Serum adiponectin levels in different types of non alcoholic liver disease. Correlation with steatosis, necroinflammation and fibrosis.

BACKGROUND AND STUDY AIMS: In recent studies adiponectin has been implicated in the pathogenesis of non alcoholic liver disease (NAFLD), a common chronic liver disease with a broad spectrum of histopathologic findings. The aim of this study was to investigate the correlation between serum adiponectin levels and steatosis, necroinflammation and fibrosis in different types of NAFLD patients. PATIENTS AND METHODS: Forty three patients with elevated liver enzymes and biopsy proven non alcoholic fatty liver disease and 38 patients with clinically diagnosed NAFLD and permanently normal liver enzymes were prospectively enrolled in the study. Patients with biopsy proven NAFLD were divided into two groups: non alcoholic steatohepatitis (NASH): 25 patients and simple steatosis: 18 patients. Serum adiponectin levels were measured with an ELISA immunoassay, and BMI, fasting serum glucose, total and HDL cholesterol, fasting triglyceride levels and insulin resistance were determined. RESULTS: Groups did not differ in age, sex, BMI, waist circumference and HOMA - IR. Only patients with confirmed NASH had lower serum adiponectin levels in comparison to NAFLD patients with both abnormal (6.6 +/- 4.7 microg/mL vs 10.8 +/- 5.6 microg/mL, p = 0.01) as well as normal liver enzymes (6.6 +/- 4.7 microg/mL vs 9.2 +/- 4.8 microg/mL, p = 0.01). For the whole NAFLD group with elevated liver enzymes no correlation was found between serum adiponectin levels and the degree of liver steatosis or fibrosis stage. Also no correlation was found between adiponectin levels and BMI, ALT, AST, gamma GT or HOMA-IR. CONCLUSIONS: Patients with established NASH have lower serum adiponectin levels than NAFLD patients with normal or abnormal liver enzymes. Adiponectin was not associated with the severity of hepatic fibrosis.

Treatment of Budd-Chiari syndrome by transjugular intrahepatic portosystemic shunt.

Patients with Budd-Chiari syndrome may require therapy with portal decompressive surgery. Transjugular intrahepatic portosystemic shunt has recently emerged as a new treatment alternative to surgical shunting, but reported literature is scarce and its long-term outcome is to be evaluated. The aim of our study was to present two patients with Budd-Chiari syndrome treated with transjugular intrahepatic portosystemic shunt. Two female patients with acute and subacute Budd-Chiari syndrome respectively, uncontrolled by medical therapy, were referred for transjugular intrahepatic portosystemic shunting. Both patients had refractory ascites, while one had hepatic insufficiency. Hepatic vein thrombosis was without definitive etiology in one patient and due to essential thrombocytosis in the other. Transjugular intrahepatic portosystemic shunt was successfully created in both patients; in one a transcaval approach was performed. Bare stents were used in one, while in the other a polytetrafluoroethylene-covered stent-graft. Technical success was 100% with no complications. Ascites resolved in both patients and liver function improved. The first patient remains asymptomatic with good shunt patency 6 months after intervention. The second patient is symptom-free two years after the shunt creation, despite stent occlusion. She remains asymptomatic due to hepatic vein collateral development. In patients with acute or subacute Budd-Chiari syndrome uncontrolled by medical therapy, transjugular intrahepatic portosystemic shunt is highly effective with no complications and an associated mid-term clinical success.

Endocarditis due to Salmonella enterica subsp. arizonae in a patient with sickle cell disease: a case report and review of the literature.

Human cases due to Salmonella enterica subsp. arizonae are especially rare, but it may affect immunocompromised patients and infants. We present a case of endocarditis in a patient with sickle cell disease and a review of earlier cases caused by this rare human pathogen. The patient was successfully treated with ceftriaxone and ciprofloxacin. There are only few cases of salmonella endocarditis reported in the last six decades and it is the first case of Salmonella enterica subsp. arizonae endocarditis in the literature to the best of our knowledge.

