Immunoblotting in the study of the antigenic structure of Leishmania

Using immunoblotting, the antigenic structure of 6 Leishmania strains has been studied: 1) MHOM/IN/80/DDS--Leishmania (Leishmania) donovani; 2) MHOM/SU/63/VL--L. sp. ZMA; 3) MHOM/SU/73/K-27--L. tropica; 4) MHOM/SU/73/5Ash--L. major; 5) MHOM/GE/84/H-132--L. mexicana amazonensis; 6) REPT/SU/83/3960-GC--L. (Sauroleishmania) gymnodactyli. Antigens of Leishmania surface membranes and rabbit antisera against them have been used. Among the agents of leishmaniasis in the Old World (1-4), the most intensive antigenic lines were found in the medium and high-molecular mass area (43-200 kD). In L. (L) mexican a amazonensis (5) species-specific lines have been identified in 10-22 kD area, which is indicative of considerable antigenic differences in Leishmania of the Old and New Worlds. The most marked antigenic differences from other types were noted in L. (S) gymnodactyli (6). It was characterized by the absence of antigenic bands in the medium and high molecular mass areas, the lines associated with species-specific determinants located in the low molecular mass area (18 and 25 kD). The results of cross-reacting c-ELISA using the same antigens and antisera correlated well with the above data of immunoblotting. Immunoblotting may be used for identification of species-specific antigenic structures which may be helpful in serological tests for more accurate leishmania identification and leishmaniasis diagnosis.     

Leishmania mexicana and Leishmania major: attenuation of wild-type parasites and vaccination with the attenuated lines

A method for attenuation of Leishmania species by culturing in vitro under gentamicin pressure has been used successfully with Leishmania mexicana, L. major, L. infantum, and L. donovani. The attenuated lines invaded but were unable to survive within bone marrow-derived macrophages in vitro, whereas wild-type parasites survived and multiplied. The attenuated lines of L. mexicana and L. major both failed to induce cutaneous lesions in the majority of BALB/c mice over a minimum 12-week observation period after subcutaneous injection of stationary phase parasites. The attenuated line of L. mexicana retained its properties in gentamicin-free medium over 40 subcultures. The attenuated lines of L. mexicana and L. major both induced significant protection in mice against challenge with wild-type parasites.     

A rapid staining technique for Leishmania parasites in splenic aspirate smears

A quick and suitable method for staining Leishmania donovani and other blood protozoa is described. The method, which could have wide applications, is a modification of Field's staining technique as used for malaria parasites. It uses the same stains in a different order. The method has been tried on smears containing malaria and trypanosomes, and the results were good. It is very cheap and requires no special apparatus or training.     

Cellular immunity of mice to Leishmania donovani in vitro: lymphokine-mediated killing of intracellular parasites in macrophages.

Leishmania donovani, an intracellular protozoan, causes kala-azar by parasitizing the macrophages of its mammalian host. Outbred NCS and CD-1 mice develop immunity to this parasite. This immunity was demonstrable when supernatant fluids from cultured splenic lymphocytes were added to infected macrophages. Only the lymphokine preparations from infected mice showed significant leishmanicidal activity. Mice receiving multiple inocula were more potent producers of leishmanicidal lymphokines than were those receiving single inocula. The expression of leishmanicidal activity in our system required continuous presence of the lymphokine preparation and was independent of trypsin- or neuraminidase-sensitive receptors of the macrophages. Light and electron microscopy revealed that, in the presence of lymphokines, macrophages appeared to be "activated," and intracellular leishmanias developed specific subcellular lesions in the kinetoplast-mitochondria. A time-course study showed that cultivation of the lymphocytes for 1 1/2 days completed the release of their leishmanicidal lymphokines which were heat-labile molecules larger than 50,000 daltons.     

利什曼原虫无鞭毛体蛋白的基因克隆化与序列分析

目的 克隆4株利什曼原虫表面无鞭毛体蛋白（amastin）的编码基因,并进行序列分析。方法 根据锥虫（T.cruzi)与利什曼原虫亲缘关系相近的原则,首先以锥虫无鞭毛体蛋白的基因为参考。对GenBank中的dbFST数据库检索,获得硕大利什曼原虫（L.major)一段309核苷酸片段,根据其序列合成探针,对硕大利什曼原虫基因组DNA文库筛选,首先获得硕大利什曼原虫无鞭毛体蛋白编码基因,再以硕大利什曼原虫无鞭 毛体蛋白编码 基因序列为依据,合成特异性引物,以多聚酶链反应（PCR）扩增获得亚马逊利会曼原虫（L.ama.)、巴西利什曼原虫（L.bra.)和墨西哥利什曼原虫（L.mex.)的无鞭毛体蛋白基因。结果 克隆了4株利什曼原虫无鞭毛体蛋白编码的基因。均为国际上首次克隆化基因,已被美国GenBank收录。结论 实现了4株利什曼原虫无鞭毛体 蛋白编码基因的克隆化。     

