Hydrazone-Functionalized trans-A2B-Corroles: Effective Synergy in Photodynamic Therapy of Lung Cancer.

A synthetic route to trans-A2B-corroles combining the macrocyclic core with a hydrazone moiety, based on the reactivity of azoalkenes toward dipyrromethanes, has been established with the aim of developing a new class of photosensitizers for photodynamic therapy of lung cancer. The study of the photophysical properties of the novel macrocycles allowed the identification of photosensitizers with absorption within the phototherapeutic window and high singlet oxygen quantum yield. Relevant structure-photodynamic activity correlations were established by studying the new corroles-based photodynamic therapy (PDT) in human lung cancer cell lines (A549 and H1299). The methyl-hydrazone corroles were more active than phenyl-hydrazone corroles, with the N-Boc and N-Ts groups being key structural features to ensure high activity. The lead photosensitizers, with IC50 values below 100 nM and no cytotoxicity per se, were significantly more active than 5,10,15-triphenylcorrole, showing that the presence of the hydrazone functional group has a strong influence on PDT activity.

Ring-Fused meso-Tetraarylchlorins as Auspicious PDT Sensitizers: Synthesis, Structural Characterization, Photophysics, and Biological Evaluation.

Novel 4,5,6,7-tetrahydropyrazolo[1,5-a]pyridine-fused meso-tetraarylchlorins, with different degrees of hydrophilicity (with methyl ester, hydroxymethyl, and carboxylic acid moieties), have been synthesized and their photophysical characterization as well as in vitro photocytotoxicity assessment against human melanoma and esophageal and bladder carcinomas was carried out. An integrated analysis of the photosensitizers' performance, considering the singlet oxygen generation data, cell internalization, and intracellular localization, allowed to establish relevant structure-photoactivity relationships and the rationalization of the observed photocytotoxicity. In the diacid and monoalcohol series, chlorins derived from meso-tetraphenylporphyrin proved to be the most efficient photodynamic therapy agents, showing IC50 values of 68 and 344 nM against A375 cells, respectively. These compounds were less active against OE19 and HT1376 cells, the diacid chlorin with IC50 values still in the nano-molar range, whereas the monohydroxymethyl-chlorin showed significantly higher IC50 values. The lead di(hydroxymethyl)-substituted meso-tetraphenylchlorin confirmed its remarkable photoactivity with IC50 values below 75 nM against the studied cancer cell lines. Subcellular accumulation of this chlorin in the mitochondria, endoplasmic reticulum, and plasma membrane was demonstrated.

Functional Impairment of Circulating FcεRI+ Monocytes and Myeloid Dendritic Cells in Hepatocellular Carcinoma and Cholangiocarcinoma Patients.

BACKGROUND: Hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA) represent the most common primary liver malignancies whose outcome is influenced by the immune response. METHODS: In this study, we have functionally characterized, by flow cytometry, circulating myeloid dendritic cells (mDCs) and FcεRI+ monocytes in a group of healthy individuals (n = 10) and in a group of patients with HCC (n = 19) and CCA (n = 8), at the time point of the surgical resection (T0) and once the patient had recovered from surgery (T1). Moreover, we proceeded to a more in depth phenotypic characterization of the FcεRI+ monocyte subpopulation. RESULTS: A significant decrease in the frequency of TNFα producing FcεRI+ monocytes and mDCs in HCC and CCA patients when compared to the group of healthy individuals was observed, and a close association between FcεRI+ monocytes and mDCs dysfunction was identified. In addition, the phenotypic characteristics of FcεRI+ monocytes from healthy individuals strongly suggest that this population drives to mDCs, which matches with the fact that both populations are functionally affected. CONCLUSIONS: The frequency and the function of circulating mDCs and FcεRI+ monocytes are affected in both HCC and CCA patients, and FcεRI+ monocytes could represent those fated to become mDCs. © 2019 International Clinical Cytometry Society.

Stroma-derived IL-6, G-CSF and Activin-A mediated dedifferentiation of lung carcinoma cells into cancer stem cells.

