Clinical indications, image acquisition and data interpretation for white blood cells and anti-granulocyte monoclonal antibody scintigraphy: an EANM procedural guideline.

INTRODUCTION: Radiolabelled autologous white blood cells (WBC) scintigraphy is being standardized all over the world to ensure high quality, specificity and reproducibility. Similarly, in many European countries radiolabelled anti-granulocyte antibodies (anti-G-mAb) are used instead of WBC with high diagnostic accuracy. The EANM Inflammation & Infection Committee is deeply involved in this process of standardization as a primary goal of the group. AIM: The main aim of this guideline is to support and promote good clinical practice despite the complex environment of a national health care system with its ethical, economic and legal aspects that must also be taken into consideration. METHOD: After the standardization of the WBC labelling procedure (already published), a group of experts from the EANM Infection & Inflammation Committee developed and validated these guidelines based on published evidences. RESULTS: Here we describe image acquisition protocols, image display procedures and image analyses as well as image interpretation criteria for the use of radiolabelled WBC and monoclonal antigranulocyte antibodies. Clinical application for WBC and anti-G-mAb scintigraphy is also described. CONCLUSIONS: These guidelines should be applied by all nuclear medicine centers in favor of a highly reproducible standardized practice.

Novel insights on the relationship between T-tubular defects and contractile dysfunction in a mouse model of hypertrophic cardiomyopathy.

Abnormalities of cardiomyocyte Ca(2+) homeostasis and excitation-contraction (E-C) coupling are early events in the pathogenesis of hypertrophic cardiomyopathy (HCM) and concomitant determinants of the diastolic dysfunction and arrhythmias typical of the disease. T-tubule remodelling has been reported to occur in HCM but little is known about its role in the E-C coupling alterations of HCM. Here, the role of T-tubule remodelling in the electro-mechanical dysfunction associated to HCM is investigated in the Δ160E cTnT mouse model that expresses a clinically-relevant HCM mutation. Contractile function of intact ventricular trabeculae is assessed in Δ160E mice and wild-type siblings. As compared with wild-type, Δ160E trabeculae show prolonged kinetics of force development and relaxation, blunted force-frequency response with reduced active tension at high stimulation frequency, and increased occurrence of spontaneous contractions. Consistently, prolonged Ca(2+) transient in terms of rise and duration are also observed in Δ160E trabeculae and isolated cardiomyocytes. Confocal imaging in cells isolated from Δ160E mice reveals significant, though modest, remodelling of T-tubular architecture. A two-photon random access microscope is employed to dissect the spatio-temporal relationship between T-tubular electrical activity and local Ca(2+) release in isolated cardiomyocytes. In Δ160E cardiomyocytes, a significant number of T-tubules (>20%) fails to propagate action potentials, with consequent delay of local Ca(2+) release. At variance with wild-type, we also observe significantly increased variability of local Ca(2+) transient rise as well as higher Ca(2+)-spark frequency. Although T-tubule structural remodelling in Δ160E myocytes is modest, T-tubule functional defects determine non-homogeneous Ca(2+) release and delayed myofilament activation that significantly contribute to mechanical dysfunction.

New approaches for imaging and therapy of solid cancer.

Radionuclide therapy is a systemic treatment that aims to deliver cytotoxic radiation to cancer cells. Due to their properties, antibodies have been considered as suitable agent for the delivery of therapeutic radioisotopes, radioimmunotherapy (RIT). This article gives an overview of new approaches for imaging and therapy of solid cancer with particular attention to strategies to enhance treatment success. Examples of increased antibody uptake by targeting stromal constituent of tumor microenvironment such as fibronectin (FN) an important tumor-associated angiogenesis targeting agent, with specifically designed antibody format will be provided. Strategies oriented to identify patients most likely to benefit from RIT including identification of radiosensitivity profiles, in vivo target identification by teragnostic approach and better prediction of dosimetric estimates would be presents. Combination regimens such as with chemo-radiotherapy and immunotherapy would be also discussed as an approach to enhance RIT success.

