The synergistic anti-nociceptive effects of nefopam and gabapentinoids in inflammatory, osteoarthritis, and neuropathic pain mouse models.

Pain is a common public health problem and remains as an unmet medical need. Currently available analgesics usually have limited efficacy or are accompanied by many adverse side effects. To achieve satisfactory pain relief by multimodal analgesia, new combinations of nefopam and gabapentinoids (pregabalin/gabapentin) were designed and assessed in inflammatory, osteoarthritis and neuropathic pain. Isobolographic analysis was performed to analyze the interactions between nefopam and gabapentinoids in carrageenan-induced inflammatory pain, mono-iodoacetate-induced osteoarthritis pain and paclitaxel-induced peripheral neuropathic pain in mice. The anti-inflammatory effect and motor performance of monotherapy or their combinations were evaluated in the carrageenan-induced inflammatory responses and rotarod test, respectively. Nefopam (1, 3, 5, 10, 30 mg/kg, p.o.), pregabalin (3, 6, 12, 24 mg/kg, p.o.) or gabapentin (25, 50, 75, 100 mg/kg, p.o.) dose-dependently reversed mechanical allodynia in three pain models. Isobolographic analysis indicated that the combinations of nefopam and gabapentinoids exerted synergistic anti-nociceptive effects in inflammatory, osteoarthritis, and neuropathic pain mouse models, as evidenced by the experimental ED50 (median effective dose) falling below the predicted additive line. Moreover, the combination of nefopam-pregabalin/gabapentin alleviated carrageenan-induced inflammation and edema, and also prevented gabapentinoids-related sedation or ataxia by lowering their effective doses. Collectively, the co-administration of nefopam and gabapentinoids showed synergistic analgesic effects and may result in improved therapeutic benefits for treating pain.

[Genotype and Phenotype of α-Thalassemia Fusion Gene in Huadu District of Guangzhou, Guangdong Province of China].

OBJECTIVE: To explore the carrier rate, genotype and phenotype of α-thalassemia fusion gene in Huadu district of Guangzhou, Guangdong province of China, and provide data reference for the prevention and control of thalassemia. METHODS: A total of 10 769 samples who were screened for thalassemia in Maternal and Child Health Hospital of Huadu District from July 2019 to November 2020 were analyzed retrospectively. Blood cell analysis and hemoglobin (Hb) electrophoresis were performed. Thalassemia genes were analyzed by gap-PCR and PCR-reverse dot blot hybridization (PCR-RDB). RESULTS: A total of 9 cases with α-thalassemia fusion gene were detected in 10 769 samples (0.08%). There were 7 cases with fusion gene heterozygote, 1 case with compound of α-thalassemia fusion gene and Hb G-Honolulu, 1 case with compound of α-thalassemia fusion gene and Hb QS. The MCV results of 4 samples of blood cell analysis were within the reference range, the Hb A2 value of 1 case was decreased, and there were no other abnormalities found. CONCLUSION: The α-thalassemia fusion gene is common in Huadu district of Guangzhou, and heterozygotes are more common, and current screening methods easily lead to misdiagnosis.

Vimentin as a potential target for diverse nervous system diseases.

Vimentin is a major type III intermediate filament protein that plays important roles in several basic cellular functions including cell migration, proliferation, and division. Although vimentin is a cytoplasmic protein, it also exists in the extracellular matrix and at the cell surface. Previous studies have shown that vimentin may exert multiple physiological effects in different nervous system injuries and diseases. For example, the studies of vimentin in spinal cord injury and stroke mainly focus on the formation of reactive astrocytes. Reduced glial scar, increased axonal regeneration, and improved motor function have been noted after spinal cord injury in vimentin and glial fibrillary acidic protein knockout (GFAP-/-VIM-/-) mice. However, attenuated glial scar formation in post-stroke in GFAP-/- VIM-/- mice resulted in abnormal neuronal network restoration and worse neurological recovery. These opposite results have been attributed to the multiple roles of glial scar in different temporal and spatial conditions. In addition, extracellular vimentin may be a neurotrophic factor that promotes axonal extension by interaction with the insulin-like growth factor 1 receptor. In the pathogenesis of bacterial meningitis, cell surface vimentin is a meningitis facilitator, acting as a receptor of multiple pathogenic bacteria, including E. coli K1, Listeria monocytogenes, and group B streptococcus. Compared with wild type mice, VIM-/- mice are less susceptible to bacterial infection and exhibit a reduced inflammatory response, suggesting that vimentin is necessary to induce the pathogenesis of meningitis. Recently published literature showed that vimentin serves as a double-edged sword in the nervous system, regulating axonal regrowth, myelination, apoptosis, and neuroinflammation. This review aims to provide an overview of vimentin in spinal cord injury, stroke, bacterial meningitis, gliomas, and peripheral nerve injury and to discuss the potential therapeutic methods involving vimentin manipulation in improving axonal regeneration, alleviating infection, inhibiting brain tumor progression, and enhancing nerve myelination.

