To determine the levels of both TSA and HD antibody in sera of patients with various malignancies and evaluate their potential role as diagnostic and/ or prognostic markers.
Laboratory based ...analysis.
Kenyatta National Hospital, Kenya Medical Research Institute and the Department of Biochemistry, University of Nairobi.
A total of 909 serum samples, 420 from cancer patients recruited at Kenyatta National Hospital and 509 from normal blood donors recruited at Nairobi Hospital.
The mean age for the patients and controls was 36 and 37 years respectively. Carcinoma patients constituted 54%, sarcoma 12.1%, lymphoma 16.4% and 17.4% had other types of tumours. The mean TSA in patients was 0.86 mg/ml +/- 0.026 compared to 0.82 mg/ml +/- 0.014 in controls. The TSA level was significantly higher in patients compared to controls (Student's t-test p = 0.031 at 0.05 confidence level). The TSA increased with age in both study groups. In patient sera, both gender gave the same mean of 0.83 mg/ml while it was 0.82 mg/ml and 0.83 mg/ml in control females and in males respectively. Sarcomas had the highest amount of 0.93 mg/ml but there was no significant statistical variation between tumour types (p = 0.076). The HD antibody mean readings were 0.004 in pathologic sera compared to 0.011 in controls. The values were significantly elevated in patients (p = 0.03) with females giving a higher value for both study groups (p = 0.628). HD antibody readings was significantly higher in carcinomas (p = 0.017) compared to those of sarcomas and lymphomas. There was no association between antibody readings and age of patient (p = 0.601).
Both TSA and HD antibody values were significantly elevated in patients compared to clinically healthy controls and while TSA levels increased with age and was independent of gender, HD antibody levels were independent of age, gender and also tumour type. The study demonstrates that although TSA is normally elevated in malignancy, most of the sialic acid shed is of N-acetyl type as some patients do not express HD antibody directed to the N-glycolyl sialic acid. The reason why some tumours would express Neu5Gc at any one time needs further evaluation.
The activity of a free N-acetylneuraminic acid (Neu5Ac)-hydroxylating enzyme which converted Neu5Ac into N-glycolyl-neuraminic acid (Neu5Gc) was demonstrated in the soluble fraction of pig mandibular ...gland. The hydroxylation was possible only with NADPH as the electron donor. The apparent Km was 4.5 mM Neu5Ac. At 0.5 mM monovalent cations had no effect on the hydroxylation of Neu5Ac whereas bivalent cations gave varied inhibition capacities ranging from 14 to 75%. EDTA gave a time-dependent enhancement of activity. It was concluded that the enzyme does not require an exogenously added inorganic cofactor. Results from salt fractionation of the soluble fraction and the use of inhibitors such as mercurials suggested that the hydroxylation of Neu5Ac to Neu5Gc may involve other, as yet unknown, component(s) and the possibility of electrons donated by NADPH being transferred to activated molecular oxygen (second substrate). We propose to name this enzyme N-acetyl-neuraminic acid hydroxylase.
Recombinant human erythropoietin (rHUEPO) was produced by Chinese hamster ovary cells and commercially distributed to hospitals by two pharmaceutical companies in Japan ('ESPO' by Kirin Brewery Co. ...Ltd., and Sankyo Co. Ltd., and 'EPOGIN' by Chugai Pharmaceutical Co. Ltd.) These products contained about 1% N-glycolylneuraminic acid (Neu5Gc) in total sialic acid content. Since humans do not synthesize Neu5Gc, successive injection of Neu5Gc-containing products was feared to lead to allergic-like symptoms. Therefore, serum levels of antibodies of Neu5Gc epitope in 90 patients who received repeated i.v. injections of ESPO or EPOGIN were determined by an enzyme immunoassay using Neu5Gc alpha 2-3Gal beta 1-4Glc-Cer, GM3(Neu5Gc), as an antigen and compared with those in 100 healthy persons. Either no or low antibody levels were detected in both groups with no significant difference. In 40 patients who received s.c. injections of ESPO or EPOGIN, serum HD antibody levels were determined before and after weekly therapeutic injections carried out for one to several months, but no significant elevations were detected in all patients. The above results indicated that therapeutic administration of rHuEPO to patients to patients with chronic renal failure is safe from allergic-like side effects associated with the production of Neu5Gc-specific antibodies, and it was concluded that Neu5Gc epitope of rHuEPO is minimally antigenic in humans.
