Hand, Foot And Mouth Disease Group Research Articles

Translocating lipopolysaccharide correlates with the severity of enterovirus A71-induced HFMD by promoting pro-inflammation and viral IRES activity

BackgroundThe increase of inflammation-inducing enterobacteria was recently observed in severe hand, foot, and mouth disease (HFMD) caused by Enterovirus A71 (EV-A71). This study aimed to verify the occurrence of bacterial translocation (BT) and further explore the contributory role of BT to severity of EV-A71-mediated HFMD cases.MethodsSerum specimens from 65 mild and 65 severe EV-A71-associated HFMD cases and 65 healthy children were collected. EV-A71 VP1 in serum, inflammatory mediators including C-reactive protein, IL-1β, IL-6, interferon-γ and tumor necrosis factor-α, BT related biomarkers including Claudin-3, intestinal fatty acid binding protein, lipopolysaccharide (LPS), soluble CD14 (sCD14) and endotoxin core antibody were measured by ELISA. Bacterial DNA (BactDNA) fragments were quantified by quantified PCR (qPCR). Rhabdomyosarcoma (RD) or SH-SY5Y cells, infected with LPS-pre-incubated EV-A71 or transfected with plasmid containing viral 2Apro or mRNA containing viral internal ribosomal entry site (IRES), were post-treated with or without LPS in vitro. EV-A71 RNA and viral or cellular proteins were determined by qPCR and western blot, respectively.ResultsCompared to mild HFMD patients, remarkably higher inflammatory mediators as well as BT-related biomarkers except BactDNA were observed in severe HFMD cases (all P < 0.05). In severe HFMD group, circulating concentrations of LPS and sCD14 showed statistical correlations with inflammation indices (all P < 0.05), serum levels of EV-A71 VP1 were found to be positively correlated with serum LPS (r = 0.341, P = 0.005) and serum sCD14 (r = 0.458, P < 0.001). In vitro, EV-A71 attachment and internalization were only slightly promoted by LPS pre-incubation; however, EV-A71 proliferation and viral 2Apro-mediated IRES activity were significantly accelerated by LPS post-treatment.ConclusionsOur results collectively indicate that gut-derived translocating LPS contributes to the severity of EV-A71-induced HFMD by driving inflammatory response and viral proliferation via viral 2Apro-mediated IRES.

Association of gene polymorphisms of CD55 with susceptibility to and severity of hand, foot, and mouth disease caused by enterovirus 71 in the Han Chinese population.

Hand, foot, and mouth disease (HFMD) caused by enterovirus 71 (EV71) can lead to high morbidity and mortality, and genetic background plays an important role during the disease process. We investigated the association between the single-nucleotide polymorphism (SNP) rs2564978 of the CD55 gene and susceptibility to and severity of HFMD using the SNPs can multiple SNP typing methods. Soluble CD55 (sCD55) expression was significantly lower in the EV71 HFMD group than in the control group and lower in severe cases than in mild cases (P < .001). Moreover, CD55 rs2564978 (C vs T OR = 1.300, 95% CI, 1.120-1.509) was associated with the risk of EV71 infection, and genotype TC was related to the severity of the infection (TC vs TT OR = 4.523, 95% CI, 2.033-10.066). Our results suggest that sCD55 expression and the CD55 polymorphism rs2564978 may influence the susceptibility to and severity of EV71 infection.

Significance of Natural Killer Cell Subsets, their Receptors, and Inflammatory Cytokines in Peripheral Blood of Children with Hand, Foot, and Mouth Disease.

