Ecografia bidimensionale e con mezzo di contrasto nello studio delle patologie gastro-intestinali del gatto

L’ecografia del tratto gastroenterico è una delle metodiche d’elezione nella valutazione diagnostica delle patologie gastrointestinali nel gatto. In questa tesi dottorale sono presentati i risultati di tre studi in cui l’ecografia convenzionale e con mezzo di contrasto è stata impiegata in gatti sani o con patologie gastroenteriche. Lo scopo del primo studio, prospettico, è stato quello di determinare lo spessore ecografico dei singoli strati di parete nell’intestino tenue in una popolazione di gatti sani. Lo strato mucoso è risultato significativamente più spesso nel duodeno e nel digiuno, per la maggiore grandezza dei villi in queste porzioni dell’intestino tenue. A livello dell’ileo, gli strati di maggior spessore sono risultati quello sottomucoso, per l’abbondante presenza di aggregati linfoidi, e quello muscolare, a causa delle caratteristiche anatomo-funzionali di sfintere che questo tratto intestinale svolge. Il secondo progetto, retrospettivo, nasce dalla collaborazione tra due centri universitari, uno italiano e uno americano, con l’obiettivo di confrontare lo spessore della tonaca muscolare intestinale in gatti affetti da Inflammatory Bowel disease (IBD) o da neoplasie intestinali. In questo studio, l’ipertrofia della tonaca muscolare (ITM) è stato maggiormente osservato in gatti con IBD rispetto a gatti con neoplasie intestinali, ma non sono state evidenziate differenze di spessore della tonaca muscolare tali da poter differenziare le due patologie. Lo scopo del terzo progetto, prospettico, è stato quello di descrivere il pattern di perfusione parietale del piccolo intestino, valutato mediante uso di mezzo di contrasto ecografico, in gatti con ITM associata a IBD. In tutti gli animali studiati, l’ITM si è associato a una modesta assunzione del mezzo di contrasto rispetto agli altri strati della parete intestinale. Questi risultati confermano che l’ITM che si osserva in gatti con IBD non è associato a significativi aumenti della vascolarizzazione di tale strato parietale.

Pathophysiology of bile acid aspiration in lung transplant recipients

Duodeno-gastroesophageal reflux aspiration is associated with chronic lung allograft dysfunction (CLAD) and aspiration of bile acids (BA), functional molecules in the gastro-intestinal tract with emulsifying properties. While links between reflux aspiration to lung disease have been identified, the relevance of bile acid as molecular ligands and outcome predictors is poorly defined. We sought to determine and quantify the various BA species in airways of the lung transplant recipients to better understand the various effects of aspirated BA that contribute to post-transplantation outcomes and to investigate their molecular effects on airway function and contractility.

Studio del microcircolo della parete gastrica affetta da patologia infiammatoria e neoplastica mediante Color-Power Doppler ed ecografia con mezzo di contrasto endovenoso

Introduzione: Negli ultimi anni, il color-power Doppler si è dimostrato un utile strumento per valutare le alterazioni della vascolarizzazione della parete intestinale nelle patologie del tratto gastro-enterico. Più di recente, i mezzi di contrasto ecografici di II generazione associati all’ecografia real-time con basso indice meccanico (CEUS) hanno permesso di valutare ecograficamente il microcircolo, consentendo la valutazione della vascolarizzazione di parete nelle patologie associate a flogosi e neoangiogenesi. Studi recenti hanno documentato i pattern Doppler e CEUS nella malattia infiammatoria intestinale. Le alterazioni della vascolarizzazione di parete nella patologia neoplastica, invece, sono state finora valutate con sola tecnica Doppler. Recenti studi basati sull’impiego di tale metodica hanno in effetti dimostrato che l’intensità del segnale vascolare di parete correla con la variante istologica della neoplasia e con il suo grado di invasione vascolare costituendo così un parametro di neoangiogenesi tumorale. Pertanto, ottenere mediante CEUS una più accurata definizione del microcircolo di parete potrebbe aiutare nella diagnosi differenziale tra patologia infiammatoria e neoplastica dello stomaco e fornire utili informazioni per valutare l’ aggressività del cancro gastrico.

