Comparison of Vascular Endothelial Growth Factor Concentration in Porcine Kidneys Removed From Living Donors After Cardiac and Brain Death.

The aim of this paper was to describe the differences in vascular endothelial growth factor (VEGF) concentration in porcine kidneys removed from living donors (group I), donors after prior induction of brain death by brain herniation (group II), and donors after cardiopulmonary arrest (group III). The groups consisted of 6 animals which underwent dual renal removal procedures; kidneys were rinsed, stored for 24 hours at 4°C and rinsed again. Renal specimens (4g) were collected before and after perfusion (time 0 and 1), after 12 hours (time 2), and after reperfusion (time 3). A Western blot was used to evaluate VEGF concentration in collected tissues homogenates. Additionally, the levels of VEGF, interleukin 1ß, tumor necrosis factor α, and endothelial nitric oxide synthase (eNOS) were detected with enzyme-linked immunosorbent assays. Directly after the removal procedure, no significant differences in VEGF levels (IOD) were observed depending on the donor (moderate levels were observed in all groups: 1.51 in group I, 1.48 in group II, and 1.35 in group III). As a consequence of perfusion and 12 hours of storage, a stable concentration in groups I and III was observed with a gradual increase of VEGF levels in group II (1.23, 2.08, and 1.67 in the respective groups at time 1; 1.49, 2.12, and 1.63 in the respective groups at time 2). After the following 12 hours, a statistically significant (P < .05) higher level of VEGF was observed in group II (2.34) in comparison to groups I and III (1.58 and 1.81, respectively). In group I, a correlation between VEGF concentration and IL-1ß was observed, while in group II there was correlation between VEGF and eNOS levels.

Caspase-3 Expression and ALT, AST, and GGT Activity After 24 Hours of Porcine Liver Cold Storage, Depending on the Type of Transgenesis.

BACKGROUND: Because of an insufficient number of human organs for transplantation, xenotransplantation may become an effective alternative. We aimed to analyze if the type of transgenesis has an influence on the hepatic caspase-3 expression, the enzyme that executes apoptosis as well as ALT, AST, and GGT activity after 24 hours of cold storage. METHODS: The experiment was carried out on the 24 livers of Polish White Landrace pigs carrying human α1,2-fucosyltransferase and/or α-galactosidase (GAL) genes and livers without this genetic modification (control). Livers were perfused, stored for 24 hours in solution, and subsequently re-flushed. Hepatic concentration of the caspase-3 protein and its mRNA expression were measured just after the animal was killed as well as after 30 minutes of perfusion and after 24 hours of cold storage followed by 30 minutes of reperfusion. Caspase-3 mRNA level was detected with the RT-PCR method. Protein concentration (capsase-3 active and inactive) was assessed with the Western blotting technique. Kinetic methods were applied for the analysis of the ALT, AST, and GGT activity. RESULTS: The highest increase of the ALT activity after cold storage was observed in the group with GAL transgenesis, whereas the GGT activity was highest in the unmodified livers. There was no difference in the caspase-3 expression and AST activity after cold storage as compared with the respective initial results (P = .57 and P = .97, respectively). CONCLUSIONS: It appears that transgenesis does not aggravate ischemic injury of the liver.

Endothelial Nitric Oxide Synthase and Endothelin-1 Expression in the Early Post-Porcine Kidney Autotransplantation Period.

