ABIN-1 is a ubiquitin sensor that restricts cell death and sustains embryonic development.

Proteins that directly regulate tumour necrosis factor receptor (TNFR) signalling have critical roles in regulating cellular activation and survival. ABIN-1 (A20 binding and inhibitor of NF-kappaB) is a novel protein that is thought to inhibit NF-kappaB signalling. Here we show that mice deficient for ABIN-1 die during embryogenesis with fetal liver apoptosis, anaemia and hypoplasia. ABIN-1 deficient cells are hypersensitive to tumour necrosis factor (TNF)-induced programmed cell death, and TNF deficiency rescues ABIN-1 deficient embryos. ABIN-1 inhibits caspase 8 recruitment to FADD (Fas-associated death domain-containing protein) in TNF-induced signalling complexes, preventing caspase 8 cleavage and programmed cell death. Moreover, ABIN-1 directly binds polyubiquitin chains and this ubiquitin sensing activity is required for ABIN-1's anti-apoptotic activity. These studies provide insights into how ubiquitination and ubiquitin sensing proteins regulate cellular and organismal survival.

Krüppel-like factor 15 activates hepatitis B virus gene expression and replication.

UNLABELLED: Hepatitis B virus (HBV) is a small DNA virus that requires cellular transcription factors for the expression of its genes. To understand the molecular mechanisms that regulate HBV gene expression, we conducted a yeast one-hybrid screen to identify novel cellular transcription factors that may control HBV gene expression. Here, we demonstrate that Krüppel-like factor 15 (KLF15), a liver-enriched transcription factor, can robustly activate HBV surface and core promoters. Mutations in the putative KLF15 binding site in the HBV core promoter abolished the ability of KLF15 to activate the core promoter in luciferase assays. Furthermore, the overexpression of KLF15 stimulated the expression of HBV surface antigen (HBsAg) and the core protein and enhanced viral replication. Conversely, small interfering RNA knockdown of the endogenous KLF15 in Huh7 cells resulted in a reduction in HBV surface- and core-promoter activities. In electrophoretic mobility shift and chromatin immunoprecipitation assays, KLF15 binds to DNA probes derived from the core promoter and the surface promoter. Introduction of an expression vector for KLF15 short hairpin RNA, together with the HBV genome into the mouse liver using hydrodynamic injection, resulted in a significant reduction in viral gene expression and DNA replication. Additionally, mutations in the KLF15 response element in the HBV core promoter significantly reduced viral DNA levels in the mouse serum. CONCLUSION: KLF15 is a novel transcriptional activator for HBV core and surface promoters. It is possible that KLF15 may serve as a potential therapeutic target to reduce HBV gene expression and viral replication.

Induction of incomplete autophagic response by hepatitis C virus via the unfolded protein response.

UNLABELLED: Autophagy is important for cellular homeostasis and can serve as innate immunity to remove intracellular pathogens. Here, we demonstrate by a battery of morphological and biochemical assays that hepatitis C virus (HCV) induces the accumulation of autophagosomes in cells without enhancing autophagic protein degradation. This induction of autophagosomes depended on the unfolded protein response (UPR), as the suppression of UPR signaling pathways suppressed HCV-induced lipidation of the microtubule-associated protein light chain 3 (LC3) protein, a necessary step for the formation of autophagosomes. The suppression of UPR or the suppression of expression of LC3 or Atg7, a protein that mediates LC3 lipidation, suppressed HCV replication, indicating a positive role of UPR and the incomplete autophagic response in HCV replication. CONCLUSION: Our studies delineate the molecular pathway by which HCV induces autophagic vacuoles and also demonstrate the perturbation of the autophagic response by HCV. These unexpected effects of HCV on the host cell likely play an important role in HCV pathogenesis.

Ubiquitin-dependent and -independent proteasomal degradation of hepatitis B virus X protein.

