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Graves' disease is one of the most common causes of hyperthyroidism. Guideline recommendations advocate the intake of thionamides for at least 1 year. If hyperthyroidism persists, subsequent radioiodine-131 treatment (RIT) is a therapeutic option. Thionamides are known to influence intra-thyroidal bio-kinetics of iodine and should therefore be discontinued at least 3 days prior to RIT if possible. However, the required therapeutic activity has to be calculated individually by pre-therapeutic measurement of the uptake prior to RIT [radioiodine-131 uptake test (RIUT)] in Germany according to national guidelines. Therefore, the aim of this study was to quantify the influence of thionamides on intra-therapeutic uptake. A cohort of 829 patients with Graves' disease undergoing RIUT and RIT was analysed. Patients were subdivided into three groups. Group A: patients with carbimazole medication (n = 312), group B: patients with methimazole medication (n = 252) and group C: patients without thionamides (n = 265). Group A and B were further subdivided depending on the reduction of dosage of thionamides. In order to analyse the influence of thionamides, the variance of the determined individual extrapolated maximum intra-thyroidal uptake (EMU) between RIUT and RIT within the single groups and within the subgroups was statistically evaluated. When administering an equal dose of thionamides or no thionamides in RIUT and RIT (groups A1, B1 and C) no significant differences were detected when comparing EMU in RIT to EMU in RIUT (p > 0.05). In the subgroups A2-A4 (reduced dosage of carbimazole prior to RIT) EMU was significantly increased in RIT compared to RIUT [21% for a reduction of 0 to < 10 mg/d (A2), 39% for a reduction of 10-15 mg/d (A3) and 80% for a reduction of > 15 mg/d (A4)]. In the subgroups B2-B4 (reduced dosage of methimazole prior to RIT) EMU was as well significantly increased in RIT compared to RIUT [26% for a reduction of 0 to < 10 mg/d (B2), 36% for a reduction of 10-15 mg/d (B3) and 59% for a reduction of > 15 mg/d (B4)]. A significant dose-dependent increase of EMU in RIT compared to EMU in RIUT in patients discontinuing or reducing thionamides was detected. Therefore, thionamides should be discontinued at least 2 days prior to RIUT in order to achieve the designated target dose more precisely and to minimize radiation exposure of organs at risk.
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Enfermedad de Graves , Hipertiroidismo , Humanos , Radioisótopos de Yodo/uso terapéutico , Metimazol , Carbimazol/uso terapéutico , Enfermedad de Graves/tratamiento farmacológico , Enfermedad de Graves/radioterapiaRESUMEN
Despite a significantly improved dietary iodine supply, solitary toxic thyroid nodules (STN) are still a common clinical problem in former iodine deficient areas. Radioiodine treatment (RIT) is a well-established therapeutic option with few side effects and high success rates. As radioiodine biokinetics are individual for every patient, the necessary activity has to be calculated individually by a pre-therapeutic measurement of the intra-therapeutic effective half-life (EHL) in a radioiodine uptake test (RIUT). A suppressive medication with triiodothyronine (T3) or tetraiodothyronine (T4) is often needed to suppress uptake in normal thyroid tissue. Therefore, the aim of this study was to quantify the possible influence of this medication on intra-therapeutic radioiodine biokinetics. A cohort of 928 patients with STN undergoing RIUT and RIT was analysed. Patients were subdivided into 3 groups. Group T3: medication with T3 (n = 274), group T4: medication with T4 (n = 184) and group NM: no additional medication (n = 470). The T3 and T4 subgroups were further subdivided depending on the dose of thyroid hormone medication. In order to analyse the influence of thyroid hormone medication on individual intra-thyroidal biokinetics, the variance of the determined individual EHL between RIUT and RIT within the single groups and within the subgroups was investigated. EHL was significantly decreased between RIUT and RIT in the T3 and T4 subgroups (EHL: T3: 5.9 ± 1.1 d in RIUT and 3.3 ± 1.4 d in RIT (- 43%) (p < 0.05); T4: 5.9 ± 1.2 d in RIUT and 3.4 ± 1.5 d in RIT (- 42%) (p < 0.05). The decrease of EHL did not differ statistically between T3 or T4. However, both showed a highly significant difference compared to the NM group (p < < 0.05). A further subgroup analysis showed a significant dependence of the decrease in EHL related to the dose of thyroid hormone medication of 35-58% (T3) and 15-67% (T4) (p < 0.05). A significantly reduced EHL compared to RIUT in patients receiving thyroid hormone medication was detected. Moreover, a significant correlation between the dose of thyroid hormone medication (T3 or T4) and the decrease of EHL was found. Therefore, an adaption of the calculated activity should be considered in RIUT to obtain the required radiation dose in RIT of patients suffering from STN.
