Objective To construct the murine allogeneic acute GVHD model.Methods C57BL/6 (H-2b) mice were used as the donors and Balb/c (H-2d) mice as the recipients in allogeneic bone marrow transplantation (BMT).Groups were set as total body radiation (TBI) control group (n =4),GVHD group (n =10),simple BM transplantation group (n =10) and normal control group (n =4).For TBI control group,mice were subjected to TBI but did not receive BMT after radiation.For GVHD group,5 days before TBI,gentamycin (320 mg/L) and erythromycin (250 mg/L) were added into the drinking water,and on the day of transplantation,mice received one total dose of 8.0 Gy 60Coγ TBI,and within 5 h,2 × 106 C57BL/6 BM cells and 1 × 107 C57BL/6 spleen cells were transfused per mouse via the tail vein.For simple BMT group,the pretreatment was the same as GVHD group,and mice received only 2 × 106 C57BL/6 BM cells per mouse via the tail vein.The mental status,activity,posture,fur,weight,and stool were observed after transplantation.Survival time of each mouse was recorded,survival rate was calculated,and survival curve was drawn.Pathological examination was done for the liver,skin,small intestine and BM on the brink of death.Results The median survival time (MST) in TBI control group,GVHD group and BMT group was (9.0 ± 0.7),(32.0 ± 3.2) and ( 17.5 ± 1.6) days respectively,and there was significant difference between every two groups (P ＜ 0.01 ).Pathological examination in TBI control group showedhematopoiesis exhaustion.GVHD group showed acute GVHD symptoms 10-13 days after allo-BMT,and the pathological changes of the skin,liver and small intestine corresponded to those of Ⅰ to Ⅱ degree of GVHD.Simple BMT group also showed acute GVHD symptoms 10-13 days after alloBMT,but their GVHD manifestation and histological changes were less serious and only 0 to Ⅰ degree of GVHD could be seen.ConclusionStable acute GVHD model can be constructed by transfusion of allogeneic BM cells and spleen cells into Balb/c mice after lethal TBI.

The graft-versus-tumor (GVT) effect of T cells induced by tumor antigen-pulsed CD8α(+) dendritic cells (DCs) in vitro was investigated in this study. Immature CD8α(+) DCs were prepared from C57BL/6 (H-2(b)) bone marrow cells by using a cytokine cocktail. On the 3rd day of culture, CD8α(+) DCs were pulsed by allogeneic (Balb/c, H-2(d)) EL9611 leukemia antigen, or RM-1 syngeneic prostate cancer antigen, with the concentration series of 0, 2.5, 5.0, 10.0, 20.0 μg/mL, respectively, then antigen-loaded immature CD8α(+) DCs were co-cultured with syngeneic T cells according to the DC/T ratio of 1:1, 2:1 and 4:1. T cell proliferation was measured by MTT assay. Cytokines including interferon gamma (IFN-γ) and interleukin-10 (IL-10) in CD8α(+) DCs and T co-culture supernatant were detected by using ELISA. Cytotoxic effect of antigen-specific T cells was tested by LDH release assay. Conventional mature DCs (mDCs) induced from C57BL/6 (H-2(b)) bone marrow cells by using granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4) served as a control. The results showed that the proliferative activity of T cells stimulated by CD8α(+) DCs loaded with allogeneic or syngeneic tumor antigen was augmented with the CD8α(+) DC/T ratio increased (P<0.05). When antigen concentration ≤ 5 μg/mL and CD8α(+) DC/T ratio ≤ 2:1, the ability of CD8α(+) DCs to stimulate T cell proliferation was higher than mDC control in allogeneic tumor antigen-pulsed groups (P<0.05), but not in syngeneic tumor antigen-pulsed groups (P>0.05). The level of IFN-γ and IL-10 in CD8α(+) DCs and T cell co-culture supernatant were increased in both allogeneic and syngeneic antigen-pulsed groups (P<0.05), and the cytokine level was higher in allogeneic antigen-pulsed groups than in syngeneic antigen groups when the CD8α(+) DC/T was 1:1 or 2:1 (P<0.05). There existed a negative correlation between the level of IL-10 and T cell proliferation. T cell cytotoxicity assay showed that when CD8α(+) DCs were pulsed with allogeneic tumor antigen, the maximal T cell killing efficiency could reach (100±7.7)%, whereas syngeneic tumor antigen-pulsed group had only (65.0±3.4)%. It was concluded that syngeneic and allogeneic tumor antigen-pulsed immature CD8α(+) DCs could stimulate T cells to exert the GVT effect in vitro, and the GVT effect was more obvious with allogeneic tumor antigen than with syngeneic tumor antigen. The optimal condition was low allogeneic tumor antigen pulsation (≤ 5 μg/mL) and low CD8α(+) DC/T ratio (1:1 and 2:1).

