We have shown a significant disruption of 24-h pattern of plasma pituitary, adrenal, and gonadal hormones in high-fat-fed rats. Our objective was to assess the effect of a high-fat diet (<em>35</em>% fat) on mean levels and 24-h pattern of several adipocytokines in rats. A normal diet-fed rats (4% fat) were used as controls. When body weight of high-fat-fed rats attained values about 25% higher than controls (after 66 days of treatment), the animals were killed at six different time intervals throughout a 24-h cycle. Plasma concentrations of insulin, adiponectin, <em>interleukin</em> (IL)-1, leptin, ghrelin, plasminogen activator inhibitor-1 (PAI-1), and monocyte chemoattractant protein-1 (MCP-1) were measured in a multianalyte profiling by using the Luminex-100 system. Tumor necrosis factor alpha (TNFalpha) and IL-6 were measured by enzyme-linked immunosorbent assay. A significant hyperglycemia developed in high-fat-fed rats, together with a significant increase in plasma insulin. Mean levels of plasma adiponectin, IL-1, IL-6, TNFalpha, and leptin augmented, and ghrelin decreased, in high-fat-fed rats. The normal daily pattern of plasma insulin, adiponectin, IL-1, IL-6, TNFalpha, leptin, ghrelin, and MCP-1 became disrupted in high-fat-fed rats. The results indicate that a high-fat diet may bring about signs of insulin resistance and mild inflammation in rats, together with the disruption in daily variations of circulating insulin and ghrelin, and of several adipocytokines including leptin, adiponectin, IL-1, IL-6, TNFalpha, and MCP-1.

BACKGROUND

Evidence of the role of cytokines produced by resident and inflammatory cells during inflammation is well established. The aim of this study was to quantify in healthy and diseased human gingiva the area fraction (AA%) occupied by collagen fibers and the amount of cytokines such as interleukin (IL)-1beta, IL-4, IL-6, tumor necrosis factor (TNF)-alpha, transforming growth factor (TGF)-beta, and epidermal growth factor (EGF) to investigate a possible correlation between such cytokines, collagen degradation, and the gingival index.

METHODS

Gingival tissue specimens from 6 healthy controls (group 1), 6 patients with mild gingival inflammation (group 2), 6 patients with moderate gingival inflammation (group 3), and 6 patients with severe gingival inflammation (group 4) were cultured for 72 hours, and the cytokines present in the culture media were quantified using an enzyme-linked immunosorbent assay (ELISA). Paraffin gingival sections from the 24 subjects were stained with sirius red F3Ba for visualization of collagen fibers, then the area fraction (AA%) occupied by the gingival fibers was determined by automated image analysis.

RESULTS

The present study revealed significant differences (P < 0.05) between means of AA% in group 1 (53%), group 2 (41%), group 3 (39.5%), and group 4 (35%) for collagen fibers. Compared to controls, there were significant increases of IL-1beta (groups 3 and 4), IL-6, and TNF-alpha (group 3); a significant decrease of IL-4 (groups 2, 3, and 4) and TGF-beta (groups-2 and, 3); and no change of EGF. The collagen AA% was significantly correlated with the amounts of IL-4 and TGF-beta, and significantly inversely correlated with the amounts of IL-1beta for all 3 inflamed groups and IL-6 and TNF-alpha for groups 2 and 3.

CONCLUSIONS

The present study showed that EGF was not changed in inflamed gingival tissue and that IL-1beta and IL-4 were particularly and intensively correlated with collagen loss. These 2 cytokines could be markers of clinical severity during active periodontitis.

OBJECTIVE

Recombinant human growth hormone (rhGH) is a salutary modulator of posttraumatic metabolic responses. However, rhGH administration is associated with deleterious side effects, such as hyperglycemia, increased free fatty acids, and triglycerides, which limit its use. Administration of beta-blocker attenuates cardiac work and resting energy expenditure after severe thermal injury and improves fat metabolism and insulin sensitivity. Therefore, the combination of rhGH plus propranolol appears ideal. The aim of the present study was to determine whether rhGH plus propranolol improves hypermetabolism and the inflammatory and acute phase response after severe burn without causing adverse side effects.

METHODS

Prospective randomized control trial.

METHODS

Shriners Hospitals for Children.

METHODS

Fifteen pediatric patients with burns>> 40% total body surface area, 0.1-16 yrs of age, admitted within 7 days after burn. Fifteen children were matched for burn size, age, gender, inhalation injury, and infection and served as controls.

METHODS

Patients in the experimental group received rhGH (0.2 mg/kg/day) and propranolol (to decrease heart rate by 15%) for>> or = 15 days.

RESULTS

Outcome measurements included resting energy expenditure, body composition, acute phase proteins, and cytokines. Both cohorts were similar in age, burn size, gender, and accompanying injuries. Percent predicted resting energy expenditure significantly decreased in patients receiving rhGH/propranolol (Delta -5% +/- 8%) compared with controls (Delta +<em>35</em>% +/- 20%) (p < .05). rhGH/propranolol administration significantly decreased serum C-reactive protein, cortisone, aspartate aminotransferase, alanine aminotransferase, free fatty acids, <em>interleukin</em>-6, <em>interleukin</em>-8, and macrophage inflammatory protein-1beta when compared with controls, while growth hormone/propranolol increased serum insulin-like growth factor-I, insulin-like growth factor binding protein-3, growth hormone, prealbumin, and <em>interleukin</em>-7 when compared with placebo (p < .05).

