Fetal and neonatal inflammation is associated with several morbidities of prematurity. Its relationship to retinopathy of prematurity (ROP) has not been investigated. Our objective was to determine the relationship between cytokine levels and ROP in the first 3 postnatal wks. Data for this study were derived from the NICHD Cytokine Study. Dried blood spots (DBS) were obtained from infants <1000 g on days 0-1, 3 +/- 1, 7 +/- 2, 14 +/- 3, and 21 +/- 3. Infants were classified into three groups-no, mild, and severe ROP. Multiplex Luminex assay was used to quantify <em>20</em> cytokines. Temporal profiles of cytokines were evaluated using mixed-effects models after controlling for covariates. Of 1074 infants enrolled, 890 were examined for ROP and 877 included in the analysis. ROP was associated with several clinical characteristics on unadjusted analyses. Eight cytokines remained significantly different across ROP groups in adjusted analyses. <em>IL</em>-6 and <em>IL</em>-17 showed significant effects in early time periods (D0-3); TGF-beta, brain-derived neurotrophic factor (BDNF), and regulated on activation, normal T cell expressed and secreted (RANTES) in later time periods (D7-21) and <em>IL</em>-18, C-reactive protein (CRP), and neurotrophin-4 (NT-4) in both early and later time periods. We conclude that perinatal inflammation may be involved in the pathogenesis of ROP.

BACKGROUND

Human papilloma virus type 16 (HPV16)-induced gynecological cancers, in particular cervical cancers, are found in many women worldwide. The HPV16 encoded oncoproteins E6 and E7 are tumor-specific targets for the adaptive immune system permitting the development of an HPV16-synthetic long peptide (SLP) vaccine with an excellent treatment profile in animal models. Here, we determined the toxicity, safety, immunogenicity and efficacy of the HPV16 SLP vaccine in patients with advanced or recurrent HPV16-induced gynecological carcinoma.

METHODS

Patients with HPV16-positive advanced or recurrent gynecological carcinoma (n = <em>20</em>) were subcutaneously vaccinated with an HPV16-SLP vaccine consisting of a mix of 13 HPV16 E6 and HPV16 E7 overlapping long peptides in Montanide ISA-51 adjuvant. The primary endpoints were safety, toxicity and tumor regression as determined by RECIST. In addition, the vaccine-induced T-cell response was assessed by proliferation and associated cytokine production as well as IFNγ-ELISPOT.

RESULTS

No systemic toxicity beyond CTCAE grade II was observed. In a few patients transient flu-like symptoms were observed. In 9 out of 16 tested patients vaccine-induced HPV16-specific proliferative responses were detected which were associated with the production of IFNγ, TNFα, IL-5 and/or IL-10. ELISPOT analysis revealed a vaccine-induced immune response in 11 of the 13 tested patients. The capacity to respond to the vaccine was positively correlated to the patient's immune status as reflected by their response to common recall antigens at the start of the trial. Median survival was 12.6 ± 9.1 months. No regression of tumors was observed among the 12 evaluable patients. Nineteen patients died of progressive disease.

CONCLUSIONS

The HPV16-SLP vaccine was well tolerated and induced a broad IFNγ-associated T-cell response in patients with advanced or recurrent HPV16-induced gynecological carcinoma but neither induced tumor regression nor prevented progressive disease. We, therefore, plan to use this vaccine in combination with chemotherapy and immunomodulation.

Our aim was to assess anti-inflammatory effects on the peripheral blood of subjects with inflammatory bowel disease (IBD) who consumed probiotic yogurt for 1 month. We studied <em>20</em> healthy controls and <em>20</em> subjects with IBD, 15 of whom had Crohn's disease and five with ulcerative colitis. All the subjects consumed Lactobacillus rhamnosus GR-1 and L. reuteri RC-14 supplemented yogurt for 30 days. The presence of putative regulatory T (T(reg)) cells (CD4(+) CD25(high)) and cytokines in T cells, monocytes and dendritic cells (DC) was determined by flow cytometry from peripheral blood before and after treatment, with or without ex vivo stimulation. Serum and faecal cytokine concentrations were determined by enzyme-linked immunosorbent assays. The proportion of CD4(+) CD25(high) T cells increased significantly (P = 0.007) in IBD patients, mean (95% confidence interval: CI) 0.84% (95% CI 0.55-1.12) before and 1.25% (95% CI 0.97-1.54) after treatment, but non-significantly in controls. The basal proportion of tumour necrosis factor (TNF)-alpha(+)/interleukin (<em>IL</em>)-12(+) monocytes and myeloid DC decreased in both subject groups, but of stimulated cells only in IBD patients. Also serum <em>IL</em>-12 concentrations and proportions of <em>IL</em>-2(+) and CD69(+) T cells from stimulated cells decreased in IBD patients. The increase in CD4(+) CD25(high) T cells correlated with the decrease in the percentage of TNF-alpha- or <em>IL</em>-12-producing monocytes and DC. The effect of the probiotic yogurt was confirmed by a follow-up study in which subjects consumed the yogurt without the probiotic organisms. Probiotic yogurt intake was associated with significant anti-inflammatory effects that paralleled the expansion of peripheral pool of putative T(reg) cells in IBD patients and with few effects in controls.

