TNF-alpha, IL-4R-alpha and IL-4 polymorphisms in mild to severe asthma from Italian Caucasians.

Asthma is a chronic airway inflammatory disease associated with airway hyperresponsiveness which affects subjects with genetic predisposition. An association has been reported between some polymorphisms in various cytokine genes and asthma. Most of them are single nucleotide polymorphisms (SNPs). These polymorphisms are detected in the protein coding sequence or in the promoter region thus influencing cytokine production. We investigated the involvement of SNP mapping in 5 cytokine genes in mild to severe asthmatics of Italian Caucasians. The frequency of alleles and genotypes, relatively to 10 allelic specificities of the cytokine genes, was defined in 57 asthmatics and in 124 control subjects by a Polymerase Chain Reaction-Sequence Specific Primer method. TNF-alpha -308A and TNF-alpha -238A allele frequencies were higher in asthmatics than in controls (p less than 0.001). Significant differences in the frequency of IL-4 -590T allele and of IL-4Ralpha +1902A allele were also detected in asthmatics in comparison with controls (pless than 0.001 and p=0.005, respectively). Similarly, IL-1alpha -889C allele was present in 84.1 percent of asthmatics and in 70.2 percent of controls (p=0.013). Furthermore, the IL-4Ralpha +1902A/A and IL-1alpha -889C/C homozygous conditions and the TNF-alpha -308G/A, TNF-alpha -238G/A, IL-4 -590T/C and IL-10 -1082G/A heterozygous conditions were significantly associated with asthma (p less than 0.05). ACA haplotype of IL-10 was observed only in asthmatic patients. This study reports, for the first time, the frequency of 10 different single nucleotide polymorphisms in 5 cytokine genes in the Italian Caucasians. Furthermore, we also indicate that in our population some single nucleotide polymorphisms are associated with mild to severe bronchial asthma.

Time course of cytokines, hemodynamic and metabolic parameters during hyperthermic intraperitoneal chemotherapy.

INTRODUCTION: Systemic response to cytoreductive surgery (CRS) and hyperthermic intraperitoneal chemotherapy (HIPEC) causes the activation of endocrine, metabolic, hemodynamic and inflammatory processes. The aim of this work is to describe and analyze the time course of the inflammatory markers concentration during CRS+HIPEC in plasma and peritoneal fluids and the association with hemodynamic and metabolic parameters. MATERIAL AND METHODS: Pre-, Intra- and Post-operative data were collected. Tumor necrosis factor (TNF), interleukine 6, procalcitonine (PCT), cancer antigen 125 (CA-125) in blood and in peritoneal fluids were evaluated. RESULTS: Thirty-eight patients included, 29 (76.3%) female. Mean/median PCI: 9.2/5. Primary malignancy: 5 colo-rectal (13.2%), 5 gastric (13.2%), 23 ovarian (60.5%) and 5 others (13.2%). CCR 0-1 reached in all patients. Cardiac Index, Heart rate and Central Venous Pressure, increased during the procedure while Stroke Volume Variation showed a decrease. Mean Arterial Pressure and Superior Vena Cava Oxygenation were stable through the whole procedure. TNF and CA-125 were steady during the whole procedure; IL-6 had a relevant increase from baseline to start of perfusion (p<0.01); PCT had a steady increase at every time point. Peritoneal sampling showed a statistically significant increase (p<0.01) between start and end of the perfusion phase for all markers but TNF. Serum and peritoneal marker concentration were similar for TNF, PCT and CA-125. IL-6 showed a sharp difference. CONCLUSION: The most significant variations are those of IL-6 and PCT. The cytokines level parallel the hemodynamic derangements. Treatment during HIPEC should mimic the established treatment during sepsis and septic shock.

Decrease of TGF-beta1 plasma levels and increase of nitric oxide synthase activity in leukocytes as potential biomarkers of Alzheimer's disease.