Serum adiponectin in chronic hepatitis C and B.

Adiponectin possesses anti-inflammatory, insulin-sensitizing and anti-atherosclerotic properties. The aim of this study was to assess the levels of serum adiponectin in patients with chronic viral hepatitis C and B and correlate them with parameters exploring insulin resistance and indices of chronic liver disease. Seventy-two patients with chronic hepatitis C virus (HCV) infection and 73 patients with chronic hepatitis B virus (HBV) infection, matched for age and sex, were studied. All individuals were examined for serum concentrations of adiponectin, insulin, C-peptide and homeostasis model assessment for insulin resistance (HOMA-IR). Viral parameters and liver histology were also evaluated. Serum adiponectin levels were significantly higher in HCV compared with HBV-infected patients. Correlation analysis in the whole group demonstrated that serum adiponectin was positively correlated with aspartate aminotransferase, alkaline phosphatase, globulins, high-density lipoprotein cholesterol and staging score, while it was negatively correlated with body mass index, insulin, C-peptide and HOMA-IR. Logistic regression analysis identified type of infection (HCV vs HBV), alcohol consumption more than 25 g daily, serum total globulin and low C-peptide as significant predictive variables associated with high adiponectin levels. Higher levels of serum adiponectin in HCV compared with HBV patients could have a role in the slower disease progression of chronic HCV infection. In addition, alcohol intake more than 25 g daily seems to be a significant predictor for hyperadiponectinaemia in patients with chronic viral hepatitis C or B. Finally, in this study, a clear positive association between adiponectin and hepatic necroinflammation or staging score was not found.

Serum lipid pattern in chronic hepatitis C: histological and virological correlations.

Lipoproteins are closely connected to the process of hepatitis C virus (HCV) infection. The aim of this study was to evaluate the lipaemic profile in patients with chronic HCV infection, and to identify any association between serum lipid levels and viral load, HCV genotype or liver histology. Total cholesterol (TC), high-density lipoprotein-cholesterol (HDL-C), low-density lipoprotein-cholesterol (LDL-C) and triglycerides (TG) were measured in the sera of 155 patients with chronic HCV infection and 138 normal subjects, matched for age and sex. Viral parameters and liver histology were evaluated in HCV-infected patients. Serum TC (P < 0.0005), HDL-C (P < 0.0005) and LDL-C (P < 0.0005) were lower in chronic hepatitis C patients compared with controls. Grading score was positively correlated with TC and LDL-C. Patients with HCV genotype 3a had significantly lower levels of TC, HDL-C, LDL-C, higher viral load and higher frequency of hepatic steatosis than those with other genotypes. Logistic regression analysis identified genotype 3a (OR, 6.96; 95% CI, 2.17-22.32, P = 0.0011) as the only significant predictive variable associated with low serum cholesterol concentration. HCV infection is associated with clinically significant lower cholesterol levels (TC, LDL and HDL) when compared with those of normal subjects. This finding is more pronounced in patients infected with HCV genotype 3a. Further studies are necessary to define the pathophysiology of the relationship between lipid metabolism and HCV infection.

Non-invasive predictors of the presence of large oesophageal varices in patients with cirrhosis.

