Foxa2-dependent hepatic gene regulatory networks depend on physiological state.

Bile acids are powerful detergents produced by the liver to aid in the absorption of dietary lipids. We recently reported a novel role for Foxa2 in bile acid metabolism. The winged helix transcription factor Foxa2 is required to prevent intrahepatic cholestasis and liver injury in mice fed a cholic acid-enriched diet. Here, we use functional genomics to study how Foxa2 regulates its targets in a cholic acid-dependent manner. We found that multiple signaling pathways essential for the hepatic response to acute liver injury are impaired in livers of Foxa2-deficient mice, suggesting that the deletion of Foxa2 in the hepatocyte affects the liver on a large scale. We also discovered distinct feed-forward regulatory loops controlling Foxa2-dependent targets in a cholic acid-dependent or -independent manner. We show that Foxa2 interacts with different transcription factors to achieve gene expression responses appropriate for each physiologic state.

Hepatocyte-specific ablation of Foxa2 alters bile acid homeostasis and results in endoplasmic reticulum stress.

Production of bile by the liver is crucial for the absorption of lipophilic nutrients. Dysregulation of bile acid homeostasis can lead to cholestatic liver disease and endoplasmic reticulum (ER) stress. We show by global location analysis ('ChIP-on-chip') and cell type-specific gene ablation that the winged helix transcription factor Foxa2 is required for normal bile acid homeostasis. As suggested by the location analysis, deletion of Foxa2 in hepatocytes in mice using the Cre-lox system leads to decreased transcription of genes encoding bile acid transporters on both the basolateral and canalicular membranes, resulting in intrahepatic cholestasis. Foxa2-deficient mice are strikingly sensitive to a diet containing cholic acid, which results in toxic accumulation of hepatic bile salts, ER stress and liver injury. In addition, we show that expression of FOXA2 is markedly decreased in liver samples from individuals with different cholestatic syndromes, suggesting that reduced FOXA2 abundance could exacerbate the injury.

Transcriptional networks in the liver: hepatocyte nuclear factor 6 function is largely independent of Foxa2.

A complex network of hepatocyte nuclear transcription factors, including HNF6 and Foxa2, regulates the expression of liver-specific genes. The current model, based on in vitro studies, suggests that HNF6 and Foxa2 interact physically. This interaction is thought to synergistically stimulate Foxa2-dependent transcription through the recruitment of p300/CBP by HNF6 and to inhibit HNF6-mediated transcription due to the interference of Foxa2 with DNA binding by HNF6. To test this model in vivo, we utilized hepatocyte-specific gene ablation to study the binding of HNF6 to its targets in the absence of Foxa2. Chromatin immunoprecipitation using anti-HNF6 antibodies was performed on chromatin isolated from Foxa2(loxP/loxP) Alfp.Cre and control mouse livers, and HNF6 binding to its target, Glut2, was determined by quantitative PCR. In contrast to the current model, we found no significant difference in HNF6 occupancy at the Glut2 promoter between Foxa2-deficient and control livers. In order to evaluate the Foxa2/HNF6 interaction model on a global scale, we performed a location analysis using a microarray with 7,000 mouse promoter fragments. Again, we found no evidence that HNF6 binding to its targets in chromatin is reduced in the presence of Foxa2. We also examined the mRNA levels of HNF6 targets in the liver using a cDNA array and found that their expression was similar in Foxa2-deficient and control mice. Overall, our studies demonstrate that HNF6 binds to and regulates its target promoters in vivo in the presence and absence of Foxa2.

Foxa2 integrates the transcriptional response of the hepatocyte to fasting.

Survival during prolonged food deprivation depends on the activation of hepatic gluconeogenesis. Inappropriate regulation of this process is a hallmark of diabetes and other metabolic diseases. Activation of the genes encoding gluconeogenic enzymes is mediated by hormone-responsive transcription factors such as the cyclic AMP response element binding protein (CREB) and the glucocorticoid receptor (GR). Here we show using cell-type-specific gene ablation that the winged helix transcription factor Foxa2 is required for activation of the hepatic gluconeogenic program during fasting. Specifically, Foxa2 promotes gene activation both by cyclic AMP, the second messenger for glucagon, and glucocorticoids. Foxa2 mediates these effects by enabling recruitment of CREB and GR to their respective target sites in chromatin. We conclude that Foxa2 is required for execution of the hepatic gluconeogenic program by integrating the transcriptional response of the hepatocyte to hormonal stimulation.

