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Literature reports suggest that the host immune system may control Malignant Pleural Mesothelioma (MPM) growth, although its activity is limited by regulatory mechanisms. In this retrospective study, we analyzed the levels of pro-inflammatory (IL-1, IL-6, TNF), immune-regulatory (IL-10) and Th1/CTL-related cytokines (IL-12p70, IFN-γ) in the pleural exudate and their relationship with overall survival (OS) in MPM. Cytokines were quantified by multiplexed immunoassay. Concentrations were dichotomized with respect to the median value. Correlation between cytokine level and OS was assessed using univariate (Kaplan–Meier curves) and multivariate (Cox regression) analyses. Regarding outcome, tumor histology, therapies undergone and IFN-γ were independent prognostic factors of OS in a 72 MPM training cohort. Notably, high concentrations of IFN-γ halved death probability (HR of high vs low IFN-γ concentration = 0.491, 95%CI 0.3–0.8, p = 0.007). Also in patients with epithelioid histology and those receiving at least one line of therapy, high IFN-γ level was an independent factor predictive of OS (HR of high vs low IFN-γ concentration were 0.497, p = 0.007 and 0.324, p = 0.006, respectively). However, these data were not confirmed in a 77 MPM validation cohort, possibly due to the low IFN-γ levels encountered in this population, and the heterogeneous distribution of disease stages between the training and the validation cohorts. None of the other cytokines showed any effect on survival. High level of IFN-γ in pleural effusion may be associated with better survival in MPM patients and potentially serve as a prognostic biomarker. Larger prospective studies are needed to ascertain this hypothesis.

Immune checkpoint inhibitors, including ipilimumab (IPI), achieve a clinical benefit in a small proportion of melanoma patients highlighting the need to investigate predictive biomarkers. In this study, we characterized tumor infiltrating lymphocytes (TILs), focusing on the CTLA-4+ subset, and evaluated their possible predictive significance. We characterized TIL density, cell type, and localization in 40 melanoma lesions from 17 patients treated with IPI. Associations of TILs with IPI timing, tissue localization, and response to IPI were estimated using a linear mixed-effects modelling approach. We found that most of TIL subsets increased in situ upon IPI therapy, with particular reference to FoxP3+ cells. TILs and TIL subsets, such as CD3+, CD45RO+, CTLA-4+, CD4+, CD8+ T cells, CD20+ B cells, and NKp46+ NK cells, showed significantly different spatial distributions in the tumor microenvironment being higher at the invasive margin (IM) as compared to the tumor center (TC) (P value

Simple Summary Malignant mesothelioma (MM), a rare and aggressive tumor, is related to asbestos exposure, which mediates a chronic inflammatory process involving the cytokine IL-6. Recent studies indicate that the PD-1/PD-L1 immune-suppressive axis is a clinically relevant target for therapy. The expression of PD-L1 in tumor cells is generally a cytokine-mediated effect. Here we show that the IL-6-related cytokine IL-27 is able to enhance PD-L1 expression and soluble (s)PD-L1 release by cultured MM cells, whereas IL-6 is ineffective. In agreement with previous findings, we found high IL-6 levels in pleural exudates from 77 MM patients which correlated with worse survival. More importantly, we also found sPD-L1 and IL-27 in the same samples. sPD-L1 and IL-27 levels showed a moderate albeit significant correlation and association with worse survival, which suggested a potential effect of IL-27 on PD-L1-mediated immune resistance in MM. Abstract Malignant mesothelioma (MM) is a rare tumor with an unfavorable prognosis. MM genesis involves asbestos-mediated local inflammation, supported by several cytokines, including IL-6. Recent data showed that targeting PD-1/PD-L1 is an effective therapy in MM. Here, we investigated the effects of IL-6 trans-signaling and the IL-6-related cytokine IL-27 on human MM cells in vitro by Western blot analysis of STAT1/3 phosphorylation. The effects on PD-L1 expression were tested by qRT-PCR and flow-cytometry and the release of soluble (s)PD-L1 by ELISA. We also measured the concentrations of sPD-L1 and, by multiplexed immunoassay, IL-6 and IL-27 in pleural fluids obtained from 77 patients in relation to survival. IL-27 predominantly mediates STAT1 phosphorylation and increases PD-L1 gene and surface protein expression and sPD-L1 release by human MM cells in vitro. IL-6 has limited activity, whereas a sIL-6R/IL-6 chimeric protein mediates trans-signaling predominantly via STAT3 phosphorylation but has no effect on PD-L1 expression and release. IL-6, IL-27, and sPD-L1 are present in pleural fluids and show a negative correlation with overall survival, but only IL-27 shows a moderate albeit significant correlation with sPD-L1 levels. Altogether these data suggest a potential role of IL-27 in PD-L1-driven immune resistance in MM.

Background Whether the extent of residual disease in the sentinel lymph node (SLN) after neoadjuvant chemotherapy (NAC) influences the prognosis in clinically node-positive breast cancer (BC) patients remains to be ascertained. Methods One hundred and thirty-four consecutive cN+/BC-patients received NAC followed by SLN biopsy and axillary lymph node dissection. Cumulative incidence of overall (OS) and disease-free (DFS) survival, BC-related recurrences and death from BC were assessed using the Kaplan-Meier method both in the whole patient population and according to the SLN status. The log rank test was used for comparisons between groups. Results The SLN was identified in 123/134 (91.8%) patients and was positive in 98/123 (79.7%) patients. Sixty-five of them (66.3%) had other axillary nodes involved. SLN sensitivity and false-negative rate were 88.0% and 2.0%, Median follow-up was 10.2 years. Ten-year cumulative incidence of axillary, breast and distant recurrences, and death from BC were 6.5%, 11.9%, 33.4% and 31.3%, respectively. Ten-year OS and DFS were 67.3% and 55.9%. When stratified by SLN status, 10-year cumulative incidence of BC-related and loco-regional events, and death from BC were similar between disease-free SLN and micrometastatic SLN subgroups (28.9% vs 30.2%, p = 0.954; 21.6% vs 13.4%, p = 0.840; 12.9 vs 24.5%, p=0.494). Likewise, 10-year OS and DFS were comparable (80.0% vs 75.5%, p=0.975 and 68.0% vs 69.8, p=0.836). Both OS and DFS were lower in patients presenting a macrometastatic SLN (60.2% and 47.5%). Conclusion Outcome of patients with micrometastatic SLN was similar to that of patients with disease-free SLN, which was more favorable as compared to that of patients with macrometastatic SLN.

