The changes of proteins fractions shares in milk and fermented milk drinks.

BACKGROUND: The aim of this research was to observe the changes which take place in the electrophoretic picture of milk proteins after pasteurisation and inoculation with different starter cultures (both traditional and probiotic). After incubation, the yoghurt, kefir, acidified milk, fermented Bifidobacterium bifidum drink and Lactobacillus acidophillus drink were chilled for 14 days to observe the changes which occurred. METHODS: The research materials were raw and pasteurised milk, as well as fermented milk- based drinks. The raw milk used for research came from Polish Holstein-Fresian black and white cows. The milk was sampled 3 times and divided into 5 parts, each of which was pasteurised at 95°C for 10 min and then cooled for inoculation: yoghurt to 45°C, kefir and acidified milk to 22°C and drinks with Bifidobacterium bifidum and Lactobacillus acidophillus to 38°C. Milk was inoculated with lyophilised, direct vat starter cultures, in an amount equal to 2% of the working starter. For the production of fermented drinks, the subsequent starters were applied: "YC-180" Christian Hansen for yoghurt, "D" Biolacta-Texel-Rhodia for kefir, CH-N--11 Christian Hansen for acidified milk, starter by Christian Hansen for the probiotic Bifidobacterium bifidum milk, starter by Biolacta-Texel-Rhodia for the probiotic Lactobacillus acidophillus milk. The analyses were conducted in raw, pasteurised and freshly fermented milk as well as in milk drinks stored for 14 days. The total solid content was estimated by the drying method; the fat content by the Gerber method; the lactose content by the Bertrand method; the protein content by the Kjeldahl method with Buchi apparatus; the density of milk was measured with lactodensimeter; acidity with a pH-meter; and potential acidity by Soxhlet-Henkl method (AOAC, 1990). The electrophoretic separation of proteins in raw and pasteurised milk, as well as in freshly produced milk drinks and those stored for 14 days, was performed with SDS-PAGE (on polyacrylamid gel) basing on procedure described by Laemmli (1970). RESULTS: It was shown that, in comparison with raw milk, the pasteurised milk had smaller amounts of αs-, ß- and κ-casein, whereas the shares of γ-casein and peptides were greater, and there were no changes in immunoglobulin, α-lactalbumin or ß-lactoglobulin levels, which indicated that hydrolysis of caseins had occurred. In all freshly fermented milk drinks, a drop in αs- and ß-casein was observed relative to raw milk. An increase in peptides and γ-casein was also noticed (with the exception of acidified milk). There were differences in α-lactalbumin and ß-lactoglobulin levels between the different drinks: raw, pasteurised or freshly fermented milk. It was shown that kefir, compared to the other drinks, had the lowest levels of αs- and ß-casein, α-lactalbumin and of peptides, as well as the highest level of γ-casein, which is evidence of an increased rate of hydrolysis in that drink. It was stated that, during the storage of fermented milk drinks, the levels of lactoferrin, serum albumin and peptides significantly increased whereas the content of κ-casein diminished. The proportions of serum albumin and lactoferrin in fermented milk drinks increased relative to raw milk and/or after storage, which is evidence of aggregation of proteins of low molecular mass into bigger conglomerates. CONCLUSIONS: The observed differences between fermented milks, including during chilled storage, in the amounts of individual proteins proves the different proteolytic abilities of starter cultures used in fermented milk production. α-lactoalbumin and ß-lactoglobulin are, besides caseins, the most allergenic milk proteins. So, kefir, because of its low α-lactoalbumin content, and Bifidobacterium bifidum milk, with the lowest content of ß-lactoglobulin, were the most advantageous and least allergenic drinks examined.

Identification and Purification of Armadillo (Dasypus novemcinctus) Immunoglobulins: Preparation of Specific Antisera to Evaluate the Immune Response in These Animals

Resumo: In this work we describe the purification and characterization of armadillo immunoglobulins. The IgM was precipitated using low-strength ionic solution and further purified by filtration through Sephadex G-200. The IgG was obtained in pure form by precipitation of serum with ammonium sulfate and DEAE-cellulose ion exchange chromatography. The purity of these immunoglobulins was evaluated by polyacrylamide gel electrophoresis. The results showed 28-kDa light chains and 55-kDa and 70-kDa heavy chains for IgG and IgM, respectively. The rabbit antibodies against these molecules were used to prepare fluorescein (FITC) and peroxidase conjugates. The FITC conjugate was used to quantify IgM-bearing lymphocytes. An average of 17% of peripheral blood lymphocytes were sIgM+ from 14 healthy animals. Additionally, in the same animals we quantified lymphocytes with the capacity to form rosettes with sheep red-blood cells; the average for this marker was 10%. Also, the production of crossreacting antibodies to BCG was evaluated in healthy and Mycobacterium leprae-inoculated animals using the peroxidase conjugates. All animals with active infection recognized BCG antigens

Evidence for B-lymphocyte mitogen activity in Borrelia burgdorferi-infected mice.

