HLA-B allele frequencies in Côte d'Ivoire defined by direct DNA sequencing: identification of HLA-B*1405, B*4410, and B*5302.

Direct automated DNA sequencing was used to analyze exons 2 and 3 of HLA-B alleles present in forty-four unrelated individuals residing in the village of Adiopodoume, Côte d'Ivoire (Ivory Coast). Of the 23 HLA-B alleles observed, the most frequently detected allele was HLA-B*5301 (22.7%), which is believed to confer resistance to severe Plasmodium falciparum malaria. B*4501 (9.1%), B*1503 (8.0%), B*0705 (5.7%), B*1510 (5.7%) and B*3501 (5.7%) occurred frequently in the population. A second allele of B53 was identified; B*5302 contains a single amino acid variation at residue 171 (Y-->H). Two additional novel alleles, B* 1405 (a single amino acid variant of B*1402) and B*4410 (a five amino acid variant of B*4403) were characterized.

Low levels of serum calcidiol in an African population compared to a North European population.

OBJECTIVE: To compare vitamin D status in an African population living at 10 degrees N with a Norwegian population living at 60 degrees N. DESIGN: Serum samples from 30 healthy young Ethiopians and 31 full term pregnant women from Addis Ababa were collected in September, and from 24 healthy Norwegians in March and 23 pregnant women from Oslo in February to June. METHODS: Serum (s) levels of calcidiol and intact parathyroid hormone (iPTH) were measured. RESULTS: The median values for s-calcidiol were significantly lower in Ethiopians compared with Norwegians (young Ethiopians 23.5nmol/l vs young Norwegians 81nmol/l, P<0.001; pregnant Ethiopians 25nmol/l vs pregnant Norwegians 36nmol/l, P<0.05) while those for s-iPTH were significantly higher (young Ethiopians 5.7pmol/l vs young Norwegians 2.4pmol/l, P<0.001; pregnant Ethiopians 4.8pmol/l vs pregnant Norwegians 2.8pmol/l, P<0.02). CONCLUSION: In spite of abundant availability of ultraviolet radiation, the population from Addis Ababa had a high rate of biochemical vitamin D deficiency compared with the Norwegian group.

Evaluation of a colorimetric assay based on 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) for rapid detection of rifampicin resistance in Mycobacterium tuberculosis.

OBJECTIVES: To standardise the colorimetric assay based on 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) for the rapid detection of rifampicin-resistant Mycobacterium tuberculosis in clinical practice and to evaluate the assay on a collection of 92 clinical isolates. DESIGN: The Bactec method was used as the reference method. Rifampicin was used for the susceptibility testing in the Bactec method at a concentration of 2 microg/ml. The MTT assay was performed in tubes containing 3 ml Dubos broth; the assay is based on the principle that live cells convert the yellow tetrazolium salt into a blue formazan. A final concentration of 2 microg/ml rifampicin was used in the assay. Optical density (OD) values at 570 nm were recorded on the third and sixth day. A strain was defined as susceptible when the relative optical density unit (RODU) (i.e., OD of rifampicin containing tube/OD of undiluted control) was < or = 0.2, and when the OD value of the rifampicin-containing tube on the sixth day was lower than the OD value on the third day. A strain was defined as resistant when the RODU was more than 0.5, and when there was an increase in OD value in the rifampicin-containing tube on the sixth day. The tubes were also read visually. RESULTS AND CONCLUSION: The result obtained by the MTT assay perfectly matched the result obtained by the Bactec method. The MTT assay was also interpretable by the naked eye. This simple, inexpensive assay could be used as a rapid screening method for identification of rifampicin-resistant strains in low-income countries.

Use of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide for rapid detection of rifampin-resistant Mycobacterium tuberculosis.

