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Abstract Background The expression levels of many genes show wide natural variation among strains or populations. This study investigated the potential for animal strain-related genotypic differences to confound gene expression profiles in acute cellular rejection (ACR). Using a rat heart transplant model and 2 different rat strains (Dark Agouti, and Brown Norway), microarrays were performed on native hearts, transplanted hearts, and peripheral blood mononuclear cells (PBMC). Results In heart tissue, strain alone affected the expression of only 33 probesets while rejection affected the expression of 1368 probesets (FDR 10% and FC ≥ 3). Only 13 genes were affected by both strain and rejection, which was < 1% (13/1368) of all probesets differentially expressed in ACR. However, for PBMC, strain alone affected 265 probesets (FDR 10% and FC ≥ 3) and the addition of ACR had little further effect. Pathway analysis of these differentially expressed strain effect genes connected them with immune response, cell motility and cell death, functional themes that overlap with those related to ACR. After accounting for animal strain, additional analysis identified 30 PBMC candidate genes potentially associated with ACR. Conclusion In ACR, genetic background has a large impact on the transcriptome of immune cells, but not heart tissue. Gene expression studies of ACR should avoid study designs that require cross strain comparisons between leukocytes.

Abstract Background Meta-analyses of N-acetylcysteine (NAC) for preventing contrast-induced nephrotoxicity (CIN) have led to disparate conclusions. Here we examine and attempt to resolve the heterogeneity evident among these trials. Methods Two reviewers independently extracted and graded the data. Limiting studies to randomized, controlled trials with adequate outcome data yielded 22 reports with 2746 patients. Results Significant heterogeneity was detected among these trials (I2 = 37%; p = 0.04). Meta-regression analysis failed to identify significant sources of heterogeneity. A modified L'Abbé plot that substituted groupwise changes in serum creatinine for nephrotoxicity rates, followed by model-based, unsupervised clustering resolved trials into two distinct, significantly different (p < 0.0001) and homogeneous populations (I2 = 0 and p > 0.5, for both). Cluster 1 studies (n = 18; 2445 patients) showed no benefit (relative risk (RR) = 0.87; 95% confidence interval (CI) 0.68–1.12, p = 0.28), while cluster 2 studies (n = 4; 301 patients) indicated that NAC was highly beneficial (RR = 0.15; 95% CI 0.07–0.33, p < 0.0001). Benefit in cluster 2 was unexpectedly associated with NAC-induced decreases in creatinine from baseline (p = 0.07). Cluster 2 studies were relatively early, small and of lower quality compared with cluster 1 studies (p = 0.01 for the three factors combined). Dialysis use across all studies (five control, eight treatment; p = 0.42) did not suggest that NAC is beneficial. Conclusion This meta-analysis does not support the efficacy of NAC to prevent CIN.

Recent experimentation with the variants of the Ebola virus that differ in the glycoprotein's poly-uridine site, which dictates the form of glycoprotein produced through a transcriptional stutter, has resulted in questions regarding the pathogenicity and lethality of the stocks used to develop products currently undergoing human clinical trials to combat the disease. In order to address these concerns and prevent the delay of these critical research programs, we designed an experiment that permitted us to intramuscularly challenge statistically significant numbers of naïve and vaccinated cynomolgus macaques with either a 7U or 8U variant of the Ebola virus, Kikwit isolate. In naïve animals, no difference in survivorship was observed; however, there was a significant delay in the disease course between the two groups. Significant differences were also observed in time-of-fever, serum chemistry, and hematology. In vaccinated animals, there was no statistical difference in survivorship between either challenge groups, with two succumbing in the 7U group compared to 1 in the 8U challenge group. In summary, survivorship was not affected, but the Ebola virus disease course in nonhuman primates is temporally influenced by glycoprotein poly-U editing site populations. [ABSTRACT FROM AUTHOR]

