Prognostic role of PPAR-γ and PTEN in the renal cell carcinoma

Objective: To explore association of peroxisome proliferator-activated receptor gamma (PPAR-γ) and phosphatase and tensin homolog (PTEN) expressions with prognosis of renal cell carcinoma (RCC). Methods: Our study subjects included 87 RCC tissues, 28 paracarcinoma tissues and 21 normal renal tissues. PPAR-γ and PTEN detection was conducted using immunohistochemistry staining. The association of PPAR-γ and PTEN with the clinical parameters and prognosis of RCC was analyzed. Kaplan-Meier method and Cox’s proportional hazards regression model were used for exploring the relation between variables and prognosis. Results: Among normal renal tissues, para-carcinoma tissues and renal cell carcinomas, positive PPAR-γ expression presented with a progressive tendency (P < 0.001), while positive PTEN expression a degressive tendency (P < 0.001). PPAR-γ expressions were closely related to tumor size, clinical stage and lymph node metastases (all P < 0.05). PTEN expressions were in close association with tumor size, Fuhrman grading, lymph node metastases (all P < 0.05). PPAR-γ expressions were in a negative relation with PTEN expressions (r = -0.417, P < 0.001). Negative PPAR-γ expressions confer a significantly higher overall survival rate than positive PPAR-γ expressions (P = 0.015), while negative PTEN expressions confer a significantly lower overall survival rate than positive PTEN expressions (P = 0.003). Clinical staging, Fuhrman grading, lymph node metastases, PPAR-γ and PTEN were independent prognostic factors for prognosis (all P < 0.05). Conclusion: PPAR-γ and PTEN expressions are related to the clinical parameters and prognosis of RCC and may be a biomarker for prognosis of RCC.     

Modulation of lipopolysaccharide-induced memory insult, γ-secretase,and neuroinflammation in triple transgenic mice by 5-lipoxygenase

Besides amyloid and tau pathology, a constant feature of Alzheimer's disease (AD) is an intense inflammatory response, which is considered an active player in its pathogenesis. The 5-Lipoxygenase (5LO) is a proinflammatory enzyme and an endogenous modulator of AD-like phenotype in mouse models of the disease. To further understand the role of 5LO in AD pathogenesis, we exposed the triple transgenic (3×Tg) and 3×Tg/5LO knockout mice to lipopolysaccharide (LPS), a known inducer of neuroinflammation, and evaluated its effect on their AD-like phenotype. 3×Tg mice treated with LPS manifested a worsening of behavior, γ-secretase up-regulation, and increased neuroinflammatory responses. These effects were completely prevented in 3×Tg mice genetically deficient for 5LO. By contrast, the absence of 5LO did not protect against increase in tau phosphorylation at specific epitopes that were mediated by the activation of the cyclin-dependent kinase 5. Our data demonstrate that the 5LO pathway affects key neuropathological features of the AD-like phenotype (behavior, abeta, microgliosis, astrocytosis) but not others (tau pathology) in the LPS-dependent neuroinflammation model. The opposite ways whereby 5LO influences the LPS-dependent effects in vivo supports the complex nature of the neuroinflammatory response in AD and its differential role in modulating amyloid and tau neuropathology.     

Agonists of PPAR-α, PPAR-γ, and RXR inhibit the formation of foam cells from macrophages in mice with inflammation

We studied the effect of agonists of peroxisome proliferator-activated receptors α and γ and retinoid X receptors on the concentration and synthesis of lipids in macrophages of C57Bl/6 mice with inflammation induced by intraperitoneal injection of zymosan. We revealed a significant increase in [1-¹⁴C]oleate incorporation into cholesterol esters and triglycerides, increase in the content of free cholesterol, cholesterol esters, and triglycerides, and formation of oil red-stained lipid inclusions in peritoneal macrophages 24 h after administration of zymosan in a dose of 50 mg/kg. Treatment with agonists of retinoid X receptors and peroxisome proliferator-activated receptors α and γ 30 min before and 12 h after zymosan injection decreased the synthesis of triglycerides and cholesterol esters, reduced the content of free cholesterol, cholesterol esters, and triglycerides in macrophages, and prevented the formation of cytoplasmic lipid inclusions in macrophage-derived foam cells during inflammation. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 144, No. 11, pp. 556–559, November, 2007     

Additive renoprotective effects of B2-kinin receptor blocker and PPAR-γ agonist in uninephrectomized db/db mice

