4« We stopped the trial at the first interim analysis because the all-cause mortality rate for patients treated with HA-1A who did not have Gram-negative bacteremia (42%; 244 of 577) exceeded that of the patients given placebo (38%; 230 of 608) by an amount greater than that in the prespecified safety stopping rule (F = 0.09; Fisher one-tailed exact test). »Objective: To compare the effectiveness of 100 mg of HA-1A and placebo in reducing the 14-day all-cause mortality rate in patients with septic shock and gram-negative bacteremia in the Centocor: HA-1A Efficacy in Septic Shock (CHESS) trial, and to assess the safety of 100 mg of HA-1 A given to patients with septic shock who did not have gram-negative bacteremia.• Design: Large, simple, group-sequential, randomized, double-blind, multicenter, placebo-controlled trial.• Setting: 603 investigators at 513 community and university-affiliated hospitals in the United States.• Patients: Within 6 hours before enrollment, the patients had been in shock with a systolic blood pressure of less than 90 mm Hg after adequate fluid challenge or had received vasopressors to maintain blood pressure. These episodes of shock began within 24 hours of enrollment. A presumptive clinical diagnosis of gram-negative infection as the cause of the shock episode and a commitment from the patients' physicians to provide full supportive care were required.• Measurements: Blood cultures were obtained within 48 hours of enrollment, and death at day 14 after treatment was recorded. Adverse events occurring within 14 days after enrollment were also tabulated.• Results: 2199 patients were enrolled; 621 (28.2%) met all enrollment criteria, received HA-1 A or placebo, and had confirmed gram-negative bacteremia. Mortality rates in this group were as follows: placebo, 32% (95 of 293) and HA-1 A, 33% (109 of 328) (P = 0.864, Fisher exact test, two-tailed; 95% CI for the difference, -6.2% to 8.6%). Mortality rates in the patients without gram-negative bacteremia were as follows: placebo, 37% (292 of 793) and HA-1 A, 41% (318 of 785) (P = 0.073, Fisher exact test, one-tailed; CI, -0.8% to 8.8%).• Conclusions: In this trial, HA-1 A was not effective in reducing the 14-day mortality rate in patients with gram-negative bacteremia and septic shock. These data do not support using septic shock as an indication for HA-1 A treatment. If HA-1 A is effective in reducing the mortality rate in patients dying from endotoxemia, these patients must be identified using other treatment criteria.
5Objective: Phase III study to confirm a trend observed in a previous phase II study showing that a single dose of lenercept,human recombinant p55 tumor necrosis factor receptor-immunoglobulinG1 (TNFR55-IgG1) fusion protein, decreased mortality inpatients with severe sepsis or early septic shock.Design: Multicenter, double-blind, phase III, placebo-controlled,randomized study.Setting: A total of 108 community and university-affiliatedhospitals in the United States (60), Canada (6) and Europe (42).Patients: A total of 1,342 patients were recruited who fulfilledthe entry criteria within the 12-hr period preceding the study drugadministration.Intervention: After randomization, an intravenous dose of 0.125mg/kg lenercept or placebo was given. The patient was monitoredfor up to 28 days, during which standard diagnostic, supportive,and therapeutic care was provided.Measurements and Main Results: The primary outcome measurewas 28-day all-cause mortality. Baseline characteristicswere as follows: a total of 1,342 patients were randomized; 662received lenercept and 680 received placebo. The mean age was60.5 yrs (range, 17–96 yrs); 39% were female; 65% had medicaladmissions, 8% had scheduled surgical admissions, and 27% hadunscheduled surgical admissions; 73% had severe sepsis withoutshock, and 27% had severe sepsis with early septic shock.Lenercept and placebo groups were similar at baseline withrespect to demographic characteristics, simplified acute physiologyscore II-predicted mortality, profiles of clinical site of infectionand microbiological documentation, number of dysfunctioningorgans, and interleukin-6 (IL-6) plasma concentration.Lenercept pharmacokinetics were similar in severe sepsis andearly septic shock patients. Tumor necrosis factor was bound ina stable manner to lenercept as reflected by the accumulation