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.

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.

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.

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.

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.

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.

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.

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.

Preliminary results of intrathymic injection of donor cells to prevent acute rejection in human heart transplantation.

In rodents, the intrathymic injection of donor cells or major histocompatibility complex peptides induces indefinite survival of a subsequent allograft with little or no immunosuppression. Here, experiments have been performed in two patients with cardiac transplantation to establish (1) the safety and tolerability of the intrathymic injection of donor leukocytes at the time of transplant surgery and (2) whether conventional immunosuppression interfered with the process of the thymic recognition of alloantigens. It was shown that the intrathymic inoculation of donor cells is safe and can be done without undesired effects. However, the procedure, as performed, did not protect from acute graft rejection. There are data enough to attribute the failure of the thymus technique in these two patients to the concomitant use of immunosuppressants. The results of this study are relevant for future trials aimed at finding the appropriate experimental conditions for the use of the thymic approach in human organ transplantation.

Fluid shear stress modulates surface expression of adhesion molecules by endothelial cells.

We investigated the effect of hemodynamic shear forces on the expression of adhesive molecules, E-selectin, and intercellular adhesion molecule-1 (ICAM-1) on human umbilical vein endothelial cells (HUVEC) exposed to laminar (8 dynes/cm2) or turbulent shear stress (8.6 dynes/cm2 average), or to a static condition. Laminar flow induced a significant time-dependent increase in the surface expression of ICAM-1, as documented by flow cytometry studies. Endothelial cell surface expression of ICAM-1 in supernatants of HUVEC exposed to laminar flow was not modified, excluding the possibility that HUVEC exposed to laminar flow synthetize factors that upregulate ICAM-1. The effect of laminar flow was specific for ICAM-1, while E-selectin expression was not modulated by the flow condition. Turbulent flow did not affect surface expression of either E-selectin or ICAM-1. To evaluate the functional significance of the laminar-flow-induced increase in ICAM-1 expression, we studied the dynamic interaction of total leukocyte suspension with HUVEC exposed to laminar flow (8 dynes/cm2 for 6 hours) in a parallel-plate flow chamber or to static condition. Leukocyte adhesion to HUVEC pre-exposed to flow was significantly enhanced, compared with HUVEC maintained in static condition (233 +/- 67 v 43 +/- 16 leukocytes/mm2, respectively), and comparable with that of interleukin-1 beta treated HUVEC. Mouse monoclonal antibody anti-ICAM-1 completely blocked flow-induced upregulation of leukocyte adhesion. Interleukin-1 beta, which upregulated E-selectin expression, caused leukocyte rolling on HUVEC that was significantly lower on flow-conditioned HUVEC and almost absent on untreated static endothelial cells. Thus, laminar flow directly and selectively upregulates ICAM-1 expression on the surface of endothelial cells and promotes leukocyte adhesion. These data are relevant to the current understanding of basic mechanisms that govern local inflammatory reactions and tissue injury.

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)

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)

Immunotherapy and recurrent abortion: a randomized clinical trial.

We conducted a randomized trial comparing expectant management versus immunotherapy with paternal leukocytes to improve obstetric outcome in women with unexplained recurrent abortion. Eligible for the study were women with unexplained recurrent abortion (three or more miscarriages and no live birth), negative findings of immunological screening and no inhibition of the mixed lymphocyte culture. These women were seen for the first time between October 1988 and March 1991 in a network of obstetric departments in Northern Italy. Subjects positive for HLA DR3 or with a partner positive for hepatitis virus B antigen were not eligible. A total of 44 women entered the study. Patients were randomly allocated to immunotherapy (22 women) or expectant management (22 women). Women allocated to immunotherapy were given 200 x 10(6) purified paternal lymphocytes before pregnancy. Median follow-up was 24 months (range 10-39) in the immunotherapy group and 25 months (range 11-38) in the expectant management group. Out of the 22 women randomized to immunotherapy, 16 became pregnant and the corresponding value was 14 in the expectant management group. Spontaneous abortion occurred in six out of the 16 pregnancies observed in the treated women. Among the 14 pregnancies observed in the expectant management group, two aborted and one late fetal death occurred. The cumulative proportions of women who became pregnant over 4 years were 37 and 45% in the immunotherapy and expectant management groups respectively; this difference was not significant. No adverse effect was observed in treated women.

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.

HLA sharing in Italian recurrent abortion couples.

We performed HLA typing in 96 couples affected by recurrent abortion "sine causa". We matched these patients with 124 fertile couples and 204 individuals random paired. No significant difference in HLA sharing was demonstrated in the three study groups. The statistical analysis denoted significant differences in regard to HLA A3, A24, B12, and DR- comparing patients and normal members of fertile couples. The frequencies of HLA Cw5, Cw6 and DR2 were different in patients when compared with their partners.