Preclinical assessment of an anti-HTLV-1 heterologous DNA/MVA vaccine protocol expressing a multiepitope HBZ protein.

BACKGROUND: Human T-lymphotropic virus 1 (HTLV-1) is associated with the development of several pathologies and chronic infection in humans. The inefficiency of the available treatments and the challenge in developing a protective vaccine highlight the need to produce effective immunotherapeutic tools. The HTLV-1 basic leucine zipper (bZIP) factor (HBZ) plays an important role in the HTLV-1 persistence, conferring a survival advantage to infected cells by reducing the HTLV-1 proteins expression, allowing infected cells to evade immune surveillance, and enhancing cell proliferation leading to increased proviral load. METHODS: We have generated a recombinant Modified Virus Vaccinia Ankara (MVA-HBZ) and a plasmid DNA (pcDNA3.1(+)-HBZ) expressing a multiepitope protein based on peptides of HBZ to study the immunogenic potential of this viral-derived protein in BALB/c mice model. Mice were immunized in a prime-boost heterologous protocol and their splenocytes (T CD4+ and T CD8+) were immunophenotyped by flow cytometry and the humoral response was evaluated by ELISA using HBZ protein produced in prokaryotic vector as antigen. RESULTS: T CD4+ and T CD8+ lymphocytes cells stimulated by HBZ-peptides (HBZ42-50 and HBZ157-176) showed polyfunctional double positive responses for TNF-α/IFN-γ, and TNF-α/IL-2. Moreover, T CD8+ cells presented a tendency in the activation of effector memory cells producing granzyme B (CD44+High/CD62L-Low), and the activation of Cytotoxic T Lymphocytes (CTLs) and cytotoxic responses in immunized mice were inferred through the production of granzyme B by effector memory T cells and the expression of CD107a by CD8+ T cells. The overall data is consistent with a directive and effector recall response, which may be able to operate actively in the elimination of HTLV-1-infected cells and, consequently, in the reduction of the proviral load. Sera from immunized mice, differently from those of control animals, showed IgG-anti-HBZ production by ELISA. CONCLUSIONS: Our results highlight the potential of the HBZ multiepitope protein expressed from plasmid DNA and a poxviral vector as candidates for therapeutic vaccine.

A systematic review of artificial intelligence algorithms for predicting acute kidney injury.

OBJECTIVE: Acute kidney injury (AKI) increases mortality and costs in hospitalized patients. New methods for early AKI identification have been developed with targeted biomarkers and electronic health records data analysis. Machine learning (ML) use in diagnostics and health data analysis has recently increased. We performed a systematic review to analyze the use of ML for AKI prediction in hospitalized adults. MATERIALS AND METHODS: Tubmed, EMBASE, Cochrane, and Web of Science databases were searched until 31st March of 2023. English-language studies using ML in adults for AKI prediction were included using predetermined eligibility search terms such as acute kidney injury, machine learning, and artificial intelligence. Two reviewers evaluated the publications' titles, abstracts, and full texts separately and obtained appropriate data. The main outcome was an area under the curve (AUC) result of at least 0.70. RESULTS: Ten studies in 102 articles were included involving 242,251 patients. Deep learning (AUC 0.907 in critical care AKI; AUC 0.797 in hospitalized patients AKI) was similar to Logistic regression (AUC 0.877 in critical care AKI; AUC 0.789 in hospitalized patients). Decision tree constructions had similar AUC. CONCLUSIONS: In this review, most ML models analyzed fulfilled the main outcome. AKI is multifactorial; however, ML performed well with different etiologies, such as cardiac-related AKI, drug-related AKI, and critical care patients. Overfitting data and constructing black box models are limitations that might jeopardize the generalization and comprehension of the results. Most studies were single-center, and three manuscripts used the same database with a predominantly Caucasian population, resulting in a lack of diversity and reducing external generalization. In conclusion, ML could effectively predict AKI in hospitalized adults. Future directions rely on including a more diverse population and completing prospective and controlled trials.

The silicone metacarpophalangeal joint arthroplasty: An in-vitro analysis.