Hand, foot, and mouth disease (HFMD) is a self-limited disease caused mainly by enterovirus 71 and coxsackievirus A16; however, some cases have severe neurological complications, pulmonary edema, and fetal death. In this study, we analyzed the changes in natural killer (NK) cell subsets, their receptors, and serum inflammatory cytokines in children with HFMD. Peripheral blood samples were collected from 70 HFMD pediatric patients admitted Department of Infectious Diseases of our hospital from July 2016 to November 2017 and from 16 healthy children receiving physical examination in the disease control and prevention center as the healthy control group. The changes in three NK cell subsets and their receptors, and serum inflammatory cytokines were detected via flow cytometry. The distribution of CD3-CD16+CD56+ and CD3-CD16+CD56- NK cell subsets in the HFMD group increased significantly compared with that in the healthy control group, while CD3-CD16-CD56+ NK cell subset showed no significant difference in the two groups. Besides, the distribution of the CD3-CD16+CD56+ NK cell subset was significantly higher than the CD3-CD16+CD56- NK cell subset. The distribution of the NKG2A receptor on the CD3-CD16+CD56+ NK cell subset in the HFMD group was significantly higher than that in the healthy control group. Moreover, the serum levels of IL-17A, IL-10, IL-6, and IL-2 in the HFMD group were higher than those in the healthy control group. The results showed that the positive distribution of NKG2A in the NK cell subset and the content of inflammatory cytokines could be of diagnostic value for early detection of HFMD in patients and prediction of the severity of the disease.

Identification of SAA and ACTB as potential biomarker of patients with severe HFMD using iTRAQ quantitative proteomics

Hand, foot and mouth disease (HFMD) is an infectious disease caused by a variety of enterovirus infections, and the most common types of virus infections are the newenterovirus71 (EV71) and coxsackievirus A group 16 (CoxA16). A small fraction of HFMD will cause further severe HFMD. A rapid and accurate diagnosis biomarker of severe HFMD is important for the timely treatment. In the study, we conducted a clinical biomarker discovery study using iTRAQ combined with MS. Serum proteome alterations in severe HFMD group (n = 32) and health control group (n = 32) were analyzed. 47 proteins were upregulated (fold change > 1.5) between the severe HFMD group and HC group. The identified proteins were classified into different groups according to the molecular function, biology processes, cellular component. During the up-regulated proteins, serum amyloid A (SAA) and human β-actin (ACTB), were confirmed in the serum of the severe HFMD and HC by ELISA assay. SAA and ACTB levels were significantly higher in the sever HFMD patients (P < .01), consistent with iTRAQ-LC-MS/MS analysis. In summary, Our results showed that SAA and human β-actin (ACTB) may be served as a potential biomarker of the clinical diagnosis of severe HFMD.

Significance of matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 levels in evaluation of severe hand, foot, and mouth disease complicated with neurogenic pulmonary edema

Objective To investigate the significance of matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1) in serum and cerebrospinal fluid for evaluation of severe hand, foot, and mouth disease (HFMD) complicated with neurogenic pulmonary edema (NPE). Methods A total of 140 patients diagnosed with HFMD in Henan Children′s Hospital were enrolled and divided into three groups including mild group, severe HFMD group without NPE , severe HFMD group with NPE .These severe HFMD patients were also divided into survival group and death group according to the 28-day prognosis. Meanwhile, 50 age-matched healthy children were selected as controls. Serum MMP-9 and TIMP-1 levels were measured in all enrolled children. At the same time, MMP-9, TIMP-1 and ratio of MMP-9/TIMP-1 in cerebrospinal fluid were measured in the severe HFMD group with and without NPE. Quantitative data were compared using one-way analysis of variance, and means comparisons between samples were conducted using LSD- t test. Results Among 140 children with HFMD, 66 were in mild group, 42 in severe HFMD without NPE group, and 32 in severe HFMD with NPE group. And 50 healthy children were in control group. After 28 days, 14 cases died in severe HFMD groups. MMP-9, TIMP-1 and MMP-9/TIMP-1 in serum of severe HFMD group with NPE increased significantly greater than those in the other three groups (F=269.356, 121.301 and 101.502, respectively, all P <0.05). MMP-9, TIMP-1 and MMP-9/TIMP-1 in cerebrospinal fluid of severe HFMD group with NPE were (57.24±8.92) μg/L, (35.26±8.14) μg/L and (1.66±0.23) μg/L, respectively, while those in cerebrospinal fluid of severe HFMD group without NPE were (30.57±3.89) μg/L, (26.25±0.32) μg/L and (1.17±0.61) μg/L, respectively. The differences between the two groups were all statistically significant (t=62.485, 37.680 and 169.387, respectively, all P<0.01). MMP-9, TIMP-1 and MMP-9/TIMP-1 in serum and cerebrospinal fluid of death group increased significantly greater than those in survival group, the difference were statistically significant (all P<0.01). The maximum area under curve (AUC) was reached when the MMP9/TIMP-1 ratio in cerebrospinal fluid was 0.890 (95% CI: 0.801-0.978). Conclusions MMP-9 and TIMP-1 may be involved in the pathogenesis of HFMD complicated with NPE. The detection of MMP-9 and TIMP-1 levels may be beneficial for the early diagnosis of severe HFMD with NPE. The imbalance of MMP-9/TIMP-1 ratio can be used as one of the predictors of severe HFMD combined with NPE. Key words: Matrix metalloproteinase 9; Hand, foot and mouth disease; Pulmonary edema; TIMP-1

