Murine natural killer cell licensing and regulation by T regulatory cells in viral responses.

Natural killer (NK) cells show differential functionality based on their capability of binding to self-MHC consistent with licensing. Here we show in vivo confirmation of the physiologic effects of licensing with differential effects of NK subsets on anti-murine cytomegalovirus (anti-MCMV) responses after syngeneic hematopoietic stem cell transplantation (HSCT) or regulatory T-cell (Treg) depletion. After HSCT, depletion of licensed NK cells led to far greater viral loads in target organs early after infection compared with nondepleted and unlicensed depleted mice. There was a preferential expansion of licensed, C-type lectin-like activating receptor Ly49H+ NK cells with increased IFNγ production after infection in nondepleted mice post-HSCT and after Treg depletion. Adoptive transfer of licensed NK subsets into immunodeficient hosts provided significantly greater MCMV resistance compared with transfer of total NK populations or unlicensed subsets. In non-HSCT mice, only concurrent depletion of Tregs or TGF-ß neutralization resulted in detection of NK licensing effects. This suggests that licensed NK cells are the initial and rapidly responding population of NK cells to MCMV infection, but are highly regulated by Tregs and TGF-ß.

Murine NK-cell licensing is reflective of donor MHC-I following allogeneic hematopoietic stem cell transplantation in murine cytomegalovirus responses.

Natural killer (NK) cells express inhibitory receptors with varied binding affinities to specific major histocompatibility complex class I (MHC-I) haplotypes. NK cells can be classified as licensed or unlicensed based on their ability or inability to bind MHC-I, respectively. The role of donor vs host MHC on their development after allogeneic hematopoietic stem cell transplantation (allo-HSCT) is not known. Following reciprocal MHC-disparate allogeneic transplants and during de novo NK-cell recovery, depletion of the licensed and not unlicensed population of NK cells as determined by the licensing patterns of donor MHC-I haplotypes, resulted in significantly increased susceptibility to murine cytomegalovirus (MCMV) infection. A corresponding expansion of the licensed Ly49H(+) NK cells occurred with greater interferon γ production by these cells than unlicensed NK cells in the context of donor MHC-I. Thus, NK licensing behavior to MCMV corresponds to the donor, and not recipient, MHC haplotype after allo-HSCT in mice.

Interleukin-10 repletion suppresses pro-inflammatory cytokines and decreases liver pathology without altering viral replication in murine cytomegalovirus (MCMV)-infected IL-10 knockout mice.

OBJECTIVE AND DESIGN: To determine the role of interleukin-10 (IL-10) in protecting against the deleterious pro-inflammatory cytokine response to murine cytomegalovirus (MCMV), we studied the impact of IL-10 repletion in MCMV-infected IL-10 knockout (KO) mice. MATERIALS AND METHODS: IL-10 KO mice were infected with a sub-lethal dose of MCMV and treated daily with 5 µg of mouse recombinant IL-10 (mrIL-10). Cytokine transcription, viral load, cytokine expression and liver histopathology were assessed in IL-10 treated and untreated mice. RESULTS: mrIL-10 repletion suppressed the exaggerated pro-inflammatory cytokine response observed in IL-10 KO mice (vs. control) both systemically and at the organ level, without affecting viral load. Levels of IFN-γ and TNF-α mRNA in livers of treated mice were ~50-70-fold lower than in untreated mice at day 5 post-infection (p ≤ 0.05). In spleens and sera, levels of IFN-γ and IL-6 were significantly lower in treated mice than in untreated mice at day 5-7 post-infection (p ≤ 0.05). IL-10 blunting of cytokine responses was accompanied by attenuation of inflammation in livers of treated mice. CONCLUSIONS: Repletion of IL-10 modulates the exaggerated pro-inflammatory cytokine responses that characterize IL-10 KO mice and protects against liver damage without altering viral load. IL-10 may be useful to control dysregulated pro-inflammatory cytokines responses during CMV infection.

