Jamil, Sarwat

Untreated and treated leprosy patients and their household contacts were screened for antibody to Toxoplasma gondii using antigen-coated latex particles. A significantly high level of seroprevalence (29·6%) was observed in the untreated leprosy patients compared to endemic controls (P < 0·01) with a mean reciprocal antibody titre of 20 007 ± 3580 (n = 98) in seropositive patients. In treated patients seroprevalence dropped to 13·5%. Seroprevalence in a group of household contacts of leprosy patients was similar to that of control subjects from an endemic area but not exposed to leprosy (7·8% and 6·1% respectively), indicating that the increased seroprevalence in leprosy patients was not merely due to increased exposure related to socioeconomic factors. Antigenic cross-reactivity between T. gondii and Mycobacterium leprae antigens was ruled out by cross inhibition experiments carried out with soluble antigens from each of the organisms. We believe these antibodies may be induced by an increase in T. gondii load in leprosy due to a transient reactivation of latent T. gondii infections, as the antibodies in these leprosy patients were not associated with any sign of eye or lymphatic pathology related to toxoplasmosis.

The assessment of chemotherapy efficacy in leprosy is difficult, since the only reliable method for determining whether the causative organism, Mycobacterium leprae, is viable depends on its growth in mouse foot pads. In an attempt to replace this expensive, time-consuming test, methods based on the polymerase chain reaction (PCR) have been developed. These methods depend on detection of DNA, which is more susceptible to degradation on cell death than are other cell components, so should be a more accurate indicator of viability. We have used a specific PCR assay to detect M lepraeDNA in skin biopsy samples from leprosy patients. By use of limiting dilution PCR (LD-PCR), the concentration of M leprae DNA in the original sample could be measured. The DNA concentration was more closely correlated with the morphological index (derived from a staining technique that distinguishes morphologically intact and damaged bacteria) than with the number of bacteria visible (bacterial index, Bl, which counts both alive and dead bacteria). In a longitudinal study of multibacillary patients on multi-drug therapy, skin biopsy samples were collected before treatment and 3, 6, 12, and 24 months after the start of therapy. While the Bl showed little or no change during treatment, the number of genomes detected by PCR fell sharply, in parallel with the Ml. We propose that PCR can be used as a rapid measure of M leprae viability and that this approach can be used for monitoring individual leprosy patients and for assessment of existing and new regimens. The method may be applicable to other infectious diseases in which culture of the causative organism is slow or impossible. [ABSTRACT FROM AUTHOR]

To study responses to Mycobacterium leprae antigens, we developed an in vitro model system in which latex particles coated with M. leprae sonic extract (MLSON) antigen were presented to monocytes. Uptake and oxidative response as measured by superoxide production to these antigens were investigated. Phagocytosis of MLSON-coated particles was greater than that of control particles in monocytes from both leprosy patients and controls from leprosy-endemic areas; uptake of MLSON-coated particles was higher in monocytes from lepromatous leprosy patients than in cells from tuberculoid leprosy patients and controls. In both patients and controls, uptake of latex particles coated with leprosy antigens triggered very little reduction of nitroblue tetrazolium although the cells were capable of mounting a respiratory burst. Antigen-coated latex particles can therefore be used as a tool to investigate monocyte responses to M. leprae and individual recombinant antigens.

Quantitative enzyme-linked immunosorbent assays detecting IgM to the soluble Mycobacterium leprae crude sonicate (CD75) and the synthetic disaccharide antigen coupled to bovine serum albumin (ND-BSA) were assessed for their ability to determine early infection in families/household contacts of leprosy patients and employees of a leprosy center working in close contact with leprosy patients. Although IgM to both antigens (CD75 and ND-BSA) correlated with the bacterial index (BI) assessed histologically on skin-biopsy samples, the level of IgM antibodies to ND-BSA was a much more sensitive indicator of low bacterial loads. A 4.4-fold difference in antibody levels was observed between the mean group levels of endemic controls (N = 116) and tuberculoid leprosy patients with a BI of 0 (N = 88), increasing to sevenfold in tuberculoid leprosy patients with a BI of 1 (N = 20). Using a statistical cut off with endemic controls (mean + 2 S.D.), household/family contacts showed 30% seropositivity (N = 180) as compared to staff contacts who showed 17% seropositivity (N = 55). Percent seropositivity in family contacts was not related to the type of leprosy of the index case (lepromatous vs. tuberculoid) or the duration of treatment of the index case. Age of the individual in the family contact group had a significant influence on seropositivity. These results support the hypothesis that, in this community, factors other than the viable bacterial load of the index case, such as genetic susceptibility, may be influencing the high rate of seropositivity in family contacts. IgM ND-BSA antibodies seem to provide a good indicator of low antigenic loads and could prove to be useful in detecting subclinical infection before the onset of dis-ease. Follow-up studies of these seropositive individuals are in progress to understand the relationship between seropositivity and the progress of clinical disease.

