Molekulska i stanična svojstva imunopatofiziološkog odgovora na klicu i cjepivo Bordetellae pertussis u mišjem modelu [Molecular and cellular properties of immunopathophysiologic response to Bordetella pertussis bacterium and vaccine in the mouse model]

Cellular and molecular response to infection and immunisation in mouse model of B. pertussis infection were studied in the disertation. The results show that B. pertussis microorganisms successfully colonize respiratory epithelium, i.e. the bacteria could be cultured from the lung and tracheal tissue taken from previously infected experimental animals. Five weeks after infection adult immunocompetent mice clear away microorganisms from respiratory tissue. Neonatal mice die due to immunological incompetence that makes them unable to control infection. When elimination of microorganisms from lung tissue ensues, specific antibodies against virulent components of B. pertussis appear in serum and bronchoalveolar lavage fluid. The presence of CD4+ T lymphocytes specific for B. pertussis that proliferate after ex vivo stimulation by antigens of microorganisms fixated in formalin could be detected in regional tracheobronchal lymph nodes of the infected animals. The production of IFN-γ, but not IL-4 nor IL-10, can be demonstrated in the supernatant of thus stimulated T lymphocytes. During the follow up of the infection, specific serum and mucosal antibodies against B. pertussis could be demonstrated only in the period of elimination of the microorganisms from respiratory tissue. Unlike this, the expression of inflammatory cytokines mRNK in lung tissue after infection correlates with the amount of B. pertussis bacteria cultured from the tissues. Immunologically defective adult animals lacking in T and B lymphocytes (SCID mice), as well as those lacking only in T lymphocytes (atymic mice, nude) which die 3-5 weeks after infection, show the importance of cells of specific immunity in the control of B. pertussis infection. The immunodeficient animals show enhanced expression of inflammatory cytokines mRNK at the death time. The expression of IFN-γ mRNK in lung tissue of immunocompetent mice appear a week after the infection. The importance of IFN-γ has been demonstrated by the in vivo neutralization of the cytokines by antibodies and by the demonstration of more microorganisms bred from lung tissue of infected IFN-γ-/-KO animals. Unlike this, IL-4 neutralization does not affect the number of bred microorganisms. Intranasal immunization of adult mice by cellular or acellular B. pertussis vaccine has protective effect in the aerosol model of infection. These two forms of immunization differ in the protective mechanism, which reflects in the lack of correlation with titer of antibodies against B. pertussis when whole microorganisms are applied. T lymphocytes from regional lymph nodes produce IFN-γ if taken from animals immunized with cellular vaccine. Unlike this, T lymphocytes from mice immunized with acellular vaccine produce IL-4 and IL-10. Mice knocked out for B lymphocytes cannot be completely immunized with cellular vaccine. Reconstitution with immune B lymphocytes has protective effect without presence of specific antibodies in the serum of reconstituted animals. Immunization of mice lacking in T lymphocytes or lacking only in CD4+ T lymphocytes is not possible. Unlike this, mice deficient in CD8+ T lymphocytes can be successfully immunized with cellular B. pertussis vaccine. Based on these evidences, immunization with whole B. pertussis microorganism requires presence of CD4+ T lymphocytes and B lymphocytes. The results indicate the presence of two different protective mechanisms of immunization against B. pertussis infection. One of them demonstrates a correlation with the titer of specific B. pertussis antibodies, while the other lacks in that relation. The results presented in the thesis could contribute in comprehension of the mechanisms of B. pertussis infection and protective mechanisms of immunisation against that bacterium, which could be practically important for production of new forms of vaccines against that infective disease

