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Ki-67, a nuclear protein expressed in all stages of cellular proliferation, is a valuable tool to assess tumor proliferation and has been linked to more aggressive tumor behavior. However, interlaboratory staining heterogeneity and inter-observer variability challenge its reproducibility. Round Robin tests are a suitable tool to standardize and harmonize immunohistochemical and molecular analyses in histopathology. The study investigates the interrater and interlaboratory reproducibility of Ki-67-scoring using both manual and automated approaches. Unstained TMA slides comprising diverse tumor types (breast cancer, neuroendocrine tumors, lymphomas, and head and neck squamous cell carcinoma) were distributed to six pathology laboratories, each employing their routine staining protocols. Manual and automated scoring methods were applied, and interrater and interlaboratory agreement assessed using intraclass correlation coefficients (ICC). The results highlight good-to-excellent reliability overall, with automated scoring demonstrating higher consistency (ICC 0.955) than manual scoring (ICC 0.871). Results were more variable when looking at the individual entities. Reliability remained good for lymphomas (ICC 0.878) and breast cancer (ICC 0.784) and was poor in well-differentiated neuroendocrine tumors (ICC 0.354). This study clearly advocates standardized practices and training to ensure consistency in Ki-67-assessment, and it demonstrates that this can be achieved in a peer-to-peer approach in local quality-circles.
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Objectives: Multipotent mesenchymal stromal cells (MSC) play a pivotal role in the bone marrow (BM) niche. Stanniocalcin 1 (STC1) secreted by MSC has been demonstrated to promote the survival of neoplastic cells and was suggested a marker for minimal residual disease of acute myeloid leukemia (AML). Therefore, we evaluated the expression of STC1 in MSC from AML patients (MSCAML) compared to MSC from healthy donors (MSCHD).Methods: Liquid culture assays of MSCAML and MSCHD were performed to compare expansion capacity. Gene expression profiles of MSCAML vs. MSCHD were established. Secretion of STC1 was tested by ELISA in MSCAML vs. MSCHD and expression of STC1 in AML- vs. HD-BM by immunohistochemistry. In addition, co-cultures of AML cells on MSC were initiated and ultrastructural intercellular communication patterns were investigated. Finally, the effect of blocking STC1 on AML cells was evaluated.Results: MSCAML showed significant decreased expansion capacity compared to MSCHD. Gene analysis revealed marked overexpression of STC1 in MSCAML. ELISA and immunohistochemical findings confirmed this observation. Electron microscopy analysis showed reciprocal stimulation between AML cells and MSC. Blockade of STC1 did not significantly affect AML cell proliferation and apoptosis.Discussion: Characteristics of MSC differ depending on whether they originate from AML patients or from HD. STC1 was mostly overexpressed in MSCAML compared to MSCHD. In vitro blockade of STC1, however, was not associated with AML cell proliferation and apoptosis.Conclusion: Differences in expression levels of glycoproteins from MSCAML compared to MSCHD not necessarily assume that these molecules are niche-relevant in leukemic disease.
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Glicoproteínas/genética , Leucemia Mieloide Aguda/genética , Células Madre Mesenquimatosas/patología , Regulación hacia Arriba , Adulto , Anciano , Células Cultivadas , Femenino , Glicoproteínas/análisis , Humanos , Leucemia Mieloide Aguda/patología , Masculino , Células Madre Mesenquimatosas/metabolismo , Persona de Mediana Edad , Células Tumorales CultivadasRESUMEN
More than half of all brain metastases show infiltrating rather than displacing growth at the macro-metastasis/organ parenchyma interface (MMPI), a finding associated with shorter survival. The lymphoid enhancer-binding factor-1 (LEF1) is an epithelial-mesenchymal transition (EMT) transcription factor that is commonly overexpressed in brain-colonizing cancer cells. Here, we overexpressed LEF1 in an in vivo breast cancer brain colonization model. It shortened survival, albeit without engaging EMT at the MMPI. By differential proteome analysis, we identified a novel function of LEF1 as a regulator of the glutathione (GSH) system, the principal cellular redox buffer. LEF1 overexpression also conferred resistance against therapeutic GSH depletion during brain colonization and improved management of intracellular ROS. We conclude that besides EMT, LEF1 facilitates metastasis by improving the antioxidative capacity of epithelial breast cancer cells, in particular during colonization of the brain parenchyma.
