In prostate cancer, race/ethnicity is the highest risk factor after adjusting for age. African Americans have more aggressive tumors at every clinical stage of the disease, resulting in poorer prognosis and increased mortality. A major barrier to identifying crucial gene activity differences is heterogeneity, including tissue composition variation intrinsic to the histology of prostate cancer. We hypothesized that differences in gene expression in specific tissue types would reveal mechanisms involved in the racial disparities of prostate cancer. We examined 17 pairs of arrays for AAs and Caucasians that were formed by closely matching the samples based on the known tissue type composition of the tumors. Using pair-wise t-test we found significantly altered gene expression between AAs and CAs. Independently, we performed multiple linear regression analyses to associate gene expression with race considering variation in percent tumor and stroma tissue. The majority of differentially expressed genes were associated with tumor-adjacent stroma rather than tumor tissue. Extracellular matrix, integrin family and signaling mediators of the epithelial-to-mesenchymal transition (EMT) pathways were all downregulated in stroma of AAs. Using MetaCore (GeneGo) analysis, we observed that <em>35</em>% of significant (p < 10(-3)) pathways identified EMT and 25% identified immune response pathways especially for <em>interleukins</em>-2, -4, -5, -6, -7, -10, -13, -15 and -22 as the major changes. Our studies reveal that altered immune and EMT processes in tumor-adjacent stroma may be responsible for the aggressive nature of prostate cancer in AAs.

BACKGROUND

Obesity is an increasingly common comorbidity in critically ill patients. Whether obesity alters sepsis outcome, susceptibility, treatment, and response is not completely understood.

METHODS

We conducted a retrospective analysis comparing three group of septic shock patients based on the intervals of actual body mass index (BMI) in patients enrolled in the VASST (Vasopressin and Septic Shock Trial) cohort. Primary outcome measurement was 28-day mortality. We tested for differences in patterns of infection by comparing the primary site of infection and organism. We also compared the treatments (fluids and vasopressors) and inflammatory response, measuring adipose tissue-related cytokine concentrations (<em>interleukin</em> [IL]-6, monocyte chemotactic protein [MCP]-1, tumor necrosis factor [TNF]-α, and resistin) in plasma in a subset of 382 patients. Of the 778 patients in VASST, 730 patients who had body weight and height measurements were analyzed. Patients with BMI<25 kg/m2 (n=276) were grouped as a reference and compared to 'overweight' (25<BMI<30 kg/m2, n=209) and 'obese' (BMI>30 kg/m2, n=245) patients.

RESULTS

Obese patients had the lowest 28-day mortality followed by overweight patients while patients with BMI<25 kg/m2 had the highest mortality (p=0.02). Compared to the patients with BMI<25 kg/m2, obese and overweight patients also had a different pattern of infection with less lung (obese 35%, overweight 45%, BMI<25 kg/m2 50%, p=0.003) and fungal infection (obese 8.2%, overweight 11%, and BMI<25 kg/m2 15.6%, p=0.03). Per kilogram, obese and overweight patients received less fluid during the first four days (p<0.05) and received less norepinephrine (obese 0.14, overweight 0.21, BMI<25 kg/m2 0.26 µg/kg/min, p<0.0001) and vasopressin (obese 0.28, overweight 0.36, BMI<25 kg/m2 0.43 µU/kg/min, p<0.0001) on day 1 compared to patients with BMI<25 kg/m2. Obese and overweight patients also had a lower plasma IL-6 concentration at baseline (obese 106 [IQR 34-686], overweight 190 [IQR 44-2339], BMI<25 kg/m2 235 [IQR 44-1793] pg/mL, p=0.046).

CONCLUSIONS

Overall obesity was associated with improved survival in septic shock and differences in pattern of infection, fluids, and vasopressors. Importantly, the magnitude of inflammatory IL-6 response is muted in the obese.

BACKGROUND

Inflammation contributes to atherogenesis. Dietary fats may be proinflammatory.

OBJECTIVE

The objective was to determine whether energy intake modulates the effects of low-fat, high-carbohydrate intakes on inflammatory markers.

METHODS

Twenty-two healthy postmenopausal women with a mean (+/-SD) age of 61 +/- 11 y, who were not receiving hormone replacement therapy, were fed eucaloric diets to reduce their fat intake from <em>35</em>% to 15% of energy. Next, the women consumed a 15%-fat ad libitum diet under free-living conditions. Serum highly sensitive C-reactive protein, <em>interleukin</em> 6, HDL serum amyloid A, and adiponectin concentrations were measured at the end of the eucaloric and ad libitum low-fat, high-carbohydrate intakes.

RESULTS

The eucaloric diet decreased adiponectin from 16.3 +/- 2.1 to 14.2 +/- 2.0 mg/L (P < 0.05) and increased triacylglycerol from 131 +/- 11 to 164 +/- 14 mg/dL (P < 0.01). The ad libitum low-fat diet caused 6 kg weight loss and decreased highly sensitive C-reactive protein from 4.3 +/- 0.6 to 2.5 +/- 0.5 mg/L (P < 0.01), decreased HDL serum amyloid A from 10.3 +/- 1.8 to 5.7 +/- 1.3 mg/L (P < 0.001), increased adiponectin from 14.2 +/- 2.0 to 16.3 +/- 1.7 mg/L (P < 0.05), and decreased triacylglycerol from 164 +/- 14 to 137 +/- 15 mg/dL (P < 0.05).

CONCLUSIONS

During the eucaloric phase, the low-fat, high-carbohydrate diet exerted unfavorable effects on the inflammatory markers. In contrast, the ad libitum low-fat, high-carbohydrate intake caused weight loss and affected inflammatory markers favorably. Thus, the energy content of a low-fat, high-carbohydrate diet determines changes in inflammatory markers.

