Purification of soluble hepatocellular carcinoma extracts.

When soluble hepatocellular carcinoma (HCC) extract prepared by 3 M KCl solution was passed through a column of Sephadex G-200, four fractions (designated as I, II, III, IV) could be obtained from the elution profile. By leukocyte migration inhibition assay, the active component of soluble HCC extract which represented tumor-associated antigen was shown to be contained in fractions I and II.     

Role of pure and biologically active amoebal RNA in assessment of lymphokines: a report of 55 ALA cases.

Amoebic liver abscess cases (55) were assessed for release of lymphokines (LMIF) using pure and biologically active amoebal RNA of axenic Entamoeba histolytica (NIH: 200) obtained with cesium chloride centrifugation. Lymphokines released by T lymphocytes in response to both amoebal RNA and whole amoebic lysate (WAL) were tested by leukocyte migration inhibition test (LMIT) on blood samples from amoebic liver abscess cases. A significant increase was observed in the release of lymphokine and 100% positivity was observed with amoebal RNA compared to whole amoebic extract with a positivity of only 78%. The difference between means leukocyte migration inhibition of the above two with regards to release of lymphokine was highly significant (P less than 0.001). This shows that patients had high degree of leukocyte sensitization to amoebal RNA of E. histolytica compared to whole amoebic lysate. These findings suggest that the amoebal RNA plays an important role as a potent antigen in the elicitation of cell mediated immune responses in amoebic liver abscess cases.     

Cellular hypersensitivity to human pancreatic B-cell clone in diabetes mellitus and its relationship to the presence of islet cell antibodies

A leukocyte migration inhibition test on the human pancreatic B-cell clone (JHPI-1) was performed in 13 IDDM patients with islet cell cytoplasmic antibody (ICCA) and/or islet cell surface antibody (ICSA), 15 IDDM patients without ICCA or ICSA, 34 NIDDM patients and 17 healthy controls. The mean values for the migration index (M.I. %) in each group were 85.4 +/- 6.9, 89.1 +/- 10.9, 98.3 +/- 7.9 and 100.0 +/- 8.5. The M.I. values were significantly decreased in IDDM patients than in NIDDM patients and controls irrespective of whether or not there were islet cell antibodies in the patients' sera. When M.I. values less than 0.83 (Mean-2 S.D.) were taken as indicative of inhibition, the percentage of IDDM and NIDDM patients with migration inhibition were 32% and 0% respectively. And the decreased M.I. values in IDDM patients proved not to be due to non-specific migration inhibition by normal M.I. values, with the human fetal lung fibroblast cells (W 138) as antigen. Our data suggested that the lymphocytes of IDDM patients might be sensitized by pancreatic B-cell antigen(s) present in the JHPI-1 cells, which promoted leukocyte migration inhibition. No correlation between the migration indices and duration of diabetes mellitus in IDDM patients was observed (r = 0.254, Y = 84.9 + 0.49 X). LMT to JHPI-1 seems to be useful in detecting the abnormal cell-mediated immunity even in patients with longstanding IDDM.     

Leukocyte migration inhibition and leukocyte adherence inhibition (LMI- and LAI-assay) in cancer patients by using preparations from human fetuses]

The sensitization of lymphocytes from patients with different tumors was tested against a 3 M KCl-extract of fetal tissue (3.--6. month) by the leukocyte adherence inhibition assay (LAI) and by the leukocyte migration inhibition assay (LMI). Sensitization was compared with the reactivity of controls without any detectable tumor. In the LAI assay the leukocytes of 13/15 patients and 2/12 controls showed an inhibition of the adherence. In the LMI-assay 11/17 tumor-bearing patients and 6/18 controls reacted positive in the presence of the antigen preparation. The two methods demonstrated that patients bearing tumors of different histology are sensitized to fetal antigens.     

