The <em>interleukin</em>-1 (IL-1) cytokines stimulate the synthesis of degradative enzymes in joint tissues and may play a role in the pathological joint destruction in osteoarthritis (OA). In this study, we have used immunohistochemistry and Western blot analysis to identify IL-1 in human OA cartilage. IL-1 alpha and IL-1 beta were evident in chondrocytes at the articular surface, as well as distributed throughout the cartilage. In many specimens, IL-1 beta but not IL-1 alpha was detected as a diffuse staining of the extracellular matrix especially surrounding superficial zone chondrocytes. Although chondrocyte-associated IL-1 alpha and IL-1 beta were detected in most specimens, cartilages exhibiting early osteoarthritic changes had the highest intensity of staining and the highest frequency of positive cells. Western blot analysis revealed intense immunoreactive bands corresponding to the <em>35</em> kDa precursor form of IL-1 alpha in all four chondrocyte lysates tested. The processed 18 kDa IL-1 beta species was present in only one of four chondrocyte lysates, and there was no clear evidence of precursor form within these cells. The results of this study indicate increased IL-1 alpha in cartilage showing early degenerative changes, suggesting an autocrine/paracrine role for this cytokine in OA pathogenesis.

Human cord blood mononuclear cells were cultured for <em>35</em> days in media containing recombinant human <em>interleukin</em> 5 (rhIL-5) supplemented with a fraction of the culture supernatant of phytohemagglutinin (PHA)-stimulated human T lymphocytes from which <em>interleukin</em> 2 (IL-2) was eliminated. Cultured cells were studied by electron microscopy and an immunogold procedure to detect subcellular site(s) of Charcot-Leyden crystal (CLC) protein. The majority of cells (greater than 70%) developing in this system were mature eosinophils, with descending frequency of other cells, including macrophages, mature basophils, eosinophilic myelocytes, and mature neutrophils. Mature eosinophils were characterized by increased numbers of primary granules, small granules, tubulovesicular structures, and decreased secondary granules. These eosinophils showed extensive piecemeal degranulation (PMD) characterized by partially empty and empty secondary granule chambers in the cytoplasm. Small, smooth vesicles were evident within empty granule chambers as well as adjacent to them. Eosinophils formed close associations with phagocytic macrophages that contained both standard-shaped and irregularly shaped CLC within phagolysosomes. Subcellular sites of CLC protein were demonstrated by immunogold in eosinophils and macrophages arising in these cultures. Charcot-Leyden crystal protein was present in the nuclear matrix and extraorganellar cytoplasm of eosinophils. Primary granules and some cytoplasmic vesicles were labeled for CLC protein, but full and empty secondary granules and the extensive network of tubulovesicles were not. Charcot-Leyden crystals were absent from eosinophils, nor were they present in the extracellular space. Charcot-Leyden crystals were absent from eosinophils, nor were they present in the extracellular space. Charcot-Leyden crystals within phagosomes of macrophages were labeled by the immunogold procedure for CLC protein. These results demonstrate that rhIL-5-supplemented, PHA-stimulated, T-cell-conditioned media induced the development of mature human eosinophils from cord blood cells. These eosinophils underwent PMD of secondary granule contents. Immunogold analysis showed eosinophil CLC protein in the cytoplasm, nucleus, and primary granules of eosinophils. Macrophages also were present in these cultures. They contained CLC protein-containing crystals in their phagosomes, suggesting active sequestration of eosinophil CLC protein by macrophages in vitro.

To assess local expression and plasma levels of pentraxin 3 (PTX3) in patients with giant cell arteritis (GCA).

Plasma and serum samples were obtained from 75 patients with GCA (20 of whom had experienced optic nerve ischemia in the previous 3 weeks and 24 of whom had experienced symptom onset in the previous 6 months and had no history of optic nerve ischemia) and 63 controls (<em>35</em> age-matched healthy subjects, 15 patients with rheumatoid arthritis, and 13 patients with chronic stable angina). In 9 patients in whom GCA was recently diagnosed, circulating levels of <em>interleukin</em>-1β (IL-1β), IL-2, IL-4, IL-6, IL-7, IL-8, IL-10, IL-12p70, CCL2/monocyte chemotactic protein 1, CCL3/macrophage inflammatory protein 1α (MIP-1α), CCL4/MIP-1β, CCL11/eotaxin, CXCL9/monokine induced by interferon-γ, CXCL10/interferon-γ-inducible 10-kd protein, tumor necrosis factor α (TNFα), interferon-γ, vascular endothelial growth factor (VEGF), granulocyte-macrophage colony-stimulating factor, and FasL were measured via a multiplexed cytometric assay. PTX3 and VEGF concentrations were assessed by enzyme-linked immunosorbent assay. PTX3 and CD68 expression were determined by immunohistochemistry and immunofluorescence on temporal artery samples.

GCA patients with very recent optic nerve ischemia had significantly higher PTX3 and VEGF levels compared to other GCA patients and controls. GCA patients with a disease duration of <6 months had significantly higher PTX3 levels compared to other GCA patients and controls. Immunohistochemistry revealed selective PTX3 expression in the wall of inflamed arteries.

Our findings indicate that local expression of PTX3 is a feature of vascular inflammation in GCA; elevated circulating levels of PTX3 identify patients with very recent optic nerve ischemia or a recent diagnosis. Optic nerve ischemia is also associated with increased circulating VEGF levels.

