BACKGROUND

Obesity leads to fat infiltration of multiple organs including the heart, kidneys, and liver. Under conditions of oxidative stress, fat-derived cytokines are released locally and result in an inflammatory process and organ dysfunction. In the liver, fat infiltration has been termed nonalcoholic fatty liver disease, which may lead to nonalcoholic steatohepatitis. No data are available, however, on the influence of obesity on pancreatic fat and cytokines, and nonalcoholic fatty pancreas disease (NAFPD) has not been described. Therefore, we designed a study to determine whether obesity is associated with increased pancreatic fat and cytokines.

METHODS

Thirty C57BL/6J lean control and 30 leptin-deficient obese female mice were fed a <em>15</em>% fat diet for 4 weeks. At 12 weeks of age all animals underwent total pancreatectomy. Pancreata from each strain were pooled for measurement of a) wet and dry weight, b) histologic presence of fat, c) triglycerides, free fatty acids (FFAs), cholesterol, phospholipids, and total fat, and d) <em>interleukin</em> (IL)-1beta and tumor necrosis factor-alpha (TNF-alpha). Data were analyzed by Student's t test and Fisher's exact test.

RESULTS

Pancreata from obese mice were heavier (p<0.05) and had more fat histologically (p<0.05). Pancreata from obese mice had more triglycerides, FFAs, cholesterol, and total fat (p<0.05). Triglycerides represented 11% of pancreatic fat in lean mice compared with 67% of pancreatic fat in obese mice (p<0.01). Cytokines IL-1beta and TNF-alpha also were elevated in the pancreata of obese mice (p<0.05).

CONCLUSIONS

These data suggest that obese mice have 1) heavier pancreata, 2) more pancreatic fat, especially triglycerides and FFAs, and 3) increased cytokines. We conclude that obesity leads to nonalcoholic fatty pancreatic disease.

The potential for <em>interleukin</em>-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1) to induce neutrophil and mononuclear phagocyte accumulation in the lungs of patients with pulmonary sarcoidosis and idiopathic pulmonary fibrosis (IPF) was investigated. Bronchoalveolar lavage (BAL) fluids from 12 patients with IPF and <em>15</em> with sarcoidosis were concentrated by reversed-phase chromatography, and their IL-8 and MCP-1 concentrations assessed by enzyme-linked immunosorbent assay (ELISA), chemotaxis, and enzyme-releasing assays with monocytes and neutrophils. ELISA revealed significantly elevated concentrations of MCP-1 (20.1 ng/mg albumin) in the BAL fluids of patients with pulmonary sarcoidosis and those with IPF (41.8 ng/mg) in comparison to 11 normal individuals (4.24 ng/mg) and <em>15</em> patients with chronic bronchitis (CB) (5.16 ng/mg). Similarly, the chemotactic activity for monocytes (MCP-1 equivalent) was strongly increased in patients with sarcoidosis (86.03 ng/mg) as well as in those with IPF (54.47 ng/mg). The chemoattractant activity of normal individuals and CB patients was 7- or 3-fold lower, respectively. Patients with IPF and sarcoidosis also had elevated IL-8 levels (<em>15</em>.5 and 26.0 ng/mg, respectively; normals: 2.14 ng/mg; and CB patients: 4.23 ng/mg) and greater neutrophil chemotaxis (60.25 and 49.68 ng/mg, respectively; normals: 0.35 ng/mg; and CB patients: 11.06 ng/mg). These data suggest that increased levels of both MCP-1 and IL-8 may be characteristic for sarcoidosis or IPF. It appears likely that both of these chemoattractants contribute to the influx of monocytes and neutrophils into the pulmonary alveolus and interstitium in these diseases.

OBJECTIVE

To evaluate the safety and pharmacokinetics of siltuximab, an anti-interleukin-6 chimeric monoclonal antibody (mAb) in patients with B-cell non-Hodgkin lymphoma (NHL), multiple myeloma, or Castleman disease.

METHODS

In an open-label, dose-finding, 7 cohort, phase I study, patients with NHL, multiple myeloma, or symptomatic Castleman disease received siltuximab 3, 6, 9, or 12 mg/kg weekly, every 2 weeks, or every 3 weeks. Response was assessed in all disease types. Clinical benefit response (CBR; composite of hemoglobin, fatigue, anorexia, fever/night sweats, weight, largest lymph node size) was also evaluated in Castleman disease.

RESULTS

Sixty-seven patients received a median of 16 siltuximab doses for a median of 8.5 (maximum 60.5) months; 29 were treated 1 year or longer. There was no dose-limiting toxicity, antibodies to siltuximab, or apparent dose-toxicity relationship. The most frequently reported possible drug-related adverse events were thrombocytopenia (25%), hypertriglyceridemia (19%), neutropenia (19%), leukopenia (18%), hypercholesterolemia (15%), and anemia (10%). None of these events led to dose delay/discontinuation except for neutropenia and thrombocytopenia (n = 1 each). No treatment-related deaths occurred. C-reactive protein (CRP) suppression was most pronounced at 12 mg/kg every 3 weeks. Mean terminal-phase half-life of siltuximab ranged 17.73 to 20.64 days. Thirty-two of 37 (86%) patients with Castleman disease improved in 1 or more CBR component; 12 of 36 evaluable Castleman disease patients had radiologic response [complete response (CR), n = 1; partial response (PR), n = 11], including 8 of 19 treated with 12 mg/kg; 2 of 14 (14%) evaluable NHL patients had PR; 2 of 13 (15%) patients with multiple myeloma had CR.

