Antidepressant potential of total flavonoids from Astragalus in a chronic stress mouse model: Implications for myelination and Wnt/ß-catenin/Olig2/Sox10 signaling axis modulation.

ETHNOPHARMACOLOGICAL RELEVANCE: Radix Astragali, a versatile traditional Chinese medicinal herb, has a rich history dating back to "Sheng Nong's herbal classic". It has been employed in clinical practice to address various ailments, including depression. One of its primary active components, total flavonoids from Astragalus (TFA), remains unexplored in terms of its potential antidepressant properties. This study delves into the antidepressant effects of TFA using a mouse model subjected to chronic unpredictable mild stress (CUMS). AIMS OF THE STUDY: The study aimed to scrutinize how TFA influenced depressive behaviors, corticosterone and glutamate levels in the hippocampus, as well as myelin-related protein expression in CUMS mice. Additionally, it sought to explore the involvement of the Wnt/ß-catenin/Olig2/Sox10 signaling axis as a potential antidepressant mechanism of TFA. MATERIALS AND METHODS: Male C57BL/6 mice were subjected to CUMS to induce depressive behaviors. TFA were orally administered at two different doses (50 mg/kg and 100 mg/kg). A battery of behavioral tests, biochemical analyses, immunohistochemistry, UPLC-MS/MS, real-time PCR, and Western blotting were employed to evaluate the antidepressant potential of TFA. The role of the Wnt/ß-catenin/Olig2/Sox10 signaling axis in the antidepressant mechanism of TFA was validated through MO3.13 cells. RESULTS: TFA administration significantly alleviated depressive behaviors in CUMS mice, as evidenced by improved sucrose preference, reduced immobility in tail suspension and forced swimming tests, and increased locomotor activity in the open field test. Moreover, TFA effectively reduced hippocampal corticosterone and glutamate levels and promoted myelin formation in the hippocampus of CUMS mice. Then, TFA increased Olig2 and Sox10 expression while inhibiting the Wnt/ß-catenin pathway in the hippocampus of CUMS mice. Finally, we further confirmed the role of TFA in promoting myelin regeneration through the Wnt/ß-catenin/Olig2/Sox10 signaling axis in MO3.13 cells. CONCLUSIONS: TFA exhibited promising antidepressant effects in the CUMS mouse model, facilitated by the restoration of myelin sheaths and regulation of corticosterone, glutamate, Olig2, Sox10, and the Wnt/ß-catenin pathway. This research provides valuable insights into the potential therapeutic application of TFA in treating depression, although further investigations are required to fully elucidate the underlying molecular mechanisms and clinical relevance.

Sex differences in a corticosterone-induced depression model in mice: Behavioral, neurochemical, and molecular insights.

Depression is characterized by a significant sex disparity, with higher rates observed in women compared to men. This study aimed to investigate the impact of sex on depressive behaviors and explore the underlying mechanisms using a corticosterone (CORT)-induced depression model in mice. Behavioral tests, Nissl staining, UPLC-MS/MS, and Western blot analysis were performed to assess behavioral changes, as well as neuronal alterations, neurotransmitter levels, and protein expressions in the hippocampus. The mice in the model group exhibited sex-specific anxiety- and depression-like behaviors. Nissl staining revealed structural abnormalities in the CA3 region of the hippocampus in females. Neurotransmitter analysis indicated decreased serotonin and norepinephrine levels in both sexes, while glutamate levels were elevated in females. Furthermore, female mice demonstrated elevated serum CORT levels. Western blot analysis revealed sex-specific alterations in specific protein expression. Female mice exhibited downregulated glucocorticoid receptor and brain-derived neurotrophic factor expression, whereas male mice showed minimal changes. Additionally, female mice displayed reduced phosphorylated AKT, phosphorylated PI3K, and phosphorylated mTOR levels. These findings enhance our understanding of sex-specific differences in the CORT-induced depression model and provide insights into the underlying mechanisms of depression. This research emphasizes sex in depression studies and supports tailored interventions.

Natural compounds as potential therapeutic candidates for multiple sclerosis: Emerging preclinical evidence.

