Helle L. Jensen

ProCNP and CNP are expressed primarily in male genital organs

Lack of knowledge about the cellular origin of C-type natriuretic peptides (CNP) in the body has hampered the understanding of their biology. We examined the tissue specific expression of proCNP and CNP in the pig. The concentration of the CNP precursor, proCNP, was measured in extracts of 32 different tissues using a newly developed RIA. In 22 tissue extracts, we also measured CNP using a commercial RIA. In selected tissues, CNP mRNA was quantified by PCR, and the cellular CNP and proCNP localization was visualized by immunocytochemistry. Extracts from selected tissues were examined by gel chromatography. The highest peptide concentrations were found in extracts from the epididymis, seminal vesicles and prostate. CNP mRNA in the seminal vesicles and epididymis was 125-fold higher than in the other tissues examined. Gel chromatography showed that a CNP-53-like peptide is the dominant CNP tissue-form. Immunocytochemistry confirmed the pattern of peptide expression measured by RIA. In conclusion most proCNP-derived peptides are synthesized in epithelial cells in the epididymis, the prostate gland and in the seminal vesicles. The expression in male genital organs suggests a role of CNP in reproduction.

Herpes simplex virus type 1-infected human embryonic lung cells studied by optimized immunogold cryosection electron microscopy.

Herpes simplex virus type 1 (HSV-1) is a common human pathogen of skin and mucous membranes and is potentially dangerous when the infection is disseminated. Viral morphogenesis, especially the mechanism of viral envelopment and the exact pathway for processing and transport of HSV-1 glycoproteins, is still unclear. We report the results of optimized immunogold-labeled cryosection electron microscopy of HSV-1-infected cultured human fibroblasts (MRC-5). The simplified method presented has proved necessary to obtain reproducible results on cellular distribution of viral glycoproteins. It is now possible to demonstrate the viral glycoprotein gD-1, but not gC-1, in the nuclear membranes and to demonstrate gD-1- and gC-1-labeled viral particles in the perinuclear space, and to show the fate of the viral particles in the endoplasmic reticulum and Golgi area in infected cells.

The effects of cell passages on the cell morphology and the outcome of herpes simplex virus type 1 infection

Because cell cultures are essential in biological research which involves the analysis of virus morphogenesis, this study focused on examining the significance of cell passages. Human embryonic lung fibroblasts (MRC-5) at passage (P) 27 were seeded twice a week to P 32, P 40, and P 48, when just at confluence and then infected with herpes simplex virus type 1 (HSV-1). The structure of the non-virus-infected (MOCK) and HSV-1 infected cells, the amount of cellular infectious virus particles and the capability to express HSV-1 glycoproteins C (gC-1) and D (gD-1) were investigated by phase-contrast and immunofluorescence light microscopy, immunogold cryosection EM, plaque assays, immunoblots, and total protein assays. Modified cell structure including fragmentation of tubulin fibers were visible in MOCK from P 38 onwards. The quantity of vimentin remained unchanged while actin accumulated and beta-tubulin decreased in HSV-1 infected late P cells compared to early P cultures. Cells of high P counts contained significantly fewer infectious virus particles, very likely of lower virulence, and their expression of gC-1 and gD-1 were concordantly reduced. These observations indicate that the number of cell P must be considered in order to reproduce results of cell biology and viral morphogenesis. The MRC-5 cells ought not to be passaged more than ten times beyond P 27 in the laboratory.

