Phage-displayed mimotopes recognizing a biologically active anti-HIV-1 gp120 murine monoclonal antibody

Antibody-dependent cellular cytotoxicity (ADCC) is a host defense mechanism in which Fc receptor-bearing effector cells in combination with antigen-specific antibodies recognize and kill antigen-expressing target cells. The authors previously described a murine monoclonal antibody (MAb-ID6) that mediated ADCC activity against HIV-infected cells. It was demonstrated that the specificity of MAb-ID6 maps to the first 204 amino acids of gp120; however, the exact epitope was not identified. In the present work, by screening phage display libraries with MAb-ID6, the authors have mapped the corresponding epitope to amino acids 86-100 (HIV-1 gp120 sequence). This epitope lies within the C1 region of gp120 and is highly conserved among all subtypes and circulating recombinant forms of HIV-1. Thus, these phage mimotopes of C1 may serve as components of a vaccine for the induction of gp120-specific antibodies mimicking MAb-ID6.     

A TaqI polymorphism in the 3'UTR of the IL-12 p40 gene correlates with increased IL-12 secretion

Interleukin-12 (IL-12) is a key cytokine for the induction of Th1 immune responses. We evaluated whether a TaqI polymorphism in the 3'UTR of the IL-12 p40 gene affects secretion of IL-12 in vitro, and whether this polymorphism is associated with susceptibility to Crohn's disease (CD). IL-12 p40 and p70 secretion by monocytes in relation to genotype was determined in 63 healthy donors. Genotype and allele frequencies of the TaqI polymorphism in 150 CD patients were compared with 145 ethnically matched healthy controls (HC). No significant association was found between genotype and IL-12 p40 secretion after stimulation of monocytes with SAC+IFNgamma. In contrast, increasing IL-12 p70 secretion was found across the categories of non-carriers, heterozygotes and homozygotes for the variant allele (median values+/-SEM: 147+/-27, 282+/-51 and 482+/-34 pg/ml, respectively; P<0.005). The allele and genotype frequencies of this polymorphism in patients with CD did not differ statistically significantly from HC. The presence of a TaqI polymorphism in the IL12 p40 3'UTR correlates with increased in vitro IL-12 p70, but not p40 secretion. While this polymorphism does not appear to be correlated with susceptibility to CD in the limited population of patients tested here, it could influence the occurrence of the disease in certain subsets of patients.     

Cellular distribution of bovine leukemia virus proteins gp51SU, Pr72(env), and Pr66(gag-pro) in persistently infected cells

Monoclonal antibodies (mAbs) against bovine leukemia virus (BLV) mature proteins and precursors were used to map the localization of these proteins in persistently infected non-lymphocytic cell lines using immunofluorescence assay (IFA) and immuno-electron microscopy. IFA staining was observed in the basolateral surface of live FLK-BLV cells. When using a mAb against Pr66(gag-pro), mottled pinpoint fluorescence was seen in the cell surface of polarized cells, but no reaction was observed in cells undergoing mitosis. However, a mAb against Pr72(env) stained only mitotic cells and cellular fragments. Additionally, in these dividing cells, this envelope (Env) precursor polyprotein was not evenly distributed but concentrated predominantly in only one daughter cell. To the best of our knowledge, this observation has not been reported previously, either for BLV or for other retroviruses. The results of immunogold electron microscopy confirmed the specificity of the mAbs in the intracellular level. In infected cells, Pr72(env) and gp51SU were seen in proximity at the plasma membrane in incipient budding sites. Additionally, the mAb against Pr72(env) also reacted with Env precursor polyproteins in the mitochondria of BLV-bat(2) ultrathin sections. These mAbs may be used as a tool for mapping virus excretion sites in the cell surface of naturally or in vitro infected cells in the different stages of the cell cycle.     