BACKGROUND: Multiple sclerosis is a chronic neurodegenerative disease, with main characteristics of pathological inflammation, neural damage and axonal demyelination. Current mainstream treatments demonstrate more or less side effects, which limit their extensive use. PURPOSE: Increasing studies indicate that natural compounds benefit multiple sclerosis without remarkable side effects. Given the needs to explore the potential effects of natural compounds of plant origin on multiple sclerosis and their mechanisms, we review publications involving the role of natural compounds in animal models of multiple sclerosis, excluding controlled trials. STUDY DESIGN AND METHODS: Articles were conducted on PubMed and Web of Science databases using the keywords ``multiple sclerosis'' and ``natural compounds'' published from January 1, 2008, to September 1, 2023. RESULTS: This review summarized the effects of natural ingredients (flavonoids, terpenoids, polyphenols, alkaloids, glycosides, and others) from three aspects: immune regulation, oxidative stress suppression, and myelin protection and regeneration in multiple sclerosis. CONCLUSION: Overall, we concluded 80 studies to show the preclinical evidence that natural compounds may attenuate multiple sclerosis progression via suppressing immune attacks and/or promoting myelin protection or endogenous repair processes. It would pave the roads for the future development of effective therapeutic regiments of multiple sclerosis.

Bear bile powder alleviates Parkinson's disease-like behavior in mice by inhibiting astrocyte-mediated neuroinflammation.

Parkinson's disease (PD) is a common neurodegenerative disease in middle-aged and elderly people. In particular, increasing evidence has showed that astrocyte-mediated neuroinflammation is involved in the pathogenesis of PD. As a precious traditional Chinese medicine, bear bile powder (BBP) has a long history of use in clinical practice. It has numerous activities, such as clearing heat, calming the liver wind and anti-inflammation, and also exhibits good therapeutic effect on convulsive epilepsy. However, whether BBP can prevent the development of PD has not been elucidated. Hence, this study was designed to explore the effect and mechanism of BBP on suppressing astrocyte-mediated neuroinflammation in a mouse model of PD. PD-like behavior was induced in the mice by intraperitoneal injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (30 mg·kg-1) for five days, followed by BBP (50, 100, and 200 mg·kg-1) treatment daily for ten days. LPS stimulated rat C6 astrocytic cells were used as a cell model of neuroinflammation. THe results indicated that BBP treatment significantly ameliorated dyskinesia, increased the levels of tyrosine hydroxylase (TH) and inhibited astrocyte hyperactivation in the substantia nigra (SN) of PD mice. Furthermore, BBP decreased the protein levels of glial fibrillary acidic protein (GFAP), cyclooxygenase 2 (COX2) and inducible nitric oxide synthase (iNOS), and up-regulated the protein levels of takeda G protein-coupled receptor 5 (TGR5) in the SN. Moreover, BBP significantly activated TGR5 in a dose-dependent manner, and decreased the protein levels of GFAP, iNOS and COX2, as well as the mRNA levels of GFAP, iNOS, COX2, interleukin (IL) -1ß, IL-6 and tumor necrosis factor-α (TNF-α) in LPS-stimulated C6 cells. Notably, BBP suppressed the phosphorylation of protein kinase B (AKT), inhibitor of NF-κB (IκBα) and nuclear factor-κB (NF-κB) proteins in vivo and in vitro. We also observed that TGR5 inhibitor triamterene attenuated the anti-neuroinflammatory effect of BBP on LPS-stimulated C6 cells. Taken together, BBP alleviates the progression of PD mice by suppressing astrocyte-mediated inflammation via TGR5.

The alteration of gut microbiota in venlafaxine-ameliorated chronic unpredictable mild stress-induced depression in mice.

Depression is associated with intestinal dysbiosis. Venlafaxine is a commonly used antidepressant in clinical practice as a serotonin and noradrenaline reuptake inhibitor. However, its effects on gut bacteria in depression remain unclear. Here, we established a mouse model of depression induced by chronic unpredictable mild stress (CUMS), and investigated the alterations of venlafaxine on the gut microbiota and potential key bacteria. Our data show that venlafaxine exerts antidepressant effects by restoring the serotonin (5-HT) system and glutamate (Glu) levels in CUMS mice. Moreover, we revealed that venlafaxine altered the diversity of gut bacteria in CUMS mice, and at genus level, Blautia, Oscillibacter, Tyzzerella, Butyricicoccus, and Enterorhabdus are the key bacteria responsible for venlafaxine-ameliorated depression in mice. Among these potential key bacteria, Blautia, Oscillibacter, and Butyricicoccus are correlated significantly with the 5-HT and 5-hydroxyindoleacetic acid levels; while Tyzzerella is correlated markedly with Glu levels. We further show that venlafaxine affected multiple functional metabolic pathways of gut bacteria in mice with CUMS-induced depression. Our results suggest that venlafaxine possibly ameliorates depression via modulating gut bacteria, and found the potential targets of its antidepressant effects.

