Indian childhood cirrhosis (ICC) & ICC-like diseases: the changing scenario of facts versus notions.

Indian childhood cirrhosis (ICC), a disease considered to have been endemic in and unique to India has now been documented in children of non-Indian origin from other countries. More recently available findings from a large multicentre study sponsored by the Indian Council of Medical Research (ICMR) have dispelled some of the generally accepted notions and have established several new facts on different aspects of the disease. All relevant reports on ICC and ICC-like diseases, till date, were reviewed to obtain a proper perspective on the current state of our understanding on this non-Wilsonian copper overload liver disease. A primary role of exogenous copper in causing the disease was earlier debated on the basis of studies in India but investigators abroad studying some sporadic cases and a series of endemic ICC-like diseases supported a hepatotoxic injury by ingested copper in genetically susceptible infants and children in ICC- like disease and in ICC. Epidemiologic and morphologic findings in the well controlled ICMR study based on 225 cases of ICC and 426 controls, all confirmed on liver biopsy, have however, convincingly refuted this concept. Additionally, this study revealed that unlike what has been believed earlier, older children more than 3 yr age can get the disease and that in its natural course the hepatic histology can transform between the characteristic one considered diagnostic and some other patterns, any one of which can be the morphologic manifestation at first presentation of the patient. Older children and cases with milder morphologic changes at presentation had longer survival. The overall inference from critical analysis of all available data is that ICC and ICC-like diseases clinically manifest in a child of any age though common in younger ones, and a clinical diagnosis must be made in any child with so-called 'cryptogenic cirrhosis'. Exposure to exogenous copper in food, milk and water should not be a prerequisite for this consideration. A liver biopsy whenever feasible should be mandatory for confirmation with the understanding that the morphologic changes in liver can present a few other patterns besides the characteristic one currently taken to be diagnostic. The ascribed current decline in encountering ICC is likely to be due partly to missing a diagnosis and partly to a true reduction in incidence consequent on time related economic and socio-cultural changes.

Cassia occidentalis poisoning as the probable cause of hepatomyoencephalopathy in children in western Uttar Pradesh.

BACKGROUND & OBJECTIVE: Recurrent annual outbreaks of acute encephalopathy illness affecting young children have been reported for several years in many districts of western Uttar Pradesh (UP). Our earlier investigations over three consecutive years (2002-2005) proved that these outbreaks were due to a fatal multi-system disease (hepatomyoencephalopathy syndrome) probably caused by some phytotoxin and not due to viral encephalitis as believed so far. We conducted a case-control study to investigate the risk, if any, from various environmental factors and also to identify the putative toxic plant responsible for development of this syndrome. METHODS: Eighteen cases with acute hepatomyoencephalopathy syndrome admitted in 2005 in a secondary care paediatric hospital of Bijnor district of western UP were included in the study. Three age-matched controls were selected for each case. A semi-structured questionnaire was developed and applied to all 18 cases and 54 controls. All interviews were conducted within one week of discharge or death of each case. Quantitative data were analyzed using the relevant established statistical tests. RESULTS: Parents of 8 (44.4%) cases gave a definite history of their children eating beans of Cassia occidentalis weed before falling ill, compared with 3 (5.6% controls), the odds ratio being 12.9 (95% CI 2.6-88.8, P<0.001). History of pica was the other associated factor with the disease, odds ratio 5.20 (95% CI 1.4-19.5, P<0.01). No other factor was found significantly associated with the disease. INTERPRETATION & CONCLUSION: Consumption of C. occidentalis beans probably caused these outbreaks, described earlier as hepatomyoencephalopathy syndrome. Public education has the potential to prevent future outbreaks.

Recurrent annual outbreaks of a hepato-myo-encephalopathy syndrome in children in western Uttar Pradesh, India.