We recently showed that the bradykinin B2 receptor (B2R) blocker icatibant (Icat) and the peroxisome proliferator-activated receptor-γ agonist rosiglitazone (Ros) exerted anti-inflammatory effects in renal tubular cells exposed to a diabetic milieu. This study aims to explore whether these effects can be translated to an experimental model of type 2 diabetic nephropathy (DN). db/db mice and their nondiabetic db/m littermates underwent sham operation or uninephrectomy (Unx) at 10 weeks and received vehicle (Veh), metformin (Met), Icat, Ros, or Icat plus Ros for 8 weeks before killing. Among the db/db group with Unx, mice that received Icat or Ros had significantly lower serum creatinine and albuminuria, which was further reduced when Icat and Ros were given in combination. These beneficial effects were not observed in the Met group that achieved similar glycemic control as Ros-treated animals. Likewise, the severity of reactive glomerular and proximal tubular hypertrophy, glomerulosclerosis, interstitial injury, cortical F4/80 and α-smooth muscle actin immunostaining, and CCL-2, ICAM-1 and TGF-β overexpression were all attenuated by Icat and Ros, and these effects were enhanced when both agents were combined. Immunohistochemical staining confirmed the proximal tubular expression of CCL-2 (inflammation) and TGF-β (fibrosis). Treatment with Icat was associated with decreased B2R, but increased, B1R expression, which was exaggerated in Unx animals. At the signaling level, Icat and Ros reduced extracellular signal-regulated kinase 1/2 and STAT1 activation, respectively. Our results suggest a deleterious role of the kallikrein-kinin system in murine-accelerated DN, which can be ameliorated by the B2R blocker Icat and enhanced by the addition of Ros. This calls for further evaluation of this novel therapeutic approach in more animal models of diabetic nephropathy.     

Immune modulation of ovalbumin-induced lung injury in mice using β-glucosylceramide and a potential role of the liver

CD1d-restricted natural killer T (NKT) cells are implicated in the pathogenesis of asthma. β-Glucosylceramide (GC), a naturally occurring lipid, was previously shown to alter NKT cell distribution in the liver. We hypothesized that GC can affect lung and liver NKT cell distribution and ameliorate asthma. Mice were sensitized by intra-peritoneal injection of ovalbumin (OVA) for 2 weeks followed by repeated intranasal OVA challenges to induce lung injury mimicking asthma. OVA induced asthma groups were either treated by intranasal instillation of normal saline, intranasal instillation of GC or inhaled budesonide. To investigate the role of the liver, hepatic fibrosis was induced using carbon tetrachloride prior to asthma induction. Allergen induced bronchoconstriction was measured prior to sacrifice. Isolated lymphocytes from lungs, livers and spleens were analyzed for OVA induced proliferation and flow cytometry. Liver and lung histology, serum aminotransferase and anti-OVA antibodies level were assessed. Treatment with GC significantly reduced OVA induced airway responsiveness (p < 0.001) similar to inhaled budesonide. GC significantly reduced the peri-bronchial and peri-vascular inflammatory infiltration mainly through an effect on T cells, as suggested by decreased T cell proliferation (p = 0.009). Liver CD4 and NKT cells significantly increased after GC treatment suggesting liver involvement. Inducing hepatic fibrosis blunted the propagation of asthma in spite of sufficient increase of serum anti-OVA titers. GC has an immunomodulatory effect on a murine model of experimental asthma. We also suggest that the liver acts as an immunomodulatory organ and might have a regulatory effect on pulmonary diseases.     

Evaluation of the expression of TLR-2, Dectin-1 and TNF-α level in invasive aspergillosis in cancer mice

Aspergillus fumigatus (A. fumigatus) is an opportunistic fungal pathogen that can cause life-threatening invasive fungal disease (invasive aspergillosis (IA)) in immuno-compromised individuals. In this study, we explored whether the innate resistance to IA in cancer mice has resulted in altered expression of TLR-2 and Dectin-1 in macrophages (flow cytometry) and production of tumour necrosis factor alpha (TNF-α; ELISA) as well as overall mortality. The data demonstrated significant increases in Dectin-1 and TLR-2 on peritoneal macrophages and mortality in cancer mice intravenously infected with A. fumigatus. TNF-α levels were not significantly increased in this group. Probably, IA causes some disorganization in inflammatory responses. We hypothesize that concomitance of IA and cancer may change the micro-environment for local or systemic immune responses. Other complementary studies are required to support our hypothesis.     