oftotal serum tumor necrosis factor a concentrations. There were369 deaths, 177 on lenercept (27% mortality) and 192 on placebo(28% mortality). A one-sided Cochran-Armitage test, stratified bygeographic region and baseline, predicted 28-day all-cause mortality(simplified acute physiology score II), gave a p value of .141(one-sided). Lenercept treatment had no effect on incidence orresolution of organ dysfunctions. There was no evidence thatlenercept was detrimental in the overall population.Conclusion: Lenercept had no significant effect on mortality inthe study population. (“Lenercept had no significant effect on mortality in the study population”
7The inflammatory and procoagulant host responses to infection are intricately linked. Infectious agents and inflammatory cytokines such as tumor necrosis factor (TNF- ) and interleukin-1 activate coagulation by stimulating the release of tissue factor from monocytes and the endothelium. The presentation of tissue factor leads to the formation of thrombin and a fibrin clot. Inflammatory cytokines and thrombin can both impair the endogenous fibrinolytic potential by stimulating the release of plasminogen-activator inhibitor 1 (PAI-1) from platelets and the endothelium. PAI-1 is a potent inhibitor of tissue plasminogen activator, the endogenous pathway for lysing a fibrin clot. In addition, the procoagulant thrombin is capable of stimulating multiple inflammatory pathways and further suppressing the endogenous fibrinolytic system by activating thrombin-activatable fibrinolysis inhibitor (TAFI). The conversion of protein C, by thrombin bound to thrombomodulin, to the serine protease activated protein C is impaired by the inflammatory response. Endothelial injury results in decreased thrombomodulin levels. The end result of the host response to infection may be the development of diffuse endovascular injury, microvascular thrombosis, organ ischemia, multiorgan dysfunction, and death. Activated protein C can intervene at multiple points during the systemic response to infection. It exerts an antithrombotic effect by inactivating factors Va and VIIIa, limiting the generation of thrombin. As a result of decreased thrombin levels, the inflammatory, procoagulant, and antifibrinolytic response induced by thrombin is reduced. In vitro data indicate that activated protein C exerts an antiinflammatory effect by inhibiting the production of inflammatory cytokines (TNF- , interleukin-1, and interleukin-6) by monocytes and limiting the rolling of monocytes and neutrophils on injured endothelium by binding selectins. Activated protein C indirectly increases the fibrinolytic response by inhibiting PAI-1.12,13,14,15,16,17NEJM 2001
12“My approach is nontraditional, but from a uniquely Western perspective.”
1319 patients/group GM-CSF (4 mg/kg/d) or placebo for 8 days. Rationale: Sustained sepsis-associated immunosuppression is associatedwith uncontrolled infection, multiple organ dysfunction, anddeath.Objectives: In the first controlled biomarker-guided immunostimulatorytrial in sepsis, we tested whether granulocyte–macrophagecolony-stimulating factor (GM-CSF) reverses monocyte deactivation,a hallmark of sepsis-associated immunosuppression (primaryendpoint), and improves the immunological and clinical course ofpatients with sepsis.Methods: In a prospective, randomized, double-blind, placebo-controlled,multicenter trial, 38 patients (19/group) with severe sepsisor septic shock and sepsis-associated immunosuppression (monocyticHLA-DR [mHLA-DR] ,8,000 monoclonal antibodies (mAb) percell for 2 d) were treated with GM-CSF (4 mg/kg/d) or placebo for8 days. The patients’ clinical and immunological course was followedup for 28 days.Measurements and Main Results: Both groups showed comparablebaseline mHLA-DR levels (5, ,628 vs. 5, ,332 mAb percell), which significantly increased within 24 hours in the GM-CSFgroup. After GM-CSF treatment, mHLA-DR was normalized in 19/19treated patients, whereas this occurred in 3/19 control subjects only(P , 0.001). GM-CSF also restored ex-vivo Toll-like receptor 2/4–induced proinflammatory monocytic cytokine production. In patientsreceiving GM-CSF, a shorter time of mechanical ventilation( vs h, P ), an improved Acute Physiologyand Chronic Health Evaluation-II score (P ), and a shorterlength of both intrahospital and intensive care unit stay wasobserved (59633 vs and vs d, respectively,both not significant). Side effects related to the intervention werenot noted.Conclusions: Biomarker-guided GM-CSF therapy in sepsis is safe andeffective for restoring monocytic immunocompetence. Use of GMCSFmay shorten the time of mechanical ventilation and hospital/intensive care unit stay. A multicenter trial powered for the improvementof clinical parameters and mortality as primary endpointsseems indicated.Clinical trial registered
