A pleiotropy scan to discover new susceptibility loci for pancreatic ductal adenocarcinoma.

Pleiotropic variants (i.e., genetic polymorphisms influencing more than one phenotype) are often associated with cancer risk. A scan of pleiotropic variants was successfully conducted ten years ago in relation to pancreatic ductal adenocarcinoma susceptibility. However, in the last decade, genetic association studies performed on several human traits have greatly increased the number of known pleiotropic variants. Based on the hypothesis that variants already associated with a least one trait have a higher probability of association with other traits, 61,052 variants reported to be associated by at least one genome wide association study (GWAS) with at least one human trait were tested in the present study consisting of two phases (discovery and validation), comprising a total of 16,055 pancreatic ductal adenocarcinoma (PDAC) cases and 212,149 controls. The meta-analysis of the two phases showed two loci (10q21.1-rs4948550 (P=6.52×10-5) and 7q36.3-rs288762 (P=3.03×10-5) potentially associated with PDAC risk. 10q21.1-rs4948550 shows a high degree of pleiotropy and it is also associated with colorectal cancer risk while 7q36.3-rs288762 is situated 28,558 base pairs upstream of the Sonic Hedgehog (SHH) gene, which is involved in the cell differentiation process and PDAC etiopathogenesis. In conclusion, none of the single nucleotide polymorphisms (SNPs) showed a formally statistically significant association after correction for multiple testing. However, given their pleiotropic nature and association with various human traits including colorectal cancer, the two SNPs showing the best associations with PDAC risk merit further investigation through fine mapping and ad hoc functional studies.

Long term effects of cigarette smoke extract or nicotine on nerve growth factor and its receptors in a bronchial epithelial cell line.

Long-term exposure to cigarette smoke induces severe injuries to respiratory system through several mechanisms, some of them are well defined, but many others are not yet elucidated. Beside its classical role in nervous system, we have previously shown that Nerve Growth Factor (NGF) and its receptors have a crucial role in airway inflammatory diseases, such as Chronic Obstructive Pulmonary Disease. To expand our knowledge about the relevance of NGF and its receptors in airway diseases induced by cigarette smoking, we exposed for 16 weeks the bronchial epithelial cell line BEAS-2B to sub-toxic concentrations of whole cigarette smoke extract or pure nicotine. Viability, cell cycle gene expression, cell morphology and migration ability were tested and compared to NGF release and gene expression. Modulation of its receptors TrKA and p75NTR was also analyzed. The present study shows that long term exposure of BEAS-2B cells to cigarette smoke extract or nicotine induces: (A) differences: in cell viability, in the expression of cell cycle-related genes, in NGF release and in gene expression of NGF and its receptors; (B) similarities: in morphology and migration ability. Taken together, our data provide new insights about the biological role of NGF and its receptors in airway diseases induced by long-term cigarette smoking and, finally, our data evidence the opportunity not to use nicotine lozenges or e-cigarettes as anti smoking replacement therapy in patients with a previous airway disease according to the ability of nicotine to increase the amount of the pro-inflammatory cytokine NGF into the bronchial environment.

Gene Expression and Localization of NGF and Its Cognate Receptors NTRK1 and NGFR in the Sex Organs of Male Rabbits.

Experiments were devised to characterize the expression of nerve growth factor, beta polypeptide (NGF), and its cognate receptors neurotrophic tyrosine kinase receptor type 1 (NTRK1) and nerve growth factor receptor (NGFR) in rabbit male sex organs, as well as the concentrations of NGF in both seminal and blood plasma of sexually mature male rabbits. Immunoreactivity and gene expression for NGF and cognate receptors were detected in testis, prostate gland and seminal vesicle. The highest levels of NGF and NTRK1 transcripts were found in the prostate, while intermediate expressions were found in the testis. NGFR transcripts were expressed at the same levels in both testis and prostate and were more abundant than in seminal vesicles. The widespread distribution of NGF in all prostate glandular cells, together with its relative high mRNA abundance, confirms that the prostate of rabbits is the main source of this neurotrophin. In conclusion, the present data suggest that the NGF system is involved in the testicular development and spermatogenesis of rabbits and that NGF may act as a potential ovulation-inducing factor being abundantly present in the seminal plasma.