CONCLUSIONS

rhGH in combination with propranolol attenuates hypermetabolism and inflammation without the adverse side effects found with rhGH therapy alone.

Pulmonary arterial hypertension (PAH) is associated with dysregulated bone morphogenetic protein receptor (BMPR)-II signaling and pulmonary vascular inflammation. We evaluated the effects of dexamethasone on monocrotaline (MCT)-induced PAH in rats for potential reversal of PAH at late time-points. Saline-treated control, MCT-exposed, MCT-exposed and dexamethasone-treated rats (5 mg·kg⁻¹·day⁻¹, 1.25 mg·kg⁻¹ and 2.5 mg·kg⁻¹·48 h⁻¹) were evaluated at day 28 and day <em>35</em> following MCT for haemodynamic parameters, right ventricular hypertrophy, morphometry, immunohistochemistry, and IL6 and BMPR2 expression. Dexamethasone improved haemodynamics and pulmonary vascular remodelling, preventing PAH development at early (day 1-14 and 1-28) and reversing PAH at late (day 14-28 and 21-<em>35</em>) time-points following MCT, as well as improving survival in MCT-exposed rats compared with controls. Both MCT-induced pulmonary IL6 overexpression and <em>interleukin</em> (IL)-6-expressing adventitial inflammatory cell infiltration were reduced with dexamethasone. This was associated with pulmonary BMPR2 downregulation following MCT, which was increased with dexamethasone, in whole lung and control pulmonary artery smooth muscle cells. Dexamethasone also reduced proliferation of rat pulmonary artery smooth muscle cells in vitro. Experimental PAH can be prevented and reversed by dexamethasone, and survival is improved. In this model, mechanisms may involve reduction of IL-6-expressing inflammatory cells, restoration of pulmonary BMPR2 expression and reduced proliferation of vascular smooth muscle cells.

Granuloma formation in response to mycobacterial infections is associated with increased expression of inducible nitric oxide synthase (NOS2) within granuloma macrophages and increased levels of nitrate/nitrite in the sera of infected mice. Continuous treatment with 5 mm or 10 mm l-N6-(1-imino-ethyl)-lysine (L-NIL), a selective NOS2-inhibitor, in acidified drinking water for up to 7 weeks consistently reduced infection-induced nitrate/nitrite to background levels in mycobacteria-infected BALB/c mice. Oral treatment with 5 mm L-NIL initiated at the time of infection significantly exacerbated growth of Mycobacterium tuberculosis, but had no effect on Mycobacterium avium colony-forming unit development in the liver, spleen and lungs of intravenously infected mice. In order to examine the role of nitric oxide in mycobacteria-induced granulomatous inflammation in the absence of any effect on the bacterial load, M. avium-infected mice were treated with 5 mm L-NIL from day 1 through 38 and the development of granulomatous lesions in the liver was assessed by histology, immunohistology and reverse-transcription-polymerase chain reaction (RT-PCR). Computer- and video-assisted morphometry performed at 4 and 7 weeks post-infection showed that treatment with L-NIL led to markedly increased number, cellularity and size of granulomatous lesions in infected mice regardless of the virulence of the M. avium isolate used for infection. Immunohistology of the liver revealed that in mice treated with L-NIL, the numbers of CD3+ T cells, CD21/<em>35</em>+ B cells, CD11b+ macrophages and RB6-8C5+ granulocytes associated with granulomatous lesions was increased. RT-PCR of the liver showed that in L-NIL-treated mice infected with M. avium, mRNA levels of tumour necrosis factor, <em>interleukin</em>-12p40, interferon-gamma, <em>interleukin</em>-10 and interferon-gamma-inducible protein-10 (IP-10) were up-regulated, while mRNA levels of <em>interleukin</em>-4, monocyte chemotactic protein-1 (MCP-1) and MCP-5 were similar to those in untreated control infected mice. When M. avium-infected mice were treated with 5 mm L-NIL between the 5th and 12th weeks of infection, similar changes in granuloma number and size were found in the absence of any effect on the bacterial load. These findings demonstrate that nitric oxide regulates the number, size and cellular composition of M. avium-induced granulomas independently of antibacterial effects by modulating the cytokine profile within infected tissues.

The human 4F2 cell surface antigen is a 120-kilodalton (kDa) disulfide-linked heterodimer which is composed of an 80- to 90-kDa glycosylated heavy chain (4F2HC) and a <em>35</em>- to 40-kDa nonglycosylated light chain (4F2LC). 4F2 belongs to a family of inducible cell surface molecules which are involved in T-lymphocyte activation and growth. To better understand the molecular mechanism(s) that controls 4F2HC gene expression in both resting and activated T cells, a 4F2HC human genomic clone was isolated and structurally characterized. The 4F2HC gene spans 8 kilobases of chromosome 11 and is composed of nine exons. The 5' upstream region of the gene displays several properties which are characteristic of housekeeping genes. It is G+C rich and hypomethylated in peripheral blood lymphocyte DNA and contains multiple binding sites for the Sp1 transcription factor while lacking TATA or CCAAT sequences. This region of the gene also displays sequence homologies with several other inducible T-cell genes, including the <em>interleukin</em>-2, <em>interleukin</em>-2 receptor alpha chain, dihydrofolate reductase, thymidine kinase, and transferrin receptor genes. A 255-base-pair fragment of the 4F2HC gene which contains 154 base pairs of the 5' flanking sequence was able to efficiently promote expression of the bacterial chloramphenicol acetyltransferase gene in human Jurkat T cells, indicating that it contains promoter or enhancer (or both) sequences. Analyses of chromatin structure in resting and lectin-activated T cells revealed the presence of stable DNase I-hypersensitive sites within both the 5' flanking and intron 1 regions of the 4F2HC gene. Although the 4F2HC gene displayed many of the structural features characteristic of a constitutively expressed gene, lectin-mediated activation of resting peripheral blood T lymphocytes resulted in a dramatic increase in steady-state levels of 4F2HC mRNA.