In vitro inhibitory effect of artesunate on the vascular endothelial growth factor secreted by mononuclear cells in hand-foot-and-mouth disease children complicated with encephalitis

Objective To investigate the dynamics of white blood cell count (WBC), prealbumin (PA), high sensitive C-reactive protein (hs-CRP), and vascular endothelial growth factor (VEGF) in hand-foot-and-mouth disease (HFMD) complicated with or without encephalitis and in vitro inhibitory effects of artesunate on VEGF secretion of mononuclear cells from HFMD patients. Methods Peripheral blood mononuclear cells from healthy control group, HFMD group and HFMD combined with encephalitis group were isolated by Ficoll density gradient centrifugation and treated with different concentrations of artesunate (25, 50, 100 mg/L). The expression of VEGF in the supernatant was examined by ELISA double antibody sandwich method. The levels of WBC, PA and hs-CRP in the three groups were also detected. Multiple samples were compared by one-way analysis of variance. Multiple comparison was performed by Dunnett T3 test. Correlation of two variables was analyzed by Spearman correlation test. Results There were significant differences in the levels of WBC, hs-CRP, PA and VEGF between the HFMD combined with or without encephalitis group and health control group (F=172.69, 366.02, 166.32 and 5 941.89, respectively, all P<0.01). There were significant differences in the levels of VEGF secreted by mononuclear cells treated with three different concentrations of artesunate between the HFMD combined with or without encephalitis groups and health control group (F=194.265 and 4 750.69, respectively, both P<0.01). Correlation analysis showed that the VEGF level secretion by mononuclear cells from HFMD patients with or without encephalitis were both negatively correlated with different concentrations of artesunate (r=-0.903 and -0.969, both P<0.01). Conclusions Compared with HFMD without encephalitis, the secretion of VEGF by mononuclear cells, WBC and hs-CRP levels in HFMD complicated with encephalitis group all increase and PA decrease significantly. Artesunate can inhibit the secretion of VEGF by mononuclear cells of patients with hand-foot-and-mouth disease in a dose-dependent manner in vitro. Key words: Hand, foot and mouth disease; Encephalitis; Artesunate; Vascular endothelial growth factors

Association between S100B gene polymorphisms and hand, foot and mouth disease caused by enterovirus 71 infection

