Identification and glycobiological characterization of circulating immune complexes in patients with visceral leishmaniasis and post kala azar dermal leishmaniasis.

Here, we investigated the quantitative and qualitative differences in antibody classes and subclasses in serum immune complexes (ICs) of Visceral Leishmaniasis (VL), Post Kala-azar Dermal Leishmaniasis (PKDL) and different cross reactive diseases like Malaria, Leprosy, Vitiligo as compared to control subjects. IC levels were measured through a newly developed PEG ELISA, using L. donovani promastigote membrane antigen coated plate. Antibody classes and subclasses were identified using polyspecific sera and monoclonal antibodies, respectively. ICs were purified using polyethylene glycol (PEG) precipitation. Conditional logistic regression showed an association between IgG1-containing ICs and increased risk of PKDL (OR=75, P <0.05) and an association of IgG-containing ICs with VL (OR=621, P=0.001). PEG ELISA demonstrated almost 13-15 fold higher IgG containing ICs titers in VL as compared to control (P <0.001). The assay further established a significant (P <0.05) difference in the IgG containing ICs titers between VL and PKDL. The isolated ICs were further analyzed by subjecting them to one-dimensional PAGE and subsequently stained with combination of periodic acid schiff (PAS) with silver. A differential banding pattern between VL and PKDL was obtained. Four distinct bands with carbohydrate rich glycoconjugates were identified in PKDL ICs, which were absent in VL and control group. It suggests the scope for developing a novel differential diagnostic assay.

Immunohistochemical localization of mycobacterium tuberculosis complex antigen with antibody to 38 kDa antigen versus Ziehl Neelsen staining in tissue granulomas of extrapulmonary tuberculosis.

BACKGROUND: Extra-pulmonary tuberculosis often presents a diagnostic challenge because of its diverse clinical manifestations and low yield of acid fast bacilli in tissue sections. AIM: The aim of the present study is immuno-histochemical localization of tuberculous bacilli or their components that persist in the granulomas but have lost the property of staining with acid fast stains and to assess the advantage of immuno-staining over conventional Ziehl Neelsen staining. MATERIAL AND METHODS: Immuno-histochemical staining using species-specific monoclonal anti-body to 38 kDa protein of Mycobacterium tuberculosis complex and Ziehl-Neelsen staining for acid fast bacilli (AFB) was done on 69; 36 cases of confirmed extra-pulmonary tuberculosis and 33 non-tuberculous cases, in archival formalin fixed paraffin embedded tissue sections OBERVATIONS: AFB positivity was observed in only 36.1% of tuberculous granulomas while immuno-histochemical staining was positive in 100% of tuberculous granulomata with zero false positivity and negativity. CONCLUSIONS: The immuno-hiostochemical localization of tuberculous bacilli and their components in tissue sections may be an efficient diagnostic adjunct to conventional ZN staining for the diagnosis of granulomas of extra-pulmonary tuberculosis. The technique is simple, sensitive and specific. It can be standardized and performed by trained technicians in routine laboratory. This will also help in clinical decision-making and in reducing the usual practice of prescribing empirical anti-tubercular treatment based on clinical suspicion alone in the absence of demonstrable evidence of tuberculous infection.

Serodiagnosis of childhood tuberculosis by ELISA.

OBJECTIVE: Diagnosis of childhood tuberculosis remains an enigma despite many recent technological developments. The present study has been taken up with the aim to assess the diagnostic potential of mycobacterium tuberculosis excretory-secretory ES-31 antigen and affinity purified anti ES-31 antibodies in the serodiagnosis of different spectrum of childhood tuberculosis. METHODS: Mycobacterium tuberculosis H37Ra excretory-secretory antigen (ES-31) and affinity purified goat anti ES-31 antibodies were used in stick penicillinase ELISA for IgG antibody detection and stick Sandwich penicillinase ELISA for detection of circulating free and immune complexed antigen in the sera of 230 children. RESULTS: Analysis of tubercular antibody, circulating free and immune complexed antigen (CIC-Ag) was done in both pulmonary and extrapulmonary form of childhood tuberculosis and overall sensitivity of 81.4% with a specificity of 93% was achieved for detection of antitubercular IgG antibodies. Of the five cases of pulmonary tuberculosis showing absence of IgG antibody, 3 showed the presence of CIC-Ag and one was found positive for both free and CIC-Ag. Similarly out of 8 cases of extrapulmonary childhood tuberculosis missed by IgG detection 5 were found to be positive for CIC-Ag and 1 showed the positive reaction for both free and immune complexed antigens. CONCLUSION: IgG antibody to excretory-secretory antigen ES-31 is found to be having good specificity with acceptable sensitivity in detecting different forms of childhood tuberculosis. Further detection of circulating free and/or immunecomplexed antigen can be used as an adjunct tool in the diagnosis of childhood tuberculosis.

