T cell receptor (TCR) engagement in apoptosis-defective, but interleukin 2 (IL-2)-producing, T cells results in impaired ZAP70/CD3-zeta association.

We have previously shown that a tyrosine to leucine replacement in the transmembrane region of T cell receptor (TCR)-beta results in a deficient induction of CD95-L and apoptosis upon TCR triggering in a transfected T cell line. By contrast, interleukin (IL)-2 production and the expression of CD25 and CD69 were normally induced. Since the mutation in TCR-beta also resulted in impaired association of CD3-zeta, it was proposed that this chain is specifically required for the induction of apoptosis. We now show that the deficient induction of CD95-L and apoptosis does not derive from a general lower production of second messengers, since intracellular Ca2+ fluxes and tyrosine phosphorylation of total proteins were elicited at wild-type levels. Unlike in T cell clones stimulated with partial agonists, both p21 and p18 forms of tyrosine-phosphorylated CD3-zeta were detected, although the overall level of tyrosine-phosphorylated CD3-zeta was low. More strikingly, inducible association of ZAP70 to CD3-zeta was strongly inhibited, despite a normal induction of ZAP70 tyrosine phosphorylation. Finally, ZAP70 was not concentrated near the plasma membrane in the apoptosis-deficient cells. These results suggest that CD3-zeta is necessary for engagement of a specific signaling pathway leading to CD95-L expression that also needs the recruitment of ZAP70.

Expression and function of a variant T cell receptor complex lacking CD3-gamma.

A T cell line termed DIL2 has been derived from an infant with a polyclonal T cell receptor (TCR)/CD3 cell surface expression defect. Indirect immunofluorescence showed that the expression of certain TCR/CD3 epitopes (like those detected by WT31 and BMA031 monoclonals) was strongly reduced (around five-fold) on DIL2, whereas other epitopes (like those detected by SP34 and Leu4) were only around two-fold lower than in normal T cell lines. Specific immunoprecipitates of surface-radioiodinated DIL2 cells contained TCR-alpha, TCR-beta, CD3-delta, CD3-epsilon and TCR-zeta chains, but lacked CD3-gamma. This structural TCR/CD3 variant was, however, capable of transducing certain activation signals, since normal proliferation and a low but significant calcium flux was observed in DIL2 cells after engagement with specific antibodies. Our data suggest that a functional TCR/CD3 complex can be expressed on the surface of T cells in the absence of CD3-gamma.

A window of opportunity for cooperativity in the T Cell Receptor.

The T-cell antigen receptor (TCR) is pre-organised in oligomers, known as nanoclusters. Nanoclusters could provide a framework for inter-TCR cooperativity upon peptide antigen-major histocompatibility complex (pMHC) binding. Here we have used soluble pMHC oligomers in search for cooperativity effects along the plasma membrane plane. We find that initial binding events favour subsequent pMHC binding to additional TCRs, during a narrow temporal window. This behaviour can be explained by a 3-state model of TCR transition from Resting to Active, to a final Inhibited state. By disrupting nanoclusters and hampering the Active conformation, we show that TCR cooperativity is consistent with TCR nanoclusters adopting the Active state in a coordinated manner. Preferential binding of pMHC to the Active TCR at the immunological synapse suggests that there is a transient time frame for signal amplification in the TCR, allowing the T cells to keep track of antigen quantity and binding time.

T lymphocyte receptor deficiencies.

Signalling through the TCR is mediated by the cytoplasmic tails of the CD3 complex. Deficiencies in the expression of different CD3 components have lead to dramatic, yet dissimilar, effects on T-cell development and to selective deficits in peripheral T-cell subsets. Recent studies of human patients and animal models with CD3 deficiencies are providing insights into the redundant and unique roles of these molecules.

Internalization and intracellular fate of TCR-CD3 complexes.