Induction of TCR Vbeta-specific CD8+ CTLs by TCR Vbeta-derived peptides bound to HLA-E.

Previous studies have identified murine and human regulatory CD8+ T cells specific for TCR-Vbeta families expressed on autologous activated CD4+ T cells. In the mouse, these regulatory CD8+ T cells were shown to be restricted by the MHC class Ib molecule, Qa-1. In the present study, we asked whether HLA-E, the human functional equivalent of Qa-1, binds Vbeta peptides and whether the HLA-E/Vbeta-peptide complex induces and restricts human CD8+ CTLs. We first created stable HLA-E gene transfectants of the C1R cell line (C1R-E). Two putative HLA-E binding nonapeptides identified in human TCR Vbeta1 and Vbeta2 chains (SLELGDSAL and LLLGPGSGL, respectively) were shown to bind to HLA-E. CD8+ T cells could be primed in vitro by C1R-E cells loaded with the Vbeta1 (C1R-E/V1) or Vbeta2 (C1R-E/V2) peptide to preferentially kill C1R-E cells loaded with the respective inducing Vbeta peptide, compared with targets loaded with the other peptides. Priming CD8+ T cells with untreated C1R-E cells did not induce Vbeta-specific CTLs. Of perhaps more physiological relevance was the finding that the CD8+ CTLs primed by C1R-E/V1 also preferentially killed activated autologous TCR Vbeta1+. Similar results were observed in reciprocal experiments using C1R-E/V2 for priming. Furthermore, anti-CD8 and anti-MHC class I mAbs inhibited this Vbeta-specific killing of C1R-E and CD4+ T cell targets. Taken together, the data provide evidence that certain TCR-Vbeta peptides can be presented by HLA-E to further induce Vbeta-specific CD8+ CTLs.

Pairing of isolated nucleic-acid bases in the absence of the DNA backbone.

The two intertwined strands of DNA are held together through base pairing--the formation of hydrogen bonds between bases located opposite each other on the two strands. DNA replication and transcription involve the breaking and re-forming of these hydrogen bonds, but it is difficult to probe these processes directly. For example, conventional DNA spectroscopy is dominated by solvent interactions, crystal modes and collective modes of the DNA backbone; gas-phase studies, in contrast, can in principle measure interactions between individual molecules in the absence of external effects, but require the vaporization of the interacting species without thermal degradation. Here we report the generation of gas-phase complexes comprising paired bases, and the spectroscopic characterization of the hydrogen bonding in isolated guanine-cytosine (G-C) and guanine-guanine (G-G) base pairs. We find that the gas-phase G-C base pair adopts a single configuration, which may be Watson-Crick, whereas G-G exists in two different configurations, and we see evidence for proton transfer in the G-C pair, an important step in radiation-induced DNA damage pathways. Interactions between different bases and between bases and water molecules can also be characterized by our approach, providing stringent tests for high-level ab initio computations that aim to elucidate the fundamental aspects of nucleotide interactions.

Fragment-Free Mass Spectrometric Analysis with Jet Cooling/VUV Photoionization.

We show that it is possible to obtain fragment-free mass spectra of large molecules by a combination of laser desorption, jet cooling, and VUV single-photon photoionization. The ability to obtain parent molecular masses is particularly important for the analysis of mixtures, such as combinations of fully saturated hydrocarbons. By varying the cooling conditions, we can also achieve partial fragmentation in order to obtain further structural information. The use of different wavelengths provides additional selectivity between aromatic and aliphatic compounds.

[Abnormal liver function tests in the primary care setting].

Results of laboratory tests ordered during a primary care encounter may reveal findings of abnormal liver function tests, including elevated liver enzymes, hyperbilirubinemia, hypoalbuminemia or abnormal coagulation tests. The object of this study was to describe the spectrum of these liver function test (LFT) abnormalities in primary care. Results of all laboratory tests ordered during 10 months in an urban primary care clinic were retrospectively reviewed and the medical charts of patients with abnormal LFTs were studied. In 217/1088 (20%) of the tests at least 1 LFT abnormality was found in 156 patients. New diagnoses were made in 104 patients. The main diagnostic groups were: non-alcoholic fatty liver changes, Gilbert's disease, acute infectious hepatitis, alcoholic liver disease and cirrhosis and hepatotoxic drug injury. In 60 patients the physician classified the abnormality as negligible and not associated with significant disease. However, an abnormal test that had been ordered for evaluation of a specific complaint, was indeed likely to represent significant disease (X2 = 29.5, p < 0.001). We conclude that finding abnormalities in liver function tests is common in the primary care clinic but does not often indicate significant liver disease.

