Characterization of genetic humanized mice with transgenic HLA DP401 or DRA but deficient in endogenous murine MHC classⅡgenes upon Staphylococcus aureus pneumonia

Background:Staphylococcus aureus can cause serious infections by secreting many superantigen exotoxins in“carrier”or“pathogenic”states.HLA DQ and HLA DR humanized mice have been used as a small animal model to study the role of two molecules during S.aureus infection.However,the contribution of HLA DP to S.aureus infection is unknown yet.Methods:In this study,we have produced HLA DP401 and HLA DRA0101 humanized mice by microinjection of C57BL/6J zygotes.Neo-floxed IAβ+/-mice were crossbred with Ella-Cre and further crossbred with HLA DP401 or HLA-DRA0101 humanized mice.After several rounds of traditional crossbreeding,we finally obtained HLA DP401-IAβ-/-and HLA DRA-IAβ-/-humanized mice,in which human DP401 or DRA0101 molecule was introduced into IAβ-/-mice deficient in endogenous murine MHC classⅡmolecules.A transnasal infection murine model of S.aureus pneumonia was induced in the humanized mice by administering 2×108CFU of S.aureus Newman dropwise into the nasal cavity.The immune responses and histopathology changes were further assessed in lungs in these infected mice.Results:We evaluated the local and systemic effects of S.aureus delivered intranasally in HLA DP401-IAβ-/-and HLA DRA-IAβ-/-transgenic mice.S.aureus Newman infection significantly increased the m RNA level of IL 12p40 in lungs in humanized mice.An increase in IFN-γand IL-6 protein was observed in HLA DRA-IAβ-/-mice.We observed a declining trend in the percentage of F4/80+macrophages in lungs in HLA DP401-IAβ-/-mice and a decreasing ratio of CD4+to CD8+T cells in lungs in IAβ-/-mice and HLA DP401-IAβ-/-mice.A decreasing ratio of Vβ3+to Vβ8+T cells was also found in the lymph node of IAβ-/-mice and HLA DP401-IAβ-/-mice.S.aureus Newman infection resulted in a weaker pathological injury in lungs in IAβ-/-genetic background mice.Conclusion:These humanized mice will be an invaluable mouse model to resolve the pathological mechanism of S.aureus pneumonia and study what role DP molecule plays in S.aureus infection.

A survey on the status of semen analysis in 118 laboratories in China

收集实验室怎么表现测试的基线信息是一合理首先向为精液分析建立 intra 实验室和内部实验室的标准化和质量控制走。我们执行了执行在大陆中国测试的实验室的调查。一张问询表， 36 镇静询问盖住精液分析的所有方面，被设计，并且一个拷贝对每 145 个实验室分布式。这些， 118 个实验室完成了问询表。调查结果证明那精液体积在 53.6% 视觉上被测量(59/110 ) 反应实验室，并且(73/103 ) 没有任何处理， 70.9% 不完全地分析的实验室液化了精液。另外，手册显微镜、帮助计算机的精液分析系统被使用分析精子集中，活动性和形态学。然而，超过五个方法在染色的平淡的精子被采用。连接酶的 immunosorbent 试金通常被使用决定 antisperm 抗体是否是在场的。几个精液的生物化学的标记被分析在仅仅 27.1%(32/118 ) 反应实验室。通常，为在对这调查作出回应的所有实验室的精液分析有 intra 实验室和内部实验室的质量控制措施的缺乏。在结论，精液分析和在调查实验室的测试结果的解释的方法显著地不同。特别地，许多实验室采用了除人的精液和 Spermcervical 粘液相互作用(1999 ) 的考试的世界健康组织实验室手册推荐的那些以外的方法。这些调查结果与可接受的质量控制为精液分析的标准化建议迫切需要让每个参数使结果可重复、有意义。