17Etude prospective randomisée double aveugle 21 patientsEtude prospective randomisée double aveugleThe efficacy and safety of high-dose intravenous polyspecific immunoglobulin G (IVIG) as adjunctive therapyin streptococcal toxic shock syndrome (STSS) were evaluated in a multicenter, randomized, double-blind,placebo-controlled trial. The trial was prematurely terminated because of slow patient recruitment, and resultswere obtained from 21 enrolled patients (10 IVIG recipients and 11 placebo recipients). The primary endpoint was mortality at 28 days, and a 3.6-fold higher mortality rate was found in the placebo group. Asignificant decrease in the sepsis-related organ failure assessment score at days 2 (Pp.02) and 3 (Pp.04)was noted in the IVIG group. Furthermore, a significant increase in plasma neutralizing activity againstsuperantigens expressed by autologous isolates was noted in the IVIG group after treatment (Pp.03).Althoughstatistical significance was not reached in the primary end point, the trial provides further support for IVIGas an efficacious adjunctive therapy in STSS.NS
18Primary challenge Relapse Clostridium difficile is the leading cause of nosocomial antibiotic-associated diarrhea, and recent outbreaksof strains with increased virulence underscore the importance of identifying novel approaches to treat andprevent relapse of Clostridium difficile-associated diarrhea (CDAD). CDAD pathology is induced by two exotoxins,toxin A and toxin B, which have been shown to be cytotoxic and, in the case of toxin A, enterotoxic. Inthis report we describe fully human monoclonal antibodies (HuMAbs) that neutralize these toxins and preventdisease in hamsters. Transgenic mice carrying human immunoglobulin genes were used to isolate HuMAbsthat neutralize the cytotoxic effects of either toxin A or toxin B in cell-based in vitro neutralization assays.Three anti-toxin A HuMAbs (3H2, CDA1, and 1B11) could all inhibit the enterotoxicity of toxin A in mouseintestinal loops and the in vivo toxicity in a systemic mouse model. Four anti-toxin B HuMAbs (MDX-1388,, 1G10, and 2A11) could neutralize cytotoxicity in vitro, although systemic toxicity in the mouse couldnot be neutralized. Anti-toxin A HuMAb CDA1 and anti-toxin B HuMAb MDX-1388 were tested in thewell-established hamster model of C. difficile disease. CDA1 alone resulted in a statistically significant reductionof mortality in hamsters; however, the combination treatment offered enhanced protection. Compared tocontrols, combination therapy reduced mortality from 100% to 45% (P < ) in the primary diseasehamster model and from 78% to 32% (P < ) in the less stringent relapse modelPrimary challengeRelapse
19BackgroundNew therapies are needed to manage the increasing incidence, severity, and high rate of recurrence of Clostridium difficile infection.MethodsWe performed a randomized, double-blind, placebo-controlled study of two neu- tralizing, fully human monoclonal antibodies against C. difficile toxins A (CDA1) and B (CDB1). The antibodies were administered together as a single infusion, each at a dose of 10 mg per kilogram of body weight, in patients with symptomatic C. difficile infection who were receiving either metronidazole or vancomycin. The primary out- come was laboratory-documented recurrence of infection during the 84 days after the administration of monoclonal antibodies or placebo.ResultsAmong the 200 patients who were enrolled (101 in the antibody group and 99 in the placebo group), the rate of recurrence of C. difficile infection was lower among pa- tients treated with monoclonal antibodies (7% vs. 25%; 95% confidence interval, 7 to 29; P<0.001). The recurrence rates among patients with the epidemic BI/NAP1/027 strain were 8% for the antibody group and 32% for the placebo group (P=0.06); among patients with more than one previous episode of C. difficile infection, recur- rence rates were 7% and 38%, respectively (P=0.006). The mean duration of the ini- tial hospitalization for inpatients did not differ significantly between the antibody and placebo groups (9.5 and 9.4 days, respectively). At least one serious adverse event was reported by 18 patients in the antibody group and by 28 patients in the placebo group (P=0.09).ConclusionsThe addition of monoclonal antibodies against C. difficile toxins to antibiotic agents significantly reduced the recurrence of C. difficile infection. (ClinicalTrials.gov num- ber, NCT )