We describe a test which uses the ability of viable cells to reduce 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) to detect resistance to a bactericidal drug, rifampin, in in vitro-cultured Mycobacterium tuberculosis. The assay shows a linear relationship between the number of viable bacteria and the ability to reduce MTT. Dead mycobacteria were unable to reduce MTT. Rifampin-sensitive M. bovis (BCG) and M. tuberculosis exposed to rifampin showed a rifampin concentration-dependent inhibition of the ability to reduce MTT, while the resistant strains were unaffected. The inhibition of MTT reduction after treatment with rifampin paralleled the reduction in the number of CFU. By using mixing experiments in which the population percentages of rifampin-sensitive and -resistant strains were varied, the assay could detect the presence of rifampin resistance in the mixture when at least 1% of the bacterial population was composed of drug-resistant strains. The assay is cheap, can be visually read, and requires less than 3 days to obtain susceptibility results. The total time required to obtain results, from the time sputum is received in the laboratory, is, in most cases, less than 4 to 5 weeks, which is the time required for primary culture of the bacteria. The MTT assay could, in combination with a test to detect resistance to isoniazid, be a cheap and rapid screening method for multidrug-resistant M. tuberculosis that is affordable even by low-income countries where tuberculosis is a major public health problem.

Immunologic responses to soluble exoantigens of Plasmodium falciparum in Gabonese children exposed to continuous intense infection.

The development of antidisease immunity in children infected with Plasmodium falciparum is thought to be related to their immunologic responses to certain soluble parasite-derived exoantigens. We have assessed both cellular and humoral responses to these antigens in a cross-sectional study of a cohort of Gabonese schoolchildren who live in an area where malaria is holoendemic and perenially transmitted, in an attempt to identify immunologic markers of this early developing protective immunity. Concurrent parasitemia was found to have a significant influence on lymphoproliferative and antibody responses to the exoantigens. Individuals with higher levels of parasitemia had significantly lower proliferative and IgG isotype responses. Higher concentrations of specific IgG1 and IgG3, in particular, were associated with lower or no parasitemia, suggesting a possible protective role for these isotypes, whereas the level of IgM antibodies showed a trend towards higher concentrations in those with parasitemia, perhaps indicative of an exoantigen-induced T cell-independent response. Cytokine responses were unaffected by either the presence or the intensity of parasitemia and were dissociated from both proliferative and antibody response to the exoantigens. However, the mitogen-stimulated production of tumor-necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL)-6 was positively correlated with the corresponding lymphoproliferative responses. At the individual level, mitogen-stimulated TNF-alpha, interferon-gamma, IL-2, and IL-6 responses were positively correlated, as were mitogen- and exoantigen-induced TNF-alpha. The results are discussed in the light of current knowledge of immune responses to the exoantigens and the development of protective immunity to P. falciparum.

In vitro lymphoproliferative responses to malaria antigens: a prospective study of residents of a holoendemic area with perennial malaria transmission.

A longitudinal, prospective study to examine the relationship between the outcome of infection with Plasmodium falciparum parasites and in vitro T-cell proliferative responses to a P. falciparum schizont extract (PfSE) was conducted in a village in south-eastern Gabon, an area where malaria is holoendemic and transmission is intense and perennial. The donor's age was found to have a strong independent influence on all malariometric indices. At the community level, the in vitro lymphoproliferative response to PfSE was bimodal with 30% of the villagers studied showing persistently low responses. The frequency of low or high responders within the study population did not show any consistent relationship with the community parasite rates or the number of either patent parasitaemic episodes or clinical malarial attacks per individual. At the individual donor level, the response was negatively correlated with P. falciparum parasite density in those donors who were parasitaemic at the time of sampling. High in vitro lymphoproliferative responses to PfSE were predictive of resistance to clinical malaria. The PfSE-induced in vitro lymphoproliferative response was dependent on antigen presenting cells, CD4+ T-cells and UCHL-1+ cells.

Cytokines in the pathogenesis of malaria: levels of IL-I beta, IL-4, IL-6, TNF-alpha and IFN-gamma in plasma of healthy individuals and malaria patients in a holoendemic area.

