Struktur- und Funktionsanalyse der zytokinbindenden Domäne des humanen Interleukin-6-Rezeptors

Die Familie der IL-6-Typ-Zytokine (IL-6, IL-11, CT-1, CNTF, LIF, OSM, BSF-3) ist durch eine vierhelikale Faltung gekennzeichnet. Alle Zytokine dieser Familie agieren über einen Rezeptorkomplex, der als gemeinsame Komponente mindestens ein Molekül gp130 enthält. IL-6 und IL-11 signalisieren über ein gp130-Homodimer, während CT-1, CNTF, LIF und OSM ein Heterodimer aus gp130 und dem strukturell verwandten LIFR oder, im Falle des OSM, auch OSMR verwenden. Die Rezeptoren der vierhelikalen Zytokine sind in ihrem extrazellulären Bereich modulartig aus Ig- und Fibronektin-Typ-III-ähnlichen Domänen aufgebaut. Sie besitzen als gemeinsame Struktureinheit ein zytokinbindendes Modul (CBM) aus zwei Fibronektin-Typ-III-ähnlichen Domänen, die durch vier konservierte Cysteine in der N-terminalen und ein konserviertes WSXWS-Motiv in der C-terminalen Domäne charakterisiert sind. Auf Zielzellen bindet IL-6 an den spezifischen IL-6 Rezeptor, worauf der Komplex aus IL-6/IL-6R mit dem Signaltransduktor gp130 assoziiert. Der IL-6R besteht in seinem extrazellulären Bereich aus drei Domänen. Die N-terminale Ig-ähnliche Domäne ist für die biologische Aktivität nicht notwendig. Die Domänen 2 und 3 bilden das CBM, welches auch in löslicher Form agonistisch wirkt. In der vorliegenden Arbeit wurden die strukturellen und funktionellen Eigenschaften der dritten extrazellulären Domäne des IL-6R untersucht. Das Protein läßt sich effizient in Bakterien exprimieren und in vitro renaturieren. Es konnte gezeigt werden, daß Domäne 3 für die Bindung an IL-6 ausreichend ist, der Komplex aus D3 und IL-6 jedoch nicht mehr mit dem gp130-Molekül assoziieren kann. Da der lösliche IL-6R (bestehend aus D2 und D3) in der Lage ist, an gp130 zu binden und ein biologisches Signal auszulösen, weisen diese Daten der C-terminalen CBM-Domäne (D3) eine ligandenbindende Funktion und der N-terminalen CBM-Domäne eine wichtige Rolle bei der Komplexbildung mit gp130 und Signalinduktion zu. Die gezeigte Expressions- und Renaturierungsstrategie für D3 wurde zur Markierung des Proteins mit 15N und 13C für die mehrdimensionale, heteronukleare NMR-Spektroskopie angewandt. Die hierdurch ermöglichte Strukturaufklärung von D3 als einer eindeutig in die Ligandenbindung involvierten Teilstruktur wird umfassendere strukturelle Informationen über den IL-6R-Komplex liefern, als es die bisherigen Mutations- bzw. Modellbaustudien konnten.