Diagnostic flowcharts in osteomyelitis, spondylodiscitis and prosthetic joint infection.

Infections of the bone, spine and prosthetic joints are serious and complex conditions to diagnose and to treat. Structured diagnostic workup may very well improve the accuracy and speed of diagnosis, thereby improving the outcome since treatment may very well be more successful and less harmful if timely management is started. Literature shows no uniform advise on diagnosis. The EANM organized a consensus meeting with representatives from the involved disciplines in order to develop common flowcharts for the diagnosis of osteomyelitis, spondylodiscitis and prosthetic joint infections. In this report the proceedings of this consensus meeting, including the proposed flowcharts for diagnosis, are published.

Common diagnostic flowcharts in infective endocarditis.

The presentation of infective endocarditis (IE) has changed over time and its diagnosis remains difficult since it relies on the conjunction of a number of criteria which have their own limitations. The Duke classification allows for a standardized approach and is now recognized as the reference method for the diagnosis of IE. The diagnostic value of the different criteria of the Duke classification can be improved by the use of transoesophageal echocardiography for the detection of endocardial involvement and the use of non-cardiac imaging for the detection of embolic events. The number of cases of IE without identified causative microorganism can be reduced due to serological analyses and broad-range polymerase chain reaction on explanted valves. Radionuclide imaging techniques are useful when the diagnosis of IE remains uncertain. [18F]FDG PET/CT can be used for the diagnosis of cardiac infection and for the detection of embolic events or metastatic infection, keeping in mind the possibility of false positive diagnosis due to its high sensitivity. Radiolabelled-leukocytes scintigraphy is more specific than [18F]FDG PET/CT and can differentiate between septic and sterile vegetations. Diagnostic flowcharts are proposed to combine the Duke classification and recent imaging techniques for the diagnostic workup of IE.

Radiolabelled leucocyte scintigraphy versus conventional radiological imaging for the management of late, low-grade vascular prosthesis infections.

PURPOSE: In this study we evaluated the diagnostic performance of (99m)Tc-HMPAO-leucocyte ((99m)Tc-HMPAO-WBC) scintigraphy in a consecutive series of 55 patients (46 men and 9 women, mean age 71 ± 9 years, range 50 - 88 years) with a suspected late or a low-grade late vascular prosthesis infection (VPI), also comparing the diagnostic accuracy of WBC with that of other radiological imaging methods. METHODS: All patients suspected of having VPI underwent clinical examination, blood tests, microbiology, US and CT, and were classified according to the Fitzgerald criteria. A final diagnosis of VPI was established in 47 of the 55 patients, with microbiological confirmation after surgical removal of the prosthesis in 36 of the 47. In the 11 patients with major contraindications to surgery, the final diagnosis was based on microbiology and clinical follow-up of at least 18 months. RESULTS: (99m)Tc-HMPAO-WBC planar, SPECT and SPECT/CT imaging identified VPI in 43 of 47 patients (20 of these also showed infection at extra-prosthetic sites). In the remaining eight patients without VPI, different sites of infections were found. The use of SPECT/CT images led to a significant reduction in the number of false-positive findings in 37% of patients (sensitivity and specificity 100 %, versus 85.1% and 62.5% for stand-alone SPECT). Sensitivity and specificity were 34% and 75% for US, 48.9% and 83.3% for CT, and 68.1% and 62.5% for the FitzGerald classification. Perioperative mortality was 5.5%, mid-term mortality 12%, and long-term mortality 27%. Survival rates were similar in patients treated with surgery and antimicrobial therapy compared to patients treated with antimicrobial therapy alone (61% versus 63%, respectively), while infection eradication at 12 months was significantly higher following surgery (83.3% versus 45.5%). CONCLUSION: (99m)Tc-HMPAO-WBC SPECT/CT is useful for detecting, localizing and defining the extent of graft infection in patients with late and low-grade late VPI with inconclusive radiological findings. (99m)Tc-HMPAO-WBC SPECT/CT might be used to optimize patient management.

