Simultaneous detection of circulating immunological parameters and tumor biomarkers in early stage breast cancer patients during adjuvant chemotherapy.

BACKGROUND: Chemotherapy-induced immune suppression has mainly been studied in patients with advanced cancer, but the influence of chemotherapy on the immune system in early stage cancer patients has so far not been studied systematically. The aim of the present study was to monitor the immune system during anthracycline- and taxane-based adjuvant chemotherapy in early stage breast cancer patients, to assess the impact of circulating tumor cells on selected immune parameters and to reveal putative angiogenic effects of circulating endothelial cells. METHODS: Peripheral blood samples from 20 early stage breast cancer patients were analyzed using a flow cytometric multi-color of antibodies to enumerate lymphocyte and dendritic cell subsets, as well as endothelial and tumor cells. An enzyme-linked immunosorbent assay (ELISA) was used to measure the levels of various serological factors. RESULTS: During chemotherapy, all immunological parameters and angiogenesis surrogate biomarkers showed significant decreases. The numbers of circulating tumor cells showed significant inverse correlations with the numbers of T helper cells, a lymphocyte subset directly related to effective anti-tumor responses. Reduced T helper cell numbers may contribute to systemic immunosuppression and, as such, the activation of dormant tumor cells. CONCLUSIONS: From our results we conclude that adjuvant chemotherapy suppresses immune function in early stage breast cancer patients. In addition, we conclude that the presence of circulating tumor cells, defined as pan-cytokeratin(+), CD326(+), CD45(-) cells, may serve as an important indicator of a patient's immune status. Further investigations are needed to firmly define circulating tumor cells as a predictor for the success of breast cancer adjuvant chemotherapy.

-295 T-to-C promoter region IL-16 gene polymorphism is associated with Whipple's disease.

Whipple's disease (WD) is a rare systemic condition caused, in genetically predisposed subjects, by Tropheryma whipplei, a common bacterium widespread in the environment. The relevance of genetic predisposition in WD is shown by the association with HLA alleles DRB1*13 and DQB1*06 and by the demonstration that, in patients with WD, the cytokine genetic profile is skewed toward a Th2 and Treg response. Since IL-16 is involved in hampering the development of a protective macrophagic response against Tropheryma whipplei, we investigated whether the genetic background of IL-16 is different between patients with WD and controls. The -295 T-to-C polymorphism of the promoter region of the IL-16 gene was studied in 90 patients with WD and 152 healthy controls. Levels of serum IL-16 protein were also tested. The frequency of the wild type T allele was significantly higher in patients with WD compared to the controls (155/180 vs. 235/304; p = 0.02 for the Chi(2) test), odds ratio 1.82 [95 % confidence interval (CI) 1.07-3.10]. The TT genotype was found in 65/90 patients with WD and 88/152 controls (p = 0.026). No relationship was found between serum levels of IL-16 and genotypes. Although the functional consequences of this genetic background on levels of IL-16 and on the course of the disease are still unknown, we found, for the first time, that the wild type T allele and the TT genotype of the -295 polymorphism are associated with WD.

Adenoid hypetrophy: definition of some risk factors.

Adenoids removed for airway obstruction and-or recurrent infections have been studied to identify a possible mechanism to explain chronicity. In this regard, macrophages may play a relevant pathogenic role as well as neutrophils during bacterial infections and eosinophils in allergic inflammation. Therefore, this study aimed at investigating some mediators as surrogate markers of inflammation in children who had to undergo to adenoidectomy. Globally, 67 children (25 females, 42 males, mean age 4.9 years), affected by persistent obstruction caused by adenoid hypertrophy were consecutively enrolled into the study. Blood samples were collected from patients and controls to determine serum CD163, Myeloperoxidase (MPO) and ECP. There were significant differences between patients and controls for serum CD163 (p less than 0.0001); MPO (p less than 0.0001); serum ECP (p less than 0.0001). This study demonstrated some risk factors for severe AH: apnoea, recurrent respiratory infections, and high serum CD163 levels.

Circulating interleukin-6 as a tumor marker for hepatocellular carcinoma.

