Genetic and phenotypic characterization of sylvatic dengue virus type 2 strains

The four serotypes of endemic dengue viruses (DENV) circulate between humans and peridomestic Aedes mosquitoes. At present endemic DENV infect 100 million people per year, and a third of the global population is at risk. In contrast, sylvatic DENV strains are maintained in a transmission cycle between nonhuman primates and sylvatic Aedes species, and are evolutionarily and ecologically distinct from endemic DENV strains. Phylogenetic analyses place sylvatic strains basal to each of the endemic serotypes, supporting the hypothesis that each of the endemic DENV serotypes emerged independently from sylvatic ancestors. We utilized complete genome analyses of both sylvatic and endemic DENV serotype 2 (DENV-2) to expand our understanding of their genetic relationships. A high degree of conservation was observed in both the 5'- and 3'-untranslated genome regions, whereas considerable differences at the nucleotide and amino acid levels were observed within the open reading frame. Additionally, replication of the two genotypes was compared in cultured cells, where endemic DENV strains produced a significantly higher output of progeny in human liver cells, but not in monkey kidney or mosquito cells. Understanding the genetic relationships and phenotypic differences between endemic and sylvatic DENV genotypes may provide valuable insight into DENV emergence and guide monitoring of future outbreaks.     

Genetic and phenotypic characterization of sylvatic dengue virus type 4 strains

Four serotypes of dengue virus (DENV 1-4) currently circulate between humans and domestic/peridomestic Aedes mosquitoes, resulting in 100 million infections per year. All four serotypes emerged, independently, from sylvatic progenitors transmitted among non-human primates by arboreal Aedes mosquitoes. This study investigated the genetic and phenotypic changes associated with emergence of human DENV-4 from its sylvatic ancestors. Analysis of complete genomes of 3 sylvatic and 4 human strains revealed high conservation of both the 5′- and 3′-untranslated regions but considerable divergence within the open reading frame. Additionally, the two ecotypes did not differ significantly in replication dynamics in cultured human liver (Huh-7), monkey kidney (Vero) or mosquito (C6/36) cells, although significant inter-strain variation within ecotypes was detected. These findings are in partial agreement with previous studies of DENV-2, where human strains produced a larger number of progeny than sylvatic strains in human liver cells but not in monkey or mosquito cells.     

Immunodeficient mice xenografted with human lymphoid cells: New models forin vivo studies of human immunobiology and infectious diseases

This review article deals with the transfer of human lymphoid or hematopoietic tissues to severely immunodeficient mice to create new small animal models for the study of human biology and disease. The degree of functional reconstitution in the three current models is discussed. SCID mice with human grafts have been infected with human immunodeficiency virus (HIV) to generate a small animal model for AIDS research. Epstein-Barr virus (EBV)-related lymphoproliferative disorders can also be modeled by the transfer of adult peripheral blood mononuclear cells to SCID mice.     

DCs-based therapies: potential strategies in severe SARS-CoV-2 infection

Pneumonia caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is spreading globally. There have been strenuous efforts to reveal the mechanisms that the host defends itself against invasion by this virus. The immune system could play a crucial role in virus infection. Dendritic cell as sentinel of the immune system plays an irreplaceable role. Dendritic cells-based therapeutic approach may be a potential strategy for SARS-CoV-2 infection. In this review, the characteristics of coronavirus are described briefly. We focus on the essential functions of dendritic cell in severe SARS-CoV-2 infection. Basis of treatment based dendritic cells to combat coronavirus infections is summarized. Finally, we propose that the combination of DCs based vaccine and other therapy is worth further study.     