Radiation leukemia virus-transformed immunocompetent T cells. II. Antigen-induced macrophage migration inhibition factor and leukocyte migration inhibition factor production

OVA-specific T cells were immortalized by infection with radiation leukemia virus (RadLV). Some clones derived from such population were shown to exhibit helper activity. We then tested clones without such function and found among them some that secreted macrophage migration inhibition factor (MIF) and leukocyte migration inhibition factor (LIF) upon exposure to the antigen in vitro. The lymphokine-producing clones, which were Thy-1+, Ly-1+ and Ly-2-, did not secrete MIF and LIF constitutively. Like other antigen-specific T cells, the immortalized clones could not be stimulated by free soluble antigen but required macrophages for presentation and for triggering the lymphokine production. The antigen-activated clones exclusively produced MIF and LIF, but not interleukin 2 or colony-stimulating factor. They neither provided helper activity nor induced delayed-type hypersensitivity. The data suggest that the T-cell clones carry the antigen receptors and that their antigen-inducible biological function is restricted to the migration inhibitory factor production.     

Detergent dissection of membrane proteins of Entamoeba histolytica and its effect on lymphokine release in in vitro.

Fifty-two amoebic liver abscess cases were assessed for the release of lymphokines (LMIF) using detergent dissected membrane proteins (DDMP) of axenic Entamoeba histolytica (NIH:200) obtained with sodium deoxycholate treatment. Lymphokines release by T lymphocytes in response to both DDMP and whole amoebic lysate (WAL) was tested by leukocyte migration inhibition test on blood samples from amoebic liver abscess cases. A significant increase was noted in the release of LMIF and 100% positivity was observed with DDMP compared to whole amoebic extract with a positivity of 73%. The difference between means of the above two with regards to release of LMIF was found to be highly significant (P less than 0.005). This shows the patients had high degree of leukocyte sensitization to surface antigens of E. histolytica compared to the whole amoebic lysate. These findings suggest that the antigens shed might have important role as a potent antigen in elicitation of CMI response in amoebic liver abscess cases.     

Leukocyte-migration inhibition in guinea pigs. I. Correlation with skin test reactivity and macrophage-migration inhibition.

Fifteen guinea pigs, immunized with one of three soluble antigens, repeatedly demonstrated inhibition of leukocyte migration and positive skin tests to the immunizing antigens. An additional five animals immunized with ovalbumin demonstrated inhibition of macrophage migration as well as direct and indirect inhibiton of leukocyte migration. Only one of fifteen animals demonstrated inhibition of leukocyte migration and none had a positive skin test with an antigen to which it had not been sensitized, indicating that the assay is antigen specific.     

Leukocyte migration inhibition reaction in trophoblastic tumor patients]

Inhibition of leukocyte migration was studied in 40 patients with trophoblastic tumours under the effect of protein extract from chorionepithelioma. A marked inhibition of leukocyte migration was revealed in patients with signs of active tumour process before and during the treatment. Four patients in whom no inhibition was revealed either before or during the treatment were an exception. In the majority of the patients the inhibition effect was absent against the background of clinical well-being achieved as a result of surgical and chemotherapeutic, or chemotherapeutic treatment alone. Leukocytes of healthy donors failed to respond to the tumour extract in all 24 cases.     

Effect of serum fractions from cancer patients on leukocyte migration out of capillary tubes]

Blood serum of patients suffering from cancer of the stomach and urinary bladder inhibited in vitro migration of autologous leukocytes, leukocytes of donors and control patients, and also guinea pig macrophages in over half of cases. In chromatography of these sera on Sephadex G-100 the activity inhibiting the leukocyte migration was revealed in fraction I (Mol. wt. over 100000) and in fractions IV and V (Mol. wt under 30 000). The blood serum and its fractions from cancer patients failed to eliminate the leukocyte migration inhibition caused by the tumour antigens in comparison with the leukocyte migration in the medium with control serum without any antigens. As suggested, the activity of fraction I inhibiting the leukocyte migration was due to the antigen-antibody complex, and of fraction IV and V--to a factor similar by its properties to the factor produced in vitro by lymphocytes stimulated by the antigens or mitogens.     

The leucocyte migration inhibition test and subpopulations of peripheral lymphocytes in insulin-dependent diabetics.

The leucocyte migration inhibition test (LMT) was performed by the agarose plate method with thyroid and pancreatic antigens in patients with insulin-dependent or independent diabetes mellitus. The mean migration indices with thyroglobulin, thyroid mitochondria and beef insulin were not significantly different in insulin-dependent diabetics from those in insulin-independent diabetics or normal controls. However, significant inhibition of leucocyte migration was observed in insulin-dependent diabetics when thyroid microsome or pancreatic extract was used as antigen. Although no significant difference was found in the percentages of T and B lymphocytes between insulin-dependent diabetics and insulin-independent diabetics or normal controls, the results of LMT strongly suggest the presence of cellular immunity against the thyroid and pancreas in insulin-dependent juvenile-onset diabetes.     

