[Cancer immunotherapy: new approaches].
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Cancer Vaccines↗
Biomedical subjects
L Alberti
Publications and source records attributed to L Alberti.
The Glu632-Leu633 deletion in cysteine rich domain of Ret induces constitutive dimerization and alters the processing of the receptor protein.
Mutations of the RET gene, encoding a receptor tyrosine kinase, have been associated with the inherited cancer syndromes MEN 2A and MEN 2B. They have also further been associated with both familial and sporadic medullary thyroid carcinomas. Missense mutations affecting cysteine residues within the extracellular domain of the receptor causes constitutive tyrosine kinase activation through the formation of disulfide-bonded homodimers. We have recently reported that a somatic 6 bp in-frame deletion, originally coding for Glu632-Leu633, potently activates the RET gene. This activation is increased with respect to the frequent MEN 2A-associated missense mutation Cys634Arg. This finding specifically correlated to the clinic behavior of the corresponding tumor, which was characterized by an unusually aggressive progression with both multiple and recurrent metastases. By examining the possibility that this deletion acts in a manner similar to cysteine substitution, we have analysed the molecular mechanism by which this oncogenic activation occurs. Phosphorylated dimers of the deleted Ret receptor were detected in immunoprecipitates separated under non-reducing conditions. Like other Cys point mutations, this 6 bp deletion affecting two amino acid residues between two adjacent Cys, is capable of activating the transforming ability of Ret by promoting receptor dimerization. These results suggest that alteration to cysteine residue position or pairing is capable of inducing ligand independent dimerization. Furthermore, we present data demonstrating that the processing and sorting of the Ret membrane receptor to the cell surface is affected by mutation type.
3T3 Cells↗
Diagnosing mental disorders in primary care: the General Health Questionnaire (GHQ) and the Symptom Check List (SCL-90-R) as screening instruments.
BACKGROUND: The treatment of mental disorders in Germany is mainly done by primary care physicians. Several studies have shown that primary care physicians have difficulty in diagnosing these disorders. Recently, several self-report questionnaires have been developed that can be used as screening instruments to identify psychopathology in primary care settings and in the community. The aim of this paper was to investigate the screening properties of the General Health Questionnaire (GHQ-12) and the Symptom Check-List (SCL-90-R) in a primary care setting in Germany. METHOD: A randomly selected sample (n = 408) of adult outpatients from 18 primary care offices in Düsseldorf was screened using the German versions of the GHQ-12 and the SCL-90-R. A structured diagnostic interview (SCID) and an impairment rating (IS) were used as a gold standard to which both questionnaires were compared. Test performance was evaluated by receiver operating characteristic (ROC) analysis. RESULTS: We found no difference in the performance of the general scores of the two questionnaires. Both instruments were able to detect cases. Complex scoring methods offered no advantages over simpler ones for the GHQ-12. ROC analysis confirmed that the SCL-90-R subscales "anxiety" and "depression" showed acceptable concurrent validity for the diagnostic groups anxiety and depression (according to DSM-III-R). CONCLUSIONS: GHQ-12 and SCL-90-R appeared to be useful tools for identifying mental disorders in primary care practice and research. The use of GHQ-12 or SCL-90-R, employed as a first step, supplemented by a second-stage interview, may enhance the detection rate of mental disorder in primary care settings.
Adult↗
[Diagnosis and management of patients with psychogenic disorders in family practice. Results of a field study].
The majority of patients with psychological disorders are seen in primary care. The diagnosis of the general practitioner (GP) influences decisively the subsequent treatment. Our study provides an insight into GP's diagnosis and treatment. 18 GP's and a psychotherapist rated 572 patients using a global diagnostic rating scale and the impairment score (BSS). The GPs noted down their treatment. The patients filled in symptom checklists (SCL-90-R, GHQ-12). Agreement between the doctor and the researchers on mental ill health was modest. GPs identified 51.4% of the patients with psychological disorders. Adjustment disorders were often ignored by GPs. They prescribed psychotherapy for 3.3% of their patients, especially patients with somatoform disorders and with high values in the impairment score. A path analysis shows, that psychotherapists' ratings agree much more with patients' self-assessment than GP ratings.
Adult↗
Grb2 binding to the different isoforms of Ret tyrosine kinase.