Coronaviruses (CoVs) possess an enveloped, single, positive-stranded RNA genome which encodes for four membrane proteins, namely spike (S), envelope (E), membrane (M) and nucleocapsid (N) proteins 3-5 [1]. With regard to pathogenicity, S proteins are essential for viral entry into host cells [2, 3]. SARS-CoV binds to the angiotensin-converting enzyme (ACE)2 which is present on nonimmune cells, such as respiratory and intestinal epithelial cells, endothelial cells, kidney cells (renal tubules) and cerebral neurons and immune cells, such as alveolar monocytes/macrophages [4-6]. Of note, CD209L or liver/lymph node special intercellular adhesion molecule-3-grabbing non-integrin (SIGN) and dendritic cell (DC)-SIGN are alternative receptors for SARS-CoV but with lower affinity [7]. In the case of MERS-CoV, S proteins bind to the host cell receptor dipeptidyl peptidase 4 (DPP4 or CD26) which is broadly expressed on intestinal, alveolar, renal, hepatic and prostate cells as well as on activated leukocytes [8]. Then, viruses replicate in target cells with release of mature virions, which, in turn, invade new target cells [9]. Evidence has been provided that SARSCoV proteins are cleaved into two subunits, S1 and S2, respectively, and the amino acids 318-510 of the S1 represent the receptor-binding domain (RBD) which binds to ACE2 [10, 11]. Quite importantly, in the context of RBD there is the receptor-binding motif (RBM) (amino acids 424- 494), which accounts for complete binding to ACE2 [11]. Moreover, by means of two residues at positions 479 and 487 RBD allows virus progression and tropism [10, 11]. In the case of MERSCoV, its RBM binds to DPP4 with residues 484-567, thus, suggesting that its RBD differs from that of SARS-CoV [12, 13]. In a very recent paper, Wan and associates [14] have investigated the receptor recognition by COVID-19 (a new term to indicate the 2019-nCoV in Wuhan) on the bases of structural studies. In this respect, the sequence of COVID-19 RBM is similar to that of SARSCoV, thus, implicating that ACE2 may represent the binding receptors for COVID-19. Furthermore, gln493 residue of COVID-19 RBM seems to allow interaction with human ACE2, thus, suggesting the ability of this virus to infect human cells. According, to Wan and associates structural analysis [14], COVID-19 binds to human ACE2 with a lesser efficiency than human SARS-CoV (2002) but with higher affinity than human SARS-CoV (2003). Furthermore, same authors predicted that a single mutation at the 501 position may enhance the COVID-19 RBD binding capacity to human ACE2 and this evolution should be monitored in infected patients [14]. These predictive findings by Wan and associates [14] are confirmed by two contemporary studies by Letko and Muster [15] and Peng and associates [16]. In particular, the report by Peng and associates [16], points out the possible origin of COVID-19 from bats [16]. From a pathogenic point of view, evidence has been provided that binding of S2 to ACE2 receptor leads to its down-regulation with subsequent lung damage in the course of SARS-CoV infection [17]. Down-regulation of ACE2 causes excessive production of angiotensin (ANG) II by the related enzyme ACE with stimulation of ANG type 1a receptor (AT1R) and enhanced lung vascular permeability [18]. In particular, same authors have reported that recombinant ACE2 could attenuate severe acute lung injury in mice [18]. Moreover, Battle and associates [19] also proposed to use already available recombinant ACE2 for intercepting COVID-19 and attenuating infection. In the previous paragraphs, the presence of ACE2 on immune cells has been pointed out and, by analogy to epithelial cells, this receptor may also be down-regulated following viral entry. Therefore, in CoV-infected animal models and in infected humans further investigations are required to clarify a possible reduced expression of ACE2 on immune cells. In fact, in the course of SARS-CoV infection, a number of immune disorders have been detected. Three reports have demonstrated the ability of CoV to inhibit interferon (IFN)- production in the course of SARS acting as IFN antagonist [20-22]. In senescent Balb/c mice, depletion of T lymphocytes is associated to more severe interstitial pneumonitis and delayed clearance of SARS-CoV, thus, suggesting a protective role played by these cells [23]. In this connection, both SARS-CoV and MERS-CoV have been shown to induce T cell apoptosis, thus, aggravating the clinical course of disease [24, 25]. Quite interestingly, memory CD8+ T cells specific for SARS-CoV M and N proteins have been detected up to 11 years post-infection [26]. As far as humoral immune responsiveness is concerned, evidence has been provided that S1 subunit from MERS-CoV is highly immunogenic in mice [27]. Moreover, monoclonal antibodies have been shown to be highly neutralizing against MERS-CoV replication and endowed with post exposure effectiveness in susceptible mice [28, 29]. Human neutralizing antibodies have also been isolated from a recovered patient, thus, suggesting the role of humoral immunity in the control of the persistence of CoV in the host [30]. In particular, IgG response occurs early in infection and its prolonged production may serve for virus clearance during recovery also in view of the absence of viremia in convalescent sera from SARS patients [31]. According to current literature, severity of COVID-19 infection correlates with lymphopenia and patients who died from COVID-19 had lower lymphocyte counts when compared to survivors [32, 33]. These data suggest that lymphocyte-mediated anti-viral activity is poorly effective against COVID-19. Despite lymphopenia, evidence for an exaggerate release of proinflammatory cytokines [<em>interleukin</em> (IL)-1 and IL-6] has been reported in the course acute respiratory syndrome in COVID19 infected patients, thus, aggravating the clinical course of disease [34]. As recently reported, during COVID-19 pandemic in both Italy and China higher frequency of fatalities have been observed in the frail elderly population with previous comorbidities [<em>35</em>]. It is well known that decline of immunity occurs in ageing and, therefore, COVID-19 may gain easier access to the respiratory tract in frail elderly patients [36]. There is evidence that ACE2 protects from severe acute lung failure and operates as a negative regulator of the renin-angiotensin system (RAS) [18, 37]. It is well known that ANG II via activation of the AT1R promotes detrimental effects on the host, such as, vasoconstriction, reactive oxygen species generation, inflammation and matrix remodelling [38]. ACE2 counterbalances the noxious effects exhibited by ANG II and AT1R via activation of AT2R which arrests cell growth, inflammation and fibrosis [39]. In this framework, Gurwitz [40] proposed to use AT1R blockers, such as losartan, as a potential treatment of COVID-19 infection. In fact, losartan as well as olmesartan, used for treating hypertension in patients, were able to increase ACE2 expression after 28 days treatment of rats with myocardial infarction [41]. Then, Gurwitz suggests to evaluate severity of symptoms in COVID-19 infected patients under previous chronic treatment with AT1R blockers in comparison to COVID-19 infected patients who did not take AT1R blockers [40]. Quite interestingly, 75% of aged COVID-19 infected patients admitted to Italian hospitals had hypertension [unpublished data]. However, the putative effects of ACE-2 down-regulation on the cardiovascular system in the course of COVID-19 pandemic need more intensive studies. Taken together, these evidences suggest that CoV-induced down-regulation of ACE2 activates RAS with collateral damage to organs, such as lungs, in the course of SARS-related pneumonia. Then, putative therapeutic measures aimed at increasing ACE2 levels on respiratory epithelial cells should be taken into serious consideration. Quite interestingly, over the past few years, three key papers have demonstrated the ability of a polyphenol, resveratrol (RES), to experimentally deactivate the RAS system in maternal and post-weaning high fat diet, arterial ageing and high fat diet, respectively [42-44]. In all these experimental models, RES led to an increase of ACE2 with reduction of organ damage, such as liver steatosis and aorta media thickness and decrease of adipose tissue mass, respectively. As far as the mechanism of action of RES is concerned, this polyphenol is able to activate sirtuin (Sirt)1 [45-47]. In turn, Sirt1 down-regulates AT1R expression via ACE2 up-regulation [43, 48]. Of importance, Lin and associates [48] have demonstrated the ability of RES to in vitro inhibit MERS-CoV infection of Vero E6 cells, thus, prolonging cell survival in virtue of an anti-apoptotic mechanism. These findings suggest a direct antiviral effect exerted by RES. It would be very interestingly to evaluate the direct effects of RES on COVID-19, in vitro. The data above discussed strongly suggest, that RES, as an activators of ACE2, should be investigated in animal models of CoV-induced severe pneumonia, also taking into account the antioxidant, anti-inflammatory and immunomodulating effects exerted by polyphenols [49]. Then, successful animal studies may pave the way for RES-based human trials in COVID-infected patients. Note added in proof During the reviewing process of this perspective other related papers have been published. Hanff and associates [50] have discussed the possible association between COVID-19-associated cardiovascular mortality and dysregulation of the Renin Angiotensin System (RAS). From a pharmacologic point of view, RAS inhibition leads to upregulation of ACE2, thus, attenuating acute respiratory syndrome and myocarditis in COVID-19-infected patients. Conversely, increase in ACE2 expression may facilitate the access into the host of COVID-19, thus, aggravating the clinical picture. Such a dilemma would be solved by clinical trials based on RAS blockade or initiation and monitoring related effects. Contemporarily, Danser and associates [51] claim that there is no evidence to stop RAS blockers in the course of COVID-19 infection. In fact, there are no available data which support that ACE inhibitors or ANG II type I receptor blockers increase COVID-19 infection via its binding to ACE2. Finally, Kuster and associates [52] write that there are no data on the strict relationship between ACE2 activity and SARS-CoV2 mortality. Moreover, in the SARSCoV2, cells expressing ACE2 were not attacked by the virus, while cells lacking ACE2 were bound by the SARS-CoV2 virus [53]. These findings suggest that also in the case of RES effects on COVID-19 infection, the dual role of ACE2 should be taken into serious consideration.

