Human papillomavirus (HPV) infects the transformation zone of the cervix and is the primary cause of cervical cancer. The infection is localized to the cervix and mucosal immunity is likely to be an important determinant for viral clearance. Previous studies of immunity to HPV have measured immune markers in the blood, but the relationship of systemic immunity to cervical immunity is poorly understood. In this study of 70 women enrolled in the ASCUS-LSIL Triage Study (ALTS), a clinical trial for management of low-grade cytologic abnormalities of the cervix, we collected paired plasma and cervical secretions to investigate the relationship between cervical concentrations of <em>interleukin</em>-10 (IL-10) and <em>interleukin</em>-12 (IL-12) and plasma levels. Neither IL-10 (p = 0.11), or IL-12 (p = -0.04) nor the ratio of IL-12 to IL-10 (p = 0.06) were correlated between blood and cervical secretions. Except for weak correlations of IL-10 among nonsmokers (p = 0.35. P = 0.019) and those in day 18-<em>27</em> of their menstrual cycle (p = 0.51, P = 0.015), this lack of correlation persisted in all subgroups defined by genital inflammation or infection, current oral contraceptive use, heme contamination and volume of collected secretions, HPV16 seropositivity, and repeat HPV infection and/or cytologic abnormalities. The lack of correlation and high concentrations in cervical secretions indicate that the cervical IL-10 and IL-12 concentrations exceed what could be expected from blood as a principle source of IL-10 and IL-12 and suggest that cytokine concentrations in cervical secretions are predominantly the result of local cytokine production.

BACKGROUND

More than 50% of the colorectal cancer (CRC) etiology has been attributed to diet. Established or suspected dietary factors modifying risk of CRC are red meat, cereals, fish, and fibre. Diet and lifestyle may be linked to cancer through inflammation. Interleukin-10 (IL-10) is an anti-inflammatory cytokine. We wanted to test if dietary factors and IL10 polymorphisms interact in relation to colorectal carcinogenesis.

METHODS

The functional IL10 polymorphism C-592A (rs1800872) and the marker rs3024505 were assessed in relation to diet and lifestyle in a nested case-cohort study of 378 CRC cases and 775 randomly selected participants from a prospective study of 57,053 persons. Genotyping data on the IL10 polymorphism C-592A, smoking and non-steroidal anti-inflammatory drugs (NSAID) was retrieved from Vogel et al. (Mutat Res, 2007; 624:88). Incidence rate ratios (IRR) and 95% Confidence Interval (95% CI) were calculated.

RESULTS

No associations were found between the IL10 rs3024505 polymorphism and risk of CRC. There was interaction between rs3024505 and dietary fibre (P-value for interaction = 0.01). IL10 rs3024505 homozygous wildtype carriers were at 27% reduced risk of CRC per 10 g fibre per day (95% CI: 0.60-0.88) whereas variant carriers had no risk reduction by fibre intake. Also, interaction between IL10 C-592A and intake of fibre was found (P-value for interaction = 0.02). Among those eating <17.0 grams of fibre per day, carriers of an C-592A variant allele had a statistically significantly higher risk of colorectal cancer compared to homozygous wildtypes. No significant differences in colorectal cancer risk were observed between the reference group (CC and <17.0 g/day) and carriers of one C-592A variant allele eating 17.0 or more grams of dietary fibre per day. This suggests that the increased risk due to carrying the variant allele can be overcome by higher fibre intake. No interactions between IL10 polymorphisms and dietary meat, cereal, or fish intake, or between IL10 rs3024505 and smoking or NSAID use were found.

CONCLUSIONS

In this northern Caucasian cohort we found interaction between IL10 and dietary fibre in CRC carcinogenesis. High intake of fibre seems to protect against CRC among individuals with IL10 related genetic susceptibility to CRC. This finding should be evaluated in other prospective and population-based cohorts with different ethnic groups.

OBJECTIVE

The proteinase-activated receptor-2 (PAR-2) is activated by serine proteases and has been demonstrated to induce proinflammatory and neuroinflammatory effects. It is considered to alter transepithelial resistance and mediates visceral hypersensitivity. This study aimed to evaluate the expression of PAR-2 in human esophageal mucosa of patients with gastroesophageal reflux disease (GERD) in relation to mucosal alterations.

METHODS

The study included 123 patients with GERD stratified to erosive reflux disease (n=50), non-erosive reflux disease (n=46), and reflux-negative patients as controls (n=<em>27</em>). Endoscopic and histopathological characterization was performed according to the Los Angeles classification and modified Ismail-Beigi criteria, respectively. PAR-2 expression was analyzed by quantitative reverse transcription (RT)-PCR and immunohistochemistry. The gene expression levels of <em>interleukin</em> (IL)-8 were determined by quantitative RT-PCR and correlated to PAR-2 expression in each patient. Performing in vitro studies, esophageal squamous cell lines (KYSE 150, KYSE 450) were incubated, adjusted to different pH (7.0, 6.0, and 5.0), and exposed to bile acids and PAR-2-activation peptide (SLIGKV-NH(2)).

