, consider these patterns to be fibrovascular septa, generated by a stromal host response. OBJECTIVE To delineate differences between vasculogenic mimicry patterns and fibrovascular septa in primary uveal melanomas. DESIGN Frequency distributions, associations with outcome, and thicknesses of trichrome-positive and PAS-positive looping patterns were determined in 234 primary uveal melanomas. Sequential sections of 13 additional primary uveal melanomas that contained PAS-positive/trichrome-negative looping patterns were stained for type I and type IV collagens, laminin, and fibronectin. Real-time quantitative polymerase chain reaction was performed on RNA from cultured uveal melanoma cells for the expression of COL1A1, COL4A2, and fibronectin. RESULTS Trichrome-positive loops were encountered less frequently than PAS-positive loops (10% vs 56%, respectively). Death from metastatic melanoma was strongly associated with PAS-positive (P < .001) but not with trichrome-positive (P = .57) loops. Trichrome-positive loops were significantly thicker than PAS-positive loops (P < .001). The PAS-positive patterns stained positive for laminin, type I and type IV collagens, and fibronectin. Type I collagen was detected within melanoma cells and focally within some PAS-positive patterns. Real-time quantitative polymerase chain reaction revealed 3-fold, 25-fold, and 97-fold increases, respectively, in expression of COL4A2, fibronectin, and COL1A1 by invasive pattern-forming primary melanoma cells compared with poorly invasive non-pattern-forming cells. CONCLUSIONS Fibrovascular septa are rare and prognostically insignificant in uveal melanomas, whereas vasculogenic mimicry patterns are associated with increased mortality. Type I collagen, seen focally in some vasculogenic mimicry patterns, may be synthesized by tumor cells, independent of a host stromal response.

MADD Knock-Down Enhances Doxorubicin and TRAIL Induced Apoptosis in Breast Cancer Cells

The Map kinase Activating Death Domain containing protein (MADD) isoform of the IG20 gene is over-expressed in different types of cancer tissues and cell lines and it functions as a negative regulator of apoptosis. Therefore, we speculated that MADD might be over-expressed in human breast cancer tissues and that MADD knock-down might synergize with chemotherapeutic or TRAIL-induced apoptosis of breast cancer cells. Analyses of breast tissue microarrays revealed over-expression of MADD in ductal and invasive carcinomas relative to benign tissues. MADD knockdown resulted in enhanced spontaneous apoptosis in human breast cancer cell lines. Moreover, MADD knockdown followed by treatment with TRAIL or doxorubicin resulted in increased cell death compared to either treatment alone. Enhanced cell death was found to be secondary to increased caspase-8 activation. These data indicate that strategies to decrease MADD expression or function in breast cancer may be utilized to increase tumor cell sensitivity to TRAIL and doxorubicin induced apoptosis.

Cell population in spleens during antibody-mediated rejection: Pathologic and clinical findings

Background In the treatment of refractory antibody-mediated rejection (AMR), splenectomy has been associated with surprisingly rapid recovery of renal function. The mechanism is still unclear. Methods We review 11 recipients, who underwent rescue splenectomy (RS) as a treatment of AMR within 3 months after kidney transplantation. At transplantation, all patients had undergone desensitization for initially positive crossmatch to their prospective donors. The cellular populations of the spleen were analyzed by immunohistochemistry. For comparison, we obtained spleen specimens from eight controls who were nontransplantation patients. Results Rejection occurred in all the patients early after transplantation (mean [SD], 7.1 [5.7] days). One graft was lost 4 weeks after kidney transplantation. A significantly higher number of plasma cells (PCs) (P=0.049) and lower number of T and B lymphocytes (P=0.02 and P=0.005, respectively) were detected in the RS group compared with the control group. By analyzing the PC variations in the RS group, significantly lower numbers of PCs were detected in the spleens of patients who received rituximab before splenectomy (P=0.0004). In contrast, a higher number of PCs were found in patients (n=3) who did not respond to splenectomy and subsequently underwent bortezomib treatment and recovered their renal function (P=0.02). Conclusions Splenectomy may reverse AMR by debulking PCs. Our analysis suggests that patients with a very high load of PCs may not be rescued by splenectomy alone and may need additional treatments.

Successful identical‐twin living donor small bowel transplant for necrotizing enterovasculitis secondary to Churg–Strauss syndrome

Churg–Strauss syndrome (CSS) is a granulomatous small‐vessel vasculitis with unknown etiology. Extra‐pulmonary manifestations of CSS are currently treated with a combination of steroids and Cyclophosphamide. Its gastrointestinal complications may be devastating, occasionally requiring extensive bowel resection resulting in short‐gut syndrome. Living‐related small bowel transplantation (LRSBTx) is a relatively standardized procedure that, not only represents a valid alternative to cadaver bowel transplant in selected cases, but also portraits excellent results when performed in experienced centers. The availability of an identical twin as a donor, which allows avoidance of immunosuppressive therapy, is a major indication for this procedure. We present the case of a young individual affected by gastrointestinal necrotizing vasculitis that lost almost his entire small bowel requiring the immediate institution of total parenteral nutrition (TPN). However, within few weeks a significant hepatic dysfunction ensued. An identical twin‐brother, not affected with CSS, became an immediate, optimal donor‐candidate for LRSBTx, the first of this kind in a patient affected with CSS. Following the procedure, two main concerns were addressed: the recipients ability to recover a regular intestinal function without immunosuppression and the possible recurrence of the primary disease. Twenty‐seven months post‐transplant, the patient enjoys a regular lifestyle without any clinical, endoscopic and histologic evidences of recurrent disease in the transplanted graft.

