Matural curcuminoids, curcumin, I, II and III isolated from turmeric (Curcuma longa) were compared for their cytotoxic, tumour reducing and antioxidant activities. Curcumin III was found to be more active than the other two as a cytotoxic agent and in the inhibition of Ehrlich ascites tumour in mice (<em>ILS</em> 74.1%). These compounds were also checked for their antioxidant activity which possibly indicates their potential use as anti-promoters. The amount of curcuminoids (I, II and III) needed for 50% inhibition of lipid peroxidation was <em>20</em>, 14 and 11 g/m. Concentrations needed for 50% inhibition of superoxides were 6.25, 4.25 and 1.9 micrograms/ml and those for hydroxyl radical were 2.3, 1.8 and 1.8 micrograms/ml, respectively. The ability of these compounds to suppress the superoxide production by macrophages activated with phorbol-12-myristate-13-acetate (PMA) indicated that all the three curcuminoids inhibited superoxide production and curcumin III produced maximum effect. These results indicate that curcumin III is the most active of the curcuminoids present in turmeric. Synthetic curcumin I and III had similar activity to natural curcumins.

OBJECTIVE

Knee osteoarthritis (OA), a common cause of chronic pain and disability, has biomechanical and inflammatory origins and is exacerbated by obesity.

OBJECTIVE

To determine whether a ≥10% reduction in body weight induced by diet, with or without exercise, would improve mechanistic and clinical outcomes more than exercise alone.

METHODS

Single-blind, 18-month, randomized clinical trial at Wake Forest University between July <em>20</em>06 and April <em>20</em>11. The diet and exercise interventions were center-based with options for the exercise groups to transition to a home-based program. Participants were 454 overweight and obese older community-dwelling adults (age ≥55 years with body mass index of 27-41) with pain and radiographic knee OA.

METHODS

Intensive diet-induced weight loss plus exercise, intensive diet-induced weight loss, or exercise.

METHODS

Mechanistic primary outcomes: knee joint compressive force and plasma <em>IL</em>-6 levels; secondary clinical outcomes: self-reported pain (range, 0-<em>20</em>), function (range, 0-68), mobility, and health-related quality of life (range, 0-100).

RESULTS

Three hundred ninety-nine participants (88%) completed the study. Mean weight loss for diet + exercise participants was 10.6 kg (11.4%); for the diet group, 8.9 kg (9.5%); and for the exercise group, 1.8 kg (2.0%). After 18 months, knee compressive forces were lower in diet participants (mean, 2487 N; 95% CI, 2393 to 2581) compared with exercise participants (2687 N; 95% CI, 2590 to 2784, pairwise difference [Δ](exercise vs diet )= <em>20</em>0 N; 95% CI, 55 to 345; P = .007). Concentrations of <em>IL</em>-6 were lower in diet + exercise (2.7 pg/mL; 95% CI, 2.5 to 3.0) and diet participants (2.7 pg/mL; 95% CI, 2.4 to 3.0) compared with exercise participants (3.1 pg/mL; 95% CI, 2.9 to 3.4; Δ(exercise vs diet + exercise) = 0.39 pg/mL; 95% CI, -0.03 to 0.81; P = .007; Δ(exercise vs diet )= 0.43 pg/mL; 95% CI, 0.01 to 0.85, P = .006). The diet + exercise group had less pain (3.6; 95% CI, 3.2 to 4.1) and better function (14.1; 95% CI, 12.6 to 15.6) than both the diet group (4.8; 95% CI, 4.3 to 5.2) and exercise group (4.7; 95% CI, 4.2 to 5.1, Δ(exercise vs diet + exercise) = 1.02; 95% CI, 0.33 to 1.71; P(pain) = .004; 18.4; 95% CI, 16.9 to 19.9; Δ(exercise vs diet + exercise), 4.29; 95% CI, 2.07 to 6.50; P(function )< .001). The diet + exercise group (44.7; 95% CI, 43.4 to 46.0) also had better physical health-related quality of life scores than the exercise group (41.9; 95% CI, 40.5 to 43.2; Δ(exercise vs diet + exercise) = -2.81; 95% CI, -4.76 to -0.86; P = .005).

CONCLUSIONS

Among overweight and obese adults with knee OA, after 18 months, participants in the diet + exercise and diet groups had more weight loss and greater reductions in IL-6 levels than those in the exercise group; those in the diet group had greater reductions in knee compressive force than those in the exercise group.

BACKGROUND

clinicaltrials.gov Identifier: NCT00381290.

Direct measurements revealed low oxygen tensions (0.5-4.5% oxygen) in murine lymphoid organs in vivo. To test whether adaptation to changes in oxygen tension may have an effect on lymphocyte functions, T cell differentiation and functions at varying oxygen tensions were studied. These studies show: 1) differentiated CTL deliver Fas ligand- and perforin-dependent lethal hit equally well at all redox conditions; 2) CTL development is delayed at 2.5% oxygen as compared with <em>20</em>% oxygen. Remarkably, development of CTL at 2.5% oxygen is more sustained and the CTL much more lytic; and 3) hypoxic exposure and TCR-mediated activation are additive in enhancing levels of hypoxia response element-containing gene products in lymphocyte supernatants. In contrast, hypoxia inhibited the accumulation of nonhypoxia response element-containing gene products (e.g., <em>IL</em>-2 and IFN-gamma) in the same cultures. This suggests that T cell activation in hypoxic conditions in vivo may lead to different patterns of lymphokine secretion and accumulation of cytokines (e.g., vascular endothelial growth factor) affecting endothelial cells and vascular permeabilization. Thus, although higher numbers of cells survive and are activated during <em>20</em>% oxygen incubation in vitro, the CTL which develop at 2.5% oxygen are more lytic with higher levels of activation markers. It is concluded that the ambient <em>20</em>% oxygen tension (plus 2-ME) is remarkably well suited for immunologic specificity and cytotoxicity studies, but oxygen dependence should be taken into account during the design and interpretation of results of in vitro T cell development assays and gene expression studies in vivo.