Embryos of most mammalian species grown in vitro would undergo developmental arrest at the approximate time of genomic activation. Stage-specific cell block and the resulting rapid loss of embryo viability in conventional culture media have limited the duration for which embryos may be cultured prior to transfer. As a result, embryos are usually transferred to the uterus at the 4-to 8-cell stage to avoid the loss of viability associated with long-term in vitro culture. Early transfer has led to asynchrony of the endometrium-trophectoderm interaction at the time of implantation and a resultant reduction in the rate of implantation. To overcome these problems, a variety of co-culture systems has been devised in which embryos can develop for a longer period prior to embryo transfer. Vero cells, derived from African green monkey kidney, share a common embryologic origin with cells from the genital tract. In addition, they are potentially safe to use, since they are highly controlled for viruses and other contaminants. Therefore, co-culture using Vero cells has been widely utilized to enhance embryo viability and development, although not without controversies. We thus designed a series of experiments to demonstrate whether Vero cells do indeed enhance mouse embryo development as well as to compare the efficacy of co-culturing mouse 1-cell embryos on Vero cell monolayer in both Ham's F-10 and human tubal fluid (HTF) culture media. 1-cell stage ICR mouse embryos were cultured either in the presence of Vero cells (Group A) or in conventional culture medium alone (Group B). In Ham's F-10 significantly more 3-to-8cell embryos developed in group A than group B (59.8 versus 10.0%; F<0.01). In contrast, there was no significant difference in embryonic development both group A and group B in HTF. However, significant differences were noted only in later embryonic stage (13 and 0%; p<0.05 of group A and B respectively, hatching or hatched). In Ham's F-10, we also could observe the beneficial effect of Vero cell on hatching process (70.7 and 42.1%; p<0.05 of group A and group B respectively).
Animals ; Cercopithecus aethiops ; Coculture Techniques* ; Culture Media ; Embryo Transfer ; Embryonic Development* ; Embryonic Structures* ; Female ; Humans ; Kidney ; Mice* ; Mice, Inbred ICR ; Pregnancy ; Uterus ; Vero Cells*

Objective To explore the sero-prevalence and risk factors for herpes simplex virus 2 (HSV-2) infection and unprotected sexual behavior in an ethnically diverse population of HIVinfected subjects in a county of Yunnan province. Methods HIV-infected individuals attending for routine follow-up by local Center for Disease Control and Prevention (CDC) were recruited to participate in the study under 'informed consent'. A face-to-face questionnaire interview was administered to each participant. Blood was drawn for HSV-2 testing by HerpeSelect HSV-2 ELISA (Focus Diagnostics) and CD4+ T counting. Results A total of 300 HIV-infected individuals participated in the study. The mean age of the subjects was 37.6 years with 76.7% as males. Ethnically, Han, Dai and Jingpo accounted for 44.3%, 37.3% and 16.0% of the sample, respectively. Half of the subjects reported HIV acquisition through injection drug use. The sero-prevalence of HSV-2 was 35.0%. Results from multiple logistic regression analysis indicated that individuals who acquired HIV through heterosexual contact were more likely to be HSV-2 positive than those who acquired HIV through injection drug use (OR=4.244,95%CI: 1.924-9.364),whereas Dai (OR=0.300,95% CI: 0.152-0.593) and Jingpo (OR=0.376, 95% CI: 0.167-0.850) were less likely to be HSV-2 positive than the Hans. Among 105 people who were co-infected with HIV/HSV-2, 60 had sexual intercourses in the past 3 months and 41.7% of them reported no or inconsistent use of condoms. Most unprotected sexual contacts occurred within married couples. Conclusion HSV-2 infection was highly prevalent among HIV-infected individuals in this county, and a significant proportion of HIV/HSV-2 co-infected subjects engaged in unprotected sex. HSV-2 testing, behavioral and biomedical interventions among HIV-infected individuals and their sexual partners should be involved in the local HIV prevention and control programs.

