Estudio clínico comparativo de 2 técnicas quirúrgicas de rizartrosis del pulgar / Comparative clinical study of 2 surgical techniques for trapeziometacarpal osteoarthritis

Objetivo. En la rizartrosis del pulgar existe gran controversia sobre la técnica quirúrgica a elegir: trapeciectomía simple, artroplastia de resección-interposición, artroplastia de resección-suspensión, artrodesis, artroplastia de suspensión-resección-interposición o artroplastia con prótesis. Estas 2 últimas son las más empleadas, sin consenso en la literatura sobre la técnica a elegir y sin suficientes estudios comparativos. El objetivo es comparar las 2 técnicas más empleadas en la actualidad: artroplastia de resección-suspensión y artroplastia con prótesis. Material y método. Presentamos un estudio prospectivo de 15 pacientes diagnosticados de rizartrosis del pulgar grados ii-iii tratados con artroplastia de resección-suspensión (grupo 1) y 15 con prótesis (grupo 2) mostrando resultados clínicos, ventajas e inconvenientes de cada una. Como variables se emplearon la escala EVA, el cuestionario DASH, la fuerza de puño y de pinza terminoterminal y terminolateral; el balance articular en aducción-abducción, en anteposición-retroposición y la oposición. Los 2 grupos son de 2 hospitales diferentes, intervenidos por un cirujano de mano de la unidad. El tiempo de seguimiento para todos los pacientes incluidos fue de 12 meses. Resultados. El EVA, DASH y fuerza de puño a los 12 meses no muestran diferencias significativas; en cuanto a la fuerza de pinza terminoterminal y terminolateral, el grupo 2 mostró los mayores valores en todos los periodos de seguimiento, con diferencias estadísticamente significativas. Conclusiones. Es fundamental la selección del paciente y la experiencia del cirujano, dados los resultados satisfactorios de ambas técnicas. La artroplastia con prótesis se reserva para grados ii y iii, pacientes de mediana edad, buena arquitectura del trapecio y cirujanos con experiencia (AU)

Objective. In trapeziometacarpal osteoarthritis (or rhizarthrosis), there is great controversy over the surgical technique to choose: simple trapeziectomy, resection-interposition arthroplasty, interposition arthroplasty suspension-or arthroplasty with implant or prosthesis. These latter 2 are the most used without consensus in the literature on the technique to choose and without sufficient comparative studies. The objective is to compare the 2 techniques most used today: suspension-interposition arthroplasty and arthroplasty with prosthesis. Material and method. A prospective study was conducted on 15 patients diagnosed with grade 2-3 rhizarthrosis treated with interposition arthroplasty-suspension (group 1) and 15 with prosthesis (group 2) showing clinical outcomes, advantages and disadvantages of each. The study variables were the visual analogue scale (VAS), the DASH questionnaire, the grip strength, the strength of end to end and end-lateral clamp, the joint balance adduction-abduction and preemption-retropositioning, and the opposition. The 2 groups are from 2 different hospitals operated on by a hand surgeon from the Hand Unit. The follow-up time for all patients included in the study was 12 months. Results. The VAS, DASH and grip strength at 12 months did not show significant differences. As regards the strength of end to end and end-lateral clamp, group 2 showed the highest values in all follow-up periods with statistically significant differences. Conclusions. Patient selection and surgical experience is essential, given the satisfactory results of both techniques. Arthroplasty prosthesis is reserved for grades 2 and 3, middle-aged patients, good trapezium architecture, and experienced surgeons (AU)

[Comparative clinical study of 2 surgical techniques for trapeziometacarpal osteoarthritis]. / Estudio clínico comparativo de 2 técnicas quirúrgicas de rizartrosis del pulgar.

