Panel Sequencing for Clinically Oriented Variant Screening and Copy Number Detection in Chronic Lymphocytic Leukemia Patients.

According to current guidelines, in chronic lymphocytic leukemia (CLL), only the TP53 molecular status must be evaluated prior to every treatment's initiation. However, additional heterogeneous genetic events are known to confer a proliferative advantage to the tumor clone and are associated with progression and treatment failure in CLL patients. Here, we describe the implementation of a comprehensive targeted sequencing solution that is suitable for routine clinical practice and allows for the detection of the most common somatic single-nucleotide and copy number variants in genes relevant to CLL. We demonstrate that this cost-effective strategy achieves variant detection with high accuracy, specificity, and sensitivity. Furthermore, we identify somatic variants and copy number variations in genes with prognostic and/or predictive value, according to the most recent literature, and the tool provides evidence about subclonal events. This next-generation sequencing (NGS) capture-based target assay is an improvement on current approaches in defining molecular prognostic and/or predictive variables in CLL patients.

Aldehyde Trapping by ADX-102 Is Protective against Cigarette Smoke and Alcohol Mediated Lung Cell Injury.

Most individuals diagnosed with alcohol use disorders smoke cigarettes. Large concentrations of malondialdehyde and acetaldehyde are found in lungs co-exposed to cigarette smoke and alcohol. Aldehydes directly injure lungs and form aldehyde protein adducts, impacting epithelial functions. Recently, 2-(3-Amino-6-chloroquinolin-2-yl)propan-2-ol (ADX-102) was developed as an aldehyde-trapping drug. We hypothesized that aldehyde-trapping compounds are protective against lung injury derived from cigarette smoke and alcohol co-exposure. To test this hypothesis, we pretreated mouse ciliated tracheal epithelial cells with 0-100 µM of ADX-102 followed by co-exposure to 5% cigarette smoke extract and 50 mM of ethanol. Pretreatment with ADX-102 dose-dependently protected against smoke and alcohol induced cilia-slowing, decreases in bronchial epithelial cell wound repair, decreases in epithelial monolayer resistance, and the formation of MAA adducts. ADX-102 concentrations up to 100 µM showed no cellular toxicity. As protein kinase C (PKC) activation is a known mechanism for slowing cilia and wound repair, we examined the effects of ADX-102 on smoke and alcohol induced PKC epsilon activity. ADX-102 prevented early (3 h) activation and late (24 h) autodownregulation of PKC epsilon in response to smoke and alcohol. These data suggest that reactive aldehydes generated from cigarette smoke and alcohol metabolism may be potential targets for therapeutic intervention to reduce lung injury.

Severe infections in patients with lymphoproliferative diseases treated with new targeted drugs: A multicentric real-world study.

BACKGROUND: Lymphoid neoplasms treatment has recently been renewed to increase antitumor efficacy and conventional chemotherapies toxicities. Limited data have been published about the infection risk associated with these new drugs, therefore this study analyzes the infectious complications in patients with lymphoproliferative diseases (LPD) treated with monoclonal antibodies (obinutuzumab, ofatumumab, brentuximab, nivolumab, or pembrolizumab), BTK inhibitors (ibrutinib and acalabrutinib), PI3K inhibitors (idelalisib) and BCL2 inhibitors (venetoclax). METHODS: Multicenter retrospective study of 458 LPD patients treated with targeted therapies in real-life setting, in 18 Spanish institutions, from the time of their commercial availability to August 2020. RESULTS: Severe infections incidence was 23% during 17-month median follow-up; cumulative incidence was higher in the first 3-6 months of targeted drug treatment and then decreased. The most frequent etiology was bacterial (54%). Nine (6%) Invasive fungal infections (IFI) were observed, in its majority in chronic lymphocytic leukemia (CLL) patients treated predominantly with ibrutinib. Significant risk factors for severe infection were: severe lymphopenia (p = 0.009, OR 4.7, range 1.3-1.7), combined targeted treatment vs single agent treatment (p = 0.014 OR 2.2 range 1.1-4.2) and previous rituximab (p = 0.03 OR 1.8, range 1.05-3.3). Infection-related mortality was 6%. In 22% of patients with severe infections, definitive discontinuation of the targeted drug was observed. CONCLUSION: A high proportion of patients presented severe infections during follow-up, with non-negligible attributable mortality, but infection incidence is not superior to the one observed during the chemotherapy era. In selected cases with specific risk factors for infection, antimicrobial prophylaxis should be considered.

