Laura Teodori

Prognostic significance of flow cytometry in lung cancer. A 5‐year study

Flow cytometrically determined cellular DNA content has been measured on specimens from 101 patients affected by lung cancer (40 epidermoid cell carcinoma, 22 adenocarcinoma, 21 large cell carcinoma, 11 small cell carcinoma, and seven undifferentiated carcinoma), and one by mesothelioma. Ninty‐eight of 102 (96%) patients with neoplastic disease evidenced the occurrence of at least one cytometrically aneuploid cell subpopulation. Fifty‐five of 102 (54%) cases evidenced the occurrence of multiclonality, that is, the presence of more than one aneuploid stem cell line. However, the incidence of multiclonality in lung carcinoma was statistically different in surgical cases (where multiple site sampling from the primary and lymph nodes was possible) in comparison to the nonsurgical ones (e.g., bronchial washing): 48/77 (62%) and six of 24 (25%), respectively. Therefore, only the 77 surgical patients were used for further analysis. The cases were classified according to the DNA index (DI) in the following way: Group A (tumors with one or more stem lines with DI ranging from 1 to 2) and Group B (tumors with at least one stem line with DI <1 or >2). A significant correlation has been found between the cytometric ploidy condition so defined (Groups A and B) and the tumor mass doubling time (DT), Group B being associated with fast growing tumors (DT lower than 90 days). A statistically better 12‐month survival rate (5‐year maximum follow‐up) was observed in Group A (88%) in respect to Group B (47%) and is evident in the patient survival time course. A better prognostic indication can be achieved by stratifying the patients according to both the cytometric ploidy condition and the tumor DT. Flow cytometric data can usefully contribute to the prognostic assessment of lung carcinoma provided that representative cellular material is collected by multiple site sampling.

DNA/protein flow cytometry as a predictive marker of malignancy in dysplasia-free Barrett's esophagus: Thirteen-year follow-up study on a cohort of patients

Intestinal metaplasia identifies Barretts esophagus (BE) and is associated with an increased risk for esophageal adenocarcinoma. Dysplasia occurs as an intermediate step. However, progression from metaplasia to neoplasia without the demonstration of dysplasia has been described. The role of dual-parameter flow cytometry (FC) as a predictor of neoplastic risk in dysplasia-free cases was evaluated. DNA/protein FC and histology were performed on 362 samples from 30 dysplasia-free BE patients, followed up since 1985 once every 1-2 years. Nine cases were aneuploid, five of which (group IV) were frankly aneuploid; in the other four cases (group III), aneuploidy was detectable by dual-parameter analysis only. Twenty-one patients were diploid. Twelve (group II) also had an abnormally high G1-phase protein content compared to group I (nine patients), which were diploid with a low-moderate protein content. In three patients of group IV an adenocarcinoma in situ was diagnosed, after 5, 6, and 10 years, respectively. In two patients of group III, a low- and a high-grade dysplasia were observed at 3 and 6 years follow-up, respectively. One patient of group I first acquired a high protein content, then an aneuploid DNA content, and then progressed to adenocarcinoma (12 years). None of the still diploid patients (17 cases) have progressed to dysplasia or cancer compared with 6 of 13 presently aneuploid patients (P < 0.01). In conclusion, DNA/protein FC is a marker of increased malignant potential and thus may be used to detect patients at higher risk in dysplasia-free BE and assist in understanding the various stages of malignant transformation in long-term follow-up studies.

Tumor Necrosis Factor-α Inhibition of Skeletal Muscle Regeneration Is Mediated by a Caspase-Dependent Stem Cell Response

