Combination of letrozole, metronomic cyclophosphamide and sorafenib is well-tolerated and shows activity in patients with primary breast cancer.

PURPOSE: To assess whether the combination of letrozole, metronomic cyclophosphamide and sorafenib (LCS) is well tolerated and shows activity in primary breast cancer (BC). METHODS: Thirteen oestrogen receptor-positive, postmenopausal, T2-4, N0-1 BC patients received the LCS combination for 6 months. In these patients we examined the pharmacokinetics of sorafenib and cyclophosphamide, toxicity of the regimen, the clinical response to therapy and changes in the levels of biologically relevant biomarkers. RESULTS: Adequate plasma concentrations of sorafenib were achieved in patients when it was dosed in combination with L+C. The mean plasma concentrations of C were consistently lower following administration of LCS, compared with administration of L+C only. The most common drug-related grade 3/4 adverse events were skin rash (69.3%), hand-foot skin reaction (69.3%) and diarrhoea (46.1%). According to RECIST Criteria, a clinical complete response was observed in 6 of 13 patients. A significant reduction in tumour size, evaluated with MRI, was also observed between baseline and 14 days of treatment in all 13 patients (P=0.005). A significant reduction in SUV uptake, measured by (18)FDG-PET/CT, was observed in all patients between baseline and 30 days of treatment (P=0.015) and between baseline and definitive surgery (P=0.0002). Using modified CT Criteria, a response was demonstrated in 8 out of 10 evaluable patients at 30 days and in 11 out of 13 evaluable patients at the definitive surgery. A significant reduction in Ki67 expression was observed in all patients at day 14 compared with baseline (P<0.00001) and in 9 out of 13 patients at the definitive surgery compared with baseline (P<0.03). There was also a significant suppression of CD31 and VEGF-A expression in response to treatment (P=0.01 and P=0.007, respectively). CONCLUSIONS: The LCS combination is feasible and tolerable. The tumour response and target biomarker modulation indicate that the combination is clinically and biologically active.

PD-L1 overexpression induces STAT signaling and promotes the secretion of pro-angiogenic cytokines in non-small cell lung cancer (NSCLC).

BACKGROUND: Monoclonal antibodies (ICI) targeting the immune checkpoint PD-1/PD-L1 alone or in combination with chemotherapy have demonstrated relevant benefits and established new standards of care in first-line treatment for advanced non-oncogene addicted non-small cell lung cancer (NSCLC). However, a relevant percentage of NSCLC patients, even with high PD-L1 expression, did not respond to ICI, highlighting the presence of intracellular resistance mechanisms that could be dependent on high PD-L1 levels. The intracellular signaling induced by PD-L1 in tumor cells and their correlation with angiogenic signaling pathways are not yet fully elucidated. METHODS: The intrinsic role of PD-L1 was initially checked in two PD-L1 overexpressing NSCLC cells by transcriptome profile and kinase array. The correlation of PD-L1 with VEGF, PECAM-1, and angiogenesis was evaluated in a cohort of advanced NSCLC patients. The secreted cytokines involved in tumor angiogenesis were assessed by Luminex assay and their effect on Huvec migration by a non-contact co-culture system. RESULTS: PD-L1 overexpressing cells modulated pathways involved in tumor inflammation and JAK-STAT signaling. In NSCLC patients, PD-L1 expression was correlated with high tumor intra-vasculature. When challenged with PBMC, PD-L1 overexpressing cells produced higher levels of pro-angiogenic factors compared to parental cells, as a consequence of STAT signaling activation. This increased production of cytokines involved in tumor angiogenesis largely stimulated Huvec migration. Finally, the addition of the anti-antiangiogenic agent nintedanib significantly reduced the spread of Huvec cells when exposed to high levels of pro-angiogenic factors. CONCLUSIONS: In this study, we reported that high PD-L1 modulates STAT signaling in the presence of PBMC and induces pro-angiogenic factor secretion. This could enforce the role of PD-L1 as a crucial regulator of the tumor microenvironment stimulating tumor progression, both as an inhibitor of T-cell activity and as a promoter of tumor angiogenesis.

Soluble PD-L1 and Circulating CD8+PD-1+ and NK Cells Enclose a Prognostic and Predictive Immune Effector Score in Immunotherapy Treated NSCLC patients.

