An account on the history of pharmacology in Spain.

Here we present an account on the history of pharmacology in Spain. Pharmacology as an independent science in Europe began with the creation of university chairs. Of particular relevance was the appointment in 1872 of Osswald Shmiedeberg as chairman of an Institute of Pharmacology at the University of Strassbourg, Germany. Teófilo Hernando pioneered in Spain the new emerging pharmacology at the beginning of the XX Century. He made a posdoctoral stay in the laboratory of Schmiedeberg, working on digitalis. In 1912 he won the chair of "Materia Médica y Arte de Recetar" at "Universidad Central of Madrid" (today, "Universidad Complutense de Madrid", UCM). He soon decided to transform such subject to the emerging modern pharmacology, with the teaching of experimental pharmacology in the third course of medical studies and clinical therapeutics (today clinical pharmacology) in the sixth course. This was the status of pharmacology in 1920, supporting the view that Hernando was a pioneer of clinical pharmacology. However, the Spanish Civil War and the II Word War interropted this division of preclinical and clinical pharmacology; only in the 1980's was clinical pharmacolgy partially developed in Spain. From a scientific point of view, Hernando directly trained various young pharmacologists that extended the new science to various Spanish universities. Some of his direct disciples were Benigno Lorenzo Velázquez, Francisco García Valdecasas, Rafael Méndez, Tomás Alday, Gabriel Sánchez de la Cuesta, Dámaso Gutiérrez or Ramón P é rez-Cirera. One of the central research subject was the analysis of the effects of digitalis on the cat and frog heart. In the initiation of the 1970 s pharmacologists trained by those Hernando's students grew throughout various universities and the "Consejo Superior de Investigaciones Científicas" (CSIC). And hence, in 1972 the "Sociedad Española de Farmacología" (SEF) emerged. Later on, in the 1990's the "Sociedad Española de Farmacología Clínica (SEFC) also emerged. The relationship between the two societies is still weak. Out of the vast scope of the pharmacological sciences, Spanish pharmacologists have made relevant contributions in two areas namely, neuropsychopharmacology and cardiovacular pharmacology. Nonetheless, in other areas such as smooth muscle, gastroenterology, pharmacogenetics and hepatic toxicity, Spanish pharmacologists have also made relevant contributions. A succint description of such contributions is made. Finally, some hints on perspectives for the further development of preclinical and clinical pharmacology in Spain, are offered.

Herbal medicines for the treatment of cardiovascular diseases: Benefits and risks - A narrative review.

Herbal medicines (HMs) have been traditionally used for the prophylaxis/treatment of cardiovascular diseases (CVDs). Their use is steadily increasing and many patients with CVDs often combine HMs with prescribed cardiovascular medications. Interestingly, up to 70% of patients do not notify cardiologists/physicians the use of HMs and up to 90% of cardiologists/physicians may not routinely inquire them about the use of HMs. There is limited scientific evidence from well-designed clinical trials supporting the efficacy and safety of HMs and because they do not reduce morbidity and mortality are not recommended in clinical guidelines for the prophylaxis/treatment of CVDs. There is also a great deal of confusion about the identification, active constituents and mechanisms of action of HMs; the lack of standardization and quality control (contaminations, adulterations) represent other sources of concern. Furthermore, the widespread perception that unlike prescription drugs HMs are safe is misleading and some HMs can cause clinically relevant adverse events and interactions, particularly when used with narrow therapeutic index prescribed cardiovascular drugs (antiarrhythmics, antithrombotics, digoxin). Cardiologists/physicians can no longer ignore the problem. They must improve their knowledge about the HMs their patients consume to provide the best advice and prevent adverse reactions and drug interactions. This narrative review addresses the putative mechanisms of action, suggested clinical uses and safety of most commonly used HMs, the pivotal role of cardiologists/physicians to protect consumers and the main challenges and gaps in evidence related to the use of HMs in the prophylaxis and treatment of CVDs.

Flavonoids: Antiplatelet Effect as Inhibitors of COX-1.