Borrelia burgdorferi produces a mitogen for murine B lymphocytes which can be measured in vitro by polyclonal stimulation of proliferation and immunoglobulin production (R. Schoenfeld, B. Araneo, Y. Ma, L. Yang, and J. J. Weis, Infect. Immun. 60:455-464, 1992). Sonicated B. burgdorferi cells also stimulated IL-6 production by splenocyte cultures. We have used the murine model for Lyme disease described by Barthold et al. (S. W. Barthold, D. S. Beck, G. M. Hansen, G. A. Terwilliger, and K. D. Moody, J. Infect. Dis. 162:133-138, 1990) to determine whether the B. burgdorferi B-cell mitogen is expressed during active infection. To correlate arthritic changes with immune events, we have studied two strains of mice injected with B. burgdorferi; one of them, C3H/HeJ, developed severe disease, and the other, BALB/c, developed only mild disease. C3H/HeJ mice displayed a persistent 10-fold increase in circulating immunoglobulin G (IgG) levels, a 2-fold increase in IgM levels, and a 15-fold increase in peripheral lymph node B-cell numbers, providing evidence of mitogenic activity. Infected BALB/c mice also had evidence for mitogen activity, since the IgG level in serum increased three- to fourfold. The bulk of the increase in circulating IgG levels was not directed against B. burgdorferi antigens, supporting the occurrence of polyclonal B-cell activation. Analysis of IgG isotypes pointed out a contrast between C3H/HeJ and BALB/c mice in that levels of all isotypes were elevated somewhat in both strains of infected mice but IgG2a levels were much more dramatically increased in the C3H/HeJ mice (28-fold) than in the BALB/c mice (4-fold). In this study, interleukin-6 levels were found to be persistently elevated in the serum of infected C3H/HeJ mice. Interestingly, interleukin-6 levels in serum were much lower in the infected BALB/c mice. These findings indicate that the B. burgdorferi mitogen is active in infected animals and may contribute to the inflammatory and immune response to infection.

Immune complexes and antibody levels in blisters over human leprosy skin lesions with or without erythema nodosum leprosum.

Erythema nodosum leprosum (ENL) is a serious complication of lepromatous leprosy, affecting skin and peripheral nerves in a large percentage of these patients, and is presumed to result from spontaneous immunologic changes. Its pathogenesis is poorly understood, although histopathologic features have suggested immune complex (IC)-mediated injury. Abundant circulating antibody is present but no convincing correlation has been established between circulating IC and ENL. We have examined cutaneous leprosy lesions in vivo using blisters induced by prolonged gentle suction to determine whether or not IC are demonstrable in lesions with or without ENL, using an IC assay based on monoclonal rheumatoid factor binding. We also examined whether antibodies involved in such IC are produced locally or reach the skin via the circulation. Surprisingly large quantities of IC were found in ENL lesions, and in some cases the quantities were significantly higher than in matching serum. Total IgG, IgA, and IgM in the skin were not higher than expected, however. Attempts to demonstrate increases in intracutaneous levels of specific anti-Mycobacterium leprae antibodies were unsuccessful. This is the first report of the demonstration of IC in suction blister fluid. The results indicate that large quantities of IC may be present in cutaneous leprosy lesions and are consistent with the hypothesis that they are formed in situ when circulating antibody combines with antigen in the skin. The nature of the antigen in these IC remains undefined.

[Serodiagnosis in pulmonary tuberculosis. Clinical evaluation]. / Serodiagnóstico en la tuberculosis pulmonar. Valoración clínica.

The IgA, IgG, IgM and IgG subsets antibodies levels were determined in 200 patients with pulmonary tuberculosis and compared to three control groups: 80 healthy individuals (50 with negative PPD skin test, 30 with positive PPD skin test), 30 leprosy patients and 20 patients with different pulmonary diseases. The technique used was an enzyme linked assay. As antigens, purified tuberculin and Ag60 from M. bovis were used. There were not statistically significant differences between antibody levels among all control groups studied, but when we compare the level in control groups with that observed in tuberculous patients, they showed higher levels of IgA, IgG, IgM, IgG2 (p less than 0.01) and IgG4 (p less than 0.05). A definite diagnosis of tuberculosis of the lung should only be established if the patient showed to be positive to IgG plus IgA or IgM and in special cases to IgG1, reaching then a diagnostic efficacy of 90% in a patient population with a 68% of positive smears for acid-fast bacilli.

