Romil Saxena

Nomenclature of the finer branches of the biliary tree: Canals, ductules, and ductular reactions in human livers

The work of liver stem cell biologists, largely carried out in rodent models, has now started to manifest in human investigations and applications. We can now recognize complex regenerative processes in tissue specimens that had only been suspected for decades, but we also struggle to describe what we see in human tissues in a way that takes into account the findings from the animal investigations, using a language derived from species not, in fact, so much like our own. This international group of liver pathologists and hepatologists, most of whom are actively engaged in both clinical work and scientific research, seeks to arrive at a consensus on nomenclature for normal human livers and human reactive lesions that can facilitate more rapid advancement of our field. (HEPATOLOGY 2004; 39:1739–1745.)

Pathologic diagnosis of early hepatocellular carcinoma : a report of the international consensus group for hepatocellular neoplasia

Advances in imaging techniques and establishment of surveillance protocols for high-risk populations have led to the detection of small hepatic nodules in patients with chronic liver diseases, particularly those with cirrhosis or chronic hepatitis caused by hepatitis B or C viruses. These nodules, comprising a broad range of diagnostic entities—some benign and some with malignant potential—are currently defined histologically, and their clinical management often depends on the ability to make a reliable histologic diagnosis. Evidence accumulated in the last two decades strongly favors the existence of a sequence of events in hepatic nodules that precedes the emergence of hepatocellular carcinoma (HCC),1-10 and these lesions are recognized as precursors of HCC. However, from the beginning of their recognition, there has been considerable confusion concerning nomenclature and diagnostic approaches to these hepatic nodules. To clarify these issues, an International Working Party (IWP) of the World Congresses of Gastroenterology proposed a consensus nomenclature and diagnostic criteria for hepatocellular nodular lesions in 1995.11 The IWP classified nodular lesions found in chronic liver disease into large regenerative nodule, lowgrade dysplastic nodule (L-DN), high-grade dysplastic nodule (H-DN), and HCC; this nomenclature has been widely adopted. In addition, the IWP introduced the concept of dysplastic focus as a cluster of hepatocytes with features of early neoplasia (in particular small cell change or iron-free foci in a siderotic background) measuring less than 0.1 cm, and defined small HCC as a tumor measuring less than 2 cm. More recent studies support the division of small HCC into two clinico-pathological groups that have been termed early HCC and progressed HCC. Early HCC has a vaguely nodular appearance and is well differentiated. Progressed HCC has a distinctly nodular pattern and is mostly moderately differentiated, often with evidence of microvascular invasion.12 Early HCC has a longer time to recurrence and a higher 5-year survival rate compared with progressed HCC.13 Small lesions with malignant potential have only subtle differences from the surrounding parenchyma, making them difficult to assess reproducibly. Differences in the application of diagnostic criteria between Western and Eastern pathologists has been a persistent difficulty in research and clinical management of these lesions.14 In order to obtain a refined and up-to-date international consensus on the histopathologic diagnosis of nodular lesions, such as dysplastic nodules and early HCC, the International Consensus Group for Hepatocellular Neoplasia (ICGHN) was convened in April 2002 in Kurume, Japan. The group has met several times up to July 2007 under the auspices of the Laennec Liver Pathology Society. The ICGHN is currently comprised of 34 pathologists and two clinicians from 13 countries. It includes most members of the original IWP who are still active and all the participants from the first ICGHN meeting. This consensus document summarizes the results of our meetings.