Flavonoids are compounds with a benzopyranic structure that exhibits multiple pharmacological activities. They are known for their venotonic activity, but their mechanism of action remains unclear. It is thought that, as this mechanism is mediated by prostaglandins, these compounds may interfere with the arachidonic acid (AA) cascade. These assays are designed to measure the antiplatelet aggregation capacity of quercetin, rutin, diosmetin, diosmin, and hidrosmin, as well as to evaluate a potential structure-activity ratio. In this paper, several studies on platelet aggregation at different concentrations (from 0.33 mM to 1.5 mM) of different flavone compounds are conducted, measuring platelet aggregation by impedance aggregometry, and the cyclooxygenase (COX) activity by metabolites generated, including the activity of the pure recombinant enzyme in the presence of these polyphenols. The results obtained showed that quercetin and diosmetin aglycones have a greater antiplatelet effect and inhibit the COX enzyme activity to a greater extent than their heterosides; however, the fact that greater inhibition of the pure recombinant enzyme was achieved by heterosides suggests that these compounds may have difficulty in crossing biological membranes. In any case, in view of the results obtained, it can be concluded that flavonoids could be useful as coadjuvants in the treatment of cardiovascular pathologies.

A New Approach to the Management of COVID-19. Antagonists of IL-6: Siltuximab.

Since the beginning of the pandemic, numerous national and international clinical trials have been conducted with a large number of drugs. Many of them are intended for the treatment of other pathologies; however, despite the great effort made, no specific drug is available for the treatment of the symptoms of respiratory disease caused by SARS-CoV-2 infection. The aim of this article is to provide data to justify the use of drugs to tackle the effects produced by IL-6 as the main inflammatory mediator in patients with COVID-19 with severe respiratory complications, considering all clinical evidence linking the poor prognosis of these patients with increased IL-6 levels in the context of cytokine release syndrome. Furthermore, data are provided to justify the proposal of a rational dosing of siltuximab, a monoclonal antibody specifically targeting IL-6, based on RCP levels, considering the limited results published so far on the use of this drug in COVID-19. A literature search was conducted on the clinical trials of siltuximab published to date as well as on the different IL-6 signalling pathways and the effects of its overexpression. Knowledge of the mechanisms of action on these pathways may provide important information for the design of drugs useful in the treatment of these patients. This article describes the characteristics, properties, mechanism of action, therapeutic uses and clinical studies conducted with siltuximab so far. The results confirm that administration of siltuximab downregulates IL-6 levels, thereby reducing the inflammatory process in COVID-19 patients with severe respiratory disease, suggesting that it can be successfully used to prevent cytokine release syndrome and death from this cause.

Binding and antiplatelet activity of quercetin, rutin, diosmetin, and diosmin flavonoids.

Platelets exert an essential role in vascular inflammation and thrombosis. Flavonoids are natural compounds employed for the clinical management of vascular disorders preventing capillary permeability, working as phlebotonics and improving the blood rheology, although their mechanism of action remains partially unknown. The effects of quercetin, rutin, diosmetin and diosmin were investigated in platelet activation utilizing blood from healthy and non-treated volunteers. The arrangement of the different activation states of platelets and GPIIb/IIIa receptor occupation was computed by flow cytometry working with calcium ionophore as pro-aggregant to provoke platelet activation and aggregation. The flavonoids studied demonstrated relevant antiplatelet activity through the blocked of GPIIb/IIIa receptors, the suppression of the platelet activation, as well as the pro-aggregate effect of calcium ionophore. Therefore, whichever of the active ingredients examined could be beneficious in the prevention of cardiovascular disease and this article also contributes to elucidate a new mechanism of action for these drugs.

Antiplatelet Activity of Coumarins: In Vitro Assays on COX-1.

