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Las enfermedades del corazón son un tema tratado frecuentemente en las investigaciones médicas, debido a que repercuten ampliamente en el funcionamiento del cuerpo humano. Por lo anterior, se destaca el estudio del tratamiento de las afecciones de este órgano, lo que resulta en una importante tarea para mantener y potenciar los conocimientos sobre los adelantos terapéuticos de las enfermedades relacionadas con el mismo. Se realiza este trabajo con el objetivo de describir los avances en la terapia de la insuficiencia cardiaca. Para ello se realizó una revisión de publicaciones científicas de los últimos dos años. A pesar de que existen diferencias respecto a la terapia propuesta para la insuficiencia cardiaca, las asociaciones americanas y europea de cardiología establecen firmemente la terapia cuádruple (con los inhibidores del receptor de la angiotensina neprilysin, los ß-bloqueadores, los antagonistas de receptor de los mineralocorticoides y los inhibidores del cotransportador-2 de glucosa/sodio) -dirigida a la insuficiencia cardiaca con fracción de eyección reducida-, la recomendación del dinitrato de isosorbide/hidralazina para los pacientes de raza negra, y la utilización de la cardioversión implantable en la prevención de muerte súbita. Se concluye que la armonización de estas guías, en ambos continentes, proporciona un tratamiento único para la insuficiencia cardiaca, aunque necesita un estudio adicional en los pacientes con fracción de eyección del ventrículo izquierdo normal.
Heart diseases are a topic frequently treated in medical researches, due to their wide repercussion on the functioning of the human body; therefore, the study of the treatment of these organ affections is highlighted, being an important task to maintain and enhance knowledge about therapeutic advances in diseases related to it. This work is carried out with the aim of describing the advances in heart failure therapy. A review of the scientific publications of the last two years was performed. Although there are differences regarding the proposed therapy for heart failure, the American and European Associations of cardiology firmly establish the quadruple therapy (with angiotensin receptor inhibitors neprilysin, ß-blockers; mineralocorticoids antagonist receptors and glucose/sodium cotransporters-2 inhibitors-targeted at heart failure with reduced ejection fraction-, the recommendation of isosorbide dinitrate/hydralazine for black patients, and the use of implantable cardioversion in the prevention of the sudden death. It is concluded that the harmonization of these guidelines, in both continents, provides a unique treatment for heart failure, although it needs additional study in patients with normal left ventricular ejection fraction.
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Esta es una revisión sobre el papel de los péptidos natriuréticos y los intentos de utilizarlos como diana terapéutica a medida que se iba comprendiendo mejor su papel en la fisiopatología de la insuficiencia cardíaca con función sistólica deprimida. Se hace un recuento de su participación en sucesivos estudios fallidos y se explican los motivos de sus fracasos, hasta lograr el éxito deseado con la combinación del sacubitrilo/valsartan, lo que produjo un cambio de paradigma en el manejo de la insuficiencia cardíaca.
This review is conducted on the role of natriuretic peptides and the attempts to use them as a treatment as their role in the pathophysiology of heart failure with depressed systolic function was better understood. A recount of their participation in successive failed studies is provided, explaining the reasons for their failures, until achieving the desired success with the combination of sacubitril/valsartan. This produced a paradigm shift in the management of heart failure.
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One of the most relevant and differentiating aspects provided by the 2021 European Society of Cardiology guidelines for the diagnosis and treatment of acute and chronic heart failure is the retraction of the historical stepped and vertical pharmacological treatment scheme for heart failure with reduced ejection fraction (HFrEF). Subsequently, it was replaced by an updated algorithm that places four therapeutic families in the same initial horizontal step with an equally high degree of recommendation (class I). In this context, these four pillars, which have demonstrated a significant reduction in mortality and hospitalizations in patients with HFrEF, include (1) angiotensin-converting enzyme inhibitors (ACEi)/angiotensin receptor blockers (ARB)/angiotensin II receptor-neprilysin inhibitors (ARNi), (2) beta blockers, (3) mineralocorticoid receptor antagonists (MRA) and (4) sodium-glucose cotransporter 2 inhibitors (SGLT2is) as the main novelty. This manuscript reviews the current therapeutic algorithm with a special focus on the therapeutic value of adding an MRA (still underused in both clinical trials and real world), changing an ACEi/ARB for an ARNi and incorporating an SGLT2i in patients with HFrEF. This article is part of the Emerging concepts in heart failure management and treatment Special Issue: https://www.drugsincontext.com/special_issues/emerging-concepts-in-heart-failure-management-and-treatment.