PI3Kγ controls leukocyte recruitment, tissue injury, and lethality in a model of graft-versus-host disease in mice

PI(3)Kγ is thought to mediate leukocyte migration to injured tissues and may be important in the pathogenesis of various T-lymphocyte-dependent pathologies, including autoimmune and inflammatory diseases. The present study evaluated the relevance of PI(3)Kγ in donor cells for the pathogenesis of acute GVHD using a model of adoptive transfer of splenocytes from WT or PI(3)Kγ(-/-) C57BL/6J mice to B6D2F1 mice, and mice that received PI(3)Kγ(-/-) cells showed reduced clinical signs of disease, bacterial translocation, tissue injury, and lethality rates. This was associated with reduced production of proinflammatory cytokines and chemokines (TNF-α, IFN-γ, CCL2, CCL3, and CCL5) and reduced infiltration of CD8(+), CD4(+), and CD11c(+) cells in the small intestine. Mechanistically, in addition to decreasing production of proinflammatory mediators, absence or pharmacological blockade of PI(3)Kγ was associated with decreased rolling and adhesion of leukocytes to the mesenteric microcirculation, as assessed by intravital microscopy. Despite decreased GVHD, there was maintained GVL activity when PI(3)Kγ(-/-) leukocytes were transferred into WT mice. In conclusion, PI(3)Kγ plays a critical role in GVHD by mediating leukocyte influx and activation in tissues. PI(3)Kγ inhibitors may be useful in the treatment of GVHD in patients undergoing BMT.     

Localization of phosphatidylinositol 4-phosphate 5-kinase (PIP5K) α confined to the surface of lipid droplets and adjacent narrow cytoplasm in progesterone-producing cells of in situ ovaries of adult mice

Phosphatidylinositol(4,5)bisphosphate (PI(4,5)P2) produced by phosphatidylinositol phosphate 5 kinase (PIP5K) plays not only as a precursor of second messengers in the phosphoinositide signal transduction, but also multiple roles influencing a variety of cellular activities. From this viewpoint, the present study attempted to localize PIP5Kα in the ovaries in situ of adult mice. PIP5Kα-immunoreactivity was confined to the surfaces of lipid droplets (LDs) and their adjacent cytoplasm in progesterone-producing cells of the interstitial glands, corpora lutea and theca interna. The LDs often contained membranous tubules/lamellae along their surfaces and within their interior whose membranes were continuous with those delineating LDs composed of a monolayer of phospholipids and were partially PIP5Kα-immunoreactive. Although granulosa cells of healthy-looking follicles were immunonegative, as the atresia progressed, PIP5Kα-immunoreactivity first appeared in sparsely dispersed dot forms in mural cells of the follicular epithelia, and then were dominant in almost all mural cells that remained after desquamation of the antral cells. The present study provides evidence suggesting that PI(4,5)P2 locally synthesized by PIP5K in LDs is involved in the lipid transfer between lipid droplets (LDs) and the endoplasmic reticulum, which eventually regulates ovarian progesterone production through control of multiple dynamic activities of LDs. It is also suggested that PIP5Kα and PI(4,5)P2 are implicated in the modulation of programmed cell death and/or acquiring the ability of progesterone production in some follicular cells surviving atresia.     

Interferon regulatory factor modulation underlies the bystander suppression of malaria antigen-driven IL-12 and IFN-γ in filaria-malaria co-infection

