Effect of enterohaemorrhagic Escherichia coli O157:H7-specific enterohaemolysin on interleukin-1ß production differs between human and mouse macrophages due to the different sensitivity of NLRP3 activation.

Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 infection in humans can cause acute haemorrhagic colitis and severe haemolytic uraemic syndrome. The role of enterohaemolysin (Ehx) in the pathogenesis of O157:H7-mediated disease in humans remains undefined. Recent studies have revealed the importance of the inflammatory response in O157:H7 pathogenesis in humans. We previously reported that Ehx markedly induced interleukin-1ß (IL-1ß) production in human macrophages. Here, we investigated the disparity in Ehx-induced IL-1ß production between human and mouse macrophages and explored the underlying mechanism regarding the activation of NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasomes. In contrast to the effects on human differentiated THP-1 cells and peripheral blood mononuclear cells, Ehx exerted no effect on IL-1ß production in mouse macrophages and splenocytes because of a disparity in pro-IL-1ß cleavage into mature IL-1ß upon caspase-1 activation. Additionally, Ehx significantly contributed to O157:H7-induced ATP release from THP-1 cells, which was not detected in mouse macrophages. Confocal microscopy demonstrated that Ehx was a key inducer of cathepsin B release in THP-1 cells but not in mouse IC-21 cells upon O157:H7 challenge. Inhibitor experiments indicated that O157:H7-induced IL-1ß production was largely dependent upon caspase-1 activation and partially dependent upon ATP signalling and cathepsin B release, which were both involved in NLRP3 activation. Moreover, inhibition of K(+) efflux drastically diminished O157:H7-induced IL-1ß production and cytotoxicity. The findings in this study may shed light on whether and how the Ehx contributes to the development of haemolytic uraemic syndrome in human O157:H7 infection.

Hemorrhagic fever caused by a novel Bunyavirus in China: pathogenesis and correlates of fatal outcome.

BACKGROUND: Hemorrhagic fever-like illness caused by a novel Bunyavirus, Huaiyangshan virus (HYSV, also known as Severe Fever with Thrombocytopenia virus [SFTSV] and Fever, Thrombocytopenia and Leukopenia Syndrome [FTLS]), has recently been described in China. METHODS: Patients with laboratory-confirmed HYSV infection who were admitted to Union Hospital or Zhongnan Hospital between April 2010 and October 2010 were included in this study. Clinical and routine laboratory data were collected and blood, throat swab, urine, or feces were obtained when possible. Viral RNA was quantified by real-time reverse-transcriptase polymerase chain reaction. Blood levels of a range of cytokines, chemokines, and acute phase proteins were assayed. RESULTS: A total of 49 patients with hemorrhagic fever caused by HYSV were included; 8 (16.3%) patients died. A fatal outcome was associated with high viral RNA load in blood at admission, as well as higher serum liver transaminase levels, more pronounced coagulation disturbances (activated partial thromboplastin time, thrombin time), and higher levels of acute phase proteins (phospholipase A, fibrinogen, hepcidin), cytokines (interleukin [IL]-6, IL-10, interferon-γ), and chemokines (IL-8, monocyte chemotactic protein 1, macrophage inflammatory protein 1b). The levels of these host parameters correlated with viral RNA levels. Blood viral RNA levels gradually declined over 3-4 weeks after illness onset, accompanied by resolution of symptoms and laboratory abnormalities. Viral RNA was also detectable in throat, urine, and fecal specimens of a substantial proportion of patients, including all fatal cases assayed. CONCLUSIONS. Viral replication and host immune responses play an important role in determining the severity and clinical outcome in patients with infection by HYSV.

The effects of interleukin (IL)-12 and IL-4 deficiency on worm development and granuloma formation in Schistosoma japonicum-infected mice.