The graft-versus-tumor (GVT) effect of T cells induced by tumor antigen-pulsed CD8α+dendritic cells (DCs) in vitro was investigated in this study.Immature CD8α+ DCs were prepared from C57BL/6 (H-2b) bone marrow cells by using a cytokine cocktail.On the 3rd day of culture,CD8α- DCs were pulsed by allogeneic (Balb/c,H-2d) EL9611 leukemia antigen,or RM-1 syngeneic prostate cancer antigen,with the concentration series of 0,2.5,5.0,10.0,20.0 μg/mL,respectively,then antigen-loaded immature CD8α+ DCs were co-cultured with syngeneic T cells according to the DC/T ratio of 1∶1,2∶1and 4∶1.T cell proliferation was measured by MTT assay.Cytokines including interferon gamma (IFN-γ)and interleukin-10 (IL-10) in CD8α+ DCs and T co-culture supernatant were detected by using ELISA.Cytotoxic effect of antigen-specific T cells was tested by LDH release assay.Conventional mature DCs　(mDCs) induced from C57BL/6 (H-2b) bone marrow cells by using granulocyte-macrophage colony　stimulating factor (GM-CSF) and interleukin-4 (IL-4) served as a control.The results showed that the　proliferative activity of T cells stimulated by CD8α+ DCs loaded with allogeneic or syngeneic tumor antigen was augmented with the CD8α+ DC/T ratio increased (P＜0.05).When antigen concentration ≤ 5μg/mL and CD8α+ DC/T ratio ≤ 2∶1,the ability of CD8α+ DCs to stimulate T cell proliferation was　higher than mDC control in allogeneic tumor antigen-pulsed groups (P＜0.05),but not in syngeneic tumor antigen-pulsed groups (P＞0.05).The level of IFN-γ and IL-10 in CD8α+DCs and T cell co-culture　supernatant were increased in both allogeneic and syngeneic antigen-pulsed groups (P＜0.05),and the　cytokine level was higher in allogeneic antigen-pulsed groups than in syngeneic antigen groups when　the CD8α+DC/T was 1∶1 or 2∶1 (P＜0.05).There existed a negative correlation between the level of　IL-10 and T cell proliferation.T cell cytotoxicity assay showed that when CD8α+ DCs were pulsed with　allogeneic tumor antigen,the maximal T cell killing efficiency could reach (100±7.7)％,whereas syngeneic tumor antigen-pulsed group had only (65.0±3.4)％.It was concluded that syngeneic and allogeneic tumor antigen-pulsed immature CD8α+ DCs could stimulate T cells to exert the GVT effect in vitro,and the GVT effect was more obvious with allogeneic tumor antigen than with syngeneic tumor antigen.The optimal condition was low allogeneic tumor antigen pulsation (≤ 5 μg/mL) and low CD8α+ DC/T　ratio (1∶1 and 2∶1).

Purpose To investigate the role of CHMP4A and TSPYL-2 in early pathogenesis of esophageal cancer.Methods Through comparison of the four subtractive libraries,early esophageal squamous cell carcinoma genes CHMP4A and TSPYL-2 were chosen for further study.Through RT-PCR and immunohistochemistry methods,CHMP4A and TSPYL-2's expression was detected in esophageal squamous cell carcinoma tissue,cancerous tissue and normal esophageal mucosa.Results CHMP4A and TSPYL-2 expression between esophageal squamous cell carcinoma and normal esophageal epithelium tissue had significant differences (P ＜ 0.05),and the CHMP4A gene expression in esophageal mucosa,field cancerization areas,esophageal squamous cell carcinoma tissue increased,while TSPYL-2 gene expression in esophageal mucosa,field cancerization areas,esophageal squamous cell carcinoma tissue decreased,which were consistent with the protein expression of CHMP4A and TSPYL-2.Conclusion CHMP4A and TSPYL-2 genes are differentially expressed in esophageal squamous cell carcinoma,which can be used as alternative genetic markers for further research.

Objective To investigate the prevalence of nutritional risk and malnutrition in hospitalized lung cancer patients in a tertiary A hospital in Chongqing.Methods From December 2013 to July 2017,2 735 consecutive lung cancer patients were admitted to the Department of Pneumology at Daping Hospital for planned anti-cancer treatment.Patients who did not complete a nutritional status assessment and who had repeated admission wcrc excluded from the study.The demographic and tumor characteristics were investigated in the 548 lung cancer inpatients who completed the study.The nutritional risk screening 2002 (NRS 2002) was used to evaluate the nutritional risk.The individual nutritional status was also evaluated using the patient-generated subjective global assessment (PG-SGA) questionnaire,anthropometry measurements and hematological measurements.The physical status was assessed by the Karnofsky performance status (KPS).Results According to the NRS 2002 score,29.56％ (162/548) of the cancer patients had nutritional risk (score ≥3).The prevalence of nutritional risk was 17.39％,15.00％,22.00％ and 36.86％,respectively,for patients with stage Ⅰ,Ⅱ,Ⅲ and Ⅳ lung cancer.Forty-four patients (9.67％) had a body mass index＜ 18.5 kg/m2 and poor general condition,and the prevalence was 6.52％,5.00％,8.67％ and 11.22％,respectively,for stages I,Ⅱ,Ⅲ and Ⅳ.A total of 107 cases (19.53％) had impaired nutritional status (indicated by a severity score of 3 in the NRS 2002).The prevalence by different stages was 10.87％ (stage Ⅰ),5.00％ (stage Ⅱ),14.67％ (stage Ⅲ) and 25.00％ (stage Ⅳ).One hundred and twenty-five patients (22.81％) had PG-SGA scores ≥ 9,with 2.19％,2.50％,12.67％,and 33.33％ of patients in stages Ⅰ,Ⅱ,Ⅲ and Ⅳ having these high scores.The KPS scores were lower in the patients with nutritional risk and malnutrition than in the patients with a normal nutritional status.Conclusions The prevalence of nutritional risk and malnutrition in patients with lung cancer were mediom.Nutritional risk screening and nutritional status assessment should be considered at the time of admission for lung cancer patients in order to ensure better outcomes of treatment.

