Neeraja Kambham

RAGE mediates a novel proinflammatory axis: a central cell surface receptor for S100/calgranulin polypeptides.

S100/calgranulin polypeptides are present at sites of inflammation, likely released by inflammatory cells targeted to such loci by a range of environmental cues. We report here that receptor for AGE (RAGE) is a central cell surface receptor for EN-RAGE (extracellular newly identified RAGE-binding protein) and related members of the S100/calgranulin superfamily. Interaction of EN-RAGEs with cellular RAGE on endothelium, mononuclear phagocytes, and lymphocytes triggers cellular activation, with generation of key proinflammatory mediators. Blockade of EN-RAGE/RAGE quenches delayed-type hypersensitivity and inflammatory colitis in murine models by arresting activation of central signaling pathways and expression of inflammatory gene mediators. These data highlight a novel paradigm in inflammation and identify roles for EN-RAGEs and RAGE in chronic cellular activation and tissue injury.

Graft-versus-host disease is enhanced by extracellular ATP activating P2X7R

Danger signals released upon cell damage can cause excessive immune-mediated tissue destruction such as that found in acute graft-versus-host disease (GVHD), allograft rejection and systemic inflammatory response syndrome. Given that ATP is found in small concentrations in the extracellular space under physiological conditions, and its receptor P2X7R is expressed on several immune cell types, ATP could function as a danger signal when released from dying cells. We observed increased ATP concentrations in the peritoneal fluid after total body irradiation, and during the development of GVHD in mice and in humans. Stimulation of antigen-presenting cells (APCs) with ATP led to increased expression of CD80 and CD86 in vitro and in vivo and actuated a cascade of proinflammatory events, including signal transducer and activator of transcription-1 (STAT1) phosphorylation, interferon-γ (IFN-γ) production and donor T cell expansion, whereas regulatory T cell numbers were reduced. P2X7R expression increased when GVHD evolved, rendering APCs more responsive to the detrimental effects of ATP, thereby providing positive feedback signals. ATP neutralization, early P2X7R blockade or genetic deficiency of P2X7R during GVHD development improved survival without immune paralysis. These data have major implications for transplantation medicine, as pharmacological interference with danger signals that act via P2X7R could lead to the development of tolerance without the need for intensive immunosuppression.

Connective Tissue Growth Factor–Specific Monoclonal Antibody Therapy Inhibits Pancreatic Tumor Growth and Metastasis

Pancreatic cancer is highly aggressive and refractory to most existing therapies. Past studies have shown that connective tissue growth factor (CTGF) expression is elevated in human pancreatic adenocarcinomas and some pancreatic cancer cell lines. To address whether and how CTGF influences tumor growth, we generated pancreatic tumor cell lines that overexpress different levels of human CTGF. The effect of CTGF overexpression on cell proliferation was measured in vitro in monolayer culture, suspension culture, or soft agar, and in vivo in tumor xenografts. Although there was no effect of CTGF expression on proliferation in two-dimensional cultures, anchorage-independent growth (AIG) was enhanced. The capacity of CTGF to enhance AIG in vitro was linked to enhanced pancreatic tumor growth in vivo when these cells were implanted s.c. in nude mice. Administration of a neutralizing CTGF-specific monoclonal antibody, FG-3019, had no effect on monolayer cell proliferation, but blocked AIG in soft agar. Consistent with this observation, anti-CTGF treatment of mice bearing established CTGF-expressing tumors abrogated CTGF-dependent tumor growth and inhibited lymph node metastases without any toxicity observed in normal tissue. Together, these studies implicate CTGF as a new target in pancreatic cancer and suggest that inhibition of CTGF with a human monoclonal antibody may control primary and metastatic tumor growth.

