Intracellular IFN-γ and IL-2 expression monitoring as surrogate markers of the risk of acute rejection and personal drug response in de novo liver transplant recipients.

Biomarker monitoring is needed in transplantation to reflect individual response to immunosuppressive drugs and graft outcome. We evaluated intracellular expression and soluble production of interferon-(IFN)-γ and interleukin-(IL)-2 as predictive biomarkers of acute rejection (AR) and personal drug response. Pharmacokinetic-pharmacodynamic profiles were determined in 47 de novo liver recipients treated with tacrolimus, mycophenolate mofetil and prednisone. Of the 47 patients, AR occurred in nine. There were no differences in drug concentrations between rejectors and non-rejectors. A pre-transplantation cut-off value of 55.80% for %CD8(+)-IFN-γ(+) identified patients at high risk of AR with a sensitivity of 75% and a specificity of 82%. In the first week post-transplantation, patients with a % inhibition for soluble IFN-γ, %CD8(+)-IFN-γ(+) and %CD8(+)-IL2(+) lower than 40% developed AR, showing low susceptibility to immunosuppressive drugs. Therefore, effector-T-cell response monitoring may help physicians to identify personal response to treatment and patients at high risk of AR.

Role of FK778 alone or in combination with tacrolimus or mTOR inhibitors as an immunomodulator of immunofunctions: in vitro evaluation of T cell proliferation and the expression of lymphocyte surface antigens.

We evaluated the in vitro capacity of FK778, alone or in combination with other immunosuppressive drugs: Tacrolimus (TRL); Sirolimus (SRL), Everolimus (EVL), to inhibit clonal expansion of T-lymphocytes and expression of lymphocyte-activation surface antigens; secondly, we compared the immunosuppressive potential of FK778 combined with TRL, SRL and EVL with the same combinations using Mycophenolic acid (MPA) as antimetabolite. Lymphocyte proliferation was assessed by 3H-Thymidine incorporation, in whole blood cultures stimulated with ConA. The effect of FK778 on alloresponse was evaluated by MLC and the expression of lymphocyte surface antigens by cytometry. FK778, TRL, SRL and EVL showed a high in vitro capacity to inhibit lymphocyte proliferation in a concentration-dependent way. Combinations of FK778 with TRL, SRL, or EVL presented an additive effect, especially FK778+TRL. Similar inhibition capacity of the clonal expansion was observed, when FK778 was combined with TRL, SRL or EVL, respecting the same combinations but using MPA instead of FK778. In addition, FK778 inhibited the expression of lymphocyte surface antigens involved in activation, co-stimulatory and apoptosis signals. In conclusion, FK778 inhibits the proliferative response induced by mitogeneic and allogeneic stimuli and the expression of surface antigens. Combinations of FK778 with TRL or mTOR inhibitors presented an additive effect and their action on T cell proliferation was similar to that of combinations with MPA. Since FK778, TRL and mTOR inhibitors present different action mechanisms and involve different cellular targets, these combinations may help prevent episodes of allorejection in organ transplants. FK778 and mTOR inhibitors may represent an alternative treatment for patients with renal failure.

Diabetes mellitus: new therapeutic approaches to treat an old disease

Diabetes mellitus is a widespread disease whose frequency increases constantly and is expected to reach alarming levels by the year 2025. Introduction of insulin therapy represented a major breakthrough; however, a very strict regimen is required to maintain blood glucose levels within the normal range and to prevent or postpone chronic complications associated with this disease. Frequent hyper- and hypoglycemia seriously affect the quality of life of these patients. Reversion of this situation can only be achieved through whole organ (pancreas) transplant or pancreatic islet transplant, the former being a high-risk surgical procedure, while the latter is a much simpler and may be accomplished in only 20-40 min. The advantages and perspectives of islet cell transplantation will be discussed, in the light of tissue engineering and gene therapy. Ongoing research carried out in our laboratory, aimed at developing clinical cell and molecular therapy protocols for diabetes will also be focused

New concepts in cyclosporine pharmacokinetic and dynamic monitoring: the impact of concomitant immunosuppression on target C2 concentrations.