[Identification of candidate genes and expression profiles, as doping biomarkers]. / Individuazione di geni candidati e profili d'espressione, come indicatori molecolari di assunzione di sostanze dopanti.

Administration of prohibited substances to enhance athletic performance represents an emerging medical, social, ethical and legal issue. Traditional controls are based on direct detection of substances or their catabolites. However out-of-competition doping may not be easily revealed by standard analytical methods. Alternative indirect control strategies are based on the evaluation of mid- and long-term effects of doping in tissues. Drug-induced long-lasting changes of gene expression may be taken as effective indicators of doping exposure. To validate this approach, we used real-time PCR to monitor the expression pattern of selected genes in human haematopoietic cells exposed to nandrolone, insulin-like growth factor I (IGF-I) or growth hormone (GH). Some candidate genes were found significantly and consistently modulated by treatments. Nandrolone up-regulated AR, ESR2 and PGR in K562 cells, and SRD5A1, PPARA and JAK2 in Jurkat cells; IGF-I up-regulated EPOR and PGR in HL60 cells, and SRD5A1 in Jurkat; GH up-regulated SRD5A1 and GHR in K562. GATA1 expression was down-regulated in IGF-1-treated HL60, ESR2 was down-regulated in nandrolone-treated Jurkat, and AR and PGR were down-regulated in GH-treated Jurkat. This pilot study shows the potential of molecular biology-based strategies in anti-doping controls.

Diffusive and convective transport through hollow fiber membranes for liver cell culture.

For an efficient membrane bioreactor design, transport phenomena determining the overall mass flux of metabolites, catabolites, cell regulatory factors, and immune-related soluble factors, need to be clarified both experimentally and theoretically. In this work, experiments and calculations aimed at discerning the simultaneous influence of both diffusive and convective mechanisms to the transport of metabolites. In particular, the transmembrane mass flux of glucose, bovine serum albumin (BSA), APO-transferrin, immunoglobulin G, and ammonia was experimentally measured, under pressure and concentration gradients, through high-flux microporous hydrophilic poly-ether-sulphone (PES-HFMs) and poly-sulphone hollow fiber membranes (PS-HFMs). These data were analyzed by means of a model based on the mechanism of capillary pore diffusion, assuming that solute spherical molecules pass through an array of solvent-filled cylindrical pores with a diffusive permeation corrected for friction and steric hindrances. Additionally, resistances to the mass transfer were taken into account. Convective permeation data were discussed in terms of morphological properties of the polymeric membranes, molecular Stokes radius, and solute-membrane interactions according to information given by contact angle measurements. The observed steady-state hydraulic permeance of PS-HFMs was 0.972 L/m2hmbar, about 15.6-fold lower than that measured for PES-HFMs (15.2 L/m2h); in general, PS-HFMs provided a significant hindrance to the transport of target species. Diffusion coefficients of metabolites were found to be similar to the corresponding values in water through PES-HFMs, but significantly reduced through PS-HFMs (D(Glucose)(Membrane)=2.8x10(-6)+/-0.6x10(-6)cm2/s, D(BSA)(Membrane)=6.4 x 10(-7)+/-1 x 10(-7)cm(/s, D(Apotransferrin)(Membrane)=2.3 x 10(-7)+/-0.25 x 10(-7)cm2/s).

New modified polyetheretherketone membrane for liver cell culture in biohybrid systems: adhesion and specific functions of isolated hepatocytes.

There has been growing interest in innovative materials with physico-chemical properties that provide improved blood/cell compatibility. We propose new polymeric membranes made of modified polyetheretherketone (PEEK-WC) as materials with potential for use in biohybrid devices. PEEK-WC exhibits high chemical, thermal stability and mechanical resistance. Owing to its lack of crystallinity this polymer can be used for preparing membranes with cheap and flexible methods. We compared the properties of PEEK-WC membranes to polyurethane membranes prepared using the same phase inverse technique and commercial membranes. The physico-chemical properties of the membranes were characterised by contact angle measurements. The different parameters acid (gamma+), base (gamma-) and Lifshitz-van der Waals (gammaLW) of the surface free energy were calculated according to Good-van Oss's model. We evaluated the cytocompatibility of PEEK-WC membranes by culturing hepatocytes isolated from rat liver. Cell adhesion and metabolic behaviour in terms of ammonia elimination, urea synthesis and protein synthesis were evaluated during the first days of culture. Liver cells adhered and formed three-dimensional aggregates on the most tested membranes. PEEK-WC membranes promoted hepatocyte adhesion most effectively. Urea synthesis, ammonia elimination and protein synthesis improved significantly when cells adhered to PEEK-WC membrane. The considerable metabolic activities of cells cultured on this membrane confirmed the good structural and physico-chemical properties of the PEEK-WC membrane that could be a promising biomaterial for cell culture in biohybrid devices.