CONCLUSIONS

No dose-related or cumulative toxicity was apparent across all disease indications. A dose of 12 mg/kg every 3 weeks was recommended on the basis of the high response rates in Castleman disease and the sustained CRP suppression. Randomized studies are ongoing in Castleman disease and multiple myeloma.

OBJECTIVE

To study the neuropsychiatric manifestations of therapy with interleukin-2 and lymphokine-activated killer cells.

METHODS

Longitudinal survey of consecutive patients who were given the treatment. Each patient was initially interviewed within 5 days before treatment, and a personal and family psychiatric history was obtained during this first session. Cognitive tests and mood self-rating instruments were administered at the beginning and end of interleukin-2 and lymphokine-activated killer cell treatments, before discharge, and at a follow-up visit 2 to 4 weeks after discharge.

METHODS

National Cancer Institute inpatient units at the National Institutes of Health.

METHODS

Sequential samples of 44 patients with metastatic cancer (age range, 28 to 69 years) who were treated systemically with recombinant interleukin-2 combined with autologous lymphokine-activated killer cells between 30 December 1985 and 31 March 1986.

RESULTS

Of the 44 patients studied, 15 developed severe behavioral changes that necessitated acute intervention, and 22 patients had severe cognitive changes (all 22 became disoriented and many also had psychometric evidence of cognitive deterioration). The neuropsychiatric side effects were dose and time related, appearing more frequently at the higher dose and almost uniformly at the end of each treatment phase. All 39 patients who were seen at follow-up had a return to their baseline cognitive scores. None of the factors investigated was found to be predictive of the development of neuropsychiatric toxicity.

CONCLUSIONS

The development of clinically significant neuropsychiatric changes during the administration of interleukin-2 and lymphokine-activated killer cells was common and may be treatment limiting. A marked latency in the appearance of neuropsychiatric changes after treatment onset was noted in almost all patients. Every patient studied recovered from the neuropsychiatric side effects.

OBJECTIVE

To determine whether natural killer (NK) cells are present within inflamed joints and whether they might play a role in amplifying the inflammatory process.

METHODS

Paired samples of peripheral blood and synovial fluid were obtained from 22 patients with inflammatory arthritis. The frequency and phenotype of the peripheral and synovial NK cells were analyzed using a panel of monoclonal antibodies. Further experiments were performed to investigate the functional capacity of the synovial NK cells.

RESULTS

The study showed that the CD3-, CD56(bright) subset of NK cells was greatly expanded within inflamed joints. Our experiments suggested that this subset of cells was preferentially recruited from the periphery and that NK cells may be further activated by cytokines present within the joint. Furthermore, synovial NK cells responded to a combination of <em>interleukin</em>-12 (IL-12) and IL-<em>15</em>, cytokines that are secreted by cells of the monocyte/macrophage lineage, by rapidly secreting interferon-gamma, a cytokine that can, in turn, activate macrophages.

CONCLUSIONS

A subset of NK cells was expanded within inflamed joints. The functional properties of these NK cells rendered them good candidates for a role in interacting with the macrophage/monocyte population within the joint, thus amplifying the production of proinflammatory cytokines.

Epithelial cells of the alimentary tract play a central role in mucosal immunophysiology. Pathogens and/or agonists that interact with mucosal surfaces often elicit epithelial responses that upregulate inflammation. Therefore, it was of interest to explore potential epithelial targeted antiinflammatory signals. Here we identified and sequenced a human enterocyte lipoxin (LX) A4 [5(S), 6(R),<em>15</em>(S)-trihydroxy-7,9,13-trans-11-cis eicosatetraenoic acid] receptor, and demonstrate that transcription of this receptor was controlled by cytokines, of which lymphocyte-derived <em>interleukin</em> (IL)-13 and interferon gamma were the most potent. When lipoxins and LXA4 stable analogs were evaluated for enterocyte functional as well as immune responses, lipoxins sharply inhibited TNF-alpha-induced IL-8 release but did not alter either barrier function or agonist-stimulated chloride secretion. <em>15</em>R/S-methyl-LXA4 and 16-phenoxy-LXA4 each attenuated (IC50 approximately 10 nM) IL-8 release. Cyclooxygenase (COX) II is emerging as an important component in wound healing and proliferation in intestinal epithelia and when acetylated by acetylsalicylic acid (aspirin) initiates the biosynthesis of a LXA4 receptor ligand. We therefore determined whether colonic cell lines (HT-29 Cl.19A, Caco-2, or T84) express the COX II isozyme. Results for RT-PCR and Western blot analysis showed that COX I as well as an IL-1beta- and TNF-alpha-inducible COX II are expressed in HT-29 Cl.19A. In addition, aspirin-treated enterocytes generated <em>15</em>R-HETE, a precursor of <em>15</em>-epi-LXA4 biosynthesis, whose potent bioactions were mimicked by the stable analog <em>15</em>R/S-methyl-LXA4. Taken together, these results identify an endogenous pathway for downregulating mucosal inflammatory events and suggest a potential therapeutic benefit for LXA4 stable analogs.