Isoliquiritigenin inhibits microglia-mediated neuroinflammation in models of Parkinson's disease via JNK/AKT/NFκB signaling pathway.

Isoliquiritigenin (ISL) is a flavonoid with numerous pharmacological properties, including anti-inflammation, yet its role in Parkinson's disease (PD) with microglia-mediated neuroinflammation remains unknown. In this study, the effects of ISL on inhibiting microglia-mediated neuroinflammation in PD were evaluated in the 1-methyl-4-phenylpyridinium (MPTP)-induced mouse model of PD and in lipopolysaccharide (LPS)-stimulated BV-2 microglia. Our results showed that ISL prevented behavioral deficits and excessive microglial activation in MPTP-treated mice. Moreover, ISL was found to prevent the elevation of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), and mitigate the phosphorylation of c-Jun N-terminal protein kinase (JNK), protein kinase B (AKT), nuclear factor kappa light-chain enhancer of activated B cells (NFκB), and inhibitor of NFκB protein ɑ (IκBɑ) in the substantia nigra and striatum of MPTP-treated mice and LPS-stimulated BV-2 cells. Meanwhile, in LPS-stimulated BV-2 cells, ISL inhibited the production of inflammatory mediators such as interleukin (IL)-1ß, IL-6 and tumor necrosis factor alpha (TNF-α). In addition, the agonist of JNK partly abolished the inhibitory effects of ISL in LPS-treated BV-2 cells. Our results demonstrated that ISL inhibits microglia-mediated neuroinflammation in PD models probably through deactivating JNK/AKT/NFκB signaling pathways. The novel findings suggest the therapeutic potential of ISL for microglia-mediated neuroinflammation in PD.

Total astragalosides promote oligodendrocyte precursor cell differentiation and enhance remyelination in cuprizone-induced mice through suppression of Wnt/ß-catenin signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Radix Astragali is a traditional Chinese medicine with various pharmacological effects. Total astragalosides (TA), the main effective ingredients in Radix Astragali, exert properties including anti-oxidative stress, anti-neuroinflammation, and neuroprotection. We previously found that TA alleviated experimental autoimmune encephalomyelitis (EAE) progression, a widely used animal model of multiple sclerosis (MS). As a chronic demyelination disease, MS generally manifests myelin loss and fails to myelin regeneration. Regulation of oligodendrocyte progenitor cells (OPCs) differentiation and remyelination is the fundamental strategy for MS treatment. However, whether TA could directly promote OPCs differentiation and remyelination is still unknown. AIMS OF THE STUDY: This study was aimed to investigate pro-differentiation and myelin regeneration effects of TA on OPCs and Cuprizone (CPZ)-induced demyelination mice, an animal model of MS, and to explore mechanism underlying from regulation of OPCs differentiation and maturation. MATERIALS AND METHODS: Mice were orally given CPZ (400 mg/kg) daily for 4 weeks to induce myelin loss, and then treated with TA (25 and 50 mg/kg) daily for 1 week. Cell proliferation assay, Western blot, RT-PCR, immunocytochemistry and immunohistochemistry were performed to explore the mechanisms. The role of TA in oligodendrocyte differentiation and maturation was evaluated using MO3.13, a human oligodendrocytic hybrid cell line. RESULTS: TA was shown to mitigate behavioral impairment in CPZ-induced mice. It markedly ameliorated myelin loss and enhanced remyelination in the corpus callosum of mice, evidenced by increased expression of myelin basic protein (MBP) and the number of CC1+ newly generated oligodendrocytes (OLs). TA also enhanced the expression of MBP at both mRNA and protein levels in MO3.13 cells. In CPZ-induced mice and MO3.13 cells, TA remarkably promoted the activation of GSK3ß, repressed the phosphorylation of ß-catenin, reduced the expression of transcription factor 4 and inhibitor of DNA binding 2. The agonist of ß-catenin, SKL2001, partially abolished the pro-differentiation effect of TA in MO3.13 cells. CONCLUSIONS: Taken together, we clarified that TA could effectively enhance the differentiation and maturation of OPCs and accelerate remyelination in CPZ-induced mice through inhibition of Wnt/ß-catenin signaling pathway. This study provides new insight into the beneficial effect of TA in the treatment of MS.