BACKGROUND: Silicone is still the gold standard implant in metacarpophalangeal arthroplasty. Whereas the clinical results are acceptable, in follow-ups with >10 years, high rates of implant fracture are common, and 5 to 7% of implants required revision. This work's purpose is to analyse the hypothesis that the joint flexion amplitude has a relevant effect on bone strain level, implant stress and bone-implant micromotion, which can reflect an increase in the risk of bone resorption/fatigue failure, implant fracture and osteolysis. METHODS: To experimentally predict the cortical loading behaviour, composite metacarpals and proximal phalanges were used in intact and implanted states. A finite element model was developed to evaluate the structural behaviour of cancellous bone and implant. This model was validated by comparing cortical strain and load-displacement curve with experimental measurements. FINDINGS: Bone strain changes between the intact and the implanted states showed a load transfer effect from the cortical to the cancellous bone that increases significantly with the flexion's amplitude rise. The peak implant stress occurred in the flexion amplitudes further away from the implant neutral angle. The highest implant pistoning motion and the highest phalanx cancellous-bone strain occurred simultaneously at the maximum flexion amplitude. INTERPRETATION: Limiting joint flexion range will be helpful to reduce the strain-shielding effect on cortical bone, minimizing the overload effect on cancellous bone and decreasing the stress levels and the pistoning motion on the implant, ultimately contributing to the longevity of silicone arthroplasty.

Social avoidance and altered hypothalamic-pituitary-adrenal axis in a mouse model of anxious depression: The role of LPA1 receptor.

Anxious depression is a prevalent disease with devastating consequences. Despite the lack of knowledge about the neurobiological basis of this subtype of depression, recently our group has identified a relationship between the LPA1 receptor, one of the six characterized G protein-coupled receptors (LPA1-6) for lysophosphatidic acid, with a mixed depressive-anxiety phenotype. Dysfunctional social behaviors, which have been related to increased activation of the hypothalamus-pituitary-adrenal (HPA) axis, are key symptoms of depression and are even more prominent in patients with comorbid anxiety and depressive disorders. Social behavior and HPA functioning were assessed in animals lacking the LPA1 receptor. For these purposes, we first examined social behaviors in wild-type and LPA1 receptor-null mice. In addition, a dexamethasone (DEX) suppression test was carried out. maLPA1-null mice exhibited social avoidance, a blunted response to DEX administration and an impaired circadian rhythm of corticosterone levels, which are features that are consistently dysregulated in many mental illnesses including anxious depression. Here, we have strengthened the previous experimental evidence for maLPA1-null mice to represent a good animal model of anxious depression, providing an opportunity to explore new therapeutic targets for the treatment of mood disorders, particularly this subtype of depression.

Membrane lipid composition of Carnobacterium maltaromaticum CNCM I-3298, a highly cryoresistant lactic bacterium.

The growing consumption of fermented products has led to an increasing demand for lactic acid bacteria (LAB), especially for LAB tolerant to freezing/thawing conditions. Carnobacterium maltaromaticum is a psychrotrophic and freeze-thawing resistant lactic acid bacterium. The membrane is the primary site of damage during the cryo-preservation process and requires modulation to improve cryoresistance. However, knowledge about the membrane structure of this LAB genus is limited. We presented here the first study of the membrane lipid composition of C. maltaromaticum CNCM I-3298 including the polar heads and the fatty acid compositions of each lipid family (neutral lipids, glycolipids, phospholipids). The strain CNCM I-3298 is principally composed of glycolipids (32%) and phospholipids (55%). About 95% of glycolipids are dihexaosyldiglycerides while less than 5% are monohexaosyldiglycerides. The disaccharide chain of dihexaosyldiglycerides is composed of α-Gal(1-2)-α-Glc chain, evidenced for the first time in a LAB strain other than Lactobacillus strains. Phosphatidylglycerol is the main phospholipid (94%). All polar lipids are exceptionally rich in C18:1 (from 70% to 80%). Regarding the fatty acid composition, C. maltaromaticum CNCM I-3298 is an atypical bacterium within the genus Carnobacterium due to its high C18:1 proportion but resemble the other Carnobacterium strains as they mostly do not contain cyclic fatty acids.

Oral amyloidosis: an update.

BACKGROUND: Amyloidosis is a disease characterized by the progressive deposition of abnormal proteins that can occur in any organ. In the oral cavity, the tongue is the most common affected site, usually causing macroglossia. Biopsy is essential for the diagnosis and the occurrence of its systemic form is mandatory to be investigated. This systematic review evaluated the existing information in the literature on Amyloidosis in the oral cavity to allow a more comprehensive and updated analysis of its clinicopathological characteristics, as well as to explore the main forms of treatment and prognostic factors. MATERIAL AND METHODS: Electronic searches were undertaken in five databases supplemented by manual scrutiny. RESULTS: A total of 111 studies were included with 158 individuals. CONCLUSIONS: The disease had a higher prevalence in women, the tongue was the most affected site, as well as the systemic form of the disease. The worst prognosis was for cases of systemic amyloidosis associated with multiple myeloma.

