A Pt(IV)-conjugated brain penetrant macrocyclic peptide shows pre-clinical efficacy in glioblastoma.

Glioblastoma (GBM) is the most aggressive primary malignant brain tumor, with a median survival of approximately 15 months. Treatment is limited by the blood-brain barrier (BBB) which restricts the passage of most drugs to the brain. We previously reported the design and synthesis of a BBB-penetrant macrocyclic cell-penetrating peptide conjugate (M13) covalently linked at the axial position of a Pt(IV) cisplatin prodrug. Here we show the Pt(IV)-M13 conjugate releases active cisplatin upon intracellular reduction and effects potent in vitro GBM cell killing. Pt(IV)-M13 significantly increased platinum uptake in an in vitro BBB spheroid model and intravenous administration of Pt(IV)-M13 in GBM tumor-bearing mice led to higher platinum levels in brain tissue and intratumorally compared with cisplatin. Pt(IV)-M13 administration was tolerated in naïve nude mice at higher dosage regimes than cisplatin and significantly extended survival above controls in a murine GBM xenograft model (median survival 33 days for Pt(IV)-M13 vs 24 days for Pt(IV) prodrug, 22.5 days for cisplatin and 22 days for control). Increased numbers of γH2AX nuclear foci, biomarkers of DNA damage, were observed in tumors of Pt(IV)-M13-treated mice, consistent with elevated platinum levels. The present work provides the first demonstration that systemic injection of a Pt(IV) complex conjugated to a brain-penetrant macrocyclic peptide can lead to increased platinum levels in the brain and extend survival in mouse GBM models, supporting further development of this approach and the utility of brain-penetrating macrocyclic peptide conjugates for delivering non-BBB penetrant drugs to the central nervous system.

Hemodynamic effect of a single dose of glucagon-like peptide 1 receptor (GLP-1R) agonist liraglutide in patients with diabetic kidney disease.

Increased peripheral resistance and autonomic nervous system is a key link in pathogenesis of arterial hypertension in diabetic kidney disease. Net effect of glucagon-like peptide 1 receptor (GLP-1R) agonists on blood pressure may result from interplay between vasodilatation, increased natriuresis, heart rate and sympathetic nervous system activity. The aim of study was to compare hemodynamic effect of single subcutaneous dose of 1.2 mg liraglutide to placebo in patients with type 2 diabetes mellitus and impaired renal function. This cross-over study included 17 patients with estimated glomerular filtration rate (eGFR) < 30 ml/min/1.73 m2 and 17 patients with eGFR > 60 ml/min/1.73 m2. Blood pressure and heart rate were monitored for 24 hours after liraglutide or placebo. Before and after each medication, systemic vascular resistance, heart rate variability, pulse wave velocity and central blood pressure were measured. Significant increases of 24 h mean heart rate and cardiac output were seen in both groups. Sympathetic predominance was observed in patients with eGFR < 30 ml/min/1.73 m2 after GLP-1R agonist compared to placebo (p = 0.005). Systemic vascular resistance decreased after liraglutide compared with placebo only in patients with eGFR > 60 ml/min/1.73 m2 (p = 0.002), whereas pulse wave velocity increased after liraglutide compared with placebo only in patients with eGFR < 30 ml/min/1.73 m2 (p = 0.0006). The 24 h mean arterial pressure after liraglutide significantly increased compared to placebo only in latter group. Liraglutide administration in patients with advanced chronic kidney disease (CKD) induces increase of blood pressure due to increase of cardiac output secondary to acceleration of heart rate associated with sympathetic predominance. The vasodilatory effect of liraglutide is preserved only in earlier CKD.

Expression of PTX3, HAS2 AND TNFAIP6 genes in relation to real-time proliferation of porcine endometrial luminal epithelial cells in primary cultivation model.

