[Research progress in immune microenvironment and immunotherapy of brain metastasis in non-small cell lung cancer].

In recent years, the rise of immunotherapy has comprehensively changed the treatment pattern of advanced non-small cell lung cancer(NSCLC). With the understanding of the immune microenvironment in brain metastasis, patients with advanced lung cancer with brain metastasis, who usually were previously excluded from clinical studies, gradually gained attention from researchers. The development from immune monotherapy to combination with chemotherapy significantly improved the survival prognosis of patients with brain metastases from lung cancer. This review aimed to briefly elucidate the mechanism of the immune microenvironment, and summarized the latest research progress of immunotherapy for NSCLC patients with brain metastasis.

Risk factors for disease progression in patients with mild to moderate coronavirus disease 2019-a multi-centre observational study.

OBJECTIVES: Since December 2019, the novel coronavirus disease 2019 (COVID-19) that emerged in Wuhan city has spread rapidly around the world. The risk for poor outcome dramatically increases once a patient progresses to the severe or critical stage. The present study aims to investigate the risk factors for disease progression in individuals with mild to moderate COVID-19. METHODS: We conducted a cohort study that included 1007 individuals with mild to moderate COVID-19 from three hospitals in Wuhan. Clinical characteristics and baseline laboratory findings were collected. Patients were followed up for 28 days for observation of disease progression. The end point was the progression to a more severe disease stage. RESULTS: During a follow up of 28 days, 720 patients (71.50%) had recovered or were symptomatically stable, 222 patients (22.05%) had progressed to severe disease, 22 patients (2.18%) had progressed to the critically ill stage and 43 patients (4.27%) had died. Multivariate Cox proportional hazards models identified that increased age (hazard ratio (HR) 2.56, 95% CI 1.97-3.33), male sex (HR 1.79, 95% CI 1.41-2.28), presence of hypertension (HR 1.44, 95% CI 1.11-1.88), diabetes (HR 1.82, 95% CI 1.35-2.44), chronic obstructive pulmonary disease (HR 2.01, 95% CI 1.38-2.93) and coronary artery disease (HR 1.83, 95% CI 1.26-2.66) were risk factors for disease progression. History of smoking was protective against disease progression (HR 0.56, 95% CI 0.34-0.91). Elevated procalcitonin (HR 1.72, 95% CI 1.02-2.90), urea nitrogen (HR 1.72, 95% CI 1.21-2.43), α-hydroxybutyrate dehydrogenase (HR 3.02, 95% CI 1.26-7.21) and D-dimer (HR 2.01, 95% CI 1.12-3.58) at baseline were also associated with risk for disease progression. CONCLUSIONS: This study identified a panel of risk factors for disease progression in individuals with mild to moderate COVID-19.

[A novel tissue-engineered bone constructed by using human adipose-derived stem cells and biomimetic calcium phosphate scaffold coprecipitated with bone morphogenetic protein-2].

