Interaction of Mycobacterium tuberculosis cell wall components with the human natural killer cell receptors NKp44 and Toll-like receptor 2.

We have previously demonstrated that a soluble form of the human NK cell natural cytotoxicity receptor NKp44, binds to the surface of Mycobacterium tuberculosis (MTB). Herein, we investigated the interaction of MTB cell wall components (CWC) with NKp44 or with Toll-like receptor 2 (TLR2) and the role of NKp44 and TLR2 in the direct activation of NK cells upon stimulation with MTB CWC. By using several purified bacterial CWC in an ELISA, we demonstrated that NKp44 was able to bind to the MTB cell wall core mycolyl-arabinogalactan-peptidoglycan (mAGP) as well as to mycolic acids (MA) and arabinogalactan (AG), while soluble TLR2 bound to MTB peptidoglycan (PG), but not to MA or AG. The mAGP complex induced NK cell expression of CD25, CD69, NKp44 and IFN-γ production at levels comparable to M. bovis Bacillus Calmette-Guérin-stimulated (BCG) cells. While AG and MA used alone failed to induce NK cell activation, mycobacterial PG-exhibited NK cell stimulatory capacity. Activation of resting NK cells by mAGP and IFN-γ production were inhibited by anti-TLR2 MAb, but not by anti-NKp44 MAb. Differently, anti-NKp44 MAb partially inhibited CD69 expression on NK cells pre-activated with IL-2 and then stimulated with mAGP or whole BCG. Overall, these results provide evidence that components abundant in mycobacterial cell wall are able to interact with NKp44 (AG, MA) and TLR-2 (PG), respectively. While interaction of TLR2 with mycobacterial cell wall promotes activation of resting NK cells and IFN-γ production, NKp44 interaction with its putative ligands could play a secondary role in maintaining cell activation.

Multipotential neural precursors transplanted into the metachromatic leukodystrophy brain fail to generate oligodendrocytes but contribute to limit brain dysfunction.

Neural stem cells appear to be best suited for regenerative therapy in neurological diseases. However, the effects of high levels of potentially toxic substances such as sulfatides--which accumulate in metachromatic leukodystrophy (MLD)--on this regenerative ability are still largely unclear. To start addressing this question, in vitro and in vivo experiments were used to examine the behavior of multipotential neural precursors exposed to abnormally high levels of sulfatides. Following transplantation of dissociated neurospheres into the brain of presymptomatic MLD pups, the majority of donor-derived cells were distributed in a caudal to rostral direction, with higher numbers in the cortex. Most if not all of the donor cells acquired an astroglial phenotype. We found no evidence of oligodendrocyte or neuronal commitment of transplanted cells in long-term-treated MLD mice (e.g. up to 1.5 years of age). This was in line with our in vitro findings of sulfatides blocking oligodendrocyte formation after induction of differentiation in sulfatide-treated epidermal growth factor/fibroblast growth factor responsive neurospheres. Transplanted MLD mice showed an improved arylsulfatase A (ARSA) activity and a significant amelioration of sulfatide metabolism, neurodegeneration and motor-learning/memory deficits. Furthermore, transplanted cells were shown to act as a source of ARSA enzyme that accumulated in endogenous brain cells, indicating the occurrence of enzyme cross-correction between transplanted and host cells. These results provide a first insight into the effect of sulfatides on the stemness properties of neural stem cells and on the effects of the MLD environment on the in vivo expectations of using neural stem cells in cell therapy.

Human CD56bright and CD56dim natural killer cell subsets respond differentially to direct stimulation with Mycobacterium bovis bacillus Calmette-Guérin.