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The aim of this study was to determine how sacubitril/valsartan compared with valsartan in an outpatient setting affects left ventricular remodeling in heart failure with reduced ejection fraction and functional (or secondary) mitral regurgitation (SMR) due to the effect of dual inhibition of the renin-angiotensin system and neprilysin. The outpatient study included 90 patients with chronic SMR who were followed up for 12 months. They received sacubitril/valsartan or valsartan instead of the more commonly used angiotensin-converting enzyme inhibitor enalapril for heart failure, in addition to standard medical therapy for heart failure. The difference in NT-proBNP change between groups was the primary endpoint. Changes in effective regurgitation orifice area, left ventricular ejection fraction, left ventricular end-systolic and end-diastolic volume indices, left atrial volume index, E/e' index, and exercise tolerance on the 6-minute walk test were secondary endpoints. In the treatment efficacy analysis, NT-proBNP levels decreased significantly by 37% in the sacubitril/valsartan group and by 11% in the valsartan group (P<0.001). Ejection fraction and exercise tolerance (increase in walking distance in the 6-min test) increased in the sacubitril/valsartan group (P<0.05). Also, in this group, the effective area of the regurgitation orifice, the left atrial volume index, the E/e' index, and the indices of the end-systolic and end-diastolic volume of the left ventricle (P<0.05) decreased more pronouncedly. Compared with valsartan, treatment with sacubitril/valsartan led to a significant improvement in cardiac remodeling in patients with SMR and heart failure with reduced ejection fraction.
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AIM: The win ratio can incorporate different types of outcomes and enhance statistical power, making it a useful method for analysing composite outcomes in cardiovascular trials. The application of this approach to the PARADISE-MI trial provides an additional perspective into understanding the effects of sacubitril/valsartan in patients with acute myocardial infarction. METHODS AND RESULTS: We conducted a post-hoc analysis of the PARADISE-MI trial, which randomly assigned patients with acute myocardial infarction complicated by a reduced left ventricular ejection fraction, pulmonary congestion, or both to receive either sacubitril/valsartan (97 mg of sacubitril and 103 mg of valsartan twice daily) or ramipril (5 mg twice daily) in addition to guideline-recommended therapy. The principal composite outcome was analysed in the hierarchical order of death due to cardiovascular causes, first hospitalization for heart failure, and first outpatient episode of symptomatic heart failure. We included events confirmed by the clinical events classification (CEC) committee as well as events identified by investigators that did not meet study definitions. Results were analysed by the unmatched win-ratio method. A win ratio that exceeds 1.00 reflects a better outcome. A total of 5661 patients underwent randomization; 2830 were assigned to receive sacubitril/valsartan and 2831 to receive ramipril. The hierarchical analysis of the principal composite outcome demonstrated a larger number of wins (1 265 767 [15.7%]) than losses (1 079 502 [13.4%]) in the sacubitril/valsartan group (win ratio of 1.17, 95% confidence interval [CI] 1.03-1.33; p = 0.015). Sensitivity analyses using alternative definitions of the composite outcome showed results similar to those of the principal analysis, except for analysis restricted to events that met CEC definitions (win ratio of 1.11, 95% CI 0.96-1.30; p = 0.16). CONCLUSION: In this post-hoc analysis of the PARADISE-MI trial using the win ratio and including investigator-identified events not having CEC confirmation, sacubitril/valsartan was superior to ramipril among high-risk survivors of acute myocardial infarction.