The purpose of this study was to evaluate the safety, tolerability, pharmacokinetics, and possible antitumor activity of a ligand fusion-protein, DAB389IL-2, in a phase I trial. This was a multicenter, open-label, dose-escalation trial. Patients with preserved organ function and histologically confirmed relapsed cutaneous T-cell lymphoma (CTCL), other non-Hodgkin's lymphomas (NHL), or Hodgkin's disease (HD) were eligible if their cancer was shown to express the <em>interleukin</em> (IL)-2 receptor by an immunohistochemical assay for the p55 or the p75 subunit. Patients received up to eight courses of DAB389IL-2 given as a short intravenous infusion daily for 5 days with subsequent courses every 21 days. The maximum tolerated dose (MTD) and tumor response was determined according to standard criteria. Seventy-three patients (44 men/29 women), aged 16 to 81 years (mean, 50.7) with CTCL (n = <em>35</em>), NHL (n = 17), and HD (n = 21) were enrolled. The patients were extensively treated, failing 0 to 15 previous therapies (median, 4). Patients received one to six courses (mean, 3.3) of DAB389IL-2 over a range of 3 to 31 micrograms/kg/day. The dose-limiting toxicity was asthenia, establishing the maximum tolerated dose of 27 micrograms/kg/day. Approximately half of all patients had significant titers of antibody to diphtheria toxin or to DAB389IL-2 at the time of enrollment compared with 92% with titers at the end of treatment. The presence of antibody did not preclude clinical response. There were five complete (CR) and eight partial (PR) remissions in patients with CTCL with one CR and two PR occurring in NHL. The median time to response was 2 months and the duration of response was 2 to 39+ months. No responses were documented in patients with HD. DAB389IL-2 is well tolerated with an MTD of 27 micrograms/kg/day. This ligand fusion-protein showed antitumor effects in patients with IL-2 receptor expressing CTCL and NHL. Additional trials in these diseases are warranted.

BACKGROUND

Traumatic brain injury (TBI) initiates interrelated inflammatory and coagulation cascades characterized by wide-spread cellular activation, induction of leukocyte and endothelial cell adhesion molecules and release of soluble pro/antiinflammatory cytokines and thrombotic mediators. Resuscitative care is focused on optimizing cerebral perfusion and reducing secondary injury processes. Hypertonic saline is an effective osmotherapeutic agent for the treatment of intracranial hypertension and has immunomodulatory properties that may confer neuroprotection. This study examined the impact of hypertonic fluids on inflammatory/coagulation cascades in isolated head injury.

METHODS

Using a prospective, randomized controlled trial we investigated the impact of prehospital resuscitation of severe TBI (GCS < 8) patients using 7.5% hypertonic saline in combination with 6% dextran-70 (HSD) vs 0.9% normal saline (NS), on selected cellular and soluble inflammatory/coagulation markers. Serial blood samples were drawn from 65 patients (30 HSD, <em>35</em> NS) at the time of hospital admission and at 12, 24, and 48-h post-resuscitation. Flow cytometry was used to analyze leukocyte cell-surface adhesion (CD62L, CD11b) and degranulation (CD63, CD66b) molecules. Circulating concentrations of soluble (s)L- and sE-selectins (sL-, sE-selectins), vascular and intercellular adhesion molecules (sVCAM-1, sICAM-1), pro/antiinflammatory cytokines [tumor necrosis factor (TNF)-alpha and <em>interleukin</em> (IL-10)], tissue factor (sTF), thrombomodulin (sTM) and D-dimers (D-D) were assessed by enzyme immunoassay. Twenty-five healthy subjects were studied as a control group.

RESULTS

TBI provoked marked alterations in a majority of the inflammatory/coagulation markers assessed in all patients. Relative to control, NS patients showed up to a 2-fold higher surface expression of CD62L, CD11b and CD66b on polymorphonuclear neutrophils (PMNs) and monocytes that persisted for 48-h. HSD blunted the expression of these cell-surface activation/adhesion molecules at all time-points to levels approaching control values. Admission concentrations of endothelial-derived sVCAM-1 and sE-selectin were generally reduced in HSD patients. Circulating sL-selectin levels were significantly elevated at 12 and 48, but not 24 h post-resuscitation with HSD. TNF-alpha and IL-10 levels were elevated above control throughout the study period in all patients, but were reduced in HSD patients. Plasma sTF and D-D levels were also significantly lower in HSD patients, whereas sTM levels remained at control levels.

CONCLUSIONS

These findings support an important modulatory role of HSD resuscitation in attenuating the upregulation of leukocyte/endothelial cell proinflammatory/prothrombotic mediators, which may help ameliorate secondary brain injury after TBI.

BACKGROUND

NCT00878631.