BACKGROUND: A large amount of evidence suggests a possible role of interleukin-6 (IL-6) in the pathogenesis of hepatocellular carcinoma (HCC). PATIENTS AND METHODS: We studied both IL-6 and A(1)FP in patients with HCC, non-neoplastic liver disease or in healthy controls. RESULTS: IL-6 titers were four-fold higher in cancer than in cirrhotic patients and 25-fold higher than in healthy controls. As for alpha1-fetoprotein (A(1)FP) titers, the highest levels were observed in cancer patients. Receiver operating characteristic (ROC) curves analysis demonstrated that IL-6 is significantly more discriminant than A(1)FP, with 'optimal' cut-off values of 7.9 pg/ml (sensitivity = 0.83, specificity = 0.83, efficiency = 0.83). The ROC curves used to distinguish HCC from cirrhotic patients only, showed higher discriminant power of IL-6 versus A(1)FP titers, with a new cut-off value of 12 pg/ml (sensitivity = 0.73, specificity = 0.87, efficiency = 0.8). Discriminant analysis on HCC and non-HCC subjects yielded sensitivity, specificity and efficiency rates of 77%, 93% and 88%, respectively. The overall efficiency of the two tests combined was 82%. CONCLUSIONS: IL-6 could be considered a promising tumor marker for HCC. In particular, the diagnostic value of the test is significantly increased when combined with A(1)FP.

Non classical HLA genes and non-HLA genes in a population of infants at familial risk of atopy.

AIM: We investigated on parental history and IgE serum level in 2588 consecutive newborns to individuate babies "at risk" of atopy at birth and we analysed the polymorphisms of class III region to evaluate the association with immunogenetic markers of HLA: C4A, C4B, LTA, RAGE and TNFA genes; we performed TNF and IgE receptor (FCERB1) physiologically related gene polymorphisms. RESULT: 791 babies/2588 (30.6%) were considered "at risk" for atopy and followed-up: 400 had familial history of atopy (at least one parent or sibling), 256 had IgE >0.35 kUA/l at birth and during the follow-up and 135 were positive for both conditions. The allele C4B2 was significantly more frequent in the sample of babies at risk (22.1% vs 10%, p< 0.001). Furthermore, the mean value of IgE at birth in babies carrying the allele C4B2 was 2.26 KUA/l versus 0.74 KUA/l in those not carrying this allele (p=0.01). No significant association emerged for RAGE at the centromeric end of class III region and for LTA, TNFA at the telomeric one. TNFRI, TNFRII and FCERB1 gene polymorphisms also seemed not implicated. CONCLUSION: Our study confirms that HLA class III region seems involved in familial predisposition to atopy, and C4B gene probably acts as a marker of a more restricted subregion.

Variations in serum levels of interleukin (IL)-1beta, IL-2, IL-6, and tumor necrosis factor-alpha during specific immunotherapy.

BACKGROUND: Cytokine production by T helper cells is essential for the induction and maintenance of allergic inflammation in the bronchial mucosa. According to recent views, specific immunotherapy (SIT) favors the differentiation of T lymphocytes into cells of the Th1 rather than those of the Th2 subset. OBJECTIVE: To determine whether or not SIT induces a decrease in the inflammatory reaction by studying eventual variations in the serum levels of IL-1beta, IL-2, IL-6, and TNF-alpha in allergic subjects during SIT. METHODS: Serum levels of IL-1beta, IL-2, IL-6, and TNF-alpha were determined before and after 3, 6, and 9 months of SIT by an immunoradiometric assay (IRMA) in 11 adults with perennial allergic asthma and/or rhinitis caused by house dust mites and in 6 nonatopic healthy volunteers. RESULTS: Median serum IL-1beta and TNF-alpha levels of the patients were significantly higher at baseline than those of the controls and decreased during SIT to values similar to or lower (P < .01) after 6 months of SIT for TNF-alpha than those of the controls. Median serum IL-2, significantly lower at baseline than in the controls, increased during SIT to a level similar to that of the controls. Although the median values of IL-1beta and TNF-alpha in the patients tended to decrease and those of IL-2 to increase during SIT, the differences were not significant; the correlation coefficients (r) of the serum levels of IL-1beta IL-6, and TNF-alpha versus duration of SIT were negative, while that of IL-2 was positive. CONCLUSIONS: Decreases in median serum IL-1beta and TNF-alpha levels during SIT, together with the increases in serum IL-2 and IL-6, compared with those of the controls furnish evidence supporting a reduction in the inflammatory response in the course of SIT.

Low serum TNF-alpha levels in subjects at risk for type 1 diabetes.

