A prospective study of salivary gland function in patients undergoing radiotherapy for squamous cell carcinoma of the oropharynx

Résumé: Le traitement du cancer avancé de la tête et du cou nécessite souvent une approche multidisciplinaire associant la chirurgie, la radiothérapie et la chimiothérapie. Chacun de ces traitements présente des avantages, des limites et des inconvénients. En raison de la localisation de la tumeur primaire et/ou des métastases ganglionnaires, les glandes salivaires majeures sont fréquemment touchées par les traitements oncologiques. La salive joue un rôle déterminant dans la cavité buccale car elle lubrifie les tissus et facilite à la fois la déglutition et l'élocution. Son contenu en électrolytes et en protéines, dont certaines possèdent un effet antibactérien, protège les dents de la déminéralisation par l'acidité. Une fonction normale, liée autant à la quantité qu'à la qualité de la salive, reste indispensable pour le maintien d'une bonne santé buccale. L'objectif de cette étude prospective a été de déterminer, dans un groupe homogène de patients, l'influence d'un traitement de radiothérapie sur divers paramètres salivaires comme la sécrétion, le pH et l'effet tampon, avant, pendant et jusqu'à un an après la fin du traitement. L'étude a aussi examiné le comportement de ces paramètres salivaires après une intervention chirurgicale seule au niveau de la tête et du cou, avec ou sans exérèse d'une glande sous- maxillaire. L'étude s'est basée sur 54 patients (45 hommes et 9 femmes) atteints d'un carcinome épidermoïde avancé avec une localisation oro-pharyngée confirmée (n = 50) ou soupçonnée (n = 4), adressés et investigués dans le Centre Hospitalier Universitaire Vaudois de Lausanne, Suisse. Tous ces patients furent traités par radiothérapie seule ou en combinaison avec une chirurgie et/ou une chimiothérapie. Trente-neuf des 54 patients parvinrent à la fin de cette étude qui s'est étendue jusqu'à 12 mois au-delà de la radiothérapie. La chirurgie de la tête et cou, en particulier après ablation de la glande sous-maxillaire, a révélé un effet négatif sur la sécrétion salivaire. Elle n'influence en revanche ni le pH, ni l'effet tampon de la salive. Cependant, l'effet sur la sécrétion salivaire lié à la chirurgie est progressivement masqué par l'effet de la radiothérapie et n'est plus identifiable après 3-6 mois. Dès le début de la radiothérapie, la sécrétion salivaire chût très manifestement pour diminuer progressivement jusqu'à 1/3 de sa capacité à la fin du traitement actinique. Une année après la fin de cette radiothérapie, la dysfonction salivaire est caractérisée par une diminution moyenne de la sécrétion salivaire, de 93 % (p < 0,0001) pour la salive au repos et de 95 % (p < 0.0001) pour la salive stimulée, par rapport aux valeurs pré-thérapeutiques. Le pH salivaire ainsi que l'effet tampon furent également influencés par le traitement actinique. L'effet tampon a présenté une diminution à 67 % à une année post-traitement en comparaison de sa valeur pré-thérapeutique. Le pH de la salive stimulée présente une légère, mais significative, diminution par rapport à sa valeur antérieure à la radiothérapie. En conclusion, la chirurgie des cancers de l'oropharynx précédant une radiothérapie a une influence négative sur la sécrétion salivaire sans aggraver l'hyposialie consécutive aux radiations ionisantes. Cette étude confirme qu'un traitement oncologique comprenant une irradiation totale des glandes salivaires majeures chez des patients atteints d'un carcinome épidermoïde avancé de la région oro-pharyngée, induit une perte sévère et à long terme de la sécrétion salivaire avec une altération du pH et de l'effet tampon Abstract: Objective. We sought to investigate the impact of head and neck cancer treatment on salivary function. Study design. The study was conducted on 54 patients with advanced squamous cell carcinoma with confirmed (n =50) or suspected (n = 4) primary oropharyngeal localization who were treated with radiation alone or in combination with surgery or chemotherapy, or both. The following groups were considered in the evaluation: 1, the entire pool of patients; 2, those undergoing surgery and those not undergoing surgery before radiation; 3, those undergoing resection and those not undergoing resection of the submandibular gland. The flow rates, pH, and buffering capacity were determined before, during, and up to 12 months after the completion of radiation. Results. Head and neck surgery, particularly when submandibular gland resection was performed, had a negative impact on salivary flow rates but did not influence pH or buffering capacity. Nonetheless, the effect of surgery on salivary flow rates decreased progressively and disappeared at 3 to 6 months after radiotherapy. More than two thirds of the salivary output was lost during radiation treatment. All patients were experiencing salivary dysfunction at 1 year after completion of radiotherapy, with average decreases of 93% (P < .0001) and 95% (P < .0001) for whole resting salivary flow and whole stimulated salivary flow, respectively, compared with the preradiotherapy values. The buffering capacity decreased to 67% of its preradiotherapy value, and whole stimulated saliva became acidic. Conclusions. The result of this study confirms that cancer treatment involving full-dose radiotherapy (RTH) to all major salivary glands for locally advanced squamous cell carcinoma of the oropharynx induces severe hyposalivation with alteration of salivary pH and buffering capacity. Head and neck surgery has a negative impact on salivary flow rates, especially when the submandibular gland is removed. However, surgery before irradiation is not a factor aggravating hyposalivation when postoperative radiotherapy includes all the major salivary glands.