Cancer stem cells (CSCs) are a small population of resistant cells inhabiting the tumors. Although comprising only nearly 3% of the tumor mass, these cells were demonstrated to orchestrate tumorigenesis and differentiation, underlie tumors' heterogeneity and mediate therapy resistance and tumor relapse. Here we show that CSCs may be formed by dedifferentiation of terminally differentiated tumor cells under stress conditions. Using a elegant co-culture cellular system, we were able to prove that nutrients and oxygen deprivation activated non-malignant stromal fibroblasts, which in turn established with tumor cells a paracrine loop mediated by Interleukine-6 (IL-6), Activin-A and Granulocyte colony-stimulating factor (G-CSF), that drove subsequent tumor formation and cellular dedifferentiation. However, by scavenging these cytokines from the media and/or blocking exosomes' mediated communication it was possible to abrogate dedifferentiation thus turning these mechanisms into potential therapeutic targets against cancer progression.

Intestinal Epithelial Stem Cells: Distinct Behavior After Surgical Injury and Teduglutide Administration.

BACKGROUND: Previous studies suggest that intestinal epithelial stem cells (IESC), critical drivers of homeostasis and regeneration, include two subpopulations: crypt-based columnar and "position +4" stem cells, identified by Lgr5 and Bmi1 biomarkers, respectively. Teduglutide is an enterotrophic counterpart of glucagon-like peptide 2. This study aimed to investigate the response of putative IESC to surgical injury and teduglutide administration on an animal model of intestinal resection and anastomosis. METHODS: Wistar rats (n = 59) were distributed into four groups: "Ileal Resection" versus "Laparotomy", subsequently subdivided into "Postoperative Teduglutide Administration" versus "No Treatment"; and sacrificed at third or seventh days, with ileal sample harvesting. Flow cytometry was used to analyze epithelial stem cells with monoclonal antibodies against Lgr5, Bmi1 and also CD44, CD24, CD166, and Grp78 surface markers. RESULTS: Surgical trauma induced an increase of epithelial stem cells population at third day (9.0 ± 0.3 versus 5.7 ± 0.3%, p = 0.0001), which was more intense and involved all subpopulations after ileal resection. At seventh day, teduglutide was significantly associated with higher proportion of Lgr5+/Bmi1- cells (5.8 ± 0.1 versus 2.9 ± 0.3%, p = 0.005) and, on the contrary, lower percentage of Lgr5-/Bmi1+ cells (0.03 ± 0.01 versus 1.9 ± 0.1%, p = 0.049) after ileal resection; and higher proportion of Lgr5+/Bmi1+ cells (1.7 ± 0.1 versus 1.1 ± 0.2%, p = 0.028) after isolated laparotomy. After surgery, Lgr5+/Bmi1- and Lgr5-/Bmi1+ subpopulations demonstrated an inverse correlation and both correlated negatively with Grp78 labeling index. Lgr5-/Bmi1+ and CD44+/CD24low/CD166+/Grp78+ cells proportions exhibited a high grade positive correlation. CONCLUSION: Those observations support the existence of two epithelial stem cells subpopulations with distinct behavior after surgical injury and teduglutide treatment.

Platinum(II) Ring-Fused Chlorins as Near-Infrared Emitting Oxygen Sensors and Photodynamic Agents.

Novel near-infrared luminescent compounds based on platinum(II) 4,5,6,7-tetrahydropyrazolo[1,5-a]pyridine-fused chlorins are described. These compounds have high photostability and display light emission, in particular simultaneous fluorescence and phosphorescence emission in solution at room temperature, in the biologically relevant 700-850 nm red and near-infrared (NIR) spectral region, making them excellent materials for biological imaging. The simultaneous presence of fluorescence and phosphorescence emission at room temperature, with the phosphorescence strongly quenched by oxygen whereas fluorescence remains unaffected, allows these compounds to be used as ratiometric oxygen sensors in chemical and biological media. Both steady-state (fluorescence vs phosphorescence intensities) and dynamic (dependence of phosphorescence lifetimes upon oxygen concentration) luminescence approaches can be used. Photocytotoxicity studies against human melanocytic melanoma cells (A375) indicate that these compounds display potential as photosensitizers in photodynamic therapy.

