Land cover and forest connectivity alter the interactions among host, pathogen and skin microbiome.

Deforestation has detrimental consequences on biodiversity, affecting species interactions at multiple scales. The associations among vertebrates, pathogens and their commensal/symbiotic microbial communities (i.e. microbiomes) have important downstream effects for biodiversity conservation, yet we know little about how deforestation contributes to changes in host microbial diversity and pathogen abundance. Here, we tested the effects of landcover, forest connectivity and infection by the chytrid fungus Batrachochytrium dendrobatidis (Bd) on amphibian skin bacterial diversity along deforestation gradients in Brazilian landscapes. If disturbance to natural habitat alters skin microbiomes as it does in vertebrate host communities, then we would expect higher host bacterial diversity in natural forest habitats. Bd infection loads are also often higher in these closed-canopy forests, which may in turn impact skin-associated bacterial communities. We found that forest corridors shaped composition of host skin microbiomes; high forest connectivity predicted greater similarity of skin bacterial communities among host populations. In addition, we found that host skin bacterial diversity and Bd loads increased towards natural vegetation. Because symbiotic bacteria can potentially buffer hosts from Bd infection, we also evaluated the bi-directional microbiome-Bd link but failed to find a significant effect of skin bacterial diversity reducing Bd infections. Although weak, we found support for Bd increasing bacterial diversity and/or for core bacteria dominance reducing Bd loads. Our research incorporates a critical element in the study of host microbiomes by linking environmental heterogeneity of landscapes to the host-pathogen-microbiome triangle.

Genetic diversity and gene flow decline with elevation in montane mayflies.

Montane environments around the globe are biodiversity 'hotspots' and important reservoirs of genetic diversity. Montane species are also typically more vulnerable to environmental change than their low-elevation counterparts due to restricted ranges and dispersal limitations. Here we focus on two abundant congeneric mayflies (Baetis bicaudatus and B. tricaudatus) from montane streams over an elevation gradient spanning 1400 m. Using single-nucleotide polymorphism genotypes, we measured population diversity and vulnerability in these two species by: (i) describing genetic diversity and population structure across elevation gradients to identify mechanisms underlying diversification; (ii) performing spatially explicit landscape analyses to identify environmental drivers of differentiation; and (iii) identifying outlier loci hypothesized to underlie adaptive divergence. Differences in the extent of population structure in these species were evident depending upon their position along the elevation gradient. Heterozygosity, effective population sizes and gene flow all declined with increasing elevation, resulting in substantial population structure in the higher elevation species (B. bicaudatus). At lower elevations, populations of both species are more genetically similar, indicating ongoing gene flow. Isolation by distance was detected at lower elevations only, whereas landscape barriers better predicted genetic distance at higher elevations. At higher elevations, dispersal was restricted due to landscape effects, resulting in greater population isolation. Our results demonstrate differentiation over small spatial scales along an elevation gradient, and highlight the importance of preserving genetic diversity in more isolated high-elevation populations.

Long-term endemism of two highly divergent lineages of the amphibian-killing fungus in the Atlantic Forest of Brazil.

The recent global spread of the amphibian-killing fungus [Batrachochytrium dendrobatidis (Bd)] has been closely tied to anthropogenic activities; however, regional patterns of spread are not completely understood. Using historical samples, we can test whether Bd was a spreading or endemic pathogen in a region within a particular time frame, because those two disease states provide different predictions for the regional demographic dynamics and population genetics of Bd. Testing historical patterns of pathogen prevalence and population genetics under these predictions is key to understanding the evolution and origin of Bd. Focusing on the Atlantic Forest (AF) of Brazil, we used qPCR assays to determine the presence or absence of Bd on 2799 preserved postmetamorphic anurans collected between 1894 and 2010 and used semi-nested PCRs to determine the frequency of rRNA ITS1 haplotypes from 52 samples. Our earliest date of detection was 1894. A mean prevalence of 23.9% over time and spatiotemporal patterns of Bd clusters indicate that Bd has been enzootic in the Brazilian AF with no evidence of regional spread within the last 116 years. ITS1 haplotypes confirm the long-term presence of two divergent strains of Bd (BdGPL and Bd-Brazil) and three spatiotemporally broad genetic demes within BdGPL, indicating that Bd was not introduced into southeast Brazil by the bullfrog trade. Our data show that the evolutionary history and pathogen dynamics of Bd in Brazil is better explained by the endemic pathogen hypothesis.