INTRODUCTION: Upfront criteria to foresee immune checkpoint inhibitors (ICIs) efficacy are far from being identified. Thus, we integrated blood descriptors of pro-inflammatory/immunosuppressive or effective anti-tumor response to non-invasively define predictive immune profiles in ICI-treated advanced non-small cell lung cancer (NSCLC). METHODS: Peripheral blood (PB) was prospectively collected at baseline from 109 consecutive NSCLC patients undergoing ICIs as first or more line treatment. Soluble PD-L1 (sPD-L1) (immunoassay), CD8+PD-1+ and NK (FACS) cells were assessed and interlaced to generate an Immune effector Score (IeffS). Lung Immune Prognostic Index (LIPI) was computed by LDH levels and derived Neutrophil-to-Lymphocyte Ratio (dNLR). All these parameters were correlated with survival outcome and treatment response. RESULTS: High sPD-L1 and low CD8+PD-1+ and NK number had negative impact on PFS (P < 0.001), OS (P < 0.01) and ICI-response (P < 0.05). Thus, sPD-L1high, CD8+PD-1+low and NKlow were considered as risk factors encompassing IeffS, whose prognostic power outperformed that of individual features and slightly exceeded that of LIPI. Accordingly, the absence of these risk factors portrayed a favorable IeffS characterizing patients with significantly (P < 0.001) prolonged PFS (median NR vs 2.3 months) and OS (median NR vs 4.1) and greater benefit from ICIs (P < 0.01). We then combined each risk parameter composing IeffS and LIPI (LDHhigh, dNLRhigh), thus defining three distinct prognostic classes. A remarkable impact of IeffS-LIPI integration was documented on survival outcome (PFS, HR = 4.61; 95%CI = 2.32-9.18; P < 0.001; OS, HR=4.03; 95%CI=1.91-8.67; P < 0.001) and ICI-response (AUC=0.90, 95%CI=0.81-0.97, P < 0.001). CONCLUSION: Composite risk models based on blood parameters featuring the tumor-host interaction might provide accurate prognostic scores able to predict ICI benefit in NSCLC patients.

Effect of inducible FHIT and p53 expression in the Calu-1 lung cancer cell line.

Loss of FHIT expression and p53 mutations are critical events in the early stages of lung carcinogenesis. The restoration of Fhit function in FHIT-negative cancer cells has been reported to cause tumour suppression by inhibition of cell proliferation and/or activation of apoptotic pathways. However, the studies designed to elucidate the biological role of Fhit and its potential interaction with p53 have produced conflicting results. We investigated here the effects of the simultaneous restoration of FHIT and p53 in Calu-1 cells by using a hormone-inducible gene expression system. We demonstrate that the restoration of FHIT expression reinforces the anti-proliferative effect associated with the simultaneous replacement of p53. Indeed, a more pronounced inhibition of cell proliferation associated with an earlier and higher induction of p21(waf1) mRNA and protein expression was observed in Fhit/p53-expressing cells compared with cells expressing p53 alone. This effect was not due to Fhit-mediated up-regulation of p53 expression; in fact p53 protein was expressed at the same level in both FHIT-positive and FHIT-negative cell clones. Consistent with this result, Fhit did not affect the expression of MDM2, a protein known to interact directly with p53 and target p53 for proteolytic degradation, thus down-regulating its activity.

Galectin-1 suppression delineates a new strategy to inhibit myeloma-induced angiogenesis and tumoral growth in vivo.

Galectin-1 (Gal-1) is involved in tumoral angiogenesis, hypoxia and metastases. Actually the Gal-1 expression profile in multiple myeloma (MM) patients and its pathophysiological role in MM-induced angiogenesis and tumoral growth are unknown. In this study, we found that Gal-1 expression by MM cells was upregulated in hypoxic conditions and that stable knockdown of hypoxia inducible factor-1α significantly downregulated its expression. Therefore, we performed Gal-1 inhibition using lentivirus transfection of shRNA anti-Gal-1 in human myeloma cell lines (HMCLs), and showed that its suppression modified transcriptional profiles in both hypoxic and normoxic conditions. Interestingly, Gal-1 inhibition in MM cells downregulated proangiogenic genes, including MMP9 and CCL2, and upregulated the antiangiogenic ones SEMA3A and CXCL10. Consistently, Gal-1 suppression in MM cells significantly decreased their proangiogenic properties in vitro. This was confirmed in vivo, in two different mouse models injected with HMCLs transfected with anti-Gal-1 shRNA or the control vector. Gal-1 suppression in both models significantly reduced tumor burden and microvascular density as compared with the control mice. Moreover, Gal-1 suppression induced smaller lytic lesions on X-ray in the intratibial model. Overall, our data indicate that Gal-1 is a new potential therapeutic target in MM blocking angiogenesis.