The predominant subclasses of mast cells in both the rat and the mouse can be distinguished from one another by their preferential synthesis of <em>35S</em>-labeled proteoglycans that contain either heparin or oversulfated chondroitin sulfate glycosaminoglycans. Although [<em>35S</em>]heparin proteoglycans have been isolated from human lung mast cells of 40-70% purity and from a skin biopsy specimen of a patient with urticaria pigmentosa, no highly sulfated chondroitin sulfate proteoglycan has been isolated from any enriched or highly purified population of human mast cells. We here demonstrate that human lung mast cells of 96% purity incorporate [<em>35S</em>] sulfate into separate heparin and chondroitin sulfate proteoglycans in an approximately equal to 2:1 ratio. As assessed by HPLC of the chondroitinase ABC digests, the chondroitin [<em>35S</em>]sulfate proteoglycans isolated from these human lung mast cells contain the same unusual chondroitin sulfate E disaccharide that is present in proteoglycans produced by <em>interleukin</em> 3-dependent mucosal-like mouse mast cells. Both the chondroitin [<em>35S</em>]sulfate E proteoglycans and the [<em>35S</em>]heparin proteoglycans were exocytosed from the [<em>35S</em>]sulfate-labeled cells via perturbation of the IgE receptor, indicating that both types of <em>35S</em>-labeled proteoglycans reside in the secretory granules of these human lung mast cells.

<em>Interleukin</em> 10 (IL-10) has the physiological role of down-regulating cell-mediated immunity. We have recently reported that mRNA for IL-10 was present in most metastatic melanoma tissues. The purpose of this investigation was to determine whether melanoma metastases produce IL-10 protein. Single-cell suspensions were prepared by enzymatic dissociation of 28 lymph node metastases and 7 s.c. metastases and cryopreserved. Of these <em>35</em> samples, 30 produced IL-10 after a 24-h incubation (median, 125.1 pg/ml). IL-10 production was slightly diminished after 25 Gy irradiation but almost completely abrogated after modification with the hapten dinitrophenyl. After 7 or 14 days in tissue culture, melanoma cells continued to produce IL-10 but only at about 10% of the levels of freshly dissociated tissues. Moreover, of eight melanoma cell lines established from these cultures, only one produced IL-10 protein. To determine whether IL-10 was produced by melanoma cells or tumor-associated leukocytes, single-cell suspensions were fractionated with anti-CD45 antibody-conjugated magnetic beads. In four of five samples, IL-10 production was increased by depletion of leukocytes, suggesting that the primary source was the melanoma cells themselves. This was confirmed by immunohistochemical staining of cytospin preparations and frozen tissue sections. Finally, 10 of 55 patients with clinically evident metastases showed elevations of circulating IL-10; three patients who had been melanoma-free developed high serum IL-10 levels, concurrent with the appearance of distant metastases. These data indicate that production of IL-10 is characteristic of metastatic melanomas and raise the possibility that this cytokine allows tumors to avoid or to modulate immunological attack.

Multiple sclerosis (MS) is a chronic debilitating disease of the central nervous system primarily mediated by T lymphocytes with specificity to neuronal antigens in genetically susceptible individuals. On the other hand, myasthenia gravis (MG) primarily involves destruction of the neuromuscular junction by antibodies specific to the acetylcholine receptor. Both autoimmune diseases are thought to result from loss of self-tolerance, which allows for the development and function of autoreactive lymphocytes. Although the mechanisms underlying compromised self-tolerance in these and other autoimmune diseases have not been fully elucidated, one possibility is numerical, functional, and/or migratory deficits in T regulatory cells (Tregs). Tregs are thought to play a critical role in the maintenance of peripheral immune tolerance. It is believed that Tregs function by suppressing the effector CD4+ T cell subsets that mediate autoimmune responses. Dysregulation of suppressive and migratory markers on Tregs have been linked to the pathogenesis of both MS and MG. For example, genetic abnormalities have been found in Treg suppressive markers CTLA-4 and CD25, while others have shown a decreased expression of FoxP3 and IL-10. Furthermore, elevated levels of pro-inflammatory cytokines such as IL-6, IL-17, and IFN-γ secreted by T effectors have been noted in MS and MG patients. This review provides several strategies of treatment which have been shown to be effective or are proposed as potential therapies to restore the function of various Treg subsets including Tr1, iTr<em>35</em>, nTregs, and iTregs. Strategies focusing on enhancing the Treg function find importance in cytokines TGF-β, IDO, <em>interleukins</em> 10, 27, and <em>35</em>, and ligands Jagged-1 and OX40L. Likewise, strategies which affect Treg migration involve chemokines CCL17 and CXCL11. In pre-clinical animal models of experimental autoimmune encephalomyelitis (EAE) and experimental autoimmune myasthenia gravis (EAMG), several strategies have been shown to ameliorate the disease and thus appear promising for treating patients with MS or MG.