CD4(+) T-helper (Th) cell is widely recognized to be capable of influencing worm development and egg granuloma formation after schistosome infection. Interleukin (IL)-12 and IL-4 play key roles in regulation of Th cell differentiation. In the present study, we subcutaneously inoculated mice with hybridoma cells secreting monoclonal antibodies to neutralize IL-12 and IL-4 and explored the effects of IL-12 and IL-4 deficiency on the worm development and granuloma formation in mice infected with cercariae of Schistosoma japonicum. It was found that deficiency of host IL-12 and IL-4 supported normal parasite survival and fecundity. However, worm development (length and female fecundity) was significantly enhanced in anti-IL-12-treated mice. Mean length of worms in anti-IL-12-treated group was significantly greater than that of intact controls on day 28 after infection (females, 11.84 ± 1.20 mm vs. 9.45 ± 1.34; males, 9.35 ± 1.21 mm vs. 8.10 ± 0.85 mm, p < 0.05). Liver egg load per pair of worms (1,770.12 ± 470.67 vs. 806.08 ± 232.37, p < 0.05) and uterine egg load of ovigerous females (93.08 ± 27.85 vs. 46.05 ± 34.24, p < 0.05) in anti-IL-12-treated mice were significantly higher than those in intact control 28 days postinfection. But these effects diminished 42 days postinfection (p > 0.05). Granuloma size in anti-IL-12-treated mice was significantly larger than that in intact mice 42 days postinfection (398.3 ± 80.7 µm vs. 294.4 ± 72.2 µm, p < 0.05). Granuloma fibrosis dramatically intensified in anti-IL-12-treated mice but diminished in anti-IL-4-treated mice. The results suggest that IL-12 may play an impeditive role in the development of S. japonicum and in granuloma formation as well as fibrosis. IL-4 may promote granuloma formation but have no effect on worm development.

Birth prevalence of orofacial clefts among perinatal infants: A register-based study in Bao'an district, Shenzhen, China.

BACKGROUND: The birth prevalence of orofacial clefts (OFCs) has been widely studied, but results are considerable varied, and epidemiological studies in southern China are few in numbers. To address this gap, we carried out a register-based study to estimate the birth prevalence of OFCs in Bao'an district, Shenzhen, China. METHODS: Data of perinatal infants born between 2003 and 2017 were extracted from Shenzhen Maternal and Child Health Management System. The overall OFCs birth prevalence with 95% confidence interval (CI) as well as subgroup analysis based on selected demographic factors was conducted. Cochran-Armitage trend tests were applied to evaluate the time trend by 5-year intervals. RESULTS: The overall birth prevalence of OFCs, cleft lip with or without cleft palate (CL/P) and cleft palate only (CPO) was 1.30 (95% CI 1.21-1.39), 1.00 (95% CI 0.92-1.08), and 0.30 (95% CI 0.25-0.34) per 1,000 births, respectively. An overall declining tendency was observed in the OFCs (from 1.83 to 1.04 per 1,000 births), specifically CL/P (from 1.53 to 0.69 per 1,000 births) birth prevalence over 5-year intervals, with statistical significance (p < 0.01). Subgroup analysis revealed that the CL/P and CPO birth prevalence was differed by infant gender, household registration, maternal age, and parity. CONCLUSION: Our findings had firstly reported the birth prevalence of OFCs in Bao'an district, and might help other researchers to plan more comprehensive public health strategies to reduce the occurrence of OFCs in further generation.

[Suppression effect of different stage antigens of Schistosoma japonicum on airway inflammation in a murine model of asthma].

OBJECTIVE: To investigate the effect of antigens of different stage Schistosoma japonicum on airway inflammation in a murine model of asthma. METHODS: 48 female BALB/c mice were randomly divided into eight groups. Mice in group A were given normal saline of equal volume as control. Group B was asthma model which was established by intraperitoneal and intranasal challenge with OVA. Mice in groups C, D and E were immunized with soluble egg antigen (SEA), soluble male worm antigen (SWA), and schistosomulum antigen (SSA) respectively 4 times in a week interval, followed by OVA sensitization as in group B 1 week after the final immunization. Mice in groups F, G, and H were immunized with SEA, SWA, and SSA respectively but sensitized and challenged with saline instead of OVA. 48 hours after asthma was induced, the mice were sacrificed. Leukocytes and eosinophils were counted in bronchoalveolar lavage fluid (BALF). The level of IL-5, IL-10 and IFN-gamma in BALF was detected. Pathologic changes in lung tissues were observed. RESULTS: Inflammation cells, especially eosinophils, appeared in airways of mice in groups B, C, D and E, but with much less number in groups C, D and E. No inflammation cells were seen in airways of group A mice. The number of leukocytes, eosinophils and level of IL-5 in BALF of group B [(98.4 +/- 16.1) x 10(4)/ml, (17.6 +/- 4.3) x 10(4)/ml, (197.9 +/- 36.5) pg/ml respectively] were significantly higher than those of group A [(8.2 +/- 1.1) x 10(4)/ml, (0.02 +/- 0.01) x 10(4)/ ml, (12.3 +/- 7.4) pg/ml], however the levels of IL-10 and IFN-gamma were significantly lower than that of group A (P < 0.05). The number of leukocytes, especially eosinophils, in BALF of groups C, D and E was significantly lower than that of group B. The level of IL-5 in BALF of groups C, D and E was significantly reduced, while that of IL-10 and IFN-gamma in BALF of the 3 groups was significantly higher than group B (P < 0.05). CONCLUSIONS: The immunization with S. japonicum antigens can effectively modulate the level of cytokines and inhibit the eosinophil infiltration and airway inflammation in asthmatic mice.