Levels of antibody, free antigen and immune-complexed antigen by ELISA in different grades of sputum positive patients of pulmonary tuberculosis.

The ES-31 (31 kDa protein) antigen was isolated from culture filtrate of Mycobacterium tuberculosis H37Ra and was shown to have potential in immunodiagnosis of pulmonary tuberculosis. Serum samples from 38 confirmed sputum positive pulmonary tuberculosis patients were grouped into AFB+, AFB++, AFB+++ based on sputum bacillary load. Analysis of tubercular antibody, circulating free and immune complexed antigen (CIC-Ag) was done in serum samples by Indirect and Sandwich ELISA using ES-31 antigen and affinity purified anti ES-31 antibody respectively. The analysis of Geometric mean titre (GMT) of all the three groups showed that GMT of tubercular antibody was considerably decreased compared to elevated levels of CIC-Ag (Antibody: 1360 to 816 and CIC-Ag: 534 to 1744) from low bacillary sample to high bacillary samples, whereas there is no significant change in the titre of circulating free antigen. Low levels of detectable antibody is observed possibly due to removal of antibody from circulation by immune complex formation as confirmed by its elevated levels in sputum AFB+++ positive patients.

Immune complex mediated lesions in experimental Kala azar: an ultrastructural study.

Immune complexes play an important role in causation of renal lesions in various diseases. Circulating immune comlexes (CIC) are described in Kala azar. Role of CIC in pathogenesis of Kalaazar is discussed in present study. BALB/C mice were experimentally infected with L. donovani promastigotes. After visceralisation of infection, sera and kidneys of infected mice were preserved. Leishmanial antigen specific CIC could be demonstrated in 100% of infected mice by PEG ELISA, while they were absent in control mice. Ultrastructural pattern of renal lesions in infected mice showed presence of focal small electron dense deposits in glomerular basement membrane and subepithelial space, resembling immune complexes (humps). Rarely subendothelial and mesangial hypercellularity was present. These findings point towards a definite role of CIC in pathogenesis of renal lesions in Kalaazar.

Presencia de inmunocomplejos circulantes y alteraciones del sistema complemento en pacientes con leucemia promielocítica y coagulación intravascular diseminada / Presence of circulating immune complexes and alterations of the complement system in patients with promyelocytic leukemia and disseminates intravascular coagulation

Se efectuó la determinación de inmunocomplejos circulantes (ICC) así como la medición del sistema complemento por la vía clásica, vía alterna, actividad del factor B y la cuantificación del tercer (C3) y cuarto (C4) componentes de complemento en 30 pacientes con leucemia promielocítica (LPM) al diagnóstico, 22 de los cuales presentaron coagulación intravascular diseminada (CID). Se demostró la existencia de niveles elevados de ICC en los enfermos con CID y una disminución significativa de la actividad de la vía clásica, los componentes C3 y C4 en los enfermos con CID, al compararlos con el grupo de pacientes que no presentaba el trastorno de la hemostasia y los controles normales, lo que sugiere la posible participación de estos parámetros en el fenómeno de la CID en estos enfermos

Immunofluorescent microscopic findings in glomerulonephritis.

Histopathology and direct immunofluorescence (DIF) microscopy were performed on renal biopsy specimens of 60 clinically suspected cases of glomerulonephritis (GN). Histopathological diagnosis was obtained in 44 (73.3%) cases and immune complex deposition were detected by DIF in 28 (46.7%) cases. Immune complex deposition were observed in all cases of membranous GN, systemic lupus erythematosus (SLE), and rapidly progressive GN (RPGN), most of the cases of diffuse proliferative GN (2 out of 3) mesangioproliferative GN (12 out of 15) and focal glomeruloscleros is (3 out of 5 cases). No immune deposits were observed in minimal change GN, chronic GN, and diabetic nephropathy. Histopathological diagnosis was not obtained in 16 (26.7%) cases, 3 (5%) of which showed immune complex deposition by DIF. Anti-GBM nephritis was demonstrated in one (3.6%) case, the rest were immune complex nephritis.

Autoantibodies, immunoglobulins, complement and circulating immune complexes in acute malaria.

