Development of a humanized HLA-A30 transgenic mouse model.

BACKGROUND: There are remarkable genetic differences between animal major histocompatibility complex (MHC) systems and the human leukocyte antigen (HLA) system. HLA transgenic humanized mouse model systems offer a much better method to study the HLA-A-related principal mechanisms for vaccine development and HLA-A-restricted responses against infection in human. METHODS: A recombinant gene encoding the chimeric HLA-A30 monochain was constructed. This HHD molecule contains the following: α1-α2 domains of HLA-A30, α3 and cytoplasmic domains of H-2Db , linked at its N-terminus to the C-terminus of human ß2m by a 15-amino-acid peptide linker. The recombinant gene encoding the chimeric HLA-A30 monochain cassette was introduced into bacterial artificial chromosome (BAC) CH502-67J3 containing the HLA-A01 gene locus by Red-mediated homologous recombination. Modified BAC CH502-67J3 was microinjected into the pronuclei of wild-type mouse oocytes. This humanized mouse model was further used to assess the immune responses against influenza A virus (H1N1) pdm09 clinically isolated from human patients. Immune cell population, cytokine production, and histopathology in the lung were analyzed. RESULTS: We describe a novel human ß2m-HLA-A30 (α1α2)-H-2Db (α3 transmembrane cytoplasmic) (HHD) monochain transgenic mouse strain, which contains the intact HLA-A01 gene locus including 49 kb 5'-UTR and 74 kb 3'-UTR of HLA-A01*01. Five transgenic lines integrated into the large genomic region of HLA-A gene locus were obtained, and the robust expression of exogenous transgene was detected in various tissues from A30-18# and A30-19# lines encompassing the intact flanking sequences. Flow cytometry revealed that the introduction of a large genomic region in HLA-A gene locus can influence the immune cell constitution in humanized mice. Pdm09 infection caused a similar immune response among HLA-A30 Tg humanized mice and wild-type mice, and induced the rapid increase of cytokines, including IFN-γ, TNF-α, and IL-6, in both HLA-A30 humanized Tg mice and wild-type mice. The expression of HLA-A30 transgene was dramatically promoted in tissues from A30-9# line at 3 days post-infection (dpi). CONCLUSIONS: We established a promising preclinical research animal model of HLA-A30 Tg humanized mouse, which could accelerate the identification of novel HLA-A30-restricted epitopes and vaccine development, and support the study of HLA-A-restricted responses against infection in humans.

Do preoperative intra-articular injections of corticosteroids or hyaluronic acid increase the risk of infection after total knee arthroplasty? A meta-analysis.

AIMS: There is conflicting evidence on the safety of intra-articular injections of hyaluronic acid (HA) or corticosteroids (CSs) before total knee arthroplasty (TKA). We performed a meta-analysis of the relationship between intra-articular injections and subsequent infection rates after TKA. METHODS: We searched PubMed, EMBASE, and the Cochrane Library for cohort studies that assessed the effect of preoperative injection of drugs into the joint cavity on the infection rate after TKA. The outcomes analyzed included the total infection rate, as well as those for different preoperative injection time periods and different drugs. RESULTS: Eight studies, including 73,880 in the injection group and 126,187 in the control group, met the inclusion criteria. The injection group had a significantly higher postoperative infection rate than the control group (risk ratio (RR) 1.16; 95% confidence interval (CI) 1.07 to 1.27; p < 0.001; I2 = 32%). For patients who received injections up to three months preoperatively, the postoperative infection risk was significantly higher than that in the control group (RR 1.26; 95% CI 1.18 to 1.35; p＜0.001; I2 = 0%). There was no significant difference in the infection rates between the four-to-six-month injection and control groups (RR 1.12; 95% CI 0.93 to 1.35; p = 0.240; I2 = 75%) or between the seven-to-12-month injection and control groups (RR 1.02; 95% CI 0.94 to 1.12; p = 0.600; I2 = 0%). CONCLUSION: Current evidence suggests that intra-articular injections of CSs or HA before TKA increase the risk of postoperative infection. Injections administered more than three months before TKA do not significantly increase the risk of infection. Cite this article: Bone Joint Res 2022;11(3):171-179.