RESULTS

PAR-2 gene expression was 7- to 10-fold upregulated (P<0.0001) in the mucosa of patients with GERD and correlated positively with IL-8 expression and with histomorphological alterations (dilated intercellular spaces, papillary elongation, basal cell hyperplasia (BCH); P<0.01). Immunohistochemistry showed an intense staining of PAR-2 throughout all epithelial layers in patients with GERD compared with controls (P=0.0005). In vitro studies revealed a 1.5- to 20-fold induction of PAR-2 gene expression in esophageal squamous cells by acidified medium (P<0.01), but not by additional bile acids. The activation of PAR-2 leads to expression and secretion of IL-8.

CONCLUSIONS

This study provides evidence of the functional importance of PAR-2-mediated pathways in the pathogenesis of GERD and GERD-associated mucosal alterations and inflammatory changes.

Obesity is a state of chronic low-grade inflammation. Limiting white adipose tissue (WAT) expansion and therefore reducing inflammation could be effective in preventing the progression of obesity and the development of associated complications. We investigated the effects of 1,2-vinyldithiin (1,2-DT), a garlic-derived organosulfur, on the differentiation and inflammatory state of human preadipocytes. Preadipocytes were prepared from subcutaneous adipose tissue of nonobese young women and differentiated in the presence of 1,2-DT. Inflammatory preadipocytes were obtained following treatment with human macrophage-secreted factors. 1,2-DT (100 micromol/L) significantly reduced gene expression of PPARgamma2 (-40%), CCAAT/enhancer binding protein-alpha (-25%), lipoprotein lipase (-22%), leptin (-30%), and adiponectin (-15%). Lipid accumulation was also significantly diminished in preadipocytes differentiated in the presence of 100 micromol/L 1,2-DT (-37%) compared with controls. Furthermore, 100 micromol/L 1,2-DT treatment for 10 d significantly reduced PPARgamma activity (-<em>27</em>%). The protein expression of perilipin and the secretion levels for 2 adipokines, leptin and adiponectin, were significantly diminished in 1,2-DT-cultured preadipocytes (-37, -51, and -43%, respectively). Moreover, the secretion of inflammatory molecules (<em>interleukin</em>-6 and monocyte chemoattractant protein-1) induced by macrophage-secreted factors was partially abolished in 100 micromol/L 1,2-DT-treated preadipocytes (-28 and -25%, respectively). In conclusion, we demonstrated that 1,2-DT, a garlic-derived organosulfur, has antiadipogenic and antiinflammatory actions on human preadipocytes and may be a novel, antiobesity nutraceutical.

The objective of this study is to investigate interactions between adipocytes and breast cancer cells, and identify the responsible factors for the observed effects. In <em>27</em> breast cancer patients undergoing mastectomy, mammary adipose tissue was obtained from the breast quadrant bearing the tumor and corresponding non-tumoral quadrant. Isolated normal breast adipocytes (NBAs) and cancer-associated adipocytes (CAAs) were cultured in collagen gels to mimic the in vivo environment. Immunohistochemistry, qRT-PCR, and cell proliferation assays were performed to analyze adipocyte phenotypes. MCF7 and MDA-MB-231 breast cancer cell lines were co-cultured with adipocytes to detect phenotypic changes. Migration of MCF7 and MDA-MB-231 cells was assessed in NBA- and CAA-conditioned media. Cytokine levels in conditioned media were measured by cytokine array. Migration assays were repeated using conditioned media containing neutralizing antibodies. NBAs and CAAs lost their morphological phenotype in culture, acquiring a spindle-like shape, and CAAs showed higher cell proliferation, suggesting reversion to an immature phenotype. In co-cultures with MCF7 or MDA-MB-231 cells, NBAs exhibited increased cell proliferation, indicating acquisition of the immature phenotype of CAAs. MCF7 and MDA-MB-231 showed higher migration in a CAA-conditioned medium than in an NBA-conditioned medium. Cytokine array analysis of conditioned media revealed higher levels of <em>interleukin</em>-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1) in the CAA-conditioned medium. Neutralization experiments using antibodies against IL-6 or MCP-1 showed abrogation of migration-enhancing effects of the CAA-conditioned medium. Adipocytes revert to an immature and proliferative phenotype in the presence of breast cancer cells, and promote cancer cell migration via adipokines including IL-6 and MCP-1.

