Ceftazidime/avibactam in the era of carbapenemase-producing Klebsiella pneumoniae: experience from a national registry study.

BACKGROUND: Infections caused by KPC-producing Klebsiella pneumoniae (Kp) are associated with high mortality. Therefore, new treatment options are urgently required. OBJECTIVES: To assess the outcomes and predictors of mortality in patients with KPC- or OXA-48-Kp infections treated with ceftazidime/avibactam with an emphasis on KPC-Kp bloodstream infections (BSIs). METHODS: A multicentre prospective observational study was conducted between January 2018 and March 2019. Patients with KPC- or OXA-48-Kp infections treated with ceftazidime/avibactam were included in the analysis. The subgroup of patients with KPC-Kp BSIs treated with ceftazidime/avibactam was matched by propensity score with a cohort of patients whose KPC-Kp BSIs had been treated with agents other than ceftazidime/avibactam with in vitro activity. RESULTS: One hundred and forty-seven patients were identified; 140 were infected with KPC producers and 7 with OXA-48 producers. For targeted therapy, 68 (46.3%) patients received monotherapy with ceftazidime/avibactam and 79 (53.7%) patients received ceftazidime/avibactam in combination with at least another active agent. The 14 and 28 day mortality rates were 9% and 20%, respectively. The 28 day mortality among the 71 patients with KPC-Kp BSIs treated with ceftazidime/avibactam was significantly lower than that observed in the 71 matched patients, whose KPC-Kp BSIs had been treated with agents other than ceftazidime/avibactam (18.3% versus 40.8%; P = 0.005). In the Cox proportional hazards model, ultimately fatal disease, rapidly fatal disease and Charlson comorbidity index ≥2 were independent predictors of death, whereas treatment with ceftazidime/avibactam-containing regimens was the only independent predictor of survival. CONCLUSIONS: Ceftazidime/avibactam appears to be an effective treatment against serious infections caused by KPC-Kp.

Mining the effect of the neonicotinoids imidacloprid and clothianidin on the chemical homeostasis and energy equilibrium of primary mouse neural stem/progenitor cells using metabolomics.

The projection of plant protection products' (PPPs) toxicity to non-target organisms at early stages of their development is challenging and demanding. Recent developments in bioanalytics, however, have facilitated the study of fluctuations in the metabolism of biological systems in response to treatments with bioactives and the discovery of corresponding toxicity biomarkers. Neonicotinoids are improved insecticides that target nicotinic acetylocholine receptors (nAChR) in insects which are similar to mammals. Nonetheless, they have sparked controversy due to effects on non-target organisms. Within this context, mammalian cell cultures represent ideal systems for the development of robust models for the dissection of PPPs' toxicity. Thus, we have investigated the toxicity of imidacloprid, clothianidin, and their mixture on primary mouse (Mus musculus) neural stem/progenitor (NSPCs) and mouse neuroblastoma-derived Neuro-2a (N2a) cells, and the undergoing metabolic changes applying metabolomics. Results revealed that NSPCs, which in vitro resemble those that reside in the postnatal and adult central nervous system, are five to seven-fold more sensitive than N2a to the applied insecticides. The energy equilibrium of NSPCs was substantially altered, as it is indicated by fluctuations of metabolites involved in energy production (e.g. glucose, lactate), Krebs cycle intermediates, and fatty acids, which are important components of cell membranes. Such evidence plausibly suggests a switch of cells' energy-producing mechanism to the direct metabolism of glucose to lactate in response to insecticides. The developed pipeline could be further exploited in the discovery of unintended effects of PPPs at early steps of development and for regulatory purposes.

Investigation of the correlation of successive earthquakes preceding main shocks in the Greek territory.

The Canonical Correlation Analysis (CCA) estimates the correlation between two vector variables by maximizing the correlation of linear combinations of their respective components. Here, the CCA is used to find correlation patterns in the last five successive, per pairs, earthquakes ( M ≥ 4.0 ) preceding 271 main shocks ( M ≥ 5.5 ) that occurred in the Greek territory during 1964-2018. The vector variables have two components, the earthquake magnitude and interevent time. The statistical significance of CCA is determined by the standard parametric test along with two proposed randomization tests, one using random shuffling of each paired dataset and one using randomly selected pairs of successive earthquakes. Simulations were designed on synthetic data from vector variables having the statistical characteristics of the real observations. The results on uncorrelated variables showed the correct size for the two randomization tests but larger type I error for the parametric significance test for small sample size. For correlated variables, the test power was equally high for both test types. The application of CCA and the significance tests to the Greek seismicity evidence the significant correlation among the last five successive preshocks, proving to be a promising tool in an a posteriori short-term earthquake forecasting.