This study investigated the presence of mRNA coding for interferon gamma (IFN gamma), tumour necrosis factor alpha (TNF alpha), and <em>interleukins</em> 2 (IL2) and 6 (IL6), in the mucosa of four coeliac patients in remission who had been challenged with either gliadin or synthetic gliadin oligopeptides. Jejunal biopsy specimens from these patients, taken before and at two, four, and six hours after challenge, were hybridised with specific <em>35S</em>-labelled DNA oligonucleotide probes. The lamina propria of all the patients contained significantly increased numbers of cytokine mRNA expressing cells four hours after challenge with gliadin or an oligopeptide corresponding to amino acids 31-49 of A-gliadin (peptide A). No significant changes were seen with the peptides corresponding to aminoacids 202-220 (peptide B) or 3-21 (peptide C) of A-gliadin, with the exception of one patient who showed a significant increase in the number of TNF alpha mRNA expressing cells four hours after challenge with peptide B. In vivo studies in coeliac disease have shown that significant histological changes occur in the mucosa of treated coeliac patients four hours after challenge with either gliadin or peptide A. These findings suggest that the histological changes seen previously in the mucosa of coeliac patients after wheat peptide challenge may be caused by increased expression of cytokines within the mucosa.

BACKGROUND

We have previously shown the long-term influence of renal ischemia/reperfusion (I/R) injury and immunosuppression on fibrotic genes and apoptosis in a rat model. For the first time, we have now investigated the effects of I/R and immunosuppression on inflammation and caspase activation.

METHODS

I/R injury was induced in the right kidney and the left was removed. Cyclosporin (CsA) (10 mg/kg), tacrolimus (0.2 mg/kg), rapamycin (1 mg/kg), or mycophenolate mofetil (MMF) (10 mg/kg) was then administered for 16 weeks. The effects of I/R and immunosuppressants on interstitial inflammation, interleukin (IL)-1beta expression, caspase-1 and caspase-3 activation, tubulointerstitial damage, and fibrosis were evaluated.

RESULTS

ED-1+ (a specific rat monocyte/macrophage marker) cells were mainly localized in the tubulointerstitium and periglomerular areas and increased in I/R group compared to controls (P < 0.01). This was further increased by CsA, but decreased by tacrolimus, rapamycin, or MMF (P < 0.05). The 17 kD active IL-1beta remained unchanged, but 35 kD IL-1beta precursor was decreased by rapamycin in comparison with I/R group (P < 0.05). The 45 kD or 20 kD caspase-1 was increased by I/R or CsA, respectively, and decreased by rapamycin (P < 0.05). The 24 kD caspase-3, which proved to be an active caspase-3 subunit, was increased in I/R and CsA groups and deceased by tacrolimus, rapamycin, or MMF (P < 0.05), but not 32 kD precursor or 17 kD active caspase-3. The activity data of caspase-1 and caspase-3 exhibited the same trend as Western blotting data. The staining of active caspase-3 was scattered in kidneys, mainly in tubular and interstitial areas, which was consistent with that of ED-1+ cells. There was a strong positive correlation between interstitial inflammation and 24 kD caspase-3 expression or caspase-3 activity (r = 0.814 or 0.484), all of which were also closely related with urinary protein (r = 0.537, 0.529, or 0.517), serum creatinine (r = 0.463, 0.573, or 0.539), tubulointerstitial damage (r = 0.794, 0.618, or 0.712) and fibrosis (r = 0.651, 0.567, or 0.469), all P < 0.01.

CONCLUSIONS

This study shows that the mechanisms of long-term I/R injury and immunosuppressants treatment include interstitial inflammation and caspase activation, most clearly demonstrated by the 24 kD active caspase-3.

The chemokine receptor CXCR6 is highly expressed on lung-derived T cells compared to blood T cells, especially in inflammatory diseases characterised by T-cell migration to the lung. This suggests that CXCR6 is a candidate lung homing receptor. The sole ligand of CXCR6, CXCL16, has previously been shown to be expressed by alveolar macrophages. The authors hypothesized that also structural lung cells express CXCL16. CXCL16 expression was detected using real-time reverse transcriptase-polymerase chain reaction (RT-PCR), Western blotting, enzyme-linked immunosorbent assay (ELISA), and flow cytometry. Chemotaxis assays were used to test functionality of the secreted protein. Human bronchial epithelial cells secreted relatively high basal levels of CXCL16 >> 1000 pg/mL). Interferon (IFN)-gamma, but not tumor necrosis factor (TNF)-alpha or <em>interleukin</em> (IL)-4, caused a modest but significant up-regulation in secretion. Airway smooth muscle and fibroblasts also expressed CXCL16, but at lower levels. Western blotting detected expression of the full-length (60-kDa) form of the chemokine in cell lysates, and the cleaved (<em>35</em>-kDa) form in culture supernatants. Concentrated supernatants from a bronchial epithelial cell line (BEAS-2B) were chemotactic for CXCR6 expressing T cells from blood. In conclusion, these results suggest that the bronchial epithelium is an important source of constitutively expressed CXCL16, which may be involved in T-cell recruitment to the lung in health and disease.

BACKGROUND

This study was performed to evaluate the attenuation of osteoarthritic (OA) pathogenesis via disruption of the stromal cell-derived factor-1 (SDF-1)/C-X-C chemokine receptor type 4 (CXCR4) signaling with AMD3100 in a guinea pig OA model.

METHODS

OA chondrocytes and cartilage explants were incubated with SDF-1, siRNA CXCR4, or anti-CXCR4 antibody before treatment with SDF-1. Matrix metalloproteases (MMPs) mRNA and protein levels were measured with real-time polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. The <em>35</em> 9-month-old male Hartley guinea pigs (0.88 kg ± 0.21 kg) were divided into three groups: AMD-treated group (n = 13); OA group (n = 11); and sham group (n = 11). At 3 months after treatment, knee joints, synovial fluid, and serum were collected for histologic and biochemical analysis. The severity of cartilage damage was assessed by using the modified Mankin score. The levels of SDF-1, glycosaminoglycans (GAGs), MMP-1, MMP-13, and <em>interleukin</em>-1 (IL-1β) were quantified with ELISA.