Skeletal muscle is susceptible to injury following trauma, neurological dysfunction, and genetic diseases. Skeletal muscle homeostasis is maintained by a pronounced regenerative capacity, which includes the recruitment of stem cells. Chronic exposure to tumor necrosis factor‐α (TNF) triggers a muscle wasting reminiscent of cachexia. To better understand the effects of TNF upon muscle homeostasis and stem cells, we exposed injured muscle to TNF at specific time points during regeneration. TNF exposure delayed the appearance of regenerating fibers, without exacerbating fiber death following the initial trauma. We observed modest cellular caspase activation during regeneration, which was markedly increased in response to TNF exposure concomitant with an inhibition in regeneration. Caspase activation did not lead to apoptosis and did not involve caspase‐3. Inhibition of caspase activity improved muscle regeneration in either the absence or the presence of TNF, revealing a nonapoptotic role for this pathway in the myogenic program. Caspase activity was localized to the interstitial cells, which also express Sca‐1, CD34, and PW1. Perturbation of PW1 activity blocked caspase activation and improved regeneration. The restricted localization of Sca‐1+, CD34+, PW1+ cells to a subset of interstitial cells with caspase activity reveals a critical regulatory role for this population during myogenesis, which may directly contribute to resident muscle stem cells or indirectly regulate stem cells through cell‐cell interactions.

A New Approach to the Study of Human Solid Tumor Cells by Means of FT-IR Microspectroscopy

Cells coming from normal and neoplastic human lung tissue were analyzed by means of FT-IR microspectroscopy. Among the various methods tested to isolate the cells, mechanical treatment alone was found to yield the best results. Monolayers of cells were homogeneously distributed by cytocentrifuge preparation on BaF2 windows, and several spectra were obtained for different circular micro-areas of the order of one hundred microns in diameter. This procedure made it possible to obtain reliable spectra and to reject those containing additional bands due to impurities arising from the isolation treatment. Spectral differences between normal and neoplastic cells reflect an increase in the intensity of the bands corresponding mainly to PO2− symmetrical and asymmetrical vibrations of DNA in pathological samples with respect to normal ones. The value of the ratio of the integrated areas of the bands at 1080 and 1540 cm−1 due to DNA and proteins, respectively, makes it possible to differentiate between normal and abnormal cells, thus suggesting the use of this parameter as an original approach in the recognition of early neoplastic transformation undetectable by means of traditional procedures.

Flow cytometry as a tool for the prognostic assessment of human neoplasia

Flow cytometry permits the quantitative description of neoplastic cell populations from the point of view of their cytogenetic and cytokinetic features. The advances in preparation of cellular monodispersed samples allow the examination not only of in vitro and hematological, but also of surgical, biopsy, endoscopic, and lavage specimens. The analysis of cytometric DNA content has evidenced the importance of (aneu)ploidy as a remarkable tumor marker. Tumors of different sites and, in some cases, stages and/or grades are characterized by a differential occurrence of diploid vs. aneuploid cell subpopulations and by the eventual presence of different stem cell lines within the same tumor. For certain classes of neoplasms, these parameters can be used for the early recognition of neoplasia and related to disease evolution and dissemination and to the results of therapy. Flow cytometry can also be used to evaluate the fraction of (cycling) cells in the S-phase and of proliferating cells (growth fraction). The percent of S cells can be extracted from cytometric DNA content histograms. Furthermore, the method of Bromodeoxyuridine (BrdUrd) incorporation has been recently introduced into flow cytometry. BrdUrd labeling in cycling cells can be detected either by the induction of quenching or enhancement of specific DNA-dye fluorescence or by fluorescent anti-BrdUrd monoclonal antibodies. This approach has been confirmed by preliminary comparative tests on cultured cells, normal and malignant bone marrow, and human solid tumor specimens. These parameters, together with other cytometric parameters of potential importance for the cellular characterization of malignancy, offer a reliable and real time-saving tool for the prognostic assessment of human tumors and the predicting and monitoring of the results of therapy.

