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.

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.

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.

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.

Evaluation of Mycobacterium leprae antigens in the serological monitoring of a clofazimine-based chemotherapeutic study of dapsone resistant lepromatous leprosy patients in Cebu, Philippines.

Thirty-one dapsone resistant lepromatous leprosy patients receiving clofazimine based therapy were serologically monitored throughout their 5-year period of treatment. Sequentially collected sera were used to examine 4 Mycobacterium leprae antigens to evaluate their usefulness in ELISA's for monitoring the progress of their therapy. The ELISA results were compared with decline in bacterial load over the treatment period and with duration of treatment. In addition the ELISA's were compared with each other. The ELISA's based on the measurement of IgM antibodies to the two neoglycoproteins (NDO and NTO) representing the phenolic glycolipid antigen of M. leprae were found to be the most effective with regard to monitoring treatment. A whole M. leprae based ELISA was less efficient in monitoring treatment because it failed to measure antibodies in 5 out of 31 patients. The ELISA-inhibition test based on the detection of antibodies to a species-specific epitope on the 36 K antigen of M. leprae was less suitable because of persistent reactivity during therapy.

Evaluation of Mycobacterium leprae antigens in the monitoring of a dapsone-based chemotherapy of previously untreated lepromatous patients in Cebu, Philippines.

Thirty-five previously untreated lepromatous patients receiving dapsone-based therapy were monitored throughout their 5-year period of treatment by serology and by pathology. Sequentially collected sera were used to evaluate the usefulness of four Mycobacterium leprae antigens as used in ELISA to monitor the progress of their therapy. ELISA results were compared with each other and with bacterial load over the treatment period and with duration of treatment. The ELISAs, based on the measurement of IgM antibody reactivity to the two neoglycoproteins (NDO and NTO) representing the phenolic glycolipid antigen of M. leprae, were found to be the most effective in monitoring treatment. A whole M. leprae based ELISA was less efficient in monitoring treatment because it failed to measure antibodies in 8 out of 35 patients and because it provided consistently lower values than either NTO or NDO. The ELISA-inhibition test based on the detection of antibodies to a species-specific epitope on the 36 K antigen of M. leprae was less suitable because of persistent reactivity during therapy, consequently resulting in no significant correlation with ELISA reactivities to NTO or NDO.

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.

Primary dapsone-resistant leprosy in Cebu, Philippines.

A survey of the prevalence of primary dapsone-resistant leprosy in Cebu, Philippines, has yielded an estimate of 3.6 per 100. Fifty-three of 55 patients proved to have M. leprae fully susceptible to dapsone. The organisms of two patients multiplied in mice administered the minimal effective dose of dapsone; and those of one of these patients also multiplied in mice administered dapsone in a 10-fold larger dose.

Evaluation of a new blood test for its potential to indicate leprosy infection prior to the appearance of clinical symptoms.

A test is described that utilized a laser nephelometric measure of the interaction between human serum and a leprosy biopsy suspension to demonstrate household contact with a leprosy index case. None of the test sera was from persons with clinical evidence of the disease. There was a 5:1 ratio of higher level reactors in sera from household contacts vs high level reactors in sera from persons in the surrounding community. This did not appear to be a result of age, sex, family relationship to the index case, or clinical character of the index case. Some index cases had high level serum reactors among their contacts; others did not. This phenomenon was not a function of the clinical classification of the index case. The reason is unknown. This test should provide another practical means to study leprosy.

Serological monitoring of previously treated lepromatous patients during a course of multiple immunotherapy treatments with heat-killed Mycobacterium leprae and BCG.

Two-hundred and seventy lepromatous patients who had completed treatment received multiple treatments with heat-killed M. leprae and BCG and were monitored for changes in humoral responses to M. leprae-specific antigens. These patients were divided into four treatment groups: placebo (n = 69); BCG (n = 68); M. leprae only (n = 71); and BCG + M. leprae (n = 62). They were monitored for 15 months, receiving five inoculations for each treatment regimen. Two ELISA systems, one measuring antibodies to M. leprae-specific epitopes of the phenolic glycolipid I (NDO-ELISA) and the other of 36-kD protein antigens (INH-ELISA) were used to measure serological changes during this period of immunotherapy. We found no significant increase in serological reactivity with the different treatments, as measured by NDO-ELISA. INH-ELISA similarly showed no significant changes, with the exception of increased values in a small group 13% (36/270) which became skin test-positive during the course of the study. The NDO-ELISA results indicate that use of heat-killed M. leprae or BCG + heat-killed M. leprae did not stimulate the humoral response to the semi-synthetic PG-I antigens of M. leprae. Thus, the NDO-ELISA may be useful in monitoring the outcome of vaccine trials in which killed M. leprae or M. leprae fractions are used, since seroconversion may indicate disease, rather than a response to the vaccine material.

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.

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.