OBJECTIVE: To construct a novel biomimetic calcium phosphate (BioCaP) scaffold loaded with bone morphogenetic protein-2 (BMP-2), and to investigate its role in the osteogenesis of human adipose-derived stem cells (hASCs) in vitro and in vivo. METHODS: The BioCaP scaffold coprecipitated with BMP-2 (BMP-2-BioCaP) was constructed in this study. Field emission scanning electron microscopy (SEM) was used to analyze the morphology of the surfaces. The release kinetics was measured to evaluate the slow-release characteristics in vitro. BMP-2-BioCaP was immersed in proliferation medium (PM) or osteogenic medium (OM), respectively. The supernatants were collected and used to culture hASCs in vitro. Cell numbers were determined using the cell-counting kit-8 (CCK-8) to assess the cell proliferation. After 7 and 14 days, alkaline phosphatase (ALP) staining and quantification were performed to test the activity of ALP. After 14 and 21 days, the calcification deposition was determined by alizarin red S (ARS) staining and quantification. The expressions of the osteoblast-related genes were tested on day 4 and day 14. In the in vivo study, 6 nude mice were used and implanted subcutaneously into the back of the nude mice for 4 groups: (1) BioCaP scaffold only, (2) BioCaP scaffold+hASCs, (3) BMP-2-BioCaP scaffold, (4) BMP-2-BioCaP scaffold+hASCs (test group). After 4 weeks of implantation, hematoxylin-eosin (HE) staining was performed to evaluate the in vivo osteogenesis of hASCs. RESULTS: SEM observations showed that BioCaP and BMP-2-BioCaP scaffold were entirely composed of straight, plate-like and sharp-edged crystal units, and the length of the crystal units varied between 5 and 10 µm. Release kinetics analysis demonstrated that BMP-2 incorporated with BioCaP could be released at certain concentration and last for more than 21 days, and the accumulative protein release could reach 20%. CCK-8 assays showed that cell proliferation was not significantly affected by BMP-2-BioCaP. ALP activity was higher by the induction of OM+BMP-2-BioCaP than of the other groups (P<0.01). More mineralization deposition and more expressions of osteoblast-related genes such as Runt-related transcription factor 2 (RUNX2), ALP, osteopontin (OPN) and osteocalcin (OC) were determined in the OM+BMP-2-BioCaP group at different time points (P<0.01). HE staining showed that, in the test group and BMP-2-BioCaP scaffold group, the extracellular matrix (ECM) with eosinophilic staining were observed around hASCs, and newly-formed bone-like tissues could be found in ECM around the scaffold materials. Moreover, compared with the BMP-2-BioCaP scaffold group, more bone-like tissues could be observed in ECM with typical structure of bone tissue in the test groups. No obvious positive results were found in the other groups. CONCLUSION: BMP-2-BioCaP scaffold could achieve slow-release of BMP-2 and promote the osteogenic differentiation of hASCs in vitro and in vivo. The novel tissue-engineered bone composed of hASCs and BMP-2-BioCaPis promising for the repair of bone defect.

Intracellular effects of the Hepatitis C virus nucleoside polymerase inhibitor RO5855 (Mericitabine Parent) and Ribavirin in combination.

Mericitabine (RG7128) is the prodrug of a highly selective cytidine nucleoside analog inhibitor (RO5855) of the hepatitis C virus (HCV) NS5B RNA-dependent RNA polymerase. This study evaluated the effects of combining RO5855 and ribavirin on HCV replication in the HCV subgenomic replicon by using two drug-drug interaction models. The effects of RO5855 and ribavirin on the intracellular metabolism of each compound, on interferon-stimulated gene (ISG) expression, and on the viability of hepatocyte-derived cells were also investigated. RO5855 and ribavirin had additive inhibitory activities against HCV subgenomic replicon replication in drug-drug interaction analyses. RO5855 did not affect the uptake or phosphorylation of ribavirin in primary human hepatocytes, human peripheral blood mononuclear cells, or genotype 1b (G1b) replicon cells. Similarly, ribavirin did not affect the concentrations of intracellular species derived from RO5855 in primary human hepatocytes or the formation of the triphosphorylated metabolites of RO5855. Ribavirin at concentrations of >40 µM significantly reduced the viability of primary hepatocytes but not of Huh7, the G1b replicon, or interferon-cured Huh7 cells. RO5855 alone or with ribavirin did not significantly alter the viability of Huh7 or G1b replicon cells, and it did not significantly affect the viability of primary hepatocytes when it was administered alone. The viability of primary hepatocytes was reduced when they were incubated with RO5855 and ribavirin, similar to the effects of ribavirin alone. RO5855 alone or with ribavirin had no effect on ISG mRNA levels in any of the cells tested. In conclusion, RO5855 did not show any unfavorable interactions with ribavirin in human hepatocytes or an HCV subgenomic replicon system.

Hepatitis C virus glycoprotein E2 binding to CD81: the role of E1E2 cleavage and protein glycosylation in bioactivity.