BACKGROUND: The aim of this study was the assessment of endothelial nitric oxide synthase (eNOS) and endothelin-1 (EDN-1) expression in porcine kidneys on the 14th and 30th days after the autotransplantation procedure. METHODS: The research was conducted on 12 animals that underwent a left renal transplantation procedure with further standardized rinsing and 24-hour storage in 4°C; subsequently, the kidneys were implanted in the right retroperitoneal space after right-sided nephrectomy. Removed kidneys were examined (group 0). Six randomly chosen animals (group 1) were under observation for 14 days and 6 others (group 2) for 30 days. RESULTS: After these observation periods, euthanasia was performed on the animals and 4-g samples were collected from the renal cortex and medulla. The Western blot technique was used to detect the eNOS and EDN-1 expression at the protein level. The obtained results are presented as absolute values of integrated optical density. Stable graft function was observed in all animals from the 2nd day after the procedure. eNOS in group 1 reached the mean value of 1.064 and was statistically significantly lower than in group 2 (2.085) or in the control group 0 (3.318). In the case of EDN-1 expression on 14th day after transplantation, the medium level was reported (0.248), which was similar to group 0 (0.216), whereas group 2 presented values 2 times higher (0.743). CONCLUSIONS: A lowered eNOS level in the organ was observed on the 14th day after autotransplantation of a pig kidney; further enzyme normalization is associated with increased EDN-1 expression.

Comparison of endothelial nitric oxide synthase and endothelin-1 levels in kidneys removed from living pigs after cardiac arrest and brain death.

OBJECTIVES: The aim of this paper was to describe differences between levels of endothelial nitric oxide synthase (NOS-3) and endothelin-1 (ET-1) in swine kidneys removed from living donors (group I) and after inducing brain death by brain herniation (group II) and cardiac arrest (group III). METHODS: Each group consisted of 3 animals who underwent dual renal removal procedure; kidneys were further rinsed according to standardized procedure with Biolasol perfusion liquid, stored for 24 hours (4°C), and rinsed again. Renal specimens of 4 g mass, including renal cortex and medulla, were collected before and after perfusion (times 0 and 1), after 12 hours (time 2), and after reperfusion (time 3). Enzyme-linked immunosorbent assay was used to describe levels of NOS-3 and ET-1 in collected tissues homogenates. Mann-Whitney U test was used to compare results in groups in relation to total protein content (ng/mg), and the correlation between the 2 substances was measured with the use of Spearman rho. RESULTS: Group I presented low and stable levels of NOS-3 in all time intervals (averages, 0.73, 0.99, 0.52, and 0.89, respectively). Level sof ET-1 were similar (0.87, 0.63, 0.69, and 0.86, respectively), and significant correlation between levels of the 2 substances was observed. Increased levels of NOS-3 (1.89 and 1.86) and ET-1 (1.38 and 1.49) were observed directly after removal in groups II and III and further maintained during organ storage. No correlation in group I was observed, and after perfusion significantly lower level of NOS-3 was observed in kidneys removed after brain death in relation to group III (1.77 vs 2.60). CONCLUSIONS: The lowest and stable levels of NOS-3 and ET1 during storage were observed in kidneys removed from living donors. Levels of analyzed substances in this group showed correlation in subsequent time intervals.

Cytochrome P450 3 A expression in pigs livers after 24-hour preservation in Biolasol solution depending on the type of transgenesis.

BACKGROUND: An insufficient number of organs for transplantation shows the need for the development of new technologies. Xenotransplantation might be the answer. OBJECTIVE: To determine if the type of transgenesis influences the level of CYP3A4, which takes an active part in xenobiotics metabolism in livers after 24-hour storage, depending on the kind of solution used for preservation. MATERIALS AND METHODS: The experiment was carried out on 30 livers of Polish White Landrace divided into 5 groups depending on transgene type. The following human genes were transferred: α1,2-fucosyltransferase (groups I and II), α-galactosidase (III), combined α1,2-fucosyltransferase/α-galactosidase transgene (IV), and livers without modification (V). The livers were perfused and subsequently stored for 24 hours in Ringer's solution (group I) or Biolasol solution (II-V). Reperfusion/reflush was performed. CYP3A29 isomer concentration was analyzed in liver specimens collected twice: 30 minutes after perfusion and 30 minutes after reperfusion/reflush. Expression of mRNA CYP3A29 was marked using RT-PCR analysis and of protein CYP3A29 using Western blotting technique. RESULTS: The most significant decrease in protein CYP3A29 expression after 24-hour preservation was observed in group I (55.88% decrease), while the least significant was observed in group IV (10.44% decrease). mRNA expression evaluation was similar: the most significant decrease was observed in group I (87.8% decrease) and the least significant in group III (4.6% decrease). CONCLUSION: α1,2-Fcosyltransferase transgene seems to influence mRNA and protein CYP3A expression in case of liver grafting and preservation for transplantation. CYP3A expression was also influenced by the kind of preservation solution used.