In areas where polyparasitism is highly prevalent, the impact of multiple parasites on the host response is underestimated. In particular, the presence of helminth infection coincident with malaria profoundly alters the production of malaria-specific IFN-γ, IL-12p70, CXCL9, CXCL10 and CXCL11, cytokines/chemokines known to be critical in mediating malaria-specific immunity. In order to elucidate the mechanisms underlying the suppression of malaria-specific cytokines/chemokines, we assessed the expression of malaria-specific IL-12Rβ1, IL-12Rβ2 and interferon regulatory factor (IRF)-1 in blood obtained from 18 filaria-infected (Fil(+)) and 17 filaria-uninfected (Fil(-)) individuals in a filaria-malaria co-endemic region of Mali. We found that Fil(+) individuals had significantly lower RNA expression of IRF-1 but not IL-12Rβ1 or IL-12Rβ2 in response to malaria antigen stimulation. We also measured the frequency of IL-12-producing DCs from these subjects and found that Fil(+) subjects had lower frequencies of IL-12(+) mDCs after malaria antigen stimulation than did the Fil(-) subjects. Modeling these data in vitro, we found that mDCs pre-exposed to live microfilariae not only produced significantly lower levels of CXCL-9, CXCL-10, IL-12p35, IL-12p40, IL-12p19 and CXCL-11 following stimulation with malaria antigen but also markedly downregulated the expression of IRF-1, IRF-2 and IRF-3 compared with microfilaria-unexposed mDCs. siRNA-inhibition of irf-1 in mDCs downregulated the production of IL-12p70 through repression of IL-12p35. Our data demonstrate that the modulation of IRFs seen in filarial (and presumably other tissue-invasive helminths) infection underlies the suppression of malaria-specific cytokines/chemokines that play a crucial role in immunity to malaria.     

Biodistribution of synthetic thymosin β4 in the serum,urine, and major organs of mice

Thymosin β₄ (Tβ₄) is a peptide of 43 amino acids that was first isolated from the thymus gland and subsequently found to be ubiquitous in nature. Tβ₄ functions mainly as an actin-sequestering molecule in non-muscle cells, where its primary role is to maintain the large pool of unpolymerized G-actin in the cell. Studies on the pharmacokinetics of Tβ₄ in human and other mammals have not been reported so far. In the present study, we have measured Tβ₄ concentrations in serum, urine, and 10 major organs of female Swiss-Webster mice following intraperitoneal administration of 400 μg synthetic Tβ₄. Using a modified enzymatic immu-noassay, our data show a significant increase of Tβ₄ in serum starting 2 min after injection and lasting for 40 min (average: 2.34±0.54μg/ml). High concentrations were found in urine (59.3 ± 7.54μg/ml) at three different time points after injection (20 min, 40 min, and 2 h). Of the 400 μg Tβ₄ administered to mice 83 % was recovered at the end of the study, 44.6% of which corresponded to urine, 1.4% to serum, and 37.5% to the organs. In 50% of the tested organs, the wet weight concentrations of Tβ₄ increased significantly from the first 40 min to 2 h after injection in comparison to their baseline wet weight concentrations. These organs were: the brain (72 μg/g vs 42 μg/g), heart (80 μg/g vs 37 μg/g), liver (15 μg/g vs 9 μg/g), kidneys (65 μg/g vs 28 μg/g), and peritoneal fat (47 μg/g vs 13 μg/g). Wet weight concentrations increased in the thymus (196 μg/g vs 147 μg/g) and muscle (45 μg/g vs 0 μg/g) after 6 h of injection. The spleen showed an increase in wet weight concentrations at the 2 min timepoint (267 μg/g vs 161 μg/g). Ovaries had a biphasic increase at 40 min(72 μg/g vs 62 μg/g) and 24 h (92 μg/g vs 62 μg/g) after Tβ₄ administration. In lungs, the highest wet weight increase after injection (149 μg/g at timepoint 6h) was not higher than its basal wet weight concentration (153 μg/g). These phar-macokinetic studies of Tβ₄ in mice have established that high levels of Tβ₄ are found in the blood following I.P. administration and the kidney rapidly removes the peptide from the circulation. The kinetics of this response should help define the proper scheduling of administration of Tβ₄ during clinical trials in disorders, such as the acute respiratory distress syndrome (ARDS), associated with actin toxicity.     

Disturbances in the secretion of neurotransmitters in IA-2/IA-2β null mice: Changes in behavior,learning and lifespan