BACKGROUND

Recent cohort studies have identified the use of large tidal volumes as a major risk factor for development of lung injury in mechanically ventilated patients without acute lung injury (ALI). We compared the effect of conventional with lower tidal volumes on pulmonary inflammation and development of lung injury in critically ill patients without ALI at the onset of mechanical ventilation.

METHODS

We performed a randomized controlled nonblinded preventive trial comparing mechanical ventilation with tidal volumes of 10 ml versus 6 ml per kilogram of predicted body weight in critically ill patients without ALI at the onset of mechanical ventilation. The primary end point was cytokine levels in bronchoalveolar lavage fluid and plasma during mechanical ventilation. The secondary end point was the development of lung injury, as determined by consensus criteria for ALI, duration of mechanical ventilation, and mortality.

RESULTS

One hundred fifty patients (74 conventional versus 76 lower tidal volume) were enrolled and analyzed. No differences were observed in lavage fluid cytokine levels at baseline between the randomization groups. Plasma interleukin-6 (<em>IL</em>-6) levels decreased significantly more strongly in the lower-tidal-volume group ((from 51 (<em>20</em> to 182) ng/ml to 11 (5 to <em>20</em>) ng/ml versus 50 (21 to 122) ng/ml to 21 (<em>20</em> to 77) ng/ml; P = 0.01)). The trial was stopped prematurely for safety reasons because the development of lung injury was higher in the conventional tidal-volume group as compared with the lower tidal-volume group (13.5% versus 2.6%; P = 0.01). Univariate analysis showed statistical relations between baseline lung-injury score, randomization group, level of positive end-expiratory pressure (PEEP), the number of transfused blood products, the presence of a risk factor for ALI, and baseline <em>IL</em>-6 lavage fluid levels and the development of lung injury. Multivariate analysis revealed the randomization group and the level of PEEP as independent predictors of the development of lung injury.

CONCLUSIONS

Mechanical ventilation with conventional tidal volumes is associated with sustained cytokine production, as measured in plasma. Our data suggest that mechanical ventilation with conventional tidal volumes contributes to the development of lung injury in patients without ALI at the onset of mechanical ventilation.

BACKGROUND

ISRCTN82533884.

BACKGROUND

Open-label oral immunotherapy (OIT) protocols have been used to treat small numbers of patients with peanut allergy. Peanut OIT has not been evaluated in double-blind, placebo-controlled trials.

OBJECTIVE

To investigate the safety and effectiveness of OIT for peanut allergy in a double-blind, placebo-controlled study.

METHODS

In this multicenter study, children ages 1 to 16 years with peanut allergy received OIT with peanut flour or placebo. Initial escalation, build-up, and maintenance phases were followed by an oral food challenge (OFC) at approximately 1 year. Titrated skin prick tests (SPTs) and laboratory studies were performed at regular intervals.

RESULTS

Twenty-eight subjects were enrolled in the study. Three peanut OIT subjects withdrew early in the study because of allergic side effects. During the double-blind, placebo-controlled food challenge, all remaining peanut OIT subjects (n = 16) ingested the maximum cumulative dose of 5000 mg (approximately <em>20</em> peanuts), whereas placebo subjects (n = 9) ingested a median cumulative dose of 280 mg (range, 0-1900 mg; P < .001). In contrast with the placebo group, the peanut OIT group showed reductions in SPT size (P < .001), <em>IL</em>-5 (P = .01), and <em>IL</em>-13 (P = .02) and increases in peanut-specific IgG(4) (P < .001). Peanut OIT subjects had initial increases in peanut-specific IgE (P < .01) but did not show significant change from baseline by the time of OFC. The ratio of forkhead box protein 3 (FoxP3)(hi): FoxP3(intermediate) CD4+ CD25+ T cells increased at the time of OFC (P = .04) in peanut OIT subjects.

CONCLUSIONS

These results conclusively demonstrate that peanut OIT induces desensitization and concurrent immune modulation. The current study continues and is evaluating the hypothesis that peanut OIT causes long-term immune tolerance.

