The bioactivity of <em>IL</em>-12 is mediated by heterodimers of disulfide-linked p35 and p40 protein subunits. Homodimeric p40 competes with heterodimer for binding to the high affinity <em>IL</em>-12R and inhibits <em>IL</em>-12 bioactivity in vitro. However, the production and significance of p40 homodimer as a cytokine antagonist in vivo have not been determined. In these studies, we observed increased amounts of both <em>IL</em>-12 p40 monomer and homodimer in the serum of C57BL/6 mice following injection of 300 microg of Salmonella enteritidis LPS. Homodimer constituted between <em>20</em> and 40% of the total circulating p40 in endotoxemic sera, as confirmed by both Sephacryl S-100 gel filtration and p40-specific immunoprecipitation analyses. Similar relative amounts of homodimer and monomer were observed in endotoxemic BALB/c, C57BL/6, IFN-gamma-deficient C57BL/6 mice and C57BL/6 mice previously infected with bacille Calmette-Guérin. To determine whether <em>IL</em>-12 p40 homodimer was capable of antagonizing <em>IL</em>-12-dependent IFN-gamma responses in vivo, we pretreated C57BL/6 mice with purified r<em>IL</em>-12 p40 homodimer before i.p. challenge with endotoxin. Mice treated with 40 to 80 microg of p40 homodimer generated 80 to 82% less circulating IFN-gamma during acute endotoxemia than saline controls (p < 0.01). We conclude that p40 homodimer is produced in vivo, functions as a cytokine antagonist in the context of the mouse model of acute endotoxemia, and may represent a novel form of self-regulating cytokine response.

Tumor necrosis factor/cachectin (TNF/C) is the principal mediator of bacterial endotoxin-induced shock and death. We found that the C3H/HeJ mouse, which is less able to produce TNF/C in response to endotoxin, has a 1,000-fold greater susceptibility to lethal infection with Escherichia coli than the TNF-responsive congenic mouse, C3H/HeN. This surprising finding suggested that this lethal peptide may also be involved in host protection. To test this hypothesis we pretreated the C3H/HeJ mouse with a combination of recombinant murine TNF/C-alpha and <em>IL</em>-1 alpha. This combination protected these mice against an intraperitoneal bacterial challenge of greater than <em>20</em> LD50S (nearly 2 x 10(2) CFU) that grew to a level of greater than 10(7) CFU/ml of blood and per gram of liver in untreated mice. This suggests a significant role for these cytokines in host defenses against invasive infections that require bacterial replication within the host. These protective mechanisms may not be important for less virulent organisms. These findings may have important implications for the proposed use of anti-TNF/C agents in the treatment of septic shock.

Stromal cell-derived factor-1 (SDF-1) provides a potent chemotactic stimulus for CD34(+) hematopoietic cells. We cultured mobilized peripheral blood (PB) and umbilical cord blood (CB) for up to 5 weeks and examined the migratory activity of cobblestone area-forming cells (CAFCs) and long-term culture-initiating cells (LTC-ICs) in a transwell assay. In this system, SDF-1 or MS-5 marrow stromal cells placed in the lower chamber induced transmembrane and transendothelial migration by 2- and 5-week-old CAFCs and LTC-ICs in 3 hours. Transmigration was blocked by preincubation of input CD34(+) cells with antibody to CXCR4. Transendothelial migration of CB CAFCs and LTC-ICs was higher than that of PB. We expanded CD34(+) cells from CB in serum-free medium with thrombopoietin, flk-2 ligand, and c-kit ligand, with or without <em>IL</em>-3 and found that CAFCs cultured in the absence of <em>IL</em>-3 had a chemotactic response equivalent to noncultured cells, even after 5 weeks. However, addition of <em>IL</em>-3 to the culture reduced this response by <em>20</em>-50%. These data indicate that SDF-1 induces chemotaxis of primitive hematopoietic cells signaling through CXCR4 and that the chemoattraction could be downmodulated by culture ex vivo.

Kawasaki syndrome (KS) is an acute febrile illness of early childhood characterized by diffuse vasculitis and marked immune activation. The present study was undertaken to determine whether the acute phase of KS is associated with circulating cytotoxic antibodies directed to target antigens induced on vascular endothelium by the monokines, <em>IL</em>-1, or tumor necrosis factor (TNF). Sera from <em>20</em> patients with acute KS, 11 patients in the convalescent phase of KS, and 17 age-matched controls were assessed for complement-dependent cytotoxic activity against 111In-labeled human endothelial cells (HEC), dermal fibroblasts, and vascular smooth muscle cells. Sera from patients with acute KS but not the other subject groups caused significant (p less than 0.01) complement-mediated killing of <em>IL</em>-1- or TNF-stimulated HEC. None of the sera tested had cytotoxicity against control HEC cultures or the other target cell types, with or without <em>IL</em>-1 or TNF pretreatment. Expression of the <em>IL</em>-1- or TNF-inducible target antigens on endothelial cells was rapid and transient, peaking at 4 h and disappearing after 24 h despite continued incubation with monokine. In contrast, we have previously shown that IFN-gamma requires 72 h to render HEC susceptible to lysis with acute KS sera. Serum adsorption studies demonstrated that <em>IL</em>-1- and TNF-inducible endothelial target antigens are distinct from IFN-gamma-inducible antigens. These observations suggest that mediator secretion by activated monocyte/macrophages could be a predisposing factor to the development of vascular injury in acute KS. Although our present observations have been restricted to KS, the development of cytotoxic antibodies directed to monokine-inducible endothelial cell antigens may also be found in other vasculitides accompanied by immune activation.