Numerous studies have suggested an important role for the Th2 cytokines <em>interleukin</em> (IL)-13 and IL-4 in the development of allergic asthma. We tested the hypothesis that IL-13 and IL-4 have direct effects on cultured airway smooth muscle cells (HASM). Using RT-PCR, we showed that HASM cells express transcripts for IL-4alpha, IL-13RalphaI, and IL-13RalphaII, but not for the common IL-2Rgamma chain. We then analyzed the capacity of the two cytokines to activate signaling pathways in HASM cells. Both IL-13 and IL-4 caused STAT-6 phosphorylation, but the time course was different between the two cytokines, with peak effects occurring <em>15</em> min after addition of IL-4 and 1 h after addition of IL-13. Effects on signaling were observed at cytokine concentrations as low as 0.3 ng/ml. IL-4 and IL-13 also caused phosphorylation of ERK MAP kinase. As suggested by the signaling studies, the biological responses of the two cytokines were also different. We used magnetic twisting cytometry to measure cell stiffness of HASM cells and tested the capacity of IL-4 and IL-13 to interfere with the reductions in cell stiffness induced by the beta-agonist isoproterenol (ISO). IL-13 (50 ng/ml for 24 h), but not IL-4, significantly reduced beta-adrenergic responsiveness of HASM cells, and the MEK inhibitor U0126 significantly reduced the effects of IL-13 on ISO-induced changes in cell stiffness. We propose that these direct effect of IL-13 on HASM cells may contribute at least in part to the airway narrowing observed in patients with asthma.

Experimental bacterial infection of the dermis induced with gram-negative microorganisms is associated with an acute inflammatory reaction, which represents the principal local defense against spread of the infection. When the inflammatory reaction is quantitated with radiolabeled cells and proteins, the kinetics resemble acute inflammation induced with other agents, such as immune complexes or chemotaxins. There is an interrelationship between the components or events of the inflammatory reaction; inasmuch as vascular injury is neutrophil-dependent, neutrophils must migrate to the site where the bacteria multiply. In neutropenic animals there is no such emigration and bacterial multiplication is not inhibited. The microorganisms shed endotoxin, which in turn induces secretion of <em>interleukin</em> 1 (IL 1) and probably tumor necrosis factor. Endotoxin is the most potent agent (10(-<em>15</em>) mol vs. 10(-12) mol of C5ades Arg) capable of inducing a neutrophil influx. Desensitization or tachyphylaxis of the tissues (probably of postcapillary venular endothelium) to IL 1 seems to control cessation of the neutrophil influx (also in vitro evidence). Phagocytosis of the bacteria by neutrophils is associated with release of oxygen radicals and lysosomal proteases from the neutrophils. These are instrumental in eliciting microvascular injury, which is characterized by enhanced vasopermeability, hemorrhage, and thrombosis.

OBJECTIVE

To investigate the effect of the histone deacetylase (HDAC) inhibitor, trichostatin A (TSA), on joint inflammation and cartilage degeneration in a collagen antibody-induced arthritis (CAIA) mouse model.

METHODS

CAIA mice were given daily subcutaneous injections of various concentrations of TSA (0, 0.5, 1.0, and 2.0 mg/kg) and various parameters were monitored for 14 days. On Day <em>15</em>, the hind paws were examined histologically. To investigate the effects of TSA on the expressions of matrix metalloproteinase (MMP)-3, MMP-13, tissue inhibitor of MMP-1 (TIMP-1), and acetyl-H4 by chondrocytes, another group of mice was sacrificed on Day 6. In vitro direct effect of TSA was examined by real-time PCR for mRNA of type II collagen, aggrecan, MMP-3, and MMP-13 in murine chondrogenic ATDC5 cells after pro-inflammatory cytokine stimulation.

RESULTS

In the TSA-treated group, clinical arthritis was significantly ameliorated in a dose-dependent manner. The severity of synovial inflammation and the cartilage destruction score were significantly lower in the TSA 2.0 mg/kg group compared to the other TSA-treated groups. On immunohistochemistry, the number of MMP-3 and MMP-13-positive chondrocytes was significantly lower in the TSA 2.0 mg/kg group than in the control group. In contrast, the number of TIMP-1-positive cells and acetyl-histone H4-positive cells was significantly higher in the TSA 2.0mg/kg group than in the control group. TSA suppressed interleukin 1-beta and tumor necrosis factor-alpha-stimulated up-regulation of MMP-3, but not MMP-13 mRNA expression by ATDC5.

CONCLUSIONS

The systemic administration of TSA ameliorated synovial inflammation in CAIA mice. Subsequently cartilage destruction was also suppressed by TSA, at least in part, by modulating chondrocyte gene expression.

