Peripheral Circulation Of Patients Research Articles

Tumor Antigen-specific T-cells are Present in the CD8αα+ T-cell Effector-memory Pool

CD8+ T-cell memory formation has recently been demonstrated to be associated with CD8alphaalpha homodimer expression by T-cells in mice. Up to now, the knowledge about the clinical significance of CD8alphaalpha+ T-cells in humans is limited. We assessed in longitudinally collected blood samples from patients with melanoma, who underwent a peptide-based vaccination, the role of CD8alphaalpha+ T-cells in tumor-specific cellular immune responses. Phenotypic analysis showed that the expression of CD8alphaalpha+ by T-cells was stable over time and associated with a CD45RA+/-CCR7- effector-memory profile. Melan-A/MART-1-specific T-cells were identified in the CD8alphaalpha+ T-cell compartment by tetramer technology. Detection of intracellular cytokine production (interleukin-2, interferon-gamma, and tumor necrosis factor-alpha) upon phorbol 12-myristate 13-acetate-ionomycin stimulation in CD8alphaalpha+ and CD8alphabeta+ T-cells revealed that CD8alphaalpha+ T-cells show a unique cytokine production pattern (tumor necrosis factor-alpha and interferon-gamma production) as compared with CD8alphabeta+ T-cells. T-cell receptor-CDR3 length analysis revealed that Melan-A/MART-1-specific CD8alphaalpha+ T-cells showed a similar T-cell receptor-repertoire as compared with Melan-A/MART-1-specific CD8alphabeta+ T-cells. Our results show that CD8alphaalpha+ T-cells represent a compartment of CD45RA+/- effector-memory cells in the peripheral circulation of patients with melanoma and suggest that CD8alphaalpha T-cells may originate from CD8+ T-cells that have down-regulated the expression of the CD8beta chain. CD8alphaalpha+ and tetramer-specific T-cells may represent a valuable marker to gauge long-term antigen-specific T-cell memory.

Vascular reactivity in patients with undifferentiated connective tissue diseases

IntroductionEndothelial dysfunction is an early event in the development of atherosclerotic disease. We investigated endothelial function in the peripheral circulation of patients with undifferentiated connective tissue diseases (UCTDs), in comparison to healthy controls. MethodsIn 15 young UCTD patients (mean age 39 years) with inactive disease and without cardiovascular risk factors and in 15 age-matched controls we evaluated endothelial function in the brachial conduit artery and in the forearm microcirculation. The first district was assessed by flow mediated dilation (FMD) and response to glyceryl trinitrate, and forearm microcirculation by blood flow changes (strain-gauge venous plethysmography) in response to intra-arterial acetylcholine (Ach) and sodium nitroprusside (SNP). Nitric oxide (NO) availability was evaluated by repeating Ach in the presence of intraarterial NG-monomethyl-l-arginine (l-NMMA), an NO synthase inhibitor. ResultsFMD and response to GTN were similar in UCTD patients and controls. UCTD patients showed a reduced response to Ach (P<0.001) and SNP (P<0.01) as compared to controls. In healthy controls l-NMMA infusion significantly (P<0.001) reduced the vascular response to Ach, while in UCTD patients l-NMMA failed to affect vasodilation to Ach. In UCTD patients, vascular responses were unrelated to clinical manifestations or autoantibody profile. ConclusionsYoung UCTD patients are characterized by reduced endothelium-dependent and -independent vasodilation in the forearm microcirculation, but not in peripheral conduit arteries. Reduced NO availability was indirectly observed. These results suggest that in the absence of traditional risk factors, inactive systemic autoimmune diseases impair NO-dependent but also endothelium-independent vasodilation selectively in the microcirculation.