<em>Interleukin</em>-6 (IL-6) was infused intravenously for 2.5 h in seven healthy human volunteers at a dose giving rise to a circulating IL-6 concentration of approximately <em>35</em> ng l(-1). The metabolic effects of this infusion were studied in subcutaneous adipose tissue on the anterior abdominal wall and in the splanchnic tissues by the Fick principle after catheterizations of an artery, a subcutaneous vein draining adipose tissue, and a hepatic vein, and measurements of regional adipose tissue and splanchnic blood flows. In control studies without IL-6 infusion subcutaneous adipose tissue metabolism was studied by the same technique in eight healthy subjects. The net release of glycerol and fatty acids from the subcutaneous abdominal adipose tissue remained constant in the control experiment. IL-6 infusion gave rise to increase in net glycerol release in subcutaneous adipose tissue while the net release of fatty acids did not change significantly. In the splanchnic region IL-6 elicited a pronounced vasodilatation, and the uptake of fatty acids and the gluconeogenic precursors glycerol and lactate increased significantly. The splanchnic net output of glucose and triacylglycerol did not change during the IL-6 infusion. It is concluded that IL-6 elicits lipolytic effects in human adipose tissue in vivo, and that IL-6 also has effects on the splanchnic lipid and carbohydrate metabolism.

Human monocytic ehrlichiosis is caused by a tick-transmitted rickettsia, Ehrlichia chaffeensis. We recently reported that E. chaffeensis grown in tick cells expresses different proteins than bacteria grown in macrophages. Therefore, we tested the hypothesis that immune responses against E. chaffeensis would be different if the mice are challenged with bacteria grown in macrophages or tick cells. We assessed the E. chaffeensis clearance from the peritoneum, spleen, and liver by C57BL/6J mice using a TaqMan-based real-time reverse transcription-PCR assay. Macrophage-grown E. chaffeensis was cleared in 2 weeks from the peritoneum, whereas the pathogen from tick cells persisted for nine additional days and included three relapses of increasing bacterial load separated by three-day intervals. Tick cell-grown bacteria also persisted in the livers and spleens with higher bacterial loads compared to macrophage-grown bacteria and fluctuated over a period of <em>35</em> days. Three-day periodic cycles were detected in T-cell CD62L/CD44 ratios in the spleen and bone marrow in response to infections with both tick cell- and macrophage-grown bacteria and were accompanied by similar periodic cycles of spleen cell cytokine secretions and nitric oxide and <em>interleukin</em>-6 by peritoneal macrophages. The E. chaffeensis-specific immunoglobulin G response was considerably higher and steadily increased in mice infected with the tick cell-derived E. chaffeensis compared to DH82-grown bacteria. In addition, antigens detected by the immunoglobulins were significantly different between mice infected with the E. chaffeensis originating from tick cells or macrophages. The differences in the immune response to tick cell-grown bacteria compared to macrophage-grown bacteria reflected a delay in the shift of gene expression from the tick cell-specific Omp 14 gene to the macrophage-specific Omp 19 gene. These data suggest that the host response to E. chaffeensis depends on the source of the bacteria and that this experimental model requires the most natural inoculum possible to allow for a realistic understanding of host resistance.

OBJECTIVE

To test the hypothesis that rheumatoid arthritis (RA) influenced levels of salivary biomarkers of periodontal disease.

METHODS

Medical assessments, periodontal examinations and pain ratings were obtained from <em>35</em> RA, <em>35</em> chronic periodontitis and <em>35</em> age- and gender-matched healthy controls in a cross-sectional, case-controlled study. Unstimulated whole saliva samples were analysed for <em>interleukin</em>-1β (IL-1β), matrix metalloproteinase-8 (MMP-8) and tumour necrosis factor-α (TNF-α) concentrations.

RESULTS

The arthritis and healthy groups had significantly less oral disease than the periodontitis group (P<0.0001), with the arthritis group having significantly more sites bleeding on probing (BOP) than matched controls (P=0.012). Salivary levels of MMP-8 and IL-1β were significantly elevated in the periodontal disease group (P<0.002), and IL-1β was the only biomarker with significantly higher levels in the arthritis group compared with controls (P=0.002). Arthritis patients receiving anti-TNF-α antibody therapy had significantly lower IL-1β and TNF-α levels compared with arthritis patients not on anti-TNF-α therapy (P=0.016, 0.024) and healthy controls (P<0.001, P=0.011), respectively.

CONCLUSIONS

RA patients have higher levels of periodontal inflammation than healthy controls, i.e., an increased BOP. Systemic inflammation appears to influence levels of select salivary biomarkers of periodontal disease, and anti-TNF-α antibody-based disease-modifying therapy significantly lowers salivary IL-1β and TNF-α levels in RA.