Activation of factor XII by tobacco glycoprotein.

A glycoprotein of mol wt ca. 18,000 daltons isolated from cured tobacco leaves (TGP-L) and from cigarette smoke condensate (TGP-CSC) activated factor XII in normal human plasma in vitro as measured by (a) shortening of the partial thromboplastin time, (b) shortening of the lysis time of euglobulin clots, and (c) generation of kinin activity. These effects were not demonstrable in plasma deficient in factor XII. The capacity of TGP-L and TGP-CSC to activate factor XII was shown to depend on the presence of rutin, a substance chemically similar to quercetin and ellagic acid, which are known activators of factor XII. Rutin and rutin coupled to bovine serum albumin, but not bovine serum albumin alone, were also demonstrated to activate factor XII. The presence in cigarette smoke of material that is both allergenic and capable of activating factor XII of the intrinsic pathway of coagulatin may be important to the pathogenesis of cardiovascular and pulmonary disease associated with cigarette smoking.

alpha2-Macroglobulin on human vascular endothelium.

alpha2-Macroglobulin (alpha2M) has been identified on the luminal surface of endothelial cells in sections of normal human arteries, veins, and lymphatics by the indirect immunofluorescent technique. The specificity of the immunofluorescent reaction was confirmed by immunoabsorption studies. Prior absorption of the anti-alpha2M antiserum by purified alpha2M at equivalence completely inhibited endothelial surface as well as hepatic parenchymal cell staining. Endothelial cells in blood vessels were not stained when sections were treated with rabbit antisera toward alpha1-antitrypsin, antithrombin III, IgG, IgA, IgM, C3, or fibrinogen. The location of alpha2M at the surface of the vessel wall suggests that this protease inhibitor may protect the vascular endothelium from potentially injurious intravascular proteases.

The experimental induction of glomerulonephritis like that in man by infection with group A streptococci.

106 rabbits received one or more courses of inoculations, spaced 1-4 months apart, with Group A streptococci, usually of strains isolated from patients with acute glomerulonephritis. 8 days to a few weeks after onset of a given infection with streptococci known to have been nephritogenic for man, marked proteinuria, often with hematuria and occasionally with azotemia, was detected in 22 of the animals. 15 of these were sacrificed a few days to a few weeks thereafter, and 10 showed renal changes like those of acute or recurrent acute glomerulonephritis in man. Such changes occurred in three other rabbits whose urine was not examined that died or were sacrificed 1-3 wk after onset of infection with streptococci of a serotype known to include a strain nephritogenic for man. The remaining seven animals in which marked proteinuria had occurred died or were sacrificed many months later, in some cases after additional infections. Two of these had become azotemic and two convulsed and died after giving birth; in these animals, there were renal changes like those that occur in man in chronic latent glomerulonephritis, toxemia of pregnancy super-imposed on chronic latent glomerulonephritis, or chronic active glomerulonephritis. Anatomical changes in the kidneys in the experimentally induced and in naturally occurring glomerulonephritis, from acute to chronic stages, are compared and illustrated. The pathogenesis of poststreptococcal glomerulonephritis is discussed.

Induction of acute cholecystitis by activation of factor XII.

