Effect of cryo preservation on Mycobacterium leprae growth in the footpads of non-immunosuppressed mice.

OBJECTIVE: To investigate using the mouse footpad system, whether the use of cryopreservants help in retaining the viability of Mycobacterium leprae samples stored at three different temperatures of 4 degrees, -20 degrees and -70 degrees C for 30 days. DESIGN: Biopsies from eight untreated lepromatous leprosy cases were homogenised and inoculated into footpads of normal Swiss White mice within 24 hours (control) and remaining homogenates in each case was divided and stored at 4 degrees C, -20 degrees C and -70 degrees C respectively for 1 month, using either 10% skimmed milk (SM) or Roswell Park Memorial Institute media + 10% glycerol (RPMI) (test). Homogenates adjusted to contain 1 x 10(4) M. leprae/footpad was inoculated into 10 mice per set. Harvestings were done at 6th, 7th, 8th and 12th months. Footpad counts showing > 1 x 10(5) M. leprae at 6th month or later were considered as positive yield. RESULTS: Control All the cases showed > 100 fold growth and 100% take. Viability at 4 degrees C: Only one case (SM) showed a 100 fold increase and 23% take. Viability at -20 degrees C: Two cases showed fold growth that was 40-60 fold less with takes of 63% (SM) and 71% (RPMI) respectively. Viability at -70 degrees C: Positivity was 45% but the fold increase was less as compared to control and takes were between 80-20%, except one RPMI where take was 100%. CONCLUSION: The viability assessed using the mouse footpad was best and consistent in the inoculas that were injected within 24 hours of harvest from the host tissue (control group). None of the storage temperatures used matched with the controls with respect to bacterial yield or % takes. Among the three storage temperatures, -70 degrees C appeared to be better with 45% of the samples showing growth. There was no significant difference noted between the two preservatives used.

Isolation of mycobacterium leprae from untreated borderline tuberculoid, mid-borderline and indeterminate cases using the mouse foot pad technique--a study of 209 cases.

Using the mouse foot pad (MFP) system, isolation of Mycobacterium leprae was attempted in 209 skin biopsies obtained from 114 borderline tuberculoid (BT), 62 mid borderline (BB) and 33 indeterminate (1) untreated cases. Unequivocal growth in the foot pads of mice was seen in 100 (47.8%) cases. Of these 100 cases that showed growth in the mouse foot pad system, in 20 cases acid fast bacilli (AFB) were detected in small numbers (1 + ) in either smear or homogenate. The remaining 80 (42%) cases were negative for AFB in both smear and homogenate. The occurrence of viable bacilli and percentage take at 12 months was highest in BB (76 and 86%) followed by BT (38 and 75%) and I (30% and 52%) cases. In most of the BT (65%) and I (60%) cases, the first peak was seen only at 12 months. These results confirm that viable bacilli can be isolated and expanded from a good proportion of negative BT-BB cases using immunocompetent Swiss White mice.

Clinical, histopathological and bacteriological study of 52 referral MB cases relapsing after MDT.

Fifty-two BB-LL relapse cases referred to our centre during 1997-2003 were investigated in detail. Twenty-four cases had been treated with extended MB-MDT [until smear negativity (NON-FDT)]. The remaining 28 cases (54%) had received one of the fixed duration regimens (FDT), of whom 11 had 24 months and 6 had 12 months of WHO MB-MDT. Eleven cases had received rifampicin/ofloxacin (RO) treatment. Follow-up slit skin smear reports were available for 41 cases, all but three cases had been smear negative at some point after release from treatment. None of the cases showed any clinical or bacteriological evidence of upgrading, i.e. LL to BT where as downgrading BB to BL occurred in five cases. The duration between cessation of treatment and reappearance of lesions (DCTR) varied from 2 to 15 years. The mean DCTR was longest (9.4 years) for the NON-FDT and 24 months MB-MDT cases. The mean DCTR was significantly lower in the 12 months MB-MDT and RO treated cases (6.8 and 6.2 years, respectively). Four of RO treated cases and four cases with multiple episodes of reaction had DCTR less than 5 years. Inadequate treatment/poor killing of Mycobacterium leprae results in early onset relapse, whereas 'persisting' or 'drug resistant mutants' contribute to late onset relapse.

Viability and drug susceptibility testing of M. leprae using mouse footpad in 37 relapse cases of leprosy.

