Complement-mediated binding of immunoglobulins to some endocrine cells of the pancreas and gut.

Unspecific binding of immunoglobulins to gastrin, somatostain and glucagon cells of the gastrointestinal mucosa or pancreas has been found to occur through a nonantigen-antibody mechanism mediated by the C14 fraction of complement. The phenomenon represents an important drawback in hormone immunohistochemistry, which can be overcome by using complement deprived, highly dilute anti-hormone sera.

[Cimetidine in the treatment of peptic ulcer]. / La cimetidina nella terapia dell'ulcera peptica.

Cimetidine, a non-thiourea-containing histamine H2-receptor antagonist, has been employed in the treatment of peptic ulcer. We studied 50 patients, partly with double-blind method, and partly in a free trial. Duodenal ulcers were completely healed in 80% of the cases, at the control endoscopy after a 4-week period of treatment with cimetidine (1000 mg/day). Peptic ulcer in general healed in 76% of the cases. Gastric secretion was significantly reduced after cimetidine. An important improvement in subjective symptoms was noted in every patient. No toxic effect was noted.

G cell distribution in normal duodenal mucosa.

G cell presence in the fundus, antrum and duodenum (bulbar, descending and horizontal parts) was investigated by an indirect immunofluorescenc technique in six asymptomatic male subjects. All subjects had normal endoscopic and histological patterns normal gastric acid secretion and normal serum gastrin levels. The results confirm the presence of G cells in the antral mucosa and the three studied duodenal portions. The G cell number of each duodenal portion was significantly lower than the antral one (P less than 0.0005). No difference could be seen between the second and third duodenal portions while each of them had a significantly lower G cell number, when compared with the bulb.

The distribution of duodenal G cells in duodenal ulcer patients.

The number of G cells is evaluated in biopsy specimens of fundic, antral and duodenal mucosa from the bulb, second and third parts in 10 patients with duodenal ulcer, and compared with that observed in 6 normal controls. G cells are absent in fundic mucosa but in the antrum their number in duodenal ulcer patients does not differ from that of controls and is strictly related to the histological pattern of the mucosa. In the second and third duodenum of duodenal ulcer patients the number of G cells is significantly higher in comparison with controls, while in the bulb the two groups do not differ significantly. Moreover, when different duodenal portions are compared no differences in the number of G cells are observed in the duodenal ulcer group; while in controls the bulbar number of G cells is higher in comparison with second and third duodenum.

Intrahepatic localization of the surface (HBsAg) and core (HBcAg) antigenic determinants associated with hepatitis B virus in biopsy samples from patients with liver disease.

109 biopsy samples from 35 HBAg serologically positive and 74 negative patients were examined by IFL for the presence of the surface and core antigenic determinants associated with the Dane particle. In no serologically negative case was specific IFL detected. Different patterns were observed in serologically positive patients: negative in acute hepatitis, strongly positive cytoplasmic HBs fluorescence in chronic HBAg carriers with normal liver, and discrete HBsAg parenchymal and mesenchymal staining and variable HBcAg staining in chronic liver disease, with HBsAg appearing more frequently in active and HBcAg in active disease. These results are compared with recent reports in this field and the clinical significance of the intrahepatic localization of HBAg is discussed.

An immunofluorescence blocking test to detect antibodies against the hepatitis B delta and core (HBcAg) antigens.

An immunofluorescence (IFL) blocking test has been developed to detect antibodies against the hepatitis B delta and core (HBcAg) antigens. The test proved useful to overcome the difficulties inherent in the detection of anti-delta by standard direct and indirect IFL procedures and provided a simple alternative to detect anti-HBc whenever serological techniques or indirect IFL, requiring purification of Dane particle cores or rare and selected HBcAg positive substrates, cannot be accomplished. The blocking test proved reliable and specific; though less sensitive than standard IFL techniques, it is simple and valuable in routine serological screening for anti-delta and anti-HBc.

Microsomal autoantibodies in chronic infection with the HBsAg associated delta (delta) agent.

