The immune response of the Chacma baboon to Bacille Calmette Guerin: development of a primate model for BCG-based vaccine research.

Mycobacterium bovis Bacilli Calmette-Guerin (BCG) is used increasingly as an efficient vector for expression of recombinant proteins to induce a strong cell-mediated immunity. Here, we tested the immune response of Chacma baboons to the Tokyo and Pasteur strains of BCG in order to obtain base-line information on the response of this primate to BCG. While a humoral immune response to BCG was detected only in some vaccinated baboons, a cellular immune response characterized by a PPD-specific delayed hypersensitivity response and BCG-specific IFN-gamma production from PBMC was a consistent finding. These responses were long-lived and could be detected beyond a year after a booster inoculation at 20 weeks. The results thus suggest that the Chacma baboon may be used as a non-human primate for the evaluation of recombinant BCG vaccines.

O(6)-alkylguanine-DNA alkyltransferase DNA repair in mycobacteria: pathogenic and non-pathogenic species differ.

SETTING: DNA repair genes assist the organism in maintaining DNA integrity in the face of environmental (mutagenic) stress. The genome sequences of M. tuberculosis and M. bovis demonstrate sequences suggestive of an O(6)-alkylguanine-DNA alkyltransferase DNA repair activity similar to that seen in almost all other bacterial and eukaryotic organisms. The near ubiquitousness of this gene implies an important function. OBJECTIVE: Our aim was to ascertain whether mycobacteria exert an alkyltransferase response to mutagen (streptozotocin) stimulation and whether alkyltransferase activity is essential for mycobacterial survival. DESIGN: Alkyltransferase activity in slow- and fast-growing mycobacterial species was determined in the presence and absence of sublethal concentrations of an alkylating agent streptozotocin. The intracellular survival and response to anti-tuberculosis drugs of an alkyltransferase knockout strain of M. bovis BCG was also determined. RESULTS: We demonstrate the presence of O(6)-alkylguanine alkyltransferase (cellular methyltransferase activity) in mycobacterial species and that there is an inducible and constitutive form in fast-growing mycobacteria (M. smegmatis), whereas only the constitutive form exists in the pathogenic or slow-growing species (M. bovis BCG) under the conditions tested. The overall activity of the constitutive form is high. We also show that intracellular growth of M. bovis BCG in macrophages is reduced when the alkyltransferase gene is absent. The presence of alkyltransferase activity appears to assist the organism in reducing the effects of isoniazid, since interruption of the gene confers sensitivity to the drug. CONCLUSIONS: We conclude that for the slow-growing mycobacteria, an inducible response is not essential as their ecological niche is stable and protected, but that the presence of the alkyltransferase activity confers a growth advantage in macrophages and offers some protection against antibiotics.

Molecular evolution of Mycobacterium tuberculosis: phylogenetic reconstruction of clonal expansion.

SETTING: M. tuberculosis isolates were collected from patients attending health clinics in a high incidence urban community and in a low incidence rural setting in South Africa. OBJECTIVE: To reconstruct the evolutionary history of a group of closely related M. tuberculosis isolates using IS6110, DRr and MTB484(1) restriction fragment length polymorphism (RFLP) data. DESIGN: Mycobacterium tuberculosis isolates containing an average of ten IS6110 elements, with a similarity index of > or = 65% were genotypically classified by DNA fingerprinting using the IS6110 derived probes IS-3' and IS-5', as well as the DRr and MTB484(1) probes, in combination with PvuII or Hinfl endonuclease digestion. These RFLP data were subjected to phylogenetic analysis using both genetic distance and parsimony algorithms. RESULTS: Phylogenetic analysis predicted the existence of two independently evolving lineages, possibly evolving from a common ancestral strain. The topology of the phylogenetic tree was supported by comprehensive bootstrapping and the specific partitioning of DNA methylation phenotypes. The observed difference in the branch lengths of the two lineages may suggest differential evolutionary rates. Isolates collected from different geographical regions demonstrate independent evolution, suggesting that it is highly unlikely that strains have been recently transmitted between the two regions. The number of evolutionary events identified in this strain family differs significantly from that of previously characterized strain families, implying that evolutionary rate may be strain family dependent. CONCLUSION: Based on this analysis we propose that the algorithm used to calculate recent epidemiological events should be revised to incorporate the evolutionary characteristics of individual strain families, thereby enhancing the accuracy of molecular epidemiological calculations.

Novel method for rapid measurement of growth of mycobacteria in detergent-free media.