Dengue virus (DV) primarily infects blood monocytes (MO) and tissue macrophages (M phi). We have shown in the present study that DV can productively infect primary human MO/M phi regardless of the stage of cell differentiation. After DV infection, the in vitro-differentiated MO/M phi secreted multiple innate cytokines and chemokines, including tumor necrosis factor alpha, alpha interferon (IFN-alpha), <em>interleukin</em>-1 beta (IL-1 beta), IL-8, IL-12, MIP-1 alpha, and RANTES but not IL-6, IL-<em>15</em>, or nitric oxide. Secretion of these mediators was highlighted by distinct magnitude, onset, kinetics, duration, and induction potential. A chemokine-to-cytokine hierarchy was noted in the magnitude and induction potential of secretion, and a chemokine-to-cytokine-to-chemokine/Th1 cytokine cascade could be seen in the production kinetics. Furthermore, we found that terminally differentiated MO/M phi cultured for more than 45 days could support productive DV infection and produce innate cytokines and chemokines, indicating that these mature cells were functionally competent in the context of a viral infection. In addition, DV replication in primary differentiated human MO/M phi was enhanced and prolonged in the presence of lipopolysaccharide (LPS), and LPS-mediated synergistic production of IFN-alpha could be seen in DV-infected MO/M phi. The secretion of innate cytokines and chemokines by differentiated MO/M phi suggests that regional accumulation of these mediators may occur in various tissues to which DV has disseminated and may thus result in local inflammation. The LPS-mediated enhancement of virus replication and synergistic IFN-alpha production suggests that concurrent bacterial infection may modulate cytokine-mediated disease progression during DV infection.

Activation of TLRs (Toll-like receptors) induces gene expression of proteins involved in the immune system response. TLR4 has been implicated in the development and progression of CVDs (cardio-vascular diseases). Innate and adaptive immunity contribute to hypertension-associated end-organ damage, although the mechanism by which this occurs remains unclear. In the present study, we hypothesize that inhibition of TLR4 decreases BP (blood pressure) and improves vascular contractility in resistance arteries from SHR (spontaneously hypertensive rats). TLR4 protein expression in mesenteric resistance arteries was higher in <em>15</em>-week-old SHR than in age-matched Wistar controls or in 5-week-old SHR. To decrease the activation of TLR4, <em>15</em>-week-old SHR and Wistar rats were treated with anti-TLR4 (anti-TLR4 antibody) or non-specific IgG control antibody for <em>15</em> days (1 μg per day, intraperitoneal). Treatment with anti-TLR4 decreased MAP (mean arterial pressure) as well as TLR4 protein expression in mesenteric resistance arteries and IL-6 (<em>interleukin</em> 6) serum levels from SHR when compared with SHR treated with IgG. No changes in these parameters were found in treated Wistar control rats. Mesenteric resistance arteries from anti-TLR4-treated SHR exhibited decreased maximal contractile response to NA (noradrenaline) compared with IgG-treated SHR. Inhibition of COX (cyclo-oxygenase)-1 and COX-2, enzymes related to inflammatory pathways, decreased NA responses only in mesenteric resistance arteries of SHR treated with IgG. COX-2 expression and TXA2 (thromboxane A2) release were decreased in SHR treated with anti-TLR4 compared with IgG-treated SHR. Our results suggest that TLR4 activation contributes to increased BP, low-grade inflammation and plays a role in the augmented vascular contractility displayed by SHR.

We have performed pulse-chase experiments to investigate the secretion and processing of <em>interleukin</em> 1 (IL-1) by human peripheral blood monocytes. Polyclonal antisera generated against either recombinant IL-1 alpha (p<em>15</em>) or IL-1 beta (p17) could distinguish the two isoelectric forms in lysates and supernatants of lipopolysaccharide-activated monocytes. In agreement with previous results, no processed IL-1 (alpha or beta) is detected in cell lysates. Both the 31-kDa precursor and 17-kDa mature forms of IL-1 were present, however, in the culture media indicating that processing is not required for secretion. The relative amounts of the secreted 31- and 17-kDa forms of IL-1 remain constant with time throughout each experiment; in addition, 31-kDa IL-1 added to monocyte cultures is not processed to the mature 17-kDa form. Precursor IL-1 beta is however, processed to 17 kDa by monocyte extracts. Therefore, the maturation and secretion of IL-1 are intimately coordinated processes. The kinetics of IL-1 secretion are unique in comparison with other secreted proteins; release of both IL-1 alpha and IL-1 beta is delayed following synthesis, and large pools of precursor IL-1 accumulate intracellularly. The intracellular half-lives of IL-1 alpha and IL-1 beta are <em>15</em> and 2.5 h, respectively. This discrepancy in half-lives is a reflection of the different kinetics with which IL-1 alpha and IL-1 beta are secreted. IL-1 beta is released continuously beginning 2 h after synthesis, whereas the secretion of IL-1 alpha is delayed for an additional 10 h. The distinct kinetics of secretion demonstrated for IL-1 alpha and IL-1 beta suggest that the release of each pI species of IL-1 is controlled by a selective mechanism(s).

Mice deficient in the Tec kinase Itk develop a large population of CD8(+) T cells with properties, including expression of memory markers, rapid production of cytokines, and dependence on <em>Interleukin</em>-<em>15</em>, resembling NKT and other innate T cell lineages. Like NKT cells, these CD8(+) T cells can be selected on hematopoietic cells. We demonstrate that these CD8(+) T cell phenotypes resulted from selection on hematopoietic cells-forcing selection on the thymic stroma reduced the number and innate phenotypes of mature Itk-deficient CD8(+) T cells. We further show that, similar to NKT cells, selection of innate-type CD8(+) T cells in Itk(-/-) mice required the adaptor SAP. Acquisition of their innate characteristics, however, required CD28. Our results suggest that SAP and Itk reciprocally regulate selection of innate and conventional CD8(+) T cells on hematopoietic cells and thymic epithelium, respectively, whereas CD28 regulates development of innate phenotypes resulting from selection on hematopoietic cells.