Endometrial cells undergo very specific changes associated with reproductive processes. Cells prepare for embryo development by increasing their volume. Then, if fertilization fails, endometrial cells are liable for apoptosis, preparing new cells that are ready for subsequent processes related to the possibility of embryo implantation and the development of pregnancy. PTX3 and TNFAIP6 are absent or reduced in cultured COCs, resulting in a functional change in COC in vitro. In this work, we want to check how PTX3, HAS2 and TNFAIP6 behave in luminal epithelium primary cell culture. Cells obtained during slaughter from porcine specimens were cultured primarily in vitro for 7 days. Their proliferation patterns were then analysed using RTCA, with the expression of genes of interest evaluated with the use of immunofluorescence and RT-qPCR. The results of these changes in the expression of the genes of interest were analysed on each of the seven days of the porcine luminal primary cell culture. Our study showed the increased level of PTX3, HAS2 and TN¬FAIP6 expression at the same hours of primary culture. Rt-qPCR showed a higher level of expression of the PTX3 gene in the first 72 h, at the end of the lag phase (in the phase of stasis in which the cells adapt to the new environment and often die). In contrast, TNFAIP6 expression increases about 96 hours when the cells are in the full log phase (logarithmic phase growth) and continue this trend in the plateau phase. We did not observe such drastic changes in the HAS2 expression pattern, which leads us to hypothesize that PTX3 and TNFAIP6 are designed to maintain a constant level of HAS2 in the cell throughout its lifetime. The obtained results could become a point of reference for further in vivo and clinical research.

Gene expression regulating lipopolysaccharide and lipid metabolic processes in porcine oral mucosal cells in long-term primary in vitro culture.

Lipids are an alternative energy source for cells and provide structural integrity in cell membrane and their metabolism is regulated with the use of different pathways, such as integrin signalling, oxidative stress, mechanical stress, and pH changes. All of those processes take place in the oral mucosa which is subject to different environmental impacts. In this study, porcine buccal pouch mucosal cells (pBPMCs) were used during long-term primary in vitro culture. The cultured cells were collected at 7, 15 and 30 days of IVC and subsequently transferred to RNA isolation. In the results of the following microarray analysis, we analyzed the genes detected, belonging to ontology groups, such as "cellular lipid metabolic process", "response to lipid" and "response to lipopolysaccharides. All of the genes involved in these ontological groups were expressed at higher levels at 7 days of IVC and substantially decreased in expression at days 15 and 30 of primary culture. We observed new genes, which may be recognized as markers in regulation of lipid metabolism in mucosal cells in vitro. The results suggested that the biochemical mechanism-involved lipids were accompanied by increased enzymatic activation and synthesis of crucial growth factors reaching high activity at day 7 of culture, which is also well documented as a stage of tissue regeneration period within oral mucosa. Therefore, this "biochemical fingerprint" may be an additional checkpoint of the integrity, resistance and easy adaptability of oral tissues, which are important conditions of success in tissue engineering and grafting for tissue reconstruction.

Genes involved in hormone metabolism and cellular response in human ovarian granulosa cells.

Endocrinal interactions are one of the most crucial regulatory mechanisms that maintain the state of homeostasis in humans. Processes such as oogenesis, folliculogenesis, menstruation and pregnancy remain under hormonal control. A key role in folliculogenesis is played by granulosa cells. Moreover, granulosa cells take part in corpus luteum formation after ovulation. Because of that, it is important to understand the ways in which the granulosa cells, associated with those processes, respond to hormonal stimulus. In the present study, a transcriptomic analysis of human granulosa cells (GCs) was carried out with the use of expression microarrays. The results were validated by RT-qPCR. The total RNA was isolated after 1st, 7th, 15th and 30th days of long-term primary cultures. The main focus of this work was placed on the genes belonging to "Response to estradiol", "Response to follicle-stimulating-hormone", "Cellular response to hormone stimulus", "Cellular hormone metabolic process" and "Hormone biosynthetic process" gene ontology groups. These groups of genes have been associated with GC hormone metabolism and cellular response to hormones. Eighty genes belonging to these groups were identified. Those that were members of more than one of the analyzed gene ontology groups, or exhibited unique expression patterns, were selected for further analysis. All of the selected genes were described, with their expression patterns detailed. In this manuscript, two gene expression patterns have been described. The first one showed large downregulation of genes in the later stages of culture, with the second one presenting upregulation of expression after day 1 of IVC. The present research was focused on six genes found to be the most important for steroidogenesis: STAR, POR, CYP11A1, ADM, GCLC, IL1B, as well as three genes of higher expression at the later stages of long-term in vitro culture: NR2F2, BMP4, COL1A1. The main goal of the presented study was to select genes involved in response to hormonal stimulus and hormone metabolism in GC long-term in vitro culture.