This study determined whether N-acetylcysteine (NAC) could affect intestinal redox status, proinflammatory cytokines, epidermal growth factor (EGF), EGF receptor (EGFR), Toll-like receptor-4 (TLR4), and aquaporin-8 in a lipopolysaccharide (LPS)-challenged piglet model. Eighteen piglets (<em>35</em>-day-old) were randomly allocated into one of the three treatments (control, LPS and NAC). The control and LPS groups were fed a basal diet, and the NAC group received the basal diet +500 mg/kg NAC. On days 10, 13, and 20 of the trial, the LPS- and NAC-treated piglets received intraperitoneal administration of LPS (100 μg/kg BW), whereas the control group received the same volume of saline. On days 10 and 20, venous blood samples were obtained at 3 h post LPS or saline injection. On day 21 of the trial, piglets were killed to obtain the intestinal mucosa for analysis. Compared with the control group, LPS challenge reduced (P < 0.05) the activities of superoxide dismutase, catalase, and glutathione peroxidase in jejunal mucosae, while increasing (P < 0.05) the concentrations of malondialdehyde, H2O2, O2 (·-) and the ratio of oxidized to reduced glutathione in jejunal mucosae, and concentrations of TNF-α, cortisol, <em>interleukin</em>-6, and prostaglandin E2 in both plasma and intestinal mucosae. These adverse effects of LPS were attenuated (P < 0.05) by NAC supplementation. Moreover, NAC prevented LPS-induced increases in abundances of intestinal HSP70 and NF-κB p65 proteins and TLR4 mRNA. NAC supplementation enhanced plasma EGF concentration and intestinal EGFR mRNA levels. Collectively, these results indicate that dietary NAC supplementation alleviates LPS-induced intestinal inflammation via regulating redox, EGF, and TLR4 signaling.

Methotrexate has been approved for the treatment of refractory rheumatoid arthritis by several regulatory agencies, including the Food and Drug Administration. The tendency is now to prescribe it at earlier stages of the disease. Methotrexate is a well known antifolate. Its exact mechanism of action in rheumatoid arthritis remains uncertain. The polyglutamated derivatives of methotrexate are potent inhibitors of various enzymes, including dihydrofolate reductase and 5-aminoimidazole-4-carboxamide ribonucleotide transformylase. Inhibitory effects on cytokines, particularly <em>interleukin</em>-1, and on arachidonic acid metabolism, as well as effects on proteolytic enzymes, have been reported. Some of them may be linked to the antifolate properties of methotrexate. Overall, the drug appears to act in rheumatoid arthritis as an anti-inflammatory agent with subtle immunomodulating properties. Direct inhibitory effects on rapidly proliferating cells in the synovium have also been suggested. Methotrexate is usually given orally. Marked interindividual variation in its bioavailability has been found. Food intake has no significant effect on the pharmacokinetics of oral methotrexate. Methotrexate undergoes significant metabolism. The functionally important metabolites are the polyglutamated derivatives of methotrexate, which are selectively retained in the cells. Less than 10% of a dose of methotrexate is oxidised to 7-hydroxy-methotrexate, irrespective of the route of administration. This metabolite is extensively (91 to 93%) bound to plasma proteins, in contrast to the parent drug (<em>35</em> to 50% bound). Methotrexate is mainly excreted by the kidneys. It undergoes tubular secretion and may thereby compete with various organic acid compounds. Early placebo-controlled trials demonstrated that weekly low dosage methotrexate produced early symptomatic improvement in most rheumatoid arthritis patients. Two meta-analyses showed that methotrexate is among the most efficacious of slow-acting antirheumatic agents, together with parenteral gold (sodium aurothiomalate), penicillamine and sulfasalazine. Furthermore, in the short term context of clinical trials, methotrexate has one of the best efficacy/toxicity ratios. There is little evidence that methotrexate, or any available slow-acting antirheumatic agent, is a true disease-modifying drug. However, the probability that a patient will continue methotrexate therapy over time appears quite favourable compared with any other slow-acting antirheumatic drug. Combination therapy with slow-acting drugs has been advised for the management of rheumatoid arthritis, but the evidence currently available does not support general use of combination therapy including methotrexate. Almost all investigations indicated that toxic effects, rather than lack of response, were the major reason for discontinuing methotrexate therapy.(ABSTRACT TRUNCATED AT 400 WORDS)