Apnea e reflusso gastroesofageo nel neonato pretermine

Objective: To document the existence of a relationship between apnea of prematurity (AOP) and gastroesophageal reflux (GER) in preterm infants. Setting: One Neonatal Intensive Care Unit Patients: Twenty-six preterm infants (gestational age<32 weeks) with recurrent apneas. Intervention: Simultaneous and synchronized recording of polysomnography and pH-impedance monitoring (pH-MII). Polysomnography detects and characterizes apneas, by recording of breathing movement, nasal airflow, electrocardiogram, pulse oximeter saturation. pH-MII is the state-of-theart methodology for GER detection in preterm newborns. Main outcome measures: Relationship between AOP and GER, which were considered temporally related if both started within 30 seconds of each other. Results: One-hundred-fifty-four apneas out of 1136 were temporally related to GER. The frequency of apnea during the one-minute time around the onset of GER was significantly higher than the one detected in the GER-free period (p=0.03). Furthermore, the frequency of apnea in the 30 seconds after GER (GER-triggered apneas) was greater than that detected in the 30 seconds before (p=0.01). A great inter-individual variability was documented in the proportion of GERtriggered apneas. A strong correlation between total number of apneas and the difference between apneas detected 30 seconds after and before GER was found (p=0.034). Conclusions: Our data show that a variable rate of apneas can be triggered by GER in very preterm infant. Further studies are needed to recognise clinical features which identify those patients who are more susceptible to GER-triggered apneas.

Validazione e applicazioni cliniche della metodica NIRA (Near Infrared Reflectance Analysis) per la valutazione del contenuto nutrizionale del latte materno in terapia intensiva neonatale

Phase 1: To validate Near-Infrared Reflectance Analysis (NIRA) as a fast, reliable and suitable method for routine evaluation of human milk’s nitrogen and fat content. Phase 2: To determine whether fat content, protein content and osmolality of HM before and after fortification may affect gastroesophageal reflux (GER) in symptomatic preterm infants. Patients and Methods: Phase 1: 124 samples of expressed human milk (55 from preterm mothers and 69 from term mothers) were used to validate NIRA against traditional methods (Gerber method for fat and Kjeldhal method for nitrogen). Phase 2: GER was evaluated in 17 symptomatic preterm newborns fed naïve and fortified HM by combined pH/intraluminal-impedance monitoring (pH-MII). HM fat and protein content was analysed by a Near-Infrared-Reflectance-Analysis (NIRA). HM osmolality was tested before and after fortification. GER indexes measured before and after fortification were compared, and were also related with HM fat and protein content and osmolality before and after fortification. Results: Phase 1: · A strong agreement was found between traditional methods’ and NIRA’s results (expressed as g/100 g of milk), both for fat and nitrogen content in term (mean fat content: NIRA=2.76; Gerber=2.76; mean nitrogen content: NIRA=1.88; Kjeldhal =1.92) and preterm (mean fat content: NIRA=3.56; Kjeldhal=3.52; mean nitrogen content: NIRA=1.91; Kjeldhal =1.89) mother’s milk. · Nitrogen content of the milk samples, measured by NIRA, ranged from 1.18 to 2.71 g/100 g of milk in preterm milk and from 1.48 to 2.47 in term milk; fat content ranged from 1.27 to 6.23 g/100 g of milk in preterm milk and from 1.01 to 6.01 g/100 g of milk in term milk. Phase 2: · An inverse correlation was found between naïve HM protein content and acid reflux index (RIpH: p=0.041, rho=-0.501). · After fortification, osmolality often exceeded the values recommended for infant feeds; furthermore, a statistically significant (p<.05) increase in non acid reflux indexes was observed. Conclusions: NIRA can be used as a fast, reliable and suitable tool for routine monitoring of macronutrient content of human milk. Protein content of naïve HM may influence acid GER in preterm infants. A standard fortification of HM may worsen non acid GER indexes and, due to the extreme variability in HM composition, may overcome both recommended protein intake and HM osmolality. Thus, an individualized fortification, based on the analysis of the composition of naïve HM, could optimize both nutrient intake and feeding tolerance.