Mycobacterium bovis bacillus Calmette-Guérin (BCG) is capable of directly stimulating several effector functions of human natural killer (NK) cells in the absence of interleukin-12 and professional antigen presenting cells. To assess the contribution of two main human NK-cell subsets (CD56(dim) and CD56(bright)) to the overall in vitro NK-cell response to BCG, peripheral blood mononuclear cells depleted of nylon wool-adherent cells or purified NK cells were stimulated with live BCG. By combining intranuclear bromodeoxyuridine (BrdU) staining and analysis of CD56 marker intensity, statistically higher percentages of BrdU(+) cells were found among the CD56(bright) subset than the CD56(dim) subset after 6 days of stimulation with BCG. Similarly, evaluation of intracellular interferon-gamma (IFN-gamma) revealed that CD56(bright) cells were those mainly involved in IFN-gamma production in response to BCG. In contrast, the CD56(dim) subset contained higher levels of perforin and granzyme A, two key molecules for exocytosis-mediated cytotoxicity, than the CD56(bright) subset. Although 16-20-h stimulation with BCG did not substantially alter the expression of cytotoxic molecules by the two subsets, a decrease in perforin content was observed in the CD56(dim), but not in the CD56(bright) subset, following 4-h incubation with the NK-sensitive target K562 cell line. This decrease in perforin content correlated with the induction by BCG-stimulated NK cells, of early markers of apoptosis on target cells to a greater extent than unstimulated cells suggesting a major role for the CD56(dim) subset in cytotoxic activity in response to BCG. Taken together, these results demonstrate that CD56(bright) and CD56(dim) human NK-cell subsets exert different functional activities in response to a live bacterial pathogen.

Purification, biochemical characterization and immunogenicity of SA5K, a secretion antigen of Mycobacterium tuberculosis.

Mycobacterium tuberculosis (MTB) secretory proteins are generally considered important antigens for immune protection against tuberculosis (TB). An 8.3-kDa secretory antigen of MTB and Mycobacterium bovis bacillus Calmette-Guérin (BCG), called SA5K, was recently identified and cloned in our laboratory. In this report, recombinant SA5K containing a histidine hexamer was expressed in Escherichia coli and purified to investigate its biochemical structure and to establish whether it was immunogenic for healthy sensitized and nonsensitized human donors and for patients infected with MTB. The protein nucleotide sequence was shown to be identical in BCG and in MTB. SA5K revealed an abnormal electrophoretic mobility in SDS-PAGE that made it look lighter than it is in Western blotting. While recombinant SA5K was poorly recognized by T lymphocytes from patients with pulmonary TB, it elicited proliferation of CD4+ T lymphocytes in the vast majority of healthy individuals sensitized to mycobacterial antigens by BCG vaccination. At a serum dilution of 1 : 80, antibodies reacting against recombinant SA5K were found in 67% of sera from TB patients and in 73% of sera from healthy subjects. The percentage of positive subjects dropped at higher serum dilutions, but no significant difference in the recognition rate was observed between TB patients and healthy donors and between healthy vaccinated and nonvaccinated subjects. Owing to the high percentage of sera from healthy subjects who recognized SA5K in Western blotting, the antigen seems to exhibit, at least in the present form, a poor specificity for an employment for a serodiagnosis of TB.

Identification, molecular cloning, and evaluation of potential use of isocitrate dehydrogenase II of Mycobacterium bovis BCG in serodiagnosis of tuberculosis.

Diagnosis of tuberculosis is time-consuming and requires infrastructures which are often not available in countries with high incidences of the disease. In the present study, an 82-kDa protein antigen was isolated by affinity chromatography and was identified by peptide mass fingerprinting as isocitrate dehydrogenase II, which is encoded by the icd2 gene of Mycobacterium bovis BCG. The icd2 gene of BCG was cloned by PCR, and the product of recombinant gene expression was purified and analyzed by two-dimensional polyacrylamide gel electrophoresis. The recombinant protein, named rICD2, was tested for its recognition by immunoglobulin G (IgG) antibodies from the sera of 16 patients with tuberculosis (TB) and 23 healthy individuals by Western blotting. The results showed that rICD2 is recognized by IgG antibodies from the sera of all TB patients tested at serum dilutions of > or = 1:640. At a serum dilution of 1:1,280, the sensitivity was 50% and the specificity was 86.9%. These results indicate that rICD2 might represent a candidate for use in a new assay for the serodiagnosis of TB.