OBJECTIVE: In trapeziometacarpal osteoarthritis (or rhizarthrosis), there is great controversy over the surgical technique to choose: simple trapeziectomy, resection-interposition arthroplasty, interposition arthroplasty suspension-or arthroplasty with implant or prosthesis. These latter 2 are the most used without consensus in the literature on the technique to choose and without sufficient comparative studies. The objective is to compare the 2 techniques most used today: suspension-interposition arthroplasty and arthroplasty with prosthesis. MATERIAL AND METHOD: A prospective study was conducted on 15 patients diagnosed with grade 2-3 rhizarthrosis treated with interposition arthroplasty-suspension (group 1) and 15 with prosthesis (group 2) showing clinical outcomes, advantages and disadvantages of each. The study variables were the visual analogue scale (VAS), the DASH questionnaire, the grip strength, the strength of end to end and end-lateral clamp, the joint balance adduction-abduction and preemption-retropositioning, and the opposition. The 2 groups are from 2 different hospitals operated on by a hand surgeon from the Hand Unit. The follow-up time for all patients included in the study was 12 months. RESULTS: The VAS, DASH and grip strength at 12 months did not show significant differences. As regards the strength of end to end and end-lateral clamp, group 2 showed the highest values in all follow-up periods with statistically significant differences. CONCLUSIONS: Patient selection and surgical experience is essential, given the satisfactory results of both techniques. Arthroplasty prosthesis is reserved for grades 2 and 3, middle-aged patients, good trapezium architecture, and experienced surgeons.

Fractura-luxación transemilunar / Translunate fracture-dislocation

Presentamos el caso de un varón deportista de profesión, que tras un traumatismo durante su práctica deportiva habitual sufre una fractura-luxación transestiloides radial-transemilunar, lesión que es poco común y que escapa de los patrones típicos. Tras reducción cerrada de urgencia fue intervenido quirúrgicamente realizando osteosíntesis del semilunar y reparación del ligamento escafolunar, con recuperación satisfactoria. Las fracturas-luxaciones carpianas son lesiones severas que pueden asociarse a múltiples patrones de lesiones ligamentosas y óseas. Bain añade el arco translunar(fractura del semilunar) para usarlo como complemento al modelo de clasificación de inestabilidades perilunares de Johnson de arco mayor-arco menor. Este tipo de lesión translunar no sigue el esquema descrito por Mayfield, aunque sí es una combinación de este concepto con una fractura del semilunar. En el caso que presentamos se produjo una afectación de los 3 arcos: fractura de la estiloides radial (arcomayor), fractura del semilunar (arco translunar) y lesión de los ligamentos carpianos (arco menor); esto aún no se ha estudiado biomecánicamente (AU)

We present the case of a 26-year-old male sportsman by profession, who suffers a radial transstyloid-translunatefracture-dislocation after a trauma during a sport practice. This is an uncommon injury that escapes from the typical injury patterns. After an urgent closed reduction, the patient undergoes surgery by osteosynthesis of the lunate fracture and scapholunate ligament repair, with a successful recovery. Carpal fracture-dislocations are severe injuries that may be associated with multiple patterns of ligamentous and bone injuries. Bain adds the translunate arc (lunatefracture) as a complement to the greater arc-lesser arc classification model of perilunate inestabilities from Johnson. This mechanism of translunate injury does not follow the pattern described by Mayfield, although it is a combination of such concept and the lunate fracture. In our clinical case, there was an involvement of the3 arches: radial styloid fracture (greater arc), lunate fracture(translunate arc) and carpal ligaments fracture (lesserarc); this has not yet been studied biomechanically (AU)

Critical role of the death receptor pathway in the antitumoral effects induced by hispanolone derivatives.