Pet toxin from enteroaggregative Escherichia coli produces cellular damage associated with fodrin disruption

Pet toxin is a serine protease from enteroaggregative Escherichia coli which has been described as causing enterotoxic and cytotoxic effects. In this paper we show that Pet produces spectrin and fodrin (nonerythroid spectrin) disruption. Using purified erythrocyte membranes treated with Pet toxin, we observed degradation of alpha- and beta-spectrin chains; this effect was dose and time dependent, and a 120-kDa protein fraction was observed as a breakdown product. Spectrin degradation and production of the 120-kDa subproduct were confirmed using specific antibodies against the alpha- and beta-spectrin chains. The same degradation effect was observed in alpha-fodrin from epithelial HEp-2 cells, both in purified cell membranes and in cultured cells which had been held in suspension for 36 h; these effects were confirmed using antifodrin rabbit antibodies. The spectrin and fodrin degradation caused by Pet is related to the Pet serine protease motif. Fluorescence and light microscopy of HEp-2 Pet-treated cells showed morphological alterations, which were associated with irregular distribution of fodrin in situ. Spectrin and fodrin degradation by Pet toxin were inhibited by anti-Pet antibodies and by phenylmethylsulfonyl fluoride. A site-directed Pet mutant, which had been shown to abolish the enterotoxic and cytotoxic effects of Pet, was unable to degrade spectrin in erythrocyte membranes or purified spectrin or fodrin in epithelial cell assays. This is a new system of cellular damage identified in bacterial toxins which includes the internalization of the protease, induction of some unknown intermediate signaling steps, and finally the fodrin degradation to destroy the cell.     

Expression of the proteoglycans versican and mel-CSPG in dysplastic nevi

Nevi with architectural disorder and cytologic atypia of melanocytes (NAD) (also called dysplastic nevi) have been controversial with regard to their relationship with melanoma risk and to their gradation in 3 degrees of atypia. Versican and the melanoma-associated proteoglycan (mel-CSPG) are 2 major proteoglycans expressed by malignant melanoma, and they have a role in the regulation of cell adhesion, migration, and differentiation. We evaluated the differences in versican and mel-CSPG expression in nevi, NAD with several degrees of atypia, and primary malignant melanoma. Immunoreactivity for versican was negative in benign melanocytic nevi, positive in NAD (ranging from weakly to intensely positive), and intensely positive in malignant melanoma. Immunostaining for mel-CSPG was negative in benign melanocytic nevi and mild to moderately positive in NAD and melanoma. Our results suggest that versican expression may be of value for distinguishing NAD from benign melanocytic nevi and for distinguishing severe NAD from mild and moderate NAD.     

The cGMP synthesis and PKG1 expression in murine lymphoid organs

Numerous reports indicate that cyclic 3',5' guanosine monophosphate (cGMP) is involved in the regulation of immune processes. However, the mechanisms responsible for the synthesis of this nucleotide and its signaling pathways in immune cells are still not well recognized. The aim of our studies was to establish: 1) which form of guanylyl cyclase (GC) synthesizes cGMP in murine lymphoid organs and 2) whether the same organs express the isoforms PKG1alpha and/or PKG1beta of protein kinase G, known as possible target for synthesized cGMP. Cells isolated from thymus, lymph nodes, and spleen were treated with activators (SNP, ANP, CNP, STa) of soluble or particulate cyclases. Sodium nitroprusside (SNP) elevated intracellular cGMP 2-fold in thymic and lymph node cells and about 10-fold in spleen cells. Atrial natriuretic peptide (ANP) caused modest but statistically significant increases of cGMP in cells of all three organs. Additionally, spleen cells elevated their cGMP content about 2-fold in response to C-type natriuretic protein (CNP). In cellular homogenates of the all analyzed organs, the antibody anti-PKG1beta stained the 78 kDa band corresponding to the molecular mass of PKG1. Only homogenates of spleen cells were stained by the antibody recognizing PKG1alpha. Our results indicate that in the investigated organs cGMP may be synthesized mainly by soluble GC in response to nitric oxide. The modest increase of cGMP upon stimulation by ANP suggests that in all these organs either exists only a small subpopulation of cells that express particulate cyclase GC-A or GC-A is expressed at very low level. In spleen cells, however, cyclase GC-B appears to be the more active enzyme. Elevated cGMP concentration may in turn activate PKG1beta in thymus, lymph node, and spleen cells and also PKG1alpha in spleen cells.     

