The effects of antiphospholipid antibodies obtained from women with SLE/APS and associated pregnancy loss on rat embryos and placental explants in culture

Recurrent fetal loss occurs in approximately 1% of women. Autoimmune causes have been suggested as a factor in some of these cases. High rates of intrauterine fetal growth retardation and increased incidence of prematurity is associated with systemic lupus erythematosus (SLE) and the antiphospholipid syndrome (APS). We found in previous studies that sera from SLE/APS patients when used as a culture medium for rat embryos were found to reduce embryonic growth and development, induce a high rate of embryonic anomalies and death and damage the yolk sac morphologically and functionally. In order to investigate the direct effect of IgG purified from women with SLE/APS on the growth and viability of embryos, we cultured 11.5-day-old rat embryos in their yolk sacs in the presence of IgG purified from SLE/APS patients with recurrent pregnancy loss (RPL). The IgG affected directly the embryo and yolk sac, reducing their growth. The purified IgG positive for anticardiolipin/anti-DNA antibodies reduced yolk sac and embryonic growth more than sera negative for these antibodies but positive for antiphosphatydilserine and for antilaminin. Monoclonal antiphosphatydilserine reduced yolk sac growth but the embryos remained intact. Following the observed damage to the yolk sac we cultured human placental explants at 5.5-8 weeks of pregnancy in sera from SLE/APS patients for 96 hours and found that these sera reduced placental trophoblastic cell growth, reduced their proliferation rate and increased their rate of apoptosis. Successful treatment of the women resulted in a correction of the damage induced in the cultured rat embryos and in the cultured placental explants.     

Human and primate malarial sera inhibit Fc receptor-mediated phagocytosis

Sera obtained from humans in P. falciparum-endemic regions and from P. vivax-infected Saimiri sciureus were assayed for their ability to inhibit Fc receptor-mediated phagocytosis. Some sera of humans exposed to P. falciparum from The Gambia, Sudan, and Thailand inhibited ingestion via the Fc receptor by normal human monocytes. In addition, sera from infected monkeys and a high molecular weight fraction of infected monkey serum inhibited ingestion of EIgG by normal monkey spleen macrophages. Generally, inhibition was correlated with higher parasitemia and higher IFA titers.     

Augmentation of lymphocyte cytotoxicity by antibody to herpesvirus saimiri associated antigens.

Sera from owl monkeys infected with Herpesvirus saimiri (HVS) mediated antibody-dependent lymphocyte cytotoxicity (ADLC) against virus-infected owl monkey kidney cells. Peripheral blood lymphocytes from rhesus monkeys served as effector cells in this cytotoxic assay. ADLC titers increased along with membrane immunofluorescence (MF) titers but among some sera, the ADLC titers were much higher than expected from the MF titers, suggesting that multiple serum factors were involved in mediating ADLC in this system. Absorption of both low and high titered sera with HVS-infected owl monkey kidney cells removed all ADLC activity. Preliminary results from serial serum samples from two infected monkeys that developed leukemia and/or lymphoma demonstrated that ADLC but not MF titers increased to high titers with progression of disease and followed essentially a different kinetic pattern than that noted by MF. The possible significance of these findings in relation to malignant disease induced by this virus is discussed.     

Extracellular matrix regulates ovine granulosa cell survival, proliferation and steroidogenesis: relationships between cell shape and function

The extracellular matrix (ECM), constituting the follicular basal lamina and present also between follicular cells and in the follicular fluid, is believed to regulate granulosa cell (GC) function during follicular development. Ovine GCs isolated from small (1-3 mm in diameter) or large (4-7 mm in diameter) antral follicles were cultured on various pure ECM components (type I collagen, fibronectin, laminin), synthetic substrata enhancing (RGD peptides) or impairing (poly 2-hydroxyethylmethacrylate (poly-hema)) cell adhesion, or in the presence of heparin. The effects of these factors, used alone or in combination with IGF-I and/or FSH, were evaluated in terms of GC spread, survival, proliferation and steroidogenesis. When grown on type I collagen (CI) gel, poly-hema or heparin, GCs from both large and small follicles exhibited a round shape and a low proliferation rate. Compared with non-coated plastic substratum as a control, these ECM or synthetic compounds enhanced estradiol secretion and reduced progesterone secretion by large-follicle GCs. In contrast, GCs from both large and small follicles spread extensively on CI coating, fibronectin, laminin and RGD peptides. Fibronectin and laminin dramatically increased the proliferation rate and enhanced survival of GCs from both origins. Moreover, fibronectin, laminin and RGD peptides reduced estradiol secretion by large-follicle GCs. Unexpectedly, CI coating increased estradiol secretion and reduced progesterone secretion by large-follicle GCs, suggesting that type I collagen was able to maintain estradiol secretion independently of GC shape. Finally, GC responsiveness to IGF-I and FSH, in terms of proliferation and steroidogenesis, was generally maintained when cells were grown on ECM components, RGD peptides and in the presence of heparin. However, when large-follicle GCs were grown as non-adherent clusters (as observed on poly-hema) basal and IGF-I- and/or FSH-stimulated progesterone secretions were totally abolished. Overall, this study shows that GC shape, survival, proliferation and steroidogenesis can be modulated in vitro by pure ECM components in a specific and coordinated manner. It is suggested that, in vivo, fibronectin and laminin would sustain follicular development by enhancing the survival and proliferation of GCs, whereas type I collagen might participate in the maintenance of estradiol secretion in large antral follicles.     