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Insuficiência Cardíaca , Infarto do Miocárdio , Humanos , Ramipril/uso terapêutico , Ramipril/farmacologia , Volume Sistólico , Antagonistas de Receptores de Angiotensina , Neprilisina , Tetrazóis/uso terapêutico , Função Ventricular Esquerda , Aminobutiratos/uso terapêutico , Valsartana/uso terapêutico , Compostos de Bifenilo/uso terapêutico , Combinação de Medicamentos , Infarto do Miocárdio/tratamento farmacológico , Infarto do Miocárdio/complicaçõesRESUMO
NPCdc is a natriuretic peptide synthesized from the amino acid sequence of the Crotalus durissus cascavella snake venom peptide, NP2Casca. NPCdc presents hypotensive and antioxidants effects. This study aimed to investigate in vivo whether angiotensin I-converting enzyme (ACE) inhibition would influence the impact of NPCdc in arterial pressure of rats submitted to 5/6 nephrectomy (Nx). Adult male Wistar rats following a 5/6 Nx were treated with enalapril (NxE group, 10 mg/kg/day, n = 9) or vehicle (Nx group, n = 8) for two weeks. On the 15th day after Nx, rats were anaesthetized and submitted to mean arterial pressure (MAP) determination before and after receiving two intravenous injections of saline (vehicle, n = 9) or NPCdc (0.3 µg/kg dissolved in saline, n = 18) separated by a 20-min interval. The kidneys were submitted to oxidative stress analysis. The basal MAP of the NxE group was nearly 20% lower (P < 0.05) than non-treated rats. NPCdc administration decreased the MAP in both groups; however, in the NxE group, the effects were observed only in the second injection. The peptide also decreased the NADPH oxidase activity in the renal cortex. Additionally, the hydrolysis of NPCdc by recombinant neprilysin (NEP) was monitored by mass spectrometry. NPCdc was cleaved by NEP at different peptides with an inhibition constant (Ki) of 1.5 µM, determined by a competitive assay using the NEP fluorescence resonance energy transfer (FRET) peptide substrate Abz-(d)Arg-Gly-Leu-EDDnp. Docking experiments confirmed the high affinity of NPCdc toward NEP. These findings provide new insights into the antihypertensive and antioxidant mechanism of action of NPCdc. Altogether, the results presented here suggest that NPCdc must be further studied as a potential therapy for cardiorenal syndromes.
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Enalapril , Neprilisina , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Animais , Pressão Sanguínea , Masculino , Peptídeos Natriuréticos , Peptídeos , Peptidil Dipeptidase A , Ratos , Ratos WistarRESUMO
Spiders are the most successful and diversified group of venomous animals. Currently, there are more than 49,000 species distributed almost all over the world. This broad distribution suggests that they have efficient strategies to improve their survival; one of them is the production of highly elaborate venoms, which are a heterogeneous mixture of molecules like inorganic salts, peptides, proteins, and enzymes. Considering this, this study aimed to analyze the venom of the spider Avicularia juruensis (Mygalomorphae: Theraphosidae) searching for proteolytic enzymes. Using zymography, electrophoresis, transcriptomics and proteomics approaches we identified one neprilysin able to degrade casein, that we named “Ajur_Neprilysin”. Neprilysins are metalloendopeptidases whose presence has already been described in animal venoms, however, its function has not yet been elucidated. Our results showed for the first time one non-bacterial neprilysin which can cleave casein and suggest that its role in envenomation is to degrade the extracellular matrix, facilitating the access of other toxins to their targets, as well as digestive fluids. Moreover, this discovery contributes to increasing the knowledge about little-studied species, since the Ajur_Neprilysin is the second neprilysin found in the venom from a mygalomorph spider.
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Humanos , Bloqueadores do Receptor Tipo 1 de Angiotensina II/uso terapêutico , Valsartana/uso terapêutico , Insuficiência Cardíaca/fisiopatologia , Ecocardiografia/métodos , Fatores de Risco de Doenças Cardíacas , Insuficiência Cardíaca/diagnóstico , Insuficiência Cardíaca/tratamento farmacológicoRESUMO
Arterial hypertension is the most prevalent chronic disease in the adult population of developed countries and it constitutes a significant risk factor in the development of cardiovascular disease, contributing to the emergence of many comorbidities, among which heart failure excels, a clinical syndrome that nowadays represents a major health problem with uncountable hospitalizations and the indolent course of which progressively worsens until quality of life decreases and lastly death occurs prematurely. In the light of this growing menace, each day more efforts are invested in the field of cardiovascular pharmacology, searching for new therapeutic options that allow us to modulate the physiological systems that appear among these pathologies. Therefore, in the later years, the study of natriuretic peptides has become so relevant, which mediate beneficial effects at the cardiovascular level such as diuresis, natriuresis, and decreasing cardiac remodeling; their metabolism is mediated by neprilysin, a metalloproteinase, widely expressed in the human and capable of catalyzing many substrates. The modulation of these functions has been studied by decades, giving room to Sacubitril, the first neprilysin inhibitor, which in conjunction with an angiotensin receptor blocker has provided a high efficacy and tolerability among patients with heart failure, for whom it has already been approved and recommended. Nonetheless, in the matter of arterial hypertension, significant findings have arisen that demonstrate the potential role that it will play among the pharmacological alternatives in the upcoming years.
