Effect of sirolimus on renal ischaemia/reperfusion injury in normotensive and hypertensive rats

Background. Renal ischaemia/reperfusion (I/R) injury and hypertension represent major alloantigen-independent factors contributing to the development of chronic allograft nephropathy of renal allografts. In the present study, we investigated the effect of the anti-proliferative immunosuppressant, sirolimus (SIR), in a model of accelerated renal injury in hypertensive transgenic rats (TGRs). Twenty anaesthetized uninephrectomized TGRs with renin overproduction [TGR(mREN2)27] and 20 normotensive Han SD (SD) rats as genetic controls had their renal pedicles clipped for 45 min and were subsequently treated with either SIR (0.5 mg/kg per day, orally) or placebo (n=10 in each group) for 16 weeks, after which time the kidneys were harvested for morphological and immunohistochemical analysis. High-renin hypertension aggravated the functional and structural changes induced by I/R in SD animals: both SIR-treated and untreated TGRs exhibited significantly greater proteinuria and suffered from more severe glomerulosclerosis (P<0.01) and vasculopathy (P<0.01), as well as compensatory renal hypertrophy (P<0.01) and tissue TGF-1 expression, than both normotensive SD groups (P<0.01). SIR-treated SD rats showed reduced proteinuria (P<0.01), glomerulosclerosis (P<0.01), and TGF-1 expression in the glomerular epithelium and proximal tubuli (P<0.05) compared with placebo-treated SD rats. SIR-treated TGRs had significantly lower proteinuria at week 4 after I/R (P<0.01) than placebo-treated TGRs, but there were no significant differences thereafter. Morphological patterns were similar in treated and untreated TGRs at week 16. High-renin-induced hypertension aggravated the renal injury induced by I/R. Sirolimus treatment ameliorated some late functional and morphological changes induced by I/R injury in hypertensive TGRs but, particularly, in normotensive SD rats.     

Commensal bacteria (normal microflora), mucosal immunity and chronic inflammatory and autoimmune diseases

Commensal microflora (normal microflora, indigenous microbiota) consists of those micro-organisms, which are present on body surfaces covered by epithelial cells and are exposed to the external environment (gastrointestinal and respiratory tract, vagina, skin, etc.). The number of bacteria colonising mucosal and skin surfaces exceeds the number of cells forming human body. Commensal bacteria co-evolved with their hosts, however, under specific conditions they are able to overcome protective host responses and exert pathologic effects. Resident bacteria form complex ecosystems, whose diversity is enormous. The most abundant microflora is present in the distal parts of the gut; the majority of the intestinal bacteria are Gram-negative anaerobes. More than 50% of intestinal bacteria cannot be cultured by conventional microbiological techniques. Molecular biological methods help in analysing the structural and functional complexity of the microflora and in identifying its components. Resident microflora contains a number of components able to activate innate and adaptive immunity. Unlimited immune activation in response to signals from commensal bacteria could pose the risk of inflammation; immune responses to mucosal microbiota therefore require a precise regulatory control. The mucosal immune system has developed specialised regulatory, anti-inflammatory mechanisms for eliminating or tolerating non-dangerous, food and airborne antigens and commensal micro-organisms (oral, mucosal tolerance). However, at the same time the mucosal immune system must provide local defense mechanisms against environmental threats (e.g. invading pathogens). This important requirement is fulfilled by several mechanisms of mucosal immunity: strongly developed innate defense mechanisms ensuring appropriate function of the mucosal barrier, existence of unique types of lymphocytes and their products, transport of polymeric immunoglobulins through epithelial cells into secretions (sIgA) and migration and homing of cells originating from the mucosal organised tissues in mucosae and exocrine glands. The important role of commensal bacteria in development of optimally functioning mucosal immune system was demonstrated in germ-free animals (using gnotobiological techniques). Involvement of commensal microflora and its components with strong immunoactivating properties (e.g. LPS, peptidoglycans, superantigens, bacterial DNA, Hsp) in etiopathogenetic mechanism of various complex, multifactorial and multigenic diseases, including inflammatory bowel diseases, periodontal disease, rheumatoid arthritis, atherosclerosis, allergy, multiorgan failure, colon cancer has been recently suggested. Animal models of human diseases reared in defined gnotobiotic conditions are helping to elucidate the aetiology of these frequent disorders. An improved understanding of commensal bacteria-host interactions employing germ-free animal models with selective colonisation strategies combined with modern molecular techniques could bring new insights into the mechanisms of mucosal immunity and also into pathogenetic mechanisms of several infectious, inflammatory, autoimmune and neoplastic diseases. Regulation of microflora composition (e.g. by probiotics and prebiotics) offers the possibility to influence the development of mucosal and systemic immunity but it can play a role also in prevention and treatment of some diseases.     