Podocyte mitosis - a catastrophe.

Podocyte loss plays a key role in the progression of glomerular disorders towards glomerulosclerosis and chronic kidney disease. Podocytes form unique cytoplasmic extensions, foot processes, which attach to the outer surface of the glomerular basement membrane and interdigitate with neighboring podocytes to form the slit diaphragm. Maintaining these sophisticated structural elements requires an intricate actin cytoskeleton. Genetic, mechanic, and immunologic or toxic forms of podocyte injury can cause podocyte loss, which causes glomerular filtration barrier dysfunction, leading to proteinuria. Cell migration and cell division are two processes that require a rearrangement of the actin cytoskeleton; this rearrangement would disrupt the podocyte foot processes, therefore, podocytes have a limited capacity to divide or migrate. Indeed, all cells need to rearrange their actin cytoskeleton to assemble a correct mitotic spindle and to complete mitosis. Podocytes, even when being forced to bypass cell cycle checkpoints to initiate DNA synthesis and chromosome segregation, cannot complete cytokinesis efficiently and thus usually generate aneuploid podocytes. Such aneuploid podocytes rapidly detach and die, a process referred to as mitotic catastrophe. Thus, detached or dead podocytes cannot be adequately replaced by the proliferation of adjacent podocytes. However, even glomerular disorders with severe podocyte injury can undergo regression and remission, suggesting alternative mechanisms to compensate for podocyte loss, such as podocyte hypertrophy or podocyte regeneration from resident renal progenitor cells. Together, mitosis of the terminally differentiated podocyte rather accelerates podocyte loss and therefore glomerulosclerosis. Finding ways to enhance podocyte regeneration from other sources remains a challenge goal to improve the treatment of chronic kidney disease in the future.

Development and testing of a new disposable sterile device for labelling white blood cells.

AIM: White blood cell (WBC) labelling requires isolation of cells from patient's blood under sterile conditions using sterile materials, buffers and disposables under good manufacturing practice (GMP) conditions. Till now, this limited the use of white blood cell scintigraphy (WBC-S) only to well equipped laboratories with trained personnel. We invented, developed and tested a disposable, sterile, closed device for blood manipulation, WBC purification and radionuclide labelling without exposing patient's blood and the operator to contamination risks. This device prototype and a final industrialized device (Leukokit®) were tested for WBC labelling and compared to standard procedure. Leukokit® was also tested in an international multi-centre study for easiness of WBC purification and labelling. METHODS: On the device prototype we tested in parallel, with blood samples from 7 volunteers, the labelling procedure compared to the standard procedure of the International Society of Radiolabeled Blood Elements (ISORBE) consensus protocol with respect to cell recovery, labelling efficiency (LE), cell viability (Trypan Blue test) and sterility (haemoculture). On the final Leukokit® we tested the biocompatibility of all components, and again the LE, erythro-sedimentation rate, cell viability, sterility and apyrogenicity. ACD-A, HES and PBS provided by Leukokit® were also compared to Heparin, Dextran and autologous plasma, respectively. In 4 samples, we tested the chemotactic activity of purified WBC against 1 mg/ml of lipopolysaccharide (LPS) and chemotaxis of 99mTc-HMPAO-labelled WBC (925 MBq) was compared to that of unlabelled cells. For the multi-centre study, 70 labellings were performed with the Leukokit® by 9 expert operators and 3 beginners from five centers using blood from both patients and volunteers. Finally, Media-Fill tests were performed by 3 operators on two different days (11 procedures) by replacing blood and kit reagents with bacterial culture media (Tryptic Soy Broth) and testing sterility of aliquots of the medium at the end of procedure. RESULTS: Tests performed with the prototype showed no significant differences with the standard procedure but a faster and safer approach. Tests performed with the final Leukokit® confirmed full biocompatibility, sterility and apyrogenicity of all reagents and plastic ware. Average WBC recovery with Leukokit® was comparable to that of the ISORBE protocol (117x106±24x106 vs. 132x106±29x106 cells, P=not significant). No differences in red blood cells and platelet content were observed. LE was 82% ± 3% for Leukokit® and 65±5% for control (P=0.0003) being PBS vs autologous plasma the main reason of such difference. Cell viability was always >99.9% in both conditions. Chemotactic tests showed no differences between all Leukokit® samples and controls. Haemocultures and Media-Fill tests were always sterile. The procedure was well accepted by expert operators and beginners, with a very fast learning curve (confidence after 2±2 labellings). CONCLUSION: The invented device offers high level of protection to operators and patients. The derived Leukokit® is safe and easy to use, and gives a high LE of WBC without affecting cell viability and function. Being a registered closed, sterile medical device, it may allow easier and faster WBC labelling that is not limited to only well equipped laboratories. Also simultaneously labelling of multiple patients is possible.

