Hybridoma growth factor (HGF) is a <em>20</em>-25 kD protein, supporting the growth of hybridoma cells in vitro and capable of replacing feeder cells. It was shown to be produced by human monocytes and a number of cultured cell lines. Recently, HGF was found to be identical to interferon-beta 2 or 26 kD protein and BSF-2, and was renamed interleukin 6 (<em>IL</em>-6). Using a sensitive bio-assay we were able to measure <em>IL</em>-6 activity in the serum and urine of healthy volunteers and renal transplant recipients. Low levels of <em>IL</em>-6 were present in the serum but not in the urine of healthy individuals. In contrast, both serum and urine of renal transplant recipients contained high levels of <em>IL</em>-6 directly after transplantation and during acute rejection episodes. On the basis of kinetic studies of the <em>IL</em>-6 response, it is concluded that serial measurement of <em>IL</em>-6, especially in urine, may be of value in monitoring renal transplant recipients. Moreover, the sensitivity of the bioassay will allow for detailed studies as to the biological significance of <em>IL</em>-6 in health and disease.

Pretreatment with recombinant interleukin 1 (<em>IL</em> 1) protects mice in a dose-dependent manner from lethal effects of ionizing radiation. Two thousand units of <em>IL</em> 1, given i.p. <em>20</em> hr before irradiation, protect 88% of C57B1/6 mice from an LD100/17 radiation dose (dose of radiation that kills 100% mice in 17 days), and 1000 U of <em>IL</em> 1 protect 100% of DBA/1 mice from an LD50/30 dose. This finding provides the first evidence that a cytokine, <em>IL</em> 1, which acts as a differentiation- and maturation-inducing agent for a variety of cells, also can serve as a signal that initiates radioprotective events in vivo. Because many of the exogenous immunomodulators that have been shown to be radioprotective also induce endogenous <em>IL</em> 1 production, our observation suggests that <em>IL</em> 1 may mediate their radioprotective effects.

BACKGROUND

Activated Stat3 is found in various types of immortal cell lines and cancers. We and others have previously demonstrated that Stat3 is constitutively activated in rat and human prostate cancer cell lines, and that Stat3 activation is involved in IL-6-mediated signaling transduction in prostate cancer cells. The aim of this study is to examine quantitative Stat3 activity in benign and malignant human prostate tissues and analyze the association between Stat3 activity levels and the clinical and pathologic parameters.

METHODS

Stat3 activity levels were analyzed in a total of 104 human primary prostate tissues using electromobility shift assay and immunohistochemical staining for phosphorylated Stat3. The tissue samples used were 42 prostate carcinomas, 42 matched normal prostate tissues from patients with prostatic adenocarcinoma (normal adjacent to tumor), and 20 normal prostate tissues from organ donors.

RESULTS

Significantly higher levels of constitutive Stat3 activity were detected in both prostate carcinomas and the matched normal prostate tissues adjacent to tumors compared to the normal prostates from donors without prostate cancer. There was no significant difference of Stat3 activity in foci of tumor and normal prostate tissue adjacent to tumor. No correlation was seen between Stat3 activity and Gleason grade or serum PSA levels in samples from prostate carcinomas.

CONCLUSIONS

These results indicate that Stat3 is constitutively activated in prostate cancer. The high level of Stat3 activity in both the prostate carcinomas and the normal prostate tissues adjacent to tumors suggests that Stat3 activation may occur before detectable histological alterations of the prostate.

BACKGROUND

Differences in cytokine/chemokine profiles among patients with neuromyelitis optica (NMO), relapsing remitting multiple sclerosis (RRMS), and primary progressive MS (PPMS), and the relationships of these profiles with clinical and neuroimaging features are unclear. A greater understanding of these profiles may help in differential diagnosis.

RESULTS

We measured 27 cytokines/chemokines and growth factors in CSF collected from <em>20</em> patients with NMO, 26 with RRMS, nine with PPMS, and 18 with other non-inflammatory neurological diseases (OND) by multiplexed fluorescent bead-based immunoassay. Interleukin (<em>IL</em>)-17A, <em>IL</em>-6, CXCL8 and CXCL10 levels were significantly higher in NMO patients than in OND and RRMS patients at relapse, while granulocyte-colony stimulating factor (G-CSF) and CCL4 levels were significantly higher in NMO patients than in OND patients. In NMO patients, <em>IL</em>-6 and CXCL8 levels were positively correlated with disability and CSF protein concentration while <em>IL</em>-6, CXCL8, G-CSF, granulocyte-macrophage colony-stimulating factor (GM-CSF) and IFN-γ were positively correlated with CSF neutrophil counts at the time of sample collection. In RRMS patients, <em>IL</em>-6 levels were significantly higher than in OND patients at the relapse phase while CSF cell counts were negatively correlated with the levels of CCL2. Correlation coefficients of cytokines/chemokines in the relapse phase were significantly different in three combinations, <em>IL</em>-6 and GM-CSF, G-CSF and GM-CSF, and GM-CSF and IFN-γ, between RRMS and NMO/NMOSD patients. In PPMS patients, CCL4 and CXCL10 levels were significantly higher than in OND patients.

