Cytokines as Mediators for or Effectors against Rotavirus Disease in Children

Rotavirus is the most common cause of severe gastroenteritisin young children, but the pathogenesis and immunity of thisdisease are not completely understood. To examine the host responseto acute infection, we collected paired serum specimens from30 children with rotavirus diarrhea and measured the levelsof nine cytokines (interleukin-1ß [IL-1ß],IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, gamma interferon [IFN- ${gamma}$ ],and tumor necrosis factor alpha [TNF- ${alpha}$ ]) using a microsphere-basedLuminex Flowmetrix system. Patients with acute rotavirus infectionhad elevated median levels of seven cytokines in serum, andof these, the levels of three (IL-6, IL-10, and IFN- ${gamma}$ ) were significantly(P < 0.05) higher than those in serum from control childrenwithout diarrhea. Patients with fever had significantly (P <0.05) higher levels of IL-6 in serum than control children,and those with fever and more episodes of diarrhea had significantly(P < 0.05) higher levels of TNF- ${alpha}$ than those without feverand with fewer episodes of diarrhea. We further demonstrateda negative association (P < 0.05) between the levels of IL-2and the number of stools on the day on which the first bloodsample was collected. Finally, patients with vomiting had significantly(P < 0.05) lower levels of IFN- ${gamma}$ than those without vomiting.Our pilot study provides evidence that the types and magnitudesof cytokine responses to rotavirus infection in children influenceor reflect the clinical outcome of disease. These findings suggestthat certain cytokines may play an important role in the pathogenesisof and the protection against rotavirus disease in childrenand, consequently, may provide directions and insights thatcould prove critical to the prevention or treatment of thisimportant disease.

INTRODUCTION

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Introduction

Group A rotavirus is the single most important cause of severedehydrating diarrhea in young children worldwide, and in developingcountries, it causes over 500,000 deaths each year (20). Rotavirusis a nonenveloped, double-stranded RNA virus that infects matureepithelial cells of the small intestine. Children with rotavirusinfections often develop symptoms of fever, vomiting, and diarrhea.Several mechanisms have been proposed to describe the pathogenesisof rotavirus diarrhea in animals (18), but it is unclear whetherthese apply to humans as well. Furthermore, the mechanisms involvedin the induction of fever and vomiting are poorly understood.This incomplete understanding of the pathogenesis of diseasein natural rotavirus infection has hampered our ability to designthe most effective strategies to prevent or treat rotavirusdiarrhea.

A host typically responds to a viral infection by developingboth innate immunity and specific adaptive immunity (35). Innateimmunity occurs in the early days of infection and plays animportant role in either the pathogenesis of or immunity tothe disease by an array of mechanisms, including activationof proinflammatory signaling pathways and the rapid productionof cytokines (34, 35, 45). Cytokines, which also include chemokines,can exhibit both pro- and anti-inflammatory activities and mediatedisease or protect the host against viral infection or disease(15, 16, 38-40, 46). In contrast, specific adaptive immunityappears later and protects the host from viral infection byantigen-specific T-cell-mediated killing of infected cells andantibody and effector cytokine responses (34, 35).

Rotavirus infection results in both cellular and humoral responsesthat protect against reinfection or severe diarrhea in youngchildren or animals when they are reinfected (5, 6, 29). However,children with a typical rotavirus infection begin recoveringfrom disease in the first week of illness, before rotavirus-specificT cells or antibodies are fully developed in the small intestine(18, 28). This observation suggests that innate immunity, suchas that provided by cytokines, participates in aiding in earlyrecovery from disease.

Only a few recent studies have demonstrated elevated levelsof several cytokines—alpha interferon (IFN- ${alpha}$ ), IFN- ${gamma}$ , interleukin-10(IL-10), and tumor necrosis factor alpha (TNF- ${alpha}$ )—in theserum or plasma of children with rotavirus diarrhea (2, 7).Little is known, however, about the role of these cytokinesin the pathogenesis of or immunity to rotavirus disease. Inorder to further our understanding of the cytokine profilesin children with rotavirus infections, we collected serum specimensfrom children with acute rotavirus diarrhea and from controlswithout gastroenteritis and measured the concentrations of ninecytokines. We further identified several cytokines that mightbe involved in innate immunity to rotavirus and the pathogenesisof rotavirus disease.