Sentinel lymph node biopsy alone, without complete axillary lymph node dissection, is the standard treatment of the axilla nodal chain in early-stage breast cancer patients presenting a negative sentinel lymph node. The updated results of the IBCSG 23-01 randomized trial recently provided evidence that this approach could be extended to early-stage breast cancer patients presenting only micrometastasis in the sentinel lymph node.On the other hand, patients with large operable or locally advanced breast cancer and clinically positive lymph nodes currently receive neoadjuvant chemotherapy and sentinel lymph node biopsy, which is then followed by complete axillary node dissection if the sentinel lymph node till contains tumor residue, regardless of the extent of nodal disease. Assuming that patients presenting only a micrometastatic sentinel lymph node after neoadjuvant chemotherapy are clinically equivalent to the IBCSG 23-01 early-breast cancer patients with only micrometastatic sentinel node, then complete axillary dissection would be unneeded also in these subset of patients in the neoadjuvant setting. The multicenter uncontrolled non-inferiority trial NEONOD 2 we here present was designed to assess this hypothesis, i.e. whether or not omission of complete axillary nodal clearance worsens prognosis in patients with sentinel node resulting only micrometastatic after neoadjuvant chemotherapy. Keywords: Infiltrating breast cancer, Clinically positive axilla, Neoadjuvant chemotherapy, Sentinel lymph node biopsy, Axillary lymph node dissection, Outcome

The expression of the immune checkpoint molecule CTLA-4 has been almost exclusively studied in the T cell lineage, but increasing evidence has shown its expression on tumors with implications for immunotherapy. To date, the degree of expression of CTLA-4 on tumor cells as a predictive biomarker of response to immune checkpoint inhibitors has not been studied. In this report, we analyzed this issue in melanoma patients treated with CTLA-4 inhibitor Ipilimumab (IPI). We show that the level of CTLA-4 expression on melanoma cells is higher than that on tumor infiltrating lymphocytes (TIL) and it is associated with clinical response to IPI therapy supporting the idea of its possible role as a predictive biomarker.

// Claudia Bighin 1 , Beatrice Dozin 2 , Francesca Poggio 1 , Marcello Ceppi 2 , Paolo Bruzzi 2 , Alessia D’Alonzo 1 , Alessia Levaggi 1 , Sara Giraudi 1 , Matteo Lambertini 1 , Loredana Miglietta 1 , Marina Vaglica 1 , Vincenzo Fontana 2 , Giuseppina Iacono 1 , Paolo Pronzato 1 and Lucia Del Mastro 1 1 Department of Medical Oncology, IRCCS Azienda Ospedaliera Universitaria San Martino – Istituto Nazionale per la Ricerca sul Cancro, Genoa, Italy 2 Department of Clinical Epidemiology, IRCCS Azienda Ospedaliera Universitaria San Martino – Istituto Nazionale per la Ricerca sul Cancro, Genoa, Italy Correspondence to: Claudia Bighin, email: // Keywords : advanced breast cancer, hormone-responsive breast cancer, first-line treatment, hormonal therapy, chemotherapy Received : November 30, 2016 Accepted : January 10, 2017 Published : January 18, 2017 Abstract Introduction Although hormonal-therapy is the preferred first-line treatment for hormone-responsive, HER2 negative metastatic breast cancer, no data from clinical trials support the choice between hormonal-therapy and chemotherapy. Methods Patients were divided into two groups according to the treatment: chemotherapy or hormonal-therapy. Outcomes in terms of clinical benefit and median overall survival (OS) were retrospectively evaluated in the two groups. To calculate the time spent in chemotherapy with respect to OS in the two groups, the proportion of patients in chemotherapy relative to those present in either group was computed at every day from the start of therapy. Results From 1999 to 2013, 119 patients received first-line hormonal-therapy (HT-first group) and 100 first-line chemotherapy (CT-first group). Patients in the CT-first group were younger and with poorer prognostic factors as compared to those in HT-first group. Clinical benefit (77 vs 81%) and median OS (50.7 vs 51.1 months) were similar in the two groups. Time spent in chemotherapy was significantly longer during the first 3 years in CT-first group (54-34%) as compared to the HT-first group (11-18%). This difference decreased after the third year and overall was 28% in the CT-first group and 18% in the HT-first group. Conclusions The sequence first-line chemotherapy followed by hormonal-therapy, as compared with the opposite sequence, is associated with a longer time of OS spent in chemotherapy. However, despite the poorer prognostic factors, patients in the CT-first group had a superimposable OS than those in the HT-first group.

Sentinel lymph node biopsy alone is the current surgical axillary treatment for early-stage breast cancer patients with a negative sentinel lymph node (SLN). The possibility to omit axillary dissection also in presence of positive SLNs has been promoted by the American College of Surgeons Oncology Group (ASOCOG) Z0011 randomized trial. Several limitations and evidences of potential selection bias made this trial fairly controversial. Stronger evidence than currently available is needed on the safety of foregoing axillary dissection in well-defined populations of patients with positive SLNs. The Italian multicentre SINODAR ONE randomized trial here presented was designed with this aim.

Background The incidence of brain metastases (BM) in melanoma patients is common and associated with poor prognosis. MAP-kinase inhibitors and immunologic checkpoint blockade antibodies led to improved survival of metastatic melanoma patients; however, patients with BM are under-represented or excluded from the majority of clinical trials and the impact of new drugs on their survival is less clear. With the present systematic review, we aimed to analyze outcomes of patients with melanoma BM treated with the new drugs, both in the setting of phase I–II–III clinical trials and in the “real world”. Methods An electronic search was performed to identify studies reporting survival outcomes of patients with melanoma BM treated with MAP-kinase inhibitors and/or immunologic checkpoint blockade antibodies, regardless of study design. Results Twenty-two studies were included for a total of 2153 patients. Median OS was 7.9 months in phase I–II–III trials and 7.7 months in “real world” studies. In clinical trials, median OS was 7.0 months for patients treated with immunotherapy and 7.9 months for patients treated with BRAF inhibitors. In “real world” studies, median OS was 4.3 months and 7.7 months for patients treated with immunotherapy and BRAF inhibitors, respectively. Evidence of clinical activity exists for both immunotherapy and MAP-kinase inhibitors. Conclusions MAP-kinase inhibitors and immunologic checkpoint blockade antibodies have clinical activity and may achieve improved OS in patients with metastatic melanoma and BM. These results support the inclusion of patients with BM in investigations of new agents and new treatment regimens for metastatic melanoma.

Sentinel lymph node biopsy (SLNB) allows for staging of the axillary node status in early-stage breast cancer (BC) patients and avoiding complete axillary lymph node dissection (ALND) when the sentinel lymph node (SLN) is proven to be free of disease. In a previous randomized trial we compared SLNB followed by ALND (ALND arm) with SLNB followed by ALND only if the SLN presented metastasis (SLNB arm). At a mid-term of ≈ 6 years median follow-up, the two strategies appeared to ensure similar survival and locoregional control. We have revised these previous findings and update the results following a 15-year observation period. Patients were randomly assigned to either the ALND or SLNB arm. The main endpoints were event-free survival (EFS), overall survival (OS), and axillary disease recurrence. EFS and OS were assessed using Kaplan–Meier analysis and the log-rank test. The ALND and SLNB arms included 115 and 110 patients, respectively. At 14.3 years median follow-up, 39 primary BC-related recurrences occurred, 22 (19 %) of which occurred in the ALND arm and 17 (16 %) occurred in the SLNB arm (p = 0.519). No axillary relapse developed in the SLNB arm, while two were observed in the ALND arm. OS (82.0 vs. 78.8 %) and EFS (72.8 vs. 72.9 %) were not statistically different between the ALND and SLNB arms (p = 0.502 and 0.953, respectively). SLNB is a safe and efficacious component of the surgical treatment of early-stage BC patients. In the long-term, SLNB is equivalent to ALND in terms of locoregional nodal disease control and survival in this subset of patients.

