[Association of Breg cells and Treg cells with the clinical effects of Infliximab in the treatment of Chinese patients with Crohn's disease].

Objective: To explore the associations of regulatory B cells (Breg cells) and regulatory T cells (Treg cells) with the clinical effect of Infliximab in the treatment of Chinese patients with Crohn's disease (CD). Methods: From January 2017 to June 2019, a total of 32 CD patients at active stage and 33 age and gender-matched healthy controls were collected from the Second Affiliated Hospital of Wenzhou Medical University in this study. Approximate 5 ml of peripheral fasting venous blood was obtained from every subject. Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood. Then multi-color flow cytometry was applied to determine the proportion of Breg (CD3-CD19+IL-10+B cells) in B cells and the proportion of Treg (CD4+CD25+Foxp3+T cells) in CD4+T cells. Infliximab (5 mg/kg) was given intravenously at week 0, 2 and 6 to induce CD remission, and then maintained with the same dose of Infliximab every 8 weeks. And the proportions of Breg and Treg were examined at week 14 of Infliximab treatment, then compared with those of week 0. Simultaneously, C-reactive protein (CRP), leucocyte count, platelet count, erythrocyte sedimentation rate were detected in CD patients to assess the clinical effect at week 0 and 14 of Infliximab treatment. Results: Before infliximab treatment, compared with healthy controls, the proportion of Breg in B cells was significantly increased [(3.15±1.17)% vs (2.64±0.38)%, P=0.024)], and the proportion of Treg in CD4+T cells was significantly decreased [(2.15±0.49)% vs (4.25±0.41)%, P<0.001] in CD patients. And the proportion of Breg was positively related with the proportion of Treg in CD patients either at week 0 or week 14 of Infliximab treatment (r=0.628, P<0.001; r=0.749, P<0.001). At week 14 of Infliximab treatment, according to symptoms, Crohn's disease activity index (CDAI) and endoscopic mucosal healing, CD patients were classified as remission group (CDAI<150 and endoscopic mucosal healing, R group) and non-remission group (CDAI≥150 or mucosal non-healing group, N group). Compared with CD patients at week 0 of Infliximab treatment, both the proportion of Breg and Treg were significantly enhanced [(5.89±2.60)% vs (3.19±1.27)%, P<0.001; (4.59±0.72)% vs (2.08±0.47)%, P<0.001], whereas CDAI and CRP was significantly reduced [CDAI: (63.19±14.69) vs (195.62±58.13), P<0.001; CRP: (3.65±2.23) mg/L vs (29.80±30.06) mg/L, P<0.001] in R group at week 14 of Infliximab treatment. The proportions of Breg and Treg were negatively related with the CRP (r=-0.279, P=0.026; r=-0.406, P=0.001) and CDAI (r=-0.409, P=0.001; r=-0.708, P<0.001) in CD patients at week 0 and 14 of Infliximab treatment. At week 14 of Infliximab treatment, ROC curve analysis showed that the predictive value of "Breg+Treg" for the effect of Infliximab was higher than the other parameters (area under ROC: 0.782, cutoff value: 0.895 5, P=0.034). Conclusions: Breg cells and Treg cells are not only significantly correlated with CD disease activity, but the combined detection of the two types of immune cells has higher clinical value for predicting the effect of Infliximab in CD patients at active stage.

[Impact of TRAIL deficiency on Th17 cells in the mice experimental colitis induced by dextran sodium sulphate].