[Oral anticoagulation therapy in the primary care setting].

The use of oral anticoagulant therapy (OAT) to prevent thromboembolism has been widespread in recent years. The concept of high- and low-intensity regimens has facilitated treatment for many, and has lowered the hazards of overly intense anticoagulation. However, a significant proportion of patients suited to the low intensity regimen are not being treated. It is not clear whether its wider use is limited by continued debate, lack of resources, lack of expertise, or other causes. We retrospectively reviewed the medical records of 32 patients treated with OAT administered in the primary care setting. The average age was 66 +/- 11 years (range 34-84). 9 were treated with high-intensity OAT: 8 due to artificial heart valves, and 1 due to a hypercoagulable syndrome with recurrent thromboembolism. 23 were treated with low-intensity OAT, 17 of whom had atrial fibrillation. 11 were also being treated continuously with other medication which interacted with OAT or interfered with other coagulation pathways. Such medication included: aspirin, dipyridamole, amiodarone, bezafibrate and allopurinol. Of 414 coagulation tests, 57% and 65% were in the therapeutic range in the high- and low-intensity OAT groups, respectively. There was no major bleeding event, but in 2 of 8 who bled, gastrointestinal bleeding led to hospitalization. Treatment was discontinued in 1 patient because of difficulties in achieving target INR, and in the 2 hospitalized for bleeding. The percentages of test results in, above and below the therapeutic range were similar to those in other large series, for both intensity regimens. We found that a significant proportion of patients were under chronic treatment with other medication which interacted with OAT. To estimate the rate of complications in primary care OAT, larger series are needed. We conclude that OAT can be given and monitored by the family physician, and that awareness of long and short term drug interactions with OAT is mandatory.

Distinct intracellular lysozyme content in normal granulocytes and monocytes: a quantitative immunoperoxidase and ultrastructural immunogold study.

Using two different immunological methods, we performed a quantitative estimation of lysozyme (LZ) in normal mature granulocytes and monocytes. An immunoperoxidase reaction for LZ in granulocytes and monocytes of 10 healthy donors measured by a scanning microdensitometer as arbitrary units showed a significantly higher LZ content in granulocytes than in monocytes. An ultrastructural immunogold reaction (IGR) for LZ performed on post-embedded thin sections showed a higher number of total gold grains in neutrophilic granulocytes than in monocytes. In monocytic granules we found a high density of gold grains per micron 2, whereas in granulocytic granules lower values were obtained. In granulocytes, LZ was found in both primary myeloperoxidase (MPO)-positive and secondary MPO-negative granules, and in monocytes the granules showed weak MPO reactivity and high LZ content. Granulocytes previously subjected to phagocytosis of bacteria and latex particles showed release of LZ on degranulation inside the phagosome, whereas in monocytes the granules remained outside the phagosome and released LZ without degranulation. Our study demonstrated a significantly higher total LZ content in granulocytes, a higher granular LZ content in monocytes, and release of LZ from intact monocyte granules during phagocytosis.

T lymphocyte T cell-B cell-activating molecule/CD40-L molecules induce normal B cells or chronic lymphocytic leukemia B cells to express CD80 (B7/BB-1) and enhance their costimulatory activity.