CTLA-4 blockade by means of ipilimumab (IPI) potentiates the immune response and improves overall survival (OS) in a minority of metastatic melanoma (MM) patients. We investigated the role of soluble CTLA-4 (sCTLA-4) as a possible biomarker for identifying this subset of patients. sCTLA-4 levels were analyzed at baseline in sera from 113 IPI-treated MM patients by ELISA, and the median value (200 pg/ml) was used to create two equally sized subgroups. Associations of sCTLA-4 with best overall response (BOR) to IPI and immune-related adverse events (irAEs) were evaluated through logistic regression. Kaplan–Meier and Cox regression methods were used to analyze OS. A remarkable association between sCTLA-4 levels and BOR was found. Specifically, the proportion of patients with sCTLA-4 > 200 pg/ml in irSD or irPD (immune-related stable or progressive disease) was, respectively, 80% (OR = 0.23; 95%CL = 0.03–1.88) and 89% (OR = 0.11; 95%CL = 0.02–0.71) and was lower than that observed among patients in irCR/irPR (immune-related complete/partial response). sCTLA-4 levels increased during IPI treatment, since the proportion of patients showing sCTLA > 200 pg/ml after 3 cycles was 4 times higher (OR = 4.41, 95%CL = 1.02–19.1) than that after 1 cycle. Moreover, a significantly lower death rate was estimated for patients with sCTLA-4 > 200 pg/ml (HR = 0.61, 95%CL = 0.39–0.98). Higher baseline sCTLA-4 levels were also associated with the onset of any irAE (p value = 0.029), in particular irAEs of the digestive tract (p value = 0.041). In conclusion, our results suggest that high sCTLA-4 serum levels might predict favorable clinical outcome and higher risk of irAEs in IPI-treated MM patients.

Background Axillary lymph node dissection (ALND) in early-breast cancer patients with positive sentinel node (SLN+) may not always be necessary. Aims To predict the finding of ≥1 metastatic axillary node in addition to SLN+(s); to discriminate between patients who would or not benefit from ALND. Methods Records of 397 consecutive patients with 1-2 SLN+s receiving ALND were reviewed. Clinico-pathological features were used in univariate and multivariate analyses to develop a logistic regression model predictive of the risk of ≥1 additional axillary node involved. The discrimination power of the model was quantified by the area under the receiver operating characteristic curve (AUC) and validated using an independent set of 83 patients. Results In univariate analyses, the risk of ≥1 additional node involved was correlated with tumor size, grade, HER-2 and Ki-67 over-expression, number of SLN+s. All factors, but Ki-67, retained in multivariate regressions were used to generate a predictive model with good discriminating power on both the training and the validation sets (AUC 0.73 and 0.75, respectively). Three patient groups were defined based on their risk to present additional axillary burden. Conclusions The model identifies SLN+-patients at low risk (≤15%) who could reasonably be spared ALND and those at high risk (>75%) who should receive ALND. For patients at intermediate risk, ALND appropriateness could be individually evaluated based on other clinico-pathological parameters.

Serum of animal origin and in particular fetal bovine serum is the most commonly utilized cell culture medium additive for in vitro cell growth and differentiation. However, several major concerns have been raised by the scientific community regarding the use of animal sera for human cell-based culture applications. Among the possible alternatives to the animal serum, platelet-derived compounds have been proposed since more than 10 years. Nevertheless, the high degree of variability between the different platelet preparations, and the lack of standardized manufacturing and quality control procedures, made difficult to reach a consensus on the applicability of this novel cell culture medium supplement. In this study, we describe the preparation of a standardized platelet-rich plasma (PRP) derivative obtained starting from human-certified buffy coat samples with a defined platelet concentration and following protocols including also freeze-drying, gamma irradiation and biological activity testing prior the product release as cell culture medium additive. Biological activity testing of the different preparations was done by determining the capability of the different PRP preparations to sustain human bone marrow mesenchymal stem cell (MSC) clone formation and proliferation. Taking advantage of a developed MSC in vitro clonogenicity test, we also determined biological activity and stability of the freeze-dried gamma-sterilized PRP preparations after their storage for different times and at different temperatures. The PRP effects on cell proliferation were determined both on primary cell cultures established from different tissues and on a cell line. Results were compared with those obtained in "traditional" parallel control cultures performed in the presence of bovine serum [10% fetal calf serum (FCS)]. Compared to FCS, the PRP addition to the culture medium increased the MSC colony number and average size. In primary cell cultures and in cell line cultures, the PRP promoted cell proliferation also in conditions where the FCS had not a proliferation stimulating effect due to either the nature of the cells and the tissue of origin (such as human articular chondrocytes from elderly patients) or to the critical low density cell seeding (such as for HeLa cells). In summary, the standardized PRP formulation would provide an "off-the-shelf" product to be used for the selection and expansion of several cell types also in critical cell culture conditions.

CTLA-4 blockade with monoclonal antibodies can lead to cancer regression in patients with metastatic melanoma (MM). CTLA-4 gene polymorphisms may influence the response to anti-CTLA-4 antibodies although few data are available regarding this issue. We analyzed six CTLA-4 single nucleotide polymorphisms (−1661A>G, −1577G>A, −658C>T, −319C>T, +49A>G, and CT60G>A) in 14 Italian MM patients and 45 healthy subjects. We found a significant association between the −1577G/A and CT60G/A genotypes and improved overall survival (Pc < 0.006, Bonferroni corrected), further confirmed by the diplotype analysis (−1577&CT60 GG-AA diplotype, p < 0.001). A positive trend toward an association between these genotypes and response to therapy was also observed.

Background Feasibility and accuracy of sentinel node biopsy (SLNB) after the delivery of neo-adjuvant chemotherapy (NAC) is controversial. We here report our experience in NAC-treated patients with locally advanced breast cancer and clinically positive axillary nodes, and compare it with the results from our previous randomized trial assessing SLNB in early-stage breast cancer patients. Patients and methods Sixty-four consecutive patients with large infiltrating tumor and clinically positive axillary nodes received NAC and subsequent lymphatic mapping, SLNB and complete axillary lymph node dissection (ALND). The status of the sentinel lymph node (SLN) was compared to that of the axilla. Results At least one SLN was identified in 60 of the 64 patients (93.8%). Among those 60 patients, 37 (61.7%) had one or more positive SLN(s) and 23 (38.3%) did not. Two of the patients with negative SLN(s) presented metastases in other non-sentinel nodes. SLNB thus had a false-negative rate, a negative predictive value and an overall accuracy of 5.1%, 91.3% and 96.7%, respectively. All these values were similar to those we reported for SLNB in the settings of early-stage breast cancer. Conclusion SLNB after NAC is safe and feasible in patients with locally advanced breast cancer and clinically positive nodes, and accurately predicts the status of the axilla.

