Ariana Gaspert

GNRH1 mutations in patients with idiopathic hypogonadotropic hypogonadism

Idiopathic hypogonadotropic hypogonadism (IHH) is a condition characterized by failure to undergo puberty in the setting of low sex steroids and low gonadotropins. IHH is due to abnormal secretion or action of the master reproductive hormone gonadotropin-releasing hormone (GnRH). Several genes have been found to be mutated in patients with IHH, yet to date no mutations have been identified in the most obvious candidate gene, GNRH1 itself, which encodes the preprohormone that is ultimately processed to produce GnRH. We screened DNA from 310 patients with normosmic IHH (nIHH) and 192 healthy control subjects for sequence changes in GNRH1. In 1 patient with severe congenital nIHH (with micropenis, bilateral cryptorchidism, and absent puberty), a homozygous frameshift mutation that is predicted to disrupt the 3 C-terminal amino acids of the GnRH decapeptide and to produce a premature stop codon was identified. Heterozygous variants not seen in controls were identified in 4 patients with nIHH: 1 nonsynonymous missense mutation in the eighth amino acid of the GnRH decapeptide, 1 nonsense mutation that causes premature termination within the GnRH-associated peptide (GAP), which lies C-terminal to the GnRH decapeptide within the GnRH precursor, and 2 sequence variants that cause nonsynonymous amino-acid substitutions in the signal peptide and in GnRH-associated peptide. Our results establish mutations in GNRH1 as a genetic cause of nIHH.

Rituximab and intravenous immunoglobulin treatment of chronic antibody-mediated kidney allograft rejection.

Kidney transplant rejections are classified into T-cell-mediated and antibody-mediated rejections (AMR). C4d staining on allograft biopsies and solid-phase assays to measure donor-specific alloantibodies have helped to precisely define the latter. Although for acute AMRs, therapy mainly relies on plasmapheresis or immunoadsorption, no studies for treatment of chronic AMR are available. Here, we report on four kidney allograft recipients suffering from chronic AMR 1 to 27 years posttransplant, who were treated with a combination of rituximab and intravenous immunoglobulin (IVIG). Rituximab/IVIG improved kidney allograft function in all four patients, whereas donor-specific antibodies were reduced in 2 of 4 patients. However, in one patient an acute rejection episode occurred 12 months after this treatment, and another patient had severe, possibly rituximab-associated lung toxicity. Thus, rituximab/IVIG may be a useful strategy for the treatment of chronic AMR, but further randomized multicenter studies are necessary to establish its efficacy and safety profile.

Apoptosis induced by ischemia and reperfusion in experimental lung transplantation.

BACKGROUND Apoptosis is a distinct form of single-cell death in response to injury. Time course of apoptosis in lung parenchymal cells during posttransplant reperfusion and the influence of oxygen content during preservation on apoptosis of parenchymal cells are studied. METHODS Orthotopic syngenic single left lung transplantation was performed in male Fischer (F344) rats after 18 hours of cold ischemia (n = 5 in all groups). Apoptotic cells were stained by the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) technique. Strictly TUNEL-positive pneumocytes were counted on anonymized slides by a pathologist on 100 fields (x400) per specimen (mean +/- SEM). RESULTS The peak of apoptotic pneumocytes occurred 2 hours after reperfusion (16.8 +/- 2.2 pneumocytes/100 fields [p/100f]; p = 0.000012 vs controls, lungs fixed after 18 hours of ischemia), whereas the lowest level of apoptotic pneumocytes was seen in lungs fixed after harvest (1.4 +/- 0.51 p/100f) and lungs not undergoing reperfusion (2.8 +/- 0.49 p/100f). Four hours after reperfusion, the number of apoptotic pneumocytes was lower than 2 hours after reperfusion (13.6 +/- 3.1 p/100f; p = 0.00032 vs controls), with a further decline at 8 hours (6.4 +/- 1.5 p/100f) and 12 hours after reperfusion (4.0 + 1.2 p/100f). Interestingly, lungs inflated with N2 before storage revealed a significantly lower level of TUNEL-positive pneumocytes 2 hours after reperfusion (8.8 2.0 p/100f) compared with lungs inflated with 100% O2 (p = 0.0052). CONCLUSIONS Apoptosis of pneumocytes after posttransplant lung reperfusion is a very early event. Prolonged hypothermic preservation without reperfusion, however, does not lead to an elevated rate of apoptotic pneumocytes in lung grafts.

