Combined effects of PPARγ<Subscript>2</Subscript> P12A and PPARα L162V polymorphisms on glucose and insulin homeostasis: the Québec Family Study

Peroxisome proliferator-activated receptors ₂ and are nuclear factors known to be important regulators of lipid and glucose metabolism. Two polymorphisms, namely PPAR₂ P12A and PPAR L162V, were investigated for their individual and interaction effects on glucose and insulin homeostasis. Genotypes were determined in 663 nondiabetic adults participating in the Québec Family Study and who underwent an oral glucose tolerance test (OGTT). The insulin and C-peptide areas under the curve (AUC) following the OGTT were higher in subjects carrying the PPAR V162 allele compared to homozygous for the L162 allele. When subjects were grouped according to both polymorphisms, higher levels of insulin and C-peptide during the OGTT were observed for those carrying the PPAR V162 allele except when they carry at the same time the PPAR₂ A12 allele. Thus, the PPAR₂ A12 allele seems protective against the deleterious effect of the PPAR V162 allele. Furthermore, a significant gene-gene interaction was observed for the acute (0–30 min) (p<0.001) and the total (p=0.05) C-peptide AUC following the OGTT. These results provide evidence of a gene-gene interaction in the regulation of plasma glucose-insulin homeostasis, and emphasize that these interactions need to be taken into account when dissecting the genetic etiology of complex disorders.     

Single nucleotide variants in the β2-adrenergic and β3-adrenergic receptor genes explained 18.3% of adolescent obesity variation

Associations of obesity with its candidate genes, -adrenergic receptor genes (ADRBs), peroxisome proliferator-activated receptor- (PPAR), and uncoupling proteins (UCPs) were studied in Korean adolescents. We analyzed the obesity-related phenotypes body mass index (BMI), percentage of body fat, plasma leptin and insulin levels, fasting glucose concentration, and plasma lipid profile in 329 teenagers to investigate the effects of seven single nucleotide variants 252G/A, 523C/A and 1053G/C in ADRB2; Trp64Arg in ADRB3; 161C/T in PPAR; Ala55Val in UCP2; and 210C/T in UCP3. The 1053G/C polymorphism (P < 0.05) in the ADRB2 gene and the Trp64Arg polymorphism (P < 0.01) in the ADRB3 gene were associated with BMI after adjustment for dietary energy intake. Trp64Arg polymorphism also influenced percentage of body fat (P < 0.01) and plasma leptin level (P < 0.05). Furthermore, significant interaction effects between the 1053G/C and Trp64Arg polymorphisms were observed on BMI (P < 0.01). The polymorphisms of the ADRB2 and ADRB3 genes explained 4.3% and 10.1% of the variation on BMI, and the two loci effect, including their epistasis, explained 18.3%. We concluded that 1053G/C and Trp64Arg polymorphisms of the ADRB genes additively and interactively contributed to the variation of complex adolescent obesity.     

Interferon-γ for treatment of severe atopic eczema in two children

Two 4- and 5-year-old children suffering from refractory atopic dermatitis were treated with recombinant interferon- (rIFN-). rIFN- was injected at 50 g subcutaneously three times a week in the first child for 3 weeks, followed by three times 25 g in week 4. In the other child two treatment courses of 4 weeks were given after a break of 2 weeks. Therapy was well tolerated. In child one reductions in eczematous body surface and severity of lesions were observed, while no beneficial effect was seen in the other. Clinical chemistry data remained unchanged. Immunological studies performed in parallel showed a decrease in total serum IgE of 50% in child 1, a decrease in spontaneous in vitro IgE production, an increase in in vitro production of interleukin-6, and a normalization of previously decreased in vitro lymphocyte responses to several mitogens. While marked immunological changes were noted during IFN- treatment, clinical benefits were not encouraging. Diminished IFN- production has been claimed to be a major pathogenic factor in atopic eczema. Our results indicate that the pathogenesis is more complex. Clinically, we were unable to confirm previous observations in adults. Further studies are needed before IFN- can be recommended for therapy of pediatric atopic eczema.Abbreviations IFN- interferon- - IL interleukin     

Monoclonal immunoglobulins on disc electrophoresis

Summary A transparent upper gel disc electrophoretic technique was applied for the detection of mono- and oligo-clonal immunoglobulins. This method allows the visualization and densitometrical recording of those proportions of IgG and IgM retained in the upper concentration gel. WhileA-globulins generally reach the lower separation gel, manyG-globulins are retained in the transparent upper gel. In several cases, two or three abnormal bands were detected in sera containing a singleM-paraprotein on immunoelectrophoresis.     

