Gabriella Misiano

Systemic Immune Responses in Alzheimer's Disease: In Vitro Mononuclear Cell Activation and Cytokine Production

To investigate the systemic signs of immune-inflammatory responses in Alzheimers disease (AD), in the present study we have analyzed blood lymphocyte subsets and the expression of activation markers on peripheral blood mononuclear cells (PBMCs) from AD patients and age-matched healthy controls (HC) activated in vitro by recombinant amyloid-beta peptide (rAbeta42). Our study of AD lymphocyte subpopulations confirms the already described decrease of the absolute number and percentage of B cells when compared to HC lymphocytes, whereas the other subsets are not significantly different in patients and controls. We report the increased expression of the activation marker CD69 and of the chemokine receptors CCR2 and CCR5 on T cells but no changes of CD25 after activation. B cells are also activated by rAbeta42 as demonstrated by the enhanced expression of CCR5. Moreover, rAbeta42 induces an increased expression of the scavenger receptor CD36 on monocytes. Some activation markers and chemokine receptors are overexpressed in unstimulated AD cells when compared to controls. This is evidence of the pro-inflammatory status of AD. Stimulation by rAbeta42 also induces the production of the pro-inflammatory cytokines IL-1beta, IL-6, IFN-gamma, and TNF-alpha, and of the anti-inflammatory cytokines IL-10 and IL-1Ra. The chemokines RANTES, MIP-1beta, and eotaxin as well as some growth factors (GM-CSF, G-CSF) are also overproduced by AD-derived PBMC activated by rAbeta42. These results support the involvement of systemic immunity in AD patients. However, our study is an observational one so we cannot draw a conclusion about its contribution to the pathophysiology of the disease.

Single nucleotide polymorphisms (SNPs) of pro-inflammatory/anti-inflammatory and thrombotic/fibrinolytic genes in patients with acute ischemic stroke in relation to TOAST subtype.

BACKGROUND The genetic basis of complex diseases like ischemic stroke probably consists of several predisposing risk factors, such as genes involved in inflammation and thrombotic pathways. On this basis the aim of our study was to evaluate the role of SNPs (single nucleotide polymorphisms) of some pro-inflammatory/anti-inflammatory and coagulation/fibrinolytic genes in patients with acute ischemic stroke. METHODS The study population consisted of 144 consecutive Caucasian adult patients who were hospitalized in the Internal Medicine Department at the University of Palermo between November 2006 and January 2008, and who met inclusion criteria. The cases were patients admitted with a diagnosis of acute ischemic stroke, and age-matched (± 3 years) control subjects: patients admitted to our Internal Medicine Department for any cause other than acute cardiovascular and cerebrovascular events and for routine checkup examinations. Molecular analysis of alleles at the -308 nucleotide (-308G/A) of TNF-α gene, -1082/-819 haplotypes of IL-10 gene, IL-1RN exon 2 VNR polymorphism, alleles at the -174 nucleotide (-174G/C) of IL-6 gene, PAI-1675 5G/4G polymorphism, alleles at the -7351 nucleotide (-7351C/T) of tPA gene was undertaken in both patient groups. RESULTS We analyzed 96 subjects with acute ischemic stroke and 48 control subjects. We observed a significantly higher frequency of IL-10 1082 AA genotype in stroke patients with a significant risk trend. We also reported a higher frequency in stroke subjects with a significant risk trend of the TPA 7351-CT genotype and of IL-1RN-VNTR 86 bp 2/2 genotype. Moreover, we observed a significant relationship with TOAST subtype only with regard to CC TPA genotype and 1/1 IL-1 VNTR 86 bp and lacunar strokes. CONCLUSIONS Ischemic stroke is a common multifactor disease, which is affected by a number of genetic mutations and environmental factors. Our findings showing a relationship between pro-inflammatory/anti-inflammatory and thrombotic/fibrinolytic genes SNPs and ischemic stroke may contribute to delineate a possible stroke risk profile in subjects with cerebrovascular risk factors.

Visceral adiposity index (VAI) is predictive of an altered adipokine profile in patients with type 2 diabetes.

