BACKGROUND

IL-23 expression is increased in psoriatic lesions and might regulate TH17 T-cell counts in patients with psoriasis.

OBJECTIVE

We sought to test a novel IL-23-specific therapeutic agent for the treatment of psoriasis.

METHODS

In this randomized, double-blind, placebo-controlled study the safety, tolerability, and clinical response of guselkumab, an anti-IL-23-specific mAb, were evaluated in patients with moderate-to-severe plaque psoriasis. A total of 24 patients were randomized to receive a single dose of placebo or 10, 30, 100, or 300 mg of guselkumab. Clinical response was assessed by using the Psoriasis Area and Severity Index (PASI). Additionally, histologic analysis and gene expression in skin biopsy specimens from guselkumab-treated patients were compared with those from placebo-treated patients.

RESULTS

At week 12, 50% (10 mg), 60% (30 and 100 mg), and 100% (300 mg) of guselkumab-treated patients, respectively, achieved a 75% improvement in PASI scores from baseline compared with 0% of placebo-treated patients. Improvements in PASI scores were generally maintained through week 24 in all guselkumab-treated patients. The proportion of patients experiencing an adverse event was comparable between the combined guselkumab (13/20 [65.0%]) and placebo (2/4 [50.0%]) groups through week 24. Analysis of lesional and nonlesional skin biopsy specimens demonstrated decreases in epidermal thickness and T-cell and dendritic cell expression in guselkumab-treated patients compared with values seen in placebo-treated patients. At week 12, significant reductions in psoriasis gene expression and serum IL-17A levels were observed in guselkumab-treated patients.

CONCLUSIONS

IL-23 inhibition with a single dose of guselkumab results in clinical responses in patients with moderate-to-severe psoriasis, suggesting that neutralization of IL-23 alone is a promising therapy for psoriasis.

The complement activation products C3a and C3a desArg are generated in the course of trauma, infection, tissue injury, and ischemia. We have investigated the effects of C3a and C3a desArg on gene expression and protein synthesis of TNF-alpha and <em>IL</em>-1 beta in PBMC. Neither C3a nor C3a desArg alone induced detectable protein or mRNA levels for TNF-alpha and <em>IL</em>-1 beta. C3a modulated LPS-induced TNF-alpha and <em>IL</em>-1 beta synthesis. In nonadherent PBMC, C3a suppressed LPS-induced synthesis of TNF-alpha (<em>20</em>-71% decrease by 0.2-10 microgram/ml of C3a, p less than 0.01) and <em>IL</em>-1 beta (19-57% decrease by 0.5-10 microgram/ml of C3a, p less than 0.01), independently of endogenous production of PGE2. C3a also suppressed LPS-induced mRNA levels for TNF-alpha and <em>IL</em>-1 beta. In contrast, in adherent PBMC, C3a at 5 to <em>20</em> microgram/ml enhanced LPS-induced TNF-alpha (75-188% increase, p less than 0.001) and <em>IL</em>-1 beta (119-274% increase, p less than 0.001) synthesis. C3a enhanced TNF-alpha and <em>IL</em>-1 beta mRNA levels in LPS-stimulated adherent cells. Furthermore, C3a desArg shared with C3a the ability to modulate LPS-induced mRNA and protein synthesis for TNF-alpha and <em>IL</em>-1 beta. These results suggest that C3a, thought to be proinflammatory, and C3a desArg, thought to be biologically inactive, are modulators of inflammation. Both C3a and C3a desArg may enhance cytokine synthesis by adherent monocytes at local inflammatory sites, while inhibiting the systemic synthesis of proinflammatory cytokines by circulating cells.

Cerebral microdialysis is a monitoring technique with expanding clinical and research utility following traumatic brain injury. This study's aim was to determine the relative recovery for 12 cytokines using both crystalloid (CNS perfusion fluid) and colloid (CNS perfusion fluid supplemented with 3.5% human serum albumin) perfusate. Six CMA71 microdialysis catheters (nominal molecular weight cut-off 100 kDa) were perfused in vitro with either crystalloid or colloid and the relative recovery (%) determined for the cytokines as follows (crystalloid/colloid perfusate): <em>IL</em>-1alpha (50.6/48), <em>IL</em>-1beta (34.6/38.4), <em>IL</em>-1ra (21.9/38.4), <em>IL</em>-2 (17.1/52.8), <em>IL</em>-4 (26/56.7), <em>IL</em>-6 (9.8/25.5), <em>IL</em>-8 (47.7/73.4), <em>IL</em>-10 (2.9/8.7), <em>IL</em>-17 (14.4/43.7), TNF-alpha (4.4/31.2), MIP-1alpha (31.8/55.6), and MIP-1beta (31.9/50.1). The colloid perfusate significantly improved relative recovery for nine of these cytokines ( p < 0.05), but not for <em>IL</em>-1alpha, <em>IL</em>-1beta, and <em>IL</em>-8. Relative recovery was related to apparent molecular weight of cytokine and to isoelectric point (pI), a surrogate marker of hydrophilicity. The mean fluid recovery for crystalloid and colloid perfusate was 92% and 145%, respectively. Scanning electron microscopy was utilized to investigate the ultrastructure of microdialysis membranes: (1) <em>20</em>-kDa membrane, (2) 100-kDa membrane, and (3) ex vivo 100-kDa membrane. The 100-kDa membranes possessed multiple large cavities and the catheter examined after use in human brain clearly demonstrated cellular debris within the pores of the membrane. While colloid perfusate improves relative recovery, it causes a net influx of fluid into the microdialysis catheter, potentially dehydrating the extracellular space. This study is the first to systematically determine relative recovery in vitro for a wide range of cytokines. The two forms of perfusion fluid require direct comparison in vivo.