The hepatitis C virus glycoproteins E1 and 2 have been expressed using recombinant baculoviruses following fusion to the carrier protein glutathione S-transferase (GST). Proteins were expressed singly and as an E1E2 polyprotein with and without an N-terminal affinity tag. Expression of the E1E2 polyprotein, even when preceded by GST, led to processing in insect cells and detection of an E1E2 complex that could be specifically purified by glutathione affinity chromatography. Baculovirus expressed E2 and a purified GST-E1E2 protein bound to the second extracellular loop of CD81 (EC2), a reported ligand for the molecule, but not to a truncated derivative of CD81 consisting of only the central domain of the loop. Purified GST-E2, however, failed to bind to CD81 suggesting a requirement for a free E2 amino terminus for biological activity. The binding to CD81 by baculovirus expressed E2 protein was comparable to that observed for E2 derived from mammalian cells when detected by a monoclonal antibody sensitive to protein conformation. Furthermore, E2 protein expressed in insect cells in the presence of N-butyldeoxynojirimycin, an inhibitor of terminal glucose residue processing, formed complexes with E1 and bound to CD81-EC2 similarly to untreated protein. Together these data suggest that although hyperglucosylation of E2 does not have a major effect on bioactivity, polyprotein processing to reveal the free amino terminus is required.

[Genetic studies of blast resistance of indica variety Zhefu 802].

One indica variety, Zhefu 802, was studied for its inheritance of blast resistance by inoculation of two strains Ken54-04 and 95-t2. The B1F1 and F2 populations from cross of Zhefu 802(R) x Lijiangxintuanheigu (S) and related parents were inoculated by spray inoculation method with the two above mentioned strains to determine R:S ratio of segregating populations of this cross. The results indicated that Zhefu 802 has two dominant resistance genes to strain Ken 54-04. One of the two genes showed resistant reaction and the other is susceptible to strain 95-t2. The allelism test indicated that one gene in Zhefu 802, which showed resistant reaction to strain 95-t2, is allelic to Pi-i gene locus and non-allelic to loci of Pi-a, Pi-sh, Pi-k, Pi-z, Pi-ta, Pi-b, Pi-t. The other gene in this variety was also estimated to be different from all of the known genes. So it may be an unknown gene. But this point needs to be confirmed further.

Immunity to St. Louis encephalitis virus by sequential immunization with recombinant vaccinia and baculovirus derived PrM/E proteins.

St. Louis encephalitis (SLE) is an important mosquito-borne disease of great public health concern in parts of the United States. South America and Canada. Protective immunogens of flaviviruses produced in different expression systems have been shown to be effective against virulent virus infection in laboratory animal models. Here we show that the pre-membrane and envelope (PrM/E) of SLE virus expressed in insect and mammalian cell systems using baculovirus and vaccinia virus, respectively, are processed correctly and showed similar antigenic characteristics as the authentic proteins. Immunization with the recombinant proteins individually or in combination resulted in neutralizing and protective immune responses. A schedule consisting of initial immunization with recombinant vaccinia virus followed by a secondary boost with recombinant baculovirus protein resulted in higher levels of neutralizing and protective immune responses. The advantages of the use of such a combined approach as a general immunization strategy are discussed.

An arbovirus cline across the northern hemisphere.

The mode and tempo of arbovirus evolution and dispersal can help to explain the dynamics of pandemics, viral outbreaks, and emerging viruses. By comparing nucleotide and deduced amino acid sequences of their envelope proteins, we describe the continuous distribution of the tick-borne encephalitis (TBE) complex viruses, the most important flaviviruses in Europe, across major geographical areas and the conditions under which mutations occur. The analyses reveal a correlation between the geographical and genetic distances of these viruses. The arthropod host appears to be a key factor for the formation and maintenance of this cline by constraining TBE dispersal and evolution. This is also illustrated by comparisons with mosquito-borne flaviviruses.

Change in phenotype of tick-borne encephalitis virus following passage in Ixodes ricinus ticks and associated amino acid substitution in the envelope protein.

