Effects of extremely low-frequency magnetic fields on L-glutamic acid aqueous solutions at 20, 40, and 60 microT static magnetic fields.

Current peaks have been observed and measured in electrolytic ionic current of L-glutamic acid aqueous solutions at room temperature, in static magnetic fields of 20, 40, and 60 muT flux densities, with a superimposed extremely low-frequency, (1/10) Hz, alternating magnetic field flux density of 40 nT. The distributions of the peaks have mean values centered at the cyclotron resonance frequency of the singly charged L-glutamic acid ion molecular mass in the corresponding static field. Amplitudes and widths of the peaks are compared and analyzed to extract their correlation. The results can be considered a contribution to the understanding of the experimental phenomenology in low-frequency electromagnetic fields on ionic currents of L-glutamic amino acid aqueous solutions. The results can be of interest in the studies on the interaction of the electromagnetic fields with some structural neurotransmitters in cellular medium.

[Prevalence of Chlamydia pneumoniae in respiratory infections in children: an ambulatory diagnostic problem]. / Prevalenza di Chlamydia pneumoniae nelle infezioni respiratorie del bambino: un problema diagnostico ambulatoriale.

It has been recently suggested that Chlamydia Pneumoniae infection is a common finding among children with acute respiratory diseases. Chlamydia cell culture is difficult and time-consuming to perform. Polymerase chain reaction (PCR) is a more rapid but also more expensive technique used to identify Chlamydia in pharyngeal swab, but it can be performed only in few specialized laboratories. We tested a rapid enzyme immuno-assay to detect Chlamydia in 20 children with respiratory infections (mean age 3.29 years; male:female ratio = 12:8) and in 21 healthy children (mean age 4.70 years male:female ratio = 15:6). Prevalence of Chlamydia isolation from pharyngeal swab was very high in both patients and healthy children without a significative difference in the two considered groups (45% vs 42%, p = 0.8). Specific Chlamydia IgG antibodies were undetectable in all patients and healthy children. Nine out of 20 patients affected by acute respiratory disease were Chlamydia-positive and 11 out 20 were Chlamydia-negative: these two groups didn't differ in regard to clinical and laboratory features, whereas duration of symptoms was significantly longer in Chlamydia-positive patients (9.3 vs 5.5 days, p = 0.014). Our study suggests a high prevalence of Chlamydia pharyngeal swab positivity in both healthy and sick children. Diagnosis of Chlamydia infection was not feasible on the basis of the considered clinical and laboratory findings.

Ciliary neurotrophic factor-induced gene expression in human neuroblastoma cell lines.

We have analyzed the response of the human neuroblastoma cell lines SK-N-SH (clone SY5Y) and SK-N-BE to the ciliary neurotrophic factor CNTF. In both cell lines CNTF induced the expression of the mRNA for two transcription factors, c-fos and NGF1A. The induction was rapid and transient reaching a maximum between 30 and 60 min after exposure to CNTF and subsequently declining. The level of induction of both c-fos and NGF1A mRNAs was much higher in SK-N-BE neuroblastoma cells compared to the SY5Y. Both cells express comparable levels of the transcript for the CNTF receptor-alpha. This mRNA was down regulated after 5 days of CNTF stimulation in both cell lines. CNTF also induced increased levels of the transcript for the growth cone associated protein GAP43 in SK-N-BE, but not in SY5Y cells. Induction followed a slower kinetic compared to that observed for c-fos and NGF1A. In fact, the GAP43 mRNA levels increased during 2 days of exposure to CNTF. Morphological analysis of CNTF treated cells showed that SK-N-BE undergo significant differentiation in response to CNTF (increased number of cells with neurites and increased neurite length) while SY5Y did not show appreciable morphological differentiation. These data shows that CNTF may elicit different response in neuroblastoma cell lines.

Nerve growth factor (NGF) in cerebrospinal fluid (CSF) from patients with various neurological disorders.