The role of Interleukin-12 in liver inflammation

Chronic liver inflammation during viral hepatitis is a major health problem worldwide. The role of proinflammatory cytokines, like IL-12, in breaking hepatic immune tolerance, and inducing acute liver inflammation and virus clearance is not clear. Nor is clear its role in uncontrolled severe inflammatory response, leading to fulminant hepatitis and hepatic failure. This work, focused in the study of the role of endogenous produced IL-12 in inducing hepatic inflammatory responses, demonstrates: In vitro, using adenovirus coding for IL-12, that hepatocytes stimulate CD4+ T cells in a tolerogenic manner, and that endogenous IL-12 is able to switch the immune response into Th1; and in vivo, that endogenous IL-12 induces hepatocyte damage and virus elimination in mice infected with adenovirus. In addition, and in order to study in vivo the relevance of IL-12 in acute inflammation, conditional IL-12 transgenic mice expressing IL-12 in the liver after cre-recombinase mediated induction were generated. For this purpose, an IL-12 fusion protein was created, which demonstrated high levels of bioactivity. Induction of IL-12 expression during embryonic development was achieved by crossbreeding with Act-Cre transgenic mice; induction of IL-12 expression in adult mice was achieved by a plasmid coding for the cre-recombinase. This study demonstrates that after induction, IL-12 is expressed in the liver of the transgenic mice. It also demonstrates that hepatic expression of IL-12 induces splenomegaly and liver inflammation, characterized by large infiltrations in portal tracts and veins, associated with hepatic damage, necrosis areas and lethality. Furthermore, constitutive hepatic IL-12 expression does not lead to abortion, but to total lethality, short after delivery. In conclusion, in this study, a transgenic mouse model has been generated, in which the expression of active IL-12 in the liver can be induced at any time; this model will be very helpful for studying hepatic pathologies. This study has also demonstrated that hepatic produced IL-12 is able of breaking liver tolerance inducing inflammation, virus elimination, severe hepatocyte damage, and lethality. These findings suggest IL-12 as a key cytokine in acute liver inflammation and fulminant hepatic failure. 5.1 Future studies Once the importance of IL-12 in inducing hepatic inflammation and virus elimination was demonstrated in this study, understanding the mechanisms of the IL-12 induced liver damage, and more important, how to avoid it will be the main focus in the future. It is very important to achieve hepatic inflammation for a more effective and faster viral elimination, but avoiding the toxicity of IL-12, which leads to massive liver injury and lethality is obviously necessary to allow IL-12 as therapy. For that purpose, future studies will be mainly base on three different points: 1. The determination of different cell populations present in the hepatic infiltration, which of them are responsible for liver injury, and as well their state of activation. 2. The measure of other pro- and anti-inflammatory cytokines and chemokines, which can play a role in IL-12-induced liver inflammation and hepatocyte damage. For these purposes, specific blocking antibodies (anti TNF-alpha, anti IL-12, anti IFN-g) will be used. The study with different transgenic mice: TNF-alpha Receptor knockout, TGF-b, will also help in determining the role of those cytokines during IL-12-induced liver damage and lethality. 3. The establishing of liver pathology models (viral infection, tumours, auto-antigens) in mice. Induction of IL-12 at any time of the pathology development will help in clarifying the role of IL-12 in those models. Finally, the transgenic mice expressing IL-23 in the liver will be generated.

Gen knockdown der Metalloproteasen Meprin alpha 1, alpha 2 und beta im Zebrabärbling Danio rerio

Die Metalloproteasen Meprin α und β übernehmen Schlüsselfunktionen in vielen (patho-) physiologischenrnProzessen. So sind sie beteiligt an der Umstrukturierung der extrazellulären Matrix, an immunologischenrnReaktionen oder an entzündlichen Gewebserkrankungen. Die beiden Enzyme kommenrnhauptsächlich in den Bürstensaummembranen von Niere und Darm sowie in der Haut von Vertebratenrnvor. Für die Erforschung der biologischen Aktivität der Meprine wurde in dieser Arbeit der ModellorganismusrnDanio rerio verwendet, der vor allem durch die Möglichkeit der gentechnischen Manipulationrnprädestiniert ist. Im Fisch konnten drei homologe Enzyme (Meprin α1, α2 und β) nachgewiesenrnwerden. Während mRNA-Analysen eine nahezu ubiquitäre Verteilung der Meprine offenbarten,rnkonnte ich mittels spezifischer Antikörper die Expression auf Proteinebene nachweisen. WährendrnMeprin α1 und β verstärkt im Darmepithel und in der Epidermis lokalisiert sind, konnte Meprinrnα2 ausschließlich in der Lamina propria des Darms identifiziert werden.rnDer Hauptteil der vorliegenden Arbeit zielt auf die spezifische Reduzierung des Expressionslevels derrnMeprine in Embryonen des Zebrabärblings. Dies wurde durch die Mikroinjektion von sogenanntenrnMorpholinos in die Zygote erzielt. Morpholinos sind RNA-Moleküle, die spezifisch an die mRNA desrnZielproteins binden können und die Translation verhindern. Die auftretenden Effekte durch das Fehlenrnder Meprine lassen so Rückschlüsse auf ihre physiologische Funktion zu. Nach der Injektion vonrnMorpholinos gegen Meprin α1 zeigten sich lediglich leichte epidermale Deformationen. Bei Meprin βrnhingegen kam es zu einer massiven Fehlbildung von Organen im Rumpf- und Schwanzbereich. Diesesrnführte zu erheblichen Defekten; die Embryonen starben innerhalb der ersten 24 Stunden nach derrnBefruchtung. Demzufolge müssen Meprin α1 und Meprin β insbesondere an der Gewebsdifferenzierungrnbeteiligt sein. Dies korreliert mit verschiedenen Experimenten, u.a. an knockout Mäusen, ausrndenen hervorgeht, dass die Prozessierung und Aktivierung der Cytokine Interleukin-1β oder Interleukin-rn18 durch Meprin β erfolgen kann.rnDie Injektion von Meprin α2-Morpholinos erbrachte ein weiteres, eindrucksvolles Ergebnis: Das Blutgefäßsystemrnvon injizierten Embryonen war vollständig unterbrochen und es sammelten sich Erythrozytenrnim Bereich der Caudalvene an. Diese Phänotypen gleichen den knockdown-Experimenten mitrndem vascular endothelial growth factor VEGF-A, dem entscheidenden Wachstumsfaktor in der Angiogenesern(Blutgefäßbildung). Eine Inkubation des humanen VEGF-A mit (humanem) rekombinantemrnMeprin α bzw. β führte zu einer differenzierten Prozessierung des Moleküls. Diese Ergebnisse legenrnnahe, dass Meprin α pro-angiogenetisch wirkt, indem es VEGF-A prozessiert und damit die Gefäßbildungrnaktiviert. Aus den Daten dieser Arbeit wird die hohe Signifikanz der Meprine für die Proliferationrnund Differenzierung spezieller Gewebe deutlich, welche somit eine wichtige Grundlage für Studienrnan höheren Vertebraten darstellt.