Objective: To investigate the impact of TNF-related apoptosis-inducing ligand (TRAIL) gene knock-out (TRAIL(-/-)) on Th17 cells in the mice colitis induced by dextran sulphate sodium (DSS). Methods: Mice were randomly assigned to 4 subgroups: wild type (WT), TRAIL(-/-), WT colitis and TRAIL(-/-)colitis (n=6/group). Colitis was induced by oral administration of 3.5% DSS for 7 consecutive days. The severity of colitis in each mouse was scored both clinically and histopathologically. Flow cytometry was performed to assess Th17 cell population in peripheral blood mononuclear cells (PBMC) and mesenteric lymph nodes (MLN). The expression levels of Th17 cell markers interleukin (IL)-17A and retinoic acid-related orphan receptor (ROR)-γt in PBMC and MLN were also examined using a quantitative polymerase chain reaction method. Results: Compared with WT group, WT colitis group displayed elevated CD4(+)IL-17A(+) Th17 cells (0.29±0.07 vs 0.08±0.03, 1.20±0.36 vs 0.40±0.11, both P<0.05) and enhanced mRNA expression of IL-17A and ROR-γt in PBMC and MLN (IL-17A: 2.43±0.87 vs 0.37±0.19, 5.03±1.77 vs 1.05±0.48, both P<0.05; ROR-γt: 2.49±0.48 vs 0.93±0.47, 23.75±7.60 vs 1.31±0.90, both P<0.05). After the DSS administration, TRAIL(-/-) group developed more severe colitis than WT group, mainly manifesting higher disease activity index, reduction of colon length and increased infiltration of inflammatory cells. In addition, TRAIL(-/-) colitis group exhibited increased proportion of CD4(+) IL-17A(+) Th17 cells (0.57±0.22 vs 0.29±0.07, P<0.001; 2.92±0.98 vs 1.20±0.36, P<0.000 1) as well as enhanced mRNA expression of IL-17A and ROR-γt in PBMC and MLN when compared with WT colitis group (IL-17A: 4.10±1.96 vs 2.43±0.87, 15.88±2.86 vs 5.03±1.77, both P<0.05; ROR-γt: 4.05±0.62 vs 2.49±0.48, 69.61±10.48 vs 23.75±7.60, both P<0.05). Conclusions: TRAIL deficiency not only promots the number and activity of Th17 cells in PBMC and in MLN, but also aggravats DSS-induced colitis in mice.

[Association of Crohn's disease with aryl hydrocarbon receptor gene polymorphisms and haplotypes].

Objective: To explore the relationship of Crohn's disease (CD) susceptibility to aryl hydrocarbon receptor (AhR) polymorphisms and haplotypes in Han population in Wenzhou city, China. Methods: A total of 310 CD patients and 573 age- and sex-matched healthy controls were enrolled in our study. Three single nucleotide polymorphisms (SNPs) of AhR(rs10249788,rs2158041,rs2066853) were determined by the improved multiple ligase detection reaction technique. Unconditional logistic regression analyses was applied to analyze the allelic and genotypic differences of each SNP between CD patients and controls, as well as their influence on the clinicopathologic characteristics in CD patients. Analyses of linkage disequilibrium and haplotype were performed by Haploview 4.2 software in all study subjects. Results: Compared with the controls, the variant allele (T) and genotype (CT+TT) of (rs2158041) were evidently decreased among CD patients (19.52% vs. 25.04%, P=0.009; 34.19% vs. 44.68%, P=0.003). According to "the Montreal Classification Standards" , CD patients were divided into different subgroups. The variant allele (T) and genotype (CT+TT) of (rs2158041) were significantly lower in patients with terminal ileum CD than in controls (16.79% vs. 25.04%, P=0.005; 28.24% vs. 44.68%, P=0.001). Similar conclusions were also drawn in patients with constricting disease when compared with the controls (15.20% vs. 25.04%, P=0.003; 28.43% vs. 44.68%, P=0.003). The three SNPs above were shown to be in a linkage disequilibrium. Compared with the controls respectively, the frequency of haplotype (CCG) was increased in CD patients (44.73% vs. 39.60%, P=0.039), whereas that of haplotype (CTG) was decreased (18.02% vs. 22.78%, P=0.047). Conclusions: AhR (rs2158041) variation might influence the risk as well as the location and behavior of CD. The haplotype (CCG) possibly increase the risk of CD development, whereas haplotype (CTG) might decrease it.

[Association of Crohn's disease with T cell immunoglobulin and mucin domain 3 gene polymorphisms in Chinese patients].

