A Semliki Forest virus expression system as a model for investigating the nuclear import and export of hepatitis B virus nucleocapsid protein.

HBV core protein (HBc), the major component of nucleocapsid is known to have an essential role in the virus life cycle as the transporter of virus genome to the host nucleus; however, molecular details of the intracellular transport of HBc remain unknown. In this study, we investigated the intracellular distribution of the HBc protein resulting from Semliki Forest virus (SFV)-driven HBc gene and HBV RNA pregenome (pgRNA) expression in BHK-21 cells. Fluorescent confocal microscopy revealed extensive HBc protein synthesis in the cytoplasm 12 hr post infection (p.i.) with recombinant pSFV1/HBc or pSFV1/HBVpgRNA viruses. Twenty-four hr p.i., the HBc protein showed asymmetric, predominantly nuclear localization in most cells. However, 42 hr p.i., the number of cells containing intranuclear HBc protein, dramatically decreased to ~ 5%, while cytoplasmic HBc protein was detected in all cells. Remarkably, 24 hr p.i. with pSFV1/HBVpgRNA virus, cytoplasmic HBc protein colocalized with HBs protein. We conclude that a HBs-HBc interaction partially prevents the transport of HBc from the cytoplasm to nucleus. The SFV-driven system can be used for identification of new factors involved in the HBV nuclear import and export.

Recombinant Semliki Forest virus vectors encoding hepatitis B virus small surface and pre-S1 antigens induce broadly reactive neutralizing antibodies.

Most hepatitis B virus (HBV) vaccines consist of viral small surface (S) protein subtype adw2 expressed in yeast cells. In spite of good efficacy, HBV-genotype and subtype differences, escape mutants and insufficient Th1 activation remain potential problems. To address these problems, we generated recombinant Semliki Forest virus (rSFV) vectors encoding S protein, subtype adw2 or ayw2, or a fragment of the large surface protein, amino acids 1-48 of the pre-S1 domain, fused to S (pre-S1.1-48/S). The antigen loop in S protein and the selected pre-S1 sequences are known targets of neutralizing antibodies. BALB/c mice were immunized intravenously with 10(7) rSFV particles and 10(8) rSFV particles 3 weeks later. Antibodies induced by rSFV encoding S proteins reacted preferentially with subtype determinants of yeast-derived S antigen but equally well with patient-derived S antigen. Immunization with rSFV encoding pre-S1.1-48/S resulted in formation of pre-S1- and S-specific immunoglobulin G (IgG), while immunization with the isogenic mutant without S start codon induced pre-S1 antibodies only. Neutralizing antibodies were determined by mixing with plasma-derived HBV/ayw2 and subsequent inoculation of susceptible primary hepatocyte cultures from Tupaia belangeri. S/adw2 antisera neutralized HBV/ayw2 as effectively as antisera raised with S/ayw2. The pre-S1 antibodies also completely neutralized HBV infectivity. The IgG1/IgG2a ratios ranged from 0.28 to 0.88 in the four immunized groups and were lowest for the pre-S1.1-48/S vector, indicating the strongest Th1 response. This vector type may induce subtype-independent and S-escape-resistant neutralizing antibodies against HBV.

Molecular characterisation and expression analysis of SEREX-defined antigen NUCB2 in gastric epithelium, gastritis and gastric cancer.

NUCB2 is an EF-hand Ca2+ binding protein that has been implicated in various physiological processes like calcium homeostasis, hypothalamic regulation of feeding and TNF receptor shedding. In our previous study we identified NUCB2 as a potential tumour antigen eliciting autoantibody responses in 5.4% of gastric cancer patients but not in the healthy individuals.The current study aimed to elucidate the molecular mechanism underlying NUCB2 immunogenicity and to gain an insight into the physiological functions of NUCB2 in the stomach. mRNA expression analysis demonstrated that NUCB2 is ubiquitously expressed in normal tissues, including lymphoid tissues, and downregulated in gastric tumours when compared with the adjacent relatively normal stomach tissues.The search for molecular alterations resulted in the identification of novel mRNA variants transcribed from an alternative promoter and expressed predominantly in gastric cancers. Western blot analysis demonstrated that the protein levels correspond to mRNA levels and revealed that NUCB2 is phosphorylated in gastric mucosa. Furthermore, a 55 kDa isoform,generated presumably by yet an unidentified post-translational modification was detected in gastric tumours and AGS gastric cancer cells but was absent in the relatively normal gastric mucosa and thereby might have served as a trigger for the immune response against NUCB2. Staining of stomach tissue microarray with anti-NUCB2 antibody revealed that it is expressed in the secretory granules of chief cells and in the cytoplasm of parietal cells in the functioning gastric glands which are lost in atrophic glands and tumour cells. Hence we propose that NUCB2 may be implicated in gastric secretion by establishing an agonist-releasable Ca2+ store in ER or Golgi apparatus, signalling via heterotrimeric Galpha proteins and/or mediating the exocytosis of the secretory granules.

Molecular characterisation and expression analysis of SEREX-defined antigen NUCB2 in gastric epithelium, gastritis and gastric cancer.