Hb Huadu [α124(H7)Ser→Thr (TCC>ACC), HBA2: c.373T>A]: A Novel Variant of the α-Globin Gene.

Here, we report a novel α chain hemoglobin (Hb) variant found during routine thalassemia screening. This Hb variant can be detected by capillary electrophoresis (CE) but cannot be recognized by high performance liquid chromatography (HPLC). Sanger sequencing revealed a heterozygous missense substitution at nucleotide 373 on the HBA2 gene, which results in the replacement of serine by threonine at codon 124 [α124(H7)Ser→Thr (TCC>ACC), HBA2: c.373T>A]. It is the first report of this variant, named Hb Huadu for the birthplace of the proband. In addition, the proband coinherited the heterozygous codons 41/42 (-TTCT) (HBB: c126_129delCTTT) on the ß-globin gene.

[Molecular Epidemiological Characteristics and Differential Diagnosis of Common 뫧-Thalassemia/HPFH].

OBJECTIVE: To investigate the molecular epidemiological characteristics of common δß-thalassemia/hereditary persistence of fetal hemoglobin(HPFH) in the prepregnant population in Huadu, and to provide a laboratory basis for prevention and control of thalassemia. METHODS: Blood samples of childbearing age people in Huadu District of Guangzhou who participated in free thalassemia testing from January 2016 to July 2021 were collected for hematological parameters analysis and hemoglobin electrophoresis. Chinese Gγ+(Aγδß)0-thalassemia, SEA-HPFH and Taiwanese deletion ß-thalassemia were detected by Gap-PCR in the samples with higher HbF(≥5%). Primers were designed for the proximal HBG1 and HBG2 promoter, and the point mutations in the proximal promoter region were detected by Sanger sequencing. Hematology parameters data were statistically analyzed. RESULTS: Among 27 088 samples, Thirteen cases of Chinese Gγ+(Aγδß)0-thalassemia and thirty-three cases of SEA-HPFH were detected, which including 3 cases of Chinese Gγ+(Aγδß)0/ßN compounded with --SEA/αα and three cases of SEA-HPFH/ßN compounded with --SEA/αα. 6 carriers with Aγ-196 C>T were also detected; No Taiwanese thalassemia genetype was detected. The total detection rate of common δß-thalassemia/HPFH was 0.19% (52/27 088). There were significant differences in the levels of MCV, MCH, HbA2, and HbF among Chinese Gγ+(Aγδß)0-thalassemia, SEA-HPFH, Aγ-196 C>T (P<0.001). The hematological parameters of Aγ-196C>T combined with α0-thalassemia were similar to those of Chinese Gγ+(Aγδß)0-thalassemia carriers, and only HbA2 was significantly lower than that of the latter, which was helpful for clinical identification. CONCLUSION: δß-thalassemia/HPFH should be included in the scope of thalassemia prevention program in the prepregnant population in Huadu District, and hematological parameters can provide some basis for identifying different types of δß-thalassemia/HPFH.

[Protective efficacy induced by dendritic cells pulsed with GST in combination with CpG oligodeoxynucleotide against Schistosoma japonicum infection].

