Inflammatory processes play essential roles in the pathogenesis of tendinitis and tendinopathy. These events are accompanied by catabolic processes initiated by pro-inflammatory cytokines such as interleukin-1β (<em>IL</em>-1β) and tumor necrosis factor-α (TNF-α). Pharmacological treatments for tendinitis are restricted to the use of non-steroidal anti-inflammatory drugs. Recent studies in various cell models have demonstrated that curcumin targets the NF-κB signaling pathway. However, its potential for the treatment of tendinitis has not been explored. Herein, we used an in vitro model of human tenocytes to study the mechanism of curcumin action on <em>IL</em>-1β-mediated inflammatory signaling. Curcumin at concentrations of 5-<em>20</em> μm inhibited <em>IL</em>-1β-induced inflammation and apoptosis in cultures of human tenocytes. The anti-inflammatory effects of curcumin included down-regulation of gene products that mediate matrix degradation (matrix metalloproteinase-1, -9, and -13), prostanoid production (cyclooxygenase-2), apoptosis (Bax and activated caspase-3), and stimulation of cell survival (Bcl-2), all known to be regulated by NF-κB. Furthermore, curcumin suppressed <em>IL</em>-1β-induced NF-κB activation via inhibition of phosphorylation and degradation of inhibitor of κBα, inhibition of inhibitor of κB-kinase activity, and inhibition of nuclear translocation of NF-κB. Furthermore, the effects of <em>IL</em>-1β were abrogated by wortmannin, suggesting a role for the phosphatidylinositol 3-kinase (PI-3K) pathway in <em>IL</em>-1β signaling. Curcumin suppressed <em>IL</em>-1β-induced PI-3K p85/Akt activation and its association with IKK. These results demonstrate, for the first time, a potential role for curcumin in treating tendon inflammation through modulation of NF-κB signaling, which involves PI-3K/Akt and the tendon-specific transcription factor scleraxis in tenocytes.

BACKGROUND

Psoriasis lesions are characterized by large-scale shifts in gene expression. Mechanisms that underlie differentially expressed genes (DEGs), however, are not completely understood. We analyzed existing datasets to evaluate genome-wide expression in lesions from 163 psoriasis patients. Our aims were to identify mechanisms that drive differential expression and to characterize heterogeneity among lesions in this large sample.

RESULTS

We identified 1233 psoriasis-increased DEGs and 977 psoriasis-decreased DEGs. Increased DEGs were attributed to keratinocyte activity (56%) and infiltration of lesions by T-cells (14%) and macrophages (11%). Decreased DEGs, in contrast, were associated with adipose tissue (63%), epidermis (14%) and dermis (4%). KC/epidermis DEGs were enriched for genes induced by <em>IL</em>-1, <em>IL</em>-17A and <em>IL</em>-<em>20</em> family cytokines, and were also disproportionately associated with AP-1 binding sites. Among all patients, 50% exhibited a heightened inflammatory signature, with increased expression of genes expressed by T-cells, monocytes and dendritic cells. 66% of patients displayed an IFN-γ-strong signature, with increased expression of genes induced by IFN-γ in addition to several other cytokines (e.g., <em>IL</em>-1, <em>IL</em>-17A and TNF). We show that such differences in gene expression can be used to differentiate between etanercept responders and non-responders.

CONCLUSIONS

Psoriasis DEGs are partly explained by shifts in the cellular composition of psoriasis lesions. Epidermal DEGs, however, may be driven by the activity of AP-1 and cellular responses to <em>IL</em>-1, <em>IL</em>-17A and <em>IL</em>-<em>20</em> family cytokines. Among patients, we uncovered a range of inflammatory- and cytokine-associated gene expression patterns. Such patterns may provide biomarkers for predicting individual responses to biologic therapy.

Inflammatory reactivity to acute laboratory stress is thought to reflect individual differences in responsivity to environmental stressors and may confer future health risk. To characterize this response, we conducted a meta-analysis of 34 studies that measured circulating inflammatory markers and 15 studies that measured stimulated production of inflammatory markers before and after exposure to laboratory challenge. Results showed significant stress-related increases in circulating interleukin (<em>IL</em>)-1β (d=0.66, p<0.001), <em>IL</em>-6 (d=0.35, p<0.001), <em>IL</em>-10 (d=0.69, p<0.001), and tumor necrosis factor(TNF)-α (d=0.28, p<0.001), but not <em>IL</em>-1ra, <em>IL</em>-2, interferon-γ, or C-reactive protein. There were sufficient data to assess the time course of <em>IL</em>-6, <em>IL</em>-1β, and TNF-α reactivity. <em>IL</em>-6 increased from baseline to measures taken 40-50, 60-75, 90, and 1<em>20</em>min following stress, with the largest effect at 90min post-stress (d=0.70, p<0.001). <em>IL</em>-1β increased from baseline to <em>20</em>-30, 40-50, and 60-70min following stress, with the largest effect between 40 and 50min post-stress (d=0.73, p=0.02). For TNF-α, there was a significant increase from baseline to 31-50min post stress (d=0.44, p=0.01), but not at later times. There was no difference in magnitude of <em>IL</em>-6 reactivity as a function of type of stress (social-evaluative versus other). For stimulated inflammatory markers, results showed stress-related increases in <em>IL</em>-1β when measured <em>20</em>-1<em>20</em>min post-stress (d=1.09, p<0.001), and in <em>IL</em>-4 and interferon-γ when measured 0-10min post stressor (d=-0.42, p<0.001 and d=0.47, p<0.001). These results extend findings from a prior meta-analysis (Steptoe et al., <em>20</em>07) to show reliable increases in circulating <em>IL</em>-6, <em>IL</em>-1β, <em>IL</em>-10 and TNF-α and stimulated <em>IL</em>-1β, <em>IL</em>-4 and interferon-γ in response to acute stress. It is possible that these responses contribute to associations between exposure to life challenges and vulnerability to inflammatory disease.

