Cytoglobin Exhibits Anti-Fibrosis Activity on Liver In Vivo and In Vitro

Cytoglobin, generated using genetic engineering method, is a kind of recombinant human stellate cell activation-associated protein. We speculate that it could influence the development of hepatic fibrosis like Sellate cell activation-associated protein which was discovered by Kawada et al. Therefore, we investigated its anti-fibrosis effect on liver both in vivo and in vitro. During our research, we found that cytoglobin showed obvious effect compared with the control group on Thioacetamide-induced liver fibrosis in SD rats, including significantly decrease in aspartate aminotransferase, Hyaluronic acid, laminin and collagen I(Col I) levels in serum and hydroxyproline in livers, which are the important indices reflecting the degree of hepatic fibrosis. Meanwhile, the viability of rat hepatic stellate cell line T6 (HSC-T6) cells was inhibited by cytoglobin and the apoptosis induced by cytoglobin in HSC-T6 cells was detected by Annexin V/PI double staining. Activation of the caspase cascade including caspase-3 for the intrinsic pathways was demonstrated. The results also showed that the expression of Bcl-2 protein decreased whereas that of Bax protein increased, leading to an increase of the Bax/Bcl-2 ratio. Our results demonstrated that cytoglobin exhibited anti-fibrosis activity on livers in vivo and in vitro, involving apoptosis induction.     

The M33 Chemokine Receptor Homolog of Murine Cytomegalovirus Exhibits a Differential Tissue-Specific Role during In Vivo Replication and Latency