Glucans are immunomodulatory carbohydrates found in the cell walls of fungi and certain bacteria. We examined the pharmacokinetics of three water-soluble glucans (glucan phosphate, laminarin, and scleroglucan) after oral administration of 1 mg/kg doses in rats. Maximum plasma concentrations for glucan phosphate occurred at 4 h. In contrast, laminarin and scleroglucan showed two plasma peaks between 0.5 and 12 h. At 24 h, 27 +/- 3% of the glucan phosphate and <em>20</em> +/- 7% of the laminarin remained in the serum. Scleroglucan was rapidly absorbed and eliminated. The liver did not significantly contribute to the clearance of plasma glucan. Biological effects were further studied in mice. Following oral administration of 1 mg, glucans were bound and internalized by intestinal epithelial cells and gut-associated lymphoid tissue (GALT) cells. Internalization of glucan by intestinal epithelial cells was not Dectin-dependent. GALT expression of Dectin-1 and toll-like receptor (TLR) 2, but not TLR4, increased following oral administration of glucan. Oral glucan increased systemic levels of interleukin (<em>IL</em>)-12 (151 +/- 15%) in mice. Oral glucan administration also increased survival in mice challenged with Staphylococcus aureus or Candida albicans. These data demonstrate that orally administered water-soluble glucans translocate from the gastrointestinal (GI) tract into the systemic circulation. The glucans are bound by GI epithelial and GALT cells, and they modulate the expression of pattern recognition receptors in the GALT, increase <em>IL</em>-12 expression, and induce protection against infectious challenge.

Interleukin-19, <em>20</em>, and 24 are new members of the <em>IL</em>-10 family binding and signaling through the <em>IL</em>-<em>20</em>R1/<em>IL</em>-<em>20</em>R2 heterodimer, while <em>IL</em>-<em>20</em> and 24 also bind to the <em>IL</em>-<em>20</em>R2/<em>IL</em>-22R1 heterodimer. Using in situ hybridization we have studied mRNA expression of <em>IL</em>-19, <em>20</em>, and 24 and their related receptor chains in skin from psoriatic patients before and during short-term treatment with either oral cyclosporine A or topical calcipotriol. In untreated lesions <em>IL</em>-19 and <em>IL</em>-<em>20</em> mRNA was expressed focally in epidermis above the dermal papillae, whereas <em>IL</em>-24 was expressed in mononuclear cells in the dermal infiltrate. The expression of <em>IL</em>-19 and <em>20</em> mRNA was confined to the basal and suprabasal keratinocytes. No expression of <em>IL</em>-19 and <em>20</em> mRNA could be detected in uninvolved psoriatic skin. Treatment with cyclosporine A and calcipotriol resulted in disappearance of the <em>IL</em>-19 and <em>20</em> mRNA. Expression of mRNA for the receptor chains <em>IL</em>-<em>20</em>R1 and <em>IL</em>-<em>20</em>R2 was found throughout the psoriatic epidermal layer, whereas <em>IL</em>-22R1 mRNA was predominantly expressed in the superficial part of the psoriatic epidermis. These findings show that <em>IL</em>-19 and <em>IL</em>-<em>20</em> are synthesized by a distinct population of keratinocytes. It remains to be clarified whether <em>IL</em>-19 and <em>IL</em>-<em>20</em> are implicated in the pathogenesis of psoriasis.

Sepsis continues to be the primary cause of death among patients in surgical intensive care units. In many cases, death does not result from the initial septic event but rather from subsequent nosocomial infection with pneumonia being the most common etiology. In addition, most deaths in patients with sepsis occur after the first 72 h. By contrast, in most animal models of sepsis, most deaths occur within the first 72 h. The purpose of this study was to develop a clinically relevant "two-hit" model of sepsis that would reflect delayed mortality because of secondary nosocomial infection. The well-accepted and widely used cecal ligation and puncture (CLP) model was used as the "first hit". Pseudomonas aeruginosa or Streptococcus pneumoniae was used to induce pneumonia in mice 72 h after CLP as a "second hit." In this study, mortality in mice undergoing CLP followed by pneumonia was significantly higher than in mice receiving pneumonia or CLP alone. S. pneumoniae pneumonia after CLP resulted in a 95% mortality compared with a <em>20</em>% mortality for pneumonia alone, P < 0.0001. Similarly, mortality of P. aeruginosa pneumonia after CLP (85%) was significantly higher than P. aeruginosa alone (<em>20</em>%), P < 0.0001. Mice undergoing CLP followed by P. aeruginosa pneumonia also had significantly higher levels of B- and T-cell apoptotic death. Finally, mice undergoing CLP followed by P. aeruginosa or S. pneumoniae pneumonia had significantly decreased concentrations of proinflammatory mediators monocyte chemoattractant protein-1 and interleukin (<em>IL</em>)-6 compared with mice undergoing CLP or pneumonia alone. In conclusion, a primary sublethal infection impairs the immune system thus rendering the host more susceptible to secondary infection and death. Double injury, that is, CLP followed by pneumonia, provides a useful tool in the study of sepsis, creating a prolonged period of infection as opposed to CLP alone. The extended duration of infection may lead to a better understanding of the mechanism of the immune dysregulation seen in clinical sepsis and therefore provides for evaluation of potential therapies that target specific stages of the immune response.

