A phase 1 antigen dose escalation trial to evaluate safety, tolerability and immunogenicity of the leprosy vaccine candidate LepVax (LEP-F1 + GLA-SE) in healthy adults.

Healthy United States-based adult volunteers with no history of travel to leprosy-endemic countries were enrolled for the first-in-human evaluation of LepVax (LEP-F1 + GLA-SE). In total 24 volunteers participated in an open-labelclinicaltrial, with 21 receiving three injections of LepVax consisting of either 2 µg or 10 µg recombinantpolyprotein LEP-F1 mixed with 5 µg of the GLA-SE adjuvant formulation. LepVax doses were provided by intramuscular injection on Days 0, 28, and 56, and safety was evaluated for one year following the final injection. LepVaxwas safe and well tolerated at both antigen doses. Immunological analyses indicated that similar LEP-F1-specific antibody and Th1 cytokine secretion (IFN-γ, IL-2, TNF) were induced by each of the antigen doses evaluated within LepVax. This clinicaltrialof the first definedvaccinecandidate for leprosy demonstrates that LepVax is safe and immunogenic in healthy subjects and supports its advancement to testing in leprosy-endemic regions.

Topical immunotherapy with diphenylcyclopropenone in vitiligo: a preliminary experience.

BACKGROUND: Despite recent significant therapeutic advances, vitiligo remains a clinical conundrum. Topical immunotherapy has been extensively tested in the treatment of various dermatologic disorders, especially those believed to have an immunologic basis. AIM: To evaluate the role of topical diphenylcyclopropenone (DPCP) in the treatment of vitiligo. METHODS: Nineteen patients with limited vitiligo lesions were enrolled in this study. After sensitization with 2% lotion of DPCP in acetone, progressively higher concentrations beginning at 0.001% up to 2% were applied weekly for 6 months to the depigmented skin lesions. RESULTS: Thirteen of the 19 patients were evaluated at the end of 6 months. Four patients with focal vitiligo, one patient with vitiligo vulgaris, and three patients with segmental vitiligo showed marked (grade 3) repigmentation. CONCLUSION: Marginal and central repigmentation with hyperpigmented borders was seen in the majority of lesions. Further controlled trials should be undertaken to evaluate the use of topical DPCP in vitiligo.

Activation of human CD4+ T cells by targeting MHC class II epitopes to endosomal compartments using human CD1 tail sequences.

Distinct CD4(+) T-cell epitopes within the same protein can be optimally processed and loaded into major histocompatibility complex (MHC) class II molecules in disparate endosomal compartments. The CD1 protein isoforms traffic to these same endosomal compartments as directed by unique cytoplasmic tail sequences, therefore we reasoned that antigen/CD1 chimeras containing the different CD1 cytoplasmic tail sequences could optimally target antigens to the MHC class II antigen presentation pathway. Evaluation of trafficking patterns revealed that all four human CD1-derived targeting sequences delivered antigen to the MHC class II antigen presentation pathway, to early/recycling, early/sorting and late endosomes/lysosomes. There was a preferential requirement for different CD1 targeting sequences for the optimal presentation of an MHC class II epitope in the following hierarchy: CD1b > CD1d = CD1c > > > CD1a or untargeted antigen. Therefore, the substitution of the CD1 ectodomain with heterologous proteins results in their traffic to distinct intracellular locations that intersect with MHC class II and this differential distribution leads to specific functional outcomes with respect to MHC class II antigen presentation. These findings may have implications in designing DNA vaccines, providing a greater variety of tools to generate T-cell responses against microbial pathogens or tumours.