We previously reported that the AUG-burdened 5' untranslated region (UTR) of <em>IL</em>-15 mRNA impedes its translation. Here we demonstrate that the nucleotide or protein sequences of the <em>IL</em>-15 signal peptide and carboxyl terminus also contribute to the poor translation of <em>IL</em>-15 transcripts. In particular, the exchange of the <em>IL</em>-15 signal peptide coding sequence with that of <em>IL</em>-2 increased cellular and secreted levels of <em>IL</em>-15 protein 15- to <em>20</em>-fold in COS cells, while <em>IL</em>-2 transcripts with the <em>IL</em>-15 signal peptide generated 30- to 50-fold less <em>IL</em>-2 protein than wild-type <em>IL</em>-2. Furthermore, the addition of an artificial epitope tag to the 3' coding sequence of <em>IL</em>-15 increased its protein production 5- to 10-fold. Combining these two <em>IL</em>-15 message modifications, in addition to removing the 5' UTR, increased <em>IL</em>-15 synthesis 250-fold compared with a wild-type construct with an intact 5' UTR. These data suggest that <em>IL</em>-15 mRNA, unlike <em>IL</em>-2 mRNA, may exist in translationally inactive pools. By storing translationally quiescent <em>IL</em>-15 mRNA, cells might respond to intracellular infections or other stimuli by rapidly transforming <em>IL</em>-15 message into one that can be efficiently translated.

Anthracyclines entrapped in small-sized, sterically stabilized liposomes have the advantage of long circulation time, reduced systemic toxicity, increased uptake into systemic tumors, and gradual release of their payload. To date, there is no information on the behavior of these liposomes in brain tumors. The objective of this study was to compare the biodistribution and clinical efficacy of free doxorubicin (F-DOX) and stealth liposome-encapsulated DOX (SL-DOX) in a secondary brain tumor model. Nine days after tumor inoculation Fischer rats with a right parietal malignant sarcoma received an intravenous dose of 6 mg/kg of either F-DOX or SL-DOX for evaluation of drug biodistribution. For therapeutic trials a single dose of 8 mg/kg was given 6 or 11 days after tumor induction, or alternatively, weekly doses (5 mg/kg) were given on Days 6, 13, and <em>20</em>. Liposome-encapsulated DOX was slowly cleared from plasma with a t1/2 of 35 hours. Free-DOX maximum tumor drug levels reached a mean value of 0.8 microgram/g and were identical in the adjacent brain and contralateral hemisphere. In contrast, SL-DOX tumor levels were 14-fold higher at their peak levels at 48 hours, declining to ninefold increased levels at 1<em>20</em> hours. A gradual increase in drug levels in the brain adjacent to tumor was noted between 72 and 1<em>20</em> hours (up to 4 micrograms/g). High-performance liquid chromatography analysis identified a small amount of aglycone metabolites within the tumor mass from 96 hours and beyond, after SL-DOX injection. Cerebrospinal fluid levels were barely detectable in tumor-bearing rats treated with F-DOX up to 1<em>20</em> hours after drug injection (< or = 0.05 microgram/ml), whereas the levels found after SL-DOX were 10- to 30-fold higher. An F-DOX single-dose treatment given 6 days after tumor inoculation increased the rats' life span (<em>ILS</em>) by 135% over controls (p < 0.05) but was not effective if given on Day 11. In contrast, SL-DOX treatment resulted in an <em>ILS</em> of 168% (p < 0.0003) with no difference when given after 6 or 11 days. Treatment with three weekly doses of SL-DOX produced an <em>ILS</em> of 189% compared to 126% by F-DOX (p < 0.0002). The authors conclude that the use of long-circulating liposomes as cytotoxic drug carriers in brain tumor results in enhanced drug exposure and improved therapeutic activity, with equal effectiveness against early small- and large-sized brain tumors.

T cells are produced through 2 mechanisms, thymopoiesis and proliferative expansion of postthymic T cells. Thymic output generates diversity of the pool, and proliferation achieves optimal clonal size of each individual T cell. To determine the contribution of these 2 mechanisms to the formation of the initial T-cell repertoire, we examined neonates of 30 to 40 weeks' gestation. Peripheral T cells were in a state of high proliferative turnover. In premature infants, 10% of T cells were dividing; the proliferation rates then declined but were still elevated in mature newborns. Throughout the third trimester, concentrations of T-cell-receptor excision circles (TRECs) were 10 per 100 T cells. Stability of TREC frequencies throughout the period of repertoire generation suggested strict regulation of clonal size to approximately 10 to <em>20</em> cells. Neonatal naive CD4+ and CD8+ T cells were explicitly responsive to <em>IL</em>-7; growth-promoting properties of <em>IL</em>-15 were selective for newborn CD8+ T cells. Neonatal T cells expressed telomerase and, in spite of the high turnover, built up a telomeric reserve. Thus, proliferative expansion, facilitated by increased cytokine responsiveness, and thymopoiesis complement each other as mechanisms of T-cell production in neonates. Maintaining optimal clonal size instead of filling the space in a lymphopenic host appears to regulate homeostatic T-cell proliferation during fetal development.

