A standardized procedure to obtain mesenchymal stem/stromal cells from minimally manipulated dental pulp and Wharton's jelly samples.

Transplantation of mesenchymal stem/stromalcells (MSCs) has emerged as an effectivemethod to treat diseased or damagedorgans and tissues, and hundreds of clinicaltrials using MSCs are currently under way todemonstrate the validity of such a therapeuticapproach. However, most MSCs used for clinicaltrials are prepared in research laboratorieswith insufficient manufacturing quality control.In particular, laboratories lack standardizedprocedures for in vitro isolation of MSCs fromtissue samples, resulting in heterogeneouspopulations of cells and variable experimentaland clinical results.MSCs are now referred to as Human CellularTissue-based Products or Advanced TherapyMedicinal Products, and guidelines fromthe American Code of Federal Regulation ofthe Food and Drug Administration (21 CFRPart 1271) and from the European MedicinesAgency (European Directive 1394/2007) definerequirements for appropriate production ofthese cells. These guidelines, commonly called"Good Manufacturing Practices" (GMP),include recommendations about laboratorycell culture procedures to ensure optimal reproducibility,efficacy and safety of the finalmedicinal product. In particular, the Food andDrug Administration divides ex vivo culturedcells into "minimally" and "more than minimally"manipulated samples, in function of theuse or not of procedures "that might alter thebiological features of the cells". Today, minimalmanipulation conditions have not beendefined for the collection and isolation ofMSCs (Torre et al. 2015)(Ducret et al. 2015).Most if not all culture protocols that have beenreported so far are unsatisfactory, becauseof the use of xeno- or allogeneic cell culturemedia, enzymatic treatment and long-termcell amplification that are known to alter thequality of MSCs.The aim of this study was to describe a standardizedprocedure for recovering MSCs withminimal handling from two promising sources,the dental pulp (DP) and the Wharton's jelly(WJ) of the umbilical cord. The quality and homogeneityof the expanded cell populationswere assessed by using flow cytometry withcriteria that go beyond the International Societyof Cellular Therapy (ISCT) guidelines forMSC characterization.

Cord blood processing by a novel filtration system.

OBJECTIVES: Availability of cord blood (CB) processing has been limited by the need for electrically aided centrifugal techniques, which often produce only low final cell product yield. Here, we describe development and characterization of a novel filter device aimed at allowing CB processing, using gentle gravity-led flow. MATERIALS AND METHODS: CB was processed with a novel filter device (CellEffic CB, consisting of non-woven fabric), without any centrifugation. Cells were harvested by flushing the filter with either HES or physiological saline solution (SALINE). Differential cell counts and viability analysis, combined with Fluorescence-Activated Cell Sorting (FACS) (total nucleated cells [TNC], mononuclear cells [MNC], CD45+ CD34+ cells, hematopoietic precursor cells [HPCs]) and clonogenic assay, were employed for analysis of CB pre- and post-processing, and after freeze/thawing. RESULTS: Processing using the novel filter yielded high quality RBC depletion while maintaining good recovery of TNC, MNC, CD34+, HPCs and colony forming unit (CFU) output. The filter performed equally well using HES or SALINE. Gravity-led flow provided gentle cell movement and protection of the stem cell compartment. Post-thaw CFU output was maintained particularly, an important indicator for CB banking. CONCLUSIONS: Geographical limitations of CB transplantation and banking have required a non-electrical, non-centrifugal solution. This novel filter CellEffic CB device revealed rapid yet gentle cell processing while maintaining the stem/progenitor cell compartment required for both haematological and regenerative medicine therapies.

[Effort to radically cure stage III and IV esophageal carcinoma with simultaneous radiotherapy and chemotherapy in standard clinical practice]. / L'intento di cura radicale del carcinoma esofageo al III e IV stadio con radioterapia e chemioterapia concomitanti nella pratica clinica comune.

