Clinical diagnosis of hyposalivation in hospitalized patients

OBJECTIVE: The aim of this study was to evaluate the effectiveness of clinical criteria for the diagnosis of hyposalivation in hospitalized patients. MATERIAL AND METHODS: A clinical study was carried out on 145 subjects (48 males; 97 females; aged 20 to 90 years). Each subject was clinically examined, in the morning and in the afternoon, along 1 day. A focused anamnesis allowed identifying symptoms of hyposalivation, like xerostomia complaints (considered as a reference symptom), chewing difficulty, dysphagia and increased frequency of liquid intake. Afterwards, dryness of the mucosa of the cheecks and floor of the mouth, as well as salivary secretion during parotid gland stimulation were assessed during oral examination. RESULTS: Results obtained with Chi-square tests showed that 71 patients (48.9%) presented xerostomia complaints, with a significant correlation with all hyposalivation symptoms (p <0.05). Furthermore, xerostomia was also significantly correlated with all data obtained during oral examination in both periods of evaluation (p<0.05). CONCLUSION: Clinical diagnosis of hyposalivation in hospitalized patients is feasible and can provide an immediate and appropriate therapy avoiding further problems and improving their quality of life.

Effects of Aedes aegypti salivary components on dendritic cell and lymphocyte biology

Abstract Background Saliva is a key element of interaction between hematophagous mosquitoes and their vertebrate hosts. In addition to allowing a successful blood meal by neutralizing or delaying hemostatic responses, the salivary cocktail is also able to modulate the effector mechanisms of host immune responses facilitating, in turn, the transmission of several types of microorganisms. Understanding how the mosquito uses its salivary components to circumvent host immunity might help to clarify the mechanisms of transmission of such pathogens and disease establishment. Methods Flow cytometry was used to evaluate if increasing concentrations of A. aegypti salivary gland extract (SGE) affects bone marrow-derived DC differentiation and maturation. Lymphocyte proliferation in the presence of SGE was estimated by a colorimetric assay. Western blot and Annexin V staining assays were used to assess apoptosis in these cells. Naïve and memory cells from mosquito-bite exposed mice or OVA-immunized mice and their respective controls were analyzed by flow cytometry. Results Concentration-response curves were employed to evaluate A. aegypti SGE effects on DC and lymphocyte biology. DCs differentiation from bone marrow precursors, their maturation and function were not directly affected by A. aegypti SGE (concentrations ranging from 2.5 to 40 μg/mL). On the other hand, lymphocytes were very sensitive to the salivary components and died in the presence of A. aegypti SGE, even at concentrations as low as 0.1 μg/mL. In addition, A. aegypti SGE was shown to induce apoptosis in all lymphocyte populations evaluated (CD4+ and CD8+ T cells, and B cells) through a mechanism involving caspase-3 and caspase-8, but not Bim. By using different approaches to generate memory cells, we were able to verify that these cells are resistant to SGE effects. Conclusion Our results show that lymphocytes, and not DCs, are the primary target of A. aegypti salivary components. In the presence of A. aegypti SGE, naïve lymphocyte populations die by apoptosis in a caspase-3- and caspase-8-dependent pathway, while memory cells are selectively more resistant to its effects. The present work contributes to elucidate the activities of A. aegypti salivary molecules on the antigen presenting cell-lymphocyte axis and in the biology of these cells.

A COCAÍNA E O CRACK: As Drogas da Morte

Diante da relevância mundial do desafio no enfrentamento do tema concernente ao uso de drogas, o presente artigo tem por escopo principal abordar alguns aspectos sobre a cocaína em suas diversas formas de consumo, isto é, como chá, em pó (inalada), como injeção (diluída) e fumo – neste caso denominado crack (“pedra”) ou merla (“pasta”). Nesse contexto, no primeiro tópico, explana-se um breve histórico sobre as drogas, com enfoque nas espécies do tipo estimulantes, no qual se ressalta os efeitos tóxicos que causam ao organismo dos usuários. A seguir, há relevo sobre os problemas sociais e psicológicos acarretados pelo uso da cocaína em qualquer de suas formas, bem como expõe apontamentos sobre métodos de tratamento

Increased SGLT1 expression in salivary gland ductal cells correlates with hyposalivation in diabetic and hypertensive rats

