Quantitative PCR reveals increased levels of tumor necrosis factor-alpha mRNA in peripheral blood mononuclear cells of multiple sclerosis patients during relapses.

Quantification of tumor necrosis factor-alpha (TNF-alpha) mRNA in peripheral blood mononuclear cells (PBMC) could provide information about disease activity in multiple sclerosis (MS); however, specific competitive methods must be utilized. A competitor cDNA, having the same sequence of the target TNF-alpha cDNA, a part from an internal 49-bp deletion, was generated and used to set-up a quantitative polymerase chain reaction (PCR) to quantify mRNA of TNF-alpha. Competitor and target were co-amplified using the same primers. The rates of generation of competitor and target TNF-alpha conformed closely to the prediction of the mathematical model, and a high level of accuracy and reproducibility was achieved. The method was applied to quantify TNF-alpha mRNA in PBMC of normal subjects and multiple sclerosis (MS) patients both during clinical relapses and remissions. A statistically significant higher level of TNF-alpha mRNA was detected during relapses than during remissions. High levels of TNF-alpha mRNA were found in 44% of relapses and 12% of samples during remissions, suggesting that TNF-alpha mRNA synthesis is abnormal in MS.

Transforming growth factor beta1 (TGFbeta1) mRNA level correlates with magnetic resonance imaging disease activity in multiple sclerosis patients.

Eight relapsing-remitting multiple sclerosis (MS) patients were tested for the level of transforming growth factor beta1 (TGFbeta1) mRNA in peripheral blood mononuclear cells every 15 days for 6 months. Disease activity was evaluated every 4 weeks by magnetic resonance imaging (MRI) and neurological examination. An inverse correlation was found between the level of TGFbeta1 mRNA and MRI disease activity. The level of TGFbeta1 mRNA predicted the presence of disease activity in the scans performed 2-4 weeks later with high sensitivity (88%) and specificity (87.5%) suggesting that TGFbeta1 mRNA quantification could be an indicator of disease activity in MS.

5D4 keratan sulfate epitope identifies a subset of ramified microglia in normal central nervous system parenchyma.

Microglia expressing keratan sulfate (KS) was studied in normal central nervous system (CNS) and in rat neonatal brain cultures. The majority of KS+ cells are ramified microglia located in the brain parenchyma; positive cells were only exceptionally found in extraparenchymal structures. KS+ cells are ubiquitous, but their density is heterogeneous throughout the CNS. Serial sections incubated with anti-KS MAb and MAb against the complement receptor type 3 (CR3) revealed a higher number of CR3+ cells and double immunofluorescence showed the presence of two microglial populations: the first expressing both KS and CR3, the second expressing only CR3. Two sets of microglial cells were found also in neonatal rat microglial cultures where only a low percentage of microglial cells expressing CR3 was also KS+. KS was not induced by microglia activation.

Morphological and biochemical investigations of mitral valve endocardiosis in pigs.

Pig endocardiosis is a pathological process affecting cardiac valves that is characterised by the accumulation of glycosaminoglycans (GAG) in the extracellular matrix. To investigate the involvement of GAG in the condition, the morphology of the mitral valves from 23 affected pigs and seven normal controls was studied and qualitative and quantitative biochemical analyses of GAG were made. Gross and histopathological lesions were characterised by valve enlargement, collagen disorganisation and myxoid degeneration. No differences between normal and diseased valves were detected by lectin histochemistry. Electron microscopy revealed myofibroblast differentiation of many fibroblasts. A statistically significant increase of total GAG and hyaluronan was detected in the mitral valves of the pigs with endocardiosis by spectrophotometric, electrophoretic and densitometric analysis of the extracted GAG. Although it is not known whether the change in hyaluronan is a primary event or a result of other changes in the extracellular matrix, its accumulation in association with myofibroblast differentiation suggests that it plays a pathogenetic role in pig endocardiosis.

Immunohistochemical mapping of perineuronal nets containing chondroitin unsulfated proteoglycan in the rat central nervous system.