23Surmortalité et SST III Infection aiguëInfection chroniqueSSTT [+]89%41%SSTT [+]SSTT [-]Mortalité21%3%The ability of Pseudomonas aeruginosa to secrete specific toxins using the type III-mediated pathway has been reported. To determine the association of this phenotype with human illness, immunoblot analysis was used to detect expression of type III secretory proteins in P. aeruginosa isolates from respiratory tract or blood cultures of 108 consecutive patients. Relative risk of mortality was 6-fold greater with expression of the type III secretory proteins ExoS, ExoT, ExoU, or PcrV. Phenotype was independently correlated with toxicity in cellular and murine models. Prevalence of this phenotype was significantly higher in acutely infected patients than in chronically infected patients with cystic fibrosis. These results suggest that the type III protein secretion system is integral to increased P. aeruginosa virulence. A positive phenotype is a predictor of poor clinical outcome. In the future, such analyses may help distinguish potentially lethal infection from colonization and help determine appropriate therapy for critically ill patients.RR décèsPcrV seule7,4PcrV + toxine(s)8,7(Roy Burman et al, J Infect Dis )
24Pneumonie à P.aeruginosa 35 patients ventilésPneumonie à P.aeruginosaProduction de ProtéinesIssues du système de sécrétionde type III+27/35-8/3522 Sévère 5 Modérées 3 Sévères 5 Modérées(81%) (19%) (38%) (62%)ExoU : 10/35 (29%) associée à 90% de formes sévèresHauser et al, Crit Care Med 2002
25Shime et al, J Immunol 2001 Les immunoglobulines polyclonales L’administration d’immunoglobulines polyclonales 1 heure ou 4 heures après l’infection pulmonaire à P. aeruginosa est capable de diminuer la mortalité. Effect lié à la portion Fab.In a mouse survival study, the i.v. administration of anti-PcrV IgG after the airspace instillation of a lethal dose of P. aeruginosa resulted in the complete survival of the animalsShime et al, J Immunol 2001
33A Phase I/II Randomized, Double-Blind, Placebo-Controlled, Single-Dose, Dose Escalation Study of KB001 in Cystic Fibrosis Patients Infected with Pseudomonas aeruginosa Milla, Carlos E; Accurso, Frank J.; Chmiel, James F.; McCoy, Karen S.; Billings, Joanne L; Atkinson, Jeffrey J; Clancy, John P; Liou, Theodore G; Acton, James D; Lynch, Susan V; Slusher, Nicole A; Burns, Jane L.; Mayer-Hamblett, Nicole; Harris, Kirk J; Patel, Rajesh; Tremblay, Thomas M; Parli, Teresa J.Twenty-seven subjects with a sputum Pa burden of >1 x 105 cfu/gmon screening and on stable CF therapy were randomized in dose-escalating cohorts to receive single IVinfusions of placebo or KB001 at either 3 mg/kg or 10 mg/kg. Clinical, pharmacokinetic and immunogenicityoutcomes were assessed. Pharmacodynamic endpoints included assessment of sputum microbial ecologyand changes from baseline in Pa burden and inflammationATS 2010
34A Phase I/II Randomized, Double-Blind, Placebo-Controlled, Single-Dose, Dose Escalation Study of KB001 in Cystic Fibrosis Patients Infected with Pseudomonas aeruginosa Milla, Carlos E; Accurso, Frank J.; Chmiel, James F.; McCoy, Karen S.; Billings, Joanne L; Atkinson, Jeffrey J; Clancy, John P; Liou, Theodore G; Acton, James D; Lynch, Susan V; Slusher, Nicole A; Burns, Jane L.; Mayer-Hamblett, Nicole; Harris, Kirk J; Patel, Rajesh; Tremblay, Thomas M; Parli, Teresa J.ATS 2010
35Vfr VqsR lasR lasI QscR RhlR RhlI RsaL RsaL LasR LasI RhlR RhlI PqsR ActivationInhibitionRsaLlasRlasIRsaLQscRLasRLasIC12C12PQSRhlRRhlIpqsHRhlRRhlIpqsRPqsRC4C4pqsApqsBpqsCpqsDpqsE
36QS Inhibition with macrolides ElastaseInsert = courbe de croissanceA: courbe de croissance avec qté croissante d’azithro. La plus faible = 2 microg/mlRhamnolipidNot bactericidalNot bacteriostaticQS InhibitionTateda K et al. AAC 2001
