Ann Rearden

The signaling adapter protein PINCH is up-regulated in the stroma of common cancers, notably at invasive edges†

PINCH is an LIM (double zinc finger domain) protein that functions as an adapter at a key convergence point for integrin and growth factor signal transduction. Because no information is available regarding its expression in vivo in human tissues, this study evaluated the distribution and abundance of PINCH in patients with breast, prostate, lung, colon, and skin carcinomas.

PEYRONIE'S DISEASE IS ASSOCIATED WITH AN HLA CLASS II ANTIGEN, HLA-DQ5, IMPLYING AN AUTOIMMUNE ETIOLOGY

PURPOSE Previous studies using serological (protein based) HLA typing methods suggested an association between various HLA antigens and Peyronies disease. We used a molecular (deoxyribonucleic acid based) HLA typing method to determine whether Peyronies disease is associated with class II HLA antigens. MATERIALS AND METHODS Class II HLA typing using the polymerase chain reaction-sequence specific primer method was performed in 31 patients with Peyronies disease, 19 age matched urological patients with disorders other than Peyronies disease and 75 major organ donor cadavers representative of the general population. RESULTS The frequency of HLA-DQ5 was significantly increased in patients with Peyronies disease (61% HLA-DQ5 positive) compared to age matched disease-free controls (11% HLA-DQ5 positive, corrected p = 0.003) and general population controls (25% HLA-DQ5 positive, corrected p = 0.005). The relative risk for Peyronies disease associated with HLA-DQ5 was 12.7 and 4.6 compared to age matched and general population controls, respectively. CONCLUSIONS This study shows a positive association for the HLA class II antigen HLA-DQ5 with Peyronies disease, suggesting that HLA-DQ5 is a risk factor for Peyronies disease and implying an autoimmune etiology for this disorder.

Detection of chimerism and early engraftment after allogeneic peripheral blood stem cell or bone marrow transplantation by short tandem repeats

Chimerism can be monitored after HLA‐matched allogeneic bone marrow transplantation (BMT) or allogeneic peripheral blood stem cell transplantation (PBSCT) by detecting polymorphisms in short tandem repeats (STR). The purpose of our study was to document early complete chimerism in BMT and PBSCT recipients using STR, and to determine whether the initial WBC recovery correlated with the days required to attain complete chimerism. A total of 5 patients (2 PBSCT and 3 BMT) were followed by STR after transplantation. Peripheral blood obtained prior to transplantation was used to determine the 2 most informative STR probes for each donor/recipient pair. STR were amplified by polymerase chain reaction (PCR) with 8 commercial probes, and PCR products were visualized with silver staining. Peripheral blood was evaluated daily post‐transplantation for WBC counts and to identify the presence of mixed or full chimerism by STR. The sensitivity of the STR technique varied from 0.05 to 1%, depending on the probe. Full chimerism was documented between day 9 and 14 in PBSCT recipients and on day 14 and 16 in BMT recipients. The initial rise in WBC occurred within 3 days of the onset of full chimerism, indicating that full chimerism is a more sensitive indicator of early engraftment. Periodic recipient monitoring using STR after complete chimerism identifies those patients who revert to mixed chimeras. The STR method may be useful in future studies to determine the significance of early engraftment and the clinical implications of sustained complete chimerism or mixed chimerism.

Targeted Ablation of PINCH1 and PINCH2 From Murine Myocardium Results in Dilated Cardiomyopathy and Early Postnatal Lethality