Atherosclerotic cardiovascular disease is the leading cause of death in developed countries. Therefore, there is an increasing interest in developing new potent and safe antiplatelet agents. Coumarins are a family of polyphenolic compounds with several pharmacological activities, including platelet aggregation inhibition. However, their antiplatelet mechanism of action needs to be further elucidated. The aim of this study is to provide insight into the biochemical mechanisms involved in this activity, as well as to establish a structure-activity relationship for these compounds. With this purpose, the antiplatelet aggregation activities of coumarin, esculetin and esculin were determined in vitro in human whole blood and platelet-rich plasma, to set the potential interference with the arachidonic acid cascade. Here, the platelet COX activity was evaluated from 0.75 mM to 6.5 mM concentration by measuring the levels of metabolites derived from its activity (MDA and TXB2), together with colorimetric assays performed with the pure recombinant enzyme. Our results evidenced that the coumarin aglycones present the greatest antiplatelet activity at 5 mM and 6.5 mM on aggregometry experiments and inhibiting MDA levels.

Monte Carlo Computation of Dose-Volume Histograms in Structures at Risk of an Eye Irradiated with Heterogeneous Ruthenium-106 Plaques.

BACKGROUND/AIMS: The aim of this work is to compare Monte Carlo simulated absorbed dose distributions obtained from 106Ru eye plaques, whose heterogeneous emitter distribution is known, with the common homogeneous approximation. The effect of these heterogeneities on segmented structures at risk is analyzed using an anthropomorphic phantom. METHODS: The generic CCA and CCB, with a homogeneous emitter map, and the specific CCA1364 and CCB1256 106Ru eye plaques are modeled with the Monte Carlo code PENELOPE. To compare the effect of the heterogeneities in the segmented volumes, cumulative dose-volume histograms are calculated for different rotations of the aforementioned plaques. RESULTS: For the cornea, the CCA with the equatorial placement yields the lowest absorbed dose rate while for the CCA1364 in the same placement the absorbed dose rate is 33% higher. The CCB1256 with the hot spot oriented towards the cornea yields the maximum dose rate per unit of activity while it is 44% lower for the CCB. CONCLUSIONS: Dose calculations based on a homogeneous distribution of the emitter substance yield the lowest absorbed dose in the analyzed structures for all plaque placements. Treatment planning based on such calculations may result in an overdose of the structures at risk.

Potential Therapeutic Anti-Inflammatory and Immunomodulatory Effects of Dihydroflavones, Flavones, and Flavonols.

Systemic inflammation, circulating immune cell activation, and endothelial cell damage play a critical role in vascular pathogenesis. Flavonoids have shown anti-inflammatory effects. In this study, we investigated the effects of different flavonoids on the production of pro-inflammatory interleukin (IL) 1ß, 6, and 8, and tumor necrosis factor α (TNF-α), in peripheral blood cells. Methods: We studied the whole blood from 36 healthy donors. Lipopolysaccharide (LPS)-stimulated (0.5 µg/mL) whole-blood aliquots were incubated in the presence or absence of different concentrations of quercetin, rutin, naringenin, naringin, diosmetin, and diosmin for 6 h. Cultures were centrifuged and the supernatant was collected in order to measure IL-1ß, TNF-α, IL-6, and IL-8 production using specific immunoassay techniques. This production was significantly inhibited by quercetin, naringenin, naringin, and diosmetin, but in no case by rutin or diosmin. Flavonoids exert different effects, maybe due to the differences between aglycons and glucosides present in their chemical structures. However, these studies suggest that quercetin, naringenin, naringin, and diosmetin could have a potential therapeutic effect in the inflammatory process of cardiovascular disease.

Monte Carlo Simulation of the Treatment of Uveal Melanoma Using Measured Heterogeneous 106Ru Plaques.

