Valutazione clinica, clinicopatologica e quantificazione di IgG e IgM in corso di leishmaniosi canina: studio retrospettivo

La leishmaniosi canina (LCan) causata da Leishmania infantum rappresenta un’importante zoonosi in molte aree del mondo ed il cane rappresenta il principale reservoir del parassita per l’uomo. Il tipo di risposta immunitaria che i soggetti colpiti mettono in atto condiziona fortemente la progressione della malattia: animali che non sviluppano un’adeguata risposta immunitaria cellulo-mediata mostrano la sintomatologia clinica nonostante abbiano una forte ma inefficace risposta umorale che contribuisce al peggioramento della sintomatologia clinica. L’obbiettivo dello studio è stato quello valutare da un punto di vista descrittivo il segnalamento, i segni clinici e clinicopatologici dei pazienti affetti da leishmaniosi portati in visita presso il Dipartimento di Scienze Mediche Veterinarie nel periodo compreso da Gennaio 2002 a Marzo 2012 con particolare attenzione sull’impatto della patologia renale e dell’anemia nel quadro clinico della LCan. In base ai risultati ottenuti è stato possibile affermare che la leishmaniosi canina è una patologia relativamente frequente nella nostra realtà clinica universitaria e che presenta caratteristiche cliniche e clinicopatologiche simili a quelle riportate in letteratura. I nostri risultati preliminari suggeriscono che in questa malattia il coinvolgimento renale e le conseguenze sistemiche che ne derivano possono essere predominanti a livello clinico e laboratoristico. La gravità del quadro clinico appare associata in maniera significativa all’entità della risposta umorale e del successivo coinvolgimento glomerulare nel contesto di una risposta infiammatoria sistemica cronica. Successivamente, sono state misurate le concentrazioni di IgG ed IgM in corso di follow-up in alcuni dei soggetti inclusi nello studio e sottoposti a differenti trattamenti anti-leishmania. Dai risultati preliminari ottenuti nel nostro lavoro è stato possibile affermare che in corso di trattamento le concentrazioni di tali immunoglobuline subiscono una riduzione progressiva confermando pertanto l’efficacia del trattamento anti-leishmania non solo nella remissione della sintomatologia clinica ma anche nel ripristino della normale risposta umorale.

Caratterizzazione clinica, eziopatogenetica e ricerca di marker diagnostici nella poliradicoloneurite acuta idiopatica del cane

La poliradicoloneurite acuta idiopatica (ACIP) è una patologia infiammatoria che interessa le radici di più nervi spinali, descritta soprattutto nel cane, più raramente nel gatto, caratterizzata da insorgenza acuta di paresi/paralisi flaccida. L’ACIP mostra notevoli similitudini con la sindrome di Guillan-Barrè dell’uomo (GBS), in cui la patogenesi è su base autoimmunitaria ed è stata correlata con la presenza di alcuni fattori scatenanti (trigger). Lo scopo di questo lavoro è stato quello di caratterizzare l’ACIP in 26 cani, descrivendone la sintomatologia, l’evoluzione clinica, i risultati degli esami diagnostici. La diagnosi si è basata sui riscontri dell’anamnesi, della visita neurologica e del decorso confermata, quando possibile, dai rilievi elettrodiagnostici. Su tutti i cani è stata valutata l’esposizione a specifici agenti infettivi (Toxoplasma gondii, Neospora canunim, Ehrlichia canis, Leishmania infantum), o altri fattori (come vaccinazioni) che potrebbero aver agito da “trigger” per l’instaurarsi della patologia; sull’intera popolazione e su 19 cani non neurologici (gruppo di controllo), si è proceduto alla ricerca degli anticorpi anti-gangliosidi. La sintomatologia di più frequente riscontro (25/26) ha coinvolto la funzione motoria (paresi/plegia) con prevalente interessamento dei 4 arti (24/25) . Sei cani hanno ricevuto una terapia farmacologica, che non ne ha influenzato il decorso, favorevole in 24/26 casi. In 9 pazienti è stata rilevata una precedente esposizione a potenziali trigger; in 10 casi si è riscontrato un titolo anticorpale positivo ad almeno un agente infettivo testato. In 17/26 cani si è ottenuto un titolo anticorpale anti-GM2 e anti-GA1; nella popolazione di controllo solo un caso è risultato positivo. Questi risultati hanno contribuito a consolidare le conoscenze di questa patologia, validando l’utilità della ricerca anticorpale anti-gangliosidica per la diagnosi di ACIP e facendo intravedere la possibilità che l’ACIP possa essere assimilate alla GBS anche dal punto di vista patogenetico, per la quale potrebbe essere considerata come modello animale spontaneo.