Lymphocyte function in human bone marrow. II. Characterization of an interleukin 2-sensitive T precursor-cell population

In the present study of human bone marrow lymphocytes, we analyze a newly recognized population of T suppressor-cell precursors which are found in marrow only and which have the potential to inhibit immunoglobulin (Ig) productionin vitro. Following exposure to interleukin 2 (IL2), suppressor precursors acquire E receptor, T3 determinants, suppressor function, and lectin responsiveness. To distinguish this population within the framework of T-cell ontogeny, it was compared to a previously described population of thymus-dependent helper T-cell precursors which express helper function following exposure to thymus-derived mediators. The two populations are completely distinct and can be separated on density gradients. Suppressor precursors expressed T8 and TAC (IL2-receptor) antigens prior toin vitro induction with IL2. The thymic hormone-dependent cells expressed T4 but not T8 or TAC determinants. In two patients with severe combined immunodeficiency disease (SCID), IL2-responsive precursor cells appeared only late after thymus epithelium transplantation, perhaps best explained by a model in which thymus-dependent differentiation pathways precede, induce, or seed pathways of extrathymic T-cell differentiation. The large pool size of over 10¹¹ suppressor and helper precursor cells present in adult bone marrow suggests that these populations may play an important role in immune homeostasis.     

人鼻咽癌CNE－2Z细胞的不同转移潜能克隆株在严重联合免疫缺陷小鼠体内的检验和比较研究

将人类鼻咽癌不同克隆株的细胞悬液移植在严重联合免疫缺陷（ＳＣＩＤ）小鼠和ＢＡＬＢ／ｃ（ｕｎ／ｕｎ）裸小鼠的颈背侧皮下，５６ｄ后处死全部动物进行观察。结果发现，在ＳＣＩＤ小鼠体内移植后ＣＮＥ２Ｌ２为高转移克隆株，其淋巴结转移率为１００％，肺转移率为７１％；而ＣＮＥ２Ｌ４为低转移克隆株，其肺转移率为１３％，淋巴结未见癌转移。这是从１个细胞母系中新筛选出的１个高转移和１个低转移的癌细胞克隆株。实验结果还显示，同ＢＡＬＢ／ｃ（ｕｎ／ｕｎ）裸小鼠相比，ＳＣＩＤ小鼠的恶性表型的表达能力高。另外，皮下移植时肿瘤细胞的数量可能与转移表型的表达有相关性，移植的瘤细胞数越多，转移率越高，反之亦然。     

Application of shRNA-containing herpes simplex virus type 1 (HSV-1)-based gene therapy for HSV-2-induced genital herpes

HSV-1-based vectors have been widely used to achieve targeted delivery of genes into the nervous system. In the current study, we aim to use shRNA-containing HSV-1-based gene delivery system for the therapy of HSV-2 infection. Guinea pigs were infected intravaginally with HSV-2 and scored daily for 100 days for the severity of vaginal disease. HSV-2 shRNA-containing HSV-1 was applied intravaginally daily between 8 and 14 days after HSV-2 challenge. Delivery of HSV-2 shRNA-containing HSV-1 had no effect on the onset of disease and acute virus shedding in animals, but resulted in a significant reduction in both the cumulative recurrent lesion days and the number of days with recurrent disease. Around half of the animals in the HSV-2 shRNA group did not develop recurrent disease 100 days post HSV-2 infection. In conclusion, HSV-2 shRNA-containing HSV-1 particles are effective in reducing the recurrence of genital herpes caused by HSV-2.     

H-2 linked genetic control of immune responsiveness to hepatitis B surface antigen (HBsAg) in mice

Recent data suggest that genes involved in the control of (1) immune responses of humans to HBsAg and (2) the susceptibility to the development of chronic hepatitis B are linked to the major HLA histocompatibility complex. Studies on the genetic regulation of anti-HBs responses and on the possible abrogation of nonresponsiveness to HBsAg in humans are difficult. In an attempt to develop a relevant animal model system, the anti-HBs response of inbred and congenic strains of mice was investigated. A great variation in anti-HBs responses among individual mice belonging to the same strains was observed. Nevertheless, it was possible to rank the inbred mouse strains studied according to their decreasing anti-HBs responses as follows: BALB/c[d] ∽ SWR/J[q] > C57BL/6J[b] ∽ DBA/2J[a] > AKR/J[k] > A/J[a] > CBA/CaJ[k] > SJL/J[s]. (Letters in brackets indicate H-2 haplotype). Only a small proportion of SJL mice had an anti-HBs response. Therefore, this strain may serve as a model for human nonresponders. Studies with the congenic strains B10.D2[d] and B10.S[s] indicated that genes conferring responsiveness to HBsAg are linked to the H-2 histocompatibility complex. However, genes not linked to H-2 also probably play a role in regulating anti-Hbs responses.     