BACKGROUND

We conducted a phase I/II randomized placebo-controlled trial with the aim of exploring whether priming with a low intradermal dose of a multiclade, multigene HIV-1 DNA vaccine could improve the immunogenicity of the same vaccine given intramuscularly prior to boosting with a heterologous HIV-1 MVA among healthy adults in Dar es Salaam, Tanzania.

METHODS

Sixty HIV-uninfected volunteers were randomized to receive DNA plasmid vaccine 1mg intradermally (id), n=<em>20</em>, or 3.8mg intramuscularly (im), n=<em>20</em>, or placebo, n=<em>20</em>, using a needle-free injection device. DNA plasmids encoding HIV-1 genes gp160 subtype A, B, C; rev B; p17/p24 gag A, B and Rtmut B were given at weeks 0, 4 and 12. Recombinant MVA (10(8)pfu) expressing HIV-1 Env, Gag, Pol of CRF01_AE or placebo was administered im at month 9 and 21.

RESULTS

The vaccines were well tolerated. Two weeks after the third HIV-DNA injection, 22/38 (58%) vaccinees had IFN-γ ELISpot responses to Gag. Two weeks after the first HIV-MVA boost all 35 (100%) vaccinees responded to Gag and 31 (89%) to Env. Two to four weeks after the second HIV-MVA boost, 28/29 (97%) vaccinees had IFN-γ ELISpot responses, 27 (93%) to Gag and 23 (79%) to Env. The id-primed recipients had significantly higher responses to Env than im recipients. Intracellular cytokine staining for Gag-specific IFN-γ/IL-2 production showed both CD8(+) and CD4(+) T cell responses. All vaccinees had HIV-specific lymphoproliferative responses. All vaccinees reacted in diagnostic HIV serological tests and 26/29 (90%) had antibodies against gp160 after the second HIV-MVA boost. Furthermore, while all of 29 vaccinee sera were negative for neutralizing antibodies against clade B, C and CRF01_AE pseudoviruses in the TZM-bl neutralization assay, in a PBMC assay, the response rate ranged from 31% to 83% positives, depending upon the clade B or CRF01_AE virus tested.

CONCLUSIONS

This vaccine approach is safe and highly immunogenic. Low dose, id HIV-DNA priming elicited higher and broader cell-mediated immune responses to Env after HIV-MVA boost compared to a higher HIV-DNA priming dose given im. Three HIV-DNA priming immunizations followed by two HIV-MVA boosts efficiently induced Env-antibody responses.

Cytokines and chemokines are soluble mediators of the immune system that play a crucial role in intercellular signaling, and in the recruitment of cells to inflammation sites. Identification of these molecules in nonhuman primates (NHP) is crucial for the understanding of complex physiological and pathological mechanisms that occur in these species, and to demonstrate whether these mechanisms function similarly in humans. The Luminex100 system is a bench-top flow cytometer that allows the user to perform up to 100 tests simultaneously in a single tube. Recently, a significant number of commercial vendors have developed kits for the simultaneous detection of multiple cytokines and chemokines of human origin with the Luminex system. These kits were tested for their capacity to recognize chemokines and cytokines of nonhuman primate origin. ELISA and ELISPOT assays were also adapted to the Luminex format, and novel assays based on new combinations of antibodies were developed. PBMC were isolated from blood from chimpanzees, rhesus macaques, baboons, cynomolgus macaques, pig-tailed macaques, and African green monkeys; these cells were stimulated in vitro and culture supernatants were used for the determination of cytokines and chemokines. Crossreactivity tables were prepared based on the ability of the reagents to detect cytokines and chemokines in NHP samples with similar intensity to the ones observed in human samples. By mixing commercially available reagents and newly developed ones, a combination has been created that allows for the detection of <em>20</em> NHP chemokines and cytokines in a single sample, including G-CSF, GM-CSF, IFN-gamma, <em>IL</em>-1beta, <em>IL</em>-1Ra, <em>IL</em>-2, <em>IL</em>-4, <em>IL</em>-5, <em>IL</em>-6, <em>IL</em>-8, <em>IL</em>-10, <em>IL</em>-12 (p40), <em>IL</em>-17, <em>IL</em>-18, MCP-1, MIP-1alpha, MIP-1beta, RANTES, TNF-alpha, and TNF-beta. These reagents may become a very useful resource for scientists working with these NHP species, which are relevant pre-clinical models for human diseases and transplantation because they approximate humans in physiology and genetics more closely than any other animal.

OBJECTIVE

Pancreatic cancer is the most deadly of all gastrointestinal (GI) malignancies, yet relatively little is known regarding mechanisms of tumor development including the role of inflammation.

BACKGROUND

Chronic pancreatitis (CP) increases the risk of developing cancer by 10- to <em>20</em>-fold; mediators of the chronic inflammatory process and the surrounding fibrotic stroma likely support a transformation to malignancy, yet the exact mechanisms remain undefined. The purpose of our present study was to determine potential inflammatory components in epithelial and stromal cells that may contribute to both CP and pancreatic cancers.

METHODS

Specimens of normal pancreas, CP, and pancreatic cancer were examined using laser-capture microdissection (LCM), gene array, and immunohistochemistry.