TCR-MHC class II interaction is required for peripheral expansion of CD4 cells in a T cell-deficient host.

It is well established that T cell-deficient nude and SCID mice can be reconstituted by i.v. injection of small numbers of purified peripheral CD4+ T cells; however, the requirements for expansion of the transferred T cells in such systems are not clear. We show here that blood and lymphoid organs of MHC class II-deficient mice (which selectively lack mature CD4+ T cells) cannot be reconstituted by transfer of purified splenic CD4+ T cells, whereas TCRalpha-deficient mice (which lack both CD4+ and CD8+ mature T cells) are readily reconstituted. The failure of CD4+ T cell reconstitution in MHC class II-deficient mice was not due to the presence of CD8+ T cells, since similar results were obtained in TCRalpha-MHC class II double-deficient mice. Consistent with most previous studies CD4+ T cells in reconstituted TCRalpha-deficient mice had a diverse TCR Vbeta repertoire and were predominantly of an activated/memory (CD44high) phenotype. Collectively our data demonstrate that the expansion of peripheral CD4+ T cells in a T cell-deficient host is dependent upon interactions of the TCR with MHC class II.

An adult thymic stromal-cell suspension model for in vitro positive selection.

Presented here is a cell-suspension model for positive selection using thymocytes from alphabeta-TCR (H-2Db-restricted) transgenic mice specific to the lymphocytic choriomeningitis virus (LCMV) on a nonselecting MHC background (H-2d or TAP-1 -/-), cocultured with freshly isolated adult thymus stromal cells of the selecting MHC type. The thymic stromal cells alone induced positive selection of functional CD4- CD8+ cells whose kinetics and efficiency were enhanced by nominal peptide. Fibroblasts expressing the selecting MHC alone did not induce positive selection; however, together with nonselecting stroma and nominal peptide, there was inefficient positive. These results suggest multiple signaling in positive selection with selection events able to occur on multiple-cell types. The ease with which this model can be manipulated should greatly facilitate the resolution of the mechanisms of positive selection in normal and pathological states.

Low density lipoprotein signaling and pancreatic beta cells protection

SummaryLow-density lipoproteins (LDLs) have an important physiological role in organism transporting cholesterol and other fatty substances to target tissues. However, elevated LDL levels in the blood are associated with the formation of arterial plaques and consequently atherosclerosis. It is therefore important to characterize the intracellular pathways induced upon LDL stimulation as they might be involved in the pathological properties of these lipoproteins. It has been previously found that LDL stimulation of mouse embryonic fibroblasts activates p38 mitogen activated protein kinases (MAPKs). This leads to cell spreading and increase in the wound healing capabilities of the cells. These two responses might occur within atherosclerotic plaques.The aim of this project is to reveal the missing links between LDL particle and activation of p38 MAPK kinase. As previously shown in our lab activation of p38 MAPK kinase by the LDL particles occur independently of classical LDL receptor (LDLR). In this study we have shown that scavenger receptor type Β class I (SR-BI) is responsible for the signal transduction from the LDLs to the p38 MAPK. We have also shown that Mitogen activated kinase kinases (MKKs) that can directly activate ρ 38 MAPK in these conditions are MKK3 and MKK6 but not MKK4. We have also tested some of the intermediate components of the pathway like Ras and PI3 kinase but found that they do not play a role.The data obtained in this study showed a part of molecular mechanism responsible for p38 MAPK activation and subsequent wound healing and can contribute to our knowledge on function of the fibroblasts in the development of the atherosclerotic plaques.Diabetes Mellitus is a condition caused by disordered metabolism of blood glucose level. It is one of the most commonly spread disease in the western world, with the incidence reaching 8% of population in United States. Two most common types of diabetes are type 1 and 2 that differs slightly in the mechanism of the development. However in the basis of both types lies the cell death of pancreatic beta cells. The aim of this work is to improve beta cells survival in different pathophysiological settings. This could be extrapolated to the conditions in which Diabetes develops in humans. We decided to use RasGAP- derived fragment Ν with its strong antiapoptotic effect in beta cells. In our lab we have demonstrated that in the mild stress conditions RasGAP can be cleaved by caspases at the position 455 producing two fragments, fragment Ν and fragment C. Fragment Ν exerts

