Different expression of HLA class I antigens in liver of children with chronic hepatitis B, evaluated by immunohistochemical method.

Studies on the quantitative expression of the Major Histocompatibility Complex (MCH) in hepatocytes chronically infected by Hepatitis B Virus (HBV) report that an increased expression of these antigens could be related to a good immunological response. In the present work we analyze the expression of the MCH antigens in cryostatic sections of liver biopsies taken from subjects (19 children) with various forms of HBsAg positive chronic hepatitis. A high expression of HLA class I antigens and a high degree of hepatocyte necrosis was evident in Chronic Active Hepatitis (CAH) and Chronic Lobular Hepatitis (CLH). On the contrary, subjects with histological diagnosis of Chronic Persistent Hepatitis (CPH) showed a low expression of such antigens. There was however, the difference that in subjects with high hepatic cytolysis and high expression of HLA class I antigens, serum HBV-DNA was clearly present in almost all the cases with CAH, but not detectable in all cases with CLH. The expression of HLA class II antigens and of Beta2 microglobulin was the same in all 19 cases. All cases with HBV-DNA positivity with high class I antigen expression had active hepatitis which seems to suggest that all attempts at viral clearance on the part of the immune system have been in vain. We hope our paper will be an additional parameter for evaluating the course of hepatitis during Interferon treatment.

[Immunofluorescence aspects of chronic hepatitis in children]. / Aspetti di immunofluorescenza di epatiti croniche in eta' pediatrica.

Liver biopsies from patients (mostly children) affected by chronic hepatitis (persistent or active, HBsAg positive or negative forms), part of them treated by immunosuppressive therapy, have been studied by means of immunofluorescence techniques. A cytoplasmic localization of HBsAg and a nuclear localization of HBcAg have been demonstrated. Moreover, a nuclear localization of immunocomplexes was demonstrated, as well as the presence of immunoglobulins. Persistent forms showed a lesser fluorescence than active forms, and so did the treated patients, if compared with recent, not yet treated subjects.

[Research on surface antigens with immunofluorescence on enterobiopsies of patients with hepatitis B]. / Ricerca dell'antigene di superficie con immunofluorescenza su enterobiopsie di soggetti affetti da epatite B.

ABsAg surface antigen was detected, on enterobiopsies at duodenal level by means of Crosby capsule, in 12 subjects with various types of hepatitis B and in 2 control subjects, by means of immunofluorescence. The antigen was present in the duodenal mucosa of a certain number of subjects with hepatitis: this case-list included subjects with a tendency to relapse or with a poor chronic course notwithstanding the treatment. Some interpretative hypotheses are set forth.

[Research on the core and delta antigens of human hepatitis virus B using immunofluorescence, with liver biopsy of pediatric subjects affected by various forms of chronic hepatitis]. / Ricerca del core e dell'antigene delta del virus B dell'epatite umana con immunofluorescenza su biopsie epatiche di soggetti in eta' pediatrica affetti da varie dorme di epatite cronica.

On liver biopsies of 20 children with different forms of chronic HBsAg-positive hepatitis, submitted to immunodepressive treatment, the presence and the distribution of core and delta antigens and their correlation with the different clinical forms have been studied with the direct immunofluorescence technique. Core antigen was present in 11 patients, delta antigen in 4 patients: both antigens in 3 cases. The percent rate of positive nuclei varied from 5 to 80%. All subjects negative for both antigens showed a favourable course. Delta antigen seems to be associated to forms with unsatisfactory course notwithstanding therapy, particularly to relapses and recurrences of chronic hepatitis in such a way as to hypothesize the possible responsibility either of another hepatitic virus (non A, non B virus) or of a different virus. Core morphological expression frequent in chronic hepatitis submitted to immunodepressive treatment, corresponded approximately to the finding thus far reported for the adults. The interpretation of these results, though difficult, shows remarkable interest for the pathogenetic and therapeutic considerations which follow.

[Immunofluorescence studies on HBsAg positive chronic hepatitis in children. Correlation with the clinical pattern]. / Studi con immunofluorescenza sull'epatite cronica HBsAg positiva nell'infanzia. Correlazioni con l'andamento clinico.

