P. Dal Monte

Complex Formation by Human Cytomegalovirus Glycoproteins M (gpUL100) and N (gpUL73)

ABSTRACT The envelope glycoproteins of human cytomegalovirus (HCMV) virions are incompletely characterized. We have analyzed complex formation between glycoprotein M (gM or gpUL100) and a second glycoprotein. gM-homologous proteins are conserved throughout the herpesvirus family and represent type III membrane proteins containing multiple hydrophobic sequences. In extracellular HCMV particles, gM was found to be complexed through disulfide bonds to a second protein with an apparent molecular mass of 50 to 60 kDa. The 50- to 60-kDa protein was found to be derived from reading frame UL73 of HCMV strain AD169. UL73-homologous genes are also conserved within herpesviruses. When transiently expressed by itself, the UL73 gene product consisted of a protein of 18 kDa. However, in the presence of gM, the UL73 gene product was posttranslationally modified to the 50- to 60-kDa species. Thus, gM and the UL73 gene product, which represents the gN homolog of herpesviruses, form a disulfide-linked complex in HCMV virions. Transient expression of gM and gN followed by fluorescence imaging with monoclonal antibodies against either protein demonstrated that complex formation was required for transport of the proteins from the endoplasmic reticulum to the Golgi andtrans-Golgi compartments. Finally, we tested the gM-gN complex for reactivity with sera from HCMV-seropositive donors. Whereas most sera failed to react with either gM or gN when expressed alone, 62% of sera were positive for the gM-gN complex. Because a murine monoclonal antibody reactive with gN in the gM-gN complex efficiently neutralizes infectious virus, the gM-gN complex may represent a major antigenic target of antiviral antibody responses.

gpUL73 (gN) genomic variants of human cytomegalovirus isolates are clustered into four distinct genotypes

Clinical isolates of human cytomegalovirus (HCMV) show differences in tissue tropism, severity of clinical manifestations and ability to establish persistent or latent infections, characteristics that are thought to be related to genomic variation among strains. This work analysed the genomic variants of a new HCMV polymorphic locus, open reading frame (ORF) UL73. This ORF encodes the envelope glycoprotein gpUL73 (gN), which associates in a high molecular mass complex with its counterpart, gM, and induces a neutralizing antibody response in the host. Detailed sequence analysis of ORF UL73 and its gene product from clinical isolates and laboratory-adapted strains shows that this glycoprotein is highly polymorphic, in the N-terminal region in particular. gpUL73 hypervariability is not randomly distributed, but the identified genomic variants are clearly clustered into four distinct genotypes (gN-1, gN-2, gN-3 and gN-4), which are not associated with the gB subtype.

Analysis of intracellular and intraviral localization of the human cytomegalovirus UL53 protein.

Human cytomegalovirus (HCMV) UL53 belongs to a family of conserved herpesvirus genes. In this work, the expression and localization of the UL53 gene product was analysed. Results obtained showed that pUL53 is a new structural protein. In infected human fibroblasts, pUL53 localizes in cytoplasmic perinuclear granular formations together with other structural viral proteins. In the nucleus, pUL53 forms patches at the nuclear periphery and co-localizes with lamin B at the internal nuclear membrane level. Immunoelectron microscopy studies have disclosed that nuclear pseudo-inclusions are labelled, whereas nucleocapsid formations within the intranuclear skein are negative. Furthermore, the mature virus particle maintains pUL53 at its tegumental level. These data suggest that pUL53 could be involved either in nucleocapsid maturation or in the egress of nucleocapsids from the nucleus to the cytoplasm through the nuclear membrane, a role compatible with the function hypothesized for UL31, its positional homologue in herpes simplex virus type 1.

Retinoids and cancer: antitumoral effects of ATRA, 9-cis RA and the new retinoid IIF on the HL-60 leukemic cell line.

Objective: To compare the antitumoral effects of all-trans retinoic acid (ATRA) and 9-cis retinoic acid (9-cis RA) with those of 5-OH,11-O-hydrophenanthrene (IIF), a new derivative of retinoic acid. Materials and Methods: The effect of retinoids was tested on cell line HL-60. Cell differentiation and apoptosis were evaluated by morphological and biochemical analysis as bcl-2 protein and by DNA fragmentation assay. The ability to activate retinoic acid receptors (RAR) and/or retinoid X receptors (RXR) and to modulate gene expression was determined by transactivation assay. Results: With cell line HL-60, the antiproliferative effect of IIF was stronger than that of ATRA and 9-cis RA. Following retinoid treatment, cells appeared to differentiate and apoptotic cells were observed. The appearance of DNA laddering and a decrease in the amount of bcl-2 protein confirmed apoptosis. IIF transcriptionally activated RXR-γ more than RAR-α. Conclusion: The findings indicate that IIF transcriptionally activates RXR-γ preferentially, induces apoptosis and has a more antiproliferative activity than ATRA and 9-cis RA on cell line HL-60.