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Yodo , Nódulo Tiroideo , Semivida , Humanos , Radioisótopos de Yodo/uso terapéutico , Nódulo Tiroideo/tratamiento farmacológico , Tiroxina , TriyodotironinaRESUMEN
Aim: Therapy success in patients with differentiated thyroid cancer (DTC) after thyroidectomy and radioiodine therapy (RIT) is proven by permanent decrease in human thyroglobulin (hTg) to <1 ng/mL. In this retrospective analysis hTg development before, during and after pregnancy were analyzed. Material and methods: A descriptive analysis of hTg courses in 47 women with 57 pregnancies under levothyroxine substitution was performed after treatment of DTC without evidence of residual or recurrent disease. We compared hTg levels before, during and after pregnancies. A median of four measurements were performed during pregnancy. Results: In five out of the 47 patients at least one hTg increase to ≥1.0 ng/mL occurred during pregnancy (P1: 1.1; P2: 1.75; P3: 1.0; P4: 1.1; P5: 1.07 ng/mL). In another three cases an increase to ≥0.5 ng/mL occurred. After delivery, all patients returned to undetectable hTg levels. Human Tg maxima during pregnancy were significantly elevated according to Friedman´s Chi2 and p Holm−Bonferroni. Conclusion: In women with ablative thyroid therapy after DTC, a temporary elevation in hTg levels during pregnancy may occur. The reason therefore remains unclear and requires further investigation.
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Adenocarcinoma , Neoplasias de la Tiroides , Femenino , Humanos , Radioisótopos de Yodo/uso terapéutico , Embarazo , Estudios Retrospectivos , Tiroglobulina/análisis , Neoplasias de la Tiroides/terapia , TiroidectomíaRESUMEN
AIM: The aim is to add a pragmatic contribution to the discussion of an algorithm to discharge patients treated with Lu-177-PSMA under the aspect of radiation protection. This also may be applied to therapies with other radioactive tracers in the future. MATERIAL AND METHODS: 478 cycles of Lu-177-PSMA-617 (140 patients) were analyzed. The remaining activity in the patient and the dose rate were correlated. From frequent intratherapeutic measurements (biexponential fit) scenarios for discharging patients are deduced. RESULTS: Thirty-four per cent of all patients treated with Lu-177-PSMA received 3 to 5 cycles per calendar year. The dose limit of 1 mSv per calendar year (German Law) at a distance of 2 m from the patient would be exceeded in 10 % and 15 % of the treated patients if discharged 72 hours after treatment given 3 and 4 cycles per calendar year, respectively. Mean specific dose rate was 0.00462µSv/(h MBq) at a distance of 1 m. A universal correlation between dose rate and the remaining activity in the patient could not be found. CONCLUSION: The multi cycle concept of the therapies with Lu-177 PSMA has to be taken into account prospectively when discharging the patients. Given the physical half-life of Lu-177 an anticipation of 4 treatment cycles per calendar year leads to a clearly arranged, conservative rule.
