Immunophilins and their ligands: insights into survival and growth of human neurons

The immunophilin receptors implicated in generating the neurotrophic effects of FK506 and rapamycin (RM) are unknown. Our studies are directed at (1) characterizing the effects of FK506 and RM on human fetal neurons and glia (2) revealing the role played by the immunophilin FKBP receptors and downstream effectors in mediating the effects of FK506 and RM on human brain cells and (3) clarifying the role of immunophilins (IP) in the normal and degenerating human brain. These studies provide the basis for the implementation of the FDA-approved immunophilin ligands (IPL) in the pharmacologic treatment of Parkinson's disease (PD). Additionally, they establish a potential link to pathogenetic and repair mechanisms associated with neurodegeneration and propose FKBP12 and FKBP52 as substrates that can be targeted by future drug design endeavors. Our studies also show for the first time that the immunophilin FKBP is present in the human brain and that its levels are altered in the brain of patients with neurodegenerative diseases. The increased levels of FKBP12 in neurons situated in areas of degeneration suggest that it may become a novel marker of pathology. Although the precise role of this immunophilin in the normal and degenerating brain awaits further clarification, this study suggests that FKBP might play a role in neuroprotection against abnormal protein aggregation, as well as participate in axonal transport and synaptic vesicle assembly. The rotamase activity of FKBP is likely to underlie these functions. If this hypothesis is confirmed, therapeutic attempts using rotamase activity-inhibiting immunophilin ligand administration in neurodegenerative disease patients need to be carefully designed.     

p53, Ki-67, and serum alpha feto-protein as predictors of hepatocellular carcinoma recurrence in liver transplant patients.

Patients with hepatocellular carcinoma who undergo orthotopic liver transplantation (OLT) are at risk for post-transplant tumor recurrence. The aim of this study was to evaluate whether expression of p53 and Ki-67 in hepatocellular carcinoma lesions present in explanted liver tissue was associated with time to tumor recurrence after OLT. Subjects consisted of 20 consecutive patients who underwent OLT and were found to have hepatocellular carcinoma in the liver explant. Immunostaining for p53 and Ki-67 was performed by standard methods. The presence of nuclear immunostaining in >10% of the tumor tissue was considered positive. Time to recurrence of hepatocellular carcinoma after OLT was compared between patients with positive and negative immunostaining by the log rank test. Multivariate analysis was performed using a Cox regression model to control for potentially confounding clinical factors. Time to post-transplant hepatocellular carcinoma recurrence was significantly more rapid in p53+ (P=0.0007) and Ki-67+ cases (P=0.001). These associations remained significant in multivariate analysis. Furthermore, time to recurrent hepatocellular carcinoma was significantly shorter in patients with a serum alpha feto-protein (AFP) level >or=100 ng/ml at time of diagnosis, compared to those with an AFP level <100 ng/ml (P=0.003). In conclusion, expression of p53 and Ki-67 in hepatocellular carcinoma lesions, and a serum AFP level >or=100 ng/ml were associated with more rapid recurrence of hepatocellular carcinoma after OLT. Identification of patients at risk for early post-transplant recurrence could be used to guide surveillance and adjuvant treatment strategies.     

Roxithromycin, Clarithromycin, and Azithromycin Attenuate the Injurious Effects of Bioactive Phospholipids on Human Respiratory Epithelium in Vitro

The effects of the bioactive phospholipids (PL), platelet-activating factor (PAF), lyso-PAF, and lysophosphatidylcholine (LPC) on the beat frequency and structural integrity of human ciliated respiratory epithelium were studied in vitro, in the presence or absence of polymorphonuclear leukocytes (PMNL), the antimicrobial agents, roxithromycin, clarithromycin, and azithromycin and the antioxidative enzymes catalase and superoxide dismutase (SOD). All three PL caused dose-dependent slowing of ciliary beat frequency (CBF) and epithelial damage (ED) at concentrations 1 g/ml, which were unaffected by inclusion of the antimicrobial agents and antioxidative enzymes. When epithelial strips were exposed to the combination of PMNL and PL, there was significant potentiation of ciliary dysfunction and ED, which was ameliorated by pretreatment of the PMNL with the antimicrobial agents or by inclusion of catalase, but not SOD. These results demonstrate that LPC, PAF, and lyso-PAF cause epithelial damage by direct mechanisms which are oxidant-independent, as well as by indirect mechanisms involving phagocyte-derived reactive oxidants. Macrolides and azalide antimicrobial agents may have beneficial effects on airway inflammation in asthma and microbial infections by protecting ciliated epithelium against oxidative damage inflicted by PL-sensitized phagocytes.     