Mycobacteria leprae isolates obtained from 37 referral relapse cases of leprosy (37 skin and 10 nerve biopsy samples) received during the years 1994-2001, were tested for viability and drug sensitivity in the mouse footpad. A significant M. leprae yield in the footpads of control mice was obtained, with 32/47 (68%) isolates (from 26 cases) thus confirming viability. Of the 28 isolates successfully drug tested, 6 (21%) were resistant to one or more drugs. All except one, were multidrug treated cases (5/24 = 21%). One of the isolates was resistant to all three drugs, i.e., dapsone (di-aminodiphenyl sulphone, DDS), rifampin (RFP), and clofazimine (CLF). Two were resistant to two drugs, i.e., DDS and RFP, and each of the others were mono resistant to DDS, RFP, or CLF. Notably, one of the isolates that showed combined resistance to DDS and RFP was derived from a borderline tuberculoid case. Also, in one case skin and nerve showed that discordance viz: M. leprae derived from skin were resistant to RFP, while those derived from nerve tested sensitive to all three drugs, indicating tissue related difference.

New structure involved in transient membrane fusion and exocytosis.

Our studies using atomic force microscopy (AFM) reveal a new group of plasma membrane structures involved in exocytosis in live pancreatic acinar cells. These studies demonstrate that "pits" and "depressions" are sites at the apical plasma membrane in live cells, where membrane-bound secretory vesicles dock and transiently fuse to release vesicular contents.

Elevada incidencia y Mycobacterium Leprae visible en lesiones recurrentes en pacientes de lepra tuberculoide Post-MDT

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Ca(2+)-dependent K(+) current and exocytosis in responses to caffeine and muscarine in voltage-clamped guinea-pig adrenal chromaffin cells.

We characterized changes in membrane currents and the cytosolic Ca(2+) concentration, [Ca(2+)](i), in response to caffeine, and compared them with those in response to muscarine using the perforated patch-clamp technique and fura-2 microfluorimetry in guinea-pig adrenal chromaffin cells. Catecholamine release from single voltage-clamped cells was monitored with amperometry using carbon microelectrodes. Caffeine produced a transient outward current (I(out)) at holding potentials over - 60 mV, increasing in amplitude with increasing the potentials. It also evoked a rapid increase of [Ca(2+)](i) at all potentials examined. The current-voltage relation revealed that the activation of K(+) channels was responsible for the I(out) evoked by caffeine. Both current and [Ca(2+)](i) responses were reversibly abolished by cyclopiazonic acid, an inhibitor of Ca(2+)-pump ATPase. At - 30 mV, the caffeine-induced I(out), but not [Ca(2+)](i), was partly inhibited by either charybdotoxin or apamin. In the majority of cells tested, caffeine induced a larger I(out) but a smaller [Ca(2+)](i) increase than muscarine. Caffeine and muscarine increased catecholamine release from voltage-clamped single cells concomitant with the transient increase of [Ca(2+)](i), and there was a positive correlation between them. These results indicate that caffeine activates Ca(2+)-dependent K(+) channels and catecholamine secretion due to the release of Ca(2+) from internal stores in voltage-clamped adrenal chromaffin cells of the guinea-pig. There seems to be a spatial difference between [Ca(2+)](i) increased by Ca(2+) release from caffeine-sensitive stores and that released from muscarine (inositol 1,4,5-trisphosphate)-sensitive ones.

Intracellular, nonreceptor-mediated signaling by adenosine: induction and prevention of neuronal apoptosis.

Inhibitory effect of adenosine on the isolated heart muscle and vascular system were first described in 1929. Since then, numerous reviews have been published on the diverse actions of this nucleoside on a wide variety of cell types. Essentially all effects of adenosine in neurons and non-neuronal cells are mediated by activation of nucleoside membrane receptors coupled to specific intracellular second messenger pathways. This brief review describes two novel actions of adenosine in peripheral sympathetic neurons, which are not mediated by adenosine receptors. First is described how adenosine and related nucleosides are able to induce apoptosis during the initial stages of neuronal growth and development in vitro and in vivo. Second is discussed how adenosine is able to prevent or delay apoptosis in more mature sympathetic neurons subjected to nerve growth factor deprivation in culture. Both the induction and prevention of apoptosis are independent of receptor activation, and totally dependent on the intracellular accumulation and subsequent phosphorylation of adenosine. The physiological significance and mechanisms by which adenosine can induce apoptosis in one situation, and rescue from apoptosis in another, are described in this article.

Primary structure and function of the catecholamine release inhibitory peptide catestatin (chromogranin A(344-364)): identification of amino acid residues crucial for activity.