The hepatitis B virus X protein (HBX) plays key regulatory roles in viral replication and the development of hepatocellular carcinoma. HBX is an unstable protein; its instability is attributed to rapid degradation through the ubiquitin-proteasome pathway. Here, we show that the middle and carboxyl-terminal domains of HBX, independently fused to GFP, render the recombinant proteins susceptible to proteasomal degradation, while the amino-terminal domain has little effect on the ubiquitination or stability of HBX. Mutation of any single or combination of up to five of six lysine residues, all located in the middle and carboxyl-terminal domain, did not prevent HBX from being ubiquitinated, ruling out any specific lysine as the sole site of ubiquitination. Surprisingly, HBX in which all six lysines were mutated and showed no evidence of ubiquitination, was still susceptible to proteasomal degradation. These results suggest that both ubiquitin-dependent and -independent proteasomal degradation processes are operative in HBX turnover.

Activation of hepatitis B virus S promoter by a cell type-restricted IRE1-dependent pathway induced by endoplasmic reticulum stress.

IRE1-alpha is an integral membrane protein of the endoplasmic reticulum (ER) that is a key sensor in the cellular transcriptional response to stress in the ER. Upon induction of ER stress, IRE1-alpha is activated, resulting in the synthesis of the active form of the transcription factor XBP1 via IRE1-mediated splicing of its mRNA. In this report, we have examined the role of IRE1-alpha and XBP1 in activation of the hepatitis B virus S promoter by ER stress. Cotransfection experiments revealed that overexpression of either IRE1-alpha or XBP1 activated this promoter. Conversely, cotransfected dominant-negative IRE1-alpha or small interfering RNA directed against XBP1 decreased the activation of the S promoter by ER stress, confirming an important role for the IRE1-alpha/XBP1 signaling pathway in activation of the S promoter. However, XBP1 does not bind directly to the S promoter; rather, a novel S promoter-binding complex that does not contain XBP1 is induced in cells undergoing ER stress in an XBP1-dependent manner. This complex, as well as transcriptional activation of the S promoter, is induced by ER stress in hepatocytes but not in fibroblasts, despite the presence of active XBP1 in the latter. Thus, the hepatitis B virus S promoter responds to a novel, cell type-restricted transcriptional pathway downstream of IRE1-alpha and XBP1.

Prominent eosinophilic intranuclear inclusions in melanocytes of a melanocytic nevus: the aftermath of an infection with molluscum contagiosum? A case report.

A 65-year-old Latino man presented to his dermatologist for the removal of two melanocytic nevi from the back. The first nevus was removed from the right scapula and contained melanocytes with prominent eosinophilic nuclear inclusion bodies. The second nevus was removed from the paravertebral region, without evidence of inclusion bodies. Ultrastructurally, the inclusions in the first nevus contained dispersed finely granular, homogenous bodies without a limiting membrane. Immunohistochemistry characterized them as ubiquitin-positive material. Reverse transcriptase in situ polymerase chain reaction analysis was positive for molluscum-specific primers, suggesting that the inclusions encountered in the first nevus were secondary to a remote, local molluscum viral infection of melanocytes.

Phaeochromocytoma crisis--a rare indication for extracorporeal membrane oxygenation.

We report on a case of phaeochromocytoma whose initial presentation mimicked an acute myocardial infarction. Veno-arterial extracorporeal membrane oxygenation was used for the management of refractory cardiogenic shock and massive lung oedema. Suspicion and diagnosis of a phaeochromocytoma were made due to its unique clinical presentation during extracorporeal membrane oxygenation. Stabilisation of the crisis and recovery of cardiopulmonary function were achieved using the support of extracorporeal membrane oxygenation. This case highlights the difficulty in the differential diagnosis of cardiogenic shock secondary to phaeochromocytoma and the important role of extracorporeal membrane oxygenation can have in the successful resuscitation and management of these patients.

Intraoperative coagulation was more interfered by HES 200/0.5 than normal saline in off-pump coronary artery bypass surgery.