Efficacy and cost effectiveness of oral ganciclovir in the prevention of cytomegalovirus disease after lung transplantation.

BACKGROUND Cytomegalovirus is the single most frequent pulmonary pathogen in lung transplant recipients who survive at least 2 weeks. Patients at increased risk are either seropositive or have received an allograft from a donor with latent infection. Morbidity and mortality caused by cytomegalovirus disease is still considerably high. METHODS In an open, comparative study, we evaluated the efficacy, tolerance, and cost effectiveness of postoperative ganciclovir prophylaxis: intravenous dose of 2x5 mg/kg/day for 14 days, followed by either intravenous doses of 5 mg/kg]day (five patients), or oral doses of 3x 1000 mg (nine patients) up to 90 days. Oral ganciclovir was continued until prednisone was tapered below 15 mg/day. Prophylaxed groups were compared with a historical control (eight patients) in respect to cytomegalovirus disease, in-hospital stay, overall costs, and survival. Follow-up times and the net state of immunosuppressive therapy between groups were comparable. RESULTS Six (75%) of the non-prophylaxed patients developed cytomegalovirus disease compared to none in the intravenous and one in the oral ganciclovir group (P=0.013). The non-prophylaxed patients had a longer cytomegalovirus-related in-hospital stay (P=0.018) and nonsignificantly higher cytomegalovirus-related costs. Bronchiolitis obliterans syndrome was less frequent with prophylaxis (P=0.039), and survival tended to be better (P=0.072). The only adverse effect was a subclavian vein thrombosis in the intravenous ganciclovir group. CONCLUSIONS In lung transplant recipients, ganciclovir prophylaxis, either intravenous or oral, is safe, well tolerated, and effective in preventing cytomegalovirus disease. Moreover, ganciclovir prophylaxis seems likely to reduce the incidence of bronchiolitis obliterans syndrome. The oral formulation might be preferable because its convenience and possibly lower costs.

Antibody-mediated kidney allograft rejection: therapeutic options and their experimental rationale

With the advent of novel therapies to directly intervene with B cell immunity and complement activation, antibody‐mediated kidney allograft rejection (AMR) has come into the focus of transplant immunologists. Intravenous immunoglobulin, rituximab, bortezomib, and eculizumab have been used to treat patients with acute AMR, apart from the standard treatment of antibody removal with plasma exchange or immunoadsorption and steroid pulses. This article describes the experimental rationale and summarizes the still limited clinical experience with these novel therapies in the transplant setting. Results with the standard treatment for acute AMR, including intense plasmapheresis, intravenous immunoglobulins, and steroids are good with a graft survival of 80% at 18 months. In contrast, patients suffering from chronic AMR have significant irreversible damage in their grafts with substantially impaired graft survival. Thus, the authors propose a step‐wise escalation of therapy in refractory cases of acute AMR and advocate an urgent need for controlled therapeutic trials for acute and chronic AMR not to inflict unnecessary harm on our patients by uncontrolled polypragmasy.

