BACKGROUND

Vitreal interleukin (IL)-1beta (IL-1beta), IL-6, IL-8, tumor necrosis factor-alpha (TNF-alpha) and nitric oxide (NO) levels have previously been determined in patients with proliferative diabetic retinopathy (PDR). However, at present there is no cohort study linking serum levels of NO and many inflammatory cytokines such as TNF-alpha, IL-1beta, soluble IL-2 receptor (sIL-2R), IL-6 and IL-8 to the grade of the microvascular complications.

OBJECTIVE

To determine the relation between the stages of DR and the levels of serum NO, TNF-alpha, IL-1beta, sIL-2R, IL-6 and chemokine IL-8 in patients with diabetes compared with healthy controls.

METHODS

Fifty-three consecutive patients with diabetes (25 men, 28 women) with or without DR and 15 non-diabetic healthy subjects (seven men, eight women) as controls were included in this prospective study. As an indicator for NO, serum total nitrite (NO2- + NO3-) levels (end-product of NO) were measured by the Griess reaction. Serum TNF-alpha, IL-1beta, sIL-2R, IL-6 and IL-8 levels were determined by a spectrophotometric technique using an Immulite chemiluminescent immunometric assay. The patients with diabetes were classified into three groups according to the stage of DR: no DR (NDR; n = 16), non-proliferative DR (NPDR; n = 18) and PDR (n = 19). The data were analysed using a Mann-Whitney U-test and the results were expressed as mean +/- SE (range).

RESULTS

The levels of IL-1beta and IL-6 were below the detection limits of the assay (for each, <5.0 pg/ml) in all patients with diabetes and controls. Soluble IL-2R levels ranged from 260 to 958 U/ml, with the highest values observed in the patients with PDR. In 47 of the 53 samples (89%) tested for diabetic patients, IL-8 levels were above the detection limits of the assay (5.0 pg/ml). IL-8 levels ranged from <5.0 to 25.0 pg/ml, with the highest mean values observed in PDR patients. TNF-alpha was detectable in 46 of 53 patients with diabetes (87%), ranging from <4.0 to 26.4 pg/ml, with again the highest values obtained in the patients with PDR. Serum NO levels ranged from 80 to 188 micromol/l, with the highest values obtained in patients with PDR. Taken together, the mean serum NO, sIL-2R, IL-8 and TNF-alpha levels increased with the stage of DR and the highest levels were found in patients with PDR. The PDR patients had significantly (for each, P < 0.001) higher serum NO (166.8 +/- 3.2 micromol/l), sIL-2R (807.9 +/- 33.3 U/ml), IL-8 (17.9 +/- 0.4 pg/ml) and TNF-alpha (15.0 +/- 0.8 pg/ml) levels compared with NPDR patients (149.5 +/- 2.1, 659.4 +/- 23.4, 12.9 +/- 1.1, 11.5 +/- 0.6, respectively), NDR patients (115.9 +/- 5.8, 373.8 +/- 15.0, 8.3 +/- 1.0, 6.6 +/- 0.9, respectively) and controls (116.6 +/- 2.3, 392.4 +/- 16.6, 7.2 +/- 0.3, 7.3 +/- 0.5, respectively). Serum levels of these parameters for NPDR patients were also significantly (for each, P < 0.01) higher compared with those of NDR patients and controls. On the other hand, serum NO, sIL-2R, IL-8 and TNF-alpha levels of patients with NDR were comparable with those of controls (for each, P>> 0.05).

CONCLUSIONS

The results of the present study suggest that NO, sIL-2R, IL-8 and TNF-alpha may play important roles in the pathophysiology and progression of DR. We think that these potentially inflammatory cytokines and NO with their endothelial implications may act together during the course and progression of DR. These molecules may serve as therapeutic targets for the treatment and/or prevention of diabetes with its systemic and ocular microvascular complications.

Although <em>interleukin</em> 2 (IL-2) and IL-<em>15</em> signal through the common gamma chain (gammac) and through IL-2 receptor beta-chain (CD122) subunits, they direct distinct physiologic and immunotherapeutic responses in T cells. The present study provides some insight into why IL-2 and IL-<em>15</em> differentially regulate T-cell function by revealing that these cytokines are strikingly distinct in their ability to control protein synthesis and T-cell mass. IL-2 and IL-<em>15</em> are shown to be equivalent mitogens for antigen-stimulated CD8(+) T cells but not for equivalent growth factors. Antigen-primed T cells cannot autonomously maintain amino acid incorporation or de novo protein synthesis without exogenous cytokine stimulation. Both IL-2 and IL-<em>15</em> induce amino acid uptake and protein synthesis in antigen-activated T cells; however, the IL-2 response is strikingly more potent than the IL-<em>15</em> response. The differential action of IL-2 and IL-<em>15</em> on amino acid uptake and protein synthesis is explained by temporal differences in signaling induced by these 2 cytokines. Hence, the present results show that cytokines that are equivalent mitogens can have different potency in terms of regulating protein synthesis and cell growth.