The peritoneal membrane function and aging

U bolesnika liječenih peritonejskom dijalizom, peritonejska membrana djelomično zamjenjuje ekskretornu funkciju bubrega. Pitanje je smanjuje li se funkcija peritonejske membrane sa starenjem slično kao i bubrežna funkcija. Da bi odgovorili na to pitanje autori su analizirali funkciju peritonejske membrane (Kt/V i klirens kreatinina) 27 anuričnih bolesnika, prosječne dobi 58,84±10,65 godina, liječenih s ukupno 8 litara dijalizata dnevno, prosječno 32,56±28,41 mjesec. U skupini kao cjelini nađena je negativna značajna korelacija dobi s tjednim Kt/V (r=-0,627, p<0,05) i negativne neznačajne korelacije s tjednim kliren-som kreatinina peritonejske membrane (r=-0,321, NS) i s nemasnom tjelesnom masom (r=-0,085, NS). Svrstavanjem bolesnika u dvije skupine, prema dobi, nije nađena značajna razlika prosječnih vrijednosti Kt/V (1,95±0,25 : 2,08±0,36; t= 1,04, NS) i tjednog klirensa kreatinina peritonejske membrane (59,85±6,03 : 64,07 ±6,35 1/1,73 m2 tjelesne površine; t=l,73, NS) između bolesnika starijih (n=16) i mlađih (n=ll) od 60 godina. Stariji bolesnici imali su značajno manje izlučivanje kreatinina dijalizatom (81,84±18,33 : 99,82±19,64 jimol/kg tjelesne težine dnevno; t=2,40, p<0,05). Autori su zaključili da u bolesnika liječenih peritonejskom dijalizom nije nađena statistički značajna razlika funkcijskih pokazatelja peritonejske membrane usporedbom mlađih i starijih od 60 godina. Utjecaj dobi na funkciju peritonejske membrane ipak se ne može negirati, jer dobiveni rezultati pokazatelji su funkcije membrane i stanja karakterističnih za stariju dob.In patients undergoing peritoneal dialysis, peritoneal membrane substitutes partially the excretory renal function. The question remains whether the function of peritoneal membrane changes significantly with age. Therefore, the authors analysed the function of peritoneal membrane (Kt/V, creatinine clearance) in 27 anuric patients, mean age 58.84±10.65 years, treated with total of 8 liters dialysate daily, during the period of 32.56±28.41 months on average. For the whole group of examines there was a significant negative correlation found between age and weekly Kt/V (r~0.627, p<0.05), but not between age and creatinine cleance (r=-0.321, NS) or age and lean body mass (r=-0.085, NS). When divided into two groups: those older than 60 (n=16), those up to 60 years of age (n=ll), no significant difference was found regarding the mean values of weekly Kt/V (1.95±0.25 : 2.08±0.36; t=1.04, NS) and weekly creatinine clearance of peritoneal membrane (59.85±6.03 : 64.07±6.35 1/1.73 m2 body surface area; t=1.73, NS). Howewer, older patients had lower creatinine excretion per kg of body weight (81.84 ±18.33 : 99.82 ±19.65 jiimol; t=2.40, p<0.05). In conclusion, no significant difference in peritoneal membrane function was found between patients older and younger than 60 years. The difference in the function of peritoneal membrane established between the two groups of peritoneal dialysis patients could not be explained as the consequence of aging solely, i.e. the influence of accompaning features of an advanced age should not be neglected