MATERIALS AND METHODS

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Materials and Methods

Study population and specimen collection. From February 1999 to June 2000, a prospective study was conductedto collect blood and fecal specimens from children <2 yearsof age seen at Children's Healthcare of Atlanta in Atlanta,Ga., and Hasbro Children's Hospital in Providence, R.I. Thirtypatients who were admitted for treatment of acute gastroenteritisin the winter or spring but who were otherwise in good healthwere enrolled in the study if their stools tested positive forrotavirus by enzyme immunoassay (EIA). Nurses constantly observedthe patients. Demographic and clinical data, including fever,vomiting, diarrhea, dehydration, and total severity scores duringthe entire period of illness and on the day on which the firstblood sample was collected, were obtained from 25 patients enrolledin Hasbro Children's Hospital. Each patient's temperature wasmeasured rectally approximately every 4 h. Total severity scoreswere based on symptom scores for diarrhea, vomiting, fever,dehydration, and hospital stay (13). Two blood samples wereobtained from each patient; the first sample was collected within72 h of enrollment and the second one was collected 21 to 28days later. The sera were aliquoted and stored at -70°C.Fecal specimens were collected from all patients and storedat -20°C.

Blood and fecal specimens were also obtained from nine childrenwho did not have gastroenteritis. Like most of the patients,the control children were enrolled in the spring (April to June).Single serum specimens were obtained from three children, andpaired serum specimens were obtained 5 weeks apart from theother six children. Finally, individual serum specimens wereobtained from healthy adults and screened for cytokines, andif they were negative, they were pooled for use in the assayas a diluent (blank) for the standard curves. Written consentwas obtained from all parents or guardians, and the protocolwas approved by the institutional review boards of the Centersfor Disease Control and Prevention and each of the hospitals.

Characterization of rotavirus strains. The G and P genotypes and serotypes of the rotavirus strainswere determined by reverse transcriptase PCR and serotypingEIA, respectively, by procedures described previously (9, 14).

Detection of cytokines. A standard capture sandwich assay was developed with the LuminexFlowmetrix system (Luminex, Austin, Tex.) to determine the levelsof cytokines in sera. Each capture antibody was coupled to adifferent bead set (microsphere beads; Luminex) (Table 1). Standards(recombinant cytokines) diluted in pooled blank serum collectedfrom healthy adults and test sera from patients or controlswere examined by one of the two multiplex assays: the 4-plexassay (with four bead sets) and the 5-plex assay (with fivebeads sets). The individual bead sets in the 4-plex assay werecoupled with monoclonal antibody to IL-1ß, IL-8, IL-10,or IL-12; and the individual bead sets in the 5-plex assay werecoupled with antibody to IL-2, IL-4, IL-6, IFN- ${gamma}$ , or TNF- ${alpha}$ . Thebeads were incubated first with diluted standards or sera andthen with detector antibodies for 30 min each at room temperature;washed twice in phosphate-buffered saline supplemented with0.02% Tween 20, 0.1% bovine serum albumin, and 0.02% NaN₃; andincubated for 15 min with fluorescent dye-conjugated streptavidin(Table 1). Cytokine levels were measured by using a flow cytometerand were analyzed with Flowmetrix software (Luminex).

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TABLE 1. Reagents used for measurement of cytokine levels with the Luminex Flowmetrix system

Analysis of cytokine data. Standard curves for each cytokine were generated on a log-logplot for each assay, and the cytokine concentrations in eachserum sample were calculated from the corresponding curve-fittingequations (8). Each specimen was tested at least twice, andthe average values were reported and analyzed. Nonparametricstatistics were used for analysis because the data were notnormally distributed (25). The cytokine levels in patients withrotavirus diarrhea and control children were compared by theWilcoxon rank-sum test. These comparisons were adjusted forsex and age by using the blocked Wilcoxon rank-sum test. Cytokinelevels in acute- and convalescent-phase sera were compared bythe Wilcoxon signed-rank test.

The Wilcoxon rank-sum test was used to examine the associationof cytokine levels with the presence of symptoms (fever, vomiting,and diarrhea) (25). Spearman's rank correlation coefficientswere used to measure the strength of the associations betweencytokine levels and symptom severity.