Aims Although there is still no clear definition of “adipose tissue dysfunction” or ATD, the identification of a clinical marker of altered fat distribution and function may provide the needed tools for early identification of a condition of cardiometabolic risk. Our aim was to evaluate the correlations among various anthropometric indices [BMI, Waist Circumference (WC), Hip Circumference (HC), Waist/Hip ratio (WHR), Body Adiposity Index (BAI) and Visceral adiposity Index (VAI)] and several adipocytokines [Visfatin, Resistin, Leptin, Soluble leptin receptors (sOB-R), Adiponectin, Ghrelin, Adipsin, PAI-1, vascular endothelial growth factor (VEGF), Hepatocyte growth factor (HGF) TNF-α, hs-CRP, IL-6, IL-18] in patients with type 2 diabetes (DM2). Materials and Methods Ninety-one DM2 patients (age: 65.25±6.38 years; 42 men and 49 women) in stable treatment for the last six months with metformin in monotherapy (1.5–2 g/day) were cross-sectionally studied. Clinical, anthropometric, and metabolic parameters were evaluated. Serum adipocytokine levels were assayed with Luminex based kits. Results At the Pearson’s correlation, among all the indices investigated, VAI showed a significant correlation with almost all adipocytokines analyzed [Visfatin, Resistin and hsCRP (all p<0.001); Adiponectin, sOb-R, IL-6, IL-18, HGF (all p<0.010); Ghrelin and VEGF (both p<0.05)]. Through a two-step cluster analysis, 55 patients were identified with the most altered adipocytokine profile (patients with ATD). At a ROC analysis, VAI showed the highest C-statistic [0.767 (95% CI 0.66–0.84)] of all the indices. Conclusions Our study suggests that the VAI, among the most common indexes of adiposity assessment, shows the best correlation with the best known adipocytokines and cardiometabolic risk serum markers. Although to date we are still far from clearly identifying an ATD, the VAI would be an easy tool for clearly mirroring a condition of cardiometabolic risk, in the absence of an overt metabolic syndrome.

Aminobisphosphonate-activated gammadelta T cells in immunotherapy of cancer: doubts no more.

Background: Activated Vγ9Vδ2 T cells are able to kill most tumour cells because of recognition by T cell receptor and natural killer receptors. Objective: We discuss the possibility that the intentional activation of γδ T cells in vivo by aminobisphosphonates may represent a promising target for the design of novel and highly innovative immunotherapy in cancer patients. Methods: The antitumoural effects of γδ T cells both in vitro and in vivo have been demonstrated suggesting a new therapeutic approach for translation into the clinical setting. Results/conclusion: Vγ9Vδ2 T lymphocytes represent a particularly interesting target for immunotherapeutic protocols based on N-aminobisphosphonate administration and several Phase I-II trials are ongoing investigating the activity of zoledronic acid plus IL-2 in solid tumours.

Combined homozygous factor H and heterozygous C2 deficiency in an Italian family

Three of four children in a family have homozygous (less than 1% of normal) deficiency of factor H of the complement system and both parents, who are first cousins, are heterozygous for the same defect. The father and two of the H-deficient siblings also have a partial C2 deficiency. One of the children with combined deficiencies is affected by systemic lupus erythematosus with nephritis. No increased susceptibility to infections has been observed in the family. H deficiency is inherited in an autosomal codominant manner and is independently transmitted from C2 deficiency and HLA haplotypes. In the homozygous state it is associated with very low serum concentrations of B and C3, barely demonstrable as activated molecules. C5 is greatly reduced (less than 5%). Also, properdin and C6–9 are decreased. The findings in this family demonstrate that the occurrence of systemic lupus erythematosus in one of the children affected by a combined deficiency of factor H and C2 raises the question whether this pathology is related to the complete factor H or to the heterozygous C2 deficiency. Complete H deficiency is not necessarily accompanied by overt illness.

