Direct Medical Costs of Four Vaccine-Preventable Infectious Diseases in Older Adults in Spain.

OBJECTIVE: Protection against vaccine-preventable diseases is especially relevant in older adults due to age-related decline in immunity (immunosenescence). However, adult vaccination remains a challenge with overall low coverage rates, which has an impact on both the patients who have these diseases and the health care system in terms of resource use and costs derived. This study aimed to estimate the direct economic impact of herpes zoster, pneumococcal disease, influenza and pertussis in Spanish adults 45 years and older. METHODS: Data from 2015 were extracted from two Spanish public databases: the Minimum Basic Data Set for Hospitalisations and the Clinical Database of Primary Care. Codes from the International Classification of Diseases and the International Classification of Primary Care were used to identify and classify the diseases analysed. The variables extracted and calculated were hospitalisation (cases, percentage, length of stay, costs, mortality), primary care (cases, percentage, costs) and referrals (cases, percentage, costs). Results were presented for the age groups 45-64 years, 65-74 years, > 74 years and all ages. RESULTS: In adults 45 years and older, total costs amounted to €134.1 million in 2015 (i.e. 63.9% of the total direct costs for all age groups): 44.4% due to pneumococcal disease, 39.5% due to influenza, 16.0% due to herpes zoster and 0.1% due to pertussis. Hospitalisations represented 58.1% (€77.9 million) of the total costs, with 15,910 admissions, 144,752 days of hospitalisation and 1170 deaths. Primary care registered 566,556 visits with a cost of €35.0 million, and 269,186 referrals with a cost of €21.1 million. CONCLUSION: The direct economic burden of herpes zoster, pneumococcal disease, influenza and pertussis in adults 45 years and older was high in Spain, and may be underestimated as it only considered medical assistance and not other applicable direct or indirect costs. Increasing vaccination rates in adults may potentially reduce the economic burden derived from these diseases, although future cost-effectiveness analysis including other disease-related costs, vaccination costs and vaccination effectiveness would be needed.

Oral secretory leukocyte protease inhibitor (SLPI): Associations with oropharyngeal cancer and treatment outcome.

BACKGROUND: Rates of oropharyngeal cancer (OPC) associated with alcohol & tobacco use have decreased, while human papillomavirus (HPV) associated OPC has increased among men in the US. Secretory leukocyte protease inhibitor (SLPI), detectable in a variety of secretions, has been implicated in cancers of the head and neck, associated with tumor progression and anti-viral activity. Using the recently verified oral gargle specimen, this study aimed to assess the association of salivary SLPI expression with risk of OPC and response to treatment. METHODS: A case-control study design compared levels of salivary SLPI among OPC cases to age and tobacco smoking matched healthy controls. Oral HPV DNA and SLPI was quantified from oral gargle specimens. Logistic regression estimated odds ratios (OR) and 95% confidence intervals (CI) for associations of oral SLPI and risk of OPC and treatment outcomes. RESULTS: In crude and adjusted analyses of 96 OPC cases and 97 age- and smoking-matched controls, OPC was not significantly associated with oral gargle SLPI levels. Among cases, oral SLPI was associated with tonsillectomy (p = 0.018) and among controls oral SLPI was associated with HPV in the oral gargle (p = 0.008). Higher concentrations of SLPI was significantly associated with increased odds of incomplete treatment response (T2: OR: 12.39; 95% CI: 1.44-106.72; T3: OR: 9.86; 95% CI: 1.13-85.90) among all cases, but not among P16+ cases. CONCLUSIONS: Salivary SLPI was not associated with OPC risk but was associated with higher odds of an incomplete treatment response.

Methylation of HPV 16 and EPB41L3 in oral gargles: Associations with oropharyngeal cancer detection and tumor characteristics.