In vivo biodistribution of antihyperglycemic biopolymer-based nanoparticles for the treatment of type 1 and type 2 diabetes.

This study aimed to assess the biodistribution of antihyperglycemic insulin-loaded alginate/dextran sulfate-based nanoparticles dual coated with chitosan and technetium-99m-albumin (99mTc-BSA) after oral administration. The oral administration of 50IU/kg insulin-loaded nanoparticles to type 1 diabetic rats showed prolonged antihyperglycemic effects up to 12h and relative pharmacological availability of 5.04% comparing to the subcutaneous administration. The oral antihyperglycemic effect was further compared between type 1 and type 2 diabetic models by the intraperitoneal glucose tolerance test, revealing that the effect lasted longer in the type 1 diabetic model. 99mTc-BSA revealed to be a good nanoparticles' tracer since there was no systemic absorption and 99mTc-BSA-nanoparticles were capable of increasing their residence time in the intestinal epithelium of balb-c mice when compared with 99mTc-BSA biodistribution. Thus, this biopolymeric-based delivery nanoparticulate system is a promising tool for the therapy of type 1 and type 2 diabetic individuals and prevention of T1D.

Novel 4,5,6,7-tetrahydropyrazolo[1,5-a]pyridine fused chlorins as very active photodynamic agents for melanoma cells.

The development of new stable 4,5,6,7-tetrahydropyrazolo[1,5-a]pyridine-fused chlorins with high absorption properties at 650 nm, and their impressive photosensitizer ability against melanotic and amelanotic cancer cells is described. Comparison between a diester-substituted chlorin with the corresponding dihydroxymethyl derivative demonstrated that the increased hydrophilicity of the latter is crucial to ensure nanomolar activity against melanoma cells. The new photosensitizer leads to death of human melanoma cells being both apoptosis and necrosis in equal parts involved in the treatment response. The dihydroxymethyl-chlorin was particularly active against human melanocytic melanoma A375 cells, which can be viewed as a solution to overcome the resistance of melanotic melanoma to photodynamic therapy.

Targeting triple-negative breast cancer cells with 6,7-bis(hydroxymethyl)-1H,3H-pyrrolo[1,2-c]thiazoles.

Further studies on 6,7-bis(hydroxymethyl)-1H,3H-pyrrolo[1,2-c]thiazoles as anticancer agents against breast cancer are reported, allowing to demonstrate the potential of these compounds for the therapy of the triple-negative breast cancer, the most challenging tumors in clinical practice. These compounds were assayed for their in vitro cytotoxicity on several human breast cancer cell lines (MCF7, HCC1954 and HCC1806 cell lines). Particularly interesting were the results obtained for 4-hydroxyphenyl substituted derivative, which proved to be the most promising compound regarding HCC1806 cell line, a triple-negative breast cancer. The effects of the two most active compounds on cell survival, viability, cell cycle, DNA damage and expression of proteins related to cell death pathways were studied. The reported results consolidate the potential of 6,7-bis(hydroxymethyl)-1H,3H-pyrrolo[1,2-c]thiazoles for the therapy of breast cancer, particularly the triple-negative.

Chiral 6,7-bis(hydroxymethyl)-1H,3H-pyrrolo[1,2-c]thiazoles with anti-breast cancer properties.

The synthesis and biological evaluation of 6,7-bis(hydroxymethyl)-1H,3H-pyrrolo[1,2-c]thiazoles as anticancer agents against MCF7 breast cancer cell lines is reported. The design of the new compounds has been guided considering (3R)-6,7-bis(hydroxymethyl)-5-methyl-3-phenyl-1H,3H-pyrrolo[1,2-c]thiazole as the lead compound due to its good performance against MCF7 breast cancer cell lines (IC(50) = 1.0 µM). The structural changes included the removal of the phenyl group at C-3, the replacement of this group by a 3,4,5-trimethoxyphenyl group, the removal of the methyl group at C-5 from the lead scaffold and the replacement of this group by a phenyl substituent. Overall, these studies showed that the combined presence of a phenyl group at C-3 and a methyl group at C-5 in the 1H,3H-pyrrolo[1,2-c]thiazole ring system is essential to ensure high cytotoxicty against MCF7 breast cancer cell lines. To probe whether the absolute configuration of the lead compound might affect the anticancer activity, its enantiomer was prepared and the activity against MCF7 cells was evaluated. (3S)-6,7-Bis(hydroxymethyl)-5-methyl-3-phenyl-1H,3H-pyrrolo[1,2-c]thiazole proved to be the most active compound so far studied, with IC(50) value of 0.5 µM.

