Potential Celiac Patients: A Model of Celiac Disease Pathogenesis

BACKGROUND AND AIM: Potential celiacs have the 'celiac type' HLA, positive anti-transglutaminase antibodies but no damage at small intestinal mucosa. Only a minority of them develops mucosal lesion. More than 40 genes were associated to Celiac Disease (CD) but we still do not know how those pathways transform a genetically predisposed individual into an affected person. The aim of the study is to explore the genetic features of Potential CD individuals. METHODS: 127 'potential' CD patients entered the study because of positive anti-tissue transglutaminase and no mucosal lesions; about 30% of those followed for four years become frankly celiac. They were genotyped for 13 polymorphisms of 'candidate genes' and compared to controls and celiacs. Moreover, 60 biopsy specimens were used for expression studies. RESULTS: Potential CD bear a lighter HLA-related risk, compared to celiac (??(2)???=???48.42; p value???=???1×10(-8)). They share most of the polymorphisms of the celiacs, but the frequency of c-REL* G allele was suggestive for a difference compared to celiac (??(2)???=???5.42; p value???=???0.02). One marker of the KIAA1109/IL-2/IL-21 candidate region differentiated potentials from celiac (rs4374642: ??2???=???7.17, p value???=???0.01). The expression of IL-21 was completely suppressed in potentials compared to celiacs (p value???=???0.02) and to controls (p value???=???0.02), in contrast IL-2, KIAA1109 and c-REL expression were over-expressed. CONCLUSIONS: Potential CD show genetic features slightly different from celiacs. Genetic and expression markers help to differentiate this condition. Potential CD is a precious biological model of the pathways leading to the small intestinal mucosal damage in genetically predisposed individuals

Inducible Slc7a7 Knockout Mouse Model Recapitulates Lysinuric Protein Intolerance Disease

Lysinuric protein intolerance (LPI) is a rare autosomal disease caused by defective cationic amino acid (CAA) transport due to mutations in SLC7A7, which encodes for the y+LAT1 transporter. LPI patients suffer from a wide variety of symptoms, which range from failure to thrive, hyperammonemia, and nephropathy to pulmonar alveolar proteinosis (PAP), a potentially life-threatening complication. Hyperammonemia is currently prevented by citrulline supplementation. However, the full impact of this treatment is not completely understood. In contrast, there is no defined therapy for the multiple reported complications of LPI, including PAP, for which bronchoalveolar lavages do not prevent progression of the disease. The lack of a viable LPI model prompted us to generate a tamoxifen-inducible Slc7a7 knockout mouse (Slc7a7-/-). The Slc7a7-/- model resembles the human LPI phenotype, including malabsorption and impaired reabsorption of CAA, hypoargininemia and hyperammonemia. Interestingly, the Slc7a7-/- mice also develops PAP and neurological impairment. We observed that citrulline treatment improves the metabolic derangement and survival. On the basis of our findings, the Slc7a7-/- model emerges as a promising tool to further study the complexity of LPI, including its immune-like complications, and to design evidence-based therapies to halt its progression

Lysinuric proetin intolerance: molecular and cellular bases of an inherited multi-system disorder