Islet-associated protein 2 (IA-2) and IA-2β are major autoantigens in type 1 diabetes and transmembrane proteins in dense core secretory vesicles (DCV) of neuroendocrine cells. The deletion of these genes results in a decrease in insulin secretion. The present study was initiated to test the hypothesis that this deletion not only affects the secretion of insulin, but has a more global effect on neuroendocrine secretion that leads to disturbances in behavior and learning. Measurement of neurotransmitters showed that norepinephrine, dopamine and 5-HT were significantly decreased in the brain of double knockout (DKO) mice (P<0.05 to <0.001). In tests evaluating anxiety-like behavior and conditioned-learning, the DKO mice showed a highly significant increase in anxiety-like behavior (P<0.01 to <0.001) and impairment of conditioned learning (P<0.01) as compared to WT mice. The DKO mice also displayed an increase in spontaneous and induced seizures (P<0.01) and age-related death. Contrary to the generally held view that IA-2 and IA-2β are expressed exclusively in DCV, subcellular fractionation studies revealed that IA-2β, but not IA-2, co-purifies with fractions rich in synaptic vesicles (SV), and that the secretion of dopamine, GABA and glutamate from the synaptosomes of the DKO mice was significantly decreased as was the number of SV (P<0.01). Taken together, these findings show that IA-2β is present in both DCV and SV, and that the deletion of IA-2/IA-2β has a global effect on the secretion of neurotransmitters. The impairment of secretion leads to behavioral and learning disturbances, seizures and reduced lifespan.     

1α,25-Dihydroxyvitamin D3 treatment does not alter neuronal cyclooxygenase-2 expression in the cerebral cortex after stroke

The inducible prostaglandin synthase, cyclooxygenase-2, is upregulated in response to cerebral ischemia and contributes to potentiation of oxidative injury. Cyclooxygenase-2 expression is regulated by retinoic acid receptors, which form heterodimers with vitamin D receptors and vitamin D. In addition, vitamin D has been reported to have neuroprotective qualities. The aim of this study was to examine whether the biologically active vitamin D₃-metabolite 1α,25-dihydroxyvitamin D₃ (1,25-D₃), influences the expression of inducible cyclooxygenase-2 in photothrombotically lesioned brain or is part of an independent neuroprotective mechanism. We compared groups of nonlesioned control rats and infarcted animals, which were treated with either 1,25-D₃ or solvent at different times postlesion. In control animals, cyclooxygenase-2 immunoreactivity was readily evident in almost all cortical neurons of layers II/III as well as in a few pyramidal cells in layer V. Following photothrombotic infarction of the right cortical hindlimb area, there was a significant, but transient, increase in cyclooxygenase-2 labeling which was restricted to neurons of the injured hemisphere in both 1,25-D₃-treated and solvent-treated rats. Highest levels of cyclooxygenase-2 immunoreactivity were seen at 12 and 24 h postlesion, followed by a gradual decrease at later time points. However, no significant differences were detected between 1,25-D₃-treated and solvent-treated lesioned rats, indicating that postischemic neuronal cyclooxygenase-2 upregulation is not influenced by 1,25-D₃. It is concluded that the neuroprotective effect of 1,25-D₃ does not depend on modulations of neuronal COX-2 expression caused by postlesional hyperexcitation.     

Activation of PPAR-γ by Carbon Monoxide from CORM-2 Leads to the Inhibition of iNOS but not COX-2 Expression in LPS-Stimulated Macrophages

The effect of CO on the expression of iNOS and COX-2 was investigated by using a CO-releasing molecule (CORM)-2 in LPS-activated RAW 264.7 cells in vitro. Interestingly, CORM-2 significantly inhibited iNOS (NO) but not COX-2 (PGE₂) expression. PPAR-γ activators such as troglitazone, GW1929, and 15-deoxy-Δ12, 14- prostaglandin J₂ showed preferential inhibitory effect on iNOS over COX-2 expression in LPS-activated macrophages. The same effect was shown in lung tissues (iNOS, COX-2) and serum (NO, PGE₂) when administered of CORM-2 in LPS-induced septic mice, indicating that CO derived from CORM-2 differentially regulates iNOS and COX-2 through PPAR-γ activation under inflammation state.     

Tagging single nucleotide polymorphisms in the PPAR-γ and RXR-α gene and type 2 diabetes risk:a case-control study of a Chinese Han population

