Search for the molecular basis of morphological variation in Mycobacterium avium

Isolates of Mycobacterium avium exhibit three different colonial variations: smooth domed (SmD), smooth transparent (SmT), and rough (Rg). Because the discrimination between morphotypes is founded on morphological rather than molecular principles and because of the absence of consensus over the relevance of morphology to pathogenesis and drug sensitivity, a comparative study at the protein level was undertaken. By direct immunization of BALB/c mice with the soluble sonicate of one of the morphotypes of M. avium serovar 2, eight monoclonal antibodies (MAbs) were identified, of which one was M. avium specific. Cross immunization of syngeneic mice with serum-absorbed antigens allowed the generation of 15 further MAbs; 11 were M. avium or M. avium complex specific, but none of them was morphotype specific. Subcellular fractions analyzed by electrophoresis showed similar profiles, with the exception of a cytosolic protein with a relative molecular mass of ca. 66 kDa (protein SmT 66), which was most highly expressed in SmT variants of M. avium serotypes 2 and 4. Because a well-known, ubiquitous stress-heat shock protein (hsp65) has a similar molecular mass, protein SmT 66 was compared with hsp65. Western blot (immunoblot) analyses using several cross-reacting MAbs and N-terminal amino acid sequencing established that this protein was not the ubiquitous stress protein. Thus, SmT 66 is the first product to be described which might be associated with the SmT morphotype.</jats:p

Puberty is associated with a marked increase of the female sex predominance in chronic autoimmune thyroiditis

BACKGROUND: Chronic autoimmune thyroiditis (CAT) displays a strong female predominance with female-to-male (F:M) ratios of 4-20:1 in adults and 2-9:1 in children and adolescents. Both genetic and hormonal factors are involved in this phenomenon. The relation between puberty and F:M ratio in CAT has never been evaluated. METHODS: The F:M ratio of 133 children with CAT (group A, age at diagnosis 2.4-17.7 years) was compared with that of 113 adult CAT patients (group B, age at diagnosis 21-79 years). Group A included 64 prepubertal (aged 2.4-13.2 years, group A1) and 69 pubertal (aged 9.2-17.4, group A2) children. RESULTS: The F:M ratio in group A was 3.0, which is significantly (p < 0.001) lower than that (10.3) found in group B patients. The F:M ratio of group A1 prepubertal children was lower (1.6) and significantly different from that of pubertal (6.7, p < 0.01) and adult patients (10.3, p < 0.0001). This phenomenon was more evident in hypothyroid as compared to euthyroid CAT. CONCLUSIONS: This study provides the first evidence that female predominance of CAT strongly increases during puberty, suggesting a major role for sex hormones in this phenomenon. Further studies are needed to clarify this point

Characterization of thyroglobulin epitopes in Sardinian adults and juveniles with Hashimoto's thyroiditis: evidence against a major effect of age and genetic background on B-cell epitopes.

Background Using recombinant human monoclonal thyroglobulin antibodies expressed as Fab molecules (TgAb-Fab), we have recently confirmed the restriction of Tg epitopes in Hashimoto’s thyroiditis (HT). Objective To investigate Tg epitopes of serum TgAb in HT adults and HT juveniles from a geographically isolated area (Sardinia). Design and Patients Serum TgAb of 39 Sardinian HT adults, 53 Sardinian HT juveniles and 45 non-Sardinian HT adults were evaluated. The binding of serum TgAb to Tg in ELISA was inhibited by four recombinant human TgAb-Fab, identifying Tg epitopic regions A–D. The percentage of Tg binding inhibition was calculated comparing the binding of serum TgAb in presence of each TgAb-Fab with that in its absence. Results In the whole cohort of 137 patients, A region TgAb-Fab induced the highest levels of inhibition (55Æ3 ± 17Æ8%) (mean ± SD). Lower levels of inhibition were induced by TgAb- Fab of regions B (27Æ8 ± 25Æ8%), C (26Æ8 ± 24Æ6%) and D (17Æ5 ± 18Æ4%). In Sardinian HT adults inhibition by TgAb-Fab of regions A, B and C were comparable to Sardinian HT juveniles; the marginal D region TgAb-Fab induced a slightly higher inhibition (22Æ1 vs. 13Æ8%; P = 0Æ034) in the former than in the latter group. In Sardinian and non-Sardinian HT adults inhibitions by the four TgAb-Fab were similar. Conclusions In HT, the Tg epitope pattern of serum TgAb was similar in juveniles and adults from a geographically restricted area and in two adult populations from different geographical areas. Thus, in HT, neither age nor genetic background appear to influence B-cell epitopes