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Lutecio , Neoplasias de la Próstata Resistentes a la Castración , Compuestos Heterocíclicos con 1 Anillo/uso terapéutico , Humanos , Lutecio/uso terapéutico , Masculino , Alta del Paciente , Radioisótopos/uso terapéutico , Radiofármacos/uso terapéuticoRESUMEN
ZIEL: Ziel der Untersuchung ist es, die Strahlenwirkung des ß--Emitters 186Re und von 662keV-Photonenstrahlung zu ermitteln, um die biologische Wirkung von Strahlung niedriger Dosisleistung (186Re) mit der hoher Dosisleistung zu vergleichen. MATERIAL UND METHODEN: Zellen der humanen Leukämie-Zelllinie BV-173 wurden mit 662keV-Photonenstrahlung respektive 186Re bestrahlt. In einem Inkubationszeitraum von 7 Tagen wurden Zahl und Vitalität der Zellen täglich bestimmt und als Dosiseffektkurven basierend auf der Vitalität dargestellt. Hierfür wurde der Zeitpunkt mit minimalem Überleben verwendet (72h 186Re und 24h Photonenstrahlung). ERGEBNISSE: Beide Strahlenarten zeigen am Auswertezeitpunkt (72h nach Versuchsbeginn für 186Re und 24h nach Versuchsbeginn für Photonenstrahlung) eine Überlebenskurve mit biexponentiellem Verlauf. Für Photonenstrahlung ist dies erklärbar durch eine Hypersensitivität im niedrigen Dosisbereich bis 1Gy, für die sich eine D0 von 3,3Gy ergibt, für Dosen über 1,0Gy liegt die D0 bei 10Gy. Für die 186Re-Inkubation ergibt sich eine D0 von 11,1Gy bei niedrigen Dosen verursacht durch die Reparatur subletaler Schäden, durch welche die biologische Wirkung abgeschwächt wird. Ab einer akkumulierten Dosis von etwa 1,6Gy zeichnet sich für 186Re ein wesentlich steilerer Kurvenverlauf mit einer D0 von 4,0Gy ab, der eine in diesem Bereich 2,5-fach stärkere biologische Wirkung als akute Photonenstrahlung wiedergibt (D0 4Gy für 186Re bzw. 10Gy für Photonen). SCHLUSSFOLGERUNG: Strahlung niedriger Dosisleistung zeigt eine geringere biologische Wirkung als eine akute Bestrahlung. Es existiert aber ein Grenzwert der akkumulierten Dosis, ab dem die biologische Wirkung von ß-Strahlung die der Photonenstrahlung sogar übertrifft.
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Línea Celular , Radiación , HumanosRESUMEN
BACKGROUND: Myelosuppression is a potential dose-limiting factor in radioligand therapy (RLT). This study aims to investigate occurrence, severity and reversibility of hematotoxic adverse events in patients undergoing RLT with 177Lu-PSMA-617 for metastatic castration-resistant prostate cancer (mCRPC). The contribution of pretreatment risk factors and cumulative treatment activity is taken into account specifically. METHODS: RLT was performed in 140 patients receiving a total of 497 cycles. A mean activity of 6.9 [Formula: see text] 1.3 GBq 177Lu-PSMA-617 per cycle was administered, and mean cumulative activity was 24.6 [Formula: see text] 15.9 GBq. Hematological parameters were measured at baseline, prior to each treatment course, 2 to 4 weeks thereafter and throughout follow-up. Toxicity was graded based on Common Terminology Criteria for Adverse Events v5.0. RESULTS: Significant (grade ≥ 3) hematologic adverse events occurred in 13 (9.3%) patients, with anemia in 10 (7.1%), leukopenia in 5 (3.6%) and thrombocytopenia in 6 (4.3%). Hematotoxicity was reversible to grade ≤ 2 through a median follow-up of 8 (IQR 9) months in all but two patients who died from disease progression within less than 3 months after RLT. Myelosuppression was significantly more frequent in patients with pre-existing grade 2 cytopenia (OR: 3.50, 95%CI 1.08-11.32, p = 0.04) or high bone tumor burden (disseminated or diffuse based on PROMISE miTNM, OR: 5.08, 95%CI 1.08-23.86, p = 0.04). Previous taxane-based chemotherapy was associated with an increased incidence of significant hematotoxicity (OR: 4.62, 95%CI 1.23-17.28, p = 0.02), while treatment with 223Ra-dichloride, cumulative RLT treatment activity and activity per cycle were not significantly correlated (p = 0.93, 0.33, 0.29). CONCLUSION: Hematologic adverse events after RLT have an acceptable overall incidence and are frequently reversible. High bone tumor burden, previous taxane-based chemotherapy and pretreatment grade 2 cytopenia may be considered as risk factors for developing clinically relevant myelosuppression, whereas cumulative RLT activity and previous 223Ra-dichloride treatment show no significant contribution to incidence rates.
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AIM: Peptide receptor radionuclide therapy (PRRT) with 177Lu-HA-DOTATATE has evolved as a new path in the treatment of somatostatin-receptor-expressing neuroendocrine tumors. The kidneys are proven as organs at risk and should be evaluated dosimetrically. Overlap with other organs will make dosimetry based on planar scintigraphy inaccurate. Aim of this study was to approximate the contribution of the kidneys to conjugated planar views without the use of a SPECT/CT. MATERIAL AND METHOD: An algorithm was developed to determine the kidney dose using an EXCEL (Microsoft) based program. Dosimetric data were drawn and merged from three modalities: an individually calibrated gamma probe, a whole-body scintigraphy (WBS) and SPECT-acquisitions. The method was evaluated for 85 kidneys. Kidney masses were obtained via CT volumetry. RESULTS: The developed algorithm combines data from the three modalities. The ratio of the events within a kidney-VOI and the events from the summed coronary SPECT views (kidney ROI) represents the contribution of the kidney to the whole-body kidney ROI. This fraction was calculated to 49 % (17 % - 78 %) and 45 % (18 % - 75 %) for the left and the right kidney, respectively. Quantification of activity was deduced from equalizing the WBS count with the concurrent gamma probe measurement. Monoexponential curves were fitted to the obtained kidney activities, with resulting doses of 0,13 to 0,77 Gy/GBq (average 0,36 and 0,39 Gy/GBq for the left and the right kidney). CONCLUSION: The presented method is suitable to perform kidney dosimetry by using a gamma probe and a gamma camera, without using SPECT/CT.