Long-term, multilineage hematopoiesis occurs in the combined absence of beta-catenin and gamma-catenin

The canonical Wnt signaling pathway plays key roles in stem-cell maintenance, progenitor cell expansion, and lineage decisions. Transcriptional responses induced by Wnt depend on the association of either beta-catenin or gamma-catenin with lymphoid enhancer factor/T cell factor transcription factors. Here we show that hematopoiesis, including thymopoiesis, is normal in the combined absence of beta- and gamma-catenin. Double-deficient hematopoietic stem cells maintain long-term repopulation capacity and multilineage differentiation potential. Unexpectedly, 2 independent ex vivo reporter gene assays show that Wnt signal transmission is maintained in double-deficient hematopoietic stem cells, thymocytes, or peripheral T cells. In contrast, Wnt signaling is strongly reduced in thymocytes lacking TCF-1 or in nonhematopoietic cells devoid of beta-catenin. These data provide the first evidence that hematopoietic cells can transduce canonical Wnt signals in the combined absence of beta- and gamma-catenin

Role of beta-1 integrin in tumor growth and dissemination

SUMMARY Cancer is one of the leading causes of disease-related mortality. In most cases, death is due to the spread of cells from the primary tumor to distant sites causing formation of metastases. To become tumorigenic, cells should acquire ability, including self-sufficiency in growth signals, insensitivity to anti-growth signals, resistance to apoptosis, sustained angiogenesis, limitless replicative potential and tissue invasion and metastasis. Tumor progression depends, in part on the relationship between tumor cells and host tissue stroma, characterized by changes of tumor cell adhesion to their microenvironment and activation of a variety of extracellular proteases that play a role in ECM degradation. integrins are adhesion proteins implicated in tumorigenesis. Their main function is to mediate cell adhesion to the ECM or to other cells and to create a link between the ECM and the cytoskeleton. Tumor cells like normal cells use integrins to attach to ECM, migrate into surrounding tissues and derive survival and growth signals. Integrin-dependent adhesion and migration are thought to play an important role in tumor dissemination. A strategy was designed to address the role of β1 integrin tumor growth and dissemination. Murine mammary carcinoma (TA3) cells were stably transfected with a soluble β1 integrin construct, which is anticipated to play a dominant negative role, being able to associate with different α-subunits expressed on the cell surface but unable to transduce signals to the nucleus. Results from studies based on soluble β1 integrin TA3 transfectants showed that 1) the integrin expression pattern at the cell surface changed with an induction of α2β1 and α5β1 heterodimers; 2) adhesion to collagens, especially collagen I was increased; 3) tumor dissemination after intrape-ritoneal injection in syngeneic mice was abolished and 4) local growth after orthotopic injection was maintained but delayed. Taken together, the data presented here suggest that β1 integrin plays a potentially important role in the regulation of tumor behavior. RESUME Le cancer est une des principales causes de mortalité suite à une maladie. Dans la plupart des cas, la mort est la conséquence de la dissémination de cellules, provenant de la tumeur primaire, dans des endroits distants et causant la formation de métastases. Afin de devenir cancéreuse, une cellule doit acquérir certaines capacités, telles qu'une auto-suffisance en facteurs de croissance, une insensibilité aux facteurs empêchant la croissance cellulaire, une résistance à l'apoptose, une angiogénèse soutenue, un potentiel de réplication illimité et une capacité à pénétrer dans les tissus et à former des colonies métastatiques. La progression d'une tumeur dépend, en partie, de la relation entre les cellules tumorales et les cellules tissulaires de l'hôte. Cette relation est caractérisée par des modifications des cellules tumorales quant à leur adhésion au microenvironnement et à l'activation de protéases qui permettent de dégrader la matrice extracellulaire. Les intégrines sont des protéines impliquées dans le développement tumoral. Leur fonction principale est de réguler l'adhésion des cellules à la matrice extracellulaire, ou à d'autres cellules, et de créer un lien entre cette matrice extracellulaire et le cytosquelette. Les cellules tumorales utilisent également les intégrines pour se lier à la matrice extracellulaire, pour migrer dans les tissus adjacents et pour induire des signaux de croissance et de survie. Ces événements d'adhésion et de migration, qui dépendent des intégrines, jouent un rôle primordial dans la dissémination des cellules cancéreuses. Une stratégie a été élaborée afin de définir le rôle de l'intégrine β1 durant la croissance et la dissémination des cellules tumorales. Des cellules provenant d'un carcinome de la glande mammaire (TA3) ont été transfectées de manière stable avec un vecteur contenant la séquence codante de la partie extracellulaire de l'intégrine β1. L'intégrine tronquée doit être capable de se lier aux sous-unités α exprimées à la surface de la cellule, mais doit être incapable de transmettre un signal à l'intérieur de la cellule. Les résultats obtenus avec les cellules TA3 transfectées contenant l'intégrine β1 soluble montrent que I) le répertoire d'expression des intégrines à la surface de la cellule a changé en faveur des hétérodimères α2β1 et α5β1; 2) l'adhésion aux collagènes, particulièrement au collagène de type I a augmenté; 3) la dissémination des cellules tumorales après une injection intrapéritonéale est empêchée; 4) la croissance tumorale après une injection orthotopique est conservée mais retardée. Ces résultats montrent que l'intégrine β1 joue un rôle primordial dans la régulation du comportement tumoral.