<em>Interleukin</em>-<em>27</em> (IL-<em>27</em>) is a pleiotropic cytokine which plays important and diverse roles in the immune system. We have previously demonstrated that IL-<em>27</em> induces potent anti-viral effects against HIV-1, HIV-2, SIV, HSV-2, KSHV and influenza viruses in macrophages. This induction occurred in an interferon (IFN) independent manner and involved down regulation of SPTBN1. MicroRNAs (miRNAs) are critical regulators of mRNA translation and turnover. There have been reports that some miRNAs inhibit viral replication. In this study, we hypothesized that IL-<em>27</em> could induce the expression of novel miRNAs in macrophages which may have functional relevance in terms of anti-viral activity and primary monocytes were differentiated into macrophages using either M-CSF (M-Mac) or a combination of M-CSF and IL-<em>27</em> (I-Mac) for seven days. Following this, total RNA was extracted from these cells and deep sequencing was performed, in parallel with gene expression microarrays. Using the novel miRNA discovery software, miRDeep, seven novel miRNAs were discovered in these macrophages. Four of which were preferentially expressed in I-Mac (miR-SX1, -SX2, -SX3 and -SX6) whilst three were detected in both M-Mac and I-Mac (miR-SX4, -SX5 and -SX7). The expression of six of the seven novel miRNAs was highly correlated with qRT-PCR using specific primer/probes designed for the novel miRNAs. Gene expression microarray further demonstrated that a number of genes were potentially targeted by these differentially expressed novel miRNAs. Finally, several of these novel miRNAs (miR-SX1, -SX4, -SX5, -SX6 and -SX7) were shown to target the open reading frames of a number of viruses (including HSV-1, HSV-2 and HHV-8) which may partially explain the anti-viral properties observed.

Background: The effectual immune response is crucial to defeat viral infections. However, exuberant immune response with features of macrophage activation syndrome (MAS) lead detrimental consequences in COVID-19 patients. Interleukin (IL)-18 is one of the leading cytokines in MAS which has not been studied in COVID-19.

Objective: To investigate the association of IL-18 with the other inflammatory markers and disease severity in COVID-19 for predicting disease prognosis.

Methods: Patients with COVID-19 who had confirmed diagnosis with SARS-CoV-2 nucleic acid RT-PCR were enrolled into the study. Data on demographic and clinical characteristics, and laboratory values of CRP, ferritin, d-dimer and procalcitonin were measured on admission. Patients were followed up prospectively with a standardized approach until hospital discharge or death. Individuals were classified as asymptomatic, mild and severe pneumonia according to their clinical, laboratory and radiological characteristics. Worse outcome was defined as requirement of intensive care unit (ICU) admission or death. Blood samples were collected at enrollment and serum levels of IL-6 and IL-18 were determined by ELISA. Association between IL-18 and other inflammatory markers and prognosis were analyzed.

Results: There were 58 COVID-19 patients (50% male) with a median age of 43 (min 22-max 81) years. Twenty age and sex matched healthy subjects were served as control group. The study population was divided into three groups according to disease severity: asymptomatic (n = 20), mild pneumonia group (n = 27) and a severe group (n = 11). During follow up nine (15.5%) patients required ICU admission and three of them were died eventually. Serum IL-18 were correlated with other inflammatory markers and biochemical markers of organ injury; creatinine, liver enzymes and troponin. Serum IL-18 levels were remarkably higher in COVID-19 patients compared to healthy subjects with being highest in severe pneumonia group (p < 0.001). IL-18 serum concentrations were almost four-fold higher in patients with worse outcome compared to good outcome (p < 0.001). Serum IL-18 above the cut off value of 576 pg/mL on admission was associated with 11.7 fold increased risk of ICU admission.

Conclusions: The serum concentrations of IL-18 correlate with other inflammatory markers and reflect disease severity. Results of the present study shed light on role of IL-18 on COVID-19 pathogenesis and might provide an evidence for the clinical trials on IL-18 antagonists for the treatment of severe COVID-19 patients.

Keywords: COVID-19; Cytokine; Intensive care unit; Interleukin-18; Prognosis.

We have studied the effects of food restriction (FR) and substitution of fish oil (FO; omega 3) for corn oil (CO; omega 6) on breast tumor incidence and survival in mouse mammary tumor virus/v-Ha-ras transgenic (Onco) mice. The diets were as follows: group 1, 5% (wt/wt) CO fed ad libitum (AL); group 2, 5% CO, restricted calories (40% fewer calories than AL; FR); group 3, 20% CO fed AL; and group 4, 20% FO fed AL. After 3 years, 40% of FR Onco (group 2) mice were alive, whereas there were no survivors in the other three groups. Similarly, tumor incidence was reduced to <em>27</em>% (5 out of 18) in FR animals (group 2), whereas it was 83% (11 out of 13) in group 1 mice, 89% (16 out of 18) in group 3 mice, and 71% (10 out of 14) in group 4 mice. These protective effects of FR on survival and tumor incidence were paralleled by higher expression of the tumor suppressor gene p53 (wild type) and free-radical scavenging enzymes (catalase and superoxide dismutase) in breast tumors. Immunoblotting showed less ras gene product, p21, and increased p53 levels in the tumors of FR mice. In addition, FR decreased RNA levels of c-erbB-2, <em>interleukin</em> 6, and the transgene v-Ha-ras in tumors. In contrast, analysis of hepatic mRNA from tumor-bearing FR mice revealed higher expression of catalase, glutathione peroxidase, and superoxide dismutase. Survival and tumor incidence were not influenced significantly by dietary supplementation with FO in place of CO. Taken together, our studies suggest that moderate restriction of energy intake significantly inhibited the development of mammary tumors and altered expression of cytokines, oncogenes, and free-radical scavenging enzymes.