Objectives: The CMFEV (cyclophosphamide, methotrexate, 5-fluorouracil, epirubicin, vincristine) regimen is an innovative schedule, designed by our Group, aimed at administering five partially or totally no cross-resistant cytotoxic agents in breast carcinoma. It was randomly compared to CMF (cyclophosphamide, methotrexate, 5-fluorouracil) as primary treatment in operable disease and demonstrated a short-term significant increase in clinical complete response rate and a long-term significant locoregional relapse-free survival in premenopausal patients. So, it seemed worth comparing this regimen with CMF as adjuvant chemotherapy in moderate risk operable breast carcinoma. Methods: Four hundred and eighty-nine patients with stage I or II moderate risk breast carcinoma were randomized to receive CMF or CMFEV regimen for 6 cycles after surgery. Main end points were overall survival (OS), invasive disease-free survival (IDFS) and recurrence-free interval (RFI), as estimated by Kaplan-Meier analyses and log-rank tests. Results: At a median observation time of 7.3 years (range 5.4 months-10.3 years), no significant differences in OS and IDFS were observed between the two arms. Deaths from breast carcinoma were more frequent with CMF (58.5%) than with CMFEV regimen (41.7%) as well as recurrences from breast carcinoma (58.8% with CMF and 41.2% with CMFEV). These differences were not statistically significant. Conclusion: CMFEV appears more effective than CMF in preventing recurrences from primary disease in patients with moderate risk stage I-II breast carcinoma. The lack of statistical significance of the observed differences was probably due to the limited number of patients enrolled which rendered the study underpowdered.

Conflicting observations have been reported about the role of CTLA-4 gene polymorphisms in the clinical outcome of allogeneic hematopoietic stem cell transplantation (HSCT). We have investigated three polymorphisms of the CTLA-4 gene (−318C>T, +49A>G, CT60G>A) in 133 donor/recipient pairs who underwent HLA-matched sibling donor HSCT for hematological malignancies. We found no association of the clinical outcome of the HSCT with either recipient or donor −318C>T and CT60G>A polymorphisms. At variance, we found a significant association of donor +49A>G G/G genotype with longer overall survival (OS; log-rank test, P = 0.04), and the number of +49A>G G-alleles in the recipient with longer OS (P = 0.027), longer disease-free survival (P = 0.036) and reduced relapse rate (P = 0.042). However, only recipient +49A>G polymorphism was retained as independent prognostic factor in a multivariate analysis, suggesting that the expression of CTLA-4 on the cells of recipient may be relevant for the clinical outcome of HSCT.

In Italy, the role of a nurse in cancer research is beginning to develop, but instruments developed to delineate the dimensions of the Italian clinical trials nursing role are lacking. This study aimed to translate into Italian the Clinical Trials Nursing Questionnaire (CTNQ) and assess its content validity, internal consistency, and stability reliability. Forward-backward translation and review by experts were performed to assess linguistic and content validation. Internal consistency reliability was computed using Cronbach alpha and by administering the translated CTNQ to a sample of 30 research nurses coming from different Italian districts. To determine the test-retest reliability, a copy of the questionnaire was given again to a subgroup of 10 research nurses. The pretest and posttest scores were compared using kappa coefficient. The comparison of the target language version with the original version allowed us to consider the translation of CTNQ as acceptable. The analyses revealed a Cronbach alpha coefficient of .98 for the frequency scale and .96 for the importance scale. The overall kappa coefficient was 0.98 for the frequency scale and 0.99 for the importance scale. The CTNQ is a valid and reliable instrument for the assessment of the research nurse's role in Italy.

We have previously reported the modulation, during chondrogenesis and/or inflammation, of two chicken genes laying in the same genomic locus and coding for two polypeptides of the lipocalin protein family, the extracellular fatty acid binding protein (ExFABP) and the chondrogenesis associated lipocalin beta (CALbeta). A third gene, located within the same cluster and coding for a new lipocalin, CALgamma, has been identified and is here characterized. Tissue distribution analyzed by real-time quantitative reverse transcriptase-polymerase chain reaction in chicken embryos shows a ubiquitous expression with predominant levels of mRNA transcripts in the liver and the brain. In the developing tibia, a high expression of CAL-gamma mRNA was evidenced by in situ hybridization within the pre-hypertrophic and the hypertrophic zones of the bone-forming cartilage. In agreement, dedifferentiated chondrocytes in vitro express the transcripts to the highest level when they redifferentiate reaching hypertrophy. Such peculiar developmental pattern of expression that is analogous to those already described for ExFABP and CALbeta suggests that all three proteins may act synergistically in the process of endochondral bone formation. Moreover, like ExFABP and CALbeta, CAL-gamma is also highly induced in dedifferentiated chondrocytes upon stimulation with lypopolysaccharides, indicating that the whole cluster quite possibly is transcriptionally activated not only in physiological morphogenic differentiation but also in pathological acute phase response. (C) 2003 Elsevier Science B.V. All rights reserved.

Chicken hypertrophic chondrocytes secrete the extracellular fatty acid binding protein (Ex-FABP), a lipocalin not expressed by their undifferentiated precursors. Genomic clones coding for the full protein are here structurally and functionally analyzed. We first determined that the promoter sequence markedly differs from that reported for the homologous p20K, and we confirmed by genomic DNA Southern analysis the exactness of our sequence. This is of relevance since we have identified another lipocalin gene within the region of discrepancy, indicating thereby the existence of a lipocalin cluster within the same chromosomal locus. By transient transfections with 5'-deletions and the chloramphenicol acetyl transferase (CAT) reporter gene, the region between nt -926 and nt -629 was shown to be strongly active, specifically in hypertrophic chondrocytes and not in dedifferentiated cells. Responsive elements for several potential transcription factors lay within this sequence. Among those, activating protein-1 (AP-1) was shown to be involved in the regulation of the Ex-FABP gene during chondrocyte differentiation, as indicated by electrophoretic mobility shift assay, AP-1 site mutagenesis and functional interference assays.

The TNM staging remains the best prognostic descriptor of lung cancer; however, new independent prognostic factors are needed, particularly for early stage disease.An evaluation of the pleural lavage cytology (PLC) was performed in 436 consecutive NSCLC patients who underwent surgical resection; clinical, pathological and follow-up data were available for 414 patients.The PLC was positive in 15 patients (3.6 %). The overall five-year survival was 35.9 % in PLC-positive and 57.8 % in PLC-negative patients (p = 0.004). To compare groups with the same prognostic characteristics, the analysis was restricted to p-stage I patients, but the survival remained worse in the PLC-positive patients (42.9 vs 69.4 %; p = 0.001). Recurrence was also observed more frequently in PLC-positive cases: 69.2 vs 34.5 %, OR 4.28 (95 % CI 1.29-14.18; p = 0.01). Among the PLC-positive patients, no difference between the local (44.4 %) and distant (55.6 %) relapse patterns was found (p = 0.82). The multivariate analysis identified four independent prognostic factors: age (p0.001), disease stage (p0.001), gender (p = 0.025) and PLC status (p = 0.012).PLC is an independent prognostic factor for NSCLC. PLC-positive NSCLC patients have a worse overall survival and a higher recurrence rate, even in stage I disease. PLC-positive patients should be considered a high risk category, who should potentially be eligible for adjuvant therapy regardless of their p-stage.