BACKGROUND: There is a correlation between cyclosporine (CsA) pharmacokinetics (PK) and pharmacodynamics (PD), especially 2 hours after drug administration. AIM: To evaluate the relationship between CsA PK and PD profiles in two groups of stable renal transplant patients treated with CsA alone or CsA plus mycophenolate mofetil (CsA+MMF), so as to define the best target for C2 and clarify the impact of concomitant immunosuppression. METHODS: Thirty-eight stable renal transplant recipients were treated with CsA (n=20) or CsA+MMF (n=18). Twelve nontreated normal healthy controls (NHC) were also included. Calcineurin activity (CNa), IL-2 production, and CsA levels were measured at 0 and 2 hours postdose. RESULTS: There were no significant differences in median CsA C2 values and CNa between the CsA alone and the CsA+MMF groups (388 microg/L and 497.5 microg/L and CNa 2h; 3.92% alkaline phosphatase [AP]; 3.94% AP, respectively). In vitro production of IL-2 was significantly lower in the CsA+MMF group than in the CsA group (median IL-2 2h: 280.52 ng/L, 169.48 ng/L, P<.001). The correlations (r) between C2 and CNa 2h were: CsA r=0.74; CsA+MMF r=0.84 (P<.001 in both cases). CONCLUSIONS: In stable renal transplant patients, median CsA C2 values below 500 microg/L were associated with inhibition of CNa and IL-2 synthesis. CNa and IL-2 production may be good biological markers of CsA immunosuppression. The measurement of CNa depends mainly on CsA concentration, whereas in vitro IL-2 production reflects the effect of both CsA and MMF. Further studies are necessary to define the optimal C2 target concentration and the possible impact of concomitant immunosuppression.

Induction of the lipocyte phenotype in murine hepatic stellate cells: reorganisation of the actin cytoskeleton.

Hepatic stellate cells (HSCs) are intralobular connective tissue cells presenting myofibroblast or lipocyte phenotypes. They participate in the homeostasis of liver extracellular matrix, repair, regeneration and fibrosis under the former phenotype, and control retinol metabolism, storage and release under the latter one. Responding to systemic or local demands, they can convert into the required phenotype with deep modifications of their structures. Using immunofluorescence microscopy and Western blots, we investigated the expression and organisation of actin filaments and of two actin-binding proteins, alpha-actinin and tropomyosin, in the cloned GRX cell line representative of murine HSCs. GRX cells expressing the myofibroblast phenotype showed typical well-organised actin stress-fibres, anchored at the focal adhesions located at the cell periphery. Retinol treatment induced active reorganisation of the cytoskeleton. The major stress fibres were reduced in length, and frequently formed a polygonal meshwork. Subsequently, they fragmented and generated diffuse or granular actin in the perinuclear area, a thin continuous layer around lipid droplets and, in fully converted lipocytes, a peripheral layer of thin actin fibres. alpha-Actinin and tropomyosin were present only in lipocytes, co-distributed with actin in a granular form. Since the cytoskeleton reorganisation preceded lipid accumulation, we conclude that the induction of the lipocyte phenotype represents a full reprogramming of cell gene expression and function. We consider that both the lipocyte and the myofibroblast phenotypes should be considered "activated states" of HSCs, each responding to specific physiological or pathological modifications of liver functions.

Intermediate filaments modulation in an in vitro model of the hepatic stellate cell activation or conversion into the lipocyte phenotype.

Hepatic stellate cells are intralobular connective tissue cells expressing the myofibroblast or the lipocyte phenotypes. They participate in homeostasis of the liver extracellular matrix, repair, regeneration, and fibrosis under the former phenotype, and control the retinol metabolism, storage, and release under the latter one. They are heterogeneous in terms of their tissue distribution, function, and expression of cytoskeletal proteins. We have studied the expressions of intermediate filaments in the cloned GRX cell line representative of murine hepatic stellate cells, by immunolabeling, reverse transcription polymerase chain reaction (RT-PCR), immunoprecipitation and Western blots. GRX cells expressed vimentin, desmin, glial fibrillary acidic protein (GFAP), and smooth muscle alpha actin (SM-alphaA). Vimentin, desmin, and SMN-alphaA were expressed in all cultures. GFAP showed a heterogeneous intensity of expression and did not form a filamentous cytoskeletal network, showing a distinct punctuate cytoplasmic distribution. When activated by inflammatory mediators, GRX cells increased expression of desmin and GFAP. Retinol-mediated induction of the lipocyte phenotype elicited a strong decrease of intermediate filament protein expression and the collapse of the filamentous structure of the cytoskeleton. Quiescent hepatic stellate precursors can respond to physiologic or pathologic stimuli, expressing activated myofibroblast or lipocyte phenotypes with distinct patterns of cytoskeleton structure, metabolic function, and interaction with the tissue environment.