The peritoneal membrane function and aging

U bolesnika liječenih peritonejskom dijalizom, peritonejska membrana djelomično zamjenjuje ekskretornu funkciju bubrega. Pitanje je smanjuje li se funkcija peritonejske membrane sa starenjem slično kao i bubrežna funkcija. Da bi odgovorili na to pitanje autori su analizirali funkciju peritonejske membrane (Kt/V i klirens kreatinina) 27 anuričnih bolesnika, prosječne dobi 58,84±10,65 godina, liječenih s ukupno 8 litara dijalizata dnevno, prosječno 32,56±28,41 mjesec. U skupini kao cjelini nađena je negativna značajna korelacija dobi s tjednim Kt/V (r=-0,627, p<0,05) i negativne neznačajne korelacije s tjednim kliren-som kreatinina peritonejske membrane (r=-0,321, NS) i s nemasnom tjelesnom masom (r=-0,085, NS). Svrstavanjem bolesnika u dvije skupine, prema dobi, nije nađena značajna razlika prosječnih vrijednosti Kt/V (1,95±0,25 : 2,08±0,36; t= 1,04, NS) i tjednog klirensa kreatinina peritonejske membrane (59,85±6,03 : 64,07 ±6,35 1/1,73 m2 tjelesne površine; t=l,73, NS) između bolesnika starijih (n=16) i mlađih (n=ll) od 60 godina. Stariji bolesnici imali su značajno manje izlučivanje kreatinina dijalizatom (81,84±18,33 : 99,82±19,64 jimol/kg tjelesne težine dnevno; t=2,40, p<0,05). Autori su zaključili da u bolesnika liječenih peritonejskom dijalizom nije nađena statistički značajna razlika funkcijskih pokazatelja peritonejske membrane usporedbom mlađih i starijih od 60 godina. Utjecaj dobi na funkciju peritonejske membrane ipak se ne može negirati, jer dobiveni rezultati pokazatelji su funkcije membrane i stanja karakterističnih za stariju dob.In patients undergoing peritoneal dialysis, peritoneal membrane substitutes partially the excretory renal function. The question remains whether the function of peritoneal membrane changes significantly with age. Therefore, the authors analysed the function of peritoneal membrane (Kt/V, creatinine clearance) in 27 anuric patients, mean age 58.84±10.65 years, treated with total of 8 liters dialysate daily, during the period of 32.56±28.41 months on average. For the whole group of examines there was a significant negative correlation found between age and weekly Kt/V (r~0.627, p<0.05), but not between age and creatinine cleance (r=-0.321, NS) or age and lean body mass (r=-0.085, NS). When divided into two groups: those older than 60 (n=16), those up to 60 years of age (n=ll), no significant difference was found regarding the mean values of weekly Kt/V (1.95±0.25 : 2.08±0.36; t=1.04, NS) and weekly creatinine clearance of peritoneal membrane (59.85±6.03 : 64.07±6.35 1/1.73 m2 body surface area; t=1.73, NS). Howewer, older patients had lower creatinine excretion per kg of body weight (81.84 ±18.33 : 99.82 ±19.65 jiimol; t=2.40, p<0.05). In conclusion, no significant difference in peritoneal membrane function was found between patients older and younger than 60 years. The difference in the function of peritoneal membrane established between the two groups of peritoneal dialysis patients could not be explained as the consequence of aging solely, i.e. the influence of accompaning features of an advanced age should not be neglected

Integrity of Gut Mucosa during Anaesthesia in Major Abdominal Surgery

The aim of the study was to examine a perfusion and integrity of small bowel in 60 subsequent patients during the major open abdominal surgery which lasted from 2 to 7 hours. Two samples of the intestinal mucosa were removed: at the beginning, and at the end of the surgical procedure in general anaesthesia. A mucosal injury was classified into 4 grades. pH, PCO2 and lactate level were measured in the blood samples from the arterial and mesenteric vein in one hour time intervals. The changes of intestinal mucosa were found in 31 patients (51.7%): in 19 patients (31.7%) grade 1 changes were recorded, in 10 patients (16.7%) grade 2, and in 2 patients (3.3%) grade 3. Grade 4 lesions were not recorded. There was a statistically significant correlation between grades of the mucosal damage and the surgery duration (p=0.001). Analysis during the one hour intervals showed that there was no exact time point when the significant aggravation of the pathohistological changes in intestinal mucosa occurred. However, when patients were allocated into two subgroups with surgical procedures lasting less than 4 hours and more than 4 hours, there was a statistically significant difference in the grades of mucosal damage between subgroups (p<0.05). More biopsies without pathohistological changes were observed in the patients whose procedure duration was < 4 hours. A significantly higher lactate concentrations in arterial and mesenteric venous blood were observed in the patients with pathohistological changes at 6 hours time point as compared to 2 hour time point in the patients without pathohistological changes (p<0.05). During the open abdominal surgery in general anaesthesia, the length of the procedure influences the grade of the intestinal mucosa injury. Deterioration of the pathohistological findings in the intestinal mucosa correlates with high lactate blood level, suggesting that the cause of these changes may result from tissue hypoxia