AIM: Rapid fluid administration is often necessary for anesthesiologists to maintain intravascular volume in off-pump coronary artery bypass (OPCAB) with acceptable hematocrits. Postoperative hypocoagulation involving postoperative bleeding and hypercoagulation involving graft patency were focused in previous studies but bleeding and blood transfusion are often peaked during vascular anastomoses during OPCAB. This study is designed to investigate the sequential effects of intraoperative coagulation with normal saline and hydroxyethyl starch (HES) solution by thromboelastography (TEG) and standard coagulation tests (SCT). METHODS: Twenty adult patients scheduled for OPCAB were enrolled in this study. After anesthetic induction, one group received HES 200/0.5 infusion up to 20 mL/kg and the other received 0.9% normal saline (NS) to maintain central venous pressure (CVP) and pulmonary artery occlusion pressures (PAOP). SCT and TEG were measured at T0 (baseline), T1 (after heparin 150 IU/kg, before vascular anastomoses), T2 (after protamine reversal), and T3 (24 hrs after the surgery) to compare the coagulation status. RESULTS: Baseline data were comparable in both groups. The number of patient who need blood components is higher in HES group. Dilutional hypocoagulation was shown by a significant prolongation of R time at T1 and T2 but also returned comparable at T3 in both groups. K, a-angle, CI and G remained unchanged in NS group but significantly affected in HES group. A statistically significant interaction between groups and treatments on maximal amplitude (MA) (P<0.01) with more blood loss in HES group 24 hours postoperatively (P=0.05). International Normalized Ratio (INR) increased significantly at T2 and T3 in both groups. CONCLUSION: A rapid infusion of either normal saline or HES solution to maintain intraoperative intravascular volume induce a significant diluted hypocoagulation during OPCAB. The use of HES solution has a prolonged dilutional hypocoagulation and a significant decrease of MA by specific platelet inhibition effects and more transfusion of blood components. All the above changes were not shown in standard coagulation tests.

Lupus-like kidney disease in mice deficient in the Src family tyrosine kinases Lyn and Fyn.

Systemic lupus erythematosus (SLE) is an autoimmune disease whose cause is poorly understood. Mice rendered deficient in specific genes have served as useful animal models in deciphering the genetic control of the disease [1]. We [2] and others [3, 4] previously demonstrated that mice deficient in the Src family tyrosine kinase Lyn developed a mild lupus-like disease with high survival rates. During the course of investigating the functional interaction of Src family kinases, we generated a mouse strain deficient in both Lyn and Fyn. The double-mutant mice died at relatively young ages and developed a severe lupus-like kidney disease. Unlike the double-mutant mice, single mutants deficient in either Lyn or Fyn lived longer and had distinct subsets of the symptoms found in the former. Lyn deficiency led to high levels of autoantibody production and glomerulonephritis, as previously reported [2--4], whereas loss of Fyn contributed to proteinuria by a B and T lymphocyte-independent mechanism. Our data suggest that the severe kidney disease in the double-mutant mice results from a combination of immunological and kidney-intrinsic defects. This new animal model may be informative about the causes of human SLE.

Role of the hepatitis B virus posttranscriptional regulatory element in export of intronless transcripts.