Bruton's tyrosine kinase (Btk), a member of the Tec family of tyrosine kinases, plays an important role in the differentiation and activation of B cells. Mutations affecting Btk cause immunodeficiency in both humans and mice. In this study we set out to investigate the potential role of Btk in Toll-like receptor 9 (TLR9) activation and the production of pro-inflammatory cytokines such as <em>interleukin</em> (IL)-6, tumour necrosis factor (TNF)-alpha and IL-12p40. Our data show that Btk-deficient B cells respond more efficiently to CpG-DNA stimulation, producing significantly higher levels of pro-inflammatory cytokines but lower levels of the inhibitory cytokine IL-10. The quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis presented in this work shows that mRNA production of one of the important new members of the IL-12 family, IL-<em>27</em>, was significantly increased in Btk-deficient B cells after CpG-DNA stimulation. In this study, we demonstrate significant differences in CpG responsiveness between transitional 1 (T1) and T2 B cells for survival and maturation. Furthermore, TLR9 expression, measured both as protein and as mRNA, was increased in Btk-defective cells, especially after TLR9 stimulation. Collectively, these data provide evidence in support of the theory that Btk regulates both TLR9 activation and expression in mouse splenic B cells.

Peripheral blood mononuclear cells from <em>27</em> healthy leprosy contacts were analyzed for lymphoproliferation and TH-1 cytokine secretion (<em>interleukin</em>-2 and gamma interferon) in response to heat shock proteins with molecular masses of 65, 18, and 10 kDa from Mycobacterium leprae and the 30-32-kDa antigen 85 (Ag 85) from Mycobacterium bovis BCG. Cells from 18 and 19 of 19 lepromin-positive contacts proliferated or produced TH-1 cytokines in response to the M. leprae 10-kDa protein and to Ag 85, respectively. Limiting-dilution analysis for two lepromin-positive contacts indicated that about one-third of M. leprae-reactive T cells displayed specificity to the M. leprae 10-kDa protein and Ag 85. The M. leprae 65- and 18-kDa proteins were less potent TH-1 response inducers: gamma interferon and <em>interleukin</em>-2 could be measured in 14 and 19 lepromin-positive contacts, respectively. In contrast, very low or undetectable proliferative and cytokine responses were found for 8 lepromin-negative contacts. Our data demonstrate that the fibronectin-binding Ag 85 and the 10-kDa GroES homolog are powerful mycobacterial TH-1 response inducers in the vast majority of lepromin-positive contacts and suggest that they might be valuable candidates for a future subunit vaccine.

OBJECTIVE

To determine cerebrospinal fluid levels of osteopontin (OPN), a proinflammatory cytokine that was found to be overexpressed in multiple sclerosis lesions and increased in plasma during relapses and in secondary progressive multiple sclerosis.

METHODS

Case series. Osteopontin, interleukin 12p40 (IL-12p40), IL-10, and matrix metalloproteinase 9 were measured by enzyme-linked immunosorbent assay by an investigator unaware of the patients' diagnoses.

METHODS

Consecutive patients with multiple sclerosis (n = 27), or other inflammatory (n = 11) or non-inflammatory (n = 23) neurological diseases, undergoing lumbar puncture, were investigated.

RESULTS

Osteopontin was significantly elevated in the cerebrospinal fluid of patients with multiple sclerosis (mean [SD], 415 [186] ng/mL) and other inflammatory diseases (563 [411] ng/mL) compared with those with noninflammatory neurological diseases (286 [150] ng/mL). Cerebrospinal fluid OPN levels were slightly higher than plasma OPN levels. Cerebrospinal fluid OPN levels positively correlated with the ability to detect cerebrospinal fluid IL-12p40.

CONCLUSIONS

Osteopontin in the cerebrospinal fluid may be, in part, of central nervous system origin, and may play an important role in central nervous system inflammation.

A newly described cytokine, <em>interleukin</em>-<em>27</em> (IL-<em>27</em>), that activates naive CD4 T cells, has recently been shown to be an anti-HIV cytokine. However, the effect of HIV infection on IL-<em>27</em> expression has not been characterized. We found that clinical characteristics, including HIV viral load, hepatitis C virus coinfection, and CD4 T cell counts, were associated with changes in serum IL-<em>27</em>. Overall, our results suggest circulating HIV may suppress IL-<em>27</em>, a critical concept in treatment development with this cytokine.

The combined effects of stress and antigen on <em>interleukin</em>-1beta (Il-1beta) have rarely been studied locally at the site of microbial challenges in vivo, so far. We here propose a model for the analysis of such effects in humans and examine its utility for acute stress trials. Twelve students (6 male, 6 female) refrained from oral hygiene in two antagonistic quadrants for 28 days to allow for increasing bacterial stimulation of the respective gingival sites due to accumulation of microbial plaque. Good oral hygiene was maintained in the remaining quadrants. At day <em>27</em> and 28 students were subjected to either stress ('public speech') or a control condition, in a cross-over design. Samples of gingival crevicular fluid (GCF) which emerges between the tooth surface and the gingival epithelium as transudate of healthy and exudate of inflamed gingival tissue, were taken immediately after stress and 60 min later for Il-1beta analysis. Salivary cortisol was assessed to prove the validity of the stress protocol. Stress induced a profound increase of salivary cortisol (p=.001). Repeated measures (stress x time x hygiene) ANOVA with gender as between factor revealed significant stress (p=.014) and hygiene (p=.038) effects on GCF-Il-1beta concentrations and tentatively significant hygiene x time (p = .097) and stress x time x hygiene x gender (p=.107) interactions. Stress induced an increase of Il-1beta as did plaque accumulation. The merits of the proposed model are discussed. It is concluded that it is well suited for the assessment of the effects of stress on inflammatory responses in vivo in humans.