Considerações acerca dos estados reacionais do portador de Hanseníase no Município de Itajaí / Considerations on reactional states of Itajaí's hansen's carriers

A pesquisa teve por objetivo, compreender e avaliar os estados reacionais de pacientes portadores de hanseníase e o tratamento das reações. O método de abordagem do estudo foi indutivo, tendo como base o referencial bibliográfico, tomou-se como fonte de dados os prontuários dos portadores de hanseníase, disponíveis no Programa de Controle de Hanseníase da Secretaria Municipal de Saúde de Itajaí. Através do trabalho com os 78 pacientes inscritos no programa, observou-se que: 60,3 por cento tem de 15-49 anos; 65,4 por cento são do sexo masculino; 70,5 por cento manifestam a forma Virchowiana da doença; 60,3 por cento apresentaram estados reacionais durante tratamento; metade destes aproximadamente apresentaram quadro clínico de neurite, tendo sido tratados com prednisona. Também foram observados outros sintomas clínicos como o eritema nodoso hansênico, e o tratamento de escolha, sempre que possível foi a talidomida. A avaliação dos estados reacionais indica que, mais da metade dos portadores em tratamento, apresentam esta manifestação imunológica. É julgada pela literatura como consequência da doença e possível reação ao esquema poliquimioterápico

Levamisole induces interleukin-18 and shifts type 1/type 2 cytokine balance.

Immune responses can be classified, according to the predominant cytokines involved, into type 1 (featuring interferon-gamma, IFN-gamma) and type 2 (featuring interleukin-4, IL-4); imbalance between type 1 and type 2 cytokine compartments has been implicated in many human diseases. Levamisole is a drug with an unknown mode of action that has been used to boost immunity in infectious diseases including leprosy, and in some cancers. To test the hypothesis that levamisole acts by inducing a shift to a type 1 immune response, we used Brown Norway (BN) rats, which are markedly biased to type 2 responses. BN rats treated with levamisole showed a dose-dependent rise in serum IFN-gamma and fall in serum immunoglobulin E (IgE) level. Detailed analysis of cytokine gene expression showed upregulation of IFN-gamma and downregulation of IL-4 messenger RNA. This coincided with marked upregulation of IL-18, a recently characterized cytokine with potent activity in stimulating IFN-gamma production. IL-12 was not induced. Further, the type 2 response induced in BN rats by mercuric chloride was markedly attenuated when rats were pretreated with levamisole: there was a 2-log reduction in maximum serum IgE level and marked attenuation of IL-4 gene upregulation. These data indicate that levamisole acts by resetting the immune balance towards a type 1 response via induction of IL-18. Our findings provide a direction for development of more specific immunomodulating therapy.

Immunomodulación en la práctica pediátrica / Immunomodulation in pediatric practice

En los últimos años la inmunoterapia ha tomado un gran auge debido, primordialmente, a la variedad de alternativas descritas, fundamentadas en una gran cantidad de estudios clínicos, lo que ha permitido que enfermedades aparentemente poco complicadas, pero cuya resolución es frecuentemente problemática, como las infecciones recurrentes de las vías respiratorias, hasta enfermedades crónicas como tuberculosis, lepra, colagenopatías y enfermedades malignas, etc., ahora puedan tener un mejor pronostico. Es importante resaltar la importancia de conocer las características farmacologicas de estos medicamentos, así como sus indicaciones precisas. con el fin de no caer en el mal uso de los mismos y poder ofrecer a los pacientes un recurso valioso y eficaz de cara al nuevo milenio

Evanescent wave fibre optic sensor for detection of L. donovani specific antibodies in sera of kala azar patients.

An easy-to-use technique for detection of antibodies specific for the parasite L. donovani in human serum sample has been developed. The method is based on an evanescent wave generated from a tapered configuration of decladded optical fibre and does not require any volumetric measurement. Tapered fibres are immobilized with the purified cell surface protein of L. donovani by covalent bonding. Treated fibres are incubated with the patient serum for 10 min followed by incubation with goat anti human IgG tagged FITC. Fluorescent intensity from the fibre has been shown to be proportional to L. donovani specific antibodies present in the test sera. Direct readings can be obtained after signal enhancement through a photomultiplier tube within 5 min. The system, when tested on 12 positive sera, did not show any false negative result. Also, no false positive result was obtained with serum samples of patients infected with leprosy, tuberculosis, typhoid and malaria, showing the specificity of the sensor and efficacy of the technique.