We describe a novel, rapid, and inexpensive method for the measurement of growth of Mycobacterium tuberculosis, Mycobacterium bovis, and Mycobacterium smegmatis in the presence or absence of detergent. The method, which employs hot NaOH treatment of mycobacterial cells to release total cellular protein, compares favorably with other methods for monitoring mycobacterial growth but is particularly useful for heavily clumped cultures grown in defined minimal medium.

Computer assisted identification and classification of streptomycete promoters.

Short sequences that were over represented in a database of Streptomyces promoter region sequences were identified. These sequences and others that were selected on the basis of the characteristics of known promoters, were tested to determine if they were found predominantly at particular distances from the transcription start site. In several cases obvious clusters were recorded. This has allowed the objective identification of potential promoter core sequences. In some cases these may define novel promoter classes. 150 Streptomyces promoters have been listed and grouped on this basis. A new and extended consensus sequence for the Streptomyces E.coli sigma 70-like promoters was determined. It showed differences from that of E.coli, both in sequence and in the spacing between the -35 and -10 regions.

Influence of ascorbic acid esters on acetaminophen-induced hepatotoxicity in mice.

Groups of male Swiss-Webster mice were gavaged with acetaminophen (APAP), APAP + ascorbyl stearate (AS), or APAP + ascorbyl palmitate (AP) at a dose of 600 mg/kg for each chemical. APAP alone caused a significant increase in liver weight/body weight ratio and hepatic glutathione (GSH) depletion. Co-administration of the ascorbate esters AP or AS with APAP prevented an increase in liver weight/body weight ratios and hepatic glutathione depletion. APAP + AS treatments caused significantly greater reductions in rectal temperature at 15-30 min post-dosing periods when compared to APAP + AP or AS treatments. Blood levels of APAP had the same relationship. The study indicates a correlation between APAP blood levels and antipyretic effect of APAP + AS and APAP + AP coadministrations. While both ascorbate esters probably afford protection against APAP-induced hepatotoxicity in mice by reducing the reactive intermediate back to the parent compound, the APAP + AS combination provides better therapeutic efficacy as an antipyretic at the 15-30 min post-dosing periods.

Effect of CGS-8216 on high-dose flurazepam convulsive threshold.

Flurazepam, a clinically proven and widely accepted sedative hypnotic, has been shown by a number of investigators to produce convulsions at toxic doses. In the present study, CGS-8216, a benzodiazepine receptor antagonist, reduced the dose of flurazepam required to produce convulsions. This suggests that the convulsant action of FLZ is exerted at a site other than the benzodiazepine receptor.

Convulsant component of a depressant benzodiazepine.

The convulsant influence of high doses of diazepam, in the presence of the benzodiazepine receptor antagonist Ro 15-1788, was studied in rats. Animals were implanted with permanent cortical screw electrodes for EEG recording. EEG spiking and accompanying clonic activity was observed in rats receiving greater than or equal to 200 mg/kg diazepam, followed 10 minutes later by Ro 15-1788 (20 mg/kg). Pentylenetetrazole and picrotoxin seizure thresholds, measured during constant rate iv infusion, were significantly lowered by pretreatment with diazepam (250 mg/kg) and Ro 15-1788 (20 mg/kg) administered 30 and 20 minutes, respectively, before seizure threshold measurement. It is proposed that this convulsive activity of diazepam is mediated through the picrotoxinin receptor.

Reduction of flurazepam convulsive threshold by Ro 15-1788.

The influence of the benzodiazepine (BZ) receptor antagonist Ro 15-1788 on the convulsant properties of flurazepam (FLZ) was studied in rats. Animals were prepared with chronic epidural electrodes for EEG recording and respiratory rates were recorded via a rubber bulb connected to a pressure transducer. FLZ convulsive thresholds were determined by continuous IV infusion in the presence and absence of Ro 15-1788 pretreatment. All animals receiving the FLZ infusion experienced convulsions preceded by dose-dependent reduction of respiratory rate. Pretreatment with Ro 15-1788 substantially reduced the FLZ convulsive threshold, suggesting a blockade of the depressant (anticonvulsant) aspect of the FLZ effect, thus, augmenting a convulsant effect at a separate receptor. An alternate hypothesis is that Ro 15-1788 may increase the affinity or intrinsic activity of the receptor responsible for the convulsant aspect of FLZ. Ro 15-1788 did not appear to alter the respiratory rate depressant effect of FLZ, although early onset of convulsions in the pretreated animals precluded measurement of respiration at the higher dose of FLZ allowable in animals that were not pretreated with the antagonist.

Increased susceptibility of audiogenic rats to barbital withdrawal convulsions.

