Cytokines in stools of children with inflammatory bowel disease or infective diarrhoea.

AIMS--To determine the concentrations of interleukin-6 (IL-6) and tumour necrosis factor alpha (TNF alpha) in stools from children. METHODS--Stool samples from 14 healthy children, 32 children with inflammatory bowel disease, and 23 children with acute diarrhoea were emulsified in an equal volume of phosphate buffered saline and then centrifuged to produce a clear supernatant fluid. IL-6 and TNF alpha were measured by enzyme linked immunosorbent assay (ELISA). RESULTS--TNF alpha was detected in the stools of all 14 healthy children (12-130 pg/g stool), but IL-6 was detected only in three. Similar results were seen in children with inactive inflammatory bowel disease. Stool TNF alpha concentrations were raised in samples from children with active inflammatory bowel disease, but in most (11/18) of these samples IL-6 was undetectable. Stool samples contained a heat-labile factor which rapidly destroyed IL-6 immunoreactivity. Most children with diarrhoea had TNF alpha concentrations similar to those of healthy controls and most were also negative for IL-6. Three children with Shigella flexneri infection had extraordinarily high concentrations of both TNF alpha and IL-6 in their stools. CONCLUSIONS--There is constant low grade production of TNF alpha in the intestine of healthy people. Raised values are indicative of mucosal inflammation, but are not specific. Stool IL-6 is of little use in assessing mucosal inflammation because immunoreactivity is rapidly lost in stool samples.     

Peripheral blood neutrophil responses in children with shigellosis.

Alterations in peripheral blood neutrophil function are known to occur in patients with colitis and may have a role in precipitating nonspecific tissue injury. It is not known whether neutrophil function is altered in patients with Shigella dysenteriae type 1 infection, during which there is extensive colitis and which may be associated with life-threatening complications in young children. Three aspects of peripheral blood neutrophil function, polarization, attachment to yeast particles, and locomotion, were therefore studied in 111 children with S. dysenteriae type 1 infection and 57 children without any infection. All children were aged 12 to 60 months. Of the children with S. dysenteriae type 1 infection, 42 had leukemoid reaction, hemolytic-uremic syndrome, or septicemia (complicated shigellosis), while the others did not (uncomplicated shigellosis). Polarization and locomotion in the absence of chemoattractants and in response to N-formylmethionyl-leucylphenylalanine (FMLP) and the lipopolysaccharide (LPS) of S. dysenteriae type 1 were determined. Attachment to unopsonized and opsonized yeast particles was also determined. Children with shigellosis (uncomplicated or complicated) had more polarized neutrophils with and without chemoattractants than uninfected children (P < 0.05). Children with complicated shigellosis had more polarized neutrophils with FMLP at 10(-7) and 10(-6) M (P < 0.05) and with LPS than children with uncomplicated shigellosis (P < 0.05). At 3 to 5 days after enrollment, the numbers of polarized neutrophils with 10(-8), 10(-6), and 10(-5) M FMLP declined in children with uncomplicated shigellosis but not in those with complicated shigellosis. Attachment to yeast particles was similar in all three groups of children. Locomotion was inhibited by LPS in children with shigellosis (P < 0.05), whether it was uncomplicated or complicated, compared with locomotion in uninfected children. Finally, neutrophil polarization in uninfected children was negatively influenced by nutritional status. Thus, poorly nourished uninfected children had more polarized neutrophils with FMLP at 10(-9) M (P < = 0.02) and 10(-5) M (P = 0.043) than their better-nourished counterparts. In summary, altered neutrophil responses are associated with both uncomplicated and complicated shigellosis.     

Detection of clostridial toxins in stools from children with diarrhoea

A cell-culture assay was used to detect toxins directly in stools from sporadic cases of infantile diarrhoea. Cytotoxins were revealed in 11 out of 58 samples from children with diarrhoea, nine of whom had no common enteric pathogens in their stools. A preliminary characterisation of the cytotoxins was obtained by neutralisation tests with clostridial antitoxins.     

Alterations in lymphocyte phenotype and function in children with shigellosis who develop complications.

