Divergent effects of the antiretroviral drugs, dolutegravir, tenofovir alafenamide, and tenofovir disoproxil fumarate, on human adipocyte function.

Combined antiretroviral therapy (cART) has been associated with increased body weight accompanied by metabolic alterations in people living with human immunodeficiency virus (PLWH). To gain insight into the combined effects of cART components on adipocyte dysfunction, we assessed whether and how treatment of human adipocytes with dolutegravir (DTG) and the nucleotide-analog reverse-transcriptase inhibitors (NRTIs), tenofovir alafenamide (TAF) and tenofovir disoproxil fumarate (TDF), alone and in combination, altered biological processes related to adipose tissue dysfunction. DTG, TAF, and TDF were applied to human Simpson-Golabi-Behmel syndrome (SGBS) adipose cells during differentiation (day 10) and ensuing differentiation (day 14). Expression of selected marker genes was determined by qPCR, the release of adipokines and inflammatory cytokines to the culture media was assessed, and cell respiration was measured. Adipogenesis was not altered by the combined treatment of human adipocytes. However, DTG at the highest dose repressed adipogenesis marker genes expression, and TAF and TDF appeared to mitigate this effect. DTG repressed the expression of adiponectin and the release of adiponectin and leptin in differentiating adipocytes, and these effects were mantained in combination with TAF and TDF. DTG plus TAF or TDF on human adipocytes enhanced inflammation and stress and increased the release of proinflammatory cytokines to the culture media. Together, our results show that combined therapy with these drugs can alter inflammation, cellular stress, and fibrosis in human adipocytes. These findings may improve our understanding and management of the effects of cART on body adiposity and metabolic dysregulation in PLWH.

Caracterización de diferentes suplementos de ácidos omega-3 en su aplicación en las edades pediátricas / Characterization of different supplements of omega-3 acids in its application in the pediatric age

La suplementación con ácidos grasos poliinsaturados, conocidos como omega-3, fundamentalmente el docosahexaenoico (DHA), parece estar indicada en la infancia con el objeto de optimizar el desarrollo neurológico, en la prevención de ciertas enfermedades y como coadyuvante de tratamientos en las de carácter inflamatorio y del desarrollo. Las presencia en el mercado de diferentes formulaciones y concentraciones, los diversos reclamos promocionales y el precio originan confusión tanto en el prescriptor como en el paciente. Objetivo: Caracterizar 23 productos comerciales con potencial utilidad en la edad pediátrica, que contienen DHA, en relación con su composición, concentración y pureza. Método: Se determina la composición lipídica, el contenido en triglicéridos (TG), diglicéridos y ésteres etílicos, así como el contenido en ácidos grasos omega-3 de cadena larga, eicosapentaenoico (EPA), docosapentanoico y DHA, sobre el total de ácidos grasos. Resultados: Existen dos presentaciones básicas, en forma de éster etílico o TG. De estos últimos la concentración de DHA y EPA es variable, y las mejores presentaciones son aquellas en que el DHA unido a TG supera los 400 mg/g de producto. Esta relación de concentración es, en general, inversa al precio del envase. Ambos aspectos son de importancia en la prescripción final si se desea aportar la dosis adecuada diaria (AU)

Polyunsaturated fatty acids supplementation, primarily docosahexaenoic acid (DHA), seems to be indicated in childhood to optimize neurological development and prevention and as adjuvant treatment of various inflammatory diseases. The presence of different formulations and concentrations, promotional claims and a wide range of prizes, cause confusion both to the prescriber and the patient. Objective: Characterize 23 commercial products containing DHA, regarding composition, concentration and purity. Methods: We determined the lipid composition, triglyceride content (TG), diglycerides and ethyl esters, and content in omega-3 fatty acids, eicosapentaenoic (EPA), docosapentaenoic and DHA on total fatty acids. Results: There are two basic presentations forms, as ethyl ester or TG. Of the latter the concentration of DHA and EPA is variable with the best presentation those in which the DHA attached to TG exceeds 400 mg/g of product. This concentration ratio is generally inversely related to the package price. Both aspects are important in the final prescription if desired to provide the appropriate daily dose (AU)

Tumour necrosis factor alpha in fat redistribution syndromes associated with combination antiretroviral therapy in HIV-1-infected patients: potential role in subcutaneous adipocyte apoptosis.