The novel chromogranin A fragment catestatin (bovine chromogranin A(344-364); RSMRLSFRARGYGFRGPGLQL) is a potent inhibitor of catecholamine release (IC50, approximately 0.2-0.3 microM) by acting as a nicotinic cholinergic antagonist. To define the minimal active region within catestatin, we tested the potencies of synthetic serial three-residue deletion (amino-terminal, carboxyl-terminal, or bidirectional) fragments to inhibit nicotine-stimulated catecholamine secretion from PC12 pheochromocytoma cells. The results revealed that a completely active core sequence of catestatin was constituted by chromogranin A(344-364). Nicotinic cationic signal transduction was affected by catestatin fragments in a manner similar to that for secretion (confirming the functional importance of the amino-terminus). To identify crucial residues within the active core, we tested serial single amino acid truncations or single residue substitutions by alanine on nicotine-induced catecholamine secretion and desensitization. Nicotinic inhibition by the active catestatin core was diminished by even single amino acid deletions. Selective alanine substitution mutagenesis of the active core revealed important roles for Met346, Leu348, Phe350, Arg351, Arg353, Gly354, Tyr355, Phe357, and Arg358 on catecholamine secretion, whereas crucial roles to inhibit desensitization of catecholamine release were noted for Arg344, Met346, Leu348, Ser349, Phe350, Arg353, Gly354, Tyr355, Gly356, and Arg358. We conclude that a small, 15-amino acid core of catestatin (chromogranin A(344-364)) is sufficient to exert the peptide's typical inhibitory effects on nicotinic cholinergic-stimulated catecholamine secretion, signal transduction, and desensitization. These studies refine the biologically active domains of catestatin and suggest that the pharmacophores for inhibition of nicotinic secretion and desensitization may not be identical.

2'-Deoxyadenosine causes cell death in embryonic chicken sympathetic ganglia and brain.

Previous work has shown that nucleosides produce apoptosis in sympathetic ganglion (SG) cells in vitro. The present study examined the effects of nucleosides on the development of the chick embryo in vivo with special attention to the SG and the optic tectum of the central nervous system. In the presence of an adenosine deaminase inhibitor, adenosine and 2'-deoxyadenosine (2'-dAdo) produced different toxicity patterns: both adenosine and 2'-dAdo were toxic to E3 embryos, but only 2'-dAdo was toxic at later stages (E6 1/2, E11). Dosage experiments on E6 1/2 embryos showed that adenosine was less toxic than 2'-dAdo and that 2'-dAdo in sublethal doses was teratogenic. We also examined the effects of 2'-dAdo on embryonic chicken SG and optic tectum in vivo to determine whether sublethal doses of 2'-dAdo produced cell death in these centers on E6 1/2 and 10. In the E6 1/2 SG, 2'-dAdo produced significant neuron loss (83%) and a decrease in SG volume (65%); however, at E10, there was only minor cell loss (7%) and no significant change in SG volume. In the optic tectum at E6 1/2, cell loss was confined mainly to the tectal ventricular zone, but there was little sign of cell loss in this organ at E10. Since cell production is vigorous in the SG and optic tectum at E6 1/2 but relatively low at E10, 2'-dAdo appears to work by stopping cell proliferation. The ineffectiveness of 2'-dAdo at E10 may result from the lethality of 2'-dAdo to the embryo at low concentrations (30 microM) in vivo, well below the apoptosis-inducing concentrations employed in vitro (100-300 microM). These data extend previous findings showing that purine and pyrimidine metabolism plays an important role in development.

Pituitary adenylyl cyclase-activating polypeptide and nerve growth factor use the proteasome to rescue nerve growth factor-deprived sympathetic neurons cultured from chick embryos.

Removal of nerve growth factor (NGF) from sympathetic neurons initiates a neuronal death program and apoptosis. We show that pituitary adenylyl cyclase-activating polypeptide (PACAP) prevents apoptosis in NGF-deprived sympathetic neurons. PACAP (100 nM) added to culture medium at the time of plating failed to support neuronal survival. However, in neurons grown for 2 days with NGF and then deprived of NGF, PACAP prevented cell death for the next 24-48 h. Uptake of [3H]norepinephrine ([3H]NE) was used as an index of survival and decreased >50% in NGF-deprived cultures within 24 h. PACAP (1-100 nM) restored [3H]NE uptake to 92 +/- 8% of that of NGF-supported controls. Depolarization-induced [3H]NE release in neurons rescued by PACAP was the same as that in NGF-supported neurons. PACAP rescue was not mimicked by forskolin or 8-bromo-cyclic AMP and was not blocked by the protein kinase A inhibitor Rp-adenosine 3',5'-cyclic monophosphothioate. Mobilization of phosphatidylinositol by muscarine failed to support NGF-deprived neurons. Thus, PACAP may use novel signaling to promote survival of sympathetic neurons. The apoptosis-associated caspase CPP32 activity increased approximately fourfold during 6 h of NGF withdrawal (145 +/- 40 versus 38 +/- 17 nmol of substrate cleaved/min/mg of protein) and returned to even below the control level in NGF-deprived, PACAP-rescued cultures (14 +/- 7 nmol/min/mg of protein). Readdition of NGF or PACAP to NGF-deprived cultures reversed CPP32 activation, and this was blocked by lactacystin, a potent and specific inhibitor of the 20S proteasome, suggesting that NGF and PACAP target CPP32 for destruction by the proteasome. As PACAP is a preganglionic neurotransmitter in autonomic ganglia, we propose a novel function for this transmitter as an apoptotic rescuer of sympathetic neurons when the supply of NGF is compromised.