Serial passage of an uncloned tick-borne encephalitis virus (strain 4387 isolated from the liver and lungs of a bank vole) in Ixodes ricinus ticks, was accompanied by gradual reduction in virulence of the virus, as indicated by transmission of virus by infected ticks feeding on laboratory mice. After the 7th serial passage in ticks (strain 4387/7), 95% of mice survived the bite of infected ticks. The surviving infected mice showed either no or only low viraemia although virus could be isolated from the brains of some mice 14 and 30 days after commencement of tick feeding, implying that the tick passaged virus might have established a persistent infection in the mice. Tests for haemagglutinating capacity were positive with TBE strain 4387 but strain 4387/7 exhibited no haemagglutinating activity over a wide pH range, suggesting that phenotypic changes, resulting from selection, had affected the site on the viral envelope protein that binds red blood cell receptors. Sequencing of the envelope protein gene of the virulent TBE strain 4387 showed 3 amino acid codon differences from western European TBE virus strain Neudorfl, which is also virulent for mice. The attenuated virus 4387/7, had an amino acid substitution that was different from 4387 and Neudorfl TBE virus (amino acid 84, E to K) and a second substitution different from 4387 but identical to Neudorfl virus (amino acid 319, I to T). Thus, the phenotypic change from virulence to attenuation was associated with a single amino acid codon change in the viral envelope gene of TBE virus. It is recognised, however, that amino acid substitutions in other parts of the viral genome have not been ruled out.

Single amino acid codon changes detected in louping ill virus antibody-resistant mutants with reduced neurovirulence.

Seven mutant viruses were derived from a Scottish strain of louping ill virus using a virus envelope-specific neutralizing monoclonal antibody. None of the mutants was neutralized and immunofluorescence microscopy confirmed that they did not bind to this antibody. Four mutants showed reduced mouse neurovirulence compared with parent virus and two mutants failed to induce protective immune responses in mice challenged with virulent tick-borne encephalitis virus. The mutants with the lowest virulence showed poor or undetectable haemagglutinating activity. The nucleotide sequence of the envelope glycoprotein gene of each of the seven mutants was determined and the deduced amino acid sequence was compared with parent virus. For each mutant, only a single amino acid codon change was detected and all the amino acid substitutions occurred within amino acid positions 308 to 311. A change from the amino acid aspartate to asparagine at amino acid position 308, which represented a potential glycosylation site, was the most effective substitution in reducing mouse neurovirulence. The results demonstrate the importance of critical sites within the envelope glycoprotein as determinants of virus virulence.

Sequencing and antigenic studies of a Norwegian virus isolated from encephalomyelitic sheep confirm the existence of louping ill virus outside Great Britain and Ireland.

We have carried out an antigenic analysis and nucleotide sequence comparison of the envelope glycoprotein of recognized louping ill virus strains isolated from Scotland with that of a Norwegian virus known to cause encephalomyelitis in sheep. Monoclonal antibodies with defined specificity for the louping ill virus envelope glycoprotein failed to distinguish between the Norwegian virus and prototype louping ill virus in indirect immunofluorescence, haemagglutination inhibition and neutralization tests. Nucleotide sequencing of the envelope glycoprotein and alignment of the deduced amino acid sequence with other known sequences revealed that the Norwegian virus closely resembles (> 95% identity for nucleotide and > 98% identity for amino acid sequences) louping ill virus. Maximum variation in identities among four strains of louping ill virus were 4.4% and 1.8% respectively for nucleotide and amino acid alignments. We conclude that sheep encephalomyelitis in Norway is caused by louping ill virus. These results imply that other viruses present in Europe and known to cause encephalitis/encephalomyelitis of sheep could be caused by louping ill virus.

Envelope protein sequences of dengue virus isolates TH-36 and TH-Sman, and identification of a type-specific genetic marker for dengue and tick-borne flaviviruses.

Complementary DNAs were synthesized from the envelope protein genes of two isolates of dengue virus (TH-36 and TH-Sman, previously suggested as possible dengue virus type 5 and dengue virus type 6 respectively) and amplified by the polymerase chain reaction using sense and antisense primers designed from conserved dengue virus gene sequences. The amplified cDNA clones were sequenced in both directions by double-stranded dideoxynucleotide sequencing. Alignment with published dengue virus sequences enabled us to assign these viruses accurately to classified serotypes, confirming that TH-36 and TH-Sman are strains of dengue virus type 2 and dengue virus type 1 respectively. Amino acid changes between the proteins encoded by these two isolates and strains of their respective serotypes may account for the significant antigenic differences observed during previous serological typing of these viruses. Moreover, sequence alignment of flavivirus envelope proteins revealed a hypervariable region, within which members of the dengue and tick-borne virus antigenic complexes show unique peptide sequences. This type-specific hypervariable domain may be useful as a genetic marker for typing dengue and tick-borne flaviviruses.