This study was designed to see whether alterations occur in peripheral blood mononuclear cell phenotype and function in children with Shigella dysenteriae 1 infection with complications (leukemoid reaction and/or hemolytic-uremic syndrome) and whether there are any alterations prior to the development of complications. The following groups of children (ages, 12 to 60 months) were compared: children without any infection (n = 51), children with uncomplicated shigellosis (n = 65), children admitted with complicated shigellosis (leukemoid reaction and/or hemolytic-uremic syndrome) (n = 29), and children with shigellosis who developed complications after enrollment (subsequently complicated shigellosis) (n = 12). Tests for the peripheral blood mononuclear cell phenotype (CD3, CD4, CD8, CD57 [corrected], CD20, and CD25), spontaneous proliferation, and the proliferative response to phytohemagglutinin, pokeweed mitogen, and the lipopolysaccharide of S. dysenteriae 1 were performed, as were skin tests for delayed-type hypersensitivity (DTH). Children who subsequently developed complications differed from other groups of children as follows: (i) the numbers of CD3+ and CD4+ cells were lower than in uninfected children (P < 0.05), (ii) the CD4/CD8 ratio was lower than in children with uncomplicated shigellosis (P < 0.05) and in uninfected children (P < 0.05), and (iii) the levels of spontaneous proliferation of peripheral blood mononuclear cells were higher and DTH responses were lower than those in children with uncomplicated shigellosis (P < 0.05 and P < 0.017, respectively). Children with complications differed by having (i) increased numbers of CD3- CD57- [corrected] CD20- cells (P < 0.05) compared with those in other groups of children and (ii) lower CD4/CD8 ratios (P < 0.05), higher levels of spontaneous proliferation (P < 0.05), and lower DTH responses (P = 0.005) than children with uncomplicated shigellosis. Three to five days after enrollment, the number of CD4+ cells increased in children who subsequently developed complications (P = 0.025), i.e., when they developed complications and at this time their CD4+ cell number was similar to that of other groups of children. Thus, lymphocyte phenotype and function are altered prior to the development of complications in children with shigellosis, and once complications develop, the pattern of alterations changes. Whether these alterations have a role in precipitating complications or whether they reflect early events underlying the development of complications remains to be elucidated.     

Viruses in the stools.

It has long been possible to isolate viruses from the stools by culture, though the viruses found are rarely implicated in disease of the gut. In contrast, only recently has it been possible to identify viruses in the stools of patients with diarrhoea. Initially, such identifications were made by electron microscopy but the unsuitability of the microscope for large-scale screening has led to the development of other methods. The new methods have concentrated on rotaviruses but other viruses are also implicated and an overall view of the significance of finding a virus in any stool specimen has to take into account the evidence about all viruses, old and new.     

Lipopolysaccharide-specific antibodies in plasma and stools of children with Shigella-associated leukemoid reaction and hemolytic-uremic syndrome.

Antibody responses to the lipopolysaccharide (LPS) of shigellae were compared between children with uncomplicated and complicated Shigella dysenteriae 1 infection. One hundred fifteen children between 12 and 60 months of age with S. dysenteriae 1 infection were studied. Of these children, 42 had complications (leukemoid reaction and/or hemolytic-uremic syndrome [complicated shigellosis] and 73 had no complications (uncomplicated shigellosis). Antibodies to the LPS of S. dysenteriae 1 and Shigella flexneri Y were measured in plasma and stools, as were total immunoglobulin A (IgA) and IgG concentrations in plasma and the total IgA concentration in stool, on enrollment and 3 to 5 days later. In the plasma, the concentrations of homologous (IgG) and heterologous (IgA) LPS antibodies on enrollment were higher in children with complicated shigellosis than in those with uncomplicated shigellosis. In stool, the concentrations on enrollment were similar between the two groups of children. There was a rise in antibody concentrations in the plasma (homologous and heterologous) and stool (homologous) between the day of enrollment and 3 to 5 days later in children with uncomplicated shigellosis but not in those with complicated shigellosis. These findings suggest that systemic stimulation is more marked in children with complications, so that a subsequent rise in plasma antibody concentrations does not occur in these children. In contrast, the lack of a rise in stool antibody concentrations in children with complicated shigellosis is suggestive of a lower-level mucosal response. Because the duration of diarrhea before enrollment influenced the homologous antibody concentrations, children were further divided into three subgroups (short [3 to 5 days], medium [6 to 9 days], and long [> 9 days] diarrhea durations before enrollment). Comparisons of homologous antibody concentrations between the two groups of children following such subdivisions showed that in children with complicated shigellosis, antibody concentrations were higher in the plasma of children in the short diarrhea duration subgroup but lower in the stool children in the medium diarrhea duration subgroup. No differences in antibody concentrations were observed in children in the other diarrhea duration subgroups. Thus, complications in shigellosis are associated with an early and strong systemic stimulation without a concomitant stimulation of the mucosal antibody response.     