Elevated Plasma Levels of 90K (Mac-2 BP) Immunostimulatory Glycoprotein in HIV-1-Infected Children

90K is a secreted serum glycoprotein with immune stimulatory activity. In this study, 90K plasma levels were determined by an enzyme-linked immunosorbent assay in 18 HIV-1-infected children and 10 uninfected control children. 90K levels in HIV-1-infected children (median, 12.5 g/ml) were higher than in HIV-1 uninfected control group (6.3 g/ml; P < 0.05). 90K levels of HIV-1-infected children classified as stage B and C (median, 15.0 g/ml and 22.7 g/ml, respectively) were higher compared to children with stage A disease (median, 7.0 g/ml; P < 0.05). A positive correlation (r = 0.5; P < 0.05) was found between 90K levels and HIV-1 RNA levels in 137 plasma samples of 18 HIV-1-infected children collected during a period of 1 year. No correlation was found between 90K levels and CD4 cell counts. These results suggest that 90K plasma levels may represent a novel marker of disease progression in HIV-1-infected children.     

Expression of a functional p75 interleukin-2 receptor on lung lymphocytes from patients with human immunodeficiency virus type 1 (HIV-1) infection

Lung involvement in patients affected by HIV-1 infection is characterized by an alveolitis sustained by the accumulation of CD8+ T lymphocytes. To investigate whetherin situ T cell growth plays a relevant role in the pooling of CD8+ lymphocytes, we have analyzed the activity of two lymphokines involved in the mechanisms of T cell proliferation, i.e., interleukin-2 (IL-2) and interleukin-4. To this aim, following appropriate triggering and blocking, the expression and the functional role of IL-2 receptors (IL-2R) (both p55 and p75 chains) and IL-4 receptors have been analyzed on T lymphocytes obtained from the bronchoalveolar lavage (BAL) of 16 HIV-1+ patients. Molecular and phenotypic studies we performed demonstrated that CD8+ lymphocytes from the BAL of HIV-1 + patients strongly expressed the p75 chain of IL-2 receptor, while neither p55 mRNA nor its surface membrane product (Tac antigen) was detectable; in addition, there was no expression of IL-4 receptors. IL-2 stimulation was able to induce T cell growth in a dose-dependent manner, whereas IL-4 did not. Finally, using mAbs which specifically block the p55 or p75 IL-2R, we showed that both subunits of IL-2R were involved in the proliferative activity of lung lymphocytes. The results obtained in the present study directly demonstrate that BAL T lymphocytes of HIV-1 + patients express a fully functional IL-2 receptor apparatus, pointing to the role for this lymphokine in maintaining the alveolitis taking place in the lungs of AIDS patients.     

Autophagy contributes to BMP type 2 receptor degradation and development of pulmonary arterial hypertension

Pulmonary arterial hypertension (PAH) is characterised by an increase in mean pulmonary arterial pressure which almost invariably leads to right heart failure and premature death. More than 70% of familial PAH and 20% of idiopathic PAH patients carry heterozygous mutations in the bone morphogenetic protein (BMP) type 2 receptor (BMPR2). However, the incomplete penetrance of BMPR2 mutations suggests that other genetic and environmental factors contribute to the disease. In the current study, we investigate the contribution of autophagy in the degradation of BMPR2 in pulmonary vascular cells. We demonstrate that endogenous BMPR2 is degraded through the lysosome in primary human pulmonary artery endothelial (PAECs) and smooth muscle cells (PASMCs): two cell types that play a key role in the pathology of the disease. By means of an elegant HaloTag system, we show that a block in lysosomal degradation leads to increased levels of BMPR2 at the plasma membrane. In addition, pharmacological or genetic manipulations of autophagy allow us to conclude that autophagy activation contributes to BMPR2 degradation. It has to be further investigated whether the role of autophagy in the degradation of BMPR2 is direct or through the modulation of the endocytic pathway. Interestingly, using an iPSC-derived endothelial cell model, our findings indicate that BMPR2 heterozygosity alone is sufficient to cause an increased autophagic flux. Besides BMPR2 heterozygosity, pro-inflammatory cytokines also contribute to an augmented autophagy in lung vascular cells. Furthermore, we demonstrate an increase in microtubule-associated protein 1 light chain 3 beta (MAP1LC3B) levels in lung sections from PAH induced in rats. Accordingly, pulmonary microvascular endothelial cells (MVECs) from end-stage idiopathic PAH patients present an elevated autophagic flux. Our findings support a model in which an increased autophagic flux in PAH patients contributes to a greater decrease in BMPR2 levels. Altogether, this study sheds light on the basic mechanisms of BMPR2 degradation and highlights a crucial role for autophagy in PAH. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