Megalin - a facultative marker of obesity-related glomerulopathy in children.

Obesity-related glomerulopathy (ORG) is an increasingly detected syndrome present in children with obesity. Megalin, a constitutive proximal tubule cell protein, when present in urine, can be considered as a biomarker indicating renal injury in these children.

Overview of the different methods used in the primary culture of oral mucosa cells.

The culture of primary cells in vitro has enabled to gain knowledge in the field of cell biology, disease mechanisms and to offer great potential in drug testing. To date, two main techniques of isolating and culturing oral mucosal cells, the direct explant method and the enzymatic method, dominate the literature and practice. In the present study, both techniques are discussed in detail, comparing the advantages and disadvantages of the two approaches in setting up a primary culture of oral mucosal cell. The direct explant technique is well-established and has been commonly used for the past 20-30 years. Although the method of setting up the cultures did not show much variations in the methodology described by authors, the culturing conditions varied according to the aims of the projects.

Human Wharton's jelly mesenchymal stem cells: properties, isolation and clinical applications.

Human Wharton's jelly mesenchymal stem cells (WJ-MSCs) exhibit CD29, CD79 and CD105 markers, characteristic for mesenchymal cell lines. Under the influence of the appropriate factors, WJ-MSCs can be dedifferentiated to osteoblasts, chondrocytes, adipocytes, myocytes, cardiomyocytes, glial cells and dopaminergic neurons. Wharton's jelly (WJ) is one of the potential sources of mesenchymal stem cells (MSCs) - obtaining these cells does not raise moral or ethical objections, because the umbilical cord (UC) is a regular waste material. The expression of the OCT-4 and Nanog proteins, which are characteristic for WJ-MSCs may indicate that these cells have retained some embryonic character. The collected data suggests that WJMSCs show increased division and telomerase activity compared to bone marrow MSCs (BM-MSCs). The published results showed no human leucocyte antigen (HLA) class II expression, with the possibility of HLA class I modification by WJ-MSCs, allowing for the transplantation of these cells both within the same and other species - which allows the use of human cells in animal models. The results of selected studies indicate that WJ-MSCs can be an essential element of regenerative medicine of the 21st century.

Insight into "nuclear-cytoplasmic shuttling" as a developmental and differentiational capability of cells in primary culture models.

Shuttling proteins are molecules that can facilitate transport through the nuclear envelope. A very large number of proteins are involved in this process that includes nuclear pore buildup, signal, receptor and enzyme proteins. There are many examples of proteins whose biological activity depends on nucleocytoplasmic transport. Very often they are largely responsible for the proper occurrence of cell division, maturation, development and differentiation. Thanks to the well mastered methods of in vitro cell culture, it is possible to trace the levels of protein expression and their distribution in cells. Advanced molecular techniques allow for precise determination of their displacement in time. Several studies are still being carried out, using primary cultures, to identify the factors that determine the maturation, development and differentiation of cells. In understanding of the detailed mechanisms controlling cell life, the key is not the level of expression of a specific protein, but its distribution in individual cellular compartments.

Transcriptomic profile of cell cycle progression genes in human ovarian granulosa cells.

The ovarian granulosa cells (GCs) that form the structure of follicle undergo substantial modification during the various stages of human folliculogenesis. These modifications include morphological changes, accompanied by differential expression of genes, encoding proteins which are mainly involved in cell growth, proliferation and differentiation. Recent data bring a new insight into the aspects of GCs' stem-like specificity and plasticity, enabling their prolonged proliferation and differentiation into other cell types. This manuscript focuses attention on emerging alterations during GC cell cycle - a series of biochemical and biophysical changes within the cell. Human GCs were collected from follicles of women set to undergo intracytoplasmic sperm injection procedure, as a part of remnant follicular fluid. The cells were primarily cultured for 30 days. Throughout this time, we observed the prominent change in cell morphology from epithelial-like to fibroblast-like, suggesting differentiation to other cell types. Additionally, at days 1, 7, 15 and 30, the RNA was isolated for molecular assays. Using Affymetrix® Human Genome U219 Array, we found 2579 human transcripts that were differentially expressed in GCs. From these genes, we extracted 582 Gene Ontology Biological Process (GO BP) Terms and 45 KEGG pathways, among which we investigated transcripts belonging to four GO BPs associated with cell proliferation: "cell cycle phase transition", "G1/S phase transition", G2/M phase transition" and "cell cycle checkpoint". Microarray results were validated by RT-qPCR. Increased expression of all the genes studied indicated that increase in GC proliferation during long-term in vitro culture is orchestrated by the up-regulation of genes related to cell cycle control. Furthermore, observed changes in cell morphology may be regulated by a presented set of genes, leading to the induction of pathways specific for stemness plasticity and transdifferentiation in vitro.

