Agnieszka Kolasa-Wołosiuk

Effects of perinatal exposure to lead (Pb) on purine receptor expression in the brain and gliosis in rats tolerant to morphine analgesia.

The aim of the present study was to investigate the molecular effects of perinatal exposure to lead (Pb) on protein and mRNA expression of purine receptors: P2X4, P2X7, adenosine receptor A1; and astrocytes (GFAP mRNA expression) and on microglia activation (Iba1 mRNA expression) in several structures of the mesolimbic system (striatum, hippocampus, prefrontal cortex) in rats expressing tolerance to the antinociceptive effect of morphine. Rat mothers were orally treated with 0.1% lead acetate from conception, through gestation, and postnatally, as well as to offspring up to day (PND) 28; subsequently molecular studies were conducted on adult (PND 60) male rats. Morphine tolerance developed more strongly in rats perinatally exposed to Pb. The analysis revealed a significant up-regulation of protein and mRNA P2X4 receptor expression in the striatum and prefrontal cortex but not in the hippocampus; P2X7 protein and mRNA receptor expression in the striatum and hippocampus, but not in the prefrontal cortex; A1 protein receptor expression in all investigated structures and A1 mRNA expression in the striatum and hippocampus; Iba1 mRNA expression in the striatum and hippocampus; and GFAP mRNA expression in the striatum and prefrontal cortex. Immunohistochemical analysis has also revealed significant alterations. Strong expressions of P2X4, P2X7, A1 receptors, astrocytes and microglia activation were observed in the hippocampus in Pb and/or morphine treated rats. The higher expression of purine receptors and glial cell activation are important markers of neuroinflammatory processes. Therefore, we conclude that Pb-induced neuroinflammation may be responsible for the intensification of morphine tolerance in the Pb-treated rats. Additionally, the dysregulation of A1 adenosine receptors, mainly in the hippocampus, may also be involved in the intensification of morphine tolerance in Pb-treated rats. Our study demonstrates the significant participation of environmental factors in addictive process; additionally, it shows the necessity of modification of addictive disorder with neuroprotective agents.

Acanthamoeba infection in lungs of mice expressed by toll-like receptors (TLR2 and TLR4).

Toll-like receptors (TLRs) play a key role in the innate immune responses to a variety of pathogens including parasites. TLRs are among the most highly conserved in the evolution of the receptor family, localized mainly on cells of the immune system and on other cells such as lung cells. The aim of this study was to determine for the first time the expression of TLR2 and TLR4 in the lung of Acanthamoeba spp. infected mice using quantitative real-time polymerase chain reaction (Q-PCR) and immunohistochemical (IHC) staining. The Acanthamoeba spp. were isolated from a patient with Acanthamoeba keratitis (AK) (strain Ac 55) and from environmental samples of water from Malta Lake (Poznań, Poland - strain Ac 43). We observed a significantly increased level of expression of TLR2 as well as TLR4 mRNA from 2 to 30 days post Acanthamoeba infection (dpi) in the lungs of mice infected with Ac55 (KP120880) and Ac43 (KP120879) strains. According to our observations, increased TLR2 and TLR4 expression in the pneumocytes, interstitial cells and epithelial cells of the bronchial tree may suggest an important role of these receptors in protective immunity against Acanthamoeba infection in the lung. Moreover, increased levels of TLR2 and TLR4 mRNA expression in infected Acanthamoeba mice may suggest the involvement of these TLRs in the recognition of this amoeba pathogen-associated molecular pattern (PAMP).

Expression and Activity of COX-1 and COX-2 in Acanthamoeba sp.-Infected Lungs According to the Host Immunological Status

Little is known about the pathomechanism of pulmonary infections caused by Acanthamoeba sp. Therefore, the aim of this study was to determine whether Acanthamoeba sp. may affect the expression and activity of cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2), resulting in the altered levels of their main products, prostaglandins (PGE2) and thromboxane B2 (TXB2), in lungs of immunocompetent or immunosuppressed hosts. Acanthamoeba sp. induced a strong expression of COX-1 and COX-2 proteins in the lungs of immunocompetent mice, which, however, did not result in significant differences in the expression of PGE2 and TXB2. Our immunohistochemical analysis showed that immunosuppression induced by glucocorticoids in Acanthamoeba sp.-infected mice caused a decrease in COX-1 and COX-2 (not at the beginning of infection) in lung tissue. These results suggest that similar to COX-2, COX-1 is an important mediator of the pathophysiology in experimental pulmonary acanthamoebiasis. We suggest that the signaling pathways important for Acanthamoeba sp. induction of lung infection might interact with each other and depend on the host immune status.