Fitness of Japanese encephalitis virus to Neuro-2a cells is determined by interactions of the viral envelope protein with highly sulfated glycosaminoglycans on the cell surface

Genetically different subpopulations were identified and purified from Japanese Encephalitis virus (JEV). Those with small plaques (SPs; <2 mm in diameter), derived from strains of T1P1, CJN, and CC27, were more competent than those with large plaques (LPs; >5 mm in diameter) when passaged in Neuro-2a cells. Differences in amino acids between SPs and LPs from each strain were shown in the viral envelope (E) protein. The amino acid at E-306 was Glu in LP but was substituted by Lys in SP in the T1P1 strain. A similar substitution occurred at E-138 in the CJN strain. However, the amino acid was Asp in LP but was substituted by Asn in SP at E-389 in the CC27 strain. All SPs were shown to have a higher affinity to the cellular membrane when compared to LPs, and this resulted in more-efficient infection of Neuro-2a cells, suggesting that the differential fitness of JEV variants to Neuro-2a cells appeared in the early phase of infection. In addition, glycosaminoglycans (GAGs) on the surface of many mammalian cells have been demonstrated to be critical for infection by JEV, especially SP variants. The present results suggest that T1P1-SP1 viruses infected Neuro-2a cells more efficiently in spite of the sparse distribution of cell surface GAGs. We conclude that highly sulfated forms of GAGs expressed by Neuro-2a cells play an important role in selecting JEV variants with specific mutations in the E glycoprotein.     

Dengue hemorrhagic fever-associated immunomediators induced via maturation of dengue virus nonstructural 4B protein in monocytes modulate endothelial cell adhesion molecules and human microvascular endothelial cells permeability

We previously demonstrated that dengue virus (DENV) nonstructural 4B protein (NS4B) induced dengue hemorrhagic fever (DHF)-associated immunomediators in THP-1 monocytes. Moreover, cleavage of NS4AB polyprotein by the NS2B3 protease, significantly increased immunomediator production to levels found after DENV infection. In this report using primary human microvascular endothelial cells (HMVEC) transwell permeability model and HMVEC monolayer, we demonstrate that the immunomediators secreted in the supernatants of DENV-infected monocytes increase HMVEC permeability and expression of ICAM-1, VCAM-1 and E-selectin. Moreover, maturation of NS4B via cleavage of 2KNS4B is sufficient to induce immunomediators that cause HMVEC phenotypic changes, which appear to be synergistically induced by TNFα and IL-8. These data suggest that therapies targeting the maturation steps of NS4B, particularly 2KNS4B processing, may reduce overall DHF-associated immunomediator levels, thereby reducing DHF-associated morbidity and mortality. Alternatively, TNFα inhibitors may be a valid intervention strategy during the later stages of infection to prevent DHF progression.     

Type I interferon supports primary CD8+ T-cell responses to peptide-pulsed dendritic cells in the absence of CD4+ T-cell help