RESULTS

SDF-1 infiltrated cartilage and decreased proteoglycan staining. Increased glycosaminoglycans and MMP-13 activity were found in the culture media in response to SDF-1 treatment. Disrupting the interaction between SDF-1 and CXCR4 with siRNA CXCR4 or CXCR4 antibody attenuated the effect of SDF-1. Safranin-O staining revealed less cartilage damage in the AMD3100-treated animals with the lowest Mankin score compared with the control animals. The levels of SDF-1, GAG, MMP1, MMP-13, and IL-1β were much lower in the synovial fluid of the AMD3100 group than in that of control group.

CONCLUSIONS

The binding of SDF-1 to CXCR4 induces OA cartilage degeneration. The catabolic processes can be disrupted by pharmacologic blockade of SDF-1/CXCR4 signaling. Together, these findings raise the possibility that disruption of the SDF-1/CXCR4 signaling can be used as a therapeutic approach to attenuate cartilage degeneration.

Primary intraocular lymphoma (PIOL) is a rare presentation of lymphoma that is particularly difficult to recognize. In our institution, 36 cases of PIOL were diagnosed between March 1997 and July 2002. The recognition of lymphoma cells by cytology with or without immunophenotyping on slides generated a strong suspicion of the diagnosis in 34 of 36 cases. The diagnosis was confirmed by measurement of <em>interleukin</em>-10 (IL-10) in the vitreous humour or aqueous humour; high levels were observed in <em>35</em> of 36 cases, all were of B-cell origin. As expected, the only case with T-cell lymphoma had a very low level of IL-10. Furthermore, IL-10 levels excluded this diagnosis in two cases that were incorrectly suspected of PIOL after cytological examination. Finally, detection of clonality by polymerase chain reaction techniques, performed in 29 cases, represented a helpful tool in diagnosing PIOL as this approach definitively confirmed the diagnosis of B- or T-cell lymphoma in 17 cases.

OBJECTIVE

To study biomarkers of angiogenesis in Parkinson disease (PD), and how these are associated with clinical characteristics, blood-brain barrier (BBB) permeability, and cerebrovascular disease.

METHODS

In this cross-sectional analysis, 38 elderly controls and 100 patients with PD (82 without dementia and 18 with dementia) were included from the prospective Swedish BioFinder study. CSF samples were analyzed for the angiogenesis biomarkers vascular endothelial growth factor (VEGF); its receptors, VEGFR-1 and VEGFR-2; placental growth factor (PlGF); angiopoietin 2 (Ang2); and <em>interleukin</em>-8. BBB permeability, white matter lesions (WMLs), and cerebral microbleeds (CMB) were assessed. CSF angiogenesis biomarkers were also measured in 2 validation cohorts: (1) 64 controls and 87 patients with PD with dementia; and (2) <em>35</em> controls and 93 patients with neuropathologically confirmed diagnosis of PD with and without dementia.

RESULTS

Patients with PD without dementia displayed higher CSF levels of VEGF, PlGF, and sVEGFR-2, and lower levels of Ang2, compared to controls. Similar alterations in VEGF, PlGF, and Ang2 levels were observed in patients with PD with dementia. Angiogenesis markers were associated with gait difficulties and orthostatic hypotension as well as with more pronounced BBB permeability, WMLs, and CMB. Moreover, higher levels of VEGF and PlGF levels were associated with increased CSF levels of neurofilament light (a marker of neurodegeneration) and monocyte chemotactic protein-1 (a marker of glial activation). The main results were validated in the 2 additional cohorts.

CONCLUSIONS

CSF biomarkers of angiogenesis are increased in PD, and they are associated with gait difficulties, BBB dysfunction, WMLs, and CMB. Abnormal angiogenesis may be important in PD pathogenesis and contribute to dopa-resistant symptoms.

The kinetics of viremia and sites of viral replication in bluetongue virus (BTV) infected calves were characterized by virus isolation, serology and immunofluorescence staining procedures. In addition, the role of the regional lymph node and lymphatics draining inoculated skin in the pathogenesis of BTV infection was determined by analyzing efferent lymph collected from indwelling cannulas. Viremia persisted for <em>35</em> to 42 days after inoculation (DAI) and virus co-circulated with neutralizing antibodies for 23 to 26 days. Virus was first isolated from peripheral blood mononuclear (PBM) cells at 3 DAI, after stimulation of PBM cells with <em>interleukin</em> 2 and mitogen. BTV was frequently isolated from erythrocytes, platelets and stimulated PBM cells but never from granulocytes and rarely from plasma during viremia. Virus was consistently isolated from erythrocytes late in the course of viremia. Interruption of efferent lymph flow by cannulation delayed the onset of viremia to 7 DAI. BTV was infrequently isolated from lymph cells, and few fluorescence positive cells were observed after lymph and PBM cells were labelled with a BTV-specific monoclonal antibody. Virus was isolated from spleen by 4 DAI and most tissues by 6 DAI, whereas virus was isolated from bone marrow only at 10 DAI. Virus was not isolated from any tissue after termination of viremia. It is concluded that primary viral replication occurred in the local lymph node and BTV then was transported in low titer to secondary sites of replication via infected lymph and PBM cells. We speculate that virus replication in spleen resulted in release of virus into the circulation and non-selective infection of blood cells which disseminated BTV to other tissues. Virus association with erythrocytes likely was responsible for prolonged viremia, although infected erythrocytes eventually were cleared from the circulation and persistent BTV infection of calves did not occur.

