To Örebro University

BACKGROUND: Contrasting findings have been reported regarding a possible constitutive expression of inducible nitric oxide synthase (iNOS) in a normal mammalian bladder. The current study was designed to further investigate such putative iNOS expression.

MATERIALS AND METHODS: The experiments were conducted with paraffin-embedded archival material from the urinary bladder of 6 normal, male Sprague-Dawley rats. In addition, two normal female mice (C57BL/6) were sacrificed and the urinary bladders were harvested. The occurrence of iNOS mRNA was examined by the RNAScope in situ hybridization method. Protein expression of iNOS and 3-nitrotyrosine (the latter used as an indicator of oxidative stress) was investigated by immunohistochemistry.

RESULTS: No expression of iNOS mRNA was observed in the bladder tissue. iNOS protein and 3-nitrotyrosine were strongly expressed in the urothelium. iNOS was also expressed perinuclearly in the detrusor.

CONCLUSIONS: Although the RNAScope methodology could not demonstrate mRNA for iNOS in the normal urinary bladder, the results by immunohistochemistry strongly suggest the occurrence of iNOS in particular, in the urothelium. Positive reactivity for 3-nitrotyrosine may indicate ongoing oxidative stress of the urothelium. The finding of perinuclear iNOS immunoreactivity could suggest an intracrine signaling function by iNOS to the nucleus.

Objective: We have previously demonstrated protein expression of the extracellular matrix degrading protein ADAMTS5 in the nuclei of urothelial cells in healthy rats. The proteoglycan versican constitutes one of the main substrates for this protease. In this follow up study we investigated a potential co-localization of versican and ADAMTS5 in the urinary bladder wall.

Material and Methods: The study was conducted with archive material (paraffin embedded bladder tissue from our previous study, i.e., 8 male Sprague-Dawley rats). Protein expression of versican was investigated by immunohistochemistry. Furthermore, the occurrence of versican mRNA was examined by in-situ hybridization.

Results: Positive immunoreactivity for versican was evident in the urothelium but also, weakly, in the detrusor. This expression was localized only in the cytoplasm, leaving the nuclei devoid of reactivity. Interestingly, versican mRNA was only sparsely observed in the urothelial cells.

Conclusions: We found by immunohistochemistry that the substrate for ADAMTS5, versican, was localized in the cytosol of urothelial cells. This demonstrates a difference regarding the expression of ADAMTS5, which was emphasized in the nuclei. This could imply an additional, non-enzymatic, function of ADAMTS5 in the urothelium.

Congo red (CR) is a histological stain used for the detection of extracellular amyloids mediating various neurodegenerative diseases. Given that damaged photoreceptors appear to degenerate similarly to other nerve cells, CR staining was evaluated in experimentally injured porcine retina. CR staining appeared mostly as discrete cytosolic deposits with no obvious plaque formation during the investigated time period. Increases of CR labeling coincided temporally with the known accumulation of mislocalized opsins and increases of cell death. Coculture, either with human retinal pigment epithelium (ARPE) or human neural progenitor (ReN) cells, was accompanied by a significant reduction of CR labeling. Of particular interest was the reduction of CR labeling in cone photoreceptors, which are important for the perception of color and fine details and afflicted in age-related macular degeneration (AMD). Electron microscopy revealed inclusions in the inner segment, cell body, and occasionally synaptic terminals of photoreceptor cells in cultured specimens. Closer examinations indicated the presence of different types of inclusions resembling protein aggregates as well as inclusion bodies. The current results indicate that injury-related response resulted in accumulation of CR deposits in photoreceptor cells, and that trophic and/or structural support attenuated this response.

OBJECTIVE: The aim of this study was to investigate whether protein expression of the extracellular matrix-degrading protease ADAMTS5 can be demonstrated in the urinary bladder of healthy rats, and, if so, to determine the localization of this enzyme.

MATERIALS AND METHODS: The experiments were conducted with eight inbred male Sprague-Dawley rats. Immunohistochemistry was used to investigate the expression of ADAMTS5 in the urinary bladder. Negative controls were established by either excluding the primary antibody or applying the antibody after it had been preabsorbed with its immunogenic peptide. Confocal microscopy was used to visualize the distribution of ADAMTS5 in the urinary bladder tissue.