Prosaposin is immunolocalized to muscle and prosaptides promote myoblast fusion and attenuate loss of muscle mass after nerve injury.

Prosaposin is the precursor of the saposins and has both neurotrophic and myelinotrophic activity in vitro and in vivo. Using an antibody specific for the holoprotein, an immunocytochemical survey demonstrated intense staining of adult rat skeletal, cardiac, and smooth muscle cells. Prosaposin immunoreactivity in muscle appears dependent on innervation, as denervated adult rat skeletal muscles showed decreased immunostaining that returned to normal levels after reinnervation. TX14(A), a peptide derived from the neurotrophic sequence of prosaposin, attenuated the decline in muscle mass loss following nerve injury induced by a constricting ligature. In vitro, both L6 myoblasts and primary chick-embryo myoblasts showed similar prosaposin immunopositivity, mainly in myotubes. TX14(A) induced a threefold increase in L6 myoblast fusion during early stages of differentiation without affecting cell proliferation. The fusion process was decreased in vitro in a dose-dependent fashion by addition of a neutralizing anti-prosaposin antibody. These data suggest that, in addition to neurotrophic and myelinotrophic activities, prosaposin has myotrophic properties.

Nerve growth factor (NGF) influences differentiation and proliferation of myogenic cells in vitro via TrKA.

Classic studies have established that muscle cells exert trophic actions on neurons of the developing peripheral nervous system through the production of neurotrophins. For this reason neurotrophins are also known as 'target-derived factors'. During differentiation, muscle cells also express some neurotrophin receptors, such as the low-affinity p75 neurotrophin receptor, which binds all neurotrophins, and the high affinity tyrosine kinase receptor TrKA, nerve growth factor (NGF) transducing receptor. The functional roles of these receptors in muscle cells are still unclear and only fragmentary and controversial data are available regarding the responsiveness of muscle cells to NGF. The aim of the present study is to investigate the effects of NGF on cells of myogenic lineage. The rat myogenic cell line L6, primary cultures of adult human myoblasts, and the human rhabdomyosarcoma cell line TE-671 were used in this study. As expected, all the three cell types expressed NGF, p75 and TrKA. NGF was expressed by L6 and primary myoblasts following differentiation, but it was constitutively expressed at high levels in the TE-671 rhabdomyosarcoma cells. In L6 myoblasts, p75 receptor was expressed in myoblasts but not in myotubes early after plating; while some primary human myoblasts expressed it at all the time-points tested. Some fusiform cells of the TE-671 rhabdomyosarcoma cell line also expressed p75. TrKA was constitutively immunodetected in all the three cell lines, suggesting that these cells may respond to NGF. Addition of exogenous NGF increased the fusion rate of both primary and L6 myoblasts, as well as the proliferation of the slowly dividing primary myoblasts. Consistently, blocking the action of endogenously produced NGF with a specific neutralizing antibody decreased the percentage of fusion in both primary and L6 myoblasts. On the contrary, blocking the binding of NGF to p75 did not affect the percentage of fusion. Furthermore, neither exogenous NGF nor NGF- or p75-neutralizing antibodies appeared to affect the rhabdomyosarcoma cells, which have a high proliferation rate and do not fuse. Pharmacological inhibition of TrKA signal transduction with K252a (in the nM range) and tyrphostin AG879 (in the low microM range) resulted in a dramatic dose-dependent decrease in proliferation of all of the myogenic cell lines tested. Interestingly, this was especially evident in the rapidly dividing rhabdomyosarcoma cell line. The TrKA inhibitors also blocked fusion of L6 and primary myoblasts and induced morphological changes characterized by the flattening of the cells and a 'spider-like' rearrangement of the intermediate filaments in all three cell lines with some minor differences. A transfection study showed that p75-overexpressing L6 cells do not fuse and present changes in their morphology similar to the TrKA-inhibitors treated L6 cells. These data support the notion that NGF expression in skeletal muscle is not only associated with a classical target-derived neurotrophic function for peripheral nervous system neurons, but also with an autocrine action which affects the proliferation, fusion into myotubes, and cell morphology of developing myoblasts. The present data also suggest that these effects of NGF are mediated by TrKA receptors and that a sustained presence of NGF is needed for increase fusion into myotubes. Lastly, the dramatic anti-proliferative effect of TrKA inhibitors on myogenic cells, and especially on the TE-671 rhabdomyosarcoma cell line, suggests that pharmacological interference with NGF signal transduction could be effective in the control of these malignancies.