[Infection and elimination of Mycobacterium leprae in SCID C.B.-17 mice (severe combined immunodeficiency)]. / Infection et élimination de Mycobacterium leprae par la souris SCID C.B.-17 (déficit immunitaire combiné sévère).

Previous studies documented that T-cell deficient nude mice failed to control M. leprae infection. In the present investigation we monitored the growth of M. leprae for up to 15 months in the SCID C.B.-17 mouse, a host deficient in both T and B lymphocytes. At 8 months post-infection 10(8) organisms/foot-pad were recovered from SCID mice vs 5 x 10(6) in normal BALB/c mice. Thereafter the number of bacilli decreased rapidly in mice infected with high-dose inoculum (10(7)); however, at all doses SCID mice eventually cleared M. leprae. During infection both T and B cells as well as serum Ig remained as low as in uninfected mice; however, in the spleen MAC-1+ cells which include macrophages and NK cells were substantially increased. These results suggest that MAC-1+ cells are involved in the anti-mycobacteria-1 defence mechanisms adopted by SCID mice to compensate their deficiency in T and B cells.

Persistent reduced solubilization of immune complexes in lepromatous leprosy patients with reactions.

It is held that immune complexes (IC) play a vital role in the pathogenesis of some of the reactions in leprosy. The complement system is known to solubilize and render IC innocuous. We have previously shown that patients undergoing lepra reactions had lowered complement-mediated IC solubilization (CMS). We, therefore, undertook a prospective study of untreated multibacillary leprosy patients and monitored their CMS levels sequentially while on therapy. In addition, the concentrations of the complement component C3d, immunoglobulins G, A and M, and circulating immune complexes (CIC) were also estimated. A total of 26 patients were included in the study and were investigated at 3-month intervals for 3 years. Thirteen of the 14 patients who did not develop reactions at all had normal CMS values, although all of them showed elevated CIC. From the inception of treatment, 10 of the 12 patients who developed lepra reactions had low CMS values which remained below normal levels even after evidence of complement activation disappeared and long after the subsidence of reaction. It is suggested that this defective CMS acts as a predisposing cause of lepra reactions.

Comparison of IgM, IgG and IgA responses to M.leprae specific antigens in leprosy.

Antibodies of IgM, IgG and IgA classes against M.leprae specific antigens (PGL-I, ND-O-BSA, and NT-O-BSA) were determined in the sera of 80 leprosy patients (28 untreated, 34 treated lepromatous and 18 tuberculoid), 25 tuberculosis patients and 33 normal individuals of Northern Thailand. No strong distinction in reactivity could be found between the three antigens. The IgM antibody assay yielded more positive results than assays for IgG and IgA. It was found that the positivity rates of IgM antibodies to all three antigens were highest in untreated lepromatous leprosy (82%). In tuberculoid leprosy, the positivity rates of IgM, IgG and IgA to the antigens were more variable, ranging from 22 to 50 percent. Patients with tuberculosis and normal individuals did not produce IgM antibodies against the antigens. The results suggested that the determination of IgM against the three antigens is a more sensitive and specific test for active leprosy than those of IgG and IgA. The relationship between the duration of treatment and IgM antibody levels in lepromatous leprosy (LL) was studied. Untreated LL patients had significantly higher IgM and IgA antibody levels than treated patients. There was no difference in IgG antibody levels between the two groups, and the levels of both groups were higher than normal controls. Serial determination of IgM antibodies in 7 LL patients revealed that treatment was strongly associated with progressive decrease in IgM antibody levels against all three antigens.

Total and anti-mycobacterial IgE levels in serum from patients with tuberculosis and leprosy.

A radioallergosorbent assay (RAST) was developed and used to determine the levels of IgE antibodies to soluble antigens of Mycobacterium tuberculosis (BCG vaccine strain) in sera from patients with tuberculosis and leprosy and in healthy control subjects. Total IgE levels in the same sera were quantitated with a commercial radioimmunoassay kit. Patients with tuberculosis and leprosy had higher total and specific IgE levels than the control groups but the overlap of levels in patients and controls was too great to render the difference diagnostically useful. Specific IgE levels were elevated in both tuberculosis and leprosy patients, suggesting that this antibody response is towards the shared mycobacterial antigens. No differences in total or specific IgE levels were found between healthy hospital workers occupationally exposed to patients with tuberculosis and factory workers who are not exposed.

