Induction of transcription factors in somatosensory cortex after tactile stimulation.

Immediate early response genes have been shown to be inducible in the central nervous system after a variety of stimuli. Induction of these transcription factors in cerebral cortex by a physiological stimulus had not previously been demonstrated. In this study, tactile stimuli induced multiple transcription factors in the somatosensory cortex. Adult male rats were lightly anesthetized with urethane. Tactile stimuli was delivered by a paint brush gently stroking an animals whiskers on one side of its face for a 15 min period. Two h later, the animals were sacrificed. Cortex contralateral to the stimulation was compared with ipsilateral cortex using antibodies raised against immediate early response gene products NGFI-A, NGFI-B, and c-fos. The different transcription factors showed slightly different patterns of response to the tactile stimulus. However, the induction of immunohistochemical staining was most prominent in layer 4 with all antibodies under study. This increase in the number of cell bodies stained was less robust than that seen in the somatosensory cortex after a seizure, and showed more of a predominance in layer 4 cells. These data demonstrate that physiologic stimulation can induce immediate early response genes in cortical cells, and that multiple immediate early response genes react to a stimulus.

Anti-leprosy protective vaccination of rhesus monkeys with BCG or BCG plus heat-killed Mycobacterium leprae: lepromin skin test results.

Groups of rhesus monkeys (RM) were vaccinated and boosted with living Mycobacterium bovis Bacillus Calmette-Guerin (BCG) or BCG + low dose (LD) heat-killed Mycobacterium leprae (HKML) or high dose (HD) HKML or were unvaccinated. Animals vaccinated with BCG + LD and HD HKML were lepromin skin tested 2 weeks after boosting. All groups were lepromin tested 37 and 46 months after challenge with live M. leprae. Fernandez (72 h) and Mitsuda (28 day) responses were recorded. Ten of 10 rhesus monkeys in each of the two BCG + HKML-vaccinated groups significantly converted to strong positive Fernandez status within 2 weeks of boosting, compared to one of six positives in the unvaccinated unchallenged normal control group. Both BCG + HKML groups were significantly protected from clinical leprosy. Six of 10 in each of the two BCG + HKML groups significantly converted to Mitsuda positivity within 2 weeks of boosting compared to zero of six in the normal control group. The sizes of the Mitsuda responses were larger in the LD group than the HD HKML vaccinated/boosted group, suggesting suppression by vaccination with higher doses of HKML in combination with BCG. Fernandez responses were negative in normal RM as well as in the unvaccinated, ML-challenged group and the BCG-vaccinated, ML-challenged group at 37 or 46 months after ML inoculation, although the BCG-vaccinated group was significantly protected from leprosy and the unvaccinated group was not. In contrast, at 37 months the Fernandez reaction was positive in the BCG plus LD and the BCG plus HD HKML-vaccinated groups, both of which were significantly protected from clinical leprosy. By 46 months, the Fernandez responses were below significance in all groups. Thus, Fernandez reactivity is not a reliable correlate to protection from experimental leprosy in RM. Mitsuda responses became strongly positive in all four ML-challenged groups by 37 months and remained strongly positive at 46 months after ML inoculation, suggesting that strong Mitsuda reactivity reflects responses to living ML. BCG or BCG + LD or HD HKML vaccination/boosting of RM produced significant clinical protection from leprosy and there was a good correlation between protection from LL forms of leprosy and positive Mitsuda skin test responses after challenge with live ML. Positive Mitsuda responses were generated in essentially all individuals after challenge with live ML, and this response was primed by prior vaccination/boosting with BCG + HKML as shown by conversion to positivity 2 weeks after boosting. The data show that resistance to clinical leprosy is reflected by Mitsuda responses in ML-exposed RM, similar to results from human studies, and confirm the suitability of RM as a model for leprosy vaccine studies.

Protective immunization of monkeys with BCG or BCG plus heat-killed Mycobacterium leprae: clinical results.