Cytokines in exhaled breath condensate of children with asthma and cystic fibrosis.

BACKGROUND: Inflammatory mediators in exhaled breath condensate (EBC) indicate ongoing inflammation in the lungs and might differentiate between asthma and cystic fibrosis (CF). OBJECTIVES: To evaluate the presence, concentration, and short-term variability of TH1- and TH2-mediated cytokines (interferon-gamma [IFN-gamma], tumor necrosis factor alpha [TNF-alpha], interleukin 10 [IL-10], IL-5, IL-4, and IL-2) in EBC of children with asthma or CF and in controls and to analyze the discriminating ability of inflammatory markers in EBC between children with asthma or CF and controls. METHODS: Expired air was conducted through a double-jacketed glass tube cooled by circulating ice water. In 33 asthmatic children, 12 children with CF, and 35 control children, EBC was collected during tidal breathing. Cytokines were measured using flow cytometry. RESULTS: Interleukin 2, IL-4, IFN-gamma, and IL-10 were detected in 16%, 16%, 11%, and 9%, respectively, of all samples in asthma and CF. Interleukin 5 and TNF-alpha were not detected in children with CF. Cytokine concentrations did not differ significantly in children with asthma vs CF. In controls, IFN-gamma, TNF-alpha, and IL-10 were detected in 9%, 14%, and 3%, respectively; IL-2, IL-4, and IL-5 were not detected in controls. CONCLUSIONS: Cytokines such as IFN-gamma, TNF-alpha, IL-10, IL-5, IL-4, and IL-2 can be detected in EBC of children with asthma or CF. However, the concentrations found are close to the detection limits of the assay used. These findings emphasize the importance of developing more sensitive techniques for the analysis of EBC and of standardizing the EBC collection method.     

Delayed and reduced adaptive humoral immune responses in children with shigellosis compared with in adults

We hypothesized that increased susceptibility to Shigella infection, increased severity of disease and high mortality in children compared with adults were consequences of insufficient adaptive immune responses. Antigen-specific immune responses were studied in paediatric patients (n = 38, 2-10 years) with shigellosis and compared with those of adult patients (n = 30, 18-45 years). Peak frequencies of antigen (invasion plasmid coded antigen B, Ipa-B; lipopolysaccharide, LPS)-specific immunoglobulin (IgM)-antibody secreting cells (ASC) were seen within 3-5 days after the onset of diarrhoea in children, while peak IgA- and IgG-ASCs were obtained 8-10 days later in line with adults. Antigen-specific ASC responses in children ranged between 2 and 4% of the total ASC responses, in contrast to 8-15% in adults. The kinetics of LPS-specific IgG subclass titres was different in younger children (2.5-5 years) (IgG1 > IgG2 > IgG4 > IgG3) compared with in older children (6-8 years) (IgG2 > IgG1 >IgG3 > IgG4) and adults. Secretory IgA levels in stool peaked 8-10 days after onset in both adults and children. However, a rapid induction of stool LPS-specific IgA, IgA1 and IgA2 occurred in adult patients within 3-5 days of onset, while in children, this was delayed by 8-10 days. Similarly, higher number of tumour necrosis factor (TNF)-alpha and interferon (IFN)-gamma expressing cells in vitro were seen in adult patients in response to antigens (LPS and Ipa-B) in the acute stage in contrast to paediatric patients. Thus, paediatric patients with shigellosis have reduced and delayed adaptive immune responses compared with adult patients.     