Hepatitis B virus S transcripts contain a region, known as the posttranscriptional regulatory element (PRE), that activates their transport from the nucleus to the cytoplasm. J. Huang and T. J. Liang (Mol. Cell. Biol. 13:7476-7486, 1993) have shown that this element can partially substitute for the human immunodeficiency virus Rev-response element (RRE) in a reporter plasmid that is dependent on the RRE and Rev protein for expression and concluded that PRE exhibits Rev-RRE-like functions by inhibiting splicing. However, we have obtained additional data which indicate that the PRE functions in a novel manner that is not dependent on inhibition of splicing. Unlike Rev-RRE, the PRE functions independently of splice donor and acceptor sites and can activate cytoplasmic expression of an intronless (so-called prespliced) beta-globin transcript. Conversely, a heterologous intron can substitute for the PRE in increasing cytoplasmic expression of hepatitis B virus S transcripts. In addition, the host nuclear factor, YL2 (p32), which enhances Rev-RRE function has no effect on PRE-dependent gene expression. Since S transcripts are not normally known to be spliced and since RNA splicing and cytoplasmic transport are tightly linked processes in higher eucaryotic cells, we conclude that the PRE functions in cis to allow the export of nuclear transcripts that do not interact efficiently with the splicing pathway and hence are normally not exported well from the nucleus. Such elements are critical for the life cycle of viruses, such as hepatitis B virus, which undergo reverse transcription during replication.

The ubiquitous transcription factor Oct-1 and the liver-specific factor HNF-1 are both required to activate transcription of a hepatitis B virus promoter.

The liver-specific transcription factor HNF-1 activates transcription of several mammalian hepatocyte-specific genes. The hepatitis B virus preS1 promoter shows hepatocyte specificity, which has been ascribed to binding of HNF-1 to a cognate DNA sequence upstream of the TATA box. We show here that there is an adjacent site that binds the ubiquitous transcription factor Oct-1. Both the Oct-1 and HNF-1 sites are necessary for liver-specific transcription of the preS1 promoter, but neither site alone activates transcription. The Oct-1 site is also necessary for activation of the preS1 promoter in HeLa cells, expressing transfected HNF-1. Our results show that while Oct-1 is not restricted to hepatocytes, it nevertheless can play a critical role in the expression of a liver-specific gene.

Hepatocyte-specific expression of the hepatitis B virus core promoter depends on both positive and negative regulation.

The core promoter of hepatitis B virus shows hepatocyte specificity, which is largely dependent on an upstream regulatory sequence that overlaps with viral enhancer II. Footprint analyses by numerous groups have shown binding by cellular proteins over a large stretch of DNA in this region, but the identity of these proteins and their role in core promoter function remain largely unknown. We present data showing that the transcription factor HNF-4 is one such factor, as it activates the core promoter approximately 20-fold via a binding site within the upstream regulatory sequence. Since HNF-4 is enriched in hepatocytes, its involvement at least partially explains the hepatocyte specificity of this promoter. In addition, however, we have found a region upstream of the HNF-4 site that suppresses activation by HNF-4 in HeLa cells but not in hepatoma cells. Therefore, the cell type specificity of the core promoter appears to result from a combination of activation by one or more factors specifically enriched in hepatocytes and repression by some other factor(s) present in nonhepatocytes, and it may provide a convenient model system for studying this type of tissue-specific transcriptional regulation in mammalian cells.

Rapid detection of cutaneous herpes simplex virus infection with the polymerase chain reaction.

A simple and specific method for detecting herpes simplex virus infection in routinely processed paraffin-embedded biopsy specimens is described. DNA is extracted from paraffin blocks, and subjected to DNA amplification with the polymerase chain reaction. After 40 rounds, an amplified band can be detected after agarose gel electrophoresis and ethidium bromide staining. This band is specific for herpes simplex virus, because tissues infected with related viruses do not give this amplified band. We have been able to detect viral DNA in small punch skin biopsies with this procedure, which can take as little as 6 h.

Enzyme histochemistry of hepatocellular neoplasms.