To evaluate the development of the neonatal immune system, we measured T lymphocyte response to Con A, intracellular <em>IL</em>-2, <em>IL</em>-4, IFN-gamma and <em>IL</em>-10 production, and natural killer cell (NKC) activity in 12 very preterm, 12 preterm and <em>20</em> term neonates, 10 children and 10 adults. Immunoproliferation to Con A was significantly lower in cord blood than in children or adults. The percentage of CD4+ lymphocytes was significantly higher in newborns while CD8+ cells were higher at older ages, with a resulting gradual decline of the CD4+/CD8+ ratio. The percentage of <em>IL</em>-2-producing CD4+ and CD8+ cells was higher in all newborn groups than in children and adults, while the percentage of <em>IL</em>-4-producing cells was higher for CD8+ and lower for CD4+ cells in cord blood than in children and adults. Neonates had substantially lower percentages of CD4+ and CD8+ IFN-gamma-producing cells. A significant negative correlation was observed between gestational age and IFN-gamma-CD4+-, <em>IL</em>-2-CD8+-, and <em>IL</em>-10- CD4+-producing cells. In addition, a positive correlation was found between gestational age and <em>IL</em>-10-CD8+-producing cells. Percentages of CD4+/CD45RA+ cells were higher and CD4+/CD45RO+ percentages were lower in newborns than in children and adults. NKC activity in infants was significantly correlated with gestational age and significantly impaired compared to children and adults. On the whole, these results suggest a gradual development of immunity during gestation and show significant immaturity of cellular immune response at birth. The reduction of NKC activity, the lower proliferative response of T cells, the reduced cytotoxic response and a dysregulated cytokine production may contribute to the neonatal increased risk of infection and to the low incidence of graft-versus-host disease after cord blood transplantation.

The vascular growth factor angiopoietin 2 (Ang-2) is known to promote inflammation and endothelial dysfunction, but its prognostic capacity and relationship to outcomes in human sepsis are unknown. This is a prospective observational cohort study of 66 patients newly admitted to a tertiary care medical intensive care unit (ICU), which included ICU patients with no sepsis (n = <em>20</em>) as well as those with sepsis (n = 10), severe sepsis (n = 12), and septic shock (n = 24). Clinical data were collected until hospital discharge, and Ang-2 and <em>IL</em>-6 levels were determined on specimens obtained after ICU admission. Serum Ang-2 correlated with <em>IL</em>-6 and severity-of-illness scores. In the septic cohort, circulating Ang-2 levels were significantly higher (P = 0.01) in those who died (24.9 ng/mL; interquartile range, 21.5-38.0 ng/mL) compared with those who survived (13.5 ng/mL; interquartile range, 8.1-21.6 ng/mL). Elevated circulating serum Ang-2 levels are associated with increased hospital mortality in patients with sepsis.

BACKGROUND

Using a model of traumatic brain injury (TBI) in the rat, this study was undertaken to characterize the short-term biochemical changes of IL-1beta, IL-10, and tumor necrosis factor TNF-alpha to determine whether injury in the brain elicits a systemic cytokine response.

METHODS

Sprague-Dawley rats were subjected to a TBI using a weight-drop model and then killed at various time points after injury. Samples of blood, brain, and liver were recovered and analyzed for concentrations of IL-1beta, IL-10, and TNF-alpha as well as IL-1beta and IL-10 mRNA expression in liver and brain.

RESULTS

In brain, IL-1beta increased in the first hour after injury, peaked at 8 hours, and declined during the final 16 hours. IL-10 quickly increased during the first 4 hours and then gradually rose over the last 20 hours. Analysis of liver showed no upregulation of these markers and plasma IL-1beta and IL-10 were unchanged compared with controls. Although not upregulated in brain, TNF-alpha showed a statistically significant (p < 0.05) rise in plasma from 14 +/- 16 pg/mL at 20 minutes to 91 +/- 28 pg/mL at 24 hours.

CONCLUSIONS

Using a model of TBI, we have demonstrated that there is a rise in both IL-1beta and IL-10 in the injured rat brain within the first 24 hours after injury without a corresponding rise in either plasma or liver. Therefore, it appears as if two strong indicators of brain injury severity are expressed and possibly carry out their actions solely in the brain.

The presence of interleukin 6 (<em>IL</em>-6), interleukin 1 (<em>IL</em>-1), interleukin 2 (<em>IL</em>-2) and tumour necrosis factor (TNF) was investigated in vitreous and aqueous aspirates from eyes undergoing vitrectomy for the treatment of different inflammatory conditions. Cadaveric vitreous from 10 normal subjects were used as controls. <em>IL</em>-6 was observed in 5 specimens from eyes with idiopathic uveitis (range = 26-264 pg/ml), in 2 specimens from eyes with uveitis complicated with retinal detachment (28 and 279 pg/ml, respectively), in 6 samples from eyes with diabetic retinopathy (range = 5-480 pg/ml), in one sample from an eye with phacolytic glaucoma (1190 pg/ml) and in one specimen from an eye with Behçet's disease (366 pg/ml). Although <em>IL</em>-1 was detected in 80% of all the samples investigated, concentrations of this cytokine greater than 3 pg/ml were only observed in 2 specimens from eyes with uveitis (5 and <em>20</em> pg/ml, respectively) and 2 samples from eyes with diabetic retinopathy (3 and 31 pg/ml, respectively). TNF was present in 3 specimens from eyes with uveitis (range = 2-24 pg/ml) and 1 sample from eyes with diabetic retinopathy (4 pg/ml), but was not detected in the eyes with phacolytic glaucoma or Behçet's disease. <em>IL</em>-2 (less than 0.1 U/ml) was detected in one sample from an eye with uveitis, one specimen from an eye with uveitis complicated with retinal detachment and 2 samples from eyes with diabetic retinopathy. None of the cytokines measured were detected in any of the control vitreous. The present observations suggest that cytokines, particularly <em>IL</em>-6 and <em>IL</em>-1, may act as local amplification signals in pathological processes associated with chronic eye inflammation.