Erosioni dentali nei bambini affetti da GERD

L’erosione dentale è definita come una perdita progressiva e irreversibile dei tessuti duri del dente, a causa di un processo chimico che non coinvolge i batteri. La sua prevalenza è accresciuta negli ultimi anni nella popolazione generale, particolarmente nei bambini e negli adolescenti e rappresenta attualmente un’enorme sfida per la cura della salute orale. L'eziologia delle erosioni è difficile da diagnosticare perché può essere il risultato di una varietà di fattori e si può manifestare in maniera differente in base alla causa che l’ha determinata. L’eziologia può essere intrinseca, cioè derivante dal contenuto acido dello stomaco associato a disordini alimentari come l’anoressia, la bulimia nervosa, il reflusso gastro-esofageo e il regurgito, o estrinseca, derivante dal contenuto acido proveniente dall’ambiente esterno (cibi dietetici, bevande analcoliche, succhi di frutta, ecc). L’erosione è particolarmente frequente nei bambini e poiché i soggetti che presentano erosioni in dentizione decidua hanno un rischio aumentato di manifestare erosioni in dentizione permanente, una diagnosi precoce e una prevenzione attuata in tenera età, aiuterà a prevenire il danno a carico dei denti permanenti. Inoltre se l’erosione dentale non è controllata e stabilizzata, il bambino potrebbe soffrire di severe perdite di superfici dentali, sensibilità dentale, malocclusione, inestetismi o anche ascessi dentali dei denti affetti. Lo studio caso-controllo è stato condotto dalla Sezione di Odontostomatologia del Dipartimento Testa - Collo, in collaborazione con l’Unità Operativa Complessa di Gastroenterologia Pediatrica dell’Azienda Ospedaliera-Univesitaria di Parma. Il gruppo di studio comprende 60 pazienti di età compresa tra i 4 e i 13 anni. Dei 60 bambini arruolati, 30 presentano sintomi e storia clinica di GERD, mentre gli altri 30 sono bambini sani senza alcun sintomo di GERD o di altri disturbi gastrointestinali. Il gruppo campione A include anche soggetti affetti, oltre che dal reflusso gastro-esofageo, da varie forme di disabilità, in alcune delle quali il GERD sembra essere parte del quadro sindromico. I risultati ottenuti dallo studio hanno dimostrato una prevalenza maggiore di erosioni dentali nel gruppo di bambini affetti da GERD, rispetto ai bambini sani, in accordo con quanto riportato dalla letteratura.

Therapeutic microbiology: characterization of Bifidobacterium strains for the treatment of enteric disorders in newborns.

Several studies support the use of probiotics for the treatment of minor gastrointestinal problems in infants. Positive effects on newborn colics have been evidenced after administration of Lactobacillus strains, whereas no studies have been reported regarding the use of bifidobacteria for this purpose. This work was therefore aimed at the characterization of Bifidobacterium strains capable of inhibiting the growth of pathogens typical of the infant gastro-intestinal tract and of coliforms isolated from colic newborns. Among the 46 Bifidobacterium strains considered, 16 showed high antimicrobial activity against potential pathogens; these strains were further characterized from a taxonomic point of view, for the presence and transferability of antibiotic resistances, for citotoxic effects and adhesion to non tumorigenic gut epithelium cell lines. Moreover, their ability to stimulate gut health by increasing the metabolic activity and the immune response of epithelial cells was also studied. The examination of all these features allowed to identify 3 B. breve strains and a B. longum subsp. longum strain as potential probiotics for the treatments of enteric disorders in newborns such as infantile colics. The formulation of a synbiotic product with an appropriate prebiotic fiber capable of supporting the growth of the selected Bifidobacterium strains was also considered in this study. In this respect the ability of the 4 selected Bifidobacterium strains to use as the sole carbon source and energy source different polisaccharide fibers was investigated The last phase of the work has been dedicated to the evaluation of the gut microbial diversity in newborns whose mothers has been subjected to antibiotic therapy a few hours before the delivery because of a Streptococcus type B infection. These newborns can represent a possible target for the probiotic strains selected in this work.