La diagnostica molecolare nel laboratorio di patologia clinica veterinaria

La prima parte del nostro studio riguarda la tecnica LAMP (Loop-mediated isothermal amplification), una tecnica di amplificazione isotermica recentemente inventata (Notomi et al., 2000). Essa presenta notevoli vantaggi rispetto alle tradizionali PCR: non necessita di strumentazioni sofisticate come i termociclatori, può essere eseguita da personale non specializzato, è una tecnica altamente sensibile e specifica ed è molto tollerante agli inibitori. Tutte queste caratteristiche fanno sì che essa possa essere utilizzata al di fuori dei laboratori diagnostici, come POCT (Point of care testing), con il vantaggio di non dover gestire la spedizione del campione e di avere in tempi molto brevi risultati paragonabili a quelli ottenuti con la tradizionale PCR. Sono state prese in considerazione malattie infettive sostenute da batteri che richiedono tempi molto lunghi per la coltivazione o che non sono addirittura coltivabili. Sono stati disegnati dei saggi per la diagnosi di patologie virali che necessitano di diagnosi tempestiva. Altri test messi a punto riguardano malattie genetiche del cane e due batteri d’interesse agro-alimentare. Tutte le prove sono state condotte con tecnica real-time per diminuire il rischio di cross-contaminazione pur riuscendo a comprendere in maniera approfondita l’andamento delle reazioni. Infine è stato messo a punto un metodo di visualizzazione colorimetrico utilizzabile con tutti i saggi messi a punto, che svincola completamente la reazione LAMP dall’esecuzione in un laboratorio specializzato. Il secondo capitolo riguarda lo studio dal punto di vista molecolare di un soggetto che presenza totale assenza di attività mieloperossidasica all’analisi di citochimica automatica (ADVIA® 2120 Hematology System). Lo studio è stato condotto attraverso amplificazione e confronto dei prodotti di PCR ottenuti sul soggetto patologico e su due soggetti con fenotipo wild-type. Si è poi provveduto al sequenziamento dei prodotti di PCR su sequenziatore automatico al fine di ricercare la mutazione responsabile della carenza di MPO nel soggetto indicato.

Valutazione dell'attività delle metalloproteinasi 2 e 9 nelle urine e nel tessuto renale di cani normali e affetti da nefropatia

Matrix metalloproteinases (MMP) are a large family of proteinases that remodel extracellular matrix (ECM) component. Recent data suggest a role for MMPs in a number of renal pathophysiologies, associated with an imbalance of ECM syntesis and degradation, which may result in an accumulation of ECM molecules and renal fibrosis. The aim of this study is to elucidate the role of pro and activated MMP-2 and 9 in urine and renal tissue of healty and nephropatic dogs. Renal tissue of 8 healty dogs and either renal tissue and urine of 9 nephropatic dogs was collected and analize using zimographic method, which is been validated in this study. Either MMPs zimographic bands were present in almost all samples. In particular, pro and activated MMP-9 zimographic bands were poorly represent in renal tissue of healty dogs, whereas were very represent in nephropatic dogs. Pro and activated MMP-2 was present in either tissue of healty and nephropatic dogs. In urine of nephropatic dogs, pro and activated MMP-9 was more evident than MMP-2, but there was not correlaction with renal tissue levels, therefore urine levels of MMPs have poorly usefulness in diagnostic pratice. The values of Pro and activated MMP-9 in nephropatic dogs were significantly higher compared with normal dogs (p < 0,05), whereas there was not statistically meaningful for Pro and activated MMP-2. In conclusion, in this study we have validated a zimographic method for renal tissue of dogs and we have illustrated the changes in nephropatic dogs, which may be useful for further study.