Characterization of thyroglobulin epitopes in Sardinian adults and juveniles with Hashimoto's thyroiditis: evidence against a major effect of age and genetic background on B-cell epitopes

BACKGROUND: Using recombinant human monoclonal thyroglobulin antibodies expressed as Fab molecules (TgAb-Fab), we have recently confirmed the restriction of Tg epitopes in Hashimoto's thyroiditis (HT). OBJECTIVE: To investigate Tg epitopes of serum TgAb in HT adults and HT juveniles from a geographically isolated area (Sardinia). DESIGN AND PATIENTS: Serum TgAb of 39 Sardinian HT adults, 53 Sardinian HT juveniles and 45 non-Sardinian HT adults were evaluated. The binding of serum TgAb to Tg in ELISA was inhibited by four recombinant human TgAb-Fab, identifying Tg epitopic regions A-D. The percentage of Tg binding inhibition was calculated comparing the binding of serum TgAb in presence of each TgAb-Fab with that in its absence. RESULTS: In the whole cohort of 137 patients, A region TgAb-Fab induced the highest levels of inhibition (55.3 +/- 17.8%) (mean +/- SD). Lower levels of inhibition were induced by TgAb-Fab of regions B (27.8 +/- 25.8%), C (26.8 +/- 24.6%) and D (17.5 +/- 18.4%). In Sardinian HT adults inhibition by TgAb-Fab of regions A, B and C were comparable to Sardinian HT juveniles; the marginal D region TgAb-Fab induced a slightly higher inhibition (22.1 vs. 13.8%; P = 0.034) in the former than in the latter group. In Sardinian and non-Sardinian HT adults inhibitions by the four TgAb-Fab were similar. CONCLUSIONS: In HT, the Tg epitope pattern of serum TgAb was similar in juveniles and adults from a geographically restricted area and in two adult populations from different geographical areas. Thus, in HT, neither age nor genetic background appear to influence B-cell epitopes

LITHIUM AND THIOCOLCHICOSIDE INTERACTION ON RAT DENTATE GYRUS NEURONAL NETWORK EXCITABILITY

The muscle relaxant thiocolchicoside (TCC) is commonly used in clinical practice also for its anti-inflammatory and analgesic effects. Several previous studies have proposed that TCC as a competitive antagonist of GABAARs, and in vivo exerts a proconvulsant and convulsant activity in rats and humans. Preliminary data suggest that pre treatment of rats with concentration of lithium similar to those found in humans under clinical conditions, markedly enhances the convulsant effects of TCC. To better understand the nature of the interaction between lithium and TCC, we performed extracellular electrophysiological recordings of fEPSPs and evaluated the neuronal excitability in rat dentate gyrus (DG) slices. After 6 h of LiCl (1 mM) incubation the I/O responses of fEPSPs were markedly enhanced compared to control. Application of 1 M TCC potentiated fEPSPs with a greater efficacy in slices after LiCl incubation. Patch-clamp recordings in DG granule cells revealed that LiCl incubation enhanced the frequency of mEPSCs and reduced paired-pulse facilitation ratio suggesting an increased probability of glutamate release. Taken together these results indicate that treatment of slices with LiCl increases fEPSP recorded from DG granule cells, and markedly potentiates the effects of TCC. The strong increase of DG neuronal excitability induced by the association of lithium and TCC is consistent with the pharmacological observation of an enhanced convulsant activity in rats and suggests that particular attention should be taken when such drug combination is used in clinical practice

Lithium potentiates the effects of thiocolchicoside on neuronal excitability in rat dentate gyrus

