Genetic Susceptibility and Predictors of Paradoxical Reactions in Buruli Ulcer.

INTRODUCTION: Buruli ulcer (BU) is the third most frequent mycobacterial disease in immunocompetent persons after tuberculosis and leprosy. During the last decade, eight weeks of antimicrobial treatment has become the standard of care. This treatment may be accompanied by transient clinical deterioration, known as paradoxical reaction. We investigate the incidence and the risks factors associated with paradoxical reaction in BU. METHODS: The lesion size of participants was assessed by careful palpation and recorded by serial acetate sheet tracings. For every time point, surface area was compared with the previous assessment. All patients received antimicrobial treatment for 8 weeks. Serum concentration of 25-hydroxyvitamin D, the primary indicator of vitamin D status, was determined in duplex for blood samples at baseline by a radioimmunoassay. We genotyped four polymorphisms in the SLC11A1 gene, previously associated with susceptibility to BU. For testing the association of genetic variants with paradoxical responses, we used a binary logistic regression analysis with the occurrence of a paradoxical response as the dependent variable. RESULTS: Paradoxical reaction occurred in 22% of the patients; the reaction was significantly associated with trunk localization (p = .039 by Χ(2)), larger lesions (p = .021 by Χ(2)) and genetic factors. The polymorphisms 3'UTR TGTG ins/ins (OR 7.19, p < .001) had a higher risk for developing paradoxical reaction compared to ins/del or del/del polymorphisms. CONCLUSIONS: Paradoxical reactions are common in BU. They are associated with trunk localization, larger lesions and polymorphisms in the SLC11A1 gene.

Acute phase proteins serum concentrations in children are related to urinary iodine excretion.

PURPOSE: The paper presents links between iodine provision and selected acute phase proteins' (APP) serum concentrations as well as their glycosylations profiles (investigated with the use of affinity immunoelectrophoresis with Concanavalin A as ligand) in children. MATERIAL AND METHOD: 116 children (58 girls and 58 boys) were enrolled. Iodine level was measured in the morning (7:30-8:30) urine portion, using Cr-As method. According to iodine level children were divided into two groups. The first one consisted of 56 children with decreased iodine level (lower than 100 micrograms/L), second--60 children with iodine level higher than 100 micrograms/L. In serum the concentration of ferritin, beta2-microglobulin (beta2-MG), thyroxin (T4), triiodothyronin (T3), thyrotrophic hormone (TSH) were measured by radioimmunoassay (BELORIS, Belarus). Concentrations of APP: C-reactive protein (CRP), alphal-acid glycoprotein (AGP), alphal-antichymotrypsin (ACT), alphal-antitrypsin (AT), haptoglobin (Hp), alpha2-macroglobulin (A2-M), ceruloplasmin (Cp) and transferrin (Tf) were measured in sera samples by rocket immunoelectrophoresis acc. to Laurell with antibodies and standard from DakoCytomation, Denmark. Microheterogeneity of AGP, ACT and Tf was estimated using affinity immunoelectrophoresis with ConA as a ligand, acc. to Bøg-Hansen. RESULTS: It was established, that CRP level was lower than upper limit of normal range. Levels of other investigated proteins were reliably dependent on the level of iodine. Especially for AGP lower level was observed for children of the group with low iodine level. In children with low iodine level along with the decrease of serum AGP concentration altered glycosylations profile was observed, namely decrease in the content of variant non-reactive to ConA (W0) and increase in content of weakly reactive (W1) and reactive (W2) variants content, which resulted in increase of the reactivity coefficient (AGP-RC). Similar tendency in alterations of distinctly glycosylated variants in relation to iodine level could be shown for ACT. Serum concentration of any investigated protein was not dependent on the concentration of the hormones of pituitary-thyroid system. CONCLUSIONS: It seems that the influence of the iodine level is direct, not via thyroid hormones. It could be suggested that in euthyroid children with low iodine excretion with urine a hidden iodine deficiency is already registered by the regulatory mechanisms and a kind of acute phase reaction is started, may be in order to increase iodine uptake and storage.

The effects of different beak trimming techniques on plasma corticosterone and performance criteria in Single Comb White Leghorn hens.

