Eastern Mediterranean Health Journal [2022; Vol.28, Issue 2]

Eastern Mediterranean Health Journal is the official health journal published by the Eastern Mediterranean Regional Office of the World Health Organization. It is a forum for the presentation and promotion of new policies and initiatives in health services; and for the exchange of ideas concepts epidemiological data research findings and other information with special reference to the Eastern Mediterranean Region. It addresses all members of the health profession medical and other health educational institutes interested NGOs WHO Collaborating Centres and individuals within and outside the Region

المجلة الصحية لشرق المتوسط هى المجلة الرسمية التى تصدرعن المكتب الاقليمى لشرق المتوسط بمنظمة الصحة العالمية. وهى منبر لتقديم السياسات والمبادرات الجديدة فى الصحة العامة والخدمات الصحية والترويج لها، و لتبادل الاراء و المفاهيم والمعطيات الوبائية ونتائج الابحاث وغير ذلك من المعلومات، و خاصة ما يتعلق منها باقليم شرق المتوسط. وهى موجهة الى كل اعضاء المهن الصحية، والكليات الطبية وسائر المعاهد التعليمية، و كذا المنظمات غير الحكومية المعنية، والمراكز المتعاونة مع منظمة الصحة العالمية والافراد المهتمين بالصحة فى الاقليم و خارجه

La Revue de Santé de la Méditerranée Orientale est une revue de santé officielle publiée par le Bureau régional de l’Organisation mondiale de la Santé pour la Méditerranée orientale. Elle offre une tribune pour la présentation et la promotion de nouvelles politiques et initiatives dans le domaine de la santé publique et des services de santé ainsi qu’à l’échange d’idées de concepts de données épidémiologiques de résultats de recherches et d’autres informations se rapportant plus particulièrement à la Région de la Méditerranée orientale. Elle s’adresse à tous les professionnels de la santé aux membres des instituts médicaux et autres instituts de formation médico-sanitaire aux ONG Centres collaborateurs de l’OMS et personnes concernés au sein et hors de la Région

Mycobacterium indicus pranii protein MIP_05962 induces Th1 cell mediated immune response in mice.

Utility of Mycobacterium indicus pranii (MIP) as a multistage vaccine against mycobacterial infections demands identification of its protective antigens. We explored antigenicity and immunogenicity of a candidate protein MIP_05962 that depicts homology to HSP18 of M. leprae and antigen1 of Mycobacterium tuberculosis. This protein elicited substantial antibody response in immunized mice along with modulation of cellular immune response towards protective Th1 type. Both CD4+ and CD8+ subsets from immunized mice produced hallmark protective cytokines, IFN-γ, TNF-α and IL-2. This protein also enhanced the CD4+ effector memory that could act as first line of defence during infections. These results point to MIP_05962 as a protective antigen that contributes, in conjunction with others, to the protective immunity of this live vaccine candidate.

A hanseníase e a vigilância em saúde

Trata-se de um e-book que aborda as práticas de articulação e integração das políticas de Vigilância em Saúde e Atenção Primária em Saúde nas ações de prevenção, controle e monitoramento dos casos de hanseníase. Aborda também o acompanhamento e monitoramento dos indicadores de saúde (SISPACTO) referente ao programa da hanseníase. Este recurso educacional compõe o curso Vigilância, prevenção e controle da hanseníase, ofertado pela UNA-SUS/UFMA. Além deste, há mais 29 e-books tratando sobre diversas questões relacionadas à Vigilância em Saúde. Todos eles fornecem conteúdos interessantes para a formação de profissionais que atuam no SUS, prioritariamente os vinculados à Vigilância em Saúde, e acadêmicos da área da saúde.

Synthesis and immunogenicity of PG-tb1 monovalent glycoconjugate.