Human immunodeficiency virus (HIV) and hepatitis C virus (HCV) cause clinically important persistent infections. The effects of virus persistence on innate immunity, including NK cell responses, and the underlying mechanisms are not fully understood. We examined the frequency, phenotype, and function of peripheral blood CD3- CD56+ NK subsets in HIV+ and HCV+ patients and identified significantly reduced numbers of total NK cells and a striking shift in NK subsets, with a marked decrease in the CD56(dim) cell fraction compared to CD56(bright) cells, in both infections. This shift influenced the phenotype and functional capacity (gamma interferon production, killing) of the total NK pool. In addition, abnormalities in the functional capacity of the CD56(dim) NK subset were observed in HIV+ patients. The shared NK alterations were found to be associated with a significant reduction in serum levels of the innate cytokine <em>interleukin</em> <em>15</em> (IL-<em>15</em>). In vitro stimulation with IL-<em>15</em> rescued NK cells of HIV+ and HCV+ patients from apoptosis and enhanced proliferation and functional activity. We hypothesize that the reduced levels of IL-<em>15</em> present in the serum during HIV and HCV infections might impact NK cell homeostasis, contributing to the common alterations of the NK pool observed in these unrelated infections.

BACKGROUND

Interleukin-2 is an important regulatory cytokine of the immune system, with potent effects on T cells, B cells, and natural killer cells. In vitro, interleukin-2 can induce the proliferation and differentiation of peripheral-blood mononuclear cells from patients infected with the human immunodeficiency virus (HIV).

METHODS

We treated 25 HIV-infected patients with interleukin-2 administered as a continuous infusion at a dosage of 6 to 18 million IU per day for 5 days every 8 weeks during a period of 7 to 25 months. All patients also received at least one approved antiviral agent. Immunologic and virologic variables were monitored monthly.

RESULTS

In 6 of 10 patients with base-line CD4 counts higher than 200 per cubic millimeter, interleukin-2 therapy was associated with at least a 50 percent increase in the number of CD4 cells. Changes ranged from -81 to +2211 cells per cubic millimeter. Interleukin-2 therapy resulted in a decline in the percentage of CD8 lymphocytes expressing HLA-DR and an increase in the percentage of CD4 lymphocytes that were positive for the p55 chain of the interleukin-2 receptor. Four patients had a transient but consistent increase in the plasma HIV RNA level at the end of each infusion. In the remaining 15 patients, who had CD4 counts of 200 or fewer cells per cubic millimeter, interleukin-2 therapy was associated with increased viral activation, few immunologic improvements, and substantial toxic effects.

CONCLUSIONS

Intermittent courses of interleukin-2 can improve some of the immunologic abnormalities associated with HIV infection in patients with more than 200 CD4 cells per cubic millimeter.

OBJECTIVE

Nonalcoholic fatty liver disease (NAFLD) and its association with insulin resistance are increasingly recognized as major health burdens. The main objectives of this study were to assess the relation between liver lipid content and serum lipids, markers of liver function and inflammation in healthy overweight subjects, and to determine whether caloric restriction (CR) (which improves insulin resistance) reduces liver lipids in association with these same measures.

METHODS

Forty-six white and black overweight men and women (BMI = 24.7-31.3 kg/m(2)) were randomized to "control (CO)" = 100% energy requirements; "CR" = 25%; "caloric restriction and increased structured exercise (CR+EX)"= 12.5% CR + 12.5% increase in energy expenditure through exercise; or "low-calorie diet (LCD)" = <em>15</em>% weight loss by liquid diet followed by weight-maintenance, for 6 months. Liver lipid content was assessed by magnetic resonance spectroscopy (MRS) and computed tomography (CT). Lipid concentrations, markers of liver function (alanine aminotransferase (ALT), alkaline phosphatase (ALK)), and whole-body inflammation (tumor necrosis factor-alpha (TNF-alpha), <em>interleukin</em>-6 (IL-6), high-sensitivity C-reactive protein (hsCRP)) were measured in fasting blood.

RESULTS

At baseline, increased liver lipid content (by MRS) correlated (P < 0.05) with elevated fasting triglyceride (r = 0.52), ALT (r = 0.42), and hsCRP (r = 0.33) concentrations after adjusting for sex, race, and alcohol consumption. With CR, liver lipid content was significantly lowered by CR, CR+EX, and LCD (detected by MRS only). The reduction in liver lipid content, however, was not significantly correlated with the reduction in triglycerides (r = 0.26; P = 0.11) or with the changes in ALT, high-density lipoprotein (HDL)-cholesterol, or markers of whole-body inflammation.

CONCLUSIONS

CR may be beneficial for reducing liver lipid and lowering triglycerides in overweight subjects without known NAFLD.