Evaluation of humoral immunity on leprosy patients in Taiwan: a preliminary report.

Twenty-four tuberculoid (T)-type and 31 lepromatous (L)-type leprosy patients from Taiwan Provincial Lo-Sheng Leprosarium were enrolled in this study. Twenty-six age- and sex- matched normal subjects were also studied as a control group. The evaluation of their general and specific humoral immunity included B-cell subpopulations, 3 major classes of immunoglobulin (G, A and M) and antibodies in the IgG class against lepromin suspension and Bacillus Calmette-Guerin (BCG) sonicate. T-type patients showed a larger B-cell percentage than L-type patients (p less than 0.01). In general, patients with leprosy, both T and L types, had higher serum immunoglobulin levels than the control group. T-type patients showed greater antibody levels than the control group (p less than 0.05 for anti-lepromin and p less than 0.0001 for anti-BCG). L-type patients demonstrated a higher anti-BCG IgG level than the control group (p less than 0.0001). The level of anti-BCG IgG was more frequently above the cutoff level than that of anti-lepromin IgG in leprosy patients (p less than 0.01 for T, p less than 0.005 for L). In conclusion, humoral immunity is not impaired in leprosy patients. Discrepancies for T- and L-type patients among B-cell subpopulation, serum immunoglobulin levels and specific antibody levels reflect different aspects of cell-mediated immunity impairment. Though leprosy patients had elevated anti-BCG IgG levels, it is impossible to differentiate L- and T-type patients; specific antigens are needed for serodiagnosis of leprosy patients in Taiwan.

Proteínas séricas e fraçöes na hanseníase / Serum proteins and fraction in leprosy

A Hanseníase está associada com alteraçöes da atividade imunológica. Algumas evidências demonstram defeitos da resposta mediada por célula. Por outro lado, pacientes com Hanseníase podem apresentar níveis normais ou aumentados da resposta humoral. Este artigo lida com a determinaçäo de IgA, IgD, IgE, IgG. IgM - (classe de imunoglobulinas) e fraçöes proteicas (alfa I, alfa II, Beta e albumina), no soro de pacientes com Hanseníase e tenta uma correlaçäo com os achados clínicos

Serum immune complexes in erythema nodosum leprosum reactions of leprosy.

Serum estimations of immunoglobulins, complement components and their presence in circulating immune complexes were carried out in 39 Lepromatous, 44 ENL and 22 Post ENL leprosy patients. Serum IgG, IgA, IgM, C3 and C4 levels were determined by single radial immunodiffusion. Serum immune complexes were precipitated with Polyethylene Glycol (PEG) and IgG, IgA, IgM, C3 and C4 were estimated by single radial immunodiffusion and expressed as % of precipitation of their serum level. Decreased IgG, IgM; increased IgA and C3; and no change in C4 levels are observed in ENL than Lepromatous and Post ENL patients. However, a gradual insignificant reduction of IgG, IgA, and IgM was found from Lepromatous to ENL and Post ENL patients in the PEG-precipitates. Similarly, C3 and C4 was found reduced insignificantly in ENL than Lepromatous and Post ENL patients. The significance of these estimations in relation to immune status of ENL reactions are discussed.

Clinical significance of changes in serum proteins, immunoglobulins, and autoantibodies in leprosy.

Changes in the level of acute phase reactants such as C-reactive protein (CRP), serum globulins, and autoantibodies have been reported previously in patients with leprosy, particularly at the lepromatous end of the spectrum. The clinical significance of these findings was investigated by comparing the same parameters of humoral immune function in populations of Australian Aboriginals with stable treated leprosy and relevant contact groups including a) noninfected European sporadic contacts and b) healthy Aboriginal relatives of patients with confirmed leprosy. Raised levels of CRP and immunoglobulins and the higher frequency of autoantibodies seen in leprosy patients compared with sporadic contacts are probably related to differences in the incidence of nonleprous infection rather than to leprosy per se. Comparable results were obtained in the leprosy patients and their family contacts. The data highlight the need to use antigen-specific assays for determining the significance of changes in acute phase reactants and for distinguishing between the primary and secondary effects of Mycobacterium leprae infection.