M2-polarized tumor-associated macrophages (TAMs) are key regulators of the link between inflammation and cancer. A negative correlation between infiltration intensity of M2-polarized TAMs and prognosis of pancreatic cancer has been reported. Epithelial-mesenchymal transition (EMT) is an important biological process in the progression of primary tumors toward metastasis. Inflammation-induced EMT has been previously shown, therefore, we hypothesized M2-polarized TAMs could induce EMT in pancreatic cancer. Toll-like receptor 4 (TLR4) signaling has an active role in tumor progression during chronic inflammation and the receptor is primarily expressed on macrophages. Activation of TLR4 on M2-polarized TAMs stimulates an increase in the cytokine interleukin-10 (<em>IL</em>-10); consequently, another aim was to investigate the potential role of TLR4/<em>IL</em>-10 signaling in the EMT of pancreatic cancer. Treatment with <em>IL</em>-4 (<em>20</em> ng/ml) for 24 h successfully induced the polarization of macrophage cell line RAW 264.7 to M2 phenotype, <em>IL</em>-10(high), <em>IL</em>-12(low), and <em>IL</em>-23(low), and high expression of CD<em>20</em>4 and CD<em>20</em>6. A coculture system allowed investigation of the roles of M2-polarized TAMs and TLR4/<em>IL</em>-10 signaling in the EMT of Panc-1 and BxPC-3 pancreatic cancer cell lines. Our results showed that coculture with M2-polarized TAMs increased fibroblastic morphology, upregulated mesenchymal markers vimentin and snail at the mRNA and protein levels, and increased proliferation, migration, and metalloproteinase (MMP)2 and MMP9 proteolytic activity in pancreatic cancer cells. Simultaneously, coculture with M2-polarized TAMs decreased the expression of the epithelial marker E-cadherin. Coculture with pancreatic cancer cells increased TLR4 mRNA and protein expression in M2-polarized TAMs. Application of TLR4 siRNA and neutralizing antibodies against TLR4 and <em>IL</em>-10 markedly inhibited E-cadherin reduction and the upregulation of snail and vimentin. Furthermore, activation of TLR4 signaling by lipopolysaccharide profoundly increased the EMT of pancreatic cancer cells. In conclusion, M2-polarized TAMs promoted EMT in pancreatic cancer cells partially through TLR4/<em>IL</em>-10 signaling, suggesting novel therapeutic strategies and enhancing our understanding of M2-polarized TAMs.

High expression of CXCR5 is one of the defining hallmarks of T follicular helper cells (T(FH)), a CD4 Th cell subset that promotes germinal center reactions and the selection and affinity maturation of B cells. CXCR5 is also expressed on <em>20</em>-25% of peripheral blood human central memory CD4 T cells (T(CM)), although the definitive function of these cells is not fully understood. The constitutive expression of CXCR5 on T(FH) cells and a fraction of circulating T(CM) suggests that CXCR5(+) T(CM) may represent a specialized subset of memory-type T(FH) cells programmed for homing to follicles and providing B cell help. To verify this assumption, we analyzed this cell population and show its specialized function in supporting humoral immune responses. Compared with their CXCR5(-) T(CM) counterparts, CXCR5(+) T(CM) expressed high levels of the chemokine CXCL13 and efficiently induced plasma cell differentiation and Ig secretion. We found that the distinct B cell helper qualities of CXCR5(+) T(CM) were mainly due to high ICOS expression and pronounced responsiveness to ICOS ligand costimulation together with large <em>IL</em>-10 secretion. Furthermore, B cell helper attributes of CXCR5(+) T(CM) were almost exclusively acquired on cognate interaction with B cells, but not with dendritic cells. This implies that a preferential recruitment of circulating CXCR5(+) T(CM) to CXCL13-rich B cell follicles is required for the promotion of a quick and efficient protective secondary humoral immune response. Taken together, we propose that CXCR5(+) T(CM) represent a distinct memory cell subset specialized in supporting Ab-mediated immune responses.

Low doses of the CD3 mAb 145 2C11 restored self tolerance to beta cell Ags in adult overtly diabetic NOD mice. Within 2 to 4 wk after treatment, complete and permanent remission of diabetes was observed. Autoreactive T cells were not deleted in CD3 Ab-protected animals as evidenced first, by the persistence of peripheral insulitis and, second, by the capacity of spleen cells from CD3 Ab-treated mice to transfer diabetes to adult irradiated syngeneic recipients. Moreover, the conferred tolerance was reproducibly reversed by a single injection of cyclophosphamide. For 5 to 7 wk after treatment, IFN-gamma production by stimulated spleen cells was significantly decreased in treated animals. One unique feature was that the CD3 Ab-induced tolerance ensued only from treatment of overtly diabetic NOD mice. Durable protection was exclusively observed when treating mice with recent onset disease (14-<em>20</em> wk old). At variance with this finding, treatment of 4- and 8-wk-old mice was without effect, and complete but transient protection followed the treatment of 12-wk-old NOD mice. The tolerogenic properties of 145 2C11 did not depend on its mitogenic capacity, since nonmitogenic F(ab')2 fragments also appeared potent at inducing durable remission in overtly diabetic NOD, although nonmitogenic CD3 F(ab')2 fragments could mediate T cell signaling, as evidenced by cytokine gene transcription (<em>IL</em>-2, IFN-gamma, <em>IL</em>-4, and <em>IL</em>-10) assessed by PCR on splenocytes from treated mice. A concomitant cyclosporine treatment abrogated the CD3 mAb-induced protection, further pointing to the crucial role of T cell signaling in the effect observed.