Peroxisome proliferator-activated receptor (PPAR-γ),which is mainly involved in adipocyte differentiation, has been suggested to play an important role in the pathogenesis of insulin resistance and atherosclerosis. We investigated the frequencies of two common tagging polymorphisms of the PPAR-γ gene and two of PPAR-α with minor allele frequency (MAF)≥ 0.05 in the Chinese Han population and analyzed the correlation between the different genotypes and the risk of type 2 diabetes mellitus (T2DM). TaqMan assay was performed to test the genotypes in T2DM patients (n = 1,105) and normal controls (n = 1,107). Serum adiponectin concentration was measured by ELISA kit. The variant genotypes rs17817276GG, rs3856806CT and rs3856806CT/TT of PPAR-γ were associated with T2DM, P = 0.023,0.037 and 0.018, respectively. Furthermore, the prevalence of haplotype GT in PPAR-γ was less frequent in the case subjects (0.3%) than in the controls (1.9%) [P < 0.001,OR(95%CI)=0.13 (0.06-0.31)]. Patients with genotype TT of rs3856806 had a higher serum level of adiponectin than those with the genotype CC and CT (P = 0.031 and 0.038, respectively). There was no statistically significant difference between patients and controls in genotype distribution of rs6537944 and rs1045570 of the RXR-α gene. The present study suggests that the variant genotypes in the PPAR-γ gene could decrease the risk for the development of T2DM in the Chinese Han population.     

Cytocomposition of the vitellarium in Khawia sinensis Hsü, 1935 (Cestoda, Caryophyllidea, Lytocestidae): another caryophyllidean species with lamellar bodies and lipids

The vitellarium of the invasive caryophyllidean tapeworm Khawia sinensis Hsü, 1935 from carp Cyprinus carpio L. was examined by means of transmission electron microscopy and cytochemical staining for glycogen with periodic acid–thiosemicarbazide–silver proteinate (PA-TSC-SP). A vitellarium consists of numerous follicles of irregular size that are interconnected by a net of vitelline ducts. Vitelline follicles are composed of vitelline cells at various stages of development that are interconnected by interstitial tissue. Vitelline follicles are surrounded by a cytoplasmic sheath associated with an intercellular matrix. Extensive development of the granular endoplasmic reticulum and Golgi complexes are both involved in the production of shell globules/shell globule clusters and characterise cytodifferentiation of vitellocytes. Nuclear and nucleolar transformation lead to the formation and storage of intranuclear glycogen, a feature specific for the Caryophyllidea. Newly observed within the mature vitellocytes of Khawia sp. is the presence of lamellar bodies and a few lipid droplets. These cytoplasmic inclusions first occur in the mature cells within the follicles and persist in the vitelline cells within vitelloducts and intrauterine eggs. Two types of lamellar bodies are detected: regular lamellar-structured body and irregular lamellar-structured body. None of the lamellar bodies are membrane bound. Results of the present study indicate that the formation of lamellar bodies may be closely related to the endoplasmic reticulum or shell globule clusters. Some of the shell globule clusters are transformed into lamellar body clusters. Ultrastructural features of vitellocytes in K. sinensis are compared with those of other monopleuroid, polypleuroid, and strobilated cestodes.     

Biodistribution of synthetic thymosin α1 in the serum,urine and major organs of mice

Thymosin fraction 5 (TF5), a thymic preparation, has been shown to be an immune-potentiating agent consisting of biologically active polypeptide components with hormone-like activities. Thymosin α₁ (Tα₁) was the first biologically active polypeptide to be purified from TF5 and completely characterized. It is an acidic peptide with an isoelectric point of 4.2 and a molecular weight of 3108. Tα₁ is considered a biological response modifier which amplifies T-cell immunity. In the present study, we have studied some pharmacokinetic properties of Tα₁ by measuring its concentrations in serum, urine and ten major organs of female Swiss-Webster mice following administration of 500 μg Tα₁ intraperitoneally. Using a modified enzymatic immunoassay, our data show a significant increase of Tα₁ in serum 2min after injection and lasting for 2 h (average: 1.55 ± 0.27μg/ml). In urine, at four different time points after injection (20min, 40 min, 2h, 6 h), increased concentrations of Tα₁ were found between 24.2 and 25.4μg/ml (average: 25 ± 0.47 μg/ml). Of the 500μg Tα₁ administered to mice, 8.97% was recovered at the end of the study, of which 2% corresponded to urine, 1.25% to serum (2 ml of serum per mouse), and 5.72% to organs. Since the urine/day volume and the serum volume of any Swiss-Webster mouse is ca 2 ml, additional extrapolation of the above mentioned values could show percentages of recovery close to 40% for urine and 2.5% for serum. In most of the organs, the wet weight concentrations of Tα₁ increased significantly during the first 40 min after injection in comparison to their baseline wet weight concentrations. These organs consisted of the following: thymus (33.1 ± 3.5 μ/g vs 18 μ/g baseline); lungs (7.7±1.1 μg/g vs 1.9 μg/g baseline); spleen (15.6±0.7 μg/g vs 5.6 μg/g); kidneys (6.2±1.1μg/g vs 3.9 μg/g); ovaries (9.2±1.4 μg/g vs 0 μg/g); and peritoneal fat (4±1 μg/g vs 0 μg/g). No significant increases were observed in the liver (1.7±0.1μg/g vs 1.4μg/g) and heart (0.7±0.5μg/g vs 0 μg/g). Increased concentrations of Tα₁ were not detected in the brain and skeletal muscle tissues. These pharmacokinetic studies of Tα₁ in mice indicate that rapid renal excretion of Tα₁ represents a major source of humoral loss following IP administration. Recent preliminary studies in humans confirm that the kidney rapidly releases high levels of Tα₁ in urine in a time frame consistent with that observed in mice.     