Adenosine is a potent endogenous regulator of tissue repair that is released from injured cells and tissues. Hepatic fibrosis results from chronic hepatic injury, and we have previously reported that endogenously generated adenosine, acting at A(2A) receptors, plays a role in toxin-induced hepatic fibrosis. Adenosine may form intracellularly and then be transported to the extracellular space or it may form extracellularly from adenine nucleotides released from injured cells. Because ecto-5'-nucleotidase (CD73) catalyzes the terminal step in extracellular adenosine formation from AMP, we determined whether CD73 plays a role in the development of hepatic fibrosis. Mice were treated overnight with PBS, CCl(4), ethanol, or thioacetamide (TAA); their livers were harvested, and slices were incubated in medium for <em>20</em> h before adenosine concentration in the supernatant was measured by HPLC. Hepatic fibrosis was induced by CCl(4) or TAA treatment in CD73 knockout (CD73KO and C57BL/6 background) and C57BL/6 control mice [wild-type (WT)] mice and quantified by digital analysis of picrosirius red stained slides and hydroxyproline content. mRNA expression was quantified by real-time polymerase chain reaction, and protein was quantified by Western blot or enzyme-linked immunosorbent assay. Livers from WT mice treated with CCl(4), ethanol, and TAA released 2- to 3-fold higher levels of adenosine than livers from comparably treated CD73KO mice. CD73KO mice were protected from fibrosis with significantly less collagen content in the livers of CD73KO than WT mice after treatment with either CCl(4) or TAA. There were far fewer alpha-smooth muscle actin positive hepatic stellate cells in CCl(4)-treated KO mice than that in WT mice. After CCl(4) treatment, the mRNA level of A(1), A(2A), A(2B), and A(3) adenosine receptors, tumor necrosis factor-alpha, interleukin (<em>IL</em>) -1beta, <em>IL</em>-13r alpha1, matrix metalloproteinase (MMP)-2, MMP-14, tissue inhibitor of metalloproteinase (TIMP) -1, and TIMP-2, and <em>IL</em>-13 level increased markedly in both CD73KO and WT mice, but Col1 alpha1, Col3 alpha1, and transforming growth factor-beta1 mRNA increased much more in WT mice than that in KO mice. Moreover, <em>IL</em>-13r alpha2, MMP-13 mRNA, and MMP-13 protein were higher in KO mice than that in WT mice. These results indicate that adenosine, formed extracellularly from adenine nucleotides, plays a major role in the pathogenesis of hepatic fibrosis and that inhibition of adenosine production or blockade of adenosine receptors may help prevent hepatic fibrosis.

The <em>IL</em>-10 gene and homologs <em>IL</em>-19, <em>IL</em>-<em>20</em>, and <em>IL</em>-24 are expressed within a highly conserved 145-kb cytokine gene cluster. Like the Th2 <em>IL</em>-4 cytokine gene cluster, it is feasible that there is coordinate regulation of these cytokines by distal regulatory elements spanning the locus. We initiated a search to characterize regulatory elements within the <em>IL</em>-10 family locus and present data herein on a conserved 40-kb region between the <em>IL</em>-19 and <em>IL</em>-10 genes. We map the location of 17 DNase I-hypersensitive sites in different murine T cell populations and identify three enhancer elements, which function in T cells in vitro. Two of these enhancer elements, located 9 kb upstream and 6.45 kb downstream of <em>IL</em>-10, display cell-specific function in the Th1-Th2 cell clones AE7 and D10 and also exhibit basic promoter activity. The downstream element, <em>IL</em>-10CNS+6.45, binds AP-1 in the absence of NFAT and expresses intergenic RNA in a Th2-specific manner, further validating its role as a Th2-specific enhancer/promoter element. We show that the five most highly conserved noncoding sequences in the 40-kb region transcribe intergenic RNA; four of these regions possess promoter activity in vitro that could account for the expression of these transcripts. Hence, we speculate that these novel regulatory elements in the <em>IL</em>-10 family gene locus function via an intermediate regulatory RNA.

OBJECTIVE

To investigate the effect of massive weight loss on (1) knee pain and disability, (2) low-grade inflammation and metabolic status and (3) joint biomarkers in obese patients with knee osteoarthritis (OA).

METHODS

140 patients involved in a gastric surgery programme were screened for painful knee OA, and 44 were included (age 44 ± 10.3 years, body mass index (BMI) 50.7 ± 7.2 kg/m(2)). Clinical data and biological samples were collected before and 6 months after surgery.