T Regulatory Type 1 Cells in Squamous Cell Carcinoma of the Head and Neck: Mechanisms of Suppression and Expansion in Advanced Disease

Regulatory T cells play a major role in tumor escape from immunosurveillance. T regulatory cells type 1 (Tr1), a subset of regulatory T cells present in the tumor and peripheral circulation of patients with head and neck squamous cell carcinoma (HNSCC), mediate immune suppression and might contribute to tumor progression. CD4+CD25-T cells were isolated from peripheral blood mononuclear cells (PBMC) or tumor-infiltrating lymphocytes (TIL) of 26 HNSCC patients and 10 normal controls. The Tr1 cell phenotype was determined before and after culture in the presence of interleukin (IL)-2, IL-10, and IL-15, each at 10 to 20 IU/mL. Suppression was measured in carboxyfluorescein diacetate succinimidyl ester-based proliferation assays with or without neutralizing anti-IL-10 or anti-transforming growth factor-beta1 (TGF-beta1) monoclonal antibodies in Transwell systems. ELISA was used to define the Tr1 cytokine profile. Tr1 cells originate from CD4(+)CD25(-) precursors present in TIL and PBMC of HNSCC patients. Cytokine-driven ex vivo expansion of Tr1 precursors yielded CD4+CD25-Foxp3lowCD132+IL-10+TGF-beta1+ populations that mediated higher suppression than Tr1 cells of normal controls (P < 0.0001). Tr1 cells suppressed proliferation of autologous responders via IL-10 and TGF-beta1 secretion. Expression of these cytokines was higher in TIL-derived than PBMC-derived Tr1 cells (P < 0.0001). The Tr1 cell frequency and suppressor function were significantly higher in patients presenting with advanced than early disease stages and in patients "cured" by oncologic therapies than in those with active disease. In HNSCC, Tr1 cell generation is promoted at the tumor site. Tr1 cells use TGF-beta and IL-10 to mediate suppression. They expand during disease progression and also following cancer therapy in patients with no evident disease.

Dehydroepiandrosterone and Dehydroepiandrosterone Sulphate in Atopic Allergy and Chronic Urticaria

The increasing amounts of evidence prove that sex hormones modulate different cells function, including mast cells and lymphocytes. Dehydroepiandrosterone (DHEA) and its sulphate ester (DHEA-S) are both the secretory products of adrenal glands and the most abundant hormones in the systemic circulation of humans, converted then into androgens and estrogens in the periphery. DHEA may affect production of Th1 and Th2- associated cytokines, suggesting their significance in diseases where imbalanced lymphocyte activity plays the essential role, also in atopic diseases. Reduced circulating concentration of these androgens has been demonstrated in patients with immune-inflammatory diseases, including atopic eczema/dermatitis syndrome and asthma. It is unknown however, whether such changes contribute in any way to etiopathogenesis of the disorders, or are merely their consequences. Chronic urticaria, not included in the atopic triad, is a mast cell-dependent disease characterised by activation of autoimmune and inflammatory processes responding to different factors. Alterations in the immune-neuro-endocrine milieu may contribute to the development of the disease. It has been demonstrated that DHEA-S concentration is significantly lower in the peripheral circulation of patients suffering from chronic urticaria, as compared with healthy subjects. On one hand, this reflects some changes in hormonal homeostasis in the course of urticarial processes, yet on the other, suggests some possible role for this hormone in pathophysiology of the disease. In this brief review we present the available data on DHEA-S involvement in events associated with atopic allergy as well as chronic urticaria.

Tumour cell dissemination following endoscopic stent insertion

This study examined whether colonoscopy or endoscopic stent insertion increases levels of carcinoembryonic antigen (CEA) and/or cytokeratin (CK) 20 mRNA expression in the peripheral circulation of patients with colorectal cancer. Peripheral venous blood samples were obtained before and after colonoscopy (38 patients) or colonic stent insertion (20). Twenty patients undergoing colonoscopy for benign conditions served as controls. Expression of mRNA was quantified using real-time reverse transcriptase-polymerase chain reaction. Circulating CK20 mRNA was detected in 13 of 38 patients who had a colonoscopy and eight of 20 patients with stent insertion. CK20 mRNA expression was increased following stent insertion (P = 0.007) but not after staging colonoscopy (P = 0.454). CEA mRNA was detected in one patient who had colonoscopy and two who had a stent inserted. Neither CEA nor CK20 mRNA was found in blood samples from controls. Endoscopic insertion of colonic stents but not staging colonoscopy results in increased levels of CK20 mRNA in the peripheral circulation.