Systemic effects on T-cell-mediated antitumor immunity, on expression of T-cell adhesion/homing receptors, and on the promotion of T-cell infiltration of neoplastic tissue may represent key steps for the efficacy of immunological therapies of cancer. In this study, we investigated whether these processes can be promoted by s.c. administration of low-dose (0.5 microg/kg) recombinant human <em>interleukin</em>-12 (rHuIL-12) to metastatic melanoma patients. A striking burst of HLA-restricted CTL precursors (CTLp) directed to autologous tumor was documented in peripheral blood by a high-efficiency limiting dilution analysis technique within a few days after rHuIL-12 injection. A similar burst in peripheral CTLp frequency was observed even when looking at response to a single tumor-derived peptide, as documented by an increase in Melan-A/Mart-1(27-<em>35</em>)-specific CTLp in two HLA-A*0201+ patients by limiting dilution analysis and by staining peripheral blood lymphocytes (PBLs) with HLA-A*0201-melanoma antigen-A/melanoma antigen recognized by T cells (Melan-A/Mart)-1 tetrameric complexes. The CTLp burst was associated, in PBLs, with enhanced expression of T-cell adhesion/homing receptors CD11a/CD18, CD49d, CD44, and with increased proportion of cutaneous lymphocyte antigen (CLA)-positive T cells. This was matched by a marked increase, in serum, of soluble forms of the endothelial cell adhesion molecules E-selectin, vascular cell adhesion molecules (VCAM)-1 and intercellular adhesion molecules (ICAM)-1. Infiltration of neoplastic tissue by CDS+ T cells with a memory and cytolytic phenotype was found by immunohistochemistry in eight of eight posttreatment metastatic lesions but not in five of five pretreatment metastatic lesions from three patients. Increased tumor necrosis and/or fibrosis were also found in several posttherapy lesions of two of three patients in comparison with pretherapy metastases. These results provide the first evidence that rHuIL-12 can boost the frequency of circulating antitumor CTLp in tumor patients, enhances expression of ligand receptor pairs contributing to the lymphocyte function-associated antigen-1/ICAM-1, very late antigen-4/VCAM-1, and CLA/E-selectin adhesion pathways, and promotes infiltration of neoplastic lesions by CD8+ memory T cells in a clinical setting.

OBJECTIVE

Monocyte adhesion to endothelial cells and subsequent secretion of matrix metalloproteinases (MMPs) by activated macrophages are key events in arteriosclerosis and restenosis. We tested the hypothesis that interleukin-10 (IL-10), a potent anti-inflammatory cytokine, inhibits monocyte-endothelial cell interactions.

METHODS

The effect of IL-10 on monocyte/endothelial cell adhesion, as well as on the expression of MMP-9 and the tissue inhibitor of MMP-9, TIMP-1, were first tested in vitro in coculture systems. In addition, we used an ex vivo binding assay to study the inhibitory effect of IL-10 on monocyte adhesion to carotid arteries obtained from either normal, or L-nitro arginine-methyl ester (L-NAME)-treated rats. The effect of IL-10 on the expression of monocyte adhesion molecules (CD18 and CD62-L) was studied by flow cytometry.

RESULTS

IL-10 (150 ng/ml) inhibits monocyte adhesion to endothelial cells (by 35%) and to carotid arteries (by 40 and 50%, in normal and L-NAME-treated rats, respectively), via direct modulation of the expression of CD18 and CD62-L. Moreover, IL-10 dose-dependently decreases MMP-9 activity and increases TIMP-1 levels in coculture systems, both at the transcriptional level.

CONCLUSIONS

Our results suggest that IL-10 is an important modulator of monocyte-endothelial cell interactions.

Mice homozygous for targeted deletion of the <em>interleukin</em> 10 gene (Il-10) have been partially characterized as a model for human frailty. These mice have increased serum <em>interleukin</em> (IL)-6 in midlife, skeletal muscle weakness, and an altered skeletal muscle gene expression profile compared to age and sex-matched C57BL/6 (B6) control mice. In order to further characterize for use as a frailty model, we evaluated the evolution of inflammatory pathway activation, endocrine change, and mortality in these mice. Serum was collected in groups of age- and sex-matched B6.129P2-Il10(tm1Cgn)/J (IL-10(tm/tm)) mice and B6 control mice at age 12, 24, 48, 72, and 90 weeks. Cytokines including IL-6, <em>interleukin</em> 1 beta (IL-1β), tumor necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ), chemokine (C-X-C motif) ligand 1 (KC), IL-12, and IL-10 were measured using electro-chemiluminescent multiplex immunoassay and insulin-like growth factor 1 (IGF-1) was measured using solid-phase enzyme-linked immunosorbent assay. A separate longitudinal cohort was monitored from age <em>35</em> weeks to approximately 100 weeks. Survival was evaluated by Kaplan-Meier survival estimates and detailed necropsy information was gathered in a subset of mice that died or were sacrificed. In IL-10(tm/tm) mice compared to B6 controls, serum IL-6, IL-1β, TNF-α, IFN-γ, KC levels were significantly elevated across the age groups, serum mean IGF-1 levels were higher in the 48-week-old groups, and overall mortality rate was significantly higher. The quadratic relationship between IGF-1 and age was significantly different between the two strains of mice. Serum IL-6 was positively associated with IGF-1 but the effect was significantly larger in IL-10(tm/tm) mice. These findings provide additional rationale for the use of the IL-10(tm/tm) mouse as a model for frailty and for low-grade inflammatory pathway activation.