The number of surface TCR-CD3 complexes is maintained by an equilibrium between the synthesis and secretion of new polypeptides, their internalization, recycling, and degradation. The different subunits of the TCR-CD3 complex do not display the same intracellular trafficking dynamics. Thus, in the absence of stimuli, TCR and zeta chains may be degraded at a higher rate than CD3 subunits, which are mostly recycled. T-cell activation by antigen, anti-TCR-CD3 antibodies, or pharmacological activators of protein kinase C, results in increased TCR-CD3 internalization, followed by the downmodulation of TCR-CD3 surface levels. Once internalized, TCR-CD3 complexes may either enter a recycling pathway or be sorted to lysosomes and degraded. Protein serine kinases and protein tyrosine kinases may influence the internalization and intracellular sorting of TCR-CD3 complexes. In line with these results TCR-CD3 ligands stimulate both TCR-CD3 internalization and degradation, whereas protein kinase C activators stimulate internalization only. Depending on the stimulus applied, internalization motifs from one or several TCR-CD3 subunits mediate endocytic routing of the complex. The involvement of signaling molecules in the intracellular fate of TCR-CD3, the nature and location of sequences for internalization and intracellular sorting, and the role of downregulation in T-cell activation are still the main open questions.

[Results of infrainguinal revascularization in patients with end-stage renal disease]. / Résultats des revascularisations sous-inguinales chez les malades en insuffisance rénale terminale.

OBJECTIVES: The aim of this retrospective study was to evaluate the efficacy of bypass in patients with endstage renal disease (ESRD) and to determine predictive factors and precise bypass indications. METHOD: Forty one patients with ESRD underwent 50 bypass, 6 limbs were stage II and 44 stage III or IV according to Leriche and Fontaine classification. Revascularisations procedures were 47 infrainguinal bypass and 3 miscellaneous. Median follow up was 17,0+/-15,7 months. RESULTS: Perioperative mortality rate was 12% (n = 6). Morbidity was as follow : 1 perioperative major imputation and 8 secondary ones. There were 26 secondary death (12 from cardiac events), cumulative survival rate declined to 42,9+/-7,7% and limb salvage rate to 77,2+/-7,5% at 2 years. Primary and secondary potency rates were 53,5+/-10,4% and 70,6+/-10%. Quality of life was good in 28% of revascularised patients. Among risk factors, myocardial events showed a statistical significance in predicting survival, good runoff and bypass occlusion showed a statistical significance in predicting limb salvage. CONCLUSION: Revascularisation can be performed in ESRD patients. However to improve the results full evaluation of myocardial risks, skin lesions and infection of the feet, available autologous vein and nutritional status may be needed in those patients.

Retroviral vector-mediated expression in primary human T cells of an endoplasmic reticulum-retained CD4 chimera inhibits human immunodeficiency virus type-1 replication.

Intracellular expression of genes that inhibit key steps in the human immunodeficiency virus (HIV-1) replicative cycle could offer an alternative therapy for AIDS treatment. One of these approaches involves the inhibition of env protein maturation through the expression of CD4 molecules with added exogenous sequences that promote their retention in the endoplasmic reticulum (ER). We have tested this strategy using a CD4 chimera (CD4epsilon10) containing an ER retention sequence derived from the TCR CD3-epsilon chain. Transfection of CD4epsilon10 in the human T cell line Jurkat made it resistant to infection with two different HIV-1 isolates, which was evaluated by measuring p24 antigen production, induction of apoptosis, and syncytia formation. Furthermore, polymerase chain reaction (PCR) analysis of genomic DNA showed no traces of the proviral HIV-1 genome in CD4epsilon10-transfected cells, suggesting it was not maintained latently in these cells. To facilitate the delivery of the CD4epsilon10 chimera to primary cells from AIDS patients, a Moloney-based retroviral vector was constructed that expresses CD4epsilon10 under the transcriptional control of the HIV-1 long terminal repeat (LTR) promoter. Transduction of the MT-2 human T cell line with this vector rendered it resistant to infection with HIV-1 by a process that involved the inhibition of gp160 proteolytic processing. Finally, transduction of the CD4epsilon10 chimera into T lymphoblasts derived from asymptomatic HIV-infected individuals demonstrated a protective effect, resulting in both an increased cellular proliferation rate and an increased percentage of CD4+ cells. These results suggest that it is feasible to use retroviral transduction of CD4epsilon10 as a gene therapy approach for AIDS treatment.

Construction of retroviral vectors carrying human CD3 gamma cDNA and reconstitution of CD3 gamma expression and T cell receptor surface expression and function in a CD3 gamma-deficient mutant T cell line.