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Complejos de Coordinación/uso terapéutico , Tumores Neuroendocrinos/diagnóstico por imagen , Tumores Neuroendocrinos/radioterapia , Octreótido/análogos & derivados , Radiometría/métodos , Tomografía Computarizada de Emisión de Fotón Único , Imagen de Cuerpo Entero , Humanos , Octreótido/uso terapéutico , Radiometría/instrumentaciónRESUMEN
PURPOSE: Existence and cause of thyroid stunning was controversially discussed for decades but the underlying mechanism remains unclear. Numerous studies describe thyroid stunning in radioiodine-131 therapy (RIT) of differentiated thyroid carcinoma. However, there are no studies evaluating thyroid stunning in benign thyroid diseases caused by the radioiodine uptake test (RIUT). Therefore, the influence of pre-therapeutic tracer radiation dose on therapeutic iodine-131 uptake was evaluated retrospectively. METHODS: A total of 914 RIT patients were included. Exclusion criteria were anti-thyroid drugs, pre- and/or intra-therapeutic effective half-lives (EHL) beyond 8.04 days and externally performed RIUT or 24 h RIUT. All patients received RIUT 1 week before RIT. Thyroid volume was estimated via ultrasound. Tracer radiation dose to the thyroid was calculated retrospectively. The dependence of changes in the pre-therapeutic to the therapeutic extrapolated-maximum-131I-uptake (EMU) from the dose in RIUT was evaluated statistically. RESULTS: EMU in RIUT ranged from 0.10 to 0.82 (median: 0.35) and EMU in RIT ranged from 0.10 to 0.74 (median: 0.33). Averaged over the whole cohort the therapeutic EMU decreased significantly (2.3% per Gray intra-thyroidal tracer radiation dose). A disease-specific evaluation showed dose-dependent thyroid stunning from 1.2% per Gray in solitary toxic nodules (n = 327) to 21% per Gray in goiters (n = 135) which was significant for the subgroups of disseminated autonomies (n = 114), multifocal autonomies (n = 178) and goiters (p < 0.05) but not for Graves' diseases (n = 160) and solitary toxic nodules (p > 0.05). CONCLUSIONS: The presented data indicate for the first time a significant dependence of pre-therapeutic radiation dose on thyroid stunning in goiter and disseminated and multifocal autonomy. To achieve the desired intra-thyroidal radiation dose, RIT activity should be adapted depending on the dose in RIUT.
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Radioisótopos de Yodo/efectos adversos , Enfermedades de la Tiroides/radioterapia , Glándula Tiroides/efectos de la radiación , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Radioisótopos de Yodo/metabolismo , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Glándula Tiroides/metabolismo , Adulto JovenRESUMEN
Germany has developed into a country with a nationwide largely sufficient iodine supply due to improved alimentary iodine supply. The reduction of iodine uptake in focal autonomies induced by reduced iodine avidity was evaluated by Gotthardt et al. 2006, showing a significant decline of pertechnetate uptake up to the year 2004. AIM: This study was intended to carry this investigation forward to the present day to analyze the course of a conjectural stabilization of iodine uptake values. PATIENTS, MATERIAL, METHODS: 283 patients who underwent radioiodine therapy for focal thyroid were analyzed retrospectively. Pertechnetate uptake was measured scintigraphically, thyroid volume sonographically and iodine uptake by iodine uptake test. The uptake percentage in the autonomous volume was correlated with autonomous volume and the resulting values tracked over a time period of seven years. RESULTS: Mean thyroid volume ranged from 24 to 29 ml, autonomous volume from 7.2 to 9.4 ml. Pertechnetate uptake ranged from 0.2 to 0.25%/ml autonomous volume. Iodine uptake values ranged from 3.2 to 4.2%/ml autonomous volume. None of the changes observed were statistically significant (all p>0.05). CONCLUSION: Prophylactic measures towards improvement of the general public´s iodine supply in Germany had led to a decline of pertechnetate and iodine uptake in the thyroid up until the turn of the millennium. The here presented data show a stabilization of 99mTc-Uptake. Our study could also show that actual iodine uptake has stabilized at a steady level over the preceding seven years.