BACKGROUND & OBJECTIVE: Outbreaks of an acute encephalopathy syndrome affecting children, with high case-fatality, have been reported in western Uttar Pradesh, India for the last many years. We investigated these cases in Bijnor district and present our findings. METHODS: Fifty five children aged 2-10 yr hospitalized from 2003 to 2005 in Bijnor, Uttar Pradesh, with features of acute encephalopathy were selected by defined clinical criteria. Various laboratory investigations were performed. RESULTS: The disease had peak incidence in early winter months. Previously healthy, 2-4 yr old rural children (mean age-3.78 yr) of very low socio-economic background were most vulnerable. Almost all had vomiting preceding unconsciousness and a majority had mild fever and abnormal behaviour/agitation. Abnormal posture of trunk and limbs were distinctive features. Fluctuation of blood pressure was seen in three-quarter cases. Serum aminotransferases, creatine phosphokinase and lactic dehydrogenase levels were found markedly raised virtually in all cases in whom the tests were performed. Serum glucose was found low (<50 mg/dl) in 47.3 per cent cases at presentation. Cerebrospinal fluid (CSF) was under normal or low pressure and without pleocytosis in all cases. No microorganism could be isolated from serum, CSF, urine and visceral specimens. Neuroimaging performed in two cases was also normal. Liver biopsy performed in 21 cases showed acute hepatotoxic injury in all with marked hydropic change and perivenular necrosis. Tibial muscle biopsy done in 8 cases showed focal necrosis while brain biopsy taken in 2 cases had mild spongiosis with focal gliosis. Forty two children succumbed to their illness (case fatality 76.4%), most within 72 h of presentation. Survivors did not show any neurological deficit. INTERPRETATION & CONCLUSION: Our findings showed that the outbreaks were due to a multi-system disease with toxic injury to liver, muscles and brain (hepato-myo-encephalopathy) and not due to viral encephalitis as believed so far. The cause remains unknown but several features suggest the possibility of phytotoxin-induced pathology.

Cassia occidentalis poisoning causes fatal coma in children in western Uttar Pradesh.

We investigated cases of the annual seasonal outbreaks of acute hepato-myo-encephalopathy in young children in western Uttar Pradesh for causal association with Cassia occidentalis poisoning, by a prospective survey in 2006. During September-October homes of 10 consecutive cases were visited and history of eating Cassia beans was obtained in all. Nine children died within 4-5 days. There appears to be an etiological association between consumption of Cassia occidentalis beans and acute hepato-myo-encephalopathy.

Kupffer cell sarcoma in rats after exposure to small doses of dimethylnitrosamine and N-2-acetylaminofluorene during hepatic regeneration.

During experiments to study the evolution of hepatocellular carcinoma in adult male Wistar rats by exposing regenerating livers to the action of small doses of dimethylnitrosamine (DMN) or N-2-acetylaminofluorene (AAF), several primary sarcomas of the liver were incidentally observed. The morphology and behavior of the tumors suggest their origin from Kupffer's cells. Kupffer cell sarcomas occurred more frequently when 70% hepatectomy was used as the regenerative stimulus. None of the 36 animals treated with AAF alone and 2 of the 38 rats given DMN only had this tumor.

Effect of protein calorie malnutrition and cell replication on aflatoxin B1-induced hepatocarcinogenesis in rats.

Malnourished and well-fed neonatal Holtzman rats 10 days of age were exposed to 3 doses of aflatoxin B1 [(AFB1) CAS: 1162-65-8] at intervals of 96 hours to study the combined effect of malnutrition and cell replication in AFB1-induced hepato-carcinogenesis. The neonatal model made use of the fact that cell replication persists in the liver for 3 weeks of postnatal life. Malnutrition during suckling was induced by adopting the techniques of Widdowson and McCance of increasing the litter size to 16. Following AFB1 administration, the malnourished animals were rehabilitated on a high-protein pellet diet given ad libitum. Preneoplastic lesions and neoplastic nodules were identified in the livers of the 2 groups. Alpha fetoprotein (AFP) was detected in the sera by immunoprecipitation. The preneoplastic lesions appeared earlier, and their progression was faster in the malnourished group as compared to the well-fed animals. By 65 weeks following AFB1 exposure, 6 of 17 (35%) animals from the malnourished group showed neoplastic nodules, whereas no such nodules were observed in the animals from the well-fed group. Neoplastic nodules showed a variable pattern of enzyme activities. Under the electron microscope the changes were again more marked in the animals of the malnourished group as compared to those of the well-fed group. In the former group serum AFP was detected as early as 46 weeks, and by 55-65 weeks almost 50% of the animals from the same group showed positivity for serum AFP. None of the animals from the well-fed group showed any positivity for serum AFP throughout the study. This study thus indicates that preneoplastic lesions-neoplastic nodules are enhanced when cell replication and malnutrition coexist during AFB1-induced hepatocarcinogenesis.

The nature and significance of liver cell vacuolation following hepatocellular injury--an analysis based on observations on rats rendered tolerant to hepatotoxic damage.