Activation-induced cell surface molecules are involved in mediating bidirectional T-B lymphocyte signaling that is important in the induction of T or B lymphocyte effector functions. In this regard, T-BAM/CD40-L is an activation-induced CD4+ T cell surface molecule known to be important in inducing B cell effector functions. This report demonstrates that T-BAM/CD40-L molecules on a Jurkat T cell leukemia subclone (D1.1) or nonlymphoid 293 kidney cell transfectants induce B cells or B-CLL cells to express CD80 (B7/BB-1) in a manner that is specifically inhibited by anti-T-BAM/CD40-L mAb 5C8. Because activation-induced B cell surface molecules, such as CD80, deliver costimulatory signals to T cells that augment T cell proliferation, the functional costimulatory capacity of T-BAM/CD40-L-primed B cells and B-CLL cells was studied. T-BAM/CD40-L-primed B cells or B-CLL cells augment the proliferative responses of allogenic T cells. Furthermore, T-BAM/CD40-L priming is specifically inhibited by mAb 5C8. Together, these studies demonstrate that T-BAM/CD40-L induces CD80 expression on resting B cells or B-CLL cells. Moreover, T-BAM/CD40-L signaling enhances B cell costimulatory capacity. These studies suggest that T-BAM/CD40-L molecules not only induce B cell differentiative processes that result in Ab secretion, but also enable B cells to prime Ag-specific T cells for subsequent clonal expansion.

Splenic Rupture in Chronic Lymphoid Disorders: Possible Role of Opportunistic Infections.

Two patients with lymphoproliferative disorders, one with chronic lymphocytic leukemia and the other hairy cell leukemia, developed spontaneous rupture of the spleen during the course of their disease. In both cases, this rare complication occurred during systemic atypical infections with Salmonella Dublin and Candida tropicalis respectively. We suggest that severe infection may sometimes play a decisive role in the development of the splenic rupture in some patients who have splenomegaly due to these disorders.

Gray platelet syndrome in the elderly.

A 68-year-old male who suffered from thrombocytopenia and mild splenomegaly for 18 years was found to present agranular gray platelets on peripheral blood smear. Bone biopsy revealed a mild, diffuse, reticular fibrosis with no collagen, and electron microscopy of the platelets showed an absence of almost all the alpha-granules. Platelet thrombospondin and fibronectin analysed by SDS-polyacrylamide gel electrophoresis and Rocket immunoelectrophoresis were absent. Follow-up of 4 years showed the same parameters with no evidence of active myeloproliferative or dysmyelopoietic disorders. Hemorrhagic diathesis was limited to ecchymoses and postprostatectomy bleeding, necessitating platelet transfusion. This led us to conclude that our patient probably had a constitutional primary alpha-granule deficiency or gray platelet syndrome. This extremely rare defect has been described in less than 10 patients, all of them very young. Our observation shows that these patients may have a long, uneventful survival.

Chediak-Higashi syndrome.

The use of cytochemical, electron microscopic, immunofluorescent, and tissue culture techniques has led to important advances in our understanding of the mechanisms underlying the pathogenesis of the Chediak-Higashi syndrome (CHS). This rare and fatal autosomal recessive disorder is clinically characterized by partial albinism, frequent pyogenic infections, and an accelerated lymphohistiocytic phase. The pathological hallmark of CHS is the presence in all white blood cells of massive lysosomal inclusions, which are formed through a combined process of fusion, cytoplasmic injury, and phagocytosis. The abnormal inclusions exhibit both azurophilic and specific granular markers, and are probably responsible for most of the impaired leukocyte and other cell functions in CHS patients. In addition, a selective profound natural killer (NK) cell function and platelet storage pool deficiencies have been described in these patients. Impaired microtubule assembly and functions, mediated by abnormal intracellular cyclic nucleotide levels, which could be corrected by treatment with ascorbic acid, were suggested to be the pathophysiological basis for CHS abnormalities. However, some recent studies have questioned this cytoskeletal model, which is suggested to be rather a secondary manifestation of CHS.

In vitro hepatitis B virus infection of human bone marrow cells.

Infection of humans with hepatitis B virus (HBV) frequently results in suppression of hematopoiesis; in some cases this may lead to severe bone marrow failure. The mechanism whereby HBV infection affects hematopoiesis is unknown. In vitro exposure of human bone marrow to HBV results in a dose-dependent inhibition of erythroid (erythroid burst forming units, BFU-E; erythroid colony-forming units CFU-E), myeloid (colony-forming units-granulocyte macrophage CFU-GM), and lymphoid (CFU-[T-lymphocytic]-TL) hematopoietic stem cells. Inactivation or immunoabsorption of HBV from sera resulted in loss of HBV-induced inhibition of hematopoietic stem cells. De novo gamma interferon was not detectable in the supernatants of cultures of bone marrow cells with HBV. Antibodies to gamma interferon did not affect the suppression of hematopoietic stem cells by HBV. Hepatitis B surface antigen (HBsAg) was detected by immune electron microscopy in nuclei of greater than 70% of immature hematopoietic cells including myeloblasts, normoblasts, and lymphoblasts; granulocytes had mostly cytoplasmic HBsAg. Hepatitis B virus core antigen (HBcAg) was also detected in about 5% of HBV infected bone marrow cells by immunoperoxidase staining. These data indicate that HBV can infect hematopoietic cells and their progenitors, thus suggesting a wider range of tropism for HBV than previously reported. These results may provide a basis to study HBV infection in vitro, and the effects of HBV on hematopoiesis.