Comparative transcriptome analyses of immune responses to LPS in peripheral blood mononuclear cells from the giant panda, human, mouse, and monkey.

Gram-negative bacteria are major pathogens that can cause illnesses in giant pandas. Lipopolysaccharides (LPS), components of Gram-negative bacteria, can activate immune responses in mammals (i.e., humans and mice) through recognition by toll-like receptors (TLRs). However, the giant pandas' immune response to LPS stimulation and the differences between the giant panda and other mammals are not fully known. In this study, we administrated peripheral blood mononuclear cells (PBMCs) from giant pandas, humans, C57BL/6 mice, and rhesus monkeys by LPS treatment at 6 h followed by RNA sequencing (RNA-seq), respectively, with control of non-stimulation. KEGG analyses of differentially expressed genes (DEGs) pathways indicated that LPS could activate the classic signaling pathway of NF-κB in PBMCs from those four tested species. Thus, similar to the other three species, NF-κB is an LPS-responsive regulator of innate immune responses in giant pandas. Furthermore, the expression patterns of adapter genes, inflammatory cytokine genes, chemokines, interferon genes, cytokine genes related to cell growth and development, costimulatory molecules, Th1/Th2 cytokine genes, Th17 cytokine genes, Th9, and Th22 cytokine genes were compared among giant pandas and three other species. Our data indicated that in addition to the similar expression patterns of certain genes among giant pandas and other species, the unique expression pattern response to LPS in giant pandas was also discovered. Furthermore, Th9, Th17, and Th22 cells might be involved in the response to LPS in giant pandas at this tested time point. This study reveals that LPS-induced immune responses have different sensitivities and response timelines in giant pandas compared with other mammals. This study facilitates further understanding of the role of the TLR signaling pathway and the immune system in giant pandas, which might be helpful for disease prevention and protection.

Generation and expression analysis of BAC humanized mice carrying HLA-DP401 haplotype.

Background: Human leukocyte antigen (HLA)-DP is much less studied than other HLA class II antigens, that is, HLA-DR and HLA-DQ, etc. However, the accumulating data have suggested the important roles of DP-restricted responses in the context of cancer, allergy, and infectious disease. Lack of animal models expressing these genes as authentic cis-haplotypes blocks our understanding for the role of HLA-DP haplotypes in immunity. Methods: To explore the potential cis-acting control elements involved in the transcriptional regulation of the HLA-DPA1/DPB1 gene, we performed the expression analysis using bacterial artificial chromosome (BAC)-based transgenic humanized mice in the C57BL/6 background, which carried the entire HLA-DP401 gene locus. We further developed a mouse model of Staphylococcus aureus pneumonia in HLA-DP401 humanized transgenic mice, and performed the analysis on the expression pattern of HLA-DP401 and immunological responses in the model. Results: In this study, we screened and identified a BAC clone spanning the entire HLA-DP gene locus. DNA from this clone was analyzed for integrity by pulsed-field gel electrophoresis and then microinjected into fertilized mouse oocytes to produce transgenic founder animals. Nine sets of PCR primers for regional markers with an average distance of 15 kb between each primer were used to confirm the integrity of the transgene in the five transgenic lines carrying the HLA-DPA1/DPB1 gene. Transgene copy numbers were determined by real-time PCR analysis. HLA-DP401 gene expression was analyzed at the mRNA and protein level. Although infection with S aureus Newman did not alter the percentage of immune cells in the spleen and thymus from the HLA-DP401-H2-Aß1 humanized mice. Increased expression of HLA-DP401 was observed in the thymus of the humanized mice infected by S aureus. Conclusions: We generated several BAC transgenic mice, and analyzed the expression of HLA-DPA1/DPB1 in those mice. A model of Saureus-induced pneumonia in the HLA-DP401-H2-Aß1-/- humanized mice was further developed, and S aureus infection upregulated the HLA-DP401 expression in thymus of those humanized mice. These findings demonstrate the potential of those HLA-DPA1/DPB1 transgenic humanized mice for developing animal models of infectious diseases and MHC-associated immunological diseases.