37no AZM (control) 2 g/ml AZM 4 g/ml AZM 8 g/ml AZM 12 g/ml AZM The consequences of O-acetylated alginate-producing Pseudomonas aeruginosa biofilms in the lungs of chronicallyinfected cystic fibrosis (CF) patients are tolerance to both antibiotic treatments and effects on the innate and theadaptive defense mechanisms. In clinical trials, azithromycin (AZM) has been shown to improve the lung functionof CF patients. The present study was conducted in accordance with previous in vitro studies suggesting that theeffect of AZM may be the inhibition of alginate production, blockage of quorum sensing (QS), and increasedsensitivity to hydrogen peroxide and the complement system. Moreover, we show that AZM may affect the polymerizationof P. aeruginosa alginate by the incomplete precipitation of polymerized alginate and high levels ofreadily dialyzable uronic acids. In addition, we find that mucoid bacteria in the stationary growth phase becamesensitive to AZM, whereas cells in the exponential phase did not. Interestingly, AZM-treated P. aeruginosa lasImutants appeared to be particularly resistant to serum, whereas bacteria with a functional QS system did not. Weshow in a CF mouse model of chronic P. aeruginosa lung infection that AZM treatment results in the suppressionof QS-regulated virulence factors, significantly improves the clearance of P. aeruginosa alginate biofilms, andreduces the severity of the lung pathology compared to that in control mice. We conclude that AZM attenuates thevirulence of P. aeruginosa, impairs its ability to form fully polymerized alginate biofilms, and increases its sensitivityto complement and stationary-phase killing, which may explain the clinical efficacy of AZM.no AZM (control)2 g/ml AZM4 g/ml AZM8 g/ml AZM12 g/ml AZM
397 patients colonized by QS-proficient isolates 320 P. aeruginosa isolates and tracheal aspirates from 29 patients, six developed VAP (20%).7 patients colonized by QS-proficient isolates57% developed VAP as compared to22 patients colonized by QS-deficient isolates9% developed VAP (p = 0.018).Thorax Aug;65(8):703-1
40With the rising development of bacterial resistance the search for new medical treatments beyond conventionalantimicrobials has become a key aim of public health research. Possible innovative strategies include the inhibition ofbacterial virulence. However, consideration must be given to the evolutionary and environmental consequences of suchnew interventions. Virulence and cooperative social behaviour of the bacterium Pseudomonas aeruginosa rely on thequorum-sensing (QS) controlled production of extracellular products (public goods). Hence QS is an attractive target foranti-virulence interventions. During colonization, non-cooperating (and hence less virulent) P. aeruginosa QS-mutants,benefiting from public goods provided by wild type isolates, naturally increase in frequency providing a relative protectionfrom invasive infection. We hypothesized that inhibition of QS-mediated gene expression removes this growth advantageand selection of less virulent QS-mutants, and maintains the predominance of more virulent QS-wild type bacteria. Weaddressed this possibility in a placebo-controlled trial investigating the anti-QS properties of azithromycin, a macrolideantibiotic devoid of bactericidal activity on P. aeruginosa, but interfering with QS, in intubated patients colonized by P.aeruginosa. In the absence of azithromycin, non-cooperating (and hence less virulent) lasR (QS)-mutants increased infrequency over time. Azithromycin significantly reduced QS-gene expression measured directly in tracheal aspirates.Concomitantly the advantage of lasR-mutants was lost and virulent wild-type isolates predominated during azithromycintreatment. We confirmed these results in vitro with fitness and invasion experiments. Azithromycin reduced growth rate ofthe wild-type, but not of the lasR-mutant. Furthermore, the lasR-mutant efficiently invaded wild-type populations in theabsence, but not in the presence of azithromycin. These in vivo and in vitro results demonstrate that anti-virulenceinterventions based on QS-blockade diminish natural selection towards reduced virulence and therefore may increase theprevalence of more virulent genotypes in the Hospital environment. More generally, the impact of intervention on theevolution of virulence of pathogenic bacteria should be assessed.