Acute, acalculous cholecystitis is seen among patients suffering with bacterial sepsis, burns, trauma, or cancer; clinical conditions that could lead to activation of factor XII-dependent pathways and result in inflammation of the gall bladder. To test this hypothesis, dogs were injected intravenously with ellagic acid or rutin, known polyphenol activators of factor XII, or with Escherichia coli endotoxin, also known to activate factor XII, and monkeys were injected intravenously with ellagic acid. In both species, in vivo activation of factor XII-dependent pathways with polyphenol activator resulted in rapid and selective development of acute vasculitis in the serosa and muscularis of the gallbladder and margination of polymorphonuclear neutrophils in pulmonary blood vessels. Intravenous injection of E. coli endotoxin in dogs resulted in necrosis and thrombosis of vessels that were especially severe in the serosa and muscularis of the gallbladder but also present in vessels of many other organs. These observations indicate that blood vessels of the gall bladder and, to a lesser degree, the lung are especially sensitive to injury consequent to in vivo activation of factor XII-dependent pathways and, in view of the common ingestion of plant polyphenols, may provide important insight into the pathogenesis of cholecystitis in man.

Synthesis of basement membrane collagen by cultured human endothelial cells.

Studies were performed to determine if cultured human endothelial cells synthesized basement membrane collagen. In culture, endothelial cells were attached to grossly visible membranous structures which on light microscopy were composed of ribbons of dense, amorphous material. On transmission electron microscopy, these membranous structures consisted of amorphous basement membrane, and material morphologically similar to microfibrils and elastic fibers. By immunofluorescence microscopy, these membranous structures stained brightly with antisera to human glomerular basement membrane. Cultured endothelial cells incorporated [3H]proline into protein; 18% of the incorporated [3H]proline was solubilized by purified collagenase. When endothelial cells were cultured with [14C]proline, 7.1% of the incorporated counts were present as [14C]hydroxyproline. Cultured endothelial cells were labeled with [3H]glycine and [3H]proline and digested with pepsin. The resulting fractions on analysis by SDS-polyacrylamide gel electrophoresis contained two radioactive protein peaks of mol wt 94,200 and 120,500. Both these peaks disappeared after digestion with purified collagenase. The peak of mol wt 120,500 corresponds to that of alpha1 (IV) collagen; the peak of the mol wt 94,200 probably corresponds to that of alpha1 (III) collagen. Thus, cultured human endothelial cells synthesize material which is morphologically and immunologically like amorphous basement membrane and biochemically like basement membrane collagen. Cultured endothelial cells probably also synthesize material which is morphologically similar to microfibrils and elastic fibers.

SmgGDS is up-regulated in prostate carcinoma and promotes tumour phenotypes in prostate cancer cells.

SmgGDS is a guanine nucleotide exchange factor with the unique ability to activate multiple small GTPases, implicating it in cancer development and progression. Here, we investigated the role of SmgGDS in prostate cancer by studying the expression of SmgGDS in benign and malignant prostatic tissues. We also probed SmgGDS function in three prostate carcinoma cell lines using small interfering RNA (siRNA). Immunohistochemical analysis revealed that SmgGDS levels were elevated in prostatic intraepithelial neoplasia (PIN), prostate carcinoma, and metastatic prostate carcinoma. In addition, expression of SmgGDS positively correlated with that of cyclooxygenase-2 (COX-2), a protein believed to promote the development of prostate carcinoma. Reduction of SmgGDS expression in prostate carcinoma cells inhibited proliferation and migration, irrespective of androgen receptor status. These effects were accompanied by a reduction in COX-2 expression and in activity of NF-kappaB, a known regulator of COX-2. Taken together, these findings suggest that SmgGDS promotes the development and progression of prostate cancer, possibly associated with NF-kappaB-dependent up-regulation of COX-2.

Cytokines profile in cystic fibrosis patients with nasal polyps.

