Evaluation of an enzyme-linked immunosorbent Raji cell assay (ELISA) in the investigation of gastrointestinal cancer

The levels of circulating immune complexes (CICs) have been estimated in a group of patients with colorectal cancer and gastric cancer, in addition to which a normal range has been established in a group of patients with benign gastrointestinal disease. A newly developed enzyme-linked immunosorbent Raji cell assay has been used in this study. Overall only 30% of patients with gastrointestinal cancer showed elevation of CIC levels outside the normal range. Elevated levels correlated with tumour differentiation bud did not correlate with site of disease or with the presence of metastases. In an attempt to define the specificity of CIC estimation, soluble tumour extract was added to sera from tumour-bearing patients. Specific IC elevations were produced by addition of allogeneic tumour extract of colon cancer in patients with colorectal cancer; this phenomenon was not seen when the same extract was added to the sera of patients with gastric cancer.     

Evaluation of an enzyme-linked immunosorbent Raji cell assay (ELISA) in the investigation of gastrointestinal cancer

Summary The levels of circulating immune complexes (CICs) have been estimated in a group of patients with colorectal cancer and gastric cancer, in addition to which a normal range has been established in a group of patients with benign gastrointestinal disease. A newly developed enzyme-linked immunosorbent Raji cell assay has been used in this study. Overall only 30% of patients with gastrointestinal cancer showed elevation of CIC levels outside the normal range. Elevated levels correlated with tumour differentiation bud did not correlate with site of disease or with the presence of metastases. In an attempt to define the specificity of CIC estimation, soluble tumour extract was added to sera from tumour-bearing patients. Specific IC elevations were produced by addition of allogeneic tumour extract of colon cancer in patients with colorectal cancer; this phenomenon was not seen when the same extract was added to the sera of patients with gastric cancer.     

Significance of detection of immune-complexed 8 kDa hydatid-specific antigen for immunodiagnosis of hydatidosis

Abstract Three micro-enzyme-linked immunosorbent assay (micro-ELISA) systems were developed and evaluated for detection of specific free circulating antigen and circulating immune-complexes (CICs) of 8 kDa antigen in the sera of patients with hydatidosis. All (100%) the sera of 30 confirmed positive cases of hydatidosis had detectable levels of antigen in the acid-treated sera. However, 23 (77%) and 26 (87%) sera of 30 confirmed cases had free as well as CICs of 8 kDa antigen in the untreated and in the polyethylene glycol (PEG) precipitated sera, respectively. None of the sera from other patients with parasitic infections or viral hepatitis had any detectable levels of 8 kDa antigen in the untreated, acid-treated or PEG-precipitated serum samples. The investigations, therefore, suggested that the demonstration of circulating antigen employing monospecific antibodies to affinity purified 8 kDa antigen in acid-treated sera is more efficient as compared to detection of free circulating antigen of CICs in the untreated or in the PEG-precipitated sera which could provide a specific immunodiagnostic tool for ongoing hydatid infection.     

Clinical importance of circulating immune complexes in human acute lymphoblastic leukemia

Summary A total of 122 sera from acute lymphoblastic leukemia (ALL) patients were analyzed for circulating immune complexes (CIC) by two methods: the ¹²⁵I-C₁q binding assay and the polyethylene glycol precipitation test (PEG). The results were correlated with induction, remission and relapse stages of the disease. Using the first method the levels of CIC in induction were 15.18±9.15, with 19/29 positive cases (65.50%), P<0.001 compared with controls. In the remission phase the levels were 9.02±5.62, 11/45 (24.49%) nonsignificant P value, and in relapse they were 16.14±11.17 28/48 (58.33%) P<0.001. The PEG precipitation test results were: 0.33±0.10, 8/22 (36.36%); 0.24±0.11, 10/48 (20.83%) and 0.28±0.10, 6/28 (21.42%), respectively. Thus the values of CIC as measured by PEG in the three clinical of phases ALL did not differ significantly from controls. This contrasts with results obtained by the radioiodinated C₁q binding assay, where the incidence of positive values was significantly higher in induction and in relapse and lower in the remission phase. These observations were extended in sequential vertical studies performed in a group of patients. These results suggest that raised CIC detected by the ¹²⁵I-C₁q method may reflect a progressive state in ALL and that quantitation of these immune complexes may provide an adequate biochemical marker for prognosis.     

