Assessment of serum copper, iron and immune complexes in potentially malignant disorders and oral cancer

Abstract Potentially malignant disorders (PMDs) of oral cavity and oral cancer remain a cause of serious concern despite intensive research and development. Diet and immunity have been identified to play a crucial role as modifying factors in these diseases. Our study intended to explore this relationship by estimating and comparing the serum levels of copper, iron and circulating immune complexes (CICs) in patients diagnosed with PMDs and oral cancer and normal healthy individuals. In this study, 40 histopathologically diagnosed cases of PMDs and oral cancer were included along with 30 healthy controls and 5 ml of venous blood was drawn using venipuncture. Serum estimation of copper, iron and CIC then followed using the colorimetric and spectrophotometric methods. The data obtained was subjected to statistical analysis using one way ANOVA and Pearson's Product-Moment Correlation Test. The mean serum copper level was measured as 138.98 ± 10.13µg/100ml in the PMD group and 141.99 ± 21.44 µg/100ml in the oral cancer as compared to 105.5 + 18.81µ/100ml in the controls. The mean serum CIC levels was highest in the oral cancer (9.65 ± 0.16OD470) followed by the PMD group (0.18 + 0.21 OD470) and least in the control group (0.048 ± 0.02OD470). Whereas, the serum levels of iron showed a significant decrease in the PMD group (110.9 ± 10.54 µg/100ml) and the oral cancer group (114.29 ± 25.83 µg/100ml) as compared with the control group (136.85 ± 14.48 µg/100ml). There was no positive correlation obtained between the three groups with respect to the chosen parameters indicating that the variables were independent of each other. It can be thus be ascertained that trace elements like copper and iron as well as humoral responses (CICs) have a close relationship with PMDs and oral cancers.

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.

Anticorpos antipeptídeos citrulinados e fator reumatoide em pacientes sudaneses com infecção por Leishmania donovani / Anti-citrullinated peptide antibodies and rheumatoid factor in Sudanese patients with Leishmania donovani infection

OBJETIVO: Este estudo avaliou a presença de anticorpos antipeptídeos citrulinados cíclicos (anti-CCP), fator reumatoide (FR) e imunocomplexos circulantes (ICC) em pacientes sudaneses infectados por Leishmania donovani. PACIENTES E MÉTODOS: Os soros foram coletados de pacientes infectados por Leishmania (n = 116) e de sudaneses saudáveis (n = 93). Dezenove pacientes sudaneses com artrite reumatoide (AR) e anti-CCP+ foram incluídos como controles positivos. Os níveis de ICC e anti-CCP foram medidos por ELISA. Para avaliar a reatividade citrulina-específica foi usada a placa-controle com peptídeos-controle cíclicos contendo arginina em vez de citrulina. RESULTADOS: Entre os pacientes infectados por Leishmania e os pacientes com AR e anti-CCP+, a maioria (86%) era positiva para FR, enquanto a frequência de positividade para ICC foi maior entre pacientes com leishmaniose visceral (LV) (LV 38%; AR e anti-CCP+ 24%). Quando foi analisada a reatividade anti-CCP, 12% dos pacientes com LV foram positivos. Os níveis de anti-CCP entre os pacientes com LV correlacionaram-se bem com os níveis de ICC encontrados (r = 0,65; P < 0,0001). No grupo de AR não foi encontrada associação entre ICC e anti-CCP. A possibilidade de que a positividade para anti-CCP se deva a reações cruzadas com ICC foi descartada experimentalmente. Ao contrário do que foi visto no soro dos sudaneses com AR, a reatividade anti-CCP não se restringiu à citrulina, mas houve reação igual com os peptídeos-controle com arginina. CONCLUSÃO: O fato de a reatividade CCP não se ter restringido à citrulina comprova tratar-se mais de um efeito de inflamação extensa e ativação imune do que de um sinal de características patogênicas compartilhadas com artrite anti-CCP. Nossos achados ressaltam a importância de se interpretar um teste CCP positivo com cuidado ao se avaliar condições não reumáticas ou em áreas onde tais infecções predominam.