Incidence studies of blood inflammatory markers as predictors of dementia in older age are few and did not take into account hyperhomocysteinemia, although this condition is associated with both inflammation and increased risk of dementia. We investigated the relationships of baseline serum C-reactive protein (CRP), serum <em>interleukin</em> 6 (IL6), plasma alpha-1-antichymotrypsin, and hyperhomocysteinemia (defined as plasma total homocysteine><em>15</em> micromol/L) with risk of incident Alzheimer's disease (AD) and vascular dementia (VaD) in a dementia-free Italian population-based elderly cohort (n=804, 53.2% women, mean age 74 years) with 4 years of follow-up. No inflammatory marker, alone or in combination, predicted AD risk whereas the combination of high CRP and high IL6 was associated with risk of VaD (HR, 2.56; 95%CI, 1.21-5.50) independently of socio-demographic confounders, traditional risk factors and hyperhomocysteinemia. By contrast, in the same model, hyperhomocysteinemia was independently associated with AD (HR, 1.91; 95%CI, 1.02-3.56) but not VaD risk. Blood inflammatory markers are associated with increased VaD risk but do not predict AD, which seems selectively associated with hyperhomocysteinemia.

OBJECTIVE

The purpose of this study was to examine the influence of a 12-wk exercise training program on inflammatory cytokine and C-reactive protein (CRP) concentrations. A secondary purpose was to determine whether training-induced changes in cytokines and CRP were influenced by age.

METHODS

Twenty-nine younger (18-35 yr) and 31 older (65-85 yr) subjects were assigned to young physically active (YPA, N = <em>15</em>; 25 +/- 5 yr), young physically inactive (YPI, N= 14; 25 +/- 4.7 yr), old physically active (OPA, N = 14; 71 +/- 4 yr), or old physically inactive (OPI, N = 17; 71 +/- 4 yr) groups. The inactive groups completed 12 wk (3 d.wk) of aerobic and resistance exercises, and the physically active control groups continued their normal exercise programs. Blood samples were collected before and after the 12-wk period, and the concentrations of serum CRP, plasma <em>interleukin</em>-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha), and <em>interleukin</em>-1 beta (IL-1beta) were determined using separate ELISA.

RESULTS

Control (YPA and OPA) estimated VO2max was unchanged. Exercise training increased estimated VO2max an average of 10.4% and increased strength by an average of 38.1% in both PI groups. Serum CRP decreased with training (YPI and OPI) groups and was not different from the YPA and OPA groups after training. Plasma IL-6 and IL-1beta did not change, whereas TNF-alpha was higher than YPI and YPA at baseline and after the intervention period.

CONCLUSIONS

These results support the use of combined aerobic/resistance training as a modality to reduce the risk of cardiovascular disease development as defined by a decrease in serum CRP concentration in healthy humans.

Obesity is a risk factor for development of insulin resistance, type 2 diabetes, cardiovascular disease, osteoarthritis, and some forms of cancer. Many of the adverse health consequences of excess fat deposition are caused by increased secretion of proinflammatory adipokines by adipose tissue. Reciprocal muscle-to-fat signaling factors, or myokines, are starting to be identified. <em>Interleukin</em>-<em>15</em> (IL-<em>15</em>) is a cytokine that is highly expressed in muscle tissue and that, on the basis of cell culture experiments, has been proposed to act as a circulating myokine that inhibits adipose tissue deposition. To test this hypothesis in vivo, two lines of transgenic mice that overexpressed IL-<em>15</em> mRNA and protein in skeletal muscle tissue were constructed. By substitution of the inefficient native IL-<em>15</em> signal peptide with a more efficient signal peptide, one of the transgenic mouse lines also exhibited elevated secretion of IL-<em>15</em> in the circulation. Overexpression of IL-<em>15</em> in muscle tissue without secretion in the bloodstream resulted in no differences in body composition. Elevated circulating levels of IL-<em>15</em> resulted in significant reductions in body fat and increased bone mineral content, without appreciably affecting lean body mass or levels of other cytokines. Elevated circulating levels of IL-<em>15</em> also inhibited adiposity induced by consumption of a high-fat/high-energy diet in male, but not female, transgenic mice. Female mice with elevated serum IL-<em>15</em> exhibited increased deposition of lean body mass on a low-fat/low-energy diet and a high-fat/high-energy diet. These findings indicate that muscle-derived circulating IL-<em>15</em> can modulate adipose tissue deposition and support addition of IL-<em>15</em> to the growing list of potential myokines that are increasingly being implicated in regulation of body composition.

BACKGROUND

Aspiration of gastroesophageal refluxate may contribute to lung transplant bronchiolitis obliterans syndrome (BOS). We investigated bile acids in bronchoalveolar lavage fluid (BALF) and studied its role in BOS.

METHODS

Surveillance pulmonary function tests and BALF were evaluated in 120 lung recipients. BOS-(0p-3) was diagnosed after 6 months' survival. BOS was defined as "early" if diagnosed within 12 months after a transplant. BALF was assayed for differential cell count, bile acids, and <em>interleukins</em> 8 and <em>15</em>. Bile acids were considered elevated if greater than normal serum levels ( or =8 micromol/L).