[The role of podocyte damage in the pathogenesis of glomerulosclerosis and possible repair mechanisms]. / Ruolo del danno podocitario nella patogenesi della glomerulosclerosi e possibili meccanismi di riparazione.

Converging evidence suggests that damage to podocytes plays a key role in progression towards glomerulosclerosis, in particular as the primary cause of all forms of focal segmental glomerulosclerosis (FSGS), the most common glomerular disease leading to end-stage renal disease. Any damage occurring to the complex architecture of specialized proteins that constitute the podocyte foot processes, essential to the highly specialized functions of podocytes, leads inevitably to loss of function in the glomerular filtration barrier, and ultimately to proteinuria. Recent studies have also highlighted that a reduction of the podocyte number in a damaged glomerulus is a critical factor for the development of proteinuria and glomerulosclerosis. As long as the podocyte loss is limited, restitution or repair is possible, which shows that the glomerular architecture can be remodeled. However, mature podocytes have limited capacity to divide and display all the phenotypic and functional features of highly specialized, terminally differentiated cells. A potential mechanism for podocyte replacement might be stem-cell-based regeneration, since it has been established that the developmental source of podocytes are resident renal progenitors. Podocyte damage could then be potentially repaired by a stem cell population resident in the kidney.

The critical role of SDF-1/CXCR4 axis in cancer and cancer stem cells metastasis.

Chemokines exert their multifunctional role in several physiologic and pathologic processes through interaction with their specific receptors. Much evidence have revealed that metastatic spread tumor cells may use chemokine-mediated mechanisms. In particular, an involvement of stromal cell-derived factor-1 (SDF-1) in growth of primary tumors and in metastatic process has been demonstrated. Indeed, it has been suggested that CXCR4 expression by tumor cells, plays a critical role in cell metastasis by a chemotactic gradient to organs expressing the ligand SDF-1. Moreover, CXCR4 overexpression correlated with poor prognosis in many types of cancer. In physiologic condition, SDF-1 also plays an essential role modulating stem cell proliferation, survival, and homing through its canonical receptor CXCR4. Recently, several studies have demonstrated the existence of a small subset of cancer cells which share many characteristics with stem cells and named cancer stem cells (CSC). They constitute a reservoir of self-sustaining cells with the ability to maintain the tumor growth. In particular, most of them express CXCR4 receptor and respond to a chemotactic gradient of its specific ligand SDF-1, suggesting that CSC probably represent a subpopulation capable of initiating metastasis. This review focuses on the role of SDF-1/CXCR4 axis in cancer and in the metastatic progression by tumoral cells, as well as the role of CSC in tumor pathogenesis and in metastatic process. A better understanding of migratory mechanism involving cancer cells and CSC provides a powerful tool for developing novel therapies reducing both local and distant recurrences.

[The role of endothelial progenitor cells in renal disease]. / Ruolo dei progenitori endoteliali nelle malattie renali.