Changes in 'A' antigenic sites in haematological disorders. An immunoautoradiographic study

Using a quantitative immunoautoradiographic technique with simultaneously exposed internal standards, antigenic sites on A1, A1B, A2 and A2B erythrocytes were quantitated. The binding of purified radioiodinated rabbit anti-A antibodies was studied by the direct incubation technique. 61 normal individuals and 44 patients suffering from leukaemia and other haematological disorders such as polycythaemia vera (PV) and aplastic anaemia (Apl.A) were investigated. Among the normal volunteers, antigenic sites gradually increased up to 3 months of life, later on they were comparable with the values of adults. A reduction of more than 50% in the antigenic sites was observed in all cases of Apl.A. 50-70% of patients with various forms of leukaemia had a decrease in antigenicity. 1 patient suffering from acute unclassified leukaemia showed two populations of red cells. This case differed from natural O/A1 chimerism where A1 erythrocytes had normal antigenicity while in the patient, A1 antigens were depressed. 2 patients (1 with acute lymphoblastic leukaemia, 1 with Apl.A) who were investigated after allogenic bone marrow transplantation had a marked decrease in antigenic sites. Reasons for A antigenic variations in haematological disorders are discussed.     

Changes in the profile of circulating HDL subfractions in severe obese adolescents following a weight reduction program

Background and aimsEpidemiological studies show that obese adolescents are candidates to suffer cardiovascular pathologies in adulthood. In order to detect subfractions with a diagnostic value for future cardiovascular disorders, we analyzed the complete lipoprotein profile of severely obese adolescents.Methods and resultsTwenty-eight obese adolescents free from comorbidities were admitted into a weight reduction program. Anthropometric parameters were monitored. The circulating lipoproteins and glycemia were measured at the beginning and at the end of the study by conventional blood analysis as well as by using lipoprotein electrophoresis. Twenty-one puberty-matched normal-weight adolescents were recruited as controls. After 4 months, participants improved anthropometric parameters. Blood analysis indicated that circulating lipoproteins were in the healthy range during intervention. Nevertheless, results obtained from lipoprotein electrophoresis showed a significant increase in the large high-density lipoprotein subfraction in the obese population at the end of intervention, but significantly lower than normal-weight counterparts. In addition, intermediate- and low-density lipoprotein subfractions were in the healthy range in controls and in obese adolescents during intervention.ConclusionsAltogether, it seems that the obese adolescents with no comorbidities do not develop a clear dyslipidemia. However, low values of large high-density lipoprotein subfractions could be considered as candidate predictors to develop cardiovascular disease in the future. For this reason, diet and exercise are key tools to fight against this pathology.Registration number for clinical trialsISRCTN99414527     

Immunization of hamsters with TLCK-killed parasites induces protection against Leishmania infection

Hamsters immunized with N-p-tosyl-L-lysine-chloromethyl ketone TLCK-treated L. brasiliensis brasiliensis (LB) from culture, infected with LB amastigotes presented: a gradual increase in T and B cell responsiveness to mitogens by lymph node lymphocytes, and an increased response to concanavalin A with no changes for dextran sulphate and pokeweed mitogen in splenocytes. Absence of parasites in lymph nodes after 6 weeks post-infection and a nodule 4 times smaller than that of infected control animals. The nodule was undetectable after 70 days of infection. Hamsters preimmunized with TLCK-treated L. donovani (LD) from culture did not show suppression of the blastogenic response to mitogens of spleen and lymph node cells after infection with LD amastigotes and survived for more than one year, whereas infected, unimmunized animals died five months after infection. Animals preimmunized with culture parasites (LB or LD) treated with phenyl-methyl-sulphonyl-fluoride (PMSF) and infected with LB or LD amastigotes did not show any protective effect.     

Cytokine regulation of female-macrophage resistance to Leishmania mexicana parasites. Role of IL-12p70

In previous studies, carried out in humans, we showed that females are resistant to Leishmania mexicana infection. We also showed that 17β-estradiol (E2) induces killing of parasites inside of murine macrophages. In this work, we compared, for the first time, L mexicana survival inside of male (male BMDM) and female (female BMDM) bone marrow-derived macrophages (BMDM) treated in vitro with E2 or dihydrotestosterone (DHT). We also compared their levels of nitric oxide (NO), interleukin (IL)-6, IL-10, IL-12p70 and tumour necrosis factor (TNF-α). We found that female BMDM are a lot less susceptible to infection as compared with male BMDM. 17β-estradiol induced killing of most parasites inside of female BMDM. Dihydrotestosterone, on the other hand, induced some parasite killing inside of some infected male BMDM. Interleukin-6 levels were higher in female BMDM treated with either hormone. Neither TNF-α nor IL-10 levels showed significant differences compared with sham controls. Interestingly IL-12p70 was more abundantly produced by sham female BMDM as compared with sham male BMDM. Only female BMDM treated with E2 trigger a robust IL-12p70 production, but it was significantly reduced in male BMDM. This suggests IL-12p70 is an important factor in female-macrophage resistance to L mexicana parasites.     