Changes in the Immune System of Female Wistar Rats After Exposure to Immunosuppressive Treatment During Pregnancy

This experimental study assessed the impact of medications frequently used after kidney transplantation on the immune system of pregnant female Wistar rats. The study evaluates medications, both approved and contraindicated during pregnancy in common therapeutic combinations. The study was conducted on 32 female Wistar rats, subjected to immunosuppressive regimens most commonly used in therapy of human kidney transplant recipients (cyclosporine A, mycophenolate mofetil and prednisone; tacrolimus, mycophenolate mofetil and prednisone; and cyclosporine A, everolimus and prednisone). The animals received drugs by oral gavage 2 weeks before pregnancy and at 3 weeks of pregnancy. We found drug regimen‐dependent differences in cytometry from spleen. Many subpopulations of lymphocytes were suppressed in rats treated with cyclosporine A, mycophenolate mofetil and prednisone and tacrolimus, mycophenolate mofetil and prednisone; the number of NK cells was increased in group of rats treated with cyclosporine A, everolimus and prednisone. We also found changes in histological examination of thymus and spleen of all treated dams. In cytokine assay, we noticed increasing levels of IL‐17 with increasing doses of concanavalin A in control group and in group of dams treated with cyclosporine A, mycophenolate mofetil and prednisone. This increase was blocked in rats treated with tacrolimus, mycophenolate mofetil and prednisone and cyclosporine A, everolimus and prednisone. Qualitative, quantitative and morphological changes of immune system in pharmacologically immunosuppressed females have been observed. Thymus structure, spleen composition and splenocytes IL‐17 production were mostly affected in drug regimen‐dependent manner.

The influence of intrauterine exposure to immunosuppressive treatment on changes in the immune system in juvenile Wistar rats

Background In our study, we assessed the impact of immunosuppressive drug combinations on changes in the immune system of juvenile Wistar rats exposed to these drugs during pregnancy. We primarily concentrated on changes in two organs of the immune system – the thymus and the spleen. Methods The study was conducted on 40 (32+8) female Wistar rats administered full and half dose of drugs, respectively, subjected to regimens commonly used in therapy of human kidney transplant recipients ([1] cyclosporine A, mycophenolate mofetil, and prednisone; [2] tacrolimus, mycophenolate mofetil, and prednisone; [3] cyclosporine A, everolimus, and prednisone). The animals received drugs by oral gavage 2 weeks before pregnancy and during 3 weeks of pregnancy. Results There were no statistically significant differences in the weight of the thymus and spleen, but changes were found in the results of blood hematology, cytometry from the spleen, and a histologic examination of the examined immune organs of juvenile Wistar rats. In the cytokine assay, changes in the level of interleukine 17 (IL-17) after increasing amounts of concanavaline A were dose-dependent; the increase of IL-17 was blocked after administration of higher doses of immunosuppressive drugs. However, after a reduction of doses, its increase resumed. Conclusion Qualitative, quantitative, and morphological changes in the immune system of infant rats born to pharmacologically immunosuppressed females were observed. Thymus structure, spleen composition, and splenocyte IL-17 production were mostly affected in a drug regimen–dependent manner.

Androgen levels and apoptosis in the testis during postnatal development of finasteride-treated male rat offspring.