Regulation of Angiogenesis Discriminates Tissue Resident MSCs from Effective and Defective Osteogenic Environments.

BACKGROUND: The biological mechanisms that contribute to atrophic long bone non-union are poorly understood. Multipotential mesenchymal stromal cells (MSCs) are key contributors to bone formation and are recognised as important mediators of blood vessel formation. This study examines the role of MSCs in tissue formation at the site of atrophic non-union. MATERIALS AND METHODS: Tissue and MSCs from non-union sites (n = 20) and induced periosteal (IP) membrane formed following the Masquelet bone reconstruction technique (n = 15) or bone marrow (n = 8) were compared. MSC content, differentiation, and influence on angiogenesis were measured in vitro. Cell content and vasculature measurements were performed by flow cytometry and histology, and gene expression was measured by quantitative polymerase chain reaction (qPCR). RESULTS: MSCs from non-union sites had comparable differentiation potential to bone marrow MSCs. Compared with induced periosteum, non-union tissue contained similar proportion of colony-forming cells, but a greater proportion of pericytes (p = 0.036), and endothelial cells (p = 0.016) and blood vessels were more numerous (p = 0.001) with smaller luminal diameter (p = 0.046). MSCs showed marked differences in angiogenic transcripts depending on the source, and those from induced periosteum, but not non-union tissue, inhibited early stages of in vitro angiogenesis. CONCLUSIONS: In vitro, non-union site derived MSCs have no impairment of differentiation capacity, but they differ from IP-derived MSCs in mediating angiogenesis. Local MSCs may thus be strongly implicated in the formation of the immature vascular network at the non-union site. Attention should be given to their angiogenic support profile when selecting MSCs for regenerative therapy.

Forecasting of the first hour aftershocks by means of the perceived magnitude.

The majority of strong earthquakes takes place a few hours after a mainshock, promoting the interest for a real time post-seismic forecasting, which is, however, very inefficient because of the incompleteness of available catalogs. Here we present a novel method that uses, as only information, the ground velocity recorded during the first 30 min after the mainshock and does not require that signals are transferred and elaborated by operational units. The method considers the logarithm of the mainshock ground velocity, its peak value defined as the perceived magnitude and the subsequent temporal decay. We conduct a forecast test on the nine M  ≥ 6 mainshocks that have occurred since 2013 in the Aegean area. We are able to forecast the number of aftershocks recorded during the first 3 days after each mainshock with an accuracy smaller than 18% in all cases but one with an accuracy of 36%.

Measles and mumps immunity in Northern Greece, 2004-2007.

A cross-sectional study was conducted in order to determine the prevalence of mumps and measles antibodies in a representative sample of the general population in Northern Greece between January 2004 and May 2007. Overall, 900 healthy individuals participated in the study. The great majority were found to be protected against measles. The total protection rate against mumps was significantly less (87% versus 72%, respectively; p<0.01). Compared to all other age groups, statistically significantly lower protection rates were found in children younger than 1.5 years (p<0.01). The lowest rates of all adult groups were found in the age group of 21 to 30 years (86% and 68% for measles and mumps, accordingly). In conclusion, protection rates against both measles and mumps seem to be lower than expected in certain age groups, such as infants and young adults.

Effect of heparin affin regulatory peptide on the expression of vascular endothelial growth factor receptors in endothelial cells.

BACKGROUND: Heparin affin regulatory peptide (HARP) is an 18-kDa secreted protein that has been implicated in tumor growth and angiogenesis, although the mechanisms involved remain largely unknown. In the present work, the effect of human recombinant HARP on the expression of the vascular endothelial growth factor (VEGF) receptors KDR, Flt-1 and neuropilin-1 was studied in cultured human umbilical vein endothelial cells (HUVEC). MATERIALS AND METHODS: The mRNA and protein levels of VEGF receptors were estimated by semi-quantitative RT-PCR and Western blot, respectively. Cell proliferation and migration were measured by MTT, direct counting of the cells and modified Boyden chamber assays. RESULTS: HARP decreased the expression of KDR but increased the expression of Flt-1 and neuropilin-1 at both the mRNA and protein level. The effect reached a maximum 4 h after the addition of HARP into the cell culture medium and was reversed at later time-points. When HARP was added to the culture medium 4 h before the addition of VEGF165, it inhibited VEGF165-induced proliferation and migration of HUVEC. CONCLUSION: These data suggest that HARP affects the expression of VEGF receptors and inhibits VEGF165-induced activation of HUVEC.