The RET proto-oncogene encodes two isoforms of a receptor tyrosine kinase which plays a role in neural crest and kidney development. Ret ligands have been recently identified as the neuron survival factor GDNF (Glial-Derived Neurotrophic Factor) and Neurturin. Somatic rearrangements of RET, designated RET/PTCs, have been frequently detected in papillary thyroid carcinomas. In addition, distinct germ-line mutations of RET gene have been associated with the inherited cancer syndromes MEN (Multiple Endocrine Neoplasia) 2A, 2B and FMTC (Familial Medullar Thyroid Carcinomas) as well as with the congenital megacolon or Hirschsprung's disease, thus enlightening a significant role of this receptor gene in diverse human pathologic conditions. In this study, by performing classical inhibition experiments using synthetic phosphopeptides and by site-directed mutagenesis of the putative docking site, we have determined that for Grb2 the latter is provided by the tyrosine 620 of Ret/ptc2 long isoform (corresponding to Tyr 1096 on proto-Ret). However, in intact cells, the interaction of Grb2 with the two short and long Ret isoforms expressed separately is of similar strength, thus suggesting that Ret short isoform interaction with Grb2 could be mediated not only by Shc but also by a molecule that binds preferentially to this isoform. This possibility is supported by the evidence that the mutant Ret/ptc2Y620F long isoform displays a weak coimmunoprecipitation with Grb2 and that this mutant, lacking the docking site for Grb2 but owing all the others phosphotyrosines, surprisingly displays a reduced transforming activity compared to that of the two WTs oncogenes. We thus conclude that in intact cells both Ret isoforms bind to Grb2, although with different modalities. In addition, the present results are in agreement with the possibility that different signal transduction pathways are associated with the two isoforms of Ret.
3T3 Cells↗
Full activation of MEN2B mutant RET by an additional MEN2A mutation or by ligand GDNF stimulation.
Germline mutations of RET gene, encoding a receptor tyrosine kinase, have been associated with the MEN2A and MEN2B inherited cancer syndromes. In MEN2A mutations affecting cysteine residues in the extracellular domain of the receptor cause constitutive activation of the tyrosine kinase by the formation of disulfide-bonded homodimers. In MEN2B a single mutation in the tyrosine kinase domain (Met918Thr) has been identified. This mutation does not lead to dimer formation, but has been shown (both biologically and biochemically) to cause ligand-independent activation of the Ret protein, but to a lesser extent than MEN2A mutations. Intramolecular activation by cis-autophosphorylation of RetMEN2B monomers has been proposed as a model for activation, although alternative mechanisms can be envisaged. Here we show that the activity of RetMEN2B can be increased by stable dimerization of the receptor. Dimerization was achieved experimentally by constructing a double mutant receptor with a MEN2A mutation (Cys634Arg) in addition to the MEN2B mutation, and by chronic exposure of RetMEN2B-expressing cells to the Ret ligand GDNF. In both cases full activation of RetMEN2B, measured by 'in vitro' transfection assays and biochemical parameters, was seen. These results indicate that the MEN2B phenotype could be influenced by the tissue distribution or concentration of Ret ligand(s).
3T3 Cells↗
[Psychosomatic disorders in dermatology--incidence and need for added psychosomatic treatment].
In a cooperative study involving the Department of Dermatology and the Department of Psychosomatic Medicine/Psychotherapy of the University of Düsseldorf, 187 patients were evaluated for psychosomatic disease and indenient psychological testing. The distribution of skin diseases and psychosomatic ICD 10 diagnoses are presented. It was found that patient evaluation-(symptom-complaint-questionnaire) and expert evaluation correlate positive. It therefore seems worthwhile examining dermatology in-patient's for psychosomatic problems in order to facilitate combined dermatological and psychosomatic approach not only in the hospital but also during the post-discharge ambulatory treatment.
Adaptation, Psychological↗
Further insights into the anti-aggregating activity of NMDA in human platelets.