Labdane diterpenoids have a broad spectrum of biological activities including antibacterial, antiviral and anti-inflammatory properties. However, little is known about their possible role in the apoptotic cell death machinery. Here, we report that hispanolone derivatives, a group of labdane diterpenoids, induce apoptosis in different tumor cell lines by activating caspase-8 with subsequent participation of mitochondrial signaling. Activation of caspase-8 by hispanolone derivatives was followed by a decrease in mitochondrial membrane potential, the release of apoptotic factors from mitochondria to the cytosol, and activation of caspases-9 and 3. Hispanolone derivatives also led to a time-dependent cleavage of Bid. Inhibition of caspase-8 abrogated these processes, suggesting that the death receptor pathway has a critical role in the apoptotic events induced by hispanolone derivatives. In addition, silencing death receptors with small interfering RNA s or pretreating cells with neutralizing antibodies to Fas ligand, tumor necrosis factor receptor 1 (TNF-R1), and TNF-α receptor 2 (TRAIL) inhibited diterpenoid-induced apoptosis, revealing it to be dependent on these death receptors. Interestingly, hispanolone derivatives had no effect on non-tumor cells. Consistently, in vivo bioluminescence imaging corroborates this antineoplasic effect, as hispanolone derivatives significantly decrease cancer growth in tumor xenograft assays. These data demostrate the antitumoral effects of hispanolone derivatives and provide relevant preclinical validation for the use of these compounds as potent therapeutic agents in cancer treatment.

Kaurane diterpenes protect against apoptosis and inhibition of phagocytosis in activated macrophages.

BACKGROUND AND PURPOSE: The kaurane diterpenes foliol and linearol are inhibitors of the activation of nuclear factor kappaB, a transcription factor involved in the inflammatory response. Effects of these diterpenes on apoptosis and phagocytosis have been analysed in cultured peritoneal macrophages and in the mouse macrophage cell line, RAW 264.7. EXPERIMENTAL APPROACH: Macrophages were maintained in culture and activated with pro-inflammatory stimuli in the absence or presence of diterpenes. Apoptosis and the phagocytosis in these cells under these conditions were determined. KEY RESULTS: Incubation of macrophages with a mixture of bacterial lipopolysaccharide (LPS)/interferon-gamma (IFN-gamma) induced apoptosis through a NO-dependent pathway, an effect significantly inhibited by foliol and linearol in the low muM range, without cytotoxic effects. Apoptosis in macrophages induced by NO donors was also inhibited. The diterpenes prevented apoptosis through a mechanism compatible with the inhibition of caspase-3 activation, release of cytochrome c to the cytosol and p53 overexpression, as well as an alteration in the levels of proteins of the Bcl-2 family, in particular, the levels of Bax. Cleavage of poly(ADP-ribose) polymerase, a well-established caspase substrate, was reduced by these diterpenes. Treatment of cells with foliol and linearol decreased phagocytosis of zymosan bioparticles by RAW 264.7 cells and to a greater extent by peritoneal macrophages. CONCLUSIONS AND IMPLICATIONS: Both diterpenes protected macrophages from apoptosis and inhibited phagocytosis, resulting in a paradoxical control of macrophage function, as viability was prolonged but inflammatory and phagocytic functions were impaired.

Ammonia prevents glutamate-induced but not low K(+)-induced apoptosis in cerebellar neurons in culture.

Cultured rat cerebellar granule neurons are widely used as a model system for studying neuronal apoptosis. Either low K(+) (5 mM) or low concentrations of glutamate (1-10 microM) induce apoptosis in cerebellar neurons in culture. However, the molecular mechanism(s) involved remain unclear. We show that long-term treatment with ammonia prevents glutamate-induced but not low K(+)-induced apoptosis in cerebellar neurons, as assessed by measuring DNA fragmentation and activation of caspase 3. Ammonia prevented glutamate-induced increase of intracellular calcium, depolarization of the inner mitochondrial membrane, release of cytochrome c to the cytosol, activation of caspase 3 and fragmentation of DNA. However, ammonia did not prevent low K(+)-induced activation of caspase 3 and fragmentation of DNA. These results indicate that the initial steps involved in the induction of apoptosis by low K(+) or by glutamate are different and that ammonia prevents glutamate-induced apoptosis by reducing glutamate-induced rise of intracellular Ca(2+), thus avoiding the activation of subsequent events of the apoptotic process.

