Povezanost koncentracije proteina akutne faze i faktora koji utiču na funkciju endotela kod pasa prirodno inficiranih protozoom Leishmania spp.

Abstract

Lajšmanioza je teško hronično oboljenje pasa i ljudi, koja je u Mediteranskom basenu izazvana protozoom Leishamania infantum. Prenosi se putem peščanih mušica iz roda Phlebotomus. Pas je glavni rezervoar ovog patogena. Crna Gora je endemsko žarište lajšmanioze pasa, pre svega opštine Bar i Ulcinj. Naročito su ugroženi psi lutalice i psi u prihvatilištima koji nisu obuhvaćeni nikakvim merama prevencije. U literaturi, lajšmanioza pasa je opisana sa aspekta kliničko-patološkog nalaza i poremećaja strukuture i funkcije tkiva i organa tokom četiri tipične progresivne faze bolesti. Manje su zastupljena savremena istraživanja vezana za različite aspekte specifičnog ćelijskog i humoralnog imunskog odgovora. Takođe, postoji i opravdana potreba da se prošire saznanja o funkcionalnom stanju vaskularnog endotela. Budući da se on nalazi na neposrednom „udaru“ specifičnog humoralnog imuniteta, dalja istraživanja bi doprinela razumevanju mehanizama III tipa preosetljivosti i vaskulitisa, odnosno lokalne i sistemske inflamacije, karakteristične za odmakle faze bolesti. Zbog toga je cilj ove disertacije bio da se ispita veza pojedinih markera oštećenja tkiva i markera oštećenja vaskularnog endotela kod lajšmanioze pasa u različitim fazama bolesti. Materijal i metode: Izvedena je opservaciona studija preseka na 50 pasa sa kliničkim znacima lajšmanioze u različitim fazama bolesti. Studija je sprovedena na višku materijala prikupljenog posle dijagnostike lajšmanioze i tokom njene terapije kod pasa u jednom od prihvatilišta za napuštene pse u opštini Bar, Crna Gora. Lajšmanioza je dijagnostikovana na osnovu kliničke slike i seroloških nalaza, dobijenih komercijalnim enzyme-linked immunosorbent assay (ELISA) paketom reagenasa. Dodatnu procenu zdravstvenog stanja omogućile se rutinske hematološke i biohemijske analize krvi. Posle postavljanja dijagnoze, na osnovu kliničkih znakova i vrednosti nivoa IgG antitela protiv Leishmania spp. psi su podeljeni u tri grupe. Prvu grupu je činilo 14 pasa sa umerenim kliničkim znacima multiorganskog karaktera i visokim nivoom antitela, koji nisu u prethodnom periodu bili na terapiji. U drugoj grupi je bilo 9 pasa sa sličnim znacima bolesti i serološkim rezultatima, ali koji koji su dobijali kombinovanu terapiju miltefosinom, alopurinolom, i vitaminima B kompleksa. Treća grupa obuhvatila je 27 pasa sa blagim znacima bolesti i niskim nivoom antitela – ni kod ovih pasa nije primenjivana terapija. U višku seruma je određena zastupljenost pojedinih proteinskih frakcija elektroforezom na agaroznom gelu; kao i nivoi proteina akutne faze (engl., acute phase proteins – APP): serum amiloida A (SAA) - komercijalnim ELISA paketom reagenasa; ceruloplazmina (CER), haptoglobina (HPT), paraoksonaze-1 (PON-1) - spektrofotometrijski. Iz odnosa koncentracija pozitivnih i negativnih APP izračunati su njihovi indeksi. Ukupne tiolske grupe (TIOL) su takođe određene spektrofotometrijski. Ispitivanja faktora koji utiču na funkciju endotela uključilo je homocistein (Hcy) i matriksne metaloproteinaze (MMP). Koncentracija Hcy određivana je automatizovanim jednostepenim hemiluminiscentnim imunometrijskim testom. Zimografskom metodom analizirane su aktivnosti MMP. Kao važan faktor u