Regulation of the p75 neurotrophin receptor in a rat myogenic cell line (L6).

Neurotrophins are expressed in muscle cells both during development and postnatally. Furthermore, during development muscle cells express high levels of the common p75 neurotrophin receptor, which binds all neurotrophins. Only fragmentary and controversial data are available regarding the responsiveness of muscle cells to neurotrophins and the importance of low-affinity p75 receptor in muscle development. The present study investigates in vitro the immunocytochemical expression of p75 in a rat myogenic cell line (L6) at various time points and in response to different coating substrates as a first step in elucidating the regulation of p75 in muscle. We found that in L6 myoblasts, p75 is expressed only at very early stages of maturation and its levels of expression are regulated by the nature of the coating substrates. p75 expression decreases in cells growing on substrates more suitable for myoblast fusion into myotubes. Time course analysis indicates a reverse correlation between myoblast fusion into myotubes and the levels of p75 expression. Myotubes were always p75 negative. Substrates not suitable for the fusion process induced a prolonged presence of p75 in myoblasts with an increase of their apoptosis. We conclude that expression of p75, at least in this in vitro condition, is regulated by the stages of myoblast differentiation and the nature of the coating substrates. According to the observed time- and substrate-related evidences, future studies should investigate in vivo both the regulation of p75 in the myoblast fusion and the effects and the importance of neurotrophins binding during myoblast differentiation.

Modulation of serotonin 5-HT3 receptor expression in injured adult rat spinal cord motoneurons.

The effects of sciatic nerve lesions on the expression of serotonin 5-HT3 receptor (5-HT3R) alpha subunit in motoneurons of the spinal cord was investigated by semi-quantitative immunohistochemistry. Following sciatic nerve crush, a significant reduction in density of staining in motoneurons was observed in longitudinal sections of the ventral horn at 3 and 15 days on the lesioned side when compared to the contralateral side (p<0.01). At 30 days after crush, after completion of sciatic nerve regeneration and reinnervation of peripheral targets, intensity of staining had returned to normal. Conversely, after sciatic nerve cut, a lesion that does not allow for target reinnervation, highly significant reductions were observed at 3, 15, 30 and 45 days. These results suggest a role for functional contacts with muscular targets in the maintenance of 5-HT3R expression in spinal motoneurons.

Effects of sciatic nerve grafts on choline acetyltransferase and p75 expression in transected adult hypoglossal motoneurons.