Expression of Complement Components Differs Between Kidney Allografts from Living and Deceased Donors

A disparity remains between graft survival of renal allografts from deceased donors and from living donors. A better understanding of the molecular mechanisms that underlie this disparity may allow the development of targeted therapies to enhance graft survival. Here, we used microarrays to examine whole genome expression profiles using tissue from 53 human renal allograft protocol biopsies obtained both at implantation and after transplantation. The gene expression profiles of living-donor kidneys and pristine deceased-donor kidneys (normal histology, young age) were significantly different before reperfusion at implantation. Deceased-donor kidneys exhibited a significant increase in renal expression of complement genes; posttransplantation biopsies from well-functioning, nonrejecting kidneys, regardless of donor source, also demonstrated a significant increase in complement expression. Peritransplantation phenomena, such as donor death and possibly cold ischemia time, contributed to differences in complement pathway gene expression. In addition, complement gene expression at the time of implantation was associated with both early and late graft function. These data suggest that complement-modulating therapy may improve graft outcomes in renal transplantation.

A Randomized, Prospective Trial of Rituximab for Acute Rejection in Pediatric Renal Transplantation

We report 1‐year outcomes of a randomized study of Rituximab versus standard‐of‐care immunosuppression (Thymoglobulin and/or pulse steroids) for treatment of biopsy confirmed, acute transplant rejection with B‐cell infiltrates, in 20 consecutive recipients (2–23 years). Graft biopsies, with Banff and CADI scores, CD20 and C4d stains, were performed at rejection and 1 and 6 months later. Peripheral blood CMV, EBV and BK viral loads, graft function, DSA, immunoglobulins, serum humanized antichimeric antibody (HACA) and Rituximab, and lymphocyte counts were monitored until 1 year posttreatment. Rituximab infusions were given with a high index of safety without HACA development and increased infections complications. Rituximab therapy resulted in complete tissue B‐cell depletion and rapid peripheral B‐cell depletion. Peripheral CD19 cells recovered at a mean time of ∼12 months. There were some benefits for the recovery of graft function (p = 0.026) and improvement of biopsy rejection scores at both the 1‐ (p = 0.0003) and 6‐month (p < 0.0001) follow‐up biopsies. Reappearance of C4d deposition was not seen on follow‐up biopsies after Rituximab therapy, but was seen in 30% of control patients. There was no change in DSA in either group, independent of rejection resolution. This study reports safety and suggests further investigation of Rituximab as an adjunctive treatment for B‐cell‐mediated graft rejection.

Characterization of intra-graft B cells during renal allograft rejection

Intra-graft CD20(+) B-cell clusters are found during acute rejection of renal allografts and correlate with graft recovery following rejection injury. Here using archived kidney tissue we conducted immunohistochemical studies to measure specific subsets of pathogenic B cells during graft rejection. Cluster-forming CD20(+) B cells in the rejected graft are likely derived from the recipient and are composed of mature B cells. These cells are activated (CD79a(+)), and present MHC Class II antigen (HLADR(+)) to CD4(+) T cells. Some of these clusters contained memory B cells (CD27(+)) and they did not correlate with intra-graft C4d deposition or with detection of donor-specific antibody. Further, several non-cluster forming CD20(-) B-lineage CD38(+) plasmablasts and plasma cells were found to infiltrate the rejected grafts and these cells strongly correlated with circulating donor-specific antibody, and to a lesser extent with intra-graft C4d. Both CD20(+) B cells and CD38(+) cells correlated with poor response of the rejection to steroids. Reduced graft survival was associated with the presence of CD20 cells in the graft. In conclusion, a specific subset of early lineage B cells appears to be an antigen-presenting cell and which when present in the rejected graft may support a steroid-resistant T-cell-mediated cellular rejection. Late lineage interstitial plasmablasts and plasma cells may also support humoral rejection. These studies suggest that detailed analysis of interstitial cellular infiltrates may allow better use of B-cell lineage specific treatments to improve graft outcomes.

Surgical Robotic Applications in Otolaryngology

Objectives To explore the feasibility of performing endo‐robotic neck surgery in a porcine model and to compare the results of robotically enhanced endoscopic surgery with those from a conventional endoscopic technique.

