In vivo expression and immunological studies of the 42-kilodalton carboxyl-terminal processing fragment of Plasmodium falciparum merozoite surface protein 1 in the baculovirus-silkworm system

The 42-kDa carboxyl-terminal processing fragment of Plasmodium falciparum merozoite surface protein 1 (MSP-1(42)) is an anti-erythrocytic stage malaria vaccine candidate. In this study, MSP-1(42) was expressed by using the Bombyx mori nuclear polyhedrosis virus-silkworm expression system, and the antigenicity and immmunogenicity of the recombinant protein, Bmp42, were evaluated. The average yield of Bmp42, as determined by a sandwich enzyme-linked immunosorbent assay (ELISA), was 379 microg/ml of infected silkworm hemolymph, which was >100-fold higher than the level attainable in cell culture medium. N-terminal amino acid sequencing revealed that Bmp42 was correctly processed in silkworm cells. Data from immunoblotting, as well as from the inhibition ELISA, suggested that the conformational B-cell epitopes of MSP-1(42) were recreated in Bmp42. Immunization of rabbits with Bmp42 in complete Freund's adjuvant generated high-titer antibody responses against the immunogen. Specificity analyses of the anti-Bmp42 antibodies using several recombinant MSP-1(19) proteins expressing variant and conserved B-cell epitopes suggested that the anti-Bmp42 antibodies recognized primarily conserved epitopes on MSP-1(19). Furthermore, the anti-Bmp42 antibodies were highly effective in inhibiting the in vitro growth of parasites carrying homologous or heterologous MSP-1(42). Our results demonstrated that the baculovirus-silkworm expression system could be employed to express biologically and immunologically active recombinant MSP-1(42) at elevated levels; thus, it is an attractive alternative for producing a protective MSP-1(42) vaccine for human use.     

天然角蛋白自身反应性B细胞亚群及功能的分析

目的:明确天然角蛋白反应性B细胞的亚群及解剖定位,初步分析其分泌天然抗角蛋白自身抗体(anti-keratinautoantibody,AKautoAb)的能力。方法:取SPF级C57BL/6小鼠的脾细胞和腹腔细胞,荧光抗体染色后用流式细胞仪分析角蛋白反应性B细胞亚群。将脾脏和腹腔淋巴细胞体外培养后,用ELISA分析AKautoAb的滴度,用ELISPOT法分析分泌AKautoAb的B细胞数。结果:腹腔中几乎所有结合角蛋白的B细胞均为B-1a细胞,脾脏中结合角蛋白的B细胞以边缘带B细胞为主。腹腔细胞中分泌AKautoAb的细胞数显著多于脾细胞,其培养上清中AKautoAb的滴度显著高于脾细胞。结论:角蛋白反应性B细胞存在于3个成熟B细胞亚群:B-1细胞、滤泡B细胞和边缘带B细胞,其中CD5 的B-1a细胞具有活跃的分泌AKautoAb的能力。     

Effects of heparin on platelet aggregation and release and thromboxane A2 production.

Heparin, when added to citrated platelet-rich plasma (PRP), caused potentiation of platelet aggregation and the release reaction induced by the aggregating agents adenosine diphosphate (ADP), arachidonic acid, collagen, and epinephrine. At low concentrations (4.7 x 10(-5) M) arachidonic acid failed to cause aggregation of platelets in citrated PRP. However, in the presence of heparin, the same concentration of arachidonic acid caused aggregation. Examination of PRP for the presence of thromboxane A2 (TxA2) by use of a bioassay revealed that heparin also stimulated release of TxA2. This finding indicated that platelets released more TxA2 when they were challenged by low concentrations of arachidonic acid in the presence of heparin than in its absence. Platelets were labeled with 3H-arachidonic acid and 14C-serotonin, and attempts were made to determine whether heparin stimulated the platelet release reaction first with subsequent increased production of TxA2, or alternatively, whether heparin stimulated TxA2 production first with subsequent enhancement of the release reaction. In view of the demonstrated simultaneous release of 14C-serotonin and 3H-arachidonic acid metabolites, it appeared that either release of 14C and 3H occurs concurrently or, even if one of these events is dependent on the other, both events take place in rapid succession. Timed sequential studies revealed that in the presence of arachidonic acid, the addition of heparin hastened the apparently simultaneous release of both 14C and 3H.     