Positive regulation of the peroxisomal beta-oxidation pathway by fatty acids through activation of peroxisome proliferator-activated receptors (PPAR).

Peroxisome proliferators regulate the transcription of genes by activating ligand-dependent transcription factors, which, due to their structure and function, can be assigned to the superfamily of nuclear hormone receptors. Three such peroxisome proliferator-activated receptors (PPAR alpha, beta, and gamma) have been cloned in Xenopus laevis. Their mRNAs are expressed differentially; xPPAR alpha and beta but not xPPAR gamma are expressed in oocytes and embryos. In the adult, expression of xPPAR alpha and beta appears to be ubiquitous, and xPPAR gamma is mainly observed in adipose tissue and kidney. Immunocytochemical analysis revealed that PPARs are nuclear proteins, and that their cytoplasmic-nuclear translocation is independent of exogenous activators. A target gene of PPARs is the gene encoding acyl-CoA oxidase (ACO), which catalyzes the rate-limiting step in the peroxisomal beta-oxidation of fatty acids. A peroxisome proliferator response element (PPRE), to which PPARs bind, has been identified within the promoter of the ACO gene. Besides the known xenobiotic activators of PPARs, such as hypolipidemic drugs, natural activators have been identified. Polyunsaturated fatty acids at physiological concentrations are efficient activators of PPARs, and 5,8,11,14-eicosatetraynoic acid (ETYA), which is the alkyne homolog of arachidonic acid, is the most potent activator of xPPAR alpha described to date. Taken together, our data suggest that PPARs have an important role in lipid metabolism.

Evidence for a TCR affinity threshold delimiting maximal CD8 T cell function.