RESULTS

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Results

Characteristics of patients and characterization of rotavirus strains. A total of 30 patients (10 girls, 20 boys) were enrolled inthe study. These patients ranged in age from 1 to 20 months,with a mean age of 8.0 ± 1.2 months and a median ageof 6.5 months. Most patients had typical clinical symptoms ofrotavirus gastroenteritis: fever, vomiting, and diarrhea. Twenty-fiveof the 30 patients were enrolled in Hasbro Children's Hospital,and detailed symptom scores during the course of illness andon the day on which the first blood sample was collected wereavailable for these patients; these included temperature, thenumber of vomiting episodes per day and the duration of vomiting,the severity of diarrhea (numbers of stools per day, durationof diarrhea, and percent dehydration), length of stay in hospital,and total symptom scores. Such detailed symptom scores werenot available for the other five patients enrolled at Children'sHealthcare of Atlanta. The nine control children (six girls,three boys) ranged in age from 1 to 7 months, with a mean ageof 4.9 ± 0.5 months and a median age of 5 months. Thismean age was not significantly (P > 0.05) different fromthat for the patients.

Of the 30 rotavirus strains examined, 23 (77%) were P[8],G1;4 (13%) were P[4],G2; and 1 (3%) each was P[6],G9, P[8],G9,and P[6],G1. Of the strains from 25 patients whose symptom scoreswere available and whose sera were examined for cytokine responses,20 (80%) were P [8],G1, 4 (17%) were P[4],G2, and 1 (4%) wasP[8],G9.

Development of multiplex Luminex assays. To simultaneously measure the levels of multiple cytokines inindividual serum specimens, we developed 4-plex and 5-plex assays.The standard curves for the cytokines in multiplex assays werecomparable to those for cytokines in single assays (data notshown). These results indicate a lack of steric hindrance andcross-reactivity between cytokines and antibodies in our multiplexassays.

Elevation of cytokine levels in sera of children with rotavirus diarrhea. Cytokine levels were measured in 30 paired serum specimens frompatients with rotavirus diarrhea and 15 serum specimens fromcontrol children, with sera from healthy adults used as theblank. Patients in the acute phase generally had higher medianlevels of seven of the nine cytokines (i.e., all cytokines exceptIL-4 and IL-12) than control children (Table 2); but the levelsof IL-6, IL-10, and IFN- ${gamma}$ were significantly elevated (P <0.05). In comparison, patients in the convalescent phase generallyhad higher median levels of six of the nine cytokines, withsignificant (P < 0.05) elevations in the levels of IL-6 andTNF- ${alpha}$ . When the concentrations of cytokines in acute- and convalescent-phasesera were compared, lower median levels of five cytokines (IL-2,IL-6, IL-8, IFN- ${gamma}$ , and TNF- ${alpha}$ ) were detected in convalescent-phasesera, but only the level of IL-6 was significantly (P < 0.05)lower. The levels of the other four cytokines in convalescent-phasesera were little changed (IL-4 and IL-10) or elevated (IL-1ßand IL-12). Because the levels of IL-1ß and IL-8 inthe sera of patients with rotavirus diarrhea were generallyhigh and were outside the linear ranges of the standard curves,the concentrations of these two cytokines were measured in thepresence of nonlabeled detector antibodies (Table 1).

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TABLE 2. Concentrations of nine cytokines in sera of rotavirus-infected patients and control children^a

Cytokines as mediators for symptom expression. We then examined possible associations between the levels ofthe nine cytokines in acute-phase sera and scores for the 10different symptoms for the 24 patients enrolled in Hasbro Children'sHospital by determining Spearman's rank correlation coefficients.Of the 90 potential correlations, we identified weak but significantly(P < 0.05) positive associations between elevated TNF- ${alpha}$ levels(median, 23.7 pg/ml; mean, 41.0 ± 10.5 pg/ml) and severalsymptom scores. For example, three patients had no fever andtheir TNF- ${alpha}$ levels in serum were lower than the median level(23.7 pg/ml). In contrast, 21 patients manifested a temperatureabove 37°C, and 14 (67%) of them had TNF- ${alpha}$ levels that weresimilar to or higher than the median, with the concentrationsranging from 20.2 to 165.6 pg/ml. There was a weak but significant(P < 0.05) dose-response relationship between elevated TNF- ${alpha}$ levels and the highest temperature during the entire periodof illness (Fig. 1A). Elevated TNF- ${alpha}$ levels were also associated,in a dose-dependent manner, with the numbers of diarrheic stoolsthat patients shed per day (P < 0.05) (Fig. 1B).

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FIG. 1. Associations of serum TNF- ${alpha}$ levels with severity of fever (A) and diarrhea (B). TNF- ${alpha}$ levels and symptom scores for 24 patients were analyzed as described in the text. Spearman's rank correlation coefficient (r_s) is presented (P < 0.05).

