Optimization of methods for intrasplenic administration of human amniotic epithelial cells in order to perform safe and effective cell-based therapy for liver diseases.

In animal experimental models the administration of stem cells into the spleen should ensure high effectiveness of their implantation in the liver due to a direct vascular connection between the two organs. The aim of this study was to update the methods of experimental intrasplenic cell transplantation using human amniotic epithelial cells (hAECs) which are promising cells in the treatment of liver diseases. BALB/c mice were administered intrasplenically with 0.5, 1, and 2 million hAECs by direct bolus injection (400 µl/min) and via a subcutaneous splenic port by fast (20 µl/min) and slow (10 µl/min) infusion. The port was prepared by translocating the spleen to the skin pocket. The spleen, liver, and lungs were collected at 3 h, 6 h, and 24 h after the administration of cells. The distribution of hAECs, histopathological changes in the organs, complete blood count, and biochemical markers of liver damage were assessed. It has been shown that the method of intrasplenic cell administration affects the degree of liver damage. The largest number of mice showing significant liver damage was observed after direct administration and the lowest after slow administration through a port. Liver damage increased with the number of administered cells, which, paradoxically, resulted in increased liver colonization efficiency. It was concluded that the administration of 1 × 106 hAECs by slow infusion via a subcutaneous splenic port reduces the incidence of complications at the expense of a slight decrease in the effectiveness of implantation of the transplanted cells in the liver.

Comparison of the Efficacy of Two Routes of Administration of Human Amniotic Epithelial Cells in Cell Therapy of Acute Hepatic Insufficiency.

The route of administration of implanted cells may affect the outcome of cell therapy by directing cell migration to the damaged site. However, the question of the relationship between the route of administration, the efficacy of colonisation of a given organ, and the efficacy of cell therapy has not been resolved. The aim of the study was to localise transplanted intravenously and intraperitoneally human amniotic epithelial cells (hAECs) in the tissues of mice, both healthy and injured, in an animal experimental model of acute liver failure (ALF). Mice intoxicated with D-Galactosamine (D-GalN) at a dose of 150 mg/100 g body weight received D-GalN alone or with a single dose of hAECs administered by different routes. Subsequently, at 6, 24, and 72 h after D-GaIN administration and at 3, 21, and 69 h after hAEC administration, lungs, spleen, liver, and blood were collected from recipient mice. The degree of liver damage and regeneration was assessed based on biochemical blood parameters, histopathological evaluation (H&E staining), and immunodetection of proliferating (Ki67+) and apoptotic (Casp+) cells. The biodistribution of the administered cells was based on immunohistochemistry and the identification of human DNA. It has been shown that after intravenous administration, in both healthy and intoxicated mice, most of the transplanted hAECs were found in the lungs, while after intraperitoneal administration, they were found in the liver. We concluded that a large number of hAECs implanted in the lungs following intravenous administration can exert a therapeutic effect on the damaged liver, while the regenerative effect of intraperitoneally injected hAECs on the liver was very limited due to the relatively lower efficiency of cell engraftment.

Impact of local delivery of allogeneic chondrocytes on the biological response and healing of the sternum bones after sternotomy.

Median sternotomy is the surgical method of choice for many procedures where one of the main problems is the long post-operative wound healing process leading to sternal dehiscence and the development of infection. This leads to prolonged hospital stay and increased mortality due to post-operative complications. A promising solution seems to be the use of allogeneic chondrocytes for wound treatment, whose properties in the field of cartilage reconstruction are widely used in medicine, mainly in orthopedics. In the present study, we investigated the effect of local delivery of allogeneic chondrocytes on the biological response and healing of the sternum after sternotomy. We optimized the culture conditions for the isolated chondrocytes, which were then applied to the sternal incision wound. Chondrocytes in the culture were assessed on the basis of the presence of chondrocyte-specific genes: Sox9, Aggrecan and Collagen II. In turn, the histopathological and immunohistochemical evaluation was used to assess the safety of implantation. In our work, we demonstrated the possibility of obtaining a viable culture of chondrocytes, which were successfully introduced into the sternal wound after sternotomy. Importantly, implantation of allogeneic chondrocytes showed no significant side effects. The obtained results open new possibilities for research on the use of allogeneic chondrocytes in the process of accelerating wound healing after median sternotomy.

