Up-regulation of TLR2 and TLR4 in dendritic cells in response to HIV type 1 and coinfection with opportunistic pathogens

The ability to trigger an innate immune response against opportunistic pathogens associated with HIV-1 infection is an important aspect of AIDS pathogenesis. Toll-like receptors (TLRs) play a critical role in innate immunity against pathogens, but in HIV-1 patients coinfected with opportunistic infections, the regulation of TLR expression has not been studied. In this context, we have evaluated the expression of TLR2 and TLR4 in monocytes, plasmacytoid dendritic cells, and myeloid dendritic cells of HIV-1 patients with or without opportunistic infections. Forty-nine HIV-1-infected individuals were classified according to viral load, highly active antiretroviral therapy (HAART), and the presence or absence of opportunistic infections, and 21 healthy subjects served as controls. Increased expression of TLR2 and TLR4 was observed in myeloid dendritic cells of HIV-1 patients coinfected with opportunistic infections (without HAART), while TLR4 increased in plasmacytoid dendritic cells, compared to both HIV-1 without opportunistic infections and healthy subjects. Moreover, TLR2 expression was higher in patients with opportunistic infections without HAART and up-regulation of TLR expression in HIV-1 patients coinfected with opportunistic infections was more pronounced in dendritic cells derived from individuals coinfected with Mycobacterium tuberculosis. The results indicate that TLR expression in innate immune cells is up-regulated in patients with a high HIV-1 load and coinfected with opportunistic pathogens. We suggest that modulation of TLRs expression represents a mechanism that promotes HIV-1 replication and AIDS pathogenesis in patients coinfected with opportunistic pathogens.     

TLR2、TLR4在肝细胞EMT现象中的表达

目的 TLR2与TLR4在大鼠肝细胞EMT现象中的表达。方法常规分离大鼠肝细胞后,以TGF-β刺激,诱导肝细胞上皮-间质转化,RT-PCR检测TLR2与TLR4在其中的表达变化。结果 TGF-β刺激细胞后细胞的形态发生了变化,RT-PCR结果显示TGF-β刺激的细胞E-cadherin mRNA表达下降(P0.15),vimentin mRNA表达上升(P0.05)。TLR2mRNA表达升高(P0.05),TLR4mRNA表达变化差异无统计学意义。TLR2mRNA与vimentin mRNA相对表达量呈线性正相关(r=0.804,P0.05),与E-cadherin mRNA相对表达量呈线性负相关(r=-0.879,P0.05)。结论肝细胞EMT现象中TLR2的表达升高,与肝细胞EMT标记物的表达有一定的相关性,提示TLR2可能促进肝细胞的EMT过程。     

TLR4激动上调内皮细胞氧化低密度脂蛋白受体LOX-1表达

目的近年研究表明介导先天免疫反应的受体Toll样受体4(TLR4)参与了动脉硬化的发生发展.业已证明氧化低密度脂蛋白受体LOX-1介导内皮细胞活化和功能失调,激发炎症过程,在动脉粥样硬化的发生和发展中起着极为重要作用.本研究观察TLR4激动是否调节内皮细胞LOX-1表达.方法应用脂多糖(LPS)刺激体外培养的人脐静脉内皮细胞(HUVECs)24 h.采用RT-PCR和流式细胞术分别检测TLR4、LOX-1 mRNA和蛋白表达水平.为了观察转录因子NF-κB在调节LOX-1表达中的作用,应用NF-κB特异性抑制剂咖啡酸苯乙酯(CAPE)预处理细胞,然后以LPS刺激,检测LOX-1 mRNA和蛋白变化.结果LPS(10～1 000 ng/mL)上调HUVECs TLR4和LOX-1 mRNA表达,LPS(1 000ng/mL)上调TLR4和LOX-1蛋白表达,CAPE(20μg/mL)可抑制LPS介导的LOX-1表达上调.结论TLR4/NF-κB信号途径可能通过上调内皮细胞LOX-1表达参与动脉粥样硬化的发生及发展.     