A PG-tb1 hapten from the West Beijing strains of Mycobacterium tuberculosis cell wall has been efficiently synthesized and conjugated to CRM197 in a simple way as linker-equipped carbohydrate by applying squaric acid chemistry for an original neoglycoprotein, creating a potent T-dependent conjugate vaccine. The intermediate monoester can be easily purified and the degree of incorporation can be monitored by MALDI-TOF mass spectrometry. After administered systemically in mice without any adjuvant, the conjugate induced high antigen-specific IgG levels in serum. Furthermore, following the third immunization, significant antibody titers frequently exceeding 0.8 million were observed in the sera of mice vaccinated with PG-CRM197 conjugate which showed the potential for preparation of TB vaccine.

Protecting people against leprosy: chemoprophylaxis and immunoprophylaxis.

Elimination of leprosy cannot be achieved by multidrug therapy alone, and new tools are needed to prevent leprosy. A randomized controlled trial with chemoprophylaxis for contacts of leprosy patients using a single dose of rifampicin (SDR) has shown an overall protective effect of approximately 60%, effective in the first 2 years after the intervention. When a contact who previously received bacillus Calmette-Guérin (BCG) vaccination also receives SDR, the protective effect is additive, approximating 80%. Vaccine trials have been conducted with BCG, often in combination with Mycobacterium leprae or related Mycobacterium vaccines as immunoprophylaxis for contacts of leprosy patients, with BCG giving the best results. Meta-analysis shows that the protective effect of BCG vaccination is larger in observational studies than in trials, 60% versus 41%, and is higher among contacts of leprosy patients than among the general population, 68% versus 53%. We believe that a future leprosy control strategy should include contact management, consisting of a contact survey, at which time preventive interventions could be added, such as chemoprophylaxis and immunoprophylaxis. Modeling studies have shown that both interventions will lower the incidence of leprosy in the population. Implementation studies of such contact-based strategy are now called for.

Protecting people against leprosy: chemoprophylaxis and immunoprophylaxis

Elimination of leprosy cannot be achieved by multidrug therapy alone, and new tools are needed to prevent leprosy. A randomized controlled trial with chemoprophylaxis for contacts of leprosy patients using a single dose of rifampicin (SDR) has shown an overall protective effect of approximately 60%, effective in the first 2 years after the intervention. When a contact who previously received bacillus Calmette-Guérin (BCG) vaccination also receives SDR, the protective effect is additive, approximating 80%. Vaccine trials have been conducted with BCG, often in combination with Mycobacterium leprae or related Mycobacterium vaccines as immunoprophylaxis for contacts of leprosy patients, with BCG giving the best results. Meta-analysis shows that the protective effect of BCG vaccination is larger in observational studies than in trials, 60% versus 41%, and is higher among contacts of leprosy patients than among the general population, 68% versus 53%. We believe that a future leprosy control strategy should include contact management, consisting of a contact survey, at which time preventive interventions could be added, such as chemoprophylaxis and immunoprophylaxis. Modeling studies have shown that both interventions will lower the incidence of leprosy in the population. Implementation studies of such contact-based strategy are now called for.

rBCG30-induced immunity and cross-protection against Mycobacterium leprae challenge are enhanced by boosting with the Mycobacterium tuberculosis 30-kilodalton antigen 85B.

Leprosy remains a major global health problem and typically occurs in regions in which tuberculosis is endemic. Vaccines are needed that protect against both infections and do so better than the suboptimal Mycobacterium bovis BCG vaccine. Here, we evaluated rBCG30, a vaccine previously demonstrated to induce protection superior to that of BCG against Mycobacterium tuberculosis and Mycobacterium bovis challenge in animal models, for efficacy against Mycobacterium leprae challenge in a murine model of leprosy. rBCG30 overexpresses the M. tuberculosis 30-kDa major secretory protein antigen 85B, which is 85% homologous with the M. leprae homolog (r30ML). Mice were sham immunized or immunized intradermally with BCG or rBCG30 and challenged 2.5 months later by injection of viable M. leprae into each hind footpad. After 7 months, vaccine efficacy was assessed by enumerating the M. leprae bacteria per footpad. Both BCG and rBCG30 induced significant protection against M. leprae challenge. In the one experiment in which a comparison between BCG and rBCG30 was feasible, rBCG30 induced significantly greater protection than did BCG. Immunization of mice with purified M. tuberculosis or M. leprae antigen 85B also induced protection against M. leprae challenge but less so than BCG or rBCG30. Notably, boosting rBCG30 with M. tuberculosis antigen 85B significantly enhanced r30ML-specific immune responses, substantially more so than boosting BCG, and significantly augmented protection against M. leprae challenge. Thus, rBCG30, a vaccine that induces improved protection against M. tuberculosis, induces cross-protection against M. leprae that is comparable or potentially superior to that induced by BCG, and boosting rBCG30 with antigen 85B further enhances immune responses and protective efficacy.