1. In the present study the effect of N-methyl-D-aspartate (NMDA) on thromboxane B2 synthesis and on [Ca2+]i was studied in human platelets. 2. NMDA (10(-7) M) completely inhibited the synthesis of thromboxane B2 from exogenous arachidonic acid (AA), while it did not interfere with the aggregating effect of the thromboxane A2 receptor agonist U-46619. 3. NMDA (0.1 microM - 10 microM) dose-dependently increased intracellular calcium in washed platelets preloaded with fura 2 AM, and this effect was not additive with that of AA. 4. NMDA shifted the dose-response curve of AA to the right. At the highest AA concentrations platelet aggregation was not inhibited. 5. The antiaggregating effect of NMDA was not antagonized by N(G)-monomethyl-L-arginine (L-NMMA), a nitric oxide synthase (NOS) inhibitor. 6. Finally, NMDA (0.01 nM - 100 nM) associated with either aspirin or indomethacin significantly potentiated the antiaggregating activity of both cyclo-oxygenase inhibitors. 7. It was concluded that NMDA is a potent inhibitor of platelet aggregation and thromboxane B2 synthesis in human platelet rich plasma (PRP).
Aspirin↗
Identification of Shc docking site on Ret tyrosine kinase.
The RET proto-oncogene encodes two isoforms of a receptor type tyrosine kinase which plays a role in neural crest and kidney development. Distinct germ-line mutations of RET have been associated with the inherited cancer syndromes MEN2A, MEN2B and FMTC as well as with the congenital disorder Hirschsprung disease (HSCR), whereas somatic rearrangements (RET/PTCs) have been frequently detected in the papillary thyroid carcinoma. Despite these findings, suggesting a relevant role for RET product in development and neoplastic processes, little is known about the signalling triggered by this receptor. In this study, we have demonstrated that the transducing adaptor molecule Shc is recruited and activated by both Ret isoforms and by the rearranged cytoplasmatic Ret/ptc2 oncoproteins as well as by the membrane bound receptor activated by MEN2A or by MEN2B associated mutations. Moreover, our analysis has identified the Ret tyrosine residue and the Shc domains involved in the interaction. In fact, here we show that both the phosphotyrosine binding domains of Shc, PTB and SH2, interact with Ret/ptc2 in vitro. However, PTB domain binds 20 folds higher amount of Ret/ptc2 than SH2. The putative binding site for either SH2 and PTB domains has been identified as Tyr586 of Ret/ptc2 (Tyr1062 on proto-Ret). In keeping with this finding, by using RET/PTC2-Y586F mutant, we have demonstrated that this tyrosine residue, the last amino acid but one before the divergence of the two Ret isoforms, is the docking site for Shc.
3T3 Cells↗
The full oncogenic activity of Ret/ptc2 depends on tyrosine 539, a docking site for phospholipase Cgamma.
RET/PTC oncogenes, generated by chromosomal rearrangements in papillary thyroid carcinomas, are constitutively activated versions of proto-RET, a gene coding for a receptor-type tyrosine kinase (TK) whose ligand is still unknown. RET/PTCs encode fusion proteins in which proto-RET TK and C-terminal domains are fused to different donor genes. The respective Ret/ptc oncoproteins display constitutive TK activity and tyrosine phosphorylation. We found that Ret/ptcs associate with and phosphorylate the SH2-containing transducer phospholipase Cgamma (PLCgamma). Two putative PLCgamma docking sites, Tyr-505 and Tyr-539, have been identified on Ret/ptc2 by competition experiments using phosphorylated peptides modelled on Ret sequence. Transfection experiments and biochemical analysis using Tyr-->Phe mutants of Ret/ptc2 allowed us to rule out Tyr-505 and to identify Tyr-539 as a functional PLCgamma docking site in vivo. Moreover, kinetic measurements showed that Tyr-539 is able to mediate high-affinity interaction with PLCgamma. Mutation of Tyr-539 resulted in a drastically reduced oncogenic activity of Ret/ptc2 on NIH 3T3 cells (75 to 90% reduction) both in vitro and in vivo, which correlates with impaired ability of Ret/ptc2 to activate PLCgamma. In conclusion, this paper demonstrates that Tyr-539 of Ret/ptc2 (Tyr-761 on the proto-RET product) is an essential docking site for the full transforming potential of the oncogene. In addition, the present data identify PLCgamma as a downstream effector of Ret/ptcs and suggest that this transducing molecule could play a crucial role in neoplastic signalling triggered by Ret/ptc oncoproteins.
3T3 Cells↗
[What is supportive psychotherapy?].