Flaxseed and its components may improve cardiovascular health because of their numerous attributes. Flaxseed contains <em>35</em>% of its mass as oil, of which 55% is alpha-linolenic acid (ALA). Flax meal, which is devoid of oil, contains the lignan secoisolariciresinol diglucoside (SDG). Flaxseed, flaxseed with very low ALA, flaxseed oil, flax lignan complex (FLC), and SDG reduce the development of hypercholesterolemic atherosclerosis by 46%, 69%, 0%, 73%, and 34%, respectively, in the rabbit model. FLC and SDG slow the progression of atherosclerosis but have no effect in regression of atherosclerosis. Suppression of atherosclerosis by flaxseed is the result of its lignan content and not the result of ALA content. Suppression of atherosclerosis is associated with lowering of serum lipids and antioxidant activity. Effects of flaxseed on serum lipids in experimental animals are variable from no change to slight reduction. Flaxseed oil does not affect serum lipids, except for a slight reduction in serum triglycerides. Lignan in general reduces serum total cholesterol and low-density lipoprotein cholesterol and raises serum high-density lipoprotein cholesterol. SDG and its metabolites have antioxidant activity. Flaxseed and flaxseed oil do not have antioxidant activity except they suppress oxygen radical production by white blood cells. Flaxseed oil/ALA has variable effects on inflammatory mediators/markers (<em>interleukin</em> [IL]-1beta, IL-2, IL-4, IL-6, IL-10, tumor necrosis factor-alpha, interferon-gamma, C-reactive protein, and serum amyloid A). Doses of ALA less than 14 g/d do not affect inflammatory mediators/markers, but 14 g/d or greater reduce inflammatory mediators/markers. Flaxseed oil decreases soluble vascular cell adhesion molecule-1 but has no effect on soluble intracellular adhesion molecule-1, soluble E-selectin, and monocyte colony-stimulating factor. Flaxseed has variable effects on IL-6, high-sensitivity C-reactive protein, and soluble vascular cell adhesion molecule-1. FLC reduces plasma levels of C-reactive protein but has no effects on IL-6, tumor necrosis factor-alpha, soluble intracellular adhesion molecule-1, soluble vascular cell adhesion molecule-1, or monocyte chemoattractant protein. Flaxseed has a very small hypotensive effect, but flaxseed oil does not lower blood pressure. However, SDG is a very potent hypotensive agent. Flaxseed oil decreases platelet aggregation and increases platelet activating inhibitor-1 and bleeding time. Flaxseed and FLC have no effect on the hemopoietic system. SDG is a potent angiogenic and antiapoptotic agent that may have a role in cardioprotection in ischemic heart disease. In conclusion, flaxseed, FLC, and SDG, but not flaxseed oil, suppress atherosclerosis, and FLC and SDG slow progression of atherosclerosis but have no effect on regression. Flaxseed oil suppresses oxygen radical production by white blood cells, prolongs bleeding time, and in higher doses suppresses serum levels of inflammatory mediators and does not lower serum lipids.

OBJECTIVE

Increased serum inflammatory markers have been found in obesity and insulin-resistant states, and could play a causative role in insulin resistance, atherosclerosis and cardiovascular disease. The polycystic ovary syndrome represents a human model of insulin resistance because both lean and obese polycystic ovary syndrome patients are insulin-resistant compared with non-hyperandrogenic women. We evaluated whether obesity, insulin resistance, or both, are related to the increased concentrations of inflammatory markers in pre-menopausal women.

METHODS

We compared <em>35</em> patients with polycystic ovary syndrome and 28 healthy women, paired for BMI, prevalence of obesity and smoking. Measurements included serum inflammatory markers, BMI, waist-to-hip ratio, blood pressure, serum glucose, insulin, lipid and hormone concentrations, and insulin sensitivity index.

RESULTS

The insulin sensitivity index was reduced in polycystic ovary syndrome patients compared with controls. However, no differences were observed between both groups in C-reactive protein, interleukin 6, tumour necrosis factor-alpha, soluble type 2 tumour necrosis factor receptor, and soluble intercellular cell adhesion molecule-1. When considering patients and controls as a whole, C-reactive protein and interleukin 6, were increased in obese subjects compared with lean women. Inverse correlations existed between insulin sensitivity index and C-reactive protein, interleukin 6, tumour necrosis factor-alpha, soluble type 2 tumour necrosis factor receptor, and soluble intercellular cell adhesion molecule-1. Only the weak correlation with C-reactive protein persisted after controlling for BMI.

CONCLUSIONS

Obesity, and not insulin resistance, is the major determinant of serum inflammatory cardiovascular risk markers in pre-menopausal women.

Culture of murine splenic B cells with <em>interleukin</em> 4 (IL-4) caused the up-regulation of the lymphocyte Fc receptor for immunoglobulin E (IgE) (Fc epsilon R) over a similar dose range as required for Ia up-regulation. However, the expression level of the Fc receptor for immunoglobulin G (Fc gamma R) did not increase, rather IL-4 caused a slight but consistent decrease in the Fc gamma R level on the B cells. Fc epsilon R+ B hybridoma cells also responded to IL-4 by exhibiting increased Fc epsilon R expression; with the hybridoma cells Fc gamma R levels were unaffected. IL-4 caused an increase in the number of Fc epsilon R per cell and the highest levels of expression were obtained by having both IgE and IL-4 present in the culture. The specificity of the increase was demonstrated by blocking IL-4-mediated actions with monoclonal anti-IL-4 (11B11). Experiments following the incorporation of [<em>35S</em>]methionine into the Fc epsilon R demonstrated that IL-4 increased the rate of Fc epsilon R biosynthesis; this provides an explanation for the IL-4-induced increase in Fc epsilon R expression. IL-4, unlike IgE, had no effect on the rate of degradation of the Fc epsilon R. Interferon-gamma (IFN-gamma) totally abrogated IL-4-mediated Fc epsilon R up-regulation; at the same concentration of IFN-gamma Ia up-regulation is also suppressed, although not as effectively. IFN-gamma was shown to directly suppress Fc epsilon R synthesis, thereby explaining the inhibitory action on Fc epsilon R levels. Finally, it was shown that 11B11 inhibited the increased expression of Fc epsilon R on B cells obtained from mice during the early, but not the late, stages of Nippostrongylus brasiliensis infection. This latter finding suggests that the high Fc epsilon R levels seen early in parasite infections are dependent upon IL-4. The results overall provide further insight into the biologic activities of IL-4.

BACKGROUND

Differential protein targeting by HIV-specific CD8 T cells is associated with disparate plasma viral loads; however, it is unclear if the quality of these responses differs depending upon the specificity of the targeted epitopes.