Dynamics of Chronic Liver Injury in Experimental Models of Hepatotoxicity.

BACKGROUND: In humans, chronic liver disease (CLD) is a serious clinical condition with many life-threatening complications. Currently, there is no therapy to stop or slow down the progression of liver fibrosis. Experimental mouse models of CLD, induced by repeated intraperitoneal injections of carbon tetrachloride (CCL4) and D-galactosamine (D-GalN), can be used to evaluate therapies that cannot be performed in humans. A major drawback of these animal models is the different dynamics of liver fibrosis progression depending on the animal strain, administered hepatotoxin, its dose, duration of intoxication, and frequency of injections. The aim of this study was to describe and compare the dynamics of progression of pathological changes in the BALB/c mouse and Sprague Dawley rat models of CLD induced by CCl4 and D-GalN. We defined the onset and duration of these changes and suggested the optimal time for therapeutic intervention in the analyzed CLD models. METHODS: CLD was induced by repeated intraperitoneal injection of CCl4 in mice (12.5 µL/100 g bw every 5 days) and rats (25-100 µL/100 g bw twice a week) and D-GalN in mice (75 mg/100 g bw twice a week) and rats (25 mg/100 g bw twice a week). Blood and liver samples were collected at weeks 2, 4, 6, 8, 10, and 12 of intoxication. Liver injury and its progression were assessed by using complete blood count and liver function blood tests as well as by analyzing histopathological changes, including fibrosis, proliferation activity, apoptosis, stellate cell activation, and gene expression. RESULTS: In mice and rats treated with CCl4, early fibrosis was observed in most pericentral areas from week 2 to 4 of intoxication. Established fibrosis developed in both rats and mice at week 6 of intoxication. Incomplete cirrhosis, defined as the presence of occasional cirrhotic nodules, was observed in rats at week 12 of intoxication. The dynamics of liver fibrosis in CCl4-treated animals were greater than in the D-GalN groups. In D-GalN-intoxicated rats and mice, the first signs of liver fibrosis were observed at weeks 4 and 10 of intoxication, respectively. The rats developed early fibrosis after 8 weeks of D-GalN intoxication. The progression of collagen deposition was accompanied by histological changes and alteration of certain genes and blood liver parameters. CONCLUSIONS: The dynamics of liver fibrosis in CCl4 treated rodents is greater than in the D-GalN treated ones. In the CCl4 models, two appropriate times for therapeutic intervention are indicated, which to varying degrees reflect the real clinical situation and may potentially differ in the obtained results: early intervention before week 4 of intoxication (early fibrosis) and late intervention after week 8 of intoxication (when signs of established fibrosis are present). Rodent models of D-GalN-induced fibrosis are not recommended due to the long incubation period and weak toxic effect.

Dynamics of Acute Liver Injury in Experimental Models of Hepatotoxicity in the Context of Their Implementation in Preclinical Studies on Stem Cell Therapy.

BACKGROUND AND AIMS: Experimental models using carbon tetrachloride (CCl4) and D-galactosamine (D-GalN) can be used in preclinical assessment of acute liver failure (ALF) therapies. Unfortunately, these models are characterized by different dynamics of liver injury depending on the animal strain, administered hepatotoxin, and its dose. The aim of this study was to compare known rat and mouse models of ALF with a view to their future introduction into preclinical cell therapy experiments. In particular, based on histopathological and molecular changes, we suggested experimental time cut-off points for an effective stem cell therapeutic intervention. METHODS: ALF was induced by a single intraperitoneal injection of CCl4 in mice (50 µL/100 g b.w.) and rats (200 µL/100 g b.w.) and D-GalN in mice (150 mg/100 g b.w.) and rats (50 mg/100 g b.w.). Blood and liver samples were collected 12 h, 24 h, 48 h and 7 days after intoxication. Blood morphology, liver function blood tests, histopathological changes, proliferation activity, apoptosis, fibrosis, and gene expression were analysed to assess liver damage. RESULTS: At 12 h, 24 h, and 48 h after CCl4 injection, mouse livers showed moderate inflammatory infiltration and massive pericentral necrosis. In rats treated with CCl4, minor lymphocytic infiltration in the liver parenchyma was seen at 12 h, followed by necrosis that appeared around central veins at 24 h and persisted to 48 h. In D-GalN-injected mice, the first histopathological signs of liver injury appeared at 48 h. In the livers of D-GalN-treated rats, moderate pericentral inflammatory infiltration occurred after 12 h, 24 h, and 48 h, accompanied by increased proliferation and apoptosis. All histological changes were accompanied by decreasing expression of certain genes. In most experimental groups of rats and mice, both histological and molecular parameters returned to the baseline values between 48 h and 7 days after intoxication. CONCLUSIONS: In mice and rats with CCl4-induced ALF, signs of liver failure can be seen as early as 12 h and develop to 48 h. In the D-GalN-induced model, mice are more resistant to the hepatotoxic effect than rats (after 12 h), and the early hepatitis phase can be observed much later, after 48 h. These cut-off points seem to be optimal for suppressing inflammation and applying effective stem cell therapy for acute liver injury.