We previously reported that a permanent transection of adult rat sciatic and hypoglossal nerves resulted in distinct changes in the levels of both low-affinity nerve growth factor receptor (p75) and choline acetyltransferase in the corresponding motoneurons as determined by immunoreactivity. Permanent axotomy of hypoglossal motoneurons induced a progressive loss of choline acetyltransferase immunoreactivity and a persistent expression of p75 immunoreactivity, phenomena that were not observed in spinal motoneurons. These observations indicated that spinal and brainstem motoneurons respond to permanent axotomy with a differential immunoreactivity for p75 and choline acetyltransferase. Such differences could be ascribed to specific intrinsic properties of each population of motoneurons or, alternatively, to different factors present in the periphery (nerve stump or target muscle). The aim of the present study was to test these two possibilities by determining if a segment of sciatic nerve transplanted to a transected hypoglossal nerve may counteract or attenuate the loss of choline acetyltransferase immunoreactivity in injured hypoglossal motoneurons. In addition, as further parameter, we analysed the presence of p75 immunoreactivity. Prior to grafting, segments of sciatic nerve were prepared by one of three methods: (i) a fresh piece; (ii) a degenerated piece; and (iii) a heated piece. Seven and 30 days following the placement of grafts, hypoglossal motoneurons were analysed for choline acetyltransferase and p75 immunolabelling. The results revealed that viable sciatic grafts (fresh and degenerated) are able to partially attenuate the loss in the number of choline acetyltransferase-positive injured hypoglossal motoneurons, even if an important decrease in choline acetyltransferase still persists with respect to the contralateral nucleus. In addition, viable sciatic grafts decreased the number of p75 immunoreactive hypoglossal motoneurons both at seven and at 30 days. In conclusion, the effects of viable sciatic grafts on the number of choline acetyltransferase and p75-labelled hypoglossal motoneurons indicate that these adult neurons are able to respond to factors released from the sciatic nerve, and that the number of injured motoneurons positive for choline acetyltransferase and p75 can be influenced by the presence of factors that may reach their proximal stumps. Furthermore, we hypothesize that the differential expression patterns between hypoglossal and sciatic motoneurons may be due, at least in part, to factors released from the nerve trunks themselves.

Axotomy induces a different modulation of both low-affinity nerve growth factor receptor and choline acetyltransferase between adult rat spinal and brainstem motoneurons.

Adult rat spinal and brainstem motoneurons re-express low-affinity nerve growth factor receptor (p75) after their axotomy. We have previously reported and quantified the time course of this reexpression in spinal motoneurons following several types of injuries of the sciatic nerve. Other studies reported the reexpression of p75 in axotomized brainstem motoneurons. Results of these previous studies differed regarding the type of the most effective triggering injury for p75 reexpression, the relative duration of this reexpression and the decrease of choline acetyltransferase (ChAT) immunoreactivity (-IR) following a permanent axotomy of spinal or brainstem motoneurons. These differences suggest that these two populations of motoneurons respond to axotomy with a different modulation of p75 and ChAT expression. The aim of the present study was to determine whether differential modulation exists. We have analyzed and quantified the presence of p75- and ChAT-IR motoneurons in the hypoglossal nucleus following the same types of injury and the same time course we previously used for sciatic motoneurons. The results show that a nerve crush is the most effective triggering injury for p75 and that it induces similar temporal patterns of p75 and ChAT expression for sciatic and hypoglossal motoneurons. In contrast, a cut injury of the sciatic and hypoglossal nerves resulted in distinct temporal courses of both p75 and ChAT expression between these two populations of motoneurons. In fact, a permanent axotomy of the hypoglossal motoneurons induced i) a much longer maintenance phase for p75 than in sciatic motoneurons and ii) a progressive loss of ChAT-IR with a successive return to normal values in contrast to the modest decrease in the sciatic motoneurons. This evidence indicates that spinal and brainstem motoneurons respond to a permanent axotomy with a different modulation of p75 and ChAT expression. Altogether, the present data and the reported evidence of a differential post-axotomy cell death support the hypothesis that these two populations of motoneurons undergo different dynamic changes after axotomy.

Modulation of low-affinity nerve growth factor receptor in injured adult rat spinal cord motoneurons.

Spinal and brainstem motoneurons of the adult rat reexpress low-affinity nerve growth factor receptor (LNGFR) and its mRNA after axotomy. We have previously reported the time courses of this reexpression after cut (no regeneration) or crush (followed by regeneration) of the sciatic nerve. We have shown that the length of the different phases of this reexpression (appearance, maintenance and disappearance) can vary according to the type of axotomy. With the present study we expand our previous data and describe and analyze the modulation the LNGFR expression in adult spinal cord motoneurons following different lesion paradigms. In one approach we have imposed three traumatic injuries that still allow regeneration of the sciatic nerve but with a different time course with respect to the crush injury (application of a silicone regeneration chamber, multiple crushes and delayed repair of ligated nerves). In a second approach, we have determined the capability of three toxic or metabolic injuries to induce LNGFR expression without any direct trauma of the nerve (experimental diabetogenesis, botulinum and alpha-bungarotoxin intoxication and 2,5-hexanedione intoxication). In a third approach, we have investigated the effect of the block of the axoplasmic transport on the LNGFR expression following different topical applications of vincristine combined with a nerve crush. The results we present are consistent with the idea that: (1) LNGFR immunoreactivity in adult motoneurons is expressed by motoneurons that are attending to an axonal outgrowth and not a generic signal of cellular damage or impairment of the motor function; (2) LNGFR expression in these motoneurons is related to and parallels the outgrowth process time frame, and (3) the signal/s that trigger and sustain this reexpression may be retrogradely transported from the periphery.