OBJECTIVE

For clinical applications of biomarkers, there is a need for multiplex assays using high throughput platforms. The objective of this study was to determine the efficacy of Luminex Multianalyte Profiling (xMAP) technology for measurement of salivary proteins and to evaluate whether multiplex assays are as effective as single-plex assays and enzyme-linked immunosorbent assay (ELISA).

RESULTS

The average levels of interleukin-8 (<em>IL</em>-8) from the single-plex assay were 3313.2 +/- 3759.8 pg ml(-1) [oral squamous cell carcinoma (OSCC), n = <em>20</em>] and 1061.7 +/- 1978.8 pg ml(-1) (control, n = <em>20</em>). The <em>IL</em>-1beta average levels from the single-plex assay were 945.2 +/- 1134.8 pg ml(-1) (OSCC, n = <em>20</em>) and 314.2 +/- 444.8 pg ml(-1) (control, n = <em>20</em>). The average levels of <em>IL</em>-8 from the multiplex assay were 2834.9 +/- 3385.6 pg ml(-1) (OSCC, n = <em>20</em>) and 947.3 +/- <em>20</em>36.8 pg ml(-1) (control, n = <em>20</em>). The <em>IL</em>-1beta average levels from the multiplex assay were 1013.5 +/- 1221.1 pg ml(-1) (OSCC, n = <em>20</em>) and 376.3 +/- 576.3 pg ml(-1) (control, n = <em>20</em>). The correlation coefficient between Luminex and ELISA assay for <em>IL</em>-8 (n = 19) and <em>IL</em>-1beta (n = 19) was 0.91 and 0.84, respectively.

CONCLUSIONS

Luminex xMAP single-plex and multiplex assays are as effective as ELISA assays for quantification of proteins in saliva. Both IL-8 and IL-1beta were expressed at significantly higher levels in OSCC subjects than in the matched healthy control subjects.

The first U.S. nationwide food sampling with measurement of dioxins, dibenzofurans, and coplanar, mono-ortho and di-ortho polychlorinated biphenyls (PCBs) is reported in this study. Twelve separate analyses were conducted on 110 food samples divided into pooled lots by category. The samples were purchased in 1995 in supermarkets in Atlanta, GA, Binghamton, NY, Chicago, <em>IL</em>, Louisville, KY, and San Diego, CA. Human milk also was collected to estimate nursing infants' consumption. The food category with highest World Health Organization (WHO) dioxin toxic equivalent (TEQ) concentration was farm-grown freshwater fish fillet with 1.7 pg/g, or parts per trillion (ppt), wet, or whole, weight. The category with the lowest TEQ level was a simulated vegandiet, with 0.09 ppt. TEQ concentrations in ocean fish, beef, chicken, pork, sandwich meat, eggs, cheese, and ice cream, as well as human milk, were in the range O.33 to 0.51 ppt, wet weight. In whole dairy milk TEQ was 0.16 ppt, and in butter 1.1 ppt. Mean daily intake of TEQ for U.S. breast-fed infants during the first year of life was estimated at 42 pg/kg body weight. For children aged 1-11 yr the estimated daily TEQ intake was 6.2 pg/kg body weight. For males and females aged 12-19 yr, the estimated TEQ intake was 3.5 and 2.7 pg/kg body weight, respectively. For adult men and women aged <em>20</em>-79 yr, estimated mean daily TEQ intakes were 2.4 and 2.2 pg/kg body weight, respectively. Estimated mean daily intake of TEQ declined with age to a low of 1.9 pg/kg body weight at age 80 yr and older. For all ages except 80 yr and over, estimates were higher for males than females. For adults, dioxins, dibenzofurans, and PCBs contributed 42%, 30%, and 28% of dietary TEQ intake, respectively. DDE was also analyzed in the pooled food samples.

Interleukin-1 (<em>IL</em>-1), tumor necrosis factor (TNF), and interferon-gamma (IFN gamma) inhibit insulin release and may be cytotoxic to isolated rodent pancreatic islets. In this study we examined the effects of <em>IL</em>-1, TNF, and IFN gamma on the viability and hormone secretion of islets isolated from adult human pancreas and maintained in monolayer culture. <em>IL</em>-1 and TNF were cytotoxic to the islet cells (<em>20</em>-30% cell lysis) in a 51Cr release cytotoxicity assay, and IFN gamma had only small effects (less than 10% lysis). Combination of maximally cytotoxic concentrations of <em>IL</em>-1 (10 U/mL) and TNF (10(3) U/mL) produced an additive cytotoxic effect. IFN gamma (10(3) U/mL) acted synergistically with <em>IL</em>-1 and TNF, and the three cytokines added together produced maximal islet cell lysis (46.4 +/- 4.3%). Assay of insulin and glucagon in the islet monolayers revealed that <em>IL</em>-1, TNF, and IFN gamma inhibited both B- and A-cell secretory functions; however, only <em>IL</em>-1 and TNF produced permanent decreases in insulin and glucagon contents in the islet cultures. These findings indicate that <em>IL</em>-1 and TNF, as single agents, are cytotoxic to human islet cells, and that this cytotoxicity can be amplified by combining the cytokines and/or adding IFN gamma. However, the lack of specificity for B-cells in vitro suggests that additional factors might be operative in vivo for the cytokine products of macrophages and lymphocytes infiltrating islets to produce the B-cell-specific damage characteristic of type 1 diabetes.