Induction of apoptosis by nitric oxide in macrophages is independent of apoptotic volume decrease.

Apoptosis occurs through a sequence of specific biochemical and morphological alterations that define the progress of cell death. The changes of the mitochondrial inner membrane potential (DeltaPsi(m)), the release of cytochrome c to the cytosol, the apoptotic volume decrease (AVD) and the activation of caspases have been measured in RAW 264.7, HeLa and Jurkat T cells incubated with molecules that induce apoptosis through the mitochondrial pathway. Our data show that NO, staurosporine, etoposide and camptothecin increased DeltaPsi(m) in macrophages but not in HeLa and Jurkat cells, that exhibited a DeltaPsi(m) decrease. Moreover, the apoptosis induced by NO in macrophages, but not that promoted by staurosporine, might occur in the absence of AVD. Analysis of the sequence of apoptotic manifestations shows that DeltaPsi(m) precedes AVD and caspase activation in RAW 264.7 cells. Inhibition of AVD abrogates apoptosis in HeLa and Jurkat T cells regardless of the stimuli used. These data suggest that the changes of DeltaPsi(m) are cell-type dependent and that AVD is dispensable for apoptosis in macrophages.

Intracellular water motion decreases in apoptotic macrophages after caspase activation.

Triggering of the macrophage cell line RAW 264.7 with lipopolysaccharide and interferon-gamma promoted apoptosis that was prevented by inhibitors of type 2 nitric oxide synthase or caspase. Using (1)H NMR analysis, we have investigated the changes of the intracellular transverse relaxation time (T(2)) and apparent diffusion coefficient (ADC) as parameters reflecting the rotational and translational motions of water in apoptotic macrophages. T(2) values decreased significantly from 287 to 182 ms in cells treated for 18 h with NO-donors. These changes of T(2) were prevented by caspase inhibitors and were not due to mitochondrial depolarization or microtubule depolymerization. The decrease of the intracellular values of T(2) and ADC in apoptotic macrophages was observed after caspase activation, but preceded phosphatidylserine exposure and nucleosomal DNA cleavage. The changes of water motion were accompanied by an enhancement of the hydrophobic properties of the intracellular milieu, as detected by fluorescent probes. These results indicate the occurrence of an alteration in the physicochemical properties of intracellular water during the course of apoptosis.

Peroxisome proliferator-activated receptor-gamma-independent inhibition of macrophage activation by the non-thiazolidinedione agonist L-796,449. Comparison with the effects of 15-deoxy-delta(12,14)-prostaglandin J(2).

The effects of L-796,449 (3-chloro-4-(3-(3-phenyl-7-propylbenzofuran-6-yloxy)propylthio)phenylacetic acid; referred to henceforth as compound G), a thiazolidinedione-unrelated peroxisome proliferator activated-receptor-gamma (PPAR-gamma) agonist, on early signaling in lipopolysaccharide-activated RAW 264.7 macrophages were analyzed and compared with those elicited by 15-deoxy-Delta(12,14)-prostaglandin J(2) and the thiazolidinedione rosiglitazone. Compound G inhibited the activation of nuclear factor kappa B through the impairment of the targeting and degradation of I kappa B proteins and promoted a redistribution of I kappa B alpha and I kappa B beta in the nucleus of activated cells. Compound G inhibited I kappa B kinase (IKK) activity both in vivo and in vitro, suggesting a direct mechanism of interaction between this molecule and the IKK complex. The effect of compound G on IKK activity was independent of PPAR-gamma engagement because RAW 264.7 cells expressed negligible levels of this nuclear receptor, and rosiglitazone failed to mimic these actions. Moreover, treatment of activated macrophages with compound G enhanced the synthesis of superoxide anion, which, in combination with the NO produced under activation conditions, triggered apoptosis through the intracellular synthesis of peroxynitrite. These results suggest that compound G might contribute to the resolution of inflammation by favoring the induction of apoptosis through mechanisms independent of PPAR-gamma engagement.

