Desley Neil

HIF-1 is expressed in normoxic tissue and displays an organ-specific regulation under systemic hypoxia

Adaptation to hypoxia is regulated by hypoxia‐inducible factor 1 (HIF‐1), a heterodimeric transcription factor consisting of an oxygen‐regulated α subunit and a constitutively expressed β subunit. Although HIF‐1 is regulated mainly by oxygen tension through the oxygen‐dependent degradation of its α subunit, in vitro it can also be modulated by cytokines, hormones and genetic alterations. To investigate HIF‐1 activation in vivo, we determined the spatial and temporal distribution of HIF‐1 in healthy mice subjected to varying fractions of inspiratory oxygen. Immunohisto‐chemical examination of brain, kidney, liver, heart, and skeletal muscle revealed that HIF‐1α is present in mice kept under normoxic conditions and is further in‐creased in response to systemic hypoxia. Moreover, immunoblot analysis showed that the kinetics of HIF‐1 α expression varies among different organs. In liver and kidney, HIF‐1 α reaches maximal levels after1hand gradually decreases to baseline levels after4hof continuous hypoxia. In the brain, however, HIF‐1α is maximally expressed after 5 h and declines to basal levels by 12 h. Whereas HIF‐1 β is constitutively expressed in brain and kidney nuclear extracts, its hepatic expression increases concomitantly with HIF‐1 α. Overall, HIF‐1 α expression in normoxic mice suggests that HIF‐1 has an important role in tissue homeostasis.—Stroka, D. M., Burkhardt, T., Desbaillets, I., Wenger, R. H., Neil, D. A. H., Bauer, C., Gassmann, M., Candinas, D. HIF‐1 is expressed in normoxic tissue and displays an organ specific regulation under systemic hypoxia. FASEB J. 15, 2445–2453 (2001)

Distinct patterns of chemokine expression are associated with leukocyte recruitment in alcoholic hepatitis and alcoholic cirrhosis

Alcoholic liver disease is associated with three histologically distinct processes: steatosis (parenchymal fat accumulation), alcoholic hapatitis (characterized by parenchymal infiltration by neutrophil polymorphs), and alcoholic cirrhosis (in which chronic inflammation and fibrosis dominate). Chemokines are cytokines that promote subset‐specific leukoycte recruitment to tissues and could therefore play a crucial role in determining which leukocyte subsets are recruited to the liver in alcoholic liver disease. This paper reports that chemokine expression is increased in the liver of patients with alcoholic liver disease and, moreover, that distinct patterns of chemokine expression are associated with the different inflammatory responses to alcohol. Interleukin‐8 (IL‐8), monocyte chemoattractant protein‐1 (MCP‐1), macrophage inflammatory protein‐1α (MIP‐1α), and MIP‐1β were all detected in the parenchyma at sites of inflammation in alcoholic hepatitis, whereas in alcoholic cirrhosis, chemokines were restricted to inflammatory cells and endothelium in the fibrous septa and portal tracts. In alcoholic hepatitis, chemokine transcription was localized to sinusoidal cells, leukocytes, and fibroblasts in areas of parenchymal inflammation, but hepatocytes, despite staining strongly for chemokine protein, were negative. In alcoholic cirrhosis, chemokine mRNA was detected in portal tract endothelium, leukocytes, and fibroblasts. Thus, alcoholic hepatitis and alcoholic cirrhosis are associated with distinct patterns of chemokine expression that are likely to be important factors in determining whether a patient develops acute parenchymal inflammation and alcoholic hepatitis, or chronic septal inflammation and alcoholic cirrhosis.

Current views on rejection pathology in liver transplantation.

Histological assessments continue to play an important role in the diagnosis and management of liver allograft rejection. The changes occurring in acute and chronic rejection are well recognized and liver biopsy remains the ‘gold standard’ for diagnosing these two conditions. Recent interest has focused on the diagnosis of late cellular rejection, which may have different histological appearances to early acute rejection and instead has features that overlap with so‐called ‘de novo autoimmune hepatitis’ and ‘idiopathic post‐transplant chronic hepatitis’. There is increasing evidence to suggest that ‘central perivenulitis’ may be an important manifestation of late rejection, although other causes of centrilobular necro‐inflammation need to be considered in the differential diagnosis. There are also important areas of overlap between rejection and recurrent hepatitis C infection and the distinction between these two conditions continues to be a problem in the assessment of liver allograft biopsies. Studies using immunohistochemical staining for C4d as a marker for antibody‐mediated damage have found evidence of C4d deposition in liver allograft rejection, but the functional significance of these observations is currently uncertain. This review will focus on these difficult and controversial areas in the pathology of rejection, documenting what is currently known and identifying areas where further clarification is required.