METHODS

We examined HIV-specific CD8 T-cell responses in HIV-infected adolescents carrying either an HLA class I allele associated with a favorable prognosis (HLA-B*57) or an allele associated with usual disease progression (HLA-B*<em>35</em> or HLA-B*53) using interferon-gamma ELISpot and ICS assays.

RESULTS

In an interferon-gamma ELISpot assay, p24 was the dominant protein targeted by B*57 carriers while responses to Nef dominated in B*<em>35</em> or B*53 positive carriers. This differential protein targeting did not change during 4 years of follow-up. In these chronically infected adolescents, there were no significant differences in the quality of the immunodominant T-cell responses between the B*57 and B*<em>35</em>/B*53 carriers as measured by peptide avidity, degranulation, and immune memory markers. There was a trend towards higher expression of interleukin-2 from B*57-KF11 restricted CD8 T cells although this difference was not significant. Nevertheless both B*57 and B*<em>35</em>/53-restricted responses were relatively potent as reflected by the propensity of CD8 T cells to escape in p24 and Nef, respectively.

CONCLUSIONS

Differential protein targeting rather than the quality of T-cell responses appears to be a major distinguishing feature of HIV-specific CD8 T cells induced in B*57 carriers. These data suggest that viral fitness costs associated with CD8 T-cell pressure is an important factor determining differences in the viral load among HIV-infected patients.

Air pollution contributes to both exacerbation and development of bronchial asthma. Studies showed that coexposure to air pollution directly promotes sensitization to inhaled allergen in neonatal mice. The aim of this study was to investigate whether prenatal exposure to air pollution could also increase susceptibility to development of asthma in early life. Pregnant female BALB/c mice were exposed to aerosolized leachate of residual oil fly ash (ROFA, 50 mg/ml, 30 min) at 5, 3, and 1 d before delivery. Offspring were treated once at 3 d of age with ovalbumin (OVA, 5 mug) and alum (ip), an intentionally suboptimal dose for sensitization, exposed to aerosolized OVA (1%, 10 min) at 12-14 d or 32-<em>35</em> d of age, and evaluated 2 d after the final exposure. The offspring of ROFA-exposed mothers (ROFA group) revealed increasing airway hyperresponsiveness (higher enhanced pause [Penh] to methacholine challenge) and elevated substantial numbers of eosinophils in the bronchoalveolar lavage flued (BALF). Histopathology revealed prominent inflammation in the lungs of ROFA group and showed increased allergen-specific IgE and IgG1 levels. Their cultured splenocytes showed an enhanced <em>interleukin</em> (IL)-4/interferon (IFN)-gamma cytokine, indicating Th2 skewed immunity. Data indicate that exposure of pregnant female mice to an air pollutant aerosol increased asthma susceptibility in their offspring.

<em>Interleukin</em> (IL)-<em>35</em> is a novel heterodimeric cytokine in the IL-12 family and is composed of two subunits: Epstein-Barr virus-induced gene 3 (EBI3) and IL-12p<em>35</em>. IL-<em>35</em> is expressed in T regulatory (Treg) cells and contributes to the immune suppression function of these cells. In contrast, we found that both IL-<em>35</em> subunits were expressed concurrently in most human cancer cell lines compared to normal cell lines. In addition, we found that TNF-α and IFN-γ stimulation led to increased IL-<em>35</em> expression in human cancer cells. Furthermore, over-expression of IL-<em>35</em> in human cancer cells suppressed cell growth in vitro, induced cell cycle arrest at the G1 phase, and mediated robust apoptosis induced by serum starvation, TNF-α, and IFN-γ stimulation through the up-regulation of Fas and concurrent down-regulation of cyclinD1, survivin, and Bcl-2 expression. In conclusion, our results reveal a novel functional role for IL-<em>35</em> in suppressing cancer activity, inhibiting cancer cell growth, and increasing the apoptosis sensitivity of human cancer cells through the regulation of genes related to the cell cycle and apoptosis. Thus, this research provides new insights into IL-<em>35</em> function and presents a possible target for the development of novel cancer therapies.

As assessed by ultrastructure, histochemical staining, and T-cell dependency, in vitro-differentiated inter-leukin 3-dependent mouse mast cells are comparable to the mast cells that reside in the gastrointestinal mucosa but not in the skin or the serosal cavity of the mouse. We now demonstrate that when cloned <em>interleukin</em> 3-dependent mast cells are cocultured with mouse skin-derived 3T3 fibroblasts in the presence of WEHI-3 conditioned medium for 28 days, the mast cells acquire the ability to stain with safranin, increase their histamine content approximately equal to 50-fold and their carboxypeptidase A content approximately equal to 100-fold, and augment approximately equal to 45-fold their biosynthesis of proteoglycans bearing <em>35S</em>-labeled heparin relative to <em>35S</em>-labeled chondroitin sulfate glycosaminoglycans. Thus, fibroblasts induce <em>interleukin</em> 3-dependent mouse mast cells to change phenotype from mucosal-like to connective tissue-like, indicating that the biochemical and functional characteristics of this mast cell type are strongly influenced by the connective tissue microenvironment.