RESULTS

Elevated BALF bile acids were measured in 20 (17%) of 120 patients. BOS was diagnosed in 36 (34%) of 107 patients and judged "early" in 21 (57%) of 36. Median BALF bile acid values were 1.6 micromol/L (range, 0-32 micromol/L) in BOS patients and 0.3 micromol/L (range, 0-16 micromol/L) in non-BOS patients ( P = .002); 2.6 micromol/L (range, 0-32 micromol/L) in early BOS patients and 0.8 micromol/L (range, 0-4.6 micromol/L) in late BOS patients, ( P = .02). Bile acids correlated with BALF IL-8 and alveolar neutrophilia (r = 0.3, P = .0004, and r = 0.3, P = .004, respectively), but not with IL-<em>15</em>. Freedom from BOS was significantly shortened in patients with elevated BALF bile acids (Cox-Mantel test, P = .0001).

CONCLUSIONS

Aspiration of duodenogastroesophageal refluxate is prevalent after lung transplantation and is associated with the development of BOS. Elevated BALF bile acids may promote early BOS development via an inflammatory process, possibly mediated by IL-8 and alveolar neutrophilia.

In the present study, it is demonstrated that cloned surface IgM-positive human B cells can be induced to proliferate and to switch with high frequencies to IgG4 and IgE production after a contact-mediated signal provided by T cell clones and <em>interleukin</em> 4 (IL-4). This T cell signal is antigen nonspecific and is provided by activated CD4+ cells, whereas activated CD8+ or resting CD4+ T cell clones are ineffective. <em>15</em>-35% of the B cell clones cultured with cloned CD4+ T cells and IL-4 produced antibodies; 35-45% of those wells in which antibodies were produced contained IgE and IgG4. In addition to B cell clones that produced IgG4 or IgE only, B cell clones producing multiple isotypes were observed. Simultaneous production of IgG4 and IgE, IgM, IgE, and IgM, or IgG4 and IgE was detected, suggesting that during clonal expansion switching might occur in successive steps from IgM to IgG4 and IgE. In addition, production of only IgM, IgG4, and IgE during clonal expansion indicates that this isotype switching is directed by the way a B cell is stimulated and that it is not a stochastic process.

The <em>interleukin</em>-2 (IL-2) receptor gamma chain is indispensable for IL-2-, IL-4-, IL-7-, IL-9-, and IL-<em>15</em>-mediated signaling. Mutations of the human gamma chain cause the X-linked severe combined immunodeficiency (XSCID), showing that T and natural killer cells absolutely require the gamma chain for their development in humans. To elucidate the roles of the gamma chain in hematopoiesis, we have generated mice, by gene targeting, that express a form of the gamma chain lacking the cytoplasmic region. Male mice carrying the truncated gamma-chain mutant, which mimics mutations in patients with XSCID, showed a decrease in the number of lymphocytes and an increase in monocytes; the number of T cells was profoundly reduced and no natural killer cells were detected, which is similar to the characteristic of human XSCID. Unlike human XSCID, the levels of B cells were also reduced. In spite of the severe decrease in CD45R+/sIgM+ B cells, the level of IgM in serum of the 8-week-old mutant mice was higher than that of control littermates. Interestingly, the stem cell population with surface phenotypes of CD34, c-kit, and Sca-1 was significantly increased. Furthermore, the colony-forming assay showed that the mutant mice had <em>15</em>-fold higher numbers of hematopoietic progenitor cells in the spleen as compared with that of controls. These results indicate that functional loss of the gamma chain causes significant effects on the immunological system in mice.

Interferon-alpha (IFN-alpha) is a pleiotropic cytokine that has antiviral, antiproliferative, and immunoregulatory functions. There is increasing evidence that IFN-alpha has an important role in T-cell biology. We have analyzed the expression of IL-2Ralpha, c-myc, and pim-1 genes in anti-CD3-activated human T lymphocytes. The induction of these genes is associated with <em>interleukin</em>-2 (IL-2)-induced T-cell proliferation. Treatment of T lymphocytes with IFN-alpha, IL-2, IL-12, and IL-<em>15</em> upregulated IL-2Ralpha, c-myc, and pim-1 gene expression. IFN-alpha also sensitized T cells to IL-2-induced proliferation, further suggesting that IFN-alpha may be involved in the regulation of T-cell mitogenesis. When we analyzed the nature of STAT proteins capable of binding to IL-2Ralpha, pim-1, and IRF-1 GAS elements after cytokine stimulation, we observed IFN-alpha-induced binding of STAT1, STAT3, and STAT4, but not STAT5 to all of these elements. Yet, IFN-alpha was able to activate binding of STAT5 to the high-affinity IFP53 GAS site. IFN-alpha enhanced tyrosine phosphorylation of STAT1, STAT3, STAT4, STAT5a, and STAT5b. IL-12 induced STAT4 and IL-2 and IL-<em>15</em> induced STAT5 binding to the GAS elements. Taken together, our results suggest that IFN-alpha, IL-2, IL-12, and IL-<em>15</em> have overlapping activities on human T cells. These findings thus emphasize the importance of IFN-alpha as a T-cell regulatory cytokine.