CONCLUSIONS

Our findings suggest distinct cytokine/chemokine alterations in CSF exist among NMO, RRMS and PPMS. In NMO, over-expression of a cluster of Th17- and Th1-related proinflammatory cytokines/chemokines is characteristic, while in PPMS, increased CCL4 and CXCL10 levels may reflect on-going low grade T cell and macrophage/microglia inflammation in the central nervous system. In RRMS, only a mild elevation of proinflammatory cytokines/chemokines was detectable at relapse.

Altered cytokine secretion as a mechanism in the etiology of depression is still obscure. The serotonin transporter (5-HTT) may play an important role in the termination of serotonergic neurotransmission by serotonin (5-HT) uptaking into presynaptic neurons and representing as an initial action site for selective 5-HTT reuptake inhibitors (SSRI). In our study, we evaluated whether cytokines and 5-HTT acted as biological markers for depression. Blood samples were collected from 42 participants. The differences in cytokine and 5-HTT mRNA expressions of leukocytes were assessed between the patients with major depression (n=<em>20</em>) and the healthy controls (n=22), along with the measurements prior and after treatment with a SSRI, fluoxetine, for 3 months in the follow-up patient group (n=8). The results revealed that the mRNA expressions of <em>IL</em>-1beta, <em>IL</em>-6, IFNgamma, TNFalpha, and 5-HTT were higher in the depressed patients than those of the healthy controls. The higher level of mRNA expressions of IFNgamma and 5-HTT diminished after fluoxetine treatment. Furthermore, we found a positive correlation between 5-HTT and cytokines mRNA expressions in total participants, which suggested that pro-inflammatory cytokines and 5-HTT might play critical roles in the pathogenesis of major depression and that their levels were affected by chronic treatment with 5-HTT inhibitors.

Sleep loss has been associated with increased sleepiness, decreased performance, elevations in inflammatory cytokines, and insulin resistance. Daytime napping has been promoted as a countermeasure to sleep loss. To assess the effects of a 2-h midafternoon nap following a night of sleep loss on postnap sleepiness, performance, cortisol, and <em>IL</em>-6, 41 young healthy individuals (<em>20</em> men, 21 women) participated in a 7-day sleep deprivation experiment (4 consecutive nights followed by a night of sleep loss and 2 recovery nights). One-half of the subjects were randomly assigned to take a midafternoon nap (1400-1600) the day following the night of total sleep loss. Serial 24-h blood sampling, multiple sleep latency test (MSLT), subjective levels of sleepiness, and psychomotor vigilance task (PVT) were completed on the fourth (predeprivation) and sixth days (postdeprivation). During the nap, subjects had a significant drop in cortisol and <em>IL</em>-6 levels (P < 0.05). After the nap they experienced significantly less sleepiness (MSLT and subjective, P < 0.05) and a smaller improvement on the PVT (P < 0.1). At that time, they had a significant transient increase in their cortisol levels (P < 0.05). In contrast, the levels of <em>IL</em>-6 tended to remain decreased for approximately 8 h (P = 0.1). We conclude that a 2-h midafternoon nap improves alertness, and to a lesser degree performance, and reverses the effects of one night of sleep loss on cortisol and <em>IL</em>-6. The redistribution of cortisol secretion and the prolonged suppression of <em>IL</em>-6 secretion are beneficial, as they improve alertness and performance.

Attention has focused on the cytokine interleukin 6 (<em>IL</em>-6) as a major mediator of acute-phase protein synthesis in hepatocytes in response to infection and tissue injury. We have evaluated the effects of <em>IL</em>-6 and <em>IL</em>-1 alpha as well as extracellular zinc and glucocorticoid hormone on metallothionein gene expression and cellular zinc accumulation in rat hepatocyte monolayer cultures. Further, we have evaluated the teleological basis for cytokine mediation by examining cytoprotection from CCl4-induced damage. Incubation of hepatocytes with <em>IL</em>-6 led to concentration-dependent and time-dependent increases in metallothionein-1 and -2 mRNA and metallothionein protein. The level of each was increased within 3 hr after the addition of <em>IL</em>-6 at 10 ng/ml (10 hepatocyte-stimulating factor units/ml). Maximal increases in metallothionein mRNA and metallothionein protein were achieved after 12 hr and 36 hr, respectively. In contrast, <em>IL</em>-1 alpha concentrations as high as <em>20</em> ng/ml (1000 lymphocyte-activating factor units/ml) had no effect. Concomitant with the up-regulation of metallothionein gene expression, <em>IL</em>-6 also increased cellular zinc. Responses to <em>IL</em>-6 required the synthetic glucocorticoid hormone dexamethasone and were optimized by increased extracellular zinc. In addition, <em>IL</em>-6 with dexamethasone, dexamethasone alone, and increased extracellular zinc each reversed, in decreasing potency, the deleterious effects of CCl4 on hepatocyte viability as measured by cell protein and lactate dehydrogenase activity of the medium. Thus, <em>IL</em>-6 is a major cytokine mediator of metallothionein gene expression and zinc metabolism in hepatocytes and provides cytoprotection from CCl4-induced hepatotoxicity via a mode consistent with dependence upon increased cellular metallothionein synthesis and zinc accumulation.