Platelet-activating factor (PAF) is a potent phospholipid with diverse physiological and pathological actions, and it is inactivated by PAF acetylhydrolase. In this study, we analyzed the tissue distribution of the plasma PAF acetylhydrolase mRNA in humans. We isolated a 3.5-kilobase fragment containing the 5' genomic sequence of the plasma PAF acetylhydrolase gene and further characterized the promoter activity. We determined the transcriptional initiation site by primer extension. We then prepared constructs containing various lengths of 5' genomic fragments fused to a luciferase reporter gene and transfected these constructs into COS-7 cells. We found that there is more than one region in the 1.3-kilobase 5' genomic sequence conferring promoter activity and that a very short 5'-flanking region (72 base pairs) is sufficient for more than 65% of the basal activity. In parallel, we examined the regulation of expression of the PAF acetylhydrolase gene. We found that interferon-gamma (IFNgamma) and lipopolysaccharide (LPS) significantly inhibited synthesis of PAF acetylhydrolase, whereas other cytokines, including IFNalpha, <em>interleukin</em> (IL) 1alpha, IL4, IL6, tumor necrosis factor-alpha, granulocyte/macrophage colony-stimulating factor, and macrophage colony-stimulating factor, had a smaller or no effect in human monocyte-derived macrophages. Furthermore, transfection of the promoter/reporter construct into macrophage RAW264.7 cells revealed that IFNgamma and LPS decreased the promoter activity by <em>35</em>% and 50%, respectively, whereas PAF stimulated it by 52% via its receptor. The promoter activity was much lower in monocytic U937 cells compared with the basal level in COS-7 cells, while the activities in P388D1 and RAW264.7 macrophagic cells were considerably higher than the basal level in COS-7 cells. There are multiple regions in the PAF acetylhydrolase promoter that contain responsive elements for signal transducer and activators of transcription-related proteins, and also for myeloid-specific transcription factors. Our data indicate that the opposite of mRNA expression in monocytes versus macrophages is due to inhibition of the promoter activity in the former and activation in the latter cells.

Mycoplasma fermentans-derived high-molecular-weight material (MDHM) was originally discovered because of its capacity to generate, through the induction of monokine synthesis, cytolytic T lymphocytes in concanavalin A-stimulated thymocyte cultures. This study shows that MDHM-activated macrophages not only released <em>interleukin</em>-6 (IL-6) but also exhibited increased synthesis of cell-associated IL-1 as well as liberation of tumor necrosis factor and prostaglandin. We determined 6-keto prostaglandin F1 alpha since it is the stable metabolite of the bioactive prostacyclin. MDHM appeared to be as potent as lipopolysaccharide in inducing the synthesis of these mediators. Priming with gamma interferon further increased MDHM-mediated IL-6 release. Since monokines can be pyrogenic, we tested the effects of an intravenous injection of MDHM on rectal temperatures and leukocyte counts in rabbits. At 1 h after a bolus injection of MDHM, leukocyte counts dropped to about <em>35</em>% of the initial values, reflecting a decrease in both lymphocytes and granulocytes. At 4 to 6 h after injection, granulocyte counts began to increase again, whereas lymphocyte counts remained low. No leukocytosis was noted during this time. The lack of leukocytosis can be explained by the failure of MDHM-stimulated macrophages to release IL-1. The property of MDHM to cause IL-6 release from macrophages and the IL-6 growth dependency of the 7TD1 hybridoma cell line were made use of in a coculture assay system to quantitate the activity of MDHM. With this method and macrophages from C3H/HeJ lipopolysaccharide-nonresponder mice, MDHM activity was found to be equally distributed in the mycoplasma growth medium and the sedimented mycoplasmas after sonication.

We investigated the potential role of different proteases in the death of cultured rat hippocampal pyramidal neurons induced by beta-amyloid (A beta) (25-<em>35</em>). Both A beta(25-<em>35</em>)- and staurosporine-induced death of these neurons appeared to involve apoptosis, as indicated using Hoechst 33342 and terminal dUDP nick end labeling staining, whereas NMDA-induced death appeared more complex. Two irreversible inhibitors of the <em>interleukin</em>-1 beta converting enzyme (ICE) and related proteases, Z-Val-Ala-Asp-CH2F and acetyl-Tyr-Val-Ala-Asp-chloromethyl ketone, blocked neuronal death produced by A beta(25-<em>35</em>), staurosporine, and NMDA to differing extents. Furthermore, MDL 28,170, a selective inhibitor of the calcium-regulated protease calpain, also inhibited death induced by all agents. A beta(25-<em>35</em>) and staurosporine stimulated the breakdown of the protein spectrin, a calpain substrate. Spectrin breakdown was inhibited by MDL 28,170 but not by ICE inhibitors. Leupeptin was only effective in preventing NMDA-induced death. These results support the role of apoptosis in neuronal death due to A beta(25-<em>35</em>) treatment and also suggest a role for calcium-regulated proteases in this process.

BACKGROUND

c-Kit and platelet-derived growth factor receptor (PDGFR) are potential molecular targets in epithelial ovarian cancer (EOC). Imatinib inhibits the kinase domain and subsequent downstream signaling of these receptor tyrosine kinases. The objective of this study was to investigate biochemical and biologic effects of imatinib on EOC.

METHODS

Patients with recurrent EOC who had received no more than 4 prior regimens and who had good end-organ function were eligible. Imatinib was administered orally at a dose of 400 mg twice daily in continuous, 28-day cycles with reassessment imaging studies obtained every other cycle. Tumor core biopsies were obtained prior to and at 4 weeks into therapy; microdissected tumor and stroma were subjected to protein lysate array analysis. Blood samples were obtained monthly for cytokine measurements.