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Radioisótopos de Yodo/farmacocinética , Tecnecio/farmacocinética , Enfermedades de la Tiroides/metabolismo , Adulto , Femenino , Humanos , Masculino , Tasa de Depuración Metabólica , Persona de Mediana Edad , Especificidad de Órganos , Radiofármacos/farmacocinética , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Enfermedades de la Tiroides/diagnóstico por imagen , Distribución TisularRESUMEN
BACKGROUND: Prior to undergoing radioiodine therapy (RIT), patients regularly have concerns about isolation on the ward (mandatory for RIT for at least 48 hours in Germany due to radiation protection legislation) as well as fear of the presence of radioactive substances. In this study, these fears were investigated before and after RIT. METHODS: A questionnaire was developed for completion both before and after radioiodine therapy. Questions included: (i) "Are you afraid of a therapy with radioactive substances?" (ii) "Do you have reservations about contact with radioactive substances?" and (iii) "Are you anxious about isolation?" Possible answers were made in a qualitative representation using a scale of 1-4 (4=full agreement, 3=mostly agreement, 2=partial agreement, and 1=no agreement). Further questions included, for example, sources of information used prior to therapy. A total of 209 patients treated by single or preplanned multiple RIT were surveyed over a period of 8 months (return 109). Analysis was done in subgroups according to age, education, disease, and number of RITs. RESULTS: Question 1, "Are you afraid of a therapy with radioactive substances?" showed a similar statistically relevant decline in each subgroup (p<0.05), except for patients with multiple RIT (p=0.81). Asked about the handling of radioactive substances and their perception about the safety in this regard, the entire collective showed a highly statistically significant (p<0.01) decrease with little variability between the groups. The question concerning fear of isolation resulted in a significant decrease (p<0.05) in all subgroups, except for patients with multiple RIT (p=0.13). Analysis of sources of information before RIT showed that older patients preferred printed material and rarely used online resources, while younger patients used the internet more frequently, in addition to printed materials. Finally, most patients would undergo radioiodine therapy again (medical indication provided), with 54% fully agreeing and only 4% not agreeing. CONCLUSIONS: The survey demonstrates a reduction in concerns about nuclear radiation, use of unsealed radioactive materials, and isolation on the ward after RIT. Surprisingly, concerns rise again before a subsequent therapy.
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Miedo/psicología , Enfermedades de la Tiroides/radioterapia , Femenino , Humanos , Radioisótopos de Yodo/uso terapéutico , Masculino , Persona de Mediana Edad , Encuestas y Cuestionarios , Enfermedades de la Tiroides/psicologíaRESUMEN
Insect stings can cause life-threatening IgE-mediated anaphylactic reactions in venom-allergic patients. Although several compounds have already been described as venom allergens, prominent allergen candidates especially in the higher m.w. range have still remained elusive. Tandem mass spectrometry-based sequencing assigned a candidate gene to the most prominent putative high m.w. allergen Api m 5 (allergen C) in honeybee (Apis mellifera) venom and also allowed identification of its homologue Ves v 3 in yellow jacket (Vespula vulgaris) venom. Both proteins exhibit a pronounced sequence identity to human dipeptidyl peptidase IV or CD26. Reactivity of a human IgE mAb verified the presence of these proteins in the venoms. Both proteins were produced in insect cells and characterized for their enzymatic activity as well as their allergenic potential using sera and basophils from insect venom-allergic patients. Both Api m 5 and Ves v 3 were recognized by specific IgE of the majority of patients even in the absence of cross-reactive carbohydrate determinants. Serologic IgE reactivity closely matched activation of human basophils by Api m 5 or Ves v 3, thus underlining their relevance in functional assays. With Api m 5 and Ves v 3, a new pair of homologous allergens becomes available for future clinical applications in diagnosis and therapy that may also contribute to the understanding of molecular mechanisms of insect venoms. Moreover, the patient IgE reactivity together with the cellular activation demonstrates for the first time the relevance of high m.w. allergens in the context of hymenoptera venom allergy.