Oropharyngeal cancer (OPC) incidence is increasing significantly among men and often requires intensive therapy causing significant morbidity. Early detection of OPC is needed, when monotherapy can be safely delivered with less treatment-associated morbidity, while maintaining high cure rates. We conducted a study of 101 pretreatment male OPC cases matched 1:1 to 101 disease-free controls for age and smoking history. Oral gargles were collected from cases and controls with additional biopsies or aspirates from cases. The HPV SPF10 -LiPA25 PCR assay was utilized for HPV genotyping. Methylation of three CpG sites (438, 427 and 425) in the EPB41L3 gene and methylation status of the L1 (6,367, 6,389), L2 (4,257, 4,262, 4,266, 4,269, 4,275, 4,282) and E2 (3,412, 3,415, 3,417, 3,433, 3,436) CpG sites of HPV 16 positive specimens was assessed by pyrosequencing. Significant correlations were observed between tumor and oral specimens for all methylation biomarkers (p < 0.01). EPB41L3 and HPV 16 L1, L2 and E2 methylation were significantly (p < 0.0001) higher among cases than controls, regardless of early vs. late disease. When HPV 16 genes and EPB41L3 methylation status were combined in a logistic regression analysis, a sensitivity of 70.3% and a specificity of 90.9% were observed for the detection of OPC from an oral gargle. Our data suggest that methylation biomarkers measured in oral gargles may have utility in identifying OPC early. Future studies are needed to replicate these findings and to inform additional biomarkers that can maximize specificity and sensitivity for early OPC detection.

Effects of Tobacco Smoking on the Tumor Immune Microenvironment in Head and Neck Squamous Cell Carcinoma.

PURPOSE: Patients with head and neck squamous cell carcinoma (HNSCC) who actively smoke during treatment have worse survival compared with never-smokers and former-smokers. We hypothesize the poor prognosis in tobacco smokers with HNSCC is, at least in part, due to ongoing suppression of immune response. We characterized the tumor immune microenvironment (TIM) of HNSCC in a retrospective cohort of 177 current, former, and never smokers. EXPERIMENTAL DESIGN: Tumor specimens were subjected to analysis of CD3, CD8, FOXP3, PD-1, PD-L1, and pancytokeratin by multiplex immunofluorescence, whole-exome sequencing, and RNA sequencing. Immune markers were measured in tumor core, tumor margin, and stroma. RESULTS: Our data indicate that current smokers have significantly lower numbers of CD8+ cytotoxic T cells and PD-L1+ cells in the TIM compared with never- and former-smokers. While tumor mutation burden and mutant allele tumor heterogeneity score do not associate with smoking status, gene-set enrichment analyses reveal significant suppression of IFNα and IFNγ response pathways in current smokers. Gene expression of canonical IFN response chemokines, CXCL9, CXCL10, and CXCL11, are lower in current smokers than in former smokers, suggesting a mechanism for the decreased immune cell migration to tumor sites. CONCLUSIONS: These results suggest active tobacco use in HNSCC has an immunosuppressive effect through inhibition of tumor infiltration of cytotoxic T cells, likely as a result of suppression of IFN response pathways. Our study highlights the importance of understanding the interaction between smoking and TIM in light of emerging immune modulators for cancer management.

Oral gargle-tumor biopsy human papillomavirus (HPV) agreement and associated factors among oropharyngeal squamous cell carcinoma (OPSCC) cases.