Background— PINCH proteins are 5 LIM domain–only adaptor proteins that function as key components of the integrin signaling pathway and play crucial roles in multiple cellular processes. Two PINCH proteins, PINCH1 and PINCH2, have been described in mammals and share high homology. Both PINCH1 and PINCH2 are ubiquitously expressed in most tissues and organs, including myocardium. Cardiac-specific PINCH1 knockout or global PINCH2 knockout mice exhibit no basal cardiac phenotype, which may reflect a redundant role for these 2 PINCH proteins in myocardium. A potential role for PINCH proteins in myocardium remains unknown. Methods and Results— To define the role of PINCH in myocardium, we generated mice that were doubly homozygous null for PINCH1 and PINCH2 in myocardium. Resulting mutants were viable at birth but developed dilated cardiomyopathy and died of heart failure within 4 weeks. Mutant hearts exhibited disruptions of intercalated disks and costameres accompanied by fibrosis. Furthermore, multiple cell adhesion proteins exhibited reduced expression and were mislocalized. Mutant cardiomyocytes were significantly smaller and irregular in size. In addition, we observed that the absence of either PINCH1 or PINCH2 in myocardium leads to exacerbated cardiac injury and deterioration in cardiac function after myocardial infarction. Conclusions— These results demonstrate essential roles for PINCHs in myocardial growth, maturation, remodeling, and function and highlight the importance of studying the role of PINCHs in human cardiac injury and cardiomyopathy.

Pinch1 Is Required for Normal Development of Cranial and Cardiac Neural Crest-Derived Structures

Pinch1, an adaptor protein composed of 5 LIM domains, has been suggested to play an important role in multiple cellular processes. We found that Pinch1 is highly expressed in neural crest cells and their derivatives. To examine the requirement for Pinch1 in neural crest development, we generated neural crest conditional Pinch1 knockout mice using the Wnt1-Cre/loxP system. Neural crest conditional Pinch1 mutant embryos die perinatally from severe cardiovascular defects with an unusual aneurysmal common arterial trunk. Pinch1 mutants also exhibit multiple deficiencies in cranial neural crest–derived structures. Fate mapping demonstrated that initial migration of neural crest cells to the pharyngeal arch region occurs normally in the mutant embryos. However, in the cardiac outflow tract of mutants, neural crest cells exhibited hyperplasia and failed to differentiate into smooth muscle. Markedly increased apoptosis is observed in outflow tract cushions of mutants between embryonic days 11.5 and 13.5, likely contributing to the observed defects in cushion/valve remodeling and ventricular septation. Expression of transforming growth factor-&bgr;2, which plays a crucial role in outflow tract development, was decreased or absent in the outflow tract of the mutants. The decrease in transforming growth factor-&bgr;2 expression preceded neural crest cell death. Together, our results demonstrate that Pinch1 plays an essential role in neural crest development, perhaps in part through transforming growth factor-&bgr; signaling.

Identification of PINCH in Schwann cells and DRG neurons: Shuttling and signaling after nerve injury

Particularly interesting new cysteine‐histidine rich protein (PINCH) is a double zinc finger domain (LIM)‐only adapter protein that functions to recruit the integrin‐linked kinase (ILK) to sites of integrin activation. Genetic studies have shown that PINCH and ILK are required for integrin signaling. Since integrin activation is associated with Schwann cell migration, neurite outgrowth and regeneration, this study examined PINCH in the normal peripheral nervous system and after chronic constriction injury (CCI) in adult Sprague‐Dawley rats. Immunohistochemistry identified PINCH immunoreactivity in cell bodies of dorsal root ganglia (DRG) neurons, axons, satellite cells, and Schwann cells. PINCH immunostaining was localized to the membrane of uninjured DRG cell bodies consistent with its localization at a site of integrin activation. In contrast, 5 days following CCI, PINCH immunostaining was diffuse throughout the DRG cell cytoplasm. Confocal microscopy of primary and transformed Schwann cells localized PINCH in cytoplasmic, perinuclear and nuclear areas. Examination of the PINCH sequence revealed a putative leucine‐rich nuclear export signal (NES) and an overlapping basic nuclear localization signal (NLS). To demonstrate nuclear export of PINCH, rabbit anti‐PINCH IgG was microinjected into Schwann cell nuclei and allowed to combine with PINCH contained within the nucleus. Immunofluorescence showed that the PINCH and anti‐PINCH IgG complex rapidly translocated to the cytoplasm. Treatment with leptomycin B caused nuclear accumulation of PINCH, indicating that the CRM1 pathway mediates nuclear export of PINCH. ILK activity in Schwann cells was enhanced by platelet‐derived growth factor (PDGF) and tumor necrosis factor α. PINCH immunoprecipitates from PDGF‐ and TNFα‐stimulated Schwann cells contained several high‐molecular‐weight threonine‐phosphorylated proteins. Taken together, these results indicate that PINCH is an abundant shuttling/signaling protein in Schwann cells and DRG neurons. GLIA 41:213–223, 2003.