The regulation by cell density of amino acid transport system L in SV40 3T3 cells.

The rate of transport of phenylalanine by System L has been measured in SV40 3T3 cells at various cell densities. When the activity of the L system was determined before any cell depletion of intracellular amino acids, a density-dependent increase in transport paralleled the decrease in cell density. This regulation was lost after cell depletion but reappeared after reloading the cells with pertinent substrates of System L. The phenylalanine transport activity modulated by cell density appeared to be related to the internal level of amino acids capable of exchange up to a definite concentration, beyond which transport activity by System L did not parallel a further increase of internal substrate level. Analysis of the relationship between influx and substrate concentration suggested that two saturable components contribute to entry of phenylalanine and leucine in depleted and in reloaded cells: a low-affinity and a high-affinity component. Both kinetic parameters of the high-affinity component appeared to be modulated by the loading treatment, but only V changed markedly. Activation energies for the high-affinity component of the amino acid transport reaction were calculated from an Arrhenius plot in reloaded cells, and were found to be different for low- and high-density cultures. This result is consistent with the interpretation that cell density modulated the rates at which the amino acid-carrier complex can move within the cell membrane.

The effect of the intracellular sodium level on the activity of amino acid transport systems L and A in SV40 3T3 cells.

The rate of transport of phenylalanine and leucine, pertinent amino acids of System L, has been measured in SV40 3T3 cells as a function of the presence of Na+ ions during the reloading phase that precedes the influx determination. The presence of Na+ ions during the reloading phase resulted in an increase of the subsequent substrate influx through System L. This effect was related to the intracellular Na+ level and was found to be independent by the presence of a chemical sodium gradient outside-inside during influx determination; furthermore, this effect could not be ascribed to a difference between control and Na+-treated cells in the internal levels of those amino acids that participate in the exchange phenomena of transport System L. The transport of phenylalanine appeared to have the ability to accept Li+ for Na+ substitution in the 'trans' position. The presence of Na+ ions in the 'trans' position was not required to optimize the transport of System A-reactive substrates, whose influxes are dependent on the presence of the cation in 'cis' position. Analysis of the relationship between influx and substrate concentration indicated that the Na+-dependent increase of substrate influx was associated with an enlarged capacity of the high-affinity component of transport System L.

Induction of amino acid transport activity in chick embryo fibroblasts by replacement of extracellular sodium chloride with disaccharide.

The activity of amino acid transport System A in avian fibroblasts was increased following incubation of the cells in a medium in which most of the NaCl normally present had been isoosmotically replaced by sucrose. This increase was detectable after 2 h of incubation, reached a maximum at about 4 h, and remained constant thereafter. Transfer of treated cells back to a normal medium resulted in decay of the induced transport activity, with a half-life of less than 2 h. Kinetic analysis revealed that the increase in transport activity arose from an increase in Vmax, with little change in Km. This induction of System A activity did not occur if an inhibitor of either RNA or protein synthesis was present in the modified medium. The use of various different solutes as replacements for NaCl in the incubation medium showed that, although each replacement caused a decrease in both cellular Na+ content and protein synthesis, only disaccharides produced the increase in amino acid transport activity. In addition, estimates of cell volume indicated that, even under iso-osmotic conditions, incubation in the sucrose-containing medium caused initial cell shrinkage, followed by swelling. It is concluded that this induction of System A activity is associated with a volume regulatory process and that this process probably accounts for the parallel responses previously observed when cells were incubated in hyperosmolar media. Induction of amino acid transport activity by this process is distinct from adaptive regulation, caused by amino acid starvation; but the two processes are not strictly additive, and so appear to converge at some step.

Multicomponent analysis of amino acid transport System L in normal and virus-transformed fibroblasts.

Amino acid transport System L in both normal Balb/c 3T3 cells and in those transformed with simian virus 40 (SV 3T3) was analysed kinetically under two different experimental conditions. Under 'zero-trans' conditions the results for both types of cell could be interpreted satisfactorily in terms of System L consisting of two components (L1 and L2) characterized by different Km values. This conclusion is in agreement with previous reports. However, under 'infinite-trans' conditions, the experimental data could not be accounted for in terms of only two components; the introduction of a third component (L3) was necessary to provide a satisfactory fit. Viral transformation affects only the L1 component, either by modification or by replacement, giving it a higher 'affinity' (lower Km) but a lower 'capacity' (lower Vmax).