Detection of circulating immune complexes by the enzyme-linked immunosorbent assay in sera from cattle infected with Fasciola hepatica

Polyethylene glycol-precipitated immune complexes (PIC) from the sera of 5 calves with Fasciola hepatica worm burdens ranging between 27 and 70 flukes were examined for parasite antigen content at 2, 4, 6, 8, 10, and 16 wk postinfection (PI) by the enzyme-linked immunosorbent assay (ELISA). Three assays were devised using an affinity-processed rabbit antibody to worm excretory/secretory (FhES) antigens. The PIC plate assay detected parasite antigen by adherence of anti-FhES antibody to PIC incubated overnight on ELISA plates, and tests were visualized using anti-rabbit peroxidase-linked antibody. The serum complex and PIC capture assay utilized the anti-FhES immunoglobulin as an antigen capture antibody linked to the solid phase. The attached complexes were then detected by the adhering bovine antibody, either soluble complexes in serum or as PIC. All assays showed circulating immune complex (CIC) values elevated at 6-8 wk PI, which generally coincided with increased host circulating antibody to FhES antigens. The greatest detection rate for all of the immune complex (IC) detection assays occurred with the PIC capture assay. It detected antigen in almost 90% of sera tested at 6 and 8 wk PI. Both the serum complex and PIC capture assay detected greatest amounts of CIC in those animals with the largest worm burdens, whereas the PIC plate assay showed no such trend. This study shows that F. hepatica antigen detection in CIC can be used to aid immunologic diagnosis of fascioliasis.     

Presence of antibodies specifically reacting with structural proteins of the mammary cancer virus among antibodies found in circulating immune complexes in patients with breast cancer

Circulating immune complexes were precipitated from breast cancer patients' sera using 2.5% polyethylene glycol. CIC isolated from 70 ml of sera from 15 patients were dissociated and immunoglobulin-containing fraction was prepared by chromatography on Sephadex G-200 column. The fraction contained IgG specific for MuMTV structural proteins, as revealed by ELISA. CIC preparations from 22 sera of breast cancer patients were digested with pepsin; Fab' fragment preparations were also analysed by ELISA, only one of them was MuMTV-specific. IgG and Fab' fragments isolated from CIC reacted specifically with MuMTV proteins, the reaction was not blocked by virus-free murine milk or other retroviruses (Ra-MuLV and MPMV).     

Circulating immune complexes and in vitro cell reactivity in paracoccidioidomycosis

The severest forms of paracoccidioidomycosis (Pcm) are associated with impaired cell-mediated immunity, a phenomenon that is reversible with therapy. It has been postulated that plasma factors could be responsible for such immune dysfunction. In this report, circulating immune complexes (CIC) were measured by the Raji cell radioimmunoassay (Raji) and by the¹²⁵I-C1q binding assay (C1q-BA) in sera from 14 patients with either active or inactive forms of Pcm and from 15 healthy controls. The C1q-B A revealed significantly elevated levels of CIC in the sera of all but one of the patients. Four of the 8 active (62%) and 2 of the 6 inactive (33%) patients had CIC levels significantly higher than the controls as determined by the Raji test. Significantly increased levels of CIC were detected only in the active patients by the Raji test. The serum of one of the patients, with a generalized infection and depressed lymphocyte responsiveness, was examined and found to contain a factor which depressed the in vitro proliferation of both homologous and normal lymphocytes. We also found that pre-culture of the patients' lymphocytes before stimulation restored their proliferative capacity, and IC were detectable in the culture supernatants. However, the subsequent addition of the patients' serum to such precultured cells did not reinduce the depression. It is suggested therefore, that the depression of T cell responses observed in Pcm is due to the presence of IC which may interact reversibly with the responding cells and/or activate a suppressor cell population whose activity is diminished by preculture.     

MicroRNA-199a targets CD44 to suppress the tumorigenicity and multidrug resistance of ovarian cancer-initiating cells