Hyodeoxycholic acid inhibits lipopolysaccharide-induced microglia inflammatory responses through regulating TGR5/AKT/NF-κB signaling pathway.

BACKGROUND: Hyodeoxycholic acid (HDCA) is a natural secondary bile acid with enormous pharmacological effects, such as modulating inflammation in neuron. However, whether HDCA could suppress microglial inflammation has not been elucidated yet. AIMS: To determine the anti-microglial inflammatory effect of HDCA in lipopolysaccharide (LPS) models and its mechanisms. METHODS: The effect of HDCA was evaluated in LPS-stimulated BV2 microglial cells in vitro and the cortex of LPS-treated mice in vivo. Immunohistochemistry and immunofluorescence were used to visualize the localization of nuclear factor kappa light-chain enhancer of activated B cells (NF-κB) and ionized calcium-binding adaptor protein-1 (Iba-1), respectively. The mRNA expression of inflammatory cytokines was measured by RT-qPCR. The protein expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), takeda G-coupled protein receptor 5 (TGR5), and the phosphorylation of protein kinase B (AKT), NF-κB, and inhibitor of NF-κB protein α (IκBα) was examined by Western blot. RESULTS: HDCA inhibited the inflammatory responses in LPS-treated BV2 cells and in the cortex of LPS-treated mice, evidenced by decreased production of inflammatory mediators such as iNOS, COX-2, tumor necrosis factor (TNF-α), interleukin (IL)-6, and IL-1ß. Further study demonstrated that HDCA repressed the phosphorylation, nuclear translocation, and transcriptional activity of NF-κB and inhibited the activation of AKT in BV-2 cells induced by LPS. Meanwhile, addition of TGR5 inhibitor, triamterene, abolished the effects of HDCA on TGR5, AKT, and NF-κB. CONCLUSION: The present study demonstrated that HDCA prevents LPS-induced microglial inflammation in vitro and in vivo, the action of which is via regulating TGR5/AKT/NF-κB signaling pathway.

Natural bear bile powder suppresses neuroinflammation in lipopolysaccharide-treated mice via regulating TGR5/AKT/NF-κB signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: According to the Tang Dynasty classics Dietetic Material Medica and the Ming Dynasty classics Compendium of Materia Medica records, bear bile powder (BBP) has been used to treat a variety of diseases, such as febrile seizures, the pathogenesis of which is associated to neuroinflammation. However, the mechanism of BBP on alleviating neuroinflammation remains unclear. AIMS OF THE STUDY: Microglia can be activated by peripheral lipopolysaccharide (LPS) and play an important role in the pathogenesis of neuroinflammation. The purpose of this study is to investigate the effects and mechanism of BBP in inhibiting LPS-induced microglia inflammation in vitro and in vivo. MATERIALS AND METHODS: The anti-microglia inflammatory effects and mechanism of BBP were assessed in LPS-treated BV2 microglial cells and in LPS-treated mice. The mRNA expression levels of the inflammatory factor and the protein expressions of cyclooxygenase-2 (COX2), inducible nitric oxide synthase (iNOS), takeda G-protein coupled receptor 5 (TGR5), nuclear factor-κB (NF-κB), inhibitor of NF-κB (IκBɑ), protein kinase B (AKT) in BV2 cells, mouse hippocampus and cortex were detected. The NF-κB transcription activity and NF-κB nuclear translocation were observed. RESULTS: Our findings showed that BBP reduces branched process retraction and NO in LPS-treated BV2 cells, inhibits the protein expression of ionized calcium binding adaptor molecule 1 in the hippocampus of LPS-treated mice. Moreover, we observed that BBP decreases tumor necrosis factor α, interleukin (IL)-6 and IL-1ß mRNA levels, deceases iNOS and COX-2 protein levels, increases TGR5 protein levels, suppresses the phosphorylation of AKT, NF-κB and IκBɑ protein in microglia both in vitro and in vivo. Further, we found that triamterene, the inhibitor of TGR5, abolishes the effects of BBP in LPS- treated BV2 cells. CONCLUSION: BBP inhibits LPS-induced microglia activation, and the mechanism of its action is partly through TGR5/AKT/NF-κB signaling pathway.