Modulation of cell growth and host protein synthesis during HIV infection in vitro.

During HIV infection of CEM cells cultured in vitro, significant differences in growth rate and protein turnover were observed with different viral preparations. There was a significant inhibition of proliferation after infection with crude HIV supernatants. On the other hand, infection with purified HIV particles obtained by filtration, differential centrifugation, and isopycnic sedimentation led to a progressively increasing stimulation of cell growth. This early stimulation was prevented by neutralizing the virus with soluble CD4 molecules. Study of cell growth in the presence of a purified membrane preparation indicated that membrane fragments contaminating the crude HIV supernatant were responsible for the observed growth inhibition. Interestingly, the stimulation of proliferation was also observed with heat-inactivated virus or after inhibition of viral replication with ZDV. In the presence of purified HIV virions, the rate of general protein synthesis was not inhibited, as is usually observed with crude viral supernatants. However, a marked reduction in protein content and increased protein degradation was found in cultures infected with either crude or purified HIV preparations.

Primer tRNA(Trp) of RSV-transformed or RAV-1-infected cells up-regulates the antiribosomal activity of gelonin.

Some ribosome-inactivating proteins (RIPs) with RNA-N-glycosidase activity on 28S rRNA require, for maximal inactivation of ribosomes, the presence of tRNA. tRNA(Trp) specifically up-regulates gelonin, the RIP from Gelonium multiflorum. The same tRNA is the primer of the reverse transcriptase of Rous sarcoma virus (RSV) and of its mutant (RAV-1) which lacks the src gene. Here we demonstrate that gelonin is more active in inhibiting endogenous protein synthesis by lysates of RSV-transformed or RAV-1-infected cells and that such increase in activity correlates with the increased amount of primer tRNA(Trp) in the cells.

An effective solution for prolonged preservation of cultured human pulmonary artery endothelial cells.

Pulmonary endothelium is considered the compartment most susceptible to preservation damage. This investigation was designed to analyze the efficacy of an original, University of Parma low-potassium-albumin solution (SPAL UP) on cultured human pulmonary artery endothelial cells (HPAEC) and to compare its effects with those of University of Wisconsin solution (UW) and Euro-Collins solution (EC). Cryopreserved HPAEC tertiary cultures were inoculated at the density of 5000 cells/cm2 in 9-cm2 well-plates; subcultures were then incubated at 10 degrees C for 6 hr and 16 hr in 2 ml/well of SPAL UP, UW, and EC. The HPAEC viability after incubation was assessed by evaluating the total protein content and the expression of cytotoxicity, and by analyzing the rate of protein synthesis and expression of cellular functionality after stress. Results after 6 hr of preservation showed that SPAL UP had a less significant cytotoxic effect than EC, exerted a less depressing effect on cellular metabolism, and enhanced functional recovery of endothelial cells compared with UW. At the second time interval (16 hr), SPAL UP provided a less cytotoxic effect than UW; besides, SPAL UP-induced cytotoxicity was similar to that of warm control. In conclusion, in vitro preliminary data regarding the use of SPAL UP in HPAEC preservation suggest its suitability as solution for prolonged lung protection.

Methodology for the assessment of lung protection. Human pulmonary artery endothelial cell preservation using haemaccel.

This investigation was designed to show an original methodology for the assessment of lung preservation and to analyze the efficacy of a low potassium polygelin solution (haemaccel [HM]) on isolated human pulmonary artery endothelial cells. The effects of HM were compared with those of low potassium dextran (LPD), Belzer (University of Wisconsin [UWS]), and Euro-Collins solutions. The viability of the endothelial cultures was assessed by means of both total protein content and recovery of metabolic cellular function expressed as the protein synthesis rate after 6 hr and 16 hr of incubation at 10 degrees C. Our results failed to show any significant difference in the total protein content for HM, LPD, and UWS, both after 6 and 16 hr of incubation; however, the Euro-Collins-preserved sample revealed a significant drop in this parameter as early as 6 hr after the start. This finding was regarded as a clear indication of cellular cytotoxicity. In contrast, the metabolism recovery capacity of the cells varied significantly between HM and UWS at 6 hr and among HM, LPD, and UWS at 16 hr; at 6 hr, however, no significant difference was observed between HM and LPD. In conclusion, HM appears to exert a more significant effect on human pulmonary artery endothelial cell metabolism recovery than do the other fluids, thus suggesting its suitability as a long-term pulmonary perfusate.