OBJECTIVE

To describe patient demographics, interventions, and outcomes in hospitalized children with macrophage activation syndrome (MAS) complicating systemic lupus erythematosus (SLE) or juvenile idiopathic arthritis (JIA).

METHODS

We performed a retrospective cohort study using data recorded in the Pediatric Health Information System (PHIS) database from October 1, 2006 to September 30, 2010. Participants had International Classification of Diseases, Ninth Revision, Clinical Modification diagnosis codes for MAS and either SLE or JIA. The primary outcome was hospital mortality (for the index admission). Secondary outcomes included intensive care unit (ICU) admission, critical care interventions, and medication use.

RESULTS

A total of 121 children at 28 children's hospitals met the inclusion criteria, including 19 children with SLE and 102 children with JIA. The index admission mortality rate was 7% (8 of 121 patients). ICU admission (33%), mechanical ventilation (26%), and inotrope/vasopressor therapy (26%) were common. Compared to children with JIA, those with SLE had a similar mortality rate (6% versus 11%, respectively; exact P = 0.6). More patients with SLE than those with JIA received ICU care (63% versus <em>27</em>%; P = 0.002), received mechanical ventilation (53% versus 21%; P = 0.003), and had cardiovascular dysfunction (47% versus 23% received inotrope/vasopressor therapy; P = 0.02). Children with SLE and those with JIA received cyclosporine at similar rates, but more children with SLE received cyclophosphamide and mycophenolate mofetil, and more children with JIA received <em>interleukin</em>-1 antagonists.

CONCLUSIONS

Organ system dysfunction is common in children with rheumatic diseases complicated by MAS, and more organ system support is required in children with underlying SLE than in children with JIA. Current treatment of pediatric MAS varies based on the underlying rheumatic disease.

OBJECTIVE

Synovial fluid glutamate concentrations increase in arthritis. Activation of kainate (KA) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate receptors (GluRs) increase interleukin-6 (IL-6) release and cause arthritic pain, respectively. We hypothesised that AMPA and KA GluRs are expressed in human arthritis, and that intra-articular NBQX (AMPA/KA GluR antagonist) prevents pain and pathology in antigen-induced arthritis (AIA).

METHODS

GluR immunohistochemistry was related to synovial inflammation and degradation in osteoarthritis (OA) and rheumatoid arthritis (RA). A single intra-articular NBQX injection was given at induction, and knee swelling and gait of AIA and AIA+NBQX rats compared over 21 days, before imaging, RT-qPCR, histology and immunohistochemistry of joints. Effects of NBQX on human primary osteoblast (HOB) activity were determined.

RESULTS

AMPAR2 and KA1 immunolocalised to remodelling bone, cartilage and synovial cells in human OA and RA, and rat AIA. All arthritic tissues showed degradation and synovial inflammation. NBQX reduced GluR abundance, knee swelling (p<0.001, days 1-21), gait abnormalities (days 1-2), end-stage joint destruction (p<0.001), synovial inflammation (p<0.001), and messenger RNA expression of meniscal IL-6 (p<0.05) and whole joint cathepsin K (p<0.01). X-ray and MRI revealed fewer cartilage and bone erosions, and less inflammation after NBQX treatment. NBQX reduced HOB number and prevented mineralisation.

CONCLUSIONS

AMPA/KA GluRs are expressed in human OA and RA, and in AIA, where a single intra-articular injection of NBQX reduced swelling by 33%, and inflammation and degeneration scores by 34% and 27%, respectively, exceeding the efficacy of approved drugs in the same model. AMPA/KA GluR antagonists represent a potential treatment for arthritis.

OBJECTIVE

In recent years, studies have been initiated to disclose the proteome of human chondrocytes and cartilage. Despite these studies, comprehensive information of the chondrocyte proteome remains limited. This study aimed to further explore the proteome expressed by human knee chondrocytes, and to study the functional aspects of heat-shock protein <em>27</em> (HSP<em>27</em>), a protein related to the previously described alphaBcrystallin, in chondrocyte biology.

METHODS

Chondrocytes isolated from human knee articular cartilage were cultured in a three-dimensional alginate culture system. To simplify the protein mixtures, proteins extracted from chondrocyte cell lysates were fractionated based on hydrophobicity and molecular weight. Proteins were digested and the resulting peptides were separated and identified by an on-line two-dimensional (2-D) nanoliquid chromatography (nanoLC)-system coupled to a quadrupole time-of-flight (Qq-TOF) mass spectrometer. Differential expression analysis of HSP<em>27</em> was performed by Western Blotting and quantitative polymerase chain reaction (QPCR). The effects of HSP<em>27</em> on chondrocyte biology were explored by suppression of HSP<em>27</em> expression induced by RNA interference (RNAi).