Protective adaptive immune responses rely on TCR-mediated recognition of Ag-derived peptides presented by self-MHC molecules. However, self-Ag (tumor)-specific TCRs are often of too low affinity to achieve best functionality. To precisely assess the relationship between TCR-peptide-MHC binding parameters and T cell function, we tested a panel of sequence-optimized HLA-A(*)0201/NY-ESO-1(157-165)-specific TCR variants with affinities lying within physiological boundaries to preserve antigenic specificity and avoid cross-reactivity, as well as two outliers (i.e., a very high- and a low-affinity TCR). Primary human CD8 T cells transduced with these TCRs demonstrated robust correlations between binding measurements of TCR affinity and avidity and the biological response of the T cells, such as TCR cell-surface clustering, intracellular signaling, proliferation, and target cell lysis. Strikingly, above a defined TCR-peptide-MHC affinity threshold (K(D) < approximately 5 muM), T cell function could not be further enhanced, revealing a plateau of maximal T cell function, compatible with the notion that multiple TCRs with slightly different affinities participate equally (codominantly) in immune responses. We propose that rational design of improved self-specific TCRs may not need to be optimized beyond a given affinity threshold to achieve both optimal T cell function and avoidance of the unpredictable risk of cross-reactivity.

Expression of T cell receptor genes in the thymus: localization of transcripts in situ and comparison of mature and immature subsets.

We have analyzed the expression of T cell receptor (TcR) genes in the thymus using in situ RNA hybridizations with probes to the constant regions of the TcR alpha, beta, gamma and delta chains. Localization of transcripts revealed low TcR alpha mRNA levels in the thymus cortex and very low levels in the subcapsular region. In contrast, TcR beta message was very abundant in the cortex. TcR gamma or delta mRNA+ thymocytes showed a scattered, predominantly cortical localization. In contrast to gamma, TcR delta transcripts were abundant in the subcapsular region. Control experiments with sorted TcR alpha/beta or gamma/delta cells revealed a detection efficiency of 75%-85% for the respective TcR mRNA and data on TcR gene expression in mature, CD3+ thymocytes were consistent with previous reports. The analysis of immature, CD3- thymocyte subsets, however, revealed a virtual absence of TcR alpha transcripts and an unexpectedly high proportion of cells (14%-29%) expressing the gene for the TcR delta chain. The data are discussed in view of current models of lineage relationships in the thymus.

Prostaglandin E2 promotes integrin alpha Vbeta 3-dependent endothelial cell adhesion, rac-activation, and spreading through cAMP/PKA-dependent signaling.

We have recently reported that the inhibition of endothelial cell COX-2 by non-steroidal anti-inflammatory drugs suppresses alpha(V)beta(3)- (but not alpha(5)beta(1)-) dependent Rac activation, endothelial cell spreading, migration, and angiogenesis (Dormond, O., Foletti, A., Paroz, C., and Ruegg, C. (2001) Nat. Med. 7, 1041-1047). Here we investigated the role of the COX-2 metabolites PGE(2) and TXA2 in regulating human umbilical vein endothelial cell (HUVEC) adhesion and spreading. We report that PGE(2) accelerated alpha(V)beta(3)-mediated HUVEC adhesion and promoted Rac activation and cell spreading, whereas the TXA2 agonist retarded adhesion and inhibited spreading. We show that the cAMP level and the cAMP-regulated protein kinase A (PKA) activity are critical mediators of these PGE(2) effects. alpha(V)beta(3)-mediated adhesion induced a transient COX-2-dependent rise in cAMP levels, whereas the cell-permeable cAMP analogue 8-brcAMP accelerated adhesion, promoted Rac activation, and cell spreading in the presence of the COX-2 inhibitor NS-398. Pharmacological inhibition of PKA completely blocked alpha(V)beta(3)-mediated adhesion. A constitutively active Rac mutant (L61Rac) rescued alpha(V)beta(3)-dependent spreading in the presence of NS398 or, but did not accelerate adhesion, whereas a dominant negative Rac mutant (N17Rac) suppressed spreading without affecting adhesion. alpha(5)beta(1)-mediated HUVEC adhesion, Rac activation, and spreading were not affected by PGE(2), 8-brcAMP, or the inhibition of PKA. In conclusion, these results demonstrate that PGE(2) accelerates alpha(V)beta(3)-mediated endothelial cell adhesion through cAMP-dependent PKA activation and induces alpha(V)beta(3)-dependent spreading via cAMP- and PKA-dependent Rac activation and may contribute to the further understanding of the regulation of vascular integrins alpha(V)beta(3) by COX-2/PGE(2) during tumor angiogenesis and inflammation.

Responsiveness of astrocytes in serum-free aggregate cultures to epidermal growth factor: dependence on the cell cycle and the epidermal growth factor concentration.