Are parenchymal changes in early post-transplant biopsies related to preservation-reperfusion injury or rejection?

Background. The progression of parenchymal changes in liver allograft biopsies due to preservation-reperfusion injury (PRI) and their differentiation from rejection related changes is poorly understood. The aim of this study was to determine which changes in a 1-week posttransplant biopsy could be attributed to PRI and which to acute rejection. Methods. One week protocol liver transplant biopsies from patients with mild PRI (day 1 AST<400 IU/L) were compared with those from patients with severe PRI (day 1 AST>2000 IU/L). Parenchymal changes (cholestasis, ballooning, steatosis, necrosis) and rejection-related inflammatory features (portal tract inflammation, bile duct inflammation, portal vein endothelial inflammation, hepatic vein endothelial inflammation, and centrilobular inflammation) were blindly assessed semiquantitatively. Results. Fat, cholestasis, and hepatocyte ballooning were significantly worse in the severe PRI group, and these features showed no correlation with histological features related to acute rejection. Centrilobular hepatocyte necrosis correlated with hepatic venular endothelial inflammation and centrilobular inflammation but not with rejection related features in portal tracts or with PRI. These findings suggest that centrilobular necrosis is a manifestation of a rejection-related parenchymal injury and may involve different pathogenetic mechanisms to rejection-related features in portal tracts. Conclusions. This study indicates that in early posttransplant biopsies, fat, cholestasis, and ballooning can largely be attributed to PRI. By contrast, centrilobular hepatocyte loss should be suspected as a rejection related phenomenon, even if typical portal tract changes are not prominent, and augmentation of immunosuppression should be considered.

CD4+CD28- T cell expansion in granulomatosis with polyangiitis (Wegener's) is driven by latent cytomegalovirus infection and is associated with an increased risk of infection and mortality.

OBJECTIVEnExpanded populations of CD4+CD28- T cells with a cytotoxic phenotype have been repeatedly reported in patients with granulomatosis with polyangiitis (Wegeners) (GPA). In healthy individuals expansion of this T cell population follows cytomegalovirus (CMV) infection. We undertook this study to investigate whether CMV infection may be responsible for driving the expansion of CD4+CD28- T cells in GPA patients and how this might relate to clinical features.nnnMETHODSnForty-eight GPA patients and 38 age-matched healthy donors were included in the study. CMV-specific IgG in serum was detected by enzyme-linked immunosorbent assay. Flow cytometric analysis was used to study T cell populations and phenotype. The presence of CMV in renal biopsy tissue from GPA patients was investigated by immunohistochemistry and polymerase chain reaction (PCR). Clinical information was obtained from patient records.nnnRESULTSnPopulations of CD4+CD28- T cells were only expanded in CMV-seropositive GPA patients and controls. In CMV-seropositive GPA patients we observed negative correlations between the percentages of CD4+CD28- T cells and both the percentage of naive T cells and the glomerular filtration rate at presentation. There was a significant association between the percentage of CD4+CD28- T cells and risk of infection and mortality. CMV could not be detected in renal tissue by PCR or immunohistochemistry. CMV seropositivity itself was not a risk factor for infection in a cohort of 182 patients with antineutrophil cytoplasmic antibody-associated vasculitis who had been recruited into clinical trials performed by the European Vasculitis Study Group.nnnCONCLUSIONnThe expansion of CD4+CD28- T cells in GPA patients is associated with CMV infection and leads to a reduction in the number of naive T cells in peripheral blood. Patients with expanded CD4+CD28- T cells have significantly increased mortality and risk of infection.