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Neoplasias Encefálicas/patología , Neoplasias Encefálicas/secundario , Neoplasias de la Mama/patología , Glutatión/metabolismo , Factor de Unión 1 al Potenciador Linfoide/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Encéfalo/patología , Línea Celular Tumoral , Movimiento Celular/fisiología , Transición Epitelial-Mesenquimal/fisiología , Femenino , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Tejido Parenquimatoso/patologíaRESUMEN
INTRODUCTION: The correct choice of a fracture-specific surgical approach with an articular accessibility in complex tibial plateau fractures to facilitate durable fracture fixation of the anatomic articular reconstruction is under debate, as the most important risk factor for malreduction in complex tibial plateau fractures is an impaired visualization of the articular surface. MATERIALS AND METHODS: Six established surgical approaches were simulated on 12 cadaver knees. The visible articular surface was labeled with an electrocautery device for each approach and subsequently analyzed with ImageJ. Areas of each hemiplateau were compared using the Student's t test. RESULTS: In the lateral tibial plateau, the dorsal 19.0 ± 5.8% of the articular surface could be exposed via the postero-lateral approach. Via the antero-lateral arthrotomy, 36.6 ± 9.4% of the anterior articular surface was visible. The additional osteotomy of the lateral femoral epicondyle significantly increased the exposure to 65.6 ± 7.7% (p < 0.001). In the medial tibial plateau, the osteotomy of the medial femoral epicondyle significantly improved visualization of the medial articular surface (62.3 ± 8.6%) compared to the postero-medial approach (14.0 ± 7.3%) and the antero-medial approach (36.9 ± 9.2%) of the articular (p < 0.001). CONCLUSIONS: Visualization of the tibial articular surface is limited through specific surgical approaches. Extension by osteotomy of the femoral epicondyle led to a significant improvement in the articular exposure without, however, obtaining sufficient visibility of the posterior joint segments, which should be included in the preoperative strategy. The proposed surgical approach-specific map of the tibial plateau may constitute an important instrument in the toolbox of an experienced surgeon to treat complex tibial plateau fractures at the highest level. LEVEL OF EVIDENCE: Level IV.
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Fijación Interna de Fracturas/métodos , Fracturas de la Tibia/cirugía , Anciano , Anciano de 80 o más Años , Cadáver , Femenino , Humanos , Articulación de la Rodilla/cirugía , Masculino , Persona de Mediana Edad , Osteotomía , Tibia/cirugíaRESUMEN
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease of complex and still poorly understood etiology. Loss of upper and lower motoneurons results in death within few years after diagnosis. Recent studies have proposed neuroprotective and disease-slowing effects of granulocyte-colony stimulating factor (G-CSF) treatment in ALS mouse models as well as humans. In this study, six ALS patients were monitored up to 3.5â¯years during continuous high-dose G-CSF administration. Repetitive analyses were performed including blood count parameters, CD34+ hematopoietic stem and progenitor cell (HSPC) and colony forming cell (CFC) counts, serum cytokine levels and leukocyte telomere length. We demonstrate that continuous G-CSF therapy was well tolerated and safe resulting in only mild adverse events during the observation period. However, no mobilization of CD34+ HSPC was detected as compared to baseline values. CFC mobilization was equally low and even a decrease of myeloid precursors was observed in some patients. Assessment of telomere length within ALS patients' leukocytes revealed that G-CSF did not significantly shorten telomeres, while those of ALS patients were shorter compared to age-matched healthy controls, irrespective of G-CSF treatment. During G-CSF stimulation, TNF-alpha, CRP, IL-16, sVCAM-1, sICAM-1, Tie-2 and VEGF were significantly increased in serum whereas MCP-1 levels decreased. In conclusion, our data show that continuous G-CSF treatment fails to increase circulating CD34+ HSPC in ALS patients. Cytokine profiles revealed G-CSF-mediated immunomodulatory and proteolytic effects. Interestingly, despite intense G-CSF stimulation, telomere length was not significantly shortened.