The role ofγδ T cells in the normal and disordered immune system

Summary A small population of T cells does not express the conventional T cell receptor characterized by the and polypeptide chains (TCR) but instead, two polypeptides termed and (TCR). This alternative receptor is able to recognize antigen. It appears early in T cell ontogeny, but its role in the thymus prior to the availability of TCR remains unclear. In selected sites such as skin or gut TCR predominates in mice which might suggest a role of T cells in the first line of defense against infection, T cells secrete lymphokines and display cytotoxic activity. However, their activation requirements may differ from what is known for T cells since MHC-nonrestricted and also CD4 and CD8 negative T cells have been described. Preferential activation by mycobacterial antigens possibly indicates a special repertoire of the T cells. In various diseases slightly increased numbers of T cells were found, but these preliminary studies have not yet provided evidence for a major pathogenetic role of T cells.List of abbreviations C constant region (immunoglobulin or TCR gene segment) - CD4 cluster of differentiation 4 (mainly on helper cells) - CD8 cluster of differentiation 8 (mainly on cytotoxic cells) - D diversity region (immunoglobulin or TCR gene segment) - DNA desoxyribonucleic acid - IL2 interleukin 2 - J joining region (immunoglobulin or TCR gene segment) - kD kiloDalton - MHC major histocompatibility complex - NK natural killer (cells) - RA rheumatoid arthritis - TCR T cell receptor - V variable region (immunoglobulin or TCR gene segment)     

Secretory characteristics of pancreatic γ-glutamyl transpeptidase

Summary In an attempt to identify the secretory mechanism of pancreatic -glutamyl transpeptidase (-GTP), constant intravenous infusions of secretin alone and in combination with caerulein were performed in anesthetized dogs prepared with a pancreatic fistula. Caerulein produced a marked increase in amylase concentration and only a slight increase in -GTP. -GTP concentration of the pancreatic juice varied from 12 to 490 mU per ml which ranged up to 188-fold higher than that of the serum. The enzyme concentration depended largely on the flow rate, revealing 3 characteristic curvlinear relationship, regardless of whether caerulein was added to the secretin infusion. No significant relation was demonstrated between amylase concentration and flow rate, amylase and -GTP concentrations, and -GTP and protein concentration. An inverse linear correlation between -GTP and chloride concentrations was obtained when flow rate was below 2.5 ml per 15 min. A significant linear relationship was demonstrated between -GTP and leucine aminopeptidase concentrations, and amylase and protein concentrations. The results presented clearly demonstrate that the mechanism of pancreatic secretion of -GTP is quite distinct from that of amylase.Supported in part by Cancer Research Grant from the Welfare Ministry of Japan.     

Association between serum-soluble CD8 levels and parameters of immune activation in patients with human immunodeficiency virus infection

Summary We compared the serum concentrations of soluble CD8 with the immune activation markers neopterin, interferon-, tumour necrosis factor-, soluble CD4, and with CD34⁺ and CD38⁺ T-cell counts in patients with human immunodeficiency virus (HIV) infection. The majority of patients had increased concentrations of soluble CD8, interferon- and neopterin, and various significant correlations existed between them. Our results support the view that enhanced soluble CD8 levels indicate activated CD8⁺ T cells in patients with HIV infection.Abbreviations sCD8 serum-soluble CD8 - sCD4 serum-soluble CD4 - IFN- interferon- - TNF =tumour necrosis factor- - HIV human immunodeficiency virus - AIDS acquired immunodeficiency virus     

Altered synthesis of interferon-γ and expression of interferon-γ receptor by peripheral blood mononuclear cells from patients with IgA nephropathy and non-IgA proliferative glomerulonephritis