Radiation pneumonitis in mice: A severe injury model for pneumocyte engraftment from bone marrow

OBJECTIVE To better understand the process by which pneumocytes can be derived from bone marrow cells, we investigated the in vivo kinetics of such engraftment following lethal irradiation. METHODS A cohort of lethally irradiated B6D2F1 female mice received whole bone marrow transplants (BMT) from age-matched male donors and were sacrificed at days 1, 3, 5, and 7 and months 2, 4, and 6 post-BMT (n = 3 for each time point). Additionally, 2 female mice who had received 200 male fluorescence-activated cell sorter (FACS)-sorted CD34(+)lin(-) cells were sacrificed 8 months post-BMT. RESULTS Lethal irradiation caused histologic evidence of pneumonitis including alveolar breakdown and hemorrhage beginning at day 3. To identify male-derived pneumocytes, simultaneous fluorescence in situ hybridization (FISH) for Y-chromosome and surfactant B messenger RNA was performed on lung tissue. Y(+) type II pneumocytes were engrafted as early as day 5 posttransplant, and eventually from 2 to 14% of the pneumocytes were donor derived in individual mice. Co-staining for epithelial-specific cytokeratins demonstrated that by 2 months, marrow-derived pneumocytes could comprise entire alveoli, suggesting that type I cells derived from type II pneumocytes. CONCLUSIONS We conclude that alveolar lining cells derive from bone marrow cells immediately after acute injury. Also, the CD34(+)lin(-) subpopulation is capable of such pulmonary engraftment.

Fatty pancreas: A factor in postoperative pancreatic fistula

Objective:To determine whether patients who develop a pancreatic fistula after pancreatoduodenectomy are more likely to have pancreatic fat than matched controls. Background:Pancreatic fistula continues to be a major cause of postoperative morbidity and increased length of stay after pancreatoduodenectomy. Factors associated with postoperative pancreatic fistula include a soft pancreas, a small pancreatic duct, the underlying pancreatic pathology, the regional blood supply, and surgeons experience. Fatty pancreas previously has not been considered as a contributing factor in the development of postoperative pancreatic fistula. Methods:Forty patients with and without a pancreatic fistula were identified from an Indiana University database of over 1000 patients undergoing pancreatoduodenectomy and matched for multiple parameters including age, gender, pancreatic pathology, surgeon, and type of operation. Surgical pathology specimens from the pancreatic neck were reviewed blindly for fat, fibrosis, vessel density, and inflammation. These parameters were scored (0–4+). Results:The pancreatic fistula patients were less likely (P < 0.05) to have diabetes but had significantly more intralobular (P < 0.001), interlobular (P < 0.05), and total pancreatic fat (P < 0.001). Fistula patients were more likely to have high pancreatic fat scores (50% vs. 13%, P < 0.001). Pancreatic fibrosis, vessel density, and duct size were lower (P < 0.001) in the fistula patients and negative correlations (P < 0.001) existed between fat and fibrosis (R = −0.40) and blood vessel density (R = −0.15). Conclusions:These data suggest that patients with postoperative pancreatic fistula have (1) increased pancreatic fat and (2) decreased pancreatic fibrosis, blood vessel density, and duct size. Therefore, we conclude that fatty pancreas is a risk factor for postoperative pancreatic fistula.

Partial hepatectomy-induced polyploidy attenuates hepatocyte replication and activates cell aging events