BACKGROUND/AIMS: The usual clinical practice is to screen all patients with established cirrhosis at the time of diagnosis by upper endoscopy for the presence of varices. Patients with large varices should be treated with non-selective beta blockers to reduce the incidence of first variceal bleeding. However, fewer than 50% of cirrhotic patients have varices at screening endoscopy and most have small sized varices, with a low risk of bleeding. The aim of the present study was to determine whether clinical or laboratory non-endoscopic parameters could predict the presence of large oesophageal varices. PATIENTS/METHODS: Seventeen variables considered relevant to the prevalence of oesophageal varices were tested in 184 patients with cirrhosis, who underwent screening endoscopy. Small varices were regarded as those which flatten with insufflation or slightly protrude into the lumen, while large varices are those which protrude into the lumen or touch each other. None of the patients was on beta blockers or other vasoactive drugs or had a history of variceal bleeding. RESULTS: Oesophageal varices were present in 92 patients (50%), and large varices in 33 patients (17.9%). Variables associated with the presence of large oesophageal varices on univariate analysis were the presence of ascites and splenomegaly either by clinical examination or by ultrasound (p < 0.01), the presence of spiders (p = 0.02), platelet count (p < 0.0001), and bilirubin (p = 0.01). Factors independently associated with the presence of large oesophageal varices on multivariate analysis were platelet count, size of spleen and presence of ascites by ultrasound. Using mean values as cut-off points, it is noteworthy that only five out of 39 patients (12.8%) with platelets > or = 18(x 10(9)/l), spleen length < or = 135 mm and no ascites had varices. Moreover, all these patients had small sized varices. On the other hand, 15 out of 18 patients (83.3%) with a platelet count < 118 x 10(9)/l, spleen length > 135 mm and ascites had varices. Moreover, five out of those 18 patients had large varices (28.3%). CONCLUSION: Thrombocytopenia, splenomegaly and ascites are independent predictors of large oesophageal varices in cirrhotic patients. We suggest that endoscopy could be avoided safely in cirrhotic patients with none of these predictive factors, as large varices are absent in this group of patients.

Keratoconjunctivitis sicca and chronic HCV infection.

BACKGROUND: The aim of this study was to determine the prevalence of keratoconjunctivitis sicca (KCS) in Greek patients with chronic hepatitis C virus (HCV) infection and its association with HCV genotypes and liver histology. PATIENTS AND METHODS: 93 HCVAb (+) patients underwent lacrimal function testing (Schirmer-1 test, break-up time test and Rose-Bengal staining test) and estimation of serum cryoglobulins and autoantibodies. 80 healthy volunteers were included in the study as controls. RESULTS: 34 out of 93 HCV patients (36.6%) and eight out of 80 healthy subjects (10%) had at least two abnormal lacrimal function tests suggestive of KCS (p < 0.001), cryoglobulinemia was evident in 20 patients (21.5%), rheumatoid factor (RF) in 43 (46.2%), antinuclear antibodies (ANA) in 19 (20.4%), antinuclear antigens (anti-SS-A and anti-SS-B) in one (1.1%) and two (2.2%) patients, respectively. Reduced prevalence of KCS was found in patients with genotype 3a compared to those with other genotypes (5/30, 16.7% vs 20/42, 47.6%, p = 0.007), probably because of their younger age. In patients with KCS a higher staging score was noted in liver biopsy compared to those without KCS (4.50 +/- 1.65 vs 3.06 +/- 1.88, p = 0.005). CONCLUSION: Greek patients with chronic HCV infection have a high prevalence of KCS (36.6%). The low frequency of anti-SS-A and anti-SS-B antibodies in these patients denotes different pathogenetic associations from primary Sjogren's syndrome.

Chronic hepatitis C virus infection without cirrhosis induces insulin resistance in patients with alpha-thalassaemia major.