Previously we reported disease-specific interaction between interferon- (IFN-) and interleukin-4 (IL-4) in patients with IgA nephropathy (IgAN), suggesting the existence of unusual T cell behavior in this disease. In the present study, we investigated characteristic synthesis of interferon- (IFN-) and expression of IFN- receptor (IFN-R) in the peripheral blood mononuclear cells (PBMC) from patients with IgAN and other chronic proliferative glomerulonephritis (PGN). Heparinized peripheral blood samples were obtained from 38 patients with chronic mesangial proliferative glomerulonephritis (CGN; including 24 with IgA nephropathy) and 20 healthy controls. PBMC were isolated by gradient centrifugation and fragments were cultured in Iscove's modified Dulbecco's medium (IMDM) supplemented with 10% fetal calf serum (FCS) for 72 hr. IFN- concentrations in supernatants were evaluated by the enzyme-linked immunosorbent assay (ELISA). Other parts of PBMC pellets were reacted with anti-human IFN-R monoclonal antibody and FITC-labeled anti-mouse second antibody for analysis of IFN-R expression on these cells by FACScan. The remaining PBMC were fractionated into CD4⁺ T cells, CD8⁺ T cells, B cells, NK, cells and macrophages using the MACS cell sorting system. The isolated cells were evaluated for IFN- or IFN-R mRNA expression by the semiquantitative RT-PCR method.In vitro IFN- synthesis was enhanced in patients with CGN, and NK cells were revealed to be responsible for such enhancement. On the other hand, the expression of IFN-R on macrophages was suppressed in CGN patients. These results suggest that impairment of regulation of the IFN- system might be involved in the development of CGN.     

Increased susceptibility of male BALB/c mice to coxsackievirus B3-induced myocarditis: role for CD1d

BALB/c male mice infected with the H3 variant of coxsackievirus B3 (CVB3) develop fulminant myocarditis. Age-matched female mice show little myocarditis due to decreased virus receptor expression on cardiac cells. TNF and IL-1 levels were increased in males by 3 days after infection. IFN levels increased more slowly throughout the 7-day observation period. CD4⁺, CD8⁺, macrophage (Mac3⁺) and ⁺ cells all accumulated in male hearts, with ⁺ cells showing early (day 3) infiltration. Females also accumulated CD4⁺ cells, but few of the other cell types. CD4⁺ cells in male hearts predominately produced IFN, indicating a Th1 cell phenotype, whereas CD4⁺ cells in females produced IL-4, but little IFN, indicating a Th2 phenotype. CD1d, a major histocompatibility complex I-like molecule often implicated in innate immunity, was increased in CVB3-infected male but not female cardiocytes both in vivo and in vitro. These results demonstrate that CVB3 infections produce gender-specific differences in both innate and adaptive immunity, which may explain the difference in disease susceptibility.     

Increase in TCRγδ T lymphocytes in synovia from rheumatoid arthritis patients with active synovitis

To determine the relative presence of TCR+ and TCR+ T cells in synovial tissue from patients with various types of inflammatory synovitis and in tissues from patients with a number of chronic T cell-mediated conditions, we stained frozen tissue sections with monoclonal antibodies in indirect immunofluorescence assays. In tissues obtained from patients with chronic T cell-mediated granulomatous conditions (Wegener's granulomatosis, lymphomatoid granulomatosis, granuloma annulare, Langerhan's cells granulomatosis, pigmented villonodular synovitis, Takayasu's arteritis, and talc granulomatosis), the T cells present were predominantly TCR+, without an increased presence of TCR+ cells. In contrast, 6 of 14 (43%) synovia from patients with rheumatoid arthritis (RA) showed increased TCR+ T cells (3–10 cells/hpf). The RA synovia with increased TCR+ cells present had an increased tissue inflammation score compared to RA synovia with few TCR+ cells [18.6±5.8 versus 11.6±4.2 (mean±SE),P<0.05]. In contrast, synovia from patients with osteoarthritis, systemic lupus erythematosus, and trauma did not show an increased presence of TCR+ T cells. Thus, in cellular inflammatory infiltrates the presence of increased TCR cells is not a component of noninfectious granulomatous inflammation but is found in approximately 40% of RA synovia with high levels of inflammation.     