BACKGROUND

Asthma is a chronic inflammatory disorder of the airways driven by T cell activation. Th2 cells and their cytokines are thought to play a role in the pathophysiology of allergic as well as non-allergic asthma.

METHODS

Airway cells were obtained by sputum induction from 15 healthy and 39 asthmatic individuals and the airway T cell cytokine profiles (interleukin (IL)-4, IL-5, IL-13, IL-10 and interferon (IFN)-gamma) at the mRNA level were studied by real time RT-PCR.

RESULTS

Asthma patients had increased expression of IL-5 (p = 0.001) and IL-13 (p = 0.03) mRNA in sputum compared with non-asthmatic controls. IL-4 mRNA and IFN-gamma mRNA were detectable in the sputum of 44% and 21% of patients, respectively, but not in controls. Sputum IL-10 mRNA levels did not differ significantly between patients and controls. Sputum mRNA expression levels of IL-4, IL-5, and IL-13 were significantly correlated with the percentage of eosinophils and were higher in subjects with allergic asthma than in those with non-allergic asthma (p = 0.03, p = 0.02 and p = 0.0002, respectively); they did not differ between mild asthmatic subjects and those with moderate to severe asthma. In contrast, IFN-gamma mRNA expression was higher in non-allergic than in allergic patients (p = 0.04) and higher in patients with moderate to severe asthma than in those with mild asthma (p<0.01). Sputum IL-5 mRNA levels (but not the other cytokine mRNA levels) were also correlated with exhaled nitric oxide (eNO) and with bronchial hyperreactivity expressed as the histamine concentration resulting in a 20% decrease in forced expiratory volume in 1 second.

CONCLUSIONS

Real time RT-PCR analysis of mRNA in induced sputum confirms a predominance of Th2 cytokines in both allergic and non-allergic asthma. IL-5 levels reflect eosinophil infiltration as well as eNO levels and hyperreactivity, and levels of the Th1 cytokine IFN-gamma indicate asthma severity. The technique is a promising tool for use in further studies of asthma severity and disease activity.

OBJECTIVE

To test the hypothesis that peripheral blood mononuclear cells in women with unexplained recurrent abortion (URA) produce T-helper 1 (TH1)-type cytokines in response to trophoblast antigens.

METHODS

Cohort study.

METHODS

Medical center.

METHODS

A total of 244 women with URA, 13 reproductively normal parous control women, and 10 men.

METHODS

Supernatants from trophoblast-activated peripheral blood mononuclear cells from all participants were tested for toxic effects on mouse embryos and by enzyme-linked immunosorbent assay (ELISA) for interferon gamma (IFN-gamma). Supernatants from <em>20</em> URA patients with embryotoxic activity and IFN-gamma, 13 reproductively normal parous women, and 10 men were further tested by ELISA for other TH1-type cytokines (interleukin-2 [<em>IL</em>-2], tumor necrosis factor-beta [TNF-beta]), TH2-type cytokines (<em>IL</em>-4, <em>IL</em>-10), and TNF-alpha.

RESULTS

Embryotoxic activity was detected in supernatants from 160 of 244 URA patients and in none of the controls. Interferon gamma was detected in supernatants from 125 of 244 URA patients and was significantly associated with embryotoxicity (121 of 160 supernatants with embryotoxicity vs four of 84 supernatants without embryotoxicity [P < .001]). Of <em>20</em> supernatants from patients chosen for further study, all were positive for TNF-alpha, 17 for TNF-beta, two for <em>IL</em>-10, and one for <em>IL</em>-4. No cytokines were detected in supernatants from unstimulated or red blood cell membrane-activated cells of women with URA. In contrast, trophoblast-activated lymphocyte supernatants from reproductively normal women and men neither were embryotoxic nor contained TH1-type cytokines, but most contained the TH2-type cytokine <em>IL</em>-10. Three supernatants from reproductively normal women also contained <em>IL</em>-4.

CONCLUSIONS

Whereas TH1-type immunity to trophoblast is associated with URA and may play a role in reproductive failure, TH2-type immunity may be a natural response to trophoblast contributing to successful pregnancy.