Nuclear factor-kappa B as potential therapeutic target in human colon cancer.

BACKGROUND: It has been proved that nuclear factor-kappa B (NF-κB) is activated in all cells, promotes proliferation of cells, regulates the immunological and inflammatory response, and contribute to the pathogenesis of many conditions, including cancer. Many studies pointed to constitutive activation of NF-κB in cells of certain malignant tumors. OBJECTIVE: The aim of the study was to analyze the role of nuclear growth factor κB as colon cancer marker and prognostic factor. MATERIALS AND METHODS: The study included 59 primary colorectal tumor patients and 15 patients in control group. The tumor samples were taken during partial colectomy and colonoscopy in control group. Tissues samples were fixed and embedded in paraffin blocks and cut. Sections were used for schedule immunohistochemical staining with the application of specific antibody for NF-κB epitope. The marker expression was compared with well-known prognostic factors in colon tumors such as tumor type, stage, and grade to establish if it might be a potential prognostic factor. RESULTS: The results showed statistically significant difference between control group and cancer group. CONCLUSIONS: The expression NF-κB did not depend on the stage and grade of colon tumors.

The relation between plasma α-synuclein level and clinical symptoms or signs of Parkinson's disease.

INTRODUCTION: Parkinson disease (PD) is the common neurodegenerative disease. α-Synuclein (ASN), main aggregating protein in neural cells of CNS in PD, was found in peripheral fluids. Testing ASN in plasma is potential test for diagnose PD, but previous studies are controversial. The aim of this study was to investigate if plasma ASN level may be a valuable biomarker, is the level of plasma ASN concentration different in various motor subtypes of diseases, is there a relation between the level of plasma ASN and the severity of motor symptoms. METHODS: Patients with PD hospitalized in Neurology Department, Medical College were performed sequencing the 8th and 9th exon of GBA gene. Next plasma ASN level was tested in 58 patients with sequenced GBA gene and in 38 healthy volunteers (HV), matched by the age (respectively 68.43 vs. 64.57 years of age) and sex (female %, respectively: 43.10 vs.44.74). Patients were assessed with the scales: UPDRS (II, III, IV), Hoehn-Yahr (HY) and qualified to PIGD or TD subtype. For homogeneity of the group patients with GBA mutation were excluded from the analysis. RESULTS: The ASN level did not differ between patients and HV (respectively: 4.53 vs. 3.73ng/ml) and between patients with different subtypes. There was inverse correlation between ASN and HY in PIGD subtype. CONCLUSIONS: Plasma ASN level is not valuable marker of the disease. It does not differ in subtypes of the disease. There is relation between plasma ASN level and the severity of the disease in PIGD subtype.

Expression of VEGF isoforms and their receptors in uterine myomas.

OBJECTIVES: The aim of the study was to determine the expression of VEGF (vascular endothelial growth factor) isoforms and their receptors in uterine myomas. MATERIAL AND METHODS: The study included 40 women with myomas of reproductive age and 40 perimenopausal women (the study group). Myometrial samples (the control group) were taken from 10 women undergoing hysterectomy for ovarian tumors and 10 older women undergoing hysterectomy for uterine prolapse. RESULTS: A significantly increased expression of VEGF-A has been found in myomas, both small and large, in the younger women, which may by a sign of increased angiogenesis and intensive tumor growth. In perimenopausal women, the increase of VEGF expression was observed only in the endothelium and vascular smooth muscle. CONCLUSION: An important conclusion of this study is that angiogenesis is independent of myoma size, which may suggest intensive tumor growth and the related increased angiogenesis. High expression of VEGF-A and VEGF-R1 receptors in large myomas can probably cause malignant transformation and more extensive growth, regardless of patient age.