OBJECTIVE: The aim of our study was to determine whether serum TNF-alpha levels in individuals at risk of developing type 1 diabetes, such as first-degree relatives of diabetic patients and children with incidental hyperglycemia, underwent alterations, and also to establish whether these levels might be used to identify individuals prior to insulin dependence. RESEARCH DESIGN AND METHOD: We studied 71 healthy first-degree relatives (FDR) of type 1 diabetic patients and 11 children with incidental hyperglycemia. We looked for immunogenetic (HLA class II serologic alleles and HLA-DQ alpha/beta genomic polymorphisms), immunologic (islet-cell and insulin autoantibodies) and metabolic (FPIR to IVGTT) markers of type 1 diabetic risk. Serum concentrations of TNF-alpha were quantified using IRMA. RESULTS: We found significantly lower serum TNF-alpha levels in FDR of type 1 diabetic patients (median: 54.3 pg/ml) (p=0.01) and in children with incidental hyperglycemia (median: 10.83 pg/ml) (p<0.0001) compared to controls (median: 76.56 pg/ml). No significant difference was observed between subjects with or without immunogenetic, immunologic and metabolic markers of type 1 diabetic risk. A negative correlation was found between serum TNF-alpha and HbA1c levels (r=-0.27, p=0.023). Two children with incidental hyperglycemia, whose TNF-alpha levels were very low, developed type 1 diabetes 6 and 8 months after this study. CONCLUSION: Our results are compatible with an impaired immune system in the prediabetic period and suggest that serum TNF-alpha concentrations may be considered as an immunological marker useful to identify subjects at risk of developing type 1 diabetes.

Cell cycle distribution of cord blood-derived haematopoietic progenitor cells and their recruitment into the S-phase of the cell cycle.

The objective of this study was to evaluate the cycling status of cord blood (CB)-derived colony-forming cells (CFC) and long-term culture-initiating cells (LTC-IC), and their recruitment into the S-phase of the cell cycle. By using the cytosine arabinoside (Ara-C) suicide approach, we found that only small proportions of both CFC and LTC-IC were in the S-phase of the cell cycle. These estimates were confirmed by flow cytometric DNA analysis, which showed that 96 +/- 2% of CB-derived CD34+ cells were in G0/G1 and only 1.6 +/- 0.4% in the S-phase. Staining of CD34+ cells with an antistatin monoclonal antibody, a marker of the G0 phase, indicated that among CD34+ cells with a flow cytometric DNA content typical of the G0/G1 phase 68 +/- 7% of cells were in the G0 phase of the cell cycle. Incubation (24 h) with interleukin 3 (IL-3), recombinant human stem cell factor (SCF) and granulocyte colony-stimulating factor (G-CSF) significantly increased the proportion of cells in the S-phase for both CFC and LTC-IC without inducing any loss in numbers. Flow cytometric DNA analysis also showed an increase in CD34+ cells in the S-phase upon continuous exposure to these cytokines. Our findings indicate that: (i) very few CB-derived CFC or LTC-IC were in the S-phase of the cell cycle; (ii) a substantial amount of CD34+ cells with a flow cytometric DNA content typical of the G0/G1 fraction was cycling, as found in the G1 phase of the cell cycle; and (iii) 24-h incubation with IL-3, SCF and G-CSF could drive a proportion of progenitor cells into the S-phase without reducing their number. These data might be useful for gene transfer protocols and the ex vivo expansion of CB-derived progenitor cells.

Eosinophils and C4 predict clinical failure of combination immunotherapy with very low dose subcutaneous interleukin-2 and interferon in renal cell carcinoma patients.

BACKGROUND AND OBJECTIVE: The clinical and immunologic activities of interleukin-2 (IL-2) in cancer patients have been extensively studied and described; however, in most of these studies, IL-2 was administered by intravenous bolus or continuous infusion, while the immunologic effects of IL-2 given by the subcutaneous (s.c.) route have not yet been well studied. DESIGN AND METHODS: The present study was aimed at evaluating the effects of IL-2, given at very low doses s.c. to patients with advanced renal cell carcinoma (RCC), on a number of immunologic parameters: number of total lymphocytes, number of CD4-, CD8-, CD25-positive cells, number of natural killer (NK) cells, titers of IL-2 soluble receptor (sIL-2R) and of C4, eosinophils, eosinophilic cationic protein (ECP) and eosinophilic protein X (EPX). Finally, a logistic regression model was performed to identify early immunologic parameters that correlate with a favorable or unfavorable treatment outcome. RESULTS: Independently from the mere report of the changes induced by immunotherapy, the analysis showed that, within the pre-treatment model, a large eosinophil number predicts the failure of IL-2 treatment; in contrast, within the post-treatment model, high C4 serum titers and, again, a large number of circulating eosinophils predict immunotherapy failure. INTERPRETATION AND CONCLUSIONS: As far as concerns C4, its negative predictive value could be related to the fact that it is an indirect index of macrophage activation; thus, even though macrophages release substances with antitumor activity, they can also stimulate the release of sIL-2R, which may compete for exogenous IL-2. Some authors have postulated that macrophages may even stimulate tumor cell growth, or impair NK activity. Despite a great amount of uncertainty concerning the role of eosinophils, in our study, blood eosinophilia predicts a poor response to immunotherapy in patients with advanced RCC, thus supporting previous observations from our own group.