OBJECTIVE: The present study evaluated the presence of anti-cyclic citrullinated peptides antibodies (anti-CCP), rheumatoid factor (RF), and circulating immune complexes (CIC) in Sudanese patients infected with the Leishmania donovani parasite. PATIENTS AND METHODS: Sera were collected from Leishmania infected patients (n = 116) and healthy Sudanese (n = 93). Nineteen Sudanese anti-CCP+ RA patients were included as positive controls. Levels of CIC and anti-CCP were measured by ELISA. Control plate with cyclic control peptides containing arginine instead of citrulline was used to evaluate citrulline specifi c reactivity. RESULTS: Among Leishmania-infected patients and anti-CCP+ RA patients, most were RF positive (86%), while the frequency of CIC positivity was higher among visceral leishmaniasis (VL) patients (VL 38%; anti-CCP+ RA 24%). When anti-CCP reactivity was analysed, 12% of VL patients were found to be positive. The levels of anti-CCP among VL patients correlated well with the CIC levels found (r = 0.65, P < 0.0001). In RA group, no association was found between CIC and anti-CCP. The possibility that anti-CCP positivity was due to cross reactions with CIC was experimentally ruled out. Contrary to what was seen in Sudanese RA sera, the CCP reactivity was not restricted to citrulline but reacted equally well with the arginine control peptide. CONCLUSION: The finding that CCP reactivity was not restricted to citrulline argues that this is more an effect of extensive inflammation and immune activation than a sign of shared pathogenic characteristics with anti-CCP arthritis. Our fi ndings stress the importance to interpret a positive CCP test carefully when evaluated in non-rheumatic conditions or in areas where such infections predominate.

Evaluation of circulating immune complexes and serum immunoglobulins in oral cancer patients - A follow up study.

Background: High serum immunoglobulins and circulating immune complexes (IgG, IgM, IgA and CIC) values in patients with cancer have been used as tumor markers. Hence, the aim of the study was to estimate these immunological markers in pre- and post-treatment phases with a follow-up of 3-24 months and to understand the prognostic significance of the same in patients with oral cancer. Materials and Methods: The malignancy group consisted of 56 patients with different stages (AJCC TNM) of oral cancer and 20 healthy control group. Samples were selected at random and subjected for sequential analysis of serum biochemical markers (IgG, IgA, IgM and CIC-circulating immune complexes levels) in the pre- and post-treatment period. Statistical method employed was the paired t test. Results: We observed significant elevated levels of all the immunological markers ( P < 0.01) when compared with the control group. Sequential analysis of these markers revealed significant reduction in immunological markers in stage I and II patients. On the contrary, stage III and IV patients showed remarkably elevated levels of IgA and CIC one year after initial treatment. Conclusions : All these immunological markers are indicative of tumor burden and Serum levels of CIC and IgA might be employed as prognostic indicators in oral cancer.

Serodiagnosis of tuberculosis: specific detection of free and complex-dissociated antibodies anti-mycobacterium tuberculosis recombinant antigens

The diagnostic test characteristics of detecting free and complex-dissociated IgG to three recombinant antigens of Mycobacterium tuberculosis (38-kDa, Ag16 and Ag85B), singly and in combination, were evaluated in sera from 161 tuberculous patients [smear-positive pulmonary TB (50), smear-negative pulmonary TB (pTBsm-) (60) and extrapulmonary TB (51)) and 214 control patients (mycobacteriosis (14), mycoses(14), leprosy(4), other underlying diseases (82) and healthy people (100)]. The individual antigens ranged from 25 percent to 42 percent in sensitivity and from 93 percent to 96 percent in specificity, while considering free IgG response. Addition of complex-dissociated antibodies against each individual antigen improved the sensitivity up to 55 percent. The number and levels of specific antibodies varied greatly from individual to individual. Combination of individual results for free and complex-dissociated IgG to 38-kDa, Ag16 and Ag85B offered 76 percent sensitivity and 83 percent specificity. When the three antigens were placed in the same well, the sensitivity was lower than that expected on the basis of single antigen (63 percent) but with a good specificity (95 percent), even in the group of mycobacteriosis or mycoses. The highest contribution of complex-dissociated IgG results to free IgG results was seen for the diagnosis of pTBsm- patients. In conclusion, although neither single recombinant antigen was reactive with most sera from TB patients even after the measurement of both free and complex-dissociated antibodies, the use of multi-antigen cocktails improved the diagnostic utility of the ELISA assay, allowing the identification of almost 70 percent of pTBsm-, with a high level of specificity; the use of additional, well selected antigens should lead to the detection of almost all patients with TB.

Characterization of entamoeba histolytica antigens in circulating immune complexes in sera of patients with amoebiasis.