Synthesis of new 2-galactosylthiazolidine-4-carboxylic acid amides. Antitumor evaluation against melanoma and breast cancer cells.

A set of 2-galactosylthiazolidine-4-carboxylic acid amides was synthesized with different length for the carbon chain amide moiety. The cytotoxicity of the molecules was evaluated against A375 melanoma and MCF7 breast cancer cell lines. For the derivatives tested, the one that contains a C(16) amide carbon chain is the most active with an IC(50) of 17.0 µM for A375 and 5.8 µM for MCF7. This compound also shows cytotoxicity in the triple negative cancer cell line HCC1806. The selectivity of the compounds was assessed by comparing the cytotoxicity in cancer cell line versus in a fibroblast cell line. Flow cytometry studies show the activation of apoptotic pathways and also DNA damages with blockage of the cell cycle in the S-phase and appearance of peaks in G0/G1-phase.

Chiral 6-hydroxymethyl-1H,3H-pyrrolo[1,2-c]thiazoles: novel antitumor DNA monoalkylating agents.

New chiral 1H,3H-pyrrolo[1,2-c]thiazoles were synthesized and screened for their in vitro activity as anti-cancer agents in three human tumor cell lines, colorectal adenocarcinoma, melanoma and breast adenocarcinoma. (R)-6-Hydroxymethyl-5-methyl-3-phenyl-1H,3H-pyrrolo[1,2-c]thiazole and the corresponding benzylcarbamate showed selectivity for breast cancer cell lines with IC(50) values of 2.4 microM and 2.2 microM, respectively. The latter also showed significant activity against colorectal adenocarcinoma cancer cell lines (IC(50) = 8.7 microM). In contrast, the 7-hydroxymethyl-5-methyl-3-phenyl-1H,3H-pyrrolo[1,2-c]thiazole gave moderate anti-cancer activity. The performance against breast cancer cell lines (IC(50) = 1.0 microM) of a potential bisalkylating agent, a (3R)-6,7-bis(hydroxymethyl)-1H,3H-pyrrolo[1,2-c]thiazole, wasn't significantly different from the one observed for the monoalkylating derivatives indicating that the main mechanism of action may in fact be the monoalkylation process.

Intermittent pringle maneuver and hepatic function: perioperative monitoring by noninvasive ICG-clearance.

BACKGROUND: Intermittent Pringle maneuver or selective portal clamping often are used to control inflow during parenchymal liver transection. This study was designed to determinate whether these maneuvers are associated with adverse hepatic function. METHODS: Resection was performed without portal clamping in 17 patients (group 1). Selective continuous portal clamping was performed in 11 patients (group 2) and the remaining 33 patients (group 3) had intermittent nonselective portal clamping (occlusion of the main portal trunk). The centers' protocol for total portal occlusion is 15-min occlusion alternated with 5-min reperfusion in patients with normal liver parenchyma or 10 min alternated with 5 min in patients with abnormal parenchyma. ICG elimination tests were conducted concurrently using a noninvasive monitor that tracks the plasma disappearance rate (PDR-ICG-%/min) and 15-min retention rate after administration (ICG-R15-%). RESULTS: There was no statistically difference between the three studied groups in terms of sequential changes of ICG-PDR (p < 0.625) or ICG-R15 (p < 0.398). CONCLUSIONS: Our study indicates that 15 min of intermittent Pringle maneuver or selective hemihepatic continuous portal clamping are safe methods of vascular control during liver resection, with no adverse effects on hepatocellular function.