DeKalb XL chicks were given a beak trim at 6 d of age (6DP) with a 2.8-mm gauge and a beak trim at 11 wk (11WB) with a block cut approximately 2 mm anterior to the nasal openings. Corticosterone (CS) levels of the 6DP treatment were (P < or = 0.01) elevated above nontrimmed CS levels at 2 h posttrim; and BW and feed consumption (FC) of the 6DP were depressed until 8 wk of age. At 11 wk of age, CS of the 11WB treatment was (P < or = 0.02) elevated above controls at 1, 2, 8, and 5 wk posttrim. The 11WB treatment resulted in a decrease in FC and a reduction in BW at 12, 14, and 16 wk of age, whereas there were no differences among treatments in livability during the pullet phase. At 72 wk of age, FC of the nontrimmed controls was greater than both beak trimmed treatments, and both beak trimmed treatments had greater hen housed eggs, percentage hen day egg production, and percentage livability. Both beak trimmed treatments resulted in better egg income, feed cost per hen, and net income (NI). The 6DP and 11WB beak trim treatments resulted in an improvement of NI per hen of 1.48 dollars and 1.86 dollars, respectively. In addition, both beak trimmed treatments exhibited better feather score and Hansen's test (fearfulness). It was concluded that pullets and hens could adapt to the physiological stress of beak trimming and out perform, during a lay phase, controls whose beaks were not trimmed.

[Immunomodulating role of endogenous thyroid hormones in mycobacterioses]. / Immunomoduliatornaia rol' éndogennykh tireoidnykh gormonov pri mikobakteriozakh.

Radionuclide tracing in peripheral blood of patients with mycobacterial infections varying in activity (lepromatous lepra, n = 98; pulmonary tuberculosis, n = 51) provided information on lymphocyte proliferative response to PHA, tuberculin, sensitin from lepra mycobacteria, on thyroid hormones concentrations. Endogenic thyroid hormones are shown to have immunomodulating properties closely related to the disease activity.

Tumor necrosis factor production in patients with leprosy.

The spectrum of host responses to Mycobacterium leprae provides a model for investigating the role of cytokines in the pathogenesis of mycobacterial disease. Of particular interest is tumor necrosis factor (TNF), a cytokine which may have both antimycobacterial and immunopathologic effects. To evaluate the potential role of TNF in leprosy, we measured TNF production in response to M. leprae and its defined constituents by peripheral blood mononuclear cells from patients across the spectrum of disease. The levels of TNF induced through the stimulation of cells with M. leprae or its dominant "lipopolysaccharide," lipoarabinomannan, were higher in patients with the tuberculoid form of the disease than in those with the lepromatous form. In patients with erythema nodosum leprosum (ENL), a reactional state of lepromatous leprosy, the levels of TNF release by peripheral blood mononuclear cells were higher than in any other form of the disease. Treatment of ENL patients with thalidomide reduced TNF secretion by more than 90%. The mycolylarabinogalactan-peptidoglycan complex of Mycobacterium species, the protein-peptidoglycan complex, and muramyl dipeptide all elicited significant TNF release. Therefore, TNF release appears to be triggered by at least two major cell wall structural constituents of M. leprae, lipoarabinomannan and segments of the cell wall skeleton. The prominent TNF release in patients with the paucibacillary tuberculoid form of the disease compared with that in patients with the multibacillary lepromatous form suggests that this cytokine contributes to a resistant immune response to mycobacterial infection. However, the marked TNF release in patients with ENL indicates that TNF may also mediate immunopathologic effects, such as fever and tissue damage.

Quantitative and qualitative studies on the major extracellular antigen of Mycobacterium tuberculosis H37Rv and Mycobacterium bovis BCG.

The Mycobacterium tuberculosis antigen 85 is a biologically important antigen. Tuberculosis patients may have strong antibodies against it, and their peripheral blood mononuclear cells respond to it with gamma-interferon production and lymphocyte proliferation. Antigen 85 is actively secreted into the culture medium during culture in vitro and is known to bind human fibronectin. A double-antibody enzyme-linked immunosorbent assay (ELISA) for quantification of antigen 85 is described. A mouse monoclonal antibody, HYT27, was used as capture antibody in the assay. HYT27 was characterized in crossed immunoelectrophoresis and found to bind all three components of the antigen 85 complex. By radioimmunoassay, HYT27 was found to bind equally well to antigens 85A and 85B. In the ELISA assay, a rabbit anti-antigen 85 antiserum was used in the second antibody layer. The specificity of the assay was tested using several different antigen preparations. The purified BCG 85A and 85B components were compared, and there was a 10 times lower sensitivity for antigen 85A due to weaker rabbit antibodies toward this component. The purified components MPT44 and MPT59 from M. tuberculosis H37Rv were compared with the components of BCG and found to correspond to BCG 85A and 85B, respectively. Mycobacterium kansasii and Mycobacterium avium both contained partially identical antigens. Small amounts of antigen 85 were detected in Mycobacterium leprae sonicates. Detecting antigen 85 by sensitive methods may be of great value in the early diagnosis of mycobacterial disease.