Density-dependent regulation of amino acid transport in a Burkitt lymphoma cell line.

Rate of proliferation and amino acid transport were assessed in the Burkitt's lymphoma-derived Namalwa cells by measurements of growth rate and proline and serine uptake. Cell density of the cultures was varied by modifying the number of cells initially seeded and growing for different periods of time. Under these experimental conditions the growth rate was not correlated with cell density. In contrast, the activity of amino acid transport through Systems A and ASC, as assessed by the uptake of proline and serine, respectively, decreased as a function of cell density. This marked decrease of transport activity cannot be explained by large alterations of cell morphology since it was observed at a cell density range where minimal change of cell volume and surface area occurred. When a constant number of cells suspended in an identical volume of medium sedimented on different settling areas, a marked effect on amino acid transport activity occurred. These results indicate that cell to cell contacts may be involved in the density-dependent regulation of transport.

Protease activation during HIV infection in a CD4-positive cell line.

The mechanism of cytopathic effects associated with HIV infection in a continuous line of CD4-positive lymphocytes (CEM cells, clone 13) has been studied. Here we report the following observations: (1) HIV infection killed a variable but always significant number of cells without a strict relationship with the syncytia formation; (2) an important decrease in the proliferation rate occurred soon after infection; (3) a marked inhibition of protein synthesis took place within the first few hours of infection and clearly before the beginning of viral protein expression. In addition, when three-day-old cultures were incubated in serum-free medium, a larger degradation of proteins was observed in infected cells in comparison to controls. An increase in protein degradation activity was observed also in vitro with extracts obtained from HIV-infected cells and incubated in the presence of endogenous- or exogenous-labeled substrates. Extracts from cells infected with heat-inactivated HIV did not show a similar degradative activity. The possible induction or activation of latent proteases during the development of the HIV infection is discussed.

Adaptive cellular response to osmotic stress in pig articular chondrocytes.

The authors studied the effects of a wide range of medium osmolarities (from 0.28 osM (physiological osmolarity of plasma and synovial fluid) to 0.58 osM) by altering Na+ concentration in high density cultures of pig articular chondrocytes in order to analyze the behaviour of some functional and structural parameters during cell adaptation to these imposed changes in the ionic environment. Biochemical and morphological results indicated that, even if isolated from the tissue matrix and cultured in vitro, chondrocytes maintained active osmoregulation systems which are present in living conditions. They showed a similar biochemical and morphological behavior when cultured at 0.28 osM and 0.38 osM but they were able, with regard to protein synthesis, aminoacid transport and proliferation rates, to respond quickly and to adapt to 0.48 osM medium as well. On the contrary, the treatment at the highest osmolarity (0.58 osM) early altered these biochemical parameters and was detrimental or even gave rise to lethal damage during long-term treatment. Furthermore, while chondrocytes cultured in 0.28-0.38 osM medium maintained phenotypic characteristics in culture, the higher osmolarities (0.48-0.58 osM) caused morphological changes in cell populations resulting in loss of phenotypic cell stability as demonstrated by their taking on a fibroblast-like shape as well as a lack of ability to assembly matrix proteoglycans.

Some questions about Eurocollins solution used for lung preservation.

Currently, Eurocollins' (EC) solution (high-potassium concentration) is the most widely clinically used pulmonary perfusate. However, recently, experimental studies have reported an increase of the lung ischemic period using low-potassium solutions. The purpose of our study, is to investigate the influence of the EC ionic composition and the effect of hyperosmolarity due to the glucose concentration on isolated alveolar type II epithelial cells. Pneumocytes type II were isolated from pathogen free Wistar rats using the modified Dobbs' method. Cells were incubated for 6 hours at 4 degrees C in EC, Collins (CL) and Ringer Lactate (RL) solutions. After that, cellular viability was evaluated by analysis of the protein synthesis assay by measuring the 35 S methionine uptake during an incorporation period of one hour at 37 degrees C (picomol 35 S met/mg proteins/h). Mean +/- standard deviation and Student "t"-test were used for data presentation and results comparison. Cellular viability at time 0 (control) before cellular incubation was 3.93 +/- 0.38. After 6 hours at 4 degrees C the results were respectively as follows: EC = 2.16 +/- 0.13; CL = 2.63 +/- 0; RL = 3.21 +/- 0.04. Our results suggest that the low-potassium extracellular type solution (RL) shows a protection on isolated type II epithelial cells statistically significant (p < 0.05) if compared with EC solution. Moreover CL solution, that has the same ionic composition EC but without glucose, presents a less cytotoxic effects on incubated cells than EC, confirming a deleterious influence of solution hyperosmolarity.