Endothelial progenitor cells in breast cancer patients

Development of new capillary blood vessels is essential for the growth of cancer. Two distinct processes, vasculogenesis and angiogenesis implement the formation of the new vascular network. Recently, it was demonstrated that vasculogenesis creates the primary network of vascular endothelial cells that will become major blood vessels in malignant tumors by the recruitment of CD34(+)/vascular endothelial growth factor receptor 2 (FLK-1)(+ )endothelial progenitor cells (EPCs) to sites of the new vessel formation with subsequent differentiation into mature endothelial cell. Therefore the aim of this study was a) to quantitate EPCs in breast cancer patients and b) to evaluate if the release of EPCs into the circulation is mainly regulated by the tumor himself. CD34(+)FLK-1(+ )EPCs were measured in the peripheral circulation of patients with breast cancer (n = 47) before and after therapy. Furthermore the potential of EPCs to differentiate into endothelial cells was investigated by late-outgrowth experiments and the metabolic uptake of dil-acetylated-LDL and immunoreactivity against von Willebrand factor. In breast cancer patients the amount of CD34(+)FLK-1(+ )EPCs (percent of peripheral blood mononuclear cells) is significantly increased in women with breast cancer. Tumors larger than 2 cm showed significantly higher values of CD34(+)FLK-1(+ )EPCs. After excision of the tumor the amount of CD34(+)FLK-1(+ )EPCs rapidly declines. Our findings lead to the tumor, as source of angiogenic chemokines, is most important for recruiting CD34(+)FLK-1(+ )EPCs during breast cancer development. Therefore circulating endothelial progenitor cells may work as a new diagnostic tool in the screening and diagnosis of breast cancer.

Invited commentary

The use of molecular techniques in the staging of malignancy (ie, molecular biologic substaging) may improve the assessment of prognosis and the assignment of therapy for patients with nonsmall cell lung cancer (NSCLC). The current staging system tends to under-stage patients, and clinical staging is even less accurate than pathologic staging. Molecular biologic substaging may be applied to the primary tumor to assess prognosis or chemoresistance, and to lymph nodes, serum, or bone marrow to assess occult systemic metastases. If it were possible to identify which patients with lung cancer will have systemic recurrence develop prior to the development of clinical or radiographic evidence, systemic therapy may be more effective in improving the outcomes of patients after resection. Molecular analysis of the primary tumor has demonstrated the ability to identify patients with increased risk of recurrence after resection for NSCLC, although this technology has not yet been investigated in phase III clinical trials, and clinical efficacy has not yet been proven. Tumor-related proteins may be secreted into the peripheral circulation of patients with cancer and are detectable by protein analysis. The measurement of serum proteins involved in the pathophysiology of invasion and metastases may be effective in providing prognostic stratification and allowing earlier therapeutic intervention. The use of serum markers has not yet reached acceptance in assessing prognosis or assigning therapy after complete resection for NSCLC. In 1997 the American Thoracic Society and The European Respiratory Society jointly published guidelines for assessment of NSCLC, indicating that no serum tumor markers had sensitivity and specificity sufficient to reliably detect occult disease or influence treatment. Most tumor proteins are elevated in a minority of patients prior to resection; using a panel of markers may allow a majority of patients to be eligible for surveillance. Although prognostic information may be attained from initial serum titers of tumor markers, these markers continue to be secreted into the peripheral circulation of patients with continued and recurrent disease. The use of serial serum determinations enables analysis of protein expression within time, and changes in protein levels may be more important than the absolute value of the protein. However, tumor proteins in the serum may be detectable prior to the point at which a recurrent tumor can be identified with conventional radiographic techniques; recently this has been demonstrated. The authors are to be commended for this important study [1Mizuguchi S. Nishiyama N. Iwata T. et al.Clinical value of serum cytokeratin 19 fragment and Sialyl-Lewis X in non-small cell lung cancer.Ann Thorac Surg. 2007; 83: 216-222Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar]. Molecular biologic substaging of patients with early stage NSLCL using proteomic and genomic strategies should be investigated to improve the accuracy of the TNM staging system to assess prognosis, to identify patients who may benefit from adjuvant therapy, and to predict recurrence. Clinical Value of Serum Cytokeratin 19 Fragment and Sialyl-Lewis X in Non-Small Cell Lung CancerThe Annals of Thoracic SurgeryVol. 83Issue 1PreviewThis study aimed to establish the clinical significance of preoperative serum cytokeratin 19 fragment (CYFRA21-1) and sialyl-Lewis x (SLex) as prognostic markers. Full-Text PDF