Response of interleukin-6 during euglycaemic and hyperglycaemic exercise in patients with type 1 diabetes mellitus

The aim of this randomized, single-blinded cross-over study was to investigate the response of interleukin-6 (IL-6) during moderate aerobic exercise in stable euglycaemia and hyperglycaemia in seven male patients with type 1 diabetes mellitus. IL-6 increased significantly over the entire study period in euglycaemia, but not in hyperglycaemia.

Delayed osteoblast differentiation and altered inflammatory response around implants placed in incisor sockets of type 2 diabetic rats

Objective: Central to the process of osseointegration is the recruitment of mesenchymal progenitor cells to the healing site, their proliferation and differentiation to bone synthesising osteoblasts. The process is under the control of pro-inflammatory cytokines and growth factors. The aim of this study was to monitor these key stages of osseointegration and the signalling milieu during bone healing around implants placed in healthy and diabetic bone. Methods: Implants were placed into the sockets of incisors extracted from the mandibles of normal Wistar and diabetic Goto-Kakizaki rats. Mandibles 1-12 weeks post-insertion of the implant were examined by histochemistry and immunocytochemistry to localise the presence of Stro-1- positive mesenchymal progenitor cells, proliferating cellular nuclear antigen proliferative cells, osteopontin and osteocalcin, macrophages, pro-inflammatory cytokines interleukin (IL)-1 , IL-6, tumour necrosis factor (TNF)- and tumour growth factor (TGF)- 1. Image analysis provided a semi-quantification of positively expressing cells. Results: Histological staining identified a delay in the formation of mineralised bone around implants placed in diabetic animals. Within the diabetic bone, the migration of Stro-1 mesenchymal cells in the healing tissue appeared to be unaffected. However, in the diabetic healing bone, the onset of cell proliferation and osteoblast differentiation were delayed and subsequently prolonged compared with normal bone. Similar patterns of change were observed in diabetic bone for the presence of IL-1 , TNF- , macrophages and TGF- 1. Conclusion: The observed alterations in the extracellular presence of pro-inflammatory cytokines, macrophages and growth factors within diabetic tissues that correlate to changes in the signalling milieu, may affect the proliferation and differentiation of mesenchymal progenitor cells in the osseointegration process. To cite this article: Colombo JS, Balani D, Sloan AJ, St Crean J, Okazaki J, Waddington RJ. Delayed osteoblast differentiation and altered inflammatory response around implants placed in incisor sockets of type 2 diabetic rats Clin. Oral Impl. Res22, 2011; 578-586 doi: 10.1111/j.1600-0501.2010.01992.x.