It has been recently shown that NGF is not only involved in the survival and development of sympathetic and neural crest-derived sensory neurons, but also in some mechanisms of the immune system. For this reason, we studied the content of NGF in CSF samples from patients with diseases in which neuroimmunological mechanisms seem to be involved (multiple sclerosis, amyotrophic lateral sclerosis, Alzheimer disease, chronic relapsing polyradiculoneuritis, Guillain-Barré syndrome, and tumors of the nervous system), as well as from a number of normal control subjects. We setup an ELISA aimed at the beta subunit of NGF, obtaining good validation tests and a detection limit of 28 pg beta NGF per ml. None of the samples was found to contain detectable levels of NGF and, when a concentration method for sample enrichment was used, only one patient was NGF-positive. This suggests that NGF is probably not involved in the neuroimmunological mechanisms underlying some inflammatory and degenerative diseases of the nervous system.

Structure-function studies of human ciliary neurotrophic factor.

Ciliary neurotrophic factor (CNTF) is a polypeptide that promotes the survival and/or differentiation of a number of neural cell types. Here we present a structural and functional analysis of the human CNTF molecule. Variant proteins were synthesized by Escherichia coli transformed with mutant cDNA constructs, and purified by SDS-polyacrylamide gel electrophoresis and reverse phase high pressure liquid chromatography. Most variant CNTF proteins lacked neurotrophic activity, but two N- and C-terminal deletions (delta 2-14 and delta 173-200, respectively) actually displayed a several-fold increase in specific activity. Loss of biological activity was accompanied by changes in the alpha-helical nature of CNTF as measured by circular dichroism. These data strengthen the proposed similarity between CNTF and the family of hematopoietic cytokines.

In vitro evaluation of nanoparticle formulations containing gangliosides.

Due to their poor bioavailability after oral administration, the use of gangliosides in medicine is limited to the parenteral route of administration. In the present study, the association with poly(alkylcyanoacrylate) nanospheres and nanocapsules of monosialoganglioside GM1 and other chemically modified gangliosides was investigated with the aim of developing a colloidal drug delivery system suitable for use by the oral route. Our results show that gangliosides can be successfully associated with a biodegradable cyanoacrylic carrier either in the form of nanospheres or as nanocapsules, avoiding any degradation of the ganglioside molecule during the polymerization process. However, the drug-loading was found to be more efficient for nanocapsules. The amount of GM1 incorporated into nanospheres appeared to be dependent on the alkyl chain length of the cyanoacrylic polymer; this amount was however too low for pharmaceutical purposes. In contrast, nanocapsules allowed the attainment of very high drug encapsulation levels, especially with lipophilic derivatives of GM1, where an increase of lipophilicity has been obtained by chemical esterification of the sialic acid residue. Drug release experiments performed in the absence of enzymes indicated that nanocapsules were stable in acid medium, in which no drug release was observed, while their behaviour in basic medium was found to be affected by the composition of the oily phase and the oil/polymer ratio.

Human neutrophil chemokinesis and polarization induced by hyaluronic acid derivatives.

Neutrophils and macrophages are known to undergo significant modifications in their morphology and basal metabolism in response to chemical factors, in particular changes in the shape, movement, phagocytic activity and degranulation. These phenomena often involve an increase in chemokinesis and cellular secretory activity, usually expressed in antimicrobial activity. Once activated, the cells can move quickly towards the source of the stimulus, where they produce and release great amounts of enzymes (e.g. proteases, hydrolases, lysozyme) and reactive oxygen metabolites (e.g. O2-., H2O2, OH.). This study has examined the ability of surfaces of selected biomaterials to influence neutrophil morphology and locomotion. The surface of two films derived from hyaluronic acid derivatives were compared with that of glass. The two hyaluronic acid derivatives, despite having a similar chemical structure, were shown to interact with human neutrophils in different ways. A hyaluronic acid ethyl ester stimulated the whole population of neutrophils to take up a non-spherical morphology (polarize) and to move with a velocity similar to that of N-formyl-methionine-leucine-phenylalanine-stimulated cells on a glass surface. In contrast, only 44% of the examined cells on the surface of hyaluronic acid benzyl ester were polarized and their mean speed was only slightly higher with respect to that found with non-stimulated cells on glass. Moreover, while on the benzyl ester and on glass a correlation between neutrophil circularity (i.e. the shape of the cell) and cell speed was found, the ethyl ester did not show any correlation.