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Antagonistas de Receptores de Angiotensina/uso terapêutico , Hipertensão/tratamento farmacológico , Neprilisina/uso terapêutico , Antagonistas de Receptores de Angiotensina/farmacologia , Humanos , Neprilisina/farmacologiaRESUMO
One of the main pathological features in the Alzheimer disease (AD) is the presence of senile plaques, primarily composed of Aß peptide aggregates, in cortex and hippocampus. AD late onset, which constitutes 90% of cases, could be mainly attributable to deficiencies in the clearance of the Aß peptide. Here we show that expression of Aß-degrading enzymes varies on a daily basis in the hippocampus. Interestingly, an intracerebroventricular injection of Aß aggregates modified temporal patterns of Aß-degrading proteases, as well as clock proteins (BMAL1 and RORα) and antioxidant enzymes (CAT and GPx) daily rhythms. Our findings showed that the increase of Aß leads to the alteration of the enzymes involved in the clearance, and, consequently, to an increase of oxidative stress and alteration of the cellular redox state, affecting the functioning of the endogenous clock and daily rhythms of BMAL1, RORα and their target genes, in this disease.
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Pholcid spiders (Araneae: Pholcidae), officially "cellar spiders" but popularly known as "daddy long-legs," are renown for the potential of deadly toxic venom, even though venom composition and potency has never formally been studied. Here we detail the venom composition of male Physocyclus mexicanus using proteomic analyses and venom-gland transcriptomes ("venomics"). We also analyze the venom's potency on insects, and assemble available evidence regarding mammalian toxicity. The majority of the venom (51% of tryptic polypeptides and 62% of unique tryptic peptides) consists of proteins homologous to known venom toxins including enzymes (astacin metalloproteases, serine proteases and metalloendopeptidases, particularly neprilysins) and venom peptide neurotoxins. We identify 17 new groups of peptides (U1-17-PHTX) most of which are homologs of known venom peptides and are predicted to have an inhibitor cysteine knot fold; of these, 13 are confirmed in the proteome. Neprilysins (M13 peptidases), and astacins (M12 peptidases) are the most abundant venom proteins, respectively representing 15 and 11% of the individual proteins and 32 and 20% of the tryptic peptides detected in crude venom. Comparative evidence suggests that the neprilysin gene family is expressed in venoms across a range of spider taxa, but has undergone an expansion in the venoms of pholcids and may play a central functional role in these spiders. Bioassays of crude venoms on crickets resulted in an effective paralytic dose of 3.9 µg/g, which is comparable to that of crude venoms of Plectreurys tristis and other Synspermiata taxa. However, crickets exhibit flaccid paralysis and regions of darkening that are not observed after P. tristis envenomation. Documented bites on humans make clear that while these spiders can bite, the typical result is a mild sting with no long-lasting effects. Together, the evidence we present indicates pholcid venoms are a source of interesting new peptides and proteins, and effects of bites on humans and other mammals are inconsequential.
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BACKGROUND: Fungal secondary metabolites are important sources for the discovery of new pharmaceuticals, as exemplified by penicillin, lovastatin and cyclosporine. Searching for secondary metabolites of the fungi Metarhizium spp., we previously identified tyrosine betaine as a major constituent. METHODS: Because of the structural similarity with other inhibitors of neprilysin (NEP), an enzyme explored for the treatment of heart failure, we devised the synthesis of tyrosine betaine and three analogues to be subjected to in vitro NEP inhibition assays and to molecular modeling studies. RESULTS: In spite of the similar binding modes with other NEP inhibitors, these compounds only displayed moderate inhibitory activities (IC50 ranging from 170.0 to 52.9 µM). However, they enclose structural features required to hinder passive blood brain barrier permeation (BBB). CONCLUSIONS: Tyrosine betaine remains as a starting point for the development of NEP inhibitors because of the low probability of BBB permeation and, consequently, of NEP inhibition at the Central Nervous System, which is associated to an increment in the Aß levels and, accordingly, with a higher risk for the onset of Alzheimer's disease.