Periostin Is Induced in Glomerular Injury and Expressed de Novo in Interstitial Renal Fibrosis

Matricellular proteins participate in the pathogenesis of chronic kidney diseases. We analyzed glomerular gene expression profiles from patients with proteinuric diseases to identify matricellular proteins contributing to the progression of human nephropathies. Several genes encoding matricellular proteins, such as SPARC, THBS1, and CTGF, were induced in progressive nephropathies, but not in nonprogressive minimal-change disease. Periostin showed the highest induction, and its transcript levels correlated negatively with glomerular filtration rate in both glomerular and tubulointerstitial specimen. In well-preserved renal tissue, periostin localized to the glomerular tuft, the vascular pole, and along Bowmans capsule; no signal was detected in the tubulointerstitial compartment. Biopsies from patients with glomerulopathies and renal dysfunction showed enhanced periostin expression in the mesangium, tubular interstitium, and sites of fibrosis. Periostin staining correlated negatively with renal function. α-smooth muscle actin-positive mesangial and interstitial cells localized close to periostin-positive sites, as indicated by co-immunofluorescence. In vitro stimulation of mesangial cells by external addition of TGF-β1 resulted in robust induction of periostin. Addition of periostin to mesangial cells induced cell proliferation and decreased the number of cells expressing activated caspase-3, a marker of apoptosis. These human data indicate for the first time a role of periostin in glomerular and interstitial injury in acquired nephropathies.

Bronchoscopic radioisotope injection for sentinel lymph-node mapping in potentially resectable non-small-cell lung cancer

OBJECTIVE Prospective study to evaluate the feasibility of a preoperative bronchoscopic radioisotope application, followed by conventional sentinel lymph-node (SLN) identification and to investigate the occurrence and distribution of micrometastases in relation to SLN activity. METHODS Twenty patients with a mean age of 63 years and proven clinical stage T1-3 N0-1 non-small-cell lung cancer (NSCLC) were included. A dosage of 80MBq radiolabeled technetium-99m nanocolloid was endoscopically administrated on intubated patients in the operation theatre. At thoracotomy, scintigraphic readings of both the primary tumor and hilar and mediastinal lymph-node stations were obtained with a hand-held gamma-counter. Patients underwent lung resection and mediastinal lymphadenectomy. Radiolabeled nodes were also examined separately on back-table. SLNs were defined as the hottest nodes or nodes with at least one-tenth of the radioactivity of the hottest nodes. SLNs pathologic assessment included standard examination using hematoxylin and eosin staining on step sections and immunohistochemistry (ICH) for cytokeratins. RESULTS Identification of SLNs was possible in 19/20 (95%) patients after bronchoscopic radioisotope application. In 7/19 (37%) patients, a unique SLN was identified, whereas in 12/19 (63%) patients, nodes from two different stations could be classified as SLNs. Metastatic nodal disease was found in 9/19 (47%) patients. ICH revealed micrometastases in 2/12 (17%) patients, initially classified nodal negative. Pathologic negative SLNs were a predictor for absence of metastatic nodal disease after mediastinal lymphadenectomy. No complication related to the procedure was observed. CONCLUSION Our preliminary results suggest that preoperative bronchoscopic radioisotope injection for SLN identification is a safe and simple method, improving accuracy of SLN detection in comparison to intraoperative technique. The absence of metastases in the SLNs seems to predict a negative nodal status accurately.