A variety of inflammatory proteins have been identified in brains of Alzheimer's disease (AD) patients, including inflammatory cytokines, acute phase proteins and complement components. In the present paper we have investigated the levels of circulating inflammatory mediators as potential biomarkers of this disease, concentrating mostly on transforming growth factor beta (TGF-beta1) in plasma and on nitric oxide synthase (NOS) activity in leukocytes. Plasma and leukocytes were isolated from 48 sporadic AD and 23 healthy control subjects of same age and sex. Since alpha2-Macroglobulin (alpha2M), an acute phase protein possibly involved in AD, is an important modulator of TGF-beta1 activity, binding and targeting this cytokine to its appropriate site of action, we have investigated the possible complex between TGF-beta1 and alpha2M in plasma of the same subjects. The results demonstrate a significant reduction of TGF-beta1 levels in plasma of AD patients. A complex between alpha2M and TGF-beta1 occurred in AD as well as healthy elderly control subjects, however, with no significant differences. Moreover, alpha2M appeared to bind only the inactive form of this cytokine. In contrast, NOS activity increased significantly in leukocytes of AD patients. Therefore, we suggest the combined determination of TGF-beta1 in the plasma and of NOS activity in the leukocytes as biomarkers of AD.

Increased plasma levels of soluble CD40, together with the decrease of TGF beta 1, as possible differential markers of Alzheimer disease.

Alzheimer's disease (AD) is a progressive neurodegenerative illness and the most frequent cause of dementia in the elderly. The identification of activated microglia within neuritic plaques, coupled with the presence of numerous inflammatory proteins, suggests that inflammation is an integral part of the pathogenetic process in AD. In the present paper we have investigated the levels of circulating inflammatory mediators as potential AD biomarkers concentrating essentially on (a) soluble CD40 (sCD40), a member of the tumor necrosis factor receptor superfamily lacking the membrane-associated endodomain by alternative splicing, and (b) transforming growth factor (TGF)-beta 1, a cytokine deeply involved in AD and playing a protective role on CNS. Decrease of TGF-beta1 in AD patients could enhance the effects of pro-inflammatory cytokines produced by activated microglia as well as the expression of factors, such as the CD40/CD40 ligand complex, by microglia and astrocytes. Total venous blood samples were obtained from 33 patients with clinical diagnosis of possible late-onset AD, 40 healthy age-matched and 11 healthy young individuals. A significant increase of sCD40 levels plasma of AD patients versus healthy controls was measured, concomitantly with a decrease in TGF-beta1 concentration. These variations, however, showed no correlation with the expression of ApoE epsilon 4 allele, which was determined in order to assess the different frequency of this risk factor between AD and control groups. Since no comparable modifications were detected in patients affected by Parkinson's disease or non-AD-based dementia, we propose that sCD40 and TGF-beta1 plasma levels might represent possible differential biomarkers of AD, and be useful pre-mortem to support the clinical diagnosis of late-onset AD.

The conundrum of HLA-DRB1*14:01/*14:54 and HLA-DRB3*02:01/*02:02 mismatches in unrelated hematopoietic SCT.

Uncertainty still exists on the role of polymorphisms outside the HLA-DRB1 binding site or inside the HLA-DRB3 binding groove in unrelated hematopoietic SCT (HSCT). The ideal model to solve the conundrum consists of the transplants mismatched for HLA-DRB1*14:01/*14:54 and/or for HLA-DRB3*02:01/*02:02. A task force was set up in Italy to recruit transplanted pairs defined as HLA-DRB1*14:01 before 2006, the year crucial for the proper definition of the HLA-DRB1*14:54 allele in molecular biology. Out of 2723 unrelated pairs, 189 transplanted in Italy from 1995 to 2006 were HLA-DRB1*14:01 positive; 103/189 pairs with good historical DNA were retyped for HLA-DRB1*14 and HLA-DRB3 at-high resolution level; 31/103 pairs had HLA-DRB1*14 and/or HLA-DRB3 mismatched; 99/103, having complete clinical data, underwent statistical analysis for OS, TRM, disease-free survival and acute and chronic GvHD. No significant involvement of HLA-DRB1*14:01/*14:54 or HLA-DRB3*02:01/*02:02 mismatches was found, either alone or combined. Our findings suggest that disparities at exon 3 of the HLA-DRB1 gene seem unlikely to influence the outcome after HSCT. The same may be envisaged for HLA-DRB3(*)02:01 and (*)02:02 alleles which, although differing in the Ag binding site, seem unable to modulate an appreciable immune response in an HSCT setting.

Neutrophilic inflammation and IL-8 levels in induced sputum of alpha-1-antitrypsin PiMZ subjects.