RESULTS

Before surgery, interleukin 6 (<em>IL</em>-6) levels were correlated with levels of high-sensitivity C reactive protein (hsCRP) (p=0.006) and Helix-II (p=0.01), a biomarker of cartilage turnover, and the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) function score (p=0.03). Surgery resulted in substantial decrease in BMI (-<em>20</em>%). Levels of insulin and insulin resistance were decreased at 6 months. Knee pain decreased after surgery (24.5 ± 21 mm vs 50 ± 26.6 mm; p<0.001), and scores on all WOMAC subscales were improved. Levels of <em>IL</em>-6 (p<0.0001), hsCRP (p<0.0001), orosomucoid (p<0.0001) and fibrinogen (p=0.04) were decreased after surgery. Weight loss resulted in a significant increase in N-terminal propeptide of type IIA collagen levels (+32%; p=0.002), a biomarker of cartilage synthesis, and a significant decrease in cartilage oligomeric matrix protein (COMP) (-36%; p<0.001), a biomarker of cartilage degradation. Changes in COMP concentration were correlated with changes in insulin levels (p=0.02) and insulin resistance (p=0.05).

CONCLUSIONS

Massive weight loss improves pain and function and decreases low-grade inflammation. Change in levels of joint biomarkers with weight loss suggests a structural effect on cartilage.

Human peripheral blood mononuclear cells analyzed immediately after isolation did not express detectable mRNA for interleukin-1 beta (<em>IL</em>-1 beta). In the strict absence of lipopolysaccharide (LPS), incubation in glass or plastic resulted in expression of <em>IL</em>-1 beta mRNA without detectable <em>IL</em>-1 beta synthesis, even when cells were incubated for <em>20</em> h. The accumulation of <em>IL</em>-1 beta mRNA was most likely due to adherence since rotating the containers reduced the amount of mRNA. However, the cells were "primed" by 3 h adherence since subsequent stimulation with LPS resulted in more <em>IL</em>-1 beta (214%) 4 h after stimulation compared to freshly obtained, LPS-stimulated cells. Ratios of <em>IL</em>-1 beta mRNA induced by LPS versus adherence were 7.8, 36, and <em>20</em> at 4, 12, and 24 h, respectively; the corresponding ratios for <em>IL</em>-1 beta protein were 18, 160, and 180. Comparing Staphylococcus epidermidis versus LPS, the ratios of <em>IL</em>-1 beta mRNA were 3.2, 0.5, and 1.2 at 4, 8, and 24 h, respectively, however, the corresponding ratios for <em>IL</em>-1 beta protein were 65, 22, and 10. The differences in transcription versus translation in these studies are unlikely due to changes in stability of mRNA since the half-life of adherence-induced <em>IL</em>-1 beta mRNA was 2.5 versus 4 h for LPS-induced mRNA. There was also no evidence of superinduction of mRNA in peripheral blood mononuclear cells stimulated with LPS, whereas, tumor necrosis factor alpha mRNA was elevated in the presence of cycloheximide. Using different methods of cell surface stimulation, our results demonstrate that synthesis of <em>IL</em>-1 beta is regulated by at least two separate mechanisms, one at the level of transcriptional activation and the other one involving translational efficiency.

Chemokine ligand <em>20</em> (CCL<em>20</em>) and human beta-defensins (HBDs) share structural and functional properties, including antiparallel beta-pleated sheet core structures, charge distribution, and signaling to adaptive immune cells via the highly selective CCR6 receptor. Because of their similarities, we hypothesized that in addition to its known adaptive immune signaling functions, CCL<em>20</em> has antimicrobial properties and participates in pulmonary innate immunity. We found that primary cultures of human airway epithelial and cultured fetal lung explants expressed CCL<em>20</em> mRNA. Expression of CCL<em>20</em> transcripts were significantly induced by interleukin (<em>IL</em>)-1beta and tumor necrosis factor-alpha, and inhibited by dexamethasone. Primary cultures of airway epithelia secreted CCL<em>20</em> both apically and basolaterally, and CCL<em>20</em> abundance was increased over 30-fold with <em>IL</em>-1beta stimulation, achieving an estimated concentration of 167 ng/ml in airway surface liquid. CCL<em>20</em> abundance in bronchoalveolar lavage fluid from patients with cystic fibrosis was nearly 90-fold higher compared with bronchoalveolar lavage fluid from healthy volunteers. Interestingly, CCL<em>20</em> exhibited salt-sensitive antimicrobial activity, mainly against Gram-negative bacteria in low mug/ml concentrations. Additionally, apical washings from <em>IL</em>-1beta-stimulated primary cultures of human airway epithelia had significantly more antimicrobial activity than unstimulated controls. CCL<em>20</em> rapidly permeabilized bacterial membranes with a time course intermediate to HBD-2 and HBD-3. Thus, CCL<em>20</em> is a bi-functional peptide with both innate and adaptive immune properties that is regulated by inflammatory mediators, expressed by airway epithelia, and increased in cystic fibrosis airway secretions.