In understanding mechanisms of liver repopulation with transplanted hepatocytes, we studied the consequences of hepatic polyploidization in the two-thirds partial hepatectomy model of liver regeneration. Liver repopulation studies using genetically marked rodent hepatocytes showed that the number of previously transplanted hepatocytes did not increase in the liver with subsequential partial hepatectomy. In contrast, recipients undergoing partial hepatectomy before cells were transplanted showed proliferation in transplanted hepatocytes, with kinetics of DNA synthesis differing in transplanted and host hepatocytes. Also, partial hepatectomy caused multiple changes in the rat liver, including accumulation of polyploid hepatocytes along with prolonged depletion of diploid hepatocytes, as well as increased senescence-associated β-galactosidase and p21 expression. Remnant hepatocytes in the partially hepatectomized liver showed increased autofluorescence and cytoplasmic complexity on flow cytometry, which are associated with lipofuscin accumulation during cell aging, and underwent apoptosis more frequently. Moreover, hepatocytes from the partially hepatectomized liver showed attenuated proliferative capacity in cell culture. These findings were compatible with decreased proliferative potential of hepatocytes experiencing partial hepatectomy compared with hepatocytes from the unperturbed liver. Attenuation of proliferative capacity and other changes in hepatocytes experiencing partial hepatectomy offer novel perspectives concerning liver regeneration in the context of cell ploidy.In understanding mechanisms of liver repopulation with transplanted hepatocytes, we studied the consequences of hepatic polyploidization in the two-thirds partial hepatectomy model of liver regeneration. Liver repopulation studies using genetically marked rodent hepatocytes showed that the number of previously transplanted hepatocytes did not increase in the liver with subsequential partial hepatectomy. In contrast, recipients undergoing partial hepatectomy before cells were transplanted showed proliferation in transplanted hepatocytes, with kinetics of DNA synthesis differing in transplanted and host hepatocytes. Also, partial hepatectomy caused multiple changes in the rat liver, including accumulation of polyploid hepatocytes along with prolonged depletion of diploid hepatocytes, as well as increased senescence-associated beta-galactosidase and p21 expression. Remnant hepatocytes in the partially hepatectomized liver showed increased autofluorescence and cytoplasmic complexity on flow cytometry, which are associated with lipofuscin accumulation during cell aging, and underwent apoptosis more frequently. Moreover, hepatocytes from the partially hepatectomized liver showed attenuated proliferative capacity in cell culture. These findings were compatible with decreased proliferative potential of hepatocytes experiencing partial hepatectomy compared with hepatocytes from the unperturbed liver. Attenuation of proliferative capacity and other changes in hepatocytes experiencing partial hepatectomy offer novel perspectives concerning liver regeneration in the context of cell ploidy.

Correlation of CT Enteroclysis With Surgical Pathology in Crohn's Disease

BACKGROUND:The response to therapy in Crohns disease (CD) depends on the inflammatory or fibrostenotic nature of the underlying pathological process. Standard diagnostic tests cannot reliably distinguish between these two entities. Although CT enteroclysis (CTE) has shown promise in the evaluation of small bowel disorders, its accuracy for the differentiation of CD phenotypes is unknown.AIMS:To determine the accuracy of CTE compared with surgical pathology in patients with CD and to assess the association of CTE variables with inflammatory or fibrostenotic pathological lesions.METHODS:CTE studies from patients who underwent resective bowel surgery for CD were reviewed and compared with the pathological specimens using a standardized scoring system. Patients were excluded if they had incomplete studies, nonresective surgeries, or a diagnosis of malignancy. CTE variables, such as mucosal and mural enhancement, wall thickness, engorgement of vasa recta (comb sign), adenopathy, and the presence and severity of strictures were compared with the pathology results using Mantel-Haenszel χ2, Spearmans rank coefficient, and logistic regression analyses.RESULTS:Of the 54 patients enrolled, 10 were excluded. The remaining patients (61% female, 84% white) underwent 44 surgical interventions generating 47 bowel segments that were included in the analysis. The indications for surgery were: bowel obstruction in 21; perforating disease in 13; and refractory, nonobstructive disease in 15. The accuracy of CTE for inflammatory and fibrostenotic lesions was 76.6% and 78.7% using a four- and three-point grading system, respectively. There was good correlation between CTE and pathology in regards to inflammation (Spearmans r = 0.7, P < 0.0001) and fibrostenosis (Spearmans r = 0.6, P < 0.0001) scores. The pathological inflammation score was significantly associated with the CTE variables mucosal enhancement, wall thickness, comb sign, and adenopathy (Mantel-Haenszel χ2 P values 0.04, 0.04, <0.0001, and 0.016, respectively). The pathological fibrostenosis score was significantly associated with the presence and severity of stenosis on CTE (P = 0.001 and 0.007, respectively). By logistic regression analysis, the strongest association was seen with the comb sign (OR 5.52, P < 0.001) for inflammation and the presence of stenosis (OR 5.87, P = 0.006) for fibrostenosis. There was no interaction between the time interval from CTE to surgery and the strength of these associations.CONCLUSIONS:CTE may reliably differentiate between inflammatory and fibrostenotic lesions and may have an important role in the management of CD. Specific CTE variables correlate with each of these phenotypes and deserve further investigations in prospective studies.