Survivin in ovary tumors.

INTRODUCTION: Survivin is a member of the inhibitor of apoptosis protein (IAP) family which are selectively overexpressed in human neoplasms, and its expression has been shown to be connected with cell proliferation. We analyzed survivin expression in ovarian epithelial neoplasms to evaluate its role in the development of ovarian tumors. MATERIAL AND METHODS: Immunohistochemistry assays were conducted in 137 cases (48 ovarian carcinoma, 43 borderline ovarian carcinoma, 46 benign ovarian tumor and 20 samples of normal ovarian tissue of ovarian epithelial neoplasms. Histological types included serous (n = 68) and mucinous (n = 69) tumors. All tumors were reviewed histopathologically and classified according to the WHO criteria. RESULTS: Survivin expression in the group of serous neoplasms was detected in 24.0% (6 of 25) of benign cases, in 60.0% (12 of 20) of borderline tumors, and 91.0% (24 of 47) of ovarian carcinomas. In the group of mucinous tumors, survivin expression was found in 33.5% (7 of 21) of benign cases, 43.5% (10 of 23) of borderline tumors, and 80.0% (20 of 25) of malignant tumors. CONCLUSIONS: Our results demonstrate that survivin overexpression may play a crucial role in the development of epithelial ovarian neoplasms and be an important prognostic factor for the influence of survivin expression on epithelial ovarian cancers.

Immunohistochemical visualization of pro-inflammatory cytokines and enzymes in ovarian tumors.

Epithelial ovarian cancer represents one of the most deadly gynaecological neoplasms in developed countries and is a highly heterogeneous disease. Epidemiological studies show that anti-inflammatory drugs reduce the incidence and mortality of several types of cancer, indicating the potential role of pro-inflammatory factors in carcinogenesis. The expression of pro-inflammatory factors in various cancer types, including ovarian cancer, was assessed in many studies, yielding in consistent results, often due to the histological heterogeneity of various cancers. The aim of the study was to investigate the expression of IL-1, IL-6, TGF-ß, TNF-α, COX-2, iNOS, and NF-kB in serous and mucinous ovarian cancers. Ninety cases of ovarian tumors classified into mucous and serous type (45 patients in each group) were selected. Each group was classified into subgroups according to the three stages of tumor differentiation, i.e. into (i) benign, (ii) borderline and (iii) malignant tumors. The presence of proteins of interest in paraffin sections was analysed by immunohistochemistry. The expression of most of the studied factors depended on the histological tumor subtype and the degree of malignancy. Expression of NF-κB appears to be related to the level of the neoplastic differentiation only in the group of serous tumors, while the presence of IL-6 in the mucinous tumor subtype was observed only in the case of benign lesions. Expression of IL-1, TNF-α and COX-2 increased with the stage of the disease in both serous and mucinous tumors. The highest level of TGF-ß expression was observed in serous borderline tumors. The different levels of iNOS immunoreactivity between the groups of serous and mucinous tumors were observed only in borderline tumors. The results of our study may be helpful in designing therapeutic strategies depending on the type of ovarian cancer.

Receptors of hypothalamic-pituitary-ovarian-axis hormone in uterine myomas.

In this study the expression of GnRH, FSH, LH, ER-α, ER-ß, and PR receptors was examined in uterine myomas of women in reproductive and perimenopausal age. In cases of GnRH and tropic hormones a membranous and cytoplasmic immunohistochemical reaction was detected, in cases of ER-α and PR the reaction was located in cell nucleus, and in the case of ER-ß it manifested also a cytoplasmic location. In some of the examined cases the expression was detected in endometrium, myocytes, and endothelium of blood vessels, in uterine glands and myoma cells. In myometrium the level of GnRH and LH receptors increases with age, whereas the level of progesterone and both estrogen receptors decreases. In myomas of women in reproductive age, independently of their size, expression of GnRH, FSH, and LH receptors was more pronounced than in myometrium. In women of perimenopausal age, independently of myoma size, expression of LH and estrogen α receptors was higher while expression of GnRH receptors was lower than in myometrium. FSH receptor expression was not observed. Expression of estrogen receptor ß was not affected by age of the woman or size of myoma. Analysis of obtained results indicates on existing in small myomas local feedback axis between GnRH-LH-progesterone.