BACKGROUND: The pathogenesis of fat redistribution syndromes (FRS) observed in the setting of highly active antiretroviral therapy (HAART) for the treatment of HIV-1-infection remains elusive. A dysregulation of the tumour necrosis factor alpha (TNF-alpha) system occurs in HIV-infected patients with FRS. MATERIALS AND METHODS: The study looked at both the in vivo and in vitro relationship between TNF-alpha and the degree of subcutaneous adipocyte apoptosis in 60 HIV-1-infected patients on HAART with FRS, another 60 HIV-1-infected patients on HAART without FRS and 60 uninfected control patients. Apoptosis was assessed by the terminal deoxynucleotidyl transferase dUTP (deoxyuridine 5'-triphosphate)-digoxigenin Nick End Labelling (TUNEL) method. Soluble receptors of TNF-alpha were determined by the sandwich enzyme immunoassay technique. The in vitro viability was assessed by staining with 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) and apoptosis by TUNEL. RESULTS: HIV-1-infected patients with FRS had significantly higher degrees of subcutaneous adipocyte apoptosis than those without FRS (P = 0.0001) and uninfected controls (P < 0.0001). There was a statistically significant association between serum levels of soluble TNF-alpha receptors #1 and #2 and the degree of subcutaneous adipocyte apoptosis in patients with and without FRS (P < 0.0001 for both receptors). In vitro, the addition of TNF-alpha (10 ng mL(-1)) to an adipocyte culture embedded with indinavir, either alone or in clinically relevant combinations with stavudine (d4T) and lamivudine (3TC), significantly decreased adipocyte viability (P = 0.0001) and increased adipocyte apoptosis (P < 0.0001) with respect to that observed with the addition of antiretrovirals alone. CONCLUSIONS: TNF-alpha plays a significant role in subcutaneous adipocyte apoptosis, which occurs in the setting of FRS in HIV-1-infected patients on highly active antiretroviral therapy.

In vitro cytotoxic study of immunoliposomal doxorubicin targeted to human CD34(+) leukemic cells.

The expression of CD34 antigen in acute myelogenous leukemias is considered an unfavourable prognosis marker for response to anticancer drugs and duration of remission. This study investigated the applicability of long-circulating immunoliposomes loaded with doxorubicin targeted to CD34 antigen present on MDR(+) human myelogenous leukemia KG-1a cell line. Immunoliposomal doxorubicin showed a higher cytotoxicity against KG-1a cells than non-targeted liposomal doxorubicin, but it did not improve over that of free drug. Although no reversal of doxorubicin resistance was found to occur through its liposomal encapsulation, a therapeutic benefit can be obtained by the selective cytotoxicity observed. Endocytosis studies demonstrated that, after binding to CD34 antigen, the immunoliposomes are not internalized by the KG-1a cells and so the cytotoxic effect might be due to drug released into the space near the cell membrane. Thus, immunotargeting of liposomal doxorubicin to CD34(+) leukemic cells may only provide an ex vivo strategy for site-selective CD34(+) leukemia cell killing.

Preparation of long-circulating immunoliposomes using PEG-cholesterol conjugates: effect of the spacer arm between PEG and cholesterol on liposomal characteristics.

Poly(ethylene glycol)-coated liposomes were prepared with two new synthesised pegylated cholesterol (Chol) derivatives linked via carbamate bond. Poly(ethylene glycol) (PEG) was directly linked to Chol (PEG-Chol) or through a space arm of diaminebutane (PEG-L-Chol). In buffer, the physicochemical properties of PC/Chol liposomes (2/1, molar ratio) containing up to 10 mol% of pegylated Chol derivatives did not change significantly and the PEG layer at liposome surface inhibited the agglutination of biotin-liposomes induced by streptavidin. On the other hand, in serum, PEG-L-Chol seemed to reduce the interactions of liposomes with serum proteins, much more than PEG-Chol. The low steric hindrance of PEG-Chol derivative may be due to the slow conformational transition rate of the polymer, since PEG may be deeper located in the membrane. The coupling efficiency of the ligand to the functionalised amino group at the polymer end was also affected, but, its antigen-binding activity was preserved. The basic physical-chemical characteristics studied in this work are relevant to assess the application of pegylated Chol liposomes as drug delivery systems.

Switching to nevirapine decreases insulin levels but does not improve subcutaneous adipocyte apoptosis in patients with highly active antiretroviral therapy-associated lipodystrophy.

Subcutaneous adipocyte apoptosis occurs in lipotrophic areas of patients with highly active antiretroviral therapy (HAART)-associated lipodystrophy. Fourteen patients with HAART-associated lipodystrophy had 2 subcutaneous biopsies for evidence of adipocyte apoptosis, the second after a randomized change to nevirapine (n=8) or after remaining on a regimen of indinavir-based HAART (n=6). Apoptosis was assessed by the terminal deoxynucleotidyl transferase dUTP-digoxigenin nick end-labeling method. Patients who were switched to nevirapine had a significant decrease in insulinemia and a significant increase in the glucose:insulin ratio. Overall, subcutaneous adipocyte apoptosis increased in 3 patients who were switched to nevirapine and in 3 who continued to receive indinavir but decreased in 2 patients switched to nevirapine and another 2 who continued to receive indinavir. Subcutaneous adipocyte apoptosis continues to occur in lipotrophic areas of patients with HAART-associated lipodystrophy despite switching from indinavir to nevirapine, suggesting that such a strategy will be useless for reversal of lipoatrophy.