Effects of Ligustrum robustum on gut microbes and obesity in rats

AIM: To investigate the anti-obesity and antibacterial effects of Ligustrum robustum(L. robustum) in vivoand in vitro and its possible mechanisms. METHODS: The effects of L. robustum aqueous extract(LR) on various gut bacteria in vitro were evaluated. The effects of LR on high-fat diet-fed(HFD) rats in vivo were also assessed. Culture methods,quantitative polymerase chain reaction,and terminalrestriction fragment length polymorphism were used to analyze the effects of LR on gut bacteria. Biochemical tests were also performed to detect the changes in obesity-related indicators after LR treatment. RESULTS: LR treatment lowered adipose weight and decreased Lee's index,blood glucose,total cholesterol,and lipid in the tested groups relative to control(P < 0.05). To determine the reasons for these changes,we assessed the potential bacteriostatic and bactericidal effects of LR on specific bacterial species in vitro. LR affected the richness,diversity,and evenness of gut bacteria,increased fecal Lactobacillus,and decreased Enterococci in HFD rats(P < 0.05). CONCLUSION: L. robustum may be a safe and effective food for weight loss and obesity control,and the effects of L. robustum might be mediated by the regulation of gut bacteria.

Induction of apoptosis in human liver carcinoma HepG2 cell line by 5-allyl-7-gen-difluoromethylenechrysin

AIM:To investigate the effect of 5-allyl-7-gen-difluoromethylenechrysin (ADFMChR) on apoptosis of human liver carcinoma HepG2 cell line and the molecular mechanisms involved. METHODS: HepG2 cells and L-02 cells were cultured in vitro and the inhibitory effect of ADFMChR on their proliferation was measured by MTT assay. The apoptosis of HepG2 cells was determined by flow cytometry (FCM) using propidium iodide (PI) fluorescence staining. DNA ladder bands were observed by DNA agarose gel electrophoresis. The influence of ADFMChR on the proxisome proliferator-activated receptor γ (PPARγ), NF-κB, Bcl-2 and Bax protein expression of HepG2 cells were analyzed by Western blotting. RESULTS: MTT assay showed that ADFMChR significantly inhibited proliferation of HepG2 cells in a dosedependent manner, with little effect on growth of L-02 cells, and when IC50 was measured as 8.45 μmol/L and 191.55 μmol/L respectively, the potency of AD- FMChR to HepG2 cells, was found to be similar to 5-fluorouracil (5-FU, IC50 was 9.27 μmol/L). The selective index of ADFMChR cytotoxicity to HepG2 cells was 22.67 (191.55/8.45), higher than 5-FU (SI was 7.05 (65.37/9.27). FCM with PI staining demonstrated that the apoptosis rates of HepG2 cells treated with 3.0, 10.0 and 30.0 μmol/L ADFMChR for 48 h were 5.79%, 9.29% and 37.8%, respectively, and were signif icantly higher when treated with 30.0 μmol/L ADFMChR than when treated with 30.0 μmol/L ChR (16.0%) (P < 0.05) and were similar to those obtained with 30.0 μmol/L 5-FU (41.0%). DNA agarose gel electrophoresis showed that treatment of HepG2 cells with 10.0 μmol/L ADFMChR for 48 h and 72 h resulted in typical DNA ladders which could be reversed by 10.00 μmol/L GW9662, a blocker of PPARγ. Western blotting analysis revealed that after 24 h of treatment with 3.0, 10.0, 30.0 μmol/L ADFM-ChR, PPARγ and Bax protein expression in HepG2 cells increased but Bcl-2 and NF-κB expression decreased; however, pre-incubation with 10.0 μmol/L GW9662 could efficiently antagonize and weaken the regulatory effect of 3.0, 30.0 μmol/L ADFMChR on PPARγ and NF-κB protein expression in HepG2 cells. CONCLUSION: ADFMChR induces apoptosis of HepG2 cell lines by activating PPARγ, inhibiting protein expression of Bcl-2 and NF-κB, and increasing Bax expression.

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-Arestricted 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-2D~b,linked at its N-terminus to the C-terminus of humanβ2m 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 humanβ2m-HLA-A30(α1α2)-H-2D~b(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.