Porcine circovirus type 2 DNA influences cytoskeleton rearrangements in plasmacytoid and monocyte-derived dendritic cells

Functional disruption of dendritic cells (DC) is an important strategy for viral pathogens to evade host defences. In this context, porcine circovirus type 2 (PCV2), a single-stranded DNA virus, impairs plasmacytoid DC (pDC) and conventional DC activation by certain viruses or Toll-like receptor (TLR) ligands. This inhibitory capacity is associated with the viral DNA, but the impairment does not affect all signalling cascades; TLR7 ligation by small chemical molecules will still induce interleukin-6 (IL-6) and tumour necrosis factor-α secretion, but not interferon-α or IL-12. In this study, the molecular mechanisms by which silencing occurs were investigated. PP2, a potent inhibitor of the Lyn and Hck kinases, produced a similar profile to the PCV2 DNA interference with cytokine secretion by pDC, efficiently inhibiting cell activation induced through TLR9, but not TLR7, ligation. Confocal microscopy and cytometry analysis strongly suggested that PCV2 DNA impairs actin polymerization and endocytosis in pDC and monocyte-derived DC, respectively. Altogether, this study delineates for the first time particular molecular mechanisms involved in PCV2 interference with DC danger recognition, which may be responsible for the virus-induced immunosuppression observed in infected pigs.

Prolonged amelioration of acute lung allograft rejection by sequential overexpression of human interleukin-10 and hepatocyte growth factor in rats

The effect of prolonged electroporation-mediated human interleukin-10 (hIL-10) overexpression 24 hours before transplantation, combined with sequential human hepatocyte growth factor (HGF) overexpression into skeletal muscle on day 5, on rat lung allograft rejection was evaluated. Left lung allotransplantation was performed from Brown-Norway to Fischer-F344 rats. Gene transfer into skeletal muscle was enhanced by electroporation. Three groups were studied: group I animals (n = 5) received 2.5 μg pCIK-hIL-10 (hIL-10/CMV [cytomegalovirus] early promoter enhancer) on day -1 and 80 μg pCIK-HGF (HGF/CMV early promoter enhancer) on day 5. Group II animals (n = 4) received 2.5 μg pCIK-hIL-10 and pUbC-hIL-10 (hIL-10/pUbC promoter) on day -1. Control group III animals (n = 4) were treated by sham electroporation on days -1 and 5. All animals received daily nontherapeutic intraperitoneal dose of cyclosporin A (2.5 mg/kg) and were sacrificed on day 15. Graft oxygenation and allograft rejection were evaluated. Significant differences were found between study groups in graft oxygenation (Pao(2)) (P = .0028; group I vs. groups II and III, P < .01 each). Pao(2) was low in group II (31 ± 1 mm Hg) and in group III controls (34 ± 10 mm Hg), without statistically significant difference between these 2 groups (P = .54). In contrast, in group I, Pao(2) of recipients sequentially transduced with IL-10 and HGF plasmids was much improved, with 112 ± 39 mm Hg (vs. groups II and III; P < .01 each), paralleled by reduced vascular and bronchial rejection (group I vs. groups II and III, P < .021 each). Sequential overexpression of anti-inflammatory cytokine IL-10, followed by sequential and overlapping HGF overexpression on day 5, preserves lung function and reduces acute lung allograft rejection up to day 15 post transplant as compared to prolonged IL-10 overexpression alone.

Impaired hepatic removal of interleukin-6 in patients with liver cirrhosis

Systemic concentrations of interleukin-6 (IL-6) are elevated in patients with liver cirrhosis, and impaired hepatic uptake of IL-6 was suggested to contribute to higher levels in these patients. To test this hypothesis IL-6 was measured in portal venous serum (PVS), hepatic venous serum (HVS) and systemic venous serum (SVS) of 41 patients with liver cirrhosis and four patients with normal liver function. IL-6 was higher in PVS than HVS of all blood donors and about 43% of portal vein derived IL-6 was extracted by the healthy liver, and 6.3% by the cirrhotic liver demonstrating markedly impaired removal of IL-6 by the latter. Whereas in patients with CHILD-PUGH stage A IL-6 in HVS was almost 25% lower than in PVS, in patients with CHILD-PUGH stage C IL-6 was similarly abundant in the two blood compartments. Ascites is a common complication in cirrhotic patients and was associated with higher IL-6 levels in all blood compartments without significant differences in hepatic excretion. Hepatic venous pressure gradient did not correlate with the degree of hepatic IL-6 removal excluding hepatic shunting as the principal cause of impaired IL-6 uptake. Furthermore, patients with alcoholic liver cirrhosis had higher IL-6 in all blood compartments than patients with cryptogenic liver cirrhosis. Aetiology of liver cirrhosis did not affect hepatic removal rate indicating higher IL-6 synthesis in patients with alcoholic liver cirrhosis. In summary, the current data provide evidence that impaired hepatic removal of IL-6 is explained by hepatic shunting and liver dysfunction in patients with liver cirrhosis partly explaining higher systemic levels.