CD8(+) T-cell responses to non-pathogen, cell-associated antigens such as minor alloantigens or peptide-pulsed dendritic cells (DC) are usually strongly dependent on help from CD4(+) T cells. However, some studies have described help-independent primary CD8(+) T-cell responses to cell-associated antigens, using immunization strategies likely to trigger natural killer (NK) cell activation and inflammatory cytokine production. We asked whether NK cell activation by MHC I-deficient cells, or administration of inflammatory cytokines, could support CD4(+) T-cell help-independent primary responses to peptide-pulsed DC. Injection of MHC I-deficient cells cross-primed CD8(+) T-cell responses to the protein antigen ovalbumin (OVA) and the male antigen HY, but did not stimulate CD8(+) T-cell responses in CD4-depleted mice; hence NK cell stimulation by MHC I-deficient cells did not replace CD4(+) T-cell help in our experiments. Dendritic cells cultured with tumour necrosis factor-α (TNF-α) or type I interferon-α (IFN-α) also failed to prime CD8(+) T-cell responses in the absence of help. Injection of TNF-α increased lymph node cellularity, but did not generate help-independent CD8(+) T-cell responses. In contrast, CD4-depleted mice injected with IFN-α made substantial primary CD8(+) T-cell responses to peptide-pulsed DC. Mice deficient for the type I IFN receptor (IFNR1) made CD8(+) T-cell responses to IFNR1-deficient, peptide-pulsed DC; hence IFN-α does not appear to be a downstream mediator of CD4(+) T-cell help. We suggest that primary CD8(+) T-cell responses will become help-independent whenever endogenous IFN-α secretion is stimulated by tissue damage, infection, or autoimmune disease.     

Some alterations of the leucoeyte β2-adrenoceptor/cAMP-system in patients with seasonal allergic rhinoconjunctivitis are related to disease activity

Background Disturbances of β₂-adrenoceptors are discussed as a pathogenic factor in atopic diseases. Methods In this study tbe expression and function of β₂-adrenoceptors on peripheral blood leucocytes (PBL) of seven atopic patients with seasonal rhinoconjunctivitis and their seven healthy controls was evaluated in relation to disease activity. Earlier reported data during pollen. season were now compared with data obtained from the same subjects after their allergic symptoms had subsided. Results The variables that had indicated a β₂-adrenoceptor subsensitivity in tbe patients during pollen season returned to control values, i.e. the reduced β₂-adrenoceptor affinity, the reduced β₂-adrenoceptor sensitivity, the reduced increase of intracellular cyclic adenosine monophosphate (cAMP) content upon stimulation with isoproterenol, and the reduced cAMP plasma concentration (values no longer significantly different from those of controls). However, the variable that had suggested an increase in activity of the cAMP degrading enzyme phosphodiesterase (PDE), i.e. the reduced basal intracellular cAMP content of the patients, remained reduced after the pollen season (4.9 ± 1.1 pmol/lO⁶’cells in patients vs 8.2 ^± 0.9pmol/10⁶ cells in controls: P < 0.05). There were no significant differences in β₂-adrenoceptor density between patients and controls at both investigations. Conclusions Atopic seasonal rhinoconjunctivitis is associated with various alterations in the PBL β_2-adrenoceptor/cAMP system that depend on disease activity. The reversible β₂-adrenoceptor subsensitivity is likely to be a consequence of the disease, whereas the irreversibly decreased basal intracellular cAMP content, suggested an elevated PDE activity, might be a basic trait of atopy.     

Macrophage migration inhibition as a correlate of cell-mediated immunity to herpes simplex virus type 2 in mice

Mice inoculated intraperitoneally or intravenously with herpes simplex virus type 2 (HSV-2) develop a focal necrotizing hepatitis. The livers show expanding foci of necrosis and increasing virus content during the first days of the infection with maximal titers achieved on day 3. The clearance of virus from the organ is manifest from day 4 onward with the most dramatic fall in virus content occurring between days 4 and 5. The development of immunity during the course of infection was assessed by adoptive transfer experiments and by measuring macrophage migration inhibition factor (MIF) production of spleen cells in an indirect agarose microdroplet assay. Antiviral activity of adoptively transferred spleen cells was demonstrable from day 4 of the infection when 50 X 10(6) spleen cells were transferred into recipient mice infected 24 h previously. MIF production in spleen cell cultures stimulated with antigen was found to be specific in that activity was only detected in cultures derived from immune mice and stimulated with the virus antigen. The response was found to be antigen-dose and cell-number dependent. Significant MIF production was demonstrable in spleen cell cultures derived from mice 3 days after the infection, i.e. concomitant with the initiation of recovery and before antiviral activity can be detected in transfer experiments. It is suggested that a delayed type hypersensitivity reaction with lymphokine production leading to recruitment of macrophages and their retention and activation in the foci of infection may be a major factor in the recovery from the infection.     