Effects of catalase and 1400W on the number of interleukin-4 and interferon-γ secreting spleen cells in mice injected with ovalbumin and alum

Context: Alum is thought to induce inflammation resulting in the release of danger signals such as uric acid (UA) which in turn enhances the immune response to an antigen. Hydrogen peroxide (H₂O₂) is produced as a byproduct in the purine catabolic pathway that leads to the production of UA. In addition, serum nitric oxide (NO) levels are increased in inflammation.

Objective: To further explore the mechanism of action of alum, this study was designed to determine the effects of catalase and 1400W on the number of interleukin-4 (IL-4) and interferon-γ (IFN-γ) secreting spleen cells in mice given ovalbumin (OVA) with alum.

Materials and methods: Groups of BALB/c mice were injected intraperitoneally with alum + OVA, alum, OVA, catalase, or 1400W. Other groups were treated with catalase or 1400W and given alum + OVA. The number of IL-4 and IFN-γ secreting spleen cells were determined at days 4 and 7 postinjection by enzyme-linked immunosorbent spot (ELISPOT).

Results: Catalase and 1400W caused a decrease in the number of IL-4 secreting spleen cells induced by alum + OVA. 1400W caused a decline in the IFN-γ secreting spleen cells induced by alum + OVA. Catalase caused an increase in IFN-γ secreting spleen cells.

Discussion and conclusion: It appears that H₂O₂ and NO are needed for alum-induced production of a T-helper 2 cytokine. NO also appears to be needed, whereas H₂O₂ appeared to inhibit an alum-induced production of a T-helper 1 cytokine. These results might explain why alum is mainly a promoter of a T-helper 2 response.     

Involvement of glutamate NMDA and AMPA receptors, glial cells and IL-1β in the spinal hyperalgesia evoked by the chemokine CCL2 in mice

We study here the involvement of excitatory amino acid receptors, glial cell activation and IL-1β release in the spinal hyperalgesia evoked by the chemokine CCL2 (MCP-1). Three hours after the intrathecal administration of CCL2 (1-100 ng), mice exhibit dose-dependent thermal hyperalgesia, that was inhibited by the coadministration of the antagonist of chemokine receptor type 2 (CCR2) RS504393 (0.3-3 μg). To assess the involvement of excitatory amino acid receptor sensitisation, CCL2 was coadministered with CPP (0.3-3 ng) and NBQX (25-250 ng), antagonists of NMDA and AMPA receptors, respectively. Both drugs blocked CCL2-evoked hyperalgesia, strongly suggesting that CCL2 evokes in vivo NMDA and AMPA receptor sensitisation, as previously described in electrophysiological studies. Furthermore, this rapid induction of CCL2-mediated hyperalgesia was blocked by the previous acute administration of the microglial inhibitor minocyclin (4.9 μg) or the astroglial inhibitor l-aminoadipate (1.6 μg). Since IL-1β can be released by activated glial cells we tested whether this cytokine could be underlying the spinal sensitisation induced by CCL2. The administration of the type I IL-1 receptor antagonist, IL-1ra (3-30 μg), partially prevented CCL2-evoked hyperalgesia. Finally, to elucidate if IL-1β could produce NMDA and AMPA receptor sensitisation by itself, we performed experiments in which this cytokine was i.t. administered. Thermal hyperalgesia induced by IL-1β (30 pg) was completely prevented by the coadministration of CPP (3 ng) but unaffected by NBQX (250 ng). Globally, our results suggest that spinal CCL2 induces thermal hyperalgesia by sensitising NMDA and AMPA receptors in a process that involves glial activation and IL-1β release.