In ovarian cancer, CD44(+) /CD117(+) stem cells, also known as cancer-initiating cells (CICs), are highly proliferative, have a low degree of differentiation, and are resistant to chemotherapeutics. Therefore, the CD44(+) /CD117(+) subpopulation is thought to be an important target for novel therapeutic strategies. In this study, we investigated the role of microRNA-199a (miR-199a) in ovarian cancer stem cells. Luciferase reporter gene assays confirmed that miR-199a targets CD44 via an miR-199a-binding site in the 3'-UTR. CD44(+) /CD117(+) ovarian CICs were enriched from human primary ovarian tumor tissues and confirmed by flow cytometric sorting. miR-199a was cloned and transfected into ovarian CICs. CD44 mRNA and protein expression was significantly decreased in miR-199a-transfected ovarian CICs as compared with miR-199a mutant-transfected and untransfected cells. Cell cycle analysis, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide proliferation assays, the colony formation assay and the transwell migration assay indicated that miR-199a significantly affected cell cycle regulation and suppressed the proliferation and invasive capacity of ovarian CICs in?vitro. miR-199a significantly increased the chemosensitivity of ovarian CICs to cisplatin, pacitaxel, and adriamycin, and reduced mRNA expression of the multidrug resistance gene ABCG2 as compared with miR-199a mutant-transfected and untransfected cells. The expression of stemness markers was also significantly reduced in miR-199a-transfected CICs as compared with miR-199a mutant-transfected and untransfected ovarian cells. Furthermore, xenograft experiments confirmed that miR-199a suppressed the growth of xenograft tumors formed by ovarian CICs in?vivo. Thus, expression of endogenous mature miR-199a may prevent tumorigenesis in human ovarian cancer by regulating expression of its target gene CD44.     

Detection of circulating immune complexes in the sera of dogs infected with Dirofilaria immitis, by Clq-binding enzyme-linked immunosorbent assay

The presence of circulating immune complexes (CIC) in the sera of dogs infected with Dirofilaria immitis was detected by using a Clq-binding enzyme-linked immunosorbent assay. Specificity of this assay with different concentrations of heat-aggravated canine IgG (ACG) was observed, i.e., the ELISA readings, expressed as microgram equivalents ACG/ml, increased with increasing amounts of ACG. The intra-assay variability was below 10%. The CIC levels of infected and uninfected dogs were 177.0 +/- 104.7 micrograms/ml and 22.8 +/- 45.8 micrograms/ml (mean +/- SD), respectively. The highest level was observed in 12 dogs with amicrofilaraemic infection. Age distribution of CIC levels in the 23 infected dogs also showed a significant positive correlation. These findings suggested that the CIC are present in the sera of dogs with dirofilariasis and may relate to canine glomerulonephritis.     

Analysis of circulating immune complexes (CIC) in tuberculosis: levels of specific antibody and antigens in CIC and relationship with serum antibody

Eighty sera from tuberculosis (TB) patients, 16 Indian and 10 American control sera were analyzed by ELISA for relative titres of antibody against mycobacterial antigens. Levels of specific antibody and mycobacterial Ag in circulating immune complexes (CIC) isolated from these sera were also studied. All these parameters were found to be elevated in TB sera as compared to control sera. Maximum increase was however noted in CIC specific antibody titres. A good correlation was observed between serum and CIC levels of specific antibody (r = 0.72) and between specific antigen (Ag) and antibody (Ab) levels within CIC (r = 0.64). In a few of the TB sera examined, CIC specific Ab contributed less than 1% to the Ab titres in sera. In order to examine the differences between different subgroups within TB patients, a statistical analysis of variance was performed. Sex of the patients had no effect on any parameter. Sputum-positive patients had significantly higher levels of CIC Ag and Ab than the sputum-negative patients, although no significant difference occurred in respect to serum Ab. All three parameters were significantly higher in patients on chemotherapy as compared to fresh untreated cases. The relevance of these observations to the development of a CIC-based immunodiagnostic assay for TB is discussed.     

Free and bound Brucella antigen and the level of circulating immune complexes in brucellosis patients

The aggregate hemagglutination test has been shown to be a highly sensitive and specific method for the detection of infectious antigenemia in different forms of brucellosis, permitting the determination of free Brucella antigen in 34% of patients with the acute form of the disease and in 53% of patients with the chronic course of brucellosis. The results of the determination of the quantitative level of circulating immune complexes (CIC) indicate that in the acute form of the disease their level exceeds the CIC level observed in the chronic course of brucellosis. The preliminary dissociation of CIC in the patients' blood sera has been found to increase the overall release of Brucella antigen to 80%.     

Hanganutziu-Deicher antigen and antibody in pathologic sera and tissues.

