安徽省流行性出血热病毒杂交瘤单克隆抗体的研制与应用

用流行性出血热病毒(EHFV)李株和陈株血凝素(HAN)分别免疫Balb/C小鼠,取其脾细胞和骨髓瘤细胞SP2/O融合,杂交瘤生长孔率为97.5％和60％,阳性率各为2.5％和22％,克隆化培养阳性率达到100％。通过筛选得2株杂交瘤细胞、一株为2B_7、另一株为1H_8。其腹水单克隆抗体(McAb)滴度均达1∶16×10~4—32×10~4。1H_8的血凝抑制滴度达1∶5120。经染色体核型分析,杂交细胞染色体2B_7为80—97条,1H_8为85—105条,每个细胞均含一条中着丝点标记染色体。McAb的Ig类别和IgG亚类检测结果2B_7为IgM,1H_8为IgG_(2b)。两者对19株不同来源的EHFV具有不同的反应,1H_8McAb与19株都具有不同程度的免疫荧光反应,滴度都较高,且对5株不同来源的EHFV-HAN具有血凝抑制活性,滴度在1∶1280—5120,故1H_8为血凝抑制抗体;2B_7只对12株EHFV起反应,对7株不起反应,且没有血凝抑制活性。这说明1H_8与2B_7具有不同的特性。     

Construction of an infectious cDNA clone of Tembusu virus isolated from breeder Peking ducks

正Dear Editor,Since April 2010,an outbreak of a new disease has elicited symptoms of high fever,loss of appetite,and reduction in egg production in layer ducks in eastern China;this phenomenon has now spread throughout China(Cao et al.,2011;Su et al.,2011).The causative agent of the disease was identified as Tembusu virus(TMUV),which was classified into the genus Flavivirus,     

Febrile response to infection in the American alligator (Alligator mississippiensis)

Temperature probes were inserted into the stomachs of juvenile American alligators (Alligator mississippiensis) maintained outdoors at ambient fluctuating temperatures. Internal body temperatures (T_b) were measured every 15 min for two days, and then the alligators were injected with bacterial lipopolysaccharide (LPS), pyrogen-free saline, or left untreated. Alligators injected intraperitoneally with LPS exhibited maximum T_bs 2.6 ± 1.1 °C and 3.5 ± 1.2 °C higher than untreated control animals on days one and two after treatment, respectively. T_bs for these animals fell to within control ranges by day three postinjection. Similarly, mean preferred body temperatures (MPBTs) were significantly higher for LPS-injected alligators on days one (4.2 ± 1.8 °C) and two (3.5 ± 1.6 °C) after treatment. Intraperitoneal injection of heat-killed Aeromonas hydrophila, a gram-negative bacterium known to infect crocodilians, resulted in a fever while injection of Staphylococcus aureus (gram positive) did not elicit a febrile response. Injection of LPS in alligators maintained indoors in a constant temperature environment resulted in no increase in internal T_b. These results indicate that alligators did not exhibit a febrile response in the absence of a thermal gradient, and suggest that febrile responses observed are probably behavioral in nature.     

Fever range temperature promotes TLR4 expression and signaling in dendritic cells

Fever improves survival and shortens disease duration in microbial infections. However, the mechanisms of these beneficial responses still remain elusive. Toll-like receptors (TLRs) play important roles in sensing microbes invading and therefore we hypothesized that fever range temperature may enhance responsiveness of dendritic cells (DCs) to lipopolysaccharide (LPS) by promoting TLR4 expression and signaling. In this study, we found that pretreatment of DCs with 39.5 degrees C temperature can up-regulate TLR4 expression in DCs and enhances LPS-induced DC production of interleukins (IL) IL-6, IL-10 and IL-12 but not tumor necrosis factor alpha (TNF-alpha). Blockade of the autocrine action of IL-10 could increase LPS-induced TNF-alpha and IL-12 production in DCs. Further experiments confirmed that TLR4 ligation activates extracellular signal-regulated kinase (ERK), p38, and nuclear factor-kappaB pathways more potently in DCs pretreated with 39.5 degrees C. We conclude that fever range temperature can promote TLR4 expression and signaling in DCs, leading to enhancement of immune responses to inflammatory stimuli. These results might reveal a possible mechanistic explanation for the significance of fever in activating innate immune responses.     