We examined a series of hepatocellular neoplasms, including 4 adenomas, 7 hepatoblastomas, and 18 hepatocellular carcinomas (HCC) with enzyme histochemistry in plastic-embedded sections. Our most striking observation was that there was a distinct difference in the staining pattern for alkaline phosphatase (Alk0) in benign and malignant tumors. Non-neoplastic controls (normal liver, reactive lesions) and benign neoplasms showed a distinctive canalicular pattern of staining with Alk0. Malignant neoplasms, however, showed a virtual absence of Alk0 staining; 6 of 7 hepatoblastomas and 17 of 18 HCCs were practically devoid of staining, while the two positive cases showed a pattern easily discernible from normal. The sensitivity of the observed Alk0 staining pattern in detecting malignant hepatocellular neoplasms was 92% and the specificity was 100%. Cytoplasmic gamma-glutamyl-transferase (GGT) was present in a minority of HCCs, but faint staining was also seen in normal liver or in adenomas. It appears that these nonmorphologic techniques may aid the surgical pathologist in the differential diagnosis of primary hepatocellular neoplasms.

Primitive polypoid granular-cell tumor and other cutaneous granular-cell neoplasms of apparent nonneural origin.

Most cutaneous and noncutaneous granular-cell tumors are currently thought to be of Schwann-cell derivation. We present seven unusual cutaneous granular-cell lesions in which Schwann-cell origin can be excluded or is inapparent. Four of these lesions are of a previously undescribed type, and, unlike conventional granular-cell tumors of the skin, show a polypoid configuration, numerous mitoses, cytologic atypia, and a primitive immunophenotype. We propose the term "primitive polypoid granular-cell tumor" for these lesions. One occurred in a child, and three in adults. There have been no metastases to date, with follow-up periods of 2, 4, 4, and 16 years, respectively, although one tumor recurred locally. Additional cases and longer follow-up may be required to rule out the possibility that primitive polypoid granular-cell tumor is a low-grade malignancy. Two other granular-cell lesions represent variants of leiomyosarcoma, one of which widely metastasized. The last case is a granular-cell form of nodular basal-cell carcinoma. Cutaneous granular-cell neoplasms can show varying differentiation and behavior. Pathologists should not equate the occurrence of cytoplasmic granularity in a cutaneous neoplasm with the diagnosis of granular-cell schwannoma.

Bacillary angiomatosis. The histopathology and differential diagnosis of a pseudoneoplastic infection in patients with human immunodeficiency virus disease.

Cutaneous vascular proliferations that clinically or pathologically resemble Kaposi's sarcoma, pyogenic granuloma, or histiocytoid (epithelioid) hemangioma may occur in patients with human immunodeficiency virus infection. These lesions, which respond well to antibiotic therapy, harbor bacilli similar to the agent of cat scratch disease. We evaluated 21 biopsy specimens from 13 patients with this condition, which we have called "bacillary angiomatosis." The architecture resembled that of lobular capillary hemangioma (pyogenic granuloma), but the endothelial cells were often larger, polygonal, and sometimes markedly atypical. The presence of neutrophils, leukocytoclastic debris, and granular material (bacteria), and the absence of either spindled cells, bizarrely shaped vascular channels, or hyaline globules help to distinguish bacillary angiomatosis from Kaposi's sarcoma. By electron microscopy, the protuberant endothelial cells were different from those of histiocytoid hemangiomas in that aggregates of intermediate filaments were absent, while numerous Weibel-Palade bodies were present. The immunophenotype of the endothelial cells was distinct from that of Kaposi's sarcoma; almost all cells showed both Factor VIII RAg and Ulex europaeus lectin positivity. Enzyme histochemistry also showed a pattern distinct from Kaposi's sarcoma. Bacillary angiomatosis presents a unique constellation of clinical and microscopic findings. It is important to be aware of these characteristics, because these lesions are easily treatable with antibiotic therapy.

A poorly differentiated lymphoma of donor origin in a renal allograft recipient.

Malignant lymphoma is a frequent complication of organ transplantation. It has been suggested that such tumors arise as a result of uncontrolled proliferation of Epstein-Barr virus-infected B lymphocytes in an immunosuppressed host. Although a few cases of posttransplant lymphomas in bone marrow transplantation have been shown to be of donor cell origin, no recipients of solid-organ transplants are known to have developed lymphomas arising from donor cells. In this report, a case of diffuse high-grade lymphoma that apparently arose in the allograft of a renal transplant recipient is described. DNA fingerprinting demonstrated the tumor to be of donor origin; Epstein-Barr sequences were absent. A therapeutic trial consisting of withdrawal of immunosuppressive agents and administration of acyclovir was unsuccessful. These data support the notion that donor cells can undergo malignant transformation in solid-organ transplant recipients, and such tumors need not carry EBV genetic material.