A longitudinal study was conducted between October 1989 and February 1990 in a malaria holoendemic area of Gabon to determine the plasma concentration of various cytokines in individuals continuously exposed to infection with malaria parasites. No cases of severe malaria were seen and fever was the main presenting symptom of clinical malaria. Parasite rates were highest in children 6-9 years old but clinical malaria was seen essentially in children below 6 years of age. The incidence of clinical malaria was highest in November and February corresponding to the beginning and end of heavy rains respectively. Parasite rates did not show any seasonal variations. Overall, there was no seasonal variation in plasma cytokine levels but both IL-6 and IL-4 levels were highest in February. Plasma concentration of TNF-alpha and IFN-gamma were higher in parasitaemic than aparasitaemic individuals and donors who had clinical malaria had higher levels of TNF-alpha, IFN-gamma and IL-6 than asymptomatic parasitaemic donors. There was a negative correlation between age of the individual and the concentration of plasma TNF-alpha and IFN-gamma suggesting that the production of these cytokines could be modulated by repeated malarial infections. Asymptomatic parasitaemic children 5-7 years of age had higher levels of plasma TNF-alpha than clinically similar children below or above this age group suggesting that refractoriness to the clinical effects of TNF-alpha may be an important factor in the ability of these children to resist clinical malaria.(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro cell-mediated immune responses to Plasmodium falciparum schizont antigens in adults from a malaria endemic area: CD8+ T lymphocytes inhibit the response of low responder individuals.

Plasmodium falciparum schizont extract and purified protein derivative were used to stimulate peripheral blood mononuclear cells obtained from healthy aparasitemic Gabonese individuals with lifelong exposure to malaria infection and non-Gabonese control subjects who have had had no clinical malaria. In vitro lymphoproliferation was measured by uptake of tritiated thymidine, while production of interleukin-2, interferon-gamma, and soluble CD8+ were measured by immunoenzymatic assays. Enumeration of interferon-gamma-producing cells was done using a modified immunoenzyme spot assay. Twenty-eight percent of Gabonese subjects were determined to be low responders in the lymphoproliferative assay, with a tritiated thymidine uptake of less than 6000 c.p.m. The proportions of T cell subsets and the kinetics of the proliferative response were similar in the low and the high responders. Removal of CD8+ T cells from mononuclear cells of low responders or culture of purified CD4+ T cells from the same individuals resulted in a 7-fold increase in the proliferative response to the schizont antigen but not to purified protein derivative (PPD). A similar increase in the proliferative response was seen in the low but not the high responder mononuclear cell cultures stimulated with the schizont antigen in the presence of exogenous interleukin 2 (IL-2) or in the presence of anti-HLA-DQ antibody. Low responder mononuclear cell cultures stimulated with schizont antigen but not PPD produced 3-fold less IL-2, 14-fold less interferon-gamma (IFN-gamma), and 3-fold more soluble CD8 than high responder mononuclear cell cultures. Removal of CD8+ T cells from low responder mononuclear cells resulted in a 2-fold increase in IL-2 production and a 4-fold increase in IFN-gamma production in response to schizont antigen. High responder mononuclear cells stimulated with schizont antigen contained four times as much IFN-gamma-producing cells as low responder cultures, with each IFN-gamma-producing cell producing three times the amount of IFN-gamma as that produced by an IFN-gamma-producing cell in low responder cultures. Removal of CD8+ T cells from low responder mononuclear cells led to a significant increase in the amount of IFN-gamma produced at the single cell level in response to schizont antigen stimulation. In such cultures, the amount of IFN-gamma produced by a single cell was similar between high and low responders. We conclude that in certain individuals, T cell responses to schizont antigen are actively down-regulated by activated schizont-specific CD8+ suppressor T cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Armadillo IgG and IgM antibody responses to phenolic glycolipid-I during experimental infection with M. leprae.

The kinetics of antibody responses of Mycobacterium leprae-infected armadillos to phenolic glycolipid-I (PGL-I) were studied by means of ELISA. The levels of both IgG and IgM antibodies to PGL-I increased with time. Some animals were less susceptible to disseminations of M. leprae infection and lived longer than others. These animals had high absorbance values (greater than 0.7) for IgG anti-PGL-I compared to more susceptible armadillos that had lower absorbance values for IgG anti-PGL-I.