[Up-expression of PD-1-PD-L induced by immunization with SEA and SMWA of Schistosoma japonicum in mice].

OBJECTIVE: To explore the expression of PD-1-PD-L pathway of mice immunized with soluble egg antigen (SEA) or soluble male worm antigen (SMWA) of Schistosoma japonicum. METHODS: Eighteen BALB/c mice were randomly divided into three groups named as control group (A), SEA immunized group (B) and SMWA immunized group (C). Mice in groups B and C were subcutaneously immunized weekly with SEA (50 microg) and SMWA (50 microg) of S. japonicum respectively. After 4 times immunization, the expression of programmed death-1 (PD-1), programmed death-ligand1 (PD-L1) and PD-L2 in splenic cells was measured with flow cytometer. The expression of IL-4 and IFN-gamma in cultural suspension of splenic cells was detected by sandwich-ELISA after stimulation with ConA. RESULTS: The expression ratio of PD-1, PD-L1 and PD-L2 was extremely low in the control group, but increased after the immunization with SEA and SMWA. The expression ratio of PD-1 was (8.24 +/- 1.31)% in SEA immunized mice, higher than the mice immunized by SMWA [(6.08 +/- 1.28)%]. PD-L2 was much more elevated in SEA immunized mice [(5.26 +/- 1.73)%] while PD-L1 more significantly increased with SMWA immunization [(10.82 +/- 2.33)%]. In addition, the up-expression of PD-L1 was associated with the level of IFN-gamma and the expression of PD-L2 was associated with IL-4 secretion. CONCLUSION: The expression of PD-1-PDL was up-regulated in BALB/c mice immunized by SEA or SMWA of S. japonicum.

[Suppression of Schistosoma japonicum eggs on TNBS-induced colitis in mice].

OBJECTIVE: To study the suppression of Schistosoma japonicum eggs against the trinitrobenzene sulfonic acid (TNBS)-induced colitis in mice. METHODS: 50 female BALB/c mice (6-8 week-old) were randomly divided into normal control group, ethanol control group, schistosome egg immunized control group, TNBS-induced colitis group and TNBS-induced colitis with egg immunization group. In TNBS-induced colitis with egg immunization group, mice were immunized 4 times with 10,000 schistosome eggs by intraperitoneal injection with one-week interval. On day 6 after the last immunization the mice were induced by TNBS and the body weight, histological change on colon and level of cytokines of mice were observed in egg-immunized and -unimmunized colitis mice. RESULTS: The unimmunized mice developed significant inflammation in colon with bloody mucus feces and decreased body weight after TNBS induction. Distinct hyperemia, edema and transmural inflammatory infiltration accompanied with ulceration were shown in colon. The level of IFN-gamma was (3.47 +/- 0.87) ng/ml and IL-4 was (146.06 +/- 45.76) pg/ml. However, in egg-immunized mice, the inflammation was suppressed greatly and the body weight recovered soon after TNBS induction. The production of IL-4 was enhanced to (598.50 +/- 135.90) pg/ml, and IFN-gamma was significantly diminished to (1.53 +/- 0.51) ng/ml. CONCLUSION: S.japonicum eggs protect mice from colitis induced by TNBS.

[Production and identification of chicken egg yolk antibodies against soluble egg antigen of Schistosoma japonicum].

OBJECTIVE: To produce and purify egg yolk immunoglobulin against soluble egg antigen (SEA) of Schistosoma japonicum, and evaluate its specificity and sensitivity. METHODS: 25-week old hen was intravenously and subcutaneously immunized with SEA of Schistosoma japonicum for 4 times. Each hen was first immunized with 60 microg SEA and subsequent injections were performed at 10-day intervals with 30 microg SEA. IgY was extracted from eggs of hen 35 d after the first inoculation by WD (water-dilution) method, eggs from non-immunized hen were used as negative control. The protein concentration of IgY was measured by BCA method, and IgY was analyzed by SDS-PAGE and Western blotting. SEA-based ELISA was used to evaluate the specificity and sensitivity of the IgY. RESULTS: 61 mg IgY was extracted from one egg. The results of SDS-PAGE and Western blotting demonstrated that the IgY contained one major protein band with molecular weight of 130,000 and could be recognized by SEA. Specific IgY could be immediately detected by SDS-PAGE and ELISA in the eggs laid by the hens from 10 days after the first immunization. On day 31 after the primary immunization, the antibody titer reached 1:1 600. 2.4 ng/ml SEA was detected by IgY based-sandwich ELISA, which indicated a high sensitivity of the purified IgY. CONCLUSION: Anti-SEA IgY with high specificity and sensitivity has been obtained and purified.