Inhibition of the nuclear factor kappa B (NF-kappa B) pathway by tetracyclic kaurene diterpenes in macrophages. Specific effects on NF-kappa B-inducing kinase activity and on the coordinate activation of ERK and p38 MAPK.

The anti-inflammatory action of most terpenes has been explained in terms of the inhibition of nuclear factor kappaB (NF-kappaB) activity. Ent-kaurene diterpenes are intermediates of the synthesis of gibberellins and inhibit the expression of NO synthase-2 and the release of tumor necrosis factor-alpha in J774 macrophages challenged with lipopolysaccharide. These diterpenes inhibit NF-kappaB and IkappaB kinase (IKK) activation in vivo but failed to affect in vitro the function of NF-kappaB, the phosphorylation and targeting of IkappaBalpha, and the activity of IKK-2. Transient expression of NF-kappaB-inducing kinase (NIK) activated the IKK complex and NF-kappaB, a process that was inhibited by kaurenes, indicating that the inhibition of NIK was one of the targets of these diterpenes. These results show that kaurenes impair the inflammatory signaling by inhibiting NIK, a member of the MAPK kinase superfamily that interacts with tumor necrosis factor receptor-associated factors, and mediate the activation of NF-kappaB by these receptors. Moreover, kaurenes delayed the phosphorylation of p38, ERK1, and ERK2 MAPKs, but not that of JNK, in response to lipopolysaccharide treatment of J774 cells. The absence of a coordinate activation of MAPK and IKK might contribute to a deficient activation of NF-kappaB that is involved in the anti-inflammatory activity of these molecules.

Protection by nitric oxide against liver inflammatory injury in animals carrying a nitric oxide synthase-2 transgene.

The effect of pre-existent hepatic NO synthesis on liver injury induced by lipopolysaccharide was studied in animals carrying a nitric oxide synthase-2 (NOS-2) transgene under the control of the phosphoenolpyruvate carboxykinase (PEPCK) promoter. These animals expressed NOS-2 in liver cells under fasting conditions. Lipopolysaccharide-induced liver injury in D-galactosamine-conditioned mice, which enhanced notably the effect of the endotoxin on the liver, was impaired in animals expressing NOS-2. This protection against inflammatory liver damage was dependent on NO synthesis and was caused by an inhibition of nuclear factor kB (NF-kB) activity and an impairment of the synthesis of the proinflammatory cytokines tumor necrosis factor a and interleukin 1b. These data indicate that intrahepatic synthesis of NO protects liver by inhibiting the release of cascades of proinflammatory mediators and suggest a beneficial role for local delivery of NO in the control of liver injury.

Contribution of cyclopentenone prostaglandins to the resolution of inflammation through the potentiation of apoptosis in activated macrophages.

Activation of the macrophage cell line RAW 264.7 with LPS and IFN-gamma induces apoptosis through the synthesis of high concentrations of NO due to the expression of NO synthase-2. In addition to NO, activated macrophages release other molecules involved in the inflammatory response, such as reactive oxygen intermediates and PGs. Treatment of macrophages with cyclopentenone PGs, which are synthesized late in the inflammatory onset, exerted a negative regulation on cell activation by impairing the expression of genes involved in host defense, among them NO synthase-2. However, despite the attenuation of NO synthesis, the percentage of apoptotic cells increased with respect to activated cells in the absence of cyclopentenone PGs. Analysis of the mechanisms by which these PGs enhanced apoptosis suggested a potentiation of superoxide anion synthesis that reacted with NO, leading to the formation of higher concentrations of peroxynitrite, a more reactive and proapoptotic molecule than the precursors. The effect of the cyclopentenone 15-deoxy-Delta(12,14)-PGJ(2) on superoxide synthesis was dependent on p38 mitogen-activated protein kinase activity, but was independent of the interaction with peroxisomal proliferator-activated receptor gamma. The potentiation of apoptosis induced by cyclopentenone PGs involved an increase in the release of cytochrome c from the mitochondria to the cytosol and in the nitration of this protein. These results suggest a role for cyclopentenone PGs in the resolution of inflammation by inducing apoptosis of activated cells.