Human T-lymphocyte subset production of immune (γ) interferon

Human T, T, and T lymphocyte subpopulations have the capacity to respond to phytohemagglutinin (PHA)in vitro with proliferation and the production of a pH 2 and heat-labile interferon. This occurs both when the subsets are isolated by direct rosetting techniques or by negative selection. Macrophages enhance the production of the interferon by each lymphocyte subset and do not themselves produce interferon in response to products of PHA-activated lymphocyte subsets. Thus our studies indicate that subpopulations of T lymphocytes known to differ with regard to morphology, surface receptors, RNA content, response to corticosteroids and X-irradiation, and other functional capabilities do not differ with regard to their capacity to produce interferon.     

Isoenzymes of γ-glutamyl transpeptidase in liver diseases

Summary A new method for the separation of isoenzymes of-glutamyl-transpeptidase is described, using electrophoresis on acetate cellulose gel and a developing solution composed by-glutamyl-naphthylamide, and a colored diazonium compound.The method permits the separation of up to four different isoenzymes, which we called-GT₁,-GT₂,-GT₃,-GT₄, the first two showing an electrophoretic migration similar to that of ₁- and ₂-globulins and the other two to that of-globulins.The present technique has proved its usefulness in detecting isoenzymes in serum with values of total-glutamyl-transpeptidase higher than 80 U/L.The application of this method in 52 patients with different types of biliary obstruction and hepatocellular damage has shown that it provides new possibilities in differential diagnosis.     

Combination therapy of cyclosporine with steroid inhibits γ-interferon and interleukin-1 gene expression at the level of mRNA synthesisin vivo

The present study examined the effect of cyclosporine (CsA) administered with steroidin vivo on the capacity of peripheral blood mononuclear cells (PBMC) from kidney transplant recipients to generate cytokines and their gene expression at the level of messenger RNA (mRNA). PBMC from CsA-prednisolone (Pred)-treated recipients displayed 66.9% inhibition (54.3±12.4 IU/ml;N=42;P<0.01) of -interferon (-IFN) production compared with normal individuals (134.6±18.6 IU/ml;N=23). Azathioprine (Az)-Pred-treated recipients displayed significantly less inhibition of -IFN generation (96.0±16.1 IU/ml;N=22;P<0.05) than CsA-treated patients. Macrophages (m) from CsA-Pred-treated recipients displayed 60.0% inhibition (5.1±0.7 U/ml;N=20;P<0.01) of interleukin-1 (IL-1) production compared with normal individuals (13.0±2.9 U/ml;N=21). These results were confirmed by the experiments using cDNA probe for -IFN or IL-1 (, ). High levels of -IFN mRNA in phytohemagglutinin (PHA)-stimulated PBMC or IL-1() mRNA in lipopolysaccharide (LPS)-stimulated m were present in normal individuals but not in CsA-treated recipients as judged by hybridization to a cloned human -IFN or IL-1() cDNA probe. These studies demonstrated that combination therapy of CsA with steroid inhibits both -IFN and IL-1 gene expression at the level of mRNA at physiological concentrations.     

Synoviocytes in chronic synovitis in situ and cytokine stimulated synovial cells in vitro neo-express α1, α3 and α5 chains of β1 integrins

The expression of the 1 integrins was examined immunohistochemically in synoviocytes from normal synovial membrane and from chronic synovitis of different aetiology and intensity. Normal synoviocytes were 61-positive but lacked 1 through 5. In mild inflammation type A synoviocytes neo-expressed 1, 3, and 5 chains. In severe inflammation both type A and B synoviocytes expressed 3, 4, 5, and 6 chains. The effects of inflammatory cytokines, as single agents or in combination, on the 1 integrin expression in cultured normal synoviocytes was determined by immunocytochemistry and flow cytometry. The 1 chain, while absent in unstimulated synoviocytes, was induced by interleukin-1 (IL-1), tumour necrosis factor- (TNF-), and interferon- (INF-). This effect was enhanced by combining IL-1 and TNF-. Expression of the 3 chain was up-regulated by IL-1 and, more intensely, by IFN-. Transforming growth factor (TGF-) inhibited the up-regulating effect of IL-1 and antagonized the effect of IFN- on 3 chain expression. Expression of the 5 chain was up-regulated significantly by co-stimulation through IL-1 together with TGF- or TNF-. Thus, the 1 integrin profile of cytokine activated synoviocytes in vitro resembled that of synoviocytes in synovitis in situ. These data suggest that IL-1, TNF-, IFN-, and TGF- are likely to be among the effectors regulating 1 integrin expression in synoviocytes in vivo.     