Epigenetic Landscapes of Single-Cell Chromatin Accessibility and Transcriptomic Immune Profiles of T Cells in COVID-19 Patients.

T cells play a critical role in coronavirus diseases. How they do so in COVID-19 may be revealed by analyzing the epigenetic chromatin accessibility of cis- and trans-regulatory elements and creating transcriptomic immune profiles. We performed single-cell assay for transposase-accessible chromatin (scATAC) and single-cell RNA (scRNA) sequencing (seq) on the peripheral blood mononuclear cells (PBMCs) of severely ill/critical patients (SCPs) infected with COVID-19, moderate patients (MPs), and healthy volunteer controls (HCs). About 76,570 and 107,862 single cells were used, respectively, for analyzing the characteristics of chromatin accessibility and transcriptomic immune profiles by the application of scATAC-seq (nine cases) and scRNA-seq (15 cases). The scATAC-seq detected 28,535 different peaks in the three groups; among these peaks, 41.6 and 10.7% were located in the promoter and enhancer regions, respectively. Compared to HCs, among the peak-located genes in the total T cells and its subsets, CD4+ T and CD8+ T cells, from SCPs and MPs were enriched with inflammatory pathways, such as mitogen-activated protein kinase (MAPK) signaling pathway and tumor necrosis factor (TNF) signaling pathway. The motifs of TBX21 were less accessible in the CD4+ T cells of SCPs compared with those in MPs. Furthermore, the scRNA-seq showed that the proportion of T cells, especially the CD4+ T cells, was decreased in SCPs and MPs compared with those in HCs. Transcriptomic results revealed that histone-related genes, and inflammatory genes, such as NFKBIA, S100A9, and PIK3R1, were highly expressed in the total T cells, CD4+ T and CD8+ T cells, both in the cases of SCPs and MPs. In the CD4+ T cells, decreased T helper-1 (Th1) cells were observed in SCPs and MPs. In the CD8+T cells, activation markers, such as CD69 and HLA class II genes (HLA-DRA, HLA-DRB1, and HLA-DRB5), were significantly upregulated in SCPs. An integrated analysis of the data from scATAC-seq and scRNA-seq showed some consistency between the approaches. Cumulatively, we have generated a landscape of chromatin epigenetic status and transcriptomic immune profiles of T cells in patients with COVID-19. This has provided a deeper dissection of the characteristics of the T cells involved at a higher resolution than from previously obtained data merely by the scRNA-seq analysis. Our data led us to suggest that the T-cell inflammatory states accompanied with defective functions in the CD4+ T cells of SCPs may be the key factors for determining the pathogenesis of and recovery from COVID-19.

Overview of the reporter genes and reporter mouse models.

Reporter genes are widely applied in biotechnology and biomedical research owning to their easy observation and lack of toxicity. Taking advantage of the reporter genes in conjunction with imaging technologies, a large number of reporter mouse models have been generated. Reporter mouse models provide systems that enable the studies of live cell imaging, cell lineage tracing, immunological research and cancers etc. in vivo. In this review, we describe the types of different reporter genes and reporter mouse models including, random reporter strains, Cre reporter strains and ROSA26 reporter strains. Collectively, these reporter mouse models have broadened scientific inquires and provided potential strategies for generation of novel reporter animal models with enhanced capabilities.

Neutralization of Zika virus by germline-like human monoclonal antibodies targeting cryptic epitopes on envelope domain III.