Patients with fever also had elevated levels of IL-6 in theacute-phase serum (median, 178.3 pg/ml; mean, 241.3 ±48.9 pg/ml). This association, however, was not in a dose-responsemanner, as the severity of fever did not clearly correlate withmore elevated levels. Nevertheless, 21 patients with fever (temperature,>37°C) had IL-6 levels (median, 174.9 pg/ml; mean, 251.2± 55.4 pg/ml) significantly (P < 0.05) higher thanthose in 15 serum specimens from control children (median, 109.5pg/ml; mean, 121.7 ± 18.4 pg/ml) (Fig. 2).

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FIG. 2. Comparison of IL-6 levels in sera of patients with fever and control children. The concentrations of IL-6 in 21 serum specimens from patients with fever and 15 specimens from controls are shown. Patients had significantly higher levels of IL-6 than control children. The statistical significance of the differences was determined by the two-tailed t test (P < 0.05).

Cytokines as effectors against symptoms. Of the nine cytokines, two (IL-2 and IFN- ${gamma}$ ) were identified tobe significantly associated with less severe symptom scoreson the day on which the first blood sample was collected. IL-2was detected in sera from 20 of the 24 patients, and the levelswere significantly (P < 0.01) higher in sera collected 4days after the onset of illness (median, 25.2 pg/ml; mean, 33.0± 9.2 pg/ml) than in sera collected in the first 3 days(median, 1.1 pg/ml; mean, 3.4 ± 1.7 pg/ml) (Fig. 3A).There was a weak but significant (P < 0.05) association betweenelevated IL-2 levels and fewer diarrheic episodes in patientswith rotavirus infection (Fig. 3B).

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FIG. 3. Concentrations of IL-2 in sera of patients with diarrhea (A) and association of serum IL-2 levels with severity of diarrhea (B). Of the 24 serum specimens from patients, 12 were collected 4 days after the onset of illness and had significantly higher levels of IL-2 than those collected in the first 3 days of illness. The statistical significance of the difference was determined by the two-tailed t test (P < 0.01). Elevated levels of IL-2 were significantly associated with fewer stools on the day of blood collection. Spearman's rank correlation coefficient (r_s) is presented (P < 0.05).

IFN- ${gamma}$ was detected in 22 (92%) of the 24 serum specimens frompatients with rotavirus diarrhea, whereas it was detected in8 (53%) of the 15 serum specimens from control children. Sevenof the 24 patients experienced at least one vomiting episodeon the day on which the first blood sample was collected andhad significantly (P < 0.05) lower IFN- ${gamma}$ levels (median, 4.7pg/ml; mean, 7.6 ± 3.4 pg/ml) than those of 17 patientswithout vomiting (median, 19.0 pg/ml; mean, 32.4 ± 8.6pg/ml) (Fig. 4A). Control children had median and mean IFN- ${gamma}$ levels of 1.4 and 18.0 ± 8.1 pg/ml, respectively. Therewas no significant difference in the levels between controlchildren and patients with or without vomiting. Of interest,12 of the 24 serum samples from patients were collected 4 daysafter the onset of illness and had median and mean IFN- ${gamma}$ levelsof 23.6 and 37.5 ± 11.3 pg/ml, respectively (Fig. 4B).Seven (58%) of these 12 patients had serum IFN- ${gamma}$ levels $>=$ 20pg/ml, and 2 (17%) experienced vomiting. In contrast, the other12 serum samples collected in the first 3 days of illness hadlower, although insignificant (P = 0.06), median and mean IFN- ${gamma}$ levels of 6.3 and 12.8 ± 5.0 pg/ml, respectively. Only2 (17%) of these 12 patients had serum IFN- ${gamma}$ levels $>=$ 20pg/ml, and 5 (42%) experienced vomiting episodes.

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FIG. 4. Concentrations of IFN- ${gamma}$ in sera of patients with rotavirus infection. (A) Concentrations of IFN- ${gamma}$ in 7 serum specimens from patients with vomiting, 17 specimens from patients without vomiting, and 15 specimens from control children. Patients with vomiting had significantly lower levels of IFN- ${gamma}$ than those without vomiting. (B) Sera collected 4 days after the onset of illness had significantly higher levels of IFN- ${gamma}$ than those collected in the first 3 days of illness. The statistical significance of the differences was determined by the two-tailed t test (P < 0.05).

Age-dependent symptom expression. We conducted additional analyses to examine if cytokine responsesand disease severity in children with rotavirus diarrhea weregender and age dependent. There were no significant differencesin cytokine responses and disease severities between girls andboys. While cytokine responses were not age related, severalsymptom scores were. Older children with rotavirus infectiontended to have fewer stools on the day of blood collection (P< 0.05) (Fig. 5A), but they were more likely than youngerpatients to vomit (P < 0.05) (Fig. 5B) and become dehydrated(P < 0.01) (Fig. 5C).