[Impact of chronic schistosomiasis japonica on the protective immunity induced by vaccine against hepatitis B virus].

OBJECTIVE: To study the impact of chronic schistosomiasis on the protective immunity induced by vaccine against hepatitis B virus. METHODS: 24 patients with chronic or advanced schistosomiasis (experimental group) and 26 healthy volunteers (control group) all without hepatitis B virus infection were selected for the study. Sera of the subjects in the two groups were collected before inoculation and on the 35th day after inoculation with yeast-derived recombinant hepatitis B vaccine. The level of anti-Hbs, IL-2 and TNF-alpha in sera was examined by ELISA respectively. RESULTS: Anti-Hbs in both groups were negative before inoculation, with an average absorbance (A value) of 0.134 and 0.150 respectively. After inoculation, positive rate of anti-Hbs was 17% (4/24, average A value 0.145 ) in experimental group and 92% (24/26, average A value was 1.210) in control group. The vaccine against hepatitis B induced significantly higher level of anti-Hbs in healthy volunteers compared with that in schistosomiasis patients (P < 0.01). The level of IL-2 and TNF-alpha increased in both groups after inoculation without significant difference compared with the level before inoculation. CONCLUSION: The results suggest that the protective immunity of patients with chronic schistosomiasis is deficient to the stimulation of hepatitis B virus and it may involve in a higher incidence of hepatitis B among schistosomiasis patients.

[Microarray DNA chip in analyzing the association between HLA-DRB and advanced hepatosplenic schistosomiasis].

OBJECTIVE: To explore possible associations between host polymorphism of HLA class II genotypes and advanced hepatosplenic schistosomiasis japonica. METHODS: 45 advanced schistosomiasis patients (experimental group) and 44 age- and sex-matched patients with chronic schistosomiasis (control group) from the same area were investigated for their HLA class II gene DRB genotypes by genotyping the alleles using microarray DNA chip. The correlation of allele frequencies to advanced hepatosplenic schistosomiasis was compared for the two groups. RESULTS: HLA-DRB1*04x exhibited markedly higher frequency in advanced patients than that in control group (P < 0.01, RR = 3.928). In contrast, the frequency of HLA-DRB1*15x in advanced patients was much lower when compared with that in control group (P < 0.01, RR = 0.050). Besides, the significant allele HLA-DRB1*15x displayed concurrence with allele DRB5*010x/020x. The linked gene haplotype DRB1*15x-DRB5*010x/020x showed significantly higher incidence in control group than in experimental group (P < 0.01). CONCLUSION: Allele HLA-DRB1*04x is positively, while HLA-DRB1*15x is negatively, correlated with advanced hepatosplenic schistosomiasis.

Maternal age, parity and isolated birth defects: a population-based case-control study in Shenzhen, China.

BACKGROUND: The etiology of birth defects has been widely studied but is not yet fully clarified, previously published data had suggested that maternal age or parity maybe involved, but without consistent conclusions. METHODS: A population-based, case-control study was nested in a cohort of perinatal infants born from 2010 to 2012 in Baoan District, Shenzhen. Four categories of isolated birth defects were defined as cases: congenital heart defects (CHD, n = 693), polydactyly (n = 352), cleft lip with or without palate (CL/P, n = 159) and equinovarus (n = 119). Controls were non-malformed infants (n = 11,307) randomly selected from the same area and period. Odds ratios (ORs) and the 95% confidence intervals (CIs) were computed by multivariable unconditional logistic regression analysis. RESULTS: Young maternal age (<25 years old) was associated with a reduced risk of CHD (adjusted OR = 0.73, 95% CI 0.59-0.90), while with an elevated risk of polydactyly (adjusted OR = 1.42, 95% CI 1.09-1.84). Increased risk of CL/P-affected pregnancy was observed in mothers older than 35 years old (adjusted OR = 2.12, 95% CI 1.26-3.57). Compared to primipara, those having their second, and third or more delivery were less likely to have infants with equinovarus, with significant adjusted ORs of 0.59 (0.40-0.89) and 0.42 (0.19-0.93), respectively. CONCLUSION: Maternal age was significantly associated with CHD, polydactyly and CL/P relevant pregnancy. Mothers with higher parity might have lower risk of equinovarus occurrence in offsprings.