OBJECTIVE

Our purpose was to determine whether endotoxin-induced preterm parturition is preceded by a change in the maternal serum and amniotic fluid concentrations of tumor necrosis factor-alpha, interleukin-6, and interleukin-1 alpha.

METHODS

C3H/HeN pregnant mice at 15 days of gestation (70% gestation) were randomized to receive an intraperitoneal injection of phosphate-buffered saline solution or lipopolysaccharide (50 micrograms/mouse). Blood (n = 93) and amniotic fluid (n = 58) were collected at 1, 4, and 10 hours after lipopolysaccharide injection. Tumor necrosis factor-alpha, interleukin-6, and interleukin-1 alpha were determined with sensitive and specific enzyme-linked immunoassays.

RESULTS

The injection-to-delivery interval was shorter in mice injected intraperitoneally with 50 micrograms lipopolysaccharide than in phosphate-buffered saline solution-treated mice (median 15.5 hours, range: 10 to 105 hours vs median 88.5 hours, range: 53 to 105 hours; p < 0.001). In comparison with phosphate-buffered saline solution-treated mice, a distinct serum cytokine pattern was observed in lipopolysaccharide-treated mice. Concentrations of tumor necrosis factor-alpha were detectable 1 and 4 hours after lipopolysaccharide injection (median 874 pg/ml, range: < 100 to 8000 pg/ml, p < 0.001; and median 263 pg/ml, range: < 100 to 927 pg/ml, p < 0.001, respectively). Concentrations of interleukin-6 were elevated at 1, 4, and 10 hours (median 11.8 ng/ml, range: 6 to 500 ng/ml, p < 0.001; median 27.1 ng/ml, range: 4.5 to 192 ng/ml, p < 0.001; median 1.95 ng/ml, range: < 0.05 to 35 ng/ml, p < 0.015, respectively). Concentrations of interleukin-1 alpha were significantly increased 4 hours after lipopolysaccharide injection (median 102 pg/ml, range: < 15 to 306 pg/ml, p < 0.001). A cytokine pattern distinct from serum was observed in amniotic fluid of lipopolysaccharide-treated mice. In comparison with controls, concentrations of interleukin-6 were significantly elevated 4 and 10 hours after treatment with lipopolysaccharide (median 0.88 ng/ml, range: 0.40 to 2.7 ng/ml, p < 0.025; and median 4 ng/ml, range: 1.9 to 33.6 ng/ml, p < 0.001, respectively). Interleukin-1 alpha was elevated 10 hours after lipopolysaccharide treatment (median 185.3 pg/ml, range: 38 to 511 pg/ml, p < 0.015). Tumor necrosis factor-alpha was not significantly increased in amniotic fluid.

CONCLUSIONS

Preterm delivery after lipopolysaccharide administration is preceded by the appearance of dramatic increases in maternal serum concentrations of tumor necrosis factor-alpha, interleukin-6, and interleukin-1 alpha and in amniotic fluid concentrations of interleukin-6 and interleukin-1 alpha.

<em>Interleukin</em>-<em>15</em> (IL-<em>15</em>) in vitro treatment of peripheral blood mononuclear cells (PBMC) from human immunodeficiency virus (HIV)-infected individuals specifically enhances the function and survival of HIV-specific CD8+ T cells, while in vivo IL-<em>15</em> treatment of mice preferentially expands memory CD8+ T cells. In this study, we investigated the in vivo effect of IL-<em>15</em> treatment in 9 SIVmac251-infected cynomolgus macaques (low dose of IL-<em>15</em>, 10 microg/kg of body weight, n = 3; high dose of IL-<em>15</em>, 100 microg/kg, n = 3; control [saline], n = 3; dose administered twice weekly for 4 weeks). IL-<em>15</em> treatment induced a nearly threefold increase in peripheral blood CD8+CD3- NK cells. Furthermore, CD8+ T-cell numbers increased more than twofold, mainly due to an increase in the CD45RA-CD62L- and CD45RA+CD62L- effector memory CD8+ T cells. Expression of Ki-67 in the CD8+ T cells indicated expansion of CD8+ T cells and not redistribution. IL-<em>15</em> did not affect CD4+ T-cell, B-cell, and CD14+ macrophage numbers. No statistically significant differences in changes from baseline in the viral load were observed when control-, low-dose-, and high-dose-treated animals were compared. No clinical adverse effects were observed in any of the animals studied. The selective expansion of effector memory CD8+ T cells and NK cells by IL-<em>15</em> further supports IL-<em>15</em>'s possible therapeutic use in viral infections such as HIV infection.

This study was designed to identify highly recurrent genetic alterations typical of Sézary syndrome (Sz), an aggressive cutaneous T-cell lymphoma/leukemia, possibly revealing pathogenetic mechanisms and novel therapeutic targets. High-resolution array-based comparative genomic hybridization was done on malignant T cells from 20 patients. Expression levels of selected biologically relevant genes residing within loci with frequent copy number alteration were measured using quantitative PCR. Combined binary ratio labeling-fluorescence in situ hybridization karyotyping was done on malignant cells from five patients. Minimal common regions with copy number alteration occurring in at least 35% of patients harbored <em>15</em> bona fide oncogenes and 3 tumor suppressor genes. Based on the function of the identified oncogenes and tumor suppressor genes, at least three molecular mechanisms are relevant in the pathogenesis of Sz. First, gain of cMYC and loss of cMYC antagonists (MXI1 and MNT) were observed in 75% and 40% to 55% of patients, respectively, which were frequently associated with deregulated gene expression. The presence of cMYC/MAX protein heterodimers in Sézary cells was confirmed using a proximity ligation assay. Second, a region containing TP53 and genome maintenance genes (RPA1/HIC1) was lost in the majority of patients. Third, the <em>interleukin</em> 2 (IL-2) pathway was affected by gain of STAT3/STAT5 and IL-2 (receptor) genes in 75% and 30%, respectively, and loss of TCF8 and DUSP5 in at least 45% of patients. In sum, the Sz genome is characterized by gross chromosomal instability with highly recurrent gains and losses. Prominent among deregulated genes are those encoding cMYC, cMYC-regulating proteins, mediators of MYC-induced apoptosis, and IL-2 signaling pathway components.