Ramipril and Risk of Hyperkalemia in Chronic Hemodialysis Patients

Angiotensin converting enzyme (ACE) inhibitors provide well known cardiorenal-protective benefits added to antihypertensive effects in chronic renal disease. These agents are underused in management of patients receiving hemodialysis (HD) because of common concern of hyperkalemia. However, few studies have investigated effect of renin angiotensin aldosterone system (RAAS) blockade on serum potassium in hemodialysis patients. We assessed the safety of ramipril in patients on maintenance HD. We enrolled 28 adult end stage renal disease (ESRD) patients treated by maintenance HD and prescribed them ramipril in doses of 1.25 to 5 mg per day. They underwent serum potassium concentration measurements before ramipril introduction and in 1 to 3 months afterwards. No significant increase in kalemia was found. Results of our study encourage the use of ACE inhibitors in chronically hemodialyzed patients, but close potassium monitoring is mandatory

Association of Cyp2c9 Gene Polymorphism with Bleeding as a Complication of Warfarin Therapy

The aim of this study was to determine the association of bleeding as a complication of warfarin therapy with polymorphism of CYP2C9 gene (alleles 1, 2 and 3). The CYP2C9 is the main enzyme for warfarin metabolism. Study included 181 patients receiving warfarin for at least one month. Allele 1 of CYP2C9 gene (in 94.5%) and genotype *1/*1 (57.5%) prevailed. Allele 3 was found in 12.7% patients. Bleeding side-effects occurred in 18 patients (10%). Patients with allele *1 needed significantly higher maintenance warfarin dose (p=0.011). Those with allele *3 had significantly lower maintenance warfarin dose (p=0.005) and higher prothrombin time (PT) at induction (p=0.034). Bleeding occurred significantly more often in those with lower maintenance warfarin dose (p=0.017). Patients with allele *3 had increased risk of bleeding, with marginal significance (p=0.05). Polymorphism of CYP2C9 could determine dose of warfarin therapy and thus it could be related to the risk of bleeding complications. Allele *3 carriers need lower warfarin dose. Therefore, initially reduced warfarin induction dose in allele *3 carriers could avoid more prolonged PT and decrease the risk of bleeding complication

Povezanost polimorfizma gena za interleukin-10 (-1082g/a) s tipom 2 šećerne bolesti i mikrovaskularnim komplikacijama u hrvatskoj populaciji