The Zika virus (ZIKV), a flavivirus transmitted by Aedes mosquitoes, has emerged as a global public health concern. Pre-existing cross-reactive antibodies against other flaviviruses could modulate immune responses to ZIKV infection by antibody-dependent enhancement, highlighting the importance of understanding the immunogenicity of the ZIKV envelope protein. In this study, we identified a panel of human monoclonal antibodies (mAbs) that target domain III (DIII) of the ZIKV envelope protein from a very large phage-display naive antibody library. These germline-like antibodies, sharing 98%-100% hoLogy with their corresponding germline IGHV genes, bound ZIKV DIII specifically with high affinities. One mAb, m301, broadly neutralized the currently circulating ZIKV strains and showed a synergistic effect with another mAb, m302, in neutralizing ZIKV in vitro and in a mouse model of ZIKV infection. Interestingly, epitope mapping and competitive binding studies suggest that m301 and m302 bind adjacent regions of the DIII C-C' loop, which represents a recently identified cryptic epitope that is intermittently exposed in an uncharacterized virus conformation. This study extended our understanding of antigenic epitopes of ZIKV antibodies and has direct implications for the design of ZIKV vaccines.

Human liver chimeric mouse model based on diphtheria toxin-induced liver injury.

AIM: To establish an inducible liver injury mouse model and transplant human hepatocytes to obtain liver-humanized mice. METHODS: We crossed three mouse strains, including albumin (Alb)-cre transgenic mice, inducible diphtheria toxin receptor (DTR) transgenic mice and severe combined immune deficient (SCID)-beige mice, to create Alb-cre/DTR/SCID-beige (ADSB) mice, which coincidentally harbor Alb-cre and DTR transgenes and are immunodeficient. As the Cre expression is driven by the liver-specific promoter Alb (encoding ALB), the DTR stop signal flanked by two loxP sites can be deleted in the ADSB mice, resulting in DTR expression in the liver. ADSB mice aged 8-10 wk were injected intraperitoneally (i.p.) with diphtheria toxin (DT) and liver damage was assessed by serum alanine aminotransferase (ALT) level. Two days later, mouse livers were sampled for histological analysis, and human hepatocytes were transplanted into the livers on the same day. A human ALB enzyme-linked immunosorbent assay was performed 7, 14, 21 and 28 d after transplantation. Human CD68 immunohistochemistry was performed 30 and 90 d after transplantation. RESULTS: We crossed Alb-cre with DTR and SCID-beige mice to obtain ADSB mice. These mice were found to have liver damage 4 d after i.p. injection of 2.5 ng/g bodyweight DT. Bodyweight began to decrease on day 2, increased on day 7, and was lowest on day 4 (range, 10.5%-13.4%). Serum ALT activity began to increase on day 2 and reached a peak value of 289.7 ± 16.2 IU/mL on day 4, then returned to background values on day 7. After transplantation of human liver cells, peripheral blood human ALB level was 1580 ± 454.8 ng/mL (range, 750.2-3064.9 ng/mL) after 28 d and Kupffer cells were present in the liver at 30 d in ADSB mice. CONCLUSION: Human hepatocytes were successfully repopulated in the livers of ADSB mice. The inducible mouse model of humanized liver in ADSB mice may have functional applications, such as hepatocyte transplantation, hepatic regeneration and drug metabolism.

The H7N9 influenza A virus infection results in lethal inflammation in the mammalian host via the NLRP3-caspase-1 inflammasome.

The avian origin influenza A virus (IAV) H7N9 has caused a considerable number of human infections associated with high rates of death since its emergence in 2013. As a vital component of the host innate immune system, the nucleotide-binding domain leucine-rich repeat containing receptor, pyrin domain containing 3 (NLRP3) inflammasome plays a critical role against H1N1 viral infection. However, the function of NLRP3 inflammasome in host immunological responses to the lethal H7N9 virus is still obscure. Here, we demonstrated that mice deficient for NLRP3 inflammasome components, including NLRP3, caspase-1, and Apoptosis-associated speck-like protein containing a CARD (ASC), were less susceptible to H7N9 viral challenge than wild type (WT) controls. Inflammasome deficiency in these animals led to significantly milder mortality and less pulmonary inflammation compared with WT mice. Furthermore, IL-1 receptor deficient mice also exhibited a higher survival rate than WT controls. Thus, our study reveals that the NLRP3 inflammasome is deleterious for the host during H7N9 infection in mice, which is due to an overwhelming inflammatory response via caspase-1 activation and associated IL-1 signal. Therefore, fine-tuning the activity of NLRP3 inflammasome or IL-1 signaling may be beneficial for the host to control H7N9 associated lethal pathogenesis.