OBJECTIVE: To study the protective effects on the infection of Schistosoma japonicum in C57BL/6 mice induced by dendritic cells DCs pulsed with GST in combination with CpG oligodeoxynucleotide. METHODS: GST was purified and used to stimulate DC2.4 cell line. Antigen loading was analyzed by immunofluorescence method. Thirty-five C57BL/6 mice were divided into seven groups(5 mice per group). Mice in groups A, B, C, D and E were immunized subcutaneously with DCs, DCs treated with PSA, DCs pulsed with GST, DCs stimulated with GST+CpG ODN, DCs stimulated with CpG ODN, respectively. For the above five groups, each mouse received 100 microl cell suspension at the density of 10(7)/ml subcutaneously for three times at 2-week intervals. Each mouse of group F was immunized subcutaneously with 50 microg GST formulated in complete Freund's adjuvant first, and 50 microg, 10 microg GST respectively in incomplete Freund's adjuvant for the last two doses. Group G received PBS and served as control. Serum samples were collected 10 days after the final immunization, and were analyzed for specific antibodies by ELISA. At two weeks after the final immunization, each mouse were challenged by 30 +/- 1 cercariae of S. japonicum. Six weeks after infection the mice were sacrificed, and number of worms was counted. RESULTS: Light green fluorescence was observed in dendritic cells under the fluoroscope after pulsing with GST which indicated the protein loaded dendritic cells. The IgG level in groups C, D and F was 0.555 2 +/- 0.078 9, 0.715 0 +/- 0.052 3, and 2.127 0 +/- 0.411 5, respectively, all higher than that of group G (P < 0.05). The worm reduction rate of group D was 53.3%, followed by group F (24.0%) and group C (21.3%). There was no significantly difference in the worm reduction rate between group D and groups F and C (P > 0.05). CONCLUSION: Dendritic cells pulsed with GST in combination with CpG oligodeoxynucleotide induce significant immunoprotection against the infection of Schistosoma japonicum.

[Cloning, expression and analysis of the heat shock protein of Cryptosporidium andersoni].

OBJECTIVE: To clone and express the partial encoding sequence of Mr 70,000 heat shock protein of Cryptosporidium andersoni (CaHSP70) in Escherichia coli and identify the recombinant protein. METHODS: Total RNA was extracted from oocysts of C. andersoni isolated from Xuzhou, Jiangsu (XZ-BOV). The CaHSP70 gene was amplified by RT-PCR. The PCR product was cloned and then subcloned into pET28a vector, and the recombinant plasmids were transformed into E.coli BL21(DE3) subsequently. The expressed protein induced by IPTG was purified and identified by SDS-PAGE and Western blotting, and was further analyzed by relevant bioinformatics softwares. The specific IgG antibodies in mice immunized by rCaHSP70 were detected by Western blotting and ELISA respectively. RESULTS: The deduced amino acid sequence showed to be identical with that of C. andersoni Mr 70,000 heat shock protein (HSP70). The recombinant protein expressed in the form of inclusion body was about Mr 43,000. It could be recognized by anti-His G labeled HRP antibodies and all the sera from mice infected with C. andersoni and children infected with C. parvum as well as sera from mice immunized with rCaHSP70 respectively. The rCaHSP70 possibly had multiple domains and potential antigenic determinants. Phylogenetic analysis showed that XZ-BOV and C. andersoni were in the same clade. ELISA showed that the level of specific antibodies against rCaHSP70 in immunized BALB/c and C57BL/6 mice was significantly higher than that of mice before immunization. CONCLUSION: The recombinant plasmid pET28a-CaHSP70 has been constructed. The purified rCaHSP70 exhibits high antigenicity and seems a potential candidate antigen for immunodiagnosis of cryptosporidiosis.

[Isolation and identification of an isolate of cow-origin Cryptosporidium sp].

OBJECTIVE: To isolate and identify Cryptosporidium oocysts from feces of naturally infected cow. METHODS: Fecal samples were collected from Cryptosporidium infected cows confirmed by modified acid-fast staining method. Oocysts were isolated and purified with Sheathed sucrose density gradient centrifugation technique. Genomic DNA was isolated with Chelex-100. Both primers were designed to amplify Cryptosporidium small subunit ribosome RNA gene (SSU rRNA) and Cryptosporidium oocyst wall protein gene (COWP), respectively. The PCR products were cloned into pGEM-T and pGEM-T Easy vector and sequenced subsequently. Homology and phylogeny were analyzed with BLASTn and MEGA software. RESULTS: The results suggested that the size of oocysts was (7.4+/-0.32) microm by (5.4+/-0.21) microm and the ratio of length and width was 1.37+/-0.07 (n=20). BLASTn revealed that the identity of SSU rRNA and COWP gene of Cryptosporidium isolated from cow to the counterparts of andersoni was 100% and 99% respectively. Phylogenetic reconstruction placed the isolated Cryptosporidium within the C. andersoni clade based on the sequence of SSU rRNA and COWP gene. CONCLUSION: What isolated from naturally infected cow feces has been identified as C. andersoni.

[Protective immunity induced by recombinant Schistosoma japonicum thioredoxin in mice].