Heterophile, Hanganutziu-Deicher (H-D) antigen was studied in pathologic sera by means of inhibition of agglutination of bovine erythrocytes by H-D antibodies. H-D antigen was demonstrated in 38% of random cancer sera, 25% of lymphoma or leukemia sera, 25% of leprosy sera, 8% of infectious mononucleosis sera, 6% of rheumatoid arthritis sera, and 27% of synovial fluids of rheumatoid arthritis patients. None of 134 normal human sera gave positive results. Some of the inhibition-positive cancer sera formed precipitation lines with H-D antibody-containing sera. Over 50% of various extracts of cancer tissues as well as spleens of lymphoma or leukemia patients were shown to contain H-D antigen by means of the inhibition test.     

Disruption of DNA synthesis and structure of mouse L1210 leukemia cells, sensitive and resistant to 1-methyl-1 nitrosourea and 1,3-bis(2-chloroethyl)-1-nitrosourea in vivo

Leukemia L1210 cells with acquired resistance to 1-methyl-1-nitrosourea (MNU) (L1210/MNU) and 1.3-bis(2-chloroethyl)-1-nitrosourea (BCNU) (L1210/BCNU) were developed from leukemia L1210 cells sensitive to these drugs (L1210/0). The modal chromosome number of leukemia L1210/MNU and L1210/BCNU cells increases from 40 (L1210/0) to 41. It was shown that in leukemia L1210/MNU cells the inhibition of DNA synthesis after MNU administration in a therapeutic dose (80 mg/kg) is lasted within 24 hours, while that in leukemia L1210/0 cell--within 96 hours. After administration of BCNU (20 mg/kg) inhibition of DNA synthesis in leukemia L1210/BCNU cells reached of 50% of control in comparison with practically complete inhibition of DNA synthesis in leukemia L1210/0 cells. Centrifugation on alkaline sucrose density gradients revealed no differences in the rate of sedimentation of leukemia L1210/0, L1210/MNU and L1210/BCNU cell lysates. After 1 hour treatment with MNU of mice bearing L1210/MNU and L1210/0 leukemia cells single-strand breaks in DNA were determined. After 4 hours these strand-breaks retained in leukemia L1210/0 cells, but were eliminated in leukemia L1210/MNU cells. Administration of BCNU to mice with leukemia L1210/0 and L1210/BCNU cells resulted in both cases in the production of DNA aggregates. There is no complete cross-resistance between MNU and BCNU which allows a substitution of these drugs providing for the increase in their therapeutic efficiency.     

In situ photoaffinity labeling of proteasome with photoactive adriamycin analogue

An intracellular adriamycin (ADM)-binding protein purified from the cytosol of L1210 mouse lymphocytic leukemia cells had a molecular weight of 700-1500 kDa and hydrolyzed Suc-LLVY-MCA. When L1210 cells were incubated with a photoactive ADM analogue, N-(p-azidobenzoyl)-adriamycin (NAB-ADM), most of the NAB-ADM was found to localize in the nuclei. In situ photoaffinity labeling of L1210 cells with NAB-ADM resulted in low protease activity in the cytosol and nuclear extracts and the cells showed selective photoincorporation of NAB-ADM into the proteasome. These results suggest that the proteasome is a translocator of ADM from the cytoplasm to the nucleus and might therefore become a new candidate for cancer chemotherapy.     

Development of enzyme-linked immunosorbent assay for detection of Mycoplasma pneumoniae antigens

The variant of enzyme-linked immunosorbent assay (ELISA) for detection of Mycoplasma pneumoniae (Mp) antigens in sera of patients with respiratory infections was developed. Sensitivity of detection of soluble antigens of Mp in modeling experiment varied from 1.5 to 1.0 ng/ml (on protein). Approbation of the assay was performed using 50 serum samples obtained from patients with confirmed diagnosis of respiratory mycoplasmosis. In the ELISA test Mp antigens were detected in 96% of samples. Obtained results were confirmed by testing of these serum samples and isolated from them circulating immune complexes (CICs) in immunoblotting using polyclonal antibodies labeled by horse-radish peroxidase. Mp antigenswere detected both in free state and as components of CICs. Specific reaction was observed with proteins, which molecular mass varied from 30 to 170 kDa (30, 37, 45, 56, 58, 72, 90, 130 and 170 (160) kDa). Obtained results point to appropriateness of use of developed assay for detection Mp antigens in sera of patients with respiratory infections.     