OBJECTIVE

To determine whether serum concentrations of 2 CXC chemokines, interleukin 8 (IL-8) and growth-regulated oncogene-alpha (GRO-alpha), which are potent chemoattractants and activators for neutrophils, are elevated and whether they correlate with clinical features in patients with systemic sclerosis (SSc).

METHODS

Serum samples from patients with diffuse cutaneous SSc (dSSc; n = 36), limited cutaneous SSc (lSSc; n = 42), systemic lupus erythematosus (SLE; n = 15), dermatomyositis (DM; n = 15), and healthy controls (n = 35) were examined by ELISA.

RESULTS

Serum IL-8 was detected significantly more frequently in patients with dSSc (61%) and lSSc (55%) relative to healthy controls (6%), patients with SLE (7%), and those with DM (13%). Similarly, serum GRO-alpha concentrations in SSc patients were significantly increased compared with controls, patients with SLE, or those with DM. Elevated IL-8 concentrations significantly correlated with decreased % DLCO and rheumatoid factor positivity, while increased GRO-alpha levels were significantly associated with decreased % DLCO and % vital capacity, involvement of kidney and muscle, the presence of anti-topoisomerase I antibody, and increased serum IgG levels.

CONCLUSIONS

Our results suggest that the elevation of serum levels of the CXC chemokines IL-8 and GRO-alpha is specific to SSc and that the elevation of CXC chemokines, particularly GRO-alpha, correlates with the involvement of internal organs, especially pulmonary damage.

OBJECTIVE

The immunopathogenic mechanisms of dry eye disease (DED), one of the most common ophthalmic conditions, is incompletely understood. Data from this prospective, double-masked, randomized trial demonstrate that targeting interleukin 1 (IL-1) by topical application of an IL-1 antagonist is efficacious in significantly reducing DED-related patient symptoms and corneal epitheliopathy.

OBJECTIVE

To evaluate the safety and efficacy of treatment with the topical IL-1 receptor antagonist anakinra (Kineret; Amgen Inc) in patients having DED associated with meibomian gland dysfunction.

METHODS

Prospective phase 1/2, randomized, double-masked, vehicle-controlled clinical trial.

METHODS

Seventy-five patients with refractory DED.

METHODS

Participants were randomized to receive treatment with topical anakinra, 2.5% (n = 30), anakinra, 5% (n = 15), or vehicle (1% carboxymethylcellulose) (n = 30) 3 times daily for 12 weeks.

METHODS

Primary outcomes were corneal fluorescein staining (CFS), complete bilateral CFS clearance, dry eye-related symptoms as measured by the Ocular Surface Disease Index, tear film breakup time, and meibomian gland secretion quality.

RESULTS

Topical anakinra was well tolerated compared with vehicle, with no reports of serious adverse reactions attributable to the therapy. After 12 weeks of therapy, participants treated with anakinra, 2.5%, achieved a 46% reduction in their mean CFS score (P = .12 compared with vehicle and P < .001 compared with baseline); participants treated with anakinra, 5%, achieved a 17% reduction in their mean CFS score (P = .88 compared with vehicle and P = .33 compared with baseline); and patients treated with vehicle achieved a 19% reduction in their mean CFS score (P = .11). Complete bilateral CFS clearance was noted in 8 of 28 patients (29%) treated with anakinra, 2.5%, vs in 2 of 29 patients (7%) treated with vehicle (P = .03). By week 12, treatment with anakinra, 2.5%, and treatment with anakinra, 5%, led to significant reductions in symptoms of 30% and 35%, respectively (P = .02 and P = .01, respectively, compared with vehicle); treatment with vehicle led to a 5% reduction in symptoms.

CONCLUSIONS

Treatment with topical anakinra, 2.5%, for 12 weeks was safe and significantly reduced symptoms and corneal epitheliopathy in patients with DED. These data suggest that the use of an IL-1 antagonist may have a role as a novel therapeutic option for patients with DED. TRIAL REGISTRATION clinicaltrials.gov Identifier: NCT00681109.

OBJECTIVE

We measured the mucosal levels of interleukin 1beta (IL-1beta), IL-2, IL-6, and IL-8 in affected segments of radiation-induced proctosigmoiditis and compared these with the levels in normal controls and inflammatory bowel disease (IBD) patients.

METHODS

Thirteen patients with histologically proven radiation proctosigmoiditis, 32 patients with active ulcerative colitis (UC), 35 patients with Crohn's disease, and 15 normal subjects undergoing routine colonoscopy were included in the study. All patients underwent colonoscopy and mucosal biopsies were obtained from both diseased and normal-appearing areas. Mucosal levels of IL-1beta, IL-2, IL-6, and IL-8 were determined by solid-phase enzyme-linked immunosorbent assay (ELISA) using the Quantikine method (R&D Systems, Minneapolis, MN). All the data were statistically analyzed using Student's t test.