TLR4激动上调内皮细胞氧化低密度脂蛋白受体LOX-1表达

目的近年研究表明介导先天免疫反应的受体Toll样受体4(TLR4)参与了动脉硬化的发生发展。业已证明氧化低密度脂蛋白受体LOX-1介导内皮细胞活化和功能失调，激发炎症过程，在动脉粥样硬化的发生和发展中起着极为重要作用。本研究观察TLR4激动是否调节内皮细胞LOX-1表达。方法应用脂多糖(LPS)刺激体外培养的人脐静脉内皮细胞(HUVECs)24h。采用RT—PCR和流式细胞术分别检测TLR4、LOX-1 mRNA和蛋白表达水平。为了观察转录因子NF—kB在调节LOX-1表达中的作用，应用NF-kB特异性抑制剂咖啡酸苯乙酯(CAPE)预处理细胞，然后以LPS刺激，检测LOX-1 mRNA和蛋白变化。结果LPS(10～1000ng／mL)上调HUVECs TLR4和LOX-1 mRNA表达，LPS(1000ng／mL)上调TLR4和LOX-1蛋白表达，CAPE(20μg／mL)可抑制LPS介导的LOX-1表达上调。结论TLR4／NF-kB信号途径可能通过上调内皮细胞LOX-1表达参与动脉粥样硬化的发生及发展。     

TLR2、TLR4和TLR9在慢性重型肝炎患者及肝衰竭大鼠中的表达

目的:观察重型肝炎患者及肝衰竭大鼠中TLR2、TLR4和TLR9的表达,为重型肝炎肝衰竭发病机制的研究提供思路.方法:免疫组织化学法检测慢性重型肝炎患者肝组织及外周血单个核细胞(PBMC)中TLR2、TLR4和TLR9表达,同时平行设立慢性乙型肝炎、肝硬化患者和正常人作为对照.免疫组织化学法检测急性肝衰竭、慢加急性肝衰竭及正常大鼠肝组织中TLR2、TLR4和TLR9表达.阳性对照为大鼠结肠组织.结果:正常人、慢性乙型肝炎和肝硬化患者肝组织中TLR2和TLR9未见表达;慢性重型肝炎患者肝组织中TLR2和TLR9有表达;正常人及慢性乙型肝炎患者PBMC中TLR2、TLR9未见表达,慢性重型肝炎患者PBMC中TLR2和TLR9有表达;正常大鼠肝组织TLR2、TLR9未见表达,急性肝衰竭和慢加急性肝衰竭大鼠肝组织中TLR2、TLR9有表达;TLR4在所有标本中均未见表达,但在阳性对照中有表达;TLR2、TLR9的表达多见于炎性细胞中,在肝实质细胞上表达很少.结论:TLR2、TLR9的高表达与机体的免疫状态密切相关,可能参与了重型肝炎和肝衰竭的发病过程.     

The Temporal Expression of T2r+ Bacteriophage Genes In Vivo and In Vitro

The kinetic order of synthesis of deoxycytidylate deaminase (EC 3.5.4.12), deoxycytidylate hydroxymethylase (EC 2.1.2.b), dihydrofolate reductase (EC 1.5.1.3), 5-hydroxymethyldeoxycytidylate kinase (EC 2.7.4.4), and thymidylate synthetase (EC 2.1.1.b) after infection of Escherichia coli with T2r(+) bacteriophage was found not to correlate with their order of synthesis in an in vitro protein-synthesizing preparation. The in vivo and in vitro synthesis of enzyme-specific messenger RNA measured in the protein-synthesizing preparation preceded each enzyme by about 1 min. Through the use of sheared DNA, it was shown that the thymidylate synthetase gene was most susceptible to a loss in template activity, which suggests that this gene is further removed from its promoter than the other genes are from theirs. With a DNA segment of 2.5 x 10(5) daltons, the synthesis of dihydrofolate reductase alone was obtained, but at a much reduced rate. Translation of the RNA from phage-infected cells treated with chloramphenicol yielded amounts of dihydrofolate reductase and deoxycytidylate hydroxymethylase activities similar to those obtained with RNA from untreated infected cells. These results suggest that the chloramphenicol RNA, which consists primarily of immediate-early RNA, may contain most, if not all, of the information required for the synthesis of phage dihydrofolate reductase and deoxycytidylate hydroxymethylase.     