Differential remodeling of extracellular matrices by breast cancer initiating cells

Cancer initiating cells (CICs) have been the focus of recent anti‐cancer therapies, exhibiting strong invasion capability via potentially enhanced ability to remodel extracellular matrices (ECM). We have identified CICs in a human breast cancer cell line, MX‐1, and developed a xenograft model in SCID mice. We investigated the CICs' matrix‐remodeling effects using Second Harmonic Generation (SHG) microscopy to identify potential phenotypic signatures of the CIC‐rich tumors. The isolated CICs exhibit higher proliferation, drug efflux and drug resistant properties in vitro; were more tumorigenic than non‐CICs, resulting in more and larger tumors in the xenograft model. The CIC‐rich tumors have less collagen in the tumor interior than in the CIC‐poor tumors supporting the idea that the CICs can remodel the collagen more effectively. The collagen fibers were preferentially aligned perpendicular to the CIC‐rich tumor boundary while parallel to the CIC‐poor tumor boundary suggesting more invasive behavior of the CIC‐rich tumors. These findings would provide potential translational values in quantifying and monitoring CIC‐rich tumors in future anti‐cancer therapies.

CIC‐rich tumors remodel the collagen matrix more than CIC‐poor tumors.     

Circulating immune complexes in cynomolgus macaques

Nonhuman primates can be used as models for the study of immune-complex-associated diseases. Recognizing that very little is known about the levels of circulating immune complexes (CICs) in normal monkeys, we have used three assays to measure the levels in serum collected from 313 adult and 106 juvenile cynomolgus macaques (Macaca fascicularis). The prevalence was higher than expected. There was a strong statistical association between CIC levels and country of origin. Monkeys from Indonesia were more likely to have elevated CICs than those from Malaya or the Philippines. This relationship was observed with all three assays. Furthermore, juvenile macaques tended to have lower levels than did adults. This study indicates that it may be important to consider genetic factors, the country of origin, or both when selecting cynomolgus macaques for research on immune-complex-associated diseases.     

A 125I-protein A-binding assay detecting antibodies to cell surface antigens

A 125I-protein A-binding assay detecting antibodies to cell surface antigens on human blood cells was developed and evaluated using sera from multitransfused nonleukemic patients sensitized against HLA antigens. The binding assay was found to be reproducible and more sensitive than conventional HLA testing. Seven patients with acute myelogenous leukemia and two patients with acute lymphoblastic leukemia successfully treated by chemotherapy were then investigated. Sera from seven of the patients studied in partial or complete remission demonstrated significant binding to autochthonous leukemic cells obtained from bone marrow or peripheral blood. In two cases sera taken during the leukemic stage demonstrated the most pronounced binding to the patients' own leukemic cells. Sera from four patients with demonstrable significant binding to autochthonous leukemic cells failed to bind to autochthonous remission cells when both types of target cells were tested in parallel. Differences in serum concentrations of IgG, IgA, and IgM were not the cause of the demonstrated increased binding of leukemic sera to autochthonous target cells. We propose that the 125I-protein A-binding assay presented in this paper detects antibodies reacting selectively with acute leukemia cells.     

The role of N-nitrosourea-induced changes in the nucleoid structure and activity of repair enzymes in the development of drug resistance in mice with leukemia L1210

Using centrifugation of the nucleoid in a neutral sucrose gradient, the damages in the secondary structure of DNA and the activity of repair enzymes, such as DNA-polymerases alpha and beta and poly(ADP-riboso) polymerase, induced by 1-methyl-nitrosourea (MNU) and 1.3-bis (2-chloroethyl)-1-nitrosourea (BCNU) injected at maximal nonlethal single doses to mice bearing parent leukemia cells (L1210/0) and resistant to MNU and BCNU leukemia L1210 cells (L1210/MNU and L1210/BCNU), were studied. The MNU-induced production of single-strand breaks in L1210/0 and L1210/MNU cells was more conspicuous in newly replicated DNA than in those in preexisting DNA. A more fast repair of the damages in newly replicated DNA was detected in L1210/BCNU and especially in L1210/MNU leukemia cells as compared with L1210/0 cells. The data obtained suggest that there are prone errors in the repair of DNA template, since most of the single-strand breaks were revealed in the newly replicated DNA synthesized on the repaired DNA. The repair of DNA damages in L1210/BCNU and especially in L1210/MNU cells was accompanied by the activation of DNA-polymerases alpha and beta and poly(ADP-riboso)polymerase. Both DNA-polymerases--alpha and beta--were shown to be involved in repair of DNA damages induced by MNU and only DNA-polymerase beta was involved in the repair of damages induced by BCNU.