A novel assay to measure B cell responses to keyhole limpet haemocyanin vaccination in healthy volunteers and subjects with systemic lupus erythematosus

The aim of the study was to characterize performance of a complementary set of assays to measure antigen-specific immune responses in subjects immunized with a neoantigen. Healthy volunteers (HV) (n = 8) and patients with systemic lupus erythematosus (SLE) (n = 6) were immunized with keyhole limpet haemocyanin (KLH) on days 1 and 29. Serum antibodies were detected using a flow cytometric bead array (CBA) that multiplexed the KLH response alongside pre-existing anti-tetanus antibodies. Peripheral blood mononuclear cells were studied by B cell ELISPOT. These assays were built upon precedent assay development in cynomolgus monkeys, which pointed towards their utility in humans. Primary anti-KLH IgG responses rose to a mean of 65–93-fold above baseline for HV and SLE patients, respectively, and secondary responses rose to a mean of 260-170-fold above baseline. High levels of anti-tetanus IgG were detected in pre-immunization samples and their levels did not change over the course of study. Anti-KLH IgG1-4 subclasses were characterized by a predominant IgG1 response, with no significant differences in subclass magnitude or distribution between HV and SLE subjects. Anti-KLH IgM levels were detectable, although the overall response was lower. IgM was not detected in two SLE subjects whodid generate an IgG response. All subjects responded to KLH by B cell ELISPOT, with no significant differences observed between HV and SLE subjects. The CBA and B cell ELISPOT assays reliably measured anti-KLH B cell responses, supporting use of this approach and these assays to assess the pharmacodynamic and potential safety impact of marketed/investigational immune-therapeutics.     

Metal Concentrations in the Radula of the Common Limpet, <Emphasis Type="Italic">Patella vulgata</Emphasis> L., from 10 Sites in the UK

Metal (Al, As, Ca, Cd, Cu, Fe, K, Mg, Mn, Na, Pb, Zn) levels in the feeding organ or radula of the common limpet Patella vulgata L. were surveyed in 10 populations over a ~150-km stretch of coastline in north-east England. The most northern population was at Beadnell in Northumberland and the most southern was at Port Mulgrave in North Yorkshire; sites included unspoilt bays and areas heavily affected by industrial contamination such as the River Tees estuary. We hypothesized that the radula might be used as an indicator of environmental contamination. There were significant differences between the sites in the ratio of radula length to shell length. Limpets from Whitburn had the smallest radula fraction (mean = 1.665), while those from Port Mulgrave the largest (mean = 1.998). Such variation is common in the literature and we detected no correlate and propose no cause. Iron was clearly the dominant metal in the radulae, with an overall of mean of 1.46% of radular weight, though this is rather low in comparison to values in the literature. Iron is naturally secreted into the developing radula as a putative hardening agent. The next most abundant metals, in descending order, were Na (at ~2000–8000 μg g⁻¹), K, Mg, Ca (~1000–1500 μg g⁻¹), Zn, Cu, Al, Pb (~7–75 μg g⁻¹), Mn, As, Cd (~0–1 μg g⁻¹). All but Al and Cd showed significant differences between the sites, but not in any consistent or convincing geographic manner. Nevertheless, the variations in metal levels between sites (e.g. Fe > 72%, Cu and Zn > 10-fold) suggest an environmental cause, but we are unable to offer any responsible factor, for example, there appeared little effect of the River Tees estuary. Cadmium is at a relatively low level in the radula in comparison to published data on pedal mucus and the flesh, but Pb is relatively high in pedal mucus and the radula and this might suggest that the radula is a detoxification route for Pb. Although the relationship between radula metal content and environmental metal content is unknown, the radula is constantly replaced and so may yet have the potential to be of use as a bioindicator, integrating metal exposure over much shorter periods than whole body burdens.     

Role of benoxaprofen and flunoxaprofen acyl glucuronides in covalent binding to rat plasma and liver proteins in vivo

Benoxaprofen (BNX) has been implicated in rare but serious hepatotoxicity which led to its withdrawal from the world market. Flunoxaprofen (FLX), a structural analog, appears to be less toxic. It has been postulated that the nonsteroidal antiinflammatory drugs associated toxicity may be related to covalent modification of proteins by their reactive acyl glucuronides, and the extent of covalent protein binding depends on both reactivity of the acyl glucuronide and the exposure to the reactive metabolite. The disposition of BNX and FLX in rats were compared upon intravenous administration of 20 mg/kg of BNX, FLX or their metabolites. Covalent binding of BNX and FLX to plasma and liver proteins were also determined, and an immunochemical approach was used to detect their hepatic targets. Similar concentrations of plasma protein adducts for BNX and FLX were detected even though the AUC of BNX-glucuronide (BNX-G) was almost twice that of FLX-glucuronide (FLX-G). Similar concentrations of liver protein adducts for BNX and FLX were also detected at 8 h, however, the hepatobiliary exposure of BNX-G was only 1/3rd that of FLX-G indicating that BNX-G was more reactive than FLX-G, which was in agreement with in vitro data. Proteins of 110 and 70 kDa were the major liver protein targets modified by covalent attachment of BNX and FLX. In conclusion, measuring covalent binding to tissue proteins in animals in addition to plasma adducts should be considered when evaluating and comparing carboxylic acid analogs that form reactive acyl glucuronides.     