A more consistent intraluminal rhesus monkey model of ischemic stroke

Endovascular surgery is advantageous in experimentally induced ischemic stroke because it causes fewer cranial traumatic lesions than invasive surgery and can closely mimic the pathophysiology in stroke patients. However, the outcomes are highly variable, which limits the accuracy of evaluations of ischemic stroke studies. In this study, eight healthy adult rhesus monkeys were randomized into two groups with four monkeys in each group: middle cerebral artery occlusion at origin segment(M1) and middle cerebral artery occlusion at M2 segment. The blood flow in the middle cerebral artery was blocked completely for 2 hours using the endovascular microcoil placement technique(1 mm × 10 cm)(undetachable), to establish a model of cerebral ischemia. The microcoil was withdrawn and the middle cerebral artery blood flow was restored. A reversible middle cerebral artery occlusion model was identified by hematoxylin-eosin staining, digital subtraction angiography, magnetic resonance angiography, magnetic resonance imaging, and neurological evaluation. The results showed that the middle cerebral artery occlusion model was successfully established in eight adult healthy rhesus monkeys, and ischemic lesions were apparent in the brain tissue of rhesus monkeys at 24 hours after occlusion. The rhesus monkeys had symptoms of neurological deficits. Compared with the M1 occlusion group, the M2 occlusion group had lower infarction volume and higher neurological scores. These experimental findings indicate that reversible middle cerebral artery occlusion can be produced with the endovascular microcoil technique in rhesus monkeys. The M2 occluded model had less infarction and less neurological impairment, which offers the potential for application in the field of brain injury research.

Nucleus Transfer Efficiency of Ear Fibroblast Cells Isolated from Bama Miniature Pigs at Various Ages

Somatic cell nucleus transfer(SCNT) has been considered the most effective method for conserving endangered animals and expanding the quantity of adult animal models. Bama miniature pigs are genetically stable and share similar biological features to humans. These pigs have been used to establish animal models for human diseases, and for many other applications. However, there is a paucity of studies on the effect of ear fibroblasts derived from different age of adult Bama miniature pigs on nucleus transfer(NT). The present study examined the NT efficiency of ear fibroblasts from fetal, newborn, 1-, 2-, 4-, 6-, 12-month-old miniature pigs by using trypan blue staining, flow cytometry and NT technique, etc., and the cell biological function and SCNT efficiency were compared between groups. The results showed that ear fibroblasts grew well after passage in each group. Spindle-shaped cells initially predominated, and gradually declined with increase of culture time and replaced by polygonal cells. Irregular cell growth occurred in the 2-month-old group and the elder groups. The growth curves of the ear fibroblasts were "S-shaped" in different age groups. The cell proliferation of postnatal ear fibroblasts, especially those from 2-, 4-, 6-, 12-month-old miniature pigs was significantly different from that of fetus ear fibroblasts(P<0.05 or P<0.01). Two-month- and 4-month-old ear fibroblasts had a significantly higher proportion of G1 stage cells(85% to 91%) than those at 6 and 12 months(66% to 74%, P<0.01). The blastocyst rate of reconstructed embryos originating from newborn, 1-, 2-, 4-month-old donor pigs was 6.06% to 7.69% with no significant difference from that in fetus fibroblast group(8.06%). It was concluded that <4-month-old adult Bama miniature pigs represent a better donor cell resource than elder pigs.