Subsets of neurons ensheathed by perineuronal nets containing chondroitin unsulfated proteoglycan have been immunohistochemically mapped throughout the rat central nervous system from the olfactory bulb to the spinal cord. A variable proportion of neurons were outlined by immunoreactivity for the monoclonal antibody (Mab 1B5), but only after chondroitinase ABC digestion. In forebrain cortical structures the only immunoreactive nets were around interneurons; in contrast, throughout the brainstem and spinal cord a large proportion of projection neurons were surrounded by intense immunoreactivity. Immunoreactivity was ordinarily found in the neuropil between neurons surrounded by an immunopositive net. By contrast, within the pyriform cortex the neuropil of the plexiform layer was intensely immunoreactive even though no perineuronal net could be found. The presence of perineuronal nets could not be correlated with any single class of neurons; however a few functionally related groups (e.g., motor and motor-related structures: motor neurons both in the spinal cord and in the efferent somatic nuclei of the brainstem, deep cerebellar nuclei, vestibular nuclei; red nucleus, reticular formation; central auditory pathway: ventral cochlear nucleus, trapezoid body, superior olive, nucleus of the lateral lemniscus, inferior colliculus, medial geniculate body) were the main components of the neuronal subpopulation displaying chondroitin unsulfated proteoglycans in the surrounding extracellular matrix. The immunodecorated neurons found in the present study and those shown by different monoclonal antibodies or by lectin cytochemistry, revealed consistent overlapping of their distribution patterns.

Keratan sulphate is a marker of differentiation of ramified microglia.

Recently we reported that the keratan sulphate epitope recognised by the monoclonal antibody 5D4 is expressed by a population of ramified microglia in adult rats. As ramified microglia is believed to differentiate from ameboid microglia during postnatal development, we studied the rat brain from birth to 90 postnatal days of life with the monoclonal antibody 5D4. Contrary to all the other microglia markers until now described, keratan sulphate is not expressed by ameboid microglia and by macrophages but appears on the surface of microglia only when the cells are differentiated and show ramified processes. The keratan sulphate positive cells become evident at different times in different central nervous system areas; the first were localised in the pyriform cortex and brainstem from the end of the second postnatal week. These observations suggest that keratan sulphate expression on microglia cells is induced by differentiation and by a resting functional state. Moreover the 5D4 monoclonal antibody showed a strong diffuse positive staining of some cortical, thalamic and white matter areas during the first two postnatal weeks. This staining was transient and it does not seem biologically correlated with the expression of the keratan sulphate on differentiated microglia.

Disappearance of the Vicia villosa-positivity from the perineuronal net containing chondroitin proteoglycan after chondroitinase digestion.

Anti-chondroitin unsulfated proteoglycan (COS-PG) 1B5 monoclonal antibody (MAb) and Vicia villosa agglutinin (VVA) recognize the same neuronal subset. Moreover, when in double immunofluorescence study the sections were digested with chondroitinase ABC (ChABC), a procedure necessary to create the epitope of 1B5 MAb, and incubated with VVA, no VVA positive neurons were detected. This finding suggests that VVA binds terminal N-acetyl-galactosamine present in the glycidic chains of COS-PG or of glycoproteins that form perineuronal molecular aggregates with COS-PG.

Localization, identification, and action of calcitonin gene-related peptide in the frog adrenal gland.

The localization of calcitonin gene-related peptide (CGRP)-like immunoreactivity in the adrenal gland of the frog, Rana ridibunda, was examined by the indirect immunofluorescence technique. Using an antiserum directed against rat alpha-CGRP, the presence of a network of positive fibers was observed in the adrenal parenchyma. The immunoreactive material has been characterized by HPLC analysis combined with RIA quantification. The elution profile revealed the existence of a single form of CGRP exhibiting the same retention time as synthetic frog CGRP. The possible involvement of CGRP in the regulation of corticosteroid secretion was studied in vitro using a perifusion system for frog adrenal slices. Graded doses of frog CGRP (from 3 x 10(-9) to 3 x 10(-6) M) increased corticosterone and aldosterone secretion in a dose-dependent manner (ED50, 4.1 x 10(-8) M). Several other forms of CGRP, i.e. rat alpha-CGRP and beta-CGRP, and human alpha-CGRP and beta-CGRP, were also capable of enhancing steroid output, but frog CGRP was the most effective stimulator of steroidogenesis. Repeated administration of rat alpha CGRP induced a reproducible stimulation of corticosteroid secretion without any tachyphylaxis. Prolonged infusion of the peptide (3 h) caused a rapid increase in corticosteroid release, followed by a gradual decline of steroid secretion, suggesting the occurrence of a desensitization phenomenon. Rat alpha-CGRP also gave rise to a significant increase in corticosteroid release from acutely dispersed adrenal cells. These results show the presence of CGRP in fibers innervating the frog adrenal gland. The data also demonstrate that synthetic CGRP exerts a direct stimulatory effect on corticosteroid secretion. Taken together, these findings suggest that CGRP, released by nerve fibers in the adrenal tissue, can locally regulate corticosteroid secretion.