The p75 neurotrophin receptor in cells of oral mucosal epithelium.

The p75 neurotrophin receptor (p75NTR) can play different roles in cells. This protein can on the one hand act in the regulation of cell growth and survival, while being an apoptosis inducing factor in different contexts. p75NTR regulates cell cycle not only in nerve cells but also in epithelial oral mucosal cells. In the former, neurotrophin-p75NTR signaling affects cell growth and survival. Recent studies showed that p75NTR is expressed in basal cells of oral mucosal epithelium and can be used as one of the markers of epithelial stem/progenitor cells. This role of p75NTR can be utilised in aspects such as tissue engineering and gene therapy. One of the examples of clinical use of cultivated oral mucosal cells is ocular surface reconstruction. p75NTR can be a significant marker of stem cells in studies of epithelial tissues, especially when the cells will exhibit other specific markers, such as CK13, CK14 and PCNA..

Balance between epithelial and stromal marker expression and distribution in primary culture model of porcine endometrium during real-time cell proliferation..

The similarity between humans and pigs, when it comes to tissue morphology, makes Sus scrofa not only a good research model, but also a potential source of cells for tissue engineering. Cell samples obtained from the pig donor, could be influenced in vitro, in order to become a source of tissue material for xenotransplantation, reconstructive and regenerative medicine. Significant amounts of data point to especially major similarities in pig and human reproductive systems. Because of that, particular scientific focus is centered on research concerning porcine COCs, theca and granulosa cells in primary cultures. One of the aspects of the reproductive process, that is still largely undiscovered, is the interaction between preimplantation blastocyst and maternal uterine tissues. In this study, we used molecular analysis techniques, such as RT-qPCR and immunocytochemistry, to analyze the expression and distribution of cytokeratin 18 and panCytokeratins 8, 18 and 19 and vimentin in porcine luminal endometrial epithelial cells, coupled with analysis of their behavior in RTCA. The results have confirmed the presence of epithelial, as well as stromal cell markers in the cells, varying in levels at different stages of culture. They have also given insight into the modes of proliferation and differentiation of studied cells in in vitro culture, as well as providing additional proof for the possible mesenchymal transdifferentiation of epithelial cells.

Enzyme linked receptor protein signaling pathway is one of the ontology groups that are highly up-regulated in porcine oocytes before in vitro maturation.

Before being able to fully participate in the processes associated with its function as a female gamete, the oocyte needs to undergo a range of changes to achieve its mature form. These morphological, biochemical and metabolomic processes are induced by the somatic tissues surrounding the oocyte, through the expression of specific transcription and growth factors. The maturation of the oocyte is highly important for the proceedings that lead to successful fertilization, early embryonic development and implantation. Domestic pigs were used as models for our study, with the cumulus-oocyte complexes obtained from the ovaries that were recovered at slaughter. After shedding of the cumulus, oocytes were assessed with BCB test, with the viable ones chosen to undergo in vitro maturation. With the use of expression microarrays, we analyzed gene expression before and after IVM and detected major changes in both genes that were proven to be associated with oocyte maturation before (FOS, VEGFA, CHRDL1, TGFBR3, FST, INSR, ID1, TXNIP, SMAD4, MAP3K1, EIF2AK3 and KIT) and genes not previously linked with reproduction associated processes (MYO1E, PHIP, KLF10 and SHOC2). All the genes were briefly described, with consideration of possible involvement of the newly discovered elements of the transcriptome in the process of oocyte maturation.

Genes involved in regulation of cellular metabolic processes, signaling and adhesion are the markers of porcine buccal pouch mucosal cells long-term primary cultured in vitro.