Epidemiologic surveillance of Clostridium difficile diarrhea in a freestanding pediatric hospital and a pediatric hospital at a university medical center.

To describe the epidemiology of Clostridium difficile in children, we cultured stool specimens from patients at the Children's Hospital Central California, Madera, CA (CHCC, n = 676) and at the University of California Davis Medical Center Pediatric Hospital, Sacramento, CA (UCDMC-PH, n = 301) for C. difficile, and toxins A and B genes and strain identity of the isolates were determined by polymerase chain reaction assays. A higher percentage of patients from UCDMC-PH were culture positive (148/301, 49%) and colonized with toxigenic strains (45/301, 15%) compared with CHCC (colonized = 178/676, 26%; toxigenic = 96/676, 14%, P < or = .001). Multiple logistic regression analysis showed decreased colonization with inpatient status (odds ratio [OR] = 0.64; 95% confidence interval [CI] = 0.46, 0.89; P = .007) and use of H-2 antagonists (OR = 0.55; 95% CI = 0.36, 0.84; P = .006), whereas underlying conditions (colonization: OR = 1.42; 95% CI = 1.02, 1.96; P = .04; toxin positive: OR = 1.60; 95% CI = 1.04, 2.44; P = .03) and exposure to > or =2 antiinfectives (colonization: OR = 1.56; 95% CI = 1.10, 2.20; P = .01; toxin positive: OR = 1.71; 95% CI = 1.10, 2.66; P = .02) increased colonization. Most isolates appear to be community acquired, although molecular analysis suggests some nosocomial transmission at UCDMC-PH. These data suggest that the epidemiology of colonization with C. difficile in children is different than previously reported.

Prevalence of enterotoxigenic Bacteroides fragilis in hospital-acquired diarrhea.

Stool specimens from 152 hospitalized patients with diarrhea were analyzed for the presence of enterotoxigenic Bacteroides fragilis (ETBF) by a nested polymerase chain reaction (PCR) assay. ETBF gene sequences were directly detected in 14/152 (9.21%) stools of patients. The prevalence of ETBF in hospital-acquired diarrhea was statistically significant when compared to a prevalence of 2.3% in control subjects (P = 0.04). B. fragilis was cultured from 19.7% (30/152) patients with diarrhea; 4 of these isolates were enterotoxigenic. To determine whether colonization with B. fragilis is heterogeneous in nature, multiple colonies from 17 individual patients were analyzed for enterotoxin gene sequences and genotyped by arbitrarily primed PCR. Of these 17 patients, 13 harbored multiple strain types suggesting heterogeneity of colonization with both enterotoxigenic and non-enterotoxigenic strains. Identification of ETBF in the stools of 10 patients in the absence of a positive culture is likely due to the noted heterogeneity and suggests that detection of enterotoxin by PCR should be performed directly in the stool. These preliminary data indicate that ETBF may play a role in hospital-acquired diarrhea of unknown origin and suggest the need for further studies.

Use of quantitative real-time PCR (qRT-PCR) to measure cytokine transcription and viral load in murine cytomegalovirus infection.

A quantitative real-time PCR (qRT-PCR) assay was developed to measure cytokine transcription profiles and viral load during sub-clinical and clinical infection with murine cytomegalovirus (MCMV). Primers/fluorogenic probes specific for mouse cytokines and for the immediate early gene 1 (IE1) of MCMV were used to quantitate cytokine responses and viral load in various organs of MCMV infected mice. Increased mRNA levels of TNF-alpha, INF-gamma and IL-10 were detected in the spleens, lungs and livers of clinically infected mice at 5 days post-infection. Transcription of these cytokines was 2-5-fold lower (p=0.07 for each cytokine) in the spleens and 10-100-fold lower in the lungs (p=0.03 for INFgamma, not significant for IL-10 and TNFalpha) and livers (p<0.05 for each cytokine) of sub-clinically infected mice. Clinical MCMV infection induced high levels of IL-6 in the lungs and spleens of infected animals, while no significant transcription of IL-6 was detected in any organ during sub-clinical infection (p<0.05). The timing of peak amounts of INF-gamma, IL-10 and IL-6 observed in the spleens of clinically infected mice correlated with high viral loads in these organs. Cytokine expression rose in the salivary glands later, at day 15, corresponding to the increase in salivary gland viral load. The qRT-PCR demonstrates that infection with MCMV induces an organ-specific cytokine response characterized by the production of TNF-alpha, INF-gamma, IL-6 and IL-10 which correlates with severity of the disease (sub-clinical versus clinical) and with viral load. In summary, qRT-PCR is a sensitive and accurate method to study MCMV infection and host responses to the virus.