Background Oral health complications in diabetes and hypertension include decreased salivary secretion. The sodium-glucose cotransporter 1 (SGLT1) protein, which transports 1 glucose/2 Na+/264 H2O molecules, is described in salivary glands. We hypothesized that changes in SGLT1 expression in the luminal membrane of ductal cell may be related to an altered salivary flow. Findings By immunohistochemistry, we investigated SGLT1 expression in ductal cells of parotid and submandibular glands from Wistar Kyoto rats (WKY), diabetic WKY (WKY-D), spontaneously hypertensive rats (SHR) and diabetic SHR (SHR-D), as well as in parotid glands from WKY subjected to sympathetic stimulation, with or without previous propranolol blockade. Diabetes and hypertension decreased the salivary secretion and increased SGLT1 expression in the luminal membrane of ductal cells, and their association exacerbated the regulations observed. After 30 min of sympathetic stimulation, SGLT1 increased in the luminal membrane of ductal cells, and that was blocked by previous injection of propranolol. Conclusions SGLT1 expression increases in the luminal membrane of salivary gland ductal cells and the salivary flow decreases in diabetic and hypertensive rats, which may be related to sympathetic activity. This study highlights the water transporter role of SGLT1 in salivary glands, which, by increasing ductal water reabsorption, may explain the hyposalivation of diabetic and hypertensive subjects

Submandibular glands and the regulation of gastric proliferation and TGFα distribution during rat postnatal development

Rodent gastric mucosa grows and differentiates during suckling-weaning transition. Among the molecules in rat milk, EGF and TGFβ are important peptides in the control of cell proliferation, and together with TGFα, they are also produced by submandibular glands. We aimed to determine the effect of saliva and milk on epithelial cell proliferation in the stomach of rat pups. We also examined the distribution of TGFα in the gastric mucosa after sialoadenectomy (SIALO) and fasting in order to determine whether this growth factor is affected by the deprivation of molecules derived from saliva and milk. SIALO was performed at 14 days and fasting was induced 3 days later. Cell proliferation was evaluated through metaphasic index and TGFα was detected by immunohistochemistry. We observed that whereas SIALO did not alter cell division, since the metaphasic index (MI) was unchanged, fasting stimulated cell proliferation (P < 0.05). After SIALO and fasting, MI was reduced when compared to the fasted group (P < 0.05). We found that TGFα is distributed along gastric gland and SIALO did not interfere in the localization and number of immunolabeled cells, but fasting increased their density when compared to the control (P < 0.05). The association of SIALO and fasting reduced TGFα immunostaining (P < 0.05). Therefore, during fasting, high MI was parallel to increased TGFα in gastric epithelium, but interestingly, this effect was found only in the presence of submandibular glands. We suggest that during suckling, peptides derived from saliva and milk are important to regulate gastric growth.

Effects of Aedes aegypti salivary components on dendritic cell and lymphocyte biology

BACKGROUND: Saliva is a key element of interaction between hematophagous mosquitoes and their vertebrate hosts. In addition to allowing a successful blood meal by neutralizing or delaying hemostatic responses, the salivary cocktail is also able to modulate the effector mechanisms of host immune responses facilitating, in turn, the transmission of several types of microorganisms. Understanding how the mosquito uses its salivary components to circumvent host immunity might help to clarify the mechanisms of transmission of such pathogens and disease establishment. METHODS: Flow cytometry was used to evaluate if increasing concentrations of A. aegypti salivary gland extract (SGE) affects bone marrow-derived DC differentiation and maturation. Lymphocyte proliferation in the presence of SGE was estimated by a colorimetric assay. Western blot and Annexin V staining assays were used to assess apoptosis in these cells. Naïve and memory cells from mosquito-bite exposed mice or OVA-immunized mice and their respective controls were analyzed by flow cytometry. RESULTS: Concentration-response curves were employed to evaluate A. aegypti SGE effects on DC and lymphocyte biology. DCs differentiation from bone marrow precursors, their maturation and function were not directly affected by A. aegypti SGE (concentrations ranging from 2.5 to 40 μg/mL). On the other hand, lymphocytes were very sensitive to the salivary components and died in the presence of A. aegypti SGE, even at concentrations as low as 0.1 μg/mL. In addition, A. aegypti SGE was shown to induce apoptosis in all lymphocyte populations evaluated (CD4+ and CD8+ T cells, and B cells) through a mechanism involving caspase-3 and caspase-8, but not Bim. By using different approaches to generate memory cells, we were able to verify that these cells are resistant to SGE effects. CONCLUSION: Our results show that lymphocytes, and not DCs, are the primary target of A. aegypti salivary components. In the presence of A. aegypti SGE, naïve lymphocyte populations die by apoptosis in a caspase-3- and caspase-8-dependent pathway, while memory cells are selectively more resistant to its effects. The present work contributes to elucidate the activities of A. aegypti salivary molecules on the antigen presenting cell-lymphocyte axis and in the biology of these cells.