Swelling with nonlipid cytoplasmic vacuolation of diffusely distributed hepatocytes is seen consistently after mild acute and subacute liver injury. Several lines of evidence point to the possibility that this change may reflect a cellular adaptation beneficial to the host, rather than a degenerative change. The nature and significance of this morphological manifestation were tested in batches of albino rats given small doses of a variety of hepatotoxins, some of which were subsequently challenged with a large highly necrogenic dose of carbon tetrachloride (CCl4). Morphological and biochemical investigations showed that cytoplasmic vacuolation of liver cells following low doses of toxins was due to excess accumulation of glycogen, predominantly of the monoparticulate form. These cells lacked features of degeneration or regeneration and were much less susceptible to injury by the large dose CCl4, as assessed by structural and serum enzyme analyses. This tolerance to toxic damage seemed to be associated with excess accumulation of intracellular glycogen. We conclude from these and other observations on animal and human livers that many of the vacuolated hepatocytes seen in liver injury are cells adaptively altered to resist further insult rather than cells undergoing hydropic degeneration, as is commonly believed.

Indian childhood cirrhosis--like liver disease in an Arab child. A brief report.

An Arab female child presented with rapidly progressive liver disease, with apparent onset in late infancy and death at 15 months. Microscopy showed panacinar hepatitis, portal and pericellular fibrosis, and diffuse Mallory bodies in the absence of steatosis or significant cholestasis. Hepatic copper concentration was moderately elevated. Known causes of early childhood cirrhosis were excluded. This case meets most of the established criteria of Indian childhood cirrhosis, yet is unusual in its occurrence in a child of Arab ancestry and in having a moderate degree of hepatocellular copper overload.

The morphology of cirrhosis. Recommendations on definition, nomenclature, and classification by a working group sponsored by the World Health Organization.

This memorandum provides guidelines on the definition, nomenclature, and classification of cirrhosis, chronic hepatitis, and hepatic fibrosis. These are considered according to morphological characteristics and aetiology. It is hoped that this system will serve as a standard for diagnostic, research, and epidemiological purposes. The relationship of cirrhosis to liver cell carcinoma is briefly discussed and the possible morphological markers of an increased risk of malignancy are defined.

A light & electron microscopic study on apoptotic bodies in acute aflatoxin B1 induced injury in the rat liver.

A subnecrogenic dose of aflatoxin B1 (AFB1) was injected intraperitoneally into 8-10 wk old male rats of the Holzman strain. Cytoplasmic and sinusoidal eosinophilic bodies were seen in the liver which appeared at 30 h and reached a maximum at 48 h. Electron microscopically some of the cytoplasmic structures were seen to be of mitochondrial origin whereas others resembled apoptotic bodies. The sinusoidal bodies were similar to apoptotic bodies.

Immunohistochemical detection of hepatitis C virus antigen in paraffin embedded liver biopsies from patients with chronic liver disease.

A simple and reproducible immunohistochemical (IHC) staining method that adequately identifies and localizes hepatitis C virus (HCV) in human livers is still not available. We performed IHC staining using both a new monoclonal antibody against HCV and a polyclonal human anti-HCV IgG to 94 liver biopsies from HCV seropositive patients with chronic hepatitis and 15 control liver biopsies. Positive nuclear staining was consistently observed predominantly in hepatocytes and much less in lymphocytes with either antibody in 57% of anti-HCV seropositive cases but in none of the controls. However, the monoclonal antibody yielded a stronger positive reaction and in a greater proportion of hepatocytes. In 78% of the positive cases, more than a quarter of the hepatocytes showed nuclear staining. The degree of hepatic HCV load as revealed by intensity and extent of positive staining did not correlate with histological changes in the liver. The new monoclonal antibody against HCV appeared to be suitable for identifying HCV in tissues by a simple IHC stain and can be used to explore the pathogenesis of liver injury induced by this virus.

Immunohistochemical study of proliferating mesenchymal spindle cells in heterogenous soft tissue lesions.

Proliferation of mesenchymal spindle cells is a main event in a variety of lesions with similar morphological features but widely divergent biological behaviour. In order to identify criteria for precise histological diagnosis, 60 human soft tissue lesions, divided into 40 cases of fibroblastic cell proliferation, 10 smooth muscle cell tumours and 10 nerve sheath cell tumours, were examined for the immunohistochemical profile of the main lesional cell in addition to other histological features. The three groups could be differentiated by determining the lineage of the constituent spindle cell on the pattern of expression of vimentin, alpha-smooth muscle actin (ASMA) and macrophage antigen CD68 (MA-CD68). Smooth muscle cells expressed ASMA and vimentin but not MA-CD68, while nerve sheath cells were negative for ASMA but positive for vimentin and MA-CD68. The fibroblastic cell lesions as a group were easily differentiated on the basis of positive reactivity for all three markers but individual lesions could only be distinguished by additional assessment of histological features. Because of consistent co-expression of ASMA, vimentin and MA-CD68 in the spindle mesenchymal cell present in all varieties of lesions in this heterogeneous group, we suggest that this proliferating "fibroblastic" cell is phenotypically a fibromyohistiocyte.