To investigate the association between rs9722 polymorphisms in the S100B gene and hand, foot and mouth disease (HFMD) caused by enterovirus 71. A total of 124 HFMD children with enterovirus 71 infection were enrolled as subjects, and 56 healthy children were enrolled as control group. The rs9722 polymorphisms in the S100B gene were detected for both groups, and the serum level of S100B protein was measured for 74 HFMD children. The rs9722 locus of the S100B gene had three genotypes, CC, CT, and TT, and the genotype frequencies were in accordance with Hardy-Weinberg equilibrium. Compared with the control group, the HFMD group had significant increases in the frequencies of TT genotype and T allele (P<0.01). Children with severe HFMD caused by enterovirus 71 infection had significantly higher frequencies of TT genotype and T allele than those with moderate or mild HFMD (P<0.05). Compared with the cured patients, the patients with poor prognosis had significant increases in the frequencies of TT genotype and T allele in the rs9722 locus of the S100B gene (P<0.05). Among the 74 children with HFMD, the children with TT genotype had the highest serum level of S100B protein, and those with CC genotype had the lowest level (P<0.01). T allele in the rs9722 locus of the S100B gene might be a risk factor for severe HFMD caused by enterovirus 71 infection.

Clinical Significance and Prognostic Effect of Serum 25-hydroxyvitamin D Concentrations in Critical and Severe Hand, Foot and Mouth Disease

Objective: To examine the association of serum 25-hydroxyvitamin D [25(OH)D] concentrations with critical and severe hand, foot and mouth disease (HFMD) and assess the clinical significance and prognostic effect of 25(OH)D concentrations in children with HFMD. Methods: This is a prospective observational study. The 138 children with HFMD were divided into common (49 cases), severe (52 cases), and critical (37 cases) HFMD groups. Another 59 healthy children undergoing outpatient medical examinations during the same period were chosen as the control group. Serum 25(OH)D concentrations were measured in all the subjects, and each group was subdivided by serum 25(OH)D concentration into 25(OH)D normal (≥30 ng/mL); insufficiency (20–29.9 ng/mL), and deficiency (<20 ng/mL) groups. The pediatric critical illness score (PCIS) was recorded for the critical and severe HFMD group upon admission to the pediatric intensive care unit (PICU). Children with critical and severe HFMD were also monitored for blood lactate (LAC), serum calcium ions (Ca++), D-dimer (DD), lactate dehydrogenase (LDH), and creatine kinase-MB (CK-MB) levels; the incidences of brainstem encephalitis, neurogenic pulmonary edema, and circulatory failure; and the 14-day mortality rate. Results: Serum 25(OH)D concentrations were generally low in all groups. The critical HFMD group showed a significantly lower serum 25(OH)D mean concentration (20.0 ± 8.4 ng/mL) and a higher proportion of deficiency (18%) compared with the control group (28.1 ± 6.6 ng/mL, 8%), common (29.5 ± 8.1 ng/mL, 10%) and severe (31.9 ± 9.7 ng/mL, 8%) HFMD groups (p < 0.05). In the critical and severe HFMD groups, the 25(OH)D deficiency group had lower PCISs than the 25(OH)D normal and insufficiency groups (p < 0.05); and had higher values than the latter two groups for LAC, LDH, CK-MB and DD; and the incidences of brainstem encephalitis, neurogenic pulmonary edema, circulatory failure, and mortality (p < 0.05). The death group showed significantly lower serum 25(OH)D concentrations and PCISs than the survival group (p < 0.05) and had higher LAC, LDH, CK-MB and DD levels and higher incidences of brainstem encephalitis, neurogenic pulmonary edema, and circulatory failure (p < 0.05). Logistic regression analysis revealed that the serum 25(OH)D concentration was an independent factor that influenced mortality in children with critical and severe HFMD. Conclusions: In this study, we find the serum 25(OH)D concentrations are substantially reduced in children with critical and severe HFMD and are associated with the severity of HFMD. The serum 25(OH)D concentrations may have clinical value for determining the progression of critical HFMD and predicting the risk of death. Further evidence is needed before it can be stated that 25(OH)D concentrations have clinical value in HMFD diagnosis.