formiranju imunskih kompleksa i patogenezi III tipa preosetljivosti određen nivo ukupnih IgA uz Western blot tehnike. Za statističku analizu korišćene su metode deskriptivne statistike, χ2 test, Kruskal-Wallis test sa post hoc analizom po Conover-u, Sperman-ova korelaciona, kao i multipla regresiona analiza. Rezultati: Obe grupe pasa sa umerenom formom bolesti su imale viši nivo IgG protiv Leishmania spp. (P<0,001), nižu hematokritsku vrednost (P<0,042), niži broj leukocita (P<0,021), višu koncentraciju uree (P<0,001), kreatinina (P<0,001), ukupnih proteina (P<0,001), višu aktivnost alanin aminotransferaze (ALT) (P<0,001) i nižu koncentraciju albumina (P<0,001) u odnosu na pse sa blagom formom bolesti. Trigliceridi su bili najviši kod pasa sa umerenom formom bolesti i terapijom (P<0,002) dok je aspartat aminotransferaza (AST) bila viša kod iste grupe pasa, ali samo u odnosu na pse sa blagom formom bolesti (P<0,001). Razdvajanjem proteinskih frakcija pokazano je da obe grupe pasa sa umerenom formom imaju poliklonsku gamapatiju (P=0,001). Koncentracija SAA se nije razlikovala između grupa, dok su koncentracije CER (P=0,010) i HPT (P=0,008) bile veće kod obe grupe pasa sa umerenom formom bolesti. Aktivnost PON-1 je bila niža kod pasa sa umerenom formom bez terapije u odnosu na pse sa blagom formom (P=0,048). Na osnovu pet indeksa APP u čijem izračunavanju su kao pozitivni APP u različitim kombinacijama korišćeni SAA, CER i HPT, a kao negativan korišćen albumin, potvrđeno je da su promene najmanje izražene u blagoj formi bolesti. Aktivnost proMMP-2 je bila niža kod pasa sa umerenom formom bez terapije u odnosu na one sa blagom formom bolesti (P=0,038), aktivnost proMMP-9 se nije razlikovala između grupa, ali je aktivnost MMP-9 bila niža u obe grupe sa umerenom formom u odnosu na blagu formu (P=0,001). Koncentracija Hcy je bila najniža (P=0,021), a odnos PON-1/Hcy najviši kod pasa koji su terapirani (P=0,023). Koncentracija TIOL-a kao i odnos TIOL/Hcy se nije razlikovao između grupa. Nivo ukupnih IgA je bio viši kod pasa sa umerenom formom i terapijom u odnosu na pse sa blagom formom bolesti (P=0,014). Takođe, aktivnost svih ispitanih MMP je negativno korelirala sa formom bolesti i aktivnošću AST, dok se kao nezavisni prediktor aktivnosti proMMP-2 i proMMP-9 pokazala alkalna fosfataza, a za MMP-9 je to bila ALT. Koncentracija Hcy je bila u negativnoj korelaciji sa formom bolesti kao i sa terapijom i sa svim biohemijskim parametrima koji pokazuju oštećenje ili poremećaj funkcije tkiva i organa. Jedini pozitivni prediktori koncentracije Hcy bili su broja eritrocita, odnosno koncentracija hemoglobina. Nivo IgA je bio u pozitivnoj korelaciji sa formom bolesti i sa većinom pokazatelja oštećenja tkiva dok je bio u negativnoj korelaciji sa koncentracijom albumina, MMP-9 i Hcy. Zaključak: Uopšteno posmatrano, izvedena ispitivanja pokazuju da je kod pasa sa umerenom formom bolesti, bez obzira na tronedeljnu terapiju, postojalo teže oštećenje tkiva i jači humoralni odgovor na infekciju sa Leishmania spp. povezan sa većim stepenom sistemske inflamacije i težim stepenom disfunkcije endotela u odnosu na pse sa blagom formom bolesti. Takođe, rezultati studije ukazuju na opravdanost daljeg proučavanja aktivnosti proMMP-2 i koncentracije Hcy, kao potencijalnih pokazatelja (dis)funkcije endotela, u smislu praćenja pozitivnih ishoda terapije.