BACKGROUND: Malaria caused by Plasmodium vivax and Plasmodium falciparum is common in the Indian subcontinent. Studies conducted elsewhere have suggested that malarial infection causes intense immunostimulation. We screened patients with malarial infection for autoantibodies and measured the immunoglobulin, circulating immune complex and complement levels to determine the extent of immunological alterations in these patients. METHODS: One hundred adults with acute malarial infection confirmed by examination of the peripheral blood smear and 25 age- and sex-matched controls were studied. An autoantibody screen and serum immunoglobulin complement (C3 and C4) and circulating immune complex levels were measured at the time of admission and 4 weeks after they became afebrile. A direct Coomb's test was also done. RESULTS: Anti-ssDNA, anti-dsDNA and rheumatoid factor were positive at the time of admission in 51, 30 and 38 patients respectively. None of the controls were positive for these autoantibodies except for one who was positive for rheumatoid factor. The IgM, IgG and IgA levels were raised in 16, 25 and 36 patients respectively. Circulating immune complex levels were raised in 32 patients and complement C3 and C4 were low in 8 and 31 patients. Follow up studies at 4 weeks in 19 patients showed that the autoantibodies were negative. However, the immunoglobulin, C4 and circulating immune complex levels remained elevated. Six per cent of patients had a positive direct Coomb's test with reticulocytosis at the time of presentation. CONCLUSION: Acute malarial infection can cause false-positive results for anti-ssDNA, anti-dsDNA and rheumatoid factor and may also cause a rise in the serum immunoglobulin, complement and circulating immune complex levels.

Deteccion de inmunocomplejos en mucosa nasal de pacientes con bloqueo nasal cronico y permanente

Se estudiaron 30 pacientes con problemas rinológicos en los que el síntoma fundamental fue el "Bloqueo Nasal Permanente". Se comprobó que los mismos presentaban un B.N.C.T. y P., que es la entidad definida por nosotros como una obstrucción permanente y crónica de las fosas nasales y que sería la expresión clínica, an tomo patológica e immunológica terminal de una "Rinitis Crónica" que conlleva una Insuficiencia Respiratoria Nasal Crónica. Estos "respiradores bucales" presentaban síntomas fundamentales: sequedad de garganta, ronquidos nocturnos, resonancia nasal de la voz, con alteraciones en los órganos de la audición y del olfato; transtornos mec nicos y secundarios derivados del respirar por la boca bajo la forma de faringitis, laringitis. bronquitis, exacerbaciones del asma bronquial y tendencia al enfisema; distrofias en el esqueleto facial; disminución de la capacidad intelectual, irritabilidad, falta de concentración, transtornos de la función cardiovascular (extrasístoloes, variaciones en la presión arterial). Fueron adolescentes y adultos jóvenes, en los que las causas alérgicas e infecciosas dominaron la etiología, demonstrada por los antecedentes alérgicos familiares y personales de atropia, pruebas intradérmicas inmediatas positivas para aeroalergenos y bacterias, y corroborada por un aumento de la IgE y eosinófilos en sangre. Como métodos de diagnóstico para confirmar el B.N.C.T. y P. se usaron: radiografías y tomografias lineales; rinomanometrias y rinofibrolaringoscopías. Los hallazgos anatomopatológicos obtenidos por biopsia nasal fueron contundentes: gran infiltrado linfomonocitario alrededor de arteriolas y vénulas; signos de vasculitis en un alto porcentaje, lo que estimuló la búsqueda de inmunocomplejos. La presencia de inmunocomplejos positivos alrededor de los vasos confirmó el curso "desfavorable" de estas rinitis, aportando progresos en el enfoque fisiopatológico, diagnóstico y en el manejo terapéutico del B.N.C.T. y P.

Humoral immunity in Hansen's disease

Introduçao: Desde a década de 60, tem-se observado alteraçoes da imunidade humoral na hanseníade. Os auto-anticorpos apresentam-se em freqüências diversas, nestes pacientes, mais habitualmente na hanseníase virchoviana, em doença de longa evoluçao e em surtos reacionais. Variaçao nas freqüências pode ser atribuída a diferença metodologias empregadas na detecçao dos anticorpos e ao grupo de doentes selecionado. Material e Métodos: Esta revisao enfoca os resultados obtidos em diversos estudos de auto-anticorpos, complexos imunes, crioglubulinas, complemento sérico na hanseníase. Destaca-se também, os anticorpos contra glicolipídeos do Mycobacterium leprae, como os antiglicolipídeos fenólitos.I, cuja magnetide é variável e depende do patrimônio genético apresentado pelo enfermo.