Rhesus and sooty mangabey monkeys (RM and SMM) were vaccinated and boosted with BCG or BCG + low dose (LD) or high dose (HD) heat-killed Mycobacterium leprae (HKML). One group was not vaccinated. Except for a group of controls, all monkeys were challenged with live M. leprae. All animals were studied longitudinally to determine antileprosy protective efficacy. BCG reduced the numbers of RM with histopathologically-diagnosed leprosy by 70% and slowed and ameliorated the appearance of symptoms. BCG + LDHKML reduced the number of RM with leprosy by 89% and BCG + HDHKML by 78%. BCG did not protect SMM from developing leprosy, but disease progress was slowed; disease in SMM was exacerbated by the addition of HKML to the vaccine. RM, as a species, are prone to paucibacillary (PB) forms of leprosy, whereas SMM are prone to multibacillary (MB) forms. Thus, BCG vaccination offers significant protection from clinical disease and slows/ameliorates the rate of progression/degree of disease at the PB end and appears to at least ameliorate symptoms at the MB end of the leprosy spectrum. BCG + HKML protects at the PB end and exacerbates disease progress at the MB end of the leprosy spectrum.

Impaired responses to Mycobacterium leprae antigens in rhesus monkeys experimentally inoculated with simian immunodeficiency virus and M. leprae.

Seven of eight rhesus monkeys (RM) coinfected with simian immunodeficiency virus (SIV) and Mycobacterium leprae harboured acid-fast bacilli (AFB) at sites of dermal inoculation and/or at disseminated sites at times of humane sacrifice (up to 270 days post-M. leprae inoculation) due to SIV-induced debilitation or, in one long term survivor's case, to date over 3 years post-M. leprae inoculation. Detectable AFB were cleared in biopsies of inoculation sites of RM inoculated with M. leprae alone after 63 days postinoculation; these sites have, so far, remained AFB-negative, thereafter. Compared to animals infected with M. leprae alone, RM coinfected with SIV plus M. leprae showed: 1, completely suppressed serum antibody responses to M. leprae-specific PGL-I antigen, but strong anti-SIV Gp120 antibody responses; 2, impaired sensitization of blood mononuclear cells (MNC) to in vitro recognition of M. leprae-specific antigens in blastogenic stimulation assays; 3, impaired in vitro responses of blood MNC to nonspecific (ConA) blastogenic stimuli; and 4, early post-M. leprae inoculation, there was a significant incremental diminution of percentages of blood CD4+CD29+ T-cells in addition to the existing SIV-induced diminished percentages of CD4+CD29+ T-cells. The results indicate that humoral and cellular immune responses to M. leprae antigens are compromised in M. leprae-inoculated RM previously infected with SIV. These results provide an immunologic basis for the demonstration of enhanced M. leprae persistence or leprosy susceptibility in SIV-M. leprae coinfected RM.

Experimental leprosy in monkeys. II. Longitudinal serological observations in sooty mangabey monkeys.

In this study, 11 SMM were grouped and inoculated with differing doses of SMM-origin Mycobacterium leprae (ML) between 4.5 x 10(8) and 1 x 10(9) by either combined IV/IC routes or by IV or IC route alone. The combined route was the most effective in eliciting progressive, disseminated LL leprosy. In all, 6 of 7 SMM inoculated by the combined routes developed leprosy requiring treatment at some point. Only 1 of 4 inoculated by a single route developed persisting leprosy requiring chemotherapy. Either no disease or spontaneous regression of initial disease occurred in the other 3 animals inoculated by a single route. Doses in excess of 1 x 10(9) ML were more effective than lesser doses. An association was observed between the development of IgG anti-PGL-I ELISA OD values and resistance to leprosy and between IgM anti-PGL-I and leprosy progression or susceptibility. Serum PGL-I antigen levels, determined by dot ELISA, paralleled disease severity longitudinally. High positive OD values of anti-LAM IgG prior to ML inoculation were observed in the majority of leprosy-susceptible SMM in contrast to negative levels in more resistant animals. Anti-LAM IgG OD values exceeded the positive cut-off point after inoculation in 5 of 11 SMM; 3 of these 5 had concurrent detectable serum levels of PGL-I antigen.