Heparin affin regulatory peptide: a new target for tumour therapy?

Heparin affin regulatory peptide (HARP), also known as pleiotrophin or heparin-binding growth-associated molecule, is an 18-kDa growth factor that has a high affinity for heparin. It constitutes with midkine and retinoic acid heparin-binding protein, a family of structurally related heparin-binding growth factors. A growing body of evidence indicates that HARP is involved in the control of cellular proliferation, migration and differentiation and plays a significant role in tumor growth and angiogenesis. HARP has a well described role in physiological as well as tumor angiogenesis, and is detected in various carcinomas, such as human breast and prostate cancer, neuroblastomas, gliomas, benign meningiomas, small cell lung cancer and mammary tumors, exhibiting a proto-oncogene function. It is also constitutively expressed in tumour cell lines and is involved in tumour growth and metastasis. Therefore, HARP appears to be a potential new target for the treatment or/and diagnosis of several types of cancer.

Measurement of cell numbers in microtiter culture plates using the fluorescent dye Hoechst 33258.

A sensitive fluorimetric assay based on DNA binding of the fluorescent dye Hoechst 33258 was developed to quantitate cell numbers in microtiter plates. The cells were fixed and incubated with the dye solution. The bound dye was extracted with ethanol and fluorescence was measured in a spectrofluorimeter. The method is sensitive for as little as a thousand cells and is particularly useful for the normalization of enzyme-linked immunoassay (ELISA) data obtained from the same cells. Another major advantage of this new technique is that in contrast to previously described methods, it requires commonly available, inexpensive equipment.

Evidence that platelets promote tube formation by endothelial cells on matrigel.

The involvement of platelets in neovascularization was investigated in the matrigel tube formation assay, an in vitro model of angiogenesis. Platelets promoted the formation of capillary-like structures (expressed as relative tube area) number- and time-dependently. Relative tube area increased from 0.98+/-0.02 (n = 8) in the presence of 6.25 x 10(4), to 3.21+/-0.12 (n=8) in the presence of 10(6) platelets/well compared to 0.54+/-0.04 (n=8) in their absence. This increase was unaffected by acetyl salicylic acid (ASA), apyrase, and hirudin. Photographs from representative experiments, showed that platelets adhered along the differentiating endothelium. Addition of alpha-thrombin (0.1-1 i.u. ml(-1)), the nitric oxide (NO) donor sodium nitroprusside (SNP; 1-100 microM) or the NO synthase inhibitor, L-NG-arginine-methylester (L-NAME, 30-300 microM) to the assay, had no effect on tube formation compared to that seen with platelets alone. Neuraminidase (0.01 i.u./10(7) platelets), which strips sialic acid residues from membrane glycoproteins, abolished the promoting effect of platelets on tube formation. The relative tube area in the presence of neuraminidase-treated platelets was 0.81+/-0.03 (n = 8), in the presence of untreated platelets 1.69+/-0.09, P<0.001 (n=8) and in the absence of platelets, 0.80+/-0.04 (n=8). The tetrapeptide Arg-Gly-Asp-Ser (RGDS; 20-200 microM) which inhibits von Willebrand factor, fibrinogen and fibronectin-mediated adhesion, had no effect on the promoting effect of platelets on tube formation. These results indicate that platelets promote angiogenesis in vitro. This effect is largely independent from activation by alpha-thrombin, is not modified by manipulating NO and prostaglandin metabolism and proceeds possibly through adhesion of the platelets to the differentiating endothelium.

Nitric oxide synthase expression, enzyme activity and NO production during angiogenesis in the chick chorioallantoic membrane.

