Serial measurement of serum cytokines, cytokine receptors and neopterin in leprosy patients with reversal reactions.

Serum levels of cytokines (IL-4, IL-5, IFN-gamma, TNF-alpha), cytokine receptors (TNFR I and II) and one monokine (neopterin) were estimated in seven leprosy patients to establish disease associated markers for reversal reactions (RR). Sera were collected at diagnosis of leprosy, at the onset of reversal reaction and at different time points during and at the end of prednisone treatment of reactions. It was expected that the serum cytokine and monokine profile before and at different time points during reactions would provide guidelines for the diagnosis and monitoring of reversal reactions in leprosy. The cytokines and cytokine receptors were measured by ELISA, whereas a radioimmunoassay was used for neopterin measurement. Six of the seven patients showed increased levels of neopterin either at the onset of RR or 1 month thereafter, and levels declined on prednisone treatment to that seen at the time of diagnosis without reactions. No consistent disease associated cytokine profile was observed in these patients. Interestingly, serum TNF-alpha levels were increased in the same patients even after completion of prednisone treatment, indicating ongoing immune activity. In conclusion, this study demonstrates that despite cytokines levels in leprosy serum being inconsistent in relation to reversal reactions, serum neopterin measurement appears to be an useful biomarker in monitoring RR patients during corticosteroid therapy.

Prospective study of serological conversion as a risk factor for development of leprosy among household contacts.

Although the prevalence of leprosy has declined over the years, there is no evidence that incidence rates are falling. A method of early detection of those people prone to develop the most infectious form of leprosy would contribute to breaking the chain of transmission. Prophylactic treatment of serologically identified high-risk contacts of incident patients should be an operationally feasible approach for routine control programs. In addition, classification of high-risk household contacts will allow control program resources to be more focused. In this prospective study, we examined the ability of serology used for the detection of antibodies to phenolic glycolipid I of Mycobacterium leprae to identify those household contacts of multibacillary leprosy patients who had the highest risk of developing leprosy. After the start of multidrug therapy for the index case, a new case of leprosy developed in one in seven of the 178 households studied. In households where new cases appeared, the seropositivity rates were significantly higher (P < 0.001) than those in households without new cases. Seropositive household contacts had a significantly higher risk of developing leprosy (relative hazard adjusted for age and sex [aRH], 7.2), notably multibacillary leprosy (aRH = 24), than seronegative contacts.

Prospective study of serological conversion as a risk factor for development of leprosy among household contacts

Although the prevalence of leprosy has decline over the years, there is no evidence that incidence rates are falling. A method of early detection of those people prone to develop the mosth infectious form of leprosy would contribute to breaking the chain of transmission. Prophylactic treatment of serologically idenfified high-risk contacts of incidend patients should be an operationally feasible approach for routine control programs. In addition, classification of high-risk household contacts will allow control program resources to be more focused. Is this prospective study, we examined the ability of serology used for the detection of antibodies to phenolic glycolipid I of Mycobacterium leprae to identify those household contacts of multibacillary leprosy patients who had the highest risk of developing leprosy. After the start of multidrug therapy for the index case, a new case of leprosy developed in one in seven of the 178 households studied. In households where new cases appeared, the seropositivity rates were significantly higher (P<0.001) than those in households without new cases. Seropositive household contacts had a significantly higher risk of developing leprosy (relative hazard adjusted for age and sex [aRH], 7.2), notably multibacillary leprosy (aRH=24), than seronegative contacts

Malignant T-cell lymphoma mimicking lepromatous leprosy.

We describe a 16-year-old Filipino boy who presented with skin lesions highly suggestive of lepromatous leprosy, but further assessment established a diagnosis of malignant T-cell lymphoma. This case emphasizes the extensive differential diagnosis of leprosy, as well as the importance of obtaining skin biopsies for diagnostic confirmation.

Detection of phenolic glycolipid I of Mycobacterium leprae in sera from leprosy patients before and after start of multidrug therapy.

A total of 100 untreated new leprosy patients were recruited prospectively and examined for the presence of phenolic glycolipid I (PGL-I) antigen in their serum specimens by dot enzyme-linked immunosorbent assay (ELISA) using rabbit anti-PGL-I antiserum. The presence of circulating PGL-I antigen was closely related to the bacterial indices (BI) of the patients. The PGL-I antigen was detectable in 27 (93.1%) of 29 patients with a BI of 4.0 or above and in 15 (68.2%) of 22 patients with a BI of 3.0 to 3.9. However, none of the 37 patients with a BI of less than 1.9 had detectable PGL-I antigen by the methods used in this study. The level of PGL-I in serum declined rapidly by about 90% 1 month after the start of multidrug therapy. This study showed clearly that anti-PGL-I IgM antibodies and circulating PGL-I antigen levels reflect the bacterial loads in untreated leprosy patients. The serological parameters based on the PGL-I antigen may therefore be useful in the assessment of leprosy patients at the time of diagnosis and possibly in monitoring patients following chemotherapy.