Comparative diagnostic efficacy of serum squamous cell carcinoma antigen in hepatocellular carcinoma

ABSTRACT: Hepatocellular carcinoma (HCC) is a common liver malignancy in Nigeria. Hepatitis B and C viruses, alcohol and Aflatoxin B are among the various aetiology. More work needs to be done in the search for markers that will aid early detection of this condition as it is uniformly fatal once advanced. Alphafetoprotein (AFP) remains the most widely used tumour marker of HCC detection in spite of its known shortcomings. The objective of this study was to determine the efficacy of serum squamous cell carcinoma antigen (SCCA) , in comparison to alphafetoprotein in the detection of HCC. METHOD: Sixty patients with HCC and thirty apparently healthy controls attending the Medical Outpatient Department(MOPD) of the University College Hospital Ibadan(UCH) Nigeria were selected for the study. Questionnaire was used to collect clinical data while AFP, SCCA levels,serum HBsAg and anti-HCV were determined using ELISA method- ( Diagnostic Automation Inc. Canada),Abdominal ultrasound scan was also done. Result:Thirty one(51.7%) out of 60 selected cases were positive for HBsAg while six(20%) out of 30 controls were positive for HBsAg(p= 0.004) .Out of the 60 cases selected for this study only 2 (3.3.%) cases were positive for hepatitis C virus, while only 1(3.3%) out of 30 control was positive for hepatitis C virus(p= 0.74). The mean AFP value for cases with HCC was 393.21ng/ml +/-386.97 compared to the control group which was 5.60 +/- 13.03 ng/ml (P value 0.001).The mean SCCA level was 0.64 +/- 0.56ng/ml and 0.71+/-0.65ng/ml for cases and controls respectively (p=0.631) CONCLUSION: Alphafetoprotein remains a good tumour marker for the diagnosis of HCC. Serum squamous cell carcinoma antigen(SCCA) has no discriminatory power and may not be useful as a tumour marker for Nigerians with hepatocellular carcinoma.     

Migration inhibition test with thyroid membrane antigen in Graves-Basedow disease and nodular goiter]

The evaluation of the migration inhibition test with thyroid membrane antigen was carried out in 20 patients with Graves' disease, 13 patients with neutral nodular goiter and 13 healthy subjects. A significant inhibition of migration by thyroid antigen as compared to the control group was demonstrated only in the patients with Graves' disease (the value of the migration index was 0.57 +/- 0.07). The respective value was 0.79 +/- 0.16 in patients with nodular goiter and 0.85 +/- 0.11 in healthy subjects. The results obtained indicate the practical value of the migration inhibition test in diagnosing the thyroid diseases having an autoimmune background.     

Tumour-associated antigens in culture medium of malignant melanoma cell strains

Summary The presence of melanoma-associated antigens naturally shed from cultured melanoma cells in spent culture medium was investigated by means of a leukocyte migration test and culture medium from four melanoma and two control cell strains.Leukocytes from 29/64 melanoma patients showed a positive reaction with spent culture medium from at least one melanoma cell strain, whereas leukocytes from only 4/25 patients with other cancers and 1/30 normal donors reacted. On the other hand, leukocytes from only 8/51 melanoma patients reacted with control culture medium. Only melanoma patients' leukocytes reacted with two or more of the melanoma cell strains used. Culture media from two melanoma cell strains were more reactive (25.3% and 29.4% positive tests with melanoma patients' leukocytes) than others (12.5% and 17.2% positive tests); this may represent either a qualitative difference (i.e., different antigens) or a quantitative one (i.e., different levels of antigen expression according to tissue culture conditions). Both inhibition and stimulation of migration were observed, but with one exception, on a given occasion, leukocytes from the same donor always reacted in the same way (i.e., either inhibition or stimulation). Migration stimulation was observed mainly with melanoma patients' leukocytes, and more especially when leukocytes were sampled from patients within a few weeks from tumour removal; migration stimulation may thus reflect a particular state of sensitization in patients.From the evidence obtained in these studies, it is concluded that spent culture medium from melanoma cell strains contains melanoma-associated antigen (s) that is (are) reactive in the leukocyte migration test and that this may contribute to the study of specific antitumour reactivity in patients and to the study and purification of tumour-associated antigens by providing an homogeneous source of antigens spontaneously released from tumour cells in conditions close to natural ones.     