BACKGROUND

Our previous studies in diabetic (DB) rats suggest that hyperlipidemia may cause a dysregulation of the cellular and local cytokine response to periodontitis (AP). The objective of the present study was to determine if diabetes has a similar dysregulatory effect on the gingival response to AP in humans.

METHODS

Peripheral blood, as well as gingival tissue (GT) and gingival crevicular fluid (GCF), was obtained from a total of <em>35</em> patients who were categorized into the following groups based on level of diabetic (type 2) control and presence or absence of adult periodontitis (AP): group 1, systemically and periodontally healthy (n = 6); group 2, systemically healthy with adult periodontitis (n = 7); group 3, well-controlled diabetes and periodontally healthy (n = 6); group 4, well-controlled diabetes with adult periodontitis (n = 5); group 5, poorly controlled diabetes and periodontally healthy (n = 5); group 6, poorly controlled diabetes and adult periodontitis (n = 6). All subjects were given a thorough periodontal examination, including probing depths (PD), clinical attachment levels (CAL), gingival index (GI), plaque index (PI), and vertical bitewing radiographs. Blood studies included levels of glycated hemoglobin (HbA1c), triglycerides (TG), cholesterol (CHL), low-density lipoproteins (LDL), and high-density lipoproteins (HDL). The levels of <em>interleukin</em>-1 beta (IL-1beta) in GCF and GT, <em>interleukin</em>-6 (IL-6), and platelet-derived growth factor AB (PDGF-AB) in GT from patients in each experimental group were analyzed by enzyme-linked immunosorbent assay (ELISA).

RESULTS

Our results indicate that all clinical indices except PI were significantly elevated in the poorly controlled and well-controlled diabetics, compared to systemically healthy patients, but only in the subjects without preexisiting AP (Tukey's multiple comparisons, P <0.05). Pairwise linear regression analysis revealed significant (P <0.01) positive associations between periodontal inflammation (PD, CAL, PI, GI) and levels of GCF IL-1beta, GT IL- 1beta GT IL-6, but not GT PDGF; moreover, GT IL-6 levels were significantly associated (P<0.05) with GT IL-1beta. As TG levels increased in the non-AP patients (group 1 < group 3 < group 5), there was a trend, not significant, for increased GCF IL-1beta levels and increased gingival inflammation. Interestingly, periodontitis resulted in increased PDGF-AB levels in the gingiva of systemically healthy and well-controlled diabetes patients, but this increase was obtunded in poorly controlled diabetes patients.

CONCLUSIONS

This confirms our earlier work in the diabetic rat model. These studies indicate that decreased metabolic control in type 2 diabetics results in increased serum triglycerides and has a negative influence on all clinical measures of periodontal health, particularly in patients without preexisting periodontitis. Levels of the cytokine IL- 1beta showed a trend for increasing as diabetic control diminished. In contrast, levels of the growth factor PDGF, which normally increase in periodontitis, decreased in poorly controlled diabetics with periodontitis. These studies suggest a possible dysregulation of the normal cytokine/growth factor signaling axis in poorly controlled type 2 diabetics that may contribute to periodontal breakdown/diminished repair.

Inflammation is a protective tissue response occurring in three distinct phases, acute, subacute and a chronic proliferative phase. We undertook the present study to understand the overall immune response of the body during adjuvant induced chronic inflammation in rat and the effect of ibuprofen and curcumin on this response. Inflammatory mediators were estimated on day 21 and day <em>35</em> after adjuvant injection. The level of C-reactive protein increased to 200% on day 21 and then reduced to 50% on day <em>35</em> compared to control. Curcumin and ibuprofen further reduced the increased levels at both the time intervals. Haptoglobin level decreased to 42% on day 21 but increased to 5 times of control on day <em>35</em>. Curcumin and ibuprofen reduced the increased levels at day <em>35</em>. No significant change was observed in Prostaglandin-E2 and Leukotriene-B4 levels and in Lymphocyte proliferation. The level of Tumor Necrosis Factor-alpha increased by three folds on day 21, but came down to 88% on day <em>35</em>. Ibuprofen treatment decreased the raised level on day 21 and increased the reduced level on day <em>35</em>. <em>Interleukin</em>-1beta increased to 2 folds on day 21 and 10 folds on day <em>35</em> which were significantly brought down by curcumin and ibuprofen. Nitric oxide level was reduced at both the time intervals, which were increased by drug treatment.

OBJECTIVE

Although myeloid-derived suppressor cells (MDSCs) have been linked to T cell tolerance, their role in autoimmune rheumatoid arthritis (RA) remains elusive. Here we investigate the potential association of MDSCs with the disease pathogenesis using a preclinical model of RA and specimen collected from patients with RA.

METHODS

The frequency of MDSCs in blood, lymphoid tissues, inflamed paws or synovial fluid and their association with disease severity, tissue inflammation and the levels of pathogenic T helper (Th) 17 cells were examined in arthritic mice or in patients with RA (n=<em>35</em>) and osteoarthritis (n=15). The MDSCs in arthritic mice were also characterised for their phenotype, inflammation status, T cell suppressive activity and their capacity of pro-Th17 cell differentiation. The involvement of MDSCs in the disease pathology and a Th17 response was examined by adoptive transfer or antibody depletion of MDSCs in arthritic mice or by coculturing mouse or human MDSCs with naïve CD4+ T cells under Th17-polarising conditions.

RESULTS

MDSCs significantly expanded in arthritic mice and in patients with RA, which correlated positively with disease severity and an inflammatory Th17 response. While displaying T cell suppressive activity, MDSCs from arthritic mice produced high levels of inflammatory cytokines (eg, interleukin (IL)-1β, TNF-α). Mouse and human MDSCs promoted Th17 cell polarisation ex vivo. Transfer of MDSCs facilitated disease progression, whereas their elimination in arthritic mice ameliorated disease symptoms concomitant with reduction of IL-17A/Th17 cells.

CONCLUSIONS

Our studies suggest that proinflammatory MDSCs with their capacity to drive Th17 cell differentiation may be a critical pathogenic factor in autoimmune arthritis.