Chediak-Higashi syndrome. Expression of the cytoplasmic defect by in vitro cultures of bone marrow progenitors.

Studies on proliferation and differentiation of granulocyte-monocyte progenitor cells in Chediak-Higashi syndrome (CHS) were done on a 1-month-old patient, using the soft-agar bone marrow culture technique. The number of granulocyte-macrophage colony-forming cells (GM-CFC) was markedly increased, but with a normal distribution into granulocyte, macrophage, or mixed colonies. Morphologic, cytochemical, and ultrastructural studies showed that 70% of the colonies consisted of cells with giant lysosomes typical of CHS, and in the remaining 30% abnormal cells were not detected. The supply of granulocyte-macrophage colony-stimulating factor (GM-CSF) by the patient's peripheral blood leukocytes was markedly decreased. Inhibition of normal in vitro granulopoiesis by the patient's lymphocytes or serum was not demonstrated. It appears that granulocyte progenitors in CHS proliferate normally, or even in excess, probably in response to intramedullary destruction of granulocytes. The majority of the progenitors are intrinsically defective and give rise to colonies that contain the abnormality. In others the defects are unidentifiable, probably due to the immaturity of the specific fusion process of the cytoplasmic granules. The abnormal leukocytes in CHS are also defective in their capacity to provide GM-CSF, and this may account in part to the overt neutropenia. These studies demonstrate that the basic cytoplasmic abnormalities of the granulocytes and monocytes in CHS are embedded in the granulocytic-monocytic committed stem cell.

A subpopulation of suppressor cells in Richter's syndrome with both monocytic and T-lymphocytic characteristics.

We evaluated T-lymphocyte functions in the peripheral blood of a patient with B-cell chronic lymphocytic leukemia after transformation to large cell lymphoma (Richter's syndrome). A subpopulation of E-rosette adherent cells were found with T-lymphocytic surface markers (OKT3+/8+/4+), monocytic characteristics (latex ingestion, nonspecific esterase staining), and suppressor activity. In contrast to the patient's nonadherent T-cells, this subpopulation suppressed PHA proliferation of autologous lymphocytes, pokeweed mitogen (PWM)-induced proliferation of normal non-T cells, and a mixed lymphocyte reaction. Further studies are warranted in patients with Richter's syndrome, in order to determine the frequency and significance of our findings.

Effect of N-methylisatin-beta-4':4'-diethylthiosemicarbazone on intracellular Moloney leukemia virus constituents.

N-Methylisatin-beta-4':4'-diethylthiosemicarbazone (M-IBDET) inhibits intracellular production of viral constituents in a mouse cell line, 3T3/MLV, chronically infected with Moloney leukemia virus. Electron microscopic observations confirmed that inhibition of virus production by the drug was not associated with any structural changes in the cell morphology or any damage to the plasma membrane, the site of viral assembly and 'budding'. Treatment of the cells with 17 microM M-IBDET for 6 h inhibited extracellular virus production by 80% but did not affect the level of viral RNA in the cytoplasm or in the plasma membrane. Intracellular reverse transcriptase activity and levels of viral structural proteins were significantly inhibited. Thus, although the drug did not affect viral RNA, it reduced viral protein synthesis.

Effect of theophylline on the proportion and function of T-suppressor cells in asthmatic children.

T-cell subpopulations and their function were studied in asthmatic children with or without theophylline therapy. A decreased proportion and function of theophylline-sensitive T-suppressor cells was found in fresh peripheral blood of asthmatic children. Following peripheral blood mononuclear cells (PBMC) incubation in theophylline-free medium, the proportion of theophylline-sensitive T-cells in asthmatic children treated with theophylline was elevated to normal values. However, their suppressive effect on control lymphocyte proliferation was unchanged.