In the present study, we have investigated the ability of human T cells to secrete <em>IL</em>-2, <em>IL</em>-4, and IFN-gamma. <em>IL</em>-4 and IFN-gamma were quantified with enzymatic immunoassays and <em>IL</em>-2 with a biologic assay by using the murine <em>IL</em>-2-dependent cell line CTLL-2. PBL, stimulated with Con A or with a combination of the phorbol ester 13-O-tetradecanoylphorbol-12-acetate and the Ca2+ ionophore A23187 secreted <em>IL</em>-2, <em>IL</em>-4, and IFN-gamma. The kinetics of the secretion of the three lymphokines was investigated with two CD4+ clones; one (GEO-2) that produced <em>IL</em>-2, <em>IL</em>-4, and IFN-gamma and another (HY640), that produced only <em>IL</em>-2 and IFN-gamma. Significant <em>IL</em>-2, <em>IL</em>-4, and IFN-gamma production was observed after only 8 h of activation. Maximal levels of <em>IL</em>-2 and <em>IL</em>-4 were found <em>20</em> h after the onset of the stimulation which subsequently decreased. In contrast, IFN-gamma levels continued to increase in a period up to 40 h and then leveled off. In spite of these differences in secretion, the kinetics of accumulation of mRNA did not differ. The <em>IL</em>-2, <em>IL</em>-4, and IFN-gamma mRNA were detectable 2 h after stimulation and continued to accumulate for a period up to <em>20</em> h. In a series of 22 CD4+ clones, 21 were able to secrete all three lymphokines upon stimulation. Almost all CD8+ clones were able to produce <em>IL</em>-2 and IFN-gamma, but only six of the 23 CD8+ T cell clones secreted <em>IL</em>-4. In addition, five CD4+ (allo)antigen-specific T cell clones were tested for <em>IL</em>-2, <em>IL</em>-4, and IFN-gamma secretion upon specific stimulation. Two alloantigen-specific and two tetanus toxoid-specific T cell clones secreted <em>IL</em>-2, <em>IL</em>-4, and IFN-gamma simultaneously, whereas one alloantigen-specific T cell clone secreted <em>IL</em>-2 and IFN-gamma, but not <em>IL</em>-4. A supernatant of the CD4+ T cell clone GEO-2, that contained high levels of IFN-gamma and <em>IL</em>-4, was unable to induce the low affinity receptor for IgE, CD23, on a Burkitt lymphoma cell line. However, after separation of <em>IL</em>-4 from IFN-gamma by using HPLC, the <em>IL</em>-4-containing fraction-induced CD23, which could be blocked by the fraction that contained IFN-gamma and by a polyclonal rabbit anti-<em>IL</em>-4 antiserum. Finally, the partly purified <em>IL</em>-4, that was devoid of <em>IL</em>-2, promoted the growth of the clone GEO-2.

In this study we describe oxazolone colitis, a new form of experimental colitis. This model is induced in SJL/J mice by the rectal instillation of the haptenating agent, oxazolone, and is characterized by a rapidly developing colitis confined to the distal half of the colon; it consists of a mixed neutrophil/lymphocyte infiltration limited to the superficial layer of the mucosa which is associated with ulceration. Oxazolone colitis is a T helper cell type 2 (Th2)-mediated process since stimulated T cells from lesional tissue produce markedly increased amounts of interleukin (<em>IL</em>)-4 and <em>IL</em>-5; in addition, anti-<em>IL</em>-4 administration leads to a striking amelioration of disease, whereas anti-<em>IL</em>-12 administration either has no effect or exacerbates disease. Finally, this proinflammatory Th2 cytokine response is counterbalanced by a massive transforming growth factor-beta (TGF-beta) response which limits both the extent and duration of disease: lesional (distal) T cells manifest a <em>20</em>-30-fold increase in TGF-beta production, whereas nonlesional (proximal) T cells manifest an even greater 40-50-fold increase. In addition, anti-TGF-beta administration leads to more severe inflammation which now involves the entire colon. The histologic features and distribution of oxazolone colitis have characteristics that resemble ulcerative colitis (UC) and thus sharply distinguish this model from most other models, which usually resemble Crohn's disease. This feature of oxazolone colitis as well as its cytokine profile have important implications to the pathogenesis and treatment of UC.

Necrotizing enterocolitis (NEC), a major cause of morbidity and mortality in premature infants, occurs after the introduction of oral feedings in conjunction with initial bacterial colonization of the gut and is hypothesized to be due to an immature (inappropriate) enterocyte response to bacterial stimuli. To test this hypothesis, we compared the enterocyte <em>IL</em>-8 response to inflammatory stimuli [lipopolysaccharide (LPS) and <em>IL</em>-1beta] in immature vs. mature human small intestine. Initial in vitro studies comparing confluent Caco-2 cells, a model for mature human enterocytes, with a primary human fetal intestinal cell line (H4 cells) demonstrated that after inflammatory stimulation fetal cells secreted more <em>IL</em>-8 (LPS, 8-fold; <em>IL</em>-1beta, <em>20</em>-fold) than Caco-2 cells. <em>IL</em>-8 mRNA activity in fetal compared to Caco-2 cells was proportionately increased by the same magnitude with both stimuli. To validate the in vitro observations, small intestinal organ cultures from fetuses vs. older children were exposed to LPS and <em>IL</em>-1beta. Again in human organ cultures from fetuses compared to older children, <em>IL</em>-8 secretion was greater (LPS, 2.5-fold; <em>IL</em>-1beta, <em>20</em>0-fold) and mRNA activity after stimulation was comparably higher, suggesting that increased transcription of the <em>IL</em>-8 gene may account for the excessive response. Using immunohistochemical staining to identify the cellular source of <em>IL</em>-8, activity was noted predominantly in villous and crypt epithelium but also in a few immunoresponsive lymphoid cells. The observation that immature human enterocytes react with excessive pro-inflammatory cytokine production after inflammatory stimulation may help in part explain why prematures exposed to initial colonizing bacteria develop necrotizing enterocolitis.

OBJECTIVE

To investigate the safety and efficacy of MRA, a recombinant human anti-interleukin-6 (anti-IL-6) receptor monoclonal antibody of the IgG1 subclass that inhibits the function of IL-6, in patients with established rheumatoid arthritis (RA).

METHODS

A randomized, double-blind, placebo-controlled, dose-escalation trial was conducted in 45 patients with active RA, as defined by the American College of Rheumatology (ACR) revised criteria. Patients were sequentially allocated to receive a single intravenous dose of either 0.1, 1, 5, or 10 mg/kg of MRA or placebo. The primary efficacy end point was meeting the ACR 20% response criteria at week 2 after treatment.