RESULTS

The mucosal levels of IL-2, IL-6, and IL-8 were significantly higher in both diseased segments (5.62 +/- 0.13, 1.60 +/- 0.31, and 21.45 +/- 4.03 pg/ml, respectively) and normal-appearing segments (3.83 +/- 0.78, 1.36 +/- 0.34, and 13.45 +/- 3.18 pg/mg) in the radiation proctitis group compared to those of normal control subjects (1.74 +/- 0.23, 0.67 +/- 0.09, and 4.99 +/- 1.39 pg/mg). These differences were statistically significant (p < 0.05). In the UC group, IL-1beta, IL-6, and IL-8 levels in diseased segments were 4.98 +/- 0.53, 2.22 +/- 0.28, and 88.85 +/- 8.05 pg/mg, respectively. In Crohn's disease patients, IL-1beta, IL-6, and IL-8 levels were 5.45 +/- 0.93, 2.88 +/- 0.58, and 61.68 +/- 10.02 pg/mg, respectively. All these levels were significantly higher (p < 0.05) compared with IL-1beta, IL-6, and IL-8 levels from normal segments of IBD patients. Compared with the radiation proctitis patients, the levels of IL-6 and IL-8 were significantly higher in the IBD group.

CONCLUSIONS

The mucosal levels of IL-2, IL-6, and IL-8 were significantly higher in both diseased and normal segments of colon in patients with radiation proctitis, compared with normal controls. Only IL-1beta levels were significantly higher in diseased segments, compared with endoscopically normal-appearing segments in radiation proctitis. These results indicate that there is a similarity in the activation of mucosal cytokines between IBD and radiation proctosigmoiditis. This may partly explain the beneficial effects of similar topical and systemic agents such as steroids and mesalamine compounds when used in radiation-induced proctosigmoiditis.

BACKGROUND

Normal epithelial cells undergo anoikis, or apoptosis on loss of anchorage to the extracellular matrix, by initiating the death receptor pathway of caspase activation. However, malignant epithelial cells with metastatic potential resist anoikis and can survive in an anchorage-independent fashion. We hypothesized that c-Fas-associated death domain-like interleukin-1-converting enzyme-like inhibitory protein (FLIP), an endogenous inhibitor of death receptor signaling, may suppress anoikis.

METHODS

We assessed viability and apoptosis of PPC-1 prostate cancer cells cultured in adherent and suspension conditions using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt and Annexin V staining assays. Expression of the death receptor Fas and activation of caspase 8 were measured using flow cytometry. Expression of Fas ligand was measured by reverse transcription-polymerase chain reaction. FLIP protein expression was measured by immunoblotting. Small-molecule inhibitors of FLIP (including the death receptor sensitizer 5809354) and small-interfering (si) RNA directed against FLIP were used to assess the effects of FLIP inhibition on anoikis of prostate cancer cells in vitro and in vivo. All statistical tests were two-sided.

RESULTS

PPC-1 cells cultured in suspension resisted anoikis, despite increased expression of Fas (0 versus 8 hours, mean relative percent expression = 100% versus 135%, difference = 35%, 95% confidence interval [CI] = 10% to 61%; P = .02) and Fas L (0 versus 24 hours, mean relative percent expression = 100% versus 208%, difference = 108%, 95% CI = 18% to 197%; P = .02). Knockdown of FLIP expression by siRNA or treatment with 5809354 sensitized prostate cancer cells to anoikis (control siRNA versus FLIP siRNA at 10 nM, mean relative percent viability = 95% versus 51%, difference = 44%, 95% CI = 34% to 54%; P<.001; control versus 5809354 at 20 microM, mean relative percent viability = 96% versus 52%, difference = 44%, 95% CI = 13% to 75%; P = .015). Inhibition of FLIP expression specifically activated caspase 8 in PPC-1 cells grown in suspension but not adherent conditions and decreased the metastatic potential of circulating PPC-1 cells in vivo.

CONCLUSIONS

FLIP may be a suppressor of anoikis and therefore a possible target for antimetastatic therapeutic strategies.

BACKGROUND

Caspase-1 is a cysteine protease that contributes to mammalian immunity through proteolytic activation of the proinflammatory cytokines, interleukin (IL)-1β and IL-18.

METHODS

To determine if caspase-1 deficiency can protect apolipoprotein E-null (Apoe(-/-)) mice from atherosclerosis, gender-matched, paired-littermate Apoe(-/-) mice with (Casp1(+/+)Apoe(-/-)) or without (Casp1(-/-)Apoe(-/-)) a functional caspase-1 (Casp1) gene were fed either a low fat diet for 26 weeks, or a saturated fat and cholesterol-enriched diet for 8 weeks. Plasma lipids and lipoproteins were determined and atherosclerosis was quantified in the aortic sinus and aortic arch.

RESULTS

On either diet, caspase-1 deficiency did not affect total serum cholesterol concentrations and lipoprotein-cholesterol distributions. However, caspase-1 deficiency significantly decreased atherosclerosis in the ascending aorta by 35%-45% in both sexes of mice fed either diet. We further examined atherosclerotic lesions for 2 indices of immune cell activation: Major Histocompatibility Complex (MHC) class II and interferon (IFN)-γ expression. There was a 40%-50% reduction in the number of lesion-associated cells expressing MHC class II from both sexes of Casp1(-/-)Apoe(-/-) mice compared with Casp1(+/+)Apoe(-/-) mice and, a significant reduction in lesion-associated IFN-γ in female Casp1(-/-)Apoe(-/-) compared with their Casp1(+/+)Apoe(-/-) counterparts.

CONCLUSIONS

We conclude that caspase-1 promotes atherosclerosis by enhancing the inflammatory status of the lesion through a mechanism likely involving activation of lesion-associated immune cells and IFN-γ expression.