Clostridium difficile and its cytotoxin in the stools of young hospitalized children. Influence of antibiotic treatment

Clostridium difficile has been searched in 153 stool samples from 138 children aged 0 to 12 months. We divided the population in two groups depending on the antibiotic treatment. We have found C difficile in 39 samples (25%). The colonization rate increases with age ranging from 5% before 1 month, to 36% between 1 and 6 months and 54% between 6 and 13 months. An environmental sampling yielded once C difficile. Contamination may be related to the environment. 29% of the isolates produced a cytopathic toxin. Toxin titers in infants' stools range from 1/160 to 1/10240. One only of these children had diarrhea. C difficile and its toxin does not seem to infer any signs of enteric illness with infants. The results obtained with the group of non treated infants are not significantly different from the ones of the other group: the colonization rates are 21% in the non treated group and 29% in the other group. The rate of strains yielding a cytophatic toxin is similar in the 2 groups. It seems reasonable to agree that antibiotics do not influence the settlement of C difficile in infants' intestine.     

The association of Blastocystis hominis and Endolimax nana with diarrheal stools in Zambian school-age children

To determine the prevalence of endoparasites and their association with diarrhea, a survey was conducted in the Southern Province of Zambia that used conventional and molecular techniques applied to stool and urine samples from school-age children (n=93). Almost half of the stools (49.5%) were diarrhetic. The overall prevalence of Endolimax nana, Schistosoma haematobium, Blastocystis hominis, Giardia lamblia, Cryptosporidium parvum, Encephalitozoon intestinalis, and Strongyloides stercoralis was 64.3, 59.1, 53.8, 19.4, 8.6, 8.6, and 1.1%, respectively. Only the associations between infection with B. hominis and E. nana with diarrhea were statistically significant. Although B. hominis and E. nana are considered to be nonpathogenic organisms, this study demonstrated that they can be associated with diarrhea in children when they occur at high prevalence and intensity. This survey supports the recent evidence that B. hominis and E. nana infections are associated with deficient sanitation and low hygiene standards and can contribute to diarrhea in children in developing countries.     

手足口病并发脑炎患儿脑电图检查的意义

目的：探讨脑电图（EEG）对小儿手足口病并发脑炎的应用及意义。方法：回顾性分析103例临床确诊为手足口病并发脑炎的患儿EEG的改变。结果：本组103例中EEG正常3例（2．9％）,异常100例（97．1％）,主要表现为弥漫性慢波,部分出现阵发性尖（棘）波或尖（棘）慢波。结论：EEG可作为小儿手足口病并发脑炎的诊断及判断小儿手足口病并发脑炎严重程度的一个指标;EEG的动态监测对判断手足口病并发脑炎患儿临床病情转归有一定的参考意义。     

Detection of Escherichia coli enterotoxins in stools.

We determined whether enterotoxigenic Escherichia coli diarrhea could be diagnosed by direct examination of stools for heat-labile (LT) and heat-stable (ST) enterotoxins. The Y-1 adrenal cell and an enzyme-linked immunosorbent assay (ELISA) detected LT in 85 and 93%, respectively, of stool specimens obtained from adults with acute diarrhea from whom an LT- and ST-producing organism had been isolated. Furthermore, the ELISA assay detected LT in 8 of 35 stool specimens from which no LT-producing E. coli had been isolated. The infant mouse assay was utilized to detect ST in these stool specimens and was found to be an insensitive method, showing positive results in only 36% of the specimens from which an ST-producing organism was isolated. Further studies are warranted to determine the diagnostic value of direct detection of LT in stools, especially by the ELISA method.     

Routine diagnosis of human rotaviruses in stools.

Electron microscopy, immune electron microscopy and complement fixation as methods of detecting rotavirus in the stools of young children with gastroenteritis were compared in a blind study during the winter of 1975-6. Complement fixation was the simplest to perform, was as sensitive as the other two, and allowed a quantitative measurement of viral excretion. Absorption of faecal extracts with fetal calf serum usually removed the anticomplementary activity of faecal extracts.     

Head-up tilt test in complicated neurocardiogenic syncope in children.