庚型肝炎病毒基因组RNA的实验转染

目的：研究庚型肝炎病毒（HGV）对恒河猴的致病性及其在恒河猴体内的复制和表达。方法：体外转录制备全长HGV基因组RNA，肝内注射感染BY1猴，9个月后取其阳性血清感染BM1猴，7个月后取BM1阳性血清感染BB1。采用RT－PCR、原位杂交、定量PCR和免疫学、血清酶学、组织病理学等方法，对HGV的感染性及致病性进行了研究。结果：BY1、BM1和BB1实验猴感染后，分别在第3、8和3周血清HGV RNA阳转，持续存在最长达21周。血清丙氨酸转移酶（ALT）均升高，最高达418IU／L。感染后血清中均检测出抗－HGV。肝组织检查呈现轻度肝炎样变，并检测到HGV mRNA和HGV E2蛋白在肝组织中的表达。结论：体外转录的HGV基因组RNA对恒河猴具有感染性，可在恒河猴中传代感染并引起肝组织轻微炎症改变。恒河猴对HGV感染敏感，可作为研究HGV的实验动物模型。     

Immunizations of monkeys with synthetic peptides disclose conserved areas on gp120 of human immunodeficiency virus type 1 associated with cross-neutralizing antibodies and T-cell recognition.

Site-directed immunization was employed to identify sites on the envelope glycoprotein gp120 for antibody-mediated neutralization of human immunodeficiency virus type 1 (HIV-1). Antisera were raised in monkeys (Macaca fascicularis) against a series of 40 overlapping synthetic peptides covering the entire amino acid sequence of gp120 from the HTLV-IIIB strain of HIV-1. Immune sera against 12 of these peptides were reactive with gp120 by immunoblotting analysis, and antisera raised against 5 peptides, corresponding to amino acids (aa) 152-176, 193-218, 206-230, 248-269, and 307-330, were highly efficient in neutralizing HIV-1 (HTLV-IIIB) infectivity in vitro. Admixture of individual neutralizing anti-peptide monkey sera resulted in increment in neutralizing antibody titer. Antisera with reactivity to the relatively conserved regions defined by aa 152-176, 193-230, and 248-269 also neutralized to different extents the infectivity of the five Swedish clinical isolates of HIV-1 tested. Only a few HIV-1-infected people were found to make antibodies to these three conserved domains of gp120 as judged by ELISA using synthetic peptides as antigens. Three of the peptides (aa 152-176, 248-269, and 307-330) that induced neutralization antibodies also induced interleukin 2 production and lymphocyte proliferation when added to cultures of peripheral blood mononuclear cells from monkeys immunized with the corresponding peptides, indicating that these domains accommodate T-cell recognition sites. The results have obvious implications for the rational design of subunit vaccines against HIV-1 infection.     

Low immunogenicity of a Plasmodium vivax circumsporozoite protein epitope bound by a protective monoclonal antibody.