The results obtained in a study of liver biopsies from children affected by different forms of chronic hepatitis during an immunodepressive treatment, accomplished by means of immunofluorescence are summarized. The different antigens correlated to B virus, localized in the nuclei (core and delta), the immunological phenomena observed in the tissue (immunoglobulins and immunocomplexes) and in serum (autoantibodies) have been investigated. The presence of HBcAg, looking finely granular, has been found in 77% of cases of CAH, similar to that found in adults. Delta Ag has been found only in 29% of cases, in the nuclei, like core Ag, but nuclei were more large and not homogeneous. The 2 antigens could be found also in the same patient, differently from adults. The absence of the two antigens has a good prognostic significance; the presence of delta antigen has on the contrary a bad prognostic meaning; the persistence of core antigen under immunodepressive treatment may correspond to a tolerance against antigen. Immunoglobulins and/or immunocomplexes, binding complement, were found in 69% of cases. The fluorescence can be found in different patterns: nuclear, due to immunocomplexes of HBV and antibodies penetrated through impaired membranes; of hepatocyte membranes, linear, probably against auto-antigens (lipoproteins liver-specific) or neo-antigens induced by the virus; in inflammatory cells (K-cells or phagocytic monocytes); or scattered granular, may be from non-eliminated immunocomplexes. Autoantibodies in serum, tested on autologous substrates, were present in 97% of cases, in prevalence antinuclear or against hepatocytary membranes, but also against cytoplasmatic components. Important variations exist between the different forms, both as for what concerns the morphological patterns and the intensity of the reactions. A statement about the severity and the evolution of the disease, with prognostic implication could be hypothetically made in the future, after further studies.

[Relation between viral antigens in hepatic tissue detected by immunofluorescence and serological markers of HBV infection in patients with chronic hepatitis]. / Rapporto tra gli antigeni virali presenti nel tessuto epatico rilevati in immunofluorescenza e i markers sierologici di infezione da HBV in soggetti affetti da epatite cronica.

In 21 subjects HBsAg positive affected by different forms of chronic hepatitis and in one carrier we compared the presence in the liver of HBsAg, HBcAg and delta studied by means of I.F. and the presence of serological markers of hepatitis B virus infection. By the I.F. test, HBsAg was present in all cases, HBcAg in 15 cases (71%), delta in 8 cases (38%). These percentages are almost the same as those of other researches in chronic hepatitis in immunodepressive treatment. About the serological markers, HBsAg and HBcAb were present in 100% of cases, while, regarding the e-anti e system, HBeAg was present in 12 cases (57%) and HBeAb in 7 (33%). In the work effected there is no relation between delta and HBeAg because in 50% of the positive delta subjects there was HBeAb. On the contrary HBcAg and HBeAg are strictly correlated. Therefore the antigens core and delta are not interdependent. We wanted to verify the existence of relation between HBcAg and HBeAg and came to the conclusion that this relation exists in 17 out of 21 subjects (81%). In our researches is clear the long persistence of the hepatic antigens without any connection with the time of treatment and the course of the various chronic hepatitis. The change in the serum from HBeAg to HBeAb occurred in many improved cases but not in all of them. We presume that the long immunodepressive treatment (about 4 years) has influenced our results.

[Chronic HBsAg negative hepatitis in childhood, Stud using the immunofluorescence method]. / Epatiti croniche HBsAg negative dell'infanzia. Studio con il metodo dell'immunofluorescenza.

13 children with chronic HBsAg negative hepatitis were examined in this work with the I.F. method. Comparison can be made with a previous work on chronic HBsAg positive hepatitis. 8 of these patients were cases of ECP, 1 was a ECA-A and 4 were ECA-B. Research has been done to point out immunoglobulins and immunocomplexes in the liver and serum. On the whole, fluorescence was less both in liver and serum than fluorescence in chronic HBsAg positive hepatitis. Reasons of it can be: the absence of anti-virus B antibodies (HBcAb, delta-Ab, HBsAb), the relative lightness of our cases and, probably, a better answer of HBsAg negative hepatitis than of HBsAg positive one to immunodepressive treatment.

Long-term evolution of chronic hepatitis B in children with antibody to hepatitis B e antigen.