Prostaglandin F2 alpha in leprosy--a preliminary study.

Prostaglandin F2 alpha was estimated in the sera of fifty patients in the leprosy spectrum to find out the status of prostaglandins in response to Mycobacterium leprae. Contrary to expectation, PGF2 alpha could be detected in only twenty-eight percent of leprosy patients. This preliminary finding is discussed in detail in the paper.

Antigenic evidence for simultaneous expression of two different lipooligosaccharides by some strains of Haemophilus influenzae type b.

Isolates of Haemophilus influenzae type b (Hib) can be divided into three antigenic groups based on their reactivities with a set of two monoclonal antibodies (MAbs) directed against epitopes in the oligosaccharide region of Hib lipooligosaccharide (LOS) (P. A. Gulig, C. C. Patrick, L. Hermanstorfer, G. H. McCracken, Jr., and E. J. Hansen, Infect. Immun. 55:513-520, 1987). Approximately 24% of Hib strains react with both of these LOS-specific MAbs. Immunoprecipitation experiments involving several of these strains indicated that the epitopes recognized by these MAbs resided in two different LOS molecules, both of which were synthesized by these particular Hib strains. In addition, Western blot (immunoblot) analysis of proteinase K-treated cell extracts of these strains that had been subjected to sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis revealed two different LOS staining patterns when they were probed independently with the two MAbs. Colony blot radioimmunoassay of hundreds of colonies of one of these Hib strains showed that each colony bound both MAbs. Immune electron microscopy confirmed that individual cells of this same Hib strain expressed both types of LOS molecule at the same time. An antibody accessibility radioimmunoassay was used to show that different Hib strains of this type varied in the relative amounts of each of the two MAbs that they could bind to their cell surfaces. These findings indicate that some Hib strains can synthesize two antigenically distinct LOS molecules simultaneously.

Appraisal of two Mycobacterium leprae-specific serological assays for monitoring chemotherapy in lepromatous (LL/BL) leprosy patients.

Two of the Mycobacterium leprae-specific assays--a serum antibody competition (for an epitope on 35-kDa protein) test (SACT) and an enzyme-linked immunosorbent assay (ELISA) for the disaccharide epitope of phenolic glycolipid-I (PGDS)--were comparatively evaluated as tools for monitoring chemotherapy in 125 lepromatous leprosy (LL/BL) patients. An adaptation of the SACT from a radioimmunoassay (RIA) to an ELISA procedure is also described. A moderate but statistically significant correlation was observed between the assays, although SACT appeared to be the more sensitive of the two. Levels of antibodies correlated better with the bacterial index (BI) than with the duration of treatment. However, wide individual variations in antibody levels (for a specific duration of treatment or BI) were seen in treated as well as untreated patients. Anti-PGDS antibody response of the IgG type was poorer than that of the IgM type and, apparently, it did not have a bearing on either treatment duration or the BI. Further studies will be needed to clarify whether the treated patients showing a negative (or low) BI and high antibody levels were harboring hidden foci of active infection, and whether treatment could safely be terminated in patients showing low values for both BI and antibody.

Effect of multidrug therapy on the levels of antibodies to Mycobacterium leprae glycolipid-1 in the leprosy spectrum.

Sera from one hundred and forty five untreated leprosy patients, ten proven cases of active pulmonary tuberculosis and twenty five healthy volunteers not exposed to M. leprae infection were assayed for PGL-1 antibodies. All available follow up samples after multidrug therapy were also assayed. A decline in the level of PGL-1 antibodies were seen in many of the post-treatment samples, giving an indirect assessment of the bacterial load and the prognosis of the disease.

Leishmania major: solid phase radioimmunoassay for antibody detection in human cutaneous leishmaniasis.