Importanza prognostica delle citochine e loro ruolo nell'immunomodulazione: risultati di studi sperimentali in vivo e in vitro

Citokines are proteins produced by several cell types and secreted in response to various stimuli. These molecules are able to modify the behaviour of other cells inducing activities like growth, differentiation and apoptosis. In the last years, veterinary scientists have investigated the role played by these factors; in fact, cytokines can act as intercellular communicative signals in immune response, cell damage repair and hematopoiesis. Up to date, various cytokines have been identified and in depth comprehension of their effects in physiology, pathology and therapy is an interesting field of research. This thesis aims to understand the role played by these mediators during natural or experimentally induced pathologies. In particular, it has been evaluated the genic and protein expressions of a large number of cytokines during several diseases and starting from different matrix. Considering the heterogeneity of materials used in experimentations, multiple methods and protocols of nucleic acids and proteins extractions have been standardized. Results on cytokines expression obtained from various in vitro and in vivo experimental studies have shown how important these mediators are in regulation and modulation of the host immune response also in veterinary medicine. In particular, the analysis of inflammatory and septic markers, like cytokines, has allowed a better understanding in the pathogenesis during horse Recurrent Airway Obstruction, foal sepsis, Bovine Viral Diarrhea Virus infection and dog Parvovirus infection and the effects of these agents on the host immune system. As experimentations with mice have shown, some pathologies of the respiratory and nervous system can be reduced or even erased by blocking cytokines inflammatory production. The in vitro cytokines expression evaluation in cells which are in vivo involved in the response to exogenous (like pathogens) or endogenous (as it happens during autoimmune diseases) inflammatory stimuli could represent a model for studying citokines effects during the host immune response. This has been analyzed using lymphocytes cultured with several St. aureus strains isolated from bovine mastitic milk and different colostrum products. In the first experiment different cytokines were expressed depending on enterotoxins produced, justifying a different behaviour of the microrganism in the mammal gland. In the second one, bone marrow cells derived incubated with murine lymphocytes with colostrum products have shown various cluster of differentiation expression , different proliferation and a modified cytokines profile. A better understanding of cytokine expression mechanisms will increase the know-how on immune response activated by several pathogen agents. In particular, blocking the cytokine production, the inhibition or catalyzation of the receptor binding mechanism and the modulation of signal transduction mechanism will represent a novel therapeutic strategy in veterinary medicine.

Valutazione del rapporto urinario proteine totali/creatinina e albumina/creatinina in cani affetti da iperadrenocorticismo e da diabete mellito