Objective: To explore the association of Crohn's disease (CD) with T cell immunoglobulin and mucin domain 3 (Tim-3) gene polymorphisms in patients of Zhejiang Han population in China. Methods: A total of 308 CD patients and 573 age- and sex-matched healthy controls were enrolled in our study. Two single nucleotide polymorphisms (SNPs) of Tim-3 (rs1036199 and rs10515746) were examined by the improved multiple ligase detection reaction technique (iMLDR). Analyses of linkage disequilibrium and haplotype were also performed by Haploview 4.2 software in all study subjects. Results: In general, the allele and genotype frequencies of Tim-3 (rs1036199 and rs10515746) were not statistically different between CD patients and the controls (all P>0.05). According to "the Montreal Classification" , CD patients were divided into different subgroups. The variant allele (C) and genotype (AC+ CC) of rs1036199 were more frequent in CD patients with penetrating diseases than in the controls (10.4% vs 1.7%, P=0.002; 20.8% vs 3.5%, P=0.023). Similar conclusions were also drawn for the variant allele (A) and genotype (CA+ AA) of rs10515746 in patients with penetrating diseases when compared with the controls (10.4% vs 2.2%, P=0.000; 20.8% vs 4.2%, P=0.033, respectively). The two SNPs of Tim-3 were in strong linkage disequilibrium (D'=1.0, r2=0.928). The haplotype (AC) formed by their wild-type alleles (A) and (C) was decreased in patients with penetrating CD compared with the controls (89.6% vs 98.3%, P=0.000). However, the haplotype (CA) formed by their variant alleles was more frequent in patients with penetrating CD than in the controls (10.4% vs 1.6%, P=0.000). Conclusions:Tim-3 (rs1036199 and rs10515746) variations might be correlated with the enhanced risk of penetrating diseases in CD patients. Furthermore, the haplotype (AC) and (CA) formed by the two SNPs might be a protective and a risky factor for penetrating CD respectively.

[Association of ulcerative colitis with fork head/winged helix transcription factor-3 gene polymorphisms in Chinese patients].

Objective: To investigate the association of ulcerative colitis (UC) with fork head/winged helix transcription factor-3 (Foxp3) polymorphisms in Han population in Zhejiang province, China. Methods: A total of 381 UC patients and 490 healthy controls were enrolled in this study.The four single nucleotide polymorphisms (SNPs) of Foxp3 (rs3761547, rs2232365, rs2294021, rs3761548) were examined by SNaPshot.The analyses of linkage disequilibrium (LD) and haplotype were also performed in all study subjects. Results: When male and female UC patients were compared with their corresponding controls respectively, the alleles and genotypes of the four SNPs were not statistically different (all P>0.05). According to severity and location of the disease, the UC patients were divided into different subgroups. The alleles (C, G, A) of (rs2232365, rs2294021, rs3761548) were more frequent in male patients with severe UC than in the male controls (69.6% vs 34.3%, P=0.001; 69.6% vs 34.3%, P=0.001; 39.1% vs 14.4%, P=0.002, respectively). As compared with the female controls, the alleles (C, G, A) and genotypes (TC+ CC, AG+ GG, CA+ AA) of (rs2232365, rs2294021, rs3761548) were significantly increased in the female patients with severe UC (51.9% vs 38.0%, 63.5% vs 39.2%, 53.8% vs 21.4%, 80.8% vs 57.7%, 84.6% vs 58.4%, 76.9% vs 34.7%, all P<0.05). The four SNPs above were shown to be in a strong LD both in male and in female subjects.When male and female UC patients were compared with their corresponding controls respectively, nevertheless, each haplotype frequency was not statistically different (all P>0.05). Conclusions:Foxp3 (rs2232365, rs2294021, rs3761548) variations might engender the increased risk of severe UC in Chinese Han patients.

[Association of Crohn's disease with transcobalamin â ¡ gene polymorphisms and serum homocysteine, folate and vitamin B12 levels in Chinese patients].