RESULTS

Gene array analysis from LCM-dissected tissues demonstrated: (i) increased expression of interleukin-8 (IL-8), an activator of the inflammatory factor nuclear factor-kappaB (NF-kappaB), and (ii) decreased expression of IkappaB (an inhibitor of NF-kappaB) in CP ductal cells compared with normal ducts. Compared with CP, cancers demonstrated: (i) increased expression of tumor related genes including S100A4, cyclin E1, and epidermal growth factor (EGF) receptor, and (ii) expression of matrix metalloproteinase 2, a pro-invasive factor for tumor cells, which was not present in the CP stroma. Increased staining of both the p50 NF-kappaB subunit and IKKalpha kinase (a protein that allows activation of NF-kappaB) was noted in CP and cancers.

CONCLUSIONS

Our results demonstrate that similar inflammatory components and downstream effectors are present in CP and pancreatic cancers. Importantly, these findings suggest that a common pathway for pancreatic cancer development may be through a chronic inflammatory process including stroma formation. These findings may lead to novel strategies for pancreatic cancer prophylaxis based on inhibition of inflammatory mediators.

Autocrine growth factors are believed to be important for maintenance of an immortalized state by Epstein-Barr virus (EBV), because cell-free supernatants of EBV-immortalized cell lines promote the proliferation of autologous cells and permit their growth at low cell density. In this study, we provide evidence for the existence of two autocrine growth factor activities produced by EBV-immortalized lines distinguished by size and biological activities. Much of the autocrine growth factor activity in lymphoblastoid cell line supernatants resided in a low-molecular-weight (less than 5,000) fraction. However, up to <em>20</em> to 30% of the autocrine growth factor activity resided in the high-molecular-weight (greater than 5,000) fraction. While the nature of the low-molecular-weight growth factor activity remains undefined, the high-molecular-weight growth factor activity was identified as interleukin-6 (<em>IL</em>-6). Culture supernatants from six EBV-induced lymphoblastoid cell lines tested contained <em>IL</em>-6 activity, because they promoted proliferation in the <em>IL</em>-6-dependent hybridoma cell line B9. In addition, a rabbit antibody to human <em>IL</em>-6 neutralized the capacity of the high-molecular-weight (greater than 5,000) fraction of a lymphoblastoid cell line supernatant to promote growth both in autologous EBV-immortalized cells and in B9 cells. Similarly, this high-molecular-weight autocrine growth factor activity was neutralized by a monoclonal antibody to human <em>IL</em>-6. Furthermore, characteristic bands, attributable to <em>IL</em>-6, were visualized in supernatants of each of four EBV-induced lymphoblastoid cell lines after immunoprecipitation with a rabbit antiserum to human <em>IL</em>-6. Thus, in addition to its previously reported properties, <em>IL</em>-6 is an autocrine growth factor for EBV-immortalized B cells cultured under serum-free conditions.

BACKGROUND

Stress during fetal life increases the risk of affective and immune disorders later in life. The altered peripheral immune response caused by prenatal stress may impact on brain function by the modification of local inflammation. In this study we have explored whether prenatal stress results in alterations in the immune response in the hippocampus of female mice during adult life.

METHODS

Pregnant C57BL/6 mice were subjected three times/day during 45 minutes to restraint stress from gestational Day 12 to delivery. Control non-stressed pregnant mice remained undisturbed. At four months of age, non-stressed and prenatally stressed females were ovariectomized. Fifteen days after surgery, mice received an i.p. injection of vehicle or of 5 mg/kg of lipopolysaccharide (LPS). Mice were sacrificed <em>20</em> hours later by decapitation and the brains were removed. Levels of interleukin-1β (<em>IL</em>1β), interleukin-6 (<em>IL</em>-6), tumor necrosis factor α (TNF-α), interferon γ-inducible protein 10 (IP10), and toll-like receptor 4 mRNA were assessed in the hippocampus by quantitative real-time polymerase chain reaction. Iba1 immunoreactivity was assessed by immunocytochemistry. Statistical significance was determined by one-way or two-way analysis of variance.

RESULTS

Prenatal stress, per se, increased <em>IL</em>1β mRNA levels in the hippocampus, increased the total number of Iba1-immunoreactive microglial cells and increased the proportion of microglial cells with large somas and retracted cellular processes. In addition, prenatally stressed and non-stressed animals showed different responses to peripheral inflammation induced by systemic administration of LPS. LPS induced a significant increase in mRNA levels of <em>IL</em>-6, TNF-α and IP10 in the hippocampus of prenatally stressed mice but not of non-stressed animals. In addition, after LPS treatment, prenatally stressed animals showed a higher proportion of Iba1-immunoreactive cells in the hippocampus with morphological characteristics of activated microglia compared to non-stressed animals. In contrast, LPS induced similar increases in expression of <em>IL</em>1β and toll-like receptor 4 in both prenatally stressed and non-stressed animals.

CONCLUSIONS

These findings indicate that prenatal stress induces long-lasting modifications in the inflammatory status of the hippocampus of female mice under basal conditions and alters the immune response of the hippocampus to peripheral inflammation.