Study of release of eosinophil cationic proteins (ECP and EPX) in the hypereosinophilic syndrome (HES) and other hypereosinophilic conditions.

BACKGROUND: Up to date, the etiology and the pathogenesis of HES are still unknown and particularly it is unclear why eosinophils in HES are more aggressive towards tissues than in other eosinophilic conditions. METHODS: We assessed the cationic proteins ECP and EPX serum concentrations, their in vitro release from polymorphonuclear cell culture, and the monoclonal antibodies EG1 and EG2 in 3 patients with HES, 6 patients with other hypereosinophilic conditions and 20 healthy control subjects. RESULTS: Serum ECP and EPX concentrations were higher in eosinophilic patients than in healthy subjects. Hypereosinophilic patients had more EG2+ cells than healthy subjects, but EG2+ rate failed to differentiate HES from other hypereosinophilic conditions (p = 0.074). Moreover, the release in vitro of ECP and EPX was significantly higher in HES patients (p < 0.05). CONCLUSIONS: Our preliminary results seem to suggest the importance of functional data, such as ECP and EPX release, in differentiating HES from other hypereosinophilic diseases. Particularly, ECP and EPX release in vitro is higher in cell cultures from HES patients than from patients with other hypereosinophilic conditions.

Modulating effect of human growth hormone on tumour necrosis factor-alpha and interleukin-1beta.

We measured serum tumour necrosis factor-alpha (TNF-alpha) as well as interleukin-1betta (IL-1beta) and GH concentrations in 15 children with isolated growth hormone deficiency (GHD), age range 5.1-13.9 years, before and 4 and 24h after the first GH injection (0.1 IU/kg s.c.). No differences were found in basal concentrations of serum TNF-alpha and IL-1beta between GHD children (10.01 +/- 1.55 pg/ml and 2.14 +/- .16 ng/ml respectively) and sex- and age-matched controls (11.57 +/- 2.16 pg/ml and 3.78 +/- 1.46 ng/ml respectively). In GHD children, serum TNF-alpha and IL-1beta values had significantly increased (P < 0.002) 4h (26.75 +/- 5.57 pg/ml and 2.99 +/- 0.21 ng/ml respectively) and decreased again 24 h after GH administration. Likewise, serum GH levels had significantly increased 4 h (from 1.29 +/- 0.69 to 48.71 +/- 13.35 ng/ml, P < 0.001) and decreased to basal values 24h after GH administration. A significant correlation was found between basal serum concentrations of GH and those of both TNF-alpha (P < 0.01) and IL-1beta (P < 0.05). However, no correlation was found between serum GH concentration and either TNF-alpha or IL-1beta levels 4 and 24h after GH administration. Our data suggest that GH plays a role in modulating TNF-alpha and IL-1beta release in humans.

Synthesis of new delta 2-isoxazoline derivatives and their pharmacological characterization as beta-adrenergic receptor antagonists.

A series of delta 2-isoxazoline derivatives structurally related to Broxaterol 1 and Falintolol 3 has been prepared and evaluated for their binding affinity to beta 1- and beta 2-adrenergic receptors. Among the tested compounds only the 3-isopropenyl anti derivative 4d is as active as the reference compounds. An electron-releasing group, probably operating through a pi-pi interaction, in the 3-position of the isoxazoline nucleus greatly enhances the affinity of the compounds. Conversely, the closest analogs of Broxaterol (3-bromo delta 2-isoxazolines 4a and 5a) are at least one order of magnitude less active than the model compound 1. Throughout the series of derivatives the anti stereoisomers are invariably more active than their syn counterparts.

Chronic tonsillitis in children: activation of polymorphonuclear cells from peripheral blood and tonsillar tissue. In vitro production of MPO, ECP and EPX.