High persistence rate of hepatitis B virus in a hydrodynamic injection-based transfection model in C3H/HeN mice.

AIM: To optimize the viral persistence rate in a hydrodynamic injection (HI) based hepatitis B virus (HBV) transfection mouse model. METHODS: (1) 5-6-wk-old male C3H/HeN and C57BL/6 mice were hydrodynamically injected with 10 µg endotoxin-free pAAV/HBV1.2 plasmid DNA via the tail vein. Hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg) and HBV DNA, both in the serum and liver, were detected at different time points post HI by ELISA, immunohistochemical staining or quantitative polymerase chain reaction (PCR); (2) male C3H/HeN and C57BL/6 mice, either hydrodynamically injected mice at 10 wk post HI or naïve mice, were all immunized subcutaneously with 5 µg HBsAg formulated in complete Freund's adjuvant three times at a 2-wk interval. Two weeks after the final immunization, splenocytes were isolated for T cell function analysis by ELISPOT assay; and (3) five weeks post HI, C3H/HeN mice were intragastrically administered 0.1 mg/kg entecavir once a day for 14 d, or were intraperitoneally injected with 1 mg/kg interferon (IFN)-α twice a week for 2 wk, or were treated with PBS as controls. The sera were collected and assayed for HBV DNA on days 0, 7 and 14 after drug treatment. RESULTS: (1) Approximately 90% (22/25) of the injected C3H/HeN mice were still HBsAg-positive at 46 wk post HI, whereas HBsAg in C57BL/6 mice were completely cleared at 24 wk. Serum levels of HBeAg in C3H/HeN mice were higher than those in C57BL/6 mice from 4 wk to 46 wk. HBV DNA levels in the hydrodynamically injected C3H/HeN mice were higher than those in the C57BL/6 mice, both in the serum (from 4 wk to 46 wk) and in the liver (detected at 8 wk and 46 wk post HI). Histology showed that hepatitis B core antigen and HBsAg were expressed longer in the liver of C3H/HeN mice than in C57BL/6; (2) HBsAg specific T cell responses after HBsAg vaccination in hydrodynamically injected C3H/HeN and C57BL/6 mice, or naive control mice were detected by ELISPOT assay. After stimulation with HBsAg, the frequencies of IFN-γ producing splenocytes in the hydrodynamically injected C3H/HeN mice were significantly lower than those in hydrodynamically injected C57BL/6 mice, control C3H/HeN and control C57BL/6 mice, which were 0, 17 ± 7, 18 ± 10, and 41 ± 10 SFCs/10(6) splenocytes, respectively, and the mean spot sizes showed the same pattern. Even just stimulated with PMA and ionomysin, T-cell responses elicited in the vaccinated control C3H/HeN were much higher than those in hydrodynamically injected C3H/HeN mice; and (3) For drug treatment experiments on the hydrodynamically injected C3H/HeN mice, serum HBV DNA levels in the entecavir treatment group declined (131.2 folds, P < 0.01) on day 7 after treatment and kept going down. In the group of IFN-α treatment, serum HBV DNA levels declined to a lowest point (6.42 folds, P < 0.05) on 7 d after treatment and then rebounded. CONCLUSION: We have developed a novel HI-based HBV transfection model using C3H/HeN mice, which had a higher HBV persistence rate than the classic C57BL/6 mouse model.

Fusion-expressed CTB improves both systemic and mucosal T-cell responses elicited by an intranasal DNA priming/intramuscular recombinant vaccinia boosting regimen.