Test and clinical significance of immunoglobulin and complements in hand foot and mouth disease combined with acute flaccid paralysis

Objective To study the serum immunoglobulin IgG, IgM, IgA and complements C3, C4 level changes in hand foot and mouth disease(HFMD) combined with acute flaccid paralysis(AFP). Methods The cases were divided into three groups in this study, including 30 cases of HFMD, 30 cases of HFMD combined with AFP, and 30 cases of healthy(normal control group). Immunoturbidimetric assay was used to test the level changes of IgG, IgM, IgA, and complements C3, C4. Results The IgG, IgA, C3 and C4 in HFMD combined with AFP group were (5.49±1.04) g/L, (0.39±0.27) g/L, (0.65.±0.19) g/L and (0.16.±0.11) g/L respectively, lower than those in HFMD group((7.07±1.63) g/L, (0.55±0.32) g/L, (0.97.±0.18) g/L, (0.23.±0.09) g/L) and normal control group((9.58±1.42) g/L, (0.81±0.33) g/L, (1.28.±0.25) g/L, (0.34.±0.16) g/L), there were statistically significant differences among groups(F=12.04, 1.84, 1.65, 1.29; P=0.031, 0.020, 0.018, 0.025). However, the expression of IgM in HFMD combined with AFP group was (1.34±0.26) g/L, higher than that in HFMD group((1.02±0.29) g/L) and normal control group((0.76±0.28) g/L), the difference was statistically significant(F=3.62, P=0.014). Conclusion HFMD combined with AFP exists severe humoral immune dysfunction, which provides a theoretical evidence for the prevention and treatment of HFMD combined with AFP. Key words: Hand foot and mouth disease; Acute flaccid paralysis; Immunoglobin; Complements

Changes of levels of serum hydrogen sulfide and interleukin-6 in children with hand, foot and mouth disease

Objective To study the significance of level changes of serum hydrogen sulfide(H2S)and interleukin-6(IL-6) in children with hand, foot and mouth disease(HFMD). Methods Nity-two cases with HFMD were enrolled and divided into severe HFMD group(48 cases) and common HFMD group(44 cases). Forty-six healthy children were enrolled as healthy control group.The serum H2S and IL-6 were detected. Results Acute phase: compared to the healthy control group[H2S(55.76±7.80) μmol/L, IL-6(61.31±13.43) ng/L], the level of serum H2S significantly reduced and the level of IL-6 significantly increased in severe HFMD group[H2S(21.72±7.52) μmol/L; IL-6(131.33±17.82) ng/L] and common HFMD group[H2S(31.86±8.41) μmol/L; IL-6(95.48±15.07) ng/L](P<0.01), and there was significantly difference between the severe HFMD group and common HFMD group(P<0.01). Recovery phase: compared to the healthy control group, the serum H2S level[(34.54±13.21) μmol/L] was lower and IL-6[(92.73±15.25) ng/L]was higher in severe HFMD group(P<0.01). The serum H2S level was negatively correlated with IL-6 in severe HFMD group and common HFMD group(r=-0.31, P<0.01; r=-0.45, P<0.01). Conclusion The serum H2S and IL-6 participate in the pathological process of HFMD, and the level change can be used as one of indicators of early diagnosis. Key words: Hand, foot and mouth disease; Hydrogen sulfide; Interleukin-6,

195: Regulatory T cells and cyclic adenosine monophosphate in enterovirus 71 infection

Background Since 1998, Taiwan has experienced several hand, foot, and mouth disease (HFMD) epidemics caused by enterovirus 71 (EV71). Two notable features of severe EV71 infection in young children are brainstem encephalitis (BE) and fatal pulmonary edema (PE). This study investigated the specific immunoregulatory characterizations of EV71 neurological complications according to the disease severity. Methods Patients younger than 18 years with virologically confirmed EV71 infections were enrolled and divided into 2 groups: the HFMD or BE group, and the autonomic nervous system (ANS) dysregulation or PE group. Laboratory findings, cytokine and cyclic adenosine monophosphate (cAMP) levels, immunophenotypes, and the regulatory T cell (Tregs) profiles of the EV71-infected patients were determined. Results Patients with ANS dysregulation or PE exhibited a significantly low frequency of CD4+ CD25+ Foxp3+ and CD4+ Foxp3+ T cells compared with patients with HFMD or BE. The expression frequency of CD4−CD8− was also significantly decreased in patients with ANS dysregulation or PE. Among patients with ANS dysregulation or PE, the expression frequency of CD4+ Foxp3+ increased markedly after milrinone treatment, and was associated with a reduction of plasma levels IL-6, IL-8 and IL-10. Plasma concentrations of cAMP were significantly decreased in patients with ANS dysregulation or PE compared with patients with HFMD or BE, however, cAMP levels increased after milrinone treatment. Conclusions These findings provided new insight into the role of regulatory T cells. Furthermore, CD4+ CD25+ Foxp3+ and cAMP expression might be related to the progression of severe EV71 infection, and its responses to milrinone treatment.