Noggin versus basic fibroblast growth factor on the differentiation of human embryonic stem cells

The difference between Noggin and basic fibroblast growth factor for the neural precursor differen-tiation from human embryonic stem cells has not been studied. In this study, 100 μg/L Noggin or 20 μg/L basic fibroblast growth factor in serum-free neural induction medium was used to differen-tiate human embryonic stem cells H14 into neural precursors using monolayer differentiation. Two weeks after induction, significantly higher numbers of neural rosettes formed in the Noggin-induced group than the basic fibroblast growth factor-induced group, as detected by phase contrast micro-scope. Immunofluorescence staining revealed expression levels of Nestin, β-III Tubulin and Sox-1 were higher in the induced cells and reverse-transcription PCR showed induced cells expressed Nestin, Sox-1 and Neurofilament mRNA. Protein and mRNA expression in the Noggin-induced group was increased compared with the basic fibroblast growth factor-induced group. Noggin has a greater effect than basic fibroblast growth factor on the induction of human embryonic stem cell differentiation into neural precursors by monolayer differentiation, as Noggin accelerates and in-creases the differentiation of neural precursors.

Efficacy of Gold Nanoparticles against Nephrotoxicity Induced by Schistosoma mansoni Infection in Mice

Objective In this study, the ameliorative effects of gold nanoparticles(gold NP) on the renal tissue damage in Schistosoma mansoni(S. mansoni)-infected mice was investigated. Methods High-resolution transmission electron microscopy was used for the characterization of NP. The gold NP at concentrations of 250, 500, and 1000 μg/kg body weight were inoculated into S. mansoni-infected mice. Results The parasite caused alterations in the histological architecture. Furthermore, it induced a significant reduction in the renal glutathione levels; however, the levels of nitric oxide and malondialdehyde were significantly elevated. The parasite also managed to downregulate KIM-1, NGAL, MCP-1, and TGF-β mR NA expression in infected animals. Notably, gold NP treatment in mice reduced the extent of histological impairment and renal oxidative damage. Gold NP were able to regulate gene expression impaired by S. Mansoni infection. Conclusion The curative effect of gold NP against renal toxicity in S. mansoni-infected mice is associated with their role as free radical scavengers.

Effectiveness of Saccharomyces boulardii in a rat model of colitis

AIM:To investigate the effects of Saccharomyces boulardii(S.boulardii) in an experimental rat model of trinitrobenzene sulfonic acid(TNBS)-induced colitis.METHODS:Thirty-two Wistar albino female rats were categorized into five groups.On the first day of the study,50 mg TNBS was administered via a rectal catheter in order to induce colitis in all rats,except those in the control group.For 14 d,the rats were fed a standard diet,without the administration of any additional supplements to either the control or TNBS groups,in addition to 1 mg/kg per day S.boulardii to the S.boulardii group,1 mg/kg per day methyl prednisolone(MP) to the MP group.The animals in the S.boulardii + MP group were coadministered these doses of S.boulardii and MP.During the study,weight loss,stool consistency,and the presence of obvious blood in the stool were evaluated,and the disease activity index(DAI) for colitis was recorded.The intestines were examined and colitis was macro-and microscopically scored.The serum and tissue levels of tumor necrosis factor-α(TNF-α) and nitric oxide(NO) were determined,and fungemia was evaluated in the blood samples.RESULTS:The mean DAI scores for the MP and S.boulardii + MP groups was significantly lower than the TNBS group(3.69 ± 0.61 vs 4.46 ± 0.34,P = 0.018 and 3.77 ± 0.73 vs 4.46 ± 0.34,P = 0.025,respectively).While no significant differences between the TNBS and the S.boulardii or MP groups could be determined in terms of serum NO levels,the level of serum NO in the S.boulardii + MP group was significantly higher than in the TNBS and S.boulardii groups(8.12 ± 4.25 μmol/L vs 3.18 ± 1.19 μmol/L,P = 0.013;8.12 ± 4.25 μmol/L vs 3.47 ± 1.66 μmol/L,P = 0.012,respectively).The tissue NO levels in the S.boulardii,MP and S.boulardii + MP groups were significantly lower than the TNBS group(16.62 ± 2.27 μmol/L vs 29.72 ± 6.10 μmol/L,P = 0.002;14.66 ± 5.18 μmol/Lvs 29.72 ± 6.10 μmol/L,P = 0.003;11.95 ± 2.34 μmol/Lvs 29.72 ± 6.10 μmol/L,P = 0.002,respectively).The tissue NO levels in the S.boulardii,MP and S.boulardii + MP groups were similar.The mean serum and tissue TNF-α levels were determined to be 12.97 ± 18.90 pg/mL and 21.75 ± 15.04 pg/mL in the control group,18.25 ± 15.44 pg/mL and 25.27 ± 11.95 pg/mL in the TNBS group,20.59 ± 16.15 pg/mL and 24.39 ± 13.06 pg/mL in the S.boulardii group,9.05 ± 5.13 pg/mL and 24.46 ± 10.85 pg/mL in the MP group,and 13.95 ± 10.17 pg/mL and 24.26 ± 10.37 pg/mL in the S.boulardii + MP group.Significant differences in terms of the levels of serum and tissue TNF-α and the macroscopic and microscopic scores were not found between the groups.S.boulardii fungemia was not observed in any of the rats.However,Candida fungemia was detected in one rat(14%) in the TNBS group,two rats(28%) in the S.boulardii group,three rats(50%) in the MP group,and three rats(42%) in S.boulardii + MP group.CONCLUSION:S.boulardii does not demonstrate considerable effects on the DAI,pathological scores,or cytokine levels but does decrease the tissue NO levels.