Although supportive psychotherapy is of great importance in psychiatry and psychosomatic medicine, supportive treatment strategies have not been paid much attention so far in literature and training. Clinical indication criteria for supportive techniques are: lack of treatment motivation and introspection, impairment of object relations and impulse control, inadequate modulation of affect and difficulties in establishing a therapeutic working alliance. After reviewing the principles of supportive psychotherapy, special aspects of supportive treatment techniques are discussed.
Humans↗
RET activation by germline MEN2A and MEN2B mutations.
The RET proto-oncogene encodes a receptor tyrosine kinase (TK). It has been shown that distinct germline mutations in the RET proto-oncogene are associated with the dominantly inherited cancer syndromes multiple endocrine neoplasia type 2A and 2B (MEN 2A and MEN 2B) and familial medullary thyroid carcinoma (FMTC) as well as Hirschsprung disease (HSCR), a congenital disorder characterised by absent enteric innervation. In this study, we have transfected NIH3T3 and PC12 phaeochromocytoma cells with MEN2A (Cys634-> Arg) and MEN2B (Met918-> Thr) RET constructs. Both caused transformation of the NIH3T3 cells and differentiation of PC12 cells. The Ret (MEN2A) and Ret (MEN2B) proteins were constitutively phosphorylated on tyrosine, and their in vitro kinase activity was significantly higher than that of the wild type protein. The MTC cell line TT carries a CYs634-> Trp MEN2A mutation, and we have shown by immunoelectronmicroscopy that Ret is clustered on the cell surface in a manner reminiscent of ligand-induced aggregation of cell surface receptors. RET is activated, as RET/PTC oncogene, by somatic rearrangements which link the TK domain to a constitutive dimerization interface in papillary thyroid carcinomas. We have compared the biological and biochemical activity of the TK domains of the wild type and MEN 2B Ret in the context of the RET/PTC. The results show that the MEN 2B mutation significantly increases the TK domain enzymatic activity suggesting that dimerization may be still necessary for MEN 2B Ret to express its full activity.
3T3 Cells↗
Loss of function effect of RET mutations causing Hirschsprung disease.
We have introduced three Hirschsprung (HSCR) mutations localized in the tyrosine kinase domain of RET into the RET/PTC2 chimaeric oncogene which is capable of transforming NIH3T3 mouse fibroblasts and of differentiating pC12 rat pheochromocytoma cells. The three HSCR mutations abolished the biological activity of RET/PTC2 in both cell types and significantly decreased its tyrosine phosphorylation. By contrast, a rare polymorphism in exon 18 does not alter the transforming capability of RET/PTC2 or its tyrosine phosphorylation. These data suggest a loss of function effect of HSCR mutations which might act through a dominant negative mechanism. Our model system is therefore capable of discriminating between causative HSCR mutations and rare polymorphisms in the tyrosine kinase domain of RET.
3T3 Cells↗
Cortisone image and emotional support by key figures in patients with bronchial asthma. An empirical study.
In a subgroup of patients with bronchial asthma, irrational fears of steroid medication can be observed beyond justified worries about side-effects. A cortisone image which involves overemphasizing the threatening aspects of cortisone often underlies non-compliant illness behaviour. In the present study, cortisone image and the relationship with a key figure was investigated in 62 patients with bronchial asthma, all of whom had participated in a structured asthma treatment and teaching programme (ATTP). The data show that (1) perceiving cortisone as threatening predicts poor adherence to ATTP guidelines how to manage severe attacks, and (2) patients having a supportive relationship with their key figure are less likely to experience threatening aspects of cortisone.
Acute Disease↗
Mesostriatal dopaminergic axons transiently express high levels of NILE during development.
The distribution of nerve growth factor-inducible large external (NILE) glycoprotein in the developing mesostriatal dopaminergic system was studied by immunolabeling prenatal, postnatal, and adult rat brains for both NILE and tyrosine hydroxylase (TH). NILE-immunoreactive (NILE-IR) material outlined the TH-immunoreactive (TH-IR) neurons in the ventral mesencephalon at ages E14, E15, and E16 but not at later ages. Fascicles of TH-IR axons in the developing mesostriatal dopaminergic tract were strongly immunoreactive for NILE from E15 through E20. In the postnatal brain, NILE-IR material no longer clearly outlined the fascicles of dopaminergic axons as they ran from the mesencephalon to the striatum. In the striatum of postnatal and adult rats, NILE-IR material was diffusely distributed through the neuropil. Our observations suggest that NILE may play a role in guidance of growing dopaminergic axons in the developing mesostriatal tract and aggregation of the dopaminergic axons into fascicles.