Ganglioside composition changes in spongiform encephalopathies: analyses of 263K scrapie-infected hamster brains.

Ganglioside composition in brains of terminally ill LVG/LAK golden Syrian hamsters infected with the 263K strain of the scrapie agent was analyzed. Results were compared to those obtained from noninfected animals matched by age, sex, and strain. Gangliosides extracted from scrapie-infected animals showed little change in major components, while an increased number of new alkali-labile species appeared. Additionally, the animal strain employed demonstrated a significant polymorphism in brain ganglioside composition. No significant changes in incubation time, clinical development or pathologic features of scrapie were associated with this polymorphism.

Production and characterization of monoclonal antibodies to human ciliary neurotrophic factor with defined epitope recognition.

Ten mouse hybridoma lines producing monoclonal antibodies (Mabs) against recombinant human ciliary neurotrophic factor (rhCNTF) have been obtained. Two monoclonal antibodies belonging to the IgG1 class were selected and characterized. Their specificity was established by ELISA and Western blotting. Epitopes recognized by the two Mabs were investigated with ELISA and Western blotting by using rhCNTF mutants, rhCNTF fragments and synthetic peptides mimicking different portions of the CNTF molecule. The carboxy-terminal part of the CNTF and particularly the sequence between aa 150 and 159 appeared to constitute the immunodominant group. The fact that certain amino acid sequences of CNTF are conserved among species was utilized to examine the crossreactivity patterns of the two Mabs with rat sciatic nerve CNTF by Western blotting and immunohistochemistry. These antibodies will be useful for studying the distribution of CNTF in the nervous system and in developing an enzyme-linked immunosorbent assay for the quantitative determinations of CNTF in various neuropathologies.

Nerve growth factor antibodies recognize neurotrophin-3.

The immunological properties of the neurotrophins NGF, BDNF, and NT-3 were compared using polyclonal and monoclonal antibodies against the beta subunit of mouse NGF. Affinity-purified anti-NGF IgG consistently recognized NGF and NT-3 on Western blots, and inhibited the trophic activity of NGF and NT-3 but not BDNF. In contrast, anti-NGF monoclonal antibodies did not block the trophic activity of either NT-3 or BDNF. These results are consistent with the greater structural overlap between NGF and NT-3 than between NGF and BDNF.

Mechanism of action of the monosialoganglioside GM1 as a modulator of CD4 expression. Evidence that GM1-CD4 interaction triggers dissociation of p56lck from CD4, and CD4 internalization and degradation.

Analyzing the mechanisms underlying the capability of the monosialoganglioside GM1 to induce CD4 modulation we observed that GM1 has a dual effect on the CD4 molecule. GM1 treatment of the lymphoma cell line MOLT-3 and CD4-transfected HeLa cells for times shorter than 30 min prevented binding of monoclonal antibodies (mAbs) recognizing epitopes located within the first NH2-terminal domains of CD4, but not of the OKT4 mAb, which binds to the region of CD4 proximal to the transmembrane domain. However, no binding of the OKT4 mAb was observed after a few hours of treatment with GM1 in both MOLT-3 cells and HeLa cells transfected with an intact CD4 molecule, but not in HeLa cells transfected with a CD4 molecule lacking the bulk of the cytoplasmic domain, suggesting that modulation of CD4 by GM1 depends on the integrity of the cytoplasmic domain. GM1 treatment blocked binding of several mAbs which recognize epitopes located within the first two NH2-terminal domains of CD4 and did not induce CD4 down-modulation if MOLT-3 cells were preincubated with the OKT4A or the OKT4 mAbs. Immunoprecipitation studies with [35S]methionine-labeled MOLT-3 cells showed that GM1-induced CD4 down-modulation was accompanied by CD4 degradation, and this was preceded by dissociation of p56lck from CD4. GM1-induced CD4 down-modulation, dissociation of p56lck from CD4, and CD4 degradation were unaffected by staurosporine, which, on the contrary, blocked these events in response to phorbol 12-myristate 13-acetate. These observations demonstrate that the first action of GM1 is to mask epitopes located within the first two NH2-terminal domains; then, GM1 triggers protein kinase C-independent signals which cause p56lck dissociation from CD4 and the delivery of the molecule to an intracellular compartment where it is eventually degraded.