AIM: The aim of this study was to investigate the cause of increased incidence of impaired glucose tolerance and diabetes mellitus in patients with alpha-thalassaemia major and chronic hepatitis C virus (HCV) infection without cirrhosis of the liver. PATIENTS AND METHODS: The study included 28 alpha-thalassaemic multi-transfused patients (14 females and 14 males; age, 25.7 +/- 6.3 years) with normal fasting glucose levels. Sixteen were seropositive for HCV and they had biopsy proven chronic hepatitis C without cirrhosis. An oral glucose tolerance test (OGTT) was performed. Glucose, insulin and C-peptide levels were measured every 30 min for 2 h. Fasting insulin resistance index (FIRI) was calculated according to the formula: FIRI = (fasting glucose x fasting insulin)/25. RESULTS: All patients had a normal OGTT except for two HCV positive and two HCV negative patients who had impaired glucose tolerance. HCV positive patients had higher fasting insulin levels (P = 0.02), higher fasting insulin/fasting glucose ratio (P = 0.017) and higher FIRI (P = 0.016) than HCV negative patients. During the OGTT, peak insulin levels occurred at 30 min in HCV negative patients but at 60 min in HCV positive. HCV infected patients had higher mean value of insulin at 60 (P = 0.017), 90 (P = 0.04), and 120 min (P = 0.04), and higher mean increment above basal at 60 (P = 0.015), 90 (P = 0.018) and 120 min (P = 0.05). The area under the curve (AUC) of insulin was also greater in HCV positive patients as compared to HCV negative (P = 0.04), although the AUC of glucose and the glucose levels at all time points of the OGTT were similar in both groups. CONCLUSIONS: The findings of this study show that alpha-thalassaemic patients with HCV infection without liver cirrhosis are more insulin resistant and have delayed insulin secretion compared to HCV negative alpha-thalassaemic patients. These changes in insulin action and secretion are evident before the development of impaired glucose tolerance and may explain the higher prevalence of diabetes mellitus in this group.

Non hypoxia-related splenic infarct in a patient with sickle cell trait and infectious mononucleosis.

Splenic infarction in patients with sickle cell trait is usually related to hypoxic conditions, while non-hypoxia-related infarcts are extremely rare. We report on a case of a 17-year-old male patient, living at sea level, who developed a severe left upper quadrant abdominal pain during the course of a febrile episode. On physical examination he had a mildly palpable but extremely painful spleen. A spleen scan revealed 2 areas of impaired radionucleide distribution. Hepatic enzymes were moderately increased and the IgM anti-EBV antibodies positive. Hemoglobin electrophoresis revealed the presence of 42% of hemoglobin S. A probable diagnosis of splenic infarction was established in a patient with sickle cell trait, during the course of infectious mononucleosis. The patient was treated symptomatically. The conditions of splenic congestion induced by the EBV infection and the high-grade fever may have contributed to splenic sequestration and subsequent infarcts.

HCV infection in the general population of a Greek island: prevalence and risk factors.

BACKGROUND/AIMS: Although HCV seroprevalence in blood donors in Greece is low (0.2-0.4%) epidemiologic characteristics of HCV infection in the general population have not been studied enough. The objective of this study was to examine the seroprevalence of HCV infection and associated risk factors in the general population of Zakinthos, a Greek island with a well-defined mixed (urban and rural) population. METHODOLOGY: A household health survey was carried out in a randomly selected sample of 718 adults. A questionnaire was completed and a blood sample was obtained from all participants. Serum samples were tested for anti-HCV antibodies by third generation enzyme-linked immunosorbent assay and supplemental test. The influence of sociodemographic characteristics and possible associated risk factors on the HCV seroprevalence was investigated by logistic regression analysis. RESULTS: The overall anti-HCV prevalence was 1.25%. A well-defined rural area with a significant higher prevalence (6.8% vs. 0.62%; P < 0.001) was identified. There was a trend of increasing prevalence with age, with a significant difference (P < 0.027) between the age groups 15-44 (0%) and over 45 (2.15%). The logistic regression analysis confirmed a significant association between anti- HCV positivity and: increasing age (P < 0.001), history of blood transfusion (0.0001), intramuscular injections (P < 0.04). CONCLUSIONS: The results of this field-survey in a well-defined general population, indicates that HCV seroprevalence (1.25%) is much higher than that of blood donors in the same area. The increasing prevalence with age and the association with parenteral exposure indicates that HCV infection can mainly be attributed to parenteral techniques in the past. The identification of a concrete rural area with particularly high seroprevalence needs further study of the whole population of the area.

Adult beta-thalassemic patients with chronic hepatitis C: long-term efficacy of alpha-IFN treatment.