Involvement of the Mycobacterium tuberculosis secreted antigen SA-5K in intracellular survival of recombinant Mycobacterium smegmatis.

A new protein (SA-5K) secreted in culture filtrates by Mycobacterium bovis, Mycobacterium tuberculosis, and few other mycobacterial species was previously identified and purified in our laboratory. In order to evaluate the putative role of SA-5K during intracellular mycobacterial growth, in the present study the SA-5K gene was cloned and expressed in Mycobacterium smegmatis, a rapid growing non-pathogenic mycobacterium which does not contain the gene for the protein. SA-5K expression in the THP-1 human macrophage cell line infected with the recombinant strain (M. smegmatis-pROL5K) was demonstrated by RT-PCR on RNA extracted from bacterial cells following 24 and 48 h of infection. Intracellular SA5K expression was associated with a higher cfu increase of M. smegmatis-pROL5K in comparison to the negative control strain (M. smegmatis recombinant for the cloning vector) (P=0.01). No significant change in SA-5K synthesis by M. smegmatis-pROL5K was observed when the recombinant strain was grown in vitro in different stress conditions such as iron deprivation, pH 4.5, presence of nitric oxide or hydrogen peroxide. The results presented in this study suggest a possible role for SA-5K in intracellular survival of recombinant M. smegmatis, though the function of the protein remains unknown.

Use of a recombinant strain of Mycobacterium avium expressing beta-galactosidase to evaluate the activities of antimycobacterial agents inside macrophages.

A reliable and low-cost method that enables rapid screening of the activity exerted by new antimicrobial agents on intracellularly growing Mycobacterium avium has been developed. To this aim, a recombinant (lacZ) strain of M. avium expressing the Escherichia coli beta-galactosidase gene was used to evaluate, in murine macrophages, the susceptibility of M. avium to common antimycobacterial agents. beta-Galactosidase levels, measured in the presence of each of the antibiotics tested, were closely correlated with the number of CFU recovered from the M. avium lacZ strain-infected macrophages.

Identification of distinct lymphocyte subsets responding to subcellular fractions of Mycobacterium bovis bacille calmette-Guérin (BCG).

In order to investigate the ability of Mycobacterium bovis BCG vaccination to induce immune responses toward different classes of mycobacterial antigens and the cell populations involved in such responses, proliferation of distinct human lymphocyte subsets from BCG-vaccinated donors in response to different subcellular fractions of BCG was analysed and compared with that of not sensitized subjects. Proliferation of different cell subsets was evaluated by flow cytometric determination of bromodeoxyuridine incorporated into DNA of dividing cells and simultaneous identification of cell surface markers. Although a certain degree of variability was observed among different donors, after 6 days of in vitro stimulation BCG-vaccinated subjects displayed, as a mean, a stronger blastogenic response to all the classes of antigens compared with non-sensitized ones. PPD, culture filtrates and membrane antigens induced a predominant proliferation of CD4+ T cells. In contrast, preparations enriched in cytosolic antigens elicited strong proliferation of gammadelta+ T cells which, as a mean, represented 55% of the proliferating cells. Although to a lesser extent, proliferation of gammadelta+ T cells was also elicited by preparations enriched in membrane and cell wall antigens. In response to the latter preparation proliferation of CD4+ T cells and CD16+/CD3- (natural killer (NK)) cells was observed, as well. In particular, cell wall antigens were found to induce significantly higher levels of proliferation of NK cells compared with all the other classes of antigens.

Molecular biology of the apteronotus NMDA receptor NR1 subunit.