An integrated method for optimized identification of effective natural inhibitors against SARS-CoV-2 3CLpro.

The current severe situation of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has not been reversed and posed great threats to global health. Therefore, there is an urgent need to find out effective antiviral drugs. The 3-chymotrypsin-like protease (3CLpro) in SARS-CoV-2 serve as a promising anti-virus target due to its essential role in the regulation of virus reproduction. Here, we report an improved integrated approach to identify effective 3CLpro inhibitors from effective Chinese herbal formulas. With this approach, we identified the 5 natural products (NPs) including narcissoside, kaempferol-3-O-gentiobioside, rutin, vicenin-2 and isoschaftoside as potential anti-SARS-CoV-2 candidates. Subsequent molecular dynamics simulation additionally revealed that these molecules can be tightly bound to 3CLpro and confirmed effectiveness against COVID-19. Moreover, kaempferol-3-o-gentiobioside, vicenin-2 and isoschaftoside were first reported to have SARS-CoV-2 3CLpro inhibitory activity. In summary, this optimized integrated strategy for drug screening can be utilized in the discovery of antiviral drugs to achieve rapid acquisition of drugs with specific effects on antiviral targets.

Evidence-Based Research Strategy of Traditional Chinese Medicine for Amyotrophic Lateral Sclerosis.

Among adult-onset motor neuron diseases, amyotrophic lateral sclerosis (ALS) is the most common. ALS involves the increasing loss of lower and upper motor neurons. Within a few years of onset, ALS causes patient death via progressive paralysis of respiratory muscles. However, the current drugs used to treat ALS, riluzole, edaravone, and dextromethorphan/quinidine, can only delay the progression of the disease and alleviate a small number of symptoms in some patients, and no completely effective treatment is available. Traditional Chinese medicine (TCM) has shown significant advantages in the treatment of ALS in China and Asia; however, the mechanism of its efficacy is unclear. This review discusses the pathogenetic hypothesis of ALS in detail from the level of neurons and glial cells and uses two current experimental animal models of ALS to design experimental strategies to study TCM treatment. We aim to provide a scientific explanation of the mechanism of the effect of TCM in the treatment of ALS, which will help clinicians and research scientists to accept the theory of TCM to treat ALS and promote the development of TCM modernization.

A combination of three plasma bile acids as a putative biomarker for schizophrenia.

The aim of the present study is to determine whether plasma bile acids (BAs) could be used as an auxiliary diagnostic biomarker to distinguish patients with schizophrenia from healthy controls. Seventeen different BAs were quantitatively measured in plasma of 12 healthy participants and 12 patients with schizophrenia. Then, the data were subjected to correlation and linear discriminant analysis (LDA). The concentrations of cholic acid (CA), taurochenodeoxycholic acid (TCDCA) and taurodeoxycholic acid (TDCA) were significantly decreased in plasma of the schizophrenia patients. Correlation analysis showed the concentrations of CA, TCDCA and TDCA were negatively correlated with schizophrenia. In addition, LDA demonstrated that combination of CA, TCDCA and TDCA with a classification formula could predict correctly classified cases and the accuracy of prediction was up to 95.83%. Combination of the three BAs may be useful to diagnose schizophrenia in plasma samples.

Hippocampal mRNA expression profiling in mice exposed to chronic unpredictable mild stress.

Depression is a state of low mood and aversion to activity, affecting a person's thoughts, behavior, motivation, feelings and sense of well-being, which is associated with dramatical gene expression changes in hippocampus. Rodents induced by chronic unpredictable mild stress (CUMS) demonstrate typical depression-like behaviors similar to clinical patients, therefore, are commonly used as a model for depression and antidepressant study. In order to enhance our understanding of the molecular mechanisms of the pathogenesis of depression, in the present study, the hippocampal mRNA expression profile of mice exposed to CUMS for 5 weeks was sequenced using Illumina HiSeq 4000 platform followed by enrichment analysis, including Hierarchical Cluster, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, and protein-protein interaction (PPI) network. Totally, 346 differently expressed mRNAs, including 208 downregulated and 138 upregulated, were identified in the hippocampus of the CUMS mice. KEGG biological pathway analysis showed that the upregulated and downregulated mRNAs were mostly enriched in 29 pathways and 8 pathways, respectively. PPI network analysis exposed that glyceraldehyde 3-phosphate dehydrogenase was the crucial node with high connectivity degree. Additionally, most of these genes in PPI network analysis have previously been linked to energy metabolism and corticosterone responses. Overall, our results indicate the possible novel molecular targets for the therapy of depression.