Combined immunostimulatory monoclonal antibodies extend survival in an aggressive transgenic hepatocellular carcinoma mouse model.

PURPOSE: Immunostimulatory monoclonal antibodies (ISmAb) that unleash antitumor immune responses are showing efficacy in cancer clinical trials. Anti-B7-H1 (PD-L1) monoclonal antibodies (mAb) block a critical inhibitory pathway in T cells, whereas anti-CD137 and OX40 mAbs provide T-cell costimulation. A combination of these ISmAbs (anti-CD137 + anti-OX40 + anti-B7-H1) was tested using a transgenic mouse model of multifocal and rapidly progressing hepatocellular carcinoma, in which c-myc drives transformation and cytosolic ovalbumin (OVA) is expressed in tumor cells as a model antigen. EXPERIMENTAL DESIGN: Flow-cytometry and immunohistochemistry were used to quantify tumor-infiltrating lymphocytes (TIL) elicited by treatment and assess their activation status and cytolytic potential. Tolerance induction and its prevention/reversal by treatment with the combination of ISmAbs were revealed by in vivo killing assays. RESULTS: The triple combination of ISmAbs extended survival of mice bearing hepatocellular carcinomas in a CD8-dependent fashion and synergized with adoptive T-cell therapy using activated OVA-specific TCR-transgenic OT-1 and OT-2 lymphocytes. Mice undergoing therapy showed clear increases in tumor infiltration by activated and blastic CD8(+) and CD4(+) T lymphocytes containing perforin/granzyme B and expressing the ISmAb-targeted receptors on their surface. The triple combination of ISmAbs did not result in enhanced OVA-specific cytotoxic T lymphocyte (CTL) activity but other antigens expressed by cell lines derived from such hepatocellular carcinomas were recognized by endogenous TILs. Adoptively transferred OVA-specific OT-1 lymphocytes into tumor-bearing mice were rendered tolerant, unless given the triple mAb therapy. CONCLUSION: Extension of survival and dense T-cell infiltrates emphasize the translational potential of combinational immunotherapy strategies for hepatocellular carcinoma. Clin Cancer Res; 19(22); 6151-62. ©2013 AACR.

Lymphatic endothelium forms integrin-engaging 3D structures during DC transit across inflamed lymphatic vessels.

Dendritic cell (DC) transmigration across the lymphatic endothelium is critical for the initiation and sustenance of immune responses. Under noninflammatory conditions, DC transit across the lymphatic endothelial cell (LEC) has been shown to be integrin independent. In contrast, there is increasing evidence for the participation of integrins and their ligands in DC transit across lymphatic endothelium under inflammation. In this sense, we describe the formation of ICAM-1 (CD54)-enriched three-dimensional structures on LEC/DC contacts, as these DCs adhere to inflamed skin lymphatic vessels and transmigrate into them. In vitro imaging revealed that under inflammation ICAM-1 accumulated on microvilli projections surrounding 60% of adhered DCs. In contrast, these structures were scarcely formed in noninflammatory conditions. Furthermore, ICAM-1-enriched microvilli were important in promoting DC transendothelial migration and DC crawling over the LEC surface. Microvilli formation was dependent on the presence of ß-integrins on the DC side and on integrin conformational affinity to ligand. Finally, we observed that LEC microvilli structures appeared in close vicinity of CCL21 depots and that their assembly was partially inhibited by CCL21-neutralizing antibodies. Therefore, under inflammatory conditions, integrin ligands form three-dimensional membrane projections around DCs. These structures offer docking sites for DC transit from the tissue toward the lymphatic vessel lumen.

Interferon producing killer dendritic cells (IKDC): a matter of controversy

La división de tareas entre las células del sistema inmunitario es undenominador común en la organización de las diferentes respuestas quelleva a cabo. Como excepción a este paradigma, una subpoblación leucocitaria de ratones parece estar equipada para matar células transformadas einfectadas con virus y, a la vez, con la capacidad de procesamiento antigé-nico y presentación a linfocitos T. Estas funciones se adscriben clásicamente de forma separada a linfocitos NK y a células dendríticas. Las IKDC (“Interferon-producing killer dendritic cells”) fueron definidas en ratón como células que expresan en su superficie CD11c con menor intensidad que las células dendríticas convencionales mientras que coexpresan B220, NK1.1, CD49b(VLA-2) y MHC de Clase II. La ausencia de CD3, CD19 y Gr1 (Ly49C) caracteriza también esta subpoblación cuando se estudia mediante citometría de (..) (AU)