OBJECTIVE: To investigate the protective immunity of the recombinant thioredoxin of Schistosoma japonicum(reSjcTrx)in mice. METHODS: Thirty 6-week old female C57BL/6 mice were randomly divided into 3 groups with 10 each: reSjcTrx with Montanide ISA720 adjuvant, adjuvant control, and infection control. Mice were vaccinated subcutaneously at week 0, 2, 4 with reSjcTrx emulsified in Montanide ISA720 adjuvant. The mice in adjuvant group was injected three times with Montanide ISA720 and saline only. Mice in infection control group were given no injection. Three weeks after final injection, each mouse was challenged with 30 +/- 1 cercariae of S. japonicum (Chinese strain). At the week six after challenge, all mice were sacrificed and perfused. The number of recovered worms and eggs from liver tissue of mice were counted. Sera were collected from mice before immunization, before challenge and before killing. The anti-SjcTrx antibodies in sera were detected with ELISA. RESULTS: ELISA showed a high level of specific IgG antibodies in mice immunized with the reSjcTrx. The worm reduction rate and egg reduction rate of reSjcTrx immunization group were 22.8% and 29.5% respectively, significantly higher than those of the control groups (P < 0.05). SDS-PAGE and Western blotting revealed that the molecular weight of expressed protein was around Mr 14 000 and could be recognized by sera from rabbit infected with S. japonicum and from mice immunized with reSjcTrx. CONCLUSION: The reSjcTrx induces certain protective immunity against schistosomiasis japonica in mice.

[Anti-schistosomiasis effect induced by full length DNA vaccine coding paramyosin of Schistosoma japonicum in C57BL/6 mice].

OBJECTIVE: To observe the anti-schistosomiasis effect in mice immunized with Sjc97 DNA vaccine. METHODS: C57BL/6 mice were vaccinated intramuscularly with the Sjc97 DNA twice at an interval of 3 weeks, and challenged with 30 +/- 2 cercariae of Schistosoma japonicum three weeks after immunization. Mice in blank plasmid vector control and infection control groups were also infected with same number of cercariae. The mice were sacrificed 7 weeks after challenge infection. The size of single egg granulomas in livers was measured with micrometer. The level of hyaluronic acid (HA) and laminin (LN) in sera of the mice was determined by ELISA. PCR-ELISA was used to examine the expression of TGF-beta1 mRNA in liver. RESULTS: The mean hepatic egg granuloma diameters of the three groups, the Sjc97 DNA, blank plasmid vector and infection control, were 183.75 +/- 42.36 microns, 303.12 +/- 37.36 microns and 304.38 +/- 53.23 microns, respectively. The hepatic granuloma was significantly smaller in the Sjc97 DNA group than that in control. The level of HA and LN in sera of Sjc97 DNA vaccinated mice was markedly lower than those in the two control groups (P < 0.01). The amount of TGF-beta1 mRNA isolated from the livers of mice in Sjc97 DNA group decreased significantly. CONCLUSION: The results showed that Sjc97 DNA vaccine may act as an effective inhibitor against formation of egg granuloma and reduce immunopathological damage caused by Schistosoma japonicum in the host.

[Preparation of DNA from Cryptosporidium parvum oocysts for PCR detection].

OBJECTIVE: To establish three methods of DNA extraction from Cryptosporidium parvum oocysts and test by PCR. METHODS: After three freeze-thaw cycles, three kinds of templates were extracted from the oocysts by Chelex-100, phenol/chloroform or genomic DNA purification system kit, and used for PCR detection. According to the sequence of a C. parrum gene (L.16996), a pair of primers was designed and synthesized, and used for PCR. The sensitivity of the template by Chelex 100 method was also tested by PCR. RESULTS: One 446 bp PCR product was observed by agarose gel electrophoresis for all three kinds of templates. The PCR sensitivity by Chelex-100 extracted DNA reached for detection of a specimen containing only 1/2 oocyst. CONCLUSION: The three kinds of extraction can all be served as templates for PCR detection of C. parvum oocysts, while Chelex 100 method is simpler, quicker and more reliable for DNA extraction of the parasite.

[Immunological characteristics induced in mice by nucleic acid vaccine harboring the gene encoding GAPDH in Schistosoma japonicum].