Infection with live mycobacteria inhibits in vitro detection of Ia antigen on macrophages.

Both antigen-specific and non-specific anergy are common features of disseminated mycobacterial infections, and the pathogenesis of such anergy is as yet not fully understood. To date, most studies have focused on the efferent limb of the immune response, and no detailed information is available on the early macrophage-T cell interaction and its consequence on T cell clonal proliferation. To gain information on this crucial phase of mycobacteriosis, we have conducted studies to evaluate the effect of M. kansasii infection on Ia expression induced by T cell-derived lymphokine and have assessed whether such cells can adequately present either mycobacterial or allogeneic antigens to T cells. In vitro infection of mouse resident peritoneal macrophages with live but not heat-killed M. kansasii resulted in a significantly reduced percentage of cells expressing monoclonal antibody detectable Ia antigen following optimal stimulation with crude lymphokine preparations or recombinant mouse gamma interferon. In parallel experiments, macrophages infected with the mycobacteria were co-cultured with syngeneic in vivo M. kansasii sensitized non-adherent, nylon-wool purified lymph node cells, and lymphoproliferation was measured by [3H]thymidine incorporation. It was shown that in co-cultures with macrophages infected with live M. kansasii, the lymphocyte proliferation was marked even in very low infection ratios. In contrast, the response to heat-killed bacilli was dose dependent, reaching peak levels only in high infection ratios. The ability of infected macrophages to present allogeneic antigens was assessed using the mixed leukocyte reaction. Macrophages infected with heat-killed M. kansasii were able to induce a mixed leukocyte reaction similar to uninfected macrophages whereas macrophages infected with live M. kansasii were unable to stimulate allogeneic T cells. These findings may have implications on immunological disturbances often seen in mycobacterial infections, such as leprosy, in which there can be large numbers of non-toxic viable intracellular bacilli.

In situ characterization of the cutaneous immune response in Ethiopian cutaneous leishmaniasis.

Cryostat sections from 10 patients with localized cutaneous leishmaniasis (LCL) and eight patients with diffuse cutaneous leishmaniasis (DCL) from Ethiopia were studied with immunofluorescence methods for the phenotypic characterization of cells in the lesions. Higher numbers of Leu 2+ and Leu 3+ cells (P less than 0.005) were found in LCL than in DCL, while the Leu 3a + b/Leu 2a ratios were the same. No differences were found in the numbers of transferrin receptor, HLA-DR, and HLA-DQ expressing cells in the granulomas. Significantly (P less than 0.0001) lower numbers of IL-2 receptors expressing (Tac+) cells were found in DCL than LCL lesions, suggesting interference in the activation of the T cells. IL-2-containing cells were absent in DCL and were found in LCL lesions. Epidermal keratinocytes above the LCL but not the DCL lesions expressed HLA-DR (but not HLA-DQ) antigen, suggesting a lower gamma-interferon production in the DCL granulomas. The number of Langerhans' cells (Leu 6+) was higher in the epidermis of DCL (P less than 0.005) than in LCL, while a lower number of Leu 6+ cells were seen in the dermal lesions (P less than 0.001). These observations could account for some of the mechanisms responsible for the disturbed immunostimulation and immunoregulation observed in the lesions of DCL.

Muramidase (lysozyme) findings in sural and radial nerve biopsies in leprosy patients after varying periods of treatment.