CD3 gamma, a subunit of the T cell receptor-CD3 (TCR/CD3) complex, helps to support surface TCR/CD3 expression and participates in signal transduction for gene induction after antigen recognition by T lymphocytes, and in TCR/CD3 down-modulation. Humans with primary immunodeficiencies caused by inherited mutations in the CD3 gamma gene or in the gene encoding epsilon CD3é, another subunit of TCR/CD3 complex, have been previously reported. To develop a gene therapy protocol for CD3-deficient patients, CD3 gamma cDNA was orientationally inserted into two retroviral vectors (LNCX and LXSN), which resulted in recombinant vectors LNCG and LGSN, respectively. Two vector producer cell lines Am12/LNCG and Am12/LGSN were established from packaging cells GP+envAm12. Their mean viral titers were 6.5 x 10(6) and 2.0 x 10(7) cfu/ml, respectively, as shown by an improved retroviral vector production and transduction method that increases titers around five-fold over conventional methods. The presence of helper virus in vector stocks was tested by marker rescue assay and found to be < 1 cfu/ml. Southern blot analysis showed that multiple copies of the vectors were present in the genome of high-titer producers and that both vectors could transfer CD3 gamma cDNA into the genome of 3T3 cells. The vectors were used to correct in vitro a CD3 gamma-deficient Jurkat mutant cell line lacking TCR/CD3 expression and termed JGN (for Jurkat gamma negative). Both vectors increased TCR/CD3 expression in JGN (normally 2% using WT31 monoclonal antibody) to 34% and 37%, respectively, in G418-selected 3-week bulk cultures. Two clones from transduced JGN cells termed JGN/LNCG13 and JGN/LNCG15, with high TCR/CD3 expression (88% and 79%, respectively), were selected for further analyses. First, CD3 gamma protein reconstitution was demonstrated by immunoprecipitation. Second, interleukin-2 production after TCR/CD3 engagement and TCR/CD3 down-modulation in response to phorbol myristate acetate were shown to be comparable to wild-type Jurkat cells. We conclude that LNCG and LGSN may be useful for gene therapy purposes.

Megalomycin C, a macrolide antibiotic that blocks protein glycosylation and shows antiviral activity.

Megalomycin C, a natural macrolide antibiotic showed a potent antiherpetic activity. At concentrations that efficiently prevented HSV-1 multiplication, the compound had no cytotoxic or antiproliferative effects. Viral DNA and protein synthesis took place at normal levels in the presence of the antibiotic, suggesting that neither the translation of viral mRNA, nor the synthesis of viral nucleic acids was affected. The incorporation of mannose and galactosamine into viral proteins was blocked and precursor, but not mature, HSV-1 glycoproteins were detected in the presence of megalomycin C. Non-infectious HSV-1 viral particles were formed when the compound was present, but their glycoproteins were not properly glycosylated.

Uridine 5'-diphosphate glucose analogues. Inhibitors of protein glycosylation that show antiviral activity.

A series of analogues of uridine 5'-diphosphate glucose and uridine 5'-diphosphate glucosamine have been synthesized by reaction of 2,3,4,6-tetra-O-benzyl-, 2,3,4,6-tetra-O-benzoyl-, 2,3,4,6-tetra-O-acetyl-, and 2,3,4,6-tetra-O-palmitoyl-alpha-D-glucopyranose and 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-alpha-D-glucopyranose with chlorosulfonyl isocyanate and 2',3'-O-isopropylideneuridine. Isopropylidene and acetyl groups of the resulting 5'-O-[[[[(alpha-D-glucopyranosyl)oxy]carbonyl]amino]sulfonyl] -2',3'-O-isopropylideneuridine derivatives were removed by reaction with a TFA/water (5:1) mixture and methanolic ammonia, respectively. The 5'-O-[[[[(2",3",4",6"-tetra-O-benzyl-and 2",3",4",6"-tetra-O-benzoyl-alpha-D-glucopyranosyl)oxy]carbonyl] amino]sulfonyl]-2',3'-O-isopropylideneuridine (13 and 19) and the corresponding deisopropylidenated derivatives showed antiviral activity as determined by the inhibition of the cytopathic effect induced by HSV-1 replication and by the plaque assay method. Compound 13 inhibited glycosylation of proteins in HSV-1 infected HeLa cells.