Estimating the net contribution of interleukin-28B variation to spontaneous hepatitis C virus clearance

The identification of associations between interleukin-28B (IL-28B) variants and the spontaneous clearance of hepatitis C virus (HCV) raises the issues of causality and the net contribution of host genetics to the trait. To estimate more precisely the net effect of IL-28B genetic variation on HCV clearance, we optimized genotyping and compared the host contributions in multiple- and single-source cohorts to control for viral and demographic effects. The analysis included individuals with chronic or spontaneously cleared HCV infections from a multiple-source cohort (n = 389) and a single-source cohort (n = 71). We performed detailed genotyping in the coding region of IL-28B and searched for copy number variations to identify the genetic variant or haplotype carrying the strongest association with viral clearance. This analysis was used to compare the effects of IL-28B variation in the two cohorts. Haplotypes characterized by carriage of the major alleles at IL-28B single-nucleotide polymorphisms (SNPs) were highly overrepresented in individuals with spontaneous clearance versus those with chronic HCV infections (66.1% versus 38.6%, P = 6 × 10(-9) ). The odds ratios for clearance were 2.1 [95% confidence interval (CI) = 1.6-3.0] and 3.9 (95% CI = 1.5-10.2) in the multiple- and single-source cohorts, respectively. Protective haplotypes were in perfect linkage (r(2) = 1.0) with a nonsynonymous coding variant (rs8103142). Copy number variants were not detected. CONCLUSION: We identified IL-28B haplotypes highly predictive of spontaneous HCV clearance. The high linkage disequilibrium between IL-28B SNPs indicates that association studies need to be complemented by functional experiments to identify single causal variants. The point estimate for the genetic effect was higher in the single-source cohort, which was used to effectively control for viral diversity, sex, and coinfections and, therefore, offered a precise estimate of the net host genetic contribution.

Adipocytokines, hepatic and inflammatory biomarkers and incidence of type 2 diabetes. the CoLaus study

Context There is contradictory information regarding the prognostic importance of adipocytokines, hepatic and inflammatory biomarkers on the incidence of type 2 diabetes. The objective was to assess the prognostic relevance of adipocytokine and inflammatory markers (C-reactive protein – CRP; interleukin-1beta – IL-1β; interleukin-6– IL-6; tumour necrosis factor-α – TNF-α; leptin and adiponectin) and gamma-glutamyl transpeptidase (γGT) on the incidence of type 2 diabetes. Methods Prospective, population-based study including 3,842 non-diabetic participants (43.3% men, age range 35 to 75 years), followed for an average of 5.5 years (2003–2008). The endpoint was the occurrence of type 2 diabetes. Results 208 participants (5.4%, 66 women) developed type 2 diabetes during follow-up. On univariate analysis, participants who developed type 2 diabetes had significantly higher baseline levels of IL-6, CRP, leptin and γGT, and lower levels of adiponectin than participants who remained free of type 2 diabetes. After adjusting for a validated type 2 diabetes risk score, only the associations with adiponectin: Odds Ratio and (95% confidence interval): 0.97 (0.64–1.47), 0.84 (0.55–1.30) and 0.64 (0.40–1.03) for the second, third and forth gender-specific quartiles respectively, remained significant (P-value for trend = 0.05). Adding each marker to a validated type 2 diabetes risk score (including age, family history of type 2 diabetes, height, waist circumference, resting heart rate, presence of hypertension, HDL cholesterol, triglycerides, fasting glucose and serum uric acid) did not improve the area under the ROC or the net reclassification index; similar findings were obtained when the markers were combined, when the markers were used as continuous (log-transformed) variables or when gender-specific quartiles were used. Conclusion Decreased adiponectin levels are associated with an increased risk for incident type 2 diabetes, but they seem to add little information regarding the risk of developing type 2 diabetes to a validated risk score.