The aim of this study was to evaluate the long-term outcome of chronic hepatitis B in 27 children who had increased alanine aminotransferase activity and antibody to hepatitis B e antigen in serum from the time of their first clinical observation. Initial histologic changes were consistent with chronic active hepatitis in 13 cases (three with associated cirrhosis) and with persistent or lobular hepatitis in the remaining cases. On the basis of virologic testing, three groups of patients were identified: (1) two children had hepatitis delta antigen in the liver and anti-delta antibody in serum, and both had severe hepatitis; (2) 10 children had hepatitis B virus DNA in serum, and 60% of them had active hepatitis; (3) 15 patients had no hepatitis B virus DNA, and 33% of them had active hepatitis. During a follow-up period of 12 months to 12 years (mean +/- SD: 6.1 +/- 2.4 years), the disease remained active in both children with anti-delta antibody, but they had no major complaints. In all eight patients who could be followed in group 2, test results became negative for hepatitis B virus DNA and alanine aminotransferase activity normalized within 4 years; biochemical remission was delayed in three patients with higher hepatitis B virus DNA levels on entry, and one of these patients had a severe exacerbation of disease activity before remission. In group 3, a total of 10 patients (71%) achieved biochemical remission within 1 year, and two within 26 months; only two patients, who were transfused at birth, had long-lasting liver damage. These results indicate a trend to early remission of liver disease in children with chronic hepatitis B with antibody to hepatitis B e antigen without delta virus infection. Antiviral therapy aimed at accelerating the termination of hepatitis B virus replication may be indicated only in those with higher levels of hepatitis B virus DNA.

A long-term study of hepatitis B virus-related antigens and immunological parameters in children with chronic hepatitis.

Forty-two children with HBsAg positive chronic hepatitis who received immunosuppressive therapy underwent multiple liver biopsies over periods ranging from one to five years. The presence of HBsAg, HBcAg, deltaAg, antinuclear antibodies and immune complexes was studied using immunofluorescent techniques. The data were correlated with the clinical status of the patients. The presence of HBsAg, HBcAg and deltaAg remained unchanged throughout the study in most positive patients. All 30 HBcAg positive cases were still positive at the end of the study. One of the negative cases became positive. Eight of the ten delta antigen positive cases showed no change. Intrahepatic immunoglobulins, immune complexes and antinuclear antibodies showed a decrease in positivity in a significant number of patients. Fifteen of the 36 patients positive for intrahepatic immunoglobulins became negative during the study, as did 12 of the 29 immune complex positive subjects and 22 of the 33 antinuclear antibody-positive cases. The persistence of HBV antigens seems to be independent of the clinical course. On the other hand, the persistence of positive immunological indices corresponds to a less favourable outcome.

[Comparison between immunofluorescent aspects of chronic HBsAG+, HBAG-hepatitis and some cases of chronic toxic-dismetabolic liver diseases]. / Confronto fra gli aspetti all'immunofluorescenza di epatiti croniche HBsAG+, HBsAG- e alcuni casi di epatopatia cronica su base tossico-dismetabolica.

In the study the aspects noticed on hepatic biopsies by I.F. in HBsAg+ and HBsAg- chronic hepatitis and in some cases of hepatitis on dismetabolic basis in Wilson's disease are compared. Great differences occurred between HBsAg+, HBsAg- and the Hepatitis in Wilson disease. In fact in the first case reactivity is often present due to immunological phenomena (presence of IgG immunocomplexes and autoantibodies) while in Wilson's disease these aspects are not found confirming the hypothesis that in the latter case is a question of an exclusively toxic damage.

[The use of an immunofluorescence technic in the control of chronic HBV hepatitis]. / L'impiego della tecnica dell'immunofluorescenza nel controllo delle epatiti croniche da HBV.

The direct and indirect immunofluorescence technique has been used to study liver biopsies in children affected by different forms of chronic HBsAg positive Hepatitis. The method allows to study the long run changes of the antigens correlated to HBV and of the related immunological phenomena both in serum (autoantibodies) and in the tissue (immunoglobulins and immunocomplexes in the liver). The results obtained are satisfactory.

[Evaluation of pituitary functional periodicity by harmonic analysis. I) Circadian somatotropin secretion in acromegaly]. / Valutazione della periodicità funzionale dell'ipofisi mediaute analisi armonica. I) - La secrezione circadiana di somatotropo nell'acromegalia.

STH circadian secretion has been investigated in 11 acromegalic patients. The physiological STH nocturnal surge is not present in acromegaly, but when data are evaluated by Fourier's cosines analysis, a rhythmicity is however found also in acromegaly. The characteristics of harmonics, namely periodicity, amplitudes and phases, are largely different in respect to pattern of normal subjects, where the rhythmicity is represented by an harmonics with 24-hour periodicity.

[Evaluation of pituitary functional periodicity by harmonic analysis. II) Circadian prolactin secretion in acromegaly]. / Valutazione della periodicitià' funzionale dell'ipofisi mediante analisi armonica. - II) La secrezione circadiana di prolattina nell'acromegalia.