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Esclerosis Amiotrófica Lateral/tratamiento farmacológico , Esclerosis Amiotrófica Lateral/genética , Factor Estimulante de Colonias de Granulocitos/administración & dosificación , Movilización de Célula Madre Hematopoyética , Homeostasis del Telómero , Adulto , Anciano , Esclerosis Amiotrófica Lateral/sangre , Antígenos CD34/metabolismo , Recuento de Células Sanguíneas , Ensayo de Unidades Formadoras de Colonias , Citocinas/sangre , Relación Dosis-Respuesta a Droga , Femenino , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/metabolismo , Humanos , Masculino , Persona de Mediana Edad , Proyectos PilotoAsunto(s)
Dasatinib/farmacología , Mielofibrosis Primaria/tratamiento farmacológico , Animales , Dasatinib/uso terapéutico , Humanos , FN-kappa B/efectos de los fármacos , Mielofibrosis Primaria/prevención & control , Inhibidores de Proteínas Quinasas , Factor de Transcripción STAT5/efectos de los fármacosRESUMEN
Intra-articular tibial plateau fractures can present a surgical challenge due to complex injury patterns and compromised soft tissue. The treatment goal is to spare the soft tissue and an anatomical reconstruction of the tibial articular surface. Depending on the course of the fracture, a fracture-specific access strategy is recommended to provide correct positioning of the plate osteosynthesis. While the anterolateral approach is used in the majority of lateral tibial plateau fractures, only one third of the joint surface is visible; however, posterolateral fragments require an individual approach, e.â¯g. posterolateral or posteromedial. If necessary, osteotomy of the femoral epicondyles can improve joint access for reduction control. Injuries to the posterior columns should be anatomically reconstructed and biomechanically correctly addressed via posterior approaches. Bony posterior cruciate ligament tears can be refixed via a minimally invasive posteromedial approach.
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Fijación Interna de Fracturas , Fracturas de la Tibia , Placas Óseas , Humanos , Articulación de la Rodilla , Osteotomía , Fracturas de la Tibia/cirugíaRESUMEN
OBJECTIVES: Multipotent mesenchymal stromal cells (MSCs) play a central role within the bone marrow (BM) niche, supporting hematopoiesis via soluble factors like cytokines and chemokines. In our study, we sought to investigate the effect of blocking transforming growth factor beta 1 (TGF-ß1) and C-X-C motif chemokine 12 (CXCL12) receptor CXCR4 on acute myeloid leukemia (AML) cells in an MSC co-culture system. METHODS: Human MSCs were obtained by BM aspirates and their phenotype and functional properties were confirmed in vitro. Co-cultures of AML cells on MSCs were initiated and compared to those on mouse fibroblasts (MS-5) and liquid cultures. Additionally, the effect of blocking CXCR4 and TGF-ß1 on AML cells was tested with and without the addition of cytarabine. RESULTS: MSCs from BM showed a typical phenotype and differentiation pattern. Co-culture of AML cells on MSCs resulted in a significantly higher proliferation capacity than on MS-5 or liquid culture. Blockade of TGF-ß1 increased AML cell proliferation and chemosensibility, while the CXCR4 antagonist plerixafor showed anti-proliferative effects and did not change cytarabine-induced cell death compared to control. DISCUSSION: Human MSCs are potent feeder cells, able to maintain AML cells in long-term culture. This favorable co-existence seems to be due in part to molecules important for communication within the niche. Blockade of TGF-ß1 and CXCL12 was associated with different effects on AML cell proliferation and chemotherapy resistance. CONCLUSION: These findings suggest a strong supporting affinity between MSCs and AML cells within the leukemic niche, where TGF-ß1 and CXCL12 pathways play an important role.
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Quimiocina CXCL12/metabolismo , Leucemia Mieloide Aguda/metabolismo , Células Madre Mesenquimatosas/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Adulto , Anciano , Animales , Antimetabolitos Antineoplásicos/farmacología , Biomarcadores , Médula Ósea/metabolismo , Médula Ósea/patología , Células de la Médula Ósea/metabolismo , Línea Celular Tumoral , Proliferación Celular , Células Cultivadas , Aberraciones Cromosómicas , Técnicas de Cocultivo , Citarabina/farmacología , Resistencia a Antineoplásicos , Femenino , Humanos , Inmunofenotipificación , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/genética , Masculino , Ratones , Persona de Mediana Edad , Clasificación del TumorRESUMEN
Myelofibrosis is a myeloproliferative neoplasm that results in cytopenia, bone marrow fibrosis and extramedullary hematopoiesis. Allogeneic hematopoietic stem cell transplantation is the only curative treatment but is associated with a risk of delayed engraftment and graft failure. In this study, patients with myelofibrosis (n=31) and acute myeloid leukemia (n=31) were analyzed for time to engraftment, graft failure and engraftment-related factors. Early and late neutrophil engraftment and late thrombocyte engraftment were significantly delayed in patients with myelofibrosis as compared to acute myeloid leukemia, and graft failure only occurred in myelofibrosis (6%). Only spleen size had a significant influence on engraftment efficiency in myelofibrosis patients. To analyze the cause for the engraftment defect, clearance of hematopoietic stem cells from peripheral blood was measured and immunohistological staining of bone marrow sections was performed. Numbers of circulating CD34+ were significantly reduced at early time points in myelofibrosis patients, whereas CD34+CD38- and colony-forming cells showed no significant difference in clearance. Staining of bone marrow sections for homing proteins revealed a loss of VCAM-1 in myelofibrosis with a corresponding significant increase in the level of soluble VCAM-1 within the peripheral blood. In conclusion, our data suggest that reduced engraftment and graft failure in myelofibrosis patients is caused by an early pooling of CD34+ hematopoietic stem cells in the spleen and a bone marrow homing defect caused by the loss of VCAM-1. Improved engraftment in myelofibrosis might be achieved by approaches that reduce spleen size and cleavage of VCAM-1 in these patients prior to hematopoietic stem cell transplantation.