OBJECTIVE: Assess oral gargle-tumor human papillomavirus (HPV) agreement among oropharyngeal squamous cell carcinoma (OPSCC) cases by several disease characteristics. MATERIALS AND METHODS: 171 treatment naïve OPSCC were enrolled 2014-2017. Tumors were categorized as early or late disease with early disease defined as T1-2 with no nodal involvement or at most a single ipsilateral positive node <3 cm. Oral gargle samples were obtained via a 30-second rinse and gargle. The RHA Kit HPV SP10-LiPA25 was utilized for HPV genotyping of tumor (FFPE) and oral gargle specimens. Sensitivity, specificity, positive and negative predictive value, percent agreement, and 95% exact binomial confidence intervals were estimated. Multivariable logistic regression models were fit to predict agreement. RESULTS: 83.0% and 93.0% of oral gargle and tumor specimens were HPV positive. Oral gargle-tumor agreement for any oncogenic HPV type and HPV 16 was 73.7%. High oncogenic HPV oral gargle-tumor agreement was observed for late disease presentation, p16 positive cases, and tumors at the tonsils (74.5-80.8%). Similar trends were observed for HPV 16. Agreement for any oncogenic HPV and HPV 16 was significantly higher for late vs. early disease (77.9% vs 57.1%, p = 0.01). Oral gargle-tumor oncogenic HPV and HPV 16 agreement was independently associated with age ≥50 years and late disease presentation. CONCLUSION: Overall, oral-tumor HPV agreement among OPSCC was relatively high. However, oral-tumor HPV agreement was significantly lower among younger cases and those diagnosed with earlier disease. Additional biomarkers are needed to improve oral HPV test characteristics to identify OPSCC early.

Human Papillomavirus Genotype Detection in Oral Gargle Samples Among Men With Newly Diagnosed Oropharyngeal Squamous Cell Carcinoma.

Importance: The most common cause of oropharyngeal squamous cell carcinoma is human papillomavirus (HPV) infection, and currently the standard of care to determine the HPV infection status in this type of carcinoma is to use p16 immunohistochemistry as a surrogate marker of high-risk HPV infection. Although p16 immunohistochemistry is limited by the inability to determine the specific HPV genotypes, oral gargle samples may be a readily available source of HPV DNA for genotyping. Objective: To determine the specific HPV genotypes present in both oral gargle samples and tumor specimens. Design, Setting, and Participants: This prospective, biomarker cohort study conducted at a single specialized cancer hospital in Florida screened approximately 800 potentially eligible participants from May 2014 through October 2017. To be eligible for participation, patients had to meet all of the following criteria: 18 years of age or older, male sex, newly diagnosed as having stage I to IV cancer of the oropharynx, a squamous cell carcinoma diagnosis, treatment naive or at least 4 weeks after chemoradiation or surgical treatment of other diseases, fully understand the study procedures and risks involved, and voluntarily agree to participate by signing an informed consent statement. Main Outcomes and Measures: Detection rate of HPV infection and HPV genotypes in oral gargle samples and tumor specimens. Results: A cohort of 204 male participants with newly diagnosed oropharyngeal squamous cell carcinoma was assessed in this prospective collection of comprehensive clinical data and oral gargle samples. Most study participants (190 [93.1%]) were white and ever smokers (114, 55.9%), with a median age of 61 years (range, 35-87 years). The HPV infection status could be assessed in 203 of 204 participants (99.5%) using oral gargle samples: 35 samples (17.2%) were negative for HPV infection, whereas 168 samples (82.8%) were positive for HPV infection. The detection rate of HPV genotypes was 93.0% in tumor specimens (160 specimens) and 82.8% (168 samples) in oral gargle samples. The oral gargle samples frequently had low-risk HPV genotypes that were not detected in tumors, but these low-risk genotypes were always a coinfection with high-risk genotypes. Conclusions and Relevance: Oral gargle samples can be used to detect the majority of clinically relevant HPV genotypes found in oropharyngeal squamous cell carcinoma, but the interpretation of HPV detected in these samples should be assessed with caution for general cancer risk assessment given that sensitive assays can concomitantly detect low-risk genotypes.

HIF-1α induction during reperfusion avoids maladaptive repair after renal ischemia/reperfusion involving miR127-3p.