Altered Endothelin-1 Vasoreactivity in Patients with Untreated Normal-Pressure Glaucoma

Vasospasm, resulting from a generalized dysfunction in the vascular endothelium, is implicated in the development of normal-pressure glaucoma (NPG). Impaired endothelium-derived nitric oxide activity and abnormalities of the endothelin system suggest systemic endothelial cell dysfunction in patients with NPG. Endothelin (ET)-1 vasoreactivity was assessed in the peripheral circulation of patients with NPG. Forearm blood flow was measured using venous occlusion plethysmography in eight patients with untreated NPG and eight age- and sex-matched healthy volunteers during intra-arterial infusion of ET-1 (5 pmol/min) and, on a separate occasion, to BQ123, a selective endothelin-A receptor antagonist, (100 nmol/min). Blood pressure and heart rate were measured in the noninfused arm, and plasma ET-1 concentrations were measured using a radioimmunoassay. Forearm blood flow fell during infusion of ET-1 (P<0.001 for both) to a similar extent in both groups (P=0.7; patients versus control subjects). In contrast, BQ123 increased forearm blood flow in both groups (P<0.001 for both), although the vasodilatation was lower in patients than in control subjects (P<0.001; patients versus control subjects). There was no difference in basal plasma ET-1 concentrations between the two groups (P=0.81; patients versus control subjects). Despite normal responses to ET-1, patients with NPG have reduced vasodilatation in response to ETA-receptor antagonism. This could be due to attenuated ETA-receptor-mediated tone, increased ETB-receptor-mediated contraction or impaired ETB-receptor-mediated release of endothelial nitric oxide. These results are consistent with the authors' previous demonstration of systemic vascular dysfunction in patients with NPG.

Alterations in the T-Cell Receptor Variable β Gene–Restricted Profile of CD8+ T Lymphocytes in the Peripheral Circulation of Patients with Squamous Cell Carcinoma of the Head and Neck

Apoptosis of activated CD8(+) T cells is often seen in tumor-infiltrating lymphocytes and circulating peripheral blood mononuclear cells (PBMC) in patients with squamous cell carcinoma of the head and neck (SCCHN). We investigated whether T-cell receptor (TCR) variable beta chain (Vbeta)-restricted T cells were more sensitive to apoptosis than non-TCR Vbeta-restricted T cells. Flow cytometry analysis with anti-TCR Vbeta antibodies was used to define expansions and contractions of Vbeta-restricted T cells in patients with SCCHN relative to normal donors. This staining was combined with Annexin V binding to indicate early T-cell apoptosis. The TCR Vbeta profiles of CD3(+) T cells in tumor-infiltrating lymphocytes and PBMCs of patients with SCCHN were altered relative to controls, with one to five expansions and numerous contractions of TCR Vbeta-restricted T cells detected. These types of alterations were significantly greater in CD8(+) than CD4(+) T cells. Enhanced Annexin V binding to CD8(+) T cells was evident in PBMCs obtained from all patients, with 3 of 13 showing preferential targeting for apoptosis of TCR Vbeta-restricted T cells. TCR Vbeta profiles of CD8(+) T cells were altered in patients with SCCHN relative to normal controls. This may reflect increased apoptosis of expanded or contracted CD8(+) T cells, which define the TCR Vbeta profile of antigen-responsive T-cell populations in patients with cancer.