In order to elucidate further the role of nitric oxide (NO) as an endogenous antiangiogenic mediator, mRNA expression of inducible nitric oxide synthase (iNOS), enzyme activity and production of NO were determined in the chick chorioallantoic membrane (CAM), an in vivo model of angiogenesis. In this model, maximum angiogenesis is reached between days 9 - 12 of chick embryo development. After that period, vascular density remains constant. Inducible NO synthase (iNOS) mRNA expression, determined by reverse transcriptase polymerase chain reaction (RT - PCR), increased from the 8th day reaching a maximum (70% increase) at days 10 - 11. NO synthase activity, determined as citrulline formation in the presence of calcium, also increased from day 8 reaching a maximum around day 10 (100% increase). Similar results were obtained in the absence of calcium suggesting that the NOS determined was the inducible form. Nitric oxide production, determined as nitrites, increased from day 8 reaching a maximum around day 10 (64% increase) and remaining stable at day 13. Finally, the bacterial lipopolysaccharide LPS (which activates transcriptionally iNOS), inhibited dose dependently angiogenesis in the CAM. These results in connection with previous findings from this laboratory, showing that NO inhibits angiogenesis in the CAM, suggest that increases in iNOS expression, enzyme activity and NO production closely parallel the progression of angiogenesis in the CAM, thus providing an endogenous brake to control this process. British Journal of Pharmacology (2000) 129, 207 - 213

Regulation of extracellular matrix remodeling and MMP-2 activation in cultured rat adrenal medullary endothelial cells.

We previously reported that short term exposure of cultured rat adrenal medullary endothelial cells (RAMEC) to thrombin enhances the subendothelial deposition of extracellular matrix (ECM) proteins fibronectin, laminin, and collagen types I (C-I) and IV (C-IV) (Papadimitriou et at., 1997). In this work, we extended our previous studies on factors that affect ECM protein deposition to include agents that activate or inhibit some of the most common intracellular signals such as cAMP, protein kinase C (PKC) and calcium. Furthemore, we investigated the possible link between the observed alterations in ECM protein deposition and the secretion of matrix metalloproteinase-2 (MMP-2). Forskolin (adenylyl cyclase activator) caused a dose-dependent increase in the deposition of all four ECM proteins studied. Isoproterenol (beta-adrenergic receptor agonist) and the membrane-permeant cAMP analogue dibutyryl-cAMP, significantly increased the deposited amounts of ECM proteins at low concentrations, and this increase was reversed at higher concentrations of both agents. All these agents had the opposite effect on MMP-2 secretion, increasing it at doses where they decreased ECM protein deposition and vice-versa. However, elevation of cAMP by the phosphodiesterase inhibitor IBMX had no effect neither on the deposited amounts of any of the ECM proteins studied nor on MMP-2 secretion. Activation of PKC by phorbol ester (PMA) resulted in a decrease in ECM protein deposition and an increase in MMP-2 secretion. Finally, chelation of intercellular calcium with BAPTA-AM resulted in an increased ECM deposition and a decrease in MMP-2 secretion, Our results show a complex pattern of regulation of ECM protein deposition by cAMP-mobilizing agents, and also indicate an inverse correlation between ECM protein deposition and secretion of MMP-2. The concerted regulation of both these processes is essential in the formation of new blood vessels and for the integrity of the vascular wall.

Regulation of extracellular matrix remodeling and MMP-2 activation in cultured rat adrenal medullary endothelial cells.

We previously reported that short-term exposure of cultured rat adrenal medullary endothelial cells (RAMEC) to thrombin enhances the subendothelial deposition of extracellular matrix (ECM) proteins fibronectin, laminin, and collagen types I (C-I) and IV (C-IV) (Papadimitriou et al. 1997). In this work, we extended our previous studies on factors that effect ECM protein deposition to include agents that activate or inhibit some of the most common intracellular signals such as cAMP, protein kinase C (PKC), and calcium. Furthermore, we investigated the possible link between the observed alterations in ECM protein deposition and the secretion of matrix metalloproteinase-2 (MMP-2). Forskolin (adenylyl cyclase activator) caused a dose-dependent increase in the deposition of all four ECM proteins studied. Isoproterenol beta-adrenergic receptor agonist) and the membrane permeant cAMP analogue dibutyryl-cAMP significantly increased the deposited amounts of ECM proteins at low concentrations, and this increase was reversed at higher concentrations of both agents. All these agents had the opposite effect on MMP-2 secretion, increasing it at doses where they decreased ECM protein deposition and vice versa. However, elevation of cAMP by the phosphodiesterase inhibitor IBMX had no effect either on the deposited amounts of any of the ECM proteins studied or on MMP-2 secretion. Activation of PKC by phorbol ester (PMA) resulted in a decrease in ECM protein deposition and an increase in MMP-2 secretion. Finally, chelation of intercellular calcium with BAPTA-AM resulted in an increased ECM deposition and a decrease in MMP-2 secretion. Our results show a complex pattern of regulation of ECM protein deposition by cAMP-mobilizing agents and also indicate an inverse correlation between ECM protein deposition and secretion of MMP-2. The concerted regulation of both of these processes is essential in the formation of new blood vessels, and for the integrity of the vascular wall.