UNLABELLED: Nasal polyposis often complicates the progress of patients with cystic fibrosis and there has been little study about the importance of cytokines in the polyps of such individuals. OBJECTIVE: To assess RNAm expression for interleukins 4, 5, 6, 8, GM-CSF and IFN-gamma by RT-PCR in eosinophilic and non-eosinophilic polyps of patients with cystic fibrosis. MATERIAL AND METHOD: A total of 124 persons were evaluated, of which thirteen patients with cystic fibrosis and nasosinusal polyps were selected--three were eosinophilic and ten were non-eosinophilic. The control group was composed of eleven individuals with normal otorhinolaryngological exam and the mean age was 18 years (3-57). The middle turbinate mucosa and nasal polyps were biopsied from the control group and the cystic fibrosis group respectively, and these were analyzed with RT-PCR. The middle turbinate mucosa was biopsied in the control group and in the cystic fibrosis group polyps that was analyzed to RT-PCR. The polyps of cystic fibrosis patients were also further anaylsed for subjected to a second biopsy in order to determine the percentage of eosinophils. IL-4, IL-5, IL-6, IL-8, IFN-gamma and GM-CSF transcriptions were analyzed. RESULTS: There was no difference in IL-5, IL-8 and GM-CSF when compared to the eosinophilic, non-eosinophilic and control groups (p>0.05). When compared to the eosinophilic and non-eosinophilic groups, higher IL-4 and IL-6 values (p=0.01 and p=0.01 respectively) were observed. When analyzed separately with the control group, IL-4 (p=0.01) expression was higher in the eosinophilic group, while IFN-gamma (p=0.03) was lower in the non-eosinophilic group. IL-5, IL-8, GM-CSF are non-specific cytokines present in the nasosinusal sinonasal polyps of cystic fibrosis patients. IL-4 and IL-6 are important mediators in the eosinophilic sinonasal polyps, while low IFN-gamma may be related to lower eosinophils in non-eosinophilic polyps.

Hybrids of amphibian chytrid show high virulence in native hosts.

Hybridization of parasites can generate new genotypes with high virulence. The fungal amphibian parasite Batrachochytrium dendrobatidis (Bd) hybridizes in Brazil's Atlantic Forest, a biodiversity hotspot where amphibian declines have been linked to Bd, but the virulence of hybrid genotypes in native hosts has never been tested. We compared the virulence (measured as host mortality and infection burden) of hybrid Bd genotypes to the parental lineages, the putatively hypovirulent lineage Bd-Brazil and the hypervirulent Global Pandemic Lineage (Bd-GPL), in a panel of native Brazilian hosts. In Brachycephalus ephippium, the hybrid exceeded the virulence (host mortality) of both parents, suggesting that novelty arising from hybridization of Bd is a conservation concern. In Ischnocnema parva, host mortality in the hybrid treatment was intermediate between the parent treatments, suggesting that this species is more vulnerable to the aggressive phenotypes associated with Bd-GPL. Dendropsophus minutus showed low overall mortality, but infection burdens were higher in frogs treated with hybrid and Bd-GPL genotypes than with Bd-Brazil genotypes. Our experiment suggests that Bd hybrids have the potential to increase disease risk in native hosts. Continued surveillance is needed to track potential spread of hybrid genotypes and detect future genomic shifts in this dynamic disease system.

Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria.

Endothelial cells were isolated from freshly obtained human umbilical cords by collagenase digestion of the interior of the umbilical vein. The cells were grown in tissue culture as a homogeneous population for periods up to 5 mo and some lines were subcultured for 10 serial passages. During the logarithmic phase of cell growth, cell-doubling time was 92 h. Light, phase contrast, and scanning electron microscopy demonstrated that cultured human endothelial cells grew as monolayers of closely opposed, polygonal large cells whereas both cultured human fibroblasts and human smooth muscle cells grew as overlapping layers of parallel arrays of slender, spindle-shaped cells. By transmission electron microscopy, cultured endothelial cells were seen to contain cytoplasmic inclusions (Weibel-Palade bodies) characteristic of in situ endothelial cells. These inclusions were also found in endothelial cells lining umbilical veins but were not seen in smooth muscle cells or fibroblasts in culture or in situ. Cultured endothelial cells contained abundant quantities of smooth muscle actomyosin. Cultured endothelial cells also contained ABH antigens appropriate to the tissue donor's blood type; these antigens were not detectable on cultured smooth muscle cells or fibroblasts. These studies demonstrate that it is possible to culture morphologically and immunologically identifiable human endothelial cells for periods up to 5 mo.