OBJECTIVE

Butyrate, produced by colonic fermentation of dietary fibers is often hypothesized to beneficially affect colonic health. This study aims to assess the effects of butyrate on inflammation and oxidative stress in subjects with chronically mildly elevated parameters of inflammation and oxidative stress.

METHODS

Thirty-five patients with ulcerative colitis in clinical remission daily administered 60 ml rectal enemas containing 100mM sodium butyrate (n=17) or saline (n=18) during <em>20</em> days (NCT00696098). Before and after the intervention feces, blood and colonic mucosal biopsies were obtained. Parameters of antioxidant defense and oxidative damage, myeloperoxidase, several cytokines, fecal calprotectin and CRP were determined.

RESULTS

Butyrate enemas induced minor effects on colonic inflammation and oxidative stress. Only a significant increase of the colonic IL-10/IL-12 ratio was found within butyrate-treated patients (p=0.02), and colonic concentrations of CCL5 were increased after butyrate compared to placebo treatment (p=0.03). Although in general butyrate did not affect colonic glutathione levels, the effects of butyrate enemas on total colonic glutathione appeared to be dependent on the level of inflammation.

CONCLUSIONS

Although UC patients in remission were characterized by low-grade oxidative stress and inflammation, rectal butyrate enemas showed only minor effects on inflammatory and oxidative stress parameters.

BACKGROUND

Hyperchloremic acidosis is common in the critically ill and is often iatrogenic. We have previously shown that hyperchloremic acidosis increases nuclear factor-kappaB DNA binding in lipopolysaccharide-stimulated RAW 264.7 cells. However, evidence that hyperchloremic acidosis leads to increased inflammation in vivo has been limited to nitric oxide.

OBJECTIVE

To determine if acidosis, induced by dilute hydrochloric acid (HCl) infusion, will increase circulating inflammatory mediator levels in an experimental model of severe sepsis in rats.

METHODS

Eighteen hours after inducing lethal sepsis by cecal ligation and puncture in <em>20</em> adult, male, Sprague-Dawley rats, we randomized animals into three groups. In groups 2 and 3, we began an IV infusion of 0.1 N HCl to reduce the standard base excess (SBE) by 5 to 10 mEq/L and 10 to 15 mEq/L, respectively. In group 1, we infused a similar volume of lactated Ringer solution. In all groups infusion continued 8 h or until the animal died.

RESULTS

We measured arterial blood gases, whole-blood lactate, and chloride, tumor necrosis factor (TNF), interleukin (IL)-6, and IL-10 levels at 0 h, 4 h, and 8 h. All measured cytokines increased over time. Compared to group 1, animals in groups 2 and 3 exhibited greater increase in all three cytokines, with the greatest increases seen with severe acidosis.

CONCLUSIONS

Moderate (SBE, - 5 to - 10) and severe (SBE, - 10 to - 15) acidosis, induced by HCl infusion, increases circulating levels of IL-6, IL-10, and TNF in normotensive septic rats.

MicroRNAs (miRNAs) are important posttranscriptional regulators in immune cells, but how viral infection regulates miRNA expression to shape dendritic cell (DC) responses has not been well characterized. We identified <em>20</em> miRNAs that were differentially expressed in primary murine DCs in response to the dsRNA agonist polyinosinic-polycytidylic acid, a subset of which were modestly regulated by influenza infection. miR-451 was unique because it was induced more strongly in primary splenic and lung DCs by live viral infection than by purified agonists of pattern recognition receptors. We determined that miR-451 regulates a subset of proinflammatory cytokine responses. Three types of primary DCs treated with antisense RNA antagomirs directed against miR-451 secreted elevated levels of <em>IL</em>-6, TNF, CCL5/RANTES, and CCL3/MIP1α, and these results were confirmed using miR-451(null) cells. miR-451 negatively regulates YWHAZ/14-3-3ζ protein levels in various cell types, and we measured a similar inhibition of YWHAZ levels in DCs. It is known that YWHAZ can control the activity of two negative regulators of cytokine production: FOXO3, which is an inhibitory transcription factor, and ZFP36/Tristetraprolin, which binds to AU-rich elements within 3'-untranslated regions to destabilize cytokine mRNAs. Inhibition of miR-451 expression correlated with increased YWHAZ protein expression and decreased ZFP36 expression, providing a possible mechanism for the elevated secretion of <em>IL</em>-6, TNF, CCL5/RANTES, and CCL3/MIP1α. miR-451 levels are themselves increased by <em>IL</em>-6 and type I IFN, potentially forming a regulatory loop. These data suggest that viral infection specifically induces a miRNA that directs a negative regulatory cascade to tune DC cytokine production.

OBJECTIVE

To measure the concentration of cytokines in the aqueous humour of eyes with exudative age-related macular degeneration (AMD).

METHODS

The clinical interventional study included a study group of 18 patients with exudative AMD and a control group of <em>20</em> patients undergoing routine cataract surgery. Age did not vary significantly (p = 0.36) between study group (80.8 ± 6.4 years) and control group (77.0 ± 9.9 years), nor did gender (p = 0.75). During the interventions, aqueous humour samples were obtained, in which the concentration of cytokines was measured using a solid-phase chemiluminescence immunoassay. Macular thickness was measured by optical coherence tomography (OCT).