Little is known about the factors that initiate and propagate tendon overuse injuries, but chronic inflammation and matrix destruction have been implicated. The purpose of this study was to evaluate the production of cyclooxygenase II (COX-2) and matrix metalloproteinases (MMPs) by tendon cells exposed to cyclic strain and inflammatory cytokines in vitro. Rabbit Achilles tendon cells were subjected to a stretching protocol with 5% elongation at 0.33 Hz for 6 h, or treated with 1000 pM interleukin-1beta (<em>IL</em>-1beta), or exposed to <em>IL</em>-1beta and stretching together. Gene expression was evaluated by RT-PCR and production of stromelysin was quantified with an ELISA. <em>IL</em>-1beta induced the expression of the collagenase-1 and stromelysin-1 genes. Production of stromelysin proenzyme by cells stimulated with <em>IL</em>-1beta was 17 times higher than production by control cells. Cells exposed to <em>IL</em>-1beta and stretching produced <em>20</em> times more stromelysin than control cells. Cells subjected to stretching alone did not produce more stromelysin than control cells. The synergistic effect of <em>IL</em>-1beta and stretching was observed at doses of <em>IL</em>-1beta ranging from 10 to 1000 pM. These data suggest that mechanical load and inflammatory cytokines can initiate a matrix destructive pathway in tendon that is more pronounced than with mechanical loading or inflammation alone.

The UL41 gene product (vhs) of herpes simplex virus (HSV) is packaged in the virion, and mediates host protein synthesis shutoff at the early stage of the virus replication cycle. In order to clarify the role of vhs in virus replication and virulence, we isolated a completely UL41-deficient mutant (the VRDelta41 strain) and its revertant (the VRDelta41R strain). In the mouse encephalitis model, the replication of strain VRDelta41 was inhibited after 2 days post-infection, resulting in low virulence, by gamma-ray-sensitive cells such as lymphocytes and/or neutrophils. The result suggested that some cytokines, produced in VRDelta41-inoculated brains, activate and induce the migration of gamma-ray-sensitive cells to the infection site. Therefore, cytokines produced by HSV-1-infected human cells were screened, and potent inductions of interleukin (<em>IL</em>)-1beta, <em>IL</em>-8 and macrophage inflammatory protein-1alpha by VRDelta41 infection were observed. Moreover, the VRDelta41 strain showed <em>20</em>- and 5-fold higher sensitivity to interferon-alpha and -beta compared to the wild-type strain, respectively. These results indicate that one important role of vhs in vivo is evasion from non-specific host defence mechanisms during primary infection through suppression of cytokine production in HSV-infected cells and reduction of the anti-HSV activity of interferon-alpha and -beta.

Development of severe sepsis is thought to result from the overproduction of cytokines, such as tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (<em>IL</em>-1beta), and nitric oxide. Recently, 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, which are antihypercholesterolemic agents, have been reported to inhibit lipopolysaccharide (LPS)-induced production of cytokines and nitric oxide in vitro. In this study, we tested these effects in vivo. After LPS administration (15 mg/kg i.p.) to CD-1 mice, serum levels of both TNF-alpha and <em>IL</em>-1beta transiently increased, and peaked at 2 h. After the peak responses of TNF-alpha and <em>IL</em>-1beta, serum levels of nitrite and nitrate increased until at least 8 h. Pretreatment of the mice with cerivastatin (<em>20</em> mg/kg i.p. 12 and 1 h before LPS injection) reduced serum levels of TNF-alpha and <em>IL</em>-1beta at 2 h, and nitrite and nitrate at 8 h, by 93, 60, and 44%, respectively. In this model of sepsis, cerivastatin significantly (P =.016) improved the rate of 7-day survival from 26.7 to 73.3%. These results cast new light on the usefulness of cerivastatin in preventing severe sepsis.