Synthesis and antiviral evaluation of nucleosides of 5-methylimidazole-4-carboxamide.

Due to the antiviral activity of certain 5-substituted imidazole nucleosides related to ribavirin, 5-methylimidazole-4-carboxamide nucleosides having beta-D-ribofuranosyl, 2-deoxy-beta- and -alpha-D-ribofuranosyl, and (2-hydroxyethoxy)methyl moieties have been prepared and tested as antiviral agents. 1-beta-D-Ribofuranosyl-5-methylimidazole-4-carboxamide was obtained by deacetylation of the corresponding tri-O-acetyl nucleoside 11 or by deacetylation and ammonolysis of the blocked ethyl 5-methylimidazole-4-carboxylate nucleoside 10, which was prepared from the stannic chloride catalyzed condensation of the trimethylsilyl derivative of ethyl 4(5)-methylimidazole-5(4)-carboxylate. Glycosylation of 4(5)-methylimidazole-5(4)-carboxamide with 3,5-di-O-p-toluoyl-2-deoxy-D-erythro-pentofuranosyl chloride via mercuric cyanide method provided an anomeric mixture of the blocked 5-methylimidazole-4-carboxamide deoxynucleoside 14 along with an anomeric mixture of the 4-methyl 5-carboxamide isomer 15. Separation of compound 14 into the corresponding beta and alpha anomers was achieved by conversion to the 3',5'-di-O-acetyl derivatives 17 and 18, which after chromatographic separation were deacetylated to give 1-(2-deoxy-beta-D-erythro-pentofuranosyl)-5-methylimidazole-4-carboxa mid e and its alpha anomer 20. 1-[(2-Hydroxyethoxy)methyl]-5-methylimidazole-4-carboxamide was prepared by alkylation of the imidazole 13 with (2-acetoxyethoxy)methyl bromide followed by treatment with methanolic ammonia. All these imidazole nucleosides were tested in HeLa cell cultures against type 1 herpes simplex and vesicular stomatitis viruses. The ribofuranosyl derivative 12 showed a significant activity against type 1 herpes simplex virus.

Screening for new compounds with antiherpes activity.

A number of compounds have been tested for antiherpes activity. Actinobolin, amicetin, carrageenan, laspartomycin, megalomycin C, pleuromutilin, suramin and tetracenomycin C showed significant protection of HeLa cell monolayers infected with herpes simplex virus type 1. The action of these new antiherpes compounds was compared with those antiherpes agents that have been described previously. Actinobolin, amicetin and tetracenomycin C were also active against viruses other than herpes simplex.

On the specificity of PCR detection of Listeria monocytogenes in food: a comparison of published primers.

A total of nine pairs of primers, seven previously published and two newly developed, have been assayed for PCR detection of Listeria monocytogenes in food. They have been tested for specificity on a total of 72 strains including reference and food isolates belonging to L. monocytogenes and other species in the genus. First of all, a polyphasic approach has been carried out in order to establish a reference strain collection. They were biochemically and genetically characterized by API-Lis and randomly amplified polymorphic DNA PCR (RAPD-PCR), respectively. Random amplification of DNA was performed with M13, T7 and T3 universal primers and a data bank was created to compile the RAPD patterns of all the analyzed strains. The UPGMA cluster analysis of RAPD profiles with primer M13 showed eight clusters at 72.3% similarity. Clusters 2 and 7 corresponded to L. monocytogenes. Clusters 1 and 6 grouped L. ivanovii strains. Clusters 3, 4, 5 and 8 corresponded to L. grayi, L. innocua, L. welshimeri and L. seeligeri, respectively. Pattern analysis revealed the existence of miss-identified reference strains which was confirmed by 16S rDNA sequence analysis. RAPD-PCR is a rapid genetic test which helped to confirm strain identity. On the basis of PCR specificity results, primers LM1-LM2 were the best combination for the detection of L. monocytogenes since they only amplified the specific fragment in strains that had been genetically and biochemically assessed as belonging to the species. Specificity of other assayed primers is discussed.

[Percutaneous transluminal angioplasty and severe limb ischaemia]. / Angioplastie transluminale percutanée et ischémie sévère des membres inférieurs.