Value of Diffusion-Weighted MRI for Assessing Liver Fibrosis and Cirrhosis

OBJECTIVE The objective of our study was to determine the usefulness of the apparent diffusion coefficient (ADC) of liver parenchyma for determining the severity of liver fibrosis. MATERIALS AND METHODS This study investigated 78 patients who underwent diffusion-weighted imaging (DWI) with 1.5-T MRI and pathologic staging of liver fibrosis based on biopsy. DWI was performed with b values of 50 and 400 s/mm(2). ADCs of liver were measured using 2.0- to 3.0-cm(2) regions of interest in the right and left lobes of the liver; the mean ADC value was used for analysis. Pathologic METAVIR scores for liver fibrosis stage were used as a reference standard. RESULTS The mean ADC values for fibrosis pathologically staged using the METAVIR classification system as F0 (n = 11), F1 (n = 16), F2 (n = 10), F3 (n = 14), and F4 (n = 27) were 125.9, 105.0, 104.5, 103.2, and 99.1 x 10(-5) s/mm(2), respectively. The correlation between the ADC values and the degree of liver fibrosis was moderate (Spearmans test, rho = -0.36). There was a significant difference in ADC values between patients with nonfibrotic liver (F0) and those with cirrhotic liver (F4) (p = 0.008). The best cutoff ADC value to distinguish between these groups was 118 x 10(-5) s/mm(2). However, ADC values were not useful for differentiating viral hepatitis patients with F2 fibrosis or higher from those with a lower degree of fibrosis (area under the receiver operating characteristic curve [AUC] = 0.66) or for differentiating low-stage fibrosis in all patients from high-stage fibrosis in all patients (AUC = 0.54). CONCLUSION The ADCs in cirrhotic livers are significantly lower than those in nonfibrotic livers. However, ADC values measured using the current generation of scanners are not reliable enough to replace liver biopsy for staging hepatic fibrosis.

Nutritional model of steatohepatitis and metabolic syndrome in the Ossabaw miniature swine

Miniature pigs residing in the Ossabaw Island (Ossabaw pigs) exhibit a thrifty genotype, and when fed a high‐calorie diet they consistently develop metabolic syndrome defined by obesity, insulin resistance, hypertension, and dyslipidemia. We conducted a study to induce steatohepatitis in Ossabaw pigs by dietary manipulation. Pigs were fed standard chow (controls, n = 15), high‐fructose diet (20% kcal from fructose and 10.5% kcal from fat) (fructose group, n = 9), atherogenic diet (20% kcal from fructose and 46% kcal from fat and 2% cholesterol and 0.7% cholate by weight) (atherogenic diet group, n = 13), and modified atherogenic diet (different source of fat and higher protein but lower choline content) (M‐Ath diet group, n = 7). All animals were sacrificed at 24 weeks after dietary intervention. The high‐fructose group had significant weight gain, hypertension, and insulin resistance but showed normal liver histology. The atherogenic diet group had metabolic syndrome and abnormal liver histology consisting of significant microvesicular steatosis and fatty Kupffer cells but no ballooning or fibrosis. The M‐Ath diet group developed severe metabolic syndrome and markedly abnormal liver histology with macrovesicular and microvesicular steatosis, fatty Kupffer cells, extensive hepatocyte ballooning, and pericellular/perisinusoidal fibrosis. Compared with controls, the M‐Ath diet group had significantly lower serum adiponectin but higher serum leptin and tumor necrosis factor (TNF) levels and higher hepatic triglyceride and malondialdehyde levels. Conclusion: Ossabaw pigs fed a modified atherogenic diet develop severe metabolic syndrome and abnormal liver histology with close resemblance to human nonalcoholic steatohepatitis (NASH). (HEPATOLOGY 2009.)