Background: The purpose of this study was to assess the effectiveness of alpha-IFN in adult beta-thalassemic patients with chronic hepatitis C. After a long-term follow-up, we describe the special pattern of biochemical and virological response of thalassemics. Methods: Thirty-two anti-HCV-positive adult thalassemic patients (19 female and 13 male, mean age 23.4+/-5.5 years) with biopsy-proven chronic hepatitis were treated with IFN alpha2beta at a dose of 3 MU thrice weekly for 6-12 months. The patients were followed up until 45-62 months after the end of treatment. Results: A sustained response was obtained in eight patients (25%). Only two of the sustained responders (25%) normalized ALT during the first 3 months of treatment. Both early and late biochemical responders cleared HCV-RNA after 6 months of treatment. Eight patients (25%) responded with ALT normalization within 2 months of treatment but relapsed soon after stopping IFN. Sixteen patients (50%) did not respond to IFN. Conclusion: The response rate in multitransfused thalassemic patients with chronic hepatitis C treated with IFN is similar to that in non-thalassemics. The special feature of thalassemics is that early biochemical response does not predict a sustained response; on the contrary, patients who normalize ALT after 6 months of IFN treatment usually do not relapse.

High prevalence of diabetes mellitus among adult beta-thalassaemic patients with chronic hepatitis C.

AIM: The aim of this study was to assess the prevalence of diabetes mellitus in patients with hepatitis C virus (HCV) chronic hepatitis and secondary haemochromatosis as a consequence of beta-thalassaemia major. This group of patients was studied in order to reveal subtle effects of early stages of HCV infection on glucose metabolism, made more apparent by the coexistence of the diabetogenic effect of haemochromatosis. PATIENTS AND METHODS: The study included 108 beta-thalassaemic multitransfused patients, 55 females and 53 males, age 26.8+/-9 years. Sixty-four patients were seropositive for HCV by ELISA-3 (61/64 HCV-polymerase chain reaction-positive by Amplicor). In 51 of these, chronic hepatitis C was documented by liver biopsy, which also showed incomplete cirrhosis for eight and cirrhosis for four patients. Diabetes was diagnosed according to the criteria of the National Diabetes Data Group of the National Institutes of Health. RESULTS: (1) Patients with thalassaemia and HCV infection were diabetic more often than thalassaemic patients without HCV infection (45.3% versus 11.3%; P<0.001). This highly significant difference was also found when patients with definite cirrhosis or incomplete cirrhosis were excluded (41% versus 11.3%; P<0.01). (2) The high frequency of diabetes in thalassaemic patients with HCV chronic hepatitis is not related to body mass index or iron load, but it seems especially evident in patients over 25 years of age (50% of HCV-positive were diabetic versus 9.5% of HCV-negative; P<0.01). CONCLUSION: The frequency of diabetes in adult thalassaemic patients is significantly increased by HCV infection, even in the absence of cirrhosis. It is probable that the coexistence of haemochromatosis makes the effect of HCV infection on glucose metabolism clinically evident, even in the stage of chronic hepatitis.

Prevalence of intrapulmonary vascular dilatations in normoxaemic patients with early liver cirrhosis.

BACKGROUND: The aim of this study was to determine the prevalence of intrapulmonary vascular dilatations (IPVD) in normoxaemic patients with early liver cirrhosis and to compare their occurrence in progressive alcoholic versus postviral hepatic insufficiency. METHODS: Pulmonary function tests and arterial blood gas measurements were performed in 75 consecutive patients with cirrhosis of alcoholic and postviral aetiology. Contrast-enhanced echocardiography was used to identify IPVD. RESULTS: All patients were grade A or B in accordance with the Child-Pugh modified classification. Arterial blood gas analyses showed normoxaemia in all patients. Eight of 75 patients (10.7%) had a positive contrast echocardiogram, all with a decreased diffusion capacity (D1CO < 75% of the expected value). The abnormality was more prominent with advancing stage of liver failure (4.5% in grade A versus 19.4% in grade B; P < 0.05) and more common in patients with alcoholic cirrhosis (17.5% in alcoholic versus 2.9% in postviral cirrhosis; P < 0.05). CONCLUSION: In normoxaemic patients with early liver cirrhosis subclinical pulmonary vasodilatation, as assessed with contrast echocardiography, can occur. The finding is more prominent in alcoholic cirrhosis and possibly reflects an advancing degree of liver insufficiency.