Expression of selected cytochrome P450 isoforms and of cooperating enzymes in colorectal tissues in selected pathological conditions.

The current interest in CYP expression in the colon results from its uniqueness as a target organ for cancer. To date, the CYP expression profiles in the colon have not yet been subject of comprehensive research. In this study, we investigated 40 patients with Crohn's disease, 40 with ulcerative colitis, and 40 healthy subjects as a control group. Colon tissues were fixed, dehydrated, cleared in xylene and embedded in paraffin. Sections were prepared from paraffin blocks for immunohistochemical staining with specific antibodies. We used antibodies to the human CYP1A1, CYP2B6, CYP2C9, CYP2E1 and CYP3A4 isoforms, as well as antibodies to the human glycoprotein P, glutathione-S transferase and antibody to the UDP-glucuronosyltransferase. The sections were stained immunohistochemically and examined using light microscopy. Cellular localization was determined, and computer image analysis was used. In all cases with Crohn's disease, the proteins studied showed at least a twofold expression. Ulcerative colitis showed a much weaker influence regarding the expression of the proteins studied but in case of CYP2C9 and UDP-glucuronosyltransferase, a decrease of expression was observed.

The TRAF2 and TRAF6 expression in myomas and myometrium of women in reproduction and perimenopausal age.

Uterine myomas represent one of the most common female diseases. Uterine myomas or fibromas are benign, hormone-responding tumours of, respectively, smooth muscles and fibroblasts and their aetiology induces a significant interest. In myomas the presence of aromatase was detected and, in addition, oestrogen was found to be synthesized in myoma cells. The studies were performed on myoma patients of generative age and those in peri-menopausal age. Expression of TRAF2 and TRAF6 proteins was examined using immunohistochemistry and Western blot approach in small and large uterine myomas isolated from women of various age. In addition, the evaluation was conducted at the periphery of every myoma. We indicated that the level of both tested proteins in myomas is higher than in control. TRAF2 level in myometrium was lower than in myomas but higher than in control. In the case of TRAF6 those changes were ambiguous. Age didn't have influence the level of expression in both tested TRAF in studied structures.

[Effect of Helicobacter pylori infection on proliferation and apoptosis in stomach antrum in smoking patients]. / Wplyw infekcji Helicobacter pylori na proliferacje i apoptoze komórek odzwiernika zoladka u pacjentów palacych papierosy.

Peptic ulcer disease develops if the balance between aggressive and defensive factors is destroyed. Among the identified risk factors are Helicobacter pylori infection (Hp), stress, cigarette smoking and taking nonsteroidal anti-inflammatory drugs. However, the exact ulcerogenic mechanism of these factors remains not fully elucidated; yet habitual smoking is the most controversial. The aim of this study was to investigate the influence of cigarette smoking and/or Hp infection on gastric mucosa expression of COX-2, iNOS and proliferation and apoptosis processes (Ki-67 and active caspase 3 expressions, respectively). In smokers without Hp all studied parameters were higher than in nonsmoker without Hp group. Hp infection increases iNOS , COX-2 and active caspase 3 expression. Highest values for iNOS, COX-2 and Ki-67 was observed in smoker with Hp infection group. Hp infection in smokers significantly decrease active caspase 3 expression.

[The influence of tobacco smoking on the occurrence of copper in hydroxiapatites of gallstones]. / Wplyw nalogu palenia tytoniu na wystepowanie miedzi w hydroksyapatytach kamieni zólciowych.

The studies were carried out on the changes in copper content in concretions of gallstones which were taken from 146 women (49 smoking and 97 no smoking) and 24 men (11 smoking and 13 no smoking), respectively. The elemental composition of gallstones was determined with ICP-AES method (Inductively Coupled Plasma - Atomic Emission Spectroscopy). Studies on copper occurrence in concretions of gallstones showed the presence of its higher concentrations in case of smoking women (9.59 microg/g) and men (8.99 microg/g) in comparison with no smoking women (7.02 microg/g) and men (7.66 microg/ g). It was shown differences in pattern of changes of copper concentration in gallstones in case of smoking and no smoking women and men.