Development of early motor learning and topical motor skills in a model of cerebellar degeneration

The development of motor learning and topical motor skills has been studied during the first month of life in neurodeficient Lurcher mutant mice derived from C3H strain. The examination of motor functions was conducted using four methods when animals were consecutively placed on: a horizontal wire, a rotating cylinder, a bridge and slanting ladder (on the rotating cylinder and slanting ladder in three different starting positions). A total of 150 animals were used, half being heterozygous mutants (+/Lc), the remaining consisting of their healthy littermates, homozygous wild-type mice (+/+). Some animals of both types were confronted with the tasks in four testing days without practice while the others were trained in all of these methods one time daily at the age of 3, 6, 9, 12, 15, 20, 25 and 30 days. The results of the research are expressed as a percentage of reaching criteria in the trained and untrained mice of both types (+/Lc, +/+), respectively on days 15, 20, 25, and 30. Because of their motor handicap, Lurcher mutants showed significantly worse results than normal mice in almost all of the tests. The most interesting observations of the study were obtained on the rotating cylinder and the slanting ladder tests where mainly untrained mutants were unable to cope promptly with the different starting position and failed. However, in 30-day-old Lurchers the effect of learning was relatively better in some tests when compared with the wild-type of mice. Experiments showed that, in spite of progressive cerebellar degeneration, the ability of motor learning in Lurcher mutants survived to a certain degree. The worsening of results after frequent training in both trained Lurchers and normal mice aged 20 days as compared with untrained ones is discussed within the context of the “overtraining reversal effect” and other possible hypotheses.     

The role of hedgehog signaling in the development of the zebrafish visual system

The vertebrate visual system is a region of the nervous system that is characterized by relative simplicity, and its development has hence been studied intensively, to serve as a paradigm for the rest of the central nervous system. The zebrafish model organism offers an impressive array of tools to dissect this process experimentally, and in recent years has helped to significantly deepen our understanding of the development of the visual system. A number of these studies have focused on the role of the Hedgehog family of secreted signaling molecules in eye development, and this is the main topic of this review. Hedgehog signaling plays an important role in all major steps of visual system development, starting with the regionalization of the eye primordium into proximal and distal territories, continuing with the control of cellular differentiation in the retina, and ending with the guidance of axonal projections from the retina to the optic centers of the brain.     

Regulation of tissue factor expression in smooth muscle cells with nitric oxide

OBJECTIVE: This study was undertaken to determine the effect of nitric oxide (NO) on tissue factor (TF) expression in vascular smooth muscle cells. STUDY DESIGN: Rat aortic smooth muscle cells (RASMCs) were exposed to NO delivered exogenously with the NO donor S-nitroso-N-acetylpenicillamine (SNAP) or produced endogenously after infection with an adenoviral vector carrying human inducible NO synthase (AdiNOS). Functional TF activity was assessed with chromogenic TF assay. TF antigen was determined with immunohistochemistry. Northern blot analysis was used to determine steady- state TF messenger RNA (mRNA). Electrophoretic mobility gel shift assay was performed to determine the nuclear binding activity of nuclear factor kappa-B (NFkappaB). NFkappaB activity was inhibited by either prior transduction of RASMCs with mutant IkappaB or treatment with pyrrolidine dithiocarbamate. RESULTS: RASMCs exposed to SNAP or infected with AdiNOS exhibited increased functional TF activity and antigen. Regardless of the source of NO, a time-dependent and concentration-dependent increase in TF activity was observed. Steady-state TF mRNA levels were also increased by NO delivered via either method. NFkappaB nuclear binding activity was also increased by NO. Inhibition of NFkappaB activity by either pyrrolidine dithiocarbamate treatment or mutant IkappaB transduction abrogated NO-induced enhancement of TF mRNA and functional activity. CONCLUSION: In RASMC, NO exposure results in upregulation of TF functional activity, antigen, and mRNA. This effect appears to be mediated by an NFkappaB-dependent pathway.     