P-glycoprotein versus MRP1 on transport kinetics of cationic lipophilic substrates: a comparative study using [99mTc]sestamibi and [99mTc]tetrofosmin.

The multidrug resistance (MDR) phenotype in cancer is closely related with the overexpression of P-glycoprotein (Pgp) and multidrug resistance protein-1 (MRP1). Although conferring resistance to a similar spectrum of drugs, these proteins present distinct transport mechanisms and have their own substrates. In this work, we compared the functional properties of Pgp and MRP1 in the transport kinetics of two cationic lipophilic tracers, [(99m)Tc]sestamibi and [(99m)Tc]tetrofosmin, in cellular models of resistance. Cellular transport kinetics of both tracers was evaluated in Small-cell lung cancer cell line H69 and in its drug-resistant sublines, H69LX4 and H69AR, overexpressing Pgp and MRP1, respectively. Studies were performed in the absence and in the presence of MDR modulators. Kinetic parameters extracted from time-activity curves were analyzed through receiver-operating characteristics curve analysis. The uptake and the efflux rate of both radiotracers were significantly higher (p < 0.05) in sensitive cells. However, MRP1 was more effective than Pgp in removing tracers from the intracellular medium. The addition of verapamil and PSC833 significantly reduced the efflux rate and restored the accumulation of both tracers in H69LX4 cells. Only verapamil was effective in the inhibition of MRP1; however, the effects were more pronounced with [(99m)Tc]sestamibi, when compared to [(99m)Tc]tetrofosmin. Outward transport of radiotracers by MRP1 was dependent on the intracellular glutathione levels. We concluded that both tracers can detect Pgp- and MRP1-mediated drug resistance, based on transport kinetics; however, MRP1 is more effective than Pgp on outward transport of radiotracers. We postulate that this finding can be useful to distinguish between the two resistance mechanisms.

Functional imaging of multidrug resistance in an orthotopic model of osteosarcoma using 99mTc-sestamibi.

PURPOSE: The purpose of this work was the development of an orthotopic model of osteosarcoma based on luciferase-expressing tumour cells for the in vivo imaging of multidrug resistance (MDR) with (99m)Tc-sestamibi. METHODS: Doxorubicin-sensitive (143B-luc(+)) and resistant (MNNG/HOS-luc(+)) osteosarcoma cell lines expressing different levels of P-glycoprotein and carrying a luciferase reporter gene were inoculated into the tibia of nude mice. Local tumour growth was monitored weekly by bioluminescence imaging and X-ray. After tumour growth, a (99m)Tc-sestamibi dynamic study was performed. A subset of animals was pre-treated with an MDR inhibitor (PSC833). Images were analysed for calculation of (99m)Tc-sestamibi washout half-life (t (1/2)), percentage washout rate (%WR) and tumour/non-tumour (T/NT) ratio. RESULTS: A progressively increasing bioluminescent signal was detected in the proximal tibia after 2 weeks. The t (1/2) of (99m)Tc-sestamibi was significantly shorter (p < 0.05) in drug-resistant MNNG/HOS-luc(+) tumours (t (1/2) = 87.3 +/- 15.7 min) than in drug-sensitive 143B-luc(+) tumours (t (1/2) = 161.0 +/- 47.4 min) and decreased significantly with PSC833 (t (1/2) = 173.0 +/- 24.5 min, p < 0.05). No significant effects of PSC833 were observed in 143B-luc(+) tumours. The T/NT ratio was significantly lower (p < 0.05) in MNNG/HOS-luc(+) tumours than in 143B-luc(+) tumours at early (1.55 +/- 0.22 vs 2.14 +/- 0.36) and delayed times (1.12 +/- 0.11 vs 1.62 +/- 0.33). PSC833 had no significant effects on the T/NT ratios of either tumour. CONCLUSION: The orthotopic injection of tumour cells provides an animal model suitable for functional imaging of MDR. In vivo bioluminescence imaging allows the non-invasive monitoring of tumour growth. The kinetic analysis of (99m)Tc-sestamibi washout provides information on the functional activity of MDR related to P-glycoprotein expression and its pharmacological inhibition in osteosarcoma.