Bacterial proteins destined for the Tat pathway are folded before crossing the inner membrane and are typically identified by an N-terminal signal peptide containing a twin arginine motif. Translocation by the Tat pathway is dependent on the products of genes which encode proteins possessing the binding site of the signal peptide and mediating the actual translocation event. In the fully virulent CO92 strain of Yersinia pestis, the tatA gene was deleted. The mutant was assayed for loss of virulence through various in vitro and in vivo assays. Deletion of the tatA gene resulted in several consequences for the mutant as compared to wild-type. Cell morphology of the mutant bacteria was altered and demonstrated a more elongated form. In addition, while cultures of the mutant strain were able to produce a biofilm, we observed a loss of adhesion of the mutant biofilm structure compared to the biofilm produced by the wild-type strain. Immuno-electron microscopy revealed a partial disruption of the F1 antigen on the surface of the mutant. The virulence of the ΔtatA mutant was assessed in various murine models of plague. The mutant was severely attenuated in the bubonic model with full virulence restored by complementation with the native gene. After small-particle aerosol challenge in a pneumonic model of infection, the mutant was also shown to be attenuated. In contrast, when mice were challenged intranasally with the mutant, very little difference in the LD50 was observed between wild-type and mutant strains. However, an increased time-to-death and delay in bacterial dissemination was observed in mice infected with the ΔtatA mutant as compared to the parent strain. Collectively, these findings demonstrate an essential role for the Tat pathway in the virulence of Y. pestis in bubonic and small-aerosol pneumonic infection but less important role for intranasal challenge. [ABSTRACT FROM AUTHOR]

The current candidate vaccine against Yersinia pestis infection consists of two subunit proteins: the capsule protein or F1 protein and the low calcium response V protein or V-antigen. Little is known of the recognition of the vaccine by the host's innate immune system and how it affects the acquired immune response to the vaccine. Thus, we vaccinated Toll-like receptor (Tlr) 2, 4, and 2/4-double deficient, as well as signal adaptor protein Myd88-deficient mice. We found that Tlr4 and Myd88 appeared to be required for an optimal immune response to the F1-V vaccine but not Tlr2 when compared to wild-type mice. However, there was a difference between the requirement for Tlr4 and MyD88 in vaccinated animals. When F1-V vaccinated Tlr4 mutant (lipopolysaccharide tolerant) and Myd88-deficient mice were challenged by aerosol with Y. pestis CO92, all but one Tlr4 mutant mice survived the challenge, but no vaccinated Myd88-deficient mice survived the challenge. Spleens from these latter nonsurviving mice showed that Y pestis was not cleared from the infected mice. Our results suggest that MyD88 appears to be important for both an optimal immune response to F1-V and in protection against a lethal challenge of Y pestis CO92 in F1-V vaccinated mice. [ABSTRACT FROM AUTHOR]

Effector cells derived from central memory CD8+ T cells were reported to engraft and survive better than those derived from effector memory populations, suggesting that they are superior for use in adoptive immunotherapy studies. However, previous studies did not evaluate the relative efficacy of effector cells derived from naïve T cells. We sought to investigate the efficacy of tumor-specific effector cells derived from naïve or central memory T-cell subsets using transgenic or retrovirally transduced T cells engineered to express a tumor-specific 1-cell receptor. We found that naïve, rather than central memory I cells, gave rise to an effector population that mediated superior antitumor immunity upon adoptive transfer. Effector cells developed from naïve I cells lost the expression of CD62L more rapidly than those derived from central memory T cells, but did not acquire the expression of KLRG-1, a marker for terminal differentiation and replicative senescence. Consistent with this KLRG-1- phenotype, naïve-derived cells were capable of a greater proliferative burst and had enhanced cytokine production after adoptive transfer. These results indicate that insertion of genes that confer antitumor specificity into naïve rather than central memory CD8+ T cells may allow superior efficacy upon adoptive transfer. [ABSTRACT FROM AUTHOR]