Molecular mimicry between HSP 65 of Mycobacterium leprae and cytokeratin 10 of the host keratin; role in pathogenesis of leprosy.

Mycobacteria are known to induce autoimmune response in the host. Anti-host keratrin antibodies (AkAbs) might be responsible for the autoimmune phenomena in leprosy patients as majority of leprosy lesions are manifested in the skin and occurrence of keratosis is not an uncommon feature. The aim of this study was to find out the level of AkAbs in leprosy patients across the spectrum and to explore its correlation with the clinical manifestation of the disease. Further, mimicking epitopes of keratin and Mycobacterium leprae components were characterized. We screened 140 leprosy patients (27 BT, 28 BL, 41 LL, 25 T1R, 19 ENL), 74 healthy controls (HC) and 3 psoriasis patients as positive control. Highest AkAbs level was observed in the psoriasis patients followed by T1R, LL, BL, ENL, TT/BT. AkAbs level was significantly (p<0.05) higher in all the groups of leprosy patients except TT/BT in comparison to HC. Significant positive correlation was found between number of lesions and level of AkAbs in leprosy patients. Highest lympho-proliferation for keratin protein was observed in T1R, followed by BL/LL, TT/BT, ENL. Lympho-proliferation was significantly (p<0.05) higher in all groups of leprosy patients except ENL in comparison to HC. Interestingly, it was noted that hyperimmunization of inbred strains of female BALB/c mice and rabbit with M. leprae soluble antigen (MLSA) induce higher level of AkAbs. The percentage of FoxP3(+) expressing Treg cells (total CD4(+)CD25(+)FoxP3(+) andCD4(+)CD25(+hi)FoxP3(+)) in splenocytes and lymph nodes of hyperimmunized mice were declined in comparison to control mice. Further, it was found that this autoimmune response can be adoptively transferred in naïve mice by splenocytes and lymph node cells as well as T cells. Comparative molecular characterization between keratin and MLSA noted a cross-reactivity/similarity between these two antigens. The cross-reactive protein of keratin was found to be in molecular weight range ≈74-51kDa and at pI 4.5 while the cross-reactive protein of MLSA was found to be in molecular weight ≈65kDa and at pI 4-4.5. Cross-reactive protein of keratin and MLSA was identified and characterized by MALDI-TOF/TOF analysis and Mascot software. It was found that the keratin (host protein) which reacted with anti-M. leprae sera is cytokeratin-10 and MLSA which reacted with anti-keratin sera is heat shock protein 65 (HSP 65). Seven B-cell epitopes of cytokeratin-10 and HSP 65 was found to be similar by multiple sequence alignment using ClustalW server and out of which 6 B-cell epitopes were found to be on the surface of HSP 65. In conclusion, our study provides evidence for the existence of molecular mimicry between cytokeratin-10 of keratin (host protein) and 65kDa HSP (groEL2) of M. leprae. Presence of heightened CMI response of leprosy patients to keratin and positive correlation of AkAbs level with number of lesions of leprosy patients showed the clinical evidence for its role in the pathogenesis in leprosy.