INTRODUCTION – In human medicine, diabetes mellitus (DM), hypertension, proteinuria and nephropathy are often associated although it is still not clear whether hypertension is the consequence or the cause of nephropathy and albuminuria. Microalbuminuria, in humans, is an early and sensitive marker which permits timely and effective therapy in the early phase of renal damage. Conversely, in dogs, these relationships were not fully investigated, even though hypertension has been associated with many diseases (Bodey and Michell, 1996). In a previous study, 20% of diabetic dogs were found proteinuric based on a U:P/C > 1 and 46% were hypertensive; this latter finding is similar to the prevalence of hypertension in diabetic people (40-80%) (Struble et al., 1998). In the same canine study, hypertension was also positively correlated with the duration of the disease, as is the case in human beings. Hypertension was also found to be a common complication of hypercortisolism (HC) in dogs, with a prevalence which varies from 50 (Goy-Thollot et al., 2002) to 80% (Danese and Aron, 1994).The aim of our study was to evaluate the urinary albumin to creatinine ratio (U:A/C) in dogs affected by Diabetes Mellitus and HC in order to ascertain if, as in human beings, it could represent an early and more sensitive marker of renal damage than U:P/C. Furthermore, the relationship between proteinuria and hypertension in DM and HC was also investigated. MATERIALS AND METHODS – Twenty dogs with DM, 14 with HC and 21 healthy dogs (control group) were included in the prospective case-control study. Inclusion criteria were hyperglycaemia, glicosuria and serum fructosamine above the reference range for DM dogs and a positive ACTH stimulation test and/or low-dose dexamethasone test and consistent findings of HC on abdominal ultrasonography in HC dogs. Dogs were excluded if affected by urinary tract infections and if the serum creatinine or urea values were above the reference range. At the moment of inclusion, an appropriate therapy had already been instituted less than 1 month earlier in 12 diabetic dogs. The control dogs were considered healthy based on clinical exam and clinicopathological findings. All dogs underwent urine sample collection by cystocentesis and systemic blood pressure measurement by means of either an oscillometric device (BP-88 Next, Colin Corporation, Japan) or by Doppler ultrasonic traducer (Minidop ES-100VX, Hadeco, Japan). The choice of method depended on the dog’s body weight: Doppler ultrasonography was employed in dogs < 20 kg of body weight and the oscillometric method in the other subjects. Dogs were considered hypertensive whenever systemic blood pressure was found ≥ 160 mmHg. The urine was assayed for U:P/C and U:A/C (Gentilini et al., 2005). The data between groups were compared using the Mann-Whitney U test. The reference ranges for U:P/C and U:A/C had already been established by our laboratory as 0.6 and 0.05, respectively. U:P/C and U:A/C findings were correlated to systemic blood pressure and Spearman R correlation coefficients were calculated. In all cases, p < 0.05 was considered statistically significant. RESULTS – The mean ± sd urinary albumin concentration in the three groups was 1.79 mg/dl ± 2.18; 20.02 mg/dl ± 43.25; 52.02 mg/dl ± 98.27, in healthy, diabetic and hypercortisolemic dogs, respectively. The urine albumin concentration differed significantly between healthy and diabetic dogs (p = 0.008) and between healthy and HC dogs (p = 0.011). U:A/C values ranged from 0.00 to 0.34 (mean ± sd 0.02 ± 0.07), 0.00 to 6.72 (mean ± sd 0.62 ± 1.52) and 0.00 to 5.52 (mean ± sd 1.27 ± 1.70) in the control, DM and HC groups, respectively; U:P/C values ranged from 0.1 to 0.6 (mean ± sd 0.17 ± 0.15) 0.1 to 6.6 (mean ± sd 0.93 ± 1.15) and 0.2 to 7.1 (mean ± sd 1.90 ± 2.11) in the control, DM and HC groups, respectively. In diabetic dogs, U:A/C was above the reference range in 11 out of 20 dogs (55%). Among these, 5/20 (25%) showed an increase only in the U:A/C ratio while, in 6/20 (30%), both the U:P/C and the U:A/C were abnormal. Among the latter, 4 dogs had already undergone therapy. In subjects affected with HC, U:P/C and U:A/C were both increased in 10/14 (71%) while in 2/14 (14%) only U:A/C was above the reference range. Overall, by comparing U:P/C and U:A/C in the various groups, a significant increase in protein excretion in disease-affected animals compared to healthy dogs was found. Blood pressure (BP) in diabetic subjects ranged from 88 to 203 mmHg (mean ± sd 143 ± 33 mmHg) and 7/20 (35%) dogs were found to be hypertensive. In HC dogs, BP ranged from 116 to 200 mmHg (mean ± sd 167 ± 26 mmHg) and 9/14 (64%) dogs were hypertensive. Blood pressure and proteinuria were not significantly correlated. Furthermore, in the DM group, U:P/C and U:A/C were both increased in 3 hypertensive dogs and 2 normotensive dogs while the only increase of U:A/C was observed in 2 hypertensive and 3 normotensive dogs. In the HC group, the U:P/C and the U:A/C were both increased in 6 hypertensive and 2 normotensive dogs; the U:A/C was the sole increased parameter in 1 hypertensive dog and in 1 dog with normal pressure. DISCUSSION AND CONCLUSION- The findings of this study suggest that, in dogs affected by DM and HC, an increase in U:P/C, U:A/C and systemic hypertension is frequently present. Remarkably, some dogs affected by both DM and HC showed an U:A/C but not U:P/C above the reference range. In diabetic dogs, albuminuria was observed in 25% of the subjects, suggesting the possibility that this parameter could be employed for detecting renal damage at an early phase when common semiquantiative tests and even U:P/C fall inside the reference range. In HC dogs, a higher number of subjects with overt proteinuria was found while only 14% presented an increase only in the U:A/C. This fact, associated with a greater number of hypertensive dogs having HC rather than DM, could suggest a greater influence on renal function by the mechanisms involved in hypertension secondary to hypercortisolemia. Furthermore, it is possible that, in HC dogs, the diagnosis was more delayed than in DM dogs. However, the lack of a statistically significant correlation between hypertension and increased protein excretion as well as the apparently random distribution of proteinuric subjects in normotensive and hypertensive cases, imply that other factors besides hypertension are involved in causing proteinuria. Longitudinal studies are needed to further investigate the relationship between hypertension and proteinuria.