Optimización de un método basado en SPE-LC-MS/MS para la determinación de multiresiduos de compuestos farmacéuticos en aguas depuradas

Universidad de Las Palmas de Gran Canaria, Facultad de Ciencias del Mar, Doctorado en Gestión Costera.

Evaluación de la presencia de compuestos farmacéuticos en aguas residuales procedentes de estaciones de depuración de aguas residuales (EDARS) de la isla de Gran Canaria

[ES]En este trabajo se ha realizado la evaluación de la concentración de compuestos farmacéuticos de uso común de diferentes grupos terapéuticos tales como, antibióticos, antiinflamatorios, analgésicos, reguladores lipídicos, betabloqueantes, antidepresivos, estimulantes y antiepilépticos, en aguas procedentes de Estaciones de Depuración de Aguas Residuales de la isla de Gran Canaria, con el fin de poner en conocimiento la situación del problema, siendo ésta un paso previo para su posible regulación y remediación. Debido a las bajas concentraciones en que estos compuestos se encuentran en las aguas residuales, para llevar a cabo el estudio, se utilizaron procesos analíticos basados en un retratamiento de la muestra utilizando la extracción en fase sólida y su posterior determinación por técnicas instrumentales de altas prestaciones

A novel phase variation mechanism in the meningococcus driven by a ligand-responsive repressor and differential spacing of distal promoter elements

Phase variable expression, mediated by high frequency reversible changes in the length of simple sequence repeats, facilitates adaptation of bacterial populations to changing environments and is frequently important in bacterial virulence. Here we elucidate a novel phase variable mechanism for NadA expression, an adhesin and invasin of Neisseria meningitidis. The NadR repressor protein binds to operators flanking the phase variable tract of the nadA promoter gene and contributes to the differential expression levels of phase variant promoters with different numbers of repeats, likely due to different spacing between operators. It is shown that IHF binds between these operators, and may permit looping of the promoter, allowing interaction of NadR at operators located distally or overlapping the promoter. The 4-hydroxyphenylacetic acid, a metabolite of aromatic amino acid catabolism that is secreted in saliva, induces nadA expression by inhibiting the DNA binding activity of the NadR repressor. When induced, only minor differences are evident between NadR-independent transcription levels of promoter phase variants, which are likely due to differential RNA polymerase contacts leading to altered promoter activity. These results suggest that NadA expression is under both stochastic and tight environmental-sensing regulatory control, and both regulations are mediated by the NadR repressor that and may be induced during colonization of the oropharynx where it plays a major role in the successful adhesion and invasion of the mucosa. Hence, simple sequence repeats in promoter regions may be a strategy used by host-adapted bacterial pathogens to randomly switch between expression states that may nonetheless still be induced by appropriate niche-specific signals.

Development of original analytical methods for the therapeutic drug monitoring of CNS druges: Antipsychotics, Antidepressants and Anxiolytics-hypnotics