M33, encoded by murine cytomegalovirus (MCMV), is a member of the UL33 homolog G-protein-coupled receptor (GPCR) family and is conserved across all the betaherpesviruses. Infection of mice with recombinant viruses lacking M33 or containing specific signaling domain mutations in M33 results in significantly diminished MCMV infection of the salivary glands. To determine the role of M33 in viral dissemination and/or infection in other tissues, viral infection with wild-type K181 virus and an M33 mutant virus, ΔM33B_T2, was characterized using two different routes of inoculation. Following both intraperitoneal (i.p.) and intranasal (i.n.) inoculation, M33 was attenuated for infection of the spleen and pancreas as early as 7 days after infection. Following i.p. inoculation, ΔM33B_T2 exhibited a severe defect in latency as measured by a diminished capacity to reactivate from spleens and lungs in reactivation assays (P < 0.001). Subsequent PCR analysis revealed markedly reduced ΔM33B_T2 viral DNA levels in the latently infected spleens, lungs, and bone marrow. Following i.n. inoculation, latent ΔM33B_T2 viral DNA was significantly reduced in the spleen and, in agreement with results from i.p. inoculation, did not reactivate from the spleen (P < 0.001). Furthermore, in vivo complementation of ΔM33B_T2 virus replication and/or dissemination to the salivary glands and pancreas was achieved by coinfection with wild-type virus. Overall, our data suggest a critical tissue-specific role for M33 during infection in the salivary glands, spleen, and pancreas but not the lungs. Our data suggest that M33 contributes to the efficient establishment or maintenance of long-term latent MCMV infection.Since the discovery of the G protein-coupled receptors (GPCRs) encoded by the betaherpesviruses, there has been intense speculation on the biological role these viral proteins play during infection (15, 16, 22). Human cytomegalovirus (HCMV), a betaherpesvirus, is a ubiquitous pathogen that asymptomatically infects humans and establishes a long-term persistent infection. HCMV is life-threatening, however, to immunocompromised individuals, such as neonates, AIDS patients, and transplant recipients. HCMV, similar to a number of herpesviruses, encodes viral genes that are predicted to impact virus-host interactions that may promote efficient long-term infection of the host. The CMVs encode genes for proteins that potentially enhance viral dissemination and replication and promote immune evasion by mimicry of host functions that influence the conditions of primary infection, the virus-specific immune response, and even long-term host control of persistent or latent infection (reviewed in references 1, 44, and 68).HCMV encodes four GPCRs (UL33, UL78, US28, and US27) which share homology to host chemokine receptors (16). This suggests that these virally encoded chemokine receptors may function similarly to their cellular receptor counterparts. Chemokines are chemoattractant cytokines that bind and activate chemokine receptors that are on the surfaces of cells. Host chemokine receptors then mediate the activation of cellular signaling pathways and cell migration to sites of inflammation by transmitting signals through G proteins (56, 70). In humans, approximately 50 chemokines and 20 chemokine receptors have been identified, many of which have close homologs in mice and other species (39). Chemokines are divided into two classes, lymphoid chemokines, which are constitutively expressed and involved in lymphoid tissue organization, and inflammatory chemokines, which are induced following infection and part of the inflammatory response (21, 39, 51). Growing evidence indicates that chemokines play a critical role in the host response to infection and inflammation during both the innate and adaptive immune responses (26), thus suggesting that the betaherpesviruses have “hijacked” the chemokine receptors from the host genome to subvert or alter these responses during infection. Besides chemokine receptors, HCMV also encodes a CXC chemokine (UL146) that induces the migration of neutrophils (48); a second CXC chemokine homolog (UL147) whose function is not yet known; a viral CC chemokine (UL131) that is critical for infection of macrophages, endothelial cells, and epithelial cells (25, 57, 73); and a RANTES decoy protein (72). A CC chemokine (vMCK or m131/129) is also encoded by murine CMV (MCMV), and a homolog in rat CMV ([RCMV] r131) that promotes monocyte-associated viremia (20, 37, 59, 60). The MCMV m131/129 chemokine was shown to recruit myelomonocytic progenitors from the bone marrow, perhaps to facilitate cell-type-specific viremia (46). Clearly, the CMVs have invested a great deal of effort into manipulating or subverting the host chemokine system, thus making it reasonable to speculate that these viral members of the chemokine system play an important role during CMV pathogenesis.Of the HCMV-encoded GPCRs, US28 has been well characterized in vitro and functions as a bona fide chemokine receptor, whereas much less is known about the receptor activity of US27, UL33, and UL78. US28 binds and sequesters CC chemokines, induces smooth-muscle cell migration, and constitutively activates signaling pathways (5, 7-9, 42, 52, 64, 67, 71). US28 and US27 are found only in primate CMVs, whereas both UL33 and UL78 are highly conserved across all betaherpesvirus genomes, suggesting an important evolutionary function for UL33 and UL78 during CMV infection. Two other betaherpesviruses, human herpesviruses 6 and 7 (HHV6 and HHV7), encode homologs to the UL33 and UL78 receptors, U12 and U51, respectively. The U12 receptors of HHV6 and HHV7 (34, 45, 66) and the HHV6-encoded U51 receptor (22) exhibit chemokine binding activity. UL33, along with its rodent CMV homologs, M33 (MCMV) and R33 (RCMV), constitutively activates signaling pathways (13, 23, 71). M33 induces smooth-muscle cell migration (39), similar to US28-mediated smooth-muscle cell migration (64). Thus, members of the UL33 family potentially function during viral infection by modulating or influencing the composition of leukocytes at sites of infection, the migration of infected cells or infiltrating leukocytes, or modulation of intracellular signaling pathways.Due to the species specificity of CMV, the in vivo role of the HCMV-encoded GPCRs cannot be addressed. However, the importance of UL33 and UL78 for viral dissemination and virulence in vivo has been indicated by disruption of the viral homologs in MCMV and RCMV (6, 19, 36, 47). Disruption of the UL33, M33, and R33 genes demonstrated that they are dispensable for replication in vitro, indicating that the UL33 family members are not required for replication or cell entry in at least some cell types (6, 19, 40). Infection of mice with M33-deficient MCMV or infection of rats with R33-deficient RCMV results in highly attenuated viruses and diminished infection of the salivary glands. The RCMV R33 protein also appears to play a role in virulence since rats infected with an R33 deletion virus had a lower mortality rate (6). More recently, constitutive M33-mediated activation of signaling pathways was shown to be essential for MCMV infection of salivary glands (14). Significantly, the UL33 protein partially rescued the defect in salivary gland infection attributed to disruption of M33, indicating the evolutionary conservation of function between the HCMV (UL33) and MCMV (M33) chemokine receptor homologs.In this paper, the role of M33 is further investigated using two routes of infection to assess viral dissemination and viral replication kinetics at different tissue sites, the numbers of infected cells following infection, and the possibility that M33 plays a role during latent infection. In addition to the critical role that M33 plays in salivary gland infection, this study reveals that M33 is important for MCMV infection of the spleen and the pancreas but not the lungs. Significantly, our studies provide preliminary evidence that disruption of M33 leads to reduced latent viral load in the spleen, lungs, and bone marrow, perhaps due to defects in the establishment and/or maintenance of latent infection. Lastly, we demonstrate that the tissue defects observed during acute infection with an M33 mutant virus (ΔM33B_T2) can be complemented in vivo when mice are coinfected with ΔM33B_T2 virus and wild-type MCMV. Taken together, our findings indicate that M33 plays a critical tissue-specific role during acute MCMV infection and, importantly, contributes to the efficient establishment or maintenance of latent MCMV infection.     