What else distinguishes cellular rejection grade 1A from 0? Annexin V and BCL in elective biopsies received from heart transplant recipients.

BACKGROUND: Despite morphologic differences of lymphocytes aggregation between nonrejection (0 on the International Society for Heart and Lung Transplantation [ISHLT] scale) and moderate focal cellular rejection (1a, ISHLT), genetic and clinical differences have not been shown in Cardiac Allograft Rejection Gene Observation (CARGO) studies. Therefore, we sought to compare the expression of selected antigens associated with apoptosis in heart transplant recipients in the context of grade 0 versus grade 1a cellular rejection episodes. We assessed the expression of annexin V, a nonspecific apoptosis marker, Bcl-2 as opposed to antiapoptotic activity of Bcl-xL and Bcl-xL/S. MATERIALS AND METHODS: We retrospectively examined 17 heart transplant patients (2 women and 15 men) of overall mean age of 46.2 +/- 13.9 years and body mass index of 25.7 +/- 3.2. Ten biopsies showed rejection grade 0 and the other 10, grade 1a on the ISHLT scale, comprising groups A and B, respectively. Endomyocardial biopsy specimens were processed using routine immunohistochemical methods. The expression of apoptotic molecules was assessed according to the IHC method: 0, the lack of expression; 1, trace; 2, distinct; and 3, strong. A correlation was analyzed between particular molecular expressions. RESULTS: We observed a significant increase in Bcl-2 expression associated with rejection. The expression of other antigens did not show a significant tendency. No correlation was noted among group A, whereas in group B those were significant strong and negative correlations with Bcl-2 and Bcl-xL/S. CONCLUSION: Bcl-2 expression corresponded to the morphologic progression of graft rejection as opposed to Bcl-xL/S activity.

Mixed cellular and humoral acute rejection in elective biopsies from heart transplant recipients.

OBJECTIVE: Acute cellular rejection in heart transplants is characterized by an active lymphocytic infiltration, whereas the humoral response shows complement deposits in myocardial tissue. Both reactions may produce hemodynamic compromise during the first months after orthotopic heart transplantation (OHT). The aim of this study was to estimate the coexistence of humoral rejection symptoms in the first posttransplant biopsy with mild/moderate cellular rejection as an additional prognostic factor. MATERIALS AND METHODS: The study group included 13 biopsies obtained from 11 men and 2 women of overall mean age of 52.6 +/- 5.3 years who displayed International Society for Heart and Lung Transplantation (ISHLT) mild/moderate rejection grades. The control group consisted of 11 biopsies obtained from 8 men and 3 women of overall mean age of 54.8 +/- 3.6 years with no signs of rejection. Complement deposits were determined immunohistochemically using anti-C4d antibodies (Quidel Corporation). RESULTS: None of the control cases showed a positive reaction, whereas 3 men in the study group of mean age of 56.1 +/- 5.8 years revealed regional positive anti-C4d expression with cellular infiltrates. This expression occurred in all myocardial components adjacent to lymphocytic infiltrations. The survival rates were comparable in both the pure cellular versus the mixed rejection groups. The relative rate of grade 3 rejection in the posttransplantation period was increased among patients with mixed types of rejection. CONCLUSIONS: The term "mixed acute rejection" should be applied to cases with coincidence of 2 forms of acute rejection. It seemed to be associated with more frequent grade 3 rejection processes upon long-term follow-up.

Frequency of Pseudomonas aeruginosa colonizations/infections in lung transplant recipients.