[Effects of signal transducers and activators of transcription 4 and 6 on development of worms and granuloma formation in mice infected with Schistosoma japonicum].

OBJECTIVE: To explore the effects of STAT4 and STAT6 on the development of worms and granuloma formation in mice infected with Schistosoma japonicum. METHODS: All the intact BALB/cJ mice and STAT4(-/-), STAT6(-/-) mice with the same genetic background were infected with 25 S. japonicum cercariae. All the mice were sacrificed on the 42nd day after infection and the worms were collected. The total number of worms and the mean number of worm pairs were counted. The liver of each mouse was removed for the count of eggs, the histological examination and the determination of the size of single-egg granulomas in the liver. RESULTS: No significant differences were found in the total number of worms, the number of worm pairs and the number of eggs per pair of worms in the liver among STAT4(-/-), STAT6(-/-) and BALB/cJ mice. The size of single-egg granulomas in the liver of STAT6(-/-) mice (213.3 +/- 68.6) microm was significantly smaller than that in the liver of normal BALB/cJ mice (319.5 +/- 71.9) microm (P < 0.05). The liver granulomas were not well formed and the liver fibrosis decreased in STAT6(-/-) mice. CONCLUSIONS: STAT4 or STAT6 deficiency has no conspicuous effect on the development and fecundity of S. japonicum. STAT6 plays an important role for the granuloma formation and liver fibrosis.

The effects of T cell deficiency on the development of worms and granuloma formation in mice infected with Schistosoma japonicum.

It is widely accepted that the immune response of the host attacks the parasite and the parasite appears to develop strategies to evade the assault. However, there is increasing evidence that the development of a parasite may be also positively influenced by the immune response of host. In this paper, we explore the effects of T cell deficiency on the development of the worms and granuloma formation in mice infected with cercariae of Schistosoma japonicum. T cell-deficient (nude) mice supported normal parasite survival and fecundity, but compared to normal mice delayed the worms' development (length and female fecundity) until 28 days after infection. However, these differences equaled out at 35 and 42 days. The nude mice apparently suppressed the size of granuloma in the livers around the eggs of S. japonicum. The granulomas were composed predominantly of neutrophils but with significantly fewer eosinophils in nude compared to normal mice. In addition, hepatocyte necrosis occurred in the vicinity of granulomas in nude but not normal mice. This is consistent with egg-granuloma formation in the host being dependent on T-lymphocyte functions and shows that the effect of T cell deficiency on the development of the worms is transitory in S. japonicum.

Schistosoma japonicum infection modulates the development of allergen-induced airway inflammation in mice.

Asthma, a chronic inflammatory disorder of the airways, is coordinated by Th2 cells in both human asthmatics and animal models of allergic asthma. It has been shown that helminth infections including Schistosoma mansoni may modulate atopic diseases including asthma. In the present study, BALB/c mice were infected with bisexual and unisexual (male) S. japonicum, respectively, prior to ovalbumin (OVA) sensitization and challenge. Compared to mice with OVA sensitization/challenge alone, S. japonicum infection led to a significant decrease of eosinophil accumulation in bronchoalveolar lavage fluid (BALF) collected 48 h postchallenge, as well as to a marked reduction in inflammatory cell infiltration around the airways and pulmonary blood vessels. Compared to OVA-immunized uninfected mice, the level of OVA-specific serum IgE as well as interleukin (IL)-4 and IL-5 in BALF were reduced, but IL-10 was strongly elevated in mice with preexisting S. japonicum infection prior to OVA immunization. These results suggest that both bisexual and male S. japonicum infections may modulate the development of allergic asthma.

Schistosoma japonicum eggs modulate the activity of CD4+ CD25+ Tregs and prevent development of colitis in mice.

Crohn's disease (CD) is considered to be caused by a disorder of the immune system and helminth infections may interact with development of the disease. We induced colitis in mice by trinitrobenzenesulfonic acid (TNBS) and observed the effects of intraperitoneally injected eggs of Schistosoma japonicum on the course of the disease. The inflammation in the colon was reduced in egg-treated mice and secretion of IFN-gamma (a Th1 cytokine) by cultured spleen cells in vitro was greatly suppressed, and of IL-4, IL-5 and IL-10 (Th2 cytokines) significantly elevated after egg injection. Also, the percentage of regulatory T-cells (Tregs) was increased in the spleens of egg-exposed mice with TNBS-induced colitis compared to non-egg exposed animals. The data suggest that Tregs may be activated by S. japonicum eggs and play a role in restoring immune disorders in TNBS-induced colitis of mice.