Recent evidence suggests that injury to the renal vasculature may play an important role in the pathogenesis of both chronic and acute ischemic kidney injury. Early alterations in peritubular capillary blood flow during reperfusion have been documented and associated with loss of normal endothelial cell function. In addition, ischemia induces alterations in endothelial cells that may promote inflammation and procoagulant activity, thus contributing to vascular congestion. Reduction of the microvasculature density increases hypoxia-mediated fibrosis and alters proper hemodynamics, which may lead to hypertension. This may play a critical role in the progression of chronic kidney disease following initial recovery from ischemia/reperfusion-induced acute kidney injury. The turnover and replacement of endothelial cells is therefore an important mechanism in the maintenance of vascular integrity also in the kidney. It is becoming clear that impaired vascular repair mechanisms as a result of a reduced number and/or impaired function of endothelial progenitor cells may contribute to renal disease. Moreover, investigators have begun to identify potential mechanisms responsible for the loss of function of endothelial progenitors in renal disease. In allografts, persistent injury results in excessive turnover of graft vascular endothelial cells. Moreover, chronic damage elicits a response that is associated with the recruitment of both leukocytes and endothelial progenitors, facilitating an overlapping process of inflammation and angiogenesis. In conclusion, angiogenesis and endothelial cell turnover play a pivotal role in renal disease and allograft rejection. Manipulation of these processes might have important implications for the development of novel therapeutic strategies in the near future.

Octreo-SPECT/CT imaging for accurate detection and localization of suspected neuroendocrine tumors.

AIM: The aim of the present study was to prospectively evaluate the add value provided by [(111)In]DTPA-octreotide single-photon emission computed tomography/computed tomography (Octreo-SPECT/CT) with respect to [(111)In]DTPA-octreotide SPECT (Octreo-SPECT) in terms of diagnostic accuracy and localization of neuroendocrine tumors (NETs). METHODS: Eighty-one consecutive patients with known or suspected NET underwent [(111)In]DTPA-octreotide scintigraphy using an integrated SPECT/low-energy-CT system (Infinia & Hawkeye; GE Medical Systems, Milwaukee, WI, USA). SPECT and fused SPECT/CT images were interpreted separately and a lesion-by-lesion analysis was performed with regard to classification (probability of NET graded on a 5-point scale) and localization of each abnormal focal tracer uptake. A subgroup analysis, distinguishing between abdominal and thoracic lesions, and a patient-by-patient analysis for likelihood of NET in each patient was also performed. Standard of reference for confirming presence or absence of NET was either histopathology or clinical/imaging follow-up data. The value of SPECT/CT imaging was assessed by ROC analysis and McNemar test. RESULTS: A final diagnosis of NET was achieved in 43 out of 81 patients and a total of 169 areas (138 NET and 31 benign/physiological) with focal tracer uptake were included in the final lesion-by-lesion analysis. SPECT/CT imaging led to a significantly higher proportion of patients (75/81=92.6% vs 64/81=79%) and lesions (163/169=96.4% vs 138/169=81.1%) correctly classified vs SPECT alone. ROC analysis confirmed that Octreo-SPECT/CT performed significantly better than Octreo-SPECT for the detection of NET on both patient- and lesion-based analysis, improving especially evaluation of abnormal tracer uptake in the abdomen. Moreover, Octreo-SPECT/CT accurately localized 160/169 (94.7%) lesions, significantly higher than SPECT alone (77/169= 45.6%). CONCLUSIONS: Octreo-SPECT/CT allows more accurate detection and localization of NETs than simple Octreo-SPECT, with major benefits for lesions located in the abdomen.

Anticlastogenic effect of Ginkgo biloba extract in Graves' disease patients receiving radioiodine therapy.