Sixteen out of 45 (36%) leprosy patients with clinical features of acute erythema nodosum leprosum (ENL) did not show the characteristic presence of neutrophils (polymorphs) in histology of the ENL lesion. The acute-phase reactants, serum amyloid A (SAA) and C-reactive protein (CRP) which are systemic markers of inflammation, and IgM and IgG antibody to Mycobacterium leprae were determined in these patients in order to understand the differences in histological diagnosis. Both SAA and CRP were elevated in ENL patients, irrespective of the presence of polymorph infiltrates, as compared to nonreactional lepromatous patients, patients with histologically confirmed reversal reactions and endemic controls, indicating that all clinically diagnosed ENL patients had ongoing inflammatory reactions. On the other hand, IgM and IgG antibodies were significantly lower (> 70%) in ENL patients as compared to nonreactional lepromatous patients. When the two ENL groups [ENL-PMN + ve (positive for neutrophils) and ENL-PMN -ve (negative for neutrophils)] were compared, there were no significant differences in the mean SAA, IgM or IgG antibody concentrations, but CRP was eightfold lower in ENL-PMN-ve as compared to the ENL-PMN+ve group. This may indicate that the timing or modulation of the reaction was different in the two ENL groups. Thus, measurement of the acute-phase response and the ratio of SAA/ CRP in particular are helpful in the clinical diagnosis of ENL reactions in leprosy.

Previous studies have found Mycobacterium leprae in nasal swabs from leprosy patients, their contacts, and persons living in endemic areas. It might be expected that M. tuberculosis would be present on nasal mucosa of pulmonary tuberculosis patients, but whether they can be detected in patients or contacts is unknown. We used the polymerase chain reaction (PCR) technique on nasal swabs from tuberculosis patients, contacts of tuberculosis patients, leprosy patients, and London controls to look for both M. tuberculosis and M. leprae. Swabs dipped in sputum specimens from smear-positive patients were used as positive controls. The PCRs were conducted in two independent laboratories. M. tuberculosis was detected in nasal swabs from 6/16 smear-positive tuberculosis patients and from 1/10 household contacts by one of the laboratories. All of the sputum swabs were positive for M. tuberculosis, and all of the London controls were negative. M. leprae were found in nasal swabs from 2/5 leprosy patients, but one laboratory also reported M. leprae in swabs from 4/21 tuberculosis patients and from one sputum specimen. The results show that M. tuberculosis can be found in the noses of some tuberculosis patients, and suggest that the bacilli also may be detected in some household contacts. The comparisons with M. leprae and between the two laboratories give further insights into the sensitivity and specificity of the technique.

PknB is a member of the newly discovered eukaryotic-like protein serine/threonine kinase (PSTK) family of proteins. The pknBgene was cloned and expressed in Escherichia coli. The active recombinant protein was purified and shown to be reactive with antiphosphoserine antibodies, as well as with antibodies to the phosphorylated eukaryotic Ser/Thr kinases mitogen-activated protein kinase kinase 3 and 6, P38, and Creb. In vitro kinase assays demonstrated that PknB is a functional kinase that is autophosphorylated on serine/threonine residues and is also able to phosphorylate the peptide substrate myelin basic protein. Analysis ofpknB expression in Mycobacterium tuberculosisindicates the presence of pknB mRNA in (i) organisms grown in vitro in bacteriological media, (ii) a murine macrophage in vitro infection model, and (iii) in vivo alveolar macrophages from a patient with tuberculosis.

We have investigated phosphatidylinositol 3-kinase (PI3K)-dependent survival signalling pathways using several cytokines in three different hemopoietic cell lines, MC/9, FDC-P1, and TF-1. Cytokines caused PI3K- and PKB-dependent phosphorylation of FOXO3a (previously known as FKHRL1) at three distinct sites. Following cytokine withdrawal or PI3K inhibition, both of which are known to lead to apoptosis, there was a loss of FOXO3a phosphorylation, and a resulting increase in forkhead transcriptional activity, along with increased expression of Fas Ligand (FasL), which could be detected at the cell surface. Concurrently, an increase in cell surface expression of Fas was also detected. Despite the presence of both FasL and Fas, there was no detectable evidence that activation of Fas-mediated apoptotic events was contributing to apoptosis resulting from cytokine starvation or inhibition of PI3K activity. Thus, inhibition of FOXO3a activity is mediated by the PI3K–PKB pathway, but regulation of FasL is not the primary means by which cell survival is regulated in cytokine-dependent hemopoietic cells. We were also able to confirm increased expression of known FOXO3a targets, Bim and p27kip1. Together, these results support the conclusion that mitochondrial-mediated signals play the major role in apoptosis of hemopoietic cells due to loss of cytokine signalling. [ABSTRACT FROM AUTHOR]