Preparation of PEG-grafted immunomagnetoliposomes entrapping citrate stabilized magnetite particles and their application in CD34+ cell sorting.

Immunomagnetic systems have been used for positive selection of a cell fraction from a mixture using appropriate surface markers with satisfactory results, as haematopoietic CD34+ cells. This work reports on the development of poly(ethylene glycol)-grafted (PEG) immunoliposomes loaded with citrate-magnetite stabilized particles as the separation vehicles for immunomagnetic separations. The magnetic ferrofluid was encapsulated into PEG-liposomes by the DRV methodology. The magnetoliposomes had a liposomal size of approximately 450 nm and a Fe/lipid molar ratio of 1.52+/-0.26, and were retained in the magnetic field created by the MiniMACS system. Anti-CD34 immunomagnetoliposomes with 100 mAb/vesicle were prepared by coupling the My10 mAb and bound specifically for CD34+ KG-1a cells in culture and in mixtures with CD34-cells (CHO or Jurkat). The magnetic cell sorting was carried out in cell mixtures KG-1a/CHO or KG-1a/Jurkat with different initial% of CD34+ Kg-1a cells. For 10(6) positive cells and 100 microM of immunomagnetoliposomes, the capture efficiency was > 85% and independent of the starting percentage of CD34+ cells. The decrease of the final purity, when the starting percentage of CD34+ cells decreases and, dependent of the CD34- cell line used, point to the degree of non-specific cell binding of My10-immunomagnetoliposomes as being crucial, among of the methodological aspects as the number of starting CD34+ cells. The CD34+ cells isolated retained the viability with an estimated recovery of 45-50%.

Preparation of immunoliposomes bearing poly(ethylene glycol)-coupled monoclonal antibody linked via a cleavable disulfide bond for ex vivo applications.

Several methods for the preparation of sterically stabilized immunoliposomes (SIL) have recently been described. This report examines an established method for coupling anti-CD34 My10 mAb to poly(ethylene glycol)-liposomes (PEG-liposomes) containing the anchor pyridyldithiopropionylamino-PEG-phosphatidylethanolamine (PDP-PEG-PE) via a cleavable disulfide bond. Efficient attachment of pyridyldithio-derivatized mAb took place (equivalent to coupling ca. 70% of total input protein) at 2 mol percent of the functionalized PEG-lipid. The My10-SIL bound specifically to CD34+ cells (human leukemic KG-1a and hematopoietic progenitor cells) and the extent of binding was a function of liposomal lipid concentration, the mAb density in the liposome surface and the CD34 cell expression. In mixtures with CD34- cells (CHO or Jurkat), CD34+KG-1a cells were determined by flow cytometry at percentages (1-4%) similar to those reported in clinical samples (such as cord blood, mobilized peripheral blood and bone marrow) using a direct immunostaining with My10-SIL. The disulfide bond was stable in cell culture medium (10% of fetal calf serum) during 8 h and cell-bound SIL can be released from cells by treatment with dithiothreitol as reducing agent under mild conditions (1 h of incubation with 50 mM DTT at 20 degrees C). SIL binding and subsequent dithiothreitol treatment did not influence the cell viability. Our approach should contribute to the development of targetable liposomal vehicles to CD34+ cells for use in ex vivo conditions as sorting of hematopoietic stem cells.

A novel strategy affords high-yield coupling of antibody to extremities of liposomal surface-grafted PEG chains.

Several methodologies for the preparation of polyethylene glycol-grafted immunoliposomes have been developed by attaching antibodies to the terminus of the polymer. Unilamellar liposomes were prepared containing a combination of a functionalized polyethylene glycol(3400) and an inert polyethylene glycol(2000) phosphatidylethanolamine derivate up to 5 mol%. The greater length of the functionalized polyethylene glycol derivate did not alter the liposomal sterical stability or the remote loading of doxorubicin. Anti-CD34 immunoliposomes were prepared by the reaction of maleimide-derivatized My10 antibody with generated thiol groups at the periphery of the liposomes and efficiencies of nearly 100% were obtained. The greater accessibility of the reactive group makes this strategy more efficient than others described. The immunoliposomes prepared bound specifically to CD34+ cells.