CANreduce-SP-adding psychological support to web-based adherence-focused guided self-help for cannabis users: study protocol for a three-arm randomized control trial.

BACKGROUND: Cannabis is the most-frequently used illicit drug in Europe. Over the last few years in Spain, treatment demand has increased, yet most cannabis users do not seek treatment despite the related problems. A web-based self-help tool, like CANreduce 2.0, could help these users to control their consumption. METHODS: This study protocol describes a three-arm randomized controlled trial (RCT) comparing the effectiveness of three approaches, in terms of reducing cannabis use among problematic cannabis users, the first two treatment arms including the Spanish version of CANreduce 2.0 (an adherence-focused, guidance-enhanced, web-based self-help tool) (1) with and (2) without psychological support; and the third group (3) treatment as usual (TAU). Study hypotheses will be tested concerning the primary outcome: change in the number of days of cannabis use over the previous week, comparing assessments at 6 weeks and 3 and 6 months follow-up between groups and against baseline. Secondary outcomes related to cannabis use will be tested similarly. Mental disorders will be explored as predictors of adherence and outcomes. Analyses will be performed on an intention-to-treat basis, then verified by complete case analyses. DISCUSSION: This study will test how effective the Spanish version of CANreduce 2.0 (CANreduce-SP) is at reducing both the frequency and quantity of cannabis use in problematic users and whether adding psychological support increases its effectiveness. TRIAL REGISTRATION: This trial is registered with the Clinical Trials Protocol Registration and Results System (PRS) number: NCT04517474 . Registered 18 August 2020, (Archived by archive.is https://archive.is/N1Y64 ). The project commenced in November 2020 and recruitment is anticipated to end by November 2022.

Gut microbiota and voluntary alcohol consumption.

Alcohol is part of the usual diet of millions of individuals worldwide. However, not all individuals who drink alcohol experience the same effects, nor will everyone develop an alcohol use disorder. Here we propose that the intestinal microbiota (IMB) helps explain the different consumption patterns of alcohol among individuals. 507 humans participated in this study and alcohol consumption and IMB composition were analyzed. On the other hand, in 80 adult male Wistar rats, behavioral tests, alcohol intoxication, fecal transplantation, administration of antibiotics and collection of fecal samples were performed. For identification and relative quantification of bacterial taxa was used the bacterial 16 S ribosomal RNA gene. In humans, we found that heavy episodic drinking is associated with a specific stool type phenotype (type 1, according to Bristol Stool Scale; p < 0.05) and with an increase in the abundance of Actinobacteria (p < 0.05). Next, using rats, we demonstrate that the transfer of IMB from alcohol-intoxicated animals causes an increase in voluntary alcohol consumption in transplant-recipient animals (p < 0.001). The relative quantification data indicate that the genus Porphyromonas could be associated with the effect on voluntary alcohol consumption. We also show that gut microbiota depletion by antibiotics administration causes a reduction in alcohol consumption (p < 0.001) and altered the relative abundance of relevant phyla such as Firmicutes, Bacteroidetes or Cyanobacteria (p < 0.05), among others. Benjamini-Hochberg false discovery rate (FDR) correction was performed for multiple comparisons. These studies reveal some of the consequences of alcohol on the IMB and provide evidence that manipulation of IMB may alter voluntary alcohol consumption.

Analgesia and pain: Dual effect of dopamine on the peripheral nociceptive system is dependent on D2-or D1-like receptor activation.

In this study, a pharmacological approach, together with the paw pressure test, was used to investigate the role of dopamine and its receptors in the peripheral processing of the nociceptive response in mice. Initially, the administration of dopamine (5, 20, and 80 ng/paw) in the hind paw of male Swiss mice (30-40 g) promoted antinociceptive effects in a dose-dependent manner. This was considered a peripheral effect, as it did not produce changes in the nociceptive threshold of the contralateral paw. The D2, D3, and D4 dopamine receptor antagonists remoxipride (4 µg/paw), U99194 (16 µg/paw), and L-745,870 (16 µg/paw), respectively, reversed the dopamine-mediated antinociception in mice with PGE2-induced hyperalgesia. The D1 and D5 dopamine receptor antagonists SKF 83566 (2 µg/paw) and SCH 23390 (1.6 µg/paw), respectively, did not alter dopamine antinociception. In contrast, dopamine at higher doses (0.1, 1, and 10 µg/paw) caused hyperalgesia in the animals, and the D1 and D5 receptor antagonists reversed this pronociceptive effect (10 µg/paw), whereas the D2 receptor antagonist remoxipride did not. Our data suggest that dopamine has a dual effect that depends on the dose, as it causes peripheral antinociceptive effects at small doses via the activation of D2-like receptors and nociceptive effects at higher doses via the activation of D1-like receptors.