OBJECTIVE

Reactive thrombocytosis is found in a number of clinical situations including infectious diseases such as pulmonary tuberculosis (PTB). To examine the possible role of interleukin (IL6) in reactive thrombocytosis and acute phase response in PTB this study measured serum IL6, C reactive protein (CRP), erythrocyte sedimentation rate (ESR), albumin concentrations in 62 PTB patients and 20 healthy volunteers.

METHODS

PTB patients were divided into two groups based on thrombocyte counts. Twenty seven PTB patients with normal thrombocyte counts constituted group 1, 35 PTB patients with thrombocytosis constituted group 2, and 20 healthy volunteers constituted group 3.

RESULTS

The median IL6 concentration of group 1 was 12.8 pg/ml (95% CI: 12.1 to 56.9 pg/ml) and group 2 was 40.6 pg/ml (95% CI: 67.1 to 168.7 pg/ml). The comparison of IL6 concentrations in the three groups was significant (p = 0.0001). Patients in group 1 had a higher concentration of CRP (p = 0.0001) and lower concentration of albumin (p = 0.002) than group 3 whereas group 2 had higher concentration of CRP (p = 0.003) and lower concentration of albumin (p = 0.002) than group 1. Serum IL6 concentrations were significantly correlated with thrombocyte counts (p = 0.004, r = 0.36), CRP (p = 0.007, r = 0.34), and albumin concentrations (p = 0.005, r = -0.34). IL6 concentrations were significantly correlated with the number of involved zones (p = 0.005, r = 0.35) and acid fast bacilli positivity (p = 0.03, r = 0.27). Patients in group 2 had weight loss (p = 0.004), fever (p = 0.038), and night sweats (p = 0.007) more frequently than group 1. Also, group 2 had more extensive radiological findings (involved zones p = 0.001, bilateral disease p = 0.0001, presence of cavity p = 0.02) than group 1.

CONCLUSIONS

IL6 might play a contributory part in reactive thrombocytosis and acute phase response in PTB.

OBJECTIVE

To monitor clinical, microbiological and host-derived alterations occurring around teeth and titanium implants during the development of experimental gingivitis/mucositis and their respective healing sequence in humans.

METHODS

Fifteen subjects with healthy or treated periodontal conditions and restored with dental implants underwent an experimental 3-week period of undisturbed plaque accumulation in the mandible. Subsequently, a 3-week period with optimal plaque control was instituted. At Days 0, 7, 14, 21, 28, <em>35</em> and 42, the presence/absence of plaque deposits around teeth and implants was assessed, (plaque index [PlI]) and the gingival/mucosal conditions were evaluated (gingival index[GI]). Subgingival/submucosal plaque samples and gingival/mucosal crevicular fluid (CF) samples were collected from two pre-determined sites around each experimental unit. CF samples were analyzed for matrix-metalloproteinase-8 (MMP-8) and <em>interleukin</em>-1beta (IL-1β). Microbial samples were analyzed using DNA-DNA hybridization for 40 species.

RESULTS

During 3 weeks of plaque accumulation, the median PlI and GI increased significantly at implants and teeth. Implant sites yielded a greater increase in the median GI compared with tooth sites. Over the 6-week experimental period, the CF levels of MMP-8 were statistically significantly higher at implants compared with teeth (P<0.05). The CF IL-1β levels did not differ statistically significantly between teeth and implants (P>0.05). No differences in the total DNA counts between implant and tooth sites were found at any time points. No differences in the detection frequency were found for putative periodontal pathogens between implant and tooth sites.

CONCLUSIONS

Peri-implant soft tissues developed a stronger inflammatory response to experimental plaque accumulation when compared with that of their gingival counterparts. Experimental gingivitis and peri-implant mucositis were reversible at the biomarker level. Clinically, however, 3 weeks of resumed plaque control did not yield pre-experimental levels of gingival and peri-implant mucosal health indicating that longer healing periods are needed.

OBJECTIVE

The associations between inflammation, diabetes and insulin resistance remain controversial. Hence, we assessed the associations between diabetes, insulin resistance (using HOMA-IR) and metabolic syndrome with the inflammatory markers high-sensitive C-reactive protein (hs-CRP), interleukin-1 beta (IL-1β), interleukin-6 (IL-6) and tumour necrosis factor-α (TNF-α).

METHODS

Cross-sectional study.

METHODS

Two thousand eight hundred and eighty-four men and 3201 women, aged 35-75, participated in this study.

METHODS

C-reactive protein was assessed by immunoassay and cytokines by multiplexed flow cytometric assay. In a subgroup of 532 participants, an oral glucose tolerance test (OGTT) was performed to screen for impaired glucose tolerance (IGT).

RESULTS

IL-6, TNF-α and hs-CRP were significantly and positively correlated with fasting plasma glucose (FPG), insulin and HOMA-IR. Participants with diabetes had higher IL-6, TNF-α and hs-CRP levels than participants without diabetes; this difference persisted for hs-CRP after multivariate adjustment. Participants with metabolic syndrome had increased IL-6, TNF-α and hs-CRP levels; these differences persisted after multivariate adjustment. Participants in the highest quartile of HOMA-IR had increased IL-6, TNF-α and hs-CRP levels; these differences persisted for TNF-α and hs-CRP after multivariate adjustment. No association was found between IL-1β levels and all diabetes and insulin resistance markers studied. Finally, participants with IGT had higher hs-CRP levels than participants with a normal OGTT, but this difference disappeared after controlling for body mass index (BMI).

CONCLUSIONS

We found that subjects with diabetes, metabolic syndrome and increased insulin resistance had increased levels of IL6, TNF-α and hs-CRP, while no association was found with IL-1β. The increased inflammatory state of subjects with IGT is partially explained by increased BMI.

OBJECTIVE

To assess local bone resorption in the context of rheumatoid synovitis and its modulation by interleukin-4 (IL-4).

METHODS

We developed an ex vivo model of bone resorption using juxtaarticular samples of bone obtained during joint surgery. We studied the histomorphometric parameters of bone resorption and the regulation of the production of IL-6, leukemia inhibitory factor (LIF), and the collagen cross-link pyridinoline, which is released during bone resorption in vivo.