A cytoplasmic autoantibody is described which gives a distinct immunofluorescence pattern in tissues of man and of varied animal species. Fluorescence is maximal in human substrates; it is strong in human hepatocytes and nephron cells and weak in thyroid adrenal and pancreatic cells. Complement fixation and fluorescence absorption studies have shown that the homologous antigen is localized in the microsomal membranes of human liver. The autoantibody was found in 13% of 81 carriers of HBsAg with chronic delta infection. It was not detected in patients with acute delta infection or in HBsAg positive and HBsAg negative patients without delta infection. The apparently exclusive occurrence of this antibody in chronic delta infection suggests that its expression is induced by persistence of the viral event.

Complement-mediated unspecific binding of immunoglobulins to some endocrine cells.

Unspecific binding of immunoglobulins to gastrin G cells, glucagon A cells and somatostatin D cells of the gastric mucosa or pancreas, as well as to the calcitonin-somatostatin cells of rabbit thyroid has been found to occur through a non antigen-antibody mechanism mediated at least in part by the C1q fraction of complement. The phenomenon represents a major drawback in hormone immunohistochemistry, which can be prevented by incubating the specific anti-hormone sera with anti-C1q antibodies or with complement-fixing immunocomplexes.

G-cell counts in antral endoscopic biopsies by immunofluorescence.

Antral gastrin-producing cell (G-cells) were counted by an immunofluorescence technique in the antral biopsies obtained at endoscopy from 67 subjects; they included patients with duodenal ulcer, gastritis, and individuals with a normal gastric mucosa. The G-cell count was significantly lower (P less than 0.01) in patients with duodenal ulcer (142 G cells per mm2) in comparison to normal subjects (327 G cells per mm2). No statistically significant correlation was found between the G-cell number and any of the other parameters tested (pentagastrin test, basal serum gastrin and its response to a standard meal).

A trichrome stain for the intrahepatic localization of the hepatitis B surface antigen (HBsAg).

A modified trichrome stain is described for the intrahepatic localization of the hepatitis B surface antigen; HBsAg containing cells exhibit specific green metachromasia contrasting with the granular brown colour of non infected hepatocytes and with the deep eosinophilic colour of ground glass cells of HBsAg-negative alcoholic or drug hepatitis. The technique is simple and reliable for routine screening of HBsAg positive material; its sensitivity is greater than H & E, similar orcein and inferior to immunohistochemistry as performed on frozen sections. Histological diagnosis can be made on the same slide, since several other morphological details are provided in the trichrome stained preparations. With this technique 387 biopsies from HBsAg seronegative individuals were negative; full cytoplasms metachromasia was mostly seen in asymptomatic HBsAg carriers, focal or partial staining in patients with histological evidence of liver cell necrosis. The presence and the staining pattern of HBsAg were of no help in predicting transition to chronicity or a transition from chronic persistent to chronic active hepatitis.

Complement fixing hepatitis B core antigen immune complexes in the liver of patients with HBs antigen positive chronic disease.

One hundred and fifty-two biopsies from serologically HBsAg positive and negative patients with liver disease were studied in immunofluorescence: for the presence of the surface (HBs) and the core (HBc) antigenic determinants foeterminants of the hepatitis B virus, of immunoglobulins and complement (C) deposits, and for the capacity to fix human C. Circumstantial evidence is presented suggesting that HBc immune-complexes are a relevant feature in the establishment and progression of chronic HBSAg liver disease. C fixation by liver cells was shown in all HBC positive patients with chronic hepatitis; an active form was present in every case, except two with a persistent hepatitis, an inverse ratio of HBc to C binding fluorescence being noted between active chronic hepatitis and cirrhotic patients. HBc without C fixation was observed in only three patients in the incubation phase of infectious hepatitis. IgG deposits were often found in HBc containing, C fixing nuclei. No C binding or IgG deposits were observed in acute self-limited type B hepatitis, in serologically positive patients with normal liver or minimal histological lesions, with and without HBs cytoplasmic fluorescence in their biopsy, or in serologically negative individuals.

Radioimmunoassay detection of IgM antibodies to the hepatitis B core antigen in HBsAg liver disease.

