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.