The role of C/EBP(epsilon) in the terminal stages of granulocyte differentiation.

As a consequence of its characterization using both in vitro and knockout mouse models, the myeloid-specific transcription factor, CCAAT/enhancer binding protein (C/EBP)epsilon, has been identified as a critical regulator of terminal granulopoiesis and one of the causative mutations in the human disease, neutrophil-specific granule deficiency. C/EBPs are a family of transcription factors sharing numerous structural and functional features and to date include C/EBPalpha, -beta, -gamma, -delta, -epsilon, and -zeta. C/EBPalpha was the first family member isolated and characterized, its essential role in hepatocyte and adipocyte differentiation demonstrated in knockout mouse models. Subsequent analysis of the hematopoietic elements in fetal mouse liver revealed its critical role in myelopoiesis. Understanding the role of C/EBPepsilon in terminal granulopoiesis in the context of other known transcription factors is ongoing with analysis of deficient and conditionally expressing cell lines and knockout models. Mouse models with targeted gene disruptions have contributed greatly to our understanding of the transcriptional regulation of granulopoiesis. Further manipulation of these models and other conditional expression systems have bypassed some of the limitations of knockout models and helped delineate the interactions of different transcription factors in affecting granulocyte development. Phenotypic expression of the loss of C/EBPepsilon in mice is extreme, resembling absolute neutropenia with systemic infection with P. aeruginosa. Future work will need to explore the regulation of C/EBPepsilon expression, its functional interactions with other transcriptional regulators such as PU.1, and its role in monocyte differentiation and function in the mouse.

Molecular analysis of the mouse S100A9 gene and evidence that the myeloid specific transcription factor C/EBPepsilon is not required for the regulation of the S100A9/A8 gene expression in neutrophils.

The genomic locus of the mouse S100A9 (MRP14) gene, a myeloid expressed gene belonging to the S100 family, is split in three exons and two introns. Insertions of B1 like and LINE elements as well as several sequence repeat structures are scattered over the gene suggesting that this region of the S100 gene cluster has been the subject of a high mutational activity in mouse evolution. The insertions may represent molecular footprints of a recently postulated inversion event, which resulted in a rearrangement of the S100 gene cluster in mouse compared to man. Deletion analysis of the promoter reveals, that a 1200 bp fragment is able to direct a cell type-specific expression of a reporter gene in granulocytic 32D cells. Unexpectedly, the myeloid-specific transcription factor C/EBPepsilon is not needed for the transcriptional upregulation of the S100A9 and S100A8 genes in neutrophils. The data described here provide further insights into the evolution of the S100 gene cluster and into the myeloid-specific regulation of the murine S100A9 gene expression.

Gamma interferon impedes the establishment of herpes simplex virus type 1 latent infection but has no impact on its maintenance or reactivation in mice.

Murine models of gamma interferon (IFN-gamma) deficiency demonstrate the role of this cytokine in attenuating acute herpes simplex virus (HSV) disease; however, the effect of IFN-gamma on the establishment and maintenance of neuronal latency and viral reactivation is not known. Using the IFN-gamma knockout (GKO) model of IFN-gamma deficiency and sensitive quantitative PCR methods, we show that IFN-gamma significantly reduces the ganglion content of latent HSV-1 in BALB/c mice, which in turn delays viral time to reactivation following UV irradiation. Similar effects were not seen in the C57BL/6 strain. These results indicate that IFN-gamma significantly attenuates latent HSV infection in the mouse model of ocular infection but has no impact on the maintenance of latency or virus reactivation.

Neutrophil-specific granule deficiency results from a novel mutation with loss of function of the transcription factor CCAAT/enhancer binding protein epsilon.

Neutrophil-specific granule deficiency (SGD) is a rare disorder characterized by recurrent pyogenic infections, defective neutrophil chemotaxis and bactericidal activity, and lack of neutrophil secondary granule proteins. CCAAT/enhancer binding protein (C/EBP)epsilon, a member of the leucine zipper family of transcription factors, is expressed primarily in myeloid cells, and its knockout mouse model possesses distinctive defects, including a lack of neutrophil secondary granule proteins. Sequence analysis of the genomic DNA of a patient with SGD revealed a five-basepair deletion in the second exon of the C/EBPepsilon locus. The predicted frame shift results in a truncation of the 32-kD major C/EBPepsilon isoform, with loss of the dimerization domain, DNA binding region, and transcriptional activity. The multiple functional defects observed in these early neutrophil progenitor cells, a consequence of C/EBPepsilon deficiency, define SGD as a defect in myelopoiesis and establish the requirement for C/EBPepsilon for the promyelocyte-myelocyte transition in myeloid differentiation.