Isolated circulating immune complexes (CICs) from sera of patients with amoebiasis were characterized to determine Entamoeba histolytica antigens that participate in the disease process. In total, 116 serum samples were collected before starting anti-amoebic therapy, and their CICs were isolated by differential polyethylene glycol precipitation. The presence of amoeba-specific antigens in CICs was detected by antigen capture enzyme-linked immunosorbent assay (ELISA) and by immunoblot assay. Antigen capture ELISA showed significantly higher optical density (p < 0.001) in all patients with amoebiasis than in the normal healthy controls and patients of non-amoebic hepatic disorder. Immunoblot assay detected amoeba-specific CICs in all 18 patients (100%) with confirmed amoebic liver abscess, 28 (80%) of 35 patients with clinically-suspected amoebic liver abscess, and 18 (78.26%) of 23 patients with amoebic colitis. No patients with non-amoebic hepatic disorders and healthy control subjects had any detectable level of amoebic antigens in CICs. Immunoblot assay revealed E. histolytica antigens of relative molecular masses of 35, 56, 70, and 90 kDa present in CICs of 64 of 76 patients with amoebiasis. The 35-kDa polypeptide was observed in 52 patients (81.25%). The results of the study suggest that the 35-kDa polypeptide antigen can be a diagnostic marker in active amoebiasis.

Comportamiento de variables inmunológicas en pacientes politraumatizados / Inmunological variables behaviour in multiple trauma patients

Se realizó el estudio descriptivo de algunas variables de la inmunidad celular y humoral que incluyen las rosetas espontáneas y activas, las inmunoglobulinas, el complemento hemolítico total y los inmunocomplejos circulantes, en 30 pacientes politraumatizados atendidos en la unidad de terapia intensiva del Instituto Superior de Medicina Militar "Dr. Luis Díaz Soto". Los pacientes fueron monitoreados mediante 3 extracciones de sangre que correspondieron a las 24 h, a las 72 h y a los 7 d después de haber sufrido el trauma. Se produjo una disminución de las rosetas, cambios de los valores de inmunoglobulinas y aumento de los inmunocomplejos circulantes; por lo que el seguimiento de estas variables sería importante en la evaluación de los politraumatizados

Significance of circulating immune complexes in myasthenia gravis.

In this study, circulating immune complexes (CICs) were isolated and characterized in the sera of myasthenia gravis (MG) patients with thymoma and MG patients without any thymic lesions. High titres of antistriational antibodies in the CICs were demonstrated by an indirect immunofluorescence (IF) method in 60 per cent (15/25) MG patients with thymoma. The CICs showed a steady decrease in these 15 patients during the post thymectomy period. Antistriational antibodies in the CICs of MG patients without thymic lesions were not detected by IF method. The results of this study emphasised the usefulness of estimation of CICs in the overall management of MG patients with thymoma.

Immunodiagnosis in bone and joint tuberculosis.

Fifty patients of bone and joint tuberculosis (age 15-70 years) and 30 healthy controls were studied for levels of immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM), T cells and circulating immune complexes (CICs) in different stages of disease. Levels of IgG, IgA, IgM, T cells and CICs were significantly raised in fresh and healed tuberculosis patients, compared to control.

Role of IgM & IgA rheumatoid factors in complement activation in patients with juvenile rheumatoid arthritis.

We studied the relationship between the degree of complement activation in juvenile rheumatoid arthritis (JRA) with the levels of circulating IgM and IgA rheumatoid factors (RF). Forty children with JRA and 25 matched controls were included in the study. Levels of C3d (a degradation product of complement component C3), circulating immune complexes (CICs), IgM RF and IgA RF were measured by ELISA. Levels of C3d, CICs, IgM RF and IgA RF were elevated in patients with JRA as compared to controls. Levels of C3d had a linear relationship with levels of CICs (P < 0.05) but not with levels of circulating IgM RF and IgA RF. Thus, complement activation occurs in children with JRA and is associated with raised levels of CICs but not with levels of circulating IgM and IgA RF. Circulating IgM and IgA RF have little, if any, role in complement activation observed in patients with JRA.

Detection of mycobacterial antigen in circulating immune complexes in patients with childhood tuberculosis.

The presence of both components (antigen and antibody) in circulating immune complexes (CIC) were detected in tuberculosis in children. Fifty two patients with pulmonary and extrapulmonary tuberculosis showed the presence of either components or both. CIC--antigen was present in 92.3% (48/52) and CIC antibody in 88.96% (46,52). Out of these 52 patients, 20 were proved cases, CIC antigen (ag) and CIC--antibody (ab) were present in 100% (20/20). In the control group both CIC-ag and CIC-ab and CIC = ab can be taken as an additional marker in diagnosis of tuberculosis.