41erythema ⁄inflammation, ulceration ⁄ granulation ⁄ polyps, - Physicianerythema ⁄inflammation,ulceration ⁄ granulation ⁄ polyps,discharge quantity,discharge type and odourPatientsDiscomfort, itchiness, wetness, smellObjectives: To evaluate the efficacy and safety of a therapeuticbacteriophage preparation (Biophage-PA) targetingantibiotic-resistant Pseudomonas aeruginosa in chronicotitis.Design: Randomised, double-blind, placebo-controlledPhase I/II clinical trial approved by UK Medicines andHealthcare products Regulatory Agency (MHRA) and theCentral Office for Research Ethics Committees (COREC)ethical review process.Setting: A single specialist university hospital.Participants: 24 patients with chronic otitis with a durationof several years (2–58). Each patient had, at the timeof entry to the trial, an ear infection because of an antibiotic-resistant P. aeruginosa strain sensitive to one or moreof the six phages present in Biophage-PA. Participantswere randomised in two groups of 12 treated with eithera single dose of Biophage-PA or placebo and followed upat 7, 21 and 42 days after treatment by the same otologist.Ears were thoroughly cleaned on each occasion andclinical and microbiological indicators measured.Main outcome measures: Physician assessederythema ⁄inflammation, ulceration ⁄ granulation ⁄ polyps,discharge quantity, discharge type and odour using aVisual Analogue Scale (VAS). Patients reported discomfort,itchiness, wetness and smell also using a VAS.Bacterial levels of P. aeruginosa and phage counts fromswabs were measured initially and at follow-up. At eachvisit patients were asked about side effects using astructured form. Digital otoscopic images were obtainedon days 0 and 42 for illustrative purposes only.Results: Relative to day 0, pooled patient- and physician-reported clinical indicators improved for the phagetreated group relative to the placebo group. Variationfrom baseline levels was statistically significant forcombined data from all clinic days only for the phagetreated group. Variation from baseline levels wasstatistically significant for the majority of the patientassessed clinical indicators only for the phage treatedgroup. P. aeruginosa counts were significantly lower onlyin the phage treated group. No treatment related adverseevent was reported.Conclusion: The first controlled clinical trial of atherapeutic bacteriophage preparation showed efficacyand safety in chronic otitis because of chemo-resistantP. aeruginosa.
45Spécifique de chaque individu 14 sujets sainsSpécifique de chaque individuZoetendal, 1998
46Probiotiques Terme introduit en 1953 par Werner Kollath Par opposition aux antibiotiquesCe sont des facteurs microbiens qui stimulent la croissance d’autres microorganismes.La nécessité du caractère vivant des souches est ajouté à la définition par Roy Fuller en 1989.
47Probiotiques Alfred Nissle Isolement en 1917 d’une souche de E. coli à partir de selles d’un soldat qui, lors d’une épidémie de Shigellose, n’a pas développé d’entérocolite.Utilisation de sa souche avec succès dans des cas de Salmonellose et Shigellose [Nissle 1959].
48Essais contrôlés et diarrhée post-antibiotique D’Souza2002BMJCremoniniAliment Pharmocol TherSzajewska2005HawrelakDigestionAdulteSzajewska2006J PediatrJohnstonCMAJ2007Cochrane Database Syst Rev.EnfantEfficacité modérée des probiotiques à prévenir la diarrhée post-ATBS. boulardii ou L. rhamnosus GG chez l’enfantL. casei DN chez la personne âgéePb dans la définition de la diarrhée et de la gravité clinique
49Essais contrôlés et colite à Clostridium difficile Lewis2007Am J GastroenterolPourDendukuri2005CMAJContreDifficulté à discerner prévention et traitement au moment de la colite
50Ampicillin (1 g/L), vancomycin (500 mg/L), neomycin sulfate (1 g/L), and metronidazole (1 g/L) in drinking water for 4 wk before PR8 virus infection (10 pfu per mouse).Antibiotic-treated mice fail to induce acquired immunityto influenza virus infection. C57BL/6 mice were givenampicillin (1 g/L), vancomycin (500 mg/L), neomycin sulfate(1 g/L), and metronidazole (1 g/L) in drinking water for 4 wkbefore PR8 virus infection (10 pfu per mouse). Two weekslater, serum and nasal wash were collected and Ag-specificantibody titers were measured (A), and T-cells were isolatedfrom spleen and restimulated with flu virion or NP peptidefor 72 h, and IFN-γ production from CD4 T cells (B) and CD8 Tcells (C) was measured. (D) Lymphocytes were collected fromthe lung of infected animals at 14 d postinfection and stainedwith flu-specific tetramer. (E) The lung washes of flu-infectedmice were harvested at 9 d postinfection, and viral titers weremeasured by plaque assay. *P < 0.05 and ***P < vs.water-fed group. Data represent the mean ± SD. Similarresults were obtained from three separate experimentsProc Natl Acad Sci USA 2011