OBJECTIVE: To investigate the association of Crohn's disease (CD) with transcobalamin Ⅱ (TCN2) gene polymorphisms and serum homocysteine (Hcy), folate, and vitamin B12 levels in Chinese patients. METHODS: In a total of 307 CD patients (CD group) and 574 healthy controls (control group) of Han ethnicity selected at 4 hospitals in Wenzhou between January 2007 and August 2015, TCN2 gene polymorphisms (rs1801198, C/G; rs9606756, A/G) in peripheral blood were determined using improved multiple ligase detection reaction (iMLDR). The Simplified Crohn's Disease Activity Index (CDAI) was applied to evaluate activity of CD. Eighty-eight CD patients and 138 age- and sex-matched controls were randomly selected from all the study subjects using computer-generated random numbers, in whom serum Hcy level was tested by enzymatic cycling assay, folate and vitamin B12 levels were examined by chemiluminescence immunoassay. RESULTS: (1) The mutant allele (G) and genotype (CG+ GG) of rs1801198 were not significantly different between the CD group and the control group (both P>0.05), but more prevalent in patients with stricturing CD than in controls (65.75% vs 56.10%, 93.15% vs 82.40%, both P<0.05). The frequencies of mutant allele (G) and heterozygous genotype (AG) of rs9606756 were higher in CD patients than in controls (2.44% vs 1.05%, 4.89% vs 2.09%, both P<0.05), also higher in patients with stricturing CD or ileocolonic CD compared with controls (stricturing CD: 4.79% vs 1.05%, 9.59% vs 2.09%; ileocolonic CD: 3.18% vs 1.05%, 6.36% vs 2.09%; all P<0.05). The two polymorphic loci (rs1801198 and rs9606756) were shown to be in a weak linkage disequilibrium. The frequency of haplotype (GG) was higher in the CD patients than in the controls (2.23% vs 0.81%, P<0.05). (2) When compared with the controls, the serum Hcy level was higher in the CD patients (P=0.023), whereas the folate and vitamin B12 levels were decreased in the CD patients (both P<0.001). The prevalence of hyperhomocysteinemia (Hcy >15 µmol/L), folate deficiency (folate <4 µg/L) and vitamin B12 deficiency (<203 ng/L) were higher in the CD patients than in the controls (18.18% vs 4.35%, 27.27% vs 5.07%, 31.82% vs 5.07%, all P<0.01). Compared to the patients at remission stage (CDAI<5), the serum Hcy level was increased in those at active stage (CDAI≥5) (P=0.005), while the folate and vitamin B12 levels were decreased in the patients ate active stage (both P<0.05). (3) According to the results of multivariate linear regression analysis, average Hcy level in the CD patients was negatively associated with folate level (ß=-0.494, P<0.001), while positively correlated with percentage of neutrophils (ß=0.294, P=0.004). Unconditional Logistic regression showed that both folate deficiency and vitamin B12 deficiency were independent risk factors for CD (OR=5.415, OR=7.112, both P<0.001). CONCLUSIONS: TCN2 rs1801198 mutation might be associated with increased risk of stricturing CD. TCN2 rs9606756 mutation might be a potential functional locus to affect CD susceptibility. Individuals carrying the haplotype (GG) formed by TCN2 (rs1801198 and rs9606756) seem to be at higher risk of developing CD. Hyperhomocysteinemia, folate deficiency, and vitamin B12 deficiency are prevalent in this cohort of CD patients of Han ethnicity from Zhejiang province. Both folate deficiency and vitamin B12 deficiency are independent risk factors for CD.

Neuroprotective effects of argatroban and C5a receptor antagonist (PMX53) following intracerebral haemorrhage.