Sensitization and reactogenicity of two doses of candidate antileprosy vaccine Mycobacterium w.

M.w vaccine is one of the antileprosy vaccines under test in an ongoing comparative vaccine trial in South India. The objective of the present study was to examine the sensitizing ability, as measured by skin test reactions to Rees' MLSA and lepromin, and reactogenicity of M.w vaccine in the local population. Two doses of M.w, 1 x 10(9) bacilli and 5 x 10(9) bacilli, were used, in two separate studies of 395 and 400 "healthy" individuals aged 1-65 years. In each study, the study subjects received either M.w vaccine or normal saline (control), by random allocation. The results showed that healing of vaccination lesions was uneventful although the healing process was somewhat prolonged with the higher dose. The mean size of lesions was 7.0 mm and 9.5 mm with the low and high doses of the vaccine, respectively. The results also showed that M.w vaccine in a dose of 1 x 10(9) bacilli, failed to induce post-vaccination sensitization as measured by reactions to Rees' MLSA and by Fernandez and Mitsuda reactions to lepromin-A. However, when the dose of the vaccine was increased to 5 x 10(9) bacilli the mean sizes of post-vaccination reactions to Rees' MLSA and lepromin-A (both early and late) were significantly larger in the vaccine group compared to that in the control group. The sensitizing effect attributable to the vaccine was of the order of 1.5 mm to 1.8 mm.

Antigen-presenting capacity of rheumatoid synovial fibroblasts.

In normal, healthy joints, synovial fibroblasts do not express major histocompatibility complex (MHC) class II molecules. However, in inflamed joints of rheumatoid arthritis (RA) patients, synovial fibroblasts show an abundant expression of MHC class II. Does this increase in expression have functional consequences for antigen presentation to T cells? To date, the precise role of synovial fibroblasts in antigen presentation has not been documented. Here, we show by three different examples that cultured synovial fibroblasts with interferon-gamma (IFN-gamma)-induced MHC class II expression are capable of processing soluble protein for presentation to CD4+ T cells. First, the antigen-presenting cell (APC) function of synovial fibroblasts was studied in an autologous model. From synovial tissue of a RA patient both a fibroblast cell line and a tetanus toxoid (TT)-specific CD4+ T-cell line were generated. A dose-dependent TT response was observed only when TT was presented by IFN-gamma-pretreated synovial fibroblasts. As more direct evidence for MHC class II-restricted antigen presentation, the response of a Mycobacterium tuberculosis-specific CD4+ T-cell clone isolated from rheumatoid synovial fluid was demonstrated in the presence of synovial fibroblasts. The response was DR4Dw4-restricted and could be inhibited by monoclonal antibody (mAb) to HLA-DR. In addition, the lymphokine secretion pattern of the synovial T-cell clone did not differ qualitatively upon antigen-specific stimulation using peripheral blood mononuclear cells (PBMC) or synovial fibroblasts as APC. In order to provide evidence for intracellular antigen processing we next examined the response of a M. leprae-specific T-cell clone with known epitope specificity. Our data suggest that synovial fibroblasts are not passive bystanders, but can become active participants in the development and maintenance of chronic inflammation.

Sensitization potential and reactogenicity of varying doses of BCG plus killed Mycobacterium leprae: an extended study.