Background: The expression levels of many genes show wide natural variation among strains or populations. This study investigated the potential for animal strain-related genotypic differences to confound gene expression profiles in acute cellular rejection (ACR). Using a rat heart transplant model and 2 different rat strains (Dark Agouti, and Brown Norway), microarrays were performed on native hearts, transplanted hearts, and peripheral blood mononuclear cells (PBMC). Results: In heart tissue, strain alone affected the expression of only 33 probesets while rejection affected the expression of 1368 probesets (FDR 10% and FC ≥ 3). Only 13 genes were affected by both strain and rejection, which was < 1% (13/1368) of all probesets differentially expressed in ACR. However, for PBMC, strain alone affected 265 probesets (FDR 10% and FC ≥ 3) and the addition of ACR had little further effect. Pathway analysis of these differentially expressed strain effect genes connected them with immune response, cell motility and cell death, functional themes that overlap with those related to ACR. After accounting for animal strain, additional analysis identified 30 PBMC candidate genes potentially associated with ACR. Conclusion: In ACR, genetic background has a large impact on the transcriptome of immune cells, but not heart tissue. Gene expression studies of ACR should avoid study designs that require cross strain comparisons between leukocytes. [ABSTRACT FROM AUTHOR]

Jacket use in NHP is a common practice and is often considered a form of refinement during experiments necessitating extended periods of catheterization. An important consideration when using jackets is the physiologic effects that jacketing has on NHP and its potential to confound research. Several studies have evaluated the stress response and habituation of NHP to various forms of restraint, but none have looked directly at the timeframe necessary for the habituation of rhesus macaques (Macaca mulatta) to jackets. We set out to determine whether 3 d was a sufficient timeframe for this species to become habituated to a jacket, with or without an undershirt, by evaluating 2 major physiologic parameters. After jacket placement, we measured food consumption and collected fecal samples to measure fecal corticosterone metabolites (FCM) daily for 2 wk. FCM measurements for NHP without undershirts were significantly increased for days 2 and 3 after jacketing before returning to baseline levels. FCM measurements for NHP with undershirts were significantly increased for only 1 d after jacketing, suggesting that the undershirt has a positive effect on jacket habituation. There were no measurable differences in food consumption during the jacket habituation period. Furthermore, no significant differences were noted between sexes. These findings suggest that FCM levels return to baseline 3 d after jacketing and could be a useful predictor of jacket habituation in rhesus macaques.

Jacketing of nonhuman primates (NHP) is a commonly used practice in the laboratory animal setting to support data collection with reduced direct human-to-animal interaction. NHP often wear jackets for several weeks, potentially leading to the formation of dermal lesions ranging from mild alopecia to severe full-thickness ulceration. We sought to evaluate the addition of a commercially available undershirt for primates as a possible refinement practice for our jacketed rhesus macaques. In this study, we compared the lesion count, location, and severity and differences in rectal body temperature between jacketed NHP with undershirts with those wearing the jackets alone. In both groups, most lesions (75%) were located at either the underarm or shoulder. The percentages of total lesions in the back and neck were lower in jacketed NHP that wore undershirts than in those that did not. In addition, the estimated odds of increased severity scores in jacketed NHP without undershirts was 1.80 times that for NHP that wore both jackets and undershirts. Both groups of NHP showed a significant decrease in dermal scores with time, indicating adaptation to the jackets with or without undershirts. However, there was no statistically significant decrease in lesion count, severity, or location in jacketed NHP that wore undershirts compared with those that did not.