[Deep lung--cellular reaction to HIV]. / Pulmão profunda--reacção celular ao VIH.

UNLABELLED: The course of HIV infection is accompanied by a wide individual variability. The complex and large interplay between host and viral factors is crucial in the disease's evolution. The lung has been recognised from the beginning of the disease as one of the main targets of infectious and non-infectious complications of AIDS. In this setting both anatomic and immunologic particularities of this organ play an important role. The hallmark of HIV is progressive immune dysfunction. Despite the intensive research into the pathogenesis, several questions remain to be answered on the dynamic effects of HIV on pulmonary cells. Previous studies in which we have participated showed the early presence of lymphocytic alveolitis from the asymptomatic phase of infection. Since then, many collected data has brought new insights into the immune and biochemical mechanisms involving HIV cell entry, as well as target cells, cytokines and other cellular mediators. In this context, the discovery that specific chemokine receptors could act as co-receptors for HIV, allowed a better understanding of the mechanisms underlying viral cellular entry and tropism. On this issue several authors have reported that in addition to the CD4 molecule, most strains of HIV use the chemokine receptor CCR5 for viral attachment and entry into the host cells. This receptor seems to be very important in disease transmission, whereas CXCR4 receptor tends to be used by the viral strains that emerge later in the disease in addition to or instead of the CCR5. AIMS: To evaluate the pulmonary cellular dynamics in AIDS patients regarding the viral load in bronchoalveolar lavage fluid (LLBA), as well as cellularity and tropism through CCR5 and CXCR4 receptors. MATERIAL: 14 AIDS patients were enrolled in this study, with a mean age of 39 +/- 14.3 years (9 males and 5 females) all HIV1, heterosexuals, 6 smokers and 8 non-smokers, none of them drug addicts. These patients were referred to bronchoscopy with BAL, for clinical suspicion of opportunistic lung infections. These patients were later divided into two groups: Group I (recent diagnosis) and Group II (non-recent diagnosis). While all patients had AIDS, group I had also recent diagnosis of opportunistic infections and had not received yet anti-retroviral therapy whilst group II had a long-term disease evolution with several opportunistic episodes and anti-retroviral therapeutic. METHODS: BAL was performed both in the middle bronchus in diffuse or in other segmentar bronchus, depending on radiographic abnormalities. Plasma viral load was performed through PCR-RT in blood samples with EDTA, centrifuged and frozen (-80 masculine Celsius) in the first 4 hours after being collected. The viral load in BALf was quantified in 9 patients using the automatized Cobas Ampliprep/Cobas Amplicor HIV1 Monitor TM Test, version 1.5 Roche Diagnostic Systems. The results were expressed in a numeric scale, with a dynamic variation of 50-750.000 copies of RNA HIV1/cm3 and later converted into a logarithmic scale. In 10 patients an immunological study was carried out in BALf and blood to quantify the lymphocyte populations and subsets (CD3, CD4, CD8, CD19, CD56 and CD56CD8) as well as the receptors CD3CCR5, CD4CCR5, CD8CCR5, CCR5Mø, CXCR4, CD3CXCR4, CXCR4CD14 and co-stimulatory molecule CD28, CD3CD28, CD4CD28, CD8CD28 through monoclonal antibodies - CD8FITC, CD19FITC, CD3PE, CD56PE, CD4PECY5-Lymphogram Cytognos; CCR5PE, CXCRFITC-R & D Systems; CD8Cy5 and CD3Cy5-DaKo, CD4PE, CD14PE, CD28FITC- Immunotech; CD4FITC-CLB, CD8Percp- Beckton Dickinson and CD3 APC - Beckton Dickinson, by flow cytometry (Facs Calibur-Beckton-Dickinson) with 3 or 4 fluorescences - FL1- -FITC, FL2-PE, FL3-PECY, FL4-APC. In the statistical analysis, we used the Student t-test, and li- near correlation. RESULTS: Presence of HIV1 in BALf (2.95 log +/- 3.08 log), in small levels compared with plasma viral loads (5.89 log +/-5.90 log) (Table IV). There was great variability of viral loads in BALf as there was in blood independent of the time elapsed between diagnosis and the exam. As for the lymphocytic populations and subsets in blood (Table V) determined in 13 patients, there was a significant fall of total lymphocytes as well as of their