Immunocytochemical investigation of hepatitis B virus-associated antigens in cases of liver cirrhosis and HBsAg antigenemia and their relationship to development of hepatocellular carcinoma

Summary Using light and ultrastructural immunoperoxidase techniques, we examined the distribution of hepatitis B virus (HBV)-associated antigens and the subcellular localization of hepatitis B surface antigen (HBsAg) in liver biopsies of HBsAg—positive patients with cirrhosis. The localization patterns of HBsAg in hepatocytes were membranous, cytoplasmic, festoon and inclusion body types. Cytoplasmic and festoon types were seen more often than the membranous type in pseudolobules, and hepatitis B core antigen (HBcAg)—positive cells with cytoplasmic type were distributed in the periphery of pseudolobules with active inflammation. Immunoelectron microscopy in the cytoplasmic or festoon type of HBsAg showed immunoreaction in the cisternae and on virus-like particles in the cisternae in patients with hepatitis B e antigen (HBeAg) antigenemia. Simultaneous staining of HBsAg and HBcAg revealed that hepatocytes with cytoplasmic or festoon type of HBsAg contained HBcAg—immunoreactivity. The inclusion body type of HBsAg was characteristic of liver cirrhosis with hepatocellular carcinoma (HCC); the subcellular localization of HBsAg was seen in clusters of the endoplasmic reticulum around the nucleus, and HBsAg—immunoreactivity was observed on many virus-like particles in most of the cisternae in those with HBeAg antigenemia. These findings suggest that the synthesis of HBsAg is active in patients with liver cirrhosis and that the formation of HBV is also active in those with HBeAg antigenemia and that HBV may be retained more in cirrhotic livers with hepatocellular carcinoma after proliferation than in those without it.     

Hepatitis B virus surface and core antigens in the liver of primary hepatocellular carcinoma cases in Virginia

Zenker-fixed paraffin-embedded sections of biopsy liver tissue from 64 cases of primary hepatocellular carcinoma (PHC) were stained for hepatitis B surface antigen (HBsAg) and for hepatitis B core antigen (HBcAg) by histochemical and/or immunohistochemical techniques in a retrospective study. PHC arose in livers with postnecrotic cirrhosis in 30 (46.9%) cases. Controls included liver biopsy sections from 123 miscellaneous liver disorders and from 67 randomly selected autopsy specimens, none of which were known to be associated with hepatitis B virus (HBV) infection. HBsAg was detected in tumorous hepatocytes in only one of the 64 cases of PHC. HBsAg was identified in nontumorous hepatocytes of 8 (20%) of 40 specimens that contained adequate nontumorous liver tissue. All of these HBsAg positive cases of PHC were associated with cirrhosis. Thus HBsAg was detected in 8 (33.3%) of 24 cases of PHC with cirrhosis, but in none of the remaining 16 cases without cirrhosis. HBcAg was not detected in the hepatocytes of those HBsAg positive PHC cases tested. Our results suggest that HBV infection may successively lead to chronic hepatitis, cirrhosis and ultimately PHC.     

Cross-reactivity of lung cancer patients' leucocytes in the leucocyte migration inhibition test

Summary Panels of 3 M KCl extracts of squamous-cell carcinomas, adenocarcinomas and oat-cell carcinomas of the lung were used for a comprehensive analysis of cross-reactivity in the leucocyte migration test. Lung cancer patients' leucocytes showed positive reactivity in 69%–100% of cases (n=353). No significant differences were observed when data were grouped with respect to the histological type of the tumours used for extraction or of the tumours of the leukocyte donors. Leukocytes of patients bearing tumours of nonpulmonary origin exposed to lung cancer extract panels and leukocytes of lung cancer patients exposed to gastrointestinal cancer extract panels were definitely less reactive (35%–47% and 6%–38%, respectively). However, a high reaction frequency was found in patients with lung metastases from different nonpulmonary tumours. This group of patients also frequently showed reactivity (52%) with normal lung tissue extracts. Patients with benign lung diseases reacted positively with lung tumour extracts in 25%–39% of cases, but donors with other benign disease and healthy controls were virtually nonreactive (0–14%).Hence, a high degree of cross-reactivity occurs in the lung cancer system and restricted cross-reactivity occurs with tumours of other organs. Possible explanations for the lung-oriented reactivity of patients with lung metastases are discussed.Abbreviations LMI leucocyte migration inhibition - MI migration index - LMT leucocyte migration test - SCC squamous-cell carcinoma - OCC oat-cell carcinoma - AC adenocarcinoma     