We have previously demonstrated the association of the chicken lipocalin Ex-FABP with cartilage formation and inflammatory responses as a marker of these processes (Descalzi Cancedda et al., Biochim. Biophys. Acta 1482, 127-135, 2000). Here we report the isolation and characterisation of a new lipocalin gene laying upstream the Ex-FABP, thus representing the second member of a possible genomic cluster. This gene contains an open reading frame coding for a polypeptide of about 19 kDa. The amino-acid sequence revealed a conserved lipocalin secondary structure. Tissue distribution of the protein in developing embryos showed a preferential expression in the heart although mRNA transcripts could be detected also in muscle, lung and liver. The lowest expression was observed in the stomach, brain and skin. During endochondral formation of long bones, the protein is differentially distributed, as the transcripts, evidenced in the tibia by in situ hybridisation, are present in the hypertrophic cone of the cartilage and mostly absent in the area of the proliferating chondrocytes. Such developmental regulation was observed also in vitro in cultured chondrocytes where the transcripts were barely detectable in dedifferentiated cells but highly expressed in hypertrophic chondrocytes. The protein was also significantly induced by lipopolysaccharide stimulation of chondrocytes, indicating a possible involvement in acute phase response. Raising specific antibodies in a rabbit allowed validating, at the protein level, all the transcriptional data. Moreover, we gained evidence that the protein is actively secreted in the extracellular matrix surrounding the chondrocytes. Because of its peculiar expression in cartilage, this new protein was named chondrogenesis-associated lipocalin beta (thereafter referred to as CAL beta). The close similarity between Ex-FABP and CAL beta expression patterns supports the hypothesis of a genomic organisation in a cluster where both genes could be co-ordinately regulated.

Extracellular Fatty Acid Binding Protein (Ex-FABP) is a 21 kDa lipocalin, expressed during chicken embryo development in hypertrophic cartilage, in muscle fibres and in blood granulocyte. The protein selectively binds with high affinity fatty acids, preferably long chain unsaturated fatty acids in chondrocyte and myoblast cultures Ex-FABP expression is increased by inflammatory-agents and repressed by anti-inflammatory-agents. In adult cartilage, Ex-FABP is expressed only in pathological conditions such as in dyschondroplastic and osteoarthritic chicken cartilage. We propose that lipocalin Ex-FABP represents a stress protein physiologically expressed in tissues where active remodelling is taking place during development and also present in tissues characterized by a stress response due to pathological conditions.

Bone marrow stromal cells (BMSC) are an attractive target for novel strategies in the gene/cell therapy of hematologic and skeletal pathologies, involving BMSC in vitro expansion/transfection and reinfusion. We investigated the effects of in vitro expansion on BMSC pluripotentiality, proliferative ability, and bone-forming efficiency in vivo. BMSC from three marrow donors were cultured to determine their growth kinetics. At each passage, their differentiation potential was verified by culture in inductive media and staining with alizarin red, alcian blue, or Sudan black, and by immunostaining for osteocalcin or collagen II. First passage cells were compared to fresh marrow for their bone-forming efficiency in vivo. Stromal cell clones were isolated from five donors and characterized for their multidifferentiation ability. The lifespan and differentiation kinetics of five of these clones were determined. After the first passage, BMSC had a markedly diminish proliferation rate and gradually lost their multiple differentiation potential. Their bone-forming efficiency in vivo was reduced by about 36 times at first confluence as compared to fresh bone marrow. Experiments on the clones yielded comparable results. Culture expansion causes BMSC to gradually lose their early progenitor properties. Both the duration and the conditions of culture could be crucial to successful clinical use of these cells and must be considered when designing novel therapeutic strategies involving stromal mesenchymal progenitor manipulation and reinfusion.

CTLA-4 expression/function can be affected by single nucleotide polymorphisms (SNPs) of CTLA-4 gene, which have been widely associated with susceptibility or progression to autoimmune diseases and cancer development. In this study, we analyzed six CTLA-4 SNPs (-1661A>G, -1577G>A, -658C>T, -319C>T, +49A>G, CT60G>A) in 197 DNA samples from 43 B-lymphoblastoid cell lines (B-LCLs), 40 systemic sclerosis (SSc) patients, 14 pre-analyzed melanoma patients and 100 Italian healthy subjects. Genotyping of -1661A>G, -1577G>A, -658C>T and CT60G>A was performed by newly developed multiplex pyrosequencing (PSQ) assays, whereas -319C>T and +49A>G by T-ARMS PCR and direct sequencing. Genotype/allele frequency were analyzed using χ(2) or Fisher exact test. Our study provides the first multiplex PSQ method that allows simultaneous genotyping of two CTLA-4 SNP pairs (i.e. -1661A>G/-658C>T and -1577G>A/CT60G>A) by two multiplex PSQ reactions. Herein, we show the CTLA-4 genotype distribution in the B-LCLs providing the first and best characterized cell line panel typed for functionally relevant CTLA-4 SNPs. We also report the significant association of the -1661A/G genotype, -1661 & -319 AC-GT diplotype and -319 & CT60 TG haplotype with susceptibility to SSc without Hashimoto's thyroiditis occurrence. Furthermore, we confirmed previous genotyping data referred to melanoma patients and provided new genotyping data for Italian healthy subjects.

The factors regulating the growth and development of mesenchymal precursor cells toward chondrogenesis are not well identified. We have developed a defined serum-free culture system that allows the proliferation of chick embryo chondrogenic cells and their maturation toward hypertrophic chondrocytes. Proliferation is obtained in adhesion in medium supplemented with insulin (Ins), Dexamethasone (Dex), and either basic fibroblast growth factor (FGF-2), platelet-derived growth factor bb, epithelial growth factor, or GH; the highest mitogenic response is induced by FGF-2 in synergy with Ins. Ins can be substituted by Ins-like growth factor I. When these cells are transferred into suspension culture in Ins/Dex and T3 without growth factor supplement, they undergo the complete chondrogenic development characterized by type X collagen synthesis and cellular hypertrophy. During differentiation, Ins cannot be substituted by Ins-like growth factor I. Chondrogenesis is also evidenced by the formation of hypertrophic cartilage when the medium is supplemented with ascorbic acid. If T3 is introduced in the proliferation phase, the cells fail to differentiate to hypertrophy in suspension unless bone morphogenetic protein-2 is added. Assays of ectopic tissue formation in nude mice, with cells implanted sc after adsorption on collagen sponge or porous hydroxyapatite ceramics, indicate that cells grown in Ins/FGF-2 reform mainly cartilage in vivo, whereas expansion in Ins/T3/Dex/FGF-2 leads to the formation of cartilage, bone, and adipose tissue.