Artificial mechanical ventilation represents a major cause of iatrogenic lung damage in intensive care. It is largely unknown which mediators, if any, contribute to the onset of such complications. We investigated whether stress caused by artificial mechanical ventilation leads to induction, synthesis, and release of cytokines or eicosanoids from lung tissue. We used the isolated perfused and ventilated mouse lung where frequent perfusate sampling allows determination of mediator release into the perfusate. Hyperventilation was executed with either negative (NPV) or positive pressure ventilation (PPV) at a transpulmonary pressure that was increased 2.5-fold above normal. Both modes of hyperventilation resulted in an approximately 1.75-fold increased expression of tumor necrosis factor alpha (TNFalpha) and interleukin-6 (<em>IL</em>-6) mRNA, but not of cyclooxygenase-2 mRNA. After switching to hyperventilation, prostacyclin release into the perfusate increased almost instantaneously from 19 +/- 17 pg/min to 230 +/- 160 pg/min (PPV) or 115 +/- 87 pg/min (NPV). The enhancement in TNFalpha and <em>IL</em>-6 production developed more slowly. In control lungs after 150 min of perfusion and ventilation, TNFalpha and <em>IL</em>-6 production was 23 +/- <em>20</em> pg/min and 330 +/- 210 pg/min, respectively. In lungs hyperventilated for 150 min, TNFalpha and <em>IL</em>-6 production were increased to 287 +/- 180 pg/min and more than 1,000 pg/min, respectively. We conclude that artificial ventilation might cause pulmonary and systemic adverse reactions by inducing the release of mediators into the circulation.

We describe a new two-step culture method for mass production in vitro of erythroid cells from either CD34+ (10(5) cells/mL) or light-density (10(6) cells/mL) cells purified from the blood of normal donors and thalassemic patients. The method includes (i) culture of the cells in the presence of dexamethasone and estradiol (10(-6) M each) and (ii) the growth factors SCF (50 ng/mL), <em>IL</em>-3 (1 ng/mL), and EPO (1 U/mL). In their proliferative phase, these cultures generated approximately 1.2 x 10(7) erythroblasts for each milliliter of blood collected from normal donors or thalassemic patients. They were composed mostly (90%) of CD45(low)/glycophorin (GPA)(neg)/CD71(1ow) cells at day 7, 50-60\% of which became CD45(neg)/GPA+/CD71high by days 15-<em>20</em>. However, when cells from days 7 to 12 of the proliferative phase were transferred in differentiation medium containing EPO and insulin, they progressed to mature erythroblasts (g90% benzidine(pos) and CD45(neg)/GPA+/CD71medium) in 4 days. Because of the high number of erythroid cells that are generated from modest volumes of blood, this method will prove useful in donor-specific studies of erythroid differentiation.

Curcumin is a naturally occurring, dietary polyphenolic phytochemical that is under preclinical trial evaluation for cancer preventive drug development and whose working pharmacological actions include anti-inflammation. With respect to inflammation, in vitro, it inhibits the activation of free radical-activated transcription factors, such as nuclear factor kappaB (NFkappaB) and AP-1, and reduces the production of pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF alpha), interleukin-1beta (<em>IL</em>-1beta), and interleukin-8. Inducible nitric oxide synthase (iNOS) is an inflammation-induced enzyme that catalyzes the production of nitric oxide (NO), a molecule that may lead to carcinogenesis. Here, we report that in ex vivo cultured BALB/c mouse peritoneal macrophages, 1-<em>20</em> microM of curcumin reduced the production of iNOS mRNA in a concentration-dependent manner. Furthermore, we demonstrated that, in vivo, two oral treatments of 0.5 mL of a 10-microM solution of curcumin (92 ng/g of body weight) reduced iNOS mRNA expression in the livers of lipopolysaccharide(LPS)-injected mice by 50-70%. Although many hold that curcumin needs to be given at dosages that are unattainable through diet to produce an in vivo effect, we were able to obtain potency at nanomoles per gram of body weight. This efficacy is associated with two modifications in our preparation and feeding regimen: 1) an aqueous solution of curcumin was prepared by initially dissolving the compound in 0.5 N NaOH and then immediately diluting it in PBS; and 2) mice were fed curcumin at dusk after fasting. Inhibition was not observed in mice that were fed ad lib., suggesting that food intake may interfere with the absorption of curcumin.