Extraordinary abilities for continuous proliferation and differentiation, associated with constant renewal triggered by stimulation from the mastication process, together with the relative lack of aesthetic complications associated with post-surgery healing, have highlighted buccal pouch mucosa as a potential source of explants that could be used in transplantation and tissue engineering. Additionally, this tissue plays a major role in the oral drug delivery process, which brings special interest to its molecular properties in the context of new drug development. There is therefore a need to analyse the exact mechanisms of oral mucosa functioning, especially when it comes to the processes that are associated with the potential clinical applications. In this study we analysed a complete transcriptome of long-term in vitro cultures of porcine buccal pouch oral mucosa cells. Using a microarray approach, we focused on genes associated with cellular metabolic processes, signalling and adhesion, from 4 gene ontology groups: "Positive regulation of cellular component movement", "Positive regulation of cellular process", "Positive regulation of intracellular signal transduction" and "Single organism cell adhesion". Nineteen genes (CCL8, CXCL2, PLK2, DUSP5, PTGS2, LIF, CCL2, ATP1B1, REL, ITGB3, SCARB1, UGCG, PDPN, LYN, ETS1, FCER1G, TGFB1, RFC4, LMO2) with fold changes higher than |2| and p value Extraordinary abilities for continuous proliferation and differentiation, associated with constant renewal triggered by stimulation from the mastication process, together with the relative lack of aesthetic complications associated with post-surgery healing, have highlighted buccal pouch mucosa as a potential source of explants that could be used in transplantation and tissue engineering. Additionally, this tissue plays a major role in the oral drug delivery process, which brings special interest to its molecular properties in the context of new drug development. There is therefore a need to analyse the exact mechanisms of oral mucosa functioning, especially when it comes to the processes that are associated with the potential clinical applications. In this study we analysed a complete transcriptome of long-term in vitro cultures of porcine buccal pouch oral mucosa cells. Using a microarray approach, we focused on genes associated with cellular metabolic processes, signalling and adhesion, from 4 gene ontology groups: "Positive regulation of cellular component movement", "Positive regulation of cellular process", "Positive regulation of intracellular signal transduction" and "Single organism cell adhesion". Nineteen genes (CCL8, CXCL2, PLK2, DUSP5, PTGS2, LIF, CCL2, ATP1B1, REL, ITGB3, SCARB1, UGCG, PDPN, LYN, ETS1, FCER1G, TGFB1, RFC4, LMO2) with fold changes higher than |2| and p value less than 0.05 were identified, described in context and analysed. While the study needs much further validation to become applicable in a clinical environment, it yields valuable information about the transcriptomic basis of oral mucosal cell functioning in vitro, that might serve as a reference for further research, aiming to apply this knowledge in clinical situations.0.05 were identified, described in context and analysed. While the study needs much further validation to become applicable in a clinical environment, it yields valuable information about the transcriptomic basis of oral mucosal cell functioning in vitro, that might serve as a reference for further research, aiming to apply this knowledge in clinical situations.

High sensitivity C-reactive protein as a cardiovascular risk marker in independent community-living elderly persons.

An alarming fact is that increasing numbers of cardiovascular disease (CVD) events occur more often among elderly individuals without previous occurrence of CVD. There are numerous modifiable risk factors such as dyslipidemia,-and inflammatory markers, as well as cardiovascular events risk charts, in the identification and prevention of cardiovascular morbidity and mortality among elderly population. Thus, the aim of this study was to analyze some CVD risk factors as well as international CVD risk charts in independent community-living elderly persons in relation to their hs-CRP concentration in serum. Of 516 elderly Caucasians, 50 were non-smoking, with no positive history of chronic or acute diseases. The patients' clinical, biochemical and CVD risk charts were recorded. The CRP values were categorized according to the known cut-off points for stratification of cardiovascular risk: low risk patients with hs-CRP of less than 1 mg/L (low hs-CRP), moderate risk with hs-CRP of 1-3 mg/L (moderate hs- CRP) and high risk with hs-CRP of >3 mg/L (high hs-CRP). The groups did not differ in terms of age, anthropometric measures, fasting glucose, creatinine and uric acid concentration or analyzed CVD risk scales. The relationship between hs-CRP levels and both lipid profile and arterial blood pressure are linearly dependent (p less than 0.02). The negative correlation for the hs-CRP and fasting glucose and DIA were found in low hs-CRP (r= -0.619; p less than 0.05 and r= -0.580; p less than 0.05 respectively) and for the hs-CRP and uric acid (r=-0.850; p less than 0.05) in the moderate hs-CRP risk group. Thus, this study should greatly simplify decision-making for clinicians around the world.