Nafamostat–Interferon-α Combination Suppresses SARS-CoV-2 Infection In Vitro and In Vivo by Cooperatively Targeting Host TMPRSS2

SARS-CoV-2 and its vaccine/immune-escaping variants continue to pose a serious threat to public health due to a paucity of effective, rapidly deployable, and widely available treatments. Here, we address these challenges by combining Pegasys (IFNα) and nafamostat to effectively suppress SARS-CoV-2 infection in cell culture and hamsters. Our results indicate that Serpin E1 is an important mediator of the antiviral activity of IFNα and that both Serpin E1 and nafamostat can target the same cellular factor TMPRSS2, which plays a critical role in viral replication. The low doses of the drugs in combination may have several clinical advantages, including fewer adverse events and improved patient outcome. Thus, our study may provide a proactive solution for the ongoing pandemic and potential future coronavirus outbreaks, which is still urgently required in many parts of the world.     

Blood-Induced Interference of Glucose Sensor Function in Vitro: Implications for in Vivo Sensor Function

Background

Although tissue hemorrhages, with resulting blood clots, are associated with glucose sensor implantation, virtually nothing known is about the impact of red blood cells and red blood cell clots on sensor function in vitro or in vivo. In these studies, we tested the hypothesis that blood can directly interfere with glucose sensor function in vitro.

Methods

To test this hypothesis, heparinized human whole blood (HWB) and nonheparinized human whole blood (WB) were obtained from normal individuals. Aliquots of HWB and WB samples were also fractionated into plasma, serum, and total leukocyte (TL) components. Resulting HWB, WB, and WB components were incubated in vitro with an amperometric glucose sensor for 24 hours at 37°C. During incubation, blood glucose levels were determined periodically using a glucose monitor, and glucose sensor function (GSF) was monitored continuously as nanoampere output.

Results

Heparinized human whole blood had no significant effect on GSF in vitro, nor did TL, serum, or plasmaderived clots from WB. Sensors incubated with WB displayed a rapid signal loss associated with clot formation at 37°C. The half-life was 0.8 ± 0.2 hours (n = 16) for sensors incubated with WB compared to 3.2 ± 0.5 (n = 12) for sensors incubated with HWB with a blood glucose level of approximately 100 mg/dl.

Conclusions

These studies demonstrated that human whole blood interfered with GSF in vitro. These studies further demonstrated that this interference was related to blood clot formation, as HWB, serum, plasma-derived clots, or TL did not interfere with GSF in vitro in the same way that WB did. These in vitro studies supported the concept that the formation of blood clots at sites of glucose sensor implantation could have a negative impact on GSF in vivo.     

HIV type 1 antigen-responsive CD4+ T-lymphocytes in exposed yet HIV Type 1 seronegative Ugandans

CD4(+) T cell help is important for the functionality of CD8(+) cytotoxic T-lymphocytes (CTLs) in limiting viral replication and may contribute to mediation of apparent resistance to HIV-1 infection in exposed seronegative (ESN) individuals. Using five HIV-1 antigens in an intracellular cytokine assay, the presence of specific antigen-responsive interferon- gamma-positive (IFN-gamma(+)) CD69(+) CD4(+) T-lymphocytes was evaluated in ESNs, their seropositive partners, and unexposed seronegative controls. Ten ESNs (five females, five uncircumcised males) were identified from 10 HIV-1 serodiscordant couples with a history of frequent unprotected sexual intercourse. All ESNs and controls were negative on two EIAs and for HIV-1 proviral DNA. The frequency of ESNs with antigen-responsive IFN-gamma(+) CD69(+) CD4(+) T-lymphocytes ranged from three to five of eight for the different HIV-1 antigens. Six of eight ESNs tested had a positive response to at least one of the five antigens. Responses were on average 3.5 times higher among seropositives compared to ESNs and absent in the five unexposed controls. A negative correlation was noted between responses in ESNs and the plasma viral load of their seropositive spouse. Clade-specific and cross-clade reactivity were noted in both ESNs and seropositive partners tested. The findings confirm that ESNs are in a state of HIV-1-specific immune activation and suggest that HIV-1-specific IFN-gamma(+) CD69(+) CD4(+) T-lymphocytes in addition to HIV-1-specific CD8(+) CTLs already described by others are potential immunological correlates of protection from persistent HIV-1 infection.     