Prevalence of the ermB gene in Clostridium difficile strains isolated at a university teaching hospital from 1987 through 1998.

We analyzed 226 strains of Clostridium difficile for the presence of erythromycin ribosomal methylase B (ermB) genes. Forty-four strains (19.4%) carried ermB genes and were resistant to erythromycin. Toxin A and toxin B gene sequences were identified in 81.9% of these 44 strains. Strains of C. difficile that carry ermB genes are common etiologic agents of C. difficile-associated diarrhea.

Spatial and temporal analysis of Clostridium difficile infection in patients at a pediatric hospital in California.

OBJECTIVE: To examine the usefulness of temporal and spatial analysis in identifying nosocomial transmission of Clostridium difficile among pediatric patients hospitalized on four wards at The Children's Hospital of Central California from September 8, 1998, to January 16, 1999. DESIGN: Stool specimens obtained from the clinical microbiology laboratory during the study period were tested by culture and latex agglutination for C. difficile. Polymerase chain reaction was used to identify toxin genes. Isolates obtained were mapped to a grid for each ward and were analyzed using the Knox test. Results were compared with DNA fingerprints generated by arbitrarily primed polymerase chain reaction. RESULTS: Total occupancy of these 4 wards was 438 during the study period. Stool specimens were available for 256 (58%) of these patients, yielding 67 C. difficile isolates and generating 2,211 case pairs for analysis by the Knox test. After stratification by toxin status, 5 clustered pairs of toxigenic isolates were identified on 1 of the wards by this method. Fingerprint analysis identified 4 clusters with indistinguishable banding patterns on 2 of the 4 wards. Two of the identified clusters were toxigenic and 2 were nontoxigenic. None of these clusters corresponded to clusters identified by the Knox test. CONCLUSIONS: The Knox test is an ineffective method for identifying cases resulting from nosocomial transmission of C. difficile in a pediatric setting due to the persistence of C. difficile spores and the unique environment of a pediatric hospital. Molecular analysis remains the most effective method.

Clindamycin resistant strains of Clostridium difficile isolated from cases of C. difficile associated diarrhea (CDAD) in a hospital in Sweden.

Fifty three strains of C. difficile recovered from the stools of 13 patients with clinical C. difficile associated diarrhea (CDAD) were analyzed for the presence of the ermB gene, for toxigenicity and fingerprinting profile by PCR based assays. Forty five percent of the isolates were resistant to clindamycin and positive for the ermB gene. All clindamycin resistant isolates were ermB positive and belonged to the same fingerprinting group, suggesting clonal spread. These preliminary results suggest that clindamycin resistant isolates may be common etiologic agents of CDAD in Sweden.

Murine cytomegalovirus (MCMV) infection upregulates P38 MAP kinase in aortas of Apo E KO mice: a molecular mechanism for MCMV-induced acceleration of atherosclerosis.