Interleukin (IL)-10 is an anti-inflammatory cytokine, and a decrease in its secretion is associated with obesity, metabolic syndrome and type 2 diabetes. However, it has not been established whether the intensity of the immune response during diabetes-associated chronic inflammation affects the devel-opment and/or progression of type 2 diabetes and its microvascular complications. The aim of this study was to investigate the role of single nucleotide polymorphism (SNP)-1082G/A for IL-10 gene in development of diabetes type 2 and its complications. DNA was extracted from blood cells of 240 overweight/obese subjects for IL-10 genotyping. Based on the presence of diabetes type 2, patients were divided in two groups: experimental group of 144 patients with diabetes type 2 and control group of 96 age- and gender-matched subjects without diabetes. Compared to control group, diabetic group had higher levels of leukocytes (p=0.012), fibrinogen (p=0.049) and plasminogen activator inhibitor-1 (PAI-1) (p=0.009), and lower levels of albumin (p=0.001). There were no differences in the frequency of SNP-1082G/A for IL-10 gene between the two groups (p=0.654). When considering diabetes related traits in all subjects in relation to specific genotype, a group with homozygous (AA) genotype had higher values of the mean fasting glucose (p<0.000001), HbA1c (p<0.000001) and HOMA-IR (p=0.003632), while the mean HOMA-B value (p=0.000178) was lower when compared to the groups with GG and GA genotypes. There was no difference in development of diabetic nephropathy, retinopathy and polyneuropathy between the IL-10 polymorphism genotypes. In conclusion, obese diabetes type 2 patients had an increased inflammation activity compared to obese non-diabetic individuals. There was no association of the investigated polymorphisms and development of type 2 diabetes and its microvascular complications. However, diabetes related traits clearly depended on the presence of specific IL-10 genotype.Smanjena sekrecija protuupalnog citokina interleukina (IL)-10 opisana je u debljini, metaboličkom sindromu i tipu 2 šećerne bolesti (T2ŠB). Međutim, nije utvrđeno može li intenzitet imunog odgovora tijekom kronične upale utjecati na razvoj i/ili progresiju dijabetesa kao i razvoj mikrovaskularnih komplikacija. Cilj ove studije bio je istražiti povezanost polimorfizma gena (SNP) -1082G/A za IL-10 s razvojem T2ŠB i kroničnih komplikacija. DNK za genotipizaciju se ekstrahirala iz krvnih stanica 240 adipoznih ispitanika koji su podijeljeni u dvije skupine: u eksperimentalnoj skupini bilo je 144 bolesnika s T2ŠB, a u kontrolnoj skupini 96 ispitanika usklađenih prema spolu i dobi. U skupini s T2ŠB nađene su više vrijednosti leukocita (p=0,012), fibrinogena (p=0,049) i inhibitora aktivacije plazminogena-1 (PAI-1) (p=0,009) te niže koncentracije albumina (p=0,001) nego u kontrolnoj skupini. Nije bilo statističke razlike u frekvencijama -1082G/A polimorfizma gena za IL-10 (p=0,654). Kada su se gledali ishodi za specifični genotip u svih ispitanika, ispitanici s homozigotnim genotipom (AA) imali su značajno više koncentracije glukoze natašte (p<0,000001), HbA1c (p<0,000001) i HOMA-IR (p=0,003632) te niži indeks HOMA-B (p=0,000178) u odnosu na ispitanike s genotipovima GG i GA. Nije bilo razlike u genotipovima polimorfizma IL-10 gena i razvoja nefropatije, retinopatije i polineuropatije. U zaključku, pretili bolesnici s T2ŠB imali su pojačanu upalnu aktivnost u usporedbi s pretilim ispitanicima bez šećerne bolesti. Nije utvrđena povezanost polimorfizma IL-10 i razvoja T2ŠB kao ni razvoja mikrovaskularnih komplikacija. Međutim, ishodi povezani s dijabetesom jasno su povezani s određenim genotipom IL-10

Effects of rHuEPO Treatment on Red Blood Cell Osmotic Resistance

Red blood cell osmotic resistance (RBCOR) is defined as resistance to osmotic changes in cell integrity after their exposure to hypotonic saline solution. The investigation examined the effect of rHuEPO on RBCOR in hemodialysed patients. The study included 58 patients aged 49±14 years, treated by hemodialysis for 59±43 months on average. Half of the patients received rHuEPO for anemia correction. RBCOR was determined in all patients as 3 values: hemolysis start point (HSP), hemolysis end point (HEP) and middle osmotic resistance (MOR). The patients underwent laboratory checkup for parameters characteristically changed in the uremic syndrome. In the control group of healthy subjects (n=16) RBCOR was only determined. No differences were found in the average values of HSP, HEP and MOR between the rHuEPO treated group of patinets and the untreated group. Compared to healthy individuals, the hemodialysed patients displayed significantly higher values of HSP, HEP and MOR. The only one significant correlation of RBCOR and routine laboratory features was found between MOR and predialytic serum concentrations of calcium (r=0.28, p<0.05) and hydrogen ions (r=0.37, p<0.05). Our results suggest that the administration of rHuEPO does not affect RBCOR in hemodialysed patients, that RBCOR is not always reduced in this population and that it correlates with a small number of laboratory parameters characteristic for the uremic syndrome

Red blood cell distribution width as a simple negative prognostic factor in patients with diffuse large B-cell lymphoma: a retrospective study