Serum-free aggregating cell cultures of fetal rat telencephalon treated with low doses (0.5 nM) of epidermal growth factor (EGF) showed a small, transient increase in DNA synthesis but no significant changes in total DNA and protein content. By contrast, treatment with high doses (13 nM) of EGF caused a marked stimulation of DNA synthesis as well as a net increase in DNA and protein content. The expression of the astrocyte-specific enzyme, glutamine synthetase, was greatly enhanced both at low and at high EGF concentrations. These results suggest that at low concentration EGF stimulates exclusively the differentiation of astrocytes, whereas at high concentration, EGF has also a mitogenic effect. Nonproliferating astrocytes in cultures treated with 0.4 microM 1-beta-D-arabinofuranosyl-cytosine were refractory to EGF treatment, indicating that their responsiveness to EGF is cell cycle-dependent. Binding studies using a crude membrane fraction of 5-day cultures showed a homogeneous population of EGF binding sites (Kd approximately equal to 2.6 nM). Specific EGF binding sites were found also in non-proliferating (and nonresponsive) cultures, although they showed slightly reduced affinity and binding capacity. This finding suggests that the cell cycle-dependent control of astroglial responsiveness to EGF does not occur at the receptor level. However, it was found that the specific EGF binding sites disappear with progressive cellular differentiation.

Tubular Dysfunction in Idiopathic Nephrotic Syndrome

Objectives: To evaluate the degree of tubular involvement in INS at various stage of the disease. Methods: 19 patients with INS were studied. 13 were steroid responders (group 1). 5 of them had biopsy which showed MCD. 6 patients were non responder to steroid or were steroid dependant with frequent relapses (group 2). Biopsies showed 3 FSGS and 3 MCD. They were treated with prednisone, ciclosporin and/ or mycofenolate mofetil. Protein, microalbumin (ALB), alpha-microglobulin (AMG), N-acetyl-beta-D-glucosaminidase (NAG) and creatinine (cr) were measured in each urine sample. Patients were considered in remission if prot/ cr ratio (g/mol) was < 20 (group 1a and 2a), and in relapse if the ratio was > 200 (group 1c and 2c). Some patients in group 1 had non nephrotic proteinuria (group 1b). Tubular dysfunction was defi ned by NAG/cr ratio (mg/mmol) > 0.86 or by AMG/cr ratio (mg/mmol) > 1.58. Results: Prot/cr ALB/cr NAG/cr AMG/cr Group 1a 10.3 ± 4.1 1.1 ± 1.0 0.19 ± 0.12 1.40 ± 0.97 Group 1b 60.4 ± 63.4 42.8 ± 66.7 0.39 ± 0.21 1.20 ± 0.56 Group 1c 713.3 ± 276.8 799.8 ± 534.9 2.25 ± 1.86* 4.25 ± 2.09* Group 2a 11.3 ± 6.1 4.7 ± 5.7 0.26 ± 0.19 1.18 ± 0.60 Group 2c 914.9 ± 718.6 682.9 ± 589.3 3.00 ± 2.72* 5.47 ± 4.30* Results are mean ± SD, p < 0.001 compared to group 1a and 2a No difference was observed between group 1 and group 2 neither in remission nor in relapse. Conclusions: These data indicate that tubular dysfunction occurs in INS but only in patients in relapse. In this population, tubular dysfunction was independent of the severity of the nephrotic syndrome, the treatment protocol and the histopathology.

Characterization and detection of naturally occurring antibodies against IL-1 alpha and IL-1 beta in normal human plasma.