Cutaneous delayed-type hypersensitivity responsiveness in lepromatous and borderline lepromatous leprosy patients as determined by MULTITEST CMI.

To assess cell mediated immune (CMI) function in patients with lepromatous and borderline lepromatous leprosy (LL and BL), 35 patients were examined with the MULTITEST CMI system to evaluate cutaneous delayed-type hypersensitivity (DTH) responsiveness to 7 recall antigens. Reactions were assessed quantitatively and qualitatively. In addition, patients were classified as "responsive" (> or = 2 positive reactions), "hypo-responsive" (1 positive reaction), or anergic. Only hyporesponsive and anergic patients were re-tested. In 23 patients tested before treatment started (Group 1), 9 were responsive, 4 hypo-responsive, and 10 anergic. Upon re-testing, 10 of the 14 hyporesponsive-anergic subjects showed improvement. In 12 patients assessed after therapy initiation (Group 2), 9 were responsive and 3 others became responsive upon re-testing. Quantitative assessment indicated variable deficiencies in cutaneous DTH reactivity that, in many cases, improved with therapy. Correlations between reactivity and disease severity (LL versus BL) or duration of disease were not observed. The MULTITEST CMI system provided a convenient, safe, and reproducible method to assess cutaneous DTH responsiveness in LL and BL patients. Our findings indicated that most LL and BL patients are able to generate detectable but generally fewer and less robust cutaneous DTH responses to recall antigens, many improving with therapy. However, a semi-quantitative classification whereby patients that reacted to 2 or more antigens were considered "responsive" showed little difference between patients and controls. Overall, the data support the contention that deficits in cutaneous DTH responsiveness probably neither predispose nor necessarily accompany lepromatous disease, a practical consideration as efforts to develop a leprosy vaccine continue.

Recombinant interleukin-2 in lepromatous leprosy lesions: immunological and microbiological consequences.

Seven patients with lepromatous leprosy (LL) were inoculated with recombinant interleukin-2 (rIL-2) at 5 lesional sites on the back, four sites receiving one dose of 10 micrograms and biopsy specimens being obtained on 4 consecutive days after the injection. At the 5th site, rIL-2 was instead administered over several days, three patients receiving a total dose of 40 micrograms and 4 patients 150 micrograms, while biopsy specimens from this site were obtained 7, 14 and 21 days after the first injection. Most injection sites developed features of a delayed-type hypersensitivity reaction, namely erythema and induration at the injection site, infiltrates rich in T helper cells, monocytes, and Langerhans cells, and at sites receiving higher doses, multinucleated Langhans giant cells and epithelioid granulomas. In some patients, there were favourable shifts in histological classification or small changes in bacterial load. Low doses of rIL-2 injected into LL lesions rapidly enhance cellular immunity and may alter the histological classification or bacterial load at the injection site.

Primary dapsone resistance in Cebu, The Philippines; cause for concern.

At a time when primary dapsone resistance was prevalent in many leprosy endemic areas, Cebu in The Philippines reported only 3.6% in the period 1975-1978 and later 8.1% in the period 1979-1982. In our current study of patients in the period 1988-1992, the number increased dramatically to 52.7%. In addition, 7.9% of the isolates are highly resistant to dapsone, a level of resistance not seen in earlier studies. This finding could have severe ramifications to the World Health Organization's multidrug therapy (WHO-MDT) mode of treatment, where dapsone is one of the principal drugs. Moreover, the increase in primary dapsone resistance may be a contributing factor in the recent finding that there has been no decline in the number of new cases found in Cebu, even after the implementation of WHO-MDT in 1985. There is a need for new drugs that could be included in the multidrug treatment for multibacillary and paucibacillary leprosy.

Minocycline in lepromatous leprosy.

Twelve patients were treated with three dose levels of minocycline for 30 days, primarily to detect the dose-related effects on Mycobacterium leprae viability, followed by another 5 months of daily minocycline for overall efficacy and persistence of clinical and antibacterial effects. Subsequently, the patients were given standard WHO/MDT chemotherapy for multibacillary leprosy. Clinical improvement was recognizable during the first month, occurring much earlier among those on minocycline 200 mg daily than those who received minocycline 100 mg daily. A similar change also was observed in one patient 11 days after three daily doses of 100 mg of minocycline. At the end of 6 months, all patients were clinically improved with a slight reduction in the average bacterial index (BI) and logarithmic index of bacilli in biopsy (LIB). The effects of minocycline on viability by mouse foot pad inoculation and palmitic acid oxidation assays were noted beginning at 10 to 14 days of daily dosing and becoming more definite after 30 days of treatment. Both tests correlated fairly well. Doses of 200 mg daily did not appear to be more efficient than minocycline 100 daily. Phenolic glycolipid-I (PGL-I) antigen determinations done on some patients during the first month remained positive and did not correlate with changes in viability results. At the end of 6 months, after 5 months of 100 mg of minocycline monotherapy, no viable organisms could be demonstrated by mouse foot pad inoculation and palmitic acid oxidation assays; assays for PGL-I antigen were all negative.(ABSTRACT TRUNCATED AT 250 WORDS)

GM-CSF activates regenerative epidermal growth and stimulates keratinocyte proliferation in human skin in vivo.