The repeat region of the Plasmodium vivax circumsporozoite (CS) protein contains 20 copies of the nine-amino acid sequence DRA A/D GQPAG. A monoclonal antibody that passively protects monkeys against sporozoite challenge recognizes a four-amino acid linear sequence AGDR included within this nonamer, but when monkeys were immunized with a vaccine, NS1(81)V20, which contains 20 copies of the nonamer, they failed to produce antibodies to AGDR. To determine if natural exposure to sporozoites induces antibodies to AGDR, we tested sera from 176 individuals from a malaria-endemic area in Flores, Indonesia. Seventy-one percent of the adults had antibodies to the P. vivax repeat region; only 18% had detectable antibodies to AGDR. None of the subjects had antibodies to the P. vivax variant repeat ANGAGNQPG. We next tested sera from six human volunteers immunized with NS1(81)V20 and found that the vaccine, despite inducing antibodies against the nonamer, as it did in the monkeys, did not induce antibodies against AGDR. To further test our ability to raise anti-AGDR antibodies using synthetic peptides, we immunized Aotus monkeys and BALB/c mice with AGDR. Sera from the mice reacted strongly with both AGDR and a recombinant protein containing the 20 copies of the nonamer. Sera from the monkeys reacted only minimally with a protein (VIVAX-1) that contains monomeric AGDR within its sequence. Sera from the mice also bound air-dried P. vivax sporozoites, while sera from the monkeys did not.(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies on trypanosomes in the Taiwan monkey

Fifty-three or 9% of 594 Taiwan monkeys, Macaca cyclopis, were found infected with a Trypanosome species. The parasites were recovered from monkey blood in diaphasic blood-agar culture medium with a Locke's solution overlay. Trypanosomes were found on only a few thick blood smears and no dividing or multiplying forms were found. Some of the organisms had a free-flagellum (4.7 microns), an undulating membrane and both ends of the body were pointed. The total length averaged 41.7 microns; nucleus was slightly anteriorly located. Blood from culture positive monkey would not infect monkeys or other animals, but cultured parasites readily infected monkeys and one mouse, and one rat treated with cortisone. Triatoma rubrofasciata and Triatoma protracta fed upon culture-positive monkeys did not become infected but one of the former became infected after membrane feeding upon cultured parasites mixed with monkey blood. Studies were also done on the development of Trypanosome conorhini in monkeys and other animals. The parasite was recovered from the hind-and mid-gut of naturally infected Taiwan Triatoma rubrofasciata (109 of 117). Some bugs also had salivary gland infections. Most laboratory animals (rats, mice, Mongolian gerbils, guinea pigs, rabbits) developed infections detected by blood-smear and culture. The parasite was serially passaged through monkeys and bugs monthly for one year but there was no change in the development pattern. Only trypmastigotes were present in blood smears; no dividing forms were found. Triatoma rubrofasciata and Triatoma protracta readily developed gut infections when fed upon monkeys, but only a few Rhodnius prolixus became infected.(ABSTRACT TRUNCATED AT 250 WORDS)     

The sialomucin CD34 is expressed on hematopoietic cells and blood vessels during murine development

The processes of angiogenesis and hematopoiesis require a high degree of coordination during embryogenesis. Whereas much is understood about the development of the vascular system in avian embryos, little information has been attained in mammals, predominantly because there are no specific markers for either blood vessels or hematopoietic cells in any developing mammalian system. We have recently shown that murine CD34 (mCD34) is expressed on the vascular endothelium in all organs and tissues of the adult mouse as well as on a small percentage of presumably hematopoietic stem cells in the bone marrow and fetal liver. Here we show that mCD34 is also expressed on the endothelium of blood vessels and on a subset of hematopoietic-like cells throughout murine development. mCD34 is first observed on the yolk sac endothelium of day 7.5 embryos and on a subset of hematopoietic cells within these yolk sacs. mCD34 expression is maintained on vessels and hematopoietic cells in all organs and tissues throughout embryogenesis. In addition, mCD34 is localized on growth conelike filopodial processes that appear at the budding edge of newly sprouted capillaries. Double staining of capillaries for mCD34 and laminin shows that these growth conelike processes seem to be free of laminin, whereas the formed capillaries seem to be coated with this extracellular matrix protein. Analysis of vessels in developing brain shows that these filopodial processes seem to be directed toward the ventricular epithelium, a previously described site of vascular endothelial growth factor synthesis. Finally, we show that the vascular structures of developing murine embryoid bodies also express mCD34. These data suggest that mCD34 is a useful marker for the analysis of the development of the blood vascular system in murine embryos.     