内镜辅助锁孔手术切除颅内蛛网膜囊肿

目的探讨内镜辅助锁孔手术切除颅内蛛网膜囊肿的临床意义。方法应用内镜辅助锁孔手术切除颅内蛛网膜囊肿19例(囊肿大部分切除加脑池交通)。结果术后3个月至2年随访,19例患者均有不同程度的改善,囊肿闭合消失15例,明显缩小4例,5例癫痫患者在小剂量抗癫痫药物辅助下均较好得到控制,头晕头痛者均获明显改善。结论内镜辅助锁孔手术切除颅内蛛网膜囊肿是一种行之有效的方法 ,疗效满意     

Epitope mapping of a PrP(Sc)-specific monoclonal antibody: identification of a novel C-terminally truncated prion fragment

Monoclonal antibodies (mAbs) against prion proteins (PrPs) are indispensable in research and diagnosis of prion diseases, however the majority of these bind both the cellular (PrP^C) and the disease-associated (PrP^Sc) isoforms. According to the widely accepted protein-only hypothesis the two isoforms share the same sequence, but differ in their conformation. In the present study we set to determine the critical binding residues of our PrP^Sc-specific mAbs with the view of discerning which residues play a key role in the conformational transition between PrP^C and PrP^Sc. Focussing on the V5B2 mAb that provided differential labelling of prion-affected tissue from individuals positive for transmissible spongiform encephalopathies, we performed alanine scanning and phage-display epitope mapping to elucidate the antigenic determinants of this mAb and gain insight into its specificity on a molecular level. We observed that instead of discriminating between the two prion protein isoforms based on conformational differences, V5B2 binds a previously uncharacterized C-terminally truncated form of PrP^Sc that ends with the residue Y226, which we named PrP226*. The addition of a single C-terminal amino-acid residue completely abolished V5B2 binding, while Western blots using recombinant full-length PrPs and PrPs terminating at Y226 confirmed that the V5B2 mAb discriminates between the two based on their difference in length.     

Keyhole limpet hemocyanin: an effective adjunct against melanoma in vivo

BACKGROUND: We have previously demonstrated the potent in vitro antiproliferative effects of keyhole limpet hemocyanin (KLH) against melanoma. Our prior studies directed us to hypothesize that KLH would be effective in vivo against melanoma, alone and in combination with conventional immunotherapy. METHODS: Mice were inoculated with 2 x 10(7) HTB68 cells and randomized to 6 groups. Treatment groups consisted of control, KLH 200 microg, alpha interferon (AIFN) 1000 IU, interleukin-2 (IL-2) 5000 IU, KLH + AIFN, and KLH + IL-2. RESULTS: KLH + IL-2 exhibited the greatest reduction in tumor volume (30%) as compared to control (P = .014), followed by KLH + AIFN (28%, P = .031). Singly treated animals had less tumor inhibition: IL-2 (30%, P = .022), KLH (18%, not significant), and AIFN (16%, not significant). CONCLUSIONS: KLH augments the effects of AIFN, one of the standard immunotherapeutic agents against melanoma in vivo. Further in vivo and early clinical studies into the effects of KLH as both a single and combined agent are warranted.     

经穹隆间第三脑室底锁孔入路的显微解剖

目的 探索经穹隆间第三脑室底锁孔入路的可行性和手术方法。 方法 设计经穹隆间第三脑室底锁孔入路(第三脑室底切口起自灰结节向后,经乳头体间,止于后穿质)。运用解剖学方法在导航辅助下在16例尸头标本上模拟经穹隆间第三脑室底锁孔入路手术,在手术显微镜下对手术显露进行观察,利用导航作解剖学测量。 结果 导航辅助下能顺利完成16例尸头标本的经穹隆间第三脑室底锁孔入路手术。冠状缝与矢状缝交点到室间孔上缘、丘脑间黏合、乳头体和中脑导水管上缘的距离分别为（68.4±4.6）mm、(66.3±6.0)mm、(86.3±5.3)mm、(82.0±7.6)mm,冠状缝与矢状缝交点到基底动脉末端分叉的操作距离为(91.8±5.0)mm。灰结节向后经乳头体间止于后穿质切开第三脑室底可获得长（9.5±2.6）mm的手术通道。术中经第三脑室底切口能清晰显露脚间池内的基底动脉末段、大脑后动脉P1段、P2段、小脑上动脉、后交通动脉以及它们的穿通支血管。向前解剖Liliequist膜可显露斜坡和鞍背,侧方可显露出动眼神经,向后显露出脚间窝。基底动脉末端分叉多偏于左侧（68.8%）,两侧大脑后动脉多向前外侧斜行（68.8%）。大部分大脑后动脉夹角上有1～4支小穿支血管自基底动脉末端分出。 结论 经穹隆间第三脑室底锁孔入路在技术上可行,深入研究可望应用于基底动脉末端动脉瘤的直接手术。     

Propolis and the immune system: a review

Propolis has been used empirically for centuries and it was always mentioned as an immunomodulatory agent. In recent years, in vitro and in vivo assays provided new information concerning its mechanisms of action, thus a review dealing with propolis and the immune system became imperative. This review compiles data from our laboratory as well as from other researchers, focusing on its chemical composition and botanical sources, the seasonal effect on its composition and biological properties, its immunomodulatory and antitumor properties, considering its effects on antibody production and on different cells of the immune system, involving the innate and adaptive immune response. In vitro and in vivo assays demonstrated the modulatory action of propolis on murine peritoneal macrophages, increasing their microbicidal activity. Its stimulant action on the lytic activity of natural killer cells against tumor cells, and on antibody production was demonstrated. Propolis inhibitory effects on lymphoproliferation may be associated to its anti-inflammatory property. In immunological assays, the best results were observed when propolis was administered over a short-term to animals. Propolis antitumor property and its anticarcinogenic and antimutagenic potential are discussed. Since humans have used propolis for different purposes and propolis-containing products have been marketed, the knowledge of its properties with scientific basis is not only of academic interest but also of those who use propolis as well. This review opens a new perspective on the investigation of propolis biological properties, mainly with respect to the immune system.