A radioimmunoassay is described for detection of IgM antibody to the hepatitis B core antigen. The assay is based on the selective absorption of IgM immunoglobulins from test serum by anti-IgM fixed on a solid phase, followed by incubation with HBcAg and radiolabeled anti-HBc of IgG type. IgM anti-HBc was found in high titers in all the patients with acute hepatitis B; in two of four patients whose acute hepatitis progressed to chronicity, IgM anti- HBc disappeared in 4-6 months despite continuing HB viremia. IgM anti-HBc was also found in low titers in 19% of the patients with chronic HBV infection. No relation was noted between the presence of IgM anti-HBc and clinical or serological categories of chronic carriers of the HBsAg. The antibody was not found in carriers with hepatitis caused by superinfection with the hepatitis A virus or the HBV-associated delta agent. IgM anti-HBc is a marker of a recent HBV infection. Its absence in HBsAg-positive individuals with acute hepatitis should rise suspicion that the patients are carriers of the HBsAg experiencing disease caused by factors other than the HBV.

Radioimmunoassay detection of IgM antibodies to the HBV-associated delta (delta) antigen:" clinical significance in delta infection.

A sensitive radioimmunoassay was developed for specific detection of IgM antibodies to the hepatitis b virus-associated delta antigen. The test is based on the selective absorption of IgM by anti-IgM fixed on a solid phase. Transient primary IgM anti-delta responses with no conversion to a secondary IgG response were observed in acute self-limited delta infection. IgM anti-delta was invariably found in hepatitis B surface antigen (HBsAg) carriers with active delta infection and liver disease, while it was absent in HBsAg-positive or negative individuals with anti-delta of IgG class but without liver damage or intrahepatic delta antigen. IgM anti-delta appears useful in defining the epidemiology of acute delta infection and in the serological diagnosis of active delta disease from nonpathogenic or past delta infection.

Dane particle-associated hepatitis B e antigen in patients with chronic hepatitis B virus infection and hepatitis B e antibody.

A commercial radioimmunoassay was adapted to detect serum Dane particle-associated HBeAg in patients whose sera contained homologous antibody. HBeAg was released from Dane particles with guanidine HCl. Dane particles were separated from anti-HBe by gel-filtration (Sepharose 4B) and ultracentrifugation of the eluate. Dane particle-HBeAg was tested in 45 HBsAg carriers with anti-HBe and was present in 8 (18%) carriers, all of whom had chronic liver disease. By contrast, HBeAg was not found in 10 carriers with normal liver histology. Serum or liver HBcAg was found in 6 of 8 patients with Dane particle-HBeAg. None of the carriers without Dane particle-HBeAg had other markers of hepatitis B virion synthesis. We conclude that Dane particle-HBeAg provides a sensitive index of active hepatitis B virus replication, a guide to the presence of chronic hepatitis in HBsAg carriers with anti-HBe, and a noninvasive method to follow infection in these patients.

Immunohistochemistry of brain 5-hydroxytryptamine.

An immunohistochemical assay for 5-hydroxytryptamine (5HT) has been developed, validated by parallel radioimmunoassay and a series of tests with monoamines or related molecules, and applied to the detection of 5HT in rat brain sections. The procedure seems to be more sensitive and specific than the classical Falck-Hillarp technique. Among amines and related compounds tested, only 5-methoxytryptamine has been found to cross-react. 5HT-immunoreactive neurons and/or fibres have been observed in the spinal cord, brain stem, hypothalamic nuclei, epiphysis and subcommissural organ, thalamus, striatum, corpus callosum, amygdala, hippocampus, olfactory tubercle, and cerebral cortex.

Enzyme-linked immunosorbent assay for detection of antibody to the hepatitis B surface antigen-associated delta antigen.

A blocking enzyme-linked immunosorbent assay (ELISA) was developed for specific detection of antibody to the hepatitis B surface antigen-associated delta antigen. The sensitivity of ELISA was intermediate between that of previously described immunofluorescence and radioimmunological assays for anti-delta. Performance of ELISA was simple and required only ordinary and inexpensive laboratory equipment.