BACKGROUND: Severe alpha-1-antitrypsin deficiency (AATD), due to homozygosity for the protease inhibitor (Pi) Z allele, is a genetic risk factor for chronic obstructive pulmonary disease (COPD). In a previous study the sputum of severe AATD subjects with airflow obstruction showed a pattern of cellular inflammation similar to COPD patients. It is uncertain whether heterozygotes for the Z allele or intermediate deficiency (PiMZ) have an increased risk of developing COPD. METHODS: Sputum cell counts and the supernatant level of the neutrophil chemoattractant interleukin (IL)-8 were investigated by sputum induction in 10 non-smoker asymptomatic PiMZ subjects with normal pulmonary function, 10 patients with stable COPD, and 10 age matched normal subjects. Data are expressed as mean (SD). RESULTS: The mean (SD) number of neutrophils was significantly higher (p<0.01) in the sputum of PiMZ subjects (84.5 (22.2) x10(4)/ml) and patients with COPD (126.9 (18.8) x10(4)/ml) than in matched normal subjects (55.0 (8.7) x10(4)/ml). IL-8 levels were increased in PiMZ subjects (828.5 (490.6) ng/ml; median 1003.0 ng/ml; range 1260-100 ng/ml) and in COPD patients (882.5 (524.3) ng/ml; median 934.9 ng/ml; range 1506-258 mg/ml) compared with normal subjects (3.5 (0.5) ng/ml; median 3.5 ng/ml; range 4.5-2.5 ng/ml). There was a significant positive correlation between IL-8 supernatant concentration and neutrophil count in PiMZ subjects (p = 0.036; r = 0.66). An inverse correlation was observed between the percentage of neutrophils and forced expiratory volume in 1 second (% predicted) in patients with COPD (p = 0.04; r = -0.43). CONCLUSIONS: These findings indicate that PiMZ subjects without airflow obstruction may have an IL-8 related neutrophilic inflammation in the airways, similar to stable COPD patients, suggesting an increased risk of developing pulmonary changes.

High serum levels of tumour necrosis factor-alpha and interleukin-8 in severe asthma: markers of systemic inflammation?

BACKGROUND: Severe asthma is characterized by elevated levels of pro-inflammatory cytokines and neutrophilic inflammation in the airways. Blood cytokines, markers of 'systemic' inflammation, may be a feature of amplified inflammation in severe asthma. OBJECTIVE: To detect differences in IL-8, TNF-alpha, IL-16 and IL-13 levels in the serum(s) of stable severe and mild-moderate asthmatics related to blood leucocytes proportion, airway calibre and exhaled nitric oxide (NO) levels. METHODS: We assessed cytokine serum levels by ELISA and blood leucocyte counts by an alkaline peroxidase method in 20 healthy controls, 22 mild-moderate [forced expiratory volume in 1 s (FEV1)(%pred): 89+/-3] and 14 severe asthmatics [FEV1(%pred): 49+/-2]. RESULTS: IL-8 and TNF-alpha levels were higher in severe asthmatics than in mild-moderate asthmatics or in controls (P<0.05). No differences in IL-16 and IL-13 levels were detected. Severe asthmatics showed higher circulating neutrophil and eosinophil number than controls (P<0.05). In severe asthmatics, exhaled NO levels were superior than in controls (P<0.05), but inferior than in mild-moderate asthmatics (P<0.05). We found positive correlation between TNF-alpha levels and exhaled NO (r=0.67; P=0.01) or circulating neutrophil counts (r=0.57; P=0.03) in severe asthmatics. CONCLUSION: sTNF-alpha and sIL-8 are markers of 'systemic' inflammation in severe asthmatics, in conjunction with augmented circulating neutrophils, suggesting the involvement of neutrophil-derived cytokine pattern in severe asthma.

The kidney triggers graft-versus-host disease in experimental combined transplantation of kidney and stem cell-enriched peripheral leukocytes.

As a preclinical step to human studies with combined stem cell-enriched peripheral leukocytes and organ transplantation from the same donor, a series of studies in rats was undertaken. These studies indicated that Lewis rats infused intravenously with major histocompatibility complex-incompatible (from Brown-Norway rats), stem cell-enriched peripheral leukocyte preparation alone never developed graft-versus-host disease (GHVD). However, GVHD invariably manifested in all animals a few days after the kidney was transplanted in rats that had been previously primed with stem cell-enriched peripheral leukocytes from the same kidney donor strain. GVHD was prevented by substituting the crude preparation of stem cell-enriched peripheral leukocytes with a purified preparation that was almost completely free of T lymphocytes. However, in these latter experiments all rats rejected their kidney graft within 10 days from the surgery. In rats previously given the crude stem cell-enriched peripheral leukocyte preparation, perioperative administration of the fusion protein CTLA4lg also prevented GVHD and prolonged kidney graft survival up to 106 to 175 days. By contrast, animals with kidney transplants, which were given CTLA4lg without stem cells, rejected their grafts within 35 days. All together, these findings may possibly contribute to the creation of rationally designed strategies of combining organ and bone marrow from the same donor to enhance mixed chimerism and prolong survival after organ transplantation.