RESULTS

Demographic features were similar between treatment groups. At week 2, a significant treatment difference was observed between the 5 mg/kg of MRA and placebo, with 5 patients (55.6%) in the MRA cohort and none in the placebo cohort achieving ACR 20% improvement. There was no statistically significant difference in the ACR 20% response between the other 3 MRA cohorts and placebo at week 2. The mean disease activity score at week 2 in those who received 5 mg/kg and 10 mg/kg of MRA was 4.8 and 4.7 (P < 0.001 and P < 0.001 by analysis of variance), respectively. These mean scores were statistically significantly lower than those in the 0.1- and 1-mg/kg MRA and the placebo cohorts (6.4, 6.2, and 7.0, respectively). The erythrocyte sedimentation rate and C-reactive protein values fell significantly in the 5- and 10-mg/kg MRA cohorts and normalized 2 weeks after treatment. Seventeen patients (5, 4, 6, 2, and 0 patients in the placebo, 0.1-, 1-, 5-, and 10-mg/kg MRA cohorts, respectively) required corticosteroid or disease-modifying antirheumatic drug treatment because of active disease before study end. They were regarded as nonresponders from the time they received these treatments. Diarrhea was the most common adverse event, occurring in 8% of patients. Seven patients (15.6%) reported a severe adverse event (3, 1, 2, and 2 patients in the placebo, 0.1-, 1-, and 10-mg/kg MRA cohorts). There were no serious adverse events that were thought to be related to the study drug.

CONCLUSIONS

This is the first randomized controlled trial showing that inhibition of IL-6 significantly improved the signs and symptoms of RA and normalized the acute-phase reactants. Further research with multiple dosing is necessary to define the most appropriate therapeutic regimen of MRA in RA.

c-kit is expressed on hematopoietic stem cells and progenitor cells, but not on lymphohematopoietic differentiated cells. Lineage marker-negative, c-kit-positive (Lin-c-kit+) bone marrow cells were fractionated by means of Ly6A/E or Sca-1 expression. Lin-c-kit+Sca-1+ cells, which consisted of 0.08% of bone marrow nucleated cells, did not contain day-8 colony-forming units-spleen (CFU-S), but 80% were day-12 CFU-S. One hundred cells rescued the lethally irradiated mice and reconstituted hematopoiesis. On the other hand, 2 x 10(3) of Lin-c-kit+Sca-1- cells formed <em>20</em> day-8 and 11 day-12 spleen colonies, but they could not rescue the lethally irradiated mice. These data indicate that Lin-c-kit+Sca-1+ cells are primitive hematopoietic stem cells and that Sca-1-cells do not contain stem cells that reconstitute hematopoiesis. Lin-c-kit+Sca-1+ cells formed no colonies in the presence of stem cell factor (SCF) or interleukin-6 (<em>IL</em>-6), and only 10% of them formed colonies in the presence of <em>IL</em>-3. However, approximately 50% of them formed large colonies in the presence of <em>IL</em>-3, <em>IL</em>-6, and SCF. Moreover, when single cells were deposited into culture medium by fluorescence-activated cell sorter clone sorting system, 40% of them proliferated on a stromal cell line (PA-6) and proliferated for more than 2 weeks. In contrast, 15% of the Lin-c-kit+Sca-1-cells formed colonies in the presence of <em>IL</em>-3, but no synergistic effects were observed in combination with SCF plus <em>IL</em>-6 and/or <em>IL</em>-3. Approximately 10% proliferated on PA-6, but most of them degenerated within 2 weeks. The population ratio of c-kit+Sca-1+ to c-kit+Sca-1- increased 2 and 4 days after exposure to 5-fluorouracil (5-FU). These results are consistent with the relative enrichment of highly proliferative colony-forming cells by 5-FU. These data show that, although c-kit is found both on the primitive hematopoietic stem cells and progenitors, Sca-1+ cells are more primitive and respond better than Sca-1- cells to a combination of hematopoietic factors, including SCF and stromal cells.

Depression and anxiety disorders are associated with increased release of peripheral cytokines; however, their functional relevance remains unknown. Using a social stress model in mice, we find preexisting individual differences in the sensitivity of the peripheral immune system that predict and promote vulnerability to social stress. Cytokine profiles were obtained <em>20</em> min after the first social stress exposure. Of the cytokines regulated by stress, <em>IL</em>-6 was most highly up-regulated only in mice that ultimately developed a susceptible behavioral phenotype following a subsequent chronic stress, and levels remained elevated for at least 1 mo. We confirmed a similar elevation of serum <em>IL</em>-6 in two separate cohorts of patients with treatment-resistant major depressive disorder. Before any physical contact in mice, we observed individual differences in <em>IL</em>-6 levels from ex vivo stimulated leukocytes that predict susceptibility versus resilience to a subsequent stressor. To shift the sensitivity of the peripheral immune system to a pro- or antidepressant state, bone marrow (BM) chimeras were generated by transplanting hematopoietic progenitor cells from stress-susceptible mice releasing high <em>IL</em>-6 or from <em>IL</em>-6 knockout (<em>IL</em>-6(-/-)) mice. Stress-susceptible BM chimeras exhibited increased social avoidance behavior after exposure to either subthreshold repeated social defeat stress (RSDS) or a purely emotional stressor termed witness defeat. <em>IL</em>-6(-/-) BM chimeric and <em>IL</em>-6(-/-) mice, as well as those treated with a systemic <em>IL</em>-6 monoclonal antibody, were resilient to social stress. These data establish that preexisting differences in stress-responsive <em>IL</em>-6 release from BM-derived leukocytes functionally contribute to social stress-induced behavioral abnormalities.

OBJECTIVE

To evaluate whether chronic therapy with probiotics affects plasma levels of cytokines and oxidative/nitrosative stress parameters, as well as liver damage, in patients with various types of chronic liver disease.