Anti-inflammatory action of type I interferons deduced from mice expressing interferon beta.

Type I interferons (IFN) are widely used for the therapeutic treatment of viral infections, tumor growth and various chronic diseases such as multiple sclerosis. Antagonism between type I IFNs and IFN-gamma has been described in cells of the immune system, in particular in the activation of macrophages. To study the systemic effects of type I IFNs we used transgenic mice carrying a human IFN-beta (hIFN-beta) gene under the control of the rat insulin I promoter. These animals expressed high levels of hIFN-beta in beta-pancreatic cells, and the ability of the macrophages to respond to pro-inflammatory stimuli was analyzed. Transgenic mice exhibited an increased extravasation of cells to the peritoneal cavity after eliciting with thioglycollate broth. The expression of the inducible form of nitric oxide synthase and cyclooxygenase-2, two enzymes involved in inflammation, was impaired in transgenic animals challenged with lipopolysaccharide and IFN-gamma. Analysis of the mechanisms leading to this attenuated inflammatory response showed a decrease in the serum levels of TNF-alpha and an inhibition of the activation of the transcription factor NF-KB in various tissues. These results indicate that systemic administration of IFN-beta might influence the response to pro-inflammatory stimuli, in particular through the antagonism of IFN-gamma signaling.

Inhibition of IkappaB kinase and IkappaB phosphorylation by 15-deoxy-Delta(12,14)-prostaglandin J(2) in activated murine macrophages.

Activation of the macrophage cell line RAW 264.7 with lipopolysaccharide (LPS) and gamma interferon (IFN-gamma) induces the expression of gene products involved in host defense, among them type 2 nitric oxide synthase. Treatment of cells with 15-deoxy-Delta(12,14)-prostaglandin J(2) (15dPGJ(2)) inhibited the LPS- and IFN-gamma-dependent synthesis of NO, a process that was not antagonized by similar concentrations of prostaglandin J(2), prostaglandin E(2), or rosiglitazone, a peroxisomal proliferator-activated receptor gamma ligand. Incubation of activated macrophages with 15dPGJ(2) inhibited the degradation of IkappaBalpha and IkappaBbeta and increased their levels in the nuclei. NF-kappaB activity, as well as the transcription of NF-kappaB-dependent genes, such as those encoding type 2 nitric oxide synthase and cyclooxygenase 2, was impaired under these conditions. Analysis of the steps leading to IkappaB phosphorylation showed an inhibition of IkappaB kinase by 15dPGJ(2) in cells treated with LPS and IFN-gamma, resulting in an impaired phosphorylation of IkappaBalpha, at least in the serine 32 residue required for targeting and degradation of this protein. Incubation of partially purified activated IkappaB kinase with 2 microM 15dPGJ(2) reduced by 83% the phosphorylation in serine 32 of IkappaBalpha, suggesting that this prostaglandin exerts direct inhibitory effects on the activity of the IkappaB kinase complex. These results show rapid actions of 15dPGJ(2), independent of peroxisomal proliferator receptor gamma activation, in macrophages challenged with low doses of LPS and IFN-gamma.

Nitric oxide induces tyrosine nitration and release of cytochrome c preceding an increase of mitochondrial transmembrane potential in macrophages.