Previous study showed that CTB (Cholera toxin subunit B) can be used as a genetic adjuvant to enhance the systemic immune responses. To further investigate whether it can also be used as a genetic adjuvant to improve mucosal immune responses, we constructed DNA and recombinant Tiantan vaccinia (rTTV) vaccines expressing OVA-CTB fusion antigen. Female C57BL/6 mice were immunized with an intranasal DNA priming/intramuscular rTTV boosting regimen. OVA specific T-cell responses were measured by IFN-γ ELISPOT and specific antibody responses were determined by ELISA. Compared to the nonadjuvant group (pSV-OVA intranasal priming/rTTV-OVA intramuscular boosting), pSV-OVA-CTB intranasal priming/rTTV-OVA-CTB intramuscular boosting group significantly improved the magnitudes of T-cell responses at spleen (1562 ± 567 SFCs/10(6) splenocytes versus 330 ± 182 SFCs/10(6) splenocytes, P < 0.01), mesenteric LN (96 ± 83 SFCs/10(6) lymphocytes versus 1 ± 2 SFCs/10(6) lymphocytes, P < 0.05), draining LNs of respiratory tract (109 ± 60 SFCs/10(6) lymphocytes versus 2 ± 2 SFCs/10(6) lymphocytes, P < 0.01) and female genital tract (89 ± 48 SFCs/10(6) lymphocytes versus 23 ± 21 SFCs/10(6) lymphocytes, P < 0.01). These results collectively demonstrated that fusion-expressed CTB could act as a potent adjuvant to improve both systemic and mucosal T-cell responses.

As a genetic adjuvant, CTA improves the immunogenicity of DNA vaccines in an ADP-ribosyltransferase activity- and IL-6-dependent manner.

Cholera toxin (CT) and its subunits (A and B) have been intensively investigated as adjuvants for protein-based vaccines. Their underlying mechanisms vary with respect to the inoculation route used. By fusing the CTA gene to either the HIV-1-derived Tat-Rev-Vif-Integrase-Nef fusion gene or the OVA gene, our study showed that the fusion of CTA in these DNA vaccines had no cytotoxic effect in vitro and significantly improved both the quantity and quality of the elicited CD8(+) T cell responses. Further experiments identified that the fusion of CTA in these DNA vaccines augmented the secretion of IL-6 in a manner that was dependent on its ADP-ribosyltransferase activity, and protein kinase A (PKA) was found to be the major mediator of its downstream signaling. By site-directed mutagenesis of the ADP-ribosyltransferase catalytic center and in vivo RNAi, we demonstrated that the ADP-ribosyltransferase activity and the upregulation of IL-6 were required for the CTA gene-mediated adjuvant effect. These findings demonstrate that when fused to an immunogen gene, the CTA gene could serve as a potent genetic adjuvant, providing new insights into the mechanisms of CTA as an adjuvant.

Boosting functional avidity of CD8+ T cells by vaccinia virus vaccination depends on intrinsic T-cell MyD88 expression but not the inflammatory milieu.

UNLABELLED: T-cell functional avidity is a crucial determinant for efficient pathogen clearance. Although recombinant DNA priming coupled with a vaccinia-vectored vaccine (VACV) boost has been widely used to mount robust CD8+ T-cell responses, how VACV boost shapes the properties of memory CD8+ T cells remains poorly defined. Here, we characterize the memory CD8+ T cells boosted by VACV and demonstrate that the intrinsic expression of MyD88 is critical for their high functional avidity. Independent of selection of clones with high-affinity T-cell receptor (TCR) or of enhanced proximal TCR signaling, the VACV boost significantly increased T-cell functional avidity through a decrease in the activation threshold. VACV-induced inflammatory milieu is not sufficient for this improvement, as simultaneous administration of the DNA vaccine and mock VACV had no effects on the functional avidity of memory CD8+ T cells. Furthermore, reciprocal adoptive transfer models revealed that the intrinsic MyD88 pathway is required for instructing the functional avidity of CD8+ T cells boosted by VACV. Taking these results together, the intrinsic MyD88 pathway is required for the high functional avidity of VACV-boosted CD8+ T cells independent of TCR selection or the VACV infection-induced MyD88-mediated inflammatory milieu. IMPORTANCE: Functional avidity is one of the crucial determinants of T-cell functionality. Interestingly, although it has been demonstrated that a DNA prime-VACV boost regimen elicits high levels of T-cell functional avidity, how VACV changes the low avidity of CD8+ T cells primed by DNA into higher ones in vivo is less defined. Here, we proved that the enhancement of CD8+ T cell avidity induced by VACV boost is mediated by the intrinsic MyD88 pathway but not the MyD88-mediated inflammatory milieu, which might provide prompts in vaccine design.