Preparation of immunoliposomes directed against CD34 antigen as target.

The My-10 monoclonal antibody has facilitated the search of haematopoietic stem cells by recognizing selectively the human CD34 antigen. In the present work, My-10 immunoliposomes directed specifically against CD34+ cells were prepared, characterized and tested in vitro. Binding to target cells at 4 degreesC of immunoliposomes containing carboxyfluorescein as aqueous marker was evaluated by flow cytometry and fluorescence microscopy. These immunoliposomes demonstrated their capacity to bind specifically to CD34+ cells. Studies have shown that 9 antibodies/vesicle were sufficient to obtain a good binding efficiency. The product was stable over one month at 4 degreesC in terms of leakage of encapsulated carboxyfluorescein, particle size and antigen binding capacity.

N-palmitoylphosphatidylethanolamine stabilizes liposomes in the presence of human serum: effect of lipidic composition and system characterization.

Liposomes containing negatively-charged phospholipid, N-palmitoylphosphatidylethanolamine (NPPE) were examined for stability in the presence of human serum, using the release of the entrapped 5,6-carboxyfluorescein as an aqueous marker. Either small unilamellar vesicles (SUV) or large unilamellar vesicles (LUV) were used. Incorporation of NPPE into PC SUV decreases leakage in the presence of serum or phosphate-buffered saline, no strictly related to size increase observed and to the surface negative charge present. The stabilizing effect of NPPE and Chol were synergistic. Inhibition of destabilization induced by serum of PC/Chol liposomes was observed when NPPE concentrations were above 12 mol%. Change in the membrane fluidity or incorporation of a monosialoganglioside into liposomes do not significantly change the half-life of liposomes in the presence of a high NPPE concentration. Incorporation of NPPE into PC/Chol liposomes increases membrane rigidity which does not change after serum incubation. The presence of NPPE in liposomes decreases lipid transfer/exchange between liposomes and lipoproteins although the same amount of serum proteins were incorporated as in PC/Chol liposomes. As expected, these proteins are accessible to trypsin digestion. In accordance with these results, the liposome agglutination assay shows no steric barrier activity. As a whole, the results obtained in this paper suggest a complex mechanism for stabilization of NPPE containing liposomes in human serum.

Role of headgroup structure in the phase behaviour of N-acylethanolamine phospholipids: hydrogen-bonding ability and headgroup size.

The physical properties of aqueous dispersions of N-acylphosphatidylethanolamine from natural origin with long N-acyl chain (NAPE) and headgroup modified analogues have been studied. N-Acylation of PE causes a significant increase in the gel-to-liquid crystalline lamellar phase transition temperature in contrast with saturated N-acyl(dipalmitoyl) PEs, and in addition it does not restrict the headgroup rotational mobility in gel phase. The results agree with the increase of hydration of the phosphate group compared with that in PE and suggest the formation of hydrogen bonds between amide groups. The modifications introduced modulate the headgroup size and their hydrogen bonding capability. An increasing number of methylene groups between the phosphate and amide groups does not modify the phase behaviour observed. N-methylation of the amide group, which prevents the possibility of intermolecular hydrogen bond formation, decreases the melting temperature and the cooperativity of the phase transition and does not change the phase behaviour, while the hydration at the ester carbonyl groups level is decreased. On the other hand, the addition of N-ethyl substituent to the amide group or substitution of an ester group for this group increases its tendency to form structures with inverted geometries. The behaviour of these compounds suggests that hydration forces must be more important than considerations of the lipid dynamic shape in predicting the relative stabilities of lamellar vs. non-lamellar phases for NAPEs with long saturated N-acyl chain.

Incorporation of N-acylethanolamine phospholipids into egg phosphatidylcholine vesicles: characterization and permeability properties of the binary systems.

We have studied the effect of the N-acylphosphatidylethanolamine (N-acylPE) on the permeability properties of liposomes composed primarily of egg phosphatidylcholine using a fluorescent anionic dye, carboxyfluorescein, as model solute. Leakage from liposomes decreased and vesicle size increased with increasing N-acylPE content. In addition, measurement of the trapped aqueous space, using the same dye marker, showed a correlation between trapped volume and vesicle size determined by dynamic light scattering. Permeability parameters were calculated according to the pseudo-first-order analysis. It appears that N-acylPE stabilizes liposomes at least in part through its ability to impart surface negative charge, in accord with the results obtained with potassium chloride as encapsulated solute. These results agreed well with osmotic response of anionic lipid vesicles. Cholesterol stabilizes N-acylPE liposomes in a proportional manner to the molar fraction of the effector.