Activation of factor XII-dependent pathways in human plasma by hematin and protoporphyrin.

Intravenous administration of hematin is effective in the treatment of acute exacerbations of the inducible porphyrias. In the course of such treatment, coagulopathies have occurred that are characterized by prolongation of prothrombin time, partial thromboplastin time, and formation of fibrin split products. In experiments in vitro with normal human plasma, we observed that hematin and protoporphyrin activated Factor XII-dependent pathways of coagulation and fibrinolysis, and that they generated kallikrein activity. Incubation of protoporphyrin with purified Factor XII resulted in activation as measured by amidolysis of a chromogenic substrate. Neither coproporphyrin, uroporphyrin, delta-aminolevulinic acid, porphobilinogen, or bilirubin activated Factor XII-dependent pathways. Exposure of serum containing added uroporphyrin, coproporphyrin, and protoporphyrin, but not hematin, to ultraviolet light (405 nm) resulted in activation of the classical pathway of the complement system. On the other hand, exposure of plasma containing uroporphyrin or coproporphyrin to ultraviolet light did not result in activation of Factor XII-dependent pathways.

Antibody to the HNK-1 glycoepitope affects fasciculation and axonal pathfinding in the developing posterior lateral line nerve of embryonic zebrafish.

The HNK-1 glycoepitope, carried by many cell recognition molecules, is present in the developing posterior lateral line nerve and on other primary axons of zebrafish. To elucidate the function of HNK-1 in vivo, the antibody 412 to HNK-1 was injected into zebrafish embryos at 16 h post fertilization (hpf). The injected antibody bound specifically to axons carrying HNK-1. This treatment selectively affected the growth of either one or both posterior lateral line nerves in 39% of the experimental cases (13 of 33 animals), which was significantly more (P<0.0002) than in uninjected, vehicle injected, and non-immune IgG injected controls (1.2% of the animals; one of 85 animals), as assessed at 27 or 33 hpf. Other HNK-1 immunoreactive nerves, such as the ventral motor nerves were unaffected, indicating that antibody binding per se did not interfere with axon growth. The primordium of the posterior lateral line was not affected in its caudal migration and in depositing differentiating neuromasts along the trunk, showing that injections did not retard development and that initial formation of lateral line organs is probably independent of contact with nerve fibers. We suggest that the HNK-1 glycoepitope is an important modulator of embryonic nerve growth.

Regenerating descending axons preferentially reroute to the gray matter in the presence of a general macrophage/microglial reaction caudal to a spinal transection in adult zebrafish.

We analyzed pathway choices of regenerating, mostly supraspinal, descending axons in the spinal cord of adult zebrafish and the cellular changes in the spinal cord caudal to a lesion site after complete spinal transection. Anterograde tracing (by application of the tracer rostral to the spinal lesion site) showed that significantly more descending axons (74%) regenerated in the spinal gray matter of the caudal spinal cord than would be expected from random growth. Retrograde tracing (by application of the tracer caudal to the spinal lesion site) showed that, rostral to the lesion, most of these axons (80%) extended into the major white matter tracts. Thus, ventral descending tracts often were devoid of labeled axons caudal to a spinal lesion but contained many axons rostral to the lesion in the same animals, indicating a pathway switch of descending axons from the white matter to the gray matter. Ascending axons of spinal neurons were not observed regrowing to the rostral tracer application site; therefore, they most likely did not contribute to the axonal populations analyzed. A macrophage/microglia response within 2 days of spinal cord transection, along with phagocytosis of myelin, was observed caudal to the transection by immunohistochemistry and electron microscopy. Nevertheless, caudal to the lesion, descending tracts in the white matter were filled with myelin debris during the time of axonal regrowth, at least up to 6 weeks postlesion. We suggest that the spontaneous regeneration of axons of supraspinal origin after spinal cord transection in adult zebrafish may be due in part to the axons' ability to negotiate novel pathways in the spinal cord gray matter.

Gradients of ephrin-A2 and ephrin-A5b mRNA during retinotopic regeneration of the optic projection in adult zebrafish.