Great strides have been made in the last few years in the pharmacological treatment of neuropsychiatric disorders, with the introduction into the therapy of several new and more efficient agents, which have improved the quality of life of many patients. Despite these advances, a large percentage of patients is still considered “non-responder” to the therapy, not drawing any benefits from it. Moreover, these patients have a peculiar therapeutic profile, due to the very frequent application of polypharmacy, attempting to obtain satisfactory remission of the multiple aspects of psychiatric syndromes. Therapy is heavily individualised and switching from one therapeutic agent to another is quite frequent. One of the main problems of this situation is the possibility of unwanted or unexpected pharmacological interactions, which can occur both during polypharmacy and during switching. Simultaneous administration of psychiatric drugs can easily lead to interactions if one of the administered compounds influences the metabolism of the others. Impaired CYP450 function due to inhibition of the enzyme is frequent. Other metabolic pathways, such as glucuronidation, can also be influenced. The Therapeutic Drug Monitoring (TDM) of psychotropic drugs is an important tool for treatment personalisation and optimisation. It deals with the determination of parent drugs and metabolites plasma levels, in order to monitor them over time and to compare these findings with clinical data. This allows establishing chemical-clinical correlations (such as those between administered dose and therapeutic and side effects), which are essential to obtain the maximum therapeutic efficacy, while minimising side and toxic effects. It is evident the importance of developing sensitive and selective analytical methods for the determination of the administered drugs and their main metabolites, in order to obtain reliable data that can correctly support clinical decisions. During the three years of Ph.D. program, some analytical methods based on HPLC have been developed, validated and successfully applied to the TDM of psychiatric patients undergoing treatment with drugs belonging to following classes: antipsychotics, antidepressants and anxiolytic-hypnotics. The biological matrices which have been processed were: blood, plasma, serum, saliva, urine, hair and rat brain. Among antipsychotics, both atypical and classical agents have been considered, such as haloperidol, chlorpromazine, clotiapine, loxapine, risperidone (and 9-hydroxyrisperidone), clozapine (as well as N-desmethylclozapine and clozapine N-oxide) and quetiapine. While the need for an accurate TDM of schizophrenic patients is being increasingly recognized by psychiatrists, only in the last few years the same attention is being paid to the TDM of depressed patients. This is leading to the acknowledgment that depression pharmacotherapy can greatly benefit from the accurate application of TDM. For this reason, the research activity has also been focused on first and second-generation antidepressant agents, like triciclic antidepressants, trazodone and m-chlorophenylpiperazine (m-cpp), paroxetine and its three main metabolites, venlafaxine and its active metabolite, and the most recent antidepressant introduced into the market, duloxetine. Among anxiolytics-hypnotics, benzodiazepines are very often involved in the pharmacotherapy of depression for the relief of anxious components; for this reason, it is useful to monitor these drugs, especially in cases of polypharmacy. The results obtained during these three years of Ph.D. program are reliable and the developed HPLC methods are suitable for the qualitative and quantitative determination of CNS drugs in biological fluids for TDM purposes.

Molekulargenetische Untersuchungen zur Reblausresistenz bei der Unterlagsrebe Börner

Dass Pflanzen gegen phytopathogene Infektionen resistent sind, ist das Ergebnis von multip-len Abwehrreaktionen. Eine solche ist auch die Hypersensitivitätsreaktion (HR). Sie ist die Folge eines Befalls von Börner mit Rebläusen und zeigt sich an Blättern und Wurzeln der resistenten Unterlagsrebe in Form von lokalen Nekrosen. Die Erzeugung von neuen, trans-genen reblausresistenten Unterlagsreben verlangt präzise Kenntnisse über die Mechanismen der Reblausresistenz. Um Resistenzgene zu identifizieren, wurden im Rahmen dieser Arbeit differenzielle Genexpressionsanalysen eingesetzt. Diese waren die Microarray Analyse mit der Geniom one Technik und die real time (RT) -PCR. Sie erlaubten eine Gegenüberstellung der Genexpression in behandeltem Wurzelgewebe mit der Expression im Normalgewebe der Unterlagsrebe Börner. Als experimenteller Induktor der HR in Börner diente die Indol-3-Essigsäure (IES), ein Bestandteil des Reblausspeichels. Frühere Untersuchungen zur Reb-lausresistenz zeigten, dass bei einer Behandlung mit IAA an Wurzeln von Börner Nekrosen entstehen, nicht jedoch an Wurzeln von der reblaustoleranten Unterlagssorte SO4 oder dem reblausanfälligem Edelreis. Das war der Grund, SO4 und Riesling als Vergleichsobjekte zu Börner für diese Studie auszuwählen. So sollte die Bedeutung der Rolle von IES als Auslö-ser der Resistenzmechanismen in Börner erklärt werden. Insgesamt konnten deutliche Unter-schiede in den Reaktionen der drei Rebsorten auf die IES Behandlung aufgedeckt werden. Während in Börner eine hohe Anzahl an Genen und diese intensiv auf den IES Reiz reagiert, fallen die Gene bei SO4 und Riesling zahlenmäßig kaum ins Gewicht und die Reaktionen der beiden Sorten auf IES zudem eher schwach aus. In der Summe waren es 27 Gene, die für die Reblausresistenz in Börner verantwortlich sein könnten. So konnte eine IES bedingte Aktivierung von Genen beobachtet werden, die bei der Produktion von Phytoalexinen be-deutsam sind, wie z.B. die phenylalanine ammonia-lyase, die lipoxygenase und die stilbene synthase. Weiter ließ sich eine Regulation von allgemein Stress assoziierten Genen und von Zellwandproteinen und eine Induktion von Signalkomponenten, etwa des Transkriptionsfak-tors ethylene response factor, nachweisen. Eine deutliche Hochregulation von Au-xintransportern in den IES behandelten Börnerwurzeln gab zudem Anhaltspunkte auf sorten-spezifische Unterschiede in der zellulären Aufnahme und Abgabe der IES. Durch die Ausar-beitung des Zusammenspiels der durch IES regulierten Gene konnten in dieser Arbeit wert-volle Hinweise auf die Mechanismen der Reblausresistenz in Börner gewonnen werden.