Diet Effects on Activation and Maturation of Feed Control over the Gastrointestinal Defence Barrier in Piglets

Weaning is an important and complex step involving many stresses that interfere deeply with feed intake, gastro-intestinal tract (GIT) development and adaptation to the weaning diet in young pigs. The health of the pig at weaning, its nutrition in the immediate post-weaning period, and the physical, microbiological and psychological environment are all factors that interact to determine food intake and subsequent growth. GIT disorders, infections and diarrhoea increase at the time of weaning, in fact pathogens such as enterotoxigenic Escherichia coli (ETEC) are major causes of mucosal damage in post-weaning disease contributing to diarrhoea in suckling and post-weaned pigs. The European ban in 2006 put on antibiotic growth promoters (AGP) has stimulated research on the mechanisms of GIT disorders and on nutritional approaches for preventing or reducing such disturbances avoiding AGPs. Concerning these aspects here are presented five studies based on the interplay among nutrition, genomic, immunity and physiology with the aim to clarify some of these problematic issues around weaning period in piglets. The first three evaluate the effects of diets threonine or tryptophan enriched on gut defence and health as possible alternatives to AGP in the gut. The fourth is focused on the possible immunological function related with the development of the stomach. The fifth is a pilot study on the gastric sensing and orexygenic signal given by fasting or re-feeding conditions. Although some results are controversial, it appears that both tryptophan and threonine supplementation in weaning diets have a preventive role in E.coli PWD and favorable effects in the gut especially in relation to ETEC susceptible genotype. While the stomach is believed as almost aseptic organ, it shows an immune activity related with the mucosal maturation. Moreover it shows an orexygenic role of both oxyntic mucosa and pyloric mucosa, and its possible relation with nutrient sensing stimuli.

Analisi proteomica dei meccanismi sottesi alla malatia da reflusso gastroesofageo

La malattia da reflusso gastroesofageo (GERD) si divide in due categorie: malattia non erosiva (NERD) ed erosiva (ERD). Questi due fenotipi di GERD mostrano caratteristiche patofisiologiche e cliniche differenti. NERD è la forma più comune. Anche se ERD e NERD sono difficili da distinguere a livello clinico, la forma NERD possiede caratteristiche fisiologiche, patofisiologiche, anatomiche, e istologiche uniche. La replicazione cellulare dello strato basale si pensa sia una delle cause implicate nella resistenza della mucosa e nella difesa strutturale dell’epitelio. Diversi studi hanno dimostrato che la proliferazione cellulare è ridotta nella mucosa esofagea esposta ad insulti acidi e peptici cronici, in pazienti GERD, in più uno studio recente ha dimostrato che il recettore per i cannabinoidi CB1 era implicato nella riparazione delle ferite nella mucosa del colon. Sulla base di questi dati abbiamo valutato la presenza del recettore CB1 in biopsie della mucosa esofagea, di pazienti ERD, NERD e di controlli sani, tramite analisi Western Blot, Immunoistochimica e Real-Time PCR, dimostrando per la prima volta la presenza di questo recettore nell’epitelio dell’esofago e una riduzione dei suoi livelli di espressione nei pazienti ERD, camparati con i NERD e con i controlli sani. Successivamente, per chiarire meglio i meccanismi molecolari che caratterizzano ERD e NERD, abbiamo effettuato un analisi proteomica con la tecnica shotgun, la quale ha evidenziato un patter proteico di 33 proteine differenzialmente espresse in pazienti NERD vs ERD, sette delle quali confermate in wester Blot, e quattro in immunoistochimica. Concludendo i nostri risultati hanno confermato che ERD e NERD sono due entità distinte a livello proteico, e hanno proposto dei candidati biomarker per la diagnosi differenziale di ERD e NERD.

Effects of different nutritional strategies on intestinal inflammation in pigs

Intestinal health is essential for the health of the body since the gastro-intestinal mucosa is the main site of interaction with the external environment, as well as the major area colonized by the microbiota. Intestinal health relies on proper barrier function, epithelial integrity and related mechanisms of protection (mucous layer, tight junctions, immune and inflammatory system). In pigs, during the weaning transition, intestinal inflammation and barrier integrity play a crucial role in regulating intestinal health and, consequently, pig’s health, growth and productivity. The aim of the project was to assess the impact of different nutritional strategies on the intestinal health of weaning piglets with reference to the inflammatory status and epithelial integrity. Therefore, in vivo trials were conducted to test the in-feed supplementation with zinc, tributyrin, or organic acids and nature-identical compounds (NIC) to weaning piglets. All the dietary interventions positively impacted the intestinal inflammatory status and, as a consequence, improved epithelial integrity by modulating tight junctions proteins (zinc or tributyrin) or by enhancing barrier properties measured with Ussing chambers (organic acids and NIC). These findings highlight that intestinal inflammation and barrier function are strictly linked, and that the control of inflammation is essential for adequate barrier function. In addition, in zinc trial and organic acids and NIC trial, better intestinal health could successfully result in better growth performance, as aimed for pig production improvement. To conclude, this work shows that dietary supplementation with bio-active substances such as zinc, tributyrin or organic acids and NIC may improve intestinal health of weaning piglets modulating intestinal inflammatory stress and barrier integrity and allowing better piglet’s health, growth and productivity.

