Detection of DNA damage in oocytes of small ovarian follicles following phosphoramide mustard exposures of cultured rodent ovaries in vitro

Healthy oocytes are critical for producing healthy children, but little is known about whether or not oocytes have the capacity to identify and recover from injury. Using a model ovotoxic alkylating drug, cyclophosphamide (CPA), and its active metabolite, phosphoramide mustard (PM), we previously showed that PM (≥ 3 μM) caused significant follicle loss in postnatal day 4 (PND4) mouse ovaries in vitro. We now investigate whether PM induces DNA damage in oocytes, examining histone H2AX phosphorylation (γH2AX), a marker of DNA double-strand breaks (DSBs). Exposure of cultured PND4 mouse ovaries to 3 and 0.1 μM PM induced significant losses of primordial and small primary follicles, respectively. PM-induced γH2AX was observed predominantly in oocytes, in which foci of γH2AX staining increased in a concentration-dependent manner and peaked 18-24 h after exposure to 3-10 μM PM. Numbers of oocytes with ≥ 5 γH2AX foci were significantly increased both 1 and 8 days after exposure to ≥ 1 μM PM compared to controls. Inhibiting the kinases that phosphorylate H2AX significantly increased follicle loss relative to PM alone. In adult mice, CPA also induced follicle loss in vivo. PM also significantly decreased primordial follicle numbers (≥ 30 μM) and increased γH2AX foci (≥ 3 μM) in cultured PND4 Sprague-Dawley rat ovaries. Results suggest oocytes can detect PM-induced damage at or below concentrations which cause significant follicle loss, and there are quantitative species-specific differences in sensitivity. Surviving oocytes with DNA damage may represent an increased risk for fertility problems or unhealthy offspring.     

The coxsackie-adenovirus receptor induces an inflammatory cardiomyopathy independent of viral infection

The coxsackie-adenovirus receptor (CAR) is a viral receptor for Group B coxsackieviruses (CVBs) and adenoviruses. CAR has been linked with the innate immune response to CVB myocarditis, and with activation of inflammatory cells in vitro. We hypothesized that CAR activates signals that promote inflammation in the myocardium independent of viral infection. To test this we conditionally overexpressed murine CAR in cardiomyocytes of adult binary transgenic mice under the control of a tetracycline-responsive (tet-off) α-myosin heavy chain (αMtTA) promoter (mCAR⁺/αMtTA⁺ mice). An inflammatory cardiomyopathy developed in both lines generated (6-mCAR⁺/αMtTA⁺ and 12-mCAR⁺/αMtTA⁺) following withdrawal of doxycycline. Cardiac CAR was upregulated at 4 weeks of age in 6-mCAR⁺/αMtTA⁺ mice and induced a mild inflammatory infiltrate (score 1.3 of 4.0 ± 0.3) at 6 weeks, with 95% of mice surviving to that time. In the second line, 12-mCAR⁺/αMtTA⁺ mice, CAR was upregulated in the majority of mice by 3 weeks of age, and by 5 weeks of age more severe cardiac inflammation (score 2.8 of 4.0 ± 0.4) developed with only 56% of mice surviving. The cardiac inflammatory infiltrate was primarily natural killer cells and macrophages in both mCAR⁺/αMtTA⁺ lines. A proinflammatory cytokine response with increased cardiac interferon-γ, interleukin (IL)-12, IL-1β, tumor necrosis factor-α and IL-6 was detected by real-time RT-PCR. CAR has been linked to signaling via the inflammatory mitogen-activated protein kinase (MAPK) cascades; therefore, we evaluated the response of these pathways in hearts with upregulated CAR. Both stress-activated JNK and p38MAPK were activated in mCAR⁺/αMtTA⁺ hearts prior to onset of inflammation and in isolated mCAR⁺/αMtTA⁺ cardiomyocytes. In conclusion, we show for the first time that CAR upregulation in the adult mouse heart induces cardiac inflammation reminiscent of early viral myocarditis. CAR-induced stress-activated MAPK signaling may contribute to the development of cardiac inflammation unrelated to viral infection per se.     

第四代HIV1/2酶联荧光免疫试验在性病门诊筛查中的应用及评价

目的探讨新的第四代可联合检测P24抗原和抗体的酶联荧光免疫试验(ELFA)技术,在性病门诊中的检测情况,评价该技术在临床上的应用价值。方法对8 495份初次筛查HIV-1/2抗体的检测者血清,采用ELFA和硒标法(金标法,DIGFA)同时进行检测,对其中任何一种方法结果为阳性的血清,送北京市疾病预防控制中心(CDC)做蛋白印迹实验(WB)确证实验。结果 79例ELFA法和DIGFA法均为阳性样本送经WB确认,结果亦为阳性;3例ELFA法检测结果阳性而DIGFA检测阴性的标本,经WB确证结果为2例不确定,1例阴性排除HIV感染。对其中2例不确定者进行定期随访,2个月后1例标本经WB确认为阳性,3月后另1例WB亦转为阳性。5例ELFA法为阴性DIGFA法为阳性的标本,经WB确认结果为4例阴性,1例不确定。该不确定者1月后随访,经WB检测而排除。结论 ELFA法因能尽早检测到刚出现的P24抗原,可以得到独立的精确的抗原抗体数值,特异性及准确性高,可将普通检测方法的窗口期缩短,可广泛应用于早期检测。     