The complete sequences and expression patterns of the NR1 (aptNR1) subunit of the N-methyl-d-aspartate (NMDA) receptor and its alternative splice isoforms have been determined for the weakly electric fish Apteronotus leptorhynchus. The deduced amino acid sequence of aptNR1 is approximately 88 % identical to the NR1 sequences of other vertebrate. Two of the three alternative splice cassettes previously described for mammalian NR1s, N1 and C1, are present in aptNR1, but the third cassette, C2, is not found. In addition, two teleost-specific splice cassettes occur on the N-terminal side of the C1 sequence. The cellular patterns of aptNR1 expression, including the patterns of N1 and C1 splicing, have been mapped using the in situ hybridization technique. High levels of aptNR1 mRNA were detected throughout the central nervous system including most neurons of the electrosensory system, with the highest levels in electrosensory lateral line lobe pyramidal cells. Expression of the N1 splice isoform was higher in more caudal regions of the brain, and expression of the C1 splice isoform was higher in more rostral regions. The N1 splice isoform was present in almost all NR1-positive cells, in contrast to the C1 splice isoform which was restricted to a subset of NR1-positive cells. These results demonstrate that the NR1 subunit of the NMDA receptor is evolutionarily conserved across species and that regulation of alternative RNA splicing modulates the properties of NR1 in different neurons of the central nervous system of A. leptorhynchus.

Identification and molecular cloning of a novel secretion antigen from Mycobacterium tuberculosis and Mycobacterium bovis BCG.

A novel protein called SA-5K was identified in Mycobacterium bovis BCG (BCG) short-term culture filtrates (CFs) by means of a recently described monoclonal antibody (mAb), L8D8. This protein had an apparent molecular mass (MM) of 5 kDa, as judged by Western blotting after sodium dodecyl sulphate-polyacrylamide gel electrophoresis in reducing conditions, and did not seem to contain any sugar or lipid substituents. In the present work, SA-5K was purified from BCG CFs by affinity chromatography. A protein that could be detected in Western blot but not by standard protein staining techniques was obtained. When SA-5K was subjected to aminoterminal sequencing, the 10 amino acids (aa) found matched the first 10-aa sequence deduced from an open reading frame (ORF) of M. tuberculosis. The ORF encodes a polypeptide, likely to include a signal for secretion, with an estimated MM of 8.3 kDa after signal peptide cleavage. The secretory nature of SA-5K was confirmed by the fact that it could only be detected in CFs, but not in other BCG subcellular fractions. After size exclusion chromatography, reactivity with mAb L8D8 was found to peak in the 45-50- and 14-16-kDa fractions. The latter MM was close to that estimated from the ORF of M. tuberculosis, implying that the 5-kDa antigen detected initially by Western blot in reducing conditions was a portion of SA-5K released after reduction of a disulphide bridge. The presence of the gene for SA-5K in BCG and its identity were confirmed by PCR (polymerase chain reaction) with specific primers and restriction analysis: the PCR product was slightly shorter in BCG than in M. tuberculosis. The gene coding for SA-5K was cloned by PCR from BCG and M. tuberculosis DNA and was expressed in Escherichia coli.

Alternative RNA splicing of the NMDA receptor NR1 mRNA in the neurons of the teleost electrosensory system.

The sequence for cDNA encoding the NMDA receptor subunit 1 (aptNR1) of the weakly electric fish Apteronotus leptorhynchus has been determined. The deduced amino acid sequence is approximately 88% identical to other vertebrate NR1 proteins, with sequence homology extending to the alternatively spliced cassettes N1 and C1. The fish and mammalian N1 and C1 splice cassettes are identical at 20 of 21 and 30 of 37 amino acid positions, respectively. We did not detect a C2 splice cassette in aptNR1 mRNA, but we did find two novel C-terminal alternative splice cassettes labeled C1' and C1". The relative levels of NR1 transcripts containing the N1 and C1 splice cassettes were determined by using RNase protection and in situ hybridization analysis. N1-containing mRNAs are more abundant in caudal brain regions, similar to the patterns reported for mammalian brain. In contrast, the relative levels of transcripts containing the C1 splice cassette are much lower in fish than in mammals, averaging only 9% for the whole brain. The levels of C1 splicing increased in more rostral brain regions. In situ hybridizations with N1- and C1-specific probes demonstrated that N1 cassette splicing occurs in most neurons but that C1 splicing is heterogeneous and is restricted to a subset of neuronal types in the electrosensory system.