Increasing evidence suggests that interleukin-1β (<em>IL</em>-1β) is a key mediator of the inflammatory response following traumatic brain injury (TBI). Recently, we showed that intracerebroventricular administration of an <em>IL</em>-1β-neutralizing antibody was neuroprotective following TBI in mice. In the present study, an anti-<em>IL</em>-1β antibody or control antibody was administered intraperitoneally following controlled cortical injury (CCI) TBI or sham injury in 105 mice and we extended our histological, immunological and behavioral analysis. First, we demonstrated that the treatment antibody reached target brain regions of brain-injured animals in high concentrations >> 11 nm) remaining up to 8 days post-TBI. At 48 h post-injury, the anti-<em>IL</em>-1β treatment attenuated the TBI-induced hemispheric edema (P < 0.05) but not the memory deficits evaluated using the Morris water maze (MWM). Neutralization of <em>IL</em>-1β did not influence the TBI-induced increases (P < 0.05) in the gene expression of the Ccl3 and Ccr2 chemokines, <em>IL</em>-6 or Gfap. Up to <em>20</em> days post-injury, neutralization of <em>IL</em>-1β was associated with improved visuospatial learning in the MWM, reduced loss of hemispheric tissue and attenuation of the microglial activation caused by TBI (P < 0.05). Motor function using the rotarod and cylinder tests was not affected by the anti-<em>IL</em>-1β treatment. Our results suggest an important negative role for <em>IL</em>-1β in TBI. The improved histological and behavioral outcome following anti-<em>IL</em>-1β treatment also implies that further exploration of <em>IL</em>-1β-neutralizing compounds as a treatment option for TBI patients is warranted.

The lack of physical activity and overnutrition in our modern lifestyle culminates in what we now experience as the current obesity and diabetes pandemic. Medical research performed over the past <em>20</em> years identified chronic low-grade inflammation as a mediator of these metabolic disorders. Hence, finding therapeutic strategies against this underlying inflammation and identifying molecules implicated in this process is of significant importance. Following the observation of an increased plasma concentration of interleukin-6 (<em>IL</em>-6) in obese patients, this protein, known predominantly as a pro-inflammatory cytokine, came into focus. In an attempt to clarify its importance, several studies implicated <em>IL</em>-6 as a co-inducer of the development of obesity-associated insulin resistance, which precedes the development of type 2 diabetes. However, the identification of <em>IL</em>-6 as a myokine, a protein produced and secreted by skeletal muscle to fulfil paracrine or endocrine roles in the insulin-sensitizing effects following exercise, provides a contrasting and hence paradoxical identity of this protein in the context of metabolism. We review here the literature considering the complex, pleiotropic role of <em>IL</em>-6 in the context of metabolism in health and disease.

BACKGROUND

Milk fat globule epidermal growth factor 8 (MFG-E8) is a potent opsonin for the clearance of apoptotic cells and is produced by mononuclear cells of immune competent organs including the spleen and lungs. It attenuates chronic and acute inflammation such as autoimmune glomerulonephritis and bacterial sepsis by enhancing apoptotic cell clearance. Ischemia-reperfusion (I/R) injury of the gut results in severe inflammation, apoptosis, and remote organ damage, including acute lung injury (ALI).

OBJECTIVE

To determine whether MFG-E8 attenuates intestinal and pulmonary inflammation after gut I/R.

METHODS

Wild-type (WT) and MFG-E8(-/-) mice underwent superior mesenteric artery occlusion for 90 minutes, followed by reperfusion for 4 hours. A group of WT mice was treated with 0.4 microg/<em>20</em> g recombinant murine MFG-E8 (rmMFG-E8) at the beginning of reperfusion. Four hours after reperfusion, MFG-E8, cytokines, myeloperoxidase activity, apoptosis, and histopathology were assessed. A 24-hour survival study was conducted in rmMFG-E8- and vehicle-treated WT mice.

RESULTS

Mesenteric I/R caused severe widespread injury and inflammation of the small intestines and remote organs, including the lungs. MFG-E8 levels decreased in the spleen and lungs by 50 to 60%, suggesting impaired apoptotic cell clearance. Treatment with rmMFG-E8 significantly suppressed inflammation (TNF-alpha, IL-6, IL-1beta, and myeloperoxidase) and injury of the lungs, liver, and kidneys. MFG-E8-deficient mice suffered from greatly increased inflammation and potentiated ALI, whereas treatment with rmMFG-E8 significantly improved the survival in WT mice.

CONCLUSIONS

MFG-E8 attenuates inflammation and ALI after gut I/R and may represent a novel therapeutic agent.

Acquisition of self-tolerance in the thymus requires T cells to discriminate strong versus weak T cell receptor binding by self-peptide-MHC complexes. We find this discrimination is reported by expression of the transcription factor Helios, which is induced during negative selection but decreases during positive selection. Helios and the proapoptotic protein Bim were coinduced in 55% of nascent CCR7(-) CD4(+) CD69(+) thymocytes. These were short-lived cells that up-regulated PD-1 and down-regulated CD4 and CD8 during Bim-dependent apoptosis. Helios and Bim were also coinduced at the subsequent CCR7(+) CD4(+) CD69(+) CD8(-) stage, and this second wave of Bim-dependent negative selection involved <em>20</em>% of nascent cells. Unlike CCR7(-) counterparts, Helios(+) CCR7(+) CD4(+) cells mount a concurrent Card11- and c-Rel-dependent activation response that opposes Bim-mediated apoptosis. This "hollow" activation response consists of many NF-κB target genes but lacks key growth mediators like <em>IL</em>-2 and Myc, and the thymocytes were not induced to proliferate. These findings identify Helios as the first marker known to diverge during positive and negative selection of thymocytes and reveal the extent, stage, and molecular nature of two distinct waves of clonal deletion in the normal thymus.