The role of afforestation species as a driver of Hg accumulation in organic horizons of forest soils from a Mediterranean mountain area in SW Europe.

Forest areas are a primary sink of atmospheric mercury (Hg) within terrestrial ecosystems, whereas forest vegetation plays a key role in atmospheric Hg transfer to soil horizons. This study assessed variations in total Hg contents (HgT) and accumulation (HgRes) in the soil organic horizons of a forest area in NE Portugal, where post-wildfire afforestation led to the substitution of the native deciduous species (Quercus pyrenaica) by fast-growing coniferous species (Pseudotsuga menziesii and Pinus nigra). The study also evaluated, for each species, the links between Hg contents and other biophilic elements of soil organic matter (C, N, S) present in organic subhorizons (OL, OF, OH). Mean HgT in the organic horizons of the different tree species follow the sequence: P. nigra (88 µg kg-1) < Q.pyrenaica (101 µg kg-1)

MutSα expression predicts a lower disease-free survival in malignant salivary gland tumors: an immunohistochemical study.

BACKGROUND: Appropriate DNA replication is vital to maintain cell integrity at the genomic level. Malfunction on DNA repair mechanisms can have implications related to tumor behavior. Our aim was to evaluate the expression of key complexes of the DNA mismatch-repair system MutSα (hMSH2-hMSH6) and MutSß (hMSH2-hMSH3) in a panel comprising the most common benign and malignant salivary gland tumors (SGT), and to determine their association with disease-free survival. MATERIAL AND METHODS: Ten cases of normal salivary gland (NSG) and 92 of SGT (54 benign and 38 malignant) were retrieved. Immunohistochemistry was performed for hMSH2, hMSH3, hMSH6. Scanned slides were digitally analyzed based on the percentage of positive cells with nuclear staining. Cases were further classified in MutSαhigh and MutSßhigh based on hMSH2-hMSH6 and hMSH3-hMSH6 expression, respectively. RESULTS: hMSH3 expression was lower in malignant SGT compared to NSG and benign cases. Adenoid cystic carcinoma (ACC) cases with perineural invasion presented a lower percentage of hMSH3 positive cells. hMSH6 was downregulated in both benign and malignant SGT compared to NSG. Malignant SGT cases with MutSαhigh expression had lower disease-free survival compared to MutSαlow cases. A 10.26-fold increased risk of presenting local recurrence was observed. CONCLUSIONS: Our findings suggest that a lack of hMSH3 protein function is associated with a more aggressive phenotype (malignancy and perineural invasion) and that MutSα overexpression predicts a poor clinical outcome in malignant SGT.

HIV-related non-Hodgkin lymphomas affecting the oral cavity: a clinicopathologic study of 11 cases.

BACKGROUND: HIV-related non-Hodgkin lymphomas of the oral cavity are rare lesions with aggressive clinical behaviour. The aim of this study is to describe the clinicopathological features of a series of HIV-related oral non-Hodgkin lymphomas. MATERIAL AND METHODS: Eleven cases of oral lymphomas affecting HIV-positive patients were retrieved from 2012 to 2019. Clinicopathological features regarding age, sex, tumour location, clinical presentation, laboratory findings, disease stage and follow-up were obtained. Histologic, immunohistochemical and in situ hybridization for EBV detection were done for diagnosis confirmation. Overall survival was estimated by Kaplan-Meier curve. RESULTS: Males predominated, with a mean age of 40.3 years-old. Maxilla and mandible were the mostly affected. Plasmablastic lymphoma and diffuse large B-cell lymphoma not otherwise specified (NOS) were the main histological types. Lesions presented as reddish ulcerated swellings, representing the first sign of AIDS in six cases. Stage IV were common (7 cases) and the mean HIV viral load was 10,557 copies/mL, with a mean of 266 CD4+ cells/mm3, 1,278 CD8+ cells/mm3 and a CD4+/CD8+ ratio of 0.26. Eight patients died of the disease (72.7%). Overall survival revealed that 78.2% of the patients died after 21 months of follow-up. CONCLUSIONS: HIV-related oral lymphomas present a poor prognosis usually diagnosed in advanced stages and in our series plasmablastic lymphoma was the most common subtype.