METHODS

A total of 22 nonalcoholic fatty liver disease (NAFLD) and <em>20</em> alcoholic liver cirrhosis (AC) patients were enrolled in the study and compared with 36 HCV-positive patients with chronic hepatitis without (<em>20</em>, CH) or with (16, CC) liver cirrhosis. All patients were treated with the probiotic VSL#3. Routine liver tests, plasma levels of tumor necrosis factor alpha (TNF-alpha), interleukin (<em>IL</em>)-6 and -10, malondialdehyde (MDA), and 4-hydroxynonenal (4-HNE), S-nitrosothiols (S-NO), were evaluated on days -30, 0, 90, and 1<em>20</em>.

RESULTS

Treatment with VSL#3 exerted different effects in the various groups of patients: in NAFLD and AC groups, it significantly improved plasma levels of MDA and 4-HNE, whereas cytokines (TNF-alpha, IL-6, and IL-10) improved only in AC patients. No such effects were observed in HCV patients. Routine liver damage tests and plasma S-NO levels were improved at the end of treatment in all groups.

CONCLUSIONS

Results of the study suggest that manipulation of intestinal flora should be taken into consideration as possible adjunctive therapy in some types of chronic liver disease.

BACKGROUND

Age-related macular degeneration (AMD) and cardiovascular disease share common risk factors. Inflammatory biomarkers, including C-reactive protein (CRP), interleukin 6 (IL-6), soluble tumor necrosis factor alpha receptor 2, soluble intercellular and vascular adhesion molecules (intercellular adhesion molecule 1 and vascular cell adhesion molecule 1), and lipid biomarkers, including lipoprotein(a) and apolipoprotein B, have all been associated with cardiovascular disease. We previously found an association between AMD and CRP in a cross-sectional analysis, but the prospective relationships between AMD, CRP, and the other cardiovascular disease markers are unknown.

OBJECTIVE

To test the hypothesis that baseline cardiovascular disease biomarkers are associated with subsequent increased risk for progression of AMD.

METHODS

This prospective cohort study involved 251 participants aged 60 years and older who had some sign of nonexudative AMD and visual acuity of 20/200 or better in at least one eye at baseline. The AMD status was assessed by standardized grading of fundus photographs, and stored fasting blood specimens obtained at baseline were analyzed for levels of the various biomarkers. The average follow-up time was 4.6 years.

METHODS

Relationship between biomarkers and incidence rates of progression of AMD.

RESULTS

Comparing the highest quartile with the lowest quartile, CRP was associated with progression of AMD, with a multivariate adjusted relative risk (RR) of 2.10 (95% confidence interval [CI], 1.06-4.18; P for trend, .046) controlling for body mass index, smoking, and other cardiovascular variables and a multivariate adjusted RR of 2.02 (95% CI, 1.00-4.04; P for trend, .06) controlling additionally for antioxidant nutrients. Interleukin 6 was also related to progression of AMD, with a multivariate adjusted RR of 1.81 (95% CI, 0.97-3.36; P for trend, .03). Comparing the highest quartile with the lowest quartile, the effect estimates for vascular cell adhesion molecule 1 (multivariate adjusted RR, 1.94) and apolipoprotein B (adjusted RR, 1.39) were in the positive direction but were not statistically significant (P for trend, .08 and .24, respectively). The CRP and IL-6 levels were both significantly related to higher body mass index and current smoking.

CONCLUSIONS

Higher levels of the systemic inflammatory markers CRP and IL-6 are independently associated with progression of AMD.

HIV-1 invades the central nervous system early during viral infection, but neurologic impairment usually occurs years later. The strongest predictor for clinical dementia is the absolute numbers of immunocompetent brain macrophages. Thus, how monocytes penetrate the brain during disease remains critical for understanding the neuropathogenic mechanisms of HIV-1 encephalitis. To these ends, we constructed an artificial blood-brain barrier (BBB) consisting of a matrix-coated membrane with brain microvascular endothelial cells (BMVEC) on one side and astrocytes on the other. Astrocyte endfeet contacted the monolayer of BMVEC that formed tight junctions. To determine the role of viral and immune factors in monocyte penetration across the BBB, HIV-infected or uninfected monocytes with or without immune stimulation were placed onto the upper chamber of the BBB model system. Placement of immune-stimulated (LPS-treated) cells onto the BBB construct elicited gaps between BMVEC, with bulging of nuclear zones and increased numbers of vesicular Golgi complexes and endoplasmic reticulum. This correlated with a profound increase (up to <em>20</em>-fold) in the number of migrating cells. Viral infection did not enhance monocyte migration. The activated monocytes showed increased numbers of philopodia, lysosomes, and vesicular Golgi complexes and expressed large levels of proinflammatory cytokines (TNF-alpha, <em>IL</em>-6, and <em>IL</em>-10). These data suggest that a major mechanism for the transendothelial migration of monocytes during HIV encephalitis is the immune activation that accompanies viral infection of the central nervous system.