The significance of Notch ligand expression in the peripheral blood of children with hand, foot and mouth disease (HFMD).

BackgroundHand, foot and mouth disease (HFMD), a virus-induced infectious disease that usually affects infants and children, has an increased incidence in China in recent years. This study attempted to investigate the role of the Notch signaling pathway in the pathogenesis of HFMD.MethodsEighty-two children diagnosed with HFMD were enrolled into this study. The HFMD group was further divided into the uncomplicated HFMD and HFMD with encephalitis groups. The control group included 40 children who underwent elective surgery for treatment of inguinal hernias.ResultsChildren with HFMD displayed significantly reduced CD3+, CD3+CD4+ and CD3+CD8+ cell subsets, but substantially enhanced CD3−CD19+ cell subset (p < 0.05 versus control subjects). The expression levels of Notch ligands Dll1 and Dll4 in the peripheral blood of the HFMD group were significantly higher than those in the control group (p < 0.05). There were statistically significant differences in CD3+, CD3+CD4+ and CD3−CD19+ cell subsets, but not in Notch ligand expression, between the uncomplicated HFMD and HFMD with encephalitis groups. Dll4 expression in HFMD subjects correlated negatively with the CD3+ and CD3+CD8+ cell subsets (p < 0.05), but positively with the CD3−CD19+ cell subset (p < 0.05). Furthermore, Dll4 expression in HFMD with encephalitis subjects correlated positively with total white blood cell (WBC) counts and total protein contents in cerebrospinal fluid (CSF) (p < 0.05).ConclusionsThe Notch ligand Dll4 exhibits a strong correlation with the CD3+, CD3+CD8+ and CD3−CD19+ cell subsets in children with HFMD, indicating that the Notch signaling may be involved in the development of HFMD by affecting the number and status of peripheral lymphocytes.

Peripheral T lymphocyte subset imbalances in children with enterovirus 71-induced hand, foot and mouth disease

Inflammatory mediators (i.e. cytokines) play a pivotal role in the regulation of pathophysiological processes during EV71-induced hand, foot and mouth disease (HFMD). Different T cell subsets have distinct cytokine secretion profiles, and alteration in the T cell subsets frequency (imbalance) during infection leads to changed cytokine patterns. However, the effects of EV71 infection on T cell subsets were not clear. The objective of this study was to determine whether EV71-induced HFMD can be explained by the emergence of particular T-cell subsets (Th1, Th2, Tc1, Tc2, Th17, Tc17 and Treg cells) and the cytokine they produced (IFN-γ, IL-4, IL-17A and TGF-β1), as well as distinct responses to EV71 infection. We found that when compared to the control group, the percentage of Th1 and Tc1 cells was significantly higher in mild and severe HFMD group. Similar results were found in the Th1/Th2 ratio and IFN-γ levels. On the other hand, the percentage of Th17 cells and IL-17A levels were the highest in severe HFMD cases, and lowest in controls. Similar trend was also found for the Th17/Treg cell ratio. An optimal cutoff value of 2.15% for Th17 cell and 6.72pg/ml for IL-17A provided a discriminatory value for differentiating the severity of HFMD cases by receiver operating characteristic curve analyses. These findings reveal that the Th1/Th2 and Th17/Treg imbalance exist in HFMD patients, suggesting their involvement in the pathogenesis of EV71 infection, which may have potential value as biomarkers.