Association of major histocompatibility complex determinants with the development of symptomatic and asymptomatic genital herpes simplex virus type 2 infections.

The clinical spectrum of herpes simplex virus (HSV) infections, ranging from asymptomatic to frequently distressing outbreaks, suggests that there may be immunologic determinants of disease severity that are associated with human leukocyte antigen (HLA) expression. A controlled, prospective study identified several major histocompatibility complex (MHC) class I and II antigens whose frequencies are associated with HSV-2 infection or with frequent symptomatic genital recurrences. Previous studies were hampered by the inability to serologically identify patients with asymptomatic HSV-2 infection. Clinical evaluation and Western blot assay were used to identify 3 subject cohorts: 1 with no prior HSV infections, 1 with HSV-2 antibodies but no recognized symptoms, and 1 with HSV-2 antibodies and frequent genital recurrences. Statistical comparisons of HLA frequencies among these cohorts showed associations of HLA-B27 and -Cw2 with symptomatic disease. Also, HLA-Cw4 was significantly associated with HSV-2 infection. These associations indicate that immunologic factors linked to the MHC influence the risk of HSV-2 infection and disease expression.

The comparative biology of latent herpes simplex virus type 1 and type 2 infections: latency-associated transcript promoter activity and expression in vitro and in infected mice.

HSV-1 and HSV-2 express abundant latency-associated transcripts (LATs) without which these viruses reactivate in animals inefficiently. To further characterize the importance of LATs to the comparative biology of latent HSV-1 and -2 infections, we assessed the relative activities of the viral LAT promoters in vitro using transient transfection assays, and the accumulation of LATs in vivo using a mouse ocular infection model. In vitro, the HSV-2 LAT promoter proved to be six to tenfold more potent than the HSV-1 promoter in driving reporter gene expression. In mice HSV-1 and -2 achieved comparable levels of virus replication in the eye, but HSV-2 grew to higher titers than HSV-1 in trigeminal ganglia and brain. Quantitative-competitive DNA and RNA (RT) PCR and in situ hybridization showed that ganglia latently infected with HSV-2 contained sixfold more copies of DNA (P=0.003), eightfold more LATS (P=0.01), and ninefold more LAT in situ-positive neurons. However, the numbers of LATs per latent genome were equivalent for both viruses. Although the HSV-2 LAT promoter is more potent than the HSV-1 promoter in transient expression assays, the accumulation of HSV-1 and 2 LATs in mouse trigeminal ganglia is comparable.

The quantity of latent viral DNA correlates with the relative rates at which herpes simplex virus types 1 and 2 cause recurrent genital herpes outbreaks.

Herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) have evolved specific anatomic tropisms and site-dependent rates of reactivation. To determine whether reactivation rates depend on distinct abilities of HSV-1 and -2 to establish latency and to express latency-associated transcripts (LATs), virulent strains of each virus were studied in the guinea pig genital model. Following infection with equivalent titers of virus, the quantities of latent HSV-2 genomes and LATs were higher in lumbosacral ganglia, and HSV-2 infections recurred more frequently and lasted longer than HSV-1 infections. In contrast, if the inoculum of HSV-1 was 10 times that of HSV-2, the quantity of HSV-1 DNA and LATs increased correspondingly and HSV-1 infections were as likely to recur as those with HSV-2. The quantity of latent virus DNA correlates with and may be a major determinant of the site-specific patterns and rates of reactivation of HSV-1 and -2.

Periodic illness associated with Epstein-Barr virus infection.

A 15-year-old boy with a 13-year history of periodic fevers, lymphadenopathy, and leukocytosis showed virological, serological, immunohistologic, and molecular evidence of persistent, active, Epstein-Barr virus (EBV) infection. Acyclovir and several other agents failed to alter his clinical course. Comprehensive immunological studies could not identify a defined immune deficiency syndrome to explain the persistent infection, although he does continue to have circulating polymeric EBV-specific immunoglobulin type A, as is seen in individuals during acute EBV infections. In vitro work suggests that this polymeric antibody prevents B cell infection by EBV. Cumulative data suggest that this patient suffers from a novel form of EBV infection.

Treatment with intralesional granulocyte instillations and interferon-gamma for a patient with chronic granulomatous disease and multiple hepatic abscesses.