Non-responsive progeny from Sprague-Dawley derived rats genetically susceptible to sound-induced (audiogenic) convulsions (AGS-negative) and non-responsive progeny from Sprague-Dawley derived rats not gentically susceptible to audiogenic convulsions (SD-negative) were subjected to a seven-day treatment regimen of sodium barbital. 125 mg/kg, every 12 hours. This represents a lower dose and shorter treatment period than that normally used in this laboratory to induce barbiturate dependence in rats. Animals were subjected to a 115 dB sound stimulus 38 hours following the last dose of sodium barbital. SD-negative rats did not become susceptible to sound-induced convulsions, but AGS-negative rats did experience convulsions when exposed to the sound stimulus during withdrawal. These results are consistent with the hypothesis that rats generally referred to as "audiogenic" may actually suffer from differences which result in an increase of susceptibility to seizures induced by any of several means.

Effect of ketamine enantiomers on sound-induced convulsions in epilepsy prone rats.

Epilepsy prone rats (Jobe et al, 1982) which had previously been determined to experience relatively mild seizures in response to a sound stimulus were used in this study in order to provide a model which would reveal either proconvulsant or anticonvulsant drug activity. Animals were administered dextrorotatory (+), levorotatory (-), or racemic (+/-) ketamine hydrochloride by intravenous (tail vein) injection and subsequently challenged with a 120 db broad spectrum sound stimulus. Evaluation of drug effect was based on presence/absence of seizure, seizure severity, and convulsive latency (time from onset of stimulus to onset of convulsive episode). Potencies relative to protection against seizure susceptibility were: (+) greater than (+/-) greater than (-), with the ratio of activity of the (+) and (-) isomers comparing to the ratio of activity found by other investigators relative to other effects produced by the isomers. Of all animals which were not fully protected, there were no instances of decrement of seizure severity. At low doses, there was a slight decrease in convulsive latency, which was the only indication of a proconvulsant effect. This effect is probably prevented at the higher doses by the anticonvulsant tendency of the drug.

Lack of influence of brain catecholamines on acute effects of barbiturates.

Rats with cerebral electrode implants were tested for sensitivity to EEG burst suppression by intravenously-infused sodium methohexital following manipulation of brain catecholamine function. Although depletion of both norepinephrine (NE) and dopamine (DA) with 6-hydroxydopamine resulted in a slight increase in methohexital sensitivity (MHS), similar depletion with alpha-methyltyrosine did not alter MHS. In addition, desipramine, an agent which selectively blocks uptake of NE did not affect MHS. The results indicate that brain NE and DA exert little, if any, effect on brain responsiveness to the acute effect of barbiturates.

Influence of treatment duration on audiogenic seizure susceptibility during barbiturate withdrawal in rats.

Barbiturate withdrawal seizure susceptibility in rats increased with increasing duration of treatment during a 15-day treatment period in which the animals were given an i.p. dose of sodium barbital every 12 h. This method of producing dependence has clear advantages over previously described methods.

Time course of audiogenic seizure susceptibility and plasma pentobarbital concentration during withdrawal.

Rats were made dependent on sodium barbital by daily oral administration of the drug over a 4 week period. At the end of this time the animals were switched to sodium pentobarbital, I.P., 30 mg/Kg every 4 hours for 3 days and withdrawn. Mean Plasma pentobarbital concentrations was observed to decline rapidly following peak concentrations which occurred approximately 1 hour after the final dose. The last samples in which pentobarbital was detectable were taken 3 hours after the last dose. Audiogenic seizure susceptibility and intensity peaked at 6 hours following the last dose, suggesting that a low concentration of barbiturate is more important in increasing seizure propensity than a sudden decrease in concentration. No electroencephalographic abnormalities were observed during the withdrawal period.

Effects of Ro 4-1284 on electrically-induced spinal cord seizures and on spinal cord norepinephrine and 5-hydroxytryptamine levels.

The time course effects of the benzoquinolizine Ro 4-1284 on spinal cord norepinephrine (NE) and 5-hydroxytryptamine (5-HT) levels were compared to the effects of this same drug on electrically-induced spinal cord seizures. The data show that a significant decrease in spinal cord NE levels and a facilitating effect on spinal cord seizures are apparent 15 minutes after Ro 4-1284 (10 mg/kg s.c.) and that both of these effects persist for at least 24 hours. Forty-eight hours after injection, the effects of Ro 4-1284 on seizure and on NE levels are completely dissipated. A significant decrease in 5-HT levels is not apparent until 1 hour after Ro 4-1284. These data suggest that noradrenergic neurons of the spinal cord act as attenuators of seizure activity. The possibility that spinal cord 5-HT also subserves a seizure attenuating function is not precluded.