Immunohistochemical study of the appearance of some markers in liver adjoining hepatocellular carcinoma

The presence and distribution of AFP, AAT and HBsAg in peritumoral non-neoplastic hepatocytes (NNH) of 27 cases and, at the same time, in the neoplastic tissue of 37 liver cell carcinoma (HCC) were studied; AFP and HBsAg were more frequently found in NNH than in HCC cells; no differences were found for AAT. The presence of HBsAg also in normal liver without cirrhosis is probably best explained by its possible role in neoplastic transformation and by the inhibition of replication of the viruses AFP, considered to be expression of dedifferentiated cells, may possible be taken up by NNH for catabolic purposes.     

Interrelationships in experiments in vitro between the migration activity of leukocytes and RNA and DNA synthesis]

The following correlations were revealed in the parallel study of leukocyte migration in vitro in the presence of a specific antigen and of spontaneous RNA and DNA synthesis in the cultured lymphocytes: 1) a direct correlation between the RNA and DNA synthesis in lymphocytes; 2) a close correlation between the antigen-induced migration and the levels of RNA and DNA synthesis. The effect of the antigen was evidenced by the inhibition or stimulation of leukocyte migration. A high ratio of RNA synthesis to DNA synthesis corresponded to the migration inhibition and a low one--to the migration stimulation. The ratio value varied mainly on account of the changes in the level of DNA synthesis. Participation of T and B cells in the regulation of the antigen-induced leukocyte mobility is discussed.     

Importance of markers of hepatitis B virus in alcoholic liver disease.

To determine the importance of the presence of serological markers of hepatitis B virus infection in patients with alcohol related liver disease we compared cumulative alcohol intake and clinical and histological features in patients with markers of hepatitis B virus infection and in those without. Hepatitis B surface antigen (HBsAg) was detected in five (2%) out of 285 patients studied and antibody to HBsAg (anti-HBs) in 41 (14%); one patient had antibody to hepatitis B core antigen alone. The combined prevalence of markers of hepatitis B virus infection was similar in patients with alcoholic cirrhosis (18%) and precirrhotic liver disease (13%). Two patients positive for HBsAg had histological features of both alcoholic liver disease and chronic active hepatitis, with stainable HBsAg. Patients with anti-HBs were, however, histologically indistinguishable from patients without markers, and the mean cumulative alcohol intake of patients with anti-HBs was similar to or even higher than that of patients with liver disease of comparable severity who had no evidence of previous infection. The presence of markers of hepatitis B virus infection was related to former residence in countries with a high prevalence of the infection and to previous parenteral treatment and blood transfusions. Infection with hepatitis B virus does not enhance the development of chronic liver disease in heavy drinkers, except in the small number who remain positive for HBsAg.     

Retrospective review of the prevalence of hepatitis B virus in several population groups

Nine different groups of individuals studied from 1969 to 1985 were tested for Hepatitis B Virus (HBV) markers. In 8 groups only HBsAg in serum was tested, in another group: tissular HBsAg, and in two of those groups: serum HBsAg, anti-HBs and anti-HBc. Mean HBsAg prevalence in groups similar to general population was 0.64%; 5% in cirrhotics; HBV prevalence in haemophiliacs was 18.87% by testing serum for HBsAg and anti-HBs; serum HBsAg prevalence in Viral Chronic Active Hepatitis was 43.24%; and Hepatocellular Cancer (HCC) group had a prevalence for HBV of 13.04% when only tissular HBsAg was tested, and 54.29% when serum HBsAg, anti-HBs and anti-HBc were tested in all patients. Costa Rica has a low HBV markers prevalence only similar to what is found in industrial developed countries.