To confirm the usefulness of head-up tilt test (HUT) in neurocardiogenic syncope (NCS) with complicating clinical features, retrospective analysis were done on 12 selected children. The age at onset was 12.7 +/- 1.9 (mean +/- SD) years. Associated clinical features were postoperative congenital heart disease (PO CHD) in 3, coexistent arrhythmia in 8 (persistent ventricular arrhythmia during exercise in 3, premature ventricular contractions in 2, ventricular couplets in 1, sinoatrial exit block in 1 and resting sinus bradycardia in 1) and ST segment depression during exercise in 1. Four of them had a history of exercise-related syncope. All 3 patients with PO CHD had arrhythmia (ventricular tachycardia in 1, sinus bradycardia in 1 and atrioventricular block in 1). HUT provoked NCS in 8 (2 during baseline tilt, 6 during isoproterenol infusion). In one each, ventricular tachycardia and loss of consciousness without hypotension and bradycardia were induced. Atenolol was tried in 5 with improvement of NCS in 4 and aggravation of dizziness in 1. During follow-up, 7 became asymptomatic (2 with atenolol) and 5 were stationary. In conclusion, HUT was valuable in diagnosing NCS even in children with complicating clinical features such as arrhythmias or PO CHD. HUT could be done as apart of initial diagnostic tests if the past history suggests NCS, regardless of associated clinical features. In some cases, the unexpected results of the test turned out useful in managing children with syncope or dizziness.     

Treatment of salmonellosis and shigellosis with the new quinolones

Sixty patients with enterocolitis (36 men, 24 females), mean age thirty seven (12 to 84) have been treated with new quinolones. Two groups have been constituted. First group included patients without bacteremia despite enterocolitis. Among them, 9 patients suffered from shigellosis (8 S. flexneri, 1 S. sonnei). Twenty-two patients had a minor salmonellosis (13 S. typhimurium, 4 S. enteritidis, 1 S. blockley, 1 S. Virchow, 1 S. london, 1 S. baildon, 1 S. C2 group). Patients in the second group underwent a septicemic salmonellosis (18 S. typhi, 3 S. paratyphi, 8 other Salmonella serotypes). All patients but one received an oral treatment. Forty-seven patients received 400 mg/day ofloxacin, 8 patients 1.500 mg/day ciprofloxacin, 5 patients 800 mg/day pefloxacin. Treatment lasted an average of 10 days (5 to 31 days). Apyrexia was observed within an average of 3 days (1 to 8 days). Blood culture have always been sterile after a two days treatment duration. No more shigella or salmonella was found in feces, five days after beginning of treatment. No relapse has been observed within a 30 days mean background (1 to 180 days). Not any intolerance needed to stop treatment. Patients have been hospitalized on an average of 11 days. Therefore, new quinolones will represent an effective, well tolerated treatment for salmonellosis or shigellosis.     

Cytokines in mice treated with amphotericin B-intralipid.

Amphotericin B (AMB) intralipid (IL) admixtures (AMB-IL) are composed of components approved for clinical use and are commercially available at low cost. They are stable and exhibit in-vitro and in-vivo efficacy against Candida infections, as well as resulting in significantly reduced toxicity in comparison with that of conventionally administered amphotericin B. We examined the production of cytokines in uninfected mice treated with AMB or AMB-IL, as evaluated by expression of mRNA corresponding to the cytokines. Expression was measured by intensity of bands in comparison to the intensity of beta-actin control bands, with the latter assigned an arbitrary standard value of 100% and other bands measured in relative percentages. We found that both in naive and compromised mice, AMB treatment caused significantly greater production of the pro-inflammatory cytokines tumour necrosis factor alpha (TNF-alpha) and interleukin 1 beta (IL-1beta) than was seen in animals treated with AMB-IL or with another lipid AMB formulation, AmBisome. We hypothesize that the superior tolerance for the AMB-IL admixtures, as compared with conventional AMB, might derive from the reduced expression of the pro-inflammatory cytokines. TNF-alpha and IL-1beta, which mediate many potentially adverse pathophysiological events similar to those seen as side-effects of AMB usage.