RESULTS

In the study group as compared to the control group, significantly higher concentrations were measured for epithelial growth factor (EGF) (p = 0.017), human growth factor (HGF) (p= 0.048), intercellular adhesion molecule-1 (ICAM1) (p = 0.028), interleukin 12p40 (IL12p40) (p = 0.009), interleukin 1a2 (IL1a2) (p = 0.01), interleukin 3 (IL3) (p = 0.02), interleukin 6 (IL6) (p = 0.006), interleukin 8 (IL8) (p = 0.02), monocyte chemoattractant protein-1 (MCP-1) (p = 0.048), monokine induced by interferon gamma (MIG) (p = 0.016), matrix metalloproteinase 9 (MMP9) (p = 0.004) and plasminogen activator inhibitor 1 (PAI1) (p = 0.006). Macular thickness was significantly associated with the concentrations of EGF (p = 0.001), HGF (p = 0.02), ICAM1 (p = 0.001), interleukin 12p40 (p = 0.006), IL 1a2 (p = 0.002), MIG (p = 0.001), MMP9 (p < 0.001) and PAI1 (p = 0.01). Interleukin 6 and MCP-1 showed significant associations with the height of retinal pigment epithelium detachment.

CONCLUSIONS

Numerous cytokines are associated with the presence and the amount of exudative AMD.

Interleukin (<em>IL</em>)-6 is a potent activator of the human hypothalamicpituitary-adrenal axis. After chronic administration of <em>IL</em>-6 in humans, there is a substantial elevation of cortisol, whereas ACTH levels are blunted. Thus, we investigated whether <em>IL</em>-6 and/or the <em>IL</em>-6 receptor (<em>IL</em>-6R) are expressed in the human adrenal gland and whether <em>IL</em>-6 could cause the release of steroid hormones by a direct action on adrenal cells in primary culture. The expression of <em>IL</em>-6 and <em>IL</em>-6R was investigated with RT-PCR and immunohistochemistry, and the effects on human adrenal steroidogenesis were tested with <em>IL</em>-6 in vitro. To avoid effects mediated by macrophages, we depleted adrenal primary cultures from macrophages using specific mouse antihuman CD68 and sheep antimouse IgG conjugated magnetic beads. The results showed that 1): <em>IL</em>-6 and <em>IL</em>-6R are expressed in adrenal cell cultures, including all cell types and those depleted of macrophages; 2) <em>IL</em>-6R is mainly expressed in the zona reticularis and the inner zona fasciculata; positive signals from the zona glomerulosa and the medulla occurred in single cells; and 3) <em>IL</em>-6 regulates adrenal synthesis of mineralocorticoids, glucocorticoids, and androgens in vitro, dependent on time and dose, in the absence of macrophages. After 24 h, aldosterone secretion increased to 172 +/- 28% SEM, cortisol to 177 +/- 27% SEM, and dehydroepiandrosterone to 153 +/- <em>20</em>% SEM of basal secretion. These findings, in combination with previous investigations, suggest that <em>IL</em>-6 exerts its acute action via the hypothalamus and the pituitary. In the adrenal gland, however, <em>IL</em>-6 seems to be a long-term regulator of stress response, integrating the responses of all cortical zones to stimuli from the immune and endocrine system.

Background: COVID-19 patients develop pneumonia generally associated to lymphopenia and severe inflammatory response due to uncontrolled cytokine release. These mediators are transcriptionally regulated by the JAK-STAT signaling pathways, which can be disabled by small molecules.

<strong class="sub-title"> Methods: </strong> A group of subjects (n = <em>20</em>) was treated with baricitinib according to an off-label use of the drug. The study was designed as an observational longitudinal trial and approved by the local ethical committee. The patients were treated with baricitinib 4 mg twice daily for 2 days, followed by 4 mg per day for the remaining 7 days. Changes in the immune phenotype and expression of pSTAT3 in blood cells were evaluated and correlated with serum-derived cytokine levels and antibodies anti-SARS-CoV-2. In a single treated patient, we evaluated also the alteration of myeloid cell functional activity.

Results: We provided evidences that baricitinib-treated patients have a marked reduction in serum levels of interleukin (IL)-6, IL-1β and tumor necrosis factor (TNF)-α, a rapid recovery in circulating T and B cell frequencies, and increased antibody production against SARS-CoV-2 spike protein, which were clinically associated with a reduction in oxygen flow need and progressive increase in the P/F.

Conclusion: Baricitinib prevented the progression towards a severe/extreme form of the viral disease by modulating the patients' immune landscape and these changes were associated with a safer and favorable clinical outcome of patients with COVID-19 pneumonia.

Trial registration: The ClinicalTrials.gov identifier of this project is protocol NCT04438629.

Funding: This work was supported by Fondazione Cariverona (ENACT Project) and Fondazione TIM.

Keywords: COVID-19; Immunology; Innate immunity.

OBJECTIVE

To determine whether inflammatory corneal neovascularization (CNV) is associated with interleukin-1 (IL-1) activity and if so, to assess the efficacy of topical interleukin-1 receptor antagonist (IL-1ra) to suppress CNV.

METHODS

Inflammatory CNV was induced on day 0 by placement of paracentral intrastromal sutures in BALB/c murine eyes. Quantification of IL-1alpha and -beta cytokine levels was done by a sandwich enzyme-linked immunosorbent assay (ELISA) on the supernatants of incubated corneas excised at specified time points after induction of CNV (n = 6 per time point studied). To study suppression of CNV by IL-1ra, animals were divided into treatment subgroups that received topical 20 mg/ml of IL-1ra mixed in 0.2% sodium hyaluronate (n = 28) or placebo (vehicle) alone (n = 22) 3 times daily during days 0-35. Other groups of animals received placebo for 1 (n = 10) or 2 (n = 14) weeks before being switched and retained on IL-1ra. Neovascularization was assessed biomicroscopically and graded by using a standardized scheme.