Division of labour among cells of the immune system is a prevailingtheme in the orchestration of immune responses. Contrary to this paradigm, a particular leukocyte population in the mouse seems to be equipped with the ability to kill transformed and virally infected cells and withthe capability to mediate antigen processing and presentation to T cells.Those two functions are classically ascribed separately to Natural Killer(NK) cells and Dendritic cells (DC). IKDC (interferon-producing killerdendritic cell) were defined in mice as cells expressing CD11c, althoughto a lesser extent than conventional DCs (cDCs), while coexpressing B220,NK1.1, CD49b (VLA-2) and MHC Class II molecules. Absence of CD3,CD19 and Gr1(Ly49c) expression also featured this minor subset uponmulticolour FACS characterization. These cells kill a variety tumor celllines in a TRAIL-dependent fashion. Furthermore, IKDC produce high (..) (AU)

Clinical development of combination strategies in immunotherapy: are we ready for more than one investigational product in an early clinical trial?

Stimulating the innate and adaptive immunity against cancer necessitates the tricking of a system evolved to fight microbial pathogens and directing its activity towards transformed self-tissue. Efficacious interventions to start and sustain the response will probably require a number of agents to tamper simultaneously or sequentially with several immune mechanisms. Although master switches controlling various functions may exist, the goal of a curative immune response will probably demand the combined actions of several therapeutic components. Synergy occurs when drugs interact in ways that enhance or magnify one or more effects or side effects. In cancer immunotherapy, two agents that have minor or no therapeutic effects as single agents can be powerful when combined. Mouse experimentation provides multiple examples of synergistic combinations. Elements to be combined include chiefly: tumor vaccines, adoptive T-cell therapies, cytokines, costimulatory molecules, molecular deactivation of immunosuppressive or tolerogenic pathways and immunostimulatory monoclonal antibodies. These novel therapies, even as single agents, are extremely complex products to be developed owing to the associated biomolecules, cell therapies or gene therapies. At present, drug-development programs are run individually for each immunotherapeutic agent and combinations are considered only at a later stage in clinical development, even in the absence of formal compulsory regulations to prevent clinical trials with combinations. As a result, instead of the search for maximal efficacy, ease of combination with standard treatments, intellectual property management, regulations and business-based decisions often guide the way. Even though the maximal effort must be made in order to prevent adverse effects in patients, it seems reasonable that combination pilot trials should be performed at an early stage, following safe completion of Phase I trials. These trials should be performed based on evidence for synergy in animal models and be simplified in terms of regulatory requirements. Such 'short-cut' combination immunotherapy trials can bring much needed efficacy earlier to the bedside.

Polly Matzinger’s “danger model” finds its predicted danger-denoting self moieties

La teoría de la “alarma” (danger) es un conjunto de postulados formalmente propuestos por Polly Matzinger hace quince años. Como teoría surgió para explicar la aparición de respuesta inmunitaria en ausencia de infección. Ha sido una teoría provocadora al proponer que el sistema inmunitario no ha evolucionado para discernir entre lo propio y lo extraño, sino para responder frente a aquello que está causando daño tisular. La propuesta del modelo coincidió en el tiempo con el descubrimiento de la potenciación de la respuesta inmunitaria por patrones moleculares presentes en microorganismos. Charles Janeway y sus colaboradores propusieron que la situación de infección se detecta mediante receptores innatos para patrones estructurales de biomoléculas microbiológicas que están ausentes en organismos superiores sanos. Se descubrió que estos agentes estimulan a las células presentadoras de antígeno para proporcionar a los linfocitos T señales coestimuladoras complementarias para las que reciben al reconocer el antígeno. La coestimulación sería crítica para que la respuesta inmunitaria se ponga en marcha y se sostenga en el tiempo. La teoría del “danger” postula la existencia de moléculas endógenas liberadas o modificadas por la destrucción o daño tisular que pueden hacer sonar la alarma en condiciones de asepsia. Hay varios hallazgos experimentales recientes que apuntan que una serie demoléculas cumplen estas condiciones: La proteína nuclear HMGB-1, el ácidoúrico, el interferon-α, el ATP extracelular, chaperonas de la familia de proteínas de stress térmico, dominios alternativos de la fibronectina y ácidos nucleicos endógenos. Algunos de estos agentes son detectados por los mismos receptores tipo Toll que detectan los patrones de biomoléculas de microbios. La identificación de estos mecanismos moleculares presta apoyo experimental a la teoría del “danger”, y sus implicaciones tienen extraordinaria importancia en inmunología tumoral y en transplante (AU)