No quantitative methods are currently available to measure different aggregation parameters in cell cultures. In this paper we describe a computer-based technique for the automatic and reliable analysis of cellular aggregates, starting from optical microscopy images of living cells grown in suspension. The method allows determination, on the same sample at different time intervals, of quantitative parameters, including aggregation percentage, average number of cells in aggregates, and aggregate size statistical distribution. To determine the number of cells in an aggregate starting from its two-dimensional microscopic profile, a model has been proposed and verified, using sphere packing theory. Algorithms have been tested on chondrocyte suspension cultures, where cell aggregation is a very early and critical event leading to cell differentiation. Using this technique for the analysis of chick embryo chondrocyte cultures, we observed that aggregate size and development kinetics depend on the culture conditions used. The method, with minor adaptations, is of potential use also in other cell systems to evaluate aggregation indexes or to study aggregation kinetics.

Loss and failure of organs and tissues are obviously major health-care problems. Living-organ and tissue transplantation have been the easiest solutions to these problems so far, but having enough donor material for all patients is unrealistic. Tissue engineering is an emerging area in biomedicine that may help solve this problem. Methods involved in tissue reconstruction include using cells alone or the association of cells with tridimensional scaffolds for more complex tissues, while organ reconstruction requires cells, scaffolds, homing systems and integration with mechanical systems and microelectronic devices. Here, the authors review the state of the art in tissue engineering and also touch upon organ engineering.

Purpose To assess the role of the Clinical Trials Nurse (CTN) and to evaluate the quality of the job performed by Clinical Trials Nurses (CTNs) in Italy. Methods The study design was descriptive. The sample included 30 CTNs in Italy who were involved in conducting clinical trials in the last years. Respondents completed the Italian Clinical Trials Nursing Questionnaire (CTNQ) developed to measure frequency and importance of clinical trials nursing activities. Data analyses included descriptive statistics, Student's t -test and Chi–Square test. Results Thirty out of 34 CTNs consented to participate. Respondents were more involved in the experimental drug management, in the protocol implementation and, partially, in the informed consent process. CTNs have a marginal position with respect to the protocol assessment and planning, subject recruitment, data management. CTNs reported high rates for the importance evaluation. Number of years in the nursing role was significantly associated with data management related activities ( p = 0.016). Items with minor response rate differences between frequency and importance were not statistically significant ( p values ranging from 0.087 to 0.911). The CTNs reported to be autonomous and competent; however, they lack and/or do not perform some nursing-related responsibilities and/or activities. Conclusions Although CTNs are not involved in all of the activities listed on the CTNQ, most of them are fully aware to be a key member of research teams. Overall, the Italian CTN role is mostly practical task-oriented and focuses little on data management and organizational activities.

Aim To investigate whether omitting intra-operative staging of the sentinel lymph node (SLN) in T1-N0 breast-cancer patients is feasible and convenient because it could allow a more efficient management of human and logistic resources without leading to an unacceptable increase in the rate of delayed axillary lymph node dissection (ALND). Methods According to the experimental procedure, T1a–T1b-patients were to not receive any intra-operative SLN evaluation on frozen sections (FS). In all T1c-patients, the SLN was macroscopically examined; if the node appeared clearly free of disease, no further intra-operative assessment was performed; if the node was clearly metastatic or presented a dubious aspect, the pathologist proceeded with analysis on FS. T2-patients, enrolled in the study as reference group, were treated according to the institutional standard procedure; they all received SLN staging on FS. Results The study included 395 T1-N0-patients. Among the 118 T1a–T1b-patients whose SLN was not analyzed at surgery, 12 (10.2%) were recalled for ALND. In the group of 258 T1c-patients, 112 received SLN analysis on FS and 146 did not. An SLN falsely negative either at macroscopic or FS examination was found in 33 (12.8%) cases. Overall, the rate of recall for ALND was 11.6% as compared to 8.4% in T2-patients. Using the experimental protocol, the institution reached a 9.6% cost saving, as compared to the standard procedure. Conclusions Omission of SLN intra-operative staging in T1-N0-patients is rather safe. It provides the institution with both management and economical advantages.

Cytotoxic T lymphocyte antigen-4 (CTLA-4) is the major negative regulator of T-cell responses, although growing evidence supports its wider role as an immune attenuator that may also act in other cell lineages. Here, we have analyzed the expression of CTLA-4 in human monocytes and monocyte-derived dendritic cells (DCs), and the effect of its engagement on cytokine production and T-cell stimulatory activity by mature DCs. CTLA-4 was highly expressed on freshly isolated monocytes, then down-modulated upon differentiation toward immature DCs (iDCs) and it was markedly upregulated on mature DCs obtained with different stimulations (lipopolysaccharides [LPS], Poly:IC, cytokines). In line with the functional role of CTLA-4 in T cells, treatment of mDCs with an agonistic anti-CTLA-4 mAb significantly enhanced secretion of regulatory interleukin (IL)-10 but reduced secretion of IL-8/IL-12 pro-inflammatory cytokines, as well as autologous CD4+ T-cell proliferation in response to stimulation with recall antigen purified protein derivative (PPD) loaded-DCs. Neutralization of IL-10 with an anti-IL-10 antibody during the mDCs-CD4+ T-cell co-culture partially restored the ability of anti-CTLA-4-treated mDCs to stimulate T-cell proliferation in response to PPD. Taken together, our data provide the first evidence that CTLA-4 receptor is expressed by human monocyte-derived mDCs upon their full activation and that it exerts immune modulatory effects.

Chondrocytes from chicken embryo tibia can be maintained in culture as adherent cells in Coon's modified Ham's F-12 medium supplemented with 10% FCS. In this condition, they dedifferentiate, losing type II collagen expression in favor of type I collagen synthesis. Their differentiation to hypertrophy can be obtained by transferring them to suspension culture. Differentiation is evidenced by the shift from type I to type II and type IX collagen synthesis and the following predominant expression of type X collagen, all markers of specific stages of the differentiation process. To identify the factors required for differentiation, we developed a serum-free culture system where only the addition of triiodothyronine (T3; 10(-11) M), insulin (60 ng/ml), and dexamethasone (10(-9) M) to the F-12 medium was sufficient to obtain hypertrophic chondrocytes. In this hormonal context, chondrocytes display the same changes in the pattern of protein synthesis as described above. For proper and complete cell maturation, T3 and insulin concentrations cannot be modified. Insulin cannot be substituted by insulin-like growth factor-I, but dexamethasone concentration can be decreased to 10(-12) M without chondrogenesis being impaired. In the latter case, the expression of type X collagen and its mRNA are inversely proportional to dexamethasone concentration. When ascorbic acid is added to the hormone-supplemented medium, differentiating chondrocytes organize their matrix leading to a cartilage-like structure with hypertrophic chondrocytes embedded in lacunae. However, this structure does not present detectable calcification, at variance with control cultures maintained in FCS. Accordingly, in the presence of the hormone mixture, the differentiating chondrocytes have low levels of alkaline phosphatase activity. This report indicates that T3 and insulin are primary factors involved in the onset and progression of chondrogenesis, while dexamethasone supports cell viability and modulates some differentiated functions.