<em>Interleukin</em>-12, a cytokine with an important role against intracellular pathogens, promotes Th1 cell development, cellmediated cytotoxicity, and interferon-gamma production. We investigated the immunoregulatory role of IL-12 in 72 chronic hepatitis B virus (HBV) carriers, 33 of whom were monitored longitudinally during interferon-alpha treatment. Serum levels of IL-12 heterodimer, IL-12 p40 subunit, IL-4, and Th1 cytokines were determined by specific ELISAs, and hepatitis B core antigen-specific T cell response by a proliferation assay. Chronic HBV carriers had higher serum levels of IL-12 and IL-12 p40 in comparison with controls (P < 0.01), suggesting that IL-12 production is not impaired. The longitudinal analysis revealed a further substantial increase >> 2.5x baseline level) of bioactive IL-12 and Th1 cytokines in patients who cleared HBV and seroconverted to anti- hepatitis B e, unlike the 23 nonresponders with persistent HBV replication (P < 0.01). The IL-12 peak followed the peak of hepatocytolysis by 9.8+/-2.8 wk and occurred either before or simultaneously with hepatitis B e seroconversion. Hepatitis B core antigen-specific T cell proliferation closely correlated with hepatocytolysis and increased significantly in all patients (8 responders and <em>15</em> nonresponders) who developed hepatitis flare, irrespective of the virological outcome. These results provide in vivo evidence that IL-12 may have an important role for viral clearance in chronic HBV infection.

BACKGROUND

The aim of the study was to better characterise the immunological origin and the behaviour of interleukin (IL)-23-responsive innate lymphoid cells (ILCs) in the gut, synovial fluid (SF) and bone marrow (BM) of patients with ankylosing spondylitis (AS).

METHODS

ILC1, ILC2 and ILC3 cells were determined and characterised by confocal microscopy and flow cytometry in ileal and BM biopsies, in peripheral blood (PB) and SF mononuclear cells obtained from patients with AS and controls. Mucosal vascular addressin cell adhesion molecule 1 (MADCAM-1), IL-7, IL-15 and aggregates of lymphoid tissue inducer cells (LTi) were evaluated by immunohistochemistry. The in vitro ability of epithelial cells in driving the differentiation of ILC3 and the effect of tumour necrosis factor inhibitors (TNFi) on the frequency of ILC3 and the expression of MADCAM1 were also assessed.

RESULTS

ILC3 characterised as Lyn(-)RORc(-)Tbet(+) NKp44(+) cells were significantly expanded in the gut, SF and BM of patients with AS compared with controls, produced high levels of IL-17 and IL-22 and expressed α4β7. MADcAM1 was overexpressed in BM and ileal high endothelial venules. IL-7 was significantly increased in AS gut, especially in the context of Paneth cells, and accompanied by the presence of aggregates of c-kit/IL-7R(+) cells (LTi). In in vitro experiments, epithelial cells from patients with AS actively induced differentiation of ILC3 from LTi. TNFi efficacy was accompanied by a significant decrease in the percentage of intestinal and circulating ILC3 and in the expression of MADCAM1.

CONCLUSIONS

Gut-derived IL-17(+) and IL-22(+)ILC3 are expanded in the peripheral blood, SF and inflamed BM of patients with AS, suggesting the presence of an active homing axis between the gut and the inflamed sacroiliac joints.

Immune evasion is now recognized as a key feature of cancer progression. In animal models, the activity of cytotoxic lymphocytes is suppressed in the tumour microenvironment by the immunosuppressive cytokine, Transforming Growth Factor (TGF)-β. Release from TGF-β-mediated inhibition restores anti-tumour immunity, suggesting a therapeutic strategy for human cancer. We demonstrate that human natural killer (NK) cells are inhibited in a TGF-β dependent manner following chronic contact-dependent interactions with tumour cells in vitro. In vivo, NK cell inhibition was localised to the human tumour microenvironment and primary ovarian tumours conferred TGF-β dependent inhibition upon autologous NK cells ex vivo. TGF-β antagonized the <em>interleukin</em> (IL)-<em>15</em> induced proliferation and gene expression associated with NK cell activation, inhibiting the expression of both NK cell activation receptor molecules and components of the cytotoxic apparatus. <em>Interleukin</em>-<em>15</em> also promotes NK cell survival and IL-<em>15</em> excluded the pro-apoptotic transcription factor FOXO3 from the nucleus. However, this IL-<em>15</em> mediated pathway was unaffected by TGF-β treatment, allowing NK cell survival. This suggested that NK cells in the tumour microenvironment might have their activity restored by TGF-β blockade and both anti-TGF-β antibodies and a small molecule inhibitor of TGF-β signalling restored the effector function of NK cells inhibited by autologous tumour cells. Thus, TGF-β blunts NK cell activation within the human tumour microenvironment but this evasion mechanism can be therapeutically targeted, boosting anti-tumour immunity.