Interactions driven by the T cell antigen receptor (TCR) determine the lineage fate of CD4(+)CD8(+) thymocytes, but the molecular mechanisms that induce the lineage-determining transcription factors are unknown. Here we found that TCR-induced transcription factors Egr2 and Egr1 had higher and more-prolonged expression in precursors of the natural killer T (NKT) than in cells of conventional lineages. Chromatin immunoprecipitation followed by deep sequencing showed that Egr2 directly bound and activated the promoter of Zbtb16, which encodes the NKT lineage-specific transcription factor PLZF. Egr2 also bound the promoter of Il2rb, which encodes the <em>interleukin</em> 2 (IL-2) receptor β-chain, and controlled the responsiveness to IL-<em>15</em>, which signals the terminal differentiation of the NKT lineage. Thus, we propose that persistent higher expression of Egr2 specifies the early and late stages of NKT lineage differentiation, providing a discriminating mechanism that enables TCR signaling to 'instruct' a thymic lineage.

Killer immunoglobulin-like receptor (KIR)-ligand mismatched natural killer (NK) cells play a key role in achieving durable remission after haplo-identical transplantation for acute myeloid leukaemia. We investigated the feasibility of transfusing haplo-identical, T-cell depleted, KIR-ligand mismatched NK cells, after conditioning therapy with melphalan and fludarabine, to patients with advanced multiple myeloma (MM) followed by delayed rescue with autologous stem cells. No graft-versus-host disease or failure of autologous stem cells to engraft was observed. There was significant variation in the number of allo-reactive NK cells transfused. However, all NK products containing allo-reactive NK cells killed the NK cell target K562, the MM cell line U266, and recipient MM cells when available. Post NK cell infusion there was a rise in endogenous <em>interleukin</em>-<em>15</em> accompanied by increasing donor chimaerism. Donor chimaerism was eventually lost, which correlated with the emergence of potent host anti-donor responses indicating that the immunosuppressive properties of the conditioning regimen require further optimization. Further, blocking of inhibitory KIR-ligands with anti-human leucocyte antigen antibody substantially enhanced killing of MM cells thus highlighting the potential for modulating NK/MM cell interaction. Encouragingly, 50% of patients achieved (near) complete remission. These data set the stage for future studies of KIR-ligand mismatched NK cell therapy in the autologous setting.

The effects of fluid percussion trauma on brain <em>interleukin</em> (IL)-6, IL-1 and tumor necrosis factor-alpha (TNF-alpha) levels have been studied. In the cortex and hippocampus of control and sham-operated rats, the levels of these cytokines were very low (below 4 units/mg protein) and constant. IL-6 and IL-1 levels in the ipsilateral cortex increased rapidly following trauma to reach a maximum of 350 and 16 units/mg protein, respectively, 8 h after the lesion, remained elevated until 18 h and decreased thereafter to basal values. TNF-alpha levels were maximally elevated (12 units/mg protein) at 3 h and 8 h and returned to basal values by 18 h. Qualitatively similar changes, but with 25-80-fold smaller amplitude, were seen in the contralateral cortex and in the ipsi- and contralateral hippocampus. The levels of IL-6 in the plasma of sham-operated and lesioned rats were only slightly elevated, whereas IL-1 and TNF-alpha were undetectable. Histological studies of brain tissue at early stages after trauma demonstrated an acute hemorrhage associated with neutrophil invasion. The administration of Ro5 4864 (0.5 mg/kg i.p.), a specific ligand of p (peripheral-type benzodiazepine) binding sites, did not result in any significant effect on the levels of IL-6, IL-1 or TNF-alpha in the brain of control or sham-operated animals. However, when administered 24 h before or <em>15</em> min after trauma, this benzodiazepine enhanced the increase of these cytokines by 2-4-fold in the ipsilateral cortex.(ABSTRACT TRUNCATED AT 250 WORDS)

Traumatic injury to the central nervous system initiates inflammatory processes that are implicated in secondary tissue damage. These processes include the synthesis of proinflammatory cytokines, leukocyte extravasation, vasogenic edema, and blood-brain barrier breakdown. <em>Interleukin</em>-10 (IL-10), a cytokine with antiinflammatory properties, negatively modulates proinflammatory cascades at multiple levels. We examined the hypothesis that IL-10 treatment can improve outcome in a clinically relevant model of traumatic brain injury (TBI). IL-10 was administered via different routes and dosing schedules in a lateral fluid-percussion model of TBI in rats. Intravenous administration of IL-10 (100 micrograms) at 30 min before and 1 h after TBI improved neurological recovery and significantly reduced TNF expression in the traumatized cortex at 4 h after injury. Such treatment was associated with lower IL-1 expression in the injured hippocampus, and to a lesser extent, in the injured cortex. Subcutaneous IL-10 administration (100 micrograms) at 10 min, 1, 3, 6, 9, and 12 h after TBI also enhanced neurological recovery. In contrast, intracerebroventricular administration of IL-10 (1 or 6 micrograms) at <em>15</em> min, 2, 4, 6, and 8 h after TBI was not beneficial. These results indicate that IL-10 treatment improves outcome after TBI and suggest that this improvement may relate, in part, to reductions in proinflammatory cytokine synthesis.