We present the case of a 16-year-old girl with p22-deficient chronic granulomatous disease in whom multiple hepatic abscesses secondary to Staphylococcus aureus infection developed. Infection persisted despite extensive surgery and aggressive antibiotic therapy. Conventional intravenous granulocyte transfusions were not tolerated because of the development of alloantibodies to HLA. Treatment with interferon-gamma and intralesional granulocyte infusions was associated with dramatic clinical and radiographic improvement. No morbidity was associated with this therapy. To our knowledge, this is the first report of treatment with intralesional granulocyte instillations. Intralesional granulocyte instillation in association with interferon-gamma administration may result in clinical improvement in the conditions of patients with chronic granulomatous disease and hepatic abscesses for whom conventional therapy has failed.

An epidemiologic study of Tourette's syndrome in a single school district.

To evaluate the frequency of Gilles de la Tourette's syndrome (TS) in children, 3034 students in three schools in a single school district in greater Los Angeles were monitored frequently over a 2-year period by a school psychologist thoroughly familiar with the symptoms of the disorder. A portion of the cases were also evaluated in a TS clinic. A total of 14 males fulfilled the Tourette Syndrome Association research criteria for definite TS. When corrected for the number of students in special education classes in the monitored schools, the frequency of definite TS in males was 1 in 152. An additional 7 males who differed only in that they were not observed for a full year were termed definite TS less than 1 year. When the two groups were combined, the frequency of definite TS was 1 in 95 for males and 1 in 759 for females. These figures do not include an additional 10 males diagnosed as having definite transient tic disorder, 2 males diagnosed as having probable TS, and 10 males diagnosed as having possible TS. In addition to tics, most of these children had problems with attention span, obsessive compulsive behavior, and learning and/or conduct disorders. Seventy percent of the students with definite TS or definite TS less than 1 year were in special education classes. Twelve percent of the children in special education classes had definite TS or definite TS less than 1 year, and 28% were in one of the diagnostic categories of definite, probable, or possible. All of the 10 definite TS patients that were seen in the clinic had attention-deficit hyperactivity disorder.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of bilirubin infusion on excretion of bilirubin and biliverdin in rabbits.

Intravenous infusion of bilirubin (BR) at 171 micrograms/min/kg into rabbits resulted in biliary concentration of BR increasing from 3.8 (control) to 243 mg/dl and BR excretion increasing from 1.7 to 66 micrograms/min/kg. BR infusion resulted in biliary concentrations of biliverdin (BV) increasing from 9.1 to 30 g/dl and BV excretion increasing from 4.2 to 8.2 micrograms/min/kg. BR infusion produced a progressive decline in bile flow. BV was the predominant bile pigment in control rabbits fed either an alfalfa-based or chlorophyll-free diet. These results imply that rabbits can oxidize BR to BV.

Hypercholeresis in turkeys following the ingestion of Crotalaria spectabilis seeds.

Young female turkeys received diets containing Crotalaria spectabilis seeds for 18 weeks. Mean values for total bile flow, biliary bile acid excretion, bile acid-dependent flow and bile acid-independent flow were significantly higher in crotalaria-fed turkeys than in controls. The hypercholeresis observed in crotalaria-fed birds involved both bile acid-dependent and bile acid-independent components of bile. Crotalaria-fed turkeys developed biliary hyperplasia. Liver weights were similar in the two groups. Since increases in 14C-erythritol clearances paralleled increases in total bile flow in crotalaria-fed turkeys (14C-erythritol bile to plasma ratios were similar in both groups), it was evident that the hypercholeresis in crotalaria-fed turkeys did not involve the ductal/ductular component of bile. Little or no ductal/ductular bile flow occurred in either group since total bile flow was equal to erythritol clearance and extrapolation to zero erythritol clearance yielded zero bile flow rates in both groups. Crotalaria-fed turkeys exhibited significantly higher biliary concentrations of chlorides (associated with lower sodium , potassium and bicarbonate concentrations) than did control birds. Validation of the use of 14C-erythritol clearance for the estimation of canalicular bile flow in turkeys was provided both by substantial decreases in bile to plasma 14C-erythritol concentration ratios following the injection of avian vaso-active intestinal peptide and the failure to demonstrate chromatographically the presence of radioactive metabolites of 14C-erythritol in plasma.

Biliverdin and bilirubin excretion in the turkey.