OBJECTIVE

Mikulicz disease has been considered to be a subtype of Sjögren's syndrome (SS). However, recent studies have suggested that Mikulicz disease is an IgG4-related disease and is distinguishable from SS. In addition, it has been reported that both interleukin-4 (IL-4) and IL-10 induce IgG4 production and inhibit IgE. This study was undertaken to examine the expression of these cytokines in patients with Mikulicz disease and patients with SS.

METHODS

Labial salivary gland (LSG) sections from 15 patients with Mikulicz disease and 18 patients with SS were examined for subsets of the infiltrating lymphocytes, expression patterns of messenger RNA (mRNA) for cytokines/chemokines, and relationships between the IgG4:IgG ratio and the expression of mRNA for IL-4 or IL-10.

RESULTS

Immunohistochemical analysis showed lymphocyte infiltration of various subsets in the LSGs of SS patients, and the selective infiltration of IgG4-positive plasma cells and Treg cells in the LSGs of Mikulicz disease patients. The levels of mRNA for both Th1 and Th2 cytokines and chemokines in LSGs from patients with SS were significantly higher than in controls, while the expression of both Th2 and Treg cells was significantly higher in the patients with Mikulicz disease than in controls. Furthermore, the expression of IL-4 or IL-10 in the LSGs was correlated with the IgG4:IgG ratio.

CONCLUSIONS

These results suggest that the pathogenesis of Mikulicz disease is different from that of SS. Mikulicz disease is a unique inflammatory disorder characterized by Th2 and regulatory immune reactions that might play key roles in IgG4 production.

<em>Interleukin</em> 13 (IL-13) shares many biological properties with IL-4, and although the receptor for IL-4 (IL-4R) has been characterized, the expression and structure of IL-13 receptor are unknown. We report here that human renal cell carcinoma (RCC) cells express large numbers of functional IL-13R. Human B lymphocytes and monocytes expressed a very small number of IL-13R, while resting or activated human T cells expressed little or no IL-13R. IL-4 did not compete for IL-13 binding, while IL-13 competed for IL-4 binding, even though IL-4R and IL-13R are structurally distinct on human RCC cells. IL-13 cross-linked with one major protein that is similar in size to the gamma c subunit of IL-2, -4, -7, -9, and -<em>15</em> receptors but was not recognized by anti-gamma c or anti-IL-4R antibodies. IL-4, on the other hand, cross-linked with two major proteins, the smaller of which appears to be similar in size to IL-13R and gamma c, but (like the IL-13R) it did not react with anti-gamma c antibody. Although as shown in this study and in previous studies, gamma c is a functional component of IL-4R in lymphoid cells, it does not appear to be associated with IL-4R on RCC cells. Even in the absence of common gamma chain IL-4 and IL-13 were able to up-regulate intracellular adhesion molecule-1 antigen on RCC cells. These data suggest that the interaction of IL-13 with IL-4R does not involve gamma c and IL-13R itself may be a novel subunit of the IL-4R.

This study aimed to examine the frequency of different subsets of circulating B and T follicular helper (Tfh) cells in patients with new-onset rheumatoid arthritis (RA) and following standard therapies. Twenty-five RA patients and <em>15</em> healthy controls (HC) were recruited for characterizing the frequency of CD27⁺, immunoglobulin (Ig)D⁺, CD86⁺, CD95⁺, Toll-like receptor (TLR)-9⁺ B cells and inducible T cell co-stimulator (ICOS) and programmed death 1 (PD-1)-positive Tfh cells and the level of serum <em>interleukin</em> (IL)-21. The potential correlation between the frequency of different subsets of B and Tfh cells and the values of clinical measures in RA patients was analysed. In comparison with HC, significantly higher percentages of circulating IgD⁺ CD27⁻ CD19⁺ naive B, CD86⁺ CD19⁺ and CD95⁺ CD19⁺ activated B, CD3⁺ CD4⁺ CXCR5⁺, CD3⁺ CD4⁺ CXCR5⁺ ICOS⁺, CD3⁺ CD4⁺ CXCR5⁺ PD-1⁺ and CD3⁺ CD4⁺ CXCR5⁺ ICOS⁺ PD-1⁺ Tfh cells but lower IgD⁺ CD27⁺ CD19⁺ preswitch memory B cells were detected, accompanied by significantly higher levels of serum IL-21 in the RA patients. Furthermore, the percentages of CD95⁺ B cells were correlated positively with the frequency of PD-1⁺ Tfh cells, but negatively with ICOS⁺ Tfh cells. The percentages of CD86⁺ B cells and ICOS⁺ Tfh cells were correlated positively with the values of disease activity score 28 (DAS28). Following the drug therapies for 1 month, the percentages of CD86⁺ B and PD-1⁺ Tfh cells were reduced significantly in the drug-responding patients. Our data suggest that activated B and Tfh cells may contribute to the pathogenesis of RA and the frequency of activated B and Tfh cells may be used as biomarkers for evaluating the therapeutic responses of individual patients with RA.