Presencia de antígeno de superficie del virus de la hepatitis B en pacientes sospechosos de hepatopatías / Presence of hepatitis B surface antigen in patients presumably having liver diseases

La determinación de pequeñas cantidades de antígeno de superficie del virus de la hepatitis B resulta de vital importancia en el tratamiento de esta afección. Se evaluó la incidencia de este marcador viral en pacientes con sospecha de afección hepática y se studió la relación entre éste con el estadio de la enfermedad y el funcionamiento hepático. Se estudiaron 838 pacientes con sospecha de hepatitis, el 9,9 por ciento fue positivo para el marcador, lo que permitió su reclasificación. El nivel de transaminasa se correspondió con la evolución del enfermo. La concentración del marcador viral en relación con el perfil hepáatico permitió la polarización de 2 subpoblaciones: 1 con valores patológicos de ambos indicadores y la otra cuya única evidencia de enfermedad era la presencia del antígeno de superficie de la hepatitis B. La determinación de este marcador por métodos inmunoenzimáticos es útil en el diagnóstico de pacientes portadores del virus, aún cuando su sintomatología no resulte evidente

Detección de inmunocomplejos circulantes mediante el método C1q-ELISA / Detection of circulating immunocomplexes by C1q-ELISA method

Se estandarizó un método inmunoenzimático (ELISA) para la determinación de inmunocomplejos circulante, tomando en cuenta la propiedad del componente C1q del sistema complemento de reconocer dichos complejos. Se empleó 1ug de C1q por pocillo y se logró un nivel de detección de 1ug equivalente de agregados de IgG humana por mL. El límite superior de detección obtenido en 35 sujetos sanos fue de 34 ug/mL con un coeficiente de variación intra e interensayo, de 8,1 y 8,9, respectivamente. Los resultados indican la utilidad del método enzimático para su empleo en el seguimiento de aquellas enfermedades asociadas con inmunocomplejos

Circulating immune complexes in leukaemias and lymphomas.

Circulating immune complexes (ClC) were estimated in 78 patients of leukaemias and lymphomas by Clq deviation ELISA and PEG assay. In all leukaemias a significant elevation in ClC was seen at the time of first presentation. While in ALL a decrease occurred on therapy as partial or complete remission was achieved, no such fall was seen in AML or CML-BC when treated. ClC levels were much higher in non-Hodgkins lymphoma than in Hodgkins disease and showed a direct correlation with B symptoms and activity of the disease. The ClC levels were highest in null-ALL followed by those in common ALL and T-ALL. The mean duration of remission in patients of ALL without elevation in ClC was much longer than in those with ClC.

Significance of pathological parameters of endomyocardial biopsy in clinical outcome of patients of dilated cardiomyopathy--II. Value of cardiac immune complexes and heart reactive antibodies.

We have evaluated the status of cardiac immune complexes and heart reactive antibodies in endomyocardial biopsies (EMB) and patients' sera from cases of dilated cardiomyopathy (DCM) using immunofluorescence. This was done with an aim to test whether this parameter can be of diagnostic and/or prognostic value in cases of DCM in its inflammatory and non inflammatory stages. Deposition of IgG was consistently observed in all cases of DCM regardless of the presence or absence of inflammation. Complement was detected in only a few while IgG and C3 together was seen to be deposited in only 4 cases. IgA and IgM were noted in an occasional case only. Heart reactive antibodies were seen in 13 of the 23 cases of DCM. Light microscopically, in 7 of the 23 biopsies mild focal lymphocytic myocarditis was detected. Presence of IgG in EMB and a low left ventricular ejection fraction (LVEF 35%) in almost all the cases, highlight the prognostic significance of IgG (in EMB) as an independent parameter. Based on this small study, it is difficult to attach significance to these observations as regards predicting the outcome of these patients. Nevertheless, the present study initiates evaluation of one of the parameters which is accessible and can be easily carried out in most routine laboratories for diagnosis, prognosis, and eventually monitoring of therapy in patients of DCM. Importance of immunofluorescence technique can be further strengthened by evaluating a larger number of cases with varying duration of symptoms and a follow up study of cases of DCM.

Circulating immune complexes in pre-eclampsia.