BACKGROUND

<em>Interleukin</em>-6 (IL-6) is a cytokine that plays a central role in host defense due to its wide range of immune and hematopoietic activities. It is found in high levels in human ejaculate, and has recently been found to regulate prostate-specific protein expression in prostate cancer cells through nonsteroidal activation of the androgen receptor. IL-6 may be a candidate mediator of morbidity in patients with metastatic disease. We attempted to evaluate the potential of circulating IL-6 levels as a marker of disease progression. MATERIALS AND METHODS Serum IL-6, prostate specific antigen (PSA), percent free PSA (%fPSA), and prostate-specific membrane antigen (PSMA) were measured using commercially available assays in 407 men, including <em>15</em> controls. The rest of the study population had clinical or histologic evidence of prostate diseases, including 41 patients with chronic prostatitis, 167 with benign prostatic hyperplasia (BPH), 8 with high-grade prostatic intraepithelial neoplasia (PIN), 88 with localized prostate cancer, 22 with local recurrence after treatment of primary tumor, 4 with advanced untreated disease (nodal or bony metastases), 23 with advanced hormone dependent disease, and 39 with advanced hormone refractory disease (PSA>> 1.0 ng/ml while on hormone treatment and/or evidence of disease progression). None had history of concurrent malignancy or acute inflammatory condition. Kruskal-Wallis analysis of variance and Spearman's correlation analysis were used for statistical analyses.

RESULTS

Serum levels of IL-6 were significantly elevated in patients with clinically evident hormone refractory disease (5.7 +/- 1.9 pg/ml) and statistical significance was seen when comparing the elevated serum IL-6 levels to those in normal controls, prostatitis, BPH, and localized and recurrent disease, (P values < 0.01). Compared to serum levels of controls and BPH, PSA was significantly elevated in advanced untreated disease and hormone refractory groups (P < 0.05). Percent fPSA was significantly lower in all cancer patients but the hormone refractory. Serum PSMA was elevated in advanced untreated prostate cancer. Serum IL-6 showed positive correlation with PSMA and negative correlation with serum PSA but did not attain statistical significance.

CONCLUSIONS

Serum IL-6 levels are significantly elevated in hormone-refractory prostate cancer patients and may be a surrogate marker of the androgen independent phenotype.

Cyclooxygenase 2 (COX-2) overexpression and production of prostaglandin E(2) (PGE(2)) by head and neck squamous cell carcinomas (HNSCC) induce type 1 regulatory T (Tr1) cells and contribute to carcinogenesis by creating a tolerogenic milieu. To test this hypothesis, CD4(+)CD25(-) T cells obtained from the peripheral blood of 10 normal donors were cocultured with autologous dendritic cells, irradiated HNSCC cells and cytokines, <em>interleukin</em> 2 (IL-2), IL-10, and IL-<em>15</em>. HNSCC cells were either COX-2 negative, constitutively expressed COX-2, were transfected with COX-2, or had COX-2 expression knocked down by small interfering RNA. Other modifications included coculture plus or minus the COX-inhibitor, Diclofenac, or synthetic PGE(2) in the absence of HNSCC. Lymphocytes proliferating in 10-day cocultures were phenotyped by flow cytometry, studied for cytokine production by ELISA and for suppressor function in CFSE inhibition assays plus or minus anti-IL-10 or anti-transforming growth factor-beta(1) (TGF-beta(1)) monoclonal antibodies (mAb). COX-2(+) HNSCC or exogenous PGE(2) induced outgrowth of Tr1 cells with the CD3(+)CD4(+)CD25(-)IL2Rbeta(+)IL2Rgamma(+)FoxP3(+)CTLA-4(+)IL-10(+)TGF-beta(1)(+)IL-4(-) phenotype and high suppressor functions (range, 46-68%). Small interfering RNA knockout of COX-2 gene in HNSCC led to outgrowth of lymphocytes with decreased IL2Rgamma (P = 0.0001), FoxP3 (P = 0.05), and IL-10 (P = 0.035) expression and low suppressor activity (range, 26-34%). Whereas COX-2(+) cocultures contained IL-10 and TGF-beta(1) (medians, 6<em>15</em> and 824 pg/mL), cytokine levels were decreased (P < 0.0001) in COX-2(-) cocultures. Inhibition of COX-2 enzymatic activity in HNSCC abrogated outgrowth of Tr1 cells. Neutralizing mAbs to IL-10 and/or TGF-beta(1) abolished Tr1-mediated suppression. COX-2 overexpression in HNSCC plays a major role in the induction of Tr1 cells in the tumor microenvironment.