Natural maternal transmission of H pylori in Mongolian gerbils

AIM: To investigate maternal H pylori infection status to determine the potential of maternal transmission. METHODS: In the present study, we examined these issues in an experimental murine model, which is a Mongolian gerbil model that has been reported as an optimal laboratory animal model to study H pylori . Pregnant Mongolian gerbils, infected experimentally with H pylori, were divided into as four groups. Following the experimental design, the stomachs of the mother and litters were isolated and assessed for transmission of H pylori at the prenatal period, parturition day, 1-wk old and 3-wk old respectively. Bacterial culture and polymerase chain reaction (PCR) were used to examine the presence of transmitted H pylori. RESULTS: All litters showed no transmission of H pylori during pregnancy and at parturition day. However, they revealed 33.3% and 69.6% at 1-wk and 3-wk of age respectively by PCR. CONCLUSION: These results suggested that vertical infection during the prenatal period or delivery procedure is unlikely as a route of mother-to-child H pylori infection. It may be that H pylori is acquired through breast- feeding, contaminated saliva and fecal-oral transmission during co-habitation.

Targeting EZH1/2 induces cell cycle arrest and inhibits cell proliferation through reactivation of p57CDKN1C and TP53INP1 in mantle cell lymphoma

Objective: To explore the effect of dysregulation of epigenetic regulator EZH1 and EZH2 on the proliferation in MCL and the underlying mechanisms.Methods: In this study, we elucidated the role of EZH1 and EZH2 overexpression by immunohistochemistry and correlated them to clinical outcome in 41 MCL patients.Quantitative real-time PCR and Western blot were applied to confirm the level of EZH1 and EZH2 in well-characterized MCL cell lines which were compared to those of na?ve B cells.Then we manipulated the expression of EZH1 and EZH2 in MCL cells using CRISPR/Cas9 system to directly investigate their functional roles in MCL.We also evaluated the effect of two small molecule selective inhibitors, EPZ005687 and UNC1999, on MCL cell proliferation, cell cycle distribution and apoptosis in vitro.Finally, we performed RNA-sequencing(RNA-Seq) and Chromatin immunoprecipitation(ChIP) assay to further gain insight into the underlying molecular mechanisms.Results: We found that EZH2 protein is overexpressed in approximately half of this cohort of MCL cases.More importantly, the overexpression of EZH2 is associated with poor OS in the patients.Nevertheless, simple EZH2 depletion in vitro has little impact on the viability of MCL cells, predominantly because of the consequent up-regulation of EZH1.Consistently, UNC1999, a dual EZH1/2 inhibitor, unlike the EZH2 selective inhibitor EPZ005687, exerts a potent inhibitory effect on MCL cells.Furthermore, we discover CDKN1C and TP53 INP1 as the two important cell cycle regulators, the expression of which are repressed by EZH1/2 mediated epigenetic regulation and are restored by EZH1/2 dual inhibition.Conclusions: Our study suggests that EZH2 participates in the pathogenesis of MCL which may serve as a potential biomarker for prognosis prediction.The dual inhibition of EZH1/2 is a promising therapeutic strategy for MCL.