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Funcionamiento Retardado del Injerto/etiología , Trasplante de Células Madre Hematopoyéticas/métodos , Mielofibrosis Primaria/terapia , Bazo/patología , Molécula 1 de Adhesión Celular Vascular/metabolismo , Adulto , Anciano , Aloinjertos , Femenino , Rechazo de Injerto/etiología , Humanos , Leucemia Mieloide Aguda/terapia , Masculino , Persona de Mediana Edad , Tamaño de los Órganos , Mielofibrosis Primaria/complicacionesRESUMEN
BACKGROUND: Currently existing classifications of tibial plateau fractures do not help to guide surgical strategy. Recently, a segment-based mapping of the tibial plateau has been introduced in order to address fractures with a fracture-specific surgical approach. The goal of the present study was to analyze incidence and fracture specifics according to a new 10-segment classification of the tibial plateau. METHODS: A total of 242 patients with 246 affected knees were included (124 females, 118 males, mean age 51.9±16.1years). Fractures were classified according to the OTA/AO classification. Fracture pattern was analyzed with respect to a 10-segment classification based on CT imaging of the proximal tibial plateau 3cm below the articular surface. RESULTS: 161 Patients suffered an OTA/AO type 41-B and 85 patients an OTA/AO type 41-C tibial plateau fracture. Females had an almost seven times higher risk to suffer a fracture due to low-energy trauma (OR 6.91, 95% CI (3.52, 13.54), p<0.001) than males. In 34% of the patients with affection of the medial tibial plateau, a fracture comminution, primarily due to low-energy trauma (p<0.001), was observed. In type B fractures, the postero-latero-lateral (65.2%), the antero-latero-lateral (64.6%) and the antero-latero-central (60.9%) segment were most frequently affected. Every second type C fracture showed an unique fracture line and zone of comminution. The tibial spine was typically involved (89.4%). A typical fracture pattern of high-energy trauma demonstrated a zone of comminution of the lateral plateau and a split fracture in the medial plateau. The most frequently affected segments were the postero-latero-central (85.9%), postero-central (84.7%), and antero-latero-central (78.8%) segment. CONCLUSION: Posterior segments were the most frequently affected in OTA/AO type B and C fractures. Acknowledging the restricted visibility of posterior segments, whose reduction and fixation is crucial for long-term success, our findings implicate the use of posterior approaches more often in the treatment of tibial plateau fractures. Also, low-energy trauma was identified as an important cause for tibial plateau fractures.