Diclofenac is a nonsteroidal anti-inflammatory drug that causes rare but serious hepatotoxicity, the mechanism of which is unclear. The purpose of the present study was to explore the potential role played by the immune processes. Antibodies to diclofenac metabolite-modified liver protein adducts were detected in the sera of seven out of seven patients with diclofenac-induced hepatotoxicity, 12 of <em>20</em> subjects on diclofenac without hepatotoxicity, and none of four healthy controls. The antibodies recognized adducts expressed in livers from rats treated with multiple doses of diclofenac, but not in those given single doses. In addition, several potential diclofenac adducts were identified in the liver of a patient with diclofenac-induced hepatic failure, but not from a normal human donor liver, by immunoblotting with an adduct-selective rabbit antiserum. To determine whether or not polymorphisms in genes encoding cytokine-related proteins influence susceptibility to hepatotoxicity, genotyping for the polymorphisms -627 in the interleukin (<em>IL</em>)-10 gene, -590 in the <em>IL</em>-4 gene, and codon 551 in the <em>IL</em>-4 receptor (<em>IL</em>-4R) were performed on DNA from 24 patients on diclofenac with hepatotoxicity, 48 subjects on diclofenac without hepatotoxicity, and healthy controls. The frequencies of the variant alleles for <em>IL</em>-10 and <em>IL</em>-4 were higher in patients (OR [odds ratio]: 2.8 for <em>IL</em>-10; 2.6 for <em>IL</em>-4; 5.3 for <em>IL</em>-10 + <em>IL</em>-4) compared with healthy controls and subjects on diclofenac without hepatotoxicity (OR: 2.8 for <em>IL</em>-10; 1.2 for <em>IL</em>-4; 5.0 for <em>IL</em>-10 + <em>IL</em>-4). In conclusion, the observed polymorphisms, resulting in low <em>IL</em>-10 and high <em>IL</em>-4 gene transcription, could favor a T helper (Th)-2 mediated antibody response to neoantigenic stimulation associated with disease susceptibility.

We investigated the relationship between transforming growth factor-beta (TGF-beta)-secreting T-regulatory (Tr) cells and anti-B16 melanoma immunity, and studied the association of early cytokines expressed at tumour sites with the generation of Tr cells. A large number of CD4(+) Tr cells producing interleukin (<em>IL</em>)-4, <em>IL</em>-10 and TGF-beta accumulated with functionally depressed CD8(+) cytotoxic T lymphocytes (CTLs) at tumour sites on day <em>20</em> after subcutaneous (s.c.) inoculation of B16 tumour cells. Tr cells consisted of two populations, which were termed T helper 3 (Th3) and Tr1 cells. B16-infiltrating Tr cells strongly inhibited the generation of B16-specific T helper 1 (Th1) cells in a TGF-beta-dependent manner and were assumed to suppress effective generation of CTLs. In addition, B16 cells markedly progressed in mice transferred adoptively by the cultured B16-infiltrating Tr cells compared with untreated mice. The capacity of these Tr cells to produce TGF-beta was hampered by neutralizing anti-<em>IL</em>-10 and partly anti-<em>IL</em>-4 monoclonal antibodies (mAbs) injected intralesionally during the early development of B16 tumours, and this treatment markedly attenuated B16 growth. Furthermore, a lesional injection of recombinant mouse <em>IL</em>-10 at an early tumour site resulted in the vigorous progression of B16 tumours. These results provide evidence that Tr cells, belonging to the T helper 3/T-regulatory 1 (Th3/Tr1) type, are activated in B16-bearing hosts under the influence of T helper 2 (Th2) cytokines, mainly <em>IL</em>-10 (produced at early tumour lesions), and that this regulatory T-cell population functions as a suppressor of anti-B16 immunity.

Interleukin 12 (<em>IL</em>-12) doses in excess of 100 ng/d have been shown to induce profound immunotoxicities in mice infected with lymphocytic choriomeningitis virus (LCMV). These immunotoxicities are characterized by almost complete inhibition of virus-induced CD8+ T cell expansion and CTL activation, and up to 2 log increases in viral replication. They are accompanied by induction of serum tumor necrosis factor (TNF). The studies presented here were undertaken to characterize mechanisms for the <em>IL</em>-12-induced toxicities and to examine expression and function of TNF in this context. Several physiological changes were induced in <em>IL</em>-12-treated uninfected and dramatically elevated in <em>IL</em>-12-treated virus-infected mice. <em>IL</em>-12 induced (a) decreases in body weights,>> 10% in uninfected and>> <em>20</em>% in LCMV-infected mice; (b) elevation of circulating glucocorticoid levels to>> 10 micrograms/dl in uninfected and>> <em>20</em> micrograms/dl in infected mice; and (c) decreases in thymic mass,>> 30% in uninfected and up to 95% in infected mice. These changes are known to be associated with circulating TNF. Northern blot and in situ hybridization analyses demonstrated that <em>IL</em>-12 induced TNF-alpha expression and that LCMV infection synergized with <em>IL</em>-12 for induction of this factor. Antibodies neutralizing TNF reversed all of the <em>IL</em>-12-induced toxicities in LCMV-infected mice including the immunotoxicities against CD8+ T cells and anti-viral defenses. The TNF-mediated immunotoxicities appeared to result from an induced cellular sensitivity to the factor, as splenic leukocytes and CD8+ T cell subsets isolated from LCMV-infected mice were more sensitive to TNF-mediated cytotoxicity in culture than were equivalent populations prepared from uninfected mice. Experiments with the glucocorticoid type II receptor antagonist, RU486, demonstrated that endogenous glucocorticoids were secondary intermediaries in <em>IL</em>-12-induced thymic atrophy. Studies in <em>IL</em>-2-deficient mice showed that the synergism was dependent upon endogenous <em>IL</em>-2. The results delineate a unique mechanism of TNF-mediated toxicity. In addition, they have significant implications concerning potential detrimental consequences of in vivo TNF induction and of <em>IL</em>-12 administration for protective anti-viral responses.