RESULTS

In this study, we identified proteins with unknown functions together with membrane proteins, transcription factors and other low abundant proteins, which have not yet been described in chondrocytes. Based on previous knowledge on the related protein alphaBcrystallin, we selected HSP<em>27</em> from the chondrocyte proteome database. Differential expression analysis revealed a decreased expression of HSP<em>27</em> in Osteoarthritic (OA) chondrocytes. RNAi experiments revealed that HSP<em>27</em> is involved in interleukin-1beta (IL-1beta) induced IL-6 secretion.

CONCLUSIONS

These findings highlight that small HSPs, especially HSP<em>27</em>, play a prominent role in the maintenance of human articular chondrocyte homeostasis.

Healing/protective responses in human visceral leishmaniasis (VL) are associated with stimulation/production of Th1 cytokines, such as interferon IFN-gamma, and conversion in the leishmanin skin test (LST). Such responses were studied for 90 days in 44 adult healthy volunteers from VL non-endemic areas, with no past history of VL/cutaneous leishmaniasis (CL) and LST non-reactivity following injection with one of four doses of Alum-precipitated autoclaved Leishmania major (Alum/ALM) +/- bacille Calmette-Guerin (BCG), a VL candidate vaccine. The vaccine was well tolerated with minimal localized side-effects and without an increase in antileishmanial antibodies or <em>interleukin</em> (IL)-5. Five volunteers (5/44; 11.4%) had significant IFN-gamma production by peripheral blood mononuclear cells (PBMCs) in response to Leishmania antigens in their prevaccination samples (P = 0.001) but were LST non-reactive. On day 45, more than half the volunteers (26/44; 59.0%) had significantly high LST indurations (mean 9.2 +/- 2.7 mm) and high IFN-gamma levels (mean 1008 +/- 395; median 1247 pg/ml). Five volunteers had significant L. donovani antigen-induced IFN-gamma production (mean 873 +/- 290; median 902; P = 0.001), but were non-reactive in LST. An additional five volunteers (5/44; 11.4%) had low IFN-gamma levels (mean 110 +/- 124 pg/ml; median 80) and were non-reactive in LST (induration = 00 mm). The remaining eight volunteers had low IFN-gamma levels, but significant LST induration (mean 10 +/- 2.9 mm; median 11). By day 90 the majority of volunteers (<em>27</em>/44; 61.4%) had significant LST induration (mean 10.8 +/- 9.9 mm; P < 0.001), but low levels of L. donovani antigen-induced IFN-gamma (mean 66.0 +/- 62 pg/ml; P>> 0.05). Eleven volunteers (11/44; 25%) had significantly high levels of IFN-gamma and LST induration, while five volunteers had low levels of IFN-gamma (<100 pg/ml) and no LST reactivity (00 mm). One volunteer was lost to follow-up. In conclusion, it is hypothesized that cellular immune responses to human VL are dichotomatous, and that IFN-gamma production and the LST response are not in a causal relationship. Following vaccination and probably cure of VL infection, the IFN-gamma response declines with time while the LST response persists. LST is a simple test that can be used to assess candidate vaccine efficacy.

We have examined the role of Ia-positive and Ia-negative accessory cells (AC) and soluble factors in Con A-stimulated murine T cell activation. Supernatant fluids containing <em>interleukin</em> 1 (IL 1) derived from the P388D1 macrophage cell line and from a lipopolysaccharide (LPS)-stimulated macrophage hybridoma provided only partial reconstitution of the response of purified T cells (18 to <em>27</em>%). The complete reconstitution obtained with gamma-irradiated spleen cells or LPS-activated B cells was inhibited by approximately 60 to 77% when anti-Ia antibody was included in the culture. Despite this apparent involvement of Ia+ spleen AC, Ia-negative L cell AC could also reconstitute the response of both Class I-restricted Lyt-2+ T cells and Class II-restricted L3T4+ T cells. When the Ia-negative AC were employed, the L3T4 antigen on L3T4+ T cells played a critical role because addition of anti-L3T4 antibody to the culture inhibited the response by 85 to 90%. In contrast, anti-L3T4 did not inhibit the response in the presence of spleen AC. These results suggest that the molecules involved in T cell-AC interactions may vary depending on the AC source. Moreover, at least one of the putative target ligands for L3T4 presumably is not Ia, because anti-L3T4 inhibited T cell stimulation when Ia-negative AC were used.