Adenosine protects chick embryonic sympathetic neurons from apoptotic death by 2'-deoxyadenosine--importance of ATP in apoptosis.

Our past work on nucleoside toxicity in sympathetic neurons has clearly revealed that adenosine and 2'-deoxyadenosine (dAdo) have different mechanisms of action in inducing apoptotic death. For example, adenosine is toxic to neurons only during early phase of growth whereas dAdo kills even mature neurons. In this study, we hypothesize that dAdo-induced apoptosis is initiated when ATP concentration of sympathetic neurons decreases below a critical level. To prove our hypothesis we used adenosine as a tool to replenish ATP levels of sympathetic neurons. We demonstrate that dAdo toxicity in mature sympathetic neurons was fully prevented by adenosine treatment. Furthermore, we demonstrate that depletion of ATP caused by dAdo was prevented by pretreatment with adenosine. These data suggest that intracellular accumulation of adenosine could play a neuroprotective role in preventing death associated with reduction in neuronal ATP concentration.

Adenosine induces apoptosis by inhibiting mRNA and protein synthesis in chick embryonic sympathetic neurons.

Our previous work has established that adenosine is toxic to chick embryonic sympathetic neurons and kills freshly plated neurons by a process of apoptosis. Although the exact mechanism remains unknown, we found that phosphorylation of adenosine was essential to the toxicity. Using markers for RNA ([3H]uridine) and protein ([35S]methionine) synthesis we demonstrate here that in freshly plated sympathetic neurons adenosine inhibits RNA and protein synthesis by about 50%. The inhibitory effects of adenosine on RNA and protein synthesis, and increased ATP synthesis were blocked by adenosine kinase inhibitor, suggesting that phosphorylated products are responsible for inhibition of RNA and protein synthesis and cell death. Adenosine-induced inhibition of RNA and protein synthesis in neuronal cells provides a new role for adenosine in the regulation of cell function.

2'-Deoxyadenosine selectively kills nonneuronal cells without affecting survival and growth of chick dorsal root ganglion neurons.

Recently, we have demonstrated that adenosine and 2'-deoxyadenosine are toxic to embryonic sympathetic neurons and proposed that purine and pyrimidine metabolism may play a critical role in the growth and development of sympathetic neurons. To extend this hypothesis further, we examined the effects of these nucleosides on two other neuronal populations in the chick embryo, sensory dorsal root ganglion neurons and parasympathetic ciliary ganglion neurons. Now, we show that 2'-deoxyadenosine and adenosine have no visible adverse effect on the viability of either sensory or parasympathetic neurons. Instead, 2'-deoxyadenosine proved to be highly toxic to the nonneuronal cells. The toxic effects of 2'-deoxyadenosine were markedly enhanced by inhibition of adenosine deaminase. In contrast, adenosine was much less toxic to nonneuronal cells than 2'-deoxyadenosine and its effect was not potentiated by inhibition of adenosine deaminase. Priming of pyrimidine pools by exogenous uridine and the specific inhibitor of the nucleoside transporter, nitrobenzylthioinosine, did not protect nonneuronal cells from 2'-deoxyadenosine toxicity. Since phosphorylation of internalized nucleosides was a key step in the initiation of toxicity in sympathetic neurons, adenosine kinase activity was compared in sensory and sympathetic neuronal cultures. The adenosine kinase activity in dorsal root ganglion cultures was only 20% of that in sympathetic ganglion cultures. Furthermore, inhibition of phosphorylation by blocking 2'-deoxyadenosine kinase with iodotubercidin and 5'-amino-5'-deoxyadenosine had no protective effect against 2'-deoxyadenosine toxicity. [3H]-thymidine incorporation was inhibited over 90% by 2'-deoxyadenosine as early as 6 h following its addition and for up to 4 days, suggesting inhibition of proliferation of nonneuronal cells by 2'-deoxyadenosine. The nucleoside was also able to wipe out already well established nonneuronal cells, leaving behind an enriched population of sensory neurons. The selective vulnerability of nonneuronal cells to 2'-deoxyadenosine offers a convenient and effective tool for removing nonneuronal cells from neuronal cultures as well as providing a new model for studying the mechanisms of nucleoside toxicity.