Circulating immune complex in murine autoimmune hepatitis.

High level of circulating immune complexes (CIC) in the serum has been reported in different forms of hepatitis particularly in complicated cases of viral hepatitis due to hepatitis B virus (HBV) infection. In this study CIC level in experimental autoimmune hepatitis were assessed by detection of polyethylene glycol (PEG) index. The sera of mice with established autoimmune hepatitis (EAH) confirmed by histopathological study showed higher PEG index (C57BL/6 mice: 34.56 +/- 6.28 and C3H mice: 31.95 +/- 28.99). The control healthy mice showed lower PEG index (C57BL/6 mice: 19.48 +/- 6.85 and C3H mice: 21.27 +/- 6.1). The high level of PEG index in EAH was found statistically significant. The role of CIC in the development of autoimmune hepatitis is discussed.

A probable explanation for mild extra-articular manifestations in Indian patients of rheumatoid arthritis: a preliminary study.

One hundred four Rheumatoid factor (RF) positive Rheumatoid Arthritis (RA) patients fulfilling ARA criteria were screened for extraarticular manifestations. Rheumatoid nodules were present in 2.8% cases. Other extraarticular manifestations such as pulmonary, cardiac, occular, renal or GI involvement were absent in these cases. However, circulating immune complexes (CICs) were highly significantly raised in all the RA patients (P < 0.001). From amongst these cases a limited number (8 cases) have been subjected to qualitative and quantitative analysis of CICs to look for whether there could be any relationship between these and mild extraarticular manifestations that were being noticed in our groups of patients. Finding showed IgG-IgG CICs in five, IgG-IgM in two and IgG-IgA in one case. Quantitative analysis revealed mean IgG 4.97 +/- 1.7 IU/ml, IgM 14.58 +/- 5.53 IU/ml and IgA 5.08 +/- 1.53 IU/ml on LD Solugen plates. Serum concentration of C3 was not reduced (94.1 +/- 8.9 mg/dl). Low IgM contents of CICs and no reduction in complement level is the likely explanation for less severe inflammatory manifestations seen in our study. The conclusion and findings have been discussed in the light of observation reported by the Western workers.

A sequential study of circulating immune complexes, complement and immunoglobulins in borderline tuberculoid leprosy patients with and without reactions.

Sequential estimates of the levels of circulating immune complexes (CIC), complement catabolic fragment C3d, complement-mediated immune complex solubilization (CMS) and immunoglobulins were made in 24 newly diagnosed with borderline tuberculoid leprosy over a 20 month period after initiation of chemotherapy. Fourteen of these patients had not suffered from reversal reactions either at the time of presentation or during the follow-up. The levels of CIC were evaluated in them from the third to the eleventh month after starting chemotherapy and immunoglobulin G (IgG) levels were evaluated up to eight months. The concentrations of C3d and immunoglobulins A (IgA) and M (IgM) were normal in these patients. The other ten patients had reversal reaction at the time of diagnosis which subsided by the third month after starting treatment. They did not have reversal reactions later. The levels of CIC and IgG were elevated and those of CMS were depressed throughout the study period. Serum C3d level was initially elevated but came down to normal by the third month while IgA and IgM levels were within normal limits. The relevance of these findings to the genesis of reversal reaction is discussed in this communication.

Alteraciones del sistema del completo en el aborto espontáneo / Alterations of the complement system in spontaneous abortion

Se efectuó la determinación de la actividad hemolítica de la vía clásica y la cuantificación de los componentes C3 y C4 del sistema del complemento en 48 mujeres con embarazos a término, 20 con antecedentes de abortos espontáneos y 28 normales, así como la determinación de inmunocomplejos circulantes y los niveles séricos de IgG, IgA e IgM. Se demostró una disminución significativa (p < 0,001) de la actividad de la vía clásica y de C3 en el grupo de mujeres abortadoras al compararlas con las normales. No se observaron alteraciones en el resto de los parámetros estudiados. Estos datos sugieren la posible participación del sistema del complemento en el mecanismo del aborto espontáneo