Presumptive Renshaw cells contain decreased calbindin during recovery from sciatic nerve lesions.

A subpopulation of calbindin-immunoreactive neurons in lamina VII of the spinal cord has been identified by its location as Renshaw cells, the anatomical substrate for recurrent inhibition. The expression of calbindin (28 kDa) in these calbindin-containing rat ventral horn interneurons was studied with immunocytochemistry after sciatic nerve injuries. One week after axotomy calbindin immunoreactivity was strongly reduced on the lesioned side between levels L4 and L6, while calbindin-containing neurons and fibers were still numerous contralaterally and cranially to the lesioned levels. With the progression of regeneration, calbindin-immunoreactive neurons reappeared, reaching a normal distribution 6-8 weeks after the crush. Similar changes could be mimicked by the intramuscular administration of botulinum toxin. These results suggest that calbindin expression in putative Renshaw cells of the spinal cord might be functionally responsive and that maintenance of calbindin expression may depend on the integrity of motoneurons and neuromuscular transmission.

Potential regulation by trophic factors of low-affinity NGF receptors in spinal motor neurons.

Developing spinal motor neurons (SMN) express low-affinity nerve growth factor receptors (LNGFR) but not high-affinity transducing NGF receptors. Moreover, SMN are not supported by NGF in vitro. In the normal adult rat most SMN are not LNGFR immunoreactive (LNGFR-IR), but they transiently reexpress LNGFR (though not the high-affinity receptor) after peripheral nerve injury. With a cut lesion of the sciatic nerve (when only a neuroma forms), the number of LNGFR-IR SMN at L4-L6 rapidly increases to a maximum between day 1 and 7 and returns to baseline levels by day 30. After a crush lesion (accompanied by regeneration to the muscle), LNGFR-IR SMN appear in about the same numbers, but they start to disappear 1 week later. We speculate that the similar appearance and differential decline of LNGFR-IR seen after the two types of lesions are regulated by the availability of a common signal such as ciliary neurotrophic factor. The adult SMN model provides a good opportunity to investigate the reexpression of LNGFR after peripheral nerve injury, and more generally, the unknown role and regulation of LNGFR.

Nerve growth factor receptor immunoreactivity in neurons of the normal adult rat spinal cord and its modulation after peripheral nerve lesions.

Motoneurons of the rat spinal cord express low-affinity nerve growth factor receptor (LNGFR) and corresponding mRNA during development, and re-express it after their axotomy by peripheral nerve injury. The present study establishes the anatomical and quantitative baseline of LNGFR immunoreactive (LNGFR-IR) neurons of the entire normal adult female rat and then investigates the temporal course for the re-expression of LNGFR-IR in lumbar motoneurons after either a crush lesion (which is followed by regeneration and reconnection to the muscle) or a cut lesion with removal of the distal stump (where a neuroma but no reconnection is formed). In the normal adult spinal cord, two types of LNGFR-IR neurons were recognized: (1) small populations of large motoneurons located in the ventral horn mainly in correspondence to the regions of the phrenic, cremasteric and dorsolateral nuclei, and (2) a more numerous and more dorsally located population of small neurons. With a sciatic cut lesion, the number of LNGFR-IR motoneurons at spinal levels L4-L6 rapidly and dramatically increased to a maximum between post-lesion days 1 and 7, apparently involving most axotomized motoneurons of the region, and returned to the baseline level by day 30. With a crush lesion, similar numbers and virtually the same time-course of LNGFR-IR appearance were seen, but the onset of progressive disappearance of LNGFR-IR neurons was delayed by one week, so that at 30 days, the most caudal motoneurons (which are last to reach their target) were still LNGFR-IR. Comparison of these two time courses gives clues to the kind of signals that may be involved in initiating and/or maintaining the LNGFR response.