INTRODUCTION The hormone-dependent events that occur throughout the first wave of spermatogenesis, such as the establishment of the number of Sertoli cells (SCs) and spermatogonial stem cells (SSCs) within the seminiferous cords and the setting up of the blood-testis barrier, are important for adult male fertility. Any changes in the T/DHT ratio can result in male subfertility or even infertility. In this study we aimed to evaluate effects of paternal exposure to 5-alpha reductase type 2 inhibitor, finasteride on litter size, androgen levels and germ cell apoptosis in male offspring during postnatal development. MATERIAL AND METHODS The subjects of the study were 7, 14, 21/22, 28, and 90-day-old Wistar male rats (F1:Fin) born from females fertilized by finasteride-treated rats. Offspring born from untreated parental animals were used as a control group (F1:Control). Animals and the collected testes were weighed, blood and intratesticular levels of T and DHT were measured by ELISA, and the apoptotic index of testicular cells was evaluated by TUNEL technique. RESULTS We observed difficulties in obtaining male newborns from female rats fertilized by finasteride-treated male rats. In the F1:Fin rats, changes in the body and testes weights occurred, and a lower number of apoptotic cells was found during postnatal maturation of the seminiferous epithelium. Changes in androgen concentrations during the first spermatogenesis wave and adult life were also evident. CONCLUSION Finasteride treatment of male adult rats may not only cause a decrease in the fertility of parental rats, but also could lead to incorrect, androgen-sensitive course of spermatogenesis in their offspring.

Effect of long-term immunosuppressive therapy on native rat liver morphology and hepatocyte- apoptosis

A negative result of therapy based on immunosuppressive drugs is its leading to pathological alterations in the organ, including liver. Use of immunosuppressive medication may also lead to organized and genetically controlled cell death - apoptosis. The aim of this study was to examine histopathological changes in the livers of rats treated with immunosuppressive drugs, and also to determine the effects of different groups of immunosuppressive drugs on apoptosis activity in the hepatocytes of rat livers. The study was conducted on archival material obtained from Department of Nephrology, Transplantology and Internal Medicine of the Independent Public Clinical Hospital No. 2 at the Pomeranian Medical University in Szczecin, Poland. Statistical comparison of the treatment groups showed that all groups with rapamycin (sirolimus)-based regimens: Tacrolimus, Rapamycin, Glucocorticosteroid (TRG); Cyclosporine, Rapamycin, Glucocorticosteroid (CRG); Mycophenolate, Rapamycin, Glucocorticosteroid (MRG) and additionally Cyclosporine, Mycophenolate, Glucocorticosteroid (CMG) exhibited significantly more pronounced apoptosis than the control group, with p < 0.01, p < 0.05, p < 0.01 and p < 0.01, respectively. Furthermore, in the TRG group, over 90% of apoptotic hepatocytes were seen in the examined classic lobules. Additionally, every liver from treatment group was pathologically altered, including dilated sinusoids, pyknotic nuclei, swollen walls of the vessels. Long-lasting immunosuppressive treatment affects the liver both in terms of histological changes within the structure of the liver and in terms of the percentage of apoptotic hepatocytes. The following study seems to be very innovating due to the duration of the experiment and used drugs-protocols, since they reflect human treatment.

Relationship between aortic wall oxidative stress/proteolytic enzyme expression and intraluminal thrombus thickness indicates a novel pathomechanism in the progression of human abdominal aortic aneurysm

The possibility that oxidative stress promotes degradation of the extracellular matrix and a relationship between intraluminal thrombus (ILT) thickness and proteolytic activity within the abdominal aortic aneurysm (AAA) wall has been suggested. In the present study, the hypothesis that thin ILT is correlated with an increase in oxidative stress–related enzymes and matrix metalloproteinase‐9 (MMP‐9) expression within the human AAA wall was investigated. We also studied the antioxidant activity of superoxide dismutases, catalase, glutathione peroxidase, glutathione reductase, and thioredoxin within the full‐thickness AAA wall and through fluoroimmunohistochemical staining of catalase and MMP‐9 expression within the inner and outer media, in relation to ILT thickness. Reactive oxygen species control the degradation and remodeling of the extracellular matrix by up‐regulating proteolytic enzymes, such as MMPs. Results showed that oxidative stress and proteolytic enzyme expression were simultaneously, significantly higher within thin thrombus (≤10 mm)‐covered aneurysm wall when compared with the wall covered by thick thrombus (≥25 mm). These findings provide the first demonstration, to our knowledge, of a causative link between oxidative stress instigating proteolytic enzyme expression at the tissue level and human AAA development. Presence of a thin circumferential thrombus should always be considered as a risk factor for the greatest increase in aneurysm growth rate and rupture, giving an indication for surgery timing.—Wiernicki, I., Parafiniuk, M., Kolasa‐Wołosiuk, A., Gutowska, I., Kazimierczak, A., Clark, J., Baranowska‐Bosiacka, I., Szumilowicz, P., Gutowski, P. Relationship between aortic wall oxidative stress/proteolytic enzyme expression and intraluminal thrombus thickness indicates a novel pathomechanism in the progression of human abdominal aortic aneurysm. FASEB J. 33, 885–895 (2019). www.fasebj.org