Minimum Requirements for Bluetongue Virus Primary Replication In Vivo

The replication mechanism of bluetongue virus (BTV) has been studied by an in vivo reverse genetics (RG) system identifying the importance of certain BTV proteins for primary replication of the virus. However, a unique in vitro cell-free virus assembly system was subsequently developed, showing that it did not require the same set of viral components, which is indicative of differences in these two systems. Here, we studied the in vivo primary replicase complex more in-depth to determine the minimum components of the complex. We showed that while NS2 is an essential component of the primary replication stage during BTV infection, NS1 is not an essential component but may play a role in enhancing BTV protein synthesis. Furthermore, we demonstrated that VP7, a major structural protein of the inner core, is not required for primary replication but appears to stabilize the replicase complex. In contrast, VP3, the other major structural core protein, is an essential component of the complex, together with the three minor enzymatic proteins (VP1, VP4, and VP6) of the core. In addition, our data have demonstrated that the smallest minor protein, VP6, which is known to possess an RNA-dependent helicase activity, may also act as an RNA translocator during assembly of the primary replicase complex.     

Cell-Free Transmission and In Vivo Replication of Marek''s Disease Virus

Marek's disease virus recovered from the feather follicle of infected chickens was found to be infectious for chickens in cell-free preparations. The virus replicated in epithelial cells of the germinative layer of the feather follicle epidermis, producing both intranuclear and round or diffuse cytoplasmic inclusion bodies in the infected cells. It was found at this site 2 weeks postinoculation and prior to the development of tumor or other gross lesions. In the nucleus, many naked and a few enveloped herpesvirions were found, whereas the cytoplasm contained predominantly enveloped herpesvirions, which were usually within the cytoplasmic inclusion bodies. Approximately 80% of the extracellular virions were enveloped. Studies with both virulent and avirulent strains of the virus revealed a relationship between virulence, contagiousness, and replication of the virus in the feather follicle.     

In Vivo Association of Ku with Mammalian Origins of DNA Replication

Ku is a heterodimeric (Ku70/86-kDa) nuclear protein with known functions in DNA repair, V(D)J recombination, and DNA replication. Here, the in vivo association of Ku with mammalian origins of DNA replication was analyzed by studying its association with ors8 and ors12, as assayed by formaldehyde cross-linking, followed by immunoprecipitation and quantitative polymerase chain reaction analysis. The association of Ku with ors8 and ors12 was also analyzed as a function of the cell cycle. This association was found to be approximately fivefold higher in cells synchronized at the G1/S border, in comparison with cells at G0, and it decreased by approximately twofold upon entry of the cells into S phase, and to near background levels in cells at G2/M phase. In addition, in vitro DNA replication experiments were performed with the use of extracts from Ku80(+/+) and Ku80(-/-) mouse embryonic fibroblasts. A decrease of approximately 70% in in vitro DNA replication was observed when the Ku80(-/-) extracts were used, compared with the Ku80(+/+) extracts. The results indicate a novel function for Ku as an origin binding-protein, which acts at the initiation step of DNA replication and dissociates after origin firing.     