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Fijación Interna de Fracturas/métodos , Fracturas Conminutas/diagnóstico por imagen , Fracturas Intraarticulares/diagnóstico por imagen , Traumatismos de la Rodilla/diagnóstico por imagen , Fracturas de la Tibia/diagnóstico por imagen , Tomografía Computarizada por Rayos X , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Fracturas Conminutas/patología , Humanos , Imagenología Tridimensional , Incidencia , Fracturas Intraarticulares/clasificación , Fracturas Intraarticulares/patología , Traumatismos de la Rodilla/fisiopatología , Masculino , Persona de Mediana Edad , Reproducibilidad de los Resultados , Estudios Retrospectivos , Fracturas de la Tibia/patología , Adulto JovenRESUMEN
OBJECTIVE: Tumor necrosis factor alpha (TNFalpha) is an old foe in allogeneic bone marrow transplantation (allo-BMT) promoting acute graft-versus-host disease (aGVHD). We investigated to what extent donor T cells contribute to TNFalpha production. METHODS: Lethally irradiated B6D2F1 mice were transplanted with bone marrow (BM) and T cells from syngeneic B6D2F1 or allogeneic B6 donors and assessed for cytokine production, aGVHD, and survival. RESULTS: Analysis of serum TNFalpha kinetics in recipients of allogeneic B6 wild-type BM and wild-type T cells revealed that TNFalpha levels peaked around day 7 after allo-BMT, whereas TNFalpha was undetectable in syngeneic controls. TNFalpha was produced by both host and donor cells. Further exploration showed that specifically donor CD4(+) but not CD8(+) T cells were the primary donor cell source of TNFalpha at this early time point; numbers of TNFalpha expressing splenic CD4(+) T cells were higher than CD8(+) T cells 7 days after allo-BMT, and maximal serum TNFalpha levels were detected following allo-BMT with only CD4(+) T cells compared to levels found in allogeneic recipients of only wild-type CD8(+) or to only CD4(+) TNFalpha(-/-) T cells. Concurrent with increased TNFalpha levels, early clinical aGVHD and mortality were more severe following allo-BMT with either wild-type CD4(+) and CD8(+) or CD4(+) T cells only. CONCLUSION: Our data demonstrate that in addition to residual host cells donor CD4(+) T cells significantly contribute to the proinflammatory cytokine milieu engendered early after allo-BMT through the production of TNFalpha. These findings support strategies focusing on TNFalpha neutralization as primary treatment for aGVHD.
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Linfocitos T CD4-Positivos/metabolismo , Enfermedad Injerto contra Huésped/etiología , Inflamación/etiología , Factor de Necrosis Tumoral alfa/biosíntesis , Factor de Necrosis Tumoral alfa/fisiología , Animales , Trasplante de Médula Ósea/efectos adversos , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/trasplante , Linfocitos T CD8-positivos , Enfermedad Injerto contra Huésped/patología , Cinética , Ratones , Ratones Endogámicos , Tasa de Supervivencia , Linfocitos T/trasplante , Donantes de Tejidos , Trasplante Homólogo , Trasplante IsogénicoRESUMEN
1,1'-Binaphthyls are prepared by a conceptually novel approach based on the photo-dehydro-Diels-Alder reaction.
RESUMEN
A series of o-alkylphenyl alkyl ketones 1 were synthesized by different methods. The presence of a leaving group X adjacent to the carbonyl group is the special peculiarity of these ketones. Upon irradiation the keto carbonyl group of these compounds undergoes an n-pi* excitation followed by a 1,5-hydrogen migration from the o-alkyl substituent to the carbonyl oxygen atom. The thus formed 1,4-diradicals are subject to a very rapid elimination of acid HX, giving 1,5-diradicals. We called this process spin center shift. After intersystem crossing these diradicals cyclize to 1-indanones 20 in good yields. Depending on the solvent and on substituents, o-alkoxyalkyl ketones 22 or benzo[c]furanes 21 are obtained as byproducts. The mechanism of the cyclization was elucidated by quantum chemical calculations and kinetic measurements.
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Little is known about the impact of cytochrome P450 polymorphisms on the metabolism of trimipramine, which is still widely used as antidepressant due to its positive effect on sleep patterns. A single oral dose of 75 mg trimipramine was given to 42 healthy volunteers selected according to their CYP2D6, CYP2C19, and CYP2C9 genotypes. The reference group included 8 subjects with homozygous active wild-type genotypes of all 3 enzymes (EM). This group was compared with 7 intermediate (IM) with 1 and 7 poor metabolizers (PM) with zero active alleles of CYP2D6 and CYP2C19, respectively, and with 4 subjects with the genotype CYP2C9*3/*3. Pharmacokinetics of trimipramine and its demethylated metabolite strongly depended on the CYP2D6 genotype. Median oral clearance of trimipramine was 276 L/h (range 180-444) in the reference group but only 36 L/h (range 24-48) in CYP2D6 PMs (P < 0.001). These differences could only be explained by an effect of CYP genotypes on both parameters, systemic clearance and bioavailability, the latter being at least 3-fold higher in CYP2D6 PMs than in the reference group. The desmethyltrimipramine area under the concentration-time curve was 40-fold greater in CYP2D6 PMs than in the reference group (1.7 vs. 0.04 mg/L x h in EMs), but below the quantification limit in most carriers of deficiencies of CYP2C19 or CYP2C9. This indicates that both CYP2C enzymes contribute to the demethylation of desmethyltrimipramine and CYP2D6 to further metabolism.