The “danger theory” is a set of postulates formally proposed by Polly Matzinger fifteen years ago. As a theory, it explains how it is possible that immune responses take place without infection. It provokingly proposed that the immune systems have not evolved for self/non-self discrimination, but to respond against what is causing tissue damage. The proposal of the “danger model” coincided in time with the discovery of the immune potentiating effects of microbial molecular patterns. Charles Janewayet al. proposed that infection would be detected by innate receptors for microbiological biomolecules that are either absent or different in mammals. These agents were found to stimulate antigen presenting cells in such a way that would provide T lymphocytes with appropriate costimulatory molecules that critically complement the signals raised by antigen recognition. This was considered absolutely critical to ignite and sustain immune responses. The danger theory predicted the existence of endogenous molecules released or modified by danger that would act in a similar fashion to the microbial molecular patterns on dendritic cell costimulatory functions. Recent evidence points to various molecules capable of sounding the alarm in aseptic conditions. These include: the nuclear protein HBGM-1, uric acid, Interferon-α, chaperones of the heat shockprotein family, alternatively spliced domains of fibronectin, and self nucleicacids. Some of these agents act through the same Toll like receptors involved in microbial pattern recognition. Identification of these mechanisms provides molecular support for the danger theory and has an extraordinary importance for tumor and transplantation immunology (AU)

Expression of enkephalin-degrading enzymes in human semen and implications for sperm motility.

OBJECTIVE: To investigate the presence of two enkephalin-degrading enzymes (aminopeptidase N [APN] and neutral endopeptidase 24.11 [NEP]) in different fractions of human semen, their distribution in sperm cells, and their effect on sperm motility. DESIGN: We performed expression assays for APN and NEP by real-time polymerase chain reaction, Western blot, and immunofluorescence techniques in sperm cells and performed motility analysis after incubation of semen samples with enzyme inhibitors and the opioid receptor antagonist naloxone. SETTING: Assisted reproduction unit and academic research laboratory. PATIENT(S): Semen from 50 normozoospermic healthy human donors. INTERVENTION(S): Spermatozoa isolated from semen on a discontinuous Percoll gradient (40%-80%), followed by swim-up, were used for all techniques except for sperm motility analysis, for which fresh semen was used. MAIN OUTCOME MEASURE(S): Immunoblotting blots, indirect immunofluorescence antibody assays, cycle threshold values for real-time polymerase chain reaction, and percentage of motile sperm. RESULT(S): We found APN in the equatorial segment of the upper post-acrosomal region of the sperm head, in the neck and along the tail of spermatozoa, in prostasomes, and in seminal fluid, whereas NEP was present in a very restricted area of a few sperm cells and in prostasomes. Messenger RNA of both enzymes was detected in spermatozoa. The inhibition of enkephalin-degrading enzymes attenuated the time-dependent decrease of sperm motility; this effect was reversed by naloxone. CONCLUSION(S): Enkephalin-degrading enzymes are present in human semen and may be involved in the control of sperm motility, mainly by the regulation of endogenous opioid peptides.

Expression and localization of delta-, kappa-, and mu-opioid receptors in human spermatozoa and implications for sperm motility.

CONTEXT: Endogenous opioid peptides signal through delta-, kappa-, and mu-opioid receptors. Some of these peptides such as endorphins and enkephalins are present in the male reproductive tract, but the presence of the corresponding receptors in human sperm cells has not yet been reported. OBJECTIVE: Our objective was to study the expression and localization of delta-, kappa-, and mu-opioid receptors on human spermatozoa and the implication in sperm motility. METHODS: The expression of receptors was studied by RT-PCR, Western blot, and immunofluorescence techniques. We evaluated the effects of activation of each opioid receptor by specific agonist and antagonist. RESULTS: Human spermatozoa express delta-, kappa-, and mu-opioid receptors. These receptors were located in different parts of the head, in the middle region, and in the tail of the sperm. Progressive motility of spermatozoa, an important parameter to evaluate male fertility, was found to be significantly reduced after incubation with the mu-receptor agonist morphine, whereas this effect was antagonized in the presence of the corresponding antagonist naloxone. The delta-receptor antagonist naltrindole significantly reduced progressive motility immediately after its addition. However, the delta-receptor agonist DPDPE had no significant effect. Finally, neither the kappa-receptor agonist U50488 nor its antagonist nor-binaltorphimine significantly affected the progressive motility of human spermatozoa. CONCLUSION: We report for first time the presence of functional delta-, kappa-, and mu-opioid receptors in human sperm membranes. These findings are indicative of a role for the opioid system in the regulation of sperm physiology.