Treatment of elicited peritoneal macrophages or the macrophage cell line RAW 264.7 with high concentrations of nitric oxide donors is followed by apoptotic cell death. Analysis of the changes in the mitochondrial transmembrane potential (DeltaPsi(m)) with specific fluorescent probes showed a rapid and persistent increase of DeltaPsi(m), a potential that usually decreases in cells undergoing apoptosis through mitochondrial-dependent mechanisms. Using confocal microscopy, the release of cytochrome c from the mitochondria to the cytosol was characterized as an early event preceding the rise of DeltaPsi(m). The cytochrome c from cells treated with nitric oxide donors was modified chemically, probably through the formation of nitrotyrosine residues, suggesting the synthesis of peroxynitrite in the mitochondria. These results indicate that nitric oxide-dependent apoptosis in macrophages occurs in the presence of a sustained increase of DeltaPsi(m), and that the chemical modification and release of cytochrome c from the mitochondria precede the changes of DeltaPsi(m).-Hortelano, S., Alvarez, A. M., Boscá, L. Nitric oxide induces tyrosine nitration and release of cytochrome c preceding an increase of mitochondrial transmembrane potential in macrophages.

Mechanisms of nitric oxide-dependent apoptosis: involvement of mitochondrial mediators.

Programmed cell death occurs in several physiopathological situations in multicellular organisms and constitutes a common mechanism of cell replacement, tissue remodelling and removal of altered cells. The effectors that induce apoptosis as well as the signalling pathways involved in the process are the subjects of current work. In addition to receptor-mediated apoptosis, highly reactive molecules, such as NO, influence cell viability either by acting as a protection against apoptogenic stimuli, or by inducing apoptosis when produced at elevated concentrations. The contribution to apoptosis of mediators released by the mitochondria and involved in the activation of caspases focused attention on the functional changes caused by NO in this organelle. NO induces mitochondrial permeability transition and promotes apoptosis in cell-free systems containing mitochondria and nuclei. Moreover, NO-dependent apoptosis can be blocked in most cases through the use of permeability transition or caspase inhibitors. The intracellular pathways activated in response to NO challenge and involved in the regulation of apoptosis are analysed.

Protective effect of cyclosporin A and FK506 from nitric oxide-dependent apoptosis in activated macrophages.

1. Activation of macrophages with lipopolysaccharide (LPS) and low doses of interferon-gamma (IFN-gamma) induced apoptotic death through a nitric oxide-dependent pathway. 2. Treatment of cells with the immunosuppressors cyclosporin A (CsA) or FK506 inhibited the activation-dependent apoptosis. 3. These drugs decreased the up-regulation of p53 and Bax characteristic of activated macrophages. Moreover, incubation of activated macrophages with CsA and FK506 contributed to maintain higher levels of Bcl-2 than in LPS/IFN-gamma treated cells. 4. The inhibition of apoptosis exerted by CsA and FK506 in macrophages was also observed when cell death was induced by treatment with chemical nitric oxide donors. 5. Incubation of macrophages with LPS/IFN-gamma barely affected caspase-1 but promoted an important activation of caspase-3. Both CsA and FK506 inhibited pathways leading to caspase-3 activation. Moreover, the cleavage of poly(ADP-ribose) polymerase, a well established caspase substrate, was reduced by these immunosuppressive drugs. 6. CsA and FK506 reduced the release of cytochrome c to the cytosol and the activation of caspase-3 in cells treated with nitric oxide donors. 7. These results indicate that CsA and FK506 protect macrophages from nitric oxide-dependent apoptosis and suggest a contribution of the macrophage to innate immunity under conditions of immunosuppression of the host.

Interferon-alpha/beta inhibits the apoptosis induced by lipopolysaccharide and interferon-gamma in murine peritoneal macrophages.