This study is an extension of a previous study on an antileprosy combination vaccine of BCG plus killed Mycobacterium leprae (KML) regarding its sensitization potential and reactogenicity. The study was extended to see if by reducing the dose of BCG in the combination vaccine the incidence of suppurative adenitis could be reduced without a significant reduction in the level of postvaccination skin-test responses. The study included 860 individuals, and three preparations of the combination vaccine [BCG 0.05 mg + 6 x 10(8) KML (I), BCG 0.05 mg + 5 x 10(7) KML (II), BCG 0.01 mg + 5 x 10(7) KML (III)] along with normal saline (i.v.) were used. Each individual received one of these four preparations by random allocation. They were also tested with Rees' M. leprae soluble antigen (MLSA) and lepromin A 12 weeks after vaccination. Reactions to the MLSA were measured after 48 hr; reactions to lepromin A after 48 hr and 3 weeks. The character and size of the local response at the vaccination site were recorded at the third, eighth, and 15th week postvaccination. The results of the study showed that by halving the dose of BCG in the combination vaccine BCG plus 6 x 10(8) KML a) the incidence of suppurative regional adenitis was reduced significantly, b) there was no significant change in the post-vaccination response at 12 weeks as measured by Rees' MLSA and lepromin A, and c) the evolution of the vaccination lesion was somewhat prolonged. This dose was found satisfactory for use in a comparative antileprosy vaccine trial in South India.

Differential production of interleukin 1 (IL-1), IL-6, tumor necrosis factor, and IL-1 receptor antagonist by human monocytes stimulated with Mycobacterium leprae and M. bovis BCG.

Human blood monocytes cultured in various serum conditions were stimulated with Mycobacterium leprae or M. bovis BCG and their cytokine-inducing abilities were compared. BCG, either live or killed, induced production of interleukin 1 (IL-1), IL-6, tumor necrosis factor (TNF), and IL-1 receptor antagonist (IL-1ra). Live BCG at a lower bacterial number was more potent than killed BCG in the induction of IL-6 and TNF. In contrast to BCG, killed M. leprae induced few cytokines except for IL-1ra. Similar results were obtained when monocytes were cultured in the presence of untreated or heat-inactivated fetal bovine serum (FBS). When FBS and human serum (HS) were compared and the effect of heat inactivation was investigated, monocytes in HS produced the most cytokines, then those in FBS, irrespective of heat inactivation, and those in heat-inactivated HS produced the least cytokines. There were no differences between live and killed M. leprae, and BCG were far more potent than M. leprae in all of our experimental conditions, indicating that the poor cytokine (IL-1, IL-6 and TNF)-inducing ability of M. leprae was not due to their viability. Cytokine production was partially in parallel with the phagocytosis of the mycobacteria. These results suggest that M. leprae favor their infection by evoking little host reaction through the induction of only low levels of immunostimulatory or proinflammatory cytokines but a substantial amount of immunosuppressive cytokine.

Functional analysis of DR17(DR3)-restricted mycobacterial T cell epitopes reveals DR17-binding motif and enables the design of allele-specific competitor peptides.

We have previously shown that p3-13 (KTIAY-DEEARR) of the 65-kDa heat shock protein (hsp65) of Mycobacterium tuberculosis and Mycobacterium leprae is selected as an important T cell epitope in HLA-DR17+ individuals, by selectively binding to (a pocket in) DR17 molecules, the major subset of the DR3 specificity. We have now further studied the interaction between p3-13, HLA-DR17 and four different TCR (V beta 5.1, V beta 1, and V beta 4) by using T cell stimulation assays, direct peptide-DR binding assays, and a large panel (n = 240) of single amino acid substitution analogs of p3-13. We find that residues 5(I) and 8(D) of p3-13 are important DR17 binding residues, whereas the residues that interact with the TCR vary slightly for each DR17-restricted clone. By using N- and C-terminal truncated derivatives of p2-20 we defined the minimal peptide length for both HLA-DR17 binding and T cell activation: the minimal peptide that bound to DR17 was seven amino acids long whereas the minimal peptide that activated T cell proliferation was eight amino acids in length. Furthermore, two new DR17-restricted epitopes were identified on hsp70 and hsp18 of M. leprae. Alignment of the critical DR17-binding residues 5(I) and 8(D) of p3-13 with these two novel epitopes and two other DR17-binding peptides revealed the presence of highly conserved amino acids at positions n and n + 3 with I, L, and V at position n and D and E at position n + 3. D and E are particularly likely to interact with the DR17-specific, positively charged pocket that we have defined earlier. Based on these results, a set of single amino acid substituted analogs that failed to activate these T cell clones but still bound specifically to DR17 was defined and tested for their ability to inhibit T cell activation by p3-13 or other DR17-restricted epitopes. Those peptides were able to inhibit the response to p3-13 as well as other DR17-restricted mycobacterial epitopes in an allele-specific manner, and are anticipated to be of potential use for immunotherapeutic and vaccine design strategies.