BACKGROUND: Lower respiratory tract infections remain a leading cause of morbidity and mortality after solid organ transplantation. The particularly increased susceptibility to infection is an especial problem in the early posttransplant period at the initial stage of immunosuppression, owing to direct contact with the hospital environment by mechanical ventilation, biopsies, injections, bronchoscopy, and bladder and vessel catheterizations exacerbated by the impaired clearance mechanisms after denervation of the transplanted lung. Airway colonization with Pseudomonas aeruginosa is common in lung transplant (LT) recipients. Therefore, we performed a retrospective analysis to address the frequency of P aeruginosa infections in our Center. MATERIALS AND METHODS: From January 2004 to December 2008, we performed 33 LT, including 4 heart-lung, 6 double, and 23 single lung transplantations. Respiratory samples were the main diagnostic material undergoing routine microbiological methods. RESULTS: P aeruginosa was isolated from 13 patients (39.4% of all 33 LT). In 10 cases (30.3%), we observed airway colonization together with lower respiratory tract infections. From 2005 to 2008, P aeruginosa was diagnosed in about 50% of LT patients each year: in 2005, 33.3%; 2006, 57.1%; 2007, 42.9%; and 2008, 40%. CONCLUSION: LT recipients in our center are at high risk for pseudomonal airway colonisation and lower respiratory tract infection that may have a significant impact on posttransplant follow-up.

Procalcitonin serum concentration during Pneumocystis jiroveci colonization or Pseudomonas aeruginosa infection/colonization in lung transplant recipients.

BACKGROUND: Allograft infection after lung transplantation (OLT) has a significant impact on outcomes and represents a diagnostic challenge. Pneumocystis jirovecii causes an opportunistic infection, life-threatening pneumonia among immunocompromised patients. Airway colonization with Pseudomonas aeruginosa is common in lung transplant recipients. The aim of the study was to evaluate procalcitonin (PCT) serum concentrations during P jiroveci and P aeruginosa colonization/infections in lung transplant recipients. MATERIALS AND METHODS: Fifteen OLT patients were retrospectively enrolled into the study (10 men and 5 women) of overall mean age of 41.4 +/- 14.6 years. In seven patients, P jiroveci cysts were diagnosed (group J) and in 13 patients, we isolated P aeruginosa (group A). In respiratory samples, P jiroveci was detected using an indirect immunofluorescence method, and P aeruginosa was isolated using routine microbiologic methods. PCT was measured using immunoluminescence assay. RESULTS: The average PCT value in group A was 0.30 +/- 0.21 and in group J, 0.88 +/- 0.43, a difference that was not significant. In group A, 3 patients (23.1%) has PCT values indicating moderate infection risk (PCT > 0.5) and one patient (7.7%), a high infection risk (PCT > 2.0 and <10). In group J, three patients (42.9%) has PCT values indicating moderate and one patient (14.3%), high infection risk. CONCLUSIONS: Bronchial tree colonization with P jiroveci as well as P aeruginosa colonization can be associated with increased PCT suggesting a general, systemic response in addition to local colonization.

Urinary iodine concentrations should be monitored to diagnose some thyroid gland diseases in heart transplant recipients.