Evaluation of contrast echocardiography and lung perfusion scan in detecting intrapulmonary vascular dilatation in normoxemic patients with early liver cirrhosis.

BACKGROUND/AIMS: Intrapulmonary vascular dilatations (IPVD) are extrahepatic complications occurring in liver transplant candidates, that can result in severe hypoxemia. The aim of this study was to compare the use of two diagnostic modalities, contrast echocardiography and lung perfusion scan, in detecting IPVD in normoxemic patients with early liver cirrhosis. METHODOLOGY: Fifty-six consecutive outpatients with biopsy-proven cirrhosis had contrast-echocardiography, a lung perfusion scan, pulmonary function tests, and arterial blood gas estimations. All patients were grade A or B according to the Child-Pugh classification. Patients with chronic intrinsic lung disease, heart failure or malignancy were excluded from the study. RESULTS: All patients had normal arterial blood-gas analyses. Eight out of 56 patients (14.3%) had a positive contrast echocardiogram, all with a decreased diffusion capacity (DLCO < 75% of the predicted value). An isolated DLCO impairment was observed in 40% of the patients with normal spirometry. None of the patients with echocardiography-proven IPVD had a positive lung perfusion scan (p<0.005). CONCLUSIONS: In normoxemic cirrhotic patients, subclinical pulmonary vasodilatation and gas-exchange abnormalities can occur. Contrast-enhanced echocardiography is the most valuable screening test in detecting IPVD in the early stages of hepatic insufficiency.

The leading role of hepatitis B and C viruses as risk factors for the development of hepatocellular carcinoma. A case control study.

A double case control study evaluated the role of hepatitis C virus (HCV) and hepatitis B virus (HBV), alcohol drinking, and tobacco smoking as potential risk factors for cepatocellular carcinoma (HCC). Fifty-one patients with HCC, 34 of whom had underlying cirrhosis, were analyzed against 51 hospital controls and 34 patients with cirrhosis, respectively. Sera from patients of all three groups were tested for HBV markers and anti-HCV antibodies. The polymerase chain reaction technique was used to detect HCV RNA in the anti-HCV-positive samples. Alcohol drinking and smoking habits were recorded for all patients. HCC risk was significantly related to the presence of hepatitis B surface antigen (HBsAg) [relative risk (RR) = 18], HCV infection (RR = 8), and alcohol abuse (RR = 4). When the presence of cirrhosis was taken into account, only HBsAg positivity was significantly associated with HCC development (RR = 6.7), indicating that HCV infection and alcohol abuse are related to HCC indirectly through the cirrhotic process. No significant interaction between HCV and HBV infection in the causation of HCC was found. Through the computation of population-attributable risk, it was found that 46% of the HCC cases in Greece could be attributed to HBsAg positivity but only 4% to HCV infection. In conclusion, HBV infection is the major risk factor in the development of HCC in Greece, either by inducing cirrhosis or by direct oncogenic effect. HCV infection is also related to HCC development, albeit indirectly through the cirrhotic process.

Sarcoidosis in a patient with myasthenia gravis. Case report and review of the literature.