Aim To determine the prognostic value of baseline red blood cell distribution width (RDW) in diffuse large B cell lymphoma (DLBCL) patients. Methods Data from 81 DLBCL patients diagnosed from 2006 to 2013 at the University Hospital Center Osijek, Osijek, Croatia, were reviewed. We evaluated disease outcome, overall survival (OS) and event-free survival (EFS), and demographic, clinical and laboratory factors affecting outcome. Univariate analysis and Cox regression analysis were used. Results Median age of patients was 64 years, 29 were men (35.8%). Higher RDW levels (%) were found in patients with advanced Ann Arbor clinical stage (14.94 ± 1.82 vs 13.55 ± 1.54, P = 0.001) and in those with poor response to therapy (14.94 ± 1.82 vs 13.55 ± 1.54, P = 0.001). Patients with RDW>15% (cut-off was calculated by receiver operating characteristics) had significantly worse OS (median [range], 33 months [20-46] vs 74 months [65-82], P < 0.001) and EFS (27 months [15-40] vs 68 months [59-77], P < 0.001). Cox regression analysis showed that RDW>15% was an independent prognostic factor for OS (HR 3.654, 95% CI 1.128-11.836) and EFS (HR 2.611, 95% CI 1.012-6-739). Conclusion High baseline RDW is an independent prognostic marker of poor outcome in patients with DLBCL. RDW could be an easily available and inexpensive marker for the risk stratification in patients with DLBCL

Sclerosing peritonitis after kidney transplantation - a case report

Sklerozirajući peritonitis (SP) rijetka je i po život opasna (smrtnost 30-93%) komplikacija peritonejske dijalize (PD). Javlja se u 0,6-3,3 % bolesnika na PD-u. Bubrežno presađivanje (transplantacija, TX) nakon PD-a povisuje rizik nastanka SP-a (prevalencija 7,5%). Etiopatogeneza nije jasna, a epidemiološki pokazatelji ukazuju na rizične čimbenike (PD per se, trajanje PD-a, hiperosmolarne otopine za PD, smanjenje ultrafiltracije-UF, beta blokatori- BB) od kojih neki mijenjaju citokinsku ravnotežu u potrbušnici, povisujući profibrotični transformirajući čimbenik -ß (TGF- ß). Pretpostavlja se da smanjena odteredba (engl. clearance) fibrina nakon prekida PD-a koji uslijedi nakon bubrežne TX uz imunosupresivne (IS) lijekove (od kojih kalcijneurinski inhibitori, CNI, djeluju profibrotično) povisuju rizik za SP. Klinički se javlja slika crijevne opstrukcije, a dijagnoza se potvrđuje kompjutoriziranom tomografijom trbuha (zadebljanje potrbušnice) i patohistološkim pregledom bioptata potrbušnice. Preporučuju se preventivni postupci u rizičnih bolesnika, uključivo specifičan izbor IS lijekova nakon TX. Prikazujemo slučaj bolesnice sa SP-om u prvoj godini nakon bubrežne TX. Rizični čimbenici za SP bili su PD u trajanju od 7 godina, smanjena UF, hiperosmolarne otopine za PD, bubrežna TX, beta blokatori i IS kalcijneurinskim inhibitorima. Liječenje je, uz kirurško, uključivalo isključivanje CNI i BB, uz primjenu IS mTOR inhibitorima i tamoksifen.Sclerosing peritonitis (SP) is rare and life threatening (mortality 30 93%) complication of peritoneal dialysis (PD). It appears in 0.6-3.3% of PD patients. Kidney transplantation (TX) increases the risk for SP (prevalence 7.5%). Ethiopathogenesis is unclear, while epidemiological data indicate risk factors (PD per se, long duration, hyperosmolar solutions for PD, reduced ultrafiltration-UF, beta blockers-BB) that change peritoneal cytokines equilibrium, increasing profibrotic transforming factor-ß (TGF- ß). Reduced fibrin clearance, considered to follow PD cessation, along with immunosuppressive (IS) drugs (calcineurin inhibitors, CNI, among them having profibrotic properties), increases the risk for PD after kidney TX. SP clinically resents by symptoms and signs of intestinal obstruction, and the diagnosis is being confirmed by abdominal computerized tomography, CT, (peritoneal thickening) and pathohistological analysis of peritoneal tissue. Preventive measures in the patients at risk are recommended, including specific IS after kidney TX. We present a case of a female patient with SP in the first year after kidney TX. Risk factors for SP in the patient were PD lasting for 7 years, reduced UF, hyperosmolar PD solutions, kidney TX, BB and CNI IS. The treatment, apart from surgery, included CNI and BB therapy cessation and mTOR inhibitor IS and tamoxifen introduction