RESULTS

This was a sensitive and dynamic model of bone resorption. The bone samples produced high levels of pyridinoline and as much cytokine as synovium pieces obtained from the same joint. IL-4 induced a 70% reduction of IL-6 and LIF production by bone pieces and reduced pyridinoline levels. Histomorphometric studies performed on bone samples indicated a 35% increase in the mean total bone area after 7 days of treatment with IL-4. More importantly, with IL-4, osteoclasts were not detectable in the bone sections.

CONCLUSIONS

The inhibitory effect of IL-4 on bone resorption extends our knowledge of its antiinflammatory properties and suggests that the inflammatory cytokine imbalance in rheumatoid synovium also contributes to defects in bone resorption in RA.

Chronic obstructive pulmonary disease (COPD) is associated with increased cardiovascular mortality. Endothelial dysfunction may underpin this association. This cross-sectional study aimed to determine the impact of airflow obstruction, systemic inflammation, oxidative stress, sympathetic activation, hypoxaemia and physical activity on endothelial function in COPD. In stable COPD patients, assessments of endothelial function by flow-mediated dilatation (FMD), cardiovascular risk (Pocock score), airflow obstruction (forced expiratory volume in 1 s (FEV1)), systemic inflammation (high-sensitivity C-reactive protein and <em>interleukin</em>-6), oxidative stress (malondialdehyde), sympathetic activation (baroreflex sensitivity), hypoxaemia (arterial oxygen tension), hypercapnia (arterial carbon dioxide tension (PaCO2)), physical activity (steps per day) and exercise capacity (6-min walking distance) were performed. Associations between FMD and potential determinants were assessed in univariate and multivariate analyses. 106 patients (Global Initiative for Chronic Obstructive Lung Disease stage I/II <em>35</em>%, stage III 25% and stage IV 40%) were included. In multivariate analysis FEV1 was positively associated with FMD, independent of other significant FMD determinants from univariate analysis (sex, smoking, combined inhaled long-acting β-adrenergic and steroid medication, heart rate, baroreflex sensitivity and PaCO2) and adjusted for potential confounders (cardiovascular risk and age). In addition, the FMD and FEV1 association was modified by physical activity. The findings of this study demonstrate that the severity of airflow obstruction is a significant determinant of endothelial function in patients with COPD. A high level of physical activity seems to have a favourable effect on this association.

Since bisphosphonates prevent bone loss in osteoporosis and rheumatoid arthritis, diseases in which the osteoclastogenic and inflammatory cytokine <em>interleukin</em>-6 plays a pathophysiologic role, we studied whether these drugs regulate the production of this cytokine by osteoblasts. Spontaneous and IL-1 + TNF-alpha stimulated IL-6 release was measured in supernatants of cultures of human osteoblastic osteosarcoma cells MG-63, pretreated for 4 hours with different doses of etidronate, clodronate or alendronate using a specific bioassay. Etidronate [from 10(-4) to 10(-8) M] or alendronate [from 10(-6) to 10(-11) M] inhibited in a dose-dependent manner the cytokine-induced IL-6 secretion [60+/-9.5% at 10(-5) M and 65+/-12% at 10(-7) M, respectively; p < 0.01]. Though significant, the inhibitory effect of clodronate was less [<em>35</em>+/-7% at 10(-5) M, p < 0.05]. These in vitro observations might have in vivo relevance in explaining at least in part the mechanisms by which bisphosphonates inhibit systemic and periarticular bone resorption.

Two colour flow cytometry techniques were used to assess the activation stages of peripheral and intraocular T lymphocytes in uveitis. Increased numbers of T lymphocytes bearing the <em>interleukin</em>-2 (IL-2) receptors were found in intraocular fluids or peripheral blood or both of <em>35</em>/51 patients with uveitis. This increased expression of IL-2 receptors on lymphocytes correlated with increased expression of other early T lymphocyte activation markers, HLA-DR and L-<em>35</em>. Both T helper cells (Leu-3A+) and suppressor cells (Leu 2A+) were activated in vivo. A positive correlation was seen between lymphocyte activation and clinical uveitis activity. In idiopathic uveitis activation of Leu-3A lymphocytes (helper/inducer) was significantly increased, and intraocular activation of the Leu-2A lymphocytes (cytotoxic/suppressor) was significantly decreased. These data show that some patients with idiopathic uveitis have a perturbation of T helper cells. Twenty-two of 31 patients with idiopathic uveitis, not associated with systemic disease, had increased peripheral T lymphocyte activation. This finding indicates that in some inflammations believed to be restricted to the eye an abnormal systemic immune activation exists.

We investigated the effects of intraarticular injections of <em>interleukin</em>-1 alpha (IL-1 alpha) into the knee joints of young (3-month-old) and old (18-month-old) C57Bl/10 mice. In this in vivo study, <em>35S</em>-sulfate incorporation and release were used to compare the effects of IL-1 on patellar cartilage proteoglycan metabolism. IL-1-induced stimulation of proteoglycan degradation was confined to the first 24 hours after injection in both young and old animals, and was only slightly lower in old cartilage than in young. In old patellar cartilage, IL-1-induced suppression of proteoglycan synthesis appeared to be more prolonged. Also, the amount of time needed to restore the cartilage matrix, characterized by proteoglycan synthesis above normal levels, was longer in old animals. Histologic analysis confirmed the retarded recovery in the cartilage of old mice. Autoradiography showed that the chondrocytes of the medial side of the femorotibial area were most vulnerable to IL-1-induced suppression of proteoglycan synthesis, especially the medial tibial plateau. As with the patellar cartilage, IL-1-induced suppression of proteoglycan synthesis in this area of articular cartilage was more prolonged in old animals. Our data indicated that the impact of IL-1 on articular cartilage is higher in old mice and that, consistent with certain loci found to be at risk in experimental osteoarthritis, there are sites at which IL-1 is more likely to act.

In the present study, MRNA for the cytokines <em>interleukin</em>-2 (IL-2), IL-4, IL-10 tumour necrosis factor-alpha (TNF-alpha) and transforming growth factor beta-1 (TGF-beta-1) were investigated in oral lichen planus (OLP) lesions using in situ hybridization with <em>35S</em>-labelled oligonucleotide probes on frozen tissue sections. In addition, the expression of interferon-gamma (IFN-gamma), IL-10 and IL-4 mRNAs was analysed in cultured lesional T lymphocytes from oral lichen planus by polymerase chain reaction. Cells expressing mRNA for IL-2, IL-4, IL-10, TNF-alpha and TGF-beta 1 were found in all the biopsies studied. Approximately 1-2% of the total number of infiltrating cells in the lesions were positive for each of the different cytokine mRNAs. Most biopsies contained basement membrane-oriented, mRNA-positive cells. In the cultured T-cell lines, message for IFN-gamma was detected in all the patients, IL-10 in all but one, and IL-4 in just one of the seven patients investigated. The results suggest that mRNA for both pro- and anti-inflammatory cytokines, i.e., mixed T-helper 1 (TH1) and TH2 cytokine profiles, are generated simultaneously by a limited number of cells in chronic lesions of OLP.