The specificities of five heterophile Hanganutziu and Deicher (HD) antibody-containing sera from four different cancer patients and one other diseased patients were compared. Three glycosphingolipids ...and one glycoprotein antigens and their chemically modified derivatives were used. The antibodies of all whole sera showed similar specificities. IgG and IgM antibody fractions of each serum were separated. Although antibodies of the same class showed similar specificities, differences were detected between the specificities of IgG and IgM. IgG antibody specificities were dependent on the hydrophobic (ceramide) group while IgM antibodies were directed more to the terminal sialic acid moiety of the glycosphingolipid antigens. The results suggested that a similar population of IgG-producing lymphocytes is stimulated in patients. Due to the similarities in specificities of HD antibodies, the results of this study will facilitate the future isolation of either IgG or IgM antibody-producing lymphocyte(s) from a patient with HD antibodies and the establishment of a monoclonal antibody through hybridization with a human myeloma cell line.
A specific, relatively sensitive, quantitative and standardized enzyme-linked immunosorbent assay (ELISA) procedure was developed for the detection of heterophile Hanganutziu and Deicher (HD) ...antibodies which are occasionally elevated in pathologic human sera. The HD antigen-active molecule used for the assay was a ganglioside (N-glycolylneuraminyllactosylceramide, abbreviated as NeuGc-LacCer) previously purified from horse erythrocyte membranes. The test used antigen-coated plastic microtiter plates and anti-human immunoglobulin G (IgG, Fab fragment) conjugated with alkaline phosphatase. Fifty-four normal human sera gave ELISA values ranging from -2 to 2%. Random sera from hospitalized patients were first screened by the horse erythrocyte hemagglutination (HA) test, whereby 5.7% (76 cases) gave abnormal HA titers of 128-4096 compared to titers in normal sera equal to or less than 64. Ninety-seven % of the patients' sera gave abnormal ELISA values (3-200%). They were classified into 3 groups: cancer (42 cases), infection (10 cases), and others (24 cases). The potential value of this ELISA method is discussed.
A relatively simple, specific and sensitive radioimmunoassay system has been developed for the detection of heterophile Hanganutziu-Deicher (H-D) antigen(s) and antibodies. The 125I-labeled H-D ...antigen-active molecule used for the assay is a bovine erythrocyte major glycoprotein previously found to have a strong H-D antigen potency. The antigen-antibody complex was precipitated with normal human serum as the carrier protein, followed by the addition of rabbit anti-human IgG F(ab')2 serum. With this method, different H-D antigen-active molecules were compared for heterophile H-D antigen potency with reasonable sensitivity detecting about 0.3 ng of cold glycoprotein. 8 different lung cancer tissues were assayed for H-D antigen. The sera from the 8 lung cancer patients were also screened by ELISA and RIA in an attempt to correlate expression of H-D antigen on tissues with elevation of H-D antibodies. The results showed that all patients' tissues expressed the antigen(s) but only 3 of them had abnormal levels of H-D antibodies. This could have been due to excess antigens in circulation or immune complexes.
A human Hanganutziu-Deicher (HD) antibody and a chicken anti-N-glycolylneuraminyllactosylceramide (HD3) antibody were compared in their reaction against HD antigen-active ganglioside (HD3) and a ...glycoprotein (GP) by radioimmunassay (RIA) . The human antibody had a 50 times higher reactivity with the glycoprotein, while the chicken antibody reacted equally with both antigens. Both antibodies had a higher reactivities with HD antigen (s) in sera of five of eight lung cancer patients than 54 normal human sera. Since four of the above five sera had no abnormal titers to GP, it was concluded that their immunological status was antigen excess. The chicken antibody may be useful in follow-up studies of cancer patients to correlate the expression of HD antigen in tissues and sera with the elevation of HD antibodies, offering alternative methods of clinical prognosis of tumor growth and/or metastases. The human HD antibody may also be useful for the detection of HD antigens of glycoprotein nature.
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