Intravenous immunoglobulin induces interferon-γ and interleukin-6in vivo

Immunoglobulin is known to be an immunomodulator. It can induce protein mediators from mononuclear cells, particularly monocytesin vitro. Intravenous immunoglobulin (IVIg) has been used as a therapy in several clinical situations. In this study, the influence of IVIg infusion on the plasma levels of two protein mediators, interferon- (IFN-) and interleukin-6 (IL-6), was assessed in patients with secondary generalized epilepsy. Compared to preinfusion levels, plasma interferon- was increased in 18 of 18 patients 20 min after the 6- to 8-hr infusion of IVIg. Plasma interferon- levels reached their peak at various times from 20 min to 3 days post IVIg infusion, dependent upon the individual patient. Plasma IL-6 levels also increased after IVIg infusion. Generally, IL-6 reached its peak level after IFN-. No activated T cells or B cells were observed as determined by the expression of surface CD25, CD23, and HLA-DR 20 min following the infusion when the IFN- and IL-6 levels were assessed. The expression of the high-affinity receptor for IgG, CD64, on monocytes was significantly enhanced after IVIg infusion, while the low-affinity receptor for IgG, CD32, was only slightly increased. Cytoplasmic staining of PBMC indicates that both CD16-positive and CD16-negative cells may contribute to the increase seen in plasma IFN-. These data raise the possibility that the therapeutic effects of intravenous immunoglobulin may be related, at least in part, to the immunomodulatory activity as demonstrated by the changes in plasma levels of IFN- and IL-6.     

Enhancement of interleukin-2 and γ-interferon productionin vitro on cord blood lymphocytes andin vivo on primary cellular immunodeficiency patients with thymic extract (Thymostimulin)

Cord blood mononuclear cells (MNC) were isolated from 20 normal full-term newborns. These MNC were preincubated with either 50, 100, or 200 µg/ml Thymostimulin or without Thymostimulin. The interleukin-2 (IL-2) and -interferon (-IFN) production, cytotoxicity, and lymphoproliferation and IL-2 receptor (Tac) expression were all significantly increased after Thymostimulin treatment. For evaluation of thein vivo effect, two combined-imimunodeficiency patients defective on the thymic level, one with progressive BCG infection, and one with DiGeorge syndrome were used. Before Thymostimulin treatment, the patient's MNC did not produce sufficient amounts of IL-2 and -IFN. The cytotoxicity and lymphoproliferation were also low. After Thymostimulin treatment, the IL-2 and -IFN production, cytotoxicity, and lymphoproliferative response were enhanced. These results suggest that Thymostimulin may be beneficial in the clinical treatment of primary cellular immunodeficiency. The improved immune reactivity including cytotoxicity and enhanced IL-2 and -IFN production in the Thymostimulin treatment also indicates that there may be a beneficial effect on the combination of chemotherapy and Thymostimulin.     

Localization of tissue transglutaminase and N (epsilon)-(gamma) -glutamyl lysine in duodenal cucosa during the development of mucosal atrophy in coeliac disease

Expression and transamidation activity of tissue transglutaminase (tTG) may be involved in the morphological modifications leading to the mucosal atrophy observed in coeliac disease (CD). We aimed to investigate the localization of tTG within the duodenal mucosa during the development of villous atrophy. The localization and level of expression of N-(-glutamyl) lysine isopeptides which could reflect the transamidation activity of tTG were also analyzed. tTG and N-(-glutamyl) lysine were localized using an immunohistochemical technique on duodenal biopsies obtained from 75 patients with CD and 51 subjects with normal mucosa (control group). The number of cases displaying tTG-expressing cells in the basement membrane and lamina propria was significantly higher in CD patients than in the control group. Moreover, the intensity of tTG staining in these areas was higher in CD. In contrast, the number of biopsies with tTG-expressing enterocytes was significantly lower in CD than in the control group. There was no difference in N-(-glutamyl) lysine between the two populations. Tissue transglutaminase was differently expressed in the various areas of the mucosa according to the stage of atrophy, whereas the localization and the intensity of the labelling of N-(-glutamyl) lysine isopeptides did not show any modification. The preferential localization in the basement membrane and lamina propria may reflect the involvement of tTG in the development of mucosal atrophy in CD.     