Easy and Reliable Double-Immunogold Labelling of Herpes Simplex Virus Type-1 Infected Cells Using Primary Monoclonal Antibodies and Studied by Cryosection Electron Microscopy

Cell biology concerns the interactions between different cellular compartments and between the cell and the environment. The mechanisms of herpes simplex virus type-1 (HSV-1) envelopment and the transport of virus particles and HSV-1 glycoproteins have not been completely investigated. It is of interest to examine the formation of complete virus particles and the cellular distribution of viral glycoproteins correlated with microtubules. The illustration of these conditions by immunocytochemistry is best done by multiple labelling techniques in the same cell. Single-staining of neighbouring serial sections or two-face double-immunolabelling methods are not technically compatible with ultrathin cryosections. The results are reported here of a simultaneous, simple and reliable immunogold double-staining technique using primary antibodies of the same species in ultrathin cryosections. Compared to other inactivation procedures, phosphate-buffered 3% paraformaldehyde plus 2% glutaraldehyde for 2 h at room temperature is an excellent and gentle method to destroy free anti-IgG binding sites on the antibodies and to prevent cross-labelling, which has proven necessary for obtaining reproducible results on cellular distribution of tubulin and viral glycoproteins gD-1 and gC-1.

A time-related study of Brefeldin A effects in HSV-1 infected cultured human fibroblasts

Glycoprotein D (gD‐1) is an essential virion envelope component of herpes simplex virus type 1 (HSV‐1) normally transported to the plasma membrane of the infected cells. In the present study, the intracellular transport of gD‐1 was inhibited in cultured HSV‐1 infected human fibroblasts by Brefeldin A (BFA) 1μg/ml medium added for 12 h after virus adsorption. Immunofluorescence light‐ and confocal microscopy revealed abolished transport of gD‐1 to the plasma membrane, juxtanuclear accumulation of gD‐1, and a disorderly arrangement of the tubulin fibres. Withdrawal of BFA influence for more than 60 min resulted in incomplete transport but increasing accumulation of gD‐1 in the plasma membrane and in Golgi‐like areas close to the nuclei. The tubulin pattern was almost normalized 6 h after removal of BFA. The egress of infectious HSV‐1 particles released 9 h post‐BFA treatment was not fully reestablished. The results indicate that BFA effects were not completely reversible and caused a sort of cytotoxic influence involving the structure of tubulin.

Thomsen-Friedenreich-related antigen in human endometrium

The Thomsen‐Friedenreich antigen (T‐antigen) Ga1β1‐3Ga1NAc, is a cryptic disaccharide structure on human erythrocytes. It is masked by sialic acid and uncovered by sialidases as neuraminidase. T‐antigen is a tumor‐associated antigen in epithelial tissues. This study reports preliminary immunohistochemical findings on the expression of the blood group related T‐antigen in human endometrium. Dewaxed sections of 10 proliferative‐, 6 interval‐, 14 secretory endometrium, and 16 endometrial carcinomas, either untreated or pretreated with neuraminidase, were subjected to an indirect immunoperoxidase technique using the monoclonal antibody 49H.8. The monoclonal antibody 49H.8 did not bind to normal cyclic endometrium, unless pretreated with neuraminidase. Neuraminidase treatment revealed staining of apical membranes of glandular‐ and surface epithelium in early or manifest secretory endometrium only. Thirteen of sixteen carcinomas stained without neuraminidase treatment, while all carcinomas stained after pretreatment with neuraminidase. Staining was more widespread than in untreated carcinomas, resembling that of secretory endometrium. Our results suggest: 1) That the sialylated 49H.8 antigen seems to be a marker of cellular differentation in the endometrium, 2) that the antigen defined by the monoclonal antibody 49H.8 might be a tumor‐associated antigen in endometrial epithelial tissue.

Assessment of response to beta-blockers by expression of βArr2 and RhoA/ROCK2 in antrum mucosa in cirrhotic patients