Recent studies have suggested that glia might play a more active role in synaptic function than previously thought. Therefore, the present studies have evaluated the potential role of spinal cord glia in acute nociceptive processing and in the thermal and mechanical hyperalgesia produced by peripheral injury. In the present experiments, we found that: (1) selective inhibition of glia metabolism with intrathecal (i.t.) administration of fluorocitrate (1 nmol) results in a marked, but reversible, attenuation of the persistent thermal and mechanical hyperalgesia produced by intraplantar zymosan (5 mg); (2) selective inhibition of the inducible form of nitric oxide synthase (iNOS) with i.t. aminoguanidine (1 pmol-1 nmol) resulted in a dose-dependent inhibition of the persistent thermal, but not mechanical hyperalgesia produced by intraplantar zymosan (5 mg); (3) i.t. coadministration of <em>interleukin</em> 1 beta (IL1 beta; 10 ng) and interferon gamma (IFN; 1000 U) resulted in expression of the message for iNOS 8 hr after administration assessed using reverse-transcription polymerase chain reaction (RT-PCR) and Southern blot analysis; and (4) i.t. administration of lipopolysaccharide (LPS; <em>15</em>0 micrograms) produced a time-dependent thermal hyperalgesia compared with saline treated-rats (<em>15</em> microliters). There was no change in mechanical withdrawal thresholds over time following any treatment, except fluorocitrate. We have previously shown that NO plays a significant role in mechanisms of hyperalgesia. In the present experiments we have extended these observations and have now shown a role for iNOS, expressed by glia, in mechanisms of hyperalgesia. These results suggest an unexplored avenue for the development of potential new and novel therapies for pain control.

Primary vitreoretinal lymphoma (PVRL), also known as primary intraocular lymphoma, is a rare malignancy typically classified as a diffuse large B-cell lymphoma and most frequently develops in elderly populations. PVRL commonly masquerades as posterior uveitis and has a unique tropism for the retina and central nervous system (CNS). Over <em>15</em>% of primary CNS lymphoma patients develop intraocular lymphoma, usually occurring in the retina and/or vitreous. Conversely, 65%-90% of PVRL patients develop CNS lymphoma. Consequently, PVRL is often fatal because of ultimate CNS association. Current PVRL animal models are limited and require further development. Typical clinical findings include vitreous cellular infiltration (lymphoma and inflammatory cells) and subretinal tumor infiltration as determined using dilated fundoscopy, fluorescent angiography, and optical coherent tomography. Currently, PVRL is most often diagnosed using both histology to identify lymphoma cells in the vitreous or retina and immunohistochemistry to indicate monoclonality. Additional adjuncts in diagnosing PVRL exist, including elevation of <em>interleukin</em>-10 levels in ocular fluids and detection of Ig(H) or T-cell receptor gene rearrangements in malignant cells. The optimal therapy for PVRL is not defined and requires the combined effort of oncologists and ophthalmologists. PVRL is sensitive to radiation therapy and exhibits high responsiveness to intravitreal methotrexate or rituximab. Although systemic chemotherapy alone can result in high response rates in patients with PVRL, there is a high relapse rate. Because of the disease rarity, international, multicenter, collaborative efforts are required to better understand the biology and pathogenesis of PVRL as well as to define both diagnostic markers and optimal therapies.

<em>Interleukin</em>-2 (IL-2) is a <em>15</em>,000 dalton glycoprotein produced naturally by human T-cells during an immune response. IL-2 has been demonstrated to have substantial activity alone or in combination with the adoptive transfer of lymphokine-activated killer cells in murine tumor models. IL-2 derived from both natural (Jurkat human T-cell tumor) and recombinant (Escherichia coli) sources has been studied in Phase I protocols designed to evaluate toxicity in patients with a variety of solid tumors and to ascertain improvement in clinical parameters and immunologic status. A total of 16 patients (7 with acquired immune deficiency syndrome [AIDS] and 9 with non-AIDS malignancies) were treated with Jurkat derived IL-2. The total maximum dose (1.3 X 10(5) U/kg) was limited only by supply of this reagent. A total of 25 patients have been treated with recombinant IL-2 (RIL-2) alone. Dose-limiting toxicity manifested by marked malaise and weight gain was achieved with doses of RIL-2 of 10(6) U/kg as a single bolus or 3000 U/kg/hr. IL-2 could be administered intraperitoneally with similar toxicity. Minimal renal or hepatic toxicity was demonstrated. Hematologic toxicity was limited to mild anemia (25/25), thrombocytopenia (10/25), and marked reversible eosinophilia (<em>15</em>/25). Pronounced weight gain greater than 2 kg (16/25) occurred in most patients, primarily those who received cumulative doses of greater than 1-3 X 10(5) U/kg of IL-2. The weight gain amounted to as much as 10% to 20% of the pretreatment weight over 3 weeks of treatment and limited our ability to give higher doses. Two partial responses (greater than 50% decrease in cross sectional diameters) were seen in two patients with melanoma metastatic to the lung.