OBJECTIVES: To determine the outcome and the place of transluminal angioplasty (ATL) in the treatment of severe limb ischaemia. MATERIALS AND METHODS: Seventy two legs at stage III and IV of Fontaine's classification have been exclusively treated by endoluminal procedures. The success was valued both on the haemodynamic post-operative improvement of the run-off flow and on the clinical statut leading to the conservation at mid-term of a functional limb. Patency and survival rate had been valued by actuarial method. RESULTS: Seventy percent of the limbs were haemodynamically improved. For the global population, a 48% clinical success rate was obtained at 6 months but 30.5% of limbs were loss. Primary patency rate was respectively 79, 71 and 68% at 6, 12 and 24 month. The quality of the run-off arteries has been the most influential factor. CONCLUSION: Endoluminal treatment of chronic limb ischaemia had lead to a clinical improvement in 48% of cases. Multi-stages and distal atherosclerotic disease of this patients limits ATL indications which results depend of run-off quality.

[Decompensation of lower limb arteritis after bone and joint surgery]. / Décompensation d'une artérite après chirurgie osseuse et articulaire.

PURPOSE OF THE STUDY: Decompensation of lower limb arteritis after bone and joint surgery is an unusual finding compared with the large number of procedures performed in both emergency and controlled settings. There is however a functional and limb-threatening risk that must not be overlooked. MATERIAL AND METHODS: We report a series of 9 patients followed in our department over the last 3 years. Emergency surgery had been required in 6 patients after trauma and 3 had undergone a planned orthopedic procedure. All the patients had at least one vascular risk factor, and 7 of them had a cardiovascular history. The inaugural sign was a trophic disorder due to a grade IV decompensated arteritis in 8 patients, including 2 with nonunion. Delay to treatment ranged from 1 to 3 months. Acute embolic ischemia required emergency care in 1 patient. RESULTS: A revascularization procedure was performed on 6 limbs and was successful in 3. There were also 6 amputations, three initially, 1 after septic shock and 2 because revascularization was impossible. Three of the amputations were required after failed revascularization. Prosthesis wearing and walking was possible in only two amputated patients. Overall rate of successful salvage was 33% (3 successful revascularizations among 9 limbs). One of the nonunions healed after revascularization; the limb was amputated for the other one. One patient died from septicemia. DISCUSSION: Our series further illustrates the severity of decompensated arteritis after bone and joint surgery, emphasizing the importance of searching for cardiovascular risk factors and functional signs suggestive of a vascular disorder. Arterial duplex Doppler and if necessary arteriography of the lower limbs should be obtained in case of doubt. Two different situations can be distinguished depending on the predictable vascular risk and the localization of the planned bone reconstruction. If the patient has an asymptomatic proximal arteritis and bone and joint surgery is planned above the knee, a revascularization procedure would not appear necessary prior to bone surgery. In other cases, it may be more advisable to treat the arteritis before attempting bone surgery. For trauma victims, the osteosynthesis technique depends greatly on knowledge of the vascular risk.

[Prognostic factors of femoropopliteal shunts. Retrospective study of a series of 52 shunts]. / Les facteurs pronostiques des pontages fémoro-poplités. Etude rétrospective d'une série de 52 pontages.

Between January 1986 and January 1991, 52 femoropopliteal shunts were carried out in 51 patients for lesions defined by the Ad Hoc Committee as grade III in 36 cases, grade II in 10 and grade I in 6. The shunt graft used was the internal saphenous vein in situ in 34 cases, inversed in 13 cases, and a PTFE prosthesis in 5 cases. The postoperative course included 12 complications and one death. At the end of the study, 10 shunts had thrombosed and 4 patients had required a major amputation. Actuarial analysis at 5 year follow up showed primary permeability of 56% and secondary permeability of 60%. Statistical analysis of secondary permeability as a function of the distal bed, an associated diabetes or the in situ or inversed venous shunt, failed to demonstrate any significant difference. These femoropopliteal venous shunts in critical ischemias reduced the incidence of major amputations, the proportion of limbs saved being higher than that for shunt permeability from the 6th postoperative month. Poor results were recorded for femoropopliteal shunts using prostheses.