Terapia chirurgica dell'acalasia scompensata: prevalenza e fattori di rischio per lo sviluppo del carcinoma esofageo, risultati clinico-funzionali a lungo termine della chirurgia conservativa e dell'esofagectomia

La chirurgia conservativa o l’esofagectomia, possono essere indicate per il trattamento della disfagia nell’acalasia scompensata. L’esofagectomia è inoltre finalizzata alla prevenzione dello sviluppo del carcinoma esofageo. Gli obiettivi erano: a) definire prevalenza e fattori di rischio per il carcinoma epidermoidale; b) confrontare i risultati clinici e funzionali di Heller-Dor con pull-down della giunzione esofagogastrica (PD-HD) ed esofagectomia. I dati in analisi, ricavati da un database istituito nel 1973 e finalizzato alla ricerca prospettica, sono stati: a) le caratteristiche cliniche, radiologiche ed endoscopiche di 573 pazienti acalasici; b) il risultato oggettivo e la qualità della vita, definita mediante questionario SF-36, dopo intervento di PD-HD (29 pazienti) e dopo esofagectomia per acalasia scompensata o carcinoma (20 pazienti). Risultati: a) sono stati diagnosticati 17 carcinomi epidermoidali ed un carcinosarcoma (3.14%). Fattori di rischio sono risultati essere: il diametro esofageo (p<0.001), il ristagno esofageo (p<0.01) e la durata dei sintomi dell’acalasia (p<0.01). Secondo l’albero di classificazione, soltanto i pazienti con esito insufficiente del trattamento ai controlli clinico-strumentali ed acalasia sigmoidea presentavano un rischio di sviluppare il carcinoma squamocellulare del 52.9%. b) Non sono state riscontrate differenze statisticamente significative tra i pazienti sottoposti ad intervento conservativo e quelli trattati con esofagectomia per quanto concerne l’esito dell’intervento valutato mediante parametri oggettivi (p=0.515). L’analisi della qualità della vita non ha evidenziato differenze statisticamente significative per quanto concerne i domini GH, RP, PF e BP. Punteggi significativamente più elevati nei domini RE (p=0.012), VT (p<0.001), MH (p=0.001) e SF (p=0.014) sono stati calcolati per PD-HD rispetto alle esofagectomie. In conclusione, PD-HD determina una miglior qualità della vita, ed è pertanto la procedura di scelta per i pazienti con basso rischio di cancro. A coloro che abbiano già raggiunto i parametri di rischio, si offrirà l’esofagectomia o l'opzione conservativa seguita da protocolli di follow-up.

The human gut microbiome in disease: Role in chemotherapy treatments and relationship with clinical outcomes

The gut microbiome (GM) is a plastic entity, capable of adapting in response to intrinsic and extrinsic factors. However, several circumstances can disrupt this homeostatic balance, forcing the GM to shift from a health-associated mutualistic configuration to a disease-associated profile. Nowadays, a new frontier of microbiome research is understanding the GM role in chemo-immunotherapies and clinical outcomes. Here, the role of the genotoxin‐producing pathogen Salmonella in colorectal carcinogenesis was characterized by in-vitro models. A synergistic effect of Salmonella and the CRC-associated mutation (APC gene) promoted a tumorigenic microenvironment by increasing cellular genomic instability. Subsequently, the GM involvement in anti-cancer therapies was investigated via next-generation sequencing in different patient cohorts. The GM trajectory during treatments was characterized for women with epithelial ovarian cancer and pediatric patients undergoing hematopoietic stem cell transplantation (HSCT). The results highlighted the loss of GM homeostasis, with diversity reduction, decrease in health-associated microorganisms and pathobiont bloom. Interestingly, a distinctive GM profile was identified in ovarian cancer patients with a poor response to chemotherapy compared to patients in remission. Moreover, maintenance of GM homeostasis through enteral feeding in pediatric HSCT patients highlighted a better prognosis, with reduced risk of clinical complications. In this context, the gut resistome – the pattern of GM antibiotic-resistance genes (ARGs) – was evaluated longitudinally in HSCT patients. The results showed new acquisitions and consolidation of ARGs already present in patients developing clinical complications. Antibiotic exposure was also evaluated in infants under low-dose antibiotic prophylaxis for vesico-ureteral reflux showing an impairment of the GM configuration with possible long-term health implications. Dramatic GM dysbiosis was finally observed in critically ill patients with COVID-19 (undergoing multiple drug therapies) and correlated with increased risk of bloodstream infection. All these findings pointed out the importance of maintaining GM homeostasis during chemotherapy treatments for improving patients’ clinical outcomes.