Mean endogenous bile flow in 11 turkeys was 0.81 plus or minus 0.52 mul/g of the liver per minute (10.4 plus or minus 5.3 mul/Kg of body weight per minute). Endogenous biliverdin and bilirubin excretory rates were 0.59 plus or minus 0.31 and 0.058 plus or minus 0.018 mug/g of liver per minute (7.6 plus or minus 3.6 and 0.76 plus or minus 0.23 mug/Kg of body weight per minute), respectively. Mean concentrations of biliverdin and bilirubin in endogenous bile were 92 plus or minus 55 and 8.9 plus or minus 5.2 mg/100 ml, respectively. Livers constituted 1.36 plus or minus 0.22 percent of the body weight. Thin layer chromatographic studies revealed a heterogeneity of bilirubin conjugates in bile.

Repeated oral administration of coumaphos in sheep: effects on erythrocyte acetylcholinesterase and other constituents.

The effects of treating adult wethers with 2 or 4 mg of coumaphos/kg of body weight each day for 6 days were investigated. The smaller dose produced a gradual decrease of erythrocyte acetylcholinesterase (AChE) activity (to maximum average reduction of approximately 45%), but without the appearance of signs of toxicosis. The larger dose appeared to be toxic. Treatment with the drug did not seem to alter significantly the anticholinesterase effects of a 2nd treatment made 6 weeks later. Coumaphos did not significantly affect serum activities of aspartate aminotransferase (glutamic oxalacetic transaminase) or isocitrate dehydrogenase (ICD) and concentrations of serum sodium and plasma calcium. A marked decrease in blood serum potassium and an increase in plasma magnesium occurred in all wethers that died after treatment with coumaphos, whereas appreciable changes did not occur in the survivors of the treatment given 6 weeks earlier. Treatment of sheep with an intravenous injection of the organophosphorous compound trichlorfon, insufficient to produce a significant effect on erythrocyte cholinesterase activity, produced additive effects with those of coumaphos.

Repeated oral administration of coumaphos in sheep: interactions of coumaphos with bishydroxycoumarin, trichlorfon, and phenobarbital sodium.

Interactions between treatments with coumaphos, bishydroxycoumarin (an anticoagulane), trichlorfon (an organophosphorous compound), and phenobarbital sodium (an inducer of microsomal enzymes) were investigated in sheep. A daily dose of 2 mg of coumaphos/kg of body weight for 6 days did not affect the plasma enzymes or the antiprothrombinemic effect of bishydroxy-coumarin in wethers. The treatment of ewes with an intravenous (IV) injection of trichlorfon, insufficient to produce significant inhibition of erythrocyte acetylcholinesterase (AChE) activity, appeared to produce additive effects with those produced by subsequent treatment with 4 mg of coumaphos/kg/day. In ewes given 40 mg of phenobarbital sodium/kg for 5 days intraperitoneally (IP), the anticholinesterase effect of 4 mg of coumaphos/kg was significantly reduced and signs of toxicity were not present. Treatment with daily doses of 2 mg of coumaphos/kg for 6 days did not modify the anticholinesterase effect of a 2nd series of treatments given 6 weeks later.

Experimental liver diseases.

The use of animal models in the experimental production of liver diseases similar to those of man is still in its infancy. There is a need to discover new models more closely related to counterpart syndromes in man in the fields of hepatorenal syndrome, neonatal jaundice, Wilson's disease, cholelithiasis, viral hepatitis, biliary atresia, and cirrhosis, to mention only a few. With the continued indiscriminate inbreeding of companion animals as well as the planned inbreeding of laboratory animals, there is little doubt that many more will soon be available. The current availability of mutant rats and sheep with bilirubin transport defects has allowed for a better understanding of how organic anions are transported by the liver. Many other currently available experimental animal models herein briefly reviewed have been only superficially studied. It is the intent of this chapter to provide for post-doctoral students an appreciation for the many animal model systems available for experimental hepatic research.

Heterogeneity of bilirubin conjugates in several animal species.

Bile was collected from various domestic and wild species (alligators, armadilirubin diazotized and subjected to thin-layer chromatography fro the separation of conjugates (as azo-dipyrroles). Diazotized canine conjugated bilirubin had Rf values in agreement with those previously reported by other; bilirubin conjugates were related to their known glycosidic composition. Variations were observed in the relative amounts of the various mono- and diconjugates among animals within species. However, it was apparent that characteristic patterns of bilirubin conjugation occur in certain animal species. Intravenous infusion of between 0.03 and 0.61 mg of bilirubin/Kg into cats resulted in little or no change in the relative percentages of the various conjugates of bilirubin to glucuronic acid in the cat continues to be the major excretory pathway under conditions of bilirubin load.