BACKGROUND: Chromosomal damage, as assessed by clastogenic factors (CFs) and micronuclei (MN) appearance, after radioiodine therapy of Graves' disease has been reported. OBJECTIVE AND METHODS: Our objective was to evaluate the effect of Ginkgo biloba extract (EGb 761) supplementation on the time course (up to 120 d) of CFs and MN appearance in lymphocytes from patients with Graves' disease after iodine-131 ((131)I) therapy. Patients were randomly assigned to EGb 761 or placebo, in a blinded manner. RESULTS: In the placebo group, MN increased early (P < 0.001) after (131)I, peaking at the 21st day (P = 0.0003) and declining thereafter. In EGb 761-treated patients, MN increased early (P < 0.05), while returning toward baseline value thereafter. Therefore, mean MN increment was significantly higher in the placebo group as compared with EGb 761-treated patients (P < 0.01). Moreover, an early (P < 0.0001) and sustained (up to 35 d; P < 0.001) MN increase induced by CFs was observed in the placebo group. Conversely, in EGb 761-treated patients, MN increase induced by CFs never reached the statistical significance; therefore, the mean of the MN increments was significantly lower than in placebo (P < 0.05). A significant positive correlation between MN maximum increment and the bone marrow dose was observed in the placebo group only (P = 0.03). No significant difference was observed in clinical outcome between the two groups. CONCLUSIONS: EGb 761 supplementation neutralized genotoxic damage induced by radioiodine treatment, without affecting the clinical outcome. Although (131)I therapy is generally safe, our data suggest that Gingko biloba extracts may prevent genetic effects of radioiodine therapy for hyperthyroid Graves' disease.

Recombinant human granulocyte colony-stimulating factor administration in a case of neutropenia due to increased neutrophil sequestration.

A 55-year-old female was admitted with fever which followed an episode of pseudomembranous colitis. Despite an accurate clinical investigation, there was no evidence for specific sites of infection. Remission of fever was not obtained with antibiotic therapy (gentamycin plus carbepenem) and progressive neutropenia was observed. Neutrophils fell to 0.3 x 10(9)/1. The diagnostic approach, including a bone marrow aspirate, excluded mechanisms leading to impaired neutrophil production, and in the suspect of increased neutrophil sequestration/destruction, whole-body scintigraphy with (99m)technetium-hexamethylpropyleneamineoxime ((99m)Tc-HMPAO)-labeled autologous leukocytes was performed. As a result, a site of leukocyte sequestration localized at the medium lobe of the right lung was detected. In an attempt to enhance neutrophil functions and achieve remission of infection, recombinant human granulocyte colony-stimulating factor (Filgrastim, Granulokine 30, Roche) at the dosage of 300 microg/day, subcutaneously, was added. As a results, fever disappeared in three days, but neutrophil recovery was slower, and normalization of the absolute neutrophil count (ANC) was obtained on day +7. The results obtained in this peculiar case of neutropenia, and the kinetics of both fever and ANC, suggest the possible combination of neutrophil function enhancement and an anti-inflammatory effect of rhG-CSF.

[Chemokines: possible therapeutic targets and useful clinical parameters in renal transplantation]. / Le chemochine nel trapianto renale: possibili bersagli terapeutici e nuovi parametri clinici.

Chemokines are a family of small, structurally related cytokines that regulate trafficking of different subsets of leukocytes, thus critically regulating inflammation. The chemokine system influences allograft biology at 3 main levels: 1) the process of ischemia-reperfusion injury, 2) the induction of transplant tolerance, and 3) the pathogenesis of acute rejection and chronic allograft nephropathy. Accordingly, following ischemia/reperfusion in a rat model, CXCR2 produced at the graft level attracts and activates granulocytes, which in turn promotes graft damage. Moreover, in some experimental models CCR4 recruits T regulatory cells and mediates transplant tolerance. Furthermore, the discovery of the involvement of CXCR3 in the induction of the alloresponse to transplant suggests that this chemokine receptor might represent an important target for treatment of both acute rejection and chronic allograft nephropathy. Indeed, CXCR3 ligands play a pivotal role in the initiation and amplification of host alloresponses and also alter vascular cell functions, which explains their critical role not only in the development of acute rejection, but also in the pathogenesis of chronic allograft nephropathy, where both immune- and nonimmune- mediated mechanisms are involved. Finally, we have recently demonstrated that the pretransplant serum level of the CXCR3 ligand IP-10/CXCL10 is a clinically useful parameter for the identification of subjects with a high risk of acute rejection, chronic allograft nephropathy, and graft failure. This simple test could contribute to the prevention of acute rejections and the individualization of immunosuppressive therapies.