Estimating the relative frequency of leukodystrophies and recommendations for carrier screening in the era of next‐generation sequencing

Leukodystrophies are a heterogeneous group of heritable disorders characterized by abnormal brain white matter signal on magnetic resonance imaging (MRI) and primary involvement of the cellular components of myelin. Previous estimates suggest the incidence of leukodystrophies as a whole to be 1 in 7,000 individuals, however the frequency of specific diagnoses relative to others has not been described. Next generation sequencing approaches offer the opportunity to redefine our understanding of the relative frequency of different leukodystrophies. We assessed the relative frequency of all 30 leukodystrophies (associated with 55 genes) in more than 49,000 exomes. We identified a relatively high frequency of disorders previously thought of as very rare, including Aicardi Goutières Syndrome, TUBB4A‐related leukodystrophy, Peroxisomal biogenesis disorders, POLR3‐related Leukodystrophy, Vanishing White Matter, and Pelizaeus‐Merzbacher Disease. Despite the relative frequency of these conditions, carrier‐screening laboratories regularly test only 20 of the 55 leukodystrophy‐related genes, and do not test at all, or test only one or a few, genes for some of the higher frequency disorders. Relative frequency of leukodystrophies previously considered very rare suggests these disorders may benefit from expanded carrier screening.     

Immunoblot analysis of c-Met expression in human colorectal cancer: Overexpression is associated with advanced stage cancer

c-Met, the receptor of hepatocyte growth factor is known to be responsible for the motility and mitogenesis of epithelial cells including cancer cells. To investigate the significance of c-Met expression in human colorectal cancer (CRC), total cellular protein, extracted from 130 CRCs were examined by Western blot analysis. The signal was quantitated by ChemiImager™ 4000 Low Light Imaging System. c-Met expression was analyzed as the ratio of tumor to matched normal tissue (T/N) and expressed as fold-increase. The cellular localization of c-Met was assessed by immunohistochemistry. The T/N fold increase of c-Met varied from 0.2 to 10.7 with a mean of 3.41 ± 0.23 (mean ± SE). 69% primary CRC showed overexpression (T/N >2.0) of c-Met. Significantly higher c-Met levels were found in CRC with blood vessel invasion (P = 0.04), and in advanced stage (P = 0.04). No relationship was noted between c-Met expression and age, tumor size, location, differentiation. C-Met immunoreactivity was observed in the membrane and cytoplasm of cancer cells. Positive staining of endothelial cells of blood vessels within normal submucosa and tumor was also evident. C-Met protein is expressed at levels significantly higher than adjacent mucosa in most primary adenocarcinomas of the colon. Our results support an important role for c-Met in human CRC progression and metastasis.     

Immunohistochemistry (S 100, KL 1) and human papillomavirus DNA hybridization on Morbus Bowen and bowenoid papulosis

Summary In this study 55 paraffin embedded samples defined as Bowen's disease or bowenoid papulosis were investigated with antibodies against S 100 protein and keratins (KL 1). S 100-positive cells were quantified and related to defined section area of the epidermal compartment by computer-assisted image analysis. The density of S 100-positive cells was compared with normal skin and was particularly related to growth patterns and keratinization of the different lesions under study. S 100-positive dendritic cells were found to be reduced overall in bowenoid lesions when compared with normal skin. Lesions with high counts of S 100-positive dendritic cells most frequentty showed a solitary growth pattern with highly conserved architecture and differentiation and no tendency to stromal invasion. In contrast, cases with low counts of S 100-positive cells very often showed multifocal development, a high degree of architectural disturbance and dedifferentiation. In this group, stromal invasion (cases of invasive carcinoma associated with Bowen's disease) was seen more often. Interestingly, this latter group of cases also revealed a peculiar keratin pattern. Frequently, the basal cell layer was decorated with KL 1 antibody, which usually recognizes only suprabasaly located keratinocytes. No differences between Bowen's disease and bowenoid papulosis were found in terms of densities of S 100-positive dendritic cells and keratin pattern. In our experience, extragenital Bowen's disease and genital Bowen's disease can not be distinguished on purely morphological grounds or with the immunocytochemical approach presented here. Interestingly, when employing in situ hybridization with HPV 16 probes three of seven samples of genital Bowen's disease harboured HPV 16 DNA, whereas six cases of extragenital disease were negative.Supported by the Deutsche Forschungsgemeinschaft (Lo 285/2-4)     

Environment modulates naloxone's suppressive effect on feeding in diabetic and non-diabetic rats

We evaluated the effect of environment on naloxone-induced suppression of feeding in streptozotocin rats and sham injected controls. Naloxone was administered to animals fasted for 24 hours and food intake was measured at 30, 60 and 120 minutes. Diabetic rats, in their home cages, were insensitive to naloxone's suppressive effect for the first 30 minutes and the 5 mg/kg dose suppressed feeding only at 120 minutes. In control rats, feeding was suppressed at 1 and 5 mg/kg naloxone during the first 30 minutes. In contrast, when animals were placed in novel plastic cages, control animals were insensitive to naloxone at all time points at doses as high as 5 mg/kg. In novel cages, diabetic rats responded to doses of 1 and 5 mg/kg during the first 30 minute period by lowering food intake. It should also be noted that basal food intake was suppressed (40-53%) when animals were placed in novel cages. These data suggest that stress of a novel environment alters the neuroregulatory system involved in inducing feeding. Lack of response of normal rats to naloxone's suppressive effect in a novel environment suggests that (1) a non-opioid feeding system operates under these conditions, or (2) opioid receptors are occupied as a result of the release of endogenous opioids due to stress. The opposite result observed in the diabetics indicates that glucose has a modulating effect on opioid effects.