Alpha-naphthylisothiocyanate-induced cholestatic mice display anxiety-like behavior closely related with enhanced serotoninergic signaling transduction in central nervous system.

Cholestasis is a pathophysiological process caused by the damage of hepatocytes or obstruction of bile flow, which often leads to emotional disorder in central nervous system. Alpha-naphthylisothiocyanate (ANIT) is the most widely used chemical to induce cholestatic models; however, the neurobehavior of ANIT-induced cholestatic model has not been investigated. The present study was designed to evaluate the anxiety-like behavior of cholestatic mice induced by a single (i.p.) injection of ANIT and its potential mechanism. For validating the model, the alanine aminotransferase, glutamic oxaloacetic transaminase, alkaline phosphatase, and total bile acid in the serum of mice were detected, and the pathological sections of hepatic lobes were also observed. After that, a series of behavioral tests were used to detect the anxiety-like behavioral changes of the ANIT-induced cholestatic mice, and then the level of 5-hydroxytryptamine and 5-hydroxyindole acetic acid in serum and prefrontal cortex were detected. Our data showed that ANIT-induced cholestatic mice exhibited increased anxiety-like behaviors in the open-field test and elevated plus maze test. Moreover, the concentration of 5-hydroxyindole acetic acid significantly decreased in the serum and the prefrontal cortex of ANIT-induced cholestatic mice compared with the control group. In addition, the expression of 5-hydroxytryptamine 1A, 5-hydroxytryptamine 2C, 5-hydroxytryptamine 3A, and 5-hydroxytryptamine 7 receptors increased in the prefrontal cortex of the model mice compared to their controls. Our results suggest that ANIT-induced cholestatic mice can display anxiety-like behavior closely related with enhanced serotoninergic signaling transduction in central nervous system.

Long-term stability and characteristics of behavioral, biochemical, and molecular markers of three different rodent models for depression.

OBJECTIVE: The present study was designed to explore the long-term differences between three mouse models for depression. METHOD: In the present study, the unpredictable chronic mild stress (UCMS) model, the glucocorticoid/corticosterone model, and the olfactory bulbectomy model were compared at two, three, and five weeks after model induction. Behavioral testing performed included forced-swimming, tail suspension, open-field and elevated plus-maze tests. In addition, 5-hydroxytryptamine (5-HT) and dopamine levels, and mRNA and protein expressions related to 5-HT synthesis, transport, and signaling were analyzed in the hippocampus of tested animals. RESULTS: Our results revealed that each model demonstrated a specific profile of markers, whereas the stability of them differed over testing time. CONCLUSIONS: Each model provided a unique set of advantages that can be considered depending on the context and aims of each study. Among the three models, the UCMS model was mostly stable and appeared to the best model for testing long-term depression-like state.

Prevalence of hepatitis C infection among intravenous drug users in Shanghai.

AIM: To characterize the prevalence of hepatitis C virus (HCV) infection among Chinese intravenous drug users (IDUs). METHODS: A total of 432 adult IDUs (95 women and 337 men) in Shanghai were included in the study. The third-generation Elecsys Anti-HCV assay (Roche Diagnostics GmbH, Sandhofer Strasse 116, D-68305, Mannheim, Germany) was used to screen for antibodies against HCV. The RIBA strip, a supplemental anti-HCV test with high specificity, was performed on all of the samples that tested positive during the initial screening. All of the anti-HCV positive samples were analyzed with a Cobas TaqMan 48 Analyzer (Roche Diagnostics) for direct detection of HCV RNA. All of the HCV RNA-positive samples were sequenced for genotype determination. RESULTS: The preliminary screening identified 262 (60.6%) subjects who were seropositive for HCV. Of the 62 females and 200 males seropositive subjects, 16 (16.7%) and 65 (19.3%), respectively, were confirmed by RIBA, yielding an overall HCV seropositive rate of 18.8%. Four female (6.5%) and 14 male (7.0%) subjects tested positive for HCV RNA, indicating an active infection rate of 4.2% for the entire study population. The 18 HCV RNA-positive serum samples were genotyped. Seven individuals were genotype 1b, and four were genotype 1a. One individual each was infected with genotypes 2a, 2b and 3a. Four subjects were co-infected with multiple strains: two with genotypes 1a and 2a, and two with genotypes 1b and 2a. The active infection rate among HCV-seropositive individuals was 22.2%, which was significantly lower than most estimates. CONCLUSION: The prevalence of HCV is relatively low among IDUs in Shanghai, with a spontaneous recovery rate much higher than previous estimates.