Many diseases are associated with catabolic conditions that induce skeletal muscle wasting. These various catabolic states may have similar and distinct mechanisms for inducing muscle protein loss. Mechanisms related to muscle wasting may also be related to muscle metabolism since glycolytic muscle fibers have greater wasting susceptibility with several diseases. The purpose of this study was to determine the relationship between muscle oxidative capacity and muscle mass loss in red and white hindlimb muscles during cancer cachexia development in the Apc(Min/+) mouse. Gastrocnemius and soleus muscles were excised from Apc(Min/+) mice at <em>20</em> wk of age. The gastrocnemius muscle was partitioned into red and white portions. Body mass (-<em>20</em>%), gastrocnemius muscle mass (-41%), soleus muscle mass (-34%), and epididymal fat pad (-100%) were significantly reduced in severely cachectic mice (n = 8) compared with mildly cachectic mice (n = 6). Circulating <em>IL</em>-6 was fivefold higher in severely cachectic mice. Cachexia significantly reduced the mitochondrial DNA-to-nuclear DNA ratio in both red and white portions of the gastrocnemius. Cytochrome c and cytochrome-c oxidase complex subunit IV (Cox IV) protein were reduced in all three muscles with severe cachexia. Changes in muscle oxidative capacity were not associated with altered myosin heavy chain expression. PGC-1α expression was suppressed by cachexia in the red and white gastrocnemius and soleus muscles. Cachexia reduced Mfn1 and Mfn2 mRNA expression and markers of oxidative stress, while Fis1 mRNA was increased by cachexia in all muscle types. Muscle oxidative capacity, mitochondria dynamics, and markers of oxidative stress are reduced in both oxidative and glycolytic muscle with severe wasting that is associated with increased circulating <em>IL</em>-6 levels.

Female and male mice deficient in <em>IL</em>-10 production by targeted disruption of the <em>IL</em>-10 gene were infected with Plasmodium chabaudi chabaudi (AS) blood-stage parasites. Both male and female mutant mice exhibited more severe signs of disease than did +/+ or heterozygous control mice. Female defective mice also displayed an increased mortality; 56% of mice died within <em>20</em> days of infection. Mortality did not appear to be due to a fulminating parasitemia as death occurred at different levels of parasitemia in the individual mice. The acute infection was accompanied by an enhanced Th1 IFN-gamma response. This response was retained in the chronic phase of infection of both male and female mutant mice, whereas in controls the responding CD4+ T cells were predominantly Th2 cells secreting <em>IL</em>-4. The data suggest that <em>IL</em>-10 regulates the inflammatory response to the parasite and that in its absence the combined effects of malaria toxins and the sustained or enhanced IFN-gamma response lead to increased pathology. In the case of female mice absence of <em>IL</em>-10 is sufficient to induce a lethal endotoxin-like reaction.

OBJECTIVE

Prophylactic administration of interleukin (IL)-10 decreases the severity of experimental pancreatitis. Prevention of post-endoscopic retrograde cholangiopancreatography (ERCP) pancreatitis in humans is a unique model to study the potential role of IL-10 in this setting.

METHODS

In a single-center, double-blind, randomized, placebo-controlled study, the effect of a single injection of 4 microg/kg (group 1) or 20 microg/kg (group 2) IL-10 was compared with that of placebo (group 0), all administered 30 minutes before therapeutic ERCP. The primary endpoint was the effect of IL-10 on serum levels of amylases and lipases measured 4, 24, and 48 hours after ERCP. The secondary objective was to evaluate changes in plasma cytokines (IL-6, IL-8, tumor necrosis factor) at the same time points and the incidence of acute pancreatitis in the 3 groups. Subjects undergoing a first therapeutic ERCP were eligible for inclusion.

RESULTS

A total of 144 patients were included. Seven were excluded based on intention to treat (n = 1) or per protocol (n = 6). Forty-five, 48, and 44 patients remained in groups 0, 1, and 2, respectively. The 3 groups were comparable for age, sex, underlying disease, indication for treatment, type of treatment, and plasma levels of C-reactive protein (CRP), cytokines, and hydrolases at baseline. No significant difference was observed in CRP, cytokine, and hydrolase plasma levels after ERCP. Forty-three patients developed hyperhydrolasemia (18 in group 0, 14 in group 1, and 11 in group 2; P = 0.297), and 19 patients developed acute clinical pancreatitis (11 in group 0, 5 in group 1, 3 in group 2; P = 0.038). Two severe cases were observed in the placebo group. No mortality related to ERCP was observed. Logistic regression identified 3 independent risk factors for post-therapeutic ERCP pancreatitis: IL-10 administration (odds ratio [OR], 0.46; 95% confidence interval [95% CI], 0.22-0.96; P = 0.039), pancreatic sphincterotomy (OR, 5.04; 95% CI, 1.53-16.61; P = 0.008), and acinarization (OR, 8.19; 95% CI, 1.83-36.57; P = 0.006).

CONCLUSIONS

A single intravenous dose of IL-10, given 30 minutes before the start of the procedure, independently reduces the incidence of post-therapeutic ERCP pancreatitis.