BACKGROUND: According to the World Health Organization, iodine excreted in urine is a measure of its supply. According to the International Council for Control of Iodine Deficiency Disorders (ICCIDD), the urinary iodine (UI) should be >100 microg I per 1 liter of urine. Severe deficiency (SID) is diagnosed when UI is <50 microg/L and a moderate deficiency (MID) when UI is <100 microg/L. MATERIALS AND METHODS: UI analysis among 32 heart transplant recipients (26 men and 6 women); of overall mean age of 50.4 +/- 12.6 years was performed using the modified Program Against Micronutrient Malnutrition method, a spectrophotometric measurement based on the Sandell-Kolthoff reaction. Results were compared with those of thyroid stimulating hormone (TSH; microIU/mL), of free tri-iodothyronine (FT3; pg/mL), and thyroxine (FT4; ng/dL). RESULTS: The average UI among the whole group was 126.4 +/- 109.6 microg/L. SID occurred in 12 patients (37.5%) and MID in 4 (12.5%); namely, mean UI of 17.0 +/- 9.6 and 79.5 +/- 5.6, respectively. In the other 16 patients (50%), the average UI was high, namely, 220.1 +/- 72.1 IU/mL. TSH, FT3, and FT4 in the whole group were within normal ranges. However, FT4 values significantly differed when SID and MID patients were compared with those displaying the recommended UI: 0.8 +/- 0.2 and 0.9 +/- 0.1 versus 1.1 +/- 0.2 respectively (P < .05). We noted decreased values of TSH in 5 patients (15.6%) and of FT3 or FT4 in 6 subjects (18.8%). CONCLUSION: There exists significant iodine deficiency among heart transplant recipients. Measurements of urinary iodine together with thyroid gland hormones may be essential to prevent thyroid gland disturbances in these patients.

Use of sildenafil in heart transplant recipients with pulmonary hypertension may prevent right heart failure.

PURPOSE: We performed a short-term outcome analysis of orthotopic heart transplantation (OHT) in patients with pulmonary hypertension (PH) treated perioperatively with oral sildenafil. METHODS: PH (pulmonary vascular resistance > 2.5 Wood units, and/or transpulmonary gradient > 12 mmHg) was diagnosed in 6 of 25 (group A) heart transplant recipients operated in 2006. This group of patients underwent a modified medication protocol including perioperative administration of oral sildenafil: 50 mg before followed by 50 or 25 mg TID after heart transplantation. Sildenafil treatment was discontinued 10 to 14 days post OHT, after stepwise dose reduction. During the ICU stay all patients underwent circulatory monitoring of pulmonary and systemic pressures and resistance as well as transthoracic echocardiogram (TTE) evaluation. RESULTS: Perioperative oral sildenafil administration in PH patients undergoing OHT was associated with good short-term outcomes in the majority of transplanted patients (4/6). Sildenafil treatment reduced pulmonary resistance and pressures with a low rate of hemodynamic instability among OHT patients. CONCLUSIONS: Pharmacologic perioperative reduction of PH improves the short-term prognosis for successful OHT. One may speculate whether sildenafil treatment transplant recipients with PH would be associated with long-term improvement of pulmonary vascular status, therefore leading to extended life-expectancy and improved outcomes.

Microbiological status of commercially available medicinal herbal drugs--a screening study.

One hundred and thirty-eight medicinal herbal drugs obtained from different suppliers were examined for microbial contaminants and for the detectability of pathogenic microorganisms. For this purpose, several microbiological standard parameters (total aerobic mesophilic count, enterobacteria, coliforms, aerobic sporeformers, yeasts and moulds, enterococci, lactobacilli, pseudomonades and aeromades) and selective methods for the detection of indicator microorganisms and pathogens (E. coli, enterohaemorrhagic E. coli [EHEC], Salmonella, Campylobacter jejuni, Pseudomonas aeruginosa, Bacillus cereus, Clostridium perfringens, Listeria, coagulase-positive staphylococci, Candida albicans, potentially aflatoxigenic moulds) were applied. The microbial load of the samples varied considerably. While none of the samples contained EHEC, Salmonellae, Pseudomonas aeruginosa, Listeriae, Staphylococcus aureus or Candida albicans, four samples were E. coli positive, two samples were presumptively Campylobacter jejuni positive and nine herbal drugs contained a potentially aflatoxigenic mould flora. Further details regarding different viable count classes as well as preparation techniques are discussed.

[Alpha 1-antitrypsin polymorphism]. / Polimorfizm alfa1-antytrypsyny.

Discovery, nomenclature and some genetical and molecular aspect of alpha 1-antitrypsin polymorphism are discussed. Particular attention has been paid to the deficiency variants and their role in human pathology.