Multiple studies suggest an association between cytomegalovirus (CMV) infection and atherogenesis; however, the molecular mechanisms by which viral infection might exacerbate atherosclerosis are not well understood. Aortas of MCMV-infected and uninfected Apo E knockout (KO) mice were analyzed for atherosclerotic lesion development and differential gene expression. Lesions in the infected mice were larger and showed more advanced disease compared to the uninfected mice. Sixty percent of the genes in the MAPK pathway were upregulated in the infected mice. p38 and ERK 1/2 MAPK genes were 5.6- and 2.0-fold higher, respectively, in aortas of infected vs. uninfected mice. Levels of VCAM-1, ICAM-1, and MCP-1 were ~2.0-2.6-fold higher in aortas of infected vs. uninfected mice. Inhibition of p38 with SB203580 resulted in lower levels of pro-atherogenic molecules and MCMV viral load in aortas of infected mice. MCMV-induced upregulation of p38 may drive the virus-induced acceleration of atherogenesis observed in our model.

Paradoxical response to prophylactic Didox (N-3, 4 trihydroxybenzamide) treatment in murine cytomegalovirus-infected mice.

BACKGROUND: In this study, we investigated the effect of Didox (DX) on the pathogenicity of and host responses to murine cytomegalovirus (MCMV) infection. METHODS: In vitro efficacy of DX against MCMV was determined using plaque reduction assays. For in vivo studies, mice infected with a sublethal dose (10(4) PFU) of MCMV were treated daily with DX (200 mg/kg) using either a prophylactic or delayed protocol. At predetermined intervals, target organs were removed for histopathology. Cytokine transcription and viral load were performed using real-time PCR. Serum cytokine levels were determined by ELISA, and T-cell markers by real-time PCR. RESULTS: DX (0.5-50 µM) inhibited MCMV plaque formation in vitro. However, in vivo, prophylactic DX treatment did not decrease viral load and prolonged hepatic proinflammatory cytokine transcription at days 3 and 5 post-infection, which corresponded with more severe histopathological changes observed in the liver. Significant CD8(+) T-cell marker suppression was seen, in accordance with DX-induced inhibition of lymphocyte proliferation observed in vitro. DX prolonged the recovery of MCMV-infected mice when given after infection was established. CONCLUSIONS: Despite promising MCMV inhibition in vitro, DX had no beneficial effect on MCMV disease in our model and paradoxically had adverse effects when administered prophylactically. The lack of correlation between in vitro activity and in vivo efficacy emphasizes the importance of selecting appropriate antiviral targets and of using animal models when testing new drugs.

In vivo characterization of cytokine profiles and viral load during murine cytomegalovirus-induced acute myocarditis.

BACKGROUND: Murine cytomegalovirus (MCMV) is an etiologic agent of acute and chronic myocarditis in BALB/c mice. Immunologic host responses appear to play a key role in pathogenesis but have been incompletely defined. METHODS: BALB/c mice were infected with a sublethal dose of MCMV. Cytokine transcription and viral load (measured by quantitative real-time polymerase chain reaction) and histopathological analyses were performed at specified time points. RESULTS: Increased tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, and interferon (IFN)-gamma, as well as IL-10 mRNA transcripts, were detected in the hearts of infected mice starting at Day 1 post-infection (p.i.), with peak levels occurring at Day 8 p.i. (7-fold, 14-fold, 41-fold, and 16-fold higher than background, respectively). Peak cytokine transcription significantly correlated with a 10-fold increase in viral load (P<.001) at Day 8 p.i. Myocarditis-related pathological changes, measured by infiltration foci, were greatest at Day 8 p.i., corresponding with peak cytokine transcription and significantly correlated with IFN-gamma levels (P<.0001). Infiltration foci were predominantly composed of CD3(+) T cells. Cardiac calcification was observed in most infected mice predominantly over the right ventricle. Histological analysis of heart sections from mice infected with recombinant enhanced green fluorescence protein-MCMV revealed a localized and sporadic pattern of virus throughout all heart layers. CONCLUSIONS: MCMV-induced myocarditis in BALB/c mice is characterized by in vivo production of proinflammatory cytokines in a pattern correlating with MCMV viral load. The infection pattern and inflammatory response is highly localized, sporadic, and involves endocardium, epicardium, as well as the myocardium, with greatest amounts of virus detected in areas of pathologic calcification.