RESULTS

Induction of CNV stimulus was associated with a significant surge in the expression of both IL-1alpha (p < 0.001) and IL-1beta (p < 0.001) as early as 2 h after the stimulus, which peaked at 24 h, before decreasing substantially in the case of IL-1beta and returning to basal levels by day 7. Topical application of IL-1ra led to a significant suppression of CNV for the duration of therapy only if initiated early after induction of the neovascular stimulus. Initiation of therapy 1 week after CNV induction was associated only with a transient suppression in the angiogenic response.

CONCLUSIONS

Our data strongly implicate IL-1 as a critical mediator in the early phase of CNV and suggest that IL-1ra can be an effective modality in suppressing CNV if initiated sufficiently early after the inflammatory neovascular stimulus.

Systemic lupus erythematosus (SLE) is characterized by the production of pathogenic autoantibodies to nucleoprotein antigens, including double-stranded DNA (dsDNA). The deposition of IgG dsDNA immune complexes in glomeruli is thought to be crucial for disease pathogenesis and complement activation. rhDNase catalyzes the hydrolysis of extracellular DNA and has been shown to delay the development of dsDNA antibodies, reduce proteinuria, and delay mortality in a lupus-prone murine model. We conducted a 40d, phase Ib, randomized, double-masked, placebo-controlled trial to determine the safety and pharmacokinetics of rhDNase, and to measure any changes in markers of disease activity in 17 patients with lupus nephritis. Patients were assigned to receive either: (1) 25 microg/kg rhDNase (n = 8); (2) 125 microg/kg rhDNase (n=6); or (3) placebo (n = 3) initial single intravenous (IV) dose followed by 10 subcutaneous (SC) doses. Skin biopsies performed on nine patients pre- and post-treatment were studied for immune complex deposition by immunofluorescence. Serum cytokine levels (s<em>IL</em>2-R, <em>IL</em>-6, <em>IL</em>-10, and TNF-alpha) were analyzed by ELISA. Cytokine secretion and antibody production were measured by ELISPOT analysis and ELISA. Serum hydrolytic activity of rhDNase was achieved after IV administration at 25 and 125 microg/kg, but not after SC administration at either dose. A t 1/2 of 3-4h was estimated from serum concentration profiles following IV administration. Serum dsDNA antibodies were unchanged (mean values: 33 IU/mL vs 39 IU/mL [pre- and post-treatment] for the 25 microg/kg group, and 74 IU/mL vs 74 IU/mL for the 125 microg/kg group, and 14 IU/mL vs <em>20</em> IU/mL for the placebo group). Complement levels (C3 and C4) and circulating immune complexes did not change appreciably during the treatment period for any of the groups. Serum cytokine profiles by ELISA revealed no changes in s<em>IL</em>-2 receptor, <em>IL</em>-6, <em>IL</em>-10, or TNF-alpha. There was no change in the number of cells secreting either Th1 or Th2 specific cytokines, nor in the number of cells secreting dsDNA antibodies. Neutralizing antibodies to rhDNase were not detected in serum at any time during the study. Immune complex deposition was unchanged in pre- and post-treatment in skin biopsies in both dose groups. rhDnase was well tolerated without significant adverse events following administration, and treatment was not associated with the development of neutralizing antibodies to rhDNase. Serum rhDNase concentrations capable of hydrolytic activity of rhDNase were achieved for a few hours following IV, but not SC administration. Serum markers of disease activity were unchanged during the study period.

BACKGROUND

Elevated plasma levels of interleukin (IL)-6, C-reactive protein (CRP), and D-dimer belong to the biological alterations of the "frailty syndrome," defining increased vulnerability for diseases and mortality with aging. We hypothesized that, compatible with premature frailty, chronic stress and age are related in predicting inflammation and coagulation activity in Alzheimer caregivers.

METHODS

Plasma IL-6, CRP, and D-dimer levels were measured in 170 individuals (mean age 73 +/- 9 years; 116 caregivers, 54 noncaregiving controls). Demographic factors, diseases, drugs, and lifestyle variables potentially affecting inflammation and coagulation were obtained by history and adjusted for as covariates in statistical analyses.

RESULTS

Caregivers had higher mean levels of IL-6 (1.38 +/- 1.42 vs 1.00 +/- 0.92 pg/mL, p =.032) and of D-dimer (723 +/- 530 vs 471 +/- 211 ng/mL, p <.001) than controls had. CRP levels were similar between groups (p =.44). The relationship between caregiver status and D-dimer was independent of covariates (p =.037) but affected by role overload. Age accounted for much of the relationship with IL-6. After controlling for covariates, the interaction between caregiver status and age was significant for D-dimer (beta =.20, p =.029) and of borderline significance for IL-6 (beta =.17, p =.090). Post hoc regression analyses indicated that, among caregivers, age was significantly correlated with both D-dimer (beta =.50, p <.001) and IL-6 (beta =.38, p =.001). Among controls, however, no significant relationship was observed between age and either D-dimer or IL-6.

CONCLUSIONS

The interaction between caregiving status and age for D-dimer and IL-6 suggests the possibility that older caregivers could be at risk of a more rapid transition to the frailty syndrome and clinical manifestations of cardiovascular diseases.