Identifying compartment-specific non-HLA targets after renal transplantation by integrating transcriptome and “antibodyome” measures

We have conducted an integrative genomics analysis of serological responses to non-HLA targets after renal transplantation, with the aim of identifying the tissue specificity and types of immunogenic non-HLA antigenic targets after transplantation. Posttransplant antibody responses were measured by paired comparative analysis of pretransplant and posttransplant serum samples from 18 pediatric renal transplant recipients, measured against 5,056 unique protein targets on the ProtoArray platform. The specificity of antibody responses were measured against gene expression levels specific to the kidney, and 2 other randomly selected organs (heart and pancreas), by integrated genomics, employing the mapping of transcription and ProtoArray platform measures, using AILUN. The likelihood of posttransplant non-HLA targets being recognized preferentially in any of 7 microdissected kidney compartments was also examined. In addition to HLA targets, non-HLA immune responses, including anti-MICA antibodies, were detected against kidney compartment-specific antigens, with highest posttransplant recognition for renal pelvis and cortex specific antigens. The compartment specificity of selected antibodies was confirmed by IHC. In conclusion, this study provides an immunogenic and anatomic roadmap of the most likely non-HLA antigens that can generate serological responses after renal transplantation. Correlation of the most significant non-HLA antibody responses with transplant health and dysfunction are currently underway.

Endometrial VEGF induces placental sFLT1 and leads to pregnancy complications

There is strong evidence that overproduction of soluble fms-like tyrosine kinase-1 (sFLT1) in the placenta is a major cause of vascular dysfunction in preeclampsia through sFLT1-dependent antagonism of VEGF. However, the cause of placental sFLT1 upregulation is not known. Here we demonstrated that in women with preeclampsia, sFLT1 is upregulated in placental trophoblasts, while VEGF is upregulated in adjacent maternal decidual cells. In response to VEGF, expression of sFlt1 mRNA, but not full-length Flt1 mRNA, increased in cultured murine trophoblast stem cells. We developed a method for transgene expression specifically in mouse endometrium and found that endometrial-specific VEGF overexpression induced placental sFLT1 production and elevated sFLT1 levels in maternal serum. This led to pregnancy losses, placental vascular defects, and preeclampsia-like symptoms, including hypertension, proteinuria, and glomerular endotheliosis in the mother. Knockdown of placental sFlt1 with a trophoblast-specific transgene caused placental vascular changes that were consistent with excess VEGF activity. Moreover, sFlt1 knockdown in VEGF-overexpressing animals enhanced symptoms produced by VEGF overexpression alone. These findings indicate that sFLT1 plays an essential role in maintaining vascular integrity in the placenta by sequestering excess maternal VEGF and suggest that a local increase in VEGF can trigger placental overexpression of sFLT1, potentially contributing to the development of preeclampsia and other pregnancy complications.

Heavy chain deposition disease: The disease spectrum

A 45-year-old white woman was found to have microscopic hematuria during her annual physical examination. After a negative urologic workup, she returned 5 months later with nephrotic syndrome, renal insufficiency, and hypocomplementemia. Renal biopsy showed a nodular sclerosing glomerulopathy that could not be further characterized because of inadequate tissue for immunofluorescence. The patient returned 8 months later with chronic renal failure. A repeat renal biopsy showed deposits composed of immunoglobulin G (IgG) heavy chain and complement components C3 and C1 along glomerular, tubular, and vascular basement membranes, with negativity for kappa and lambda light chains, findings consistent with heavy chain deposition disease (HCDD). The heavy chain subclass was exclusively IgG3. Staining with monoclonal antibodies to epitopes of the constant domains of IgG heavy chain showed a CH1 deletion, indicating a truncated heavy chain. On review of the previously reported cases of HCDD, common clinical presentations include nephrotic syndrome, renal insufficiency, hematuria, and, in some cases, hypocomplementemia. In most patients, the hematologic disorder is mild, without overt myeloma. Light microscopy shows a nodular sclerosing glomerulopathy, and heavy chain deposits are detectable within basement membranes throughout the kidney by immunofluorescence and electron microscopy. There is no effective treatment for this condition, and virtually all patients progress to chronic renal failure.