Retinol uptake and metabolism, and cellular retinol binding protein expression in an in vitro model of hepatic stellate cells.

Liver is a major site of retinoid metabolism and storage, and more than 80% of the liver retinoids are stored in hepatic stellate cells. These cells represent less than 1% of the total liver protein, reaching a very high relative intracellular retinoid concentration. The plasma level of retinol is maintained close to 2 microM, and hepatic stellate cells have to be able both to uptake or to release retinol depending upon the extracellular retinol status. In view of their paucity in the liver tissue, stellate cells have been studied in primary cultures, in which they loose rapidly the stored lipids and retinol, and convert spontaneously into the activated myofibroblast phenotype, turning a long-term study of their retinol metabolism impossible. We have analyzed the retinol metabolism in the established GRX cell line, representative of stellate cells. We showed that this cell line behaves very similarly, with respect the retinol uptake and release, to primary cultures of hepatic stellate cells. Moreover, we showed that the cellular retinol binding protein (CRBP-I) expression in these cells, relevant for both uptake and esterification of retinol, responds to the extracellular retinol status, and is correlated to the retinol binding capacity of the cytosol. Its expression is not associated with the overall induction of the lipocyte phenotype by other agents. We conclude that the GRX cell line represents an in vitro model of hepatic stellate cells, and responds very efficiently to wide variations of the extracellular retinol status by autonomous controls of its uptake, storage or release.

Complicaciones de la salpingoclasia por minilaparotomía en una población de pacientes / Complications of the salpingoclasia made minilaparatomy in a population of patients

Con el propósito de conocer la incidencia, diagnóstico y pronóstico de las complicaciones de la salpingoclasia por minilaparotomía, en el Hospital Maternidad Nuestra Señora de la Altagracia, Santo Domingo, República Dominicana, se realizó un estudio descriptivo, transversal y retrospectivo, durante el período comprendido entre Febrero 1993 a Febrero 1994, se estudiaron un total de 2,424 pacientes que se sometieron al procedimiento de salpingoclasia, de las cuales 64 tuvieron complicaciones para un 2.70//: la edad promedio fue entre los 25-34 años. La complicación principal fue la infección del área quirúrgica (61//); de las pacientes 17 requirieron manejo quirúrgico 47 de ellas manejo clínico. De todos los casos, sólo tuvimos uno con pronóstico reservado, los restantes un buen pronóstico. Se determina que la salpingloclasia por minilaparotomia es un procedimiento con baja incidencia de morbilidad

[Current assessments of electroconvulsive therapy]. / Estimari actuale privind terapia electroconvulsivanta.

As the therapeutical effects of the current psychopharmacotherapy did not live up the expectations, in the last 10-15 years a tendency towards up-to-dating some older therapeutical methods, restrained for various reasons, was noticed. The up-to-dated electroconvulsive therapy proves to be highly efficient in some major mental affections. This paper is intended as a review of the literature and seems to reveal some advantages of the electroconvulsive therapy as compared to some chemotherapeutic agents, but only if used in precisely restricted specific conditions.

[The dynamics of ethanol metabolism in patients with chronic alcoholism]. / Dinamica metabolizarii etanolului, referinte cazuistice la bolnavii cu etilism cronic.

The results of the investigations carried out in alcoholics and nonalcoholics regarding certain aspects of ethanol metabolism are presented. The marked interindividual differences in alcohol blood levels and intergroup differences in acetaldehyde levels, through a projection in dynamic process, are commented upon. The programmed ethanolic load induced changes in blood alcohol, within comparable limits, irrespective of subjects' state. At all intervals of postintake determinations, the acetaldehyde concentrations were higher in the alcoholic subjects. No dose-effect relation between the involved elements (ethanol metabolite) was found.