Growing evidence suggests that interleukin (<em>IL</em>)-17 and <em>IL</em>-17-secreting CD4(+)T (Th17) cells are involved in the pathogenic mechanisms of multiple sclerosis (MS). <em>IL</em>-17-secreting CD8(+)T cells were recently identified as a novel subset of CD8(+)T cells. We aimed to analyze the role of Th17 and <em>IL</em>-17 secreting CD8(+)T cells in the pathogenesis of neuromyelitis optica (NMO) as well as MS. Fourteen patients with NMO, <em>20</em> with MS and 16 control participants (CTL) were enrolled between November <em>20</em>08 and December <em>20</em>09. The proportion of Th17 cells and <em>IL</em>-17 secreting CD8(+)T cells were counted using flow cytometry, and serum levels of <em>IL</em>-6, <em>IL</em>-17, <em>IL</em>-21, <em>IL</em>-23, and transforming growth factor-beta (TGF-β) were measured by enzyme-linked immunosorbent assay. Patients with NMO had a larger proportion of Th17 cells than patients with MS (3.72% versus [vs.] 2.58%, p=0.02) and CTL (3.72% vs. 1.36%, p<0.001). The proportion of Th17 cells in patients with MS was also markedly higher than in the CTL (2.58% vs. 1.36%, p<0.001). <em>IL</em>-17-secreting CD8(+)T cell counts in NMO patients were markedly higher than in MS patients (1.61% vs. 1.09%, p=0.036) and CTLs (1.61% vs. 0.58%, p<0.001). The proportion of <em>IL</em>-17-secreting CD8(+)T cells in MS patients was also higher than in CTLs (1.09% vs. 0.58%, p=0.002). Serum <em>IL</em>-17 and <em>IL</em>-23 levels were increased in patients with NMO and MS, while serum <em>IL</em>-21 concentration was higher only in NMO patients compared to CTL. We concluded that Th17 cells were highly activated in patients with NMO. <em>IL</em>-17-secreting CD8(+)T cells were increased in patients with NMO and MS during relapse and have an important role in the pathological mechanism of NMO and MS.

Previous studies have shown that T cells from human alcoholics overexpress activation or memory markers such as human leukocyte antigen-DR, CD45RO, CD57, and CD11b and may have reduced levels of CD62L. In those studies, we demonstrated that the increased CD57(+) T cell population rapidly produces interferon-gamma (IFN-gamma) and tumor necrosis factor alpha, independent of a second signal requirement, consistent with an increased effector T cell population. In contrast to the length of alcohol abuse by human alcoholics, most work with mice has involved 2-week ethanol exposures or less, which result in decreased IFN-gamma responses. In the present work, we have evaluated C57Bl/6 or BALB/c mice, which were administered <em>20</em>% w/v ethanol in water for 3-13 weeks. In these mice, rapid cytoplasmic IFN-gamma expression by T cells after stimulation through the T cell receptor was significantly increased versus normals. Studies of surface-activation markers showed that T cells from chronically ethanol-fed mice had reduced CD62L expression and an increased percentage of CD44(hi) T cells. The CD44(hi) subset was largely second signal-independent for secreted IFN-gamma and interleukin (<em>IL</em>)-4 production at early times after stimulation. The enriched T cells of chronic ethanol mice secreted more IFN-gamma and <em>IL</em>-4 than controls and equivalent <em>IL</em>-2 at early times after stimulation (6-24 h). The overall results support the concept that in humans and mice, chronic alcohol exposure of sufficient duration results in T cell activation or sensitization in vivo and an increased percentage of the effector/memory subset.

BACKGROUND

The magnitude of surgical trauma after laparoscopic and open colonic resection was evaluated by examining postoperative serum values of interleukin-6 (IL-6), IL-10, C-reactive protein (CRP), and granulocyte elastase (GE) for further evidence of the benefit realized with minimally invasive approaches in colonic surgery.

METHODS

Altogether, 42 patients with Crohn's disease (n = 20) or colon carcinomas/adenomas (n = 22) were matched by age, gender, body mass index (BMI), and Crohn's Disease Activity Index for either a laparoscopic (n = 21) or an open colonic resection (n = 21). In both groups the postoperative serum levels of IL-6, IL-10, C-RP, and granulocyte elastase were determined, as indicators of surgical stress.

RESULTS

Laparoscopic and open colonic resection caused a significant increase in serum IL-6, IL-10, CRP, and granulocyte elastase levels. The comparison between laparoscopic and open colonic resections, however, showed significantly lower serum IL-6, IL-10, CRP, and granulocyte elastase levels after laparoscopic colonic resection, which was most evident for IL-6 and granulocyte elastase.

CONCLUSIONS

Our study demonstrated that IL-6 and granulocyte elastase may be appropriated particularly to monitor surgical stress. By using these parameters, we found a significant reduction in surgical trauma after laparoscopic surgery, was compared with the open procedure. This supports the clinical findings of a clear benefit for patients undergoing laparoscopic colonic surgery.

Brain metastasis of breast cancer profoundly affects the cognitive and sensory functions as well as morbidity of patients, and the 1 year survival rate among these patients remains less than <em>20</em>%. However, the pathological mechanism of brain metastasis is as yet poorly understood. In this report, we found that metastatic breast tumour cells in the brain highly expressed <em>IL</em>-1β which then 'activated' surrounding astrocytes. This activation significantly augmented the expression of JAG1 in the astrocytes, and the direct interaction of the reactivated astrocytes and cancer stem-like cells (CSCs) significantly stimulated Notch signalling in CSCs. We also found that the activated Notch signalling in CSCs up-regulated HES5 followed by promoting self-renewal of CSCs. Furthermore, we have shown that the blood-brain barrier permeable Notch inhibitor, Compound E, can significantly suppress the brain metastasis in vivo. These results represent a novel paradigm for the understanding of how metastatic breast CSCs re-establish their niche for their self-renewal in a totally different microenvironment, which opens a new avenue to identify a novel and specific target for the brain metastatic disease.