This study was designed to identify highly recurrent genetic alterations typical of Sézary syndrome (Sz), an aggressive cutaneous T-cell lymphoma/leukemia, possibly revealing pathogenetic mechanisms and novel therapeutic targets. High-resolution array-based comparative genomic hybridization was done on malignant T cells from <em>20</em> patients. Expression levels of selected biologically relevant genes residing within loci with frequent copy number alteration were measured using quantitative PCR. Combined binary ratio labeling-fluorescence in situ hybridization karyotyping was done on malignant cells from five patients. Minimal common regions with copy number alteration occurring in at least 35% of patients harbored 15 bona fide oncogenes and 3 tumor suppressor genes. Based on the function of the identified oncogenes and tumor suppressor genes, at least three molecular mechanisms are relevant in the pathogenesis of Sz. First, gain of cMYC and loss of cMYC antagonists (MXI1 and MNT) were observed in 75% and 40% to 55% of patients, respectively, which were frequently associated with deregulated gene expression. The presence of cMYC/MAX protein heterodimers in Sézary cells was confirmed using a proximity ligation assay. Second, a region containing TP53 and genome maintenance genes (RPA1/HIC1) was lost in the majority of patients. Third, the interleukin 2 (<em>IL</em>-2) pathway was affected by gain of STAT3/STAT5 and <em>IL</em>-2 (receptor) genes in 75% and 30%, respectively, and loss of TCF8 and DUSP5 in at least 45% of patients. In sum, the Sz genome is characterized by gross chromosomal instability with highly recurrent gains and losses. Prominent among deregulated genes are those encoding cMYC, cMYC-regulating proteins, mediators of MYC-induced apoptosis, and <em>IL</em>-2 signaling pathway components.

OBJECTIVE

We evaluated the gastrointestinal permeability and bacterial translocation in cirrhotic patients with portal hypertension (PHT) prior to and after non-selective betablocker (NSBB) treatment.

METHODS

Hepatic venous pressure gradient (HVPG) was measured prior to and under NSBB treatment. Gastroduodenal and intestinal permeability was assessed by the sucrose-lactulose-mannitol (SLM) test. Anti-gliadin and anti-endomysial antibodies were measured. Levels of LPS-binding protein (LBP) and interleukin-6 (IL-6) were quantified by ELISA, and NOD2 and toll-like receptor 2 (TLR2) polymorphisms were genotyped.

RESULTS

Fifty cirrhotics were included (72% male, 18% ascites, 60% alcoholic etiology). Abnormal gastroduodenal and intestinal permeability was found in 72% and 59% of patients, respectively. Patients with severe portal hypertension (HVPG ≥20 mm Hg; n=35) had increased markers of gastroduodenal/intestinal permeability (urine sucrose levels p=0.049; sucrose/mannitol ratios p=0.007; intestinal permeability indices p=0.002), and bacterial translocation (LBP p=0.002; IL-6 p=0.025) than patients with HVPG <20 mm Hg. A substantial portion of patients showed elevated levels of anti-gliadin antibodies (IgA: 60%, IgG: 34%) whereas no anti-endomysial antibodies were detected. A significant correlation of portal pressure (i.e., HVPG) with all markers of gastroduodenal/intestinal permeability and with LBP and IL-6 levels was observed. NOD2 and TLR2 risk variants were associated with abnormal intestinal permeability and elevated markers of bacterial translocation. At follow-up HVPG measurements under NSBB, we found an amelioration of gastroduodenal/intestinal permeability and a decrease of bacterial translocation (LBP - 16% p=0.018; IL-6 - 41% p<0.0001) levels, which was not limited to hemodynamic responders. Abnormal SLM test results and higher LBP/IL-6 levels were associated with a higher risk of variceal bleeding during follow-up but not with mortality.

CONCLUSIONS

Abnormal gastroduodenal/intestinal permeability, anti-gliadin antibodies, and bacterial translocation are common findings in cirrhotic patients and are correlated with the degree of portal hypertension. NSBB treatment ameliorates gastroduodenal/intestinal permeability and reduces bacterial translocation partially independent of their hemodynamic effects on portal pressure, which may contribute to a reduced risk of variceal bleeding.