Interleukin-17A stimulates granulocyte-macrophage colony-stimulating factor release by murine osteoblasts in the presence of 1,25-dihydroxyvitamin D(3) and inhibits murine osteoclast development in vitro

OBJECTIVE To investigate the effects of interleukin-17A (IL-17A) on osteoclastogenesis in vitro. METHODS Bone marrow cells (BMCs) were isolated from the excised tibia and femora of wild-type C57BL/6J mice, and osteoblasts were obtained by sequential digestion of the calvariae of ddY, C57BL/6J, and granulocyte-macrophage colony-stimulating factor-knockout (GM-CSF(-/-)) mice. Monocultures of BMCs or cocultures of BMCs and osteoblasts were supplemented with or without 1,25-dihydroxyvitamin D(3)(1,25[OH](2)D(3)), recombinant human macrophage colony-stimulating factor (M-CSF), RANKL, and IL-17A. After 5-6 days, the cultures were fixed with 4% paraformaldehyde and subsequently stained for the osteoclast marker enzyme tartrate-resistant acid phosphatase (TRAP). Osteoprotegerin (OPG) and GM-CSF expression were measured by enzyme-linked immunosorbent assay, and transcripts for RANK and RANKL were detected by real-time polymerase chain reaction. RESULTS In both culture systems, IL-17A alone did not affect the development of osteoclasts. However, the addition of IL-17A plus 1,25(OH)(2)D(3) to cocultures inhibited early osteoclast development within the first 3 days of culture and induced release of GM-CSF into the culture supernatants. Furthermore, in cocultures of GM-CSF(-/-) mouse osteoblasts and wild-type mouse BMCs, IL-17A did not affect osteoclast development, corroborating the role of GM-CSF as the mediator of the observed inhibition of osteoclastogenesis by IL-17A. CONCLUSION These findings suggest that IL-17A interferes with the differentiation of osteoclast precursors by inducing the release of GM-CSF from osteoblasts.

Prolonged amelioration of acute lung allograft rejection by overexpression of human interleukin-10 under control of a long acting ubiquitin C promoter in rats

BACKGROUND: The prolonged effect of electroporation-mediated human interleukin-10 (hIL-10) overexpression in skeletal muscle under the control of the constitutional polyubiquitin C promoter (pUb hIL-10) on rat lung allograft rejection was evaluated. METHODS: Left lung allotransplantation was performed from Brown-Norway to Fischer-F344 rats. Either 2.5 mug pCIK hIL-10 (hIL-10/cytomegalovirus early promoter enhancer) alone (Group I/sacrifice Day 5 and II/sacrifice Day 10) or in combination with 2.5 mug pUb hIL-10 (hIL-10/UbC promoter; Group III/sacrifice Day 10) were injected into the tibialis anterior muscle of the recipient, followed by electroporation 24 hours before transplantation. Animals in Control Groups IV and V without gene transfer were euthanized on Day 5 and 10, respectively. All animals received a daily non-therapeutic dose of cyclosporine A (2.5 mg/kg). RESULTS: In Control Group IV, complete rejection (median A3B3) was noted on Day 5 with a Pao(2) of 43 +/- 9 mm Hg. In recipients of Control Group V, measurement of gas exchange on Day 10 and rejection grading was impossible because of complete destruction of the allograft. Group I animals on Day 5 (233 +/- 123 mm Hg; p = 0.02 vs Group IV) and Group II animals on Day 10 (150 +/- 139 mm Hg; p = 0.15 vs Group IV) demonstrated improved graft function. Graft function in Group III was further improved on Day 10 (299 +/- 123 mm Hg; p = 0.002 vs Group IV; p = 0.05 vs Group II; p = 0.36 vs Group I). Rejection was significantly reduced in Group III (median, A2B2) compared with Group II (median, A4B3; p < 0.05). CONCLUSIONS: Interleukin-10 overexpression under control of the constitutive ubiquitin C promoter ameliorates acute rejection and preserves lung graft function for a prolonged time.