OBJECTIVE: To study the immunological characteristics induced in C57BL/6 mice by nucleic acid vaccine harboring the gene encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of Schistosoma japonicum. METHODS: The gene encoding GAPDH of S. japonicum from screening of cDNA library was amplified using universal primer T3 promoter and T7 promoter, the PCR product was cloned into the T-A vector pT-Adv. GAPDH-pT-Adv and eukaryotic expression vector pcDNA3 were digested by restriction endonucleases Hind III and Xho I, and the nucleic acid vaccine harboring the gene encoding GAPDH was then constructed by ligating the digested products. C57BL/6 mice were immunized using the purified pcDNA3-SjGAPDH. The expression of GAPDH in local muscle in mice was examined by immunofluorescence assay. The specific characteristics induced by pcDNA3-SjGAPDH were analyzed using SDS-PAGE, Western blotting, and ELISA respectively. RESULTS: Light green fluorescence was observed in local muscle cell under the fluoroscope 24 and 48 hours after immunization, which indicated the expression of GAPDH. The recombinant plasmid induced specific anti-GAPDH IgG, predominantly IgG2a, IgG2b and IgG3. Cytokines IFN-gamma, IL-2 but IL-4 were detected in C57BL/6 mice vaccinated with the vaccine. CONCLUSION: The DNA vaccine, pcDNA3-SjGAPDH induces Th1 type immune response in mice.

[Screening of cDNA library of Schistosoma japonicum with sera from rabbits vaccinated with ultraviolet-attenuated schistosomula].

OBJECTIVE: To search new potential schistosomiasis vaccine by screening cDNA library with sera of rabbits vaccinated with attenuated larvae. METHODS: Schistosoma japonicum (Sj) adult worm cDNA library was screened with sera of rabbits vaccinated with ultraviolet-attenuated schistosomula and by sera of infected rabbits, and the inserts of positive clones were amplified and sequenced. RESULTS: Six kinds of Sj genes were obtained after three rounds of screening. Among the genes were glyceraldehyde 3-phosphate dehydrogenase (GAPDH), serine protease inhibitors (serpin), mitochondrion coding region, part of myosin heavy chain gene and two new genes, respectively. CONCLUSION: Screening cDNA library with sera of animals vaccinated with attenuated larvae is an effective way to search new vaccine candidates for schistosomiasis.

[Schistosoma japonicum: screening of cDNA library with sera from self-cured water buffaloes (Bos buffelus)].

OBJECTIVE: To understand and identify the molecules related to the self-cure of Schistosoma japonicum infection in water buffaloes. METHODS: The S. japonicum adult worm cDNA library was immunologically screened with the sera of self-cured water buffaloes. The positive clones were identified, cloned, sequenced and analysed with software. RESULTS: Three genes encoding antigens relevant to sera antibodies in water buffaloes were cloned and sequenced. These antigens included paramyosin (Sj97), GST, carbonyl reductase-like 20-beta-hydroxysteroid dehydrogenase(CR/20-beta-HSD). CONCLUSION: Sera from self-cured S. japonicum infected water buffaloes can be used to screen adult worm cDNA library for vaccine development.

[Preliminary observation on specific antibody level and reduction of ovulation induced by recombinant Schistosoma japonicum 26 kDa GST antigen in water buffaloes].

OBJECTIVE: To observe the specific antibody level and reduction of egg-laying induced by a recombinant Schistosoma japonicum 26 kDa GST antigen (rSjc26 GST) in water buffaloes. METHODS: 20 water buffaloes were randomly divided into two groups, the vaccination group and control group, with 10 buffaloes each. The subjects in vaccination group were immunized with rSjc26 GST antigen while the control received adjuvant only. After challenged with S. japonicum cercariae, the anti-rSjc26 GST antibody level and the numbers of eggs and miracidia in stool were detected. RESULTS: The anti-rSjc26 GST antibody appeared 1 month after immunization with rSjc26 GST antigen and maintained a high level for 12 months. Numbers of eggs (EPG) and miracidia (MPG) in vaccination group were significantly lower than those in control group during the period of day 50 to day 90 post-challenge. However, EPG and MPG tended to decrease starting from day 100 post challenge in both groups. The difference of EPG and MPG between the two groups diminished progressively, and both groups showed zero egg count from day 330 on post challenge. CONCLUSION: The specific anti-rSjc26 GST antibody was detected in vaccinated water buffaloes and maintained a high level for 12 months. The vaccination showed a significant effect to the reduction of ovulation in the first three months after S. japonicum cercariae challenge.