Using the immunoperoxidase staining method, tissue muramidase (lysozyme) activity was studied in 34 nerve biopsies from leprosy patients and compared to findings in the skin. In a majority of lepromatous and borderline-lepromatous leprosy patients, the enzyme was seen to form a saccular pattern within the cells; whereas a granular pattern was found at the tuberculoid end of the leprosy spectrum, as well as during reversal reactions. Indeed, the most intense enzymatic activity was found in four patients with reversal reactions. Compared to the skin, muramidase activity was found to be more intense and persisted longer in the nerves. Successful antileprosy treatment reduced the enzymatic activity in both the nerves and the skin, but more so in the skin. Schwann cells and axons did not show muramidase activity, indicating that the muramidase-positive cells are not of neuronal origin. Our results suggest that a high percentage of mononuclear cells infiltrating the peripheral nerves in leprosy are derived from blood monocytes. The function of tissue muramidase in leprosy is not yet clear. Its peculiar intracellular distribution pattern in the different forms of leprosy, however, warrants further study to elucidate its role in the pathogenesis of the disease.

Sural nerve biopsy in leprosy patients after varying periods of treatment: histopathological and bacteriological findings on light microscopy.

As part of a larger study of nerve biopsies from leprosy patients in Ethiopia for the presence of muramidase (lysozyme), sections were also examined by light microscopy after staining with hematoxylin and eosin for cellular infiltrate and a modification of the Ziehl-Neelsen stain for leprosy bacilli. The muramidase findings will be reported separately. This paper describes the infiltrative and bacterial findings in a group of 18 patients, including four with nonlepromatous forms of leprosy who were suffering from delayed hypersensitivity reaction at the time of biopsy. The findings were unexpectedly interesting and revealing. Lepromatous and borderline-lepromatous patients all showed endoneurial and perineurial infiltration of considerable extent and, in several instances, bacilli were wide-spread from one end of the biopsy to the other; in two patients, solid-staining bacilli and globi were found, indicating relapse. In all except two of the nonlepromatous patients (mainly borderline-tuberculoid) there was an extensive and severe granulomatous infiltration, and in one case there was marked caseation in the endoneurial zone. Within the limits of the present study, the findings indicate that biopsy of a peripheral nerve, even when it is not obviously associated with a skin lesion, may reveal pathological changes which are greater in degree than those suggested by skin biopsy or clinical examination. These observations in a somewhat heterogeneous group of patients treated for varying periods of time, and in a study which was not prospectively planned, suggest that similar observations in a larger group of untreated and treated patients, including those who have relapsed, may be of value.

Demonstration of mycobacterial antigens in nerve biopsies from leprosy patients using peroxidase-antiperoxidase immunoenzyme technique.

Peripheral nerve biopsies from patients with leprosy were stained with anti-Mycobacterium bovis (BCG) in a peroxidase-antiperoxidase (PAP) system to demonstrate intraneural mycobacterial antigens. Most M. leprae antigens have been shown to cross-react with BCG. Of the 30 biopsies from borderline tuberculoid (BT) patients 18 had acid-fast bacilli while 26 of them had demonstrable mycobacterial antigens in their nerves. All borderline lepromatous (BL) and lepromatous leprosy (LL) nerve biopsies had both M. leprae and mycobacterial antigens within them. Most of the antigens in the BT patients were seen to be extracellular. In BL and LL patients antigens were seen both extracellularly and intracellularly in Schwann cells and infiltrating macrophages. Mycobacterial antigens in BT nerves were always seen to be surrounded by a mononuclear cell reaction while in the BL and LL patients antigens could be seen with minimal cellular infiltrate and the neural architecture was more or less preserved. While bacilli could not be seen in BT patients who had been released from treatment for more than 4 years, mycobacterial antigens could still be seen in some patients who had been released from treatment for up to 5 years. Patients with no skin lesions but with large, painful, or tender nerves were found to have intraneural inflammation surrounding mycobacterial antigens, while those with a similar clinical picture but without tender or painful nerves showed no marked inflammation within their nerves despite the presence of mycobacterial antigens. From these findings it was concluded that immunologically mediated inflammatory response toward intraneurally located M. leprae antigens in conjunction with other host factors may be necessary for nerve damage in the BT leprosy patients. In the BL and LL patients the mechanisms of nerve damage are still unknown with certainty but local effects and immune-complex damage secondary to abundant M. leprae antigens are worth exploring. The use of immunohistological techniques should offer a new approach in the study of the immunopathology of leprosy.