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FIG. 5. Age and disease severity by selected symptoms. Demographic and clinical data for 25 patients were analyzed as described in the text. Spearman's rank correlation coefficient (r_s) is presented. +, P < 0.05; #, P < 0.01.

DISCUSSION

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Discussion

Cytokines comprise a diverse group of small proteins with bothpro- and anti-inflammatory properties and are increasingly recognizedto play critical roles in the pathogenesis and immunity of infectiousdiseases (4, 34). To examine cytokine profiles and their potentialfunctions in children with natural rotavirus infections, wedetermined cytokine concentrations in serum using a newly developedbead-based immunoassay. This assay provides an easy, rapid,sensitive, and accurate means by which cytokine levels can bemeasured in the sera of children with rotavirus diarrhea. Theability to simultaneously measure the levels of multiple cytokinesin a single reaction makes this assay even more appealing becausea significantly lower volume of serum is required compared withthe volume needed in traditional EIAs or bioassays, which measurethe levels of only one cytokine at a time and also require 20times more specimen (17, 44).

We demonstrated an overall increased cytokine response in childrenwith acute rotavirus diarrhea compared with those in controlchildren. The levels of three of the nine cytokines (IL-6, IL-10,and IFN- ${gamma}$ ) in acute-phase sera were significantly elevated. Theseresults indicate that both the Th1 and the Th2 types of cytokinesare produced in young children with natural rotavirus infection,and Th1-like anti-inflammatory cytokines (IL-2 and IFN- ${gamma}$ ) werepredominately produced in the late phase of acute infection.Our results agree with those of a recent study performed inBangladesh (2), in which the levels of three cytokines (IL-10,TNF- ${alpha}$ , and IFN- ${gamma}$ ) were found to be elevated in the sera of childrenwith acute or persistent rotavirus diarrhea compared with thelevels in uninfected, control children.

We present, for the first time, evidence for cytokine mediationof disease expression in children with rotavirus infection,as demonstrated by associations between elevated TNF- ${alpha}$ and IL-6levels and expression of fever in the acute phase of infection.Both TNF- ${alpha}$ and IL-6 are multifunctional cytokines and are consideredendogenous pyrogens. Once they are overproduced and presentin the circulation, these cytokines can act as humoral signalsand contribute to the acute febrile reaction by different mechanisms,including induction of the acute-phase protein response andinflammatory stimulation of the hypothalamus or afferent sensorynerves (11, 22, 36). Elevated levels of cytokines in serum orlocal secretions have also been documented to mediate diseaseexpression or respiratory tract inflammation in children oradults infected with influenza A virus or respiratory syncytialvirus (19, 27, 39, 40). In addition, we observed an associationbetween the overproduction of TNF- ${alpha}$ and more diarrheal episodesin patients with rotavirus infection, although the mechanismfor this relationship is unknown. It could be due to the abilityof TNF- ${alpha}$ to induce increased levels of Cl^- secretion in intestinalepithelial cells, as has been reported for patients with otherinfections or inflammatory processes (21, 31). We could notidentify cytokines that might mediate the expression or severityof vomiting. A previous study, however, demonstrated that IFN- ${alpha}$ levels were highly elevated and significantly correlated withthe severity (the number of episodes) of vomiting in childrenwith rotavirus diarrhea (7).

We demonstrated a delayed activation of IL-2 and IFN- ${gamma}$ in childrenwith rotavirus infection, as evidenced by their predominantproduction 4 days after the onset of illness. Because IL-2 issecreted exclusively by antigen-activated T cells and IFN- ${gamma}$ isproduced mainly by T cells, NK cells, and macrophages, thesetwo cytokines constitute a second wave of the host responseto infection in adaptive immunity (32, 41). The observationthat IL-2 and IFN- ${gamma}$ were negatively associated with symptoms(diarrheal or vomiting episodes) on the day on which the firstblood sample was collected indicates that these cytokines maybe important components of host protective immunity and, consequently,mediate antiviral activities and help patients recover fromsevere rotavirus disease. Our results concur with those of previousstudies that exogenously administered IFNs are protective againstdiarrhea in calves and pigs (23, 37), but they do not agreewith results showing a lack of protection against disease inmice (1). IFN- ${gamma}$ was previously found to be secreted by rotavirus-stimulatedperipheral blood mononuclear cells of healthy adults and toinhibit rotavirus entry into human intestinal epithelial cells(3, 47). Of note, IL-2 and IFN- ${gamma}$ have been demonstrated to conferresistance to other infectious diseases of humans and animals(12, 42).