Detection of antibodies to muramyl dipeptide, the adjuvant moiety of streptococcal cell wall, in patients with rheumatic fever

The presence of antibodies to muramyl dipeptide (MDP), an adjuvant structure of streptococcal peptidoglycan, was investigated in patients with acute rheumatic fever (ARF). The detection of these antibodies was done by an enzyme-linked immunosorbent assay with a synthetic multivalent MDP conjugate as an antigen. Sera from 33 of 54 children with ARF had detectable levels of antibodies to MDP at the time of diagnosis. Such antibodies could only be detected in sera from two of 52 healthy children and one of 21 children with acute poststreptococcal glomerulonephritis. The specificity of the antibodies to MDP was demonstrated by inhibition, with free MDP, of binding of the sera to the MDP conjugate. The possible use of these antibodies in the diagnosis of ARF and in screening for safe synthetic muramyl peptides in immunization of humans is discussed.     

Phage displaying peptides mimic schistosoma antigenic epitopes selected by rat natural antibodies and protective immunity induced by their immunization in mice

AIM: To obtain the short peptides mimic antigenic epitopes selected by rat natural antibodies to schistosomes, and to explore their immunoprotection against schistosomiasis in mice. METHODS: Adults worm antigens (AWA) were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and enzyme-linked transferred immunoblotting methods with normal SD rat sera (NRS). The killing effects on schistosomula with fresh and heat-inactivated sera from SD rats were observed. Then the purified IgG from sera of SD rats was used to biopan a phage random peptide library and 20 randomly selected positive clones were detected by ELISA and 2 of them were sequenced. Sixty female mice were immunized thrice with positive phage clones (0, 2nd, 4th wk). Each mouse was challenged with 40 cercariae, and all mice were killed 42 d after challenge. The worms and the liver eggs were counted. RESULTS: NRS could specifically react to the molecules of 75 000, 47 000, 34 500 and 23 000 of AWA. Sera from SD rats showed that the mortality rate of schistosomula was 76.2%, and when the sera were heat-inactivated in vitro, the mortality rate was decreased to 41.0% after being cultured for 48 h. The specific phages bound to IgG were enriched about 300-folds after three rounds of biopanning. Twenty clones were detected by ELISA, 19 of them bound to the specific IgG of rat sera. Immunization with these epitopes was carried out in mice. Compared with the control groups, the mixture of two mimic peptides could induce 34.9% (P = 0.000) worm reduction and 67.6% (P = 0.000) total liver egg reduction in mice. Two different mimic peptides could respectively induce 31.0% (P= 0.001), 14.5% (P= 0.074) worm reduction and 61.2% (P= 0.000), 35.7% (P = 0.000) total liver egg reduction. The specific antibody could be induced by immunization of the mimic peptides, and the antibody titer in immunized mice reached more than 1:6 400 as detected by ELISA. CONCLUSION: Specific peptides mimic antigenic molecules can be obtained by biopanning the phage random peptide library and a partially protective immunity against schistosome infection can be stimulated by these phage epitopes in mice.     

抗幽门螺杆菌粘附素HpaA卵黄抗体的制备

目的研制高效价特异性的抗幽门螺杆菌黏附素鸡卵黄抗体免疫球蛋白抗体。方法用纯化重组幽门螺杆菌黏附素(Helicobacter pyloriadhesin A,rHpaA)抗原免疫22周龄罗曼鸡,母鸡免疫应答后,在卵黄中产生抗幽门螺杆菌黏附素抗体;经硫酸铵法初步纯化浓缩后;以间接ELISA鉴定抗体效价及免疫学活性;采用Bradford法测定蛋白含量;并用SDS-PAGE法测定分子量及鉴定抗体纯度。结果母鸡初次免疫第10d即有抗体产生,初次免疫后75d左右抗体效价超过1∶10000;最高可达为1∶12800。卵黄抗体经硫酸铵法纯化后,用SDS-PAGE分析,出现两条蛋白带,纯度达95%以上。其含量为10.56mg/ml蛋黄。结论本研究首次研制并鉴定抗黏附素卵黄抗体,开拓了我国预防幽门螺杆菌感染的新领域,分析了抗体在产蛋期卵黄液中表达的进度,为今后该抗体的持续、高质量诱导奠定基础。有望获得高效价、高产量的抗黏附素的IgY抗体(IgY-HpaA),为制备口服制剂开辟了广阔前景。     

Serological differences between acute and chronic schistosomiasis mansoni detected by enzyme-linked immunosorbent assay (ELISA).