Idiopathic Noncirrhotic Intrahepatic Portal Hypertension is Associated with Sustained ADAMTS13 Deficiency

BackgroundADAMTS13 deficiency leading to excess ultralarge von Willebrand factor (VWF) multimers and platelet clumping is typically found in thrombotic thrombocytopenic purpura (a type of thrombotic microangiopathy). Idiopathic noncirrhotic intrahepatic portal hypertension (NCIPH) is a microangiopathy of portal venules associated with significant thrombocytopenia and predisposing gut disorders.AimTo determine whether the portal microangiopathy in NCIPH is associated with ADAMTS13 deficiency.MethodsPlasma levels of ADAMTS13, anti-ADAMTS13 antibodies, and VWF were compared between cases (NCIPH patients) and controls (with chronic liver diseases of other etiology) matched for severity of liver dysfunction. Eighteen NCIPH patients [median (range) MELD score 12 (7–25)] and 25 controls [MELD score 11 (4–26)] were studied.ResultsADAMTS13 activity was reduced in all 18 NCIPH patients and significantly lower than controls (median, IQR: 12.5%, 5–25% and 59.0%, 44–84%, respectively, Pxa0<xa00.0001) [normal range for plasma ADAMTS13 activity (55–160%)]. ADAMTS13 activity was <5% in 5/18 NCIPH patients (28%) and 0/25 controls (Pxa0=xa00.009). ADAMTS13 antigen levels were also decreased. Sustained low ADAMTS13 levels were seen in four NCIPH patients over 6xa0weeks to 11xa0months (highest ADAMTS13 level in each patient: <5%, 6%, 6%, and 25%), despite two patients having MELD score 12. Although nine cases had low titer anti-ADAMTS13 antibodies, there was no significant difference between cases and controls. Abnormally large VWF multimers were observed in 4/11 NCIPH patients (36%) and in 0/22 controls (Pxa0=xa00.008).ConclusionsSustained deficiency of ADAMTS13 appears characteristic of NCIPH, irrespective of severity of liver disease.

Intrahepatic complement activation, sinusoidal endothelial injury, and lactic acidosis are associated with initial poor function of the liver after transplantation.

Background. Changes in glucose metabolism in the liver during transplantation have been recently described using microdialysis. Here, these findings are correlated with histopathologic, immunohistochemical, and ultrastructural changes in liver. Methods. Microdialysis catheters were inserted into 15 human livers, which were perfused with isotonic solution, and samples of perfusate were analyzed before harvest, after storage, and after reperfusion. At each stage Menghini needle biopsy samples were taken and each studied using light and electron microscopy. Results. Six livers showed serum biochemical evidence of initial poor function. These livers had significantly more staining for complement fragment 4d (C4d) of both lobular and periportal hepatocytes. C4d-positive hepatocytes were also found in the liver during cold storage (3 of 15). These periportal hepatocytes also showed evidence of necrosis and were found to have intracellular neutrophils. Hepatocyte rounding in zone III, necrosis, and C4d staining in recipient were also significantly correlated with the degree of lactic acidosis during this phase. Intrahepatic lactic acidosis at all time points was significantly associated with sinusoidal endothelial cell injury after reperfusion. There were no correlations between glucose, pyruvate, and glycerol levels and histopathologic changes in the liver. Discussion. In the patients studied, the degree of C4d staining correlated with initial poor function and was associated with intrahepatic lactic acidosis in the donor during cold storage and after reperfusion. Complement activity in the liver during cold storage may be after in situ activation. Intrahepatic lactic acidosis is associated with sinusoidal endothelial cell and hepatocyte injury. The role of intrahepatic neutrophils is uncertain and could possibly be in response to cell necrosis.