Regeneration of optic axons in the continuously growing optic system of adult zebrafish was analyzed by anterograde tracing and correlated with the mRNA expression patterns of the recognition molecules ephrin-A2 and ephrin-A5b in retinal targets. The optic tectum and diencephalic targets are all reinnervated after a lesion. However, the rate of erroneous pathway choices was increased at the chiasm and the bifurcation between the ventral and dorsal brachium of the optic tract compared to unlesioned animals. Tracer application to different retinal positions revealed retinotopic reinnervation of the tectum within 4 weeks after the lesion. In situ hybridization analysis indicated the presence of rostral-low to caudal-high gradients of ephrin-A2 and ephrin-A5b mRNAs in unlesioned control tecta and after a unilateral optic nerve lesion. By contrast, the parvocellular superficial pretectal nucleus showed retinotopic organization of optic fibers but no detectable expression of ephrin-A2 and ephrin-A5b mRNAs. However, a row of cells delineating the terminal field of optic fibers in the dorsal part of the periventricular pretectal nucleus was intensely labeled for ephrin-A5b mRNA and may thus provide a stop signal for ingrowing axons. Ephrin-A2 and ephrin-A5b mRNAs were not detectable in the adult retina, despite their prominent expression during development. Thus, given a complementary receptor system in retinal ganglion cells, expression of ephrin-A2 and ephrin-A5b in primary targets of optic fibers in adult zebrafish may contribute to guidance of optic axons that are continuously added to the adult projection and of regenerating axons after optic nerve lesion.

Axonal regrowth after spinal cord transection in adult zebrafish.

Using axonal tracers, we characterized the neurons projecting from the brain to the spinal cord as well as the terminal fields of ascending spinal projections in the brain of adult zebrafish with unlesioned or transected spinal cords. Twenty distinct brain nuclei were found to project to the spinal cord. These nuclei were similar to those found in the closely related goldfish, except that additionally the parvocellular preoptic nucleus, the medial octavolateralis nucleus, and the nucleus tangentialis, but not the facial lobe, projected to the spinal cord in zebrafish. Terminal fields of axons, visualized by anterograde tracing, were seen in the telencephalon, the diencephalon, the torus semicircularis, the optic tectum, the eminentia granularis, and throughout the ventral brainstem in unlesioned animals. Following spinal cord transection at a level approximately 3.5 mm caudal to the brainstem/spinal cord transition zone, neurons in most brain nuclei grew axons beyond the transection site into the distal spinal cord to the level of retrograde tracer application within 6 weeks. However, the individually identifiable Mauthner cells were never seen to do so up to 15 weeks after spinal cord transection. Nearly all neurons survived axotomy, and the vast majority of axons that had grown beyond the transection site belonged to previously axotomized neurons as shown by double tracing. Terminal fields were not re-established in the torus semicircularis and the eminentia granularis following spinal cord transection.

Immunohistological localization of tenascin-C in the developing and regenerating retinotectal system of two amphibian species.

The expression pattern of the extracellular matrix molecule tenascin-C was investigated in the retinotectal system of the frog Discoglossus pictus and the salamander Pleurodeles waltl during development and optic nerve regeneration in the adult. In both species, the retina was devoid of tenascin-C immunoreactivity at all ages studied. During development, tenascin-C was distributed in a gradient in the optic nerve, with the highest immunoreactivity in the eye near part of the optic nerve. The myelin-associated glycoprotein was distributed in a gradient with opposite polarity. In Discoglossus, but not Pleurodeles, tenascin-C was detected in the anterior chiasm. In the tectum of both species, tenascin-C was observed in deep cellular and fiber layers but not in the layers receiving optic fibers or proliferative zones. The distribution patterns of tenascin-C were the same during development and in the adult, except for a disappearance of the molecule from the intraocular part of the optic nerve. After lesioning the optic nerve of adult animals, tenascin-C was strongly reexpressed in the intraocular part of the optic nerve but was only weakly upregulated in the distal optic nerve stump. In contrast, a chondroitin sulfate epitope was strongly upregulated in the distal optic nerve stump. These observations suggest that during development, tenascin-C serves as an attenuating barrier for myelinating cells in the optic nerve and contributes to the guidance of growing retinal ganglion cell axons. Due to its sustained expression in the adult, tenascin-C may have similar functions during regeneration of the lesioned adult retinotectal system.