Electroporation-mediated interleukin-10 overexpression in skeletal muscle reduces acute rejection in rat cardiac allografts

OBJECTIVES: Human interleukin 10 (hIL-10) may reduce acute rejection after organ transplantation. Our previous data shows that electroporation-mediated transfer of plasmid DNA to peripheral muscle enhances gene transduction dramatically. This study was designed to investigate the effect of electroporation-mediated overexpression of hIL-10 on acute rejection of cardiac allografts in the rat. METHODS: The study was designed to evaluate the effect of hIL-10 gene transfer on (a) early rejection pattern and (b) graft survival. Gene transfer was achieved by intramuscular (i.m.) injection into the tibialis anterior muscle of Fischer (F344) male recipients followed by electroporation 24 h prior to transplantation. Heterotopic cardiac transplantation was performed from male Brown Norway rat to F344. Four groups were studied (n = 6). Treated animals in groups B1 and B2 received 2.5 microg of pCIK hIL-10 and control animals in groups A1 and A2 distilled water. Graft function was assessed by daily palpation. Animals from group A1 were sacrificed at the cessation of the heart beat of the graft and those in group B1 were sacrificed at day 7; blood was taken for ELISA measurement of hIL-10 and tissue for myeloperoxidase (MPO) measurement and histological assessment. To evaluate graft survival, groups A2 and B2 were sacrificed at cessation of the heart beat of the graft. RESULTS: Histological examination revealed severe rejection (IIIB-IV) in group A1 in contrast to low to moderate rejection (IA-IIIA) in group B1 (p = 0.02). MPO activity was significantly lower in group B1 compared to group A1 (18 +/- 7 vs. 32 +/- 14 mU/mg protein, p = 0.05). Serum hIL-10 levels were 46 +/- 13 pg/ml in group B1 vs. 0 pg/ml in group A1. At day 7 all heart allografts in the treated groups B1 and B2 were beating, whereas they stopped beating at 5 +/- 2 days in groups A1 and A2 vs. 14 +/- 2 days in group B2 (p = 0.0012). CONCLUSIONS: Electroporation-mediated intramuscular overexpression of hIL-10 reduces acute rejection and improves survival of heterotopic heart allografts in rats. This study demonstrates that peripheral overexpression of specific genes in skeletal muscle may reduce acute rejection after whole organ transplantation.

Poor sleep is associated with higher plasma proinflammatory cytokine interleukin-6 and procoagulant marker fibrin D-dimer in older caregivers of people with Alzheimer's disease

OBJECTIVES: To determine whether objective measures of sleep correlate with plasma levels of the proinflammatory cytokine interleukin (IL)-6 and the procoagulant marker fibrin D-dimer in caregivers of patients with dementia. DESIGN: Cross-sectional study. SETTING: Subjects' homes. PARTICIPANTS: Sixty-four community-dwelling spousal caregivers (69% women, mean age+/-standard deviation 72+/-9) and 36 sex-matched noncaregiving controls. MEASUREMENTS: All participants underwent in-home full-night polysomnography. Demographic and lifestyle factors, depression, diseases, and medication that could affect inflammation, coagulation, and sleep were controlled for in analyses regressing sleep variables and caregiver status and their interaction on plasma levels of IL-6 and D-dimer. RESULTS: Caregivers had higher levels of D-dimer (781+/-591 vs 463+/-214 ng/mL, P=.001) and IL-6 (1.42+/-1.52 vs 0.99+/-0.86 pg/mL, P<.06) and lower levels of total sleep time (369+/-70 vs 393+/-51 minutes, P=.049) and sleep efficiency (77+/-11 vs 82+/-9%, P=.04) than controls. After controlling for age and body mass index, longer wake time after sleep onset (change in coefficient of determination (DeltaR2)=0.039, P=.04) and the interaction between caregiver status and higher apnea-hypopnea index (DeltaR2=0.054, P=.01) were predictors of IL-6. Controlling for age, caregiver status independently predicted D-dimer levels (DeltaR2=0.047, P=.01). Controlling for age and caregiver status, lower sleep efficiency (DeltaR2=0.032, P=.03) and the interaction between caregiver status and more Stage 2 sleep (DeltaR2=0.037, P=.02) independently predicted plasma D-dimer levels. CONCLUSION: Poor sleep was associated with higher plasma IL-6 and D-dimer levels. These effects were most pronounced in caregivers of subjects with Alzheimer's disease. The findings suggest a mechanism that may explain how disturbed sleep might be associated downstream with cardiovascular risk, particularly in older people under chronic stress.