The systemic cytokine response to major surgical trauma was studied in <em>20</em> patients undergoing elective aortic surgery and five patients after inguinal hernia repair. Tumour necrosis factor alpha and interferon gamma were not detected in these patients. An early and short-lived interleukin 1 beta (<em>IL</em>-1 beta) response to major surgery was detected only by intensive sampling in the perioperative period. The <em>IL</em>-1 beta peak preceded a more marked interleukin 6 (<em>IL</em>-6) response that peaked 4-48 h after surgery. <em>IL</em>-6 levels had fallen sharply by 48-72 h in all patients who had an uneventful postoperative course. The <em>IL</em>-6 peaks were significantly lower after hernia surgery than after major aortic operations (P < 0.001); <em>IL</em>-1 beta was not detected in any samples. Three patients undergoing aortic surgery developed unexpected major postoperative complications. <em>IL</em>-6 levels in this group were significantly higher than those of the other patients undergoing aortic surgery within 6-8 h of skin incision, and remained elevated for longer. These rises in plasma <em>IL</em>-6 levels preceded the clinical onset of major complications by 12-48 h. The systemic <em>IL</em>-1 beta and <em>IL</em>-6 response to surgical trauma increased with the severity of the surgical insult. An early, exaggerated <em>IL</em>-6 response was associated with the subsequent clinical development of major complications.

To isolate a stable tumor cell line capable of producing human interleukin 2 (<em>IL</em>-2; formerly referred to as T cell growth factor), 16 human T and B leukemia cell lines were screened for constitutive and mitogen-stimulated <em>IL</em>-2 production. We found that the T cell leukemia line designated Jurkat-FHCRC produced>> <em>20</em>0 U/ml of <em>IL</em>-2 activity after a 24-h stimulation with T cell mitogens. Peak mitogen-induced <em>IL</em>-2 activity was found in supernates harvested from 24-h Jurkat-FHCRC cell cultures stimulated with either 1% phytohemagglutinin or <em>20</em> microgram/ml concanavalin A. Addition of the fatty acid derivative phorbol myristate acetate to mitogen-stimulated cultures increased Jurkat-FHCRC <em>IL</em>-2 production to concentrations>> 400 U/ml. <em>IL</em>-2 activity observed in such cases represented between 100--300 times that produced in conventional cultures of mitogen- or alloantigen-stimulated normal human peripheral blood or splenic lymphocytes. Jurkat-FHCRC-derived conditioned medium demonstrated equal capacity to promote the sustained in vitro proliferation of either murine or human activated T cell lines confirming the ability of Jurkat-FHCRC cells to produce human <em>IL</em>-2. These studies identify a new source of human <em>IL</em>-2 and establish a valuable reagent for the isolation and further molecular characterization of this immunoregulatory molecule.

We have generated primary effector populations from naive CD8 T cells in response to antigen and determined their patterns of cytokine secretion upon restimulation. The effect of exogenous factors on the effector generation was examined and compared with responses of antigen-specific CD4 effectors generated under comparable conditions. CD8 cells from bm1 mice were stimulated with C57BL/6 (B6) antigen presenting cells (APCs) bearing allogeneic class I and CD8 cells from female severe combined immunodeficiency (SCID) B6 mice, transgenic for a T cell receptor alpha/beta (TCR-alpha/beta) that recognizes H-Y on Db, were stimulated with APCs from male mice. In parallel, CD4 cells from bm12 mice were stimulated with alloantigen and CD4 cells from V beta 3/V alpha 11 TCR transgenics were stimulated with a peptide of pigeon cytochrome c on IEk. T cells from both transgenic mice were of naive phenotype whereas normal mice contained 10-<em>20</em>% memory cells. Effector CD8 populations generated were L-selectin low, CD45RB high, and CD44 high. Naive CD8 cells from SCID anti-H-Y mice made little or no cytokine immediately upon stimulation in contrast to naive CD4 which produced large amounts of interleukin 2 (<em>IL</em>-2). Both populations, however, generated primary effectors over 4-5 d that made substantial quantities of many cytokines upon restimulation. Both CD8 and CD4 effectors produced similar patterns of cytokines with alloantigen or specific antigen. Cytokines present during naive CD8 stimulation influenced the cytokine secretion profile of the effectors, as previously shown for CD4 cells, although secretion by CD8 effectors was generally lower than that of CD4 effectors. CD8 cells cultured with <em>IL</em>-2 alone made predominantly interferon gamma (IFN-gamma) and no <em>IL</em>-4 or <em>IL</em>-5, similar to CD4 cells. Priming with IFN-gamma increased IFN-gamma secretion from CD4 effectors, but had little if any effect on CD8 cells. In contrast, priming with <em>IL</em>-12 generated CD8 effectors, as well as CD4 effectors, producing elevated quantities of IFN-gamma, with similar levels from both the CD4 and CD8 populations. The presence of <em>IL</em>-4 during effector cell generation promoted synthesis of <em>IL</em>-4 and <em>IL</em>-5 from both CD8 and CD4 cells while downregulating IFN-gamma secretion. CD8 cells made only small amounts of <em>IL</em>-4, more than 100-fold less than CD4 cells, whereas significant levels of <em>IL</em>-5 were induced, only 3-10-fold lower than from CD4.(ABSTRACT TRUNCATED AT 400 WORDS)

BACKGROUND

There is little knowledge about clinical variables associated with vitamin D (VitD) insufficiency in asthmatic children.

OBJECTIVE

We sought to investigate disease variables associated with VitD insufficiency in patients with childhood asthma and interaction of VitD with corticosteroid-mediated anti-inflammatory responses.

METHODS

We analyzed 25-hydroxyvitamin D serum levels in 100 asthmatic children to investigate relationships between 25-hydroxyvitamin D levels and patients' characteristics. We determined VitD's effects on dexamethasone (DEX) induction of mitogen-activated protein kinase phosphatase 1 and IL-10 in PBMCs.