PURPOSES: To investigate the viral etiology of community-acquired pneumonia in Korean adults, we have detected respiratory viruses (Respiatory syncytial virus, adenovirus, influenza virus and parainfluenza virus) in the way of prospective, multi-center study. METHODS: From July 1997 to April 2000, nasal aspirates or sputum were obtained from adults patients with community pneumonia admitted to the participating hospitals and transferred immediately to the central laboratory in the Seoul National University Children's Hospital. The specimens were divided into three parts. One part was used for indirect immunofluorescent test for respiratory viruses, the other part for the culture of RSV and adenovirus in HEp-2 cell monolayer. Another part was used for the culture of influenza virus and parainfluenza virus in MDCK or LLC- MK2 cell monolayers. RESULTS: Of 317 samples, 32 (10.1%) specimens were positive for viral isolation by indirect IF staining or culture, including one dual-infected specimen (adenovirus and parainfluenza virus). Influenza virus was most commonly detected (16 specimens). Parainfluenza virus, adenovirus and RSV were detected in 10, 4 and 3 patients, respectively. All isolated influenza viruses were type A (H3N2 in 9 patients, H1N1 in 2 and unspecified in 5), and 8 out of 10 parainfluenza virus isolates were type 3. CONCLUSION: Similar to previous foreign reports, a significant portion of community-acquired pneumonia in Korean adult is caused by respiratory viruses. Our data empathized the need of referral system for viral diagnosis and of nationwide investigation on respiratory virus infections.
Adenoviridae ; Adult* ; Diagnosis ; Humans ; Orthomyxoviridae ; Paramyxoviridae Infections ; Pneumonia* ; Prospective Studies ; Referral and Consultation ; Seoul ; Sputum

Oxygen levels are unequal in different living geographical locations of human and related to normal physiology of health. The reduction of oxygen level in the body can lead to a variety of diseases, such as stroke caused by cerebral ischemia and hypoxia. In the recent years, many studies have elucidated the molecular and cellular mechanisms of organism response to different oxygen concentrations by using the nematode Caenorhabditis elegans (C. elegans) as model organism. C. elegans can escape hypoxia or hyperoxia and adapt to the ambient oxygen environments, and there are different response and regulation mechanisms in different degrees of hypoxia environment. In this paper, recent advances in the reaction of nematodes to different oxygen concentrations and the underlying mechanism were reviewed.
Animals ; Caenorhabditis elegans ; Caenorhabditis elegans Proteins ; Humans ; Hypoxia ; Oxygen

Background: The Omicron variant has become the most prevalent SARS-CoV-2 variant. Omicron is known to induce milder lesions compared to the original Wuhan strain. Fatal infection of the Wuhan strain into the brain has been well documented in COVID-19 mouse models and human COVID-19 cases, but apparent infections into the brain by Omicron have not been reported in human adult cases or animal models. In this study, we investigated whether Omicron could spread to the brain using K18-hACE2 mice susceptible to SARS-CoV-2 infection. Results: K18-hACE2 mice were intranasally infected with 1 × 105 PFU of the original Wuhan strain and the Omicron variant of SARS-CoV-2. A follow-up was conducted 7 days post infection. All Wuhan-infected mice showed > 20% body weight loss, defined as the lethal condition, whereas two out of five Omicron-infected mice (40%) lost > 20% body weight. Histopathological analysis based on H&E staining revealed inflammatory responses in the brains of these two Omicron-infected mice. Immunostaining analysis of viral nucleocapsid protein revealed severe infection of neuron cells in the brains of these two Omicron-infected mice. Lymphoid depletion and apoptosis were observed in the spleen of Omicron-infected mice with brain infection. Conclusion Lethal conditions, such as severe body weight loss and encephalopathy, can occur in Omicron-infected K18-hACE2 mice. Our study reports, for the first time, that Omicron can induce brain infection with lymphoid depletion in the mouse COVID-19 model.