N-methyl-D-aspartate receptor 1 mRNA distribution in the central nervous system of the weakly electric fish Apteronotus leptorhynchus.

We have isolated a partial cDNA for the N-methyl-D-aspartate (NMDA) receptor 1 (NMDAR1) subunit from an Apteronotus leptorhynchus brain cDNA library. The A. leptorhynchus cDNA fragment, which corresponds to nucleotides 135-903 within the 5' region of the rat NR1 mRNA, encodes 252 amino acids that are >80% identical to the homologous segments of the rat, human, and duck NR1 proteins. RNAse protection assays revealed that the A. leptorhynchus NR1 mRNA was highly enriched in the forebrain and hypothalamus, with lesser amounts in the brainstem, and very low levels in the cerebellum. In situ hybridization also demonstrated that neurons in the pallial forebrain were highly enriched in NR1 transcripts. High levels of NR1 mRNA were found in pyramidal cells within the optic tectum and octavolateral regions. Pyramidal cells of the electrosensory lateral line lobe had the highest levels of expression, and the NR1 mRNA was found to be selectively enriched in their apical dendrites.

Repetitive treatment with serotonin modifies protein synthesis and protein phosphorylation in the central nervous system of Hirudo medicinalis.

Serotonin (5HT) is the neurotransmitter involved in some forms of short-term memory in the leech. Behavioral experiments have demonstrated that long-term memory requires new protein synthesis. With the aim of studying the molecular mechanism underlying memory processes in the leech, we have analyzed the effect of 5HT on protein synthesis and protein phosphorylation. Segmental ganglia of the leech central nervous system have been labeled, proteins have been separated by two-dimensional-electrophoresis and labeled proteins detected by autoradiography. Our findings indicate that repetitive treatment with 5HT produces either the persistence of phosphorylation or changes in protein synthesis in several proteins.

Interleukin-1 and interleukin-6 modify protein phosphorylation in the central nervous system of Hirudo medicinalis.

Recent data suggest that IL-1-like molecules have been conserved during evolution. The signal transduction mechanism of IL-1 is not known, but kinase activation has been reported. With the aim of studying if human IL-1 has any effect on the leech nervous system, we have added this cytokine to segmental ganglia labeled with [32P]ortophosphoric acid; proteins have been separated by electrophoresis and phosphoproteins detected by autoradiography. In the present paper we show that human IL-1 and IL-6 are able to induce changes on protein phosphorylation in the leech central nervous system and that these changes are similar to those ones induced by the neurotransmitter serotonin.

Effect of serotonin on protein phosphorylation in the central nervous system of the leech Hirudo medicinalis.

1. Phosphoproteins of different regions of the Hirudo medicinalis central nervous system have been analysed by means of two-dimensional electrophoresis. 2. Serotonin, 8-Br-cAMP and phorbol 12,13-dibutyrate stimulate phosphorylation of a number of proteins whose isoelectric points and molecular weights are presented. 3. A group of proteins of 78 kDa and pI = 6-6.5, whose level of phosphorylation increases in the presence of serotonin, 8-Br-cAMP and phorbol ester, is observed only in segmental but not in cephalic or caudal ganglia. 4. The putative roles of these phosphoproteins are discussed.

Effect of phorbol ester on protein phosphorylation in the central nervous system of the leech Hirudo medicinalis: a two-dimensional electrophoretical analysis.

1. Proteins of different regions of the Hirudo medicinalis central nervous system have been analyzed by means of two-dimensional electrophoresis. 2. Subcellular distribution of phosphoproteins has been studied in leech segmental ganglia. 3. Phorbol 12,13-dibutyrate, a protein kinase C activator, stimulates the phosphorylation of a number of proteins whose isoelectric points and mol. wts are presented. 4. Putative roles for these phosphoproteins are discussed.