Murine mast-cell and T-cell growth factor activities, distinct from interleukins 3 and 2 (<em>IL</em>-3 and <em>IL</em>-2), have been identified and partially purified from the supernatant of the activated helper T-cell line Cl.Ly1+2-/9. This mast-cell growth factor (MCGF) activity supports only low levels of proliferation of several <em>IL</em>-3-dependent mast-cell lines and synergistically enhances the growth of mast cells in the presence of <em>IL</em>-3. The T-cell growth factor (TCGF) stimulates the proliferation of several T-cell lines, but to a lesser extent than recombinant <em>IL</em>-2. The MCGF and TCGF activities were not separable despite multiple biochemical fractionations, suggesting that both activities reside in the same protein. The MCGF/TCGF was separated from endogenous <em>IL</em>-3 by cation-exchange chromatography at neutral pH and could be distinguished from <em>IL</em>-2 by unique elution conditions from reverse-phase columns. Two bands of MCGF/TCGF activity were eluted from gels after sodium dodecyl sulfate/PAGE; under nonreducing conditions, the activities corresponded to molecular masses of <em>20</em> and 15 kDa, while after reduction, the molecular masses were 21 and 16 kDa. Thus, both activities may correspond to single polypeptide chains. The majority of the MCGF/TCGF activity appears to reside in the <em>20</em>-kDa species, which displays a pI of 6.2 on chromatofocusing.

BACKGROUND

Paracoccidioides brasiliensis is a facultative intracellular dimorphic fungus that causes paracoccidioidomycosis (PCM), the most important deep mycosis in Latin America. Only a small percentage of individuals infected by P. brasiliensis develop clinical PCM, possibly in part because of genetically determined interindividual variability of host immunity. However, no primary immunodeficiency has ever been associated with PCM.

METHODS

We describe the first patient, to our knowledge, with PCM and a well-defined primary immunodeficiency in the beta 1 subunit of the interleukin (IL)-12/IL-23 receptor, a disorder previously shown to be specifically associated with impaired interferon (IFN)-gamma production, mycobacteriosis, and salmonellosis.

RESULTS

Our patient had a childhood history of bacille Calmette-Guérin disease and nontyphoid salmonellosis and, at the age of 20 years, presented to our clinic with a disseminated (acute) form of PCM. He responded well to antifungal treatment and is now doing well at 24 years of age.

CONCLUSIONS

This unique observation supports previous studies of PCM suggesting that IL-12, IL-23, and IFN-gamma play an important role in protective immunity to P. brasiliensis. Tuberculosis and PCM are thus not only related clinically and pathologically, but also by their immunological pathogenesis. Our study further expands the spectrum of clinical manifestations of inherited defects of the IL-12/IL-23-IFN-gamma axis. Patients with unexplained deep fungal infections, such as PCM, should be tested for defects in the IL-12/IL-23-IFN- gamma axis.

Inflammation involving the retina and choroid is a common clinical problem, but the mechanisms that elicit and maintain ocular inflammation remain poorly understood. Interposed between the sensory retina and the systemic blood circulation within the choroid is the neural-derived retinal pigment epithelium (RPE), which forms part of the blood-retina barrier. The RPE is actively phagocytic and shares several features with mononuclear phagocytes of bone marrow origin, including the production of a neutrophil chemotactic factor, interleukin 8, after stimulation with interleukin 1 beta (<em>IL</em>-1 beta) or tumor necrosis factor alpha (TNF-alpha). Because monocyte-derived macrophages are present in retinal lesions of many common and blinding diseases, we monitored human RPE cells or monocyte chemotactic protein (MCP) mRNA expression and activity following cytokine stimulation. Cultured human RPE cells were left unstimulated or exposed to recombinant human <em>IL</em>-1 beta, TNF-alpha, or lipopolysaccharide. MCP mRNA expression in RPE cells and biologically active MCP in RPE cell supernatants were present 1 hour after stimulation and maintained for 24 hours. Conditioned media from RPE cells stimulated with <em>20</em> ng/ml of <em>IL</em>-1 beta or TNF-alpha for 24 hours contained biologically active monocyte chemotactic activity that rose rapidly from baseline levels over 4 hours and plateaued over the subsequent <em>20</em> hours. RPE chemotactic activity was dose dependent using concentrations of these cytokines ranging from <em>20</em> pg/ml to <em>20</em> ng/ml of 4-hour assays. Time- and concentration-dependent expression of RPE cell MCP mRNA was also found in the same cultures. Peak MCP mRNA expression occurred after 8 hours of stimulation with <em>IL</em>-1 beta or TNF-alpha. Maximal steady-state MCP mRNA expression occurred at <em>20</em> ng/ml for <em>IL</em>-1 beta. Immunohistochemical staining using specific anti-MCP antibodies resulted in distinctive RPE cell staining, confirming the presence of MCP in human RPE cells. These findings demonstrate that cytokine-stimulated RPE cells may evoke or augment mononuclear phagocyte-mediated ocular inflammation by synthesizing MCP.

