RESUMO
Frameshifts lead to complete alteration of the intended amino acid sequences, and therefore may affect the biological activities of protein therapeutics and pose potential immunogenicity risks. We report here the identification and characterization of a novel -1 frameshift variant in a recombinant IgG1 therapeutic monoclonal antibody (mAb) produced in Chinese hamster ovary cells during the cell line selection studies. The variant was initially observed as an atypical post-monomer fragment peak in size exclusion chromatography. Characterization of the fragment peak using intact and reduced liquid chromatography-mass spectrometry (LC-MS) analyses determined that the fragment consisted of a normal light chain disulfide-linked to an aberrant 26 kDa fragment that could not be assigned to any HC fragment even after considering common modifications. Further analysis using LC-MS/MS peptide mapping revealed that the aberrant fragment contained the expected HC amino acid sequence (1-232) followed by a 20-mer novel sequence corresponding to expression of heavy chain DNA sequence in the -1 reading frame. Examination of the DNA sequence around the frameshift initiation site revealed that a mononucleotide repeat GGGGGG located in the IgG1 HC constant region was most likely the structural root cause of the frameshift. Rapid identification of the frameshift allowed us to avoid use of a problematic cell line containing the frameshift as the production cell line. The frameshift reported here may be observed in other mAb products and the hypothesis-driven analytical approaches employed here may be valuable for rapid identification and characterization of frameshift variants in other recombinant proteins.
Assuntos
Anticorpos Monoclonais Humanizados/genética , Mutação da Fase de Leitura , Regiões Constantes de Imunoglobulina/genética , Imunoglobulina G/genética , Cadeias Pesadas de Imunoglobulinas/genética , Animais , Anticorpos Monoclonais Humanizados/biossíntese , Células CHO , Cricetinae , Cricetulus , Humanos , Regiões Constantes de Imunoglobulina/biossíntese , Imunoglobulina G/biossíntese , Cadeias Pesadas de Imunoglobulinas/biossíntese , Proteínas RecombinantesRESUMO
BACKGROUND: To perform a meta-analysis evaluating the diagnostic ability of fecal lactoferrin (FL) to distinguish inflammatory bowel disease (IBD) from irritable bowel syndrome (IBS). METHODS: The Medline, EMBASE, Web of Science, Cochrane library and CNKI databases were systematically searched for studies that used FL concentrations to distinguish between IBD and IBS. The sensitivity, specificity, and other diagnostic indexes of FL were pooled using a random-effects model. RESULTS: Seven studies, involving 1012 patients, were eligible for inclusion. In distinguishing IBD from IBS, FL had a pooled sensitivity of 0.78 (95% confidence interval [CI]: 0.75, 0.82), a specificity of 0.94 (95% CI: 0.91, 0.96), a positive likelihood ratio of 12.31 (95% CI: 5.93, 29.15), and a negative likelihood ratio of 0.23 (95% CI: 0.18, 0.29). The area under the summary receiver-operating characteristic curve was 0.94 (95% CI: 0.90, 0.98) and the diagnostic odds ratio was 52.65 (95% CI: 25.69, 107.91). CONCLUSIONS: FL, as a noninvasive and simple marker, is useful in differentiating between IBD and IBS.
Assuntos
Fezes/química , Doenças Inflamatórias Intestinais/diagnóstico , Síndrome do Intestino Irritável/diagnóstico , Lactoferrina/análise , Biomarcadores/análise , Diagnóstico Diferencial , Humanos , Sensibilidade e EspecificidadeRESUMO
OBJECTIVE: To compare efficacy and tolerability of 7-day standard triple therapy versus 7-day levofloxacin-based triple therapy in first-line treatment for Helicobacter pylori (H. pylori) infection. METHODS: Three hundred consecutive H.pylori positive patients were randomized to receive: clarithromycin, amoxicillin, lansoprazole (Group A: n = 150); or amoxicillin, levofloxacin, lansoprazole (Group B: n = 150). H. pylori status was rechecked by (13)C-urea breath test 4 weeks after the end of therapy. RESULTS: The eradication rates in intention to treat (ITT) and per protocol (PP) analyses were: Group A, 74.5% (111/149) and 78.2% (111/142); and Group B, 82.4% (122/148) and 83.0%(122/147). Although the eradication rate achieved with levofloxacin-based triple therapy was higher than that with standard therapies in either ITT or PP analysis, but no significantly difference was found between the two triple therapies. The incidence of side effects was similar among groups. CONCLUSIONS: A 7-day levofloxacin-based triple therapy can achieve higher H.pylori eradication rate than standard regimen. The levofloxacin-based regimen can be one effective therapy for the first-line anti-H.pylori treatment.