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Insuficiência Cardíaca/tratamento farmacológico , Neprilisina/antagonistas & inibidores , Inibidores de Proteases/farmacologia , Tirosina/análogos & derivados , Cristalografia por Raios X , Insuficiência Cardíaca/metabolismo , Humanos , Modelos Moleculares , Estrutura Molecular , Neprilisina/metabolismo , Inibidores de Proteases/síntese química , Inibidores de Proteases/química , Tirosina/síntese química , Tirosina/química , Tirosina/farmacologiaRESUMO
Chronic hypoxia has been postulated as one of the mechanisms involved in salt-sensitive hypertension and chronic kidney disease (CKD). Kidneys have a critical role in the regulation of arterial blood pressure through vasoactive systems, such as the renin-angiotensin and the kallikrein-kinin systems, with the angiotensin-converting enzyme (ACE) and kallikrein being two of the main enzymes that produce angiotensin II and bradykinin, respectively. Neutral endopeptidase 24.11 or neprilysin is another enzyme that among its functions degrade vasoactive peptides including angiotensin II and bradykinin, and generate angiotensin 1-7. On the other hand, the kidneys are vulnerable to hypoxic injury due to the active electrolyte transportation that requires a high oxygen consumption; however, the oxygen supply is limited in the medullary regions for anatomical reasons. With the hypothesis that the chronic reduction of oxygen under normobaric conditions would impact renal vasoactive enzyme components and, therefore; alter the normal balance of the vasoactive systems, we exposed male Sprague-Dawley rats to normobaric hypoxia (10% O2) for 2 weeks. We then processed renal tissue to identify the expression and distribution of kallikrein, ACE and neutral endopeptidase 24.11 as well as markers of kidney damage. We found that chronic hypoxia produced focal damage in the kidney, mainly in the cortico-medullary region, and increased the expression of osteopontin. Moreover, we observed an increase of ACE protein in the brush border of proximal tubules at the outer medullary region, with increased mRNA levels. Kallikrein abundance did not change significantly with hypoxia, but a tendency toward reduction was observed at protein and mRNA levels. Neutral endopeptidase 24.11 was localized in proximal tubules, and was abundantly expressed under normoxic conditions, which markedly decreased both at protein and mRNA levels with chronic hypoxia. Taken together, our results suggest that chronic hypoxia produces focal kidney damage along with an imbalance of key components of the renal vasoactive system, which could be the initial steps for a long-term contribution to salt-sensitive hypertension and CKD.
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ABSTRACT The discovery of arteannuin (qinghaosu) in the 20th Century was a major advance for medicine. Besides functioning as a malaria therapy, arteannuin is a pharmacological agent in a range of other diseases, but its mechanism of action remains obscure. In this study, the reverse docking server PharmMapper was used to identify potential targets of arteannuin. The results were checked using the chemical-protein interactome servers DRAR-CPI and DDI-CPI, and verified by AutoDock Vina. The results showed that neprilysin (also known as CD10), a common acute lymphoblastic leukaemia antigen, was the top disease-related target of arteannuin. The chemical-protein interactome and docking results agreed with those of PharmMapper, further implicating neprilysin as a potential target. Although experimental verification is required, this study provides guidance for future pharmacological investigations into novel clinical applications for arteannuin.
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Simulação por Computador/classificação , Neprilisina/farmacologia , Artemisininas/análise , Reposicionamento de Medicamentos/estatística & dados numéricosRESUMO
Heart failure is a global problem with elevated prevalence, and it is associated with substantial cardiovascular morbidity and mortality. Treating heart-failure patients has been a very challenging task. This review highlights the main pharmacological developments in the field of heart failure with reduced ejection fraction, giving emphasis to a drug that has a dual-acting inhibition of the neprilysin and renin-angiotensin-aldosterone system. Neprilysin is an enzyme that participates in the breakdown of biologically active natriuretic peptides and several other vasoactive compounds. The inhibition of neprilysin has been a therapeutic target for several drugs tested in cardiovascular disease, mainly for heart failure and/or hypertension. However, side effects and a lack of efficacy led to discontinuation of their development. LCZ696 is a first-in-class neprilysin- and angiotensin-receptor inhibitor that has been developed for use in heart failure. This drug is composed of two molecular moieties in a single crystalline complex: a neprilysin-inhibitor prodrug (sacubitril) and the angiotensin-receptor blocker (valsartan). The PARADIGM-HF trial demonstrated that this drug was superior to an angiotensin-converting enzyme inhibitor (enalapril) in reducing mortality in patients with heart failure with reduced ejection fraction. The ability to block the angiotensin receptor and augment the endogenous natriuretic peptide system provides a distinctive mechanism of action in cardiovascular disease.