Low In Vitro Production of Interferon-γ and Tumor Necrosis Factor-α in HIV-Seronegative Patients with Pulmonary Disease Caused by Nontuberculous Mycobacteria

We studied 32 HIV-seronegative patients with pulmonary disease caused by nontuberculous mycobacteria (NTM). Immunologic studies included lymphocyte subset analysis by flow cytometry, measurement of interferon- (IFN-) and tumor necrosis factor- (TNF-) production followingin vitro stimulation of diluted whole blood (DWB) and peripheral blood mononuclear cells (PBMC) by phytohemagglutinin (PHA), anti-CD3 as well as purified protein derivative of tuberculin (PPD), and in four cases with different amounts of the very mycobacterium, which caused disease in these patients. Data were compared to those of 30 HIV-seronegative patients with disease byMycobacterium tuberculosis (MTb). Following -CD3-stimulation of PBMC, NTM patients showed lower IFN-(P < 0.00005) and lower TNF-(P < 0.02). For a subgroup of tuberculin skin test-positive NTM patients we found significantly lower PPD-induced IFN- releases in cultured DWB(P < 0.0002) and PBMC(P < 0.0004) compared to MTb patients. Data for PPD-induced TNF- release for this subgroup were also significant(P < 0.001 andP < 0.05, respectively). The four NTM patients with poor PPD-induced IFN- response hardly showed increased cytokine production on stimulation with their specific mycobacterium. The lower production capacity of IFN- and TNF- of NTM patients compared to the MTb patients points to an immunologic imbalance forming the basis for their increased susceptibility to pulmonary infections by nontuberculous mycobacteria.     

Two types of potassium channel regulated by ATP in pancreatic B cells isolated from a type-2 diabetic human

Two types of K channel regulated by ATP were observed in pancreatic cells from a type-2 diabetic man. One type had a conductance of 67 pS at-70 mV in symmetrical 140 mM KCl and was inhibited by intracellular ATP with a half-maximal concentration of 40 M. ATP inhibition was antagonised by ADP. Tolbutamide inhibited the whole-cell K currents half-maximally at 25 M. This channel has properties similar to those found for the ATP-sensitive K channel in rodent and normal human cells. The second channel type observed was an ATP-activated K channel. It had a conductance of 37 pS at -70 mV in symmetrical 140 mM KCl and was activated half-maximally by 9 M intracellular ATP. This channel was unaffected by 1 mM tolbutamide. In cell-attached patches, one cell out of four tested responded to 20 mM glucose with depolarization. The role of the ATP-activated K channel with respect to the (patho)physiology of the cell is uncertain.     

γ-Glutamyltranspeptidase-Aktivität im Urin bei Gesunden und Nierenkranken

Zusammenfassung Die -Glutamyltranspeptidase-Aktivität (-GT) in der Niere ist bei chronischen Nephropathien und beim akuten Nierenversagen vermindert. Bei Gesunden wurde im Urin eine -GT-Aktivität von 12,7–37,7 mU/min ermittelt. Serum- und Urinaktivität verhielten sich unabhängig voneinander. Eine erhöhte -GT-Ausscheidung fand sich bei 75% der Nierenkranken mit normaler GFR, aber nur bei 11% mit eingeschränkter GFR.Zwischen -GT-Ausscheidung und Kreatininclearance bestand bei Gesunden (N=68,r=0,67) und Nierenkranken (N=114,r=0,71) eine statistisch hochsignifikante Korrelation. Bei Gesunden wurden deshalb die Toleranzgrenzen für beide Parameter gemeinsam berechnet. Bei Nierenkranken nahm die Enzymausscheidung proportional zur GFR und damit dem funktionsfähig verbleibenden Nierenparenchym ab. Die Relation -GT/C_Kreatinin erlaubt eine pathologisch gesteigerte Enzymabgabe aus den Tubuluszellen bei noch im Normalbereich liegender Gesamtausscheidung zu erkennen.