Intracerebral haemorrhage (ICH) is a subtype of stroke that associated with neurological dysfunction and inflammation, which may be ameliorated by a neuroprotective strategy targeting the complement cascade. The protective effect of C5a-receptor antagonist (PMX53) solely and in combination with thrombin antagonist (argatroban) was investigated in the ICH mouse model, respectively. Adult male C57BL/6J wild-type (WT) mice and C3(-/-) mice were randomized to receive PMX53/argatroban 1, 3 and 5 days after ICH. A double injection technique was used to infuse 25 µl of autologous whole blood into the right striatum. Mice in the sham group received only needle insertion. Brain water content and mRNA of inflammatory factors were measured on the first, third and fifth days after ICH, respectively. Neurological dysfunction was assessed using a 28-point neurological scoring system in the three cohorts, namely, on days 1, 3 and 5 following ICH. Animals treated with PMX53/argatroban demonstrated significant improvements in neurological function and fewer neurological apoptosis detected by TUNEL [terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labelling] and ßIII-tubulin dual-staining compared with vehicle-treated animals. Compared with sham-treated mice, the brain water content in argatroban/PMX53-treated mice was decreased significantly in both the ipsilateral cortex and ipsilateral striatum. Administration of PMX53/argatroban provided a synergistic neuroprotective effect via reducing inflammatory factors and brain oedema, leading to improvements in neurofunctional outcome. The results of this study indicated that simultaneous blockade of the thrombin and C5a receptors represent a promising neuroprotective strategy in haemorrhagic stroke.

The release of cupric ion in simulated uterine: New material nano-Cu/low-density polyethylene used for intrauterine devices.

With the development of IUDs, a number of copper-bearing devices are now commercially available, including the copper-T, the Multiload and the copper-T in various other forms, so-called "the second-generation" IUDs. In this article, we report on nano-Cu/low-density polyethylene composite as a potential copper carrier in IUD. Two issues for the new material are addressed: the effectiveness of polymers in reducing the initial burst in cupric ion release and the amount and pattern of continuing release. The aim of this study was to investigate copper ion release from this composite as a basis for considering its used in an IUD.

[An experimental study of the ocular hypotensive action of dopaminergic antagonists].

The ocular hypotensive action of a group of dopaminergic antagonists was observed in the low intraocular pressure model of albino rabbits infused with 20% NaCl. It was found that haloperidol lowered IOP significantly in both eyes, though the drug was administered in one eye. Compared with timolol and pilocarpine, the strength in lowering IOP was in the order haloperidol greater than timolol greater than pilocarpine. Haloperidol did not affect the pupil. It may be a promising antiglaucoma agent.

[A cytological study of inhibition of secondary cataract with heparin].

Secondary cataract is caused primarily by migration and proliferation of fibroblasts on the posterior capsular surface after extracapsular cataract extraction. Human fetus skin fibroblast cell culture was used, by measurement of cell's life cycle, cellular DNA content, increase of cell number and density, to investigate the heparin inhibitory action on the growth of the cells. It is discovered that the growth rate of the fibroblasts is lowered in the culture medium with heparin and the inhibition takes place at the interval between G1 and S stage and at M stage of the cell's life cycle.

[A clinical study of inhibition of secondary cataract with heparin].

Extracapsular cataract extraction with posterior chamber lens implantation was performed on 47 eyes with senile cataract of which 25 eyes were in the control group and 22 eyes were irrigated with heparin solution (25U/ml) in the operation, the drug group. Postoperatively, 2 eyes with hyphema were found in the drug group, but the deposition of fibrin and cells on the anterior lenticular surface and the posterior capsular opacification were significantly less than those in the control group. 3 months after the operation, the mean visual acuity in the drug group was 0.70 +/- 0.26 markedly higher than that in the control group, 0.54 +/- 0.28 (P < 0.05).

[Ocular hypotensive action of l-stepholidine].

Ocular hypotension was made by iv 20% NaCl 2.5 ml/kg in rabbits. On this model, l-stepholidine (l-SPD) lowered intraocular pressure when administered topically to the eyes. The ocular hypotensive action of 0.5% l-SPD was weaker than that of 0.5% timolol, but stronger than that of 0.5% pilocarpine. The maximal actions of the 3 drugs were similar. There were no significant changes of pupil diameter and carbonic anhydrase activity after l-SPD administered, but which suppressed aqueous humor formation. As the ciliary process of the eye behaves much like the renal glomerulus, dopamine increases the glomerular blood flow and dopaminergic antagonist decreases it, the mechanism of l-SPD in lowering intraocular pressure may be related to the dopaminergic receptor blocking effect of it.