CD4+ T cells support host defence against herpesviruses and other viral pathogens. We identified that CD4+ T cells from systemic and mucosal tissues of hosts infected with the β-herpesviridae human cytomegalovirus (HCMV) or murine cytomegalovirus (MCMV) express the regulatory cytokine <em>interleukin</em> (IL)-10. IL-10+CD4+ T cells co-expressed TH1-associated transcription factors and chemokine receptors. Mice lacking T cell-derived IL-10 elicited enhanced antiviral T cell responses and restricted MCMV persistence in salivary glands and secretion in saliva. Thus, IL-10+CD4+ T cells suppress antiviral immune responses against CMV. Expansion of this T-cell population in the periphery was promoted by IL-<em>27</em> whereas mucosal IL-10+ T cell responses were ICOS-dependent. Infected Il<em>27</em>rα-deficient mice with reduced peripheral IL-10+CD4+ T cell accumulation displayed robust T cell responses and restricted MCMV persistence and shedding. Temporal inhibition experiments revealed that IL-<em>27</em>R signaling during initial infection was required for the suppression of T cell immunity and control of virus shedding during MCMV persistence. IL-<em>27</em> production was promoted by type-I IFN, suggesting that β-herpesviridae exploit the immune-regulatory properties of this antiviral pathway to establish chronicity. Further, our data reveal that cytokine signaling events during initial infection profoundly influence virus chronicity.

OBJECTIVE

We evaluated the expression of epithelial cell adhesion molecule (EpCAM) and the potential of MT201 (adecatumumab), a human-monoclonal-antibody that targets EpCAM against chemotherapy-resistant ovarian disease.

METHODS

EpCAM expression was evaluated by real-time polymerase chain reaction and flow cytometry. Sensitivity to MT201 antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity was tested in 4-hour chromium-release assays. The effect of interleukin-2 on MT201 ADCC was also studied.

RESULTS

High messenger RNA expression by real-time polymerase chain reaction and high EpCAM surface expression by flow cytometry was detected in 71% of ovarian cancers (5 of 7 cell lines). Although these cell lines were highly resistant to complement-dependent cytotoxicity and natural killer-dependent cytotoxicity in vitro (range of killing, 0-7%), EpCAM-positive cell lines showed high sensitivity to MT201 ADCC (range of killing, 27-66%). Incubation with interleukin-2 further increased the cytotoxic activity against EpCAM-positive ovarian cancer cell lines.

CONCLUSIONS

MT201 may represent a novel, potentially highly effective treatment option for patients with ovarian carcinoma whose body is harboring disease refractory to chemotherapy.

In mice and women, terminal differentiation of uterine natural killer (uNK) cells commences during endometrial decidualization. Both proliferation and interferon (IFN)-gamma are induced. Uterine NK cell precursors appear to home from secondary lymphoid organs to decidualizing uteri and localize mesometrially to the central decidua basalis, the site of maternal arterial modification at Gestation Days (gd) 9.5-10. In mice, genetic absence of uNK cells results in absence of pregnancy-induced spiral artery modification. Administration of IFN-gamma to uNK-negative pregnant females induces arterial modifications without fetal loss. In this study, we investigated the roles of cytokines, known in other tissues to differentiate and activate NK cells, in induction of IFN-gamma production in normal mouse implantation sites. Fecundity evaluation, implantation site morphometry, and IFN-gamma quantification in <em>interleukin</em> (IL)-12p40(0/0), IL-18(0/0), dual IL-12p40(0/0)/IL-18(0/0) and congenic strains revealed the importance of both IL-12 and IL-18 in the induction of spiral artery modification and IFN-gamma synthesis. Immediately after implantation, IL-18 was localized transiently to decidual cells, but by gd8, IL-18 was produced solely by uNK cells, suggesting that early uNK cells are activated by stroma and lymphocyte-derived signals maintain later uNK cell activation. Mesometrial tissue of C57Bl/6J mice was examined by reverse transcription polymerase chain reaction assay in virgin, early postimplantation, and midgestation females for expression of the heterodimeric cytokines IL-23 (composed of IL-12p40 and a novel alpha chain), IL-<em>27</em> (composed of two IL-12-related chains) and IL-<em>27</em>R. No expression was detected in virgin uteri. The four genes were induced by gd6, and uNK cells isolated from midgestation transcribed IL-23alpha and IL-<em>27</em>R. This study advances the understanding of uNK cell activation during normal pregnancy.