The role of the NadR regulator during infection and its implication for the coverage of a new Meningococcus B vaccine

Background: Neisseria meningitides represents a major cause of meningitis and sepsis. The meningococcal regulator NadR was previously shown to repress the expression of the Neisserial Adhesin A (NadA) and play a major role in its phase-variation. NadA is a surface exposed protein involved in epithelial cell adhesion and colonization and a major component of 4CMenB, a novel vaccine to prevent meningococcus serogroup B infection. The NadR mediated repression of NadA is attenuated by 4-HPA, a natural molecule released in human saliva. Results: In this thesis we investigated the global role of NadR during meningogoccal infection, identifying through microarray analysis the NadR regulon. Two distinct types of NadR targets were identified, differing in their promoter architectures and 4HPA responsive activities: type I are induced, while type II are co-repressed in response to the same 4HPA signal. We then investigate the mechanism of regulation of NadR by 4-HPA, generating NadR mutants and identifying classes or residues involved in either NadR DNA binding or 4HPA responsive activities. Finally, we studied the impact of NadR mediated repression of NadA on the vaccine coverage of 4CMenB. A selected MenB strains is not killed by sera from immunized infants when the strain is grown in vitro, however, in an in vivo passive protection model, the same sera protected infant rats from bacteremia. Finally, using bioluminescent reporters, nadA expression in the infant rat model was induced in vivo at 3 h post-infection. Conclusions: Our results suggest that NadR coordinates a broad transcriptional response to signals present in the human host, enabling the meningococcus to adapt to the relevant host niche. During infectious disease the effect of the same signal on NadR changes between different targets. In particular NadA expression is induced in vivo, leading to efficient killing of meningococcus by anti-NadA antibodies elicited by the 4CMenB vaccine.

Hair: a tool to evaluate the HPA axis activity

Hair cortisol is a novel marker to measure long-term secretion cortisol free from many methodological caveats associated with other matrices such as plasma, saliva, urine, milk and faeces. For decades hair analysis has been successfully used in forensic science and toxicology to evaluate the exposure to exogenous substances and assess endogenous steroid hormones. Evaluation of cortisol in hair matrix began about a decade ago and have over the past five years had a remarkable development by advancing knowledge and affirming this method as a new and efficient way to study the hypothalamic-pituitary-adrenal (HPA) axis activity over a long time period. In farm animals, certain environmental or management conditions can potentially activate the HPA axis. Given the importance of cortisol in monitoring the HPA axis activity, a first approach has involved the study on the distribution of hair cortisol concentrations (HCC) in healthy dairy cows showing a physiological range of variation of this hormone. Moreover, HCC have been significantly influenced also by changes in environmental conditions and a significant positive correlation was detected between HCC and cows clinically or physiologically compromised suggesting that these cows were subjected to repeated HPA axis activation. Additionally, Crossbreed F1 heifers showed significantly lower HCC compared to pure animals and a breed influence has been seen also on the HPA axis activity stimulated by an environmental change showing thus a higher level of resilience and a better adaptability to the environment of certain genotypes. Hair proved to be an excellent matrix also in the study of the activation of the HPA axis during the perinatal period. The use of hair analysis in research holds great promise to significantly enhance current understanding on the role of HPA axis over a long period of time.