Diagnostic efficacy of (13)C-urea breath test for Helicobacter pylori infection in hemodialysis patients.

The noninvasive urea breath test (UBT) avoids the discomforts and risks of invasive endoscopic methods of Helicobacter pylori detection. This study investigated the diagnostic efficacy of carbon 13 ((13)C)-labeled UBT for H pylori detection in 70 patients with end-stage renal disease (ESRD) undergoing hemodialysis (HD) and 70 dyspeptic controls without renal impairment. With H pylori infection defined as a positive result on either histological examination or culture of gastric biopsy specimen, we evaluated the reliability of the (13)C-UBT in detecting H pylori infection in both groups. To ascertain whether HD therapy affects the diagnostic efficacy of the UBT, the test was performed twice in patients with ESRD (before and after HD) at least 72 hours apart. In each UBT session, the baseline, 10-minute, and 15-minute (Delta15) gas samples were obtained to analyze excess (13)CO(2)/(12)CO(2) ratio (ECR). Histological stain and/or culture studies found that 33 of the patients with ESRD (47. 1%) and 42 of the control patients (60%) had H pylori infection. (13)C-UBT for H pylori detection in patients with ESRD was found to be only 93.8% sensitive and 85.3% specific. These results were achieved by gas sampling (Delta15) after HD therapy with a cutoff ECR value greater than 5. Conversely, the UBT in the control group achieved the greatest diagnostic efficacy (sensitivity, 97.6%; specificity, 96.4%) with a comparatively lower ECR cutoff value of 4. We conclude that the diagnostic accuracy for H pylori detection in patients with ESRD could be improved by performing (13)C-UBT (Delta15) after HD therapy and assessing the UBT with a cutoff ECR value greater than 5. However, the diagnostic efficacy of the UBT for patients with ESRD remained less accurate than that for dyspeptic patients without renal impairment.

Use of the polymerase chain reaction in the diagnosis of unsuspected herpes simplex viral pneumonia: report of a case.

A 30-year-old apparently immunocompetent woman presented with acute respiratory failure (acute respiratory distress syndrome). No etiologic agent was found, and she died 2 weeks later despite antibiotic therapy. Postmortem examination of the lungs showed diffuse organizing alveolar damage with superimposed focal necrotizing peribronchiolar pneumonia. Cultures obtained from lung tissue were negative for virus, fungi, and bacteria. Histopathologic and electron microscopic studies showed that the necrotizing changes were consistent with herpesvirus infection. With the use of a new diagnostic tool, the polymerase chain reaction, a specific diagnosis of herpes simplex virus pneumonia was made, and other viral agents were excluded. The polymerase chain reaction is a sensitive, specific, and rapid technique that may greatly facilitate establishing an infectious etiology in cases of pneumonia.

Benign lymphoepithelial cysts of the parotid gland. A histologic, cytologic, and ultrastructural study.

Five cases of benign lymphoepithelial cysts (BLCs) of the parotid gland are reported, and the histologic, aspiration cytologic, and ultrastructural findings are described in detail. These uncommon parotid lesions contain epithelium-lined cystic spaces encased by abundant lymphoid tissue with germinal centers. The epithelium was "mucoepidermoid" in three of our cases and squamous in two. Familiarity with the morphologic features of BLCs should make it possible to distinguish them from similar-appearing cystic lesions, especially low-grade cystic mucoepidermoid carcinoma and cystic types of benign lymphoepithelial lesion (so-called Mikulicz's disease). These distinctions, however, are difficult on aspiration cytology specimens.