Severe hemolytic disease of the newborn due to anti-Vw and detection of glycophorin A antigens on the Miltenberger I sialoglycoprotein by Western blotting.

Abstract. Anti‐Vw detecting an antigen on Miltenberger I (Mi I) variant glycophorin A (GPA) has rarely been reported as a cause of hemolytic disease of the newborn (HDN). We report an infant with severe HDN due to anti‐Vw. Examination of the Vw+ erythrocytes of the father and paternal grandmother by sodium dodecylsulphate polyacrylamide gel electrophoresis showed an extra trypsin‐sensitive, periodic‐acid‐Schiff staining band, consistent with Mi I variant GPA. Staining of Western blots by monoclonal antibodies showed that normal paternal GPA expressed blood group M, while Mi I variant GPA expressed blood group N. Mi I variant GPA expressed the trypsin‐sensitive antigenic determinant detected by MoAb 10F7, indicating that the alterations known to occur in the trypsin‐sensitive fragment of Mi I variant GPA do not affect expression of the antigen detected by 10F7.

Effect of Crossmatching on Outcome in Organ Transplantation

The complement-dependent cytotoxicity (CDC) crossmatch and the flow cytometry crossmatch (FCXM) are both used prospectively in renal transplantation, and their use is under evaluation in other types of major organ transplantation. The FCXM is the more sensitive method and better predicts outcome in second and subsequent renal allografts. Improved survival has unmasked the detrimental effect of a positive crossmatch on outcome in liver transplantation. Because of the urgent need of liver transplant candidates, it is unrealistic to defer transplantation until a crossmatch-negative donor is found; however, additional therapeutic measures may be taken to improve outcome for crossmatch-positive liver recipients. Some reports suggest that prospective crossmatching may improve outcome for sensitized heart recipients, and, additionally, recent studies have demonstrated that HLA compatibility between donor and recipient is an independent variable affecting survival after heart transplantation, prompting a reassessment of the current practice of transplanting hearts without consideration of the HLA match.

Evolution of the Glycophorin Gene Family in the Hominoid Primates

Analysis of nucleotide sequences of the human glycophorin A (GPA) and glycophorin B (GPB) genes has indicated that the GPA gene most closely resembles the ancestral gene, whereas the GPB gene likely arose from the GPA gene by homologous recombination. To study the evolution of the glycophorin gene family in the hominoid primates, restricted DNA on Southern blots from man, pygmy chimpanzee, common chimpanzee, gorilla, orangutan, and gibbon was probed with cDNA fragments encoding the human GPA and GPB coding and 3′-untranslated regions. This showed the presence in all of the hominoid primates of at least one GPA-like gene. In addition, at least one GPB-like gene was detected in man, both chimpanzee species, and gorilla, strongly suggesting that the event that produced the GPB gene occurred in the common ancestor of man-chimpanzee-gorilla. An unexpected finding in this study was the conservation ofEcoRI restriction sites relative to those of the other four enzymes used; the significance of this observation is unclear, but raises the question of nonrandomness ofEcoRI restriction sites in noncoding regions. Further analysis of the evolution of this multigene family, including nucleotide sequence analysis, will be useful in clarification of the evolutionary relationships of the hominoid primates, in correlation with the structure and function of the glycophorin molecules, and in assessment of the role of evolution in the autogenicity of glycophorin determinants.

Phospholipid Dependence of Wrb Antigen Expression in Human Erythrocyte Membranes

Phospholipid depletion of human erythrocyte membranes by phospholipase A2 modification markedly decreased binding of two monoclonal antibodies to the Wrb (Wright) blood group determinant on glycophorin A. Binding of seven monoclonal antibodies to other glycophorin A determinants, including anti‐M and anti‐N, was unaffected by phospholipid depletion. These results indicate that Wrb antigen expression is phospholipid‐dependent in human erythrocyte membranes.