Gender and age-related variability of macrophage representation in the internal thoracic artery wall: does it matter?

Some recent reports suggested that elderly and female patients did not benefit from implantation of the second internal thoracic artery (ITA) during coronary artery bypass surgery (CABG). Macrophages, among other cells, were described to be involved in both atherosclerosis and aortocoronary grafts failure. The aim of the study was to examine the age and gender association with different distribution of CD68+ cells within the layers of ITA wall. This study involved 158 consecutive patients (95 male and 63 female), with the mean age of 64.5±9.5 years, who underwent elective CABG procedures. During surgery, the surplus distal segments of ITA were harvested for immunohistochemical analysis. The number and distribution of CD68+ cells was calculated and plotted against the age and gender of the study participants. CD68+ cells were present in all of the harvested ITA fragments (median 44), more in women (55) than in men (42) (p less than 0.001). However, this difference was of statistical significance exclusively in the tunica intima. Approximately 70% of macrophages were found in the tunica adventitia. The total number of CD68+ cells the in arterial wall as well as in the tunica intima and adventitia correlated positively with the age of patients (r=0.544, r=501 and r=0.462, respectively). The lack of significant advantages of the use of two thoracic arteries, in elderly patients and women, might have resulted from the larger population of CD68+ cells in their walls, especially the tunica intima. However, this result from immunohistochemical analysis needs validation in long-term clinical research on a larger cohort of patients.

Up-regulated autophagy: as a protective factor in adipose tissue of WOKW rats with metabolic syndrome.

BACKGROUND: Wistar Ottawa Karlsburg W (RT1u) rats (WOKW) are a model of the metabolic syndrome (MetS). Adipose tissue (AT) and peripheral nerves of WOKW rats exhibit up-regulated autophagy and inflammation corresponding with decreased apoptosis rate. The aim of this study was to characterize AT in WOKW rats in relation to autophagic activity. METHODS: mRNA and protein expression of adiponectin, pro-inflammatory and pro-apoptotic markers including MCP1, TNFα, cleaved caspase-3 and RNF157, a new candidate gene regulated through autophagy, were analyzed in adipocytes isolated from visceral and subcutaneous AT of 5-month old WOKW rats with MetS and LEW.1W controls in response to pharmacological inhibition of autophagy. Immunohistochemistry was performed to detect adiponectin and RNF157 protein in cultured adipocytes. RESULTS: Inhibition of autophagy by LY294002 was associated with a fourfold up-regulation of adiponectin expression and a decrease of RNF157 protein and pro-inflammatory markers-MCP-1 and TNFα predominantly in visceral adipocytes of obese WOKW rats compared to LEW.1W rats. Moreover, inhibition of autophagic activity correlates with an activation of cleaved caspase-3 apoptotic signaling pathway. CONCLUSIONS: Up-regulated autophagy in obese WOKW rats contributes to the regulation of visceral AT function and involves an altered balance between pro-inflammatory and protective adipokine expression. Our data suggest that activation of AT autophagy protects against adipocyte apoptosis at least under conditions of obesity related MetS in WOKW rats.

Haematopoiesis: “living in the shadow” of stem cell differentiation.

Haematopoiesis is one of the most well understood stem-cell associated processes. It is a process in which pluripotent hematopoietic stem cells (HSCs) self-proliferate and differentiate into all types of blood cells. The process takes place in marrow of the flat bones in adults, however its location changes several times through embryonic and foetal development. Given the broad range of blood cells and the major differences in their build and function, together with the fact that their numbers need to be maintained within relatively narrow margins in order to maintain homeostasis despite changing environmental conditions, makes the whole process of haematopoiesis highly regulated and depending on a variety of growth factors. When influenced by those, HSCs undergo several irreversible steps, with every next one committing them to an even more specialised fate, ending with all the specific types of mostly short-lived blood cells, that are unable to proliferate on their own and need constant replenishment from the HSC pool. Because the process of haematopoiesis is the only source of all the members of the group of cells performing a range of highly important roles in functioning of the organism, significant damage to the underlying stem cells can cause a range of severe diseases. Many treatments are suggested for managing their symptoms or slowing progress, with bone marrow transplant being one of the only ones that offer possible permanent solution and, despite being a relatively risky procedure, is being widely performed, with the methods constantly improving in order to achieve progressively better results in both treatability and survivability of the patients.