subsets, although more marked in CD4 cells; 42.9% had CD4 levels < 50 cels/mm3 and only 2 patients (n masculine 12, 13) had CD4> 250 cels/mm3. The CD19 was reduced with an individual distribution similar to the CD4 subset. In most cases, the fall of CD8 accompanied the decrease of CD4 and CD19 (patients-n masculine 7 and 8). The lymphocyte populations and subsets in BALf (10 patients) (Table VI) showed a percentual distribution similar to that observed in blood (Table VII) for CD3, CD19, CD4 and CD8 lymphocytes, although the percentage of T cells was higher than in blood (94.5 +/- 5 /84.1 +/-10.4) as opposed to B cells (2.2 +/-3 /10.4 +/- 9.6). In BALf CD8 T cells were higher than in blood (77.7 +/- 17.6 /67.6 +/- 4.2), which was not observed for the CD4 lymphocytes (8.1 +/- 9.5 BALf vs.10.4 +/- 9.6 in blood). The natural killer activity expressed by CD56 T cells had important individual variations in both biological fluids: higher levels in blood than in BALf (9.1 +/-8 /2.9 +/-1.9). The cytotoxic activity of CD56CD8 was similar in blood and in BALf (2.2 +/- 2 / 1.7+/- 1.2) while the individual distribution seemed more homogeneous in BALf (Table VI) than in blood (Table VII). The double-negative (DN) cells had slightly higher values in BALf (7.6 +/- 4.5 vs 5.6 +/- 5.3). Curiously, in BALf we observed a higher percentage of less differentiated cells (13 +/- 13.6) (Table VI). The analysis of the receptors CCR5 and CXCR4 showed in general terms different behaviour concerning the two biological means (Tables VI and VII). Thus, the CCR5 CD3 was higher in blood (10.9+/- 13.2) than in BALf (8.4 +/- +/- 3.5) while the CCR5 CD4 and CCR5 CD8 had an increased expression in BALf in relation to blood ( 2 +/- 2.3 and 4.9 +/- 3.7 / 0.9 +/- 0.7 and 4.1 +/- 4.0 respectively). Concerning the expression of this receptor on monocyte macrophage lineage a marked higher value was attained in BALft (77.8 +/- +/- 41 in BALf vs. 18.7 +/- 15 in blood). On the contrary the total expression of CXCR4 was higher in BALf (31 +/- 19.9) than in blood (16.4 +/- +/- 8.1). This tendency extended equally to the T lymphocytes (26.6 +/- 19.8 vs. 10.7 +/-7.6) and also to the monocyte-macrophage lineage in an exuberant manner (84.5 +/- 30.2 / 4.8 +/- 4.6). The co- stimulatory activity of CD28 showed higher values in blood (22.8 +/- 16.2) than in BALf (15.9 +/- +/- 10.1) for total T cells, CD4 and CD8 lymphocytes 22.5 +/-16.7; 7.8 +/- 8.3; 13.3 +/- 8.3 / 16.5 +/- +/- 10.5; 2.9 +/- 2.8; 10.8 +/- 8.0 respectively). CONCLUSIONS: 1. HIV infection is responsible for important and extensive abnormalities in lung host defences. 2. The complex interaction between host and aggressor as well as the immune response particularly represented by natural killer and cytotoxic activities, apoptosis, and opportunistic diseases or others, therapeutics and other factors may contribute to the difficulty in obtaining homogenous medical samples within research. There are also ethical issues that restrict a purely scientific approach to these patients. 3. These results point to a pulmonary response to HIV in a compartmentalised fashion according to the dynamic cellular elements involved and receptors in which the latter had distinct profiles related to the biological fluids. In this context, the lung compartimental response is particularly dependent on alveolar macrophages activity which is from the beginning the cornerstone of this process and is the last cellular defense mechanism in this territory when all others are profoundly affected. 4. The dynamics of chemokines receptors may be very important in therapeutic approach as the blockage of the CCR5 receptor does not seem to trigger an increased expression of CXCR4 strains. In fact, we found that CXCR4 remained high in monocyte-macrophage cells throughout infection and its expression was increased in T-lymphocytes in Group II patients as opposed to CCR5 behavior in BALf which significantly decreases. However, in blood, CCR5 expression increased, unlike CXCR4. 5. Due to high co-existing opportunistic infections (71.4%) we cannot ignore the hypothesis that this increased expression of CXCR4 was a result of the modulation induced by opportunistic agents. 6. Finally, this striking individual variability undoubtly has clinical implications. This makes a case-by-case management strategy the correct approach.