RESULTS

Twenty-three patients were enrolled, including 16 patients who received imatinib 600 mg daily because of gastrointestinal (GI) toxicity and fluid accumulation at the starting dose. The median time to disease progression was 2 months (range, 2-14 months). Common grade 3 toxicities included edema/ascites/pleural effusions in 11 patients (48%), GI complaints in 8 patients (<em>35</em>%), fatigue in 3 patients (13%), and grade 2 and 3 cytopenias in 10 patients and 3 patients (43% and 13%), respectively. Increased circulating levels of <em>interleukin</em> 6 were associated with grade>>/=2 fluid collection (P = .02). A statistically significant trend was observed between pretreatment phosphorylated-kit levels in microdissected tumor and stroma and GI toxicity (P < .01), between tumor levels of epidermal growth factor receptor (EGFR) and PDGFR with grade of fatigue (P </= .005), and EGFR and phosphorylated-AKT levels with grade of ascites and edema (P </= .01).

CONCLUSIONS

The results of this study indicated imatinib had minimal activity as a single agent in EOC. Its ability to modulate its molecular targets suggests that it may be considered in combinatorial therapy.

OBJECTIVE

To determine the regulating role of interleukin-1 alpha and beta (IL-1 alpha, beta) and tumor necrosis factor alpha (TNF-alpha) on inhibition of proteoglycan synthesis and proteoglycan degradation in early immune complex arthritis (ICA) in the mouse.

METHODS

In the early phases of arthritis, IL-1 and TNF were measured using cytokine specific bioassays, the NOB.1 EL-4 and L929 assay, respectively. The impact of IL-1 in proteoglycan synthesis was studied by neutralizing the formed IL-1 during early arthritis either by giving anti-IL-1 specific antibodies intravenously or IL-1 receptor antagonist (IL-1ra) intraperitoneally by osmotic pumps. TNF-alpha was neutralized by giving monoclonal antibodies directed against murine TNF-alpha. Synthesis of proteoglycans was measured ex vivo by uptake of 35S-sulfate by patellae derived from inflamed and control, noninflamed knee joints. In vivo formation of 35S-sulfate labeled proteoglycans was studied by autoradiography. Degradation of proteoglycans was measured by labeling patellae in vivo with 35S-sulfate before arthritis induction.

RESULTS

High levels of IL-1 are formed during the first phase of immune complex arthritis (ICA). Neutralization of either IL-1 alpha or beta with specific polyclonal antibodies resulted only in partial blocking, whereas a combination fully blocked inhibition of proteoglycan synthesis. Full blocking was also found after systemic treatment with high amounts of IL-1 receptor antagonist (1.2 mg/day during 3 days). Influx of cells was also significantly reduced both in the anti-IL-1 as well as in the IL-1ra treated groups. Whether infiltrating cells are involved in inhibition of proteoglycan synthesis was further investigated in neutropenic mice. Significantly higher levels of IL-1 were found in arthritic joints of neutropenic compared with control mice. Suppression of proteoglycan synthesis was similar in arthritic knee joints of normal and neutropenic mice. However, only minor proteoglycan degradation was found in the latter. TNF-alpha was undetectable in the bioassay in early ICA and neutralization of TNF-alpha did not change either swelling, cell influx, proteoglycan synthesis or proteoglycan degradation.

CONCLUSIONS

Local production of IL-1 in ICA in knee joints seems directly responsible for inhibition of proteoglycan synthesis. A direct role of IL-1 in proteoglycan loss is unlikely, but indirectly IL-1 may be involved in proteoglycan breakdown by attracting inflammatory leukocytes and activating synovial cells. TNF-alpha seemed to have no effect on either cell influx, proteoglycan synthesis or proteoglycan degradation in this model.

Signals propagated via the gp130 subunit of the <em>interleukin</em>-6 (IL-6)-type cytokine receptors mediate, among various cellular responses, proliferation of hematopoietic cells and induction of acute-phase plasma protein (APP) genes in hepatic cells. Hematopoietic growth control by gp130 is critically dependent on activation of both STAT3 and protein tyrosine phosphatase 2 (SHP-2). To investigate whether induction of APP genes has a similar requirement for SHP-2, we constructed two chimeric receptors, G-gp130 and G-gp130(Y2F), consisting of the transmembrane and cytoplasmic domains of gp130 harboring either a wild-type or a mutated SHP-2 binding site, respectively, fused to the extracellular domain of the granulocyte colony-stimulating factor (G-CSF) receptor. Rat hepatoma H-<em>35</em> cells stably expressing the chimeric receptors were generated by retroviral transduction. Both chimeric receptors transmitted a G-CSF-induced signal characteristic of that triggered by IL-6 through the endogenous gp130 receptor; i.e., both activated the appropriate JAK, induced DNA binding activity by STAT1 and STAT3, and up-regulated expression of the target APP genes, those for alpha-fibrinogen and haptoglobin. Notwithstanding these similarities in the patterns of signaling responses elicited, mutation of the SHP-2 interaction site in G-gp130(Y2F) abrogated ligand-activated receptor recruitment of SHP-2 as expected. Moreover, the tyrosine phosphorylation state of the chimeric receptor, the associated JAK activity, and the induced DNA binding activity of STAT1 and STAT3 were maintained at elevated levels and for an extended period of time in G-gp130(Y2F)-expressing cells following G-CSF treatment compared to that in cells displaying the G-gp130 receptor. H-<em>35</em> cells ectopically expressing G-gp130(Y2F) were also found to display an enhanced sensitivity to G-CSF and a higher level of induction of APP genes. Overexpression of the enzymatically inactive SHP-2 enhanced the signaling by the wild-type but not by the Y2F mutant G-gp130 receptor. These results indicate that gp130 signaling for APP gene induction in hepatic cells differs qualitatively from that controlling the proliferative response in hematopoietic cells in not being strictly dependent on SHP-2. The data further suggest that SHP-2 functions normally to attenuate gp130-mediated signaling in hepatic (and, perhaps, other) cells by moderating JAK action.