Challenge of elicited peritoneal macrophages with lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) was followed by an apoptotic response. These cells expressed cytokine-inducible nitric oxide synthase (iNOS) in response to these stimuli, and the NO released contributed markedly to the apoptotic death, as deduced from the increased viability observed when iNOS activity was inhibited. The antiviral type I IFN (IFN-alpha/beta) down-regulated the high levels of NO produced when cells were stimulated with suboptimal doses of LPS and IFN-gamma. Moreover, IFN-alpha/beta also decreased cell death in LPS/IFN-gamma-activated cells, as determined by the reduction in the content of oligonucleosomal DNA fragments, in the binding of annexin V to the plasma membrane, and in the amount of hypodiploid cells when analyzed by flow cytometry after in vivo staining with propidium iodide. Kinetic analysis of the protection exerted by IFN-alpha/beta) against the apoptosis induced by treatment with LPS and IFN-gamma showed that type I IFNs were very effective when added up to 1 h after IFN-gamma/LPS stimulation. Addition of IFN-alpha/beta 4 h after stimulation with IFN-gamma/LPS failed completely to prevent apoptosis. This inhibition of apoptosis elicited by IFN-alpha/beta suggests the existence of a mechanism intended to improve macrophage viability in the course of certain viral infections.

Suppression of HIV-1 infection in linomide-treated SCID-hu-PBL mice.

BACKGROUND: Proinflammatory cytokine overproduction, as well as synthesis of the inducible form of nitric oxide synthase (iNOS), are known to play a major role in HIV-1-triggered disease. AIDS patients show increased serum tumour necrosis factor (TNF)-alpha and interferon (IFN)-gamma levels, which synergize with HIV-1-produced nitric oxide (NO) to augment viral replication. Linomide has strong immunomodulatory effects in animals and humans, yielding promising clinical benefits in several pathological disorders including septic shock and autoimmune disease, processes largely mediated by overproduction of these cytokines. In peripheral T cells, linomide also prevents apoptosis triggered by a variety of stimuli, including superantigens, dexamethasone and vaccinia virus. DESIGN AND METHODS: Linomide inhibits production of proinflammatory cytokines such as TNF-alpha, interleukin-1 beta and IFN-gamma, as well as iNOS synthesis. The SCID-hu-PBL mouse model was used to analyse the effect of linomide on HIV-1 infection. T-cell frequency was characterized in reconstituted animals, and the frequency of infected mice and viral load of infected animals were studied. RESULTS: Linomide promotes an increase in human CD4+ T-cell counts in the peritoneal cavity of HIV-1-infected, linomide-treated mice. Linomide also prevents human TNF-alpha and IFN-gamma production, as well as iNOS expression and affects the viral load, promoting potent suppression of HIV-1 infectivity as detected in peritoneal cavity and spleen. CONCLUSIONS: The combination of linomide's properties, namely, blockage of proinflammatory cytokine and NO production, as well as prevention of apoptosis, is of paramount interest, making linomide a potential candidate for combating HIV-1 infection or preventing some of its associated pathological manifestations.

Triggering of peritoneal macrophages with IFN-alpha/beta attenuates the expression of inducible nitric oxide synthase through a decrease in NF-kappaB activation.

Triggering peritoneal macrophages with IFN-gamma and a low concentration of LPS induced the expression of the inducible form of nitric oxide synthase (iNOS). This process was significantly inhibited when IFN-alpha/beta was added during the initial 2 h after the start of IFN-gamma/LPS activation. Evaluation of the transcriptional activity using run-on assays indicated that IFN-alpha/beta inhibited the transcription of iNOS. Transfection experiments using a 1.7-kb promoter sequence corresponding to the 5' flanking region of the murine iNOS gene showed decreased promoter activity in the presence of type I IFNs. Analysis of the transcription factors that participate in iNOS expression revealed a marked decrease of NF-kappaB activation, a nuclear factor required for the transcription of this gene. The degradation of IkappaB alpha and IkappaB beta, which is required for the translocation of NF-kappaB to the nucleus, was inhibited in the presence of IFN-alpha/beta. However, the activity of other transcription factors such as IFN regulatory factor 1, which is involved in the expression of iNOS in response to IFN-gamma, was not affected by IFN-alpha/beta stimulation. These results suggest that in the presence of IFN-alpha/beta, the activity of the iNOS promoter is impaired, and this attenuated nitric oxide synthase expression could be important in pathophysiologic situations in which secretion of type I IFNs occurs.