Granulocyte/macrophage-colony-stimulating factor (GM-CSF), an immunomodulator of hematopoietic cells, has also been shown to stimulate human keratinocyte proliferation in vitro and speed healing of wounds in the skin of lepromatous leprosy patients. In this study we have examined the in vivo effects of recombinant human GM-CSF on epidermal keratinocyte proliferation and on expression of proteins marking regenerative epidermal growth. Skin biopsies from GM-CSF injected cutaneous sites were obtained between 1 and 6 d following administration of 7.5 or 15 micrograms of the growth factor. Activation of keratinocyte proliferation, quantified as the expression of the Ki67+ nuclear antigen, was noted 1 d following GM-CSF administration. A regenerative epidermal phenotype, demonstrated by immunohistochemical staining of cellular proteins involucrin, filaggrin, and keratin 16, was similarly noted as early as 1 d following GM-CSF injection. This phenotype persisted as late as 6 d post-injection. These results suggest that GM-CSF injection into human skin induces keratinocyte proliferation as well as regenerative differentiation of the epidermis. To date no other cytokine has been shown to be mitogenic for human keratinocytes both in vivo and in vitro or to alter keratinocyte differentiation along the "alternate" or regenerative pathway.

T cells bearing V beta 6 T cell receptors in the cell-mediated immune response to Mycobacterium leprae.

The skin lesions of leprosy provide a window into the immunoregulatory events involved in the human immune response to infection. T cells are thought to play a vital role in the pathogenesis of different forms of the disease. To identify predominant specific T cell subpopulations in leprosy lesions, the TCR-beta chain repertoire was simultaneously studied in skin biopsy specimens and PBMC from both immunologically resistant tuberculoid leprosy and susceptible lepromatous leprosy patients. This was accomplished by obtaining RNA from lesions and PBMC, synthesizing cDNA, and performing the polymerase chain reaction. We found that TCR gene subfamilies V beta 6.1 through V beta 6.4 (V beta 6.1-4) were strikingly overrepresented in lesions vs PBMC of seven of nine tuberculoid patients but only one of nine lepromatous patients. Similarly, V beta 6.5/6.8/6.9 subfamilies were predominant in four of nine tuberculoid patients, but none of the nine lepromatous patients. To explore the influence of the complementarity-determining region 3 (CDR3) in selection of T cells expressing V beta 6 TCR, we sequenced the V beta 6.1-4-C beta polymerase chain reaction products derived from the lesions and PBMC of two tuberculoid patients. From the analysis of deduced amino acid sequences, we found conserved amino acid residues and amino acid motifs in the CDR3 region of the lesion-derived sequences from each patient. Our data suggest that the nominal Ag select T cells bearing V beta 6 TCR in the cell-mediated immune response to Mycobacterium leprae.

Cross-sectional assessment of ELISA reactivity in leprosy patients, contacts, and normal population using the semisynthetic antigen natural disaccharide octyl bovine serum albumin (ND-O-BSA) in Cebu, The Philippines.

An indirect enzyme-linked immunosorbent assay (ELISA) using natural disaccharide octyl bovine serum albumin (ND-O-BSA) as antigen was used in testing leprosy patients, contacts and a normal population in Cebu, The Philippines, from 1985 to 1989. A total of 1413 persons were studied. The results suggested that ELISA reactivity and the bacterial index (BI) correlate in a general way. In multibacillary (MB) leprosy, positivity ranges from 54.2% to 92.3% among patients with a BI of < 2+ to > 4+ on the Ridley scale, with an overall average of 84.5%. Paucibacillary (PB) leprosy patients have a low degree of reactivity, with only 15.0% ELISA positive. The test is more efficient in detecting MB than PB leprosy. The contacts of MB leprosy showed 6.5% positivity; contacts of PB leprosy, 7.0% positivity. The normal population showed 1.7% positive ELISA or 17 per thousand population, which is very much less than that of the household contacts. However, because the normal population is a much larger population than the household contact population in a community, more new leprosy cases would emanate from it. Leprosy workers are concerned about the transmission of the disease to household contacts. However, for the reason stated above, we should be more concerned with the silent spread of the disease to the normal population in the community.(ABSTRACT TRUNCATED AT 250 WORDS)

Evaluation of polymerase chain reaction amplification of Mycobacterium leprae-specific repetitive sequence in biopsy specimens from leprosy patients.