McArdle's disease: two clinical expressions in the same pedigree.

Two patients with McArdle's disease within the same pedigree and with two different clinical forms are presented. The first patient suffered from progressive muscle weakness and atrophy. Muscle morphology was that of myopathy. Residual activity of phosphorylase was 28% and sodium dodecyl sulphate electrophoresis showed decreased protein. The second case was typical of McArdle's disease, clinically and biochemically. It was concluded that the first patient was a heterozygote (residual activity 28% of normal) and the second was a homozygote, the genetic transmission being autosomal recessive.

HIV-1 co-receptor CCR5 and CCR2 mutations among Greeks.

The frequency of CCR5 and CCR2 alleles in human immunodeficiency virus (HIV)-positive and HIV-negative populations of Northern Greece was investigated. The frequency of the CCR5Delta32 allele among the HIV-negative subjects was 0.052, while it was approximately two-fold lower among the seropositives, suggesting that the heterozygous genotype confers a partial resistance to the HIV infection. No significant difference in CCR2 allele frequency between the two groups was observed.

Donor lung pretreatment with surfactant in experimental transplantation preserves graft hemodynamics and alveolar morphology.

In experimental lung transplantation, the reduction of endogenous surfactant properties occurs after graft preservation and transplant reperfusion. The aim of this study was to evaluate the efficacy of donor lung pretreatment with exogenous surfactant on graft damage after ischemia and reperfusion. Fourteen (control group A, n = 8; study group B, n= 6) young female white pigs (mean weight 27 +/- 3.5 kg) were used in a newly developed autotransplantation model within situcold ischemia. In study group B, before thoracotomy, 1.5 ml/kg surfactant apoprotein-A-free surfactant was administrated into the left main bronchus via flexible bronchoscopy. Belzer UW solution was used for lung preservation. Cold ischemia was achieved for 3 hr with interlobar lung parenchyma temperature at 8 +/- 1.3 degrees C, and central temperature maintained at 37.20 +/- 0.5 degrees C. Animals were sacrificed after 3 hr of graft reperfusion. At the end of reperfusion, pulmonary vascular resistance index (was 447.80 dyn/sec.cm(5).m(2)(+/-66.8) in group A vs 249.51 in group B (P< 0.001) and serum nitric oxide was adequately preserved. The mean alveolar surface area estimated by computerized morphometry was 5280.84 (4991.1) microm(2)(group A) vs 3997.89 (3284.70) microm(2)(group B;P< 0.005). Histology revealed milder macrophage and lymphocyte infiltration in group B at the end of reperfusion. Pretreatment of donor lung with an surfactant apoprotein-A -free surfactant agent appears to be beneficial in terms of maintaining serum NO and reducing hemodynamic disturbances. Furthermore, alveolar histology and stereomorphology are better preserved.

X-rays affect the expression of genes involved in angiogenesis.

BACKGROUND: We have previously shown, using the chicken embryo chorioallantoic membrane (CAM) model of in vivo angiogenesis, that X-rays act on the extracellular matrix and enhance normal and tumor-induced angiogenesis. In the present work, we studied the effect of X-rays on the gene expression of three proteins that are important regulators of angiogenesis: vascular endothelial growth factor (VEGF), heparin affin regulatory peptide (HARP) and inducible nitric oxide synthase (iNOS). MATERIALS AND METHODS: An area of 1 cm2 of the CAM, restricted by a plastic ring was irradiated at room temperature. The expression of the genes was studied using RT-PCR and the amounts of the mRNAs were quantified using image analysis of the corresponding agarose gels of the RT-PCR products. RESULTS: VEGF mRNA was decreased 6 h after irradiation. However, at later time points, VEGF expression was significantly increased compared with the nonirradiated tissue. Similarly, X-rays down-regulated both HARP and iNOS expression 6 h after irradiation and the effect was reversed at later time points, similarly to the effect of X-rays on VEGF. CONCLUSION: These data support the notion that X-rays increase the expression of genes that favor angiogenesis.

Interactions of PC/Chol and PS/Chol liposomes with human cells in vitro.