Background: Sentinel lymph node (SLN) staging is currently used to avoid complete axillary dissection in breast cancer patients with negative SLNs. Evidence of a similar efficacy, in terms of survival and regional control, of this strategy as compared with axillary resection is based on few clinical trials. In 1998, we started a randomized study comparing the two strategies, and we present here its results. Materials and methods: Patients were randomly assigned to sentinel lymph node biopsy (SLNB) and axillary dissection [axillary lymph node dissection (ALND arm)] or to SLNB plus axillary resection if SLNs contained metastases (SLNB arm). Main end points were overall survival (OS) and axillary recurrence. Results: One hundred and fifteen patients were assigned to the ALND arm and 110 to the SLNB arm. A positive SLN was found in 27 patients in the ALND arm and in 31 in the SLNB arm. Overall accuracy of SLNB was 93.0%. Sensitivity and negative predictive values were 77.1% and 91.1%, respectively. At a median follow-up of 5.5 years, no axillary recurrence was observed in the SLNB arm. OS and event-free survival were not statistically different between the two arms. Conclusions: The SLNB procedure does not appear inferior to conventional ALND for the subset of patients here considered.

Investigation is lacking regarding the clinical impact of human leukocyte antigen (HLA) class I downregulation in breast cancer and results are inconsistent. In this study, we investigated the expression of HLA class I, the heavy chain, and beta2-microglobulin (beta2-m) by immunohistochemistry in 67 breast carcinomas (BC) and correlated results with clinical-pathologic parameters and patient outcomes. Seventy-six percent of BC were downregulated for HLA class I, whereas downregulation of heavy chain and beta2-m was observed in 57 and 46% of BC, respectively. A significant association existed between the absence of tumor necrosis and downregulation of class I and beta2-m and between the absence of lymphovascular invasion and patient's age and downregulated class I and heavy chain, respectively. Among the lymph node-positive BC patients, a significantly improved overall survival was observed in those showing beta2-m downregulation compared with patients with normal beta2-m. This result may correlate with the role of beta2-m in regulating cancer cell growth.

ObjectiveThe role of surgery in the treatment of preoperatively diagnosed N2 non–small cell lung cancer remains controversial. This study sought significant prognostic factors to select candidates for surgery and assess prognosis.MethodsThe study population included 277 patients who underwent primary resection (192) or induction chemotherapy followed by surgery (85) for preoperatively diagnosed, potentially resectable N2 non–small cell lung cancer. N2 descriptors were prospectively recorded. Kaplan–Meier curves were used to evaluate survival, and statistical significance of differences between curves was assessed by log-rank test. Cox regression was used for multivariate analyses.ResultsPreoperative significant prognostic factors were number of mediastinal node levels involved (P < .001), symptom severity (P = .013), clinical T (P = .041), and induction chemotherapy (P = .001). Three groups with different prognoses were based on individual prognostic score. The group that did best had a median survival of 29.6 months. Postoperative predictors of survival were pathologic T (P = .003), tumor residue (P = .034), and number of mediastinal nodes involved (P < .001). Of 3 groups with different prognoses, the most favorable had a median survival as long as 42 months.ConclusionThis study provides a practical tool that uses significant prognostic factors to predict which patients with preoperatively diagnosed N2 non–small cell lung cancer have better prognoses. Because patients with the favorable prognostic factors showed good long-term survival and excellent local disease control, surgery should still play an important role in the multimodality treatment of these patients.

OBJECTIVES The therapeutic value of pulmonary middle lobectomy (PML) has been questioned. PML is currently regarded as a standard form of lobectomy, even so it shares some surgical features with segmentectomies (SEG) more than with lobectomies. The present study's aim was to assess the therapeutic value of PML with respect to other lobectomies (LOBs) and SEGs. METHODS A total of 902 consecutive patients who underwent lobectomy or SEG with mediastinal lymph node dissection for Stage I-IIIa non-small-cell lung cancer were analysed. Patients with pT4 tumours and/or pathologically incomplete resection were excluded. RESULTS PML was performed in 50 patients, SEG in 44 and LOBs were performed in 808. The three study groups were homogeneous, except for gender, pT and grade: females, pT1 and G1 tumours were more frequent in the PML and SEG groups. The lymph node dissection yield was poorer in PML (P < 0.007) and SEG (P < 0.001) groups when compared with LOB group. Five-year overall survival (OS) was 45.3% for PML, 54.0% for SEG and 60.2% for LOB (P = 0.793). When limiting the analysis to G2-3 right-sided tumours, 5-year survival was lower in the PML group than in the LOB group: 41.3 vs 59.0% (P = 0.057). Similar results were found when analysing pT2-3 right-sided tumours: 27.3 vs 57.3% (P = 0.049). Multivariable analysis showed four independent prognostic factors: age (P = 0.001), pathological stage (P < 0.001), gender (P = 0.005) and the type of surgical resection (P = 0.029). PML (hazard ratio, HR = 1.63) and SEG (HR = 1.64) were detrimental in comparison with LOB. After adjusting for baseline differences between groups (propensity score), a trend towards a worse OS in PML group when compared with LOB group was observed (HR = 1.38, P = 0.150). CONCLUSIONS Both the lymphadenectomy yield and prognosis make PML more similar to SEG than lobectomy, especially for pT2-3 or G2-3 tumours.

To compare the respective performance and effectiveness of autologous chondrocyte implantation (ACI) and mosaicplasty at resurfacing local full-thickness chondral defects of the knee.Randomized clinical trial.Multicenter trial at orthopedic clinics and university hospitals conducted from 1997 to 2000.A population of patients selected according to eligibility criteria of age, traumatic origin of the defect, its localization, size, and gravity, and above all, no previous surgical treatment of the lesion. Forty-seven patients were randomly assigned to ACI or mosaicplasty and subjected to arthroscopic debridement of the lesion at the time of enrollment. They were called for surgery 6 months after the initial debridement.Improved knee functionality as assessed by repeated clinical evaluation based on the International Knee Documentation Committee Scale and the Lysholm Knee Scoring Scale.Fourteen patients (31.8%) experienced substantial improvement following the initial debridement and, being clinically cured, received no further treatment. Seven patients (15.9%) were lost to follow-up. Among the 23 patients (52.3%) who could effectively be evaluated, a complete recovery (ie, Lysholm Knee Scoring Scale score, 90-100) was observed upon clinical examination in 88% of the mosaicplasty-treated patients and in 68% of the ACI-treated ones (P = 0.093).Although the low power of our study prevents definitive conclusions, ACI and mosaicplasty are cartilage repair techniques that are clinically equivalent and similar in performance. The high percentage of spontaneous improvement ((1/3) of the patients) observed after simple debridement calls into question the need for prompt surgical treatment of patients with lesions similar to those included in this clinical trial. Moreover, this finding warrants further investigation, ideally through randomized clinical trials in which patients subjected to debridement alone are compared with patients undergoing reconstructive surgery.