BACKGROUND/AIMS: Ruthenium plaques are used for the treatment of ocular tumors. The aim of this work is the comparison between simulated absorbed dose distributions tallied in an anthropomorphic phantom, obtained from ideal homogeneous plaques, and real eye plaques in which the actual heterogeneous distribution of 106Ru was measured. The placement of the plaques with respect to the tumor location was taken into consideration to optimize the effectiveness of the treatment. METHODS: The generic CCA and CCB, and the specific CCA1364 and CCB1256 106Ru eye plaques were modeled with the Monte Carlo code PENELOPE. To compare the suitability of each treatment for an anterior, equatorial and posterior tumor location, cumulative dose-volume histograms for the tumors and structures at risk were calculated. RESULTS: Eccentric placements of the plaques, taking into account the inhomogeneities of the emitter map, can substantially reduce the dose delivered to structures at risk while maintaining the prescribed dose at the tumor apex. CONCLUSIONS: The emitter map distribution of the plaque and the computerized tomography of the patient used in a Monte Carlo simulation allow an accurate determination of the plaque position with respect to the tumor with the potential to reduce the dose to sensitive structures.

Monte Carlo Estimation of Absorbed Dose Distributions Obtained from Heterogeneous 106Ru Eye Plaques.

BACKGROUND: The distribution of the emitter substance in 106Ru eye plaques is usually assumed to be homogeneous for treatment planning purposes. However, this distribution is never homogeneous, and it widely differs from plaque to plaque due to manufacturing factors. METHODS: By Monte Carlo simulation of radiation transport, we study the absorbed dose distribution obtained from the specific CCA1364 and CCB1256 106Ru plaques, whose actual emitter distributions were measured. The idealized, homogeneous CCA and CCB plaques are also simulated. RESULTS: The largest discrepancy in depth dose distribution observed between the heterogeneous and the homogeneous plaques was 7.9 and 23.7% for the CCA and CCB plaques, respectively. In terms of isodose lines, the line referring to 100% of the reference dose penetrates 0.2 and 1.8 mm deeper in the case of heterogeneous CCA and CCB plaques, respectively, with respect to the homogeneous counterpart. CONCLUSIONS: The observed differences in absorbed dose distributions obtained from heterogeneous and homogeneous plaques are clinically irrelevant if the plaques are used with a lateral safety margin of at least 2 mm. However, these differences may be relevant if the plaques are used in eccentric positioning.

Antiplatelet activity of flavonoid and coumarin drugs.

Polyphenols are used as phlebotonic drugs, but their mechanism of action remains unknown. Since platelet activity and platelet-endothelial cell interactions are involved in the pathogenesis of cardiovascular disease, this work examines whether different flavonoid and coumarin drugs are able to inhibit platelet aggregation. This specific case of coumarins, the antiplatelet effect is not linked with a possible interaction over blood coagulation since this effect only dicoumarols have it. The antiplatelet capacity of polyphenols was assayed using peripheral blood platelets from healthy controls. The distribution of the different platelets subsets was quantified by flow cytometry, using the calcium ionophore as a pro-aggregant. The number of GPIIb/IIIa receptors occupied by the drugs was assayed by flow cytometry using two CD61 surface fluorescein antibodies. All the polyphenols tested inhibited platelet aggregation. A percentage antiplatelet activity of 88.91±7.98% was recorded for naringin, 48.43±8.84% for naringenin, 53.83±7.87% for esculetin, 54.65±6.91% for fraxetin, and 25.75±4.12% for coumarin. Naringin showed significantly greater percentage occupation of GPIIb/IIIa receptors than did naringenin (14.82±0.81% vs. 3.90±0.55%), and esculetin returned significantly higher values than fraxetin and coumarin (12.47±0.97 vs. 7.53±0.49 and 7.90±0.69 respectively). All drugs show important antiplatelet activity. Naringin was the best antiplatelet compound, showing the greatest antiplatelet activity and the highest percentage binding of GPIIb/IIIa receptors. However, any of the compounds used could be used in the prevention of cardiovascular disease.

Monte Carlo Simulation of the Treatment of Eye Tumors with (106)Ru Plaques: A Study on Maximum Tumor Height and Eccentric Placement.