Preeclampsia (PE) affects 5-7% of all pregnancies in the United States and is the leading cause of maternal and prenatal morbidity. PE is associated with hypertension after week <em>20</em> of gestation, decreased renal function and small-for-gestational-age babies. Women with PE exhibit chronic inflammation and production of autoantibodies. It is hypothesized that during PE, placental ischaemia occurs as a result of shallow trophoblast invasion which is associated with an immune imbalance where pro-inflammatory CD4(+) T-cells are increased and T regulatory cells (Tregs) are decreased. This imbalance leads to chronic inflammation characterized by oxidative stress, pro-inflammatory cytokines and autoantibodies. Studies conducted in our laboratory have demonstrated the importance of this immune imbalance in causing hypertension in response to placental ischaemia in pregnant rats. These studies confirm that increased CD4(+) T-cells and decreased Tregs during pregnancy leads to elevated inflammatory cytokines, endothelin (ET-1), reactive oxygen species (ROS) and agonistic autoantibodies to the angiotensin II (Ang II), type 1 receptor (AT1-AA). All of these factors taken together play an important role in increasing the blood pressure during pregnancy. Specifically, this review focuses on the decrease in Tregs, and their associated regulatory cytokine interleukin (<em>IL</em>)-10, which is seen in response to placental ischaemia during pregnancy. This study will also examine the effect of regulatory immune cell repopulation on the pathophysiology of PE. These studies show that restoring the balance of the immune system through increasing Tregs, either by adoptive transfer or by infusing <em>IL</em>-10, reduces the blood pressure and pathophysiology associated with placental ischaemia in pregnant rats.

BACKGROUND

The objectives of this study were to measure levels of gingival crevicular fluid (GCF) biomarkers and subgingival bacterial species in periodontally healthy subjects and subjects with periodontitis to explore the relationships among these biomarkers, the subgingival microbiota, and the clinical parameters of periodontal disease.

METHODS

Clinical periodontal parameters were measured at six sites per tooth in <em>20</em> subjects with periodontitis and <em>20</em> periodontally healthy subjects. GCF and subgingival plaque samples were obtained from the mesio-buccal aspect of every tooth. GCF levels of interleukin (<em>IL</em>)-1beta and <em>IL</em>-8 and matrix metalloproteinase 8 were measured using checkerboard immunoblotting, and the levels of 40 bacterial taxa were quantified using checkerboard DNA-DNA hybridization. A subset of "clinically healthy" sites from each group was analyzed separately. The significance of the differences between groups was determined using the unpaired t test or the Mann-Whitney test. Correlations among immunologic, microbiologic, and clinical data were determined using the Spearman rank correlation coefficient.

RESULTS

There were positive correlations among mean clinical parameters, mean levels of the three biomarkers, and the proportions of orange and red complex species (P <0.05). Clinically healthy sites from subjects with periodontitis had higher levels of IL-1beta and IL-8 and higher proportions of orange and red complex species (P <0.05) than clinically healthy sites from periodontally healthy subjects. Red complex species were positively associated with the expression of all biomarkers (P <0.05), whereas purple and yellow complex species had negative correlations with IL-1beta and IL-8 (P <0.05).

CONCLUSIONS

Clinically healthy sites from subjects with periodontitis have higher levels of GCF biomarkers and periodontal pathogens than clinically healthy sites from periodontally healthy subjects. Different microbial complexes demonstrated distinct associations with specific GCF biomarkers.

OBJECTIVE

The clinical onset and severity of intestinal disorders in humans and animals can be profoundly impacted by early life stress. Here we investigated the impact of early weaning stress in pigs on intestinal physiology, clinical disease, and immune response to subsequent challenge with enterotoxigenic F18 E. coli (ETEC).

METHODS

Pigs weaned from their dam at 16 d, 18 d, and <em>20</em> d of age were given a direct oral challenge of F18 ETEC at 26 d of age. Pigs were monitored from days 0 to 4 post-infection for clinical signs of disease. On Day 4 post-ETEC challenge, ileal barrier function, histopathologic and inflammatory cytokine analysis were performed on ileal mucosa.

RESULTS

Early weaned pigs (16 d and 18 d weaning age) exhibited a more rapid onset and severity of diarrhea and reductions in weight gain in response to ETEC challenge compared with late weaned pigs (<em>20</em> d weaning age). ETEC challenge induced intestinal barrier injury in early weaned pigs, indicated by reductions in ileal transepithelial electrical resistance (TER) and elevated FD4 flux rates, in early weaned pig ileum but not in late weaned pigs. ETEC-induced marked elevations in IL-6 and IL-8, neutrophil recruitment, and mast cell activation in late-weaned pigs; these responses were attenuated in early weaned pigs. TNF levels elevated in ETEC challenged ileal mucosa from early weaned pigs but not in other weaning age groups.

CONCLUSIONS

These data demonstrate the early weaning stress can profoundly alter subsequent immune and physiology responses and clinical outcomes to subsequent infectious pathogen challenge. Given the link between early life stress and gastrointestinal diseases of animals and humans, a more fundamental understanding of the mechanisms by which early life stress impacts subsequent pathophysiologic intestinal responses has implications for the prevention and management of important GI disorders in humans and animals.

BACKGROUND

Exposure to ambient ultrafine particles has been associated with cardiopulmonary toxicity and mortality. Adverse effects specifically linked to ultrafine particles include loss of sympathovagal balance and altered hemostasis.

OBJECTIVE

To characterize the effects of acute exposure to ambient ultrafine particles in young healthy humans.

METHODS

Nineteen healthy nonsmoking male and female subjects between the ages of 18 and 35 were exposed to filtered air or to an atmosphere in which captured ultrafine (<0.16 microm) particles were concentrated by a factor of up to <em>20</em>-fold over ambient levels with the use of particle concentrators fitted with size-selective outlets (ultrafine concentrated ambient particles [UFCAPs]). Subjects underwent bronchoalveolar lavage 18 hours after each exposure. Cardiovascular endpoints measured included pulmonary function, clinical chemistry, and hematological parameters, as well as heart rate variability and repolarization indices.