[ITALIANO] Introduzione: L’ intolleranza alle proteine con lisinuria, ereditata come carattere autosomico recessivo, è causata da un difettivo trasporto di aminoacidi cationici (CAA) alla membrana basolaterale delle cellule epiteliali di intestino e rene. I pazienti sono usualmente asintomatici durante il periodo dell'allattamento; dopo lo svezzamento, allorché il contenuto dell’ apporto proteico aumenta, si manifestano i segni clinici della malattia: vomito, diarrea, epatosplenomegalia, osteoporosi, episodi di coma iperammoniemico, ritardo mentale, coinvolgimento polmonare (principalmente come proteinosi alveolare), anomalie ematologiche, alterata risposta immunologica, malattia cronica renale. L' alterazione metabolica nella LPI è caratterizzata da: ridotto assorbimento intestinale di CAA, aumentata escrezione renale di CAA e una disfunzione del ciclo dell'urea che porta ad iperammonemia ed oroticoaciduria. La LPI è causata da mutazioni del gene SLC7A7, che codifica per la proteina y+LAT1, appartenente alla famiglia dei trasportatori eterodimerici. Questo trasportatore di aminoacidi è composto da una catena pesante, 4F2hc, codificata dal gene SLC3A2, e da una catena leggera, y+LAT1, unite mediante un ponte disolfuro. La co-espressione di 4F2hc e di y+LAT1 induce il trasporto di CAA di tipo y+L. Specifici obiettivi: Il principale obiettivo della ricerca è stato la conoscenza delle basi cellulari e molecolari della fisiopatologia della LPI. Le strategie di ricerca utilizzate sono state: 1. Analisi mutazionale di pazienti affetti da LPI; 2. Analisi funzionale di mutanti del gene SLC7A7; 3. Creazione di modelli animali e cellulari di LPI. Risultati: Analisi mutazionale di pazienti affetti da LPI. Lo studio ha evidenziato la presenza di un elevato numero di mutazioni, distribuite lungo tutta la sequenza codificante del gene SLC7A7. Analisi funzionale di mutanti del gene SLC7A7. Gli studi funzionali hanno apportano nuove informazioni sulla patogenesi della LPI: a) un nuovo modello del trasportatore di CAA con struttura multieterodimerica e b) una possibile interferenza tra la proteina y+LAT1 e la proteina y+LAT2 (gene SLC7A6), proteina altamente omologa a y+LAT1 ed appartenente alla stessa famiglia dei trasportatori eterodimerici. Quest’ interferenza potrebbe spiegare perché sia possibile un meccanismo compensatorio, noto nei linfoblasti di pazienti LPI, ove è stata dimostrata una over-espressione di SLC7A6. Creazione di modelli animali di LPI. E’ stato generato il primo modello animale della LPI come topo knock-out (KO) costitutivo per il gene Slc7a7 mediante il sistema d’inserzione casuale gene-trap in cellule ES. Lo studio del fenotipo di tale animale non ha fornito i contributi auspicati alla comprensione dei meccanismi patogenetici della LPI; infatti, per la letalità dell’ animale, si sta procedendo alla creazione di un modello murino di tipo condizionale mediante la delezione del gene Slc7a7 in specifici tessuti e con temporale controllo dello spegnimento del gene. Creazione di un modello cellulare di LPI. Il primo modello cellulare di LPI è stato ottenuto mediante isolamento di cellule di tubulo renale dalle urine dei pazienti affetti da LPI. Tale modello cellulare è stato utilizzato per valutare la relazione tra l’interessamento renale riscontrato in molti pazienti affetti da LPI ed il metabolismo dell’ossido nitrico (NO). I nostri dati preliminari confermano una possibile relazione tra la produzione di NO, l’apoptosi indotta ed il coinvolgimento renale. Conclusioni: Il lavoro descritto in questa tesi ha portato alle seguenti conclusioni: 1. nuova organizzazione multieteromerica del trasportatore di aminoacidi cationici; 2. generazione di un modello knock-out (KO) costitutivo del gene Slc7a7, il cui fenotipo è caratterizzato da un grave ritardo di crescita che non si accompagna ad alterazioni strutturali durante lo sviluppo embrionale. 3. lo studio delle cellule di tubulo renale ha indicato una chiara relazione tra il coinvolgimento renale nell’uomo e l’apoptosi indotta da elevati livelli di NO sintetizzato in tali cellule. 4. questi risultati suggeriscono che i protocolli convenzionali dovrebbero essere rivalutati in quanto l’eccesso di citrullina, che giornaliermente è somminstrata ai pazienti, potrebbe indurre un danno cellulare maggiore dovuto alla neo-sintesi di arginina. / [ENGLISH] Background: Lysinuric protein intolerance (LPI; MIM 222700) is caused by a defective transport of cationic amino acids (CAA for Cationic Amino Acids) to the basolateral membrane of the epithelial cells of intestine and kidney. Clinical manifestations of LPI include:vomiting, diarrhea, stunted growth, visceromegaly, osteoporosis, episodes of coma, mental delay, severe pulmonary (pulmonary alveolar proteinosis, PAP) and renal involvements. The metabolic derangement of this disease includes: reduced intestinal absorption of CAA, increased CAA renal excretion and impairment of the urea cycle that brings to hyperammonemia and increased orotic aciduria. The molecular basis of LPI is a defective transport of CAA normally exerted by system y+L. The y+L activity is induced by a heterodimer composed of: • the heavy chain 4F2hc, encoded by the SLC3A2 gene; • a light chain that can be either y+LAT1 (encoded by the SLC7A7 gene) or y+LAT2 (encoded by the SLC7A6 gene). We found that the SLC7A7 gene is mutated in LPI patients and we also characterized most of the mutations found so far in Italian and non-Italian patients. Specific objectives: The overall aim of the present thesis is to contribute to the elucidation of the pathophysiology of LPI, using three different approaches: 1. Mutational analysis of LPI patients; 2. Functional studies of SLC7A7 mutants; 3. Creation of animal and cellular models of LPI. Results: Our functional results revealed a new scenario in the knowledge on LPI disorder. In fact, our results provide further insight into the molecular pathogenesis of LPI: a putative multiheteromeric structure of both [4F2hc/y+LAT1] and [4F2hc/y+LAT2], and the interference between y+LAT1 and y+LAT2 proteins. This interference can explain why the compensator mechanism (via an increased expression of SLC7A6 seen in lymphoblasts and renal tubular cells from LPI patients) may not be sufficient to restore the y+L system activity. The first animal model of LPI, a constitutive knock-out of Slc7a7, has been generated to study the pathophysiology of LPI and explore new therapeutic protocols. At the moment, we have succeeded in generating this model but it turned out to be lethal in the perinatal period. This lethality limited our studies. To circumvent the problem, we are generating a conditional Slc7a7 gene mouse model, using the Cre/loxP recombination system. With this mouse model, single aspects of the disease can be investigated without the risk of a full-blown clinical phenotype, which caused the lethality in the constitutive knock-out mouse. The first cellular model of LPI was obtained by isolation of renal tubular cells from urine of LPI patients. Using this cellular model we studied the relation between renal compliance and the nitric oxide metabolism. Our preliminary results confirm a possible relation between production of nitric oxide and renal involvement known in LPI. Conclusions: The work described in this thesis has led to the following results: 1. putative new model of y+L transporter with a putative multiheteromeric structure; 2. ablation of Slc7a7 causes severe prenatal growth retardation with no gross developmental abnormalities but with unbalanced NO metabolism; 3. unbalanced NO metabolism might be the key to understand the pathophysiology of many complications of LPI in man; 4. renal involvement in human LPI has a clear relationship with apoptosis induced by high levels of NO synthesis in renal tubular cells; 5. conventional therapeutic protocols of LPI should be revised immediately in order to avoid excessive citrulline intake and possible iatrogenic complications arising from disease