[Serological survey of Toxoplasma gondii infection among public health practitioners in Xuhui District of Shanghai].

448 public health practitioners in the district were selected randomly from Dec. 2010 to Mar. 2011. Blood specimens were collected and tested for anti-T. gondii IgG by ELISA. The result showed that the positive rate was 10.3% (46/448). No significant difference was found between males and females, so as different cities of origin (P > 0.05). The positive rate was higher in > or = 30 age group (14.9%, 29/195) than that in < 30 age group (6.7%, 17/253)(P < 0.05), and the highest sero-prevalence was recorded in 30-39 age group (15.8%, 16/101). The positive rate was higher in subjects engaged in the food production and processing enterprises (12.6%, 36/286) than those in other industries (6.2%, 10/162) (P < 0.05).

Modification of a human monoclonal antibody Fab fragment specific for Plasmodium falciparum 19-kDa C-terminal merozoite surface protein 1 by site-directed mutagenesis.

We recently produced human monoclonal antibody Fab fragments specific for the 19-kDa C-terminal merozoite surface protein 1 of Plasmodium falciparum in a bacterial expression system. The effect of single amino acid modifications in the third complementarity-determining regions of the heavy and light chains on affinity was examined in one of the Fab fragments, Pf25. Recombination polymerase chain reaction was used to modify Tyr(92) or Ile(97) in the light chain and Val(101) or Trp(107) in the heavy chain. No effective replacements for Tyr(92) and Val(101) were found, but possible substitutions of Ile(97) with Gly, Leu, Glu, Ala and Ser, and of Trp(107) with Arg and Ser were demonstrated. Of these modified Fab fragments, the affinities of Fabs with Ile(97)-Leu and Trp(107)-Ser mutations were slightly higher than that of the original Fab. The effects of these modifications on the antigen-antibody interaction are discussed.

Production of high-affinity human monoclonal antibody fab fragments to the 19-kilodalton C-terminal merozoite surface protein 1 of Plasmodium falciparum.

A combinatorial immunoglobulin gene library was constructed from peripheral blood lymphocytes of eight patients infected with Plasmodium falciparum and was screened for the production of human monoclonal antibody Fab fragments to the C-terminal 19-kDa fragment of P. falciparum merozoite surface protein 1 (MSP-1(19)). Three Fab clones recognized recombinant MSP-1(19) under nonreducing conditions. Indirect immunofluorescence microscopy demonstrated that three Fab clones stained the surfaces of late trophozoites/schizonts and merozoites of the FCR3 and 3D7 strains, suggesting the Fabs' reactivities to a conserved epitope. Sequence analysis of the heavy-chain genes revealed that the closest germ line V segments were VH1-8 and VH7-81, with 91% to 98% homology. The closest germ line D segment was D3-10, and the closest germ line J segment was JH4 or JH5, with 90% to 97% homology. In the light-chain genes, the closest germ line V segment was A27 for the Jkappa2, Jkappa4, and Jkappa5 segments. The dissociation constants of these Fab fragments for recombinant MSP-1(19) ranged from 1.09 x 10(-9) to 2.66 x 10(-9) M. The binding of the three Fab fragments to MSP-1(19) was competitively inhibited by the anti-MSP-1(19) mouse monoclonal antibody 12.8, which inhibits erythrocyte invasion by merozoites. However, the human Fab fragment with the highest affinity did not inhibit in vitro growth of P. falciparum. This is the first report of gene analysis and bacterial expression of human monoclonal antibodies to P. falciparum MSP-1(19). The combinatorial immunoglobulin gene library derived from malaria patients provides a potential tool for producing high-affinity human antibodies specific for P. falciparum.