Laboratory diagnosis of late-onset sepsis in newborns by multiplex real-time PCR.

Bloodstream infections (BSIs) are an important cause of neonatal morbidity and mortality, and often result in prolonged hospitalization of infants who are admitted to neonatal intensive care units (VerboonMaciolek et al., 2006). Late-onset neonatal sepsis (occurring in newborns aged older than 3 days) occurs in approximately 0.1 % of all newborns and in up to ~25 % of very low birth weight infants (birth weight ,1500 g) (Kaufman & Fairchild, 2004). Early diagnosis of sepsis and prompt treatment are critical in preventing severe and life-threatening complications in these patients (Harbarth et al., 2003; Kollef, 2003; Lodise et al., 2003). The clinical recognition of sepsis in neonates is difficult, however, because the signs and symptoms are often non-specific (Gerdes, 1991; Verboon-Maciolek et al., 2006) and blood cultures (BCs) are rarely positive.

IgG subclass distribution of anti-HBs antibodies following vaccination with cDNA HBsAg

Serum samples were obtained during follow-up of nine young adults vaccinated over 1 year with cDNA hepatitis B antigen. The absolute amounts of anti-HBs IgG subclass antibodies present in the sera were determined by comparing the optical densities (OD) obtained using an antigen-specific ELISA with those obtained by serial dilutions of known amounts of human IgG1-4. The calibration curves for each IgG subclass were corrected for the corresponding coating efficiency. Our data suggest that HBs antibody responses of vaccinated subjects occur in all IgG subclasses but IgG1 and IgG2 are the major subclasses involved.

HUMAN CYTOMEGALOVIRUS INFECTION: A COMPLEX DIAGNOSTIC PROBLEM IN WHICH MOLECULAR BIOLOGY HAS INDUCED A RAPID EVOLUTION

Human cytomegalovirus (HCMV) is associated with several diseases in immunocompromised individuals. CMV infection can be diagnosed directly by demonstration of the virus or virus components in pathological materials or indirectly through serology. Molecular biology has allowed detailed studies of the viral genome and its antigenic gene products and has led to major advances in CMV diagnosis providing new tools for both the analysis of the CMV-specific immune response and the detection of virus-specific antigens or genetic material. In an attempt to provide a guide for the correlation between laboratory findings and clinical interpretation, we discuss in this work the clinical settings in which the presence of CMV needs to be diagnosed, and how a CMV diagnosis should be asked for or interpreted by the clinician in view of the variety of new or improved laboratory tests now available.

An International Multi-Clinic Study Comparing the Therapeutic Efficacy of Colloidal Bismuth Subcitrate Coated Tablets with Chewing Tablets in the Treatment of Duodenal Ulceration

The results of a randomized, single-blind, multi-clinic study comparing the therapeutic efficacy and degree of oral staining of new colloidal bismuth subcitrate (CBS) coated tablets over 4 weeks of treatment in patients suffering from duodenal ulceration are reported. The data were collected from 9 clinics in the Netherlands, Belgium, Ireland, the United Kingdom, and Italy. The results from 94 patients treated with CBS coated tablets and 95 patients treated with CBS chewing tablets were statistically evaluated. Healing rates after 4 weeks of therapy appeared to be 76% for CBS coated tablets and 72% for CBS chewing tablets, so no statistically significant difference in therapeutic efficacy was seen. A highly significant degree of discolouration of all parts of the oral cavity was observed in patients treated with CBS chewing tablets, whereas only a few patients treated with CBS coated tablets experienced a slight staining of the tongue. Blood bismuth concentrations during the study had a range of less than or equal to 3 to 33 micrograms/l. The new CBS coated tablet form has an excellent patient compliance as compared to the chewing tablets.

Prokaryotic expression of a large fragment of the most antigenic cytomegalovirus DNA-binding protein (ppUL44) and its reactivity with human antibodies.

We isolated and characterized from a lambda gt11 expression library clones expressing portions of human cytomegalovirus (HCMV)-p52. This nonstructural viral protein is encoded by UL44 and is known to be one of the best IgM reactive antigens. The reactivity of these clones was studied with human antibody and the gene fragment coding for the most immune-reactive portion of p52 (aa 202-434) was cloned in a prokaryotic expression vector, pROS, which overexpresses the antigen as a fusion protein to a truncated molecule of beta-galactosidase.

Fibrinogen storage disease without hypofibrinogenaemia associated with acute infection

Aims:  The presence of ground glass hepatocytes in a liver biopsy may be related to different conditions, including fibrinogen storage disease. Three types of fibrinogen storage disease have been described, namely types I, II and III. Type I is an hereditary hypofibrinogenaemia genetically characterized by a mutant variant of the fibrinogen molecule designated as fibrinogen Brescia, consistent with a γ284 Gly→Arg mutation. Only rare cases of types II and III fibrinogen storage disease have been described. The purpose of the present paper is to describe two cases of fibrinogen storage disease without associated hypofibrinogenaemia, which appeared during acute infectious diseases.