Etiologic spectrum of acute sporadic viral hepatitis in children in India.

The relative magnitude by hepatitis A virus (HAV), hepatitis B virus (HBV) and hepatitis Non-A, Non-B virus (HNANBV) was determined in 496 children from three different parts of India suffering from acute viral hepatitis by tests for specific IgM class anti-HAV and anti-HBV antibodies in the serum. HAV, HBV and NANB infections accounted for 55.8 per cent, 20.2 per cent and 23.2 per cent of cases respectively. Hepatitis A largely (59.5%) affected younger children of 1-5 yr. Nearly a third of children affected by NANB hepatitis were additionally positive for HBsAg. The proportions of HAV and HBV infected cases respectively decreased and increased with increasing age whereas the incidence of HNANBV infection remained almost constant throughout childhood. Acute NANB hepatitis, a major health problem in the adults of India is also common throughout childhood. This study suggests that this infection does not impart long lasting protective immunity.

Relationship between histology and aetiological factors in primary liver cancer.

A review of 205 primary hepatic cancers from different geographic areas reveals that HCC which forms the vast majority (86%) of these, commonly presents as the trabecular variety (76%). The compact and pseudoglandular forms are rare (18% and 6%). Relatively fewer cases of undifferentiated cancer, hepatoblastoma and cholangiocarcinoma are encountered. HCC is often associated with the presence of hepatitis B virus surface antigen (HBsAg) in the liver. This is particularly true of the trabecular variety (81%). A proportion of the compact variety as well as undifferentiated cancers do not appear to be related to HBV infection. Cholangio carcinomas are considered to be related to infection by liver flukes. Hepatoblastomas occur in infancy and childhood and show no known aetiologic association. Aflatoxin may be aetiologically related to same cases of HCC. Continued use of oral contraceptives can occasionally induce HCC.

Liver specific autoimmunity and chronic liver disease--an enigmatic relationship.

Refinements in techniques for identifying liver specific immune responses have currently aroused considerable interest in the role of such immunity in the pathogenesis of chronic hepatic disorders. Presence of antibodies against liver cell membrane was tested for in 242 samples of serum and in 86 liver biopsies from patients with liver diseases as well as from controls. Indirect immunofluorescence tests on isolated rabbit hepatocytes and on human liver cells from biopsies respectively were carried out. In the serum, antibody was detected in 16 per cent, several of these cases belonging to the category of recovering acute viral hepatitis and some to chronic hepatitis or to other liver disorders. Serum HBsAg status in these antibody carriers vary. Antibodies on liver cell membrane were detected in approximately 25 per cent cases of hepatitis. The distribution among different cases were like in case of serum positivity. The pathogenetic role of these antibodies in initiation or progression of disease appears doubtful. It is possible that presence of these antibodies merely represents an immune response following liver injury and may not be responsible in causing acute or chronic liver disease.

Synovial fluid changes in mycoplasma induced septicaemic polyarthritis of goat kids.

Synovial fluid samples of goat kids inoculated (ip) with 5 ml of 48 hr log phase culture of Mycoplasma mycoides sub sp. mycoides (large colony type) containing 10(7) cfu/ml were analysed for physical, cytological and biochemical properties. The synovial effusions were exudative in nature with increased volume. Gross appearances were serofibrinous, haemorrhagic and turbid containing flocculent materials with immediate clot formation. Mucinous precipitate quality was very poor having friable precipitates with cloudy supernatant. There were high total leucocytic and erythrocytic counts with significant high numbers of both neutrophils and lymphocytes. Synovial fluid sugar contents were significantly reduced, whereas total protein contents were significantly increased with concomitant reduction in albumin:globulin ratio. The alkaline phosphatase and transaminase values were also markedly increased in the synovial fluids of mycoplasma induced polyarthritic goat kids. The results may provide a clinical guideline for diagnosis, chemotherapy and prognosis of different joint diseases in domesticated animals.