The Effect of Prostaglandin E1 on Platelet Function in Vitro and in Vivo

S ummary Low concentrations of prostaglandin E₁ (PGE₁) inhibit ADP-induced aggregation in pig and rabbit citrated platelet-rich plasma and in suspensions of washed platelets. Higher concentrations also inhibit the initial change in shape induced by ADP and the release of platelet ATP, ADP and serotonin caused by stimuli such as collagen, thrombin, antigen-antibody complexes and gamma-globulin-coated polystyrene particles. PGE₁ is not taken up by platelets and its effects can be removed by resuspending platelets in fresh medium. Immediately following an intra-arterial injection, ADP-induced platelet aggregation is suppressed, but after 5 min the response returns to normal. PGE₁ inhibits haemostasis in rabbits when given as a continuous infusion. It is concluded that the effect of PGE₁ on haemostasis involves inhibition of the release of ADP from platelets exposed to collagen and thrombin, and inhibition of ADP-induced aggregation.     

Toll样受体(TLR)2、TLR4和TLR9在大鼠结肠炎模型结肠组织中的表达及其意义

Toll样受体（TLRs）家族可能在一系列免疫性疾病中发挥重要作用。目的：观察TLR2、TLR4和TLR9在大鼠结肠炎模型结肠组织中的表达，探讨三者在炎症性肠病（IBD）发病机制中的作用。方法：以三硝基苯磺酸（TNBS）＋乙醇灌肠制备大鼠结肠炎模型，观察和评估结肠黏膜的大体和组织学变化。分别以黄嘌呤氧化酶法和紫外分光光度法测定超氧化物歧化酶（SOD）和髓过氧化物酶（MPO）活性；以免疫组化方法检测TLR2、TLR4和TLR9的表达，以逆转录聚合酶链反应（RT-PCR）检测三者mRNA的表达。结果：造模后结肠组织中可见大量炎性细胞浸润，累及黏膜下层和固有层。与正常对照组相比，模型组结肠组织SOD活性显著降低（P〈0．01），MPO活性显著升高（P〈0．01）。正常对照组结肠黏膜下层和固有层炎性细胞胞膜和胞质仅有少量TLR2、TLR4表达，未见TLR9表达；模型组三者表达均显著增加（P〈0．01），此外还可见TLR2表达于肠上皮近肠腔侧胞膜，TLR4表达于肠上皮近肠腔侧胞膜和腺上皮近腺腔侧胞膜。模型组结肠组织可见TLR2、TLR4、TLR9 mRNA表达，而正常对照组未检出三者mRNA的表达。TLR2、TLR4、TLR9的表达与MPO活性呈正相关（P〈0．05），与SOD活性呈负相关（P〈0．01）。结论：大鼠结肠炎模型结肠组织中TLR2、TLR4和TLR9表达明显增加，可能与结肠的自身免疫损伤有关。     

Toll样受体和核苷酸结合寡聚蛋白2与克罗恩病

Toll样受体(TLR)和核苷酸结合寡聚蛋白2(NOD2)在结构上都富含亮氨酸重复序列(LRR),并在肠道黏膜的多种细胞表达,介导微生物与肠道黏膜之间的固有免疫反应。近年来研究发现,TLR和NOD2介导的免疫反应紊乱对克罗恩病的发生起重要作用。     

3-Indoleacetonitrile Is Highly Effective in Treating Influenza A Virus Infection In Vitro and In Vivo

Influenza A viruses are serious zoonotic pathogens that continuously cause pandemics in several animal hosts, including birds, pigs, and humans. Indole derivatives containing an indole core framework have been extensively studied and developed to prevent and/or treat viral infection. This study evaluated the anti-influenza activity of several indole derivatives, including 3-indoleacetonitrile, indole-3-carboxaldehyde, 3-carboxyindole, and gramine, in A549 and MDCK cells. Among these compounds, 3-indoleacetonitrile exerts profound antiviral activity against a broad spectrum of influenza A viruses, as tested in A549 cells. Importantly, in a mouse model, 3-indoleacetonitrile with a non-toxic concentration of 20 mg/kg effectively reduced the mortality and weight loss, diminished lung virus titers, and alleviated lung lesions of mice lethally challenged with A/duck/Hubei/WH18/2015 H5N6 and A/Puerto Rico/8/1934 H1N1 influenza A viruses. The antiviral properties enable the potential use of 3-indoleacetonitrile for the treatment of IAV infection.