PRL levels over 24 hours have been studied in 9 acromegalic patients (4 men and 4 women). Hormone levels were found significantly higher than age and sex matched normal subjects in 4 cases (2 males and 2 females). PRL and STH levels were significantly correlated in men, but not in women. A 24-hour periodicity of PRL secretion was found in 4 cases, however in 2 of them the phase was anticipated in respect to the control group. In 4 of the remaining subjects the hormone secretion is made up by a succession of episodes with higher frequency than in the control group. Only in one case no significant periodicity of secretion was observed.

[Evaluation of functional periodicity of the pituitary gland by harmonic analysis.--IV) Circadian prolactin secretion in women with breast cancer]. / Valutazione della periodicita'funzionale dell'ipofisi mediante analisi armonica.--IV) La secrezione circadiana della prolattina in donne affette da carcinoma mammario.

Secretory periodicity of prolactin (PRL) is preserved in women with breast cancer in spontaneous or surgical menopause. Hormone titres occurring during the day may be occasionally significantly higher in respect to the ones observe in normal controls at the same hours. The periodicity features calculated by the Fourier's method show however normal indices, namely mesor (mean PRL titres in the 24 hours) phase, amplitude and frequency of oscillations.

[Evaluation of functional periodicity of the pituitary gland by harmonic analysis.--III Circadian prolactin secretion in subjects with prolactin-secreting adenoma]. / Valutazione della periodicita' funzionale dell'ipofisi mediante analisi armonica.--III) La secrezione circadiana di prolattina in soccetti con adenoma prolattino-secermente.

The 24-hour periodicity of PRL secretion in 7 prolactinoma subjects of both sexes has been studied by means of the Fourier's harmonic analysis. In all cases elevated PRL titres have been found, that did not show the physiological changes due to sleep and wake. In most of the cases could not be demonstrated either a significant secretory periodicity or secretory episodes occurred with a frequency higher than in the control groups. Such secretory features may depend on changes in the neoplastic cells' receptors for regulating neurotransmitters.

Long-term outcome of chronic type B hepatitis in patients who acquire hepatitis B virus infection in childhood.

Seventy-six children aged 1-13 years who were known to be positive for hepatitis B surface antigen and hepatitis B e antigen in serum for at least 6 months and who had biopsy-proven chronic hepatitis have been followed longitudinally for 1-12 years (mean, 5 years). Twenty-three of them are now young adults. Eight patients had acute type B hepatitis 12-24 months before entering the study, while 68 patients came to observation during a chronic phase. At the beginning of follow-up, all 76 children were positive in serum for hepatitis B virus DNA, and 44 (58%) had chronic active hepatitis, associated with cirrhosis in two cases. During follow-up, 23 (30%) patients remained hepatitis B e antigen-positive, most with unchanged biochemical and histological features. The other 53 (70%) cases seroconverted to hepatitis B e antibody and cleared hepatitis B virus DNA from serum, including 7 of 8 (87%) patients with acute hepatitis at presentation. After seroconversion, alanine aminotransferase levels normalized in all patients and remained normal in 49 patients (92.5%) throughout a mean observation period of 3 years. Five of these children, including 2 of 7 (29%) with previous acute hepatitis, eventually cleared hepatitis B surface antigen from their sera. Finally, 4 (7.5%) patients experienced a mild increase of alanine aminotransferase levels several months after seroconversion in the absence of hepatitis B virus replication or of delta virus superinfection. Clinical and virological parameters did not significantly differ between patients with or without acute onset; however, seroconversion occurred earlier, and the rate of hepatitis B surface antigen clearance was greater in the former than in the latter group. The present data indicate that approximately two thirds of children with hepatitis B e antigen- and hepatitis B virus DNA-positive chronic hepatitis clear hepatitis B virus DNA from their sera before reaching adulthood. After termination of viral replication, most patients achieve a sustained biochemical remission, suggesting the disappearance of disease activity. Reactivation of virus replication after hepatitis B e antibody seroconversion has never been observed in this series, although mild alanine aminotransferase level abnormalities could be detected in a minority of cases.

Liver cirrhosis associated with chronic hepatitis B virus infection in childhood.

We evaluated the prevalence and the clinical features of liver cirrhosis associated with chronic hepatitis B virus (HBV) infection in a prospective study of 292 consecutive children who were chronic HBsAg carriers with increased aminotransferase activity. Liver histologic changes at presentation were consistent with cirrhosis in 10 (3.4%) patients (100% boys, mean age 4.0 +/- 3.3 years). In none of the remaining children, including 166 with histologic evidence of chronic active hepatitis, did the condition progress to cirrhosis during an observation period of 1 to 10 years. This lack of progression suggests that cirrhosis is an early complication of chronic HBV disease in some patients. A higher prevalence of delta infection and increased incidence of blood transfusions were observed in patients with cirrhosis, supporting the hypothesis that superinfection with delta or non-A, non-B agents may play a synergistic role. Eight of 10 patients had histologic features of disease activity at presentation, although only two had symptoms. During follow-up, persistence of disease activity was observed only in the three delta antigen-positive patients. None of the patients with inactive cirrhosis have developed signs of liver failure or portal hypertension.