Nonalcoholic fatty pancreas disease

BACKGROUND Obesity leads to fat infiltration of multiple organs including the heart, kidneys, and liver. Under conditions of oxidative stress, fat-derived cytokines are released locally and result in an inflammatory process and organ dysfunction. In the liver, fat infiltration has been termed nonalcoholic fatty liver disease, which may lead to nonalcoholic steatohepatitis. No data are available, however, on the influence of obesity on pancreatic fat and cytokines, and nonalcoholic fatty pancreas disease (NAFPD) has not been described. Therefore, we designed a study to determine whether obesity is associated with increased pancreatic fat and cytokines. MATERIALS AND METHODS Thirty C57BL/6J lean control and 30 leptin-deficient obese female mice were fed a 15% fat diet for 4 weeks. At 12 weeks of age all animals underwent total pancreatectomy. Pancreata from each strain were pooled for measurement of a) wet and dry weight, b) histologic presence of fat, c) triglycerides, free fatty acids (FFAs), cholesterol, phospholipids, and total fat, and d) interleukin (IL)-1beta and tumor necrosis factor-alpha (TNF-alpha). Data were analyzed by Students t test and Fishers exact test. RESULTS Pancreata from obese mice were heavier (p<0.05) and had more fat histologically (p<0.05). Pancreata from obese mice had more triglycerides, FFAs, cholesterol, and total fat (p<0.05). Triglycerides represented 11% of pancreatic fat in lean mice compared with 67% of pancreatic fat in obese mice (p<0.01). Cytokines IL-1beta and TNF-alpha also were elevated in the pancreata of obese mice (p<0.05). CONCLUSIONS These data suggest that obese mice have 1) heavier pancreata, 2) more pancreatic fat, especially triglycerides and FFAs, and 3) increased cytokines. We conclude that obesity leads to nonalcoholic fatty pancreatic disease.

Autotaxin expression and its connection with the TNF-alpha-NF-κB axis in human hepatocellular carcinoma

BackgroundAutotaxin (ATX) is an extracellular lysophospholipase D that generates lysophosphatidic acid (LPA) from lysophosphatidylcholine (LPC). Both ATX and LPA have been shown to be involved in many cancers. However, the functional role of ATX and the regulation of ATX expression in human hepatocellular carcinoma (HCC) remain elusive.ResultsIn this study, ATX expression was evaluated in tissues from 38 human HCC and 10 normal control subjects. ATX was detected mainly in tumor cells within tissue sections and its over-expression in HCC was specifically correlated with inflammation and liver cirrhosis. In addition, ATX expression was examined in normal human hepatocytes and liver cancer cell lines. Hepatoma Hep3B and Huh7 cells displayed stronger ATX expression than hepatoblastoma HepG2 cells and normal hepatocytes did. Proinflammtory cytokine tumor necrosis factor alpha (TNF-α) promoted ATX expression and secretion selectively in Hep3B and Huh7 cells, which led to a corresponding increase in lysophospholipase-D activity. Moreover, we explored the mechanism governing the expression of ATX in hepatoma cells and established a critical role of nuclear factor-kappa B (NF-κB) in basal and TNF-α induced ATX expression. Further study showed that secreted enzymatically active ATX stimulated Hep3B cell invasion.ConclusionsThis report highlights for the first time the clinical and biological evidence for the involvement of ATX in human HCC. Our observation that links the TNF-α/NF-κB axis and the ATX-LPA signaling pathway suggests that ATX is likely playing an important role in inflammation related liver tumorigenesis.