The gelatin-degrading matrix metalloproteinase (MMP) activities and their inhibitors produced by rabbit articular chondrocytes have been characterized by gel substrate analysis ('zymography') after electrophoresis on non-reducing sodium dodecyl sulfate-polyacrylamide gels containing gelatin. Differentiated chondrocytes in confluent primary culture produced constitutively only one gelatinase which presented the main characteristics of proMMP-2 ('72 kDa type IV procollagenase'). It had an apparent Mr of 66,000 (unreduced), which was partially or totally converted to 61,000 by, respectively, trypsin or APMA treatment; exogenous TIMP (tissue inhibitor or metalloproteinases) inhibited the conversion triggered by APMA but not that induced by trypsin. This proMMP-2 was also the predominant gelatinase found, together with its 61 kDa activation product, in extracts of articular cartilage. Differentiated chondrocytes simultaneously produced MMP inhibitors which on reverse zymograms were distributed over two bands with Mr of 27,500 and 20,400, resistant to both pH 2 and 100 degrees C, corresponding, respectively, presumably, to TIMP and TIMP-2. <em>Interleukin</em>-1 (IL1) and tumor necrosis factor alpha (TNF alpha) did not affect the production of the proMMP-2 nor of the two species of TIMP. However, IL1 induced the coordinated production of 91 and 55 kDa gelatinases. The 91 kDa activity is likely to correspond to proMMP-9. It could be converted to a 81 kDa gelatinase by trypsin or APMA treatment, in a process that was inhibited in both cases by exogenous TIMP. The 55 kDa gelatinolytic activity most probably represents the sum of the activities of proMMP-1 (procollagenase) and proMMP-3 (prostromelysin). It was sequentially converted to lower size forms (49 to <em>35</em> kDa) by either trypsin or APMA; that conversion was inhibited by TIMP, with the exception, however, of the first steps (from 55 to 49, then to 42 kDa) induced by trypsin. The 55 kDa and its conversion forms were all active on both gelatin and casein. TNF alpha did also stimulate the production of proMMP-9, although less efficiently than IL1, but it did not induce, or very poorly, that of the 55 kDa proMMP-1/proMMP-3 activity. Low levels of proMMP-9 and of its 81 kDa product of activation were also found in extracts of cartilage. With increasing passage number and cell dedifferentiation, confluent chondrocytes produced increasing amounts of proMMP-2 and of the two species of TIMP. A spontaneous low production of proMMP-9 and proMMP-1/proMMP-3 was only occasionally observed in cultures of dedifferentiated chondrocytes, accompanying a spontaneous low production of procollagenase.(ABSTRACT TRUNCATED AT 400 WORDS)

Lipopolysaccharide (LPS)-binding protein (LBP) is a normal plasma protein and an acute phase reactant important for host responses to Gram-negative bacteria and LPS. LBP forms high affinity complexes with LPS which bind to CD14, a monocyte surface protein, to initiate the release of inflammatory mediators. We found that human primary hepatocytes synthesize LBP and that the synthesis is up-regulated by <em>interleukin</em> (IL)-6. To examine this phenomenon in more detail, we evaluated the capacity of IL-6, IL-1, and tumor necrosis factor to induce LBP synthesis in HepG2 cells in the presence or absence of dexamethasone. IL-6 induced LBP synthesis. Dexamethasone, IL-1, and tumor necrosis factor had a synergistic effect when combined with IL-6, but demonstrated minimal effect independently. LBP biosynthesis was evaluated by immunoprecipitation of <em>35S</em>-labeled LBP from HepG2 supernatants, measurement of steady-state LBP mRNA levels, and analysis of LBP-dependent LPS binding to CD14 positive cells. An <em>35S</em>-labeled, 60-kDa protein was immunoprecipitated with anti-LBP antibody from IL-6-stimulated HepG2 cell supernatants. Northern blot analysis of cellular RNA revealed an increase in LBP mRNA in IL-6-stimulated cells. CD14 expressing cells bound fluoresceinated LPS in the presence of supernatants from HepG2 cells treated with IL-6. These data provide the first information about specific cytokine and dexamethasone regulation of LBP expression in HepG2 cells. LBP behaves like a Type 1 acute phase protein.

OBJECTIVE

The purpose of this study was to determine if a bedside test, the MMP-8 PTD Check, can be of value in the antenatal identification of funisitis. This test can be performed in 15 min without any laboratory equipment.

METHODS

The relationship between the presence or absence of funisitis and the results of an MMP-8 PTD Check was examined in 139 patients who delivered preterm singleton neonates (gestational age (<em>35</em> weeks) within 72 h of amniocentesis. Amniotic fluid (AF) was cultured for aerobic and anaerobic bacteria and for genital mycoplasmas. AF was analyzed for white blood cell (WBC) count, <em>interleukin</em>-6 (IL-6) and an MMP-8 PTD Check. The IL-6 concentration was also determined in umbilical cord plasma collected at birth. Funisitis was diagnosed in the presence of neutrophil infiltration into the umbilical vessel walls or Wharton's jelly.

RESULTS

1) Funisitis was present in 27% (38/139) of cases; 2) A positive MMP-8 PTD Check had a sensitivity of 97% (37/38), a specificity of 63% (64/101), a positive predictive value of 50% (37/74) and a negative predictive value of 99% (64/65) in the identification of funisitis; 3) Among cases without funisitis, patients with a positive MMP-8 PTD Check had a significantly higher median AF IL-6 concentration, AF WBC count, and umbilical cord plasma IL-6 concentration at birth than those with a negative MMP-8 PTD Check (P<0.05 for each).

CONCLUSIONS

The MMP-8 PTD Check is a rapid, simple and sensitive bedside test which allows assessment of the risk of funisitis.