A label-free approach by infrared spectroscopic imaging for interrogating the biochemistry of diabetic nephropathy progression

Routine histology, the current gold standard, involves staining for specific biomolecules. However, untapped biochemical information in tissue can be gathered using biochemical imaging. Infrared spectroscopy is an emerging modality that allows label-free chemical imaging to derive biochemical information (such as protein, lipids, DNA, collagen) from tissues. Here we employed this technology in order to better predict the development of diabetic nephropathy. Using human primary kidney biopsies or nephrectomies, we obtained tissue from 4 histologically normal kidneys, 4 histologically normal kidneys from diabetic subjects, and 5 kidneys with evidence of diabetic nephropathy. A biochemical signature of diabetic nephropathy was derived that enabled prediction of nephropathy based on the ratio of only 2 spectral frequencies. Nonetheless, using the entire spectrum of biochemical information, we were able to detect renal disease with near-perfect accuracy. Additionally, study of sequential protocol biopsies from 3 transplanted kidneys showed biochemical changes even prior to clinical manifestation of diabetic nephropathy. Thus, infrared imaging can identify critical biochemical alterations that precede morphologic changes, potentially allowing for earlier intervention.

Differential expression of laminin isoforms in diabetic nephropathy and other renal diseases

Laminin a non-collagenous glycoprotein is a major component of the renal glomerular basement membrane and mesangium. Thus far eleven distinct chains have been described, permutations of which make up 15 laminin isoforms. Laminin molecules interact with cells and other matrix molecules during organ development and differentiation. We studied the distribution of laminin isoforms in patients with type 1 diabetic nephropathy, membranous nephropathy, membranoproliferative glomerulonephritis and IgA nephropathy/ Henoch–Schönlein purpura. Immunofluorescence microscopic studies with laminin-chain-specific antibodies to the α1, α2, α5, β1, β2 and γ1 chains detected α2, β1 and γ1 chain expression in the normal mesangium and α5, β2 and γ1 in normal glomerular basement membrane. Significantly, constituents of the glomerular basement membrane, α5, β2 and γ1 chains were overexpressed in kidneys with diabetic nephropathy. Initially the constituents of the mesangium increased commensurate with the degree of mesangial expansion and degree of diabetic nephropathy. Reduction in α2 chain intensity was observed with severe mesangial expansion and in the areas of nodular glomerulosclerosis. In addition, with late disease aberrant expression of α2 and β2 chains was observed in the mesangium. Glomerular basement membrane in renal disease overexpressed molecules normally present in that location. In summary, the alterations in basement membrane composition in various renal diseases seem to not only reflect the balance between synthesis and degradation of normal basement membrane constituents, but also their aberrant expression.

Squamous cell carcinoma in a chronically rejected renal allograft

The malignant degeneration of a chronically rejected kidney allograft has been rarely reported. Almost invariably such malignancies originated in the transitional epithelium. We herein present the first occurrence of squamous cell carcinoma (SCC), originating from occult donor cells, in a chronically rejected renal allograft. Nearly 20 years after chronic rejection and loss of function of a cadaver renal graft, our patient developed increasing abdominal discomfort, decrease in appetite and weight loss. A CT‐scan of the abdomen showed an abnormally enlarged and irregularly contoured mass at the level of the rejected allograft. Given the clinical and radiologic picture suggestive of either an infectious or intraparenchymal hemorrhagic process, a transplant nephrectomy was performed. At surgery, it was immediately evident that a malignant degenerative process had affected the graft. The histological features of the specimen were diagnostic for a well‐differentiated SCC. The donor origin of the tumor was established through a DNA microchimerism assay performed on the operative specimens. The patient did well after resection of the malignancy, although he died 5 months later owing to a myocardial infarction. In summary, even several years following the transplant, the possibility of a malignancy of donor origin developing within a failed allograft should always be considered as part of the differential diagnosis in unusual post‐transplant settings.

Electrolyte imbalances in pediatric living related small bowel transplantation

Background. Pediatric small bowel transplantations are associated with pronounced electrolyte disturbances in the postoperative period. We investigated the pattern of electrolyte disturbances with regard to enteral malabsorption, renal compensation, and the influence of immunosuppression. Methods. We reviewed 11 small bowel transplantations between October 2002 and February 2007. The data collected included frequent serum, ostomy, and urine electrolyte profiles, renal function parameters, and FK 506 levels in the postoperative period up until either discharge or graft loss. Results. Our results show enteral losses most prominent during the first 4 weeks postoperatively that are only partially compensated by the kidneys. Subsequently, enteral losses improved, although renal function remained challenged, particularly glomerular filtration and phosphorus, magnesium losses, which correlated with high FK 506 levels. Conclusion. Our data reveal several electrolyte imbalances different and unique to postoperative small bowel transplants. Although enteral losses improve along with graft villi formation, electrolyte abnormalities continue, to which FK 506-mediated renal toxicity might contribute.