Sangrado variceal duodenal secundario a trombosis venosa mesentérica: a propósito de un caso / Duodenal variceal bleeding secondary to mesenteric vein thrombosis

La trombosis venosa mesentérica corresponde a un factor obstructivo del sistema venoso intestinal intraluminal, trayendo consecuencias clínicas dadas por isquemia intestinal y aumento de la circulación colateral debido a una dificultad del drenaje sanguíneo. Raramente, esto podría generar várices ectópicas, pero esta descrito en la literatura la existencia de várices duodenales producto de este mecanismo. En este articulo se presenta un reporte de un caso, que presenta un sangrado variceal de origen duodenal secundaria a una trombosis venosa portal y mesentérica, sin causa aparente.

Mesenteric vein thrombosis is an intraluminal obstruction of the intestinal venous system with clinical consequences due to intestinal ischemia and an increase in collateral circulation caused by compromised venous return. It very rarely generates ectopic varicose veins but duodenal varicose veins caused by this mechanism have been described in the literature. In this article we report the case of duodenal variceal bleeding secondary to a portal and mesenteric venous thrombosis with no apparent cause.

Enfermedad de Still del Adulto, a propósito de un caso / Still's disease, a clinical case

La enfermedad de Still del Adulto es una variante sistémica del universo llamado "artritis idiopática juvenil", cuya diferencia es la edad de aparición, recibiendo este nombre cuando el cuadro se manifiesta en personas mayores de 15 años. Es una enfermedad autoinflamatoria caracterizada por fiebre intermitente, artritis, rash evanescente y linfadenopatías, asociado a manifestaciones de compromiso sistémico. Es una patología de baja prevalencia, pero es importante considerarla como uno de los principales diagnósticos etiológicos de la fiebre de origen desconocido.

Adult-onset Still's disease is a systemic variant of Still's disease distinguished from juvenile idiopathic arthritis by its onset age of over 15. It is an auto-inflammatory disease characterized by intermittent fever, arthritis, short-lived rash and lymphadenopathies. It has low prevalence but is important to consider as one of the principle etiological diagnoses of fever of unknown origin.

Contribución de la temperatura fría y el sabor ácido en la intervención fonoaudiológica de la disfagia orofaríngea / Contribution of cold temperature and sour taste in speech and language intervention of oropharyngeal dysphagia

El fonoaudiólogo es el principal profesional en la rehabilitación no farmacológica y no quirúrgica del usuario con disfagia. Su participación es fundamental tanto para el aminoramiento del riesgo de aspiración o penetración laríngea, como para mejorar o restaurar la función deglutoria. Para este fin, posee opciones terapéuticas directas e indirectas, cuya elección y aplicación dependerá de la patología que curse el usuario, las redes que posea para su recuperación y la motivación intrínseca del mismo. Entre las estrategias de intervención indirecta se encuentra el Tratamiento Sensorio-Motor Oral (OSMT, por sus siglas en inglés), el cual pretende producir una aceleración en el desencadenamiento del proceso deglutorio mediante la ejercitación de los músculos orofaciales en conjunto con diferentes estímulos sensoriales (específicamente la temperatura fría y el sabor ácido). La presente revisión tiene por objetivo dilucidar si la utilización de la temperatura fría y el sabor ácido son útiles como mecanismo de intervención indirecta de la disfagia. Se concluye que las acciones propuestas son efectivas simplemente como mecanismos compensatorios en el proceso deglutorio, puesto que modifican las características del bolo alimenticio e incrementan momentáneamente las sensaciones intraorales.

The speech-language pathologist (SLP) is the main professional in the nonpharmacological and non-surgical rehabilitation of patients with dysphagia. Their role is essential for both reducing the risk of aspiration or laryngeal penetration and improving or restoring the swallowing function. To this end, the SLP has direct and indirect therapeutic options, whose choice and application will depend on the patient’s condition, support networks, and their intrinsic motivation. As part of the indirect intervention strategies, the oral sensorymotor treatment (OSMT) aims to exercise the orofacial muscles, and introduce sensory input by the application of cold temperature and sour taste to increase the triggering speed of the swallowing reflex. This review seeks to determine whether the use of cold temperature and sour taste are effective indirect mechanisms for treating patients with dysphagia. It is concluded that the proposed actions in this review are useful simply as compensatory mechanisms in the swallowing process, as they modify the bolus properties and increase, temporarily, the intra-oral sensations.