Trigonella foenum-graecum seed extract modulates expression of lipid metabolism-related genes in HepG2 cells

Objective:To investigate anti-dyslipidemic effects of hydroalcoholic fenugreek seed extracts,diosgenin,and 4-OH-Ile on HepG2 cell line.Methods:HepG2 cells were treated with hydroalcoholic fenugreek seed extracts,diosgenin,4-OH-Ile,and orlistat.IC20 was calculated using the MTT method.The cells were then pretreated with IC20 concentrations for 24 and 48 h.Real time PCR was employed to measure expression of liver X receptor alpha(LXR a),sterol regulatory element-binding protein-1 C(SREBP-1 C),acetyl-CoA carboxylase(ACC),fatty acid synthase(FAS),fibroblast growth factor 21(FGF21),peroxisome proliferator-activated receptor gamma(PPAR γ),and lowdensity lipoprotein receptor(LDLR).Results:The results showed that LXR α(P=0.003,P<0.001).,SREBP-1 C(P<0.001,P<0.001),ACC(P=0.002,P=0.006),and FAS(P<0.001,P<0.001)were downregulated significantly,while FGF21(P<0.001,P<0.001),PPAR γ(P=0.004,P<0.001),and LDLR(P<0.001,P<0.001)were upregulated significantly in HepG2 cells treated with the IC20 of hydroalcoholic fenugreek seed extracts,diosgenin,4-OH-Ile,and orlistat in 24 and 48 h,respectively.Conclusions:Hydroalcoholic fenugreek seed extracts,diosgenin,and 4-OH-Ile significantly modulate the expression of some important lipid metabolism related genes,which is similar to orlistat.Trigonella foenum.-graecum seed extract or its derivatives should be further investigated for their dyslipidemia effects and its complications.

Effects of Walnut on Lipid Profile as Well as the Expression of Sterol-Regulatory Element Binding Protein-1c(SREBP-1c) and Peroxisome Proliferator Activated Receptors <i>α</i>(PPAR<i>α</i>) in Diabetic Rat

Diabetes Mellitus has appeared as a universal burden. Studies have reported that mortality from Coronary Heart Disease (CHD) in diabetic patients is 2 - 4 times higher than nondiabetics. In this respect, walnut is a treatment which has beneficial effects on CHD risk factors. PPARα and SREBP-1c play an important role in the regulation of lipid metabolism. This study was aimed to evaluate the effects of walnut on lipid profile as well as SREBP-1c and PPARα protein levels in rats. Animals were randomly divided into 3 groups (n = 6);Group 1: Received chow only (control), Group 2: Diabetic rats + chow, Group 3: Diabetic rats + chow supplemented with 4% of whole walnuts. After four weeks rats were sacrificed, blood was collected;lipid profiles as well as SREBP-1c and PPARα protein levels were determined. Compared with diabetic rats walnut significantly decreased serum cholesterol (P < 0.01), LDL-c (P < 0.01), triglyceride (P < 0.001) and VLDL-c (P < 0.001) and also increased HDL-c (P < 0.05) compared with diabetic. Moreover, SREBP-1c protein level significantly decreased (P < 0.05) and PPARα significantly increased in walnut group compared with diabetic group (P < 0.05). The findings showed that walnut administration in diet clinically decreases atherosclerosis risk factors. Lipid profile reduction might be due to the rise of PPARα and the reduction of SREBP-1c by this medical treatment in liver.