The aromatase expression in myomas and myometriums of women in reproduction and perimenopausal age.

Uterine myomas represent one of the most common female pathologies. Uterine smooth muscle myomas or fibromas are benign tumours which respond to hormones and their etiology induces wide interest. The myomas were found to contain aromatase and, in addition, cells of the myomas were found to synthesize estrogen. This study was conducted on patients with the myomas, in either generative age or in the perimenopausal period. Expression of aromatase was detected in patients of various age, with large or small uterine myomas, using an immunohistochemical technique. In addition expression of the enzyme was examined at the periphery of every myoma.

[Chromium in hydroxiapatites of gallstones from active and passive smoking and non smoking women]. / Chrom w hydroksyapatytach kamieni zólciowych u kobiet palacych aktywnie i biernie oraz niepalacych tyton.

The studies were carried out on the changes in chromium content in concretions of gallstones taken from 93 women (24 active smoking, 18 passive smoking, 51 non smoking women), respectively. Chromium content was determined with ICP-AES method (Inductively Coupled Plasma - Atomic Emission Spectroscopy). Studies on chromium occurrence in concretions of gallstones showed the presence of its higher concentrations in passive smoking women (3.15 microgCr/g) in comparison with active smoking (1.20 microgCr/g) and non smoking women (0.89 microgCr/g). Separate patterns of changes in chromium in active, passive and no smoking women gallstones were demonstrated.

[Lead content in blood of children living near zinc smelter plant exposure on environmental tobacco smoking (ETS)]. / Wystepowanie olowiu w krwi dzieci zamieszkalych w zasiegu oddzialywania Huty Cynku "Miasteczko Slaskie" narazonych na bierne palenie tytoniu.

In this paper the results of concentration of lead in the blood of children living near and background the plant smelter are presented. 510 girls and 567 boys from schools located at Tarnowskie Góry and Miasteczko Slaskie took part in the study. Concentration of lead content in the blood of children was determined by the ASA method with the apparatus Pye Unicam SP-9. Smoking of parents increased the level of lead in the blood. Higher concentration of lead was show in blood girls (8.09 microg/100 ml) and boys (9.78 microg/100 ml) exposure to tobacco smoke in comparison to girls (6.19 microg/100 ml) and boys (6.81 microg/100 ml) living in no smoking conditions.

[Aluminum in hydroxyapatites of gallstones from smoking and non smoking women]. / Glin w hydroksyapatytach zlogów pecherzyka zólciowego kobiet palacych i niepalacych.

The investigation on the aluminum changes in gallstones from 149 women, including 49 smokers and 79 non-smokers women was carried out. All patients were inhabitants of Southern Poland. Aluminum concentration was determined using Inductively Coupled Plasma - Atomic Emission Spectroscopy (ICP-AES) with the measurement precision of 1-3% and the detectability of 0.01 microg/g. Arithmetical mean was statistically significantly higher for non-smoking women (33.39 microgAI/g) in comparison to smoking women (20.30 microgAI/g). The changes of given elements were different for smoking women in comparison to non-smoking women.

[The changes of barium and strontium content in gallstones from smoking and non-smoking women]. / Zmiany zawartosci baru i strontu w zlogach kamieni zólciowych kobiet palacych i niepalacych tyton.

The problem of statistical characteristics of occurrence barium and strontium content in gallstones from smoking and non-smoking women living in southern Poland is presented in the work. The subjects of the research were gallstones, gained intraoperatively from 146 women (49 smoking, 97 non-smoking). The content of barium and strontium was determined using atomic emission spectroscopy with inductive coupled plasma (ICP-AES). The statistical characteristic of barium and strontium content in gallstones shows that smoking does not decide about content level of these elements in gallstones. Content of barium (2.32 microgBa/g) as well as strontium (3.23 microgSr/g) in gallstones from non smoking women are higher in comparison to content of these elements in gallstones from smoking women (1.91 microgBa/g and 2.76 microgSr/g).