Colic surgery in the equine patient: novel biomarkers, postoperative management, microbiotal influence and epidemiological analysis on the italian territory

The aim of this work was to investigate novel diagnostic and prognostic tools, postoperative treatments and epidemiologic factors impacting the outcome of surgical cases of colic. To make a more accurate diagnosis and establish a prognosis, several biomarkers have been investigated in colic patients. In this study we evaluated peritoneal PCT and blood ADMA and SDMA in SIRS positive and negative colic patients to be used as prognostic biomarkers. Our results highlighted the limits of these biomarkers in detection and the lack of specificity. In fact PCT was not detectable and even if ADMA and SDMA significantly increased in colic horses, they are not diagnostic nor prognostic markers for SIRS. Fluid therapy has been described to be crucial for the outcome of colic patients, nevertheless no guidelines have been established. Overhydration was the common practice in post surgical management. We compared cases with an extended fluid therapy protocol and cases with a restricted protocol. Results showed that survival rate and postoperative complications were similar between the groups, despite costs being significantly lower in the restricted group. The possible correlation between intestinal microbiota and colics has gained interest. In this study, cecal and colonic content from horses undergoing laparotomy were collected, and the microbiota analized. Results showed some differences in microbiota between discharged and non discharged patients, and between strangulating and non strangulating types of colic, that might suggest some influence of hind gut microbiota on the disease. A multicentric study involving three veterinary teaching hospitals on the italian territory was conducted investigating factors affecting postoperative survival and complications in colics. Results showed that the influence of age, PCV, TPP, blood lactate, reflux, type of disease, type of lesion, presence of anastomosis, duration of surgery and surgeons, were in line with literature. Amount of crystalloids used could affected the outcome.

Ultra-hypofractionated stereotactic radiotherapy for prostate cancer in 5 fractions: current evidences and our clinical experience

AIMS: The present is a retrospective evaluation of acute genito-urinary (GU) and gastro-intestinal (GI) toxicity, in addition to biochemical recurrence rate in 57 prostate cancer patients treated at our Institution with ultra-hypofractionated RT (UHRT) schedule. METHODS: From January 2021 to December 2022 we have treated 57 patients with prostate cancer, using an UHRT scheme of 5-fractions every other day for a total dose delivered of 36.25 Gy, according to the PACE-B trial treatment schedule. Good urinary function, assessed by International Prostate Symptom Score (IPSS), were required. The simulation CT scans were acquired in supine position and fused with MRI for CTVs definition for every patient. Each treatment was performed by Accuray's TomoTherapy with daily IGRT. The evaluation of the set-up was very restrictive before daily treatment delivery. RESULTS: According to RTOG toxicity scale, the acute GU toxicity at 3 months from RT, GU toxicity was G0 for 30 patients (52.6%), G1 for 26 (45.6%) and G2 for one only (1.75%); rectal toxicity was G0 for 56 patients (98.25%) and G1 for one only (1.75%). The median follow-up (FU) was 9 months (2-24 months). In the following FU months, we observed progressively lower urinary and rectal toxicity, except for one patient who showed G2 GU toxicity at 12 months. All but one patient had a progressive PSA value decrease. CONCLUSIONS: In our experience, UHRT appears to be safe and well tolerated even without the use of rectal spacer devices. A longer FU is necessary to evaluate late toxicity and disease control rate.