A pleiotropy scan to discover new susceptibility loci for pancreatic ductal adenocarcinoma.

Pleiotropic variants (i.e., genetic polymorphisms influencing more than one phenotype) are often associated with cancer risk. A scan of pleiotropic variants was successfully conducted ten years ago in relation to pancreatic ductal adenocarcinoma susceptibility. However, in the last decade, genetic association studies performed on several human traits have greatly increased the number of known pleiotropic variants. Based on the hypothesis that variants already associated with a least one trait have a higher probability of association with other traits, 61,052 variants reported to be associated by at least one genome wide association study (GWAS) with at least one human trait were tested in the present study consisting of two phases (discovery and validation), comprising a total of 16,055 pancreatic ductal adenocarcinoma (PDAC) cases and 212,149 controls. The meta-analysis of the two phases showed two loci (10q21.1-rs4948550 (P=6.52×10-5) and 7q36.3-rs288762 (P=3.03×10-5) potentially associated with PDAC risk. 10q21.1-rs4948550 shows a high degree of pleiotropy and it is also associated with colorectal cancer risk while 7q36.3-rs288762 is situated 28,558 base pairs upstream of the Sonic Hedgehog (SHH) gene, which is involved in the cell differentiation process and PDAC etiopathogenesis. In conclusion, none of the single nucleotide polymorphisms (SNPs) showed a formally statistically significant association after correction for multiple testing. However, given their pleiotropic nature and association with various human traits including colorectal cancer, the two SNPs showing the best associations with PDAC risk merit further investigation through fine mapping and ad hoc functional studies.

A practical approach for gmp-compliant validation of real-time PCR method for mycoplasma detection in human mesenchymal stromal cells as advanced therapy medicinal product.

BACKGROUND: Manufacturing of human Mesenchymal Stromal Cells as advanced therapy medicinal product (ATMP) for clinical use involves an ex vivo expansion, which leads to a risk of contamination by microbiological agents. Even if manufacturing under Good Manufacturing Practice (GMP) license minimizes this risk, contamination of cell cultures by mycoplasmas still represents a widespread problem. Furthermore, the absence of mycoplasma contamination represents one of ATMPs release criteria. Since July 2007, European Pharmacopoeia (EuPh) offers the possibility to replace official mycoplasma detection methods with Nucleic Acid Amplification techniques, after suitable validation. As an Italian authorized Cell Factory, we developed an in-house GMP-compliant validation of real-time PCR method for mycoplasma detection in human Mesenchymal Stromal Cells, according to EuPh sec. 2.6.7 and International Conference on Harmonization Q2. MATERIALS AND METHODS: The study was performed in compliance with GMP international requirements with MycoSEQ™ Mycoplasma Detection Assay (Thermofisher) on QuantStudio5 real-Time PCR (Applied Biosystems). Assay validation was developed to evaluate sensitivity, interferences matrix-related, specificity and robustness. RESULTS: MycoSEQ™ Mycoplasma Detection Assay has been successfully validated on human Mesenchymal Stromal Cells as results comply with validation protocol acceptance criteria. CONCLUSIONS: MycoSEQ™ Mycoplasma Detection Assay is a fast, sensitive and specific PCR-based Nucleic Acid Test assay that can be used as an alternative to official mycoplasma test methods for lot release of human Mesenchymal Stromal Cells as advanced therapy medicinal product (ATMP). Moreover, our study underlines the presence of interference on real-time PCR reaction due to matrix composition, pointing out a practical approach for method validation (i.e interference removal).

Extracorporeal photopheresis as an immunomodulatory agent: Haematocrit-dependent effects on natural killer cells.

The GvHD is a major cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (HSCT). Extracorporeal photopheresis (ECP) represents an alternative therapeutic strategy to immunosuppressive therapy. Although ECP is used since 1990s, the mechanism of action has not yet been completely clarified. We analyzed cells collected from 20 ECP procedures of 4 patients affected by chronic GvHD and, for comparison, Peripheral Blood Mononuclear Cells (PBMCs) of 10 healthy donors undergoing from same type of photochemiotherapy, evaluating by flow cytometry, the effects before and after photoactivation with 8-MOP. The analysis showed a significant increase in cell death after ECP in particular in CD4 T lymphocytes as described in literature correlated with haematocrit value. Most interesting data emerge from the analysis of cytotoxic activity of NK cells, using flow cytometry analysis of surface expression of CD107a in the presence of target cells (K562). In all analyzed samples it was possible to document a statistically significant reduction of the cytotoxic activity of NK cells after photoactivation. The decrease of the cytotoxic activity was related to hematocrit value of leukoapheresis: in fact, lower HCT values were associated with a more marked reduction of cytotoxic activity. The study confirms literature data about the increase of cellular mortality induce by ECP. Furthermore, for the first time it is demonstrated that the ECP exerts a marked and significant inhibitory effect on the cytotoxic activity of NK cells. Our study suggests that lower values of hematocrit are associated with better treatment outcome.