Based on previous studies showing that strontium ranelate (S12911) modulates bone loss in osteoporosis, it could be hypothesized that this drug also is effective on cartilage degradation in osteoarthritis (OA). This was investigated in vitro on normal and OA human chondrocytes treated or not treated with <em>interleukin</em>-1beta (IL-1beta). This model mimics, in vitro, the imbalance between chondroformation and chondroresorption processes observed in vivo in OA cartilage. Chondrocytes were isolated from cartilage by enzymatic digestion and cultured for 24-72 h with 10(-4)-10(-3) M strontium ranelate, 10(-3) M calcium ranelate, or 2 x 10(-3) M SrCl2 with or without IL-1beta or insulin-like growth factor I (IGF-I). Stromelysin activity and stromelysin quantitation were assayed by spectrofluorometry and enzyme amplified sensitivity immunoassay (EASIA), respectively. Proteoglycans (PG) were quantified using a radioimmunoassay. Newly synthesized glycosaminoglycans (GAGs) were quantified by labeled sulfate (Na2(<em>35</em>)SO4) incorporation. This method allowed the PG size after exclusion chromatography to be determined. Strontium ranelate, calcium ranelate, and SrCl2 did not modify stromelysin synthesis even in the presence of IL-1beta. Calcium ranelate induced stromelysin activation whereas strontium compounds were ineffective. Strontium ranelate and SrCl2 both strongly stimulated PG production suggesting an ionic effect of strontium independent of the organic moiety. Moreover, 10(-3) M strontium ranelate increased the stimulatory effect of IGF-I (10(-9) M) on PG synthesis but did not reverse the inhibitory effect of IL-1beta. Strontium ranelate strongly stimulates human cartilage matrix formation in vitro by a direct ionic effect without stimulating the chondroresorption processes. This finding provides a preclinical basis for in vivo testing of strontium ranelate in OA.

OBJECTIVE

We attempted to augment immunity to melanoma antigens using interleukin-12 (IL-12) with aluminum hydroxide (alum) for sustained release or granulocyte macrophage colony-stimulating factor (GM-CSF) added to a multipeptide vaccine.

METHODS

Sixty patients with high-risk resected melanoma were randomized to receive melanoma peptides gp100(209-217) (210M), MART-1(26-35) (27L), and tyrosinase(368-376) (370D) with adjuvant Montanide ISA 51 and either IL-12 at 30 ng/kg with alum (group A), IL-12 at 100 ng/kg with alum (group B), or IL-12 at 30 ng/kg with 250 mug GM-CSF (group C).

RESULTS

Three patients had stage IIC (5%), 50 had stage III (83%), and 7 had stage IV (12%) melanoma. Most toxicities were grade 1/2 and resolved rapidly. Significant toxicity included grade 3 colitis and visual changes and grade 3 headache resolving after stopping IL-12 but continuing peptide vaccine. A higher rate of post-vaccine 6-month immune response to gp100 and MART-1 was observed in group A (15 of 19) or B (19 of 20) that received IL-12 plus alum versus group C with IL-12/GM-CSF (4 of 21; P < 0.001). Post-vaccine enzyme-linked immunospot response rates to peptide analogues in group B were higher than group A (P = 0.031 for gp100 and P = 0.010 for MART-1); both were higher than group C (P < 0.001 for gp100 and P < 0.026 for MART-1). With a median of 24 months of follow-up, 23 patients have relapsed. Post-vaccine immune response to MART-1 was associated with relapse-free survival (P = 0.012).

CONCLUSIONS

IL-12 with alum augmented an immune response to melanoma antigens compared with IL-12 with GM-CSF. Immune response was associated with time to relapse.

Cytokines coordinate the activities of innate and adaptive immune systems and the <em>Interleukin</em> 12 (IL-12) family of cytokines has emerged as critical regulators of immunity in infectious and autoimmune diseases. While some members (IL-12 and IL-23) are associated with the pathogenesis of chronic inflammatory diseases, others (IL-27 and IL-<em>35</em>) mitigate autoimmune diseases. Unlike IL-12, IL-23 and IL-27 that are produced mainly by antigen presenting cells, IL-<em>35</em> is predominantly secreted by regulatory B (i<em>35</em>-Bregs) and T (iTR<em>35</em>) cells. The discovery that IL-<em>35</em> can induce the conversion or expansion of lymphocytes to regulatory B and T cells has considerable implications for therapeutic use of autologous regulatory B and T cells in human diseases. Although our current understanding of the immunobiology of IL-<em>35</em> or its subunits (p<em>35</em> and Ebi3) is still rudimentary, our goal in this review is to summarize what we know about this enigmatic cytokine and its potential clinical use, particularly in the treatment of CNS autoimmune diseases.

The serum concentration of the inter-alpha trypsin inhibitor heavy chain 4 protein (ITIH4) increases (from 1.4-3 times) in male patients suffering of different acute-phase processes (myocardial infarction, unstable angina or programmed surgery). The concentration of C-reactive protein (CRP) in these samples ranged from 15 microg/ml to 133 microg/ml. Using the hepatocarcinoma HepG2 cell line we have observed up-regulation of ITIH4 mRNA expression upon dose-response treatments with <em>interleukin</em>-6 (IL-6). This effect correlates with the increase of radiolabeled ITIH4 in the cellular media of (<em>35</em>)S-labeled HepG2 cells treated with the cytokine. A similar effect was observed for haptoglobin mRNA, used as a control for acute-phase protein expression. IL-1beta, although up-regulating the expression of alpha(1)-acid glycoprotein in these cells, did not induce any effect in the expression of ITIH4. No changes were observed after TNF-alpha treatments. The results presented here indicate that ITIH4 is a type II acute-phase protein in humans.