A new extracellular type solution for lung preservation: "in vitro" comparison with Beltzer, low potassium Dextran and Euro-Collins solutions by means of human lung fibroblasts.

UNLABELLED: The attempt to synthesise efficacious solution to prolong lung preservation is, at present one of the most interesting challenges in transplant research. Recently, several issues emphasise the central role of ionic composition of lung-flush solutions, underlining, however, that colloid-free solutions are clearly detrimental. We have been studying a complex extracellular type solution (SPAL UP) synthesised to minimise the pathological events that occur during both preservation and reperfusion period. We report the results of toxicity of SPAL UP on normal human fibroblasts obtained from foetal lung (WI-38). WI-38 cells were seeded at 1.4 x 10(4)/cm2 in disposable plastic 12-well plates. After 3 days, cells were incubated in SPAL UP, Beltzer (UWS), Low Potassium Dextran (LPD) and Eurocollins (ECS) solutions for 6 hours at 10 degrees C. Cellular viability was evaluated by the rate of protein synthesis exploiting the incorporation of 35S-Methionine (2 microCi/ml) in growth medium with 10 mM unlabelled Methionine during 30 minutes incubation at 37 degrees C. The results were expressed as nmol. 35S-Methionine/mg of proteins/minute, and presented as means +/- SD of data of three (n = 3) well for each solution studied. RESULTS: the viability at time 0 before incubation (considered as control) was 1.65 +/- 0.1; after hypothermic preservation the data were respectively as follow: SPAL UP 0.51 +/- 0.09; UW 0.24 +/- 0.02; ECS 0.19 +/- 0.01; LDP 0.19 +/- 0.05. CONCLUSIONS: in this "in vitro" model SPAL UP solution provides a significantly (p < 0.05) better cell preservation than regular UW, ECS and LPD solutions.

An original application of plasma expanders: heart-lung preservation.

The lack of an ideal heart-lung preservation solution is one of the principal factor that limits the wide spread of transplantation. The aim of this work was to investigate the efficacy of Haemaccel (HM) on isolated human pulmonary artery endothelial cells comparing its effects with those of University of Wisconsin (UWS). Subcultures of HPAEC were inoculated at the density of 5,000 cells per cm2 in 9 cm2 well-plates. Cells were incubated with HM and UWS for 6 hrs at 10 degrees C. Cellular viability was analysed by the total protein content (cytotoxicity index) and by the rate of protein synthesis (metabolic index). The results showed that HM and UWS did no show a significant differences in the toxicity when compared with the control; on the contrary, HM seems to determine a less inhibitory effect on cellular metabolism permitting a more rapid cellular metabolic recovery than UWS. Thus, HM appears to be more suitable for the preservation of isolated HPAEC than UWS.

The effect of dextran for hypothermic (10 degrees C) preservation of human fetal lung fibroblasts.

BACKGROUND: Low-potassium (LP) solution with Dextran (Dx) improves lung preservation. Nevertheless, the role of Dx in simple cold storage is not established yet. This study was designed to investigate the relationship between molecular weight and concentration of Dx in LP solutions and its effects on cell viability after prolonged hypothermic preservation. METHODS: Human fetal lung fibroblasts (WI-38) were preserved at 10 degrees C for 16 hrs in five solutions containing respectively Dx11, Dx17, Dx39.2, Dx71, Dx178 at 2% and at 5% concentrations and in LP solution without Dx. Cell viability was assessed by means of both the analysis of the total protein content (cytotoxicity index) and the rate of protein synthesis (index of cellular functioning). RESULTS: No differences were recorded in total protein content among the solutions tested. By contrast, the index of cellular functioning was significantly higher using LPDx178 at both concentrations. However, LPDx178 exerted a more significant cytotoxic effect than did LP alone. CONCLUSIONS: These effects were not mediated by the variation of osmolarity; two factors probably influenced this protection: the low oncotic pressure of the LPDx178 solution and an effect chemically specific due to the increased molecular weight of Dx still unknown. Nevertheless, during 10 degrees C preservation, WI-38 cells were better preserved with LP solution without Dx confirming, thus, that during simple cold storage the presence of an oncotic pressure might be harmful.