We have used an efficient cDNA subtraction library procedure to identify newly induced genes in human B lymphocytes infected for 6 h with Epstein-Barr virus (EBV). Among the genes identified by automated sequencing of a random subset of clones from this library, one coded the EBV BCRF1 open reading frame, which specifies the viral interleukin 10 gene (v<em>IL</em>-10). This molecule is highly homologous to human (h)<em>IL</em>-10 and was previously thought to represent a "late" viral gene expressed only during the lytic phase of virus replication. Using gene amplification by reverse transcriptase polymerase chain reaction of B cell RNA obtained at varying times after infection, we detected v<em>IL</em>-10 expression within a few hours of EBV infection, followed, <em>20</em>-30 h later by expression of h<em>IL</em>-10. Expression of both genes continued beyond the initial transformation phase (5-10 d) and was present in all transformed cell lines tested. When added at the time of viral infection, antisense (but not sense) oligonucleotides for v<em>IL</em>-10 mRNA (cytosolic half-life, approximately 6 h) prevented subsequent B cell transformation. The antisense effect was highly specific, leaving the expression levels of other transformation-related genes intact. Addition of exogenous (h)<em>IL</em>-10 rescued the transformation process in antisense-treated cells. Our observations establish v<em>IL</em>-10 as a new latency gene with a directly transformation-prerequisite function.

mAbs previously reported to be specific for <em>IL</em>-8R type A (<em>IL</em>-8R-A) and mAbs specific for <em>IL</em>-8R type B (<em>IL</em>-8R-B), which are described in this paper, were used to investigate the expression of each receptor on various types of cells. We generated mAbs specific for <em>IL</em>-8R-B, 4D1, and 10H2 by immunizing mice with 293 cells that expressed <em>IL</em>-8R-B and by selecting hybridoma cell lines that secreted mAbs that bind to human neutrophils. Flow cytometry showed that mAbs 4D1 and 10H2 were specific for <em>IL</em>-8R-B, as determined by their exclusive binding to 293-27 cells that expressed <em>IL</em>-8R-B, but not to 293-71 cells that expressed <em>IL</em>-8R-A. Epitopes recognized by these <em>IL</em>-8R-B-specific mAbs were shown to be within the N-terminal residues 1-18 of the <em>IL</em>-8R-B on the basis of their binding to various N-terminal peptides, as measured by ELISA. These <em>IL</em>-8R-B-specific mAbs were able to inhibit up to 90 and 50% of the 125I-labeled <em>IL</em>-8 binding to 293-27 cells and human neutrophils, respectively. The combination of mAb 9H1 (anti-<em>IL</em>-8R-A) and mAb 10H2 (anti-<em>IL</em>-8R-B) inhibited approximately 70% of 125I-labeled <em>IL</em>-8 binding to human neutrophils. Flow cytometry showed a wide range of donor variation in the expression levels of <em>IL</em>-8R-A and <em>IL</em>-8R-B on various human peripheral blood leukocytes. All neutrophils, all monocytes, and 5 to 25% of total lymphocytes (CD8+ T cells and CD56+ NK cells) expressed <em>IL</em>-8R. Neutrophils expressed the highest level of both <em>IL</em>-8R-A and <em>IL</em>-8R-B, at an approximately equal ratio, whereas monocytes and <em>IL</em>-8R+ lymphocytes expressed higher levels of <em>IL</em>-8R-B than <em>IL</em>-8R-A. Double-color flow cytometric analysis showed that 7 to 42% of CD8+ T cells and 39 to 76% of CD56+ NK cells, but no CD <em>20</em>+ B cells or CD4+ T cells, expressed <em>IL</em>-8R.

Neutrophil-activating peptide 1/interleukin 8 (NAP-1/<em>IL</em>-8), neutrophil-activating peptide 2 (NAP-2), and gro/melanoma growth-stimulatory activity (gro/MGSA) are potent inflammatory cytokines with homologous structure and similar neutrophil-activating properties. Receptors on human neutrophils that interact with these peptides were studied. Analysis of 125I-NAP-1/<em>IL</em>-8 binding at 0-4 degrees C revealed 64,500 +/- 14,000 receptors/cell with an apparent Kd of 0.18 +/- 0.07 nM (mean +/- S.D. of six independent experiments). Unlabeled NAP-1/<em>IL</em>-8, NAP-2, and gro/MGSA competed with 125I-NAP-1/<em>IL</em>-8 for binding to human neutrophils. Competition with increasing concentrations of unlabeled NAP-2 and gro/MGSA resolved two classes of NAP-1/<em>IL</em>-8 binding sites: about 70% of them bound NAP-2 and gro/MGSA with high affinity (Kd: 0.34 +/- 0.2 and 0.14 +/- 0.02), while 30% were of low affinity (Kd: 100 +/- <em>20</em> and 130 +/- 10 nM). Different binding sites, however, were not apparent upon competition with unlabeled NAP-1/<em>IL</em>-8, suggesting that both classes of receptors have similar affinities for NAP-1/<em>IL</em>-8. The existence of two receptors was also suggested by ligand cross-linking and cross-desensitization experiments. Two neutrophil membrane proteins with apparent Mr of 66,000-74,000 and 42,000-46,000 became cross-linked to 125I-NAP-1/<em>IL</em>-8, and the labeling was decreased when excess NAP-1/<em>IL</em>-8, NAP-2, or gro/MGSA was present. Stimulation of neutrophils with NAP-1/<em>IL</em>-8 resulted in desensitization toward a subsequent challenge with NAP-2 or gro/MGSA as shown by the rise in cytosolic free calcium. By contrast, following primary stimulation with NAP-2 or gro/MGSA, responses to NAP-1/<em>IL</em>-8 were only moderately attenuated, supporting the existence of NAP-1/<em>IL</em>-8 receptors which bind NAP-2 or gro/MGSA with low affinity. In conclusion, our results demonstrate that NAP-2 and gro/MGSA act upon human neutrophils by directly interacting with two classes of receptors for NAP-1/<em>IL</em>-8.