A strategy to improve the efficacy of vaccination against tuberculosis and leprosy.

The pathogens responsible for leprosy, tuberculosis and the leishmaniases can induce different classes of immunity, but protection is provided only by a cell-mediated response. Here, Peter Bretscher proposes a strategy to achieve an immunological imprint that ensures a stable cell-mediated response upon natural infection.

Limiting dilution analysis in leprosy.

Peripheral blood mononuclear cells (PBM) obtained from leprosy patients and healthy controls were cultured with Mycobacterium leprae and the control antigens, BCG and SKSD. Parallel cultures were supplemented with additional interleukin-2 (IL-2). On the basis of the level of response to M. leprae, leprosy patients could be divided into low, intermediate and high responders. The addition of IL-2 resulted in enhanced proliferation to antigen only by cells from intermediate responders. This effect was neither antigen specific nor was it confined to cells from leprosy patients. When limiting dilution analyses were performed on cells from 26 patients across the leprosy spectrum, no M. leprae-reactive lymphocytes were detected in cells from subjects with lepromatous disease. The precursor frequency for cultures containing M. leprae plus IL-2 was no greater than that of cultures containing IL-2 alone, thereby excluding the possibility of clonal anergy reversible with IL-2. This was observed in both untreated patients and those on long-term treatment, which made sequestration of antigen-reactive cells within leprosy lesions an unlikely explanation. On the other hand, M. leprae-reactive lymphocytes were detected in patients with tuberculoid and borderline tuberculoid disease and in two subjects with borderline lepromatous leprosy in type I reversal reaction. IL-2 reactive cells were detected in all patients regardless of clinical classification. Three 'suppressor' curves were obtained but were not confined to cells from lepromatous patients. Taken together, these findings suggest that the non-responsiveness to M. leprae characteristic of the great majority of multibacillary patients is due to an absence of antigen-sensitive T cells.

Cellular immune responses to mycobacterial heat shock proteins in Nepali leprosy patients and controls.

Sixty-three leprosy patients, representing the entire leprosy spectrum from tuberculoid to lepromatous disease, and 17 healthy Nepali subjects were tested for their T-cell responses to the purified p65 and p70 protein antigens of Mycobacterium bovis BCG using a lymphocyte proliferative assay. There was strong correlation between the responses to BCG and M. leprae and the responses to the two antigens. Patients and controls lacking a response to either of the whole cell preparations failed to respond to the purified antigens, while BCG responders at the lepromatous pole of the disease did mount a response to both antigens. Significant differences in the magnitude of the responses to these antigens were obtained between controls and the disease groups. In individual subjects, responses to the two antigens were significantly correlated to each other.

M. leprae- and BCG-induced chemiluminescence response of monocytes from leprosy patients and healthy subjects: effects of gamma-interferon and GM-CSF.

Mycobacterium leprae, in contrast to BCG, failed to trigger any chemiluminescence (CL) response in mononuclear cells from either leprosy patients or healthy subjects, a deficit not reversed by either interferon-gamma or GM-CSF. Chemiluminescence responses induced without mycobacteria or with BCG were found to be lower in leprosy patients than in controls. M. leprae were also less well phagocytosed than BCG. However, there was a significant difference in phagocytosis between healthy and tuberculoid leprosy subjects. Phagocytosis was not altered by the addition of either lymphokine, and no major differences between healthy subjects and patients were observed. Preincubating mononuclear cells with anti-mycobacteria antibodies (lepromatous patients' sera) did not increase the CL response nor the phagocytosis of M. leprae or BCG.