Animals↗
Mutations that impair swarming motility in Serratia marcescens 274 include but are not limited to those affecting chemotaxis or flagellar function.
Serratia marcescens exists in two cell forms and displays two kinds of motility depending on the type of growth surface encountered (L. Alberti and R. M. Harshey, J. Bacteriol. 172:4322-4328, 1990). In liquid medium, the bacteria are short rods with few flagella and show classical swimming behavior. Upon growth on a solid surface (0.7 to 0.85% agar), they differentiate into elongated, multinucleate, copiously flagellated forms that swarm over the agar surface. The flagella of swimmer and swarmer cells are composed of the same flagellin protein. We show in this study that disruption of hag, the gene encoding flagellin, abolishes both swimming and swarming motility. We have used transposon mini-Mu lac kan to isolate mutants of S. marcescens defective in both kinds of motility. Of the 155 mutants obtained, all Fla- mutants (lacking flagella) and Mot- mutants (paralyzed flagella) were defective for both swimming and swarming, as expected. All Che- mutants (chemotaxis defective) were also defective for swarming, suggesting that an intact chemotaxis system is essential for swarming. About one-third of the mutants were specifically affected only in swarming. Of this class, a large majority showed active "swarming motility" when viewed through the microscope (analogous to the active "swimming motility" of Che- mutants) but failed to show significant movement away from the site of initial inoculation on a macroscopic scale. These results suggest that bacteria swarming on a solid surface require many genes in addition to those required for chemotaxis and flagellar function, which extend the swarming movement outward. We also show in this study that nonflagellate S. marcescens is capable of spreading rapidly on low-agar media.
Cell Division↗
[Affective-cognitive coping with attacks and illness behavior in patients with bronchial asthma].
The purpose of this study was to investigate attack-related cognitive coping styles in 80 patients with bronchial asthma and to relate them to outcome parameters of illness behaviour. All patients had participated in a 5-day-lasting asthma treatment and teaching programme (ATTP). Outcome variables to be predicted were: The number of days in hospital due to asthma during the year following the training programme and the patients' adherence to the recommendations of the programme one year later. Independent predictor variables were: pre-training attack frequency, hospitalization due to asthma, medication intensity, pulmonary function, asthma symptom anxiety, trait anxiety and attack-related cognitive coping styles. Multiple regression analyses show the importance of a "minimizing/self-confident" coping style in predicting days in hospital, while a "diverting" style predicts best a poor management of attacks in patients on oral steroids.
Adaptation, Psychological↗
Molecular cloning of NILE glycoprotein and evidence for its continued expression in mature rat CNS.
The NILE glycoprotein is a rat neuronal cell adhesion molecule which has been reported to be very similar in structure, function, and distribution to the mouse L1 glycoprotein. Here we report the complete nucleotide sequence of the NILE message (5,208 nucleotides) and the deduced amino acid sequence of the NILE polypeptide (1,257 amino acids). The predicted NILE protein is 96% identical to L1 at the amino acid level, confirming that the two molecules are homologues. The sequence information shows that NILE is a transmembrane molecule with an extensive ectodomain and a much smaller cytoplasmic domain. The extracellular portion of the molecule contains six immunoglobulin C-2 type domains followed by five fibronectin type III repeats. These two structural motifs are characteristic of several other cell adhesion molecules. The cytoplasmic tails of NILE and L1 are identical to each other and distinct from the cytoplasmic regions of all other cell adhesion molecules except Ng-CAM and neuroglian. Several possible sites for phosphorylation are present in the cytoplasmic tail of NILE. Antisera were produced against two NILE-beta-galactosidase fusion proteins containing distinct segments of the NILE polypeptide: the cytoplasmic domain and the segment containing fibronectin type III repeats. Immunoblots with these antisera and Northern blots with a NILE cDNA probe indicate that NILE continues to be expressed in most areas of the mature rat brain. This contradicts previous immunofluorescence data, which suggested that NILE was substantially down-regulated in maturing nerve fiber tracts. This raises the possibility that NILE could be masked in situ by interactions with other cell surface molecules.
Aging↗