Interactions between phosphatidylcholine (PC) or phosphatidylserine (PS) liposomes and human umbilical vein endothelial cells (HUVEC) or human promyelocytic leukemia cells (HL60) were investigated. Pyramine encapsulating or rhodamine incorporating small unilamellar liposomes with mean diameters around 80 nm (demonstrated to retain encapsulated material and to be nontoxic under experimental conditions) were used. Liposome uptake by both types of cells increased when increasing amounts of vesicles were co-incubated. For both lipid compositions, the interaction with HUVEC was very fast (association reached a plateau within 5 min) and so was the release of internalized vesicles (90% within 10 min at 37 degrees C). The reduced association values at 4 degrees C and the punctuate fluorescence observed in the cell cytoplasm after interaction, were indicative of whole liposome internalization. This internalization was clathrin-independent, since it was not inhibited by sodium azide and deoxyglucose. Pre-treatment of HUVEC with filipin or NEM resulted in modification of the interaction, something that could be due to alterations in the biochemical characteristics of HUVEC membranes that inhibit vesicular processes. In HL-60 cells, a slower association and faster release of PC/Chol liposomes was demonstrated, while association of both liposomes with these cells was energy-and temperature-independent. Nevertheless, morphological studies revealed differences in the interactions: A bright fluorescent rim observed after interaction with PC/Chol liposomes, suggests that these liposomes were adsorbed on the surface of HL60 cells, while the uniform cytoplasmic fluorescence observed after incubation with PS/Chol liposomes was indicative of fusion as the interaction mechanism.

Incidence and other epidemiological characteristics of sudden cardiac death in northwest Greece.

Sudden cardiac death (SCD) has not been investigated separately in Greece. The aim of this study is to describe the epidemiological characteristics of people dying suddenly out of hospital in an area of Greece. In 1990, a population based study was started to detect the cases of people dying suddenly out of hospital (< 1 h after onset of acute symptoms or < 6 h after being seen alive) in a closed population in Northwest Greece (Ioannina area: 160,000 inhabitants). During a 3.5 year period, 283 potential cases aged 30-70 years were identified by monitoring the mortality in the emergency rooms of the two hospitals of the area, the coroner's office and the death certificates from the Government Department of Statistics. The diagnosis of SCD was established in 223 (183 men, 40 women; mean ages 59 and 61 years respectively) after visiting and interviewing the relatives and/or the family doctors within 12 days (range 1-28) after the death. SCD in the study accounts for 50% of all cardiovascular deaths and is the most common cause of death after neoplasia. The most common place of death was home (151 cases, 68%), and in 174 cases (78%) deaths occurred while the patients were relaxing or during routine activities. Prodromal symptoms were reported in 57 cases (26%). The time of day of death showed a circadian variation, with a peak in the late morning from 9:00 to 12:00. Ninety four (42%) had a prior history of heart disease. One hundred and ninety one cases (86%) occurred in the subgroup of age 50-70 years.(ABSTRACT TRUNCATED AT 250 WORDS)

UPTAKE OF LIPOSOMES WHICH INCORPORATE A GLYCOPEPTIDE FRACTION OF ASIALOFETUIN BY HepG(2) CELLS.

We examined the interaction between liposomes which incorporate a fraction triantennary glycopeptide (AF(2)) of asialofetuin and human hepatoma cells (HepG(2)) in vitro. HepG(2) cells are known to express the asialoglycoprotein receptor. For liposome preparation AF(2) was cleaved from asialofetuin, purified and conjugated with different length (C(12),C(16) and C(18)) fatty acids (FA). The conjugates were subsequently incorporated into pre-formed sonicated liposomes using a mild cholate incubation method. Interactions between AF(2)/FA-liposomes as well as control-liposomes (with no ligand) and cells (in the presence of serum) were measured at different lipid doses after incubating HepG(2) cells with liposomes at 4 degrees C and 37 degrees C, in the absence and presence of galactose, and also evaluated by fluorescence microscopy. More extensive studies were performed with the AF(2)/C(18)-liposomes which were previously found to incorporate higher amounts of ligand and be the most stable of the formulations prepared. Results from both, morphological and quantitative studies, demonstrate that AF(2)/C(16) and especially AF(2)/C(18)-liposomes are bound and taken up by the cells by a galactose specific mechanism. The AF(2)/C(12)-liposomes-which were previously found to incorporate low amounts of ligand in a non-stable way- were taken up by the cells in amounts similar to those of the control liposomes (without ligand) while this uptake was not reduced by galactose and therefore possibly non-specific. The intracellular localization of AF(2)/C(18)-liposomes was further evidenced by intracellular acidification using NH(4)Cl. These conclusions, justify the importance of further in vivo studies in order to demonstrate the capability of the proposed system to target hepatocytes.