Serum samples from 20 non-pregnant women, 30 women with normal pregnancy and 50 women with pregnancy associated with pre-eclampsia were tested for circulating immune complexes using the polyethyleneglycol precipitation method. A highly significant positive correlation was found between circulating immune complexes and severe pre-eclampsia (BP greater than 140/90 mm Hg, albuminuria greater than 0.25 g/l). In contrast to this the difference in immune complex levels between non-pregnant subject, normal pregnancy cases and patients with mild pre-eclampsia was not statistically significant. A significant positive correlation was found between the level of circulating immune complexes and the severity of albuminuria. These findings suggest that circulating immune complexes, though not seeming to play an aetiological role in pre-eclampsia may very well be involved in its pathogenesis.

Immunologic parameters in infective endocarditis: a prospective study.

To evaluate the role of immunoglobulins, complement, circulating immune complexes (CIC), heart antibodies (HAb) and rheumatoid factor (RF) in infective endocarditis, we studied 28 consecutive patients before and after therapy. Statistically significant elevation was seen in IgG (p less than 0.001) and IgA (P less than 0.001) level prior to initiation of therapy as compared to a control group. Following drug treatment a fall was noted in IgA (P less than 0.01) and IgM (p less than 0.01) level as compared to basal values. Low C3 levels were seen in those with renal involvement (p less than 0.05). CIC levels estimated by 4% PEG precipitation assay were found to be elevated in 64% of patients. Patients with shorter duration of illness (less than three months) had higher levels of CIC containing IgG (P less than 0.005), IgA (P less than 0.05) and IgM (P less than 0.05), as compared to those with a longer duration. Initial CIC levels did not predict the clinical course and were found to be of no value in prognosis, although an improvement in congestive heart failure was associated with a rise in C3 (P less than 0.05) and IgM (P less than 0.05) containing CIC and an overall clinical improvement with a rise in IgA (p less than 0.05) containing CIC. There was no statistically significant difference in CIC level, for the entire group studied, before and after therapy. Patients who had rheumatoid factor in their initial serum sample demonstrated a fall in IgG, IgA and IgM containing CIC and a rise in C3 with therapy. The converse was true for those who lacked RF.(ABSTRACT TRUNCATED AT 250 WORDS)

Certain immunological parameters in subacute cold stress.

The effect of subacute cold swimming stress on the immune system of albino rats was investigated. Subacute cold stressed animals showed an increase in total WBC count, eosinophils and basophils. Phagocytic index and avidity index were also increased indicating hyperactive phagocytic process. On the other hand NBT reduction and soluble immune complex levels decreased significantly in stressed animals. There were no significant changes in the weight of the lymphoid organs.

Pre-operative and post-operative evaluation of circulating immune complexes in patients with gastric adenocarcinoma.

Circulating immune complexes (CICs) in the sera of patients with histologically proven adenocarcinoma of stomach were sequentially studied. Serial CICs levels, quantitated using a sensitive method F(ab')2 anti-C3 ELISA, were measured before surgery and in a post-operative follow up. CICs could be detected in 85% of the patients pre-operatively, while ten days after surgery positivity decreased to 71%. Thirty days after surgery, the mean CIC levels decreased significantly and positivity fell to 46%. The results indicate that removal of primary tumor mass results in a sharp decline of CIC levels.

Anticuerpos antilipoproteinas de baja densidad e inmunocomplejos APO (B) lipoproteina-anticuerpo en pacientes con enfermedad vascular perifèrica: empleo de anticuerpos monoclonales / Anti LDL anbodies and B apolipoprotein-antibody immunocomplexes in patients with peripheral vascular disease: use of monoclonal antibodies

Se estudiò la presencia de anticuerpos anti-LDL a IC apo-B-lipoproteinas anticuerpo en un total de 60 pacientes con aterosclerosis perifèrica y en 60 con angiopatìa diabètica. Los resultados se compararon con un grupo de 41 individuos no diabèticos y sin sìntomas clìnicos de enfermedad cardiovascular. Entre algunos pacientes diabèticos y ateroscleròticos el nivel de anticuerpos anti-LDL fue significativamente màs elevado, aunque la proporción de casos que tuvieron valores por encima del valor correspondiente al 90 percentil del grupo control, tomado èste como valor lìmite, no fue diferente. Tampoco se encontrò diferencia en el nivel de IC apo-B lipoproteina-anticuerpo cuando se empleó para su determinaciòn un anticuerpo policlonal. Sin embargo, se detectaron niveles altos de estos complejos y una proporciòn significativamente elevada de pacientes ateroscleròticos por encima del valor lìmite fijado cuando se empleò para estos fines el anticuerpo monoclonal 12E6E8. Se discute la importancia que puede tener el empleo de anticuerpos monoclonales para detectar lipoproteinas de estructura alterada de alto potencial aterogènico y de los inmunocomplejos que pueden originar