Pharmacological modulation of stem cell function.

The discovery of stem cells (SC) has shed new light on the understanding of mechanisms responsible for ischemic and degenerative disorders, and opened a new field for regenerative medicine. Furthermore, dysregulation of SC self-renewal and their transformation seem to be involved also in the development of cancer, suggesting that pharmacological treatment devoted to regulate SC genomic and phenotypic functions might represent a potential new strategy even for the treatment of neoplastic disorders. SC display a promiscuous set of transcription factors and an open chromatin structure which are required to maintain their multipotentiality, while they are progressively quenched during differentiation into specific multiple lineages. The mechanisms that govern stem cell fate decisions are under tight control but remain potentially alterable. Recent studies have shown that several currently used drugs such as colony stimulating factors, statins, angiotensin-II receptor antagonists/ACE-inhibitors, Erythropoietin, nitric oxide donors, estrogens and glitazones, have modulatory activity on SC functions. These drugs mostly enhance SC survival and mobilization. Furthermore, a series of new pharmacological agents such as the chemokine receptor antagonist AMD3100, glycogen synthase kinase-3 (GSK-3) inhibitors and histone deacetylase inhibitors (HDACi), that modulate the growth, differentiation and mobilization of SC, have been recently discovered and are currently under evaluation in both in vivo experimental models and preliminary clinical trials. Thus, modulation of SC properties through pharmacological treatment represents a new field of investigation which may lead to the development of novel strategies for the treatment not only of ischemic and degenerative disorders, but also of cancer.

CXCR3 and alphaEbeta7 integrin identify a subset of CD8+ mature thymocytes that share phenotypic and functional properties with CD8+ gut intraepithelial lymphocytes.

BACKGROUND: We previously demonstrated the existence of two distinct subsets of T cell receptor (TCR)alphabeta+CD8alphabeta+ single positive (SP) cells in human postnatal thymus which express the chemokine receptor CCR7 or CXCR3 and migrate in vitro in response to their specific ligands. AIM: To investigate whether these two CD8+ thymocyte subsets had distinct peripheral colonisation. METHODS: TCRalphabeta+CD8+ SP cells were obtained from normal postnatal thymus, mesenteric lymph node (LNs), small bowel, and peripheral blood (PB) specimens. Cells were then evaluated for expression of surface molecules, cytolytic potential, telomere length, and profile of cytokine production. RESULTS: CD8+CCR7+CXCR3- thymocytes exhibited CD62L, in common with those which localise to LNs. In contrast, CD8+CCR7-CXCR3+ thymocytes lacked CD62L but exhibited CD103, similar to intraepithelial lymphocytes (IELs) present in the gut mucosa where the CXCR3 ligand, CXCL10, and the CD103 ligand, E-cadherin, are highly and consistently expressed. In addition, thymocytes and gut CD8+CXCR3+CD103+ cells showed comparable telomere length, which was higher than that of PB CXCR3+CD8+ T cells. However, both of these populations contained perforin and granzyme A, and displayed the ability to produce interferon gamma and interleukin 2. Of note, CXCR3 deficient, in comparison with wild-type C57Black/6, mice showed decreased proportions of CD3+CD8alphabeta+ and increased proportions of CD3+CD8alphaalpha+ lymphocytes at gut level. Moreover, adoptive transfer of CD3+CD8alphabeta+ thymocytes from wild-type into CXCR3 deficient mice resulted in a significant increase in CD3+CD8alphabeta+ T cells in the gut mucosa but not in other tissues. CONCLUSIONS: The results of this study demonstrate the existence of a previously unrecognised subset of TCRalphabeta+CD8alphabeta+ SP CXCR3+CD103+ thymocytes which share phenotypic and functional features with CD8+ IELs, thus suggesting the possibility of their direct colonisation of the gut mucosa.