Rituximab is a chimeric murine/human monoclonal antibody that binds to CD<em>20</em> on B lymphocytes. Although binding of the Fab domain may induce apoptosis, the Fc domain recruits immune effector functions to mediate cell lysis. Interleukin-12 (<em>IL</em>-12) facilitates cytolytic T-cell responses, enhances the lytic activity of natural killer (NK) cells, and induces the secretion of interferon gamma (IFN-gamma) by both T and NK cells. Therefore, the hypothesis was considered that combining <em>IL</em>-12 with rituximab would augment the immune-mediated cell lysis induced by rituximab. A phase 1 study of <em>IL</em>-12 in combination with rituximab was conducted in 43 adults with B-cell lymphoma to determine the optimal immunologic dose of this combination. Rituximab was administered at a dose of 375 mg/m(2) by intravenous infusion weekly for 4 weeks, and <em>IL</em>-12 was given subcutaneously twice weekly. The starting dose of <em>IL</em>-12 was 30 ng/kg and this was escalated to 500 ng/kg. Constitutional symptoms and liver enzyme elevations at 500 ng/kg of <em>IL</em>-12 were dose limiting. A greater than <em>20</em>-fold increase in the serum levels of IFN-gamma and a 2.5- to 5-fold increase in inducible protein 10 (IP-10) levels was seen at <em>IL</em>-12 doses of 100 ng/kg or greater. Objective responses occurred in 29 of the 43 patients (69%), with 8 of 11 complete responses seen at <em>IL</em>-12 doses of 300 ng/kg or greater. The optimal immunologic dose of <em>IL</em>-12 in combination with rituximab was determined to be 300 ng/kg subcutaneously twice weekly starting on day 2. These data suggest that <em>IL</em>-12 and rituximab is an active combination and further studies of this combination in B-cell non-Hodgkin lymphoma are warranted.

OBJECTIVE

Most mechanistic studies of pancreatitis in mice employ the secretagogue-induced model. The currently reported studies were designed to develop an alternative, and possibly more clinically relevant, mouse model of pancreatitis.

METHODS

Na-taurocholate (10-50 microl, 1-5%) in saline, or saline alone, was retrogradely infused into the mouse pancreatic duct. The animals were killed 6-24 hours later and the severity of pancreatitis in the pancreatic head and tail was examined by quantitating hyperamylasemia, pancreatic edema, acinar cell necrosis, and pancreatic inflammation. In addition, intrapancreatic activation of trypsinogen, generation of IL-6, intrapulmonary sequestration of neutrophils, and alterations in lung compliance were evaluated. The effects of Na-taurocholate on in-vitro acinar cell calcium transients, viability, and trypsinogen activation were examined.

RESULTS

Little or no evidence of pancreatitis was observed in mice infused with saline alone or in the tail of pancreata removed from animals infused with Na-taurocholate. In the head of the pancreas, evidence of pancreatitis was observed 12-24 hours after infusion of 20-50 microl 2-5% Na-taurocholate and the earliest morphological changes involved terminal duct and acinar cells. Intrapancreatic trypsin activity was transiently elevated within 5 minutes of Na-taurocholate infusion and pancreatic IL-6 levels were elevated 24 hours later. Under in-vitro conditions, Na-taurocholate triggered pathological acinar cell calcium transients, cell death, and calcium-dependent trypsinogen activation.

CONCLUSIONS

This clinically relevant model of acute biliary pancreatitis yields reproducible results and its severity can be easily manipulated. It is ideally suited for use in mechanistic studies employing genetically modified mouse strains.

A major goal of experimental and clinical hematology is the identification of mechanisms and conditions that support the expansion of transplantable hematopoietic stem cells. In normal marrow, such cells appear to be identical to (or represent a subset of) a population referred to as long-term-culture-initiating cells (LTC-ICs) so-named because of their ability to produce colony-forming cell (CFC) progeny for>> or = 5 weeks when cocultured with stromal fibroblasts. Some expansion of LTC-ICs in vitro has recently been described, but identification of the factors required and whether LTC-IC self-renewal divisions are involved have remained unresolved issues. To address these issues, we examined the maintenance and/or generation of LTC-ICs from single CD34+ CD38- cells cultured for variable periods under different culture conditions. Analysis of the progeny obtained from cultures containing a feeder layer of murine fibroblasts engineered to produce steel factor, interleukin (<em>IL</em>)-3, and granulocyte colony-stimulating factor showed that approximately <em>20</em>% of the input LTC-ICs (representing approximately 2% of the original CD34+ CD38- cells) executed self-renewal divisions within a 6-week period. Incubation of the same CD34+ CD38- starting populations as single cells in a defined (serum free) liquid medium supplemented with Flt-3 ligand, steel factor, <em>IL</em>-3, <em>IL</em>-6, granulocyte colony-stimulating factor, and nerve growth factor resulted in the proliferation of initial cells to produce clones of from 4 to 1000 cells within 10 days, approximately 40% of which included>> or = 1 LTC-IC. In contrast, in similar cultures containing methylcellulose, input LTC-ICs appeared to persist but not divide. Overall the LTC-IC expansion in the liquid cultures was 30-fold in the first 10 days and 50-fold by the end of another 1-3 weeks. Documentation of human LTC-IC self-renewal in vitro and identification of defined conditions that permit their extensive and rapid amplification should facilitate analysis of the molecular mechanisms underlying these processes and their exploitation for a variety of therapeutic applications.