Microculture for the isolation of Leishmania parasites from cutaneous lesions -- Sri Lankan experience

Novy, McNeal and Nicolle (NNN) medium and Evans' modified Tobie's medium are two conventional media for the isolation of Leishmania parasites in in-vitro cultures. Both are biphasic, with a solid layer of blood agar, and are normally prepared in glass test-tubes. In Sri Lanka at least, a monophasic microcapillary culture, based solely on RPMI 1640 medium supplemented with foetal calf serum, has been found simpler, more economical and more sensitive, for the isolation of L. donovani from skin lesions, than the use of Evans' modified Tobie's medium.     

Changes in the cellular profile of induced sputum after allergen-induced asthmatic responses.

Allergen inhalation causes airway inflammation and an increase in histamine airway responsiveness. We have used cell counts in sputum induced by hypertonic saline aerosol to assess airway inflammation before and 32 h after asthmatic responses to allergen. Twelve asthmatic subjects (mean age, 27.4 yr; range, 20-38 yr) had an inhalation test with D. farinae, ragweed pollen, or cat extract. All of them developed an early response with a fall in FEV1, of 24.8% (SD, 6.3%); nine of 12 had a definite late response (fall in FEV1 greater than or equal to 15%), and 10 of 12 had an increase in airway responsiveness to histamine at 32 h (PC20 reduced by greater than twofold). Sputum was induced by hypertonic saline after the histamine test, before and 32 h after the allergen challenge, at the same time of day. The quality of the sample was scored according to visual inspection and inverted microscopy and by salivary contamination. Plugs arising from the lower respiratory tract were selected for further evaluation. Differential cell counts of eosinophils (Eo) and metachromatic cells (MCC) (mast cell and basophils) were obtained from direct smears, blind to the clinical procedures. The mean fall in FEV1 after hypertonic saline was 6.4% (range, zero to 28%). The sputum samples were adequate in 79.5% of attempts. Eo and MCC increased significantly from 3.8 (4.4) to 18.2 (22.8)% (p = 0.01) and from 0.05 (0.17) to 0.25 (0.76)% (p = 0.04), respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

亚马逊利什曼原虫无鞭毛体蛋白基因的克隆化与序列分析

目的：克隆亚马逊利什曼原虫（L.ama)无鞭毛蛋白（amastin)的编码基因,并对其同源基因序列进行分析,方法：根据我们首次克隆的硕大利什曼原虫（L.major)无鞭毛体蛋白的编码基因,设计并合成核苷酸序列特异性引物,以亚马逊利什曼原虫基因组DNA为模板,以多聚酶链反应PCR技术扩增无鞭毛体的编码基因DNA片段,并进行核苷酸 列测定以及核苷酸序列的同源性分析。结果：克隆了亚马逊利什曼原虫无鞭毛体蛋白的编码基因,含有单一开放读框,长度为552bp,编码的无鞭毛体蛋白由183个氨基酸残基（aa)组成,亚马逊利什曼原虫与硕大利什曼原虫无鞭毛体蛋白编码基因之间高度同源,在核苷酸与氨基酸残基序列水平上的同源性分别为96％和94％,结论：首次实现亚马逊利什曼原虫无鞭毛体蛋白基因的克隆化。     

Changes of the cytokine profile in inflammatory bowel diseases

Cytokines are indispensable signals of the mucosa-associated immune system for maintaining normal gut homeostasis. An imbalance of their profile in favour of inflammation initiation may lead to disease states, such as that is observed in inflammatory bowel diseases (IBD). Although Crohn’s disease (CD) is often described as a prototype of T-helper 1-type diseases, and ulcerative colitis (UC) is traditionally viewed as a T-helper 2-mediated condition, the classic paradigm, which categorises cytokines into pro- and anti-inflammatory groups, has recently been changed. The inflammation regulatory pathways may not be mutually exclusive as individual cytokines can have diverse and even opposing functions in various clinical and immunological settings. None the less there are many common immunological responses in IBD that are mediated by cytokines. Although they regulate and influence the development, course and recurrence of the inflammatory process, the concrete pathogenic role of these small signaling molecules is sometimes not unambiguous in the subtypes of the disease. Our aim is to review the current information about pro- and anti-inflammatory effects of traditionally studied and recently discovered cytokines in the pathogenesis of UC and CD. The better understanding of their production and functional activity may lead to the development of new therapeutic modalities.