CXCR3-binding chemokines: novel multifunctional therapeutic targets.

The goal to attenuate inflammation without inducing generalized immunosuppression has focused the attention on chemokines, a family of chemotactic peptides that regulate the leukocyte traffick into tissues. However, the development of drugs that block ckemokine activity may be hampered by the observation that some chemokines display pleiotropic biologic functions. For example, the chemokines CXCL9/Mig, CXCL10/IP-10, and CXCL11/I-TAC exhibit the ability to recruit different leukocytes subsets, the capacity to induce the proliferation of vascular pericytes as well as powerful anti-tumor effects, which are mediated by a common receptor, named CXCR3. Because of their pleiotropic biologic effects, these chemokines have been proposed as possible therapeutic targets in cancer, allograft rejection, glomerulonephritis, diabetes, multiple sclerosis, and autoimmune disorders of the thyroid. The chemokine CXCL4/PF4 shares several activities with CXCL9, CXCL10, and CXCL11, including angiostatic effects, although its specific receptor has remained unknown for a long time. Recently, we provided evidence that the different functions of CXCL9, CXCL10, and CXCL11 on distinct cell types can be at least partly explained by the interaction of these chemokines with two distinct receptors. Indeed, in addition to the classic form of CXCR3 receptor, which we have renamed as CXCR3-A, a novel CXCR3 receptor variant (CXCR3-B) was identified, that not only mediates the angiostatic activity of CXCR3 ligands, but also acts as functional receptor for CXCL4. In this review, we focus on the accumulating evidence demonstrating the pivotal role of CXCR3-binding chemokines in several human diseases. Studies based on CXCR3 targeting have shown its importance in different pathologic conditions and orally active small molecules capable of inhibiting this receptor are now being developed in order to be tested for their activity in humans.

Measurement of somatostatin receptor subtype 2 mRNA in breast cancer and corresponding normal tissue.

Somatostatin analogs are effective in inhibiting growth of human breast cancer cell lines. These antiproliferative effects are mediated by specific receptors located on cell membranes. The somatostatin receptor subtype 2 (sst2) is the principal mediator of somatostatin effects in normal and cancer cells, and its presence has already been demonstrated in breast cancer. The purpose of our study was to evaluate the clinical relevance of the expression of sst2 by quantifying its mRNA in a large group of infiltrating breast cancers and their corresponding normal tissues. The expression of sst2 mRNA was measured with quantitative real time RT-PCR in 169 breast cancers and in their corresponding unaffected tissues. We evaluated the association of sst2 expression with the commonest clinical-pathologic features of breast cancer. The correlation with a marker of cell proliferation (Ki-67) and with receptor concentration was also evaluated. In cancer tissues, we found that the absolute concentrations of sst2 mRNA were significantly higher in estrogen receptor (ER)-positive samples (P=0.002) as well as in lymph-node-negative cancers (P=0.04) (Student's t-test or one-way ANOVA). In addition, sst2 mRNA was significantly higher in breast cancers than in corresponding unaffected tissues (P=0.0002). However, when the clinical-pathologic parameters were considered, this gradient maintained its statistical significance only in tumors expressing positive prognostic markers, such as the presence of ER (P=0.0005) and progesterone receptors (PgR) (P=0005), and the lack of lymph-node involvement (P=0.0003). The same difference was also significant in postmenopausal women (P=0.001) and in T1 patients (P=0.001). In addition, sst2 mRNA expression was significantly higher (P=0.008) in low-proliferating breast cancers. Finally, we found that the quantitative expression of sst2 mRNA was directly related to the PgR concentration in breast cancer tissues (P<0.001). Our data seem to indicate that an upregulation of sst2 gene expression is a common feature of breast cancers which, on the basis of conventional predictive parameters, are expected to have a better prognosis. Featuring a possible role of somatostatin analogs in combined endocrine therapies for breast cancer, our results seem to confirm that the sst2 status of the tumor should be previously investigated.