The case of a 38-year-old white female, with a long history of myasthenia gravis, who developed sarcoidosis is presented. The diagnosis of myasthenia gravis had been established on the basis of typical clinical symptoms with severe myastenic crises necessitating respiratory support, characteristic electromyographic findings, positive anti-acetylcholine receptor antibodies and response to appropriate medications. After 9 years of disease and while in apparent remission, she presented with ankle arthritis and bilateral hilar adenopathy. A transbronchial lung biopsy showed noncaseating granuloma, typical of sarcoidosis. To our knowledge, the combination of myasthenia gravis and sarcoidosis is extremely rare and emphasizes the intriguing tendency of some immunologic disorders to appear together in certain individuals.

The diagnostic value of gastrocnemius muscle biopsy in sarcoidosis presenting with erythema nodosum and hilar adenopathy.

Five cases of sarcoidosis were identified among twenty cases of erythema nodosum, admitted to the Department of Medicine, during a two-year period. The clinical picture was dominated by hilar adenopathy and erythema nodosum in all cases. All five patients had gastrocnemius muscle biopsy performed, which revealed noncaseating epithelioid granuloma in four. Diagnosis in the fifth patient was made by transbronchial lung biopsy. The present study suggests, that gastrocnemius muscle biopsy might be considered as the initial diagnostic procedure of choice in this, more commonly seen in Caucasians, syndrome of sarcoidosis, presenting with hilar adenopathy and erythema nodosum, based on its safety, ease of performance and relatively high sensitivity and specificity.

The reproductive potential of normal mouse embryo fibroblasts during culture in vitro.

A direct estimate of the reproductive potential of mouse embryo fibroblasts through their entire lifespan has been made using the mini-clone technique, which permits the direct observation of the growth fraction in a bulk population by inspection of the growth behaviour of individual cells. We have measured the colony size on each island that contained one or two cells at the beginning and the fraction of islands which, starting from one or two cells failed to divide even once. We observed that even in a young culture there are individual cells that can only reproduce two or three times. With each succeeding passage the distribution of colony sizes shifts to a greater proportion of small colonies. The median colony size decreases with each passage. Furthermore, the fraction of non-dividers directly observed increases smoothly with time; the fraction of non-dividers is quite small at the first passage but increases steadily to reach 0.6 at the last passage, after about 30 generations. These smooth changes in the growth behaviour of this cell strain are accurately described by the mortalization theory of Shall and Stein, in which the single parameter gamma, describes the change in reproductive potential over the entire lifespan. The parameter gamma describes the rate at which the doubling time of the culture increases; it is the number of generations at which half of the newborn cells are themselves reproductively sterile. Our present data provide an estimate of gamma for this cell strain equal to 21.2 generations, which compares well with a previous estimate of 20.3 generations.

Kinetics of in vitro ageing of mouse embryo fibroblasts.

The kinetics of ageing of normal mouse embryo fibroblast cells in culture have been determined. The growth pattern during every passage was established. It was observed that the growth pattern was not exponential, but that the growth rate declined progressively both within and with every passage. We also estimated the cell cycle parameters using the Fraction of Labelled Mitoses method at every passage. We found that the cell cycle duration was constant throughout the lifespan of this cell strain; the median value of the cell cycle duration was found to be 15.5 +/- 0.5 h (S.D., n = 8). From these two sets of observations we infer that the fraction of dividing cells declines smoothly from the beginning of the culture. Our data exclude quite positively any description of ageing of the fibroblast population in terms of a catastrophe or any abrupt change in the population. Our data are also inconsistent with a linear decline in growth fraction. On the contrary, we observed that there was a gradual and smooth decline in the growth rate of the strain, due to a smoothly declining growth fraction. This smooth change in the growth behaviour of this cell strain is accurately described by the mortalization theory of Shall & Stein in which the single parameter gamma (gamma), describes the change in reproductive potential over the entire lifespan. The parameter gamma describes the rate at which the doubling time of the culture increases. It is the number of generations at which half of the newborn cells are themselves reproductively sterile. Our present data provided an estimate of gamma for this cell strain, which was consistent during the entire lifespan of the strain; the best estimate of gamma for this cell strain was 20.3 +/- 0.6 generations (S.D., n = 19).