Thiocolchicoside (TCC) is used clinically for its muscle relaxant, anti-inflammatory, and analgesic properties. Our previous electrophysiological studies have indicated that TCC is a competitive antagonist of GABAARs, an action that results in a proconvulsant and convulsant activity in rats and humans. Recent preliminary evidence has suggested that pre-treatment of rats with lithium, at concentrations found in humans treated with lithium under clinical conditions, markedly enhances the convulsant effects of TCC. We thus aimed at evaluating the effects of the interaction between lithium and TCC on neuronal excitability in the rat dentate gyrus (DG). Hippocampal slices were incubated in the absence or presence of 1 mM LiCl for 6 h, and fEPSP were then recorded. LiCl incubation induced a significant increase of the I/O responses of fEPSPs compared to control conditions. Patch-clamp analysis in DG granule cells revealed that LiCl incubation enhanced the frequency of mEPSCs and reduced paired-pulse facilitation ratio suggesting that lithium increases the probability of glutamate release. Application of 1 uM TCC potentiated fEPSPs with a greater extent in slices incubated with LiCl compared with those with vehicle. These results indicate that treatment of slices with LiCl increases the excitatory postsynaptic responses in DG granule cells, and markedly potentiates the effects of TCC. The strong increase of DG neuronal excitability induced by the association of lithium and TCC is consistent with the pharmacological observation of an enhanced convulsant activity in rats and suggests that some caution should be taken when such drug combination is used in clinical practice

Inactivation of Mycobacterium tuberculosis mannosyltransferase pimB reduces the cell wall lipoarabinomannan and lipomannan content and increases the rate of bacterial-induced human macrophage cell death

The Mycobacterium tuberculosis (M.tb) cell wall contains an important group of structurally related mannosylated lipoglycans called phosphatidyl-myo-inositol mannosides (PIMs), lipomannan (LM), and mannose-capped lipoarabinomannan (ManLAM), where the terminal α-[1→2] mannosyl structures on higher order PIMs and ManLAM have been shown to engage C-type lectins such as the macrophage mannose receptor directing M.tb phagosome maturation arrest. An important gene described in the biosynthesis of these molecules is the mannosyltransferase pimB (Rv0557). Here, we disrupted pimB in a virulent strain of M.tb. We demonstrate that the inactivation of pimB in M.tb does not abolish the production of any of its cell wall mannosylated lipoglycans; however, it results in a quantitative decrease in the ManLAM and LM content without affecting higher order PIMs. This finding indicates gene redundancy or the possibility of an alternative biosynthetic pathway that may compensate for the PimB deficiency. Furthermore, infection of human macrophages by the pimB mutant leads to an alteration in macrophage phenotype concomitant with a significant increase in the rate of macrophage death

Biosynthesis of mycobacterial lipoarabinomannan: Role of a branching mannosyltransferase

Lipoarabinomannan (LAM), one of the few known bacterial glycosylphosphoinositides (GPIs), occurs in various structural forms in Mycobacterium species. It has been implicated in key aspects of the physiology of Mycobacterium tuberculosis and the immunology and pathogenesis of tuberculosis. Yet, little is known of the biosynthesis of LAM. A bioinformatics approach identified putative integral membrane proteins, MSMEG4250 in Mycobacterium smegmatis and Rv2181 in M. tuberculosis, with 10 predicted transmembrane domains and a glycosyltransferase (GT) motif (DID), features that are common to eukaryotic mannosyltransferases (ManTs) of the GT-C superfamily that rely on polyprenyl-linked rather than nucleotide-linked sugar donors. Inactivation of M. smegmatis MSMEG4250 by allelic exchange resulted in altered growth and inability to synthesize lipomannan (LM) but accumulation of a previously uncharacterized, truncated LAM. MALDI-TOF/MS and NMR indicated a structure lower in molecular weight than the native molecule, a preponderance of 6-linked Manp residues, and the absence of 2,6-linked and terminal Manp. Complementation of the mutant with the corresponding ortholog of M. tuberculosis H37Rv restored normal LM/LAM synthesis. The data suggest that MSMEG4250 and Rv2181 are ManTs that are responsible for the addition of α(1→2) branches to the mannan core of LM/LAM and that arrest of this branching in the mutant deters formation of native LAM. The results allow for the presentation of a unique model of LM and LAM biosynthesis. The generation of mutants defective in the synthesis of LM/LAM will help define the role of these GPIs in the immunology and pathogenesis of mycobacterial infections and physiology of the organism