Experimental leprosy in monkeys. I. Sooty mangabey monkeys: transmission, susceptibility, clinical and pathological findings.

A total of 31 sooty mangabey monkeys (SMM) (Cercocebus torquatus atys) inoculated by various routes with differing numbers of SMM-origin Mycobacterium leprae (ML) and 4 SMM inoculated with human-origin ML were observed for 4-12 years. SMM-origin ML was more pathogenic in SMM than human-origin ML. The spectrum of disease ranged from indeterminate to borderline and lepromatous in different animals. Some animals developed pure neural leprosy. Erythema nodosum leprosum (SNL) was also observed. Combined intravenous/intracutaneous (IV/IC) routes of inoculation more effectively induced advancing, disseminated lepromatous forms of leprosy; IV or IC routes alone were less effective at comparable doses. Total IV/IC doses of SMM-origin ML equal to or greater than 5 x 10(8), with morphologic indices (MIs) ranging from 5 to 10%, produced advancing, disseminated LL leprosy in 92% of SMM. Lower IV/IC doses and inoculations by a single IV or IC route produced fewer leprosy infections and more spontaneous regressions. As a species, captive SMM are highly susceptible to experimental leprosy and provide an excellent model for the longitudinal study of leprosy.

Experimental leprosy in rhesus monkeys: transmission, susceptibility, clinical and immunological findings.

A total of 46 Rhesus monkeys (RM) was inoculated with Mycobacterium leprae (ML) and followed clinically and immunologically for extended periods. Twenty-one (45.7%) of the RM developed leprosy spanning the known leprosy spectrum, with six of 21 (28.6%) having disease in the borderline lepromatous to lepromatous area of the spectrum. RM with paucibacillary forms of leprosy produced predominantly IgG anti-phenolic glycolipid (PGL-I) antibodies and positive lepromin skin test and/or in vitro blastogenesis responses; IgM anti-PGL-I predominated in animals with BB-LL leprosy and correlated with negative immune responses to lepromin. IgG anti-PGL-I antibodies persisted in a number of RM for several years without histopathological evidence of leprosy, suggesting possible persisting subclinical infection. The data show that RM are a valuable model for the study of leprosy. Eleven of the 46 RM were inoculated with ML from sources infected with simian immunodeficiency virus (SIV), the monkey counterpart to the human immunodeficiency virus (HIV). The possible effect of SIV on the clinical outcome of ML infection could not be determined due to insufficient numbers of animals to yield statistically significant results.

N-acetyl-beta-D-glucosaminidase activity within BAL from macaques exposed to generic coal dusts.

N-acetyl-beta(beta)-D-glucosaminidase is a lysosomal enzyme secreted by alveolar macrophages in response to phagocytosis of particulate material. Alveolar macrophages participate in the degradation and fibrosis of pulmonary tissue that results in pneumoconiosis. Known quantities of four characterized respirable dusts were bronchoscopically placed into the right caudal lung lobe of macaque monkeys. Bronchoalveolar lavage (BAL) samples were collected from dust-exposed right lung and unexposed left lung of the same individuals at 2-week intervals for 12 weeks after dust instillation. The samples were tested for N-acetyl-beta-D-glucosaminidase activity to determine if the enzyme levels could serve as an indicator of pulmonary injury induced by generic coal dusts when compared to known fibrogenic and nuisance dusts. Installation of generic quartz, anthracite, or TiO2 dusts produced significant elevations of enzyme activity and increased numbers of macrophages in the dust-exposed lobes. Elevations in enzymatic activity and macrophage numbers were greatest in response to generic quartz dust. These results suggest that quantitative levels of N-acetyl-beta-D-glucosaminidase activity may be a useful indicator of acute and chronic lung injury following exposure to fibrogenic and nonfibrogenic dusts.

Antileprosy protective vaccination of rhesus monkeys with BCG or BCG plus heat-killed Mycobacterium leprae: immunologic observations.