脑缺血大鼠海马超微结构的形态定量研究

对大鼠双侧颈总动脉夹闭2小时,经透射电镜观察海马,用Weibel氏形态定量法分别测量了海马1区和3区的线粒体和突触的表面积密度(Svi)、表面积—体积比(Si/Vi),并与对照组进行了比较。结果表明:缺血时海马1区受损较严重,表现为线粒体体积缩小,数量减少,Svi下降;突触数量有增多的趋势,但突触的Svi与Si/Vi变化不明显。缺血组海马3区受损较轻,线粒体和突触的结构基本正常,其Svi、Si/Vi与对照组差别不显著,提示了3区对缺血有较强的耐受性。     

小儿热性惊厥的脑电图

本文对296例小儿热性惊厥脑电图进行分析。结果:小儿热性惊厥脑电图(EEG)异常率48.65％,主要以阵性高幅慢波为主(77.78％)。小儿癫痫脑电图异常率(89.61％)。主要以阵发性棘慢波和局灶性改变为主(62.78％)。二周后EEG复查热性惊厥EEG大部分恢复正常(83.35％)。而小儿癫痫EEG未有恢复正常的。热性惊厥发作次数愈多,发作持续时间愈长EEG改变愈明显,二周后脑电图恢复正常的愈少。这对小儿热性惊厥的诊断和预后判定有重要价值。     

Clinical and genetic characteristics of Stargardt disease in a large Western China cohort: Report 1

Stargardt disease 1 (STGD1) is the most prevalent retinal dystrophy caused by pathogenic biallelic ABCA4 variants. Forty‐two unrelated patients mostly originating from Western China were recruited. Comprehensive ophthalmological examinations, including visual acuity measurements (subjective function), fundus autofluorescence (retinal imaging), and full‐field electroretinography (objective function), were performed. Next‐generation sequencing (target/whole exome) and direct sequencing were conducted. Genotype grouping was performed based on the presence of deleterious variants. The median age of onset/age was 10.0 (5–52)/29.5 (12–72) years, and the median visual acuity in the right/left eye was 1.30 (0.15–2.28)/1.30 (0.15–2.28) in the logarithm of the minimum angle of resolution unit. Ten patients (10/38, 27.0%) showed confined macular dysfunction, and 27 (27/37, 73.7%) had generalized retinal dysfunction. Fifty‐eight pathogenic/likely pathogenic ABCA4 variants, including 14 novel variants, were identified. Eight patients (8/35, 22.8%) harbored multiple deleterious variants, and 17 (17/35, 48.6%) had a single deleterious variant. Significant associations were revealed between subjective functional, retinal imaging, and objective functional groups, identifying a significant genotype–phenotype association. This study illustrates a large phenotypic/genotypic spectrum in a large well‐characterized STGD1 cohort. A distinct genetic background of the Chinese population from the Caucasian population was identified; meanwhile, a genotype–phenotype association was similarly represented.     