BACKGROUND/AIMS: Ruthenium plaques are used for the treatment of ocular tumors. There is, however, a controversy regarding the maximum treatable tumor height. Some advocate eccentric plaque placement, without a posterior safety margin, to avoid collateral damage to the fovea and optic disc, but this has raised concerns about marginal tumor recurrence. There is a need for quantitative information on the spatial absorbed dose distribution in the tumor and adjacent tissues. We have overcome this obstacle using an approach based on Monte Carlo simulation of radiation transport. METHODS: CCA and CCB (106)Ru plaques were modeled and their geometry embedded in a computerized tomography scan of the eye of a patient. Different tumor sizes and locations were simulated with the general-purpose Monte Carlo code PENELOPE. RESULTS: Cumulative dose-volume histograms were obtained for the tumors and the tissues at risk considered. Plots of isodose lines for both plaques were obtained in a computerized tomography study. CONCLUSIONS: Ruthenium eye plaques are an adequate treatment option for tumors up to around 5 mm in height. According to our results, assuming a correct placement of the plaque, a tumor of 6.5 mm apical height is about the maximum size that can be treated safely with the large CCB plaque.

Breakthrough cancer pain - still a challenge.

Breakthrough cancer pain is defined as transient pain exacerbation in patients with stable and controlled basal pain. Although variable, the prevalence of breakthrough cancer pain is high (33%-95%). According to the American Pain Foundation, breakthrough pain is observed in 50%-90% of all hospitalized cancer patients, in 89% of all patients admitted to homes for the elderly and terminal-patient care centers, and in 35% of all ambulatory care cancer patients. The management of breakthrough cancer pain should involve an interdisciplinary and multimodal approach. The introduction of new fentanyl formulations has represented a great advance and has notably improved treatment. Among these, the pectin-based intranasal formulation adjusts very well to the profile of breakthrough pain attacks, is effective, has a good toxicity profile, and allows for convenient dosing - affording rapid and effective analgesia with the added advantage of being easily administered by caregivers when patients are unable to collaborate.

Electron irradiation of conjunctival lymphoma--Monte Carlo simulation of the minute dose distribution and technique optimization.

PURPOSE: External beam radiotherapy is the only conservative curative approach for Stage I non-Hodgkin lymphomas of the conjunctiva. The target volume is geometrically complex because it includes the eyeball and lid conjunctiva. Furthermore, the target volume is adjacent to radiosensitive structures, including the lens, lacrimal glands, cornea, retina, and papilla. The radiotherapy planning and optimization requires accurate calculation of the dose in these anatomical structures that are much smaller than the structures traditionally considered in radiotherapy. Neither conventional treatment planning systems nor dosimetric measurements can reliably determine the dose distribution in these small irradiated volumes. METHODS AND MATERIALS: The Monte Carlo simulations of a Varian Clinac 2100 C/D and human eye were performed using the penelope and penEasyLinac codes. Dose distributions and dose volume histograms were calculated for the bulbar conjunctiva, cornea, lens, retina, papilla, lacrimal gland, and anterior and posterior hemispheres. RESULTS: The simulated results allow choosing the most adequate treatment setup configuration, which is an electron beam energy of 6 MeV with additional bolus and collimation by a cerrobend block with a central cylindrical hole of 3.0 cm diameter and central cylindrical rod of 1.0 cm diameter. CONCLUSIONS: Monte Carlo simulation is a useful method to calculate the minute dose distribution in ocular tissue and to optimize the electron irradiation technique in highly critical structures. Using a voxelized eye phantom based on patient computed tomography images, the dose distribution can be estimated with a standard statistical uncertainty of less than 2.4% in 3 min using a computing cluster with 30 cores, which makes this planning technique clinically relevant.

[Pharmacology]. / Farmaecología.