Detection of antibodies to human nerve antigens in sera from leprosy patients by ELISA.

Anti-neural antibodies have been implicated to play a role in the pathogenesis of nerve damage in leprosy patients. To find the relationship between anti-neural antibodies and clinical findings, we attempted to detect antibodies against neurofilament-enriched proteins by ELISA in sera from leprosy patients. Of 289 sera from leprosy patients, 74 (25.6%) had significant anti-neural antibodies; in contrast, 1 (5.0%) of 20 tuberculosis patients and 11 (7.1%) of 154 controls were seroreactive to nerve antigen. When clinical types were considered, a significant level of anti-neural IgG antibodies was detectable in 53 (30.1%) of 176 sera from lepromatous patients compared with 21 (18.6%) of 113 sera from tuberculoid patients, indicating that lepromatous patients were more likely to be seropositive to nerve antigens in ELISA. Some of the ELISA-reactive sera showed antibody reactivity with 38-kD, 40-kD and 43-kD nerve antigens in Western blotting analysis. There was no apparent correlation between seroreactivity to nerve antigens and bacterial load in leprosy patients. Although there was no statistical significance, anti-neural antibodies were detectable more often among the patients on chemotherapy than the untreated and among the patients with erythema nodosum leprosum than without. The results, therefore, suggest that anti-neural antibodies are elicited during the course of leprosy and may be associated with the extensiveness of nerve involvement in the patients.

Multiple lesions of sporotrichoid leishmaniasis in a Filipino expatriate.

Sporotrichoid cutaneous leishmaniasis (CL) was diagnosed in a Filipino man who had worked in Saudi Arabia for 2 years. Two primary lesions-one on the forearm and one on the abdomen-were characterized by "satellite" papules and subcutaneous nodules extending proximally in a sporotrichoid pattern. Leishmania organisms were found in both primary lesions and a subcutaneous nodule. There was no evidence of systemic involvement. Recognition that Old World CL may disseminate through lymphatic channels has diagnostic and therapeutic importance.

Detection of phenolic glycolipid-I antigen and antibody in sera from new and relapsed lepromatous patients treated with various drug regimens.

Since phenolic glycolipid-I (PGL-I) is an unequivocal marker of Mycobacterium leprae, the antigen has been a good candidate for the serodiagnosis and monitoring the effectiveness of leprosy chemotherapy. As an effort to define the kinetics of the PGL-I antigen and its antibodies in leprosy patients, this study was initiated to examine the serum specimens obtained serially from lepromatous patients under chemotherapy trials. PGL-I was detectable in 64 (94.1%) of 68 new lepromatous (bacterial index, BI = 3.2 to 5.8) and in 26 (78.8%) of 33 relapsed lepromatous patients (BI = 3.0 to 5.3). Meanwhile, virtually all of the new and relapsed patients were strongly seropositive to PGL-I. PGL-I was not detectable in any of the patients about 18 months after chemotherapy was initiated; however, anti-PGL-I reactivity declined by 50% at 2 years and by about 70% at 5 years after chemotherapy regardless of the drug regimens under study. Considering the rapid disappearance of the PGL-I antigen and steady decrease in anti-PGL-I IgM antibodies following chemotherapy, the PGL-I-based serology may be useful for monitoring the effectiveness of treatment, at both the early and late stages, in leprosy patients whose initial sera contain a significant level of PGL-I antigen or antibodies.

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.

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.

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.

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)

Improvement in psoriasis after intradermal administration of heat-killed Mycobacterium vaccae.

BACKGROUND: New treatments for psoriasis are being developed, but many are associated with limited efficacy, side-effects, or rapid recurrence after discontinuation. Thus, the aim of new agents is to induce longer term remissions with fewer side-effects. Preliminary studies have shown that Mycobacterium vaccae, a nonpathogenic organism prepared as a heat-killed suspension, may induce periods of remission in some psoriasis patients when administered intradermally. METHODS: To further assess the efficacy and tolerability of M. vaccae in patients with moderate to severe psoriasis (psoriasis area and severity index (PASI) of 12-35), we conducted an open label study whereby 24 patients received two intradermal inoculations of M. vaccae in lesion-free deltoid skin, separated by a period of 3 weeks. RESULTS: Twelve weeks after starting treatment, 14 of 24 patients (58%) showed marked improvement in the PASI score (greater than 50% reduction), two had moderate improvement (25-50% reduction), six were unchanged (< 25% reduction), and two had worsened (> 5% increase). By 24 weeks, 11 of 22 patients continued to show greater than 50% improvement. Five patients had complete clearance of skin lesions that lasted for at least 6 months. CONCLUSIONS: Intradermal administration of heat-killed M. vaccae suspension was well tolerated and induced clinically significant improvement in a majority of psoriasis patients in this cohort. Placebo-controlled testing to further define the efficacy of this treatment is warranted.