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Aminobutiratos/farmacologia , Antagonistas de Receptores de Angiotensina/química , Antagonistas de Receptores de Angiotensina/farmacologia , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Enalapril/análise , Insuficiência Cardíaca/tratamento farmacológico , Insuficiência Cardíaca/fisiopatologia , Neprilisina/antagonistas & inibidores , Tetrazóis/farmacologia , Valsartana/farmacologia , Aminobutiratos/química , Inibidores da Enzima Conversora de Angiotensina/química , Compostos de Bifenilo , Combinação de Medicamentos , Enalapril/química , Humanos , Neprilisina/química , Tetrazóis/químicaRESUMO
The scorpion Tityus serrulatus venom comprises a complex mixture of molecules that paralyzes and kills preys, especially insects. However, venom components also interact with molecules in humans, causing clinic envenomation. This cross-interaction may result from homologous molecular targets in mammalians and insects, such as (NEP)-like enzymes. In face of these similarities, we searched for peptides in Tityus serrulatus venom using human NEP as a screening tool. We found a NEP-inhibiting peptide with the primary sequence YLPT, which is very similar to that of the insect neuropeptide proctolin (RYLPT). Thus, we named the new peptide [des-Arg(1)]-proctolin. Comparative NEP activity assays using natural substrates demonstrated that [des-Arg(1)]-proctolin has high specificity for NEP and better inhibitory activity than proctolin. To test the initial hypothesis that molecular homologies allow Tityus serrulatus venom to act on both mammal and insect targets, we investigated the presence of a NEP-like in cockroaches, the main scorpion prey, that could be likewise inhibited by [des-Arg(1)]-proctolin. Indeed, we detected a possible NEP-like in a homogenate of cockroach heads whose activity was blocked by thiorphan and also by [des-Arg(1)]-proctolin. Western blot analysis using a human NEP monoclonal antibody suggested a NEP-like enzyme in the homogenate of cockroach heads. Our study describes for the first time a proctolin-like peptide, named [des-Arg(1)]-proctolin, isolated from Tityus serrulatus venom. The tetrapeptide inhibits human NEP activity and a NEP-like activity in a cockroach head homogenate, thus it may play a role in human envenomation as well as in the paralysis and death of scorpion preys.
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Inibidores Enzimáticos/farmacologia , Neuropeptídeos/química , Neuropeptídeos/farmacologia , Oligopeptídeos/química , Oligopeptídeos/farmacologia , Venenos de Escorpião/química , Animais , Western Blotting , Baratas/enzimologia , Avaliação Pré-Clínica de Medicamentos/métodos , Inibidores Enzimáticos/química , Cabeça , Humanos , Hidrólise , Neprilisina/antagonistas & inibidores , Escorpiões/química , Tiorfano/farmacologiaRESUMO
Adequate management of phyllodes tumors of the breast (PTB) remains a challenge because of the difficulty in correctly establishing preoperative diagnosis. The aim of this study was to evaluate the usefulness of Ki-67, CD10, CD34, p53, CD117, and of the number of mast cells in the differential diagnosis of benign PTB and cellular fibroadenomas (CFs) as well as in the grading of PTB. Fifty-one primary PTB and 14 CFs were examined by immunohistochemistry.When evaluating CD117 expression, higher epithelial expression was present in CF as well as an increased number of mast cells in benign PTB. Stromal expression of Ki-67, CD10, CD34, and p53 were relevant to PTB grading, of which the first 3 showed significance in the distinction of benign and borderline PTB, as well as between benign and malignant PTB. P53 was relevant only for the discrimination between benign and malign PTB. None of the markers showed significance in distinguishing between borderline and malign PTB.