Groups of rhesus monkeys were vaccinated and boosted with Mycobacterium bovis bacillus Calmette Guerin (BCG) or BCG plus low-dose (LD) or high-dose (HD) heat-killed M. leprae (HKML), or were unvaccinated. Prior to and following vaccination-boosting and subsequent M. leprae (ML) challenge, these and unvaccinated, unchallenged control monkeys were observed longitudinally for approximately 3 years. Vaccination with BCG plus HKML initially stimulated significant in vitro blood mononuclear cell blastogenic responses to lepromin, which returned to baseline post-boosting and post-live-ML-challenge, minimally reappearing significantly 2 years post-ML-challenge. Vaccination with BCG failed to stimulated positive blastogenic responses to lepromin before ML-challenge but small, marginally positive, intermittent responses were seen post-ML-challenge. Compared to the unvaccinated ML-challenged group, significant increases in the numbers of blood CD4+ and CD8+ T-cell subsets and an increased CD4+:CD8+ ratio were observed in both BCG plus HKML-vaccinated, ML-challenged groups, but not in the BCG-only-vaccinated, ML-challenged group. CD4+CD29+ and CD4+CD45RA+ subset numbers increased significantly over time in only the BCG plus LD HKML-vaccinated, ML-challenged group. Compared to unvaccinated, ML-challenged groups, vaccination with BCG or BCG plus HKML followed by ML-challenge produced lower IgM:IgG antiphenolic glycolipid-I (PGL-I) serum antibody ratios and protected rhesus monkeys from clinical leprosy, consistent with prior observations that low IgM:IgG anti-PGL-I responses correlated with resistance to and protection from leprosy.

Antileprosy protective vaccination of sooty mangabey monkeys with BCG or BCG plus heat-killed Mycobacterium leprae: immunologic observations.

Groups of sooty mangabey monkeys (SMM) were vaccinated and boosted with Mycobacterium bovis bacillus Calmette-Guerin (BCG), or BCG + low-dose (LD) or high-dose (HD) heat-killed M. leprae (HKML), or were unvaccinated. Prior to and following vaccination-boosting and subsequent M. leprae (ML) challenge, these and unvaccinated, unchallenged control monkeys were immunologically observed longitudinally for approximately 3 years. SMM [multibacillary (MB) leprosy-prone as a species] were not protected clinically by BCG or BCG + HKML, although the disease progress was slowed by vaccination with BCG alone. The longitudinal immune response profiles to BCG or BCG + HKML in SMM showed that: 1) vaccination with BCG or BCG + HKML initially stimulated significant in vitro blood mononuclear cell blastogenic responses to ML antigens, which returned to baseline post-boosting and post-live ML challenge; 2) BCG + LD HKML-vaccinated groups gave the largest blastongenic response (SI = 23) followed by the BCG + HD HKML group (SI = 14.5) and by the BCG-only vaccinated group (SI = 3.6); 3) significantly diminished numbers of blood CD4+ (helper) and CD4+CD29+ (helper-inducer) T-cell subsets were observed longitudinally in all ML-challenged groups compared to controls regardless of whether they had been vaccinated or not; 4) CD8+ (suppressor) T-cell numbers remained longitudinally constant, on average, in all ML-challenged groups (vaccinated or not) compared to controls; 5) there was a significant decrease in the CD4+:CD8+ ratio over time in all ML-challenged groups (vaccinated or not); 6) vaccination with BCG or BCG + LD or HD HKML resulted in significantly increased numbers of CD4+CD45RA+ (suppressor-inducer) T cells longitudinally compared to the unvaccinated, ML-challenged control group; and 7) over time, vaccination with BCG + HKML followed by live ML-challenge produced higher IGM:IgG antiphenolic glycolipid-I (PGL-I) serum antibody response ratios than BCG-only vaccinated, ML-challenged monkeys or unvaccinated, ML-challenged SMM, consistent with prior observations that IgG anti-PGL-I responses correlate with resistance to and protection from clinical leprosy and IgM anti-PGL-I responses correlate with increased susceptibility.