Production and post-surgical modification of VEGF, tPA and PAI-1 in patients with glioma

Malignant gliomas are associated with risk of thromboembolism, but the molecular link between tumor and peripheral pro-coagulant status has not been elucidated. Vascular Endothelial Growth Factor (VEGF), tissue-type Plasminogen Activator (tPA), Plasminogen Activator Inhibitor-1 (PAI-1) and lipoprotein (lp) (a) influence the pro-coagulant status. To assess whether the presence of the tumor influenced the peripheral levels of VEGF, tPA, PAI-1 and lp(a), we studied the expression and secretion of VEGF, tPA, PAI-1 and lp(a) in glioma specimens, in peripheral blood and in primary glioma-derived cultures. We also measured lp(a), VEGF, tPA and PAI-1 in the peripheral circulation of patients, before and after surgery for glioma.VEGF, tPA and PAI-1 were expressed in glioma specimens. Glioma cells were indeed a major source of tPA and PAI-1; these molecules were significantly more expressed in glioma than in patient’s blood cells. Lp(a) was rarely expressed in glioma specimens and not expressed in blood cells. In glioma, VEGF, tPA and PAI-1 were localized mainly in tumor cells; tPA was localized also in the extracellular matrix and PAI-1 in tumor vascular lumen. Glioma cells were indeed able to produce and release VEGF, tPA and PAI-1. After surgery, peripheral levels of VEGF and PAI-1 were increased, while tPA and lp(a) were unchanged.The great amount of VEGF, tPA and PAI-1 produced by glioma could influence peripheral levels of these molecules. The partial resection of the tumor by surgery was not able to decrease plasma levels of these molecules.

Spontaneous Apoptosis of Epitope-Specific Tetramer+ CD8+ T Lymphocytes in the Peripheral Circulation of Patients With Cancer

s: Abstracts for the 20th Annual Scientific Meeting of the International Society for Biological Therapy of Cancer (Primary Authors are Italicized): IMMUNE MONITORING

Chemokine C Receptor 7 Expression and Protection of Circulating CD8+ T Lymphocytes from Apoptosis

Chemokine C receptor 7 (CCR7) expression is important for lymphocyte homing to tissues. We hypothesized that CCR7 also plays a role in CD8(+) T-cell protection from apoptosis. Its expression was determined on circulating T cells in patients with cancer and related to that of molecules responsible for lymphocyte susceptibility/resistance to apoptosis. Peripheral blood mononuclear cells were obtained from 36 patients with squamous cell carcinoma of the head and neck and 16 normal controls. Multicolor flow cytometry was used to evaluate CCR7, Fas, Bax, and Bcl-2 expression in CD8(+) T cells. Annexin V binding to CD8(+)CCR7(+) and CD8(+)CCR7(-) T-cell subsets was compared. Fewer CD8(+)CCR7(+) T cells bound Annexin V than CD8(+)CCR7(-) T cells in normal control and patients (P < 0.0001). CCR7 expression correlated with higher Bcl-2 but lower Bax and Fas expression levels in CD8(+) T cells in both normal control and patients (P < 0.0001). In patients, the CD8(+)CCR7(+) subset was reduced relative to normal control (P = 0.008) and replaced with an excess of apoptosis-sensitive CD8(+)CCR7(-) T cells. To study CCR7 signaling, CD8(+) T cells were stimulated with CCR7 ligands, chemokine C ligands 19 or 21. Ligand binding to CCR7 resulted in phosphorylation of Akt and increased Bcl-2 expression in CD8(+)CCR7(+) T cells, suggesting that CCR7 protects effector T cells from apoptosis through the phosphatidylinositol 3-kinase/Akt pathway. The absence of CCR7 expression on the majority of CD8(+) T cells in the peripheral circulation of patients with squamous cell carcinoma of the head and neck contributes to apoptosis and a rapid turnover of these effector cells.