Sera from patients with acute and chronic schistosomiasis mansoni, and from laboratory-infected monkeys, were examined by an enzyme-linked immunosorbent assay technique using antigens prepared from eggs, cercariae, and adult worms. Sera from patients with acute schistosomiasis and from monkeys 2 months post-infection reacted more positively to cercarial antigen than to adult worm antigen whereas sera from both patients with chronic schistosomiasis and monkeys infected for longer than 4 months reacted more positively to adult worm antigen. These differential responses to antigen serologically differentiated between acute and chronic schistosome infections.     

Tocilizumab, a humanized anti-interleukin-6 receptor antibody, improved anemia in monkey arthritis by suppressing IL-6-induced hepcidin production

We have reported that serum IL-6 level was related with the degree of anemia in monkey collagen-induced arthritis (CIA). In this study, we examined whether IL-6 blockade ameliorated an anemia in monkey CIA. CIA was induced by twice immunization of bovine type II collagen with adjuvant. When anemia became evident, anti-IL-6 receptor antibody, tocilizumab was intravenously injected once a week for 4 weeks. Controls received PBS in a same manner. Hematological and biochemical parameters were measured regularly and serum hepcidin-25 levels were measured by SELDI-TOF mass spectrometry. Moreover, hepcidin mRNA induction in Hep3B cells by serum from arthritic monkeys was examined by real-time PCR. Administration of tocilizumab rapidly decreased CRP levels and improved iron-deficient anemia within 1 week. Tocilizumab induced rapid but transient reduction in serum hepcidin-25. Hepcidin mRNA expression was more potently induced by serum from arthritic monkey and this was inhibited by the addition of tocilizumab. Blockade of IL-6 signaling rapidly improved anemia in monkey arthritis via the inhibition of IL-6-induced hepcidin production.     

Experimental horizontal transmission of Herpesvirus saimiri from squirrel monkeys to an owl monkey.

Herpesvirus saimiri was naturally transmitted from squirrel monkeys excreting the virus to one of two owl monkeys housed in the same cage. The owl monkey became infected approximately three months after contact was initiated. H. saimiri was consistently isolated from the peripheral lymphocytes until this animal died eight months later. During this period the owl monkey developed specific antibody to H. saimiri to a maximal neutralization index of 5.5 logs. The other monkey remained uninfected for an ovservation period of one year. The documentation of this horizontal transmission of H. saimiri infection from squirrel monkeys to an owl monkey suggests that owl monkeys developing spontaneous malignant lymphomas associated with H. saimiri infection may also have acquired the infection in this manner.     

鼠疫活菌苗接种人群和豚鼠血清学反应及豚鼠自动保护力试验观察

用PHA、PHA-SPA和ELISA-SPA法检测138人和40只豚鼠免疫接种后血清中FI抗体滴度。结果显示:在接种后的3个月内,人和豚鼠血清中抗体滴度分别介于1:20～1:2560和1:20～1:5120之间,免疫接种后6个月从人血清中未检出FI抗体,豚鼠的FI抗体滴度为1:20。人和豚鼠血清抗体阳转率在免疫接种后1个月分别为24～38％和100％,在免疫接种后3个月分别为2.1％和5～15％。免疫接种后3个月和6个月,用250和500MLP141鼠疫菌株攻毒的豚鼠的保护率分别为100％和50～80％。     

Ontogeny of the haemopoietic system: yolk sac origin of in vivo and in vitro colony forming cells in the developing mouse embryo

Summary . The mouse yolk sac has been shown to contain in-vivo colony forming cells capable of producing granulocytic, megakaryocytic and erythroid spleen colonies; in-vitro colony forming cells producing granulocytic and mononuclear-macrophage colonies in agar; and cells capable of repopuiating the lymphoid and myeloid tissue of lethally irradiated hosts. Similar haemopoietic precursor cells were also demonstrated in the blood at the time of initiation of the circulation and in the early embryonic liver. Organ cultures of 7 day embryos with intact yolk sacs, and embryos or yolk sacs after separation have shown the autonomous nature of the development of haemopoiesis in the yolk sac and the dependence of intra-embryonic haemopoiesis, particularly in embryonic liver, on colonization by yolk sac haemopoietic cells. Both in-vivo and in-vitro colony forming cells have been involved in the first migration stream, between yolk sac and embryonic liver, and evidence has been presented for the role of local environmental factors in controlling the differentiation of these cell types. These results support the view that development of haemopoietic organs in both embryo and adult is dependent on colonization by circulating cells and that these circulating stem cells originate initially in the yolk sac. This indicates that the yolk sac is the only site of genuine de novo formation of haemopoietic stem cells.