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 tran-scriptional 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 expres-sion 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-Aβ1 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 expres-sion of HLA-DPA1/DPB1 in those mice.A model of S aureus-induced pneumonia in the HLA-DP401-H2-Aβ1^(-/-)humanized mice was further developed,and S aureus in-fection 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.

Vibration in mice: A review of comparative effects and use in translational research

Sound pressure waves surround individuals in everyday life and are perceived by animals and humans primarily through sound or vibration. When sound pressure waves traverse through a solid medium, vibration will result. Vibration has long been considered an unwanted variable in animal research and may confound scientific endeavors using animals. Understanding the characteristics of vibration is required to determine whether effects in animals are likely to be therapeutic or result in adverse biological effects. The eighth edition of the "Guide for the Care and Use of Laboratory Animals" highlights the importance of considering vibration and its effects on animals in the research setting, but knowledge of the level of vibration for eliciting these effects was unknown. The literature provides information regarding therapeutic use of vibration in humans, but the range of conditions to be of therapeutic benefit is varied and without clarity. Understanding the characteristics of vibration(eg, frequency and magnitude) necessary to cause various effects will ultimately assist in the evaluation of this environmental factor and its role on a number of potential therapeutic regimens for use in humans. This paper will review the principles of vibration, sources within a research setting, comparative physiological effects in various species, and the relative potential use of vibration in the mouse as a translational research model.

A functional study of low molecular weight IgM from patients with autoimmune disease

High levels of low molecular weight (LMW) IgM in certain diseases are associated with clinical and laboratory indices which reflect the severity of the disease. These associations suggest that LMW IgM may play an important role in the immunopathogenesis of these diseases. To further approach the question concerning the functional activity of LMW IgM in disease, a panel of LMW IgM and high molecular weight (HMW) IgM preparations with or without rheumatoid factor (RF) activity were used to investigate their antibody binding activity and their effector function. It was found that LMW IgM-RF and HMW IgM-RF had a similar binding capacity to Fc fragment as there was no significant difference in the affinity index between them. It further showed that the rate of activation and total amount of utilization of complement by LMW IgM and HMW IgM was similar, although the mean fluorescence of C3 deposition by IgM-RF and HMW IgM-RF was slightly higher than that of LMW IgM-RF and other control RF antibodies. However, the current study demonstrated that LMW IgM had strong neutrophil activating properties when compared with HMW IgM. These findings suggest that one mechanism of LMW IgM contributing to the immunopathogenesis of RA may be due to the formation of circulating immune complex ( CIC) by LMW IgM with subsequent activation of neutrophils. Whether LMW IgM has other functional activity in disease is unclear and needs further investigation.

Flavonoid-rich beverage effects on lipid profile and blood pressure in diabetic patients

AIM: To compare freeze-dried strawberry(FDS) beverage and strawberry-flavored drink effects on lipid profile and blood pressure in type 2 diabetic(T2D) patients.METHODS: In a randomized, double-blind, controlled trial, 36 subjects with T2D(23 females; mean ± SE age: 51.57 ± 10 years) were randomly divided into two groups. Participants consumed two cups of either pure FDS beverage(each cup containing 25 g freeze-dried strawberry powder equivalent to one serving of fresh strawberries; intervention group) or an iso-caloric drinkwith strawberry flavoring(similar to the FDS drink in fiber content and color; placebo group) daily for 6 wk. Anthropometric measurements, 3 d, 24 h dietary recall, and fasting blood samples were collected at baseline and at weeks 6 intervention. After lying down and relaxing for approximately 10 min, each participant's blood pressure was recorded in triplicate with 5 min intervals; recordings were made at baseline and the trial end-point. Each participant's lipid profile was assessed before and after intervention.RESULTS: Assessment at the weeks 6 intervention showed a significant reduction from baseline in total cholesterol levels and total cholesterol to high-density lipoprotein cholesterol(HDL-C) ratio in the intervention group(179.01 ± 31.86 to 165.9 ± 32.4 mg/L; P = 0.00 and 3.9 ± 0.88 to 3.6 ± 0.082 mg/L; P = 0.00 respectively), but the change was not significantly different between the two groups(P = 0.07, P = 0.29 respectively). Systolic blood pressure levels were significantly reduced from baseline in both the FDS and placebo drink groups(129.95 ± 14.9 to 114.3 ± 27.5 mm Hg; P = 0.02 and 127.6 ± 15.6 to 122.9 ± 14.47 mm Hg; P = 0.00 respectively), but the reduction was not significantly different between the two groups. Diastolic blood pressure was significantly reduced post-intervention in the FDS drink group compared to placebo group(78.7 ± 7.2 vs 84.4 ± 5.8; P = 0.01), the reduction was also significant within the FDS drink group(84.2 ± 8.03 to 78.7 ± 7.2; P = 0.00). Triglycerides, HDL-C concentrations and anthropometric indices showed no significant differences between or within groups. CONCLUSION: Short-term FDS supplementation improved selected cardiovascular risk factors in subjects with T2 D. Long-term effects on other metabolic biomarkers need to be investigated in future trials.