Efecto de Debaryomyces hansenii en la respuesta antioxidante de juveniles de camarón blanco Litopenaeus vannamei / Effect of Debaryomyces hansenii on the antioxidant response of juvenile white shrimp Litopenaeus vannamei

Objetivo. Determinar la respuesta antioxidante [actividad de superóxido dismutasa (SOD) y catalasa (CAT)] así como la cuenta total de hemocitos (CTH) y el contenido de proteínas (CP) en camarones (Litopenaeus vannamei) expuestos a diferentes dosis y cepas de la levadura Debaryomyces hansenii (DH5, DH6, LL1), y un inmunoestimulante comercial (LAM). Materiales y métodos. Las levaduras fueron cultivadas y suministradas diariamente en concentraciones diferentes (104 – 106 UFC/mL) directamente a los tanques de cultivo de los camarones (8 ± 0.2 g) mientras que LAM fue aplicado una vez a la semana (0.5 mg/L). Los organismos fueron mantenidos bajo condiciones de laboratorio (28°C, 35%, 80% de recambio diario de agua, dieta comercial para camarón ad libitum). Los tratamientos fueron distribuidos por duplicado y los resultados evaluados a los 15 días con un análisis de varianza y una prueba de Tukey. Resultados. Se registró un CTH significativo (p<0.05) en los tratamientos con DH6 y LL1 (106 UFC/mL) comparada con el control, mientras que las cepas DH5 y DH6 revelaron un incremento significativo (p<0.05) de CP con la dosis de 104 UFC/mL. Los camarones tratados con LAM incrementaron significativamente (p<0.05) los valores de SOD y CAT. Conclusiones. Los resultados obtenidos demuestran que D. hansenii incrementa la respuesta antioxidante y CTH en camarones.

A Mycobacterium leprae Hsp65 mutant as a candidate for mitigating lupus aggravation in mice.

Hsp60 is an abundant and highly conserved family of intracellular molecules. Increased levels of this family of proteins have been observed in the extracellular compartment in chronic inflammation. Administration of M. leprae Hsp65 [WT] in [NZBxNZW]F(1) mice accelerates the Systemic Lupus Erythematosus [SLE] progression whereas the point mutated K(409)A Hsp65 protein delays the disease. Here, the biological effects of M. leprae Hsp65 Leader pep and K(409)A pep synthetic peptides, which cover residues 352-371, are presented. Peptides had immunomodulatory effects similar to that observed with their respective proteins on survival and the combined administration of K(409)A+Leader pep or K(409)A pep+WT showed that the mutant forms were able to inhibit the deleterious effect of WT on mortality, indicating the neutralizing potential of the mutant molecules in SLE progression. Molecular modeling showed that replacing Lysine by Alanine affects the electrostatic potential of the 352-371 region. The number of interactions observed for WT is much higher than for Hsp65 K(409)A and mouse Hsp60. The immunomodulatory effects of the point-mutated protein and peptide occurred regardless of the catalytic activity. These findings may be related to the lack of effect on survival when F(1) mice were inoculated with Hsp60 or K(409)A pep. Our findings indicate the use of point-mutated Hsp65 molecules, such as the K(409)A protein and its corresponding peptide, that may minimize or delay the onset of SLE, representing a new approach to the treatment of autoimmune diseases.

IL-9 promotes anti-Mycobacterium leprae cytotoxicity: involvement of IFNgamma.

Interleukin 9 (IL-9) is a T-cell derived factor preferentially expressed by CD4+ Th2 cells and it has been characterized both in human and murine systems. It is a pleiotropic cytokine with multiple functions on cells of the lymphoid, myeloid and mast cell lineages, as well as on lung epithelial cells. Other activities described for IL-9 support its contribution to asthma and its important role in helminthic infections, where a Th2 response can be protective and IL-9 enhances resistance or is responsible for elimination of the nematode. Nevertheless, until recently there were no studies on its role in bacterial infections in man. We have demonstrated that cytokines can modulate the specific cytotoxicity generation in peripheral blood mononuclear cells from leprosy patients and normal controls. In the present report we studied the effect of IL-9 in this experimental model. Our results indicate that IL-9 can counteract the negative effect mediated by IL-4 on the generation of M. leprae-induced cytotoxic T lymphocytes. Moreover, it can increase this lytic activity in controls and enhance the stimulatory effect of IL-2 or IL-6 in cells from leprosy patients and controls. IL-9 is also able to revert the inhibitory effect of IL-10 and IL-13 on the M. leprae-induced cytotoxic activity. Although the exact mechanism of action of IL-9 remains to be determined, interferon gamma seems to be required for the effect of IL-9 in this experimental model. These data suggest that IL-9 may have an atypical Th2 behaviour and play a role in the modulation of the immune response to mycobacterial infections.