Biopsy specimens were obtained from 102 leprosy patients before chemotherapy and examined by polymerase chain reaction (PCR) using the primers amplifying the 372-bp DNA of a repetitive sequence of Mycobacterium leprae. The PCR results were then compared with bacterial indices (BI) of slit-skin smears and biopsy specimens. The intensities of DNA bands were in general correlated with the numbers of acid-fast bacilli, and even a sample with only one organism gave a PCR positive result. Ten 5-micron sections from each frozen tissue sample were pooled and processed for DNA preparation. PCR was positive for 11 (73.3%) of 15 biopsy specimens with BI of 0 determined for the paraffin sections from the same biopsy samples. PCR also gave positive results for 84 (96.6%) of 87 BI positive biopsy samples. Although the difference in overall results between the two methods was not statistically significant, PCR seemed to have an advantage over microscopic examination in detecting M. leprae in biopsy specimens negative for acid-fast bacilli. Further evaluation of PCR using more specimens from leprosy patients who are bacteriologically negative is warranted to ensure PCR's advantage over the conventional microscopic examination for the diagnosis of leprosy.

Novel responses of human skin to intradermal recombinant granulocyte/macrophage-colony-stimulating factor: Langerhans cell recruitment, keratinocyte growth, and enhanced wound healing.

Recombinant granulocyte/macrophage-colony-stimulating factor (rGM-CSF), prepared from Chinese hamster ovary (CHO) cells and Escherichia coli, was administered to 35 patients with the borderline and polar lepromatous forms of leprosy by the intradermal and subcutaneous routes at doses of 7.5-45.0 micrograms/d for 10 d. With each of these doses and routes, increases in the number of circulating eosinophils were noted. After the intradermal injection, the local skin sites demonstrated zones of roughening and micronodularity that appeared within 24-48 h and persisted for more than 6 d. Reinjection of sites led to enhanced areas of epidermal reaction. GM-CSF prepared from CHO cells was a more potent inducer of this effect. GM-CSF given by the subcutaneous route, at higher doses, failed to initiate these changes. At the microscopic level, the epidermis became thickened (+75%) with increased numbers and layers of enlarged keratinocytes. These contained increased numbers of ribosomes and prominent nucleoli, and were imbedded in a looser meshwork of the zona Pellucida. The modified keratinocytes remained MHC class II antigen negative throughout the course of the response. A major change in the dermis was the progressive accumulation of CD1+, Birbeck granule-positive cells. These Langerhans were recognizable at 48 h after intradermal injection and reached maximum numbers by 4 d. During this period the number of epidermal Langerhans cells remained relatively constant. No increment in dermal Langerhans cells occurred when GLM-CSF was injected by the subcutaneous route. No appreciable increase in the numbers of T cells and monocytes was noted, and granulocytes and eosinophils were largely present within the dermal microvasculature. 4-mm punch biopsies taken from injected sites and adjacent controls were compared in terms of the rapidity of wound healing. 22 of 26 sites demonstrated more rapid filling and hemostasis, whereas four were equivalent to controls. We conclude that rGM-CSF, when introduced into the skin, leads to enhanced keratinocyte growth, the selective recruitment of Langerhans cells into the dermis, and enhanced wound healing of the prepared site. There was no evidence of an enhanced cell-mediated response to Mycobacterium leprae, and bacillary numbers remained unchanged.

Prevalence of IgM antibodies to phenolic glycolipid I among household contacts and controls in Korea and the Philippines.

Phenolic glycolipid I (PGL-I) is a Mycobacterium leprae-specific antigen and the antibodies to the antigen may suggest an M. leprae infection. To compare the M. leprae transmission among the populations, we compared the prevalence of anti-PGL-I IgM antibodies among household contacts and controls between Korea and the Philippines. In Korea (prevalence of leprosy--0.04: 1000), the prevalence of anti-PGL-I antibodies were 4.8% among controls and 8.0% among contacts, respectively. On the other hand, the seroprevalence rate was 10.8% among controls and 13.4% among contacts in the Philippines (prevalence of leprosy--0.70: 1000). Interestingly, a marked difference was noted in the prevalance of anti-PGL-I antibodies among children between the countries; 10-14% among children under 10 years old and 15-18% among those aged between 10 and 19 in the Philippines compared to 0% and 2.9-6.4% in Korea, respectively. This study, therefore suggests that a high prevalance of anti-PGL-I IgM antibodies among children may indicate an active transmission of M. leprae, resulting in a higher incidence of leprosy in the population.