Synthesis and purification of biologically active rat brain-derived neurotrophic factor from Escherichia coli.

The cDNA for rat brain-derived neurotrophic factor was cloned as the prepro and mature sequences into two independent expression vectors under control of the T7 promoter. When these vectors were transfected into Escherichia coli the prepro and mature forms of brain-derived neurotrophic factor accounted for about 20% and 25% of total E. coli proteins, and displayed molecular sizes of 26 kDa and 15 kDa, respectively. Mature brain-derived neurotrophic factor was extracted from E. coli inclusion bodies, refolded in the presence of CuCl2 and purified. The resulting protein had an ED50 of 3 ng/ml in supporting survival of cultured embryonic dorsal root ganglion neurons.

Synthesis and preliminary characterisation of new esters of the bacterial polysaccharide gellan.

Under the appropriate experimental conditions, ethyl, propyl, and methylprednisolon-21-yl esters of gellan can be obtained without significant degradation. At low degrees of esterification (de), depending on the ester moiety, the products are water-soluble, which allows the influence of hydrophilicity and charge density on their ability to assume an ordered conformation in dilute aqueous solution to be studied. With high de, the products were soluble only in organic solvents (e.g., methyl sulphoxide) with good film-forming capacity. The methylprednisolon-21-yl esters have been characterised in a preliminary manner in terms of drug-release kinetics.

Production of novel monoclonal antibodies against rabbit platelet factor four.

Ten hybridomas producing monoclonal antibodies (Mabs) against rabbit platelet factor 4 (PF4) were obtained from the fusion of splenocytes from mice immunized with purified rabbit PF4 and NSO mouse myeloma cells. When the reactivities of these monoclonal antibodies were determined by enzyme-linked immunosorbent assay and immunoblotting with human and rabbit PF4, they showed a high degree of specificity. Only one Mab recognized an epitope common to the human and rabbit molecules, the other nine reacted only with the rabbit protein. All the antibodies recognized, in crude platelet lysates, a band that comigrates with the purified PF4 protein. None of these antibodies cross-reacted with major rabbit or human platelet-poor plasma proteins. The significance of the Mabs in immunological and physiological studies is discussed.

Competitive binding assay for quantitative determination of GM1 ganglioside in plasma and cerebrospinal fluid.

A competitive binding assay for the quantitative determination of GM1 ganglioside is described. After extraction from biological fluids, GM1 was incubated with a known amount of cholera toxin B-subunit conjugated with horseradish peroxidase, and exposed to GM1 adsorbed onto polystyrene microwells. Since GM1 in solution blocks the binding of toxin B-subunit to GM1 adsorbed onto the solid phase, enzyme activity serves as a reciprocal measure of GM1 concentration in the sample. The assay was used to determine the basal level of GM1 in plasma and cerebrospinal fluid in different populations.

Interleukin-1 beta regulates proenkephalin gene expression in astrocytes cultured from rat cortex.