Use of the immunodominant 18-kiloDalton small heat shock protein as a serological marker for exposure to Mycobacterium ulcerans.

While it is well established that proximity to wetlands is a risk factor for contracting Buruli ulcer, it is not clear what proportion of a population living in an area where the etiologic agent, Mycobacterium ulcerans, is endemic is actually exposed to this disease. Immunological cross-reactivity among mycobacterial species complicates the development of a specific serological test. Among immunodominant proteins recognized by a panel of anti-M. ulcerans monoclonal antibodies, the M. ulcerans homologue of the M. leprae 18-kDa small heat shock protein (shsp) was identified. Since this shsp has no homologues in M. bovis and M. tuberculosis, we evaluated its use as a target antigen for a serological test. Anti-18-kDa shsp antibodies were frequently found in the sera of Buruli ulcer patients and of healthy household contacts but rarely found in controls from regions where the infection is not endemic. The results indicate that only a small proportion of M. ulcerans-infected individuals contract the clinical disease.

The combination of plasmid interleukin-12 with a single DNA vaccine is more effective than Mycobacterium bovis (bacille Calmette-Guèrin) in protecting against systemic Mycobacterim avium infection.

Sub-unit vaccines utilizing purified mycobacterial proteins or DNA vaccines induce partial protection against mycobacterial infections. For example, immunization with DNA vaccines expressing the gene for the immunodominant 35000 MW protein, common to Mycobacterium avium and Mycobacterium leprae but absent from the Mycobacterium tuberculosis complex, conferred significant protection against infection with either virulent M. avium or M. leprae in mice. However, the level of protection was equivalent to that obtained with the viable, attenuated vaccine, Mycobacterium bovis, bacille Calmette-Guèrin (BCG). The cytokine, interleukin (IL)-12, is essential for priming naïve CD4+ T lymphocytes to differentiate into interferon-gamma (IFN-gamma)-secreting T cells. We have used a novel self-splicing vector expressing both chains of murine IL-12 to determine if plasmid IL-12 would increase the efficacy of a vaccine expressing the M. avium 35000 MW protein (DNA-Av35). Co-immunization with p2AIL-12 and DNA-Av35 led to a significant increase in the number of antigen-specific IFN-gamma secreting cells and total amount of IFN-gamma released, but a concomitant fall in the antibody response to the 35000 MW protein. This pattern of response was associated with enhanced clearance of M. avium from the liver and spleen of coimmunized mice, and was significantly more effective than BCG or DNA-Av35. alone. Following M. avium challenge there was significant increase in the expansion of the 35000 MW antigen-reactive T cells in the coimmunized mice. Therefore, plasmid-delivered IL-12 acts as an effective adjuvant to increase the protective efficacy of a single DNA vaccine against M. avium infection above that achieved by BCG, and this strategy may improve the efficacy of subunit vaccines against M. leprae and M. tuberculosis.

Effect of hsp65 DNA vaccination carrying immunostimulatory DNA sequences (CpG motifs) against Mycobacterium leprae multiplication in mice.