Aflatoxin B1 induced hepatocarcinogenesis in neonatal rats.

Role of cell replication on aflatoxin B1 (AFB1) induced hepatocarcinogenesis was investigated in neonatal rats showing persistence of cell replication in the liver for 21 days of post natal life. Adult (8-10 weeks old) rats displaying no hepatocytic proliferation served as controls. Three doses of AFB1 were administered to both the groups at intervals of 48 hr with the doses starting on 10th day of age in the neonatal group. Appearance of phenotypically altered preneoplastic hepatocytes was quantitated in both the groups. A significantly higher incidence of preneoplastic foci was recorded in neonatal rats as compared to adult animals. The results suggest that presence of cell replication in neonatal rats at the time of AFB1 administration enhances the process of hepatocarcinogenesis.

Multicentric clinical trials for safety and efficacy evaluation of alpha;beta arteether in complicated P. falciparum malaria.

OBJECTIVE: To evaluate efficacy of alpha;beta arteether in patients of P. falciparum malaria presenting with complications was undertaken in a multicentric clinical trial. METHOD: Each patient who consented to undergo clinical trial with parenteral Arteether was treated with a fixed dose schedule of Arteether given intramuscularly in a dose of 150 mg once a day on three consecutive days. Every patient was followed upto 28 days with clinical, haematological and parasitological monitoring every day upto one week and thereafter at 14, 21 and 28 days. The response was assessed in terms of fever clearance time, parasite clearance time, cure rate and parasite reappearance rate. RESULTS: A total of 211 patients of P. falciparum malaria were included in the study from four centres (Bhilai, Guwahati, Jamshedpur and Rourkela). Results of this study showed that fever clearance time ranged between 24-168 hours, parasite clearance time ranged between 24-120 hours and overall mortality ranged between 4-8.5%. Out of 211, only 14 patients expired during the study, of these, 10 patients expired within first two days i.e. before completing the three day schedule of arteether therapy. Tolerability to arteether injection was good in all these patients and no untoward effects were experienced or reported during the study. Overall cure rate observed in these studies was 93%. CONCLUSION: This study shows a rapid parasite and fever clearance in patients of complicated P. falciparum malaria.

A multicentric study with arteether in patients of uncomplicated falciparum malaria.

Two hundred and sixty seven patients of uncomplicated P. falciparum malaria completed study in a multicentric phase III clinical trial of Arteether. Arteether was given intramuscularly in a dose of 150 mg daily for three consecutive days. Each patient was followed upto 28 days of alpha, beta arteether therapy. The cure rate was 97% with fever clearance time between 1-7 days (24-168 hours) and parasite clearance time between 1-3 days (24-72 hours). Parasite reappearance rate was found to be 3% and reported at only three of the centres. Following the treatment no adverse effect was observed on haematological, biochemical and vital clinical parameters.

Hepatocellular carcinoma--a model of human cancer: clinico-pathological features, etiology and pathogenesis.

Hepatocellular carcinoma (HCC), a unique human neoplasm, has interested many in several fields of biological and health sciences. This cancer is credited as the first that can be largely eliminated by a safe anti-viral vaccine and other transmission control measures. It is also the first cancer for which a reliable diagnostic tumour marker was identified and studies on this tumor in humans and animals have provided a large body of information on causation and step-wise evolution of cancers. Being a common and rapidly fatal tumour, mainly affecting males in the more populous developing countries, HCC may well be the commonest cancer of the human male. Clinical features are not specific for HCC but manifestations represent varying combinations of those due to cirrhosis which is a very frequently associated and pre-existent disease, those due to tumour and those due to malignancy. This tumour commonly takes a massive form with satellite nodules or a scattered multinodular form. A fibrolamellar variant is biologically and clinically quite different from the usual HCC and the small capsulated variant is seen in some geographic areas. Microscopically the trabecular variety is common and differentiation from metastatic cancers and benign lesions may need use of newly described immunohistochemical markers in addition to clinical evidence of cirrhosis. Hepatitis B and C viruses, dietary aflatoxin B1 and cirrhosis from any cause are common risk factors in that order of importance. Several lines of evidence including molecular biology and animal studies support these etiological linkages. Studies in experimental animals using chemical carcinogens, different hepatotropic viruses and aflatoxin have greatly helped our understanding of carcinogenesis in general and hepatocarcinogenesis in particular. Individual HCC risk factors may contribute to summation of mutations subsequent to repeated and continued liver cell injury act as carcinogen/co-carcinogen or be involved in both capacities.