RESULTS

Exposure to UFCAPs was statistically associated with an increase in frequency domain markers of heart rate variability, specifically indicative of elevated vagal input to the heart. Consistent with this finding were increases in the variance associated with the duration of the QT interval. In addition, UFCAP exposure resulted in a significant increase in blood levels of the fibrin degradation product D-dimer as well as a modest elevation in the inflammatory chemokine IL-8 recovered in the lavage fluid.

CONCLUSIONS

These findings show mild inflammatory and prothrombic responses and are suggestive of alterations in cardiac repolarization induced by UFCAP inhalation.

The activation of mononuclear phagocytes (M phi) and their generation of oxidative products is influenced by various cytokines as well as by normal maturational changes. We examined the effects of <em>IL</em>-4 on superoxide (O2-) production (cytochrome c reduction) by cultured M phi and the modulation of these effects by IFN-gamma and <em>IL</em>-1. Incubation of <em>IL</em>-4 (<em>20</em>0 U/ml) with M phi inhibited M phi PMA (100 ng/ml)-stimulated O2-. production by 23% at 24 h, 34% at 48 h, and 70 to 85% at 72 to 96 h. <em>IL</em>-4 similarly inhibited M phi O2-. production in response to zymosan. <em>IL</em>-4 did not affect M phi viability, adherence to microtiter plates, or ability to phagocytose boiled yeast. In comparison with M phi, neutrophil O2-. production was not inhibited after 4 to <em>20</em> h incubation with <em>IL</em>-4. When <em>IL</em>-4 was washed out as early as 1 h after the initiation of M phi culture, significant inhibition of O2-. production was observed 4 days later. Sequential addition of either <em>IL</em>-4 or IFN-gamma to cultures demonstrated reciprocal cytokine effects on M phi; <em>IL</em>-4 partially inhibited O2-. production by M phi previously treated with rIFN-gamma whereas rIFN-gamma partially augmented O2-. production by M phi previously treated with <em>IL</em>-4. Because <em>IL</em>-4 has been reported to inhibit <em>IL</em>-1 production, add-back experiments were performed; addition of <em>IL</em>-1 only partly reconstituted O2-. production in <em>IL</em>-4-treated cells. Further characterization showed that although M phi protein synthesis was enhanced by both rIFN-gamma and <em>IL</em>-4 treatment, acid phosphatase, a marker of maturation to the macrophage phenotype, was markedly increased at an earlier time point in <em>IL</em>-4-treated M phi, and correlated with a decline in O2-. production. The ability of <em>IL</em>-4 to suppress M phi O2-. production implicates <em>IL</em>-4 as an important regulator of this aspect of the inflammatory response.

Cortisol and inflammatory proteins are released into the blood in response to stressors and chronic elevations of blood cortisol and inflammatory proteins may contribute to ongoing disease processes and could be useful biomarkers of disease. How chronic circadian misalignment influences cortisol and inflammatory proteins, however, is largely unknown and this was the focus of the current study. Specifically, we examined the influence of weeks of chronic circadian misalignment on cortisol, stress ratings, and pro- and anti-inflammatory proteins in humans. We also compared the effects of acute total sleep deprivation and chronic circadian misalignment on cortisol levels. Healthy, drug free females and males (N=17) aged <em>20</em>-41 participated. After 3weeks of maintaining consistent sleep-wake schedules at home, six laboratory baseline days and nights, a 40-h constant routine (CR, total sleep deprivation) to examine circadian rhythms for melatonin and cortisol, participants were scheduled to a 25-day laboratory entrainment protocol that resulted in sleep and circadian disruption for eight of the participants. A second constant routine was conducted to reassess melatonin and cortisol rhythms on days 34-35. Plasma cortisol levels were also measured during sampling windows every week and trapezoidal area under the curve (AUC) was used to estimate 24-h cortisol levels. Inflammatory proteins were assessed at baseline and near the end of the entrainment protocol. Acute total sleep deprivation significantly increased cortisol levels (p<0.0001), whereas chronic circadian misalignment significantly reduced cortisol levels (p<0.05). Participants who exhibited normal circadian phase relationships with the wakefulness-sleep schedule showed little change in cortisol levels. Stress ratings increased during acute sleep deprivation (p<0.0001), whereas stress ratings remained low across weeks of study for both the misaligned and synchronized control group. Circadian misalignment significantly increased plasma tumor necrosis factor-alpha (TNF-α), interleukin 10 (<em>IL</em>-10) and C-reactive protein (CRP) (p<0.05). Little change was observed for the TNF-α/<em>IL</em>-10 ratio during circadian misalignment, whereas the TNF-α/<em>IL</em>-10 ratio and CRP levels decreased in the synchronized control group across weeks of circadian entrainment. The current findings demonstrate that total sleep deprivation and chronic circadian misalignment modulate cortisol levels and that chronic circadian misalignment increases plasma concentrations of pro- and anti-inflammatory proteins.

OBJECTIVE

To determine if statin therapy reduces the incidence of severe sepsis and the levels of inflammatory cytokines in patients with acute bacterial infection.

METHODS

Double-blind placebo controlled randomized clinical trial.

METHODS

Department of medicine and medical intensive care unit in a tertiary university medical center.

METHODS

A total of 83 patients with suspected or documented bacterial infection were enrolled. We randomly assigned 42 patients to receive 40 mg of simvastatin orally, followed by <em>20</em> mg of simvastatin, and 41 to receive matching placebo.

RESULTS

The study was prematurely terminated due to slow recruitment rate. Here we report the analysis of the secondary outcome: change in cytokines levels at 72 h. Both groups were evenly matched in terms of co-morbidity and severity of illness on admission. Four of the 83 patients enrolled developed severe sepsis, two in each group. No difference was observed in other clinical variables and there were no mortalities. Cytokine levels were randomly assessed in 40 patients (<em>20</em> in each group). Both TNF-alpha and IL-6 levels were significantly reduced in the simvastatin group (p = 0.02 and p = 0.02, respectively), while no such difference was observed in the placebo group (p = 0.35 and 0.39, respectively).