Delta hepatitis in inapparent carriers of hepatitis B surface antigen. A disease simulating acute hepatitis B progressive to chronicity.

Infection with the hepatitis B surface antigen (HBsAg)-associated delta agent (delta) was determined in a series of Italian patients with a diagnosis of acute hepatitis B (HBsAg-positive) progressive to chronicity. Twenty-two of 27 (81%) and 12 of 18 (67%) patients collected, respectively, in Naples and Cagliari, where delta is highly endemic, developed immunoglobulin M antibody to delta and/or rising titers of immunoglobulin G anti-delta during the initial acute phase of the disease. In each of them, anti-delta increased to a high-titered plateau indicative of chronic delta infection. Delta markers were found in none of the 13 patients collected in Siena, where the prevalence of delta infection is low. The great majority of the patients with anti-delta and a progressive form of HBsAg-positive hepatitis lacked the IgM antibody to hepatitis B core antigen. They were presumably unrecognized carriers of HBsAg who became infected by delta and developed hepatitis induced by this agent. In areas where delta is endemic, it may represent the true cause of seemingly type B hepatitis progressing to chronic HBsAg-positive liver disease.

Cell-mediated cytotoxicity to autologous hepatocytes in HBsAg positive liver disease: an analysis of the killing specificity and of the clinical use of the test.

The specificity of a system measuring cell-mediated cytotoxicity as effector-induced target cell detachment from plastic recently adopted to study autologous hepatocyte killing in liver disease, was examined in 17 HBsAg positive liver patients whose hepatocytes (after biopsy digestion with collagenase) were incubated in Terasaki plates with the corresponding blood lymphocytes over two days. The hepatocyte viability and the specificity of the effectors were evaluated as determinants of the clinical value of the test. We found that: (a) hepatocytes in all experiments showed membrane damage owing to the lytic action of collagenase on the small liver core; (b) patients' lymphocytes detached diseased autologous hepatocytes more efficiently than did normal lymphocytes with healthy hepatocytes; (c) in eight patients cytotoxicity appeared equally distributed between a population enriched in T cells and one enriched in non-T cells; yet the mean cytotoxic index of the latter subset was higher than that of the former; (d) cytotoxicity was not blocked by the addition of either aggregated IgG or purified HBsAg; (e) protein synthesis seemed required to promote hepatocyte detachment, for lymphocytes treated with Actinomycin D were no longer active. Poor target viability detracts from the specificity and the clinical value of the test, that therefore turns out to be a major problem of liver cell culture.

Antral G- and D-cell counts in chronic renal failure.

Antral somatostatin- and gastrin-producing cells (D and G cells) were studied in a group of patients with chronic renal failure (CRF) in comparison with a control group. Gastric acid secretion and serum gastrin, phosphate, and parathormone (PTH) levels were also evaluated in every patient. The group with CRF showed a mild increase both in G- and in D-cell denisty. In this group serum phosphate and PTH levels were higher than normal, showing hyperparathyroidism in every patient. A direct correlation was found between G-cell density and parathyroid function in patients with CRF. Hyperparathyroidism, therefore, seems to play a role in the mechanism of increased serum gastrin levels in CRF.

Immunofluorescence detection of new antigen-antibody system (delta/anti-delta) associated to hepatitis B virus in liver and in serum of HBsAg carriers.

A new antigen-antibody system associated with the hepatitis B virus and immunologically distinct from the HB surface, core, and e systems is reported. The new antigen, termed delta, was detected by direct immunofluorescence only in the liver cell nuclei of patients with HBsAg positive chronic liver disease. At present, the intrahepatic expression of HBcAg and delta antigen appears to be mutually exclusive. No ultrastructural aspect corresponding to the delta antigen could be identified under the electron microscope. delta antibody was found in the serum of chronic HBsAg carriers, with a higher prevalence in patients with liver damage. The nuclear fluorescence patterns of HBcAg and delta antigen were similar; it is only possible to discriminate between the two antigens by using the respective specific antisera.