In this study, we explored whether thrombopoietin (Tpo) has a direct in vitro effect on the proliferation and differentiation of long-term repopulating hematopoietic stem cells (LTR-HSC). We previously reported a cell separation method that uses the fluorescence-activated cell sorter selection of low Hoescht 33342/low Rhodamine 123 (low Ho/low Rh) fluorescence cell fractions that are highly enriched for LTR-HSC and can reconstitute lethally irradiated recipients with fewer than <em>20</em> cells. Low Ho/low Rh cells clone with high proliferative potential in vitro in the presence of stem cell factor (SCF) + interleukin-3 (<em>IL</em>-3) + <em>IL</em>-6 (90% to 100% HPP-CFC). Tpo alone did not induce proliferation of these low Ho/low Rh cells. However, in combination with SCF or <em>IL</em>-3, Tpo had several synergistic effects on cell proliferation. When Tpo was added to single growth factors (either SCF or <em>IL</em>-3 or the combination of both), the time required for the first cell division of low Ho/low Rh cells was significantly shortened and their cloning efficiency increased substantially. Moreover, the subsequent clonal expansion at the early time points of culture was significantly augmented by Tpo. Low Ho/low Rh cells, when assayed in agar directly after sorting, did not form megakaryocyte colonies in any growth condition tested. Several days of culture in the presence of multiple cytokines were required to obtain colony-forming units-megakaryocyte (CFU-Mk). In contrast, more differentiated, low Ho/high Rh cells, previously shown to contain short-term repopulating hematopoietic stem cells (STR-HSC), were able to form megakaryocyte colonies in agar when cultured in Tpo alone directly after sorting. These data establish that Tpo acts directly on primitive hematopoietic stem cells selected using the Ho/Rh method, but this effect is dependent on the presence of pluripotent cytokines. These cells subsequently differentiate into CFU-Mk, which are capable of responding to Tpo alone. Together with the results of previous reports of its effects on erythroid progenitors, these results suggest that the effects of Tpo on hematopoiesis are greater than initially anticipated.

From the early 1900s, visceral leishmaniasis (VL; kala-azar) has been among the most important health problems in Sudan, particularly in the main endemic area in the eastern and central regions. Several major epidemics have occurred, the most recent--in Western Upper Nile province in southern Sudan, detected in 1988--claiming over 100,000 lives. The disease spread to other areas that were previously not known to be endemic for VL. A major upsurge in the number of cases was noted in the endemic area. These events triggered renewed interest in the disease. Epidemiological and entomological studies confirmed Phlebotomus orientalis as the vector in several parts of the country, typically associated with Acacia seyal and Balanites aegyptiaca vegetation. Infection rates with Leishmania were high, but subject to seasonal variation, as were the numbers of sand flies. Parasites isolated from humans and sand flies belonged to three zymodemes (MON-18, MON-30 and MON-82), which all belong to the L. donovani sensu lato cluster. Transmission dynamics have not been elucidated fully; heavy transmission in relatively scarcely populated areas such as Dinder national park suggested zoonotic transmission whereas the large numbers of patients with post kala-azar dermal leishmaniasis (PKDL) in heavily affected villages may indicate a human reservoir and anthroponotic transmission. Clinical presentation in adults and in children did not differ significantly, except that children were more anaemic. Fever, weight loss, hepato-splenomegaly and lymphadenopathy were the most common findings. PKDL was much more common than expected (56% of patients with VL developed PKDL), but other post-VL manifestations were also found affecting the eyes (uveitis, conjunctivitis, blepharitis), nasal and/or oral mucosa. Evaluation of diagnostic methods showed that parasitological diagnosis should still be the mainstay in diagnosis, with sensitivities for lymph node, bone marrow and spleen aspirates of 58%, 70% and 96%, respectively. Simple, cheap serological tests are needed. The direct agglutination test (DAT) had a sensitivity of 72%, specificity of 94%, positive predictive value of 78% and negative predictive value of 92%. As with other serological tests, the DAT cannot distinguish between active disease, subclinical infection or past infection. The introduction of freeze-dried antigen and control sera greatly improved the practicality and accuracy of the DAT in the field. An enzyme-linked immunosorbent assay using recombinant K39 antigen had higher sensitivity than DAT (93%). The polymerase chain reaction using peripheral blood gave a sensitivity of 70-93% and was more sensitive than microscopy of lymph node or bone marrow aspirates in patients with suspected VL. The leishmanin skin test (LST) was typically negative during active VL and converted to positive in c. 80% of patients 6 months after treatment. Immunological studies showed that both Th1 and Th2 cell responses could be demonstrated in lymph nodes from VL patients as evidenced by the presence of messenger ribonucleic acid for interleukin (<em>IL</em>)-10, interferon gamma and <em>IL</em>-2. Treatment of peripheral blood mononuclear cells from VL patients with <em>IL</em>-12 was found to drive the immune response toward a Th1 type response with the production of interferon gamma, indicating a potential therapeutic role for <em>IL</em>-12. VL responded well to treatment with sodium stibogluconate, which is still the first line drug at a dose of <em>20</em> mg/kg intravenously or intramuscularly per day for 15-30 d. Side effects and resistance were rare. Liposomal amphotericin B was effective, with few side effects. Control measures have not been implemented. Based on observations that VL does not occur in individuals who have a positive LST, probably because of previous cutaneous leishmaniasis, a vaccine containing heat-killed L. major promastigotes is currently undergoing a phase III trial.

OBJECTIVE

To measure levels of various inflammatory cytokines in the aqueous humor of patients with primary open-angle glaucoma (POAG), exfoliation glaucoma (EXG), and senile cataract.