GLUT-4 (glucose transporter) receptor, tumor necrosis factor-alpha (TNF-alpha), <em>interleukins</em>-6 (IL-6), daf-genes and PPARs (peroxisomal proliferation activator receptors) play a role in the development of insulin resistance syndrome and associated conditions. But, the exact interaction between these molecules/factors and the mechanism(s) by which they produce insulin resistance syndrome is not clear. I propose that a defect in the activity of the enzymes Delta6 and Delta5 desaturases that are essential for the formation of long chain metabolites of essential fatty acids, linoleic acid and alpha-linolenic acid, is a factor in the development of insulin resistance syndrome. Long chain polyunsaturated fatty acids (LCPUFAs) increase cell membrane fluidity and enhance the number of insulin receptors and the affinity of insulin to its receptors; suppress TNF-alpha, IL-6, macrophage migration inhibitory factor (MIF) and leptin synthesis; increase the number of GLUT-4 receptors, serve as endogenous ligands of PPARs, modify lipolysis, and regulate the balance between pro- and anti-oxidants, and thus, play a critical role in the pathogenesis of insulin resistance. In the nematode, Caenorhabditis elegans, the protein encoded by daf-2 is <em>35</em>% identical to the human insulin receptor; daf-7 codes a transforming growth factor-beta (TGF-beta) type signal and daf-16 enhances superoxide dismutase (SOD) expression. Melatonin has anti-oxidant actions similar to daf-16, TGF-beta and SOD. Calorie restriction enhances the activity of Delta6 and Delta5 desaturases, melatonin production, decreases daf-2 signaling, free radical generation, and augments anti-oxidant defenses that may explain the beneficial effect of diet control in the management of obesity, insulin resistance, and type II diabetes mellitus. These evidences suggest that the activities of Delta6 and Delta5 enzymes play a critical role in the expression and regulation of GLUT-4, TNF-alpha, IL-6, MIF, daf-genes, melatonin, and leptin by modulating the synthesis and tissue concentrations of LCPUFAs. Caloric restriction delays ageing by activating Sir 2 deacetylase in yeast, and expression of Sir 2 (SIRT1) in human cells. Both insulin and insulin-like growth factor-1 (IGF-1) attenuated this response. SIRT1 sequesters the proapoptotic factor Bax, prevents stress-induced apoptosis of cells, and thus, prolongs survival. In addition, SIRT1 repressed PPAR-gamma, and overexpression of SIRT1 attenuated adipogenesis, and upregulation of SIRT in differentiated fat cells triggered lipolysis and loss of fat, events that are known to attenuate insulin resistance and prolong life span. It remains to be seen whether LCPUFAs have a regulatory role in SIRT1 expression and control Sir 2 deacetylase activity. Thus, calorie restriction or reduced food intake has a role not only in the pathobiology of insulin resistance, but also in other associated conditions such as obesity, type II diabetes mellitus, ageing, and longevity.

OBJECTIVE

To investigate possible causes of hypocalcemia and to assess parathyroid hormone (PTH) secretion in intensive care unit (ICU) patients.

METHODS

Combined cross-sectional and prospective study.

METHODS

ICU in a university hospital.

METHODS

Thirteen patients with sepsis and 13 patients who underwent major surgery.

METHODS

None.

RESULTS

Calcium metabolic indices were investigated during the first 24 hrs in the ICU and after 2 days. Eight of the surgical patients and five of the septic patients were subjected to a citrate/calcium infusion on day 1 in the ICU, to study the dynamics of PTH secretion. The blood ionized calcium (Ca2+) concentration was generally low in the septic patients (mean +/- SD, 1.03+/-0.08 mmol/L; reference value, 1.10-1.30) and increased, but not normalized, after 2 days. Hypocalcemia was only occasionally seen in the surgical patients. In the septic patients, urinary excretion of calcium was low; and, in both patient groups, elevated concentrations of two markers of bone resorption, deoxypyridinoline and ICTP (serum carboxy-terminal cross-linked telopeptide of type I collagen), were found. In cases of sepsis, the concentrations of proinflammatory cytokines were high (394+/-536 pg/mL for tumor necrosis factor-alpha and 5676+/-5190 pg/mL for <em>interleukin</em>-6, both normally <10-20). The Ca2+ concentration was inversely related to tumor necrosis factor-alpha and <em>interleukin</em>-6 (r2 = .<em>35</em>-.42; p<.01), as well as to procalcitonin (r2 = .71; p<.01). Despite normocalcemia in the surgical patients, serum PTH concentrations were elevated in both patient groups (97 and 109 ng/L) (reference value, <55 ng/L), both on day 1 and day 3 in the ICU. The citrate/calcium infusion revealed an increased secretory response of PTH to lowered Ca2+ concentrations in both groups of patients (p<.05), when compared with matched healthy controls.

CONCLUSIONS

Hypocalcemia was common in septic ICU patients and was not the result of an increased urinary excretion of calcium or of an attenuated bone resorption, but seemed related to the inflammatory response. An increased PTH secretion was found in both patient groups.