Fibronectin binding protein and rheumatoid arthritis

Rheumatoid arthritis autoimmune disease, is thought to be caused by an interaction between constitutional and environmental [possibly microbial] factors. Mycobacterium tuberculosis is considered one of causative agent of Rheumatoid arthritis. The Fibronectin - binding protein [FnBp] antigens [30/31] kDa antigen] of M. tuberculosis is one of the major secretory molecules and is probably routinely recognized by the host immune system in the early stage of tuberculosis in infection serum immune complexes, prepared from Rheumatoid arthritis [RA], Tuberculosis [TB] patients and normals, were analyzed for the presence of FnBp by ELISA using CF8 [anti - FnBp monoclonal antibody] and by western blotting using Fibronectin - HRP. Significant differences were noted between normal and patient sera by using the monoclonal antibody XF8 [p < 0.001]. We have detected a 37 kDa Fibronectin - binding protein in RA patients' serum immune complexes, which are reported to play an important role in bacterial diseases. We have shown that the 30/31kDa FnBp is a characteristic feature of tuberculosis. This was a quite interesting finding that suggests there are different forms of Fibronectin - binding protein antigens probably involved in pathogenesis of RA

Role of circulating immune complex in aspirin-sensitive asthma

BACKGROUND & OBJECTIVES: The pathogenic mechanism of aspirin-sensitive asthma (ASA-BA) remains to be further defined. To evaluate the role of circulating immune complex (CIC) in ASA-BA. SUBJECTS & METHODS: We measured IgG- and IgA-IC level by ELISA using anti-C3 antibody in 33 ASA-BA patients whose sensitivity was confirmed by lysine-aspirin bronchoprovocation test, and compared with those of 14 allergic, 14 intrinsic asthma patients and 7 healthy controls. RESULTS: There was no significant difference in IgG-IC level among the four groups (p > 0.05), while IgA-IC levels of aspirin-sensitive asthma were higher than those of other groups (p = 0.0035). Patients with nasal polyp had significantly higher IgG-IC than those without it (p = 0.02). No differences were found according to medication and symptom scores, and presence of atopy, rhino-sinusitis, urticaria or concurrent sensitivity to sulfite (p > 0.05). Insignificant correlation was found between IgG-IC level and asthma duration, total IgE level, or circulating eosinophil count. CONCLUSION: These findings suggest a possible contribution of IgG-IC to the development of nasal polyp in ASA-BA. Further study will be needed to clarify the role of IgA-IC in the pathogenesis of ASA-BA.

Role of circulating immune complexes in prognostic evaluation and management of genitourinary cancer patients.

Circulating immune complexes (CIC) were estimated in 48 Patients of genitourinary cancer by polyethylene glycol precipitation (PEG pptn.) test and latex agglutination inhibition (LAI). The results were compared with 25 healthy control volunteers. Pathological levels of CIC were observed in 79.18 percent patients of genitourinary cancer by combination of PEG pptn. and LAI tests, while no seropositivity for CIC was observed in control group (p < 0.001). Sequential increase in seropositivity for CIC was observed with advancing stage of genitourinary cancer i.e. number of seropositive patients in cancer stage I were 60 percent, stage II-71.42 percent, stage III-85.71 percent and stage IV-100 percent. Variation IN CIC levels in different patients within the same stage are compared. Circulating antigen antibody complexes have a significant role as prognostic monitors in management of genitourinary cancer patients. Statistical evaluation of data on intra- and inter-assay variation has been given. CIC levels rise with increases in tumor burden in vivo hence variation in CIC levels within the same stage in different patient have a significant role as prognostic monitors in management of individual patients.

Fibronectin binding protein and rheumatiod arthritis

Rheumatoid arthritis autoimmune disease, is thought to be caused by an interaction between constitutional and environmental [possibly microbial] factors. Mycobacterium tuberculosis is considered one of the causative agent of Rheumatoid arthritis. The Fibronectin - binding protein [FnBp] antigens [30/31] kDa antigen] of M tuberculosis is one of the major secretory molecules and is probably routinely recognized by the host immune system in the early stage of tuberculosis infection. Serum immune complexes, prepared from Rheumatoid arthritis [RA], Tuberculosis [TB] patients and normals, were analyzed for the presence of FnBp by ELISA using CF8 [anti-FnBp monoclonal antibody] and by western blotting using Fibronectin - HRP. Significant differences were noted between normal and patient sera by using the monoclonal antibody CF8 p < 0.001]. We have detected a 37 kDa Fibronectin - binding protein in RA patients' serum immune complexes, which are reported to play an important role in bacterial disease. We have shown that the 30/31 kDa FaBp is a characteristic feature of tuberculosis. This was a quite interesting finding that suggests there are different forms of Fibronectin - binding protein antigens probably involved in pathogenesis of RA