Carbon monoxide (CO) is an endogenous messenger that suppresses inflammation, modulates apoptosis and promotes vascular remodeling. Here, microarrays were employed to globally characterize the CO (250 ppm) suppression of early (1 h) LPS-induced inflammation in human monocytic THP-1 cells. CO suppressed 79 of 101 immediate-early genes induced by LPS; 19% (15/79) were transcription factors and most others were cytokines, chemokines and immune response genes. The prototypic effects of CO on transcription and protein production occurred early but decreased rapidly. CO activated p38 MAPK, ERK1/2 and Akt and caused an early and transitory delay in LPS-induced JNK activation. However, selective inhibitors of these kinases failed to block CO suppression of LPS-induced IL-1beta, an inflammation marker. Of CO-suppressed genes, 81% (64/79) were found to have promoters with putative NF-kappaB binding sites. CO was subsequently shown to block LPS-induced phosphorylation and degradation of IkappaBalpha in human monocytes, thereby inhibiting NF-kappaB signal transduction. CO broadly suppresses the initial inflammatory response of human monocytes to LPS by reshaping proximal events in TLR4 signal transduction such as stress kinase responses and early NF-kappaB activation. These rapid, but transient effects of CO may have therapeutic applications in acute pulmonary and vascular injury.

Background: Prefractionation techniques such as serum albumin depletion are useful precursors to proteomic analysis, but they may introduce preanalytical bias if the depletion is not reproducible. We examined the reproducibility of albumin immunodepletion and describe a method of QC for this process., Methods: Depletion of albumin from pooled serum, performed using IgY immunoaffinity spin columns, was assessed for 21 runs on each of 4 columns. We measured albumin concentrations, after albumin depletion, by use of an immunoturbidimetric assay on the Beckman LX 20 analyzer and assessed mass spectra of albumin-depleted samples by use of SELDI-TOF mass spectrometry., Results: There was substantial run-to-run variation in efficiency of albumin depletion, with systematic decline in efficiency after multiple uses of the columns. Mean depletion efficiency was >95% for 15 of the 1st 17 runs and <90% for runs 18 to 21. We evaluated the 10 highest-intensity peaks present in all spectra from runs 1, 8, 17, and 21 and assessed the effect of albumin depletion on SELDI-TOF mass spectrometry reproducibility. Comparing the %CV of relative intensities for low and high m/z measurements revealed a significant difference of run 21 compared with runs 1, 8, and 17 (P <0.0001). Six-fold more peaks were found in albumin-depleted than unfractionated serum at both high and low m/z., Conclusions: Sporadic and systematic variation in efficiency of albumin depletion by spin columns may contribute significant preanalytical bias to proteomic approaches of biomarker discovery. This variation requires ongoing QC of the albumin depletion process by quantification of albumin concentration to assess depletion efficiency.

A shock canine pneumonia model that permitted relief of discomfort with the use of objective criteria was developed and validated. After intrabronchial Staphylococcus aureus challenge, mechanical ventilation, antibiotics, fluids, vasopressors, sedatives, and analgesics were titrated based on algorithms for 96 h. Increasing S. aureus (1 to 8 x 10(9) colony-forming units/kg) produced decreasing survival rates (P = 0.04). From 4 to 96 h, changes in arterial-alveolar oxygen gradients, mean pulmonary artery pressure, IL-1, serum sodium levels, mechanical ventilation, and vasopressor support were ordered based on survival time [acute nonsurvivors (< or =24 h until death, n = 8) > or = subacute nonsurvivors (>24 to 96 h until death, n = 8) > or = survivors (> or =96 h until death, n = 22) (all P < 0.05)]. In the first 12 h, increases in lactate and renal abnormalities were greatest in acute nonsurvivors (all P < 0.05). Compared with survivors, subacute nonsurvivors had greater rises in cytokines and liver enzymes and greater falls in platelets, white cell counts, pH, and urine output from 24 to 96 h (all P < 0.05). Importantly, these changes were not attributable to dosages of sedation, which decreased in nonsurvivors [survivors vs. nonsurvivors: 5.0 +/- 1.0 vs. 3.8 +/- 0.7 ml x h(-1) x (fentanyl/midazolam/ medetomidine)(-1); P = 0.02]. In this model, the pain control regimen did not mask changes in metabolic function and lung injury or the need for more hemodynamic and pulmonary support related to increasing severity of sepsis. The integration into this model of both specific and supportive titrated therapies routinely used in septic patients may provide a more realistic setting to evaluate therapies for sepsis.