Immunolocalization of ciliary neuronotrophic factor in adult rat sciatic nerve.

Two rabbit polyclonal antibodies were raised against synthetic peptides corresponding to residue numbers 45-59 and 181-200 of rat ciliary neuronotrophic factor (CNTF). The resulting antibodies were purified by affinity chromatography and both purified antibodies reacted by enzyme-linked immunoassay (ELISA) and immunoblotting with rat sciatic nerve CNTF. The anti-CNTF peptide antibodies were used to immunostain sections of adult rat sciatic nerve, previously known as the richest tissue source of CNTF. By light microscopy both antibodies appeared to stain exclusively Schwann cells and axons and both did so with the same pattern of specific staining. Immunostaining was eliminated by absorption of the anti-peptide antibodies with either their corresponding peptide or with purified rat nerve CNTF or by using purified nonspecific IgG. Schwann cells were stained and in semi-thin sections this staining appeared to be in the Schwann cell cytoplasm. Axons could be stained in addition to Schwann cells providing higher concentrations of antibodies were used. Epineurial, endoneurial and endothelial cells appeared unstained. Since all Schwann cells and axons appear to contain CNTF and since CNTF is known to act in vitro to support sensory and sympathetic ganglionic and motor neurons, we suggest that Schwann cells may normally provide CNTF to those neurons contributing axons to the peripheral nerve.

Accuracy of reinnervation by peripheral nerve axons regenerating across a 10-mm gap within an impermeable chamber.

The axon regeneration following a peripheral nerve injury often fails to restore a complete functional recovery. One of the causes of this unsatisfactory result has been attributed to regrowth of regenerating fibers to inappropriate peripheral targets. The accuracy of reinnervation by axons regenerating across a 10-mm gap within an impermeable chamber has been studied by using a sequential retrograde double-labeling technique. Despite the long gap between the nerve stumps, at 4 weeks a mean of 30.5% of the regenerating axons can reinnervate the original muscular area. These data confirm previous studies in which a preferential reinnervation is reported not to be absolutely dependent on the axon's mechanical alignment.

Immunohistochemical study of neuropeptide Y-containing nerve fibers in the human clitoris and penis.

The occurrence and distribution of neuropeptide Y in the human clitoris and penis was investigated by light immunohistochemistry. Neuropeptide Y-containing nerve fibers were detected in the tunicae of arteries and veins as well as among trabecular smooth muscle. The distribution pattern of the peptide was similar in both organs although a higher density of immunoreactive nerve fibers was detected in the penis. The immunolocalization of neuropeptide Y was also compared with that of neuron-specific enolase, a neuronal marker which labels the entire nerve network. It is suggested that neuropeptide Y is involved in the physiology of the penis and the clitoris, affecting vascular and nonvascular smooth muscle activity.

Immunocytochemical localization of S-100b protein in degenerating and regenerating rat sciatic nerves.

We studied the cellular and subcellular distribution of S-100b protein in normal, crushed, and transected rat sciatic nerves by an immunocytochemical procedure. In uninjured nerves, S-100b protein was restricted to the cytoplasm and membranes of Schwann cells, with no reaction product present in the nucleus or in axons. Similar images were seen from the first to the thirtieth day after the crush in activated Schwann cells during the degeneration period, i.e., up to the seventh post-lesion day, and in normal Schwann cells reappearing during the regeneration period, i.e., after the seventh post-lesion day, in the zone of the crush and proximal and distal to it. By the technique employed, there seemed to be no differences in the intensity of the immune reaction product in normal and activated Schwann cells. Also, similar images were seen in the proximal stump of transected nerves. Only a slight S-100b protein immune reaction product could be observed in the rare activated Schwann cells present in the distal stump around the seventh post-lesion day, the majority of cell types being represented by fibroblasts and elongated cells at this stage and thereafter. By immunochemical assays, similar results as those presented here have been reported and interpreted as indicative of the presence of S-100 protein in axons or, alternatively, of axonal control over expression of S-100 protein in Schwann cells. Our immunocytochemical data clearly show that the strong reduction in the S-100 protein content of the distal stump of transected nerves is owing to the paucity of Schwann cells and to the decrease in the S-100 protein content of these cells, rather than to degeneration of axons.