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Alérgenos/química , Alérgenos/inmunología , Venenos de Abeja/química , Venenos de Abeja/inmunología , Dipeptidil Peptidasa 4/química , Dipeptidil Peptidasa 4/inmunología , Venenos de Avispas/química , Venenos de Avispas/inmunología , Alérgenos/genética , Secuencia de Aminoácidos , Animales , Venenos de Abeja/genética , Abejas/enzimología , Abejas/genética , Abejas/inmunología , Dipeptidil Peptidasa 4/genética , Humanos , Inmunoglobulina E/biosíntesis , Mordeduras y Picaduras de Insectos/inmunología , Mordeduras y Picaduras de Insectos/terapia , Proteínas de Insectos/química , Proteínas de Insectos/genética , Proteínas de Insectos/inmunología , Datos de Secuencia Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Spodoptera/genética , Spodoptera/inmunología , Venenos de Avispas/genética , Avispas/enzimología , Avispas/genética , Avispas/inmunologíaRESUMEN
BACKGROUND: Acid phosphatase (Api m 3) is a major allergen in honeybee (Apis mellifera) venom, and its availability as a recombinant protein may facilitate the development of improved diagnostic tests and immunotherapies. OBJECTIVE: One objective is the determination of the complete primary structure of Api m 3 and to obtain recombinant Api m 3 on the basis of expression in insect cells. Another objective is the quantitative analysis of patient serum IgE antibody reactive to recombinant Api m 3. METHODS: The cloning of Api m 3 from venom gland cDNA and its expression as a full-length protein in eukaryotic insect cells is described. The immunoreactivity of serum IgE antibodies of honeybee venom-sensitized patients to recombinant Api m 3 was determined in an enzyme immunoassay. RESULTS: PCR amplification generated a 1122-bp DNA fragment whose identity as the coding sequence of Api m 3 was verified by several means. Recombinant Api m 3, expressed in Trichoplusia ni cells, showed an expected molecular weight and enzymatic activity at pH 4.5. Analysis of tryptic fragments of purified recombinant Api m 3 by mass spectrometry confirmed its identity. In immunoassays, recombinant Api m 3 is specifically recognized by IgE antibodies of pooled serum in Western blots and by 37% of the individual sera of honeybee venom-sensitized patients in ELISA analysis. CONCLUSION: The availability of recombinant Api m 3 provides a tool for both the development of improved diagnostic tests and the design of safer and more effective immunotherapeutic approaches for honeybee venom allergy. CLINICAL IMPLICATIONS: The recombinant venom allergen Api m 3 is a key element in the search for an optimized component-resolved approach to honeybee venom allergy with regard to both the development of superior diagnostic tests and the improvement of allergen immunotherapy.
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Fosfatasa Ácida/genética , Fosfatasa Ácida/inmunología , Alérgenos/genética , Venenos de Abeja/genética , Venenos de Abeja/inmunología , Abejas/genética , Abejas/inmunología , Secuencia de Aminoácidos , Anafilaxia/etiología , Anafilaxia/terapia , Animales , Secuencia de Bases , Abejas/enzimología , Línea Celular , Clonación Molecular , ADN Complementario/genética , Desensibilización Inmunológica , Expresión Génica , Genes de Insecto , Humanos , Inmunoglobulina E/sangre , Técnicas In Vitro , Mordeduras y Picaduras de Insectos/complicaciones , Mordeduras y Picaduras de Insectos/inmunología , Datos de Secuencia Molecular , Mariposas Nocturnas , Homología de Secuencia de AminoácidoRESUMEN
We have designed and evaluated novel DNA stem-loop structured probes for enzymatic detection of nucleic acid targets. These probes constitute a novel class of conformational switches for enzymatic activity, which in the absence of a target sterically shield an affinity label and upon hybridization of the target to the recognition sequence that forms the loop of the probe restore accessibility of the label for the binding of a reporter enzyme. Analysis of probe characteristics revealed stem stability as the most important parameter governing detection functionality, while other factors such as the length of linker molecules attaching the label to the stem-loop structure and the nature of the solid support proved to be less critical. Apparently, the bulky nature of the reporter enzyme facilitates shielding of the label in the absence of the target, thereby conferring considerable structural tolerance to the conformational switch system. The stem-loop structured probes allow sensitive detection of unlabeled nucleic acid targets. Employing a microtiter assay format, 4 ng of bacterial 16S ribosomal RNA corresponding to 8 fmol could be detected, which can be compared favorably with current immobilized molecular beacon concepts based on fluorescence detection.