Ischemia/reperfusion (I/R) leads to Acute Kidney Injury. HIF-1α is a key factor during organ response to I/R. We previously demonstrated that HIF-1α is induced during renal reperfusion, after ischemia. Here we investigate the role of HIF-1α and the HIF-1α dependent mechanisms in renal repair after ischemia. By interference of HIF-1α in a rat model of renal I/R, we observed loss of expression and mis-localization of e-cadherin and induction of α-SMA, MMP-13, TGFß, and collagen I. Moreover, we demonstrate that HIF-1α inhibition promotes renal cell infiltrates by inducing IL-1ß, TNF-α, MCP-1 and VCAM-1, through NFkB activity. In addition, HIF-1α inhibition induced proximal tubule cells proliferation but it did not induce compensatory apoptosis, both in vivo. In vitro, HIF-1α knockdown in HK2 cells subjected to hypoxia/reoxygenation (H/R) promote cell entry into S phase, correlating with in vivo data. HIF-1α interference leads to downregulation of miR-127-3p and induction of its target gene Bcl6 in vivo. Moreover, modulation of miR-127-3p in HK2 cells subjected to H/R results in EMT regulation: miR127-3p inhibition promote loss of e-cadherin and induction of α-SMA and collagen I. In conclusion, HIF-1α induction during reperfusion is a protector mechanism implicated in a normal renal tissue repair after I/R.

MicroRNA let-7d is a target of cannabinoid CB1 receptor and controls cannabinoid signaling.

Cannabinoid CB1 receptor, the molecular target of endocannabinoids and cannabis active components, is one of the most abundant metabotropic receptors in the brain. Cannabis is widely used for both recreational and medicinal purposes. Despite the ever-growing fundamental roles of microRNAs in the brain, the possible molecular connections between the CB1 receptor and microRNAs are surprisingly unknown. Here, by using reporter gene constructs that express interaction sequences for microRNAs in human SH-SY5Y neuroblastoma cells, we show that CB1 receptor activation enhances the expression of several microRNAs, including let-7d. This was confirmed by measuring hsa-let-7d expression levels. Accordingly, knocking-down CB1 receptor in zebrafish reduced dre-let-7d levels, and knocking-out CB1 receptor in mice decreased mmu-let-7d levels in the cortex, striatum and hippocampus. Conversely, knocking-down let-7d increased CB1 receptor mRNA expression in zebrafish, SH-SY5Y cells and primary striatal neurons. Likewise, in primary striatal neurons chronically exposed to a cannabinoid or opioid agonist, a let-7d-inhibiting sequence facilitated not only cannabinoid or opioid signaling but also cannabinoid/opioid cross-signaling. Taken together, these findings provide the first evidence for a bidirectional link between the CB1 receptor and a microRNA, namely let-7d, and thus unveil a new player in the complex process of cannabinoid action.

A Pilot Study Identifying a Set of microRNAs As Precise Diagnostic Biomarkers of Acute Kidney Injury.

In the last decade, Acute Kidney Injury (AKI) diagnosis and therapy have not notably improved probably due to delay in the diagnosis, among other issues. Precocity and accuracy should be critical parameters in novel AKI biomarker discovery. microRNAs are key regulators of cell responses to many stimuli and they can be secreted to the extracellular environment. Therefore, they can be detected in body fluids and are emerging as novel disease biomarkers. We aimed to identify and validate serum miRNAs useful for AKI diagnosis and management. Using qRT-PCR arrays in serum samples, we determined miRNAs differentially expressed between AKI patients and healthy controls. Statistical and target prediction analysis allowed us to identify a panel of 10 serum miRNAs. This set was further validated, by qRT-PCR, in two independent cohorts of patients with relevant morbi-mortality related to AKI: Intensive Care Units (ICU) and Cardiac Surgery (CS). Statistical correlations with patient clinical parameter were performed. Our results demonstrated that the 10 selected miRNAs (miR-101-3p, miR-127-3p, miR-210-3p, miR-126-3p, miR-26b-5p, miR-29a-3p, miR-146a-5p, miR-27a-3p, miR-93-3p and miR-10a-5p) were diagnostic biomarkers of AKI in ICU patients, exhibiting areas under the curve close to 1 in ROC analysis. Outstandingly, serum miRNAs estimated before CS predicted AKI development later on, thus becoming biomarkers to predict AKI predisposition. Moreover, after surgery, the expression of the miRNAs was modulated days before serum creatinine increased, demonstrating early diagnostic value. In summary, we have identified a set of serum miRNAs as AKI biomarkers useful in clinical practice, since they demonstrate early detection and high diagnostic value and they recognize patients at risk.