OBJECTIVE

Tissue degradation in corneal thinning disorders, such as keratoconus (KC), involves the expression of inflammatory mediators. The purpose of this study was to determine the levels of proinflammatory cytokines and matrix metalloproteinase 9 (MMP-9) in tears from both eyes of unilateral keratoconus (KC) patients.

METHODS

Thirty patients diagnosed as having asymmetrical KC (30 KC eyes, and 30 subclinical KC eyes) and <em>20</em> normal control subjects (one eye) were studied in a prospective, cross-sectional study. Keratoconus screening programmes were performed on these participants. Ten microlitres of tears was collected from each eye. The concentrations of cytokines (interleukin-6 (<em>IL</em>-6) and tumour necrosis factor alpha (TNF-alpha)) and MMP-9 were measured by ELISA.

RESULTS

Mean values for <em>IL</em>-6 levels were similar in KC and subclinical KC samples (5.5 (4.9 to 6.9) vs 5.7 (4.5 to 6.2) pg/ml, p = 0.131), but significantly higher in relation to the control group (2.2 (1.0 to 4.1) pg/ml, p<0.0001). Significant differences were found in TNF-alpha levels between KC and subclinical KC eyes (5.4 (4.1 to 6.8) vs 4.8 (4.2 to 6.0) pg/ml, p = 0.032) and control group (1.8 (1.5 to 2.3) pg/ml, p<0.0001). Increased values of MMP-9 were found in KC (59.4 (50.6 to 66.1) ng/ml) vs subclinical KC eye (7.0 (4.8 to 8.6) ng/ml) (p<0.0001). MMP-9 levels in the control group (6.1 (3.9 to 8.3) ng/ml) and subclinical KC were similar (p = 0.<em>20</em>3).

CONCLUSIONS

IL-6 and TNF-alpha are overexpressed in the tears of subclinical and KC eyes. Increased MMP-9 levels were found only in the KC eye. These results indicate that the pathogenesis of KC may involve chronic inflammatory events.

<strong class="sub-title"> Background: </strong> A recent increase in children admitted with hypotensive shock and fever in the context of the COVID-19 outbreak requires an urgent characterization and assessment of the involvement of SARS-CoV-2 infection. This is a case series performed at 4 academic tertiary care centers in Paris of all the children admitted to the pediatric intensive care unit (PICU) with shock, fever and suspected SARS-CoV-2 infection between April 15th and April 27th, <em>20</em><em>20</em>.

<strong class="sub-title"> Results: </strong> <em>20</em> critically ill children admitted for shock had an acute myocarditis (left ventricular ejection fraction, 35% (25-55); troponin, 269 ng/mL (31-4607)), and arterial hypotension with mainly vasoplegic clinical presentation. The first symptoms before PICU admission were intense abdominal pain and fever for 6 days (1-10). All children had highly elevated C-reactive protein (> 94 mg/L) and procalcitonin (> 1.6 ng/mL) without microbial cause. At least one feature of Kawasaki disease was found in all children (fever, n = <em>20</em>, skin rash, n = 10; conjunctivitis, n = 6; cheilitis, n = 5; adenitis, n = 2), but none had the typical form. SARS-CoV-2 PCR and serology were positive for 10 and 15 children, respectively. One child had both negative SARS-CoV-2 PCR and serology, but had a typical SARS-CoV-2 chest tomography scan. All children but one needed an inotropic/vasoactive drug support (epinephrine, n = 12; milrinone, n = 10; dobutamine, n = 6, norepinephrine, n = 4) and 8 were intubated. All children received intravenous immunoglobulin (2 g per kilogram) with adjuvant corticosteroids (n = 2), <em>IL</em> 1 receptor antagonist (n = 1) or a monoclonal antibody against <em>IL</em>-6 receptor (n = 1). All children survived and were afebrile with a full left ventricular function recovery at PICU discharge.

Conclusions: Acute myocarditis with intense systemic inflammation and atypical Kawasaki disease is an emerging severe pediatric disease following SARS-CoV-2 infection. Early recognition of this disease is needed and referral to an expert center is recommended. A delayed and inappropriate host immunological response is suspected. While underlying mechanisms remain unclear, further investigations are required to target an optimal treatment.

Keywords: Acute myocarditis; Children; Multisystem inflammatory syndrome; SARS-CoV-2; Shock.

OBJECTIVE

The aim of this study was to investigate the precise clinical characteristics and to analyse the association between the anti-MDA5 antibody (anti-MDA5ab) titre and disease status in patients with anti-MDA5ab-positive DM.