Drug susceptibility profile and pathogenicity of H7N9 influenza virus (Anhui1 lineage) with R292K substitution.

Neuraminidase inhibitors (NAIs) are the only available licensed therapeutics against human H7N9 influenza virus infections. The emergence of NAI-resistant variants of H7N9viruses with an NA R292K mutation poses a therapeutic challenge. A comprehensive understanding of the susceptibility of these viruses to clinically available NAIs, non-NAIs and their combinations is crucial for effective treatment. In this study, by using limited serial passage and plaque purification, an R292K variant of the Anhui1 lineage was isolated from a patient with clinical evidence of resistance to oseltamivir. In vitro and cell-based assays confirmed a high level of resistance conferred by the R292K mutation to oseltamivir carboxylate and a moderate level of resistance to zanamivir and peramivir. Non-NAI antivirals, such as T-705, ribavirin and NT-300, efficiently inhibited both the variant and the wild-type in cell-based assays. A combination of NAIs and non-NAIs did not exhibit a marked synergistic effect against the R292K variant. However, the combination of two non-NAIs (T-705 and ribavirin) exhibited significant synergism against the mutant virus. In experimentally infected mice, the variant showed delayed onset of symptoms, a reduced viral load and attenuated lethality compared with the wild-type. Our study suggested non-NAIs should be tested clinically for H7N9 patients with a sustained high viral load. Possible drug combination regimens, such as T-705 plus ribavirin, should be further tested in animal models. The pathogenicity and transmissibility of the R292K H7N9 variant should be further assessed with genetically well-characterized pairs of viruses and, most-desirably, with competitive fitness experiments.

The average IFN-γ secreting capacity of specific CD8(+) T cells is compromised while increasing copies of a single T cell epitope encoded by DNA vaccine.

Previous studies suggested that both the frequency and the mean fluorescence intensity (MFI) of cytokine secreting T cells could be of great value for immunogenicity evaluation of a vaccine. In this study, by constructing epitope-based DNA vaccines encoding a previously identified CD8(+) T cell epitope, we investigated the influence of multiplying epitope copies on both the frequency and the MFI of specific IFN-γ secreting CD8(+) T cells. We found that frequencies of specific CD8(+) T cell could be improved by multiplying epitope copies, while the MFI of IFN-γ secreted by epitope-specific CD8(+) T cells decreased synchronously. And further analysis showed that the decrease of MFI was not caused by the functional avidity variation of CD8(+) T cell receptor.

[Immunogenicities and comparison of DNA vaccines encoding pol genes derived from B`/C and A/E recombinant HIV-1 strains].

OBJECTIVE: To construct and compare the immunogenicities of DNA vaccines expressing pol genes derived from B`/C and A/E recombinant subtypes of HIV-1 in China. METHODS: Two DNA vaccines were constructed by inserting the codon optimized pol genes derived from B'/C and A/E subtypes of HIV-1 into mammalian expression vector pSV1.0. In vitro expression efficiencies of the two DNA vaccines were determined by Western blotting and their immunogenicities were compared by i.m. immunizing female BALB/c mice. After immunization, mice splenocytes were isolated sterilely and IFN-γ based enzyme linked immunospot assay (ELISPOT) was employed to read out the specific T cell immunity. RESULTS: The constructed DNA vaccines were validated by restriction enzyme digestion and DNA sequencing. Western blotting result showed both of the two DNA vaccines could be expressed at appreciable levels in vitro. Under the stimulation of Consensus B Pol peptide pools, specific T cell frequency elicited by pSVAE-Pol was (636±178) SFCs/10(6) splenocytes; specific T cell frequency elicited by pSVCN-Pol was (468±265)SFCs/10(6) splenocytes (P=0.412). Under the stimulation of HIV-1 AE2f Pol peptide pools, specific T cell frequency elicited by pSVAE-Pol was (1378±611) SFCs/10(6) splenocytes; specific T cell frequency elicited by pSVCN-Pol was (713±61) SFCs/10(6) splenocytes (P=0.134). Further analysis suggested pSVAE-Pol induced specific T cell responses mainly focused on Pol 1 peptide pool, while, in addition to induce Pol 1 specific T cell responses, pSVCN-Pol could also elicit T cell responses against consensus B Pol 2 peptide pool. CONCLUSION: Although pSVAE-Pol was more immunogenic, pSVCN-Pol could induce T cell responses against broader epitope spectrum. Rational vaccine design may need combine them together.