Role of microRNAs in the immunity process of the flat oyster Ostrea edulis against bonamiosis.

MicroRNAs (miRNAs) are small (∼22nt) non-coding regulatory single strand RNA molecules that reduce stability and/or translation of sequence-complementary target. miRNAs are a key component of gene regulatory networks and have been involved in a wide variety of biological processes, such as signal transduction, cell proliferation and apoptosis. Many miRNAs are broadly conserved among the animal lineages and even between invertebrates and vertebrates. The European flat oyster Ostrea edulis is highly susceptible to infection with Bonamia ostreae, an intracellular parasite able to survive and proliferate within oyster haemocytes. Mollusc haemocytes play a key role in the immune response of molluscs as main cellular effectors. The roles of miRNAs in the immune response of O. edulis to bonamiosis were analysed using a commercial microarray platform (miRCURY LNA™ v2, Exiqon) for miRNAs. Expression of miRNAs in haemocytes from oysters with different bonamiosis intensity was compared. Differential expression was detected in 63 and 76 miRNAs when comparing heavily-affected with non-affected oysters and with lightly-affected ones, respectively. Among them, 19 miRNAs are known to be linked to immune response, being responsible of proliferation and activation of macrophages, inflammation, apoptosis and/or oxidative damage, which is consistent with the modulation of their expression in oyster haemocytes due to bonamiosis.

Cloning and characterization of neoplasia-related genes in flat oyster Ostrea edulis.

Bonamiosis and disseminated neoplasia (DN) are the most important diseases affecting cultured flat oysters Ostrea edulis in Galicia (NW Spain). Previous research using suppresive substraction hybridisation that had been performed addressing the molecular basis of DN as well as the induction and development of the disease in oysters, yielded the whole open reading frame of nine genes: XBP-1, RACK, NDPk, C1qTNF, RPA3, SAP18, p23, ubiquitin and ferritin. These nine genes were characterized in this study. The phylogenetic relationships for each gene were studied using minimum-evolution methods. Quantitative-PCR assays were also developed to analyse the modulation of the expression of these genes by bonamiosis and disseminated neoplasia. Gene expression profiles were studied in haemolymph cells and in various organs (gill, gonad, mantle and digestive gland) of oysters affected by bonamiosis, disseminated neoplasia, both diseases and in non-affected oysters (control). The expression of XBP-1, NDPk, RPA3, SAP18 and ferritin increased in haemolymph cells of oysters with heavy bonamiosis. The expression of C1qTNF; SAP18 and p23 increased in haemolymph cells of oysters with DN. The expression of XBP-1, RACK, NDPk, RPA3 and p23 significantly increased in haemolymph cells of oysters affected by both diseases. There were changes in the expression of a number of genes in different organs depeding on disease stage: RACK expression increased in gills of oysters with bonamiosis, XBP-1 increased in mantle and digestive organs of oysters with light DN and RPA3 expression increased in gonads of oysters with heavy bonamiosis and heavy neoplasia.

Molecular characterisation of TNF, AIF, dermatopontin and VAMP genes of the flat oyster Ostrea edulis and analysis of their modulation by diseases.