Ligilactobacillus salivarius functionalities, applications, and manufacturing challenges.

Ligilactobacillus salivarius is a lactic acid bacteria that has been gaining attention as a promising probiotic. Numerous strains exhibit functional properties with health benefits such as antimicrobial activity, immunological effects, and the ability to modulate the intestinal microbiota. However, just a small number of them are manufactured at an industrial scale and included in commercial products. The under exploitation of L. salivarius strains that remain in the freezer of companies is due to their incapacity to overcome the environmental stresses induced by production and stabilization processes.The present study summarizes the functionalities and applications of L. salivarius reported to date. It aims also at providing a critical evaluation of the literature available on the manufacturing steps of L. salivarius concentrates, the bacterial quality after each step of the process, and the putative degradation and preservation mechanisms. Here, we highlight the principal issues and future research challenges for improving the production and long-term preservation at the industrial scale of this microorganism, and probably of other probiotics.Key points• L. salivarius beneficial properties and commercialized products.• Production conditions and viability of L. salivarius after stabilization processes.• Prospects for identifying preservation mechanisms to improve L. salivarius stability.

A comparative evaluation of methods for estimating the colostrum quality in sows / Avaliação comparativa de métodos para estimar a qualidade do colostro em porcas

Colostrum is the main source of immunoglobulins (Ig) for neonate piglets and plays a crucial role within the health and growth of the piglet. Currently in pig farming, there are still no widespread practical methods for measuring the Ig concentration in colostrum at herd level. We evaluated sows' colostrum IgG concentration using an optical and a digital Brix refractometer and their performance was correlated to an IgG ELISA test, and flow cytometry. Colostrum concentrations of IgG and IgA averaged 74.05 ± 21.37mg/mL and 20.2 ± 5.32mg/mL respectively. The mean value of the Brix percentages for optical refractometer was 26.32%, and for digital was 28.32%. The Brix refractometer measurements of colostrum samples presented high correlation for IgG content analyzed by ELISA (Optical = 0.74, Digital = 0.87; P <0.001). Considering the immunophenotyping, the values for IgG and IgA lymphoblasts indicated a highly significant relationship to ELISA (IgG=0.77, IgA=0.84; P<0.001). The Brix refractometer can be considered a useful tool to be included in a colostrum monitoring program to improve potentially neonatal health. In addition, we demonstrated that flow cytometry can be an important tool to analyze and characterize the immunological potential of sow colostrum.(AU)

O colostro é a principal fonte de imunoglobulinas (Ig) para leitões recém-nascidos e desempenha um papel crucial na saúde e no crescimento dos leitões. Atualmente, na suinocultura, ainda não existem métodos amplamente utilizados na prática de produção para medir a concentração de imunoglobulinas no colostro suíno. Avaliou-se a concentração de IgG no colostro de porcas usando refratômetros Brix óptico e digital, e o desempenho foi comparado com ELISA e citometria de fluxo. As concentrações de IgG e IgA no colostro foram 74,05 ± 21,37mg/mL e 20,2 ± 5,32mg/mL, respectivamente. A percentagem de Brix média das amostras de colostro para o refratômetro óptico foi 26,32%, e para o digital foi 28,32%. As medições dos refratômetros de Brix apresentaram elevada correlação com a concentrações de IgG medidas por ELISA (óptico=0,74, digital=0,87; P<0,001). Considerando a imunofenotipagem, os valores dos linfoblastos IgG e IgA apresentaram alta correlação com o ELISA (IgG=0,77, IgA=0,84; P<0,001). O refratômetro Brix pode ser considerado uma ferramenta útil para ser incluída em um programa de monitoramento de colostro para melhorar a saúde neonatal. Além disso, foi demonstrado que a citometria de fluxo pode ser uma ferramenta importante para analisar e caracterizar o potencial imunológico do colostro de porcas.(AU)

Decreased levels of cathepsin Z mRNA expressed by immune blood cells: diagnostic and prognostic implications in prostate cancer.