CONCLUSIONS

Statin therapy may be associated with a reduction in the levels of inflammatory cytokines in patients with acute bacterial infections. Large controlled trials will determine if this reduction will translate into a clinical benefit.

Current procedures for the genetic manipulation of hematopoietic stem cells are relatively inefficient due, in part, to a poor understanding of the conditions for ex vivo maintenance or expansion of stem cells. We report improvements in the retroviral transduction of human stem cells based on the SCID-repopulating cell (SRC) assay and analysis of Lin(-) CD34(+)CD38(-) cells as a surrogate measure of stem cell function. Based on our earlier study of the conditions required for ex vivo expansion of Lin(-)CD34(+) CD38(-) cells and SRC, CD34(+)-enriched lineage-depleted umbilical cord blood cells were cultured for 2 to 6 days on fibronectin fragment in MGIN (MSCV-EGFP-Neo) retroviral supernatant (containing 1.5% fetal bovine serum) and <em>IL</em>-6, SCF, Flt-3 ligand, and G-CSF. Both CD34(+)CD38(-) cells (<em>20</em>.8%) and CFC (26.3%) were efficiently marked. When the bone marrow of engrafted NOD/SCID mice was examined, 75% (12/16) contained multilineage (myeloid and B lymphoid) EGFP(+) human cells composing as much as 59% of the graft. Half of these mice received a limiting dose of SRC, suggesting that the marked cells were derived from a single transduced SRC. Surprisingly, these culture conditions produced a large expansion (166-fold) of cells with the CD34(+)CD38(-) phenotype (n = <em>20</em>). However, there was no increase in SRC numbers, indicating dissociation between the CD34(+)CD38(-) phenotype and SRC function. The underlying mechanism involved apparent downregulation of CD38 expression within a population of cultured CD34(+)CD38(+) cells that no longer contained any SRC function. These results suggest that the relationship between stem cell function and cell surface phenotype may not be reliable for cultured cells. (Blood. <em>20</em>00;95:102-110)

The effect of CNS-targeted <em>IL</em>-6 gene expression has been thoroughly investigated in the otherwise nonperturbed brain but not following brain injury. Here we examined the impact of astrocyte-targeted <em>IL</em>-6 production in a traumatic brain injury (cryolesion) model using GFAP-<em>IL</em>6 transgenic mice. This study demonstrated that transgenic <em>IL</em>-6 production significantly increased wound healing following the cryolesion. Thus, at <em>20</em> days postlesion (dpl) the GFAP-<em>IL</em>6 mice showed almost complete wound healing compared to litter mate nontransgenic controls. It seems likely that a reduced inflammatory response in the long term could be responsible for this <em>IL</em>-6-related effect. Thus, while in the acute phase following cryolesion (1-6 dpl) the recruitment of macrophages and T lymphocytes was higher in GFAP-<em>IL</em>6 mice, at 10-<em>20</em> dpl it was significantly reduced compared to controls. Reactive astrogliosis was also significantly increased up to but not including <em>20</em> dpl in the GFAP-<em>IL</em>6 mice. Oxidative stress as well as apoptotic cell death was significantly decreased throughout the time period studied in the GFAP-<em>IL</em>6 mice compared to controls. This could be linked to the altered inflammatory response as well as to the transgenic <em>IL</em>-6-induced increase of the antioxidant, neuroprotective proteins metallothionein-I + II. These results indicate that although in the brain the chronic astrocyte-targeted expression of <em>IL</em>-6 spontaneously induces an inflammatory response causing significant damage, during an acute neuropathological insult such as following traumatic injury, a clear neuroprotective role is evident.

Human eosinophils are cytokine-producing cells that are prominent in IgE-dependent allergic tissue reactions. <em>IL</em>-4 promotes the development of the Th2-type phenotype in T cells and is an essential cofactor for IgE production by B cells. We detected mRNA for <em>IL</em>-4 by reverse transcription-PCR in blood eosinophils from atopic asthmatics. By specific ELISA, 108 +/- <em>20</em> pg of <em>IL</em>-4 protein/10(6) cells could be extracted from whole cells, and approximately 30% of the <em>IL</em>-4 was released after incubation with serum-coated particles. Using immunocytochemistry, eosinophils from atopic asthmatics and nonatopic controls showed <em>IL</em>-4 immunoreactivity using an anti-<em>IL</em>-4 mAb. <em>IL</em>-4 was located predominantly in the eosinophil granules, as shown by both immunogold electron microscopy and a cell fractionation technique that dissociated cell granules from membrane and cytosolic components. <em>IL</em>-4 mRNA colocalized with eosinophils (using sequential immunocytochemistry with an eosinophil-specific (EG2) mAb and in situ hybridization using an <em>IL</em>-4-specific antisense riboprobe) in both cell cytospins from bronchoalveolar lavage fluid from asthmatics as well as skin biopsies obtained from allergen-induced late phase (6-h) reactions in atopic subjects. Using double immunocytochemistry on skin biopsies with eosinophil- and <em>IL</em>-4-specific mAb, 83.5 +/- 3.5% of eosinophils were <em>IL</em>-4+. Conversely, eosinophils accounted for 46.5 +/- 3.9% of the total cells expressing <em>IL</em>-4 immunoreactivity. Thus, human eosinophils express mRNA for <em>IL</em>-4, and the translated product is contained within the crystalloid granule from which it is released after stimulation with serum-coated particles. These observations are consistent with the hypothesis that eosinophils contribute to the development of the Th2 phenotype by T cells infiltrating atopic allergic reactions as well as to IgE synthesis.