Correlation between e/anti e system and evolution of B virus chronic hepatitis in pediatric patients.

The authors have performed a longitudinal study of 118 children affected with B virus chronic hepatitis. Our first observation revealed 92 children with HBeAg positive (26 CPH, 66 CAH), 22 children with anti HBe positive (6CPH, 15 CAH, 1 cirrhosis), 4 children (CAH) with e/anti-e negative. A correlation between the severity of clinical forms and the behaviour of the e/anti-e system was not observed. Seroconversion was observed during the follow up period in 37 of 92 subjects in an average time of 59.83 +/- 32 months, time rather prolonged in patients under immunosuppressive therapy. To compare the clinical progress and the evolution of CPH and CAH respectively, always with regard to the e/anti-e system, statistically significant differences did not result. Only anti HBe positive recovered subjects, inclusive of seroconverted patients and those anti HBe from the first observation, showed significant results to the statistical analysis. Still, seroconversion corresponds frequently to a stable improvement of hepatitis. On the contrary evolution into cirrhosis was observed in 5 patients that had anti HBe antibodies.

Chronic hepatitis in childhood: the spectrum of the disease.

During a multicentre study of chronic hepatitis in childhood diagnosed by biopsy, the spectrum of the disease has been evaluated in 196 consecutive patients, including 157 from Northern Italy and 39 from Southern Italy. Only 31% of patients in the former group and 27% in the latter were symptomatic when first seen: the majority of cases being seen after familial screenings for hepatitis B virus (HBV) markers or during intercurrent infections, thus suggesting that the frequency of chronic hepatitis in childhood might be largely underestimated in our area. In Southern and Northern Italy 83% of symptomatic and 95% of asymptomatic patients were hepatitis B surface antigen (HBsAg) positive in serum; only 15 (8.3%) of these children were born to mothers known to be HBsAg positive at delivery, but a high circulation of HBV was found in their families: in fact more than 65% of household contacts in Northern Italy and more than 90% in Southern Italy had serological evidence of past or ongoing HBV infection. These data indicate that, although familial screenings for HBV could have enhanced the percentage of HBsAg positive asymptomatic cases, chronic hepatitis in Italian children is mainly caused by HBV infection acquired in the familial setting through horizontal transmission. Such findings also emphasise the importance of mass vaccination of infants as the most effective means to prevent chronic type B hepatitis in childhood in our area. Among HBsAg positive children 55% had histological features of chronic active hepatitis and 85% were hepatitis Be antigen (HBeAg) positive in serum. Anti-HBe positive hepatitis was significantly more frequent in Southern than in Northern Italy in parallel with the significantly higher prevalence (17%) of hepatitis delta virus infection in that area. Of the 16 HBsAg negative cases included in the study three had autoimmune hepatitis, three Wilson's disease, one alpha1 antitrypsin deficiency, and nine had cryptogenic hepatitis, often associated to mild liver lesions resembling those seen in our adult patients with chronic non-A, non-B hepatitis unrelated to percutaneous exposure.

Chronic hepatitis in carriers of hepatitis B surface antigen, with intrahepatic expression of the delta antigen. An active and progressive disease unresponsive to immunosuppressive treatment.

To assess the characteristics of chronic hepatitis in hepatitis B surface antigen (HBsAg) carriers with intrahepatic delta antigen, the hepatic histologic findings of 137 patients were reviewed; 101 patients were followed for 2 to 6 years. The predominant liver disease was chronic active hepatitis in 93 patients or cirrhosis in 32; minor forms of chronic persistent or lobular hepatitis were seen in 12 patients. Eight of the 26 patients with an initial diagnosis of cirrhosis died during the follow-up period. Cirrhosis developed in 31 of 75 patients (41%) without nodular regeneration seen in the first biopsy specimen; 5 of these patients died. Treatment with prednisone or azathioprine did not induce histologic amelioration of delta hepatitis or prevent cirrhosis. Chronic HBsAg hepatitis with intrahepatic expression of the delta antigen is an active, progressive disease unresponsive to conventional immunosuppressive treatment.