Ten patients with ovarian cancer refractory to conventional therapy were treated with intraperitoneal (i.p.) recombinant <em>interleukin</em>-2 (rIL-2) and lymphokine-activated killer cells (LAK). The 28-day protocol consisted of 6 priming i.p. rIL-2 infusions on days 0, 4, 6, 8, 10, and 12. Leukapheresis was performed for mononuclear cell collection on days 15, 16, 17, and 18 and lymphokine-activated killer cells were given i.p. with the rIL-2 on days 19 and 21. Three additional i.p. rIL-2 infusions were given on days 23, 25, and <em>27</em>. Three dose levels of rIL-2 were tested: 5 X 10(5), 2 X 10(6), and 8 X 10(6) units/m2 body surface area. The dose-limiting toxicity was abdominal pain secondary to ascites accumulation with significant weight gain. Other toxic effects included decreased performance status, fever, nausea and vomiting, diarrhea, and anemia. Peripheral lymphocytosis and eosinophilia were seen at all dose levels. The maximum tolerated dose is 8 X 10(6) units/m2/dose. Peripheral and peritoneal IL-2 levels were measured with a bioassay using an IL-2-dependent cell line. At the highest dose level, serum IL-2 was greater than 10 units/ml for 18 h. After the first infusion, a 2-log dilution of the i.p. IL-2 was measured in the serum. In the postleukapheresis i.p. IL-2-dosing period less IL-2 was detected in the serum than in the earlier i.p. IL-2-priming period. The induction and persistence of LAK activity were studied. Peritoneal LAK activity was detected as early as 4 days after the first i.p. infusion, by day 11 in all evaluable patients, and persisted for the 6-day interval between priming IL-2 and LAK/IL-2 infusion. Peritoneal lytic activity persisted until day 28 in 5 tested patients. These peritoneal cells retained lytic activity 48 h in culture medium without rIL-2 present. Peritoneal LAK activity correlated with the percentage of mononuclear cells and the percentage of CD56-positive mononuclear cells in the peritoneum. The yield of peripheral lymphocytes after the six i.p. priming doses of rIL-2 correlated with the dose level of rIL-2 infused. Peripheral blood LAK activity showed a minimal, however progressive, increase during the treatment protocol. LAK activity could be enhanced if rIL-2 was present during the 4-h assay. These studies indicate that i.p. rIL-2 infusion induced durable regional LAK activity and primes peripheral blood cells for LAK activity if exposed briefly to additional IL-2.

Cytokines from the <em>interleukin</em>-6 (IL-6) family have been reported to play an important synergistic role with angiotensin II in the development of pathological cardiac hypertrophy. Whether their expression pattern changes in vivo, in an angiotensin I-dependent hypertrophied myocardium has not been reported. In this study, we addressed that issue using two animal models of angiotensin II-dependent cardiac hypertrophy. Heterozygous transgenic TGR(mRen2)<em>27</em> (TGR) with an overactive cardiac renin angiotensin system and the closely related spontaneously hypertensive rats (SHR) were compared to their respective control rats. The mRNA levels of IL-6, leukemia inhibitory factor (LIF), ciliary neurotrophic factor (CNTF) and cardiotrophin-1 (CT-1) as well as their receptor subunits, glycoprotein 130 (gp130), IL-6 receptor (IL-6R), LIFR, and CNTFR, were measured by semi-quantitative RT-PCR. The protein levels of IL-6, LIF and CT-1 were investigated by western blot. TGR and SHR both displayed significant over expression of mRNA and protein levels for IL-6 and LIF. In TGR, the increased level of LIF was accompanied by a decrease in mRNA levels for LIFR and CNTFR. In SHR, a higher level of mRNA IL-6R was observed. By contrast, the mRNA and protein levels for CT-1 and the mRNA level for gp130 did not vary in these two models. These findings suggest that IL-6 and LIF, but not CT-1, contribute to angiotensin II-dependent left ventricular hypertrophy in the two hypertensive rat models, TGR(mRen2)<em>27</em> and SHR.

<em>Interleukin</em>-12 (IL-12) and the more recently discovered IL-23 and IL-<em>27</em> constitute a unique family of structurally related, heterodimeric cytokines that regulate cell-mediated immune responses and T helper 1 (Th1)-type inflammatory reactions. Not surprisingly, the potentiality of treating conditions such as multiple sclerosis (MS) and rheumatoid arthritis (RA) through pharmacological interference with IL-12 pathways has received widespread attention. In this review we have examined over 50 substances with reported IL-12 inhibitory effects. We demonstrate that a majority of these belong to a limited number of major functional classes, each of which targets discrete events in the IL-12 biological pathway. Thus, most IL-12 inhibitory substances appear to work either through inhibition of transcription factor NF-kappa B activation, up-regulation of intracellular cAMP, blockage of posttranslational processing or interference with signal transduction pathways. In addition, cyclophilin-binding drugs, and generic inhibitors of nuclear histone deacetylases, and of ion channels, pumps and antiporters are emerging as potential leads to novel targets for interference with IL-12 production. Many inhibitors of NF-kappa B and of IL-12 signal transduction have been proven effective in limiting or preventing disease in experimental autoimmune encephalomyelitis (EAE) models of MS. The sharing of the p40 subunit, the IL-12R beta 1 and components of the signal transduction pathways between IL-12 and IL-23 raises the question as to whether the beneficial effects of various drugs previously ascribed to inhibition of IL-12 may, in fact, have been due to concurrent blockage of both cytokines, or of IL-23, rather than IL-12. Moreover, the homodimeric beta(2)-form of IL-12, though originally considered to display only antagonistic effects, is now emerging as a pronounced agonist in a variety of inflammatory processes. Reassessment of IL-12 inhibitory compounds is therefore needed to scrutinize their effects on IL-12 alpha beta, beta(2) and IL-23 formation. This is likely to open exciting perspectives to the identification of drugs that target these cytokines either indiscriminately or selectively. The functional diversity of presently available inhibitors should facilitate an unprecedented flexibility in designing future trials for the treatment of IL-12- and IL-23-mediated disorders.