[Effect of early high-loading-dose tirofiban on platelet activity in patients with acute ST-segment elevation myocardial infarction undergoing primary percutaneous coronary intervention].

OBJECTIVE: To investigate the effect of early high-loading-dose tirofiban on platelet activity for patients with acute ST-segment elevation myocardial infarction (STEMI) undergoing primary percutaneous coronary intervention. METHODS: A total of 120 acute STEMI patients were treated with 300 mg aspirin and 600 mg loading dose clopidogrel and randomized to high-dose tirofiban (25 µg/kg bolus followed by 0.15 µg×kg(-1)×min(-1) infusion for 36 hours, n = 40), standard-dose tirofiban (10 µg/kg bolus followed by 0.15 µg×kg(-1)×min(-1) infusion for 36 hours, n = 40) or control (no tirofiban, n = 40) before angiography. Inhibition of platelet aggregation (IPA) was assessed before angiography, at 10 min and 24 hours after tirofiban infusion, and at 12 and 24 hours after stopping tirofiban infusion by the thrombelastography assay. RESULTS: There was no significant difference in baseline of IPA between the 3 groups (P > 0.05). IPA was significantly higher in high-dose tirofiban group compared with standard-dose tirofiban and no tirofiban group at 10 minutes after tirofiban infusion [(84.2 ± 12.0)% vs. (67.8 ± 26.8)% and (31.5 ± 21.9)%, all P < 0.01]. At 24 hours after tirofiban infusion, the IPA of high-dose and standard-dose tirofiban was similar [(93.0 ± 9.8)% vs. (88.5 ± 18.1)%, P > 0.05] and was significantly higher than no tirofiban group [(40.4 ± 22.8)%, all P < 0.01]. IPA was similar at 12 and 24 hours after stopping tirofiban use among the 3 groups (all P > 0.05). The maximum amplitude of high-dose tirofiban and standard-dose tirofiban groups at different time points was similar (all P > 0.05), and maximum amplitude in both tirofiban groups was significantly lower than in no tirofiban group at 10 min [(47.2 ± 7.6) mm and (50.0 ± 9.8) mm vs. (57.7 ± 6.5) mm, all P < 0.01] and at 24 hours after stopping tirofiban infusion [(54.6 ± 5.6) mm and (54.3 ± 9.0) mm vs. (59.6 ± 4.0) mm, all P < 0.01]. CONCLUSION: Early use of high-loading-dose of tirofiban on top of 600 mg loading dose clopidogrel is more efficient on inhibiting platelet activity than standard dose of tirofiban in patients with acute STEMI undergoing primary primary percutaneous coronary intervention.

[Study on optical energy transmission in biotic tissues by Monte Carlo method].

Biotic tissues are a kind of highly scattering random media; studies on laser light propagation in biotic tissues play an important role in bio-medical diagnostics and therapeutics. The propagation and distribution of infinitely narrow photon beam in tissues are simulated by Monte Carlo method in this paper. Also presented are the energy distribution with regard to depths, light distribution in tissues, reflection and transmittance on the upper and lower surface. The optical parameters adopted in this study are g, albedo and microa, which have influence on energy distribution. The results show: The energy distribution decreases more quickly with the increase of depths and reveals a peak value close to the surface; g factor plays an important part in the lost energy on the upper surface and lower surface; the decrease of g factor causes weaking of the forward moving ability, so the penetration depth becomes smaller and the energy becomes dispersives variation of albedo has distinct effect on the shallow and deep tissues.