Silencing HIV-1 In Vivo

RNA interference holds great promise for antiviral therapy, but delivering effective quantities of small interfering RNAs (siRNAs) into the right target cells in vivo represents a major challenge. Kumar et al. (2008) now report a technique to target siRNAs specifically to T cells to suppress viral infection in a humanized mouse model of HIV/AIDS.     

乙型肝炎病毒(HBV)体内外对单个核细胞白细胞介素-1和白细胞介素-2诱生活性影响的研究

本研究用PAP法、胸腺细胞增殖法、脾细胞增殖法,分别检测16例体外HBV感染的骨髓单个核细胞与16例慢性乙型肝炎患者体内感染的骨髓单个核细胞(MNCs)中的HBcAg和白细胞介素-1(IL-1)、白细胞介素-2(IL-2)的诱生活性(以△cpm值表示)。结果显示,体外HBV感染组与体内HBV感染组骨髓MNCs中HBcAg检出率分别为50％和43.7％。本实验结果表明,HBV在体外感染骨髓MNCs,且与体内自然感染相符,但光镜下未观察到致细胞病变效应(CPE)。体外感染组与体内感染组IL-I和IL-2活性均较对照组明显下降(P<0.01)。且细胞中HBcAg检出阳性者较阴性者下降更为明显(P<0.01)。IL-1和IL-2诱生活性降低与HBV侵染免疫细胞及其在细胞内复制有密切关系,从而提示,IL-1和IL-2降低可能影响HBV的清除而引起慢性化过程。     

Multiple Lytic Origins of Replication Are Required for Optimal Gammaherpesvirus Fitness In Vitro and In Vivo

An unresolved question in herpesvirus biology is why some herpesviruses contain more than one lytic origin of replication (oriLyt). Using murine gammaherpesvirus 68 (MHV-68) as model virus containing two oriLyts, we demonstrate that loss of either of the two oriLyts was well tolerated in some situations but not in others both in vitro and in vivo. This was related to the cell type, the organ or the route of inoculation. Depending on the cell type, different cellular proteins, for example Hexim1 and Rbbp4, were found to be associated with oriLyt DNA. Overexpression or downregulation of these proteins differentially affected the growth of mutants lacking either the left or the right oriLyt. Thus, multiple oriLyts are required to ensure optimal fitness in different cell types and tissues.     

An In Vivo Giemsa Chromosome Banding Technique

An in vivo chromosome banding technique has been developed. Swiss albino mice were injected with the DNA alkylating agents ethyl methanesulfonate, methyl methanesulfonate, or methyl ethanesulfonate 12, 24, 48 or 72 hours prior to cell harvesting. After harvesting, the cells were fixed with 3:1 methanol-acetic acid and slides were prepared by air drying. The slides were stained 2¹/₂ minutes in 3% Giemsa in pH 6.8 Sorensen's buffer. All three alkylating agents induced chromosome bands similar to the Giemsa bands induced by other banding techniques which involve postfixation treatments.     

Posaconazole Exhibits In Vitro and In Vivo Synergistic Antifungal Activity with Caspofungin or FK506 against Candida albicans

The object of this study was to test whether posaconazole, a broad-spectrum antifungal agent inhibiting ergosterol biosynthesis, exhibits synergy with the β-1,3 glucan synthase inhibitor caspofungin or the calcineurin inhibitor FK506 against the human fungal pathogen Candida albicans. Although current drug treatments for Candida infection are often efficacious, the available antifungal armamentarium may not be keeping pace with the increasing incidence of drug resistant strains. The development of drug combinations or novel antifungal drugs to address emerging drug resistance is therefore of general importance. Combination drug therapies are employed to treat patients with HIV, cancer, or tuberculosis, and has considerable promise in the treatment of fungal infections like cryptococcal meningitis and C. albicans infections. Our studies reported here demonstrate that posaconazole exhibits in vitro synergy with caspofungin or FK506 against drug susceptible or resistant C. albicans strains. Furthermore, these combinations also show in vivo synergy against C. albicans strain SC5314 and its derived echinocandin-resistant mutants, which harbor an S645Y mutation in the CaFks1 β-1,3 glucan synthase drug target, suggesting potential therapeutic applicability for these combinations in the future.