A DNA expressing hsp65 of Mycobacterium leprae (pACB/hsp65) was constructed by using a vector containing immunostimulatory DNA sequences (pACB). At 12 weeks post-immunization, spleen cells from BALB/cA mice immunized with pACB/hsp65, produced a significantly higher amount of IFN-gamma than mice immunized with pACB in the absence of any in vitro stimulation, and further enhanced its production upon secondary in vitro stimulation with M. leprae lysate and hsp65. On the other hand, while production of IL-12 was observed in mice immunized with pACB/hsp65 12 weeks before, the cytokine production was inhibited by in vitro secondary stimulation with M. leprae or hsp65. At 18 weeks post-immunization, the production of both IFN-gamma and IL-12 was apparently down-regulated, but that of IL-10 was up-regulated. IL-10 seemed to suppress the IFN-gamma and IL-12 productions, because their production was recovered by neutralization of IL-10 with anti-IL-10 mAb. Furthermore, when the efficiency of pACB/hsp65 as a vaccine against M. leprae was evaluated in vivo, the mice immunized with pACB/hsp65 suppressed the multiplication of subsequently challenged M. leprae. These results suggest that a DNA containing M. leprae-derived hsp65 and immunostimulatory sequences might be a potent vaccine candidate against M. leprae infection.

Prevention of diabetes in non-obese diabetic mice by a single immunization with Mycobacterium leprae.

The incidence of overt diabetes was completely prevented by a single intradermal inoculation of Mycobacterium leprae (M. leprae) into Non-obese diabetic (NOD) mice as young as 6-7 weeks. Partial prevention was also observed in cases when 65 kD heat-shock protein (hsp65) with Freund's incomplete adjuvant (FIA) was injected, and no prevention was observed by 38 kD with FIA immunization. Histological examination of pancreata demonstrated that control and M. leprae-immunized mice at 24 weeks of age developed the insulitis even though the number of lymphocytes infiltrated in the treated ones were less than the controls. However, later, at 47 weeks of age, even the immunized mice become to develop very severe insulitis. Thus, M. leprae-immunization did not prevent the incidences of insulitis. The spontaneous development of serum antibody against hsp65 and 38 kD protein preceded the onset of diabetes in NOD mice. Lymphocytes response, IFN-gamma and IL-10 production of splenocytes cultures stimulated with hsp65 were examined to clue the reasons for the prevention of IDDM incidence by M. leprae immunization. The spontaneous development of anti-hsp65 T lymphocytes preceded the outbreak of overt IDDM in control NOD mice, but also appeared in M. leprae immunized cases in which the IDDM incidence was prevented, and both control and M. leprae immunized groups produced IFN-gamma and IL-10 by stimulation with hsp65.

Evaluation of B7-1 (CD80) and B7-2 (CD86) costimulatory molecules and dendritic cells on the immune response in leprosy.

The cell activation depends on T cell antigen receptor binding to antigen plus MHC and costimulation. The binding of CD28, expressed on the T cell surface to B7 (B7-1 or CD80/B7-2 or CD86) present on the antigen--presenting cells (APCs), determines, in several T cell function models, if activation or anergy follows antigenic stimulation. In leprosy, the role of CD80 and CD86 as costimulatory signal in M. leprae-specific cellular immunity has not yet been defined. We investigated the role of B7-CD28 pathway of T cell activation in the in vitro response to M. leprae, following stimulation in the presence of monocytes or dendritic cells (DCs) as APCs. Monocytes were purified, by cold aggregation, from peripheral blood mononuclear leukocytes (PBMC), isolated from leprosy patients. In order to obtain DCs, the monocytes were cultured in the presence of IL-4 and GM-CSF. T cells were purified from PBMC by negative selection with mABs and C'. The phenotype of the cell populations was monitored by FACS. Lymphoproliferative assays were performed with T cells, in the presence of monocytes or DCs. The cells were stimulated by M. leprae in the presence of anti-CD80 antibody (Ab) and/or anti-CD86 antibody (Ab) (Innogenetics). In some experiments Il-10, Il-12 and anti-Il-12 Ab were also added to the culture. We observed a significantly more efficient APC function for DCs when compared to monocytes in T cell in vitro responses to M. leprae. Regardless of the clinical form of Leprosy, the M. leprae-specific immune response was markedly reduced in the presence of anti-CD86 Ab. Il-12 increase the immune response to M. leprae while IL-10 or anti-IL-12 Ab reduce this response when monocytes or DCs were used as APCs.