Tetracycline inhibits the nitric oxide synthase activity induced by endotoxin in cultured murine macrophages

Here we investigate the effects of tetracycline base and of a semi-synthetic tetracycline derivative, doxycycline, on the induction of inducible nitric oxide synthase and, hence, on the production of nitric oxide (NO) by lipopolysaccharide in J774 macrophage cultured in vitro. The treatment of J774 line with tetracycline base (6.25-250 microM) or doxycycline (5-50 microM) dose-dependently decreased the lipopolysaccharide-stimulated (1 microg/ml) inducible NO synthase activity and, consequently, nitrite formation. For instance, the inhibition was 70% for tetracycline base at 250 microM and 68% for doxycycline at 50 microM. The inhibitory effect of tetracyclines was due neither to a reduction in the viability of the cells, studied as colorimetric 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide (MTT) reduction assay, nor to an indiscriminate inhibition of total protein synthesis, but to a specific decrease in inducible NO synthase protein content in the cells, as attested by the significant reduction of the expression of inducible NO synthase, assayed by sodium-dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. However, no effect of tetracyclines on inducible NO synthase mRNA accumulation could be demonstrated in lipopolysaccharide-stimulated macrophage line, suggesting that the inhibitory effect of tetracyclines on NO synthesis involves post-transcriptional events. The reduction in lipopolysaccharide-stimulated nitrite accumulation produced by tetracyclines was significantly less when they were applied 6 h after lipopolysaccharide and absent 12 h after lipopolysaccharide, indicating that tetracyclines modify an early event in inducible NO synthase activation operating after mRNA transcription. The findings presented in this study indicate that the modulation of NO synthesis is another possible pathway by which tetracyclines may function as anti-inflammatory compounds.

Analysis of IL-6, IL-10 and IL-17 genetic polymorphisms as risk factors for sepsis development in burned patients

Infection risk, sepsis and mortality after severe burn are primarily determined by patient age, burn size, and depth. Whether genetic differences contribute to otherwise unexpected variability in outcomes is unknown. We sought to determine whether there was an association between IL-6, IL-10 and IL-17 polymorphisms with cytokine production and development of sepsis. We evaluated 71 patients with burns ≥15% TBSA and 109 healthy subjects. The genotypes of IL-6 (-174C/G), IL-10 (-819C/T and -1082A/G) and IL-17 (7488T/C) polymorphisms were identified applying polymerase chain reaction protocols. The cytokine levels in serum were determined with enzyme-linked immunoabsorbent assays. Our results demonstrated no significant differences in the genotype frequencies studied between burn patients and healthy subjects. No significant associations were found among IL-6 and IL-17F genotypes and the related cytokine serum levels. Only IL-10 promoter -1082GG genotype was related to an increased IL-10 production in burned patients. In addition, septic subjects bearing -1082G/G genotype have shown the highest and non-septic bearing -1082A/* genotypes the lowest IL-10 serum levels. All together these data seem to indicate that genetically determined individual difference in IL-10 production might influence the susceptibility to septic complications in burned patients and suggest that these markers might be useful in burned patient management.

Relevance of gamma interferon, tumor necrosis factor alpha, and interleukin-10 gene polymorphisms to susceptibility to Mediterranean spotted fever.

ABSTRACT The acute phase of Mediterranean spotted fever (MSF) is characterized by dramatic changes in cytokine production patterns, clearly indicating their role in the immunomodulation of the response against the microorganism, and the differences in cytokine production seem to influence the extent and severity of the disease. In this study, the single nucleotide polymorphisms (SNPs) of tumor necrosis factor alpha (TNF-α) −308G/A (rs1800629) and interleukin-10 (IL-10) −1087G/A (rs1800896), −824C/T (rs1800871), and −597C/A (rs1800872) and the gamma interferon (IFN-γ) T/A SNP at position +874 (rs2430561) were typed in 80 Sicilian patients affected by MSF and in 288 control subjects matched for age, gender, and geographic origin. No significant differences in TNF-α −308G/A genotype frequencies were observed. The +874TT genotype, associated with an increased production of IFN-γ, was found to be significantly less frequent in MSF patients than in the control group (odds ratio [OR], 0.18; 95% confidence interval [95% CI], 0.06 to 0.51; P corrected for the number of genotypes [Pc], 0.0021). In addition, when evaluating the IFN-γ and IL-10 genotype interaction, a significant increase of +874AA/−597CA (OR, 5.31; 95% CI, 2.37 to 11.88; Pc, 0.0027) combined genotypes was observed. In conclusion, our data strongly suggest that finely genetically tuned cytokine production may play a crucial role in the regulation of the immune response against rickettsial infection, therefore influencing the disease outcomes, ranging from nonapparent or subclinical condition to overt or fatal disease.