RESULTS

The median 25-hydroxyvitamin D serum level was 31 ng/mL. Forty-seven percent of subjects had VitD levels in the insufficient range (<30 ng/mL), whereas 17% were VitD deficient (<20 ng/mL). Log(10) IgE (P = .01, rho = -0.25) and the number of positive aeroallergen skin prick test responses (P = .02, rho = -0.23) showed a significant inverse correlation with VitD levels, whereas FEV(1) percent predicted (P = .004, rho = 0.34) and FEV(1)/forced vital capacity ratio (P = .01, rho = 0.30) showed a significant positive correlation with VitD levels. The use of inhaled steroids (P = .0475), use of oral steroids (P = .02), and total steroid dose (P = .001) all showed significant inverse correlations with VitD levels. The amount of mitogen-activated protein kinase phosphatase 1 and <em>IL</em>10 mRNA induced by VitD plus DEX was significantly greater than that induced by DEX alone (P < .01). In an experimental model of steroid resistance in which DEX alone did not inhibit T-cell proliferation, addition of VitD to DEX resulted in significant dose-dependent suppression of cell proliferation.

CONCLUSIONS

Corticosteroid use and worsening airflow limitation are associated with lower VitD serum levels in asthmatic patients. VitD enhances glucocorticoid action in PBMCs from asthmatic patients and enhances the immunosuppressive function of DEX in vitro.

The results of this study, in which CD4 T cells were harvested from mice at various times during the course of a virulent Mycobacterium tuberculosis infection and examined for their secretion of cytokines during culture in vitro with bone marrow-derived macrophages presenting mycobacterial Ag, provide evidence for the existence of two separate waves of cytokine-producing CD4 cells. The first, which peaks at the time at which protective immunity was maximally expressed, was characterized as a IFN-gamma-secreting cell population that preferentially recognized macrophages presenting mycobacterial culture filtrate proteins, or that were infected with the live organism. A second population, which emerged <em>20</em> to 40 days later at a time when the infection had been contained, secreted <em>IL</em>-4 in response to the filtrate proteins, but also reacted particularly strongly to the 65-kDa (hsp60) heat shock protein molecule of M. tuberculosis. These data indicate that acquired immunity to tuberculosis infection involves the production of both Th1- and Th2-like cell populations that differ in terms of their kinetics of emergence and loss, and in terms of their Ag specificity.

Purified recombinant human interleukin 2 (R<em>IL</em> 2) derived from E. coli containing the inserted gene encoding for <em>IL</em> 2 was administered to <em>20</em> patients with a variety of malignancies. Toxicity was dose related and included fever, chills, malaise, arthralgias, myalgias, and unexpectedly, weight gain related to marked fluid retention. All patients receiving more than 10(5) U/kg total cumulative dose developed evidence of fluid retention, and all patients requiring discontinuance of R<em>IL</em> 2 (11/<em>20</em>) received total doses of between 2.54 X 10(5) U/kg to 15.4 X 10(5) U/kg. The limiting dose with this preparation was 3000 U/kg/hr by continuous administration or 10(6) U/kg by bolus administration. <em>IL</em> 2 was rapidly cleared from the plasma, with a half life of 6.9 min, and a later delayed clearance was consistent with a two-compartment model, with slower release from the extravascular space back into the plasma compartment. A marked change in lymphoid cells in the periphery was noted with an early depletion of all lymphoid cells, followed by an expansion of such cells with continuous <em>IL</em> 2 administration. A twofold to 16-fold expansion of total lymphoid cells in the peripheral blood could be demonstrated. TAC+ cells representing up to 25% of the circulating peripheral blood mononuclear cells could be demonstrated with 3 wk of continuous R<em>IL</em> 2 administration. Interferon-gamma levels increased in patients treated with <em>IL</em> 2. Precursors of lymphokine-activated killer cells generated under standard conditions were depleted within 2 to 3 min after <em>IL</em> 2 administration, but repopulated the peripheral blood after 7 to 10 days of continuous <em>IL</em> 2 administration. No tumor regression was seen in any of the cancer patients treated with <em>IL</em> 2 alone.

The cell surface density of high-affinity membrane receptors for the T-lymphocytotrophic hormone interleukin 2 (<em>IL</em>-2) determines the rate of T-cell-cycle progression. Since 10-fold greater numbers of <em>IL</em>-2 receptor molecules were found by using a radiolabeled monoclonal antibody reactive with <em>IL</em>-2 receptors (anti-Tac) compared with binding of <em>IL</em>-2, the functional relationship of the binding sites recognized by both of these ligands was assessed. In the presence of cycloheximide, <em>IL</em>-2 binding sites declined with a half-time (t1/2) of 2.6 hr, whereas the decay of anti-Tac binding sites was much slower (t 1/2 = 6.4 hr). Moreover, after limited membrane proteolysis, the half-time for the reappearance of <em>IL</em>-2 binding sites was remarkably similar to its decay (t 1/2 = 2.2 hr), while Tac antigen reappearance was markedly retarded, returning to only <em>20</em>% of original levels within 5 hr after proteolysis. Addition of homogeneous immunoaffinity-purified <em>IL</em>-2 to cell populations that expressed equivalent <em>IL</em>-2 and anti-Tac binding sites resulted in a time- and temperature-dependent 8- to 10-fold enhancement of Tac epitope expression and, simultaneously, a <em>20</em>-30% diminishment of detectable high-affinity <em>IL</em>-2 binding sites. As the magnitude of the <em>IL</em>-2-dependent proliferative response correlated with the density of high-affinity <em>IL</em>-2 binding sites, rather than Tac antigen levels, quantitation of Tac epitope density does not provide a reliable indication of <em>IL</em>-2-responsiveness among activated T-cell populations. Instead, <em>IL</em>-2-receptor interactions actually promote the loss of <em>IL</em>-2 responsiveness by diminishing the density of high-affinity binding sites at the time that Tac antigen levels are increased.