Glial cells execute essential functions in central nervous system (CNS) development and are also believed to play important roles during gliosis in response to trauma or disease. These developmental and pathological states have also been associated with elevated expression of opioid genes. Because levels of the cytokine interleukin-1 beta (IL-1 beta) increase following CNS lesions, we examined the possible influence of IL-1 beta on the expression of opioid genes in astrocytes cultured from rat cortex. Proenkephalin mRNA expression was stimulated by IL-1 beta in a time- and concentration-dependent manner, being maximal with 5 U/ml IL-1 beta at 4 h. Although the beta-adrenergic agonist isoproterenol was also active, interferon, glutamate, and carbachol were not. Unlike isoproterenol, the actions of IL-1 beta were not associated with a cyclic adenosine monophosphate (AMP)-dependent pathway. Interleukin-1 beta also regulated a proenkephalin-chloramphenicol acetyltransferase fusion gene transiently transfected into astrocytes, with a dose-response similar to that active in proenkephalin mRNA. These effects of IL-1 beta were region-specific, not being observed with either cerebellar or hippocampal astrocytes; however, isoproterenol was active in the latter cell populations. Proenkephalin mRNA in cortical astrocytes was stimulated following a temperature stress. These results suggest that enhanced proenkephalin gene expression in astrocytes by IL-1 beta may be important in neuroimmune interactions and in trauma-induced CNS injury or stress.

Effect of parenteral administration of GM1 on cytokines and anti-ganglioside antibody patterns. Preliminary report in normal human individuals.

To assess the effects of monosialoganglioside GM1 on some immunological parameters, 12 healthy men were treated with 100 mg GM1 i.m. daily for 15 days. Before and after treatment, the following were studied: (1) serum levels of antibodies against GM1, asialo-GM1 (aGM1), GM2 and GD1b; (2) serum levels of interleukin (IL)-1 beta, IL-2, soluble IL-2 receptor (sIL-2R), tumor necrosis factor-alpha (TNF-alpha), and interferon-gamma (IFN-gamma); (3) IL-1 beta and TNF-alpha production by peripheral blood monocytes (PBMO). Anti-ganglioside antibody and cytokine serum levels were not affected by exogenous GM1 administration with the exception of a transient increase in anti-GM1 antibody titer observed in one subject. In addition, no inhibition of IL-1 beta and TNF-alpha production by PBMO was observed. These preliminary data do not support a potential immunogenic or immunomodulatory function for in vivo administered GM1.

Synthesis of the biologically active beta-subunit of human nerve growth factor in Escherichia coli.

The gene (NGFB) encoding the beta subunit of mature human nerve growth factor (hNGFB) was subcloned into the pJLA503 expression vector under the control of bacteriophage promoters PR and PL, and expressed in Escherichia coli. The recombinant protein represented approximately 3% of the total cellular protein. Biologically active hNGFB was solubilized (0.2% total NGFB) and purified by cation-exchange chromatography and it yielded two bands on polyacrylamide-gel electrophoresis under nonreducing conditions, corresponding to the monomeric (14 kDa) and homodimeric (26.5 kDa) forms of the molecule. Both hNGFB forms were immunopositive on Western blots with rabbit anti-NGFB antibodies; however, following additional purification, only the species corresponding to the hNGFB homodimer was biologically active on cultured chicken dorsal root ganglion neurons. These results demonstrate the feasibility of synthesizing the biologically active form of hNGFB in E. coli.

A simple method to detect contaminating peptides and amino acids in large-scale ganglioside preparations.

A reverse-phase liquid chromatography method was developed to analyse the presence of contaminating peptides and amino acids in large-scale monosialoganglioside preparations. Samples were hydrolysed under controlled conditions and derivatized with phenylisothiocyanate. PTC-amino acids were then separated and identified by HPLC. The sensitivity of the method allowed detection of a least 50 pmoles of amino acid per mg of ganglioside with excellent reproducibility and little or no interference of by-products derived from hydrolysis of the glycosphingolipid. The response was consistently linear for each amino acid within the examined analytical range. The ease and speed of performance make this method a useful tool for rapidly monitoring large-scale extraction and purification processes of biologicals obtained from natural sources.