Identification of 26 new constitutional RB1 gene mutations in Spanish, Colombian, and Cuban retinoblastoma patients

Constitutional mutations in the RB1 gene predispose to retinoblastoma development. Hence genetic screening of retinoblastoma patients and relatives is important for genetic counseling purposes. In addition, RB1 gene mutation studies may help decipher the molecular mechanisms leading to tumors with different degrees of penetrance or expressivity. In the course of genetically screening of 107 hereditary and non-hereditary retinoblastoma patients (11 familiar bilateral, 4 familiar unilateral, 49 sporadic bilateral and 43 sporadic unilateral) and kindred from Spain, Colombia and Cuba, using direct PCR sequencing, we observed 45 distinct mutations and four RB1 deletions in 53 patients (9 familiar bilateral, 2 familiar unilateral, 31 sporadic bilateral and 11 sporadic unilateral). Most of these mutations (26/45, 57%) have not been reported before. In 32 patients, the predisposing mutations correspond to nonsense (mainly CpG transitions) and small insertions or deletions whose expected outcome is a truncated Rb protein that lacks the functional pockets and tail. Five single aminoacid replacements and seventeen mutations affecting splicing sites were also observed in retinoblastoma patients. Two of these sixteen mutations are of unclear pathogenic nature.     

HPLC analysis of 16 compounds from Artemisia ordosica

Objective: To establish a high-performance liquid chromatographic method (HPLC) for the simultaneous determination of sixteen compounds from Artemisia ordosica. Methods: HPLC was used to analyze 16 quality indicators of A. ordosica. The HPLC conditions were as follows: Agilent Eclipse Plus C18 column (250 mm × 4.6 mm, 5 μm) with acetonitrile (A)-water (B) as mobile phase, gradient elution: 0?10 min, 75%?65% B; 10?30 min, 65%?35 % B; and finally 30?40 min, 35%?15% B. The flow rate was 1.0 mL/min, the column temperature was 40 °C, the injection volume was 10 μL, and monitored by absorbance at 285 nm for compounds 1?10, 12 and 225 nm for compounds 11, 13?16. Results: Under the selected experimental chromatographic conditions, compounds 1?16 showed good linearity (r > 0.9993) in a wide concentration range. Their average recoveries were 99.50%, 95.38%, 97.75%, 96.00%, 98.20%, 97.50%, 95.50%, 99.33%, 96.75%, 96.50%, 98.50%, 97.83%, 99.20%, 95.33%, 97.33% and 96.30%, respectively, and the RSD were 1.99%, 1.81%, 1.63%, 1.98%, 1.67%, 1.92%, 1.74%, 1.67%, 1.90%, 1.72%, 1.88%, 1.83%, 1.79%, 1.76%, 1.81% and 1.96%, respectively. Conclusion: Based on the results of the HPLC analysis, it was concluded that p-hydroxycinnamic acid (1), O-hydroxycinnamic acid (2), coniferyl alcohol (5), 5,4''-dihydroxy-7,3''-dimethoxyflavanone (8), 5,4''-dihydroxy-7-methoxyflavanone (9), 5-hydroxy-7,4''-dimethoxyflavanone (12), dehydrofalcarindiol (13), arteordoyn A (14), dehydrofalcarinol (15) and capillarin (16) are best suited for the role of quality indicators of A. ordosica grown in different ecological environments.     

胆道术后严重大出血54例临床分析

目的 分析胆道术后严重出血的临床病程和治疗效果。方法 回顾性分析2016-2018年海军军医大学附属东方肝胆外科医院胆道外科行手术治疗的胆道疾病病人中需要行介入治疗（内镜和血管造影治疗）或再次手术的严重大出血者54例临床资料。结果 胆道术后严重出血的发生率为1.7%（54/3183）。胆道术后严重出血相关的总体死亡率为0.53%（17/3183）。54例病人中,有3例行内镜检查,仅1例行内镜治疗成功止血;34例接受介入血管造影检查的病人中,23例接受了介入性栓塞治疗（包括介入性钢圈栓塞、覆膜支架等血管介入治疗）,18例止血成功,成功率为78.3%（18/23）。35例行再次剖腹止血手术,其中19例将手术作为一线治疗方案,16例为内镜或介入治疗失败后行补救性治疗。结论 胆道术后严重出血原因较多,临床表现多样,早期发现和治疗至关重要。应综合考虑发病时间、伴发并发症情况和初次手术方案制定治疗决策。介入治疗临床安全性和止血成功率较高,但在介入治疗止血失败的情况下应紧急中转手术治疗。