The production, manipulation and rescue of a bacterial artificial chromosome clone of Vaccinia virus (VAC-BAC) in order to expedite construction of expression vectors and mutagenesis of the genome has been described (Domi & Moss, <em>20</em>02, PNAS99 12415-<em>20</em>). The genomic BAC clone was 'rescued' back to infectious virus using a Fowlpox virus helper to supply transcriptional machinery. We apply here a similar approach to the attenuated strain Modified Vaccinia virus Ankara (MVA), now widely used as a safe non-replicating recombinant vaccine vector in mammals, including humans. Four apparently full-length, rescuable clones were obtained, which had indistinguishable immunogenicity in mice. One clone was shotgun sequenced and found to be identical to the parent. We employed GalK recombination-mediated genetic engineering (recombineering) of MVA-BAC to delete five selected viral genes. Deletion of C12L, A44L, A46R or B7R did not significantly affect CD8(+) T cell immunogenicity in BALB/c mice, but deletion of B15R enhanced specific CD8(+) T cell responses to one of two endogenous viral epitopes (from the E2 and F2 proteins), in accordance with published work (Staib et al., <em>20</em>05, J. Gen. Virol.86, 1997-<em>20</em>06). In addition, we found a higher frequency of triple-positive IFN-gamma, TNF-alpha and <em>IL</em>-2 secreting E3-specific CD8+ T-cells 8 weeks after vaccination with MVA lacking B15R. Furthermore, a recombinant vaccine capable of inducing CD8(+) T cells against an epitope from Plasmodium berghei was created using GalK counterselection to insert an antigen expression cassette lacking a tandem marker gene into the traditional thymidine kinase locus of MVA-BAC. MVA continues to feature prominently in clinical trials of recombinant vaccines against diseases such as HIV-AIDS, malaria and tuberculosis. Here we demonstrate in proof-of-concept experiments that MVA-BAC recombineering is a viable route to more rapid and efficient generation of new candidate mutant and recombinant vaccines based on a clinically deployable viral vector.

BACKGROUND

Increased intracranial pressure (ICP) is a serious, life-threatening, secondary event following traumatic brain injury (TBI). In many cases, ICP rises in a delayed fashion, reaching a maximal level 48-96 hours after the initial insult. While pressure catheters can be implanted to monitor ICP, there is no clinically proven method for determining a patient's risk for developing this pathology.

METHODS

In the present study, we employed antibody array and Luminex-based screening methods to interrogate the levels of inflammatory cytokines in the serum of healthy volunteers and in severe TBI patients (GCS<or=8) with or without incidence of elevated intracranial pressure (ICP). De-identified samples and ELISAs were used to confirm the sensitivity and specificity of <em>IL</em>-6 as a prognostic marker of elevated ICP in both isolated TBI patients, and polytrauma patients with TBI.

RESULTS

Consistent with previous reports, we observed sustained increases in <em>IL</em>-6 levels in TBI patients irrespective of their ICP status. However, the group of patients who subsequently experienced ICP>>or= 25 mm Hg had significantly higher <em>IL</em>-6 levels within the first 17 hours of injury as compared to the patients whose ICP remained <or=20 mm Hg. When blinded samples (n = 22) were assessed, a serum <em>IL</em>-6 cut-off of <5 pg/ml correctly identified 100% of all the healthy volunteers, a cut-off of >128 pg/ml correctly identified 85% of isolated TBI patients who subsequently developed elevated ICP, and values between these cut-off values correctly identified 75% of all patients whose ICP remained <or=20 mm Hg throughout the study period. In contrast, the marker had no prognostic value in predicting elevated ICP in polytrauma patients with TBI. When the levels of serum <em>IL</em>-6 were assessed in patients with orthopedic injury (n = 7) in the absence of TBI, a significant increase was found in these patients compared to healthy volunteers, albeit lower than that observed in TBI patients.

CONCLUSIONS

Our results suggest that serum IL-6 can be used for the differential diagnosis of elevated ICP in isolated TBI.

The extent to which DNA methylation contributes to proper regulation of murine T cell effector function is unclear. In this study, we show that in the absence of the maintenance DNA methyltransferase Dnmt1, silencing of <em>IL</em>-4, <em>IL</em>-5, <em>IL</em>-13, and <em>IL</em>-10 in CD8 T cells was abolished, and expression of these Th2 cytokines increased as much as 1000-fold compared with that of control CD8 T cells. Th2 cytokine expression also increased in Dnmt1(-/-) CD4 T cells, but the increase ( approximately <em>20</em>-40-fold for <em>IL</em>-4 and <em>IL</em>-10, </=5-fold for <em>IL</em>-5 and <em>IL</em>-13) was less than for CD8 T cells. As a result, both Dnmt1(-/-) CD4 and CD8 T cells expressed high and comparable amounts of Th2 cytokines. Loss of Dnmt1 had more subtle effects on <em>IL</em>-2 (</=5-fold increase) and IFN-gamma ( approximately 5-10-fold increase) expression and did not affect the normal bias for greater <em>IL</em>-2 expression by CD4 T cells and greater IFN-gamma expression by CD8 T cells, nor the exclusive expression of perforin and granzyme B by the CD8 T cells. These results indicate that Dnmt1 and DNA methylation are necessary to prevent cell autonomous Th2 cytokine expression in CD8 T cells but are not essential for maintaining proper T cell subset-specific expression of Th1 or CTL effectors. We also found that the expression of Th2 cytokines by Dnmt1(-/-) T cells was appropriately up-regulated in Th2 conditions and down-regulated in Th1 conditions, indicating that transcription factors and DNA methylation are complementary and nonredundant mechanisms by which the Th2 effector program is regulated.