Lymphocyte homeostasis and the antitumor immune response

Lymphocyte homeostasis is regulated by proliferation of antigen-responsive T-cells in the peripheral circulation and their apoptosis. Patients with cancer have altered lymphocyte homeostasis. Spontaneous apoptosis of circulating CD8+ antigen-responding effector T-cells contributes to rapid lymphocyte turnover and depressed absolute numbers of T-cell subsets observed in patients with cancer. A rapid transit of naive CD8+ T-cells to the expanded memory pool and enhanced apoptosis of antitumor effector T-cells in the peripheral circulation of patients with cancer are partly responsible for this rapid lymphocyte turnover. Future strategies for restoration of normal lymphocyte homeostasis in cancer will involve therapies with survival cytokines and factors selected for extending survival of antitumor effector cells and establishing long-term immunologic memory.

Impact of simvastatin on hemostatic and fibrinolytic regulators in Type 2 diabetes mellitus

Simvastatin, a widely used 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase inhibitor, effectively reduced cardiac death and ischemic events in patients with coronary heart disease (CHD) and diabetes mellitus (DM). The mechanism of cardiovascular benefits of statins in DM remains unclear. We examined how simvastatin influences the levels of several in vivo markers for coagulation and fibrinolysis in 26 Type 2 DM patients. The diabetic patients received 20 mg/day of simvastatin up to 12 months. The levels of total cholesterol (TC), low density lipoprotein–cholesterol (LDL-c) and triglycerides in peripheral circulation of patients were significantly reduced after ≥6 weeks of simvastatin treatment. Levels of prothrombin fragment 1 + 2 (F1 + 2), factor VII, plasminogen activator inhibitor-1 (PAI-1) and tissue factor pathway inhibitor (TFPI) antigens, but not tissue plasminogen activator (tPA) antigen, in the pre-simvastatin plasmas of the diabetic patients were significantly higher than the levels found in plasmas of healthy subjects. Significant reductions in F1 + 2 and PAI-1 levels were evident ≥6 weeks after the diabetic patients received simvastatin. Levels of total tPA, TFPI, FVII, hemoglobin A1c, fasting blood glucose, and insulin in the diabetic patients’ plasma were not significantly altered by simvastatin treatment. Positive correlations were found between PAI-1 versus TC, PAI-1 versus LDL-c, and FVII versus triglycerides in the plasmas of simvastatin-treated patients. The results suggest that simvastatin reduces in vivo prothrombinase activity and PAI-1 levels in type 2 DM patients. These actions may contribute to the protective properties of simvastatin against ischemic events in diabetic patients.

Characteristics of CD4+CD25+ regulatory T cells in the peripheral circulation of patients with head and neck cancer