Toward novel antirejection strategies: in vivo immunosuppressive properties of CTLA4Ig.

Allograft rejection is a process that depends on T cell receptor-ligand interaction and costimulatory signals generated when accessory molecules binds to their ligands, such as CD28 to the B7 molecules. We investigated the possibility that B7 blockade in vivo by the soluble CD28 receptor homolog CTLA4Ig modulates rejection process in a rat model of kidney allograft. Lewis rats orthotopically transplanted with MHC incompatible kidney from Brown-Norway rats were given an intraperitoneal injection of CTLA4Ig (0.2 or 0.5 mg/day) or a nonspecific immunoglobulin for seven days, starting the day of transplant. While control rats rejected the graft within 10 days, all animals given CTLA4Ig had a prolonged kidney allograft survival, independently from the dose of the fusion protein employed. Actually, at the dose of 0.2 mg/day kidney grafts survived 36 to 50 days (median 44 days), while with the highest dose graft survival was 40 to 60 days (median 50 days). In all CTLA4Ig-treated rats renal grafts were well functioning as documented by serum creatinine concentrations comparable to age- and sex-matched control rats 30 days after transplant. At this time in vitro mixed lymphocyte culture (MLR) experiments showed a significant reduction of proliferation of peripheral blood lymphocytes from CTLA4Ig-treated rats when challenged with BN but not third party Wistar Furth lymphocytes. We have also shown that combining a short course of CTLA4Ig (0.2 mg/day) with a dose of cyclosporine (CsA) low enough to fail to inhibit graft rejection allowed indefinite engraftment of kidney allograft without the need of continuous immunosuppression.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunosuppressive therapy abrogates unresponsiveness to renal allograft induced by thymic recognition of donor antigens.

This laboratory and others have previously shown that the intrathymic injection of donor cells or major histocompatibility complex allopeptides induced indefinite survival of a subsequent graft without immunosuppression. This approach may open interesting new perspectives for transplant medicine. Studies to explore the feasibility of the technique in humans can only be designed with some form of concomitant immunosuppression to avoid the risk of irreversible rejection in the case that the thymus approach fails. Thus, one of the first issue to address is whether conventional immunosuppression interfered with the process of thymus tolerance. This study was designed to investigate the above issue. In transplanted Lewis control rats, cyclosporin A (CsA) (10 mg/kg per day) and methylprednisolone (MP) (10 mg/kg twice daily) for 3 days were invariably followed by kidney graft rejection within 10 days. In subsequent experiments, five groups of Lewis rats were injected with medium alone or Brown-Norway (BN) leukocytes into the thymus, and 24 h later, they were orthotopically transplanted with major histocompatibility complex-incompatible kidneys from BN rats. At the time of transplantation, Lewis rats received MP (10 mg/kg twice daily) CsA (10 mg/kg per day), or the combination of the two (MP+CsA at the same dose) for 3 days. Lewis rats injected intrathymically with BN leukocytes but not receiving immunosuppressants had indefinite survival of their kidney graft. The effect of the intrathymic injection of donor cells of inducing unresponsiveness to a subsequent kidney graft was abolished by concomitant immunosuppression. All animals given immunosuppressants rejected their graft within 12 days after surgery.(ABSTRACT TRUNCATED AT 250 WORDS)

Colchicine allows prolonged survival of highly reactive renal allograft in the rat.