METHODS

Aqueous humor samples were obtained from 64 eyes of 64 Japanese subjects (POAG, <em>20</em> eyes; EXG, 23 eyes; and cataract, 21 control eyes). The levels of eight cytokines including interleukin (<em>IL</em>)1-β, <em>IL</em>-6, <em>IL</em>-8, transforming growth factor (TGF)-β1, tumor necrosis factor (TNF)-α, serum amyloid A (SAA), migration inhibitory factor (MIF), and vascular endothelial growth factor (VEGF)-A were estimated using the multiplex bead immunoassay technique.

RESULTS

Compared with the cataract group, the levels of TGF-β1, IL-8, and SAA were significantly higher in aqueous humor samples from the POAG (5.0-fold, 2.3-fold, and 11.9-fold, respectively) and EXG (12.5-fold, 4.0-fold, and 18.3-fold, respectively) groups. Except for a significant decrease in the IL-6 level in the POAG (0.23-fold) group, no other cytokine levels differed in the POAG and EXG groups compared with the cataract group. The levels of TGF-β1, IL-8, and SAA were positively correlated with each other (ρ = 0.723-0.786; P < 0.0001), the intraocular pressure (IOP) (ρ = 0.392-0.662; P < 0.0001-0.0019), and the number of glaucoma medications (ρ = 0.478-0.659; P < 0.0001-0.0001).

CONCLUSIONS

Cytokine networks including TGF-β1, IL-8, and SAA in aqueous humor may have critical roles in IOP elevations in patients with open-angle glaucoma.

Systemic sclerosis (SSc) is a connective tissue disorder characterized by excessive collagen deposition in the skin and internal organs. Several cytokines and chemokines have been implicated in the induction of fibrosis, but a definitive relationship between specific cytokines and organ involvement has not been established yet. Serum samples, PBMC and T cell lines (TCL) obtained from 54 patients affected by SSc and <em>20</em> healthy donors (HD) were examined by ELISA for Interferon-gamma (IFN-gamma ), interleukin (<em>IL</em>)-4, <em>IL</em>-6, <em>IL</em>-10, <em>IL</em>-18, Transforming growth factor (TGF)-beta1, Tumour necrosis factor (TNF)-alpha, sCD30, Macrophage derived chemokine (MDC), Monocyte chemoattractant protein (MCP)-1, Macrophage inflammatory protein (MIP)-1alpha and Regulated on activation normal T-cell expressed and secreted (RANTES). In all the SSc serum samples, we found significantly increased levels of <em>IL</em>6, TNFalpha and MCP-1 but reduced amounts of gamma-IFN and MDC. <em>IL</em>6, <em>IL</em>10, <em>IL</em>18, MIP-1alpha and TNFalpha measured in supernatants from PHA-stimulated PBMC and <em>IL</em>6, MCP-1 and RANTES in supernatants from stimulated TCL were also increased in patients. MDC was decreased in all the biological SSc sources studied. TGF-beta1, <em>IL</em>10, and sCD30 were produced at a significantly lower level by SSc TCL. Serum <em>IL</em>6 and sCD30 levels were significantly increased in dc-SSc patients compared to lc-SSc as were levels of MCP-1 produced by PBMC and <em>IL</em>10 from TCL. We observed a strict relationship between pulmonary fibrosis and <em>IL</em>10, MCP-1 (both from TCL) and serum <em>IL</em>6. Kidney involvement was related to serum MCP-1 levels and <em>IL</em>18 production from PBMC. Oesophageal involvement correlated with MDC production from PBMC and <em>IL</em>10 synthesis by TCL. We showed that <em>IL</em>-6, <em>IL</em>-10, MDC and MCP-1 are variably associated with internal organ involvement and allow the discrimination between limited and diffuse forms of the disease.

BACKGROUND

Atopic dermatitis (AD) is classified as extrinsic and intrinsic, representing approximately 80% and <em>20</em>% of patients with the disease, respectively. Although sharing a similar clinical phenotype, only extrinsic AD is characterized by high serum IgE levels. Because most patients with AD exhibit high IgE levels, an "allergic"/IgE-mediated disease pathogenesis was hypothesized. However, current models associate AD with T-cell activation, particularly TH2/TH22 polarization, and epidermal barrier defects.

OBJECTIVE

We sought to define whether both variants share a common pathogenesis.

METHODS

We stratified 51 patients with severe AD into extrinsic AD (n = 42) and intrinsic AD (n = 9) groups (with similar mean disease activity/SCORAD scores) and analyzed the molecular and cellular skin pathology of lesional and nonlesional intrinsic AD and extrinsic AD by using gene expression (real-time PCR) and immunohistochemistry.

RESULTS

A significant correlation between IgE levels and SCORAD scores (r = 0.76, P < 10(-5)) was found only in patients with extrinsic AD. Marked infiltrates of T cells and dendritic cells and corresponding epidermal alterations (keratin 16, Mki67, and S100A7/A8/A9) defined lesional skin of patients with both variants. However, higher activation of all inflammatory axes (including TH2) was detected in patients with intrinsic AD, particularly TH17 and TH22 cytokines. Positive correlations between TH17-related molecules and SCORAD scores were only found in patients with intrinsic AD, whereas only patients with extrinsic AD showed positive correlations between SCORAD scores and TH2 cytokine (IL-4 and IL-5) levels and negative correlations with differentiation products (loricrin and periplakin).