During the development and testing of a radioreceptor assay (RRA) for human IL-1, we have detected and identified the presence of auto-antibodies to IL-1 in normal human plasma (NHP). The RRA is based on the competition between human 125I-labeled rIL-1 alpha and standard or unknown quantities of IL-1 alpha or IL-1 beta for binding to a limited amounts of IL-1 receptor (IL-1R) isolated from the EL4 mouse thymoma cell line. NHP from 20 out of 100 unselected blood donors were found to completely inhibit the binding of 125I-labeled IL-1 alpha to its receptor, suggesting the presence in these NHP samples of either abnormal amounts of IL-1 or of a factor binding to the 125I-labeled IL-1 alpha. Special care was taken to ascertain that the inhibitory factors were antibodies and not soluble IL-1 receptor antagonist. When plasma samples with inhibiting activity were incubated with labeled IL-1 alpha and chromatographed on a Sephadex G200 column, they were found to contain 125I-labeled complexes with an apparent molecular weight of 150-200kD. The IL-1 binding factor could be eliminated from plasma by incubation with protein A-Sepharose, suggesting that it consisted in IgG antibodies directed against IL-1. Furthermore, the antibody nature of the inhibiting factor was confirmed by its binding to purified rIL-1 coupled to Sepharose. Screening of 200 NHP samples by incubation with 100 pg of 125I-labeled IL-1 followed by precipitation with 12% of polyethylene glycol (PEG) confirmed that about 25% of NHP contain detectable IgG antibodies to IL-1 alpha, while only 2% of NHP contain antibodies to IL-1 beta. No correlation between the presence of these anti-IL-1 antibodies and any particular major histocompatibility complex or any pathological conditions was detected. We suggest that all serum samples assayed for IL-1 alpha or IL-1 beta content should be pretested with the PEG precipitation assay described here.

Assessment of vaccine-induced CD4 T cell responses to the 119-143 immunodominant region of the tumor-specific antigen NY-ESO-1 using DRB1*0101 tetramers.

Abstract: Purpose: NY-ESO-1 (ESO), a tumor-specific antigen of the cancer/testis group, is presently viewed as an important model antigen for the development of generic anticancer vaccines. The ESO119-143 region is immunodominant following immunization with a recombinant ESO vaccine. In this study, we generated DRB1*0101/ESO119-143 tetramers and used them to assess CD4 T-cell responses in vaccinated patients expressing DRB1*0101 (DR1). Experimental Design: We generated tetramers of DRB1*0101 incorporating peptide ESO119-143 using a previously described strategy. We assessed ESO119-143-specific CD4 T cells in peptide-stimulated post-vaccine cultures using the tetramers. We isolated DR1/ESO119-143 tetramer(+) cells by cell sorting and characterized them functionally. We assessed vaccine-induced CD4(+) DR1/ESO119-143 tetramer(+) T cells ex vivo and characterized them phenotypically. Results: Staining of cultures from vaccinated patients with DR1/ESO119-143 tetramers identified vaccine-induced CD4 T cells. Tetramer(+) cells isolated by cell sorting were of T(H)1 type and efficiently recognized full-length ESO. We identified ESO123-137 as the minimal optimal epitope recognized by DR1-restricted ESO-specific CD4 T cells. By assessing DR1/ESO119-143 tetramer(+) cells using T cell receptor (TCR) beta chain variable region (V beta)-specific antibodies, we identified several frequently used V beta. Finally, direct ex vivo staining of patients' CD4 T cells with tetramers allowed the direct quantification and phenotyping of vaccine-induced ESO-specific CD4 T cells. Conclusions: The development of DR1/ESO119-143 tetramers, allowing the direct visualization, isolation, and characterization of ESO-specific CD4 T cells, will be instrumental for the evaluation of spontaneous and vaccine-induced immune responses to this important tumor antigen in DR1-expressing patients

Synthesis of a radioiodinated photoreactive MAGE-1 peptide derivative and photoaffinity labeling of cell-associated human leukocyte antigen-A1 molecules.

The synthesis of a photoreactive derivative of the human leukocyte antigen-A1 (HLA-A1)-restricted MAGE-1 peptide 161-169 (EADPTGHSY) is described. Using conventional automated solid-phase peptide synthesis, a photoreactive derivative of this peptide was synthesized by replacing histidine-167 with photo-reactive N-beta-4-azidosalicyloyl-L-2,3-diaminopropionic acid. The C-terminal tyrosine was incorporated as phosphotyrosine. This peptide derivative was radioiodinated in the presence of chloramine T. This iodination took place selectively at the photoreactive group, because the phosphate ester prevented tyrosine iodination. Following dephosphorylation with alkaline phosphatase and chromatographic purification, the radiolabeled peptide derivative was incubated with cells expressing HLA-A1 or other HLA molecules. Photoactivation resulted in efficient photoaffinity labeling of HLA-A1. Other HLA molecules or other cellular components were not detectably labeled. This labeling was inhibited by HLA-A1 but not by HLA-A2-binding peptides. This synthesis is generally applicable and can also be adapted to the synthesis of well-defined radiolabeled nonphotoreactive peptide derivatives.