Envelope gene sequences of human T-lymphotropic virus type I in Taiwan

Summary Three major types of HTLV-I had been proposed, the Melanesian type, the Zairian type, and the cosmopolitan type, which was further divided into subtypes A, B and C, according to the phylogenetic tree constructed from LTR sequences of current HLTV-I isolates. In this study, the envelope gene sequences of HTLV-I from 9 Taiwanese were analyzed. Based on the phylogenetic tree constructed by unweighed pair group method and the sequence homology analysis by GCG computer programs, the envelope gene sequences of HTLV-I proviruses from these 9 Taiwanese belonged to subtype A or subtype B of the cosmopolitan type and were closely related to HTLV-I from Japan. Twelve subtype-specific nucleotide variations were deduced from the comparison of complete or partial envelope gene sequences of 16 HTLV-I isolates of known subtypes as well as those of 9 Taiwanese. These data provided the basis for subtyping the cosmopolitan type of HTLV-I by amplification of envelope gene sequences and restriction fragment length polymorphism studies. A more extensive survey based upon this proposal was warranted.     

Synergistic antimicrobial effect of cefotaxime and minocycline on proinflammatory cytokine levels in a murine model of Vibrio vulnificus infection.

BACKGROUND AND PURPOSE: Vibrio vulnificus causes primary bacteremia and necrotizing wound infection, leading to high morbidity and mortality in humans. This study aimed to evaluate the antimicrobial effect of cefotaxime and minocycline on proinflammatory cytokine levels in a murine model of V. vulnificus infection. METHODS: We investigated the dynamics of proinflammatory cytokines and their modulation by antimicrobial agents using a murine model of V. vulnificus infection. The change in cytokine levels was followed over a time course to identify the antimicrobial activity of the drugs against V. vulnificus. BALB/c female mice were challenged with an intraperitoneal infection using a clinical invasive isolate of Vv05191, and their cytokine levels were assayed over various time points. RESULTS: Serum levels of tumor necrosis factor-alpha, interleukin (IL)-1 beta, and IL-6 post-infection were found to be inoculum dose-dependent and positively correlated to the subsequent fatality rate in the infected mice. With an inoculum of 6.6 x 10(6) colony-forming units and intraperitoneal administration of cefotaxime, minocycline, or both, the serum and peritoneal fluid cytokine levels increased and then declined gradually. Comparison of the 3 antimicrobial regimens revealed that the magnitude of reduction in cytokine levels was greatest in mice treated with cefotaxime-minocycline combination. Moreover, the peritoneal fluid cytokine level in the combination group was significantly lower than that in the groups treated with minocycline or cefotaxime alone. CONCLUSIONS: The current results support the superiority of the combination therapy in treating invasive V. vulnificus infections.     

Febrile effects of polyriboinosinic acid: polyribocytidylic acid and interferon: relationship to somatostatin in rat hypothalamus

The changes in thermoregulatory effectors produced by an injection of polyriboinosinic acid: polyribocytidylic acid (Poly I:C) or interferon were assessed and compared in control rats, in rats with hypothalamic somatostatin (SS) receptor blockade and in rats with hypothalamic SS depletion. Intrahypothalamic (i.h., 0.05–0.50 μg) or intraperitoneal (i.p., 100–600 μg) administration of Poly I:C caused a dose-related rise in colon temperature in control rats at all ambient temperatures (T_a) studied. A Poly I:C-induced fever was produced by increased metabolism at a T_a of 8 °C, whereas at 30 °C, it was caused by cutaneous vasoconstriction. At a T_a of 22 °C, the fever was caused by increased metabolism and cutaneous vasoconstriction. On the other hand, i.h. administration of SS-14 antagonist (0.1–0.5 ng) caused a dose-related fall in colon temperature at T_a of 8 °C or 22 °C. At a T_a of 8 °C, the hypothermia was caused by decreased metabolism, whereas at 22 °C, it was caused by decreased metabolism and cutaneous vasodilation. At a T_a of 30 °C, the thermoregulatory effectors were not affected by SS-14 antagonist treatment. Furthermore, the fever induced by Poly I:C or interferon was significantly reduced by pretreatment of rats with an i.p. dose of cysteamine (30 mg. kg⁻¹) or an i.h. dose of SS-14 antagonist (0.1 ng). The results indicate that a somatostatinergic pathway in rat hypothalamus may mediate the fever induced by interferon or its inducer Poly I:C.