Endotoxin is a potent inflammatory stimulus and induces polymorphonuclear leukocyte (PMNL) infiltration into tissues. Macrophage (M phi) derived <em>IL</em>-1 has been proposed as a mediator of this response. TNF alpha is also produced by M phi s in response to endotoxin and both <em>IL</em>-1 and TNF enhance PMNL adhesion to vascular endothelium in vitro. We investigated the activity of recombinant human <em>IL</em>-1 alpha, <em>IL</em>-1 beta, and TNF alpha in inducing PMNL infiltration into the skin of rabbits using a quantitative 51Cr labelled blood leukocyte assay. <em>IL</em>-1 alpha and <em>IL</em>-1 beta induced progressive PMNL accumulation, the 50% maximal response being induced by approximately equal to <em>20</em> units. In comparison, TNF alpha even at 100,000 U, induced only mild PMNL accumulations, although <em>IL</em>-1 alpha and TNF alpha were similarly active in inducing PMNL adherence to human umbilical vein endothelium. The human TNF alpha preparation was pyrogenic and induced acute, transient neutropenia in rabbits upon i.v. infusion, <em>IL</em>-1 alpha, <em>IL</em>-1 beta and TNF alpha are often secreted simultaneously by M phi s, therefore we investigated their action in combination. The combination of <em>IL</em>-1 alpha with <em>IL</em>-1 beta was nearly additive in inducing PMNL accumulation, i.e., 87% of predicted result based on the sum of the responses to individual components. The combination of TNF alpha with either <em>IL</em>-1, each in submaximal doses, resulted in 65-125% greater than the additive response. No such effect was observed when these monokines were injected in combination with PMNL chemotactic stimuli. These results indicate a complex interaction between inflammatory monokines in the regulation of PMNL accumulation in vivo.

The steady-state kinetic properties of SH-PTP1 (PTP1C, SHP, HCP), a Src homology 2 (SH2) domain-containing protein tyrosine phosphatase (PTPase), were assessed and compared with those of three truncation mutants, using p-nitrophenyl phosphate, phosphotyrosyl (pY) peptides, and reduced, carboxyamido-methylated, maleylated, and tyrosyl-phosphorylated lysozyme as substrates. At physiological pH (7.4), truncation of the two N-terminal SH2 domains [SH-PTP1(delta SH2)] or the last 35 amino acids of the C-terminus [SH-PTP1(delta C35)] activated the phosphatase activity by 30-fold and <em>20</em>-34-fold relative to the wild-type enzyme, respectively. Truncation of the last 60 amino acids resulted in a mutant [SH-PTP1(delta C60)] with wild-type activity. SH-PTP1 and SH-PTP1(delta C60) displayed apparent saturation kinetics toward pNPP only at acidic pH (pH < or = 5.4); as pH increased above 5.5, their apparent KM values increased dramatically. In contrast, SH-PTP1(delta SH2) obeyed normal Michaelis-Menten kinetics at all pH values tested (pH 5.1-7.4) with a constant KM (10-14 mM). Furthermore, two synthetic pY peptides corresponding to known and potential phosphorylation sites on the erythropoietin (EPOR pY429) and interleukin-3 (<em>IL</em>-3R pY628) receptors bound specifically to the N-terminal SH2 domain of SH-PTP1 (KD = 1.8-10 microM) and activated the catalytic activity of SH-PTP1 and SH-PTP1(delta C60) but not SH-PTP1(delta SH2), in a concentration-dependent manner. Maximal activation (25-30-fold) of SH-PTP1 was achieved at 70 microM EPOR pY429, and the maximally activated enzyme approached the activity of SH-PTP1(delta SH2). Addition of EPOR pY429 peptide, which corresponds to the recently identified in vivo binding site for SH-PTP1, at 40 microM also completely restored the saturation kinetic behavior of SH-PTP1 (at pH 7.4) toward pNPP, with catalytic parameters (KM = 12.8 mM, kcat = 3.2 s-1) similar to those of SH-PTP1(delta SH2). These data suggest that the SH2 domains of SH-PTP1 serve to autoinhibit the phosphatase activity of the PTPase domain. A model is proposed in which the SH2 domains interact with the PTPase domain in a pY-independent fashion and drive the PTPase domain into an inactive conformation.

The active form of vitamin D (1,25-dihydroxycholecalciferol) is a potent immune system regulator. Treating mice with 1, 25-dihydroxycholecalciferol and feeding them diets high in calcium can completely suppress the induction of experimental autoimmune diseases such as experimental autoimmune encephalomyelitis (EAE). Experiments described here were carried out on mice in which development of EAE was induced. Mice were fed diets containing various amounts of calcium and 1,25-dihydroxychole-calciferol. Variables measured were as follows: 1) incidence and severity of EAE; 2) serum calcium concentrations; 3) body weight; 4) total number of cells in the lymph nodes; and 5) interleukin-4 (<em>IL</em>-4) and transforming growth factor-beta1 (TGF-beta1) mRNA levels. When calcium was removed from the diet, the incidence of EAE was reduced <em>20</em>% in both males and females. Further, the lower the dietary level of calcium, the higher was the dose of 1,25-dihydroxycholecalciferol required to prevent the symptoms. Thus, 1, 25-dihydroxycholecalciferol was found most effective in mice fed a diet adequate or high in calcium. 1,25-Dihydroxycholecalciferol treatment of mice fed high dietary calcium resulted in a decreased number of lymphocytes in the lymph nodes and increased <em>IL</em>-4 and TGF-beta1 mRNA levels. When calcium was omitted from the diet, 1, 25-dihydroxycholecalciferol supplementation increased TGF-beta1 mRNA. Increased <em>IL</em>-4 mRNA and decreased lymphocytes in the lymph nodes in response to 1,25-dihydroxycholecalciferol occurred only when dietary calcium was adequate or high. Our results suggest that dietary calcium and 1,25-dihydroxycholecalciferol are both involved in the prevention of symptomatic EAE.