Chronic Kidney Disease After Nonrenal Solid Organ Transplantation: A Histological Assessment and Utility of Chronic Allograft Damage Index Scoring

Background. It is proposed that chronic calcineurin inhibitor (CNI) nephrotoxicity has a central role in chronic kidney disease after nonrenal solid organ transplantation (NRSOT), although there are little data on renal histology in this setting. The aim of this study was to assess the histological features and renal outcomes of a cohort of patients with chronic kidney disease after NRSOT. Methods. Renal biopsies of 62 NRSOT recipients were evaluated for histological diagnoses. Biopsies were graded for chronic allograft damage index parameters and for arteriolar hyalinosis. The sum of all chronic allograft damage index parameters and arteriolar hyalinosis scores was called chronic damage index. Results. The biopsies were performed at a median of 4 (range: 0.3–15.9) years after NRSOT and at serum creatinine of 318±17.7 &mgr;mol/L (mean±standard deviation). Twenty-two (35.5%) biopsies showed predominant features of chronic CNI nephrotoxicity, 27 (43.5%) predominant features of hypertensive nephropathy, and 12 (19.3%) an alternative primary renal pathology. Twenty-four (38.7%) patients had progression to end-stage renal disease, at a median of 1.5 (0–10.1) years after biopsy and 6.9 (0.3–19.2) years after NRSOT. The risk of renal progression was associated with in situ damage measured by chronic damage index. Conclusions. Although CNI nephrotoxicity is an important cause of renal failure after NRSOT, many patients do not have overt histological evidence of CNI toxicity. Quantitative parameters of chronic damage can stratify renal prognosis.

The Use of an Acellular Oxygen Carrier in a Human Liver Model of Normothermic Machine Perfusion

Background Normothermic machine perfusion of the liver (NMP-L) is a novel technique that preserves liver grafts under near-physiological conditions while maintaining their normal metabolic activity. This process requires an adequate oxygen supply, typically delivered by packed red blood cells (RBC). We present the first experience using an acellular hemoglobin-based oxygen carrier (HBOC) Hemopure in a human model of NMP-L. Methods Five discarded high-risk human livers were perfused with HBOC-based perfusion fluid and matched to 5 RBC-perfused livers. Perfusion parameters, oxygen extraction, metabolic activity, and histological features were compared during 6 hours of NMP-L. The cytotoxicity of Hemopure was also tested on human hepatic primary cell line cultures using an in vitro model of ischemia reperfusion injury. Results The vascular flow parameters and the perfusate lactate clearance were similar in both groups. The HBOC-perfused livers extracted more oxygen than those perfused with RBCs (O2 extraction ratio 13.75 vs 9.43 % ×105 per gram of tissue, P = 0.001). In vitro exposure to Hemopure did not alter intracellular levels of reactive oxygen species, and there was no increase in apoptosis or necrosis observed in any of the tested cell lines. Histological findings were comparable between groups. There was no evidence of histological damage caused by Hemopure. Conclusions Hemopure can be used as an alternative oxygen carrier to packed red cells in NMP-L perfusion fluid.

ABO-incompatible kidney transplantation is a novel risk factor for BK nephropathy.

Variable ABOi BKVAN+ (n = 6) ABOi BKVAN− (n = 53) P A link between ABO-incompatible kidney transplantation and the risk of BK virus allograft nephropathy (BKVAN) has been identified in a report from JohnsHopkins Hospital. The 11 (17.7%) of 62 cases of BKVAN were identified by protocol (n = 5) or indication (n = 6) biopsies. The incidence of BKVAN in HLA–incompatible allograft recipients (transplantation after the removal of donor-specific HLA antibodies) and compatible allograft recipients was 5.9% and 3.0%, respectively. In a multivariable analysis, ABO-incompatible kidney transplantation was identified as an independent risk factor for the development of BKVAN. To date, no other report has corroborated this apparent overrepresentation of BKVAN in ABO-incompatible kidney transplant recipients. Between 2007 and 2013 with a median follow-up of 46.7 months (22.9–57.9), 59 ABO-incompatible kidney transplantations have been performed using antigenspecific immunoadsorption and rituximab induction (more recently basiliximab alone) at our center. Maintenance immunosuppression consists of tacrolimus, mycophenolate mofetil, and corticosteroids. In this cohort, 6 cases of BKVAN have been diagnosed on indication biopsy in the context of allograft dysfunction (10.2%, diagnosed 3–11 months after transplantation; Table 1). This incidence is similar to the indication biopsy cohort at Johns Hopkins Hospital and significantly greater than the frequency of detection in contemporaneous blood group–compatible transplants at our center (10.2% vs 3.2%, respectively, P = 0.014). In those with BKVAN, there is an overrepresentation of donor blood group B (5/6 vs 16/53, P = 0.018) and lower