Antibacterial potential of fresh fruit juices against multi-drug resistant pathogens

Objective: To explore the antibacterial activity of fresh fruit juices against drug-resistant pathogens. Methods: Fresh juices were prepared by squeezing 7 fruits [Citrus sinensis, Citrus aurantifolia (C. aurantifolia), Punica granatum (P. granatum), Malus domestica, Ananas comosus, Fragaria ananasia, and Actinidia deliciosa]. The antibacterial activities were studied by using well-diffusion, minimum inhibitory (MIC), and minimum bactericidal (MBC) assays against 7 clinical pathogens: Gram-positive, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Staphylococcus epidermidis;Gram-negative, 2 strains of Pseudomonas aeruginosa, 2 strains of Klebsiella pneumoniae. Results: The diffusion test revealed that Malus domestica juice had no antibacterial activity against the tested pathogens;Citrus sinensis and Ananas comosus revealed low antibacterial activity;Fragaria ananasia and Actinidia deliciosa revealed moderate antibacterial activity;P. granatum and C. aurantifolia exhibited high antibacterial activity against most of the clinical strains. MIC and MBC tests exhibited that P. granatum and C. aurantifolia had noticeable bactericidal effects with MBC/MIC values ranging between 2 to 4. Conclusions: The crude fresh juices of P. gronatum and C. aurantifolia have potential as natural therapeutic agents against some multi-drug resistant bacteria and they could prevent pathogenic diseases.

Prevalence and genotype distribution of Enterobius vermicularis among kindergarteners in Shiraz and Khorramabad cities, Iran

Objective: To study the prevalence and genotype of Enterobius(E.)vermicularis from adhesive tape samples in the cities of Shiraz and Khorramabad, Iran. Methods: A total of 1 000 adhesive tape samples from kindergartens in Shiraz(500 samples) and Khorramabad(500 samples) were collected and tested using a microscope to find E. vermicularis egg/s. A questionnaire was filled out for each sample. In order to characterize the genotype of E. vermicularis, the PCR-sequencing method of the mitochondrial cytochrome C oxidase subunit 1(cox1) gene was used. Genomic DNA was extracted from the positive scotch tape samples of E. vermicularis. The cox1 gene was amplified by the polymerase chain reaction and sequenced. The sequence data were aligned using the BioEdit software and compared with the published sequences in GenBank. Phylogenetic analysis was performed using the maximum likelihood method. Results: The parasitological method showed that 15 out of the 500 samples from Shiraz(3.00%) and 12 out of the 500 samples from Khorramabad(2.40%) were infected with E. vermicularis eggs. BLAST analysis indicated that the sequenced isolates belonged to E. vermicularis genotype B while three different haplotypes were also identified. Conclusions: This is the first study on genotyping E. vermicularis in the cities of Shiraz and Khorramabad. Considering the public health importance of the disease, further studies are necessary to characterize the genotype of E. vermicularis in human populations from other regions of Iran.