Amphibian-specific regulation of polysialic acid and the neural cell adhesion molecule in development and regeneration of the retinotectal system of the salamander Pleurodeles waltl.

Antibodies specific to the neural cell adhesion molecule (NCAM-total), the 180 x 10(3) M(r) component of NCAM (NCAM-180) and polysialic acid (PSA) were used in immunohistochemistry and Western blots to detect the spatiotemporal dynamics of these molecules in development and regeneration of the retinotectal system of Pleurodeles waltl. NCAM-total and NCAM-180 are continuously expressed in the retina, optic nerve, and tectum of the developing and adult salamander. This is also found for the 140 x 10(3) M(r) component of NCAM in Western blots of the retina. In the larval retina, PSA is present in the inner plexiform layer (IPL) and a few cells in all nuclear layers. At metamorphosis, PSA expression in the retina strongly increases in the layer of cone photoreceptor somata. Several cells in the inner nuclear layer and Müller cell processes also begin to express PSA. This pattern persists into adulthood. The optic nerve and the tectum are strongly PSA-immunoreactive throughout development. In the adult optic nerve and optic fiber pathway in the brain, PSA expression is selectively downregulated. In the crush-lesioned adult optic nerve, regenerating fibers are NCAM-180-positive but PSA-negative. This demonstrates a molecular difference between growing nerve fibers of Pleurodeles in development and in regeneration. PSA regulation is closely correlated with metamorphosis, thus suggesting that PSA expression may be under hormonal control. Some aspects of PSA and NCAM isoform expression patterns in the retinotectal system of salamanders differ considerably from that of other vertebrates. The sustained expression of NCAM isoforms in adult salamanders might be due to secondary simplification (paedomorphosis).

Transforming growth factor (TGF)-beta mimics and anti-TGF-beta antibody abrogates the in vivo effects of cyclosporine: demonstration of a direct role of TGF-beta in immunosuppression and nephrotoxicity of cyclosporine.

BACKGROUND: Cyclosporine (CsA) has been shown to induce the expression of transforming growth factor (TGF)-beta both in vitro and in vivo. It is hypothesized that the efficacy as well as the side effects of CsA are mediated by TGF-beta. This study was planned to investigate whether anti-TGF-beta mitigated and TGF-beta reproduced the in vivo effects of CsA to directly prove this hypothesis. METHODS: B6AF1 (H2b/k.d) mice were divided into groups and received the following: CsA, vehicle (olive oil), CsA + anti-TGF-beta1 antibody, TGF-beta1, or vehicle phosphate-buffered saline/bovine serum albumin. All studies were carried out at 10 and 28 days after the last day of CsA administration with the exception of the exogenous TGF-beta experiments, which were performed 5 days after exogenous TGF-beta administration. The efficacy was studied by the anti-CD3-induced ex vivo proliferation of splenocytes measured by [3H]thymidine uptake; TGF-beta protein levels were quantified by ELISA. TGF-beta, collagen, and fibronectin gene expression was studied using reverse transcriptase-polymerase chain reaction, and histopathological analysis was made on periodic acid-Schiff- and trichrome C-stained thin kidney sections. RESULTS: CsA treatment resulted in decreased ex vivo proliferation of splenocytes, an increase in TGF-beta protein in the sera, and renal histopathological changes including tubular swelling, vacuolization, thrombotic microangiopathy, and increased expression of TGF-beta, collagen and fibronectin genes. All of these findings were blocked by anti-TGF-beta antibody. CONCLUSION: The study demonstrates the in vivo modulation of the effects of CsA by manipulating TGF-beta levels and suggests that TGF-beta at least in part mediates CsA's beneficial and detrimental effects.