Static magnetic fields enhance skeletal muscle differentiation in vitro by improving myoblast alignment

Static magnetic field (SMF) interacts with mammal skeletal muscle; however, SMF effects on skeletal muscle cells are poorly investigated. The myogenic cell line L6, an in vitro model of muscle development, was used to investigate the effect of a 80 ± mT SMF generated by a custom‐made magnet. SMF promoted myogenic cell differentiation and hypertrophy, i.e., increased accumulation of actin and myosin and formation of large multinucleated myotubes. The elevated number of nuclei per myotube was derived from increased cell fusion efficiency, with no changes in cell proliferation upon SMF exposure. No alterations in myogenin expression, a modulator of myogenesis, occurred upon SMF exposure. SMF induced cells to align in parallel bundles, an orientation conserved throughout differentiation. SMF stimulated formation of actin stress‐fiber like structures. SMF rescued muscle differentiation in the presence of TNF, a muscle differentiation inhibitor. We believe this is the first report showing that SMF promotes myogenic differentiation and cell alignment, in the absence of any invasive manipulation. SMF‐enhanced parallel orientation of myotubes is relevant to tissue engineering of a highly organized tissue such as skeletal muscle. SMF rescue of muscle differentiation in the presence of TNF may have important therapeutic implications.

Static magnetic fields affect cell size, shape, orientation, and membrane surface of human glioblastoma cells, as demonstrated by electron, optic, and atomic force microscopy

It is common knowledge that static magnetic fields (SMF) do not interact with living cells; thus, fewer studies of SMF compared with variable magnetic fields are carried out. However, evidence demonstrated that SMF affect cellular structures. To investigate the effect of exposure to increasing doses of SMF on cell morphology, human glioblastoma cells were exposed to SMF ranging between 80 and 3,000 G (8 and 300 mT).

Native extracellular matrix: A new scaffolding platform for repair of damaged muscle

Effective clinical treatments for volumetric muscle loss resulting from traumatic injury or resection of a large amount of muscle mass are not available to date. Tissue engineering may represent an alternative treatment approach. Decellularization of tissues and whole organs is a recently introduced platform technology for creating scaffolding materials for tissue engineering and regenerative medicine. The muscle stem cell niche is composed of a three-dimensional architecture of fibrous proteins, proteoglycans, and glycosaminoglycans, synthesized by the resident cells that form an intricate extracellular matrix (ECM) network in equilibrium with the surrounding cells and growth factors. A consistent body of evidence indicates that ECM proteins regulate stem cell differentiation and renewal and are highly relevant to tissue engineering applications. The ECM also provides a supportive medium for blood or lymphatic vessels and for nerves. Thus, the ECM is the natures ideal biological scaffold material. ECM-based bioscaffolds can be recellularized to create potentially functional constructs as a regenerative medicine strategy for organ replacement or tissue repopulation. This article reviews current strategies for the repair of damaged muscle using bioscaffolds obtained from animal ECM by decellularization of small intestinal submucosa (SIS), urinary bladder mucosa (UB), and skeletal muscle, and proposes some innovative approaches for the application of such strategies in the clinical setting.

Use of multiparameter analysis for Vibrio alginolyticus viable but nonculturable state determination

Vibrio alginolyticus is known to enter into a viable but nonculturable (VBNC) state in response to environmental conditions unfavorable to the growth. Cells in VBNC condition pose a public health threat because they are potentially pathogenic.

Considerations in the Design of Possible Cell Cycle Effective Drugs

Antineoplastic agents are known to induce differential cytotoxic and cytostatic effects throughout the cell cycle. Many drugs have greater toxicity for cycling cells and act selectively at one or more phases of the cycle and may cause partial synchrony of surviving cells. However, these observations have been generally carried out on in vitro systems only and present a variety of complexities and pitfalls. Furthermore, human tumours are often characterized by a relatively low fraction of proliferating cells and present a large cellular heterogeneity as far as their cytogenetic, cytokinetic, and clonogenic features and their responses to drugs are concerned. Therefore, resistance to chemotherapy is due to various factors characterizing, in some instances, each individual tumour. In spite of the advent of technological advances such as flow cytometry, it is still difficult to design kinetic-orientated therapies especially for the treatment of solid tumours. Consequently, it is also difficult to design protocols based on cell cycle effective drugs. The possibility remains, at least for the moment, to stratify tumours according to their cellular heterogeneity. Different protocols could then be assigned to classes of tumours. Such an approach could be completed by further advances in the cellular monitoring of individual tumours.