Effect of partially modified retro-inverso analogues derived from C-reactive protein on the induction of nitric oxide synthesis in peritoneal macrophages

The ability of three modified tetrapeptides, representing fragments of the C‐reactive protein (CRP) sequence and stabilized in the first peptide bond by retro‐inverso modification, to affect the secretion of nitric oxide (NO) was studied in macrophages of BALB/c mice. These tetrapeptides, resembling the aminoacid sequence of tuftsin (CRP I, H‐gThr‐(R,S)mLys‐Pro‐Leu‐OH, ITF 1192; CRP II, H‐gGly‐(R, S)mLys‐Pro‐Arg‐OH, ITF 1127; CRP III, H‐gThr‐(R,S)mLys‐Pro‐Gln‐OH, ITF 1193), were able to induce NO synthesis by peritoneal macrophages in a dose‐dependent manner; the most stimulating dose was 1000 ng ml−1 for CRP II and 100 ng ml−1 for CRP I and CRP III. NO synthesis was not strictly dependent on lipopolysaccharide (LPS) activation. The enhanced effect of retro‐inverso CRP‐related analogues on the expression of iNOS (inducible NO synthase) was confirmed by higher levels of iNOS activity in the cytosol and by the increase in iNOS protein, as evaluated by Western blot analysis, in macrophages stimulated by CPR compared with untreated ones. The production of NO by retro‐inverso CRP‐peptide analogues was significantly inhibited by dexamethasone (20 μm), NG‐monomethyl‐l‐arginine (l‐NMMA) (500 μm) and pyrrolidine dithiocarbamate (PDTC) (100 μm). Retro‐inverso CRP‐peptide analogues stimulated macrophages to produce high levels of interleukin‐1 (IL‐1) and tumour necrosis factor‐α (TNF‐α) in the presence of LPS. Retro‐inverso CRP‐peptide analogues stimulated NO synthesis by the enhancement of endogenously produced IL‐1 and TNF‐α, as the treatment of peritoneal macrophages with LPS in the presence of neutralizing anti‐IL‐1 and anti‐TNF monoclonal antibodies (mAbs) reduced retro‐inverso analogue‐induced NO secretion. Data indicate a predominant role for IL‐1α in the induction of NO secretion by retro‐inverso analogues. These results suggest that retro‐inverso CRP derived analogues act as costimulators of NO and cytokine synthesis in macrophages. The mechanisms by which they cause iNOS induction appear to be strongly dependent on the activation of nuclear factor‐κB (NF‐κB).

Cytokine serum profile in a group of Sicilian Nonagenarians

The aim of our study was to evaluate the possibility of using multiplex analysis of the cytokine profile as a marker for successful aging by comparing cytokine plasmatic levels of a group of Sicilian nonagenarians with those of young controls. We analyzed a panel of 17 cytokines, comprehensive of haematopoietic factors T helper 1 (Th1), Th2, inflammation regulatory cytokines, and chemokines. The assay was carried out using the Luminex system. Interleukin (IL)-6 levels (p = 0.01) were increased in nonagenarians, whereas no modifications of other proinflammatory cytokines and chemokines were observed. Interferon-gamma (IFN-γ) and IL-2 levels are unmodified, suggesting a substantial maintenance of relevant T cell functions. In addition, a significant increase of IL-12 serum levels in nonagenarians versus young controls that might be related to the increase of natural killer (NK) cell functions characterizing aging processes was observed. The analysis of Th2 cytokines show an increase of IL-13 and a reduction of IL-4 levels mirroring the maintenance of some effectors mechanisms of the immunoresponse in advanced ages. Our results suggest that the multiplex analysis of cytokine levels might be useful in defining a successful aging profile.