Chemosensitization by a non-apoptogenic heat shock protein 70-binding apoptosis-inducing factor mutant

Heat shock protein 70 (HSP70) inhibits apoptosis and thereby increases the survival of cells exposed to a wide range of lethal stimuli. HSP70 has also been shown to increase the tumorigenicity of cancer cells in rodent models. The protective function of this chaperone involves interaction and neutralization of the caspase activator apoptotic protease activation factor-1 and the mitochondrial flavoprotein apoptosis-inducing factor (AIF). In this work, we determined by deletional mutagenesis that a domain of AIF comprised between amino acids 150 and 228 is engaged in a molecular interaction with the substrate-binding domain of HSP70. Computer calculations favored this conclusion. On the basis of this information, we constructed an AIF-derived protein, which is cytosolic, noncytotoxic, yet maintains its capacity to interact with HSP70. This protein, designated ADD70, sensitized different human cancer cells to apoptosis induced by a variety of death stimuli by its capacity to interact with HSP70 and therefore to sequester HSP70. Thus, its chemosensitizing effect was lost in cells in which inducible HSP70 genes had been deleted. These data delineate a novel strategy for the selective neutralization of HSP70.     

Hippocampal negative event‐related potential recorded in humans during a simple sensorimotor task occurs independently of motor execution

A hippocampal‐prominent event‐related potential (ERP) with a peak latency at around 450 ms is consistently observed as a correlate of hippocampal activity during various cognitive tasks. Some intracranial EEG studies demonstrated that the amplitude of this hippocampal potential was greater in response to stimuli requiring an overt motor response, in comparison with stimuli for which no motor response is required. These findings could indicate that hippocampal‐evoked activity is related to movement execution as well as stimulus evaluation and associated memory processes. The aim of the present study was to investigate the temporal relationship between the hippocampal negative potential latency and motor responses. We analyzed ERPs recorded with 22 depth electrodes implanted into the hippocampi of 11 epileptic patients. Subjects were instructed to press a button after the presentation of a tone. All investigated hippocampi generated a prominent negative ERP peaking at ～420 ms. In 16 from 22 cases, we found that the ERP latency did not correlate with the reaction time; in different subjects, this potential could either precede or follow the motor response. Our results indicate that the hippocampal negative ERP occurs independently of motor execution. We suggest that hippocampal‐evoked activity, recorded in a simple sensorimotor task, is related to the evaluation of stimulus meaning within the context of situation. © 2013 Wiley Periodicals, Inc.     

Alzheimer's Therapeutics: Translation of Preclinical Science to Clinical Drug Development

Over the past three decades, significant progress has been made in understanding the neurobiology of Alzheimer''s disease. In recent years, the first attempts to implement novel mechanism-based treatments brought rather disappointing results, with low, if any, drug efficacy and significant side effects. A discrepancy between our expectations based on preclinical models and the results of clinical trials calls for a revision of our theoretical views and questions every stage of translation—from how we model the disease to how we run clinical trials. In the following sections, we will use some specific examples of the therapeutics from acetylcholinesterase inhibitors to recent anti-Aβ immunization and γ-secretase inhibition to discuss whether preclinical studies could predict the limitations in efficacy and side effects that we were so disappointed to observe in recent clinical trials. We discuss ways to improve both the predictive validity of mouse models and the translation of knowledge between preclinical and clinical stages of drug development.