Human monoclonal antibody to tumor-associated ganglioside GD2: suppressed growth of human melanoma in nude mice

Human IgM kappa monoclonal antibody (anti-GD2) to a tumor antigen, ganglioside GD2, has been produced by Epstein-Barr virus-transformed human B lymphoblasts. In the present study, we demonstrated the anti-tumor effects of passively administered anti-GD2 against an ascites-form human melanoma cell line, M14-A, transplanted into athymic nude mice. M14-A expresses GD2 in vivo. Cells (5 X 10(5) were inoculated intraperitoneally (i.p.) or subcutaneously (s.c.) into CD-1 nude mice that had received i.p. injections of 200 micrograms anti-GD2 and rabbit complement. Significant tumor-free intervals were observed in the treated mice (P less than 0.005) for i.p. tumors and P less than 0.025 for s.c. tumors). M14-A formed well-vascularized s.c. tumors if injected into young nude mice. Three-wk-old CD-1 nude mice bearing 2-3 mm M14-A s.c. tumor nodules were treated i.p. with anti-GD2 and rabbit complement. Tumor growth was delayed for 25 days. On day 15, treated tumors were 20% the size of control tumors. Because most biopsied or autopsied melanomas express GD2, and because patients with melanoma produce autoantibodies to GD2, the results in this study may provide important information for future passive immunization with human monoclonal antibody and for active specific immunization with GD2 antigen.     

Isolation of complementary DNA unique to the genome of avian myeloblastosis virus (AMV)

Avian myeloblastosis virus (AMV) can transform avian cells of hemopoietic origin, such as bone marrow, embryonic yolk sac, and circulating macrophages. The AMV is defective in its replication and can only replicate in the presence of helper viruses. This defectiveness in replication is probably due to a deletion or substitution of nucleotide sequences in its genome. The AMV genome contains no sequences homologous to the src gene, which is responsible for the transforming function of the sarcoma viruses. We attempted to identify the AMV sequences that may contain sequences which are responsible for its transforming function. We isolated a complementary DNA (cDNA_AMV) that hybridized preferentially to the RNA of the transforming AMV but not to the RNA of the helper virus. Using this cDNA_AMV as a probe, we determined the size of the AMV genome to be 33–34 S with a molecular weight of 2.6 × 10⁶. A similar molecular weight estimation of the AMV genome size was obtained by methylmercury-agarose gel electrophoresis of AMV RNA. In AMV-producer myeloblasts we can detect about 6000–7000 copies per cell of AMV-specific RNA, whereas fewer than 2 copies per cell of AMV RNA are found in helper virus-infected cells. In AMV nonproducer myeloblasts, about 2000 copies of AMV-specific RNA are detected. Furthermore, we find that RNA of AMV NP myeloblasts can only hybridize to 55% of cDNA complementary to helper virus genome. In uninfected hemopoietic cells, e.g., bone marrow cells, about 20 copies per cell of AMV-specific RNA are present, whereas in uninfected chick embryo fibroblasts less than 1 copy per cell is found.     

Structure,expression pattern and biological activity of molecular complex TREM-2/DAP12

DNAX-activating protein of 12 kDa (DAP12) is a member of type I transmembrane adapter proteins containing immunoreceptor tyrosine-based activation motifs (ITAMs). In humans DAP12 gene is located on chromosome 19q13.1. DAP12 forms a molecular complex with triggering receptor expressed on myeloid cells two (TREM-2). TREM-2 ligation leads to the activation of Src family kinases, phosphorylation of tyrosine residues in the ITAM of DAP12, recruitment of the Syk and ZAP70 tyrosine kinases and initiation of an intracellular signaling cascade. Depending on the cell type, DAP12/TREM-2 activation plays an important role in activation and differentiation of osteoclasts, phagocytosis of bacteria, brain and bone homeostasis and inhibition of the toll-like receptor (TLR) signaling in macrophages and dendritic cells. A proper understanding of the function of this complex receptor has been restrained because of the elusive nature of TREM-2 ligands.