Cathepsin Z (CTSZ) is a cysteine protease responsible for the adhesion and migration of both immune and tumor cells. Due to its dual role, we hypothesized that the site of CTSZ expression could be determinant of the pro- or anti-tumorigenic effects of this enzyme. To test this hypothesis, we analyzed CTSZ expression data in healthy and tumor tissues by bioinformatics and evaluated the expression levels of CTSZ mRNA in the blood cells of prostate cancer (PCa) patients by qRT-PCR compared with healthy subjects, evaluating its diagnostic and prognostic implications for this type of cancer. Immune cells present in the blood of healthy patients overexpress CTSZ. In PCa, we found decreased CTSZ mRNA levels in blood cells, 75% lower than in healthy subjects, that diminished even more during biochemical relapse. CTSZ mRNA in the blood cells had an area under the curve for PCa diagnosis of 0.832, with a 93.3% specificity, and a positive likelihood ratio of 9.4. The site of CTSZ mRNA expression is fundamental to determine its final role as a protective determinant in PCa, such as CTSZ mRNA in the blood cells, or a malignant determinant, such as found for CTSZ expressed in high levels by different types of primary and metastatic tumors. Low CTSZ mRNA expression in the total blood is a possible PCa marker complementary to prostate-specific antigen (PSA) for biopsy decisions, with the potential to eliminate unnecessary biopsies.

Corrigendum to 'Dental pathology of the wild Iberian wolf (Canis lupus signatus): the study of a 20th century Portuguese museum collection': Veterinary and animal science 9 (2020) 100100.

[This corrects the article DOI: 10.1016/j.vas.2020.100100.].

Clinical impact of COVID-19 on people with substance use disorders.

BACKGROUND: People with substance use disorders are considered at increased risk of COVID-19 and its more serious complications, however data on the impact of COVID-19 are lacking. The study aimed to describe the clinical characteristics and outcomes of COVID-19 on people with substance use disorders. METHODS: an observational study was carried out including patients aged ≥ years with COVID-19 pneumonia admitted to an urban hospital during March 12 to June 21,2020. RESULTS: Among 2078 patients admitted, 27 (1.3%) were people with substance use disorders: 23(85.2%) were men with a median age of 56.1 + 10.3 years and. The main SUD were alcohol in 18(66.7%) patients, heroine in 6(22.2%) and cocaine in 3(11.1%) and 24(88.8%) patients were on ongoing substance use disorder treatment. One or more comorbidities associated to COVID-19 risk were observed in 18(66.6%) of patients. During a median length of stay of 10 days (IQR:7-19), severe pneumonia developed in 7(25.9%) patients, acute respiratory distress syndrome in 5 (18.5%) and none died. CONCLUSION: Larger sample sizes and sero-epidemiological studies are needed to confirm the low incidence of severe COVID-19 on patients with SUD.

Salivary gland cancer in Southern Brazil: a prognostic study of 107 cases.

BACKGROUND: Salivary gland cancers (SGC) represent an uncommon group of heterogeneous tumors. We performed a retrospective survey of SGC diagnosed in a reference center for treatment of malignant tumors from the south of Brazil aiming to determine the prognostic value of demographic, clinic and pathologic features. MATERIAL AND METHODS: Cases diagnosed as SGC between 2006 and 2016 were retrospectively collected. Medical records were examined to extract demographic, clinic, pathologic and follow-up information. RESULTS: One-hundred and seven cases of SGC were identified. The most common SGC were mucoepidermoid carcinoma (MEC) (n = 39) followed by adenoid cystic carcinoma (AdCC) (n = 29). Among AdCCs, 55.2% of cases were classified as cribriform, 27.6% as tubular and 17.2% as solid. The tubular subtype had the highest percentage of cases with perineural invasion (p=0.01). Among MEC, 61.5% of cases were classified as low grade, 15.4% as intermediate grade and 19.9% as high grade. Low grade MEC had the lowest percentage of cases with perineural invasion (p=0.04). The 5-year survival for loco-regional control, disease-free survival (DFS) and disease-specific survival were 75%, 70% and 84%, respectively. The following features were associated with poor DFS: advanced age (p=0.03), rural residency (p=0.01), being a smoker or former smoker (p=0.01), pain (p=0.03), nodal metastasis (p<0.001), need for chemotherapy (p=0.02), neck dissection (p=0.04), perineural invasion (p=0.01), and being diagnosed with AdCC compared to MEC (p=0.02). CONCLUSIONS: The clinco-demographic and pathologic features identified as prognostic factors reveal the profile of patients at increased risk of recurrence and who would benefit from closer follow-up.