NUCB2 is an EF-hand Ca2+ binding protein that has been implicated in various physiological processes like calcium homeostasis, hypothalamic regulation of feeding and TNF receptor shedding. In our previous study we identified NUCB2 as a potential tumour antigen eliciting autoantibody responses in 5.4% of gastric cancer patients but not in the healthy individuals. The current study aimed to elucidate the molecular mechanism underlying NUCB2 immunogenicity and to gain an insight into the physiological functions of NUCB2 in the stomach. mRNA expression analysis demonstrated that NUCB2 is ubiquitously expressed in normal tissues, including lymphoid tissues, and downregulated in gastric tumours when compared with the adjacent relatively normal stomach tissues. The search for molecular alterations resulted in the identification of novel mRNA variants transcribed from an alternative promoter and expressed predominantly in gastric cancers. Western blot analysis demonstrated that the protein levels correspond to mRNA levels and revealed that NUCB2 is phosphorylated in gastric mucosa. Furthermore, a 55 kDa isoform, generated presumably by yet an unidentified post-translational modification was detected in gastric tumours and AGS gastric cancer cells but was absent in the relatively normal gastric mucosa and thereby might have served as a trigger for the immune response against NUCB2. Staining of stomach tissue microarray with anti-NUCB2 antibody revealed that it is expressed in the secretory granules of chief cells and in the cytoplasm of parietal cells in the functioning gastric glands which are lost in atrophic glands and tumour cells. Hence we propose that NUCB2 may be implicated in gastric secretion by establishing an agonist-releasable Ca2+ store in ER or Golgi apparatus, signalling via heterotrimeric Gα proteins and/or mediating the exocytosis of the secretory granules.

Analysis of the immune status in Latvian Chernobyl clean-up workers with nononcological thyroid diseases.

The aim of the present work was to characterize the immune status of 385 individuals who participated in the 1986-90 clean-up work of the after effects of the Chernobyl nuclear power plant explosion. Fifty-nine Chernobyl clean-up workers developed the most common thyroid diseases; euthyroid nodular and diffuse goiter; 47 healthy blood donors were taken as controls. The levels of immunoglobulins (IgA, IgG and IgM), the numbers of peripheral blood leukocytes, lymphocytes, monocytes, T lymphocytes and their subpopulations (CD3+, CD4+, CD8+), B lymphocytes (CD19+), natural killer (NK) cells (CD16+), classical and alternative pathway activity of complement (CH50, APH50), the C3 split product C3d, and neutrophil phagocytosis were determined in the peripheral blood. We found a significantly decreased number of CD16+ cells (natural killer), of CD4+ and CD8+ T lymphocytes, a reduced neutrophil phagocytic activity as well as a significant complement activation in Chernobyl clean-up workers with and without thyroid diseases when compared with normal levels and those in the control group. In addition, the number of CD3+ and CD4+ cells was significantly higher in patients with nodular goiter when compared with that in patients with diffuse goiter. Levels of IgG and numbers of monocytes were significantly decreased in persons who worked in Chernobyl in 1986 during the first 2 months after the accident (with maximal radiation exposure) but were without correlation to thyroid disorders. Our results clearly reflect an impaired immune system in the Chernobyl clean-up workers even 10-14 years after the nuclear accident.

Retrospective dosimetry for Latvian workers at Chernobyl.

Between 1986 and 1991 approximately 6500 Latvian inhabitants were recruited for clean-up work at the Chernobyl nuclear power plant. Their absorbed doses are usually unknown, because less than half of them had their external exposure officially documented. Clinical investigations show a high morbidity rate for these clean-up workers when compared with that of the general population. In order to understand the causes of their diseases and the impact of ionising radiation, electron spin resonance (ESR) has been used to measure the absorbed doses in human tooth enamel. The doses estimated by ESR were between two and three times higher than previously documented and are in accord with the results of immunological and biological tests. The results may be explained by considering the effects of irradiation caused by long-lived incorporated radionuclides.

[The cellular immunity indices of patients with malignant melanoma using the viral immunomodulator rigvir]. / Pokazateli kletochnogo immuniteta u bol'nykh zlokachestvennoi melanomoi pri primenenii virusnogo immunomoduliatora rigvira.

The effect of rigvir, an immunomodulator of the viral origin, on cell-mediated immunity was studied in patients with skin malignant melanoma. Rosette formation and monoclonal antibody techniques were used to measure blood immunocompetent cell levels in patients with the above pathology, cases of benign skin tumors and healthy subjects. Rigvir was shown to influence natural resistance by raising blood monocyte and large granule-containing lymphocyte levels. It potentiated recruitment of pre-T-lymphocytes and young active T-lymphocytes to the peripheral blood.

[The reaction of the T-immunity system in patients with malignant skin melanoma and stomach cancer to active nonspecific immunotherapy]. / Reaktsiia T-sistemy immuniteta bol'nykh zlokachestvennoi melanomoi kozhi i rakom zheludka na aktivnuiu nespetsificheskuiu immunoterapiiu.

Changes in E-receptor-bearing T-lymphocyte level (total and that of active T-lymphocytes) were studied in peripheral blood and resected material obtained from skin malignant melanoma and gastric cancer patients treated with rigvir, an original immunomodulator of the viral origin. Injection of rigvir into peripheral blood was followed by an increase in active T-lymphocyte level and stimulated their migration into tumor. The latter was determined by stage and rate of tumor advancement.

[Immunomodulation mechanisms in the anti-tumor effect of the ECHO-7 enterovirus]. / Mekhanizm immunomoduliatsii v protivoopukholevom deistvii énterovirusa ECHO-7.

It has been established that the complex systemic and local administration of human ECHO-7 virus exerts an inhibitory effect on the growth of MX-17 tumour in the BALB/c mice without the signs of viral oncolysis. The antitumour effect is due to a modulation of the antitumour immunity (immunological regression) against a background of the viral immunogenesis.