Human beta-defensin-1 (hBD-1) is a member of the family of small cationic antimicrobial peptides that have been identified in several mucosal epithelia. Because human gingival epithelium is a site that is constantly challenged by oral microorganisms, we examined the expression of hBD-1 in human gingival epithelial and fibroblast cell cultures and tissue samples. Cell cultures were challenged with cell wall extracts of Porphyromonas gingivalis or Fusobacterium nucleatum, Escherichia coli lipopolysaccharide, tumor necrosis factor alpha, or phorbol myristate acetate. hBD-1 mRNA was detected in unstimulated and stimulated cultures by reverse transcription (RT)-PCR using several primer sets specific for hBD-1. Gingival epithelial cells, but not gingival fibroblasts, expressed a product of the predicted size for hBD-1 mRNA. The sequence of the PCR product was identical to that of hBD-1. hBD-1 mRNA expression was not significantly modulated by any of the stimulants tested. Human gingival tissues from noninflamed and inflamed sites were also analyzed by RT-PCR. hBD-1 mRNA was expressed in all tissue samples. The relative expression of hBD-1 mRNA was similar in noninflamed and inflamed tissues obtained from each of four patients undergoing treatment for periodontitis. However, the relative expression of hBD-1 mRNA varied in gingival biopsies obtained from <em>15</em> different normal individuals, and the relative hBD-1 expression was unrelated to <em>interleukin</em>-8 expression. Our findings show the constitutive expression of hBD-1 mRNA in cultured epithelial cells and gingival tissues but not gingival fibroblasts. These findings suggest that expression of hBD-1 may play a role as part of the innate host defenses in maintaining normal gingival health.

BACKGROUND

The current paradigm for the pathogenesis of COPD includes an ultimately maladaptive local inflammatory response to environmental stimuli. We examined the hypothesis that systemic inflammatory biomarkers are associated with impaired lung function, particularly among those with extensive cigarette smoking.

METHODS

Using data from the Framingham Heart Study, we examined cross-sectional associations of systemic inflammatory biomarkers (CD40 ligand [CD40L], intercellular adhesion molecule [ICAM]-1, interleukin [IL]-6, monocyte chemoattractant protein-1, P-selectin, and myeloperoxidase, in addition to C-reactive protein) to impaired lung function.

RESULTS

IL-6 was consistently associated with impaired lung function; a 1-SD higher concentration of IL-6 was associated with a 41-mL lower FEV(1) (95% confidence interval [CI], - 61 to - 20) and a borderline 15% higher odds of COPD (odds ratio, 1.15; 95% CI, 0.99 to 1.34). Additionally, P-selectin was associated with lower FEV(1) levels; after adjusting for the other biomarkers, a 1-SD higher concentration of P-selectin predicted an FEV(1) that was on average 19 mL lower (95% CI, - 37 to 0). Including the biomarkers individually as sole exposures in the models generally strengthened the impaired lung function/biomarker association; the relations of ICAM-1 to FEV(1), and ICAM and CD40L to COPD became significant. The observed associations did not vary significantly with smoking history, except that the association between CD40L and COPD appeared greater in individuals with more extensive smoking histories.

CONCLUSIONS

Among participants in the Framingham Heart Study, systemic inflammation was associated with lower levels of pulmonary function. Further research into the role of systemic inflammation in the development of pulmonary dysfunction is merited.

OBJECTIVE

A phase I study of patients with metastatic malignant melanoma (MM) and renal cell carcinoma (RCC) evaluated the safety and maximum tolerated dose (MTD), pharmacokinetics, pharmacodynamics, and preliminary antitumor activity of recombinant human interleukin-21 (rIL-21).

METHODS

Patients who had one or fewer prior systemic treatments for metastatic MM or RCC were treated with rIL-21 administered for two 5-day cycles on days 1 through 5 and 15 through 19 of a treatment course; rIL-21 was administered by rapid intravenous infusion in an outpatient setting. Cohorts of patients received doses ranging from 3 to 100 microg/kg/dose, and an expanded cohort was treated at the MTD. Patients with stable disease (SD) or better could receive additional treatment cycles.

RESULTS

Forty-three patients were treated (24 MM; 19 RCC), including 28 in the expanded cohort. Dose-limiting toxicities consisted primarily of transient grade 3 laboratory abnormalities. The MTD was estimated to be 30 microg/kg. The most common adverse events included flu-like symptoms, pruritus, and rash. Twelve patients received up to five additional two-cycle courses of treatment without cumulative toxicity, except for one patient with reversible grade 4 hepatotoxicity. Serum concentrations of rIL-21 increased in a dose-proportional manner. Dose-dependent increases in soluble CD25 reflected lymphocyte activation. Antitumor activity was observed in both MM (one complete response and 11 SD) and RCC (four partial responses, 13 SD).

CONCLUSIONS

Outpatient therapy with rIL-21 at 30 microg/kg was well tolerated, had dose-dependent pharmacokinetics and pharmacodynamics, and was associated with antitumor activity in patients with MM and RCC.