Autologous chondrocyte implantation is currently applied in clinics as an innovative tool for articular cartilage repair. Animal models have been and still are being used to validate and further improve the technique. However, in various species, the outcome varies from hyaline-like cartilage to fibrocartilage. This may be due partly to the spontaneous dedifferentiation of chondrocytes once cultured in vitro. Here we assessed whether the extent of dedifferentiation varies between species and we hypothesized that the level of chondrocyte phenotype stability during expansion may contribute to the maintenance of their chondrogenic commitment and redifferentiation potential. Condyle chondrocytes were harvested from sheep, dog, and human, and expanded for 1, 6, or 12 cell duplications. At each interval, cell phenotype was monitored (morphology and biosynthesis of cartilage markers) and redifferentiation was assessed by an in vitro assay of chondrogenesis in micromass pellet and an in vivo assay of ectopic cartilage formation in immunodeficient mice. Results indicate that, during culture, the sheep chondrocyte phenotype is maintained better than that of human chondrocytes, which in turn dedifferentiate to a lesser extent than dog chondrocytes Accordingly, after expansion, sheep chondrocytes spontaneously reform hyaline-like cartilage; human chondrocytes redifferentiate only under stimulation with chondrogenic inducers whereas, after a few passages, dog chondrocytes lose any capacity to redifferentiate regardless of the presence of inducers. Thus, conditions allowing cartilage formation in one species are not necessarily transposable to other species. Therefore, results with animal models should be cautiously applied to humans. In addition, for tissue-engineering purposes, the number of cell duplications must be, for each species, carefully monitored to remain in the range of amplification allowing redifferentiation and chondrogenesis.

Human articular cartilage heals poorly in adults and current surgical procedures do not provide long-term repair. Cell therapy and tissue engineering could become the treatment of choice, but suffer a major limitation as chondrocytes in vitro lose the differentiated phenotype. In vivo, the chondrogenic lineage is specified by transcription factor Sox9. Thus, cell-based therapy could be successful if Sox9 expression and chondrogenic commitment of the expanded cells were preserved. To achieve this goal, we developed a serum-free medium that supports cell proliferation and preserves the differentiation potential. Indeed, expression of Sox9 is maintained when the conventionally used serum is substituted for by this defined supplement. Spontaneous cartilage formation after expansion in serum-free medium is obtained in vitro in a high-density pellet culture and confirmed in vivo in a functional assay in immunodeficient mice. By contrast, cells grown in serum lose the expression of Sox9 and fail to reform cartilage both in vitro and in vivo unless they are rescued by chondrogenic inducers such as transforming growth factor beta(1) and dexamethasone. Our data emphasize the importance of the microenvironment in modulating commitment, plasticity, and phenotype of chondrocytes, and provide an experimental system to study their physiological or pathological metabolism in a controlled context.

A novel lipocalin gene is here reported that represents the fourth member of a cluster we have identified in the chicken genome. This cluster also includes Chondrogenesis-Associated Lipocalins β and γ (CALβ, CALγ) and Extracellular Fatty Acid Binding Protein (Ex-FABP). The new gene codes for a 22-kDa secreted protein with three cysteine residues and a series of sequence features well conserved in the lipocalin family. All the genes in the cluster are structurally similar presenting comparable exon/intron boundary positions and exon sizes. A phylogenetic analysis indicates the monophyletic grouping of these genes, and their relationship with the lipocalins α-1-microglobulin (A1mg), complement factor 8γ chain (C8GC), prostaglandin D synthase (PGDS), and neutrophil-gelatinase-associated lipocalin (NGAL). The new cluster gene appears to be the ortholog of the mammalian C8GC and was thus named Ggal-C8GC. This orthology also suggests that this lipocalin was present in the ancestor common to reptiles and mammals. In addition to other expressing tissues, Ex-FABP, CALβ and CALγ genes are highly transcribed in chondrocytes at late stages of chondrogenesis during endochondral bone formation and/or upon inflammatory stimulation. Here, we show that they are also transcriptionally induced when chondrocytes are subjected to various biological events as cell quiescence, cell shape transition, and hormonal stimulation. By contrast, Ggal-C8GC transcripts are only barely detectable in chondrocytes, but are more abundant in liver, kidney, brain, heart, skeletal muscle and particularly in skin. Moreover, no expression induction was observed neither during chondrocyte differentiation, nor upon any of the stimulations mentioned above. This indicates that the Ggal-C8GC gene was co-opted for a novel function after the duplication events that gave rise to the cluster. The peculiar coordinated regulation of Ex-FABP, CALβ and CALγ, and the apparent divergent role of Ggal-C8GC suggest that these gene duplications may have been maintained during evolution by a sub-functionalization mechanism where some common function(s) are shared by several members of the cluster and some other specialized function(s) are unique to other members. © 2004 Elsevier B.V. All rights reserved., This work was partially supported by funds from the Associazione Italiana per la Ricerca sul Cancro (AIRC, Italy) and MURST.

Trauma and disease of bones and joints, frequently involving structural damage to both the articular cartilage surface and the subchondral bone, result in severe pain and disability for millions of people worldwide and represent major challenges for the orthopedic surgeons. Therapeutic repair of skeletal tissues by tissue engineering has raised the interest of the scientific community, providing very promising results in preclinical animal models and clinical pilot studies. In this review, we discuss this approach. The choice of a proper cell type is addressed. The use of terminally differentiated cells, as in the case of autologous chondrocyte implantation, is compared with the advantages/disadvantages of using more undifferentiated cell types, such as stem cells or early mesenchymal progenitors that retain multi-lineage and self-renewal potentials. The need for proper scaffold matrices is also examined, and we provide a brief overview of their fundamental properties. A description of the natural and biosynthetic materials currently used for reconstruction purposes, either of cartilage or bone, is given. Finally, we highlight the positive aspects and the remaining problems that will drive future research in articular cartilage and bone repair.

Extracellular Fatty Acid Binding Protein (Ex-FABP) is a 21 kDa lipocalin, expressed during chicken embryo development in hypertrophic cartilage, in muscle fibres and in blood granulocyte. The protein selectively binds with high affinity fatty acids, preferably long chain unsaturated fatty acids in chondrocyte and myoblast cultures Ex-FABP expression is increased by inflammatory-agents and repressed by anti-inflammatory-agents. In adult cartilage, Ex-FABP is expressed only in pathological conditions such as in dyschondroplastic and osteoarthritic chicken cartilage. We propose that lipocalin Ex-FABP represents a stress protein physiologically expressed in tissues where active remodelling is taking place during development and also present in tissues characterized by a stress response due to pathological conditions.