Explorative Randomized Phase II Clinical Study of the Efficacy and Safety of Finafloxacin versus Ciprofloxacin for Treatment of Complicated Urinary Tract Infections.

The broad-spectrum C-8-cyano-fluoroquinolone finafloxacin displays enhanced activity under acidic conditions. This phase II clinical study compared the efficacies and safeties of finafloxacin and ciprofloxacin in patients with complicated urinary tract infection and/or pyelonephritis. A 5-day regimen with 800 mg finafloxacin once a day (q.d.) (FINA05) had results similar to those of a 10-day regimen with 800 mg finafloxacin q.d. (FINA10). Combined microbiological and clinical responses at the test-of-cure (TOC) visit were 70% for FINA05, 68% for FINA10, and 57% for a 10-day ciprofloxacin regimen (CIPRO10) in 193 patients (64 for FINA05, 68 for FINA10, and 61 for CIPRO10) of the microbiological intent-to-treat (mITT) population. Additionally, the clinical effects of ciprofloxacin on patients with an acidic urine pH (80% of patients) were reduced, whereas the effects of finafloxacin were unchanged. Finafloxacin was safe and well tolerated. Overall, 43.4% of the patients in the FINA05 group, 42.7% in the FINA10 group, and 54.2% in the CIPRO10 group experienced mostly mild and treatment-emergent but unrelated adverse events. A short-course regimen of 5 days of finafloxacin resulted in high eradication and improved clinical outcome rates compared to those for treatment with ciprofloxacin for 10 days. In contrast to those of ciprofloxacin, the clinical effects of finafloxacin were not reduced by acidic urine pH. Hospitalized adults were randomized 1:1:1 to finafloxacin treatment (800 mg q.d.) for either 5 or 10 days or to ciprofloxacin treatment (400 mg/500 mg b.i.d.) for 10 days with an optional switch from intravenous (i.v.) to oral administration at day 3. The primary endpoint was the combined microbiological and clinical response at the TOC visit in the microbiological intent-to-treat population. (This study has been registered at ClinicalTrials.gov under identifier NCT01928433.).

Expression of genes responsible for cell morphogenesis involved in differentiation in porcine buccal pouch mucosal cells during long-term primary culture and real-time proliferation in vitro.

Recently, using experimental animal model, we demonstrated that porcine buccal pouch mucosal cells reflect increased proliferation capability during primary cultivation in vitro. Although the histological structure and morphogenesis in oral cavity is well recognized, the molecular mechanisms which regulate this process still need further investigation. This study was aimed to analyze the molecular marker expression profile involved in morphogenesis and differentiation capacity of porcine buccal pouch mucosal cells during their long-term primary cultivation in vitro. The experiment was performed on buccal pouch mucosal cells isolated from 80 pubertal crossbred Landrace gilts. After collection, the cells were treated enzymatically and transferred into a primary in vitro culture (IVC) system and cultured for 30 days. The cells were collected for RNA isolation after 7, 15 and 30 days of IVC and were checked for their real-time proliferative status using the RTCA system. We found an increased expression of FN1 and SOX9 genes when calculated against ACTB after 7, and 30 days of IVC, (P less than 0.01, P less than 0.001, respectively). The CXCL12 mRNA was down-regulated after 7, 15 and 30 days of IVC, but not statistically significant. Similar expression profile was observed when calculated against HPRT, however, DAB2 was found to be higher expressed at day 15 of IVC, (P less than 0.05). The cell index measured during real-time cell proliferation was substantially increased between 96 h and 147h of IVC and reached the log phase. Since FN1 and SOX9 revealed significant increase of expression after long-term culture in vitro, it is suggested that expression of these differentiation and stemness genes is accompanied by cell proliferation. Moreover, FN1 and SOX9 might be recognized as new markers of buccal pouch mucosal cell proliferation and differentiation in pigs in in vitro primary culture model.