Patients with squamous cell carcinoma of the head and neck (SCCHN) have depressed antitumour immunity. The presence of CD4+CD25+ (Treg) cells in these patients might be, in part, responsible for downregulation of antitumour immune responses. To evaluate the frequency and characteristics of Treg in the peripheral circulation of patients with SCCHN, we used multicolour flow cytometry. Expression of CCR7, CD62L, ζ chain and Annexin V binding to Treg and non-Treg CD4+ lymphocyte populations were evaluated. Treg were confirmed to be Foxp3+ and GITR+. The Treg frequency was significantly elevated in patients with active disease and those with no evidence of disease (NED) following curative therapies. Both Treg and non-Treg CD4+ T cells in patients were significantly enriched in CCR7− and CD62L− cell subsets. Although Treg in patients contained a higher proportion of double negative (CCR7−CD62L−) cells, the majority of Tregs were CCR7−CD62L+. The proportion of Annexin V+CD4+ T cells was higher in patients (P<0.00005) than normal controls (NC), and Treg were significantly more sensitive to apoptosis than non-Treg in patients and NC. Expression of ζ was reduced in all subsets of CD4+ T cells obtained from patients vs NC. The data suggest that Treg in patients with SCCHN largely contain T cells with the ‘effector’ phenotype, which bind Annexin V and have low ζ expression, consistent with their activation state and a rapid turnover in the peripheral circulation.

Elevated serum procollagen type III peptide in splanchnic and peripheral circulation of patients with inflammatory bowel disease submitted to surgery

BackgroundIn the hypothesis that the increased collagen metabolism in the intestinal wall of patients affected by inflammatory bowel disease (IBD) is reflected in the systemic circulation, we aimed the study to evaluate serum level of procollagen III peptide (PIIIP) in peripheral and splanchnic circulation by a commercial radioimmunoassay of patients with different histories of disease.MethodsTwenty-seven patients, 17 with Crohn and 10 with ulcerative colitis submitted to surgery were studied. Blood samples were obtained before surgery from a peripheral vein and during surgery from the mesenteric vein draining the affected intestinal segment. Fifteen healthy age and sex matched subjects were studied to determine normal range for peripheral PIIIP.ResultsIn IBD patients peripheral PIIIP level was significantly higher if compared with controls (5.0 ± 1.9 vs 2.7 ± 0.7 μg/l; p = 0.0001); splanchnic PIIIP level was 5.5 ± 2.6 μg/l showing a positive gradient between splanchnic and peripheral concentrations of PIIIP. No significant differences between groups nor correlations with patients' age and duration of disease were found.ConclusionsWe provide evidence that the increased local collagen metabolism in active IBD is reflected also in the systemic circulation irrespective of the history of the disease, suggesting that PIIIP should be considered more appropiately as a marker of the activity phases of IBD.

Down-regulation of zeta-chain expression in T cells: a biomarker of prognosis in cancer?

The zeta chain is a 16-kDa molecule associated with the T-cell receptor (TCR)-CD3 complex in T lymphocytes and FcgammaRIII in CD3(-)CD56(+)CD16(+) natural killer (NK cells). The zeta chain functions as a transmembrane signaling molecule in lymphocytes. Expression of zeta was found to be decreased in CD4(+) and CD8(+) T lymphocytes isolated from the tumor site or from the peripheral circulation of patients with cancer. A quantitative flow cytometry-based assay for zeta-chain expression allows for reproducible serial evaluations of disease- or therapy-associated changes in expression of this signaling molecule in phenotypically defined subsets of immune cells. Semiquantitative evaluation of zeta expression in paraffin-embedded tissue specimens can link it to the conventional markers of prognosis or survival. Several distinct mechanisms may be responsible for decreased/absent zeta in T cells of patients with cancer. Monitoring for zeta expression is useful for assessing immune competence in these patients and for following changes in immune competence during anticancer therapies. Correlations made between clinical findings, pathologic results, and zeta expression in immune cells suggest that low/absent zeta is predictive of poor prognosis and survival in patients with cancer. Thus, zeta is emerging as a clinically relevant signaling molecule, which also seems to predict a favorable response to biologic therapies and could be helpful in a selection of patients for immunotherapy trials. Validation studies have yet to be performed for this putative immunologic biomarker. Its consistent use for monitoring under standardized conditions of cancer patients treated with biotherapies may help in confirming a role for zeta as a correlate of prognosis or survival.