Two of the main characteristics of western societies in the last fifty years have been the medicalization of the human life and the environmental degradation. The first one has forced human being to consider medicines use related to what would be rational, reasonable and well-reasoned. The second one brought us to a new ecologist conscience. In relation to the "human social system", the effects of medication can be considered very positive as a whole, particularly those related to the amazing increase of expectative and quality of life. But, along with those unquestionable beneficial effects, medicines have also caused some negative effects for other biotic and abiotic systems, such as microbian alterations and their undesirable consequences which have involved the massive use of antibiotics in medicine and veterinary, the uncontrolled elimination of millions of doses of all kind of drugs, additives and excipients, etc., as well as atmospheric contamination and degradation of forests and deep oceans which can have been caused by investigation and production of determinated drugs. In this context Pharmacology appears as a scientific discipline that studies the research (R), development (D), production (P), and utilization (U) of drugs and medical substances in relation to the environment. From a farmaecologic perspective the drugs utilization has its development in three main contexts, all of them closely related: prescription quality, farmaceutical care, and patient's active participation in his own disease and treatment.

In vitro starch digestibility and expected glycemic index of pound cakes baked in two-cycle microwave-toaster and conventional oven.

Bread baking technology has an important effect on starch digestibility measured as its predicted glycemic index tested in vitro. The aim of this work was to evaluate the changes in predicted glycemic index of pound cake baked in a two-cycle microwave toaster and a conventional oven. The glycemic index was calculated from hydrolysis index values by the Granfeldt method. Non-significant differences (P > 0.05) were found in hydrolysis index (60.67 ± 3.96 for the product baked in microwave oven and 65.94 ± 4.09 for the product baked in conventional oven) and predicted glycemic index content (60.5 for product baked in microwave oven and 65 for the product baked in conventional oven) in freshly-baked samples. Results clearly demonstrate that the baking pound cake conventional process could be replicated using a two-cycle multifunction microwave oven, reducing the traditional baking time. Further research is required in order to achieve pound cake crumb uniformity.

Applying pattern recognition methods plus quantum and physico-chemical molecular descriptors to analyze the anabolic activity of structurally diverse steroids.

The great cost associated with the development of new anabolic-androgenic steroid (AASs) makes necessary the development of computational methods that shorten the drug discovery pipeline. Toward this end, quantum, and physicochemical molecular descriptors, plus linear discriminant analysis (LDA) were used to analyze the anabolic/androgenic activity of structurally diverse steroids and to discover novel AASs, as well as also to give a structural interpretation of their anabolic-androgenic ratio (AAR). The obtained models are able to correctly classify 91.67% (86.27%) of the AASs in the training (test) sets, respectively. The results of predictions on the 10% full-out cross-validation test also evidence the robustness of the obtained model. Moreover, these classification functions are applied to an "in house" library of chemicals, to find novel AASs. Two new AASs are synthesized and tested for in vivo activity. Although both AASs are less active than some commercially AASs, this result leaves a door open to a virtual variational study of the structure of the two compounds, to improve their biological activity. The LDA-assisted QSAR models presented here, could significantly reduce the number of synthesized and tested AASs, as well as could increase the chance of finding new chemical entities with higher AAR.

CD4 and CD8 T cell response to the rHSP60 from Klebsiella pneumoniae in peripheral blood mononuclear cells from patients with ankylosing spondylitis.

OBJECTIVE: To determine the processing pathways used by peripheral blood mononuclear cells (PBMC) and present the rHSP60Kp, and the T cell subpopulations involved in the response, in patients with ankylosing spondylitis (AS) METHODS: The lymphoproliferative response to the rHSP60Kp in PBMC from 14 HLA-B27+ AS patients and 15 B27- healthy controls was assessed by 3H-TdR incorporation. The processing pathways for the rHSP60Kp were analyzed by 3H-TdR incorporation in fresh PBMC from patients using homologous PBMC preincubated with the antigen and specific inhibitors: chloroquine, N-acetyl-L-leucil-L-leucil-L-nor-leucinal (LLnL) or brefeldin A (BFA), fixed with p-formaldehyde (fixed APC). The CD4+/CD8+ T cell subpopulation activated with the antigen was determined by three colours flow cytometry in PBMC from patients. RESULTS: Eight out of fourteen patients showed positive lymphoproliferative responses to the rHSP60Kp while none of the healthy controls responded (p < 0.012). In five patients S.I. was above 4.0. In these patients lymphoproliferation was lower when chloroquine and LLnL was used and it became negative with BFA, indicating that both pathways are used. CD4+ and CD8+ T cells populations expressed CD69 when activated by the rHSP60Kp. CONCLUSIONS: Our results suggest that CD4 and CD8 T cells participate in the response to the rHSP60Kp in B27+ AS patients.