Induction of caspase activation and cleavage of the viral nucleocapsid protein in different cell types during Crimean-Congo hemorrhagic fever virus infection

Regulation of apoptosis during infection has been observed for several viral pathogens. Programmed cell death and regulation of apoptosis in response to a viral infection are important factors for host or virus survival. It is not known whether Crimean-Congo hemorrhagic fever virus (CCHFV) infection regulates the apoptosis process in vitro. This study for the first time suggests that CCHFV induces apoptosis, which may be dependent on caspase-3 activation. This study also shows that the coding sequence of the S segment of CCHFV contains a proteolytic cleavage site, DEVD, which is conserved in all CCHFV strains. By using different recombinant expression systems and site-directed mutagenesis, we demonstrated that this motif is subject to caspase cleavage. We also demonstrate that CCHFV nucleocapsid protein (NP) is cleaved into a 30-kDa fragment at the same time as caspase activity is induced during infection. Using caspase inhibitors and cells lacking caspase-3, we clearly demonstrate that the cleavage of NP is caspase-3-dependent. We also show that the inhibition of apoptosis induced progeny viral titers of ～80-90%. Thus, caspase-3-dependent cleavage of NP may represent a host defense mechanism against lytic CCHFV infection. Taken together, these data suggest that the most abundant protein of CCHFV, which has several essential functions such as protection of viral RNA and participation in various processes in the replication cycle, can be subjected to cleavage by host cell caspases.     

The immune system as key to cancer treatment: Triggering its activity with microbial agents

Traditional methods such as chemotherapy and radiation therapy offer only limited success in treating cancer. Part of the reason is related to our misunderstanding of what cancer is: it is not the cause but the consequence of a weakened living system. Localized cellular stress, caused by toxins, mutagens or radiation, coupled with a weakened systemic response or inability to support or defend the cells that are under attack, cause these cells to revert to an ancient, unicellular mode of survival, therefore cutting links with the overarching organism and defend themselves from the threat as if they were individual entities. We hypothesize that strengthening the organism, specifically the immune system, is a more promising approach toward a cure for cancer than attempting to exterminate cancer cells. The hypothesis can be tested by experiments that are designed to strengthen the immune system by both traditional means (e.g., ingestion of natural substances known to increase the activity of the immune system, such as fruits, vegetables, and nuts), diminish immune system inhibitors released by cancer cells (e.g., TGF-β), and by the injection of heat-killed or genetically altered pathogenic bacteria to trigger a massive response (fever response) of the immune system into the affected area and compare those results to traditionally used methods.     

HFRS患者特异性IgA,IgE抗体及其免疫复合物测定

为进一步研究ＨＦＲＳ免疫损伤机制,用ＥＬＩＳＡ法同步测定了１０８例不同临床型、不同病日、病期ＨＦＲＳ患者血清中特异性ＩｇＡ、ＩｇＥ抗体以及ＨＦＲＳ病毒特异性ＩｇＡ、ＩｇＥ型ＣＩＣ的水平及检出率。发现ＨＦＲＳＩｇＡ型抗体在轻型病例高于中、重型病例;ＨＦＲＳＩｇＥ型抗体及ＩｇＥ型ＣＩＣ在重型病例高于中、轻型病例。上述差异在病程早期（发热、休克少尿期,或是３～８病日）尤为突出。ＩｇＡ型ＣＩＣ则未见到上述差异。     

Peripheral TNFalpha, but not peripheral IL-1, requires endogenous IL-1 or TNFalpha induction in the brain for the febrile response

It is known that peripherally administered IL-1 and TNFα induce fever through mechanisms involving prostaglandin (PG)E₂. In this report, we compared the signaling cascade induced in the brain by TNFα and IL-1. Peripheral administration of TNFα-induced enhanced fever in IL-1 Receptor antagonist KO mice, suggesting that IL-1 is involved in the TNFα mediated fever. IL-1α, but not TNFα, induced fever in IL-1α/β/TNFα KO mice, although central administration of TNFα-induced fever. Only IL-1α, but not TNFα, induced IL-6 in the IL-1α/β/TNFα KO mouse brain, while both cytokines induced cyclooxygenase (Cox)-2. Icv administration of PGE₂ induced only transient fever in contrast to the TNFα- or IL-1α-induced fever that lasted longer. Taken together, either IL-1 or TNFα induction in the brain is required for the response induced by TNFα but not by IL-1α, and that both Cox-2 and IL-6 induction are required for prolonged febrile response against these cytokines.