Non-stochastic quadratic fingerprints and LDA-based QSAR models in hit and lead generation through virtual screening: theoretical and experimental assessment of a promising method for the discovery of new antimalarial compounds.

In order to explore the ability of non-stochastic quadratic indices to encode chemical information in antimalarials, four quantitative models for the discrimination of compounds having this property were generated and statistically compared. Accuracies of 90.2% and 83.3% for the training and test sets, respectively, were observed for the best of all the models, which included non-stochastic quadratic fingerprints weighted with Pauling electronegativities. With a comparative purpose and as a second validation experiment, an exercise of virtual screening of 65 already-reported antimalarials was carried out. Finally, 17 new compounds were classified as either active/inactive ones and experimentally evaluated for their potential antimalarial properties on the ferriprotoporphyrin (FP) IX biocrystallization inhibition test (FBIT). The theoretical predictions were in agreement with the experimental results. In the assayed test compound C5 resulted more active than chloroquine. The current result illustrates the usefulness of the TOMOCOMD-CARDD strategy in rational antimalarial-drug design, at the time that it introduces a new family of organic compounds as starting point for the development of promising antimalarials.

Quantitative Structure Antitumoral-Activity Relationships of Thiadiazinthione Derivatives Using the Novel Hybrid Molecular Index pMRc / s. t

The recently defined molar-refractivity-partition index was applied to a family of 1,3,5- thiadiazin-2-thione derivatives in order to establish quantitative structure-antitumoral models. The goal of this effort is to establish the relationships between the structure and biological response of these compounds. Method. After the splitting of the sample in two sets, their indices were correlated against the measured biological activity. The combined use of our index with others had been able to describe not only the topologic but also the London dispersive forces of any fragment in relation to the biological response of the sets. Results. The obtained models showed correlation coefficients of 0,87 and 0,81 respectively linking structural and biological features of the molecules. The mean relative error values were less than 7 percent. According to the models, the activity of the first sample is related mostly to molecular topology and dispersive forces. Sample two activity was associated to the size and branching of the substituents, and also to the London forces. Conclusion. The index was able to discriminate between pure topological features and those related to dispersive forces(AU)

A linear discrimination analysis based virtual screening of trichomonacidal lead-like compounds: outcomes of in silico studies supported by experimental results.

A computational (virtual) screening test to identify potential trichomonacidals has been developed. Molecular structures of trichomonacidal and non-trichomonacidal drugs were represented using stochastic and non-stochastic atom-based quadratic indices and a linear discrimination analysis (LDA) was trained to classify molecules regarding their antiprotozoan activity. Validation tests revealed that our LDA-QSAR models recognize at least 88.24% of trichomonacidal lead-like compounds and suggest using this methodology in virtual screening protocols. These classification functions were then applied to find new lead antitrichomonal compounds. In this connection, the biological assays of eight compounds, selected by computational screening using the present models, give good results (87.50% of good classification). In general, most of the compounds showed high activity against Trichomonas vaginalis at the concentration of 100 microg/ml and low cytotoxicity to this concentration. In particular, two heterocyclic derivatives (VA7-67 and VA7-69) maintained their efficacy at 10 microg/ml with an important trichomonacidal activity (100.00% of reduction), but it is remarkable that the compound VA7-67 did not show cytotoxic effects in macrophage cultivations. This result opens a door to a virtual study considering a higher variability of the structural core already evaluated, as well as of other chemicals not included in this study.

Synthesis and biological properties of new 5-nitroindazole derivatives.

A series of new 3-alkoxy- or 3-hydroxy-1-[omega-(dialkylamino)alkyl]-5-nitroindazoles have been synthesized and their trichomonacidal, antichagasic and antineoplastic properties studied. Five derivatives (5, 6, 8, 9 and 17) showed remarkable trichomonacidal activity against Trichomonas vaginalis at 10 microg/mL concentration. Three compounds (8, 10, 11) exhibited interesting antichagasic activity and these same compounds moderate antineoplastic activity against TK-10 and HT-29 cell lines. Unspecific cytotoxicity against macrophages has also been evaluated and only compounds 9, 10 and 11 resulted cytotoxic at the higher dose evaluated (100 microg/mL), loosing cytotoxicity at lower doses. QSAR studies have been carried out. X-ray crystallographic study of compound 8 has been performed.

Synthesis and antiparasitic properties of new 4-N-benzylamino-4-hetarylbut-1-enes.