We demonstrated no significant associations of elevated cytokinelevels in the acute phase with total severity scores, whichare a combined assessment of symptoms (fever, vomiting, diarrhea,and dehydration) and length of hospital stay. Furthermore, wefound no significant associations between elevated cytokinelevels in the convalescent phase and symptom scores. These datasuggest that the associations between elevated levels of severalcytokines in patients with acute diarrhea and individual symptomscores are specific.

Another important finding of this study is the significant elevationof IL-10 levels in the sera of children with rotavirus diarrhea.Like most other cytokines, IL-10 exhibits numerous effects onthe modulation of immune responses and ion transport in thesmall intestine, and it presents both potential benefits (anti-inflammatoryeffect) and severe risks (association with fatality) to patientswith infectious diseases (24, 26, 30). However, in our study,IL-10 was not associated with either protection against rotavirusdisease or the expression or severity of fever, vomiting, ordiarrhea. Whether IL-10 is associated with other systemic symptomsor even fatal outcomes, as reported in humans and animals withmeningococcal or pneumococcal disease (24, 43), remains to bedetermined.

We demonstrated no distinct age-related differences in cytokineresponses to rotavirus infection but found a positive associationbetween age and disease severity, as evidenced by less severevomiting and dehydration among infants in the first severalmonths of life. Our finding that older children have fewer stoolsthan younger infants suggests that age itself is a deterrentof diarrhea severity, an observation reported previously (10,33). Further studies are needed to examine and compare the cytokineand other host responses between younger and older children.

Our present study has several limitations. First, the standardantigens used in our bead-based Luminex assay were recombinantcytokines expressed in Escherichia coli or insect cells, andantibodies were raised against these recombinant cytokines.Despite their specificity, affinity, and ability to recognizemost isoforms of recombinant cytokines, it is not clear if theseantibodies bind to all isoforms of the native cytokines. Consequently,the use of these recombinant cytokines, together with the inclusionof nonlabeled detector antibodies (Table 1), in the assay mayresult in underestimation of the levels of some cytokines. Second,the relatively small numbers of children and the large variationsin cytokine concentrations may have affected the accuracy ofour results. Future studies will need to examine larger numbersof age- and sex-matched control and patient populations to substantiateassociations between cytokine levels and disease severity, examinepossible differential cytokine profiles in children infectedwith various rotavirus serotypes, and subsequently identifypotential associations between serotypes and disease severity.In addition, because the half-lives of most cytokines are believedto be short, a series of serum specimens collected at varioustime points would be needed to examine the kinetics of the cytokineprofiles. However, we were able to collect only one serum specimenduring each of the acute and convalescent phases of infectionfrom young children with rotavirus diarrhea. Finally, the originsof the serum cytokines detected in our study are not clear.They could be secreted by NK cells, lymphocytes, monocytes,or macrophages in the circulation or in intestinal tissues.Since most cytokines exhibit their function locally in a paracrineor even autocrine fashion, further studies are needed to examinethe cytokine profiles present in intestinal fluids or expressedin lymphoid or epithelial cells of the small intestine fromchildren with rotavirus diarrhea.

In summary, we have examined the cytokine responses to rotavirusin children with diarrhea and identified several cytokines thatare potential mediators for or effectors against the expressionof clinical symptoms or severity of disease. These new findingsshould help us redefine our research direction to include thecontributions of host factors to disease initiation and progression,a proposition that has not yet received much attention. We mayalso potentially use early cytokine profiles to predict diseaseoutcome or vaccine efficacy in hosts infected with or vaccinatedagainst rotavirus, as recently reported for other virus infections(48). Finally, these findings may allow us to design new therapeuticstrategies and vaccines against rotavirus disease in children.

ACKNOWLEDGMENTS

We thank Kathi Kellar and Ralph Tripp for help with the Luminexassays and Claudia Chesley for editorial assistance.

FOOTNOTES

* Corresponding author. Mailing address: Viral Gastroenteritis Section (MS G04), National Center for Infectious Diseases, 1600 Clifton Rd., Atlanta, GA 30333. Phone: (404) 639-2861. Fax: (404) 639-3645. E-mail: bjiang{at}cdc.gov.

REFERENCES

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