The Role of Transthoracic Ultrasound in the Study of Interstitial Lung Diseases: High-Resolution Computed Tomography Versus Ultrasound Patterns: Our Preliminary Experience

Transthoracic ultrasound (TUS) is a readily available imaging tool that can provide a quick real-time evaluation. The aim of this preliminary study was to establish a complementary role for this imaging method in the approach of interstitial lung diseases (ILDs). TUS examination was performed in 43 consecutive patients with pulmonary fibrosis and TUS findings were compared with the corresponding high-resolution computed tomography (HRCT) scans. All patients showed a thickened hyperechoic pleural line, despite no difference between dominant HRCT patterns (ground glass, honeycombing, mixed pattern) being recorded (p > 0.05). However, pleural lines’ thickening showed a significant difference between different HRCT degree of fibrosis (p 3 B-lines and subpleural nodules was also assessed in a large number of patients, although they did not demonstrate any particular association with a specific HRCT finding or fibrotic degree. Results allow us to suggest a complementary role for TUS in facilitating an early diagnosis of ILD or helping to detect a possible disease progression or eventual complications during routine clinical practice (with pleural line measurements and subpleural nodules), although HRCT remains the gold standard in the definition of ILD pattern, disease extent and follow-up

Improving the estimation of celiac disease sibling risk by non-HLA genes.

Celiac Disease (CD) is a polygenic trait, and HLA genes explain less than half of the genetic variation. Through large GWAs more than 40 associated non-HLA genes were identified, but they give a small contribution to the heritability of the disease. The aim of this study is to improve the estimate of the CD risk in siblings, by adding to HLA a small set of non-HLA genes. One-hundred fifty-seven Italian families with a confirmed CD case and at least one other sib and both parents were recruited. Among 249 sibs, 29 developed CD in a 6 year follow-up period. All individuals were typed for HLA and 10 SNPs in non-HLA genes: CCR1/CCR3 (rs6441961), IL12A/SCHIP1 and IL12A (rs17810546 and rs9811792), TAGAP (rs1738074), RGS1 (rs2816316), LPP (rs1464510), OLIG3 (rs2327832), REL (rs842647), IL2/IL21 (rs6822844), SH2B3 (rs3184504). Three associated SNPs (in LPP, REL, and RGS1 genes) were identified through the Transmission Disequilibrium Test and a Bayesian approach was used to assign a score (BS) to each detected HLA+SNPs genotype combination. We then classified CD sibs as at low or at high risk if their BS was respectively < or ≥ median BS value within each HLA risk group. A larger number (72%) of CD sibs showed a BS ≥ the median value and had a more than two fold higher OR than CD sibs with a BS value < the median (O.R = 2.53, p = 0.047). Our HLA+SNPs genotype classification, showed both a higher predictive negative value (95% vs 91%) and diagnostic sensitivity (79% vs 45%) than the HLA only. In conclusion, the estimate of the CD risk by HLA+SNPs approach, even if not applicable to prevention, could be a precious tool to improve the prediction of the disease in a cohort of first degree relatives, particularly in the low HLA risk groups

Could transthoracic ultrasound be useful to suggest a small airways disease in severe uncontrolled asthma?

Transthoracic ultrasound (TUS) is an accepted complementary tool in the diagnostic process of several pleuro-pulmonary diseases. However, to the best of our knowledge, TUS findings in patients with severe asthma have never been systematically described