Assuntos
Antibacterianos/uso terapêutico , Infecções por Helicobacter/tratamento farmacológico , Levofloxacino , Ofloxacino/uso terapêutico , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Antibacterianos/administração & dosagem , Quimioterapia Combinada , Feminino , Helicobacter pylori , Humanos , Masculino , Pessoa de Meia-Idade , Ofloxacino/administração & dosagem , Adulto JovemRESUMO
Pisatin is an isoflavonoid phytoalexin synthesized by pea (Pisum sativum L.). Previous studies have identified two enzymes apparently involved in the synthesis of this phytoalexin, isoflavone reductase (IFR), which catalyzes an intermediate step in pisatin biosynthesis, and (+)6a-hydroxymaackiain 3-O-methyltransferase (HMM), an enzyme catalyzing the terminal step. To further evaluate the involvement of these enzymes in pisatin biosynthesis, sense- and antisense-oriented cDNAs of Ifr and Hmm fused to the 35s CaMV promoter, and Agrobacterium rhizogenes, were used to produce transgenic pea hairy root cultures. PDA, a gene encoding pisatin demethylating activity (pda) in the pea-pathogenic fungus Nectria haematococca, also was used in an attempt to reduce pisatin levels. Although hairy root tissue with either sense or antisense Ifr cDNA produced less pisatin, the greatest reduction occurred with sense or antisense Hmm cDNA. The reduced pisatin production in these lines was associated with reduced amounts of Hmm transcripts, HMM protein, and HMM enzyme activity. Hairy roots containing the PDA gene also produced less pisatin. To evaluate the role of pisatin in disease resistance, the virulence of N. haematococca on the transgenic roots that produced the lowest levels of pisatin was tested. Hairy roots expressing antisense Hmm were more susceptible than the control hairy roots to isolates of N. haematococca that are either virulent or nonvirulent on wild-type pea plants. This appears to be the first case of producing transgenic plant tissue with a reduced ability to produce a phytoalexin and demonstrating that such tissue is less resistant to fungal infection: these results support the hypothesis that phytoalexin production is a disease resistance mechanism.
Assuntos
Genes Fúngicos/genética , Genes de Plantas/genética , Hypocreales/patogenicidade , Pisum sativum/genética , Raízes de Plantas/genética , Pterocarpanos/biossíntese , Clonagem Molecular , DNA Complementar/química , DNA Complementar/genética , Genes Fúngicos/fisiologia , Genes de Plantas/fisiologia , Imunidade Inata/genética , Imunidade Inata/fisiologia , Metiltransferases/genética , Metiltransferases/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Pisum sativum/metabolismo , Pisum sativum/microbiologia , Doenças das Plantas/microbiologia , Raízes de Plantas/metabolismo , Raízes de Plantas/microbiologia , Plantas Geneticamente Modificadas , Rhizobium/genética , Rhizobium/metabolismo , Análise de Sequência de DNARESUMO
OBJECTIVE: To explore the characteristics of Helicobacter pylori (H. pylori) antigen in serum and to evaluate its clinical diagnostic value. METHODS: Enzyme-linked immunosorbant assay (ELISA) was developed to detect the soluble H. pylori antigen (S-Hp) and circulatory specific H. pylori antigen immunocomplexes (Hp-IC) in serum. RESULTS: The positive rate of S-Hp was 90.91% from 66 patients with H. pylori infection, which was much greater than 0% found in 28 controls (P < 0.001). Moreover, its concentration closely reflected the number of H. pylori in the gastric mucosa layer. We also found that Hp-IC existed bound with IgG and/or IgA in patients with positive S-Hp. However, there is no evidence to show the concentration of S-Hp reduced significantly in followed-up subjects after effective therapy. CONCLUSIONS: These methods as newly and noninvasive complementary tools can be used for clinical diagnosis of H. pylori infection. In addition, S-Hp and Hp-IC may be of importance in H. pylori pathogenesis.