Colchicine, with its immunosuppressive properties, has been used with beneficial effects in autoimmune diseases. Whether colchicine, by virtue of the above properties, could attenuate the process of kidney allograft rejection in the rat is investigated in this report. Untreated Lewis rats (N = 6) given an incompatible kidney allograft from Brown-Norway rats rejected the graft within 12 days. Colchicine at a daily ip dose of 40 (N = 6) or 10 (N = 4) micrograms/kg promoted long-term survival (> 170 days) of major histocompatibility complex-incompatible kidney grafts. Animals (N = 4) given 4 micrograms of colchicine per kilogram had a graft failure within 10 days. Experiments have also been performed to evaluate the effect of colchicine withdrawal at different time intervals from transplantation on subsequent allograft survival. Colchicine (40 micrograms/kg per day ip) was given for 12, 6, or 1 mo or for 15 days to an additional four groups of six animals each without any other immunosuppressants. The withdrawal of colchicine did confer long-term inhibition of the immune system in animals treated for at least 1 mo, as documented by a graft survival of more than 80 days. By contrast, those animals who discontinued colchicine after only 15 days of treatment had graft rejection within the next 8 days. Mixed lymphocyte culture experiments showed a significant (P < 0.01) reduction of the proliferation of peripheral blood lymphocytes taken from all groups of animals 30 days after colchicine withdrawal when challenged with Brown-Norway lymphocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Intrathymic transplantation of donor cells allows a stabilized, incompatible renal transplant to survive indefinitely after cyclosporine withdrawal.

Recent experiments have shown that exposure of maturing rat thymocytes to donor cells induces a condition of donor-specific unresponsiveness in the recipient that allows indefinite survival of a subsequent kidney transplant without the need for long-term immunosuppressants. Here, studies were performed in Lewis (RT1(1)) rats to determine whether (a) the process of unresponsiveness to kidney allograft induced by intrathymic donor cell inoculation occurred also with a noninbred strain of donor animals, and (b) this technique could allow the elimination of the need for daily immunosuppressive therapy in animals already transplanted with an incompatible kidney. Kidneys from noninbred Sprague-Dawley rats transplanted in incompatible Lewis (RT1(1)) rats, previously injected intrathymically with cells from the same donor, survived indefinitely. Intrathymic inoculation of donor cells into Lewis rats allowed a stabilized, incompatible renal allograft from Brown-Norway (RT1n) rats to survive indefinitely after discontinuation of immunosuppressive treatment with cyclosporine. These findings provide an approach for renal transplantation without immunosuppressive therapy and a potential strategy to overcome side effects related to the use of immunosuppressants in animals already transplanted.

[HBV serologic markers in the chronic alcoholism (author's transl)]. / I marcatori sierici dell'HBV nell'etilismo cronico.

The prevalence of serum markers, HBsAg, anti-HBsAg, HBcAg and anti-HBeAg in a group of subjects affected by chronic alcoholism with fatty liver or with cirrhotic stages is reported. An incidence of chronic HBsAg carriers, similar to that found in healthy subjects, was noticed, while an elevated incidence of other serological markers of previous contact with HBV were found. The Authors discuss the significance of this report, on the basis of a greater possibility of contact with HBV, for social-economic situations of those patients. The etiopathogenetic role of HBV in the cirrhotic evolution of alcoholic liver diseases was excluded.

Peripheral T-cell subsets in patients with alopecia areata in different clinical phases.

An analysis of T-cell subpopulations was carried out in the peripheral blood of 21 subjects with alopecia areata (AA) of the scalp in various phases of its evolution and in 18 healthy control subjects by means of different monoclonal antibodies of OKT series (T3, T4, T8, T11). Patients with AA in active phase showed a significant reduction of OKT 8+ cells (p less than 0.002) and a significant increase of OKT 4+ cells (p less than 0.002) versus controls. On the contrary, patients with regrowing hair showed a significant increase of circulating OKT 8+ cells compared with controls (p less than 0.002). No abnormality in the distribution of T-cell subsets in patients with AA in stable phase has been observed.

Mycosis fungoides. Peripheral T cell subsets defined by specific monoclonal antibodies.

Peripheral blood lymphocytes from 16 mycosis fungoides (MF) patients were studied using OKT series monoclonal antihuman T cell antibodies. The percentage of OKT3+ cells was in normal range for all MF patients compared with controls; the percentage of OKT4+ cells was significantly increased (p less than 0.002) in MF patients versus controls; the percentage of OKT8+ and OKT11+ cells in the MF group did not differ from controls. The OKT4+ cell expansion was apparently not dependent from the clinical stage of disease. These findings suggest that in MF patients there is a circulating OKT4+ cell expansion and, indirectly, that MF could be regarded as a helper T cell neoplasm.