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Hidrocarburo de Aril Hidroxilasas/genética , Citocromo P-450 CYP2D6/genética , Oxigenasas de Función Mixta/genética , Polimorfismo Genético , Trimipramina/farmacocinética , Adulto , Anciano , Área Bajo la Curva , Intervalos de Confianza , Citocromo P-450 CYP2C19 , Citocromo P-450 CYP2C9 , Femenino , Humanos , Masculino , Tasa de Depuración Metabólica/efectos de los fármacos , Tasa de Depuración Metabólica/genética , Persona de Mediana Edad , Estadísticas no ParamétricasRESUMEN
In the present article the anatomy and histology of the male genital system of an endeostigmatid mite are described for the first time. The Endeostigmata probably are a paraphyletic group supposed to include the most primitive actinotrichid mites. In Nanorchestes amphibius, the testis comprises a paired germinal region connected with an unpaired glandular region. In the germinal region, spermiogenesis takes place in cysts of a somatic cell containing germ cells representing the same developmental stage. In the lumen of the glandular region, the spermatozoa are stored together with secretions of the glandular epithelium. These secretions are probably involved in the formation of spermatophores. From the glandular region, spermatozoa and secretions are released into the vasa deferentia that histologically can be divided into three sections, beginning with a short paired region with strong circular muscles serving as a sphincter, continuing with a paired proximal zone, followed by a short unpaired distal section. The distal vas deferens leads into the chitinous, unpaired ductus ejaculatorius which is followed by the progenital chamber. The ductus ejaculatorius is composed of a proximal section and a proximal, central, and anterior chamber. It is accompanied by a complex system of muscles and sclerites probably involved in the formation and ejaculation of the spermatophore. A similar organization can also be found in Prostigmata, but not in Oribatida. Anterior to the progenital chamber is located a paired accessory gland that probably produces a lipid secretion. Spermiogenesis is characterized by disintegration of the nuclear envelope, condensation of chromatin, and extensive reduction of the amount of sperm cell cytoplasm. The mature aflagellate, U-shaped spermatozoa are simple in structure and lack mitochondria and an acrosome complex. The results do not support the current view that Nanorchestidae are more closely related to Sarcoptiformes, i.e., Oribatida and Astigmata, than to Prostigmata.
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Genitales Masculinos/anatomía & histología , Ácaros/anatomía & histología , Ácaros/fisiología , Espermatogénesis , Animales , Genitales Masculinos/ultraestructura , Técnicas Histológicas , Masculino , Microscopía Electrónica , FilogeniaRESUMEN
In-vitro data indicated a contribution of cytochrome P450 enzymes 1A2, 3A4, 2C9, 2C19 and 2D6 to biotransformation of doxepin. We studied the effects of genetic polymorphisms in CYP2D6, CYP2C9 and CYP2C19 on E- and Z-doxepin pharmacokinetics in humans. Doxepin kinetics was studied after a single oral dose of 75 mg in healthy volunteers genotyped as extensive (EM), intermediate (IM) and poor (PM) metabolizers of substrates of CYP2D6 and of CYP2C19 and as slow metabolizers with the CYP2C9 genotype *3/*3. E-, Z-doxepin and -desmethyldoxepin were quantified in plasma by HPLC. Data were analyzed by non-parametric pharmacokinetics and statistics and by population pharmacokinetic modeling considering effects of genotype on clearance and bioavailability. Mean E-doxepin clearance (95% confidence interval) was 406 (390-445), 247 (241-271), and 127 (124-139) l h(-1) in EMs, IMs and PMs of CYP2D6. In addition, EMs had about 2-fold lower bioavailability compared with PMs indicating significant contribution of CYP2D6 to E-doxepin first-pass metabolism. E-doxepin oral clearance was also significantly lower in carriers of CYP2C9*3/*3 (238 l h(-1) ). CYP2C19 was involved in Z-doxepin metabolism with 2.5-fold differences in oral clearances (73 l h(-1) in CYP2C19 PMs compared with 191 l h(-1) in EMs). The area under the curve (0-48 h) of the active metabolite -desmethyldoxepin was dependent on CYP2D6 genotype with a median of 5.28, 1.35, and 1.28 nmol l h(-1) in PMs, IMs, and EMs of CYP2D6. The genetically polymorphic enzymes exhibited highly stereoselective effects on doxepin biotransformation in humans. The CYP2D6 polymorphism had a major impact on E-doxepin pharmacokinetics and CYP2D6 PMs might be at an elevated risk for adverse drug effects when treated with common recommended doses.