Multidrug resistance mediated by ABC transporters in osteosarcoma cell lines: mRNA analysis and functional radiotracer studies.

Drug resistance remains a significant impediment to successful chemotherapy and constitutes a major prognostic factor in osteosarcoma (OS) patients. This study was designed to identify the role and prognostic significance of multidrug-resistance (MDR)-related transporters, such as multidrug resistance protein 1 (MDR1), multidrug-resistance-associated protein (MRP1) and breast-cancer-related protein (BCRP), in OS using cationic lipophilic radiotracers. We evaluated the chemosensitivity of four OS cell lines (Saos-2, 143B, MNNG/HOS and U-2OS) to doxorubicin (DOX), cisplatin (CIS) and methotrexate. The expression of MDR-related transporters was analyzed at mRNA level by quantitative polymerase chain reaction and at functional level by 99mTc sestamibi and 99mTc tetrofosmin. The effectiveness of MDR modulators [cyclosporin A (CsA) and imatinib] on transporter inhibition and on the reversal of resistance was also assessed. MNNG/HOS and U-2OS cells expressing high levels of MDR1 were highly resistant to DOX and showed reduced accumulation and higher efflux for radiotracers. Although MRP1 was uniformly expressed in all cells, only U-2OS was resistant to CIS. CsA restored sensitivity to DOX and CIS, and enhanced the accumulation and efflux half-life of radiotracers in MDR1-expressing cell lines. The chemosensitivity of OS cells to DOX was strongly dependent on mRNA MDR1 expression and could be circumvented by adding CsA. The kinetic parameters of radiotracers correlated with MDR1 expression levels, hence predicting DOX resistance. We concluded that sensitivity to chemotherapy is strongly dependent on the expression of MDR1 transporter and that radiotracer studies could prove clinically useful in predicting chemotherapy response and in evaluating the efficacy of MDR-reversing agents.

Comparison of the predicted in vivo behaviour of the Sn(II)-APDDMP complex and the results as studied in a rodent model.

In a quest for more effective radiopharmaceuticals for pain palliation of metastatic bone cancer, this paper relates results obtained with ((117m)Sn labelled) Sn(II) complexed to the bone seeking bisphosphonate, N,N-dimethylenephosphonate-1-hydroxy-3-aminopropylidenediphosphonate (APDDMP). APDDMP is synthesised from the known bone cancer pain palliation agent 1-hydroxy-3-aminopropylidenediphosphonate (APD, Pamindronate). This work is performed to utilise the idea that the low bone marrow radio toxicity of (117m)Sn could afford a highly effective radiopharmaceutical in pain palliation but also in the curative treatment of bone metastasis. Complex-formation constants of APDDMP with the important blood plasma metal-ions, Ca(2+), Mg(2+), Zn(2+) as well as the added metal ion, Sn(2+) were measured by glass electrode potentiometry at 25 degrees C and I = 150 mM. Blood plasma models were constructed using the computer code ECCLES and the results compared with those gathered from tests on a rodent model. The ((117m)Sn-labelled) Sn(II)-APDDMP complex was found to have only some liver and bone uptake although a high trabecular to normal bone ratio was recorded. From the blood plasma model this was shown to be primarily due to the high affinity of APDDMP for Ca(II) causing some of the Sn(II)-APDDMP complex to dissociate. High kidney uptake and excretion as well as high bladder uptake was recorded which was shown to be due to the dissociation of the Sn(II)-APDDMP complex in blood plasma. Animal model observations could be explained by the blood plasma modelling.