Unmethylated CpG dinucleotides (CpG motif) in bacterial DNA or synthetic oligodeoxynucleotides (CpG DNA) rapidly activate murine B cells to secrete <em>IL</em>-6 and IgM, as well as to proliferate. Within 30 min after CpG DNA stimulation in vivo, <em>IL</em>-6 mRNA levels were increased in liver, spleen, and thymus cells. Serum <em>IL</em>-6 protein was markedly increased within 1 h of stimulation. Treatment of a B cell line with CpG DNA led to an increase in the transcriptional activity of the <em>IL</em>-6 promoter. This CpG DNA-induced <em>IL</em>-6 production was not mediated via either a protein kinase C (PKC)-, protein kinase A (PKA)-, or nitric oxide (NO.)-dependent pathway but was inhibited by an antioxidant. In addition, the level of intracellular reactive oxygen species was increased within <em>20</em> min after CpG DNA, but not control non-CpG DNA, treatment. These results suggest that CpG DNA-induced <em>IL</em>-6 production is mediated through a reactive oxygen intermediate-dependent pathway. CpG DNA-mediated <em>IL</em>-6 production was enhanced by simultaneous signals delivered through the Ag receptor. The addition of neutralizing Abs against <em>IL</em>-6 to B cell cultures along with CpG oligodeoxynucleotides essentially abolished the CpG DNA-induced increased IgM secretion but had no significant effect on the B cell proliferation induced by the CpG motif. Our results suggest that the induction of <em>IL</em>-6 expression in response to CpG motifs in bacterial DNA may be an important immune defense mechanism that facilitates a rapid response to microbial infection.

<em>IL</em>-6/IFN-beta 2 is a family of phosphoglycoproteins ranging in size from 19 to 30 kDa which elicits a broad range of physiologic and immune responses. Several cytokines, including TNF, have been shown to stimulate <em>IL</em>-6 production in cell culture. In this report, we describe the rapid induction of circulating biologically active <em>IL</em>-6 by the systemic administration of rTNF to patients with cancer. Low levels of <em>IL</em>-6 activity could be detected in the sera of patients as early as 5 min after rTNF infusion. <em>IL</em>-6 levels peaked approximately 2 to 3 h after rTNF bolus administration and were undetectable in most cases within 8 h. <em>IL</em>-6 was detected in two separate bioassays--the hybridoma B9 proliferation and the hepatocyte-stimulating factor assay. Maximum detectable levels of <em>IL</em>-6 ranged from 160 to 310 hybridoma growth factor units and 11-82 ng/ml in the hepatocyte-stimulating factor assay. <em>IL</em>-6 induction decreased after serial, daily doses of rTNF. Serial serum samples of patients receiving <em>IL</em>-2 or IFN-alpha were also assayed for <em>IL</em>-6 production. <em>IL</em>-2-treated but not IFN-alpha-treated patients generated low levels of <em>IL</em>-6 (range less than <em>20</em> to 95 hybridoma growth factor units/ml). Interestingly, in patients treated with <em>IL</em>-2, serum levels of TNF were detectable and peak TNF activity preceded measurable <em>IL</em>-6 levels. Serum levels of acute phase plasma proteins and of corticosteroid rose in response to rTNF administration. C-reactive protein increased (2.5 to 4.0-fold) within 8 h of rTNF administration and cortisol levels rose (10- to <em>20</em>-fold) within 4 h after rTNF injection. We conclude that rTNF administration in man leads to the induction of circulating <em>IL</em>-6 which, due to its broad range of activities, may be an important physiologic signal regulating the immune response.

<em>IL</em>-10 is a monocyte/lymphocyte derived cytokine which has been shown to inhibit certain cellular immune responses such as delayed hypersensitivity. In particular, the production of tumour necrosis factor (TNF), <em>IL</em>-1 and <em>IL</em>-6, which are involved in malaria pathology, are strongly inhibited by <em>IL</em>-10. Accordingly, we examined whether <em>IL</em>-10 could be involved in a human acute parasitic infection such as Plasmodium falciparum malaria. Human <em>IL</em>-10 levels in plasma were determined by two-site ELISA method, taking care to avoid non-specific reactions due to autoantibodies. Fourteen cerebral, 11 severe, and <em>20</em> mild malaria cases had mean <em>IL</em>-10 levels of 2812, 2882 and 913 pg/ml, respectively, while 98% of healthy individuals had undetectable (less than 100 pg/ml) circulating <em>IL</em>-10. Thirteen of the 25 cerebral/severe cases had>> <em>20</em>00 pg/ml. In 11 hospitalized patients, circulating <em>IL</em>-10 levels were found to return to virtually normal levels 7 days after antimalarial chemotherapy when biological and clinical malaria features had disappeared (mean levels fell from 3880 to 333 pg/ml). Further studies are required to determine whether these elevated levels of <em>IL</em>-10 play a beneficial role by reducing the parasite-induced inflammatory response, or a detrimental one by decreasing the cellular immune responses.