Caratterizzazione microbiologica ed epidemiologica del microbiota del cavo orale mediante metodiche colturali e molecolari

La ricerca si è focalizzata su due degli aspetti di interesse odontoiatrico più diffusi: la carie dentaria e la parodontite cronica. Il problema della carie dentaria è stato studiato in una popolazione di 39 soggetti affetti da cardiopatia congenita in cui la scarsa igiene orale è fattore di rischio per problematiche di salute generale e soprattutto per lo sviluppo di endocardite infettiva. I dati osservati e confrontati con quelli di un omogeneo gruppo di controllo dimostrano che nella dentatura decidua questi bambini hanno più denti cariati, come dimostrato dalla significativa differenza dell'indice dmft. Nella dentatura permanente non si osservano differenze tra i due gruppi. La carica microbica totale rilevata nella saliva e la presenza di Streptococcus mutans non mostrano differenze tra i due gruppi. I problemi di parodontite cronica sono stati studiati in un gruppo di 352 soggetti italiani adulti in cui si è definita la prevalenza dei 6 più importanti patogeni parodontali e la possibile correlazione con parametri clinici (pus, sanguinamento al sondaggio - BOP, profondità di sondaggio della tasca parodontale – PPD). Tra le 6 specie batteriche ricercate, quello di più frequente riscontro è stato Fusobacterium nucleatum (95%), mentre quello con carica batterica più alta è stato Tannerella forsythia. La carica batterica di Porphyromonas gingivalis, Treponema denticola, Tannerella forsythia e Fusobacterium nucleatum ha mostrato una correlazione diretta con il BOP e la presenza di pus. Inoltre, si è riscontrato che la carica batterica di tutte le specie (tranne Aggregatibacterium actinomycetemcomitans) aumenta all'aumentare del PPD. Tra le variabili studiate, PPD rappresenta il più importante fattore di rischio per la presenza di parodontopatogeni, mentre BOP è un indicatore di rischio per la ricerca del complesso rosso.

Confronto fra periodizzazione Tradizionale e a Blocchi per l’allenamento della forza in atleti maschi e partecipanti di genere femminile.

La prima parte dello studio riguarda la descrizione dell’origine e delle caratteristiche che differenziano la periodizzazione tradizionale e quella a blocchi per l’allenamento della forza. L’obiettivo della seconda parte del lavoro è stato quello di confrontare gli adattamento ormonali e prestativi ad un programma di allenamento della forza periodizzato secondo il modello tradizionale o secondo quello a blocchi in un campione di atleti di forza. Venticinque atleti maschi sono stati assegnati con procedura randomizzata al gruppo con programmazione tradizionale (TP) o a quello a blocchi (BP). Prelievi di saliva sono stati effettuati prima e dopo 6 diverse sedute di allenamento durante il programma al fine di rilevare i livelli di testosterone (T) e cortisolo (C). Le valutazioni dei parametri antropometrici e prestativi sono state effettuate prima e dopo le 15 settimane di allenamento previste. In nessuno dei due gruppi vi sono state variazioni significative nei livelli ormonali. I risultati indicano che il gruppo BP ha ottenuto incrementi superiori a quello TP riguardo alla forza massima (p = 0,040) ed alla potenza (p = 0,035) espressa alla panca piana. Nella terza parte dello studio, la periodizzazione tradizionale e quella a blocchi sono state confrontate riguardo agli effetti sulla forza massima e sull’ipertrofia in donne allenate di livello amatoriale. Diciassette donne hanno partecipato all’esperimento allenandosi 3 volte a settimana per 10 settimane. I risultati dimostrano che entrambe le periodizzazioni hanno portato a miglioramenti significativi di forza e potenza; il gruppo TP ha tuttavia ottenuto incrementi superiori di forza massima (p = 0,039) e ipertrofia degli arti inferiori (p = 0,004). La periodizzazione tradizionale quindi si è dimostrata più efficace per aumentare la forza massima e la sezione muscolare della coscia in partecipanti di genere femminile. I risultati contrastanti nei due generi potrebbero essere legati a rapporti diversi fra processi anabolici e catabolici.