Leukapheresis for autologous stem cell transplantation: Comparative study of two different thawing methods WSCFD(®) Stem Cell Fast Thawer KW versus 37 °C thermostatic bath.

BACKGROUND: Leukapheresis for autologous stem cell transplantation represents an efficient technique for the reconstitution of haematopoietic system in patients subjected to a high-dose chemotherapy for the treatment of haematological malignancies. The current regulations emphasise first steps of leukapheresis procedure but do not recommend methods for thawing, only suggesting that it must be performed as soon as possible in a 37 °C thermostatic bath. AIM OF THE STUDY: We compared the classic method of thawing with an innovative and fully traceable method that uses WSCFD(®) Stem Cell Fast Thawer KW. MATERIALS AND METHODS: The first part of the study was focused on the thermodynamic process of the two methods, thawing 6 "simulated" leukapheresis (buffy coats of healthy donors cryopreserved with saline solution, 5% HSA and 5% DMSO) and analysing the thawing curve obtained, by using an inside probe. In the second part, we focused on the recovery of viable CD34+ cells and leukocytes, thawing 20 real leukapheresis from paediatric patients. In this phase we also analyse final core bag temperature, time of procedure, cellular recovery with ISHAGE single platform flow cytometry assay and clonogenic potential performing a CFU assay. RESULTS: We found no significant differences between the two methods, both for thermodynamic aspect and cellular recovery. Thawing curves were similar and the paired Student's-t test used for statistical analysis showed a CD34+ cells recovery of 92.2% ± 11.4 using WSCFD(®) versus 90% ± 11.1 of thermostatic bath. Data were similar even for leukocytes recovery (80.8% ± 9.5 with WSCFD(®) and 79.2% ± 14.4 with thermostatic bath). All thawed products never exceeded the core temperature of 30 °C and no differences were found about the post-thaw clonogenic potential (614 × 10(4) ± 98.3 total CFU using WSCFD(®) versus 592 × 10(4) ± 78.5 using thermostatic bath). The only difference observed was about the thawing time: WSCFD method requires a slightly longer time but, on the other hand, it correlates with reduced mean increase in temperature per minute, as a result of a more linear thawing curve. CONCLUSIONS: WSCFD(®) can replace the 37 °C thermostatic bath thawing procedure for leukapheresis, providing more security and fully traceable process data.

Hemodynamics of the sapheno-femoral complex: an operational diagnosis of proximal femoral valve function.

AIM: A significant relationship between great saphenous vein (GSV) caliber at the thigh and function of the terminal valve of the sapheno-femoral junction (SFJ) has already been demonstrated. Yet, the function of the proximal common femoral valve (FV), which is missing in 20-24% of cases, might also play a significant role in SFJ reflux. The aim of this paper was to verify whether GSV caliber also predicts the function/presence of FV. METHODS: Using a high-resolution duplex scanner we selected 572 GSVs showing clear-cut SFJ incompetence. Then, by positioning the probe on the inguinal skin fold and orientating the probe upward, we tested FV function. Valve incompetence was diagnosed when a retrograde flow lasting longer than 0.5 s was elicited by both calf squeezing with sudden release and Valsalva maneuver, with the patient standing. Finally, in all patients we measured GSV caliber 15 cm below the groin in the standing position. RESULTS: GSV caliber =7 was not predictive of FV function/presence (51.9% competence vs 48.1% incompetence/absence). In contrast, GSV caliber < or = 6 mm and GSV caliber > or = 8 mm were highly predictive of FV competence and incompetence/absence, respectively (sensibility 98.6%, specificity 80.4%, positive predictive power 88.2%, negative predictive power 97.4%, diagnostic accuracy 91.3%). CONCLUSIONS: Our data strengthen the relationship between GSV caliber at the thigh and hemodynamics of the whole sapheno-femoral complex, including in this definition the FV also.

Hemodynamics of the sapheno-femoral junction. Patterns of reflux and their clinical implications.