A radioimmunoassay for the quantitative determination of antileishmanial antibody in sera from patients suffering from cutaneous leishmaniasis was developed. The assay, using as antigen either the soluble fraction from freeze-thawed sonicated Leishmania major (LRC-L137) promastigotes or a carbohydrate-lipid containing fraction obtained by extraction with hexane-isopropanol, was shown to be sensitive and reproducible. The sera of 95 patients were examined. These were from patients with cutaneous leishmaniasis (26 from the Jordan Valley and 13 from Sinai), kala-azar (9), malaria (24), schistosomiasis (10), toxoplasmosis (5), and leprosy (8); controls were 37 normal human sera. No significant antigen dependent differences were observed using sera from cutaneous leishmaniasis patients, although differences in the immunological response were observed between the two populations of these patients. Antileishmanial activity was not detected in sera from patients with malaria, schistosomiasis, or toxoplasmosis. Although sera from leprosy patients crossreacted with the carbohydrate-lipid containing fraction, it was nevertheless more strain specific than freeze thawed sonicated L. major.

Leprosy: altered complement receptors in disseminated disease.

We have studied the expression of the C3b receptor (CR1) on erythrocytes of 55 patients with Hansen's disease. We developed a radioimmunoassay utilizing a monoclonal antibody that recognized an epitope different from the C3b binding site, which therefore enabled us to measure total number of CR1 regardless of receptor occupancy. We observed that patients in the lepromatous pole of the disease had a mean of 310 CR1/erythrocyte, whereas the ones in the tuberculoid pole showed a mean of 577 CR1/erythrocyte; 77 normal controls had a mean of 512 CR1/erythrocyte. The number of C3b receptors on the cells of lepromatous patients was significantly decreased (p less than .001) when compared to the normal population or tuberculoid patients. The presence of receptors for the C3b fragment of complement (CR1) on the surface of human erythrocytes enables these cells to participate in a number of immune functions including the clearance of circulating immune complexes. These findings could bear importance in the ability of the host to clear immune complexes from the circulation in patients with lepromatous leprosy.

Immunoassays of field isolates of Mycobacterium bovis and other mycobacteria by use of monoclonal antibodies.

Antigen extracts obtained by sonication of 22 strains of Mycobacterium bovis from cattle and badgers together with extracts of strains of M. tuberculosis, M. paratuberculosis, M. avium, M. africanum, M. kansasi, M. leprae and BCG were examined with a panel of 10 monoclonal antibodies to M. tuberculosis or M. leprae. Antigen extracts were coated in aqueous solution (wet coating) and the extracts were also dried on to the polyvinyl plates (dry coating). When dry coating was compared to wet coating, there was a major increase in the binding of monoclonal antibody ML03 to M. avium and M. paratuberculosis, monoclonal antibody ML02 to M. paratuberculosis, and monoclonal antibodies TB71 and TB72 to the majority of M. bovis isolates. The study confirmed that on wet-coated plates, monoclonal antibodies TB71 and TB72 bind poorly or not at all to M. bovis and that monoclonal antibodies TB68, TB78, TB77 and TB23 each bind to field strains of M. bovis while TB23 binds poorly to BCG in wet-coating conditions. Antibodies TB72 and TB71, originally thought to be specific for M. tuberculosis, each reacted with M. africanum. Antibody TB78 bound to M. paratuberculosis but did not react with M. avium, and M. avium and M. paratuberculosis were distinguished from M. bovis and M. tuberculosis by the binding of antibody ML03 to dry-coated plates. When wet-coated plates were used, ML03 bound strongly only to M. leprae. The panel of monoclonal antibodies did not demonstrate distinct serotype differences between the field isolates of M. bovis.

Antibody response to phenolic glycolipid I in inbred mice immunized with Mycobacterium leprae.

The level of circulating antibody to phenolic glycolipid I of Mycobacterium leprae was determined in 18 inbred strains of mice after immunization with M. leprae organisms. By using a solid-phase radioimmunoassay with phenolic glycolipid I as test antigen, a continuous distribution of antibody levels ranging from high to low was observed. The level was found to be controlled by multiple genes, including both H-2 complex- and Igh allotype complex-linked genes. Low antibody response to phenolic glycolipid I was shown to be inherited as a dominant trait in three combinations of high X low responder F1 progeny.