METHODS

Twenty-seven patients who presented with DM and were positive for the anti-MDA5ab were enrolled. The association between the clinical manifestations and the clinical parameters, including the anti-MDA5ab, was analysed.

RESULTS

The complication of rapidly progressive interstitial lung disease (RP-<em>IL</em>D) occurred in <em>20</em> (74%) patients. The frequencies of fatal outcome, relapse and malignancy were 33, 4 and 4%, respectively. Remarkably, a fatal outcome occurred within the first 6 months. Compared with six non-RP-<em>IL</em>D patients, elderly age at onset, severely involved pulmonary function and high levels of serum ferritin were present in <em>20</em> RP-<em>IL</em>D patients with anti-MDA5ab. Alveolar-arterial oxygen difference (AaDO(2)) ≥32 mmHg and ferritin ≥828 ng/ml at admission were poor prognostic factors in RP-<em>IL</em>D patients with anti-MDA5ab-positive DM. The median value of the anti-MDA5ab titre on admission was higher in patients who later died than in those who survived. The efficacy of treatment was indicated by the anti-MDA5ab, ferritin and <em>IL</em>-18 concentrations. The decline index of the anti-MDA5ab titre after treatment was lower in the subset of patients who died than in the subset of patients who lived. Sustained high levels of anti-MDA5ab, ferritin and <em>IL</em>-18 were present in the patients who died.

CONCLUSIONS

Anti-MDA5ab titre and ferritin and IL-18 concentrations are useful for the evaluation of the response to treatment and the status of ILD in patients with anti-MAD5ab-positive DM.

Interleukin-12 (<em>IL</em>-12) has been used in numerous immunotherapy protocols against melanoma. However, delivery of <em>IL</em>-12 in the form of recombinant protein can result in severe toxicity, and gene therapy has had limited success against B16.F10 murine melanoma. The purpose of this study was to examine the effectiveness of in vivo electroporation for the delivery of plasmid DNA encoding <em>IL</em>-12 as an antitumor agent against B16.F10 melanoma. We treated mice bearing established B16.F10 melanoma tumors with intratumoral (i.t.) or intramuscular (i.m.) injections of a plasmid encoding <em>IL</em>-12, followed by in vivo electroporation. For i.t. treatments, we used an applicator containing six penetrating electrodes to deliver 1500-V/cm, 100-micros pulses. We administered i.m. pulses with an applicator containing four penetrating electrodes delivering 100-V/cm, <em>20</em>-ms pulses. The i.t. treatment resulted in the cure of 47% of tumor-bearing mice, and 70% of cured mice were resistant to challenge with B16.F10 cells. The i.m. treatment did not result in tumor regression. We found that i.t. treatment resulted in increased levels of <em>IL</em>-12 and interferon-gamma (IFN-gamma) within the tumors, the influx of lymphocytes into the tumors, and reduction in vascularity. Neither i.m. nor i.t. treatment was successful against B16.F10 tumors in a nude mouse model, supporting a role for T cells in regression of this tumor model.

<em>IL</em>-17-producing CD4(+) T cells, so called T(h)17 cells, constitute a newly identified inflammatogenic cell population, which is critically involved in some inflammatory diseases. To explore the role of T(h)17 cells in murine experimental autoimmune uveoretinitis (EAU), a model of human autoimmune uveitis where T(h)1 responses predominantly participate in the pathogenesis, <em>IL</em>-17(-/-) mice were immunized with interphotoreceptor retinoid-binding protein peptide 1-<em>20</em> for disease induction. Funduscopic examination revealed that EAU was induced in <em>IL</em>-17(-/-) mice just like in wild-type (WT) mice at early phases of the disease. However, at later/maintenance phases, the severity was significantly reduced in <em>IL</em>-17(-/-) mice. Expression of IFN-gamma and MCP-1 was comparable between WT and <em>IL</em>-17(-/-) mice during the time course. In vivo blockade of IFN-gamma and <em>IL</em>-4 resulted in exacerbation of EAU at later phases with augmented <em>IL</em>-17 production. Taken together, our data demonstrated that <em>IL</em>-17/T(h)17 participates in the late phases of EAU and also that T(h)1 and T(h)17 responses are differentially required for EAU.