AIM: The detection of reflux elicited by the compression/release test with the PW Doppler sample at the level of the sapheno-femoral arch might not be sufficient by itself to diagnose the incompetence of the whole sapheno-femoral junction (SFJ). The aim of this study was to further refine the diagnosis by positioning the PW Doppler sample at different levels of SFJ and eliciting reflux both by squeezing and with the Valsalva manoeuvre. In addition, the relationship of the findings with the vein diameter was taken into consideration. METHODS: By using a high resolution duplex scanner, 1 294 great saphenous veins (GSV) found to be incompetent by the compression/release test at duplex investigation of the saphenous arch, were also tested at the same level by the Valsalva manoeuvre. Subsequently, the tests were repeated by positioning the PW Doppler sample at the femoral side of the terminal valve, at the saphenous arch tributaries, and at the pre-terminal valve level. Furthermore, the GSV diameter in the standing position was measured at 15 cm from the groin in all patients, and correlated with the hemodynamic patterns found at the junction level. RESULTS: Comparing to compression/release test at the level of the saphenous arch, the Valsalva manoeuvre was negative in 259 (20%) lower limbs and positive in 1 035 (80%). Among the 1 294 GSV found to be incompetent at compression/release test at the level of the saphenous arch, only 710 (55%) lower limbs showed incompetence of the terminal valve. A total of 124 patients (10%), presenting with a competent terminal valve but with a positive Valsalva manoeuvre in the arch, showed a downward flow from a pelvic tributary of the GSV. Finally, a significant statistical correlation between the presence of a competent terminal valve and a GSV diameter <5 mm has been found (p<0.001). CONCLUSION: Our data show that the detection of reflux elicited by compression/release test at the level of the saphenous arch is insufficient to diagnose the incompetence of the terminal valve. Our results, together with the correlation between the saphenous trunk diameter at the thigh and the competence or the incompetence of the terminal valve, present significant clinical implications when sapheno-femoral surgical disconnection is contemplated.

Ambulatory conservative hemodynamic management of varicose veins: critical analysis of results at 3 years.

This report describes the results of our 3-year experience using ambulatory conservative hemodynamic management (ACHM) for lower extremity venous insufficiency involving the greater saphenous vein (GSV), with specific analysis of recurrence due to neoformation of vessels. We performed 289 ACHM procedures in 259 consecutive patients with GSV-related varicose veins. Follow-up clinical examination and Doppler ultrasound imaging was carried out at 3, 6, 12, 24, and 36 months in all cases to assess formation of neovessels supplied either by the superficial (A) or deep (B) venous system. Our data showed that ACHM achieved excellent improvement, with complete disappearance of varicose veins in 41.2% of cases, good improvement in 43%, fair improvement in 14.1%, and no improvement in 1.7%. The only predictor of outcome was the quality of drainage from the GSV vein. Poor drainage leads to neoformation of vessels supplied by the superficial (A) venous system. In about 50% of cases, drainage appeared spontaneously within 1 year, with a subsequent reduction in formation of neovessels. Neoformation of vessels supplied by the deep (B) venous system (10%) was independent of the quality drainage. This finding suggests that formation of these neovesseis is unrelated to the surgical method used to treat varicose veins. In patients with poor drainage of the saphenous network, neoformation of vessels supplied by the superficial (A) venous system is predictable with regard to both topography and delay. ACHM is a good tool for treatment of varicose veins, as reliable statistical prediction of mid-term results is possible using available models.

Epidermal growth factor signaling and mitogenesis in Plcg1 null mouse embryonic fibroblasts.

Gene targeting techniques and early mouse embryos have been used to produce immortalized fibroblasts genetically deficient in phospholipase C (PLC)-gamma1, a ubiquitous tyrosine kinase substrate. Plcg1(-/-) embryos die at embryonic day 9; however, cells derived from these embryos proliferate as well as cells from Plcg1(+/+) embryos. The null cells do grow to a higher saturation density in serum-containing media, as their capacity to spread out is decreased compared with that of wild-type cells. In terms of epidermal growth factor receptor activation and internalization, or growth factor induction of mitogen-activated protein kinase, c-fos, or DNA synthesis in quiescent cells, PLcg1(-/-) cells respond equivalently to PLcg1(+/+) cells. Also, null cells are able to migrate effectively in a wounded monolayer. Therefore, immortalized fibroblasts do not require PLC-gamma1 for many responses to growth factors.

[Comparison between the CHIVA cure and stripping in the treatment of varicose veins of the legs: follow-up of 3 years]. / Comparaison entre cure C.H.I.V.A. et stripping dans le traitement des veines variqueuses des membres inférieurs: suivi de 3 ans.

OBJECTIVE: To assess three-year results following "CHIVA". DESIGN: Comparison between our results and data in the literature following stripping. SETTING: Phlebologic Surgery, 46 Datini Street, Florence, Italy. PATIENTS: 148 consecutive operated patients suffering from varicose veins (166 procedures). INTERVENTIONS: "CHIVA", as described by Franceschi and Bailly. MAIN OUTCOME MEASURES: The presence of visible varicose veins, graded in three categories, according to Hobbs and Einarsson, as shown by clinical examination. RESULTS: Very good results were obtained in 100 cases, who presented without any finding of varicose veins throughout the follow-up. Of the remaining 66, in 1 case we found a new varicose network completely developed, whereas in 65 cases only some visible residual or recurrent vessels were present. CONCLUSIONS: Significantly better results were observed in the CHIVA group compared with stripping groups (P < 0,001).