Leishmaniosis is a severe chronic disease of dogs and humans which is in the Mediterranean region caused by the protozoan Leishmania infantum. It is transmitted by sandflies belonging to the genus Phlebotomus. Dogs are the main reservoir of this pathogen. Montenegro is an endemic focus of canine leishmaniosis, particularly in the municipalities of Bar and Ulcinj. Stray dogs and dogs in animal shelters, for which there are no preventive measures, are particularly at risk. Canine leishmaniosis has been described in terms of clinicopathological findings and disturbances in the structure and function of tissues and organs during four typical progressive stages of the disease. Current research on various aspects of specific cellular and humoral immune responses is less well-represented. In addition, there is a need to increase the knowledge regarding the functional state of the vascular endothelium. Because it is directly "hit" by specific humoral immunity, further research would contribute to the understanding of the mechanisms of type III hypersensitivity and vasculitis, that is, local and systemic inflammation characteristic of the advanced stages of the disease. Therefore, the aim of this dissertation was to investigate the relationship between specific markers of tissue damage and vascular endothelial damage in canine leishmaniosis at different stages of the disease. Material and methods: An observational cross-sectional study was performed on 50 dogs with clinical signs of leishmaniosis in different stages of the disease. The study was conducted on excess material collected after the diagnosis of leishmaniosis and during its therapy in dogs in one of the shelters for abandoned dogs in the municipality of Bar, Montenegro. Leishmaniosis was diagnosed based on the clinical picture and serological findings, obtained with a commercial enzyme-linked immunosorbent assay (ELISA) reagent package. Routine hematological and biochemical blood analyzes enabled an additional assessment of the state of health. After diagnosis, on the basis of clinical signs and values of IgG antibodies against Leishmania spp. the dogs are divided into three groups. The first group consisted of 14 dogs with moderate clinical signs of a multiorgan character and a high level of antibodies, which had not received therapy in the previous period. In the second group, there were 9 dogs with similar disease signs and serological results, but which received combined therapy with miltefosine, allopurinol, and B vitamins. The third group included 27 dogs with mild signs of the disease and a low level of antibodies - no therapy was applied to these dogs either. In the excess serum, the representation of certain protein fractions was determined by electrophoresis on agarose gel; as well as levels of acute phase proteins (APP): serum amyloid A (SAA) - with a commercial ELISA package of reagents; ceruloplasmin (CER), haptoglobin (HPT), paraoxonase-1 (PON-1) - spectrophotometrically. Their indices were calculated from the ratio of positive and negative APP concentrations. Total thiol groups (TIOL) were also determined spectrophotometrically. Examination of factors affecting endothelial function included homocysteine (Hcy) and matrix metalloproteinases (MMPs). The concentration of Hcy was determined by an automated one-step chemiluminescent immunometric test. MMP activities were analyzed using the zymographic method. As an important factor in the formation of immune complexes and the pathogenesis of type III hypersensitivity, the level of total IgA was determined using Western blot techniques. Descriptive statistics, χ2 test, Kruskal-Wallis test with post hoc analysis according to Conover, Spearman's correlation and multiple regression analysis were used for statistical analysis. Results: Both groups with moderate leishmaniosis had higher levels of IgG against Leishmania spp. (P<0.001), lower hematocrit (P<0.042) and leukocyte count (P<0.021), higher concentrations of urea (P<0.001), creatinine (P<0.001), total protein (P<0.001), alanine aminotransferase (ALT) (P<0.001), and lower albumin levels (P<0.001) than dogs affected with the mild form of the disease. Triglycerides were the highest in the group consisting of treated dogs (P<0.002), while the same group of dogs had higher aspartate aminotransferase activity (AST), but only in comparison with the group of dogs with the mild form of leishmaniosis (P<0.001). Serum protein electrophoresis revealed the presence of polyclonal gammopathy in dogs with moderate leishmaniosis, regardless of whether they received therapy (P=0.001). The concentration of SAA did not differ between the groups, while both groups with the moderate form had higher CER (P=0.010) and HPT (P=0.008) levels than the dogs with the mild form. The PON-1 activity measured in untreated dogs with moderate leishmaniosis was lower than that recorded in dogs with mild disease (P=0.048). The five APP indexes, calculated by combining SAA, CER, and HPT, as the positive and albumin, as the negative APP, additionally confirmed that the changes were the least protruding in the mild form. Lower proMMP-2 levels were measured in untreated dogs with the moderate disease than in those with mild leishmaniosis (P=0.038). The groups had similar proMMP-9 activity; nevertheless, MMP-9 activity was lower in both groups with moderate disease than in the group with mild leishmaniosis (P=0.001). The Hcy concentration was the lowest (P=0.021), and the PON-1/Hcy ratio was the highest among the treated dogs (P=0.023). Serum TIOL levels and the TIOL/Hcy ratio did not differ between the groups. Total IgA was higher in the treated dogs with the moderate disease than in the dogs with the mild form (P=0.014). Furthermore, the activity of all tested MMPs was negatively correlated with the disease severity and AST activity; alkaline phosphatase appeared as an independent predictor of proMMP-2 and proMMP-9 activities, while in the case of MMP9, it was ALT. Hcy was in a negative correlation with the stage of the disease, treatment, and the biochemistry parameters indicating the damage or dysfunction of tissues and organs. The only positive predictors of Hcy concentration were erythrocyte number and hemoglobin concentration. IgA levels were positively correlated with the disease stage and the majority of the laboratory indicators of tissue damage, while a negative correlation existed with albumin, MMP-9, and Hcy levels. In summary, the tests performed show that dogs with a moderate form of the disease had more severe tissue damage and a stronger humoral response to Leishmania spp. infection associated with a higher degree of systemic inflammation and a more severe degree of endothelial dysfunction compared with dogs with a mild form of the disease, regardless of the three-week therapy. In addition, the results of this study indicate that further investigation of proMMP-2 activity and Hcy levels are warranted as potential indicators of endothelial (dys)function in terms of monitoring positive therapeutic outcomes.