BACKGROUND & AIMS Non-selective beta-blockers (NSBB) are first choice for prevention of variceal bleeding. But possible deleterious effects in refractory ascites and frequent non-response are clinical drawbacks. Since levels of vasoactive proteins in antrum mucosa reflect vascular dysfunction in cirrhosis, these expression levels might also reflect hemodynamic response to NSBB. METHODS Biopsies from the gastric and duodenal mucosa of 25 patients with cirrhosis were collected and the hepatic venous pressure gradient (HVPG) was measured before and after an acute propranolol challenge. Transcription and protein expression of Ras homolog family member A (RhoA), Rho-kinase (ROCK)2, beta-arrestin2 (βArr2), endothelial nitric oxide synthase (eNOS) and the phosphorylation of downstream effectors VASP and moesin were analyzed using PCR and Western blot. Further 21 patients on NSBB were evaluated on their follow up for events of variceal bleeding defined as non-response. RESULTS Ten patients showed HVPG <10mmHg, further seven patients showed significant hemodynamic response to NSBB, whereas eight patients were non-responders. The mucosal transcription of vasoactive proteins was higher in antrum mucosa compared to corpus and duodenum. The transcriptional levels of vasoactive proteins were higher in patients with HVPG >10mmHg and HVPG >16mmHg. Interestingly, mRNA levels of RhoA and ROCK2 were lower in patients with large varices at endoscopy. Moreover, RhoA and ROCK2 transcription correlated with the decrease of HVPG after acute NSBB challenge. Finally, acute and long-term non-responders showed lower expression of βArr2 in antrum mucosa. CONCLUSION This study shows for the first time that the expression of βArr2 in antrum mucosa biopsies might reflect the hemodynamic response to NSBB and their long-term protective effect. This finding might offer an easy approach at upper endoscopy to facilitate the decision to treat with NSBB if varices are present.

Morphologic, immunohistochemical, immunologic, ultrastructural, and time-related study of herpes simplex virus type 1-infected cultured human fibroblasts.

Membrane glycoproteins of enveloped animal viruses are synthesized, processed, and transported inside infected cells. Expression of viral glycoproteins on the surface of viral particles and host cells are essential for many biologic functions. In the case of herpes simplex virus, the glycoprotein molecules may act as nucleation points for virus assembly and budding at the nuclear membrane. The temporal distribution of herpes simplex virus type 1 particles and glycoproteins in cultured human fibroblasts was studied by titration plaque assay, immunoblots, immunofluorescence light microscopy, and immunogold cryosection electron microscopy to describe the virus-cell interactions. These concordant analyses revealed significant release of infectious viral particles to the medium at 6 hours postinfection, that the capacity of the host cells to make infectious viral particles was complete at 18 hours postinfection, and that the infection brought time-related modifications of tubulin, cell morphology, and viral glycoproteins. The data presented is in accord with the theory of envelopment at the nuclear membranes containing immature glycoproteins followed by multiple deenvelopments and reenvelopments of the virus particles during the transport and maturation in the endoplasmic reticulum and the Golgi complex.

Metastasizing malignant pleomorphic adenoma in a young man

We present the case of a younger man with metastasizing carcinoma of unknown primary site, where autopsy revealed a malignant pleomorphic adenoma (MPA) of the submandibular gland. MPA is very rare in young persons.

The morphogenesis of herpes simplex virus type 1 in infected parental mouse L fibroblasts and mutant gro29 cells.

Mutants of cell lines and viruses are important biological tools. The pathway of herpesvirus particle maturation and egress are contentious issues. The mutant gro29 line of mouse L cells is defective for egress of herpes simplex virus type 1 (HSV‐1) virions, and a candidate for studies of virus‐cell interactions. The properties of uninfected and HSV‐1‐infected L fibroblasts and gro29 cells investigated by protein assay, immunoblot, titration assay, immunofluorescence light microscopy and immunogold cryosection electron microscopy are reported. The ultrastructure of both HSV‐1‐infected L and gro29 cells confirmed primary envelopment of virions at the nuclear membranes followed by maturing multiple de‐envelopments and re‐envelopments in the endoplasmic reticulum and in the Golgi complex. The gro29 cells presented changed cytoskeleton, abolished egress of virions, and were defective in the trafficking of glycoproteins, giving rise to accumulation of viral particles and glycoproteins in the endoplasmic reticulum and the Golgi complex. The results suggest that gro29 cells harbour a causal underlying defect of the cytoskeleton in addition to the HSV‐1‐induced cytoskeletal changes.