PURPOSE: Chemotherapy and concurrent irradiation, intended to cure, are presently standard treatments for non metastatic, unresectable oesophageal cancer. The results of the combined therapy are superior to those of radiotherapy alone, attaining 25-35% 2-year survival rates. However these results mainly refer to stage I and II tumours as most of the available literature has focussed on these groups. The aim of our report is to present our experience with Stage III and IV patients. MATERIAL AND METHODS: Sixty-four Stage III and IV oesophageal cancer patients were referred to our Departments from January 1, 1990 to December 31, 1996. Diagnosis was obtained through oesophagoscopy and biopsy, stage was assessed by physical examination, chest CT scan, bronchoscopy, barium X-ray examination, upper abdomen ultrasonography and bone nuclide scan. Thirty-four patients, with no signs of blood-born metastases and in satisfactory medical conditions (i.e. age not exceeding 70 years, weight loss not exceeding 10% of body weight, normal serum values of BUN and creatinine, no other severe disease), were submitted to concurrent chemo-radiotherapy. The case features were as follows: histology of squamous cell carcinoma in 32 cases, of adenocarcinoma in 2; tumour in the upper third of the oesophagus in 11 (32.5%), in the middle third in 18 (53%), in the lower third in 5 (14.5%); male/female ratio 29/5, age 48-68 years (mean 56), Karnofsky performance status of 60% or higher. On referral, 18 out of 34 (53%) had a weight loss more than 5% of body weight and 22 (64.5%) had dysphagia. Twenty-one had Stage III (61.75%) and 13 stage IV (38.25%) cancer, with metastasis limited to the supraclavicular or coeliac nodes, which could be included in the radiation volume. In all cases chemotherapy consisted of 5-Fluoruracil (administered in a continuous i.v. infusion, from day 1 to 5, with a 750-1.000 mg/n.sq daily dose) and Cisplatin (75-100 mg/n.sq on the first day, or 20 mg/n.sq for 5 consecutive daily doses, administered by i.v. bolus). Three to 5 cycles were administered, one every 21 days. Irradiation started with the first cycle of chemotherapy in 5 patients, with the second or third cycle in 29. At least two cycles of chemotherapy were administered during the course of radiation. Radiotherapy was performed with 4 to 18 MeV linear accelerator X-rays, or telecobalt, through opposite anterior and posterior treatment portals or more complex field arrangements. The doses were in the range of 44-66 Gy, with fractionation of 5x180-200 cGy weekly sessions. After treatment, periodic follow-up controls were carried out in all cases. Thorough restaging was performed only in selected cases, thus a systematic evaluation of objective responses was not possible. Data on improvement of swallowing were always available, however, and the early therapeutic results were analysed accordingly. Toxicity was recorded according to the WHO parameters. Two-year survival after conclusion of the treatment was calculated according to Kaplan and Maier. Survival was analysed (log-rank test) according to stage, Performance Status, oesophagectomy and body weight loss. RESULTS: After treatment, subjective symptomatic relief occurred in 17 of the 22 patients presenting dysphagia (77.5%). Acute toxicity (Grade III or IV WHO) of the treatment accounted for 47% of hematologic adverse effects, 40% of mucositis, 20.5% of vomiting or diarrhoea not responding to drug treatment. Treatment delays of more than one week, due to toxicity, occurred in 23.5%. Moreover, we observed 20.5% of mild cardiotoxicity and 6% of mild nephrotoxicity. No symptomatic lung fibrosis was observed. No death could be related to toxicity. Overall 2 year survival was 13%, with a median value of 10 months. Survival analysis, according to stage, showed 2 year values of 24% in Stage III and 0% in Stage IV (p=0.09). No significant difference was related to Performance Status and weight loss. Six patients showed a remarkable improvement in symptoms and general conditions after treatment, and were restaged with oesophagoscopy, thoracic CT scan and bronchoscopy, which evidenced resectable residual tumors, and they were then operated. Although histologic examination showed tumour in all the resected specimens, 2 patients survived more than two years (33.5% survival, median 14 months). Due to the small number of operated patients, no attempt was made to assess the significance of this result, in comparison with the other cases. DISCUSSION AND CONCLUSIONS: Many Stage III and IV patients, selected for an aggressive chemo-radiation approach on the grounds of satisfactory medical conditions, can obtain relief of dysphagia. Toxicity can be severe, but is rarely life-threatening. Some cases, without extrathoracic spread of the tumor can achieve long term survival (in our experience 24% 2-year survival in Stage III, in our experience which favourably compares with the results obtained by other authors). Whether surgery may improve the therapeutic results of chemo-radiotherapy in patients whose tumour has become resectable, is an issue that cannot be satisfactorily addressed on the basis of our experience, nor are the results from the available literature exhaustive to this regard.

[Assessment of primary renal lymphoma with computerized tomography]. / Valutazione dei linfomi renali primitivi con Tomografia Computerizzata.

Renal involvement during lymphoma can be extrinsic, i.e., renal compression or displacement due to lymph node masses, or intrinsic, i.e., parenchymal involvement secondary to blood or lymphatic spread, or primary, as initial neoplastic site. Primary renal lymphoma is very rare (3% of all renal lymphomas) for the absence of lymphatic tissue in the kidney. The disease might be due to parapyelic lymph nodes or to blood spreading from an unknown site. In our study we reviewed the CT findings of five cases of primary non-Hodgkin's renal lymphoma with surgical or histologic confirmation. Renal alterations due to lymphomatous involvement were classified according to macroscopic pathologic findings: type I (single nodular disease, 2 patients), type II (multinodular disease, 1 patient), and type III (infiltrating disease with retroperitoneal involvement, 2 patients). In the two patients with single nodular involvement (type I), CT showed a solid, hypodense and clear-cut nodule. In the only patient with multinodular disease (type II), renal tissue was replaced by multiple hypodense nodular masses, which were partially confluent. In the two infiltrating forms with retroperitoneal involvement (type III), renal structure was diffusely disorganized, with thickening of soft tissues and perirenal fasciae, peripyelic infiltration and, in one case, urinary tract obstruction. To conclude, CT always allowed the accurate assessment of the presence, site and size of renal lesions and of perirenal and urinary involvement. However, CT findings were completely aspecific, not allowing an unquestionable differential diagnosis with other conditions, e.g., hypernephroma, transitional cell carcinoma, metastatic lesions or chronic inflammations. Therefore, a biopsy specimen is necessary to make an unquestionable diagnosis.

Biliary and pancreatic secretions in abdominal irradiation.

The biliary and pancreatic secretions have been determined in patients given pelvic or para-aortic irradiation, with a dose of 50 Gy in the former group and between 36 and 40 Gy in the latter. A test meal containing polyethylene glycol (PEG) as reference substance was used. Each sample of the duodenal content was assayed for volume, PEG content, amylase and trypsin activity, pH and biliary secretion. No significant modifications of biliary and pancreatic secretions were demonstrated after irradiation, suggesting that these functions are not involved in the pathogenesis of the malabsorption radiation syndrome.