CD4 and CD8 T cell response to the rHSP60 from Klebsiella pneumoniae in peripheral blood mononuclear cells from patients with ankylosing spondylitis / Respuesta de linfocitos T CD4 y CD8 contra la rHSP60 de Klebsiella pneumoniae en células mononucleares de sangre periférica de pacientes con espondilitis anquilosa

Objective. To determine the processing pathways used by peripheral blood mononuclear cells (PBMC) and present the rHSP60Kp, and the T cell subpopulations involved in the response, in patients with ankylosing spondylitis (AS) Methods. The lymphoproliferative response to the rHSP60Kp in PBMC from 14 HLA-B27 + AS patients and 15 B27 healthy controls was assessed by ³H-TdR incorporation. The processing pathways for the rHSP60Kp were analyzed by ³H-TdR incorporation in fresh PBMC from patients using homologous PBMC preincubated with the antigen and specific inhibitors: chloroquine, N-acetyl-L-leucil-L-leucil-L-nor-leucinal (LLnL) or brefeldin A (BFA), fixed with p-formaldehyde (fixed APC). The CD4+/CD8+ T cell subpopulation activated with the antigen was determined by three colours flow cytometry in PBMC from patients. Results. Eight out of fourteen patients showed positive lymphoproliferative responses to the rHSP60Kp while none of the healthy controls responded (p < 0.012). In five patients S.I. was above 4.0. In these patients lymphoproliferation was lower when chloroquine and LLnL was used and it became negative with BFA, indicating that both pathways are used. CD4+ and CD8+ T cells populations expressed CD69 when activated by the rHSP60Kp. Conclusions. Our results suggest that CD4 and CD8 T cells participate in the response to the rHSP60Kp in B27+ AS patients.

Objetivo.Determinar las vías utilizadas por las células mononucleares de sangre periférica (CMSP) de pacientes con espondilitis anquilosante para procesar a la rHSPGO de Klebsiella pneumoniae (rHSPGOKp) y las subpoblaciones de linfocitos T involucrados en la activación. Métodos. Se determinó la respuesta linfoproliferativa, por incorporación de ³H-TdR en CMSP, en presencia de la rHSPGOKp, en 14 pacientes con EA HLA-B27+y en 15 sujetos sanos HLA-B27-. La ruta de procesamiento y presentación de la rHSPGOKp se determinó por incorporación de ³H-TdR en las CMSP de los pacientes utilizando como células presentadoras a las CMSP homologas, preíncubadas con el antígeno y los inhibidores específicos: cloroquína, brefeldína A y N-acetil-L-leucil-L-leucil-L-nor-leucinal (LLnL), y fijadas con p-formaldehído. Se evaluaron las subpoblaciones de linfocitos T CD4+ y CD8+ que expresaron CD69, frente al antígeno, por citometría de flujo. Resultados. Ocho de los 14 pacientes y ninguno de los sujetos sanos, tuvo respuesta linfoproliferativa positiva (IE > 3.0) contra la rHSPGOKp (p < 0.012). En cinco de los pacientes el I.E. fue superior a 4.0. En estos pacientes la linfoproliferación disminuyó cuando se utilizó cloroquína y LLnL, y se hizo negativa cuando se utilizó BFA, lo que indica que ambas vías son empleadas. Las subpoblaciones de linfocitos T (CD4+ y CD8+) expresaron CD69 frente al antigeno. Conclusiones. Nuestros resultados sugieren que ambas poblaciones de linfocitos T: CD4+ y CD8+ participan en la respuesta a la rHSPGOKp.