Activation of polymorphonuclear cells (PMNs), neutrophils and eosinophils, occurs in response to infections. Myeloperoxidase (MPO) is an enzyme contained in neutrophils with toxic activity for many microorganisms including bacteria. Fosinophil cationic protein (ECP) and eosinophil X protein (EPX) are released by activated eosinophils in response to different inflammatory stimuli. Determination of serum levels of MPO, ECP and EPX permits assessment of the state of activation of these cells. The purpose of this study was to evaluate the state of activation of neutrophils and eosinophils present in tonsillar tissue and peripheral blood from 30 children (18 boys and 12 girls) undergoing tonsillectomy for chronic tonsillitis. For determination of serum levels of MPO, ECP and EPX, peripheral blood samples were obtained at the time of surgery: PMNs were separated by erythrosedimentation from peripheral blood and from tonsillar tissue after surgery and cultured for 7 days. The cells were plated (10(5) well) and the plates were incubated in 5% CO(2), 7% O(2), 80% N(2). After 2, 24, 48, 72, 96, 120, 144 h, supernatants were removed from the cultures of both tonsillar tissue and peripheral blood PMNs for determination of in vitro release of MPO, ECP and EPX. The assays were carried out using a radioimmunotechnique (RIA, Pharmacia) and the concentrations of the three proteins (mean/6 wells) for each time were expressed in mu g/l. The data suggest activation of PMNs in children with chronic tonsillitis.

Effects of 3'-hydroxyfarrerol (IdB 1031), a novel flavonoid agent, on phagocyte products.

The novel flavonoid compound 3'-hydroxyfarrerol (IdB 1031) was tested in a number of in vitro experiments in order to ascertain its effects on some functions and products of human phagocytes. We found that IdB 1031 did not depress neutrophil phagocytosis and chemotaxis, whereas at a concentration of 10(-4) M it significantly (p < 0.05) reduced the fMLP-triggered neutrophil production of superoxide anion. At the same concentration, the compound decreased the release of neutrophil elastase and myeloperoxidase from neutrophils (p < 0.05). We also found evidence that IdB 1031 is a non competitive, reversible inhibitor of human neutrophil elastase (Ki 200 microns). Finally, IdB 1031 at the concentration of 10(-5) M significantly reduced the release of tumor necrosis factor-alpha and interleukin-8 from monocytes (p < 0.05). We conclude that, in spite of the moderate activity displayed by IdB 1031, these findings add to our current knowledge on the spectrum of the antiinflammatory activities of flavonoids.

Benign idiopathic hypereosinophilia: a feeble masquerader or a smouldering form of the hypereosinophilic syndrome?

In a patient with long-standing idiopathic hypereosinophilia with no apparent organ damage we measured serum eosinophil cationic protein (ECP) and eosinophil protein X (EPX) titers, activated circulating eosinophil rates (by means of monoclonal antibodies EG1 and EG2), and the release of ECP and EPX in vitro by leukocytes at different cultures stages in order to detect possible functional abnormalities associated with hypereosinophilia. Our patient had elevated serum levels of both ECP and EPX, together with a high EG2 count, which would suggest eosinophil activation. However, serum levels of ECP and EPX were not significantly high in relation to the total number of eosinophil cells, although they were more numerous than in healthy controls. Moreover, the release of intracytoplasmic basic proteins by the patient's eosinophils was poor even after in vitro stimulation. Since hypereosinophilic syndrome (HES) with organ damage can appear as long as 8-9 years after the presence of a hypereosinophilic state, the absolutely benign nature of our patient's condition still cannot be defined. Thus, there is the possibility it could be slow-onset or smoldering HES.

Low serum levels of tumor necrosis factor-alpha in insulin-dependent diabetic children.

We measured serum levels of tumor necrosis factor-alpha (TNF-alpha) in 48 children with insulin-dependent diabetes mellitus (IDDM), divided into two groups according to disease duration (group I < 6 months and group II > 3 years): group I 15 patients, aged 2.2-13.7 years, and group II 33 patients, aged 4.5-25.5 years. Thirty-six age- and sex-matched healthy subjects served as controls. TNF-alpha levels were measured by immunoradiometric assay. We found that TNF-alpha levels were lower in all IDDM patients (29.65 +/- 3.83 pg/ml) than in controls (74.74 +/- 10.17 pg/ml) (p < 0.0001), as well as in group I (24.07 +/- 3.65 pg/ml) and group II (32.16 +/- 5.29 pg/ml) as compared to controls (p < 0.001). TNF-alpha levels were significantly lower in patients with antibodies than in those without antibodies and in controls. Similar results were found in longstanding IDDM patients. No correlation was found between serum TNF-alpha and chronologic age, duration of disease, metabolic control, insulin requirement and HLA typing. During a 1-year follow-up study in 12 group I patients no significant variations in TNF-alpha levels were observed. It has been reported that the administration of exogenous TNF suppresses the development of diabetes in nonobese diabetic mice, low producers of endogenous TNF. The results suggest that aberrant TNF-alpha synthesis may contribute to immune dysregulation thus favoring the development of autoimmune diseases.