Impairment in forearm endothelium-dependent vasodilation after heart transplantation

Background Endothelial dysfunction has been found in the peripheral circulation of patients with severe heart failure. However, whether the endothelial dysfunction improves after heart transplantation remains unknown. Our aim was to assess the forearm endothelium-dependent vasoreactivity one and six months after heart transplantation. Methods We studied 12 patients using high resolution brachial artery ultrasound to assess flow-mediated dilation induced by reactive hyperemia and nitroglycerin induced dilation (NTGdil). Results One month after heart transplantation, endothelium-dependent vasodilation was significantly impaired in 10 patients (83%), while it was preserved in the remaining two (17%) (0.4% ± 2.4% vs 9.9% ± 4.6%, respectively, P = .001). NTGdil was normal in both groups (12% ± 10% vs 23% ± 5%, respectively, P = NS). At six months, endothelial dysfunction was present in all patients including the two patients without endothelial dysfunction at the first study. Conclusions The present study demonstrates that peripheral endothelial dysfunction is present after heart transplantation despite the improvement in left ventricular function. More studies are needed to prove if endothelial dysfunction reversion may improve survival in heart transplantation.

Role of prolactin receptor and CD25 in protection of circulating T lymphocytes from apoptosis in patients with breast cancer.

Prolactin (PRL) has been reported to inhibit apoptosis in various cell types and to serve as a cofactor in the upregulation of CD25 on T cells during activation. We investigated a possible relation between prolactin receptor (PRL-R) or IL-2 receptor alpha (IL-2Rα, CD25) expression on circulating T lymphocytes and their apoptosis in patients with breast cancer. Peripheral blood mononuclear cells obtained from 25 patients, 25 normal controls (NC) and three cord blood samples were evaluated for Annexin V binding and expression of CD95, CD25, and PRL-R on CD3+ T cells by multicolour flow cytometry. Plasma levels of PRL, sCD95L, and sIL-2R were determined in patients and controls and related to T-cell apoptosis. The ability of PRL to protect T cells from apoptosis induced by various agents was also studied. Expression of PRL-R on the surface of T cells was comparable in patients with breast cancer and NC, but PRL plasma levels in patients were significantly lower (P<0.05). In patients, 18±11% (mean±s.d.) of CD3+ cells bound Annexin V, compared to 9±6% in NC (P<0.0004). Percentages of CD3+Fas+ and CD3+CD25+ cells were higher in the peripheral circulation of patients than NC (P<0.0001 and <0.04, respectively). Levels of sFasL were lowest in plasma of the patients with the highest proportions of CD3+Fas+ T cells. Most T cells undergoing apoptosis were CD3+CD25− in patients, and the proportion of CD3+CD25− Annexin V+ cells was significantly increased in patients compared to NC (P<0.006). Ex vivo PRL protected T cells from starvation-induced or anti-CD3Ab-induced but not from Fas/FasL-dependent apoptosis. These results indicate that expression of CD25 but not of PRL-R on the surface of activated T lymphocytes appears to be involved in modulating Fas/Fas – ligand interactions, which are, in part, responsible for apoptosis of T lymphocytes and excessive turnover of immune cells in the circulation of patients with breast cancer.

Apoptosis of immune cells in the tumor microenvironment and peripheral circulation of patients with cancer: implications for immunotherapy

Rapid turnover of lymphocytes observed in patients with cancer appears to be driven by increased apoptosis of T lymphocytes or insufficient thymic output of recent thymic emigrants (RTE). Using multicolor flow cytometry and apoptosis assays, we found that CD8 +CD95 +Annexin + T cells are dying at a rate that is significantly higher in patients with cancer than in normal controls (NC). CD8 + effector subsets of T cells were particularly vulnerable to apoptosis. Thymic excision circle (TREC) analysis of peripheral blood lymphocytes showed a decreased number of RTE in these patients. Together, the data suggest that a high rate of T-cell turnover might contribute to immunologic imbalance in patients with cancer and have unfavorable effects on immunotherapy, including therapeutic antitumor vaccines.