Bonamiosis and disseminated neoplasia (DN) are the most important diseases affecting cultured flat oysters (Ostrea edulis) in Galicia (NW Spain). Previous research of the response of O. edulis against bonamiosis by suppression subtractive hybridisation yielded a partial expressed sequence tag of tumour necrosis factor (TNF) and allograft inflammatory factor (AIF), as well as the whole open reading frame for dermatopontin and vesicle-associated membrane (VAMP). Herein, the complete open reading frames of TNF and AIF genes were determined by the rapid amplification of cDNA, and the deduced amino acid sequences of the four genes were characterised. Phylogenetic relationships for each gene were studied using maximum likelihood parameters. Quantitative-PCR assays were also performed in order to analyse the modulation of the expression of these genes by bonamiosis and disseminated neoplasia. Gene expression profiles were studied in haemolymph cells and in various organs (gill, gonad, mantle and digestive gland) of oysters affected by bonamiosis, DN, and both diseases with regard to non-affected oysters (control). TNF expression in haemolymph cells was up-regulated at heavy stage of bonamiosis but its expression was not affected by DN. AIF expression was up-regulated at heavy stage of bonamiosis in haemolymph cells and mantle, which is associated with heavy inflammatory response, and in haemolymph cells of oysters affected by DN. AIF expression was, however, down-regulated in other organs as gills and gonads. Dermatopontin expression was down-regulated in haemolymph cells and digestive gland of oysters affected by bonamiosis, but DN had no significant effect on its expression. Gills and gonads showed up-regulation of dermatopontin expression associated with bonamiosis. There were significant differences in the expression of TNF and VAMP depending on the bonamiosis intensity stage whereas no significant differences were detected between light and heavy severity degrees of DN for the studied genes. VAMP expression showed also differences among haemolymph cells and the organs studied. The occurrence of both diseases in oysters involved haemolymph cell gene expression patterns different from those associated to each disease separately: no significant effect was observed in TNF expression, dermatopontin was up-regulated and marked up-regulation of AIF and VAMP was recorded, which suggests a multiplier effect of the combination of both diseases for the latter two genes.

Identification of relevant cancer related-genes in the flat oyster Ostrea edulis affected by disseminated neoplasia.

Disseminated neoplasia (DN), an oyster disease resembling leukaemia, has been reported in a number of species of marine bivalve molluscs. The disease is characterised by a proliferation of abnormal circulating cells of unknown origin resulting in the invasion of tissues and organs, frequently with a fatal end of the affected individuals. To obtain a more comprehensive view of bivalve cancer processes, suppressive subtracted hybridisation (SSH) and quantitative RT-PCR (q-PCR) approaches were combined to investigate changes in the transcriptome of Ostrea edulis haemolymph cells associated to DN. Two SSH libraries were constructed and 587 expressed sequence tags (ESTs) were sequenced, obtaining 329 ESTs which showed expression changes in neoplastic process. Transcription expression analyses (q-PCR) were done for a total of 24 genes that could be relevant in neoplastic process, including genes with role in the regulation of cell cycle, apoptosis or chromosomal defects. Most of those genes had not been reported in association with cancer in non-vertebrate organisms. The over-expression and under-expression of some of those genes in DN-affected oysters was in agreement with observations in vertebrate cancer. The results herein reported contribute to cancer understanding in bivalve molluscs.

Identification and expression of immune genes in the flat oyster Ostrea edulis in response to bonamiosis.

The European flat Ostrea edulis is highly susceptible to infection by the protozoan Bonamia ostreae and Bonamia exitiosa, intracellular parasites able to survive and proliferate within the oyster haemocytes. The parasite, once phagocytosed by the haemocyte, the main cellular effector of the immune system, appears to have some counter mechanism that turns off the haemocyte's metabolic destructive capacity, so that the parasite survives within the cell. To further understand the molecular basis of the immune response of the flat oyster against the bonamiosis, suppression subtractive hybridization and Q-PCR approaches were combined to identify genes involved in the development of the infection both in early and advanced phases. Four subtractive cDNA libraries were constructed and sequenced, obtaining a high number of ESTs that were seen to be up or down-regulated in the infection. A group of ESTs that play a role in the immune response, such as cytokines, stress proteins, eicosanoids, proteins implicated in phagocytosis and cell junction as well as in transcription signalling were identified and their expression was analysed at different infection levels by Q-PCR. The results here reported can help to enrich our understanding about the immune response of O. edulis against bonamiosis and improve our knowledge of the immune mechanisms of oysters.