Levels of mRNA for IFN-beta 2/B cell differentiation factor2/hepatocyte-stimulating factor (IFN-beta 2) in confluent quiescent cultures of human diploid fibroblasts (FS-4 strain) are enhanced by TNF, <em>IL</em>-1 alpha and beta, platelet-derived growth factor (PDGF) and IFN-beta 1. Of these cytokines, <em>IL</em>-1 alpha and beta cause a particularly strong increase in the accumulation of IFN-beta 2 mRNA in fibroblasts. We have evaluated whether the IFN-beta 2 gene is regulated at the transcriptional level by using nuclear run-on transcription assays. We observed that the IFN-beta 2 gene is transcribed at a low level in uninduced FS-4 cells and that this transcriptional activity is increased 2- to 3-fold in cycloheximide-treated cells, <em>20</em>- to 35-fold in <em>IL</em>-1 alpha-treated cells, and 5- to 15-fold in TNF-treated cells. PDGF and IFN-beta 1 enhance transcription across the IFN-beta 2 gene 2- to 3-fold. The enhancing effect of <em>IL</em>-1 alpha on IFN-beta 2 gene transcription, but not that of TNF, PDGF, or IFN-beta 1, is inhibited by cycloheximide, suggesting that newly-synthesized protein is involved in the increase in IFN-beta 2 transcription in response to <em>IL</em>-1 alpha but not in the response to the other stimuli. Furthermore, the enhancement of IFN-beta 2 transcription is sustained for up to 14 h after <em>IL</em>-1 alpha induction but is transient and declines to base line levels within 6 h after TNF addition. These observations suggest that there are important differences in the mechanisms by which <em>IL</em>-1 alpha and TNF increase IFN-beta 2 gene transcription in fibroblasts.

Methemoglobin is a form of hemoglobin that does not bind oxygen. When its concentration is elevated in red blood cells, functional anemia and tissue hypoxia may occur. We performed a retrospective case series to describe the cases of acquired methemoglobinemia (methemoglobin level >2%) detected and the clinical circumstances under which they occurred at 2 tertiary care hospitals and affiliated outpatient clinics over 28 months. We surveyed co-oximetry laboratory data to identify patients with methemoglobinemia. We reviewed these patients' medical records to extract the clinical information and context. One hundred thirty-eight cases of acquired methemoglobinemia were detected over the 28 months. There was no gender predisposition, and the condition occurred over a wide range of ages (patients aged 4 days to 86 years). Cases occurred in many areas of the hospital, including outpatient clinics. One fatality and 3 near-fatalities were directly attributable to methemoglobinemia. Dapsone was the most common etiology of acquired methemoglobinemia, accounting for 42% of all cases. The mean peak methemoglobin level among these individuals was 7.6%. In 5 of the patients with the most severely elevated levels, <em>20</em>% benzocaine spray (Hurricaine Topical Anesthetic spray, Beutlich Pharmaceuticals, Waukegan, <em>IL</em>) was the etiology, associated with a mean peak methemoglobin level of 43.8%. Eleven pediatric patients developed methemoglobinemia either from exogenous exposure, such as drugs, or due to serious illness, such as gastrointestinal infections with dehydration. Almost all (94%) patients with methemoglobinemia were anemic. Drugs that cause acquired methemoglobinemia are ubiquitous in both the hospital and the outpatient setting. Acquired methemoglobinemia is a treatable condition that causes significant morbidity and even mortality. We hope that a heightened awareness of methemoglobinemia will result in improved recognition and treatment. Primary prevention efforts have the potential to reduce the morbidity and mortality associated with this condition.

A well defined model of T cell-mediated hypersensitivity-type granulomatous inflammation induced by Schistosoma mansoni eggs was used to assess the role of <em>IL</em>-4 and IFN-gamma in granuloma development. Synchronized pulmonary granulomas were induced and isolated from S. mansoni-infected mice during vigorous (8 wk) and modulated (<em>20</em> wk) stages of the disease. The sequential production of <em>IL</em>-4 and IFN was determined and related to temporal changes in granuloma macrophage production of <em>IL</em>-1, TNF, and superoxide anion (O2-). During the vigorous stage, <em>IL</em>-4 was produced on days 1 and 2 of granuloma formation, whereas IFN was released in greatest amounts on days 4 to 8. The peak of <em>IL</em>-4 occurred in a window between the peak of <em>IL</em>-1 (1 day) and maximal TNF production (8 to 16 days). Maximal O2- release tended to parallel IFN production. During the modulated stage when the inflammatory response is attenuated, <em>IL</em>-4 production was dramatically reduced as were levels of <em>IL</em>-1 and TNF, but IFN production persisted and maximum O2(-)-producing capacity was only delayed in onset. mAb specific for <em>IL</em>-4 and IFN were used to examine the effect of in vivo depletion of these cytokines on granuloma development. Administration of a single 1.0-mg dose of anti-<em>IL</em>-4 antibodies to mice with synchronously developing granulomas dramatically reduced granuloma size (40 to 50% suppression of area) during an 8-day study period, whereas antibodies to IFN had no effect on size. However, the latter treatment reduced giant cell formation. Our results indicate that granuloma development involves an orchestrated production of cytokines possibly resulting from sequential participation of different Th cell populations. Moreover, <em>IL</em>-4 is a pivotal cytokine in anamnestic cellular recruitment and subject to endogenous regulation.