Identification of recombinant antibodies against multiple distinct toll-like receptors by homolog mining a single immune scFv phage library

The generation of recombinant single-chain antibodies from either non-immune or immune phage display antibody libraries is an effective means to obtain high affinity antibodies against a specific target. Non-immune libraries contain a wide variety of antibodies but these are often low affinity. Immune libraries contain a high frequency of high affinity antibodies, but are typically limited to a single antigen. Due to the V_H and V_L recombination that occurs during antibody library construction, we hypothesized that an immune antibody library produced against one member of a protein family would contain antibodies specific for other members of the same protein family. Here, we tested this hypothesis by mining an existing anti-human Toll-like receptor-2 (hTLR2) antibody library for antibodies specific for other members of the TLR family. This procedure, referred to as homolog mining, proved to be effective. Using a cell-based system to pan and screen the anti-TLR2 library, we identified single chain antibodies specific for three of the four hTLR2 homologs we targeted. The antibodies identified, anti-murine TLR2, anti-hTLR5, and anti-hTLR6, bind specifically to their target, with no cross-reactivity to hTLR2 or other TLRs tested. These results demonstrate that combinatorial re-assortment of V_H and V_L fragments from multiple sources during Ab library construction increases Ab repertoire complexity, allowing antibody libraries produced by immunization with one antigen to be used to obtain antibodies specific to related antigens. The principle of homolog mining may be extended to other protein families and will facilitate and accelerate antibody production processes.     

Improved T cell assay for identification of type 1 diabetes patients

Diabetes mellitus is comprised primarily of two clinically separate diseases: type 1 (T1D) and type 2 diabetes (T2D). T1D is a cell-mediated autoimmune disease directed against the beta cells and characterized by autoantibody (Ab) and T cell reactivity to islet proteins whereas, T2D is non-autoimmune. Despite the fact that the pathological process in autoimmune diabetes involves T cells, immune markers of diabetes have primarily centered on the presence of circulating serum islet autoantibodies. In two masked NIH sponsored workshops, our cellular immunoblotting T cell assay, which uses isolated human islets separated into 18 molecular weight fractions, has been validated to be able to distinguish T1D patients from controls with excellent specificity and sensitivity. In this study, we utilized the first workshop to select eight molecular weight fractions of human islets that were the most discriminatory between T1D patients and controls. Using these eight molecular weight fractions identified in the first workshop, we validated the preferential recognition of these 8 blot sections in a second workshop. We then re-calculated the sensitivity and specificity of the cellular immunoblotting assay for both workshops using only the data from these 8 blot sections. We observed increases in both sensitivity and specificity compared to the original workshop data for both workshops. The use of 8 instead of 18 molecular weight regions allows for a significant reduction in the amount of blood needed from patients, thus allowing cellular immunoblotting to be performed on pediatric patients participating in immunomodulatory studies. This improved T cell assay, which directly measures islet reactive T cell responses in autoimmune diabetes patients with excellent sensitivity and specificity, will likely improve patient follow-up during intervention studies.     

Subcellular fractionation of human eosinophils: Isolation of functional specific granules on isoosmotic density gradients

Subcellular fractionation has been an important tool in investigating human eosinophil structure and function, including localizing of cytokine/chemokines within granules, investigating granule protein translocation and intracellular transport during eosinophil secretion, and studying secretory mechanisms of granules. The resolution of organelles obtained by subcellular fractionation was improved considerably after the introduction of nonionic iodinated density-gradient metrizamide and Nycodenz media that, unlike sucrose, exhibit relatively low tonicity throughout the gradient. However, the structure and membrane preservation of isolated organelles were still compromised due to the lack of gradient isoosmolarity. This paper describes a detailed protocol of subcellular fractionation of nitrogen cavitated eosinophils on an isoosmotic iodinated density gradient (iodixanol - OptiPrep) and the isolation of well preserved and functional membrane-bound specific granules.     

Inhibition of HIV replication by a CD4‐reactive Fab of an IgM clone isolated from a healthy HIV‐seronegative individual

HIV replication is restricted by some anti‐CD4 mouse mAb in vitro and in vivo. However, a human monoclonal anti‐CD4 Ab has not been isolated. We screened EBV‐transformed peripheral B cells from 12 adult donors for CD4‐reactive Ab production followed by functional reconstitution of Fab genes. Three independent IgM Fab clones reactive specifically to CD4 were isolated from a healthy HIV‐seronegative adult (～0.0013% of the peripheral B cells). The germ line combinations for the V_H and V_L genes were V_H3‐33/_L6, V_H3‐33/_L12, and V_H4‐4/_L12, respectively, accompanied by somatic hypermutations. Genetic analysis revealed a preference for V‐gene usage to develop CD4‐reactive Ab. Notably, one of the CD4‐reactive clones, HO538‐213, with an 1×10^?8 M dissociation constant (Kd) to recombinant human CD4, limited the replication of R5‐tropic and X4‐tropic HIV‐1 strains at 1–2.5 μg/mL in primary mononuclear cells. This is the first clonal genetic analysis of human monoclonal CD4‐reactive Ab. A mAb against CD4 isolated from a healthy individual could be useful in the intervention of HIV/AIDS.