New derivatives of 4-N-benzylamino-4-hetarylbut-1-ene containing a pyridyl nucleus were synthesized from benzylamines and pyridine aldehydes. N-oxide derivatives were obtained from these homoallylamines. Study of the antiparasitic properties of obtained pyridine derivatives as well as of four related benzazepines previously described, was carried out using cytotoxicity assays against Trichomonas vaginalis and epimastigote form of Trypanosoma cruzi protozoa. Compounds showing activity against epimastigote T. cruzi were tested against the amastigote form; unspecific cytotoxicity against macrophages was also studied.

A computer-based approach to the rational discovery of new trichomonacidal drugs by atom-type linear indices.

Computational approaches are developed to design or rationally select, from structural databases, new lead trichomonacidal compounds. First, a data set of 111 compounds was split (design) into training and predicting series using hierarchical and partitional cluster analyses. Later, two discriminant functions were derived with the use of non-stochastic and stochastic atom-type linear indices. The obtained LDA (linear discrimination analysis)-based QSAR (quantitative structure-activity relationship) models, using non-stochastic and stochastic descriptors were able to classify correctly 95.56% (90.48%) and 91.11% (85.71%) of the compounds in training (test) sets, respectively. The result of predictions on the 10% full-out cross-validation test also evidenced the quality (robustness, stability and predictive power) of the obtained models. These models were orthogonalized using the Randic orthogonalization procedure. Afterwards, a simulation experiment of virtual screening was conducted to test the possibilities of the classification models developed here in detecting antitrichomonal chemicals of diverse chemical structures. In this sense, the 100.00% and 77.77% of the screened compounds were detected by the LDA-based QSAR models (Eq. 13 and Eq. 14, correspondingly) as trichomonacidal. Finally, new lead trichomonacidals were discovered by prediction of their antirichomonal activity with obtained models. The most of tested chemicals exhibit the predicted antitrichomonal effect in the performed ligand-based virtual screening, yielding an accuracy of the 90.48% (19/21). These results support a role for TOMOCOMD-CARDD descriptors in the biosilico discovery of new compounds.

Quantitative structure antitumoral-activity relationships of thiadiazinthione derivatives using the novel hybrid molecular index pMRchi.

PURPOSE: The recently defined molar-refractivity-partition index was applied to a family of 1,3,5- thiadiazin-2-thione derivatives in order to establish quantitative structure-antitumoral models. The goal of this effort is to establish the relationships between the structure and biological response of these compounds. METHOD: After the splitting of the sample in two sets, their indices were correlated against the measured biological activity. The combined use of our index with others had been able to describe not only the topologic but also the London dispersive forces of any fragment in relation to the biological response of the sets. RESULTS: The obtained models showed correlation coefficients of 0.87 and 0.81 respectively linking structural and biological features of the molecules. The mean relative error values were less than 7%. According to the models, the activity of the first sample is related mostly to molecular topology and dispersive forces. Sample two activity was associated to the size and branching of the substituents, and also to the London forces. CONCLUSION: The index was able to discriminate between pure topological features and those related to dispersive forces.

Pyroglutamyl peptidase I and prolyl endopeptidase in human semen: increased activity in necrozoospermia.

Thyrotropin-releasing hormone (TRH) and its analogues have been reported to have important functions in human semen. In the present paper, we have characterized the activity of the TRH-degrading enzymes pyroglutamyl peptidase I and prolyl endopeptidase in the fluid and prostasomes of human semen and in subcellular fractions of the corresponding sperm. Enzymatic activities were measured fluorimetrically using beta-naphthylamine derivatives as substrate. Activity associated with both enzymes was detected in seminal fluid and in the prostasome fraction, as well as in soluble and particulate sperm subcellular fractions. Pyroglutamyl-peptidase I activity presented highest levels in the particulate sperm fraction, whereas the activity of prolyl endopeptidase was maximal in the soluble sperm fraction. In addition, we compared the activity of both enzymes in different seminal fractions in normozoospermic, fertile men and in subfertile patients with different abnormalities revealed by spermiogram analysis (astenozoospermia, necrozoospermia and teratozoospermia). The activities of pyroglutamyl peptidase I and prolyl endopeptidase in necrozoospermia were found to be higher in the corresponding soluble and particulate sperm fractions, respectively, with respect to those measured in normozoospermic semen. The results of the present study indicate that these enzymes may participate in regulating the levels of seminal TRH analogues and in mediating sperm death associated with necrozoospermia.