Identification of Stanniocalcin 2 as Prognostic Marker in Renal Cell Carcinoma

BACKGROUND For an individualized therapy in renal cell carcinoma (RCC), there is a clear need for novel prognostic biomarkers to ensure adequate risk stratification and help with the choice of therapy options. OBJECTIVE To identify new secreted biomarkers for diagnosis and estimation of prognosis in RCC. DESIGN, SETTING, AND PARTICIPANTS A meta-analysis of published microarray data was performed. Stanniocalcin 2 (STC2), a glycoprotein hormone that is involved in regulatory effects on calcium and phosphate transport in the kidney, was found overexpressed in tumors and hence analyzed in detail. Kidney tissue samples derived from 108 patients with RCC undergoing radical nephrectomy between July 2003 and January 2006 were used to validate and estimate the potential of STC2 as a biomarker for RCC. MEASUREMENTS STC2, found upregulated in clear cell RCC, was analyzed in detail using real-time reverse transcription-polymerase chain reaction (RT-PCR), western blotting, and immunohistochemistry. Furthermore, STC2 protein expression determined on a tissue microarray was correlated to clinical pathologic parameters, including patient survival. RESULTS AND LIMITATIONS STC2 was upregulated at the mRNA and protein levels in RCC. In normal renal tissue, STC2 expression was limited to distal tubuli and glomeruli, whereas in tumor a strong cytoplasmic and also membranous staining was detected. STC2 expression was found in clear cell, chromophobe, and papillary RCC. Strong cytoplasmic STC2 expression was significantly associated with shorter patient survival in Kaplan-Meier analyses. In the group of patients without metastases, cytoplasmic STC2 expression was also found as a significant independent risk factor in multivariate analysis. A limitation of the study is the small number of patients. CONCLUSIONS Increased cytoplasmic STC2 expression correlated with conventional indicators of aggressiveness of RCC and shorter overall patient survival times. STC2 could become an adjunct tissue biomarker that may be useful in the postoperative risk stratification of RCC patients.

Podoplanin-positive cells are a hallmark of encapsulating peritoneal sclerosis

BACKGROUND Encapsulating peritoneal sclerosis (EPS) and simple peritoneal sclerosis are important complications of long-term peritoneal dialysis (PD). Podoplanin is expressed by mesothelial cells and lymphatic vessels, which are involved in inflammatory reactions in the peritoneal cavity. METHODS We studied 69 peritoneal biopsies from patients on PD (n = 16), patients with EPS (n = 18) and control biopsies taken at the time of hernia repair (n = 15) or appendectomy (n = 20). Immunohistochemistry was performed to localize podoplanin. Additionally, markers of endothelial cells, mesothelial cells, myofibroblasts (smooth muscle actin), proliferating cells, and double labelling for smooth muscle actin/podoplanin were used on selected biopsies. RESULTS Podoplanin was present on the endothelium of lymphatic vessels in the submesothelial fibrous tissue and on mesothelial cells. In patients on PD and in biopsies with appendicitis, the mesothelial cells demonstrated a cuboidal appearance and circumferential podoplanin staining, with gaps between the cells. The number of lymphatic vessels was variable, but prominent at sites of fibrosis. In patients with EPS, a diffuse infiltration of podoplanin-positive cells with a fibroblastic appearance was present in 15 out of 18 biopsies. This pattern was focally present in 3 out of 16 on PD and none in the 35 controls. The podoplanin-positive cells did not express the endothelial marker or the mesothelial marker (calretinin). CONCLUSIONS EPS is characterized by a population of podoplanin and smooth muscle actin double-positive cells. Podoplanin might be a suitable morphological marker supporting the diagnosis and might be involved in the pathogenesis of EPS.

FAT1 mutations cause a glomerulotubular nephropathy

Steroid-resistant nephrotic syndrome (SRNS) causes 15% of chronic kidney disease (CKD). Here we show that recessive mutations in FAT1 cause a distinct renal disease entity in four families with a combination of SRNS, tubular ectasia, haematuria and facultative neurological involvement. Loss of FAT1 results in decreased cell adhesion and migration in fibroblasts and podocytes and the decreased migration is partially reversed by a RAC1/CDC42 activator. Podocyte-specific deletion of Fat1 in mice induces abnormal glomerular filtration barrier development, leading to podocyte foot process effacement. Knockdown of Fat1 in renal tubular cells reduces migration, decreases active RAC1 and CDC42, and induces defects in lumen formation. Knockdown of fat1 in zebrafish causes pronephric cysts, which is partially rescued by RAC1/CDC42 activators, confirming a role of the two small GTPases in the pathogenesis. These findings provide new insights into the pathogenesis of SRNS and tubulopathy, linking FAT1 and RAC1/CDC42 to podocyte and tubular cell function.