CONCLUSIONS

Although differences in TH17 and TH22 activation exist between patients with intrinsic AD and those with extrinsic AD, we identified common disease-defining features of T-cell activation, production of polarized cytokines, and keratinocyte responses to immune products. Our data indicate that a TH2 bias is not the sole cause of high IgE levels in patients with extrinsic AD, with important implications for similar therapeutic interventions.

OBJECTIVE

To evaluate the effects of SB 242235, a potent and selective inhibitor of p38 mitogen-activated protein (MAP) kinase, on joint integrity in rats with adjuvant-induced arthritis (AIA).

METHODS

Male Lewis rats with AIA were orally treated either prophylactically (days 0-<em>20</em>) or therapeutically (days 10-<em>20</em>) with SB 242235. Efficacy was determined by measurements of paw inflammation, dual-energy x-ray absorptiometry for bone-mineral density (BMD), magnetic resonance imaging (MRI), microcomputed tomography (CT), and histologic evaluation. Serum tumor necrosis factor alpha (TNFalpha) in normal (non-AIA) rats and serum interleukin-6 (<em>IL</em>-6) levels in rats with AIA were measured as markers of the antiinflammatory effects of the compound.

RESULTS

SB 242235 inhibited lipopolysaccharide-stimulated serum levels of TNFalpha in normal rats, with a median effective dose of 3.99 mg/kg. When SB 242235 was administered to AIA rats prophylactically on days 0-<em>20</em>, it inhibited paw edema at 30 mg/kg and 10 mg/kg per day by 56% and 33%, respectively. Therapeutic administration on days 10-<em>20</em> was also effective, and inhibition of paw edema was observed at 60, 30, and 10 mg/kg (73%, 51%, and 19%, respectively). Significant improvement in joint integrity was demonstrated by showing normalization of BMD and also by MRI and micro-CT analysis. Protection of bone, cartilage, and soft tissues was also shown histologically. Serum <em>IL</em>-6 levels were decreased in AIA rats treated with the 60 mg/kg dose of compound.

CONCLUSIONS

Symptoms of AIA in rats were significantly reduced by both prophylactic and therapeutic treatment with the p38 MAP kinase inhibitor, SB 242235. Results from measurements of paw inflammation, assessment of BMD, MRI, and micro-CT indicate that this compound exerts a protective effect on joint integrity, and thus appears to have disease-modifying properties.

BACKGROUND

Hyper-IgD and periodic fever syndrome (HIDS) is an hereditary autoinflammatory syndrome, characterised by recurrent inflammatory attacks. Treatment of HIDS is difficult, although simvastatin is beneficial and etanercept might be effective. Studying the treatment of a rare periodic syndrome is complicated by the varying frequency and severity of symptoms and low prevalence. Our aim was to develop a system of clinical observations to evaluate effectiveness of treatment-on-demand.

METHODS

Seven fever episodes in three HIDS patients were monitored, with and without administration of etanercept or anakinra. We developed a clinical score, which includes 12 symptoms. In one patient, inflammatory attacks were provoked by vaccination.

CONCLUSIONS

At the onset of an attack, all patients reported a clinical score between <em>20</em> and 25. The score was used to quantify severity and define the end of an attack. Reproducible monitoring of inflammatory episodes was difficult, even in this pilot study. The effect of early administration of etanercept was variable. In one patient, a fever episode could be readily provoked within 12 to 24 hours by vaccination. In this patient, the <em>IL</em>-1ra analogue anakinra was more successful in aborting the inflammatory attack than etanercept. We propose that this vaccination model will allow evaluation of treatment-on-demand in a controlled setting.

The contribution of accessory toxins to the acute inflammatory response to Vibrio cholerae was assessed in a murine pulmonary model. Intranasal administration of an El Tor O1 V. cholerae strain deleted of cholera toxin genes (ctxAB) caused diffuse pneumonia characterized by infiltration of PMNs, tissue damage, and hemorrhage. By contrast, the ctxAB mutant with an additional deletion in the actin-cross-linking repeats-in-toxin (RTX) toxin gene (rtxA) caused a less severe pathology and decreased serum levels of proinflammatory molecules interleukin (<em>IL</em>)-6 and murine macrophage inflammatory protein (MIP)-2. These data suggest that the RTX toxin contributes to the severity of acute inflammatory responses. Deletions within the genes for either hemagglutinin/protease (hapA) or hemolysin (hlyA) did not significantly affect virulence in this model. Compound deletion of ctxAB, hlyA, hapA, and rtxA created strain KFV101, which colonized the lung but induced pulmonary disease with limited inflammation and significantly reduced serum titers of <em>IL</em>-6 and MIP-2. 100% of mice inoculated with KFV101 survive, compared with <em>20</em>% of mice inoculated with the ctxAB mutant. Thus, the reduced virulence of KFV101 makes it a prototype for multi-toxin deleted vaccine strains that could be used for protection against V. cholerae without the adverse effects of the accessory cholera toxins.