Changes in the immune system in experimental acanthamoebiasis in immunocompetent and immunosuppressed hosts

BackgroundAcanthamoebiasis is most often found in patients with immune deficiency, with infections facilitated by the intake of immunosuppressive drugs. The host immune response to Acanthamoeba spp. infection is poorly understood. Thus, in this study, we aimed to examine the course of Acanthamoeba spp. infection taking into account the host’s immunological status, including assessment of the hematological parameters, cytokine analysis, immunophenotypic changes in spleen populations, and histological spleen changes, which could help clarify some aspects of the immune response to acanthamoebiasis. In our experimental study, we used Acanthamoeba strain AM 22 isolated from the bronchoaspirate of a patient with acute myeloid leukaemia (AML) and atypical pneumonia symptoms.ResultsAcanthamoeba spp. affected the hematological parameters in immunocompetent and immunosuppressed mice and induced a change in spleen weight during infection. Moreover, analysis of anti-inflammatory (IL-4 and IL-10) and pro-inflammatory (IL-17A and IFN-γ) cytokines produced by splenocytes stimulated with concanavalin A demonstrated that Acanthamoeba spp. induced a selective Th1, Th2 and Th17 response at later stages of the infection in immunocompetent hosts. In the case of hosts with low immunity, Acanthamoeba elicited robust Th1 cell-mediated immunity without the participation of Th17. We observed suppression of CD8+ and CD4+ T lymphocytes and CD3+CD4-CD8- double-negative (DN) T lymphocyte populations in the beginning, and in the case of CD3+/CD4+/CD8+ double-positive (DP) T cells in the final phase of Acanthamoeba spp. infection in hosts with low immunity. Also, CD4+T lymphocytes and CD3+/CD4+ and CD3+/CD8+ lymphocyte counts during each stage of acanthamoebiasis were shown to be upregulated.ConclusionsWe demonstrated that analysis of the immune response and pathogenesis mechanisms of clinical isolates of Acanthamoeba spp. in an animal model not only has purely cognitive significance but above all, may help in the development of effective methods of pharmacological therapy especially in patients with low immunity.

Immunosuppressive treatment affects morphology and apoptotic intensity of the liver in pregnant Wistar rats

Introduction: Immunosuppressive drugs are crucial for patients who have undergone transplantation. These medicines prolong proper functioning of the transplanted organ, but, on the other hand, they lead to various disorders, including pathological alterations within native organs and apoptosis. The aim of our study was to investigate the influence of immunosuppressive drugs on morphology, physiology and hepatocyte apoptosis in the liver of pregnant female Wistar rats. Material and methods: The study was conducted on 32 female rats exposed to immunosuppressive drugs before (2 weeks) and during pregnancy (3 weeks). The regimens included drugs commonly used in therapy following kidney transplantation (cyclosporine A, mycophenolate mofetil, prednisone, tacrolimus and everolimus). Results: We observed a decreased liver weight in all treated dams in comparison to the control rats. No statistically significant differences among all examined groups were found regarding the concentration of either alanine or aspartate aminotransferase. The most pronounced apoptosis and the most severe morphological abnormalities were found in rats treated with cyclosporine A, prednisone and everolimus; but moderate changes were visible in other treatment groups. The alterations that we observed included dilated sinusoids, inflammation foci, the disappearance of boundaries between hepatocytes, ruptured blood vessels, degradation of cytoplasm, and hemosiderosis. Conclusions: It seems possible that the alterations seen here are due to the early stage of liver damage, and that the biochemical parameters of the liver were not affected yet. Immunosuppressive therapy during pregnancy disturbs the physiology of the liver, and causes morphological changes in this organ.