Purpose T cells genetically modified to express chimeric antigen receptors (CARs) targeting CD19 (CAR-19) have potent activity against acute lymphoblastic leukemia, but fewer results supporting treatment of lymphoma with CAR-19 T cells have been published. Patients with lymphoma that is chemotherapy refractory or relapsed after autologous stem-cell transplantation have a grim prognosis, and new treatments for these patients are clearly needed. Chemotherapy administered before adoptive T-cell transfer has been shown to enhance the antimalignancy activity of adoptively transferred T cells. Patients and Methods We treated 22 patients with advanced-stage lymphoma in a clinical trial of CAR-19 T cells preceded by low-dose chemotherapy. Nineteen patients had diffuse large B-cell lymphoma, two patients had follicular lymphoma, and one patient had mantle cell lymphoma. Patients received a single dose of CAR-19 T cells 2 days after a low-dose chemotherapy conditioning regimen of cyclophosphamide plus fludarabine. Results The overall remission rate was 73% with 55% complete remissions and 18% partial remissions. Eleven of 12 complete remissions are ongoing. Fifty-five percent of patients had grade 3 or 4 neurologic toxicities that completely resolved. The low-dose chemotherapy conditioning regimen depleted blood lymphocytes and increased serum <em>interleukin</em>-<em>15</em> (IL-<em>15</em>). Patients who achieved a remission had a median peak blood CAR+ cell level of 98/μL and those who did not achieve a remission had a median peak blood CAR+ cell level of <em>15</em>/μL ( P = .027). High serum IL-<em>15</em> levels were associated with high peak blood CAR+ cell levels ( P = .001) and remissions of lymphoma ( P < .001). Conclusion CAR-19 T cells preceded by low-dose chemotherapy induced remission of advanced-stage lymphoma, and high serum IL-<em>15</em> levels were associated with the effectiveness of this treatment regimen. CAR-19 T cells will likely become an important treatment for patients with relapsed lymphoma.

From the early 1900s, visceral leishmaniasis (VL; kala-azar) has been among the most important health problems in Sudan, particularly in the main endemic area in the eastern and central regions. Several major epidemics have occurred, the most recent--in Western Upper Nile province in southern Sudan, detected in 1988--claiming over 100,000 lives. The disease spread to other areas that were previously not known to be endemic for VL. A major upsurge in the number of cases was noted in the endemic area. These events triggered renewed interest in the disease. Epidemiological and entomological studies confirmed Phlebotomus orientalis as the vector in several parts of the country, typically associated with Acacia seyal and Balanites aegyptiaca vegetation. Infection rates with Leishmania were high, but subject to seasonal variation, as were the numbers of sand flies. Parasites isolated from humans and sand flies belonged to three zymodemes (MON-18, MON-30 and MON-82), which all belong to the L. donovani sensu lato cluster. Transmission dynamics have not been elucidated fully; heavy transmission in relatively scarcely populated areas such as Dinder national park suggested zoonotic transmission whereas the large numbers of patients with post kala-azar dermal leishmaniasis (PKDL) in heavily affected villages may indicate a human reservoir and anthroponotic transmission. Clinical presentation in adults and in children did not differ significantly, except that children were more anaemic. Fever, weight loss, hepato-splenomegaly and lymphadenopathy were the most common findings. PKDL was much more common than expected (56% of patients with VL developed PKDL), but other post-VL manifestations were also found affecting the eyes (uveitis, conjunctivitis, blepharitis), nasal and/or oral mucosa. Evaluation of diagnostic methods showed that parasitological diagnosis should still be the mainstay in diagnosis, with sensitivities for lymph node, bone marrow and spleen aspirates of 58%, 70% and 96%, respectively. Simple, cheap serological tests are needed. The direct agglutination test (DAT) had a sensitivity of 72%, specificity of 94%, positive predictive value of 78% and negative predictive value of 92%. As with other serological tests, the DAT cannot distinguish between active disease, subclinical infection or past infection. The introduction of freeze-dried antigen and control sera greatly improved the practicality and accuracy of the DAT in the field. An enzyme-linked immunosorbent assay using recombinant K39 antigen had higher sensitivity than DAT (93%). The polymerase chain reaction using peripheral blood gave a sensitivity of 70-93% and was more sensitive than microscopy of lymph node or bone marrow aspirates in patients with suspected VL. The leishmanin skin test (LST) was typically negative during active VL and converted to positive in c. 80% of patients 6 months after treatment. Immunological studies showed that both Th1 and Th2 cell responses could be demonstrated in lymph nodes from VL patients as evidenced by the presence of messenger ribonucleic acid for <em>interleukin</em> (IL)-10, interferon gamma and IL-2. Treatment of peripheral blood mononuclear cells from VL patients with IL-12 was found to drive the immune response toward a Th1 type response with the production of interferon gamma, indicating a potential therapeutic role for IL-12. VL responded well to treatment with sodium stibogluconate, which is still the first line drug at a dose of 20 mg/kg intravenously or intramuscularly per day for <em>15</em>-30 d. Side effects and resistance were rare. Liposomal amphotericin B was effective, with few side effects. Control measures have not been implemented. Based on observations that VL does not occur in individuals who have a positive LST, probably because of previous cutaneous leishmaniasis, a vaccine containing heat-killed L. major promastigotes is currently undergoing a phase III trial.