RESULTS: Immunoreactivity for ADAMTS5 was demonstrated in the urothelium and in the detrusor. This expression was localized not only in the cytoplasm, but also in the nuclei. Confocal microscopy corroborated these findings.

CONCLUSION: Expression of ADAMTS5 was demonstrated in the cytoplasm as well as in the nuclei of the urothelium and detrusor cells, suggesting that it may play a role at the transcriptional level.

Malignant tumors, including breast cancers, are frequently infiltrated with innate immune cells and tumor-associated macrophages (TAMs) represent the major inflammatory component in stroma of many tumors. In this study, we examined the immunoreactivity of the macrophage markers CD68 and CD163 as well as the hormone receptors estrogen receptor alpha (ER alpha), progesterone receptor (PR), estrogen receptor beta 1 (ER beta 1), human epidermal growth factor receptor 2 (HER-2), matrix metalloproteinase 9 (MMP-9), urokinase-type plasminogen activator receptor (uPAR) and the proliferations marker Ki67 in 17 breast cancer biopsies. The quantitative score for CD68(+) and CD163(+) strongly indicate M2 phenotype dominance in the currently investigated biopsies. We found that an increasing level of macrophages was negatively associated with ER alpha or PR, whereas a positive association was observed for Ki-67 or uPAR. No significant association could be seen between the level of macrophage and HER-2, ER beta 1 or MMP-9 expression. Effect of conditioned media (CM) generated from cultured human M1 and M2 macrophage phenotypes were investigated on the proliferation and expression of selected markers in the T47D breast cancer cell line. We found that in contrast to the in vivo situation, in particularly the CM from M1 macrophages decreased the growth and Ki67 expression in T47D, and significantly increased ER beta 1 mRNA levels. Moreover, in accordance to the in vivo situation the CM from the macrophages decreased the expression of ER alpha protein as well as ER alpha or PR mRNA. In conclusion our results show that macrophages alone have the capability to decrease the tumor cell expression of ER alpha and PR in vitro. In the tumor environment in vivo macrophages also contribute to an increase in tumor cell expression of uPAR and Ki67, suggesting that macrophages are involved in impairing the prognosis for breast cancer patients.

Background: Macromolecules, when used as intraperitoneal volume markers, have the disadvantage of leaking into the surrounding tissue. Therefore, Cr-51-labeled erythrocytes were evaluated as markers of intraperitoneal volume and used in combination with I-125-labeled bovine serum albumin to study albumin transport into peritoneal tissues in a rat model of peritoneal dialysis (PD).

Methods: Single dwells of 20 mL of lactate-buffered filter-sterilized PD fluid at glucose concentrations of 0.5%, 2.5%, and 3.9% were performed for 1 or 4 hours. Tissue biopsies from abdominal muscle, diaphragm, liver, and intestine, and blood and dialysate samples, were analyzed for radioactivity.

Results: The dialysate distribution volume of labeled erythrocytes, measured after correction for lymphatic clearance to blood, was strongly correlated with, but constantly 3.3 mL larger than, drained volumes. Erythrocyte activity of rinsed peritoneal tissue biopsies corresponded to only 1 mL of dialysate, supporting our utilization of erythrocytes as markers of intraperitoneal volume. The difference between the distribution volumes of albumin and erythrocytes was analyzed to represent the albumin loss into the peritoneal tissues, which increased rapidly during the first few minutes of the dwell and then leveled out at 2.5 mL. It resumed when osmotic ultrafiltration turned into reabsorption and, at the end of the dwell, it was significantly lower for the highest osmolarity PD fluid (3.9% glucose). Biopsy data showed the lowest albumin accumulation and edema formation in abdominal muscle for the 3.9% fluid.

Conclusion: Labeled erythrocytes are acceptable markers of intraperitoneal volume and, combined with labeled albumin, provided novel kinetic data on albumin transport in peritoneal tissues.

Resistance of tumor cells to chemotherapy, such as 5-fluorouracil (5-FU), is an obstacle for successful treatment of cancer. As a follow-up of a previous study we have investigated the effect of conditioned media (CM) from macrophages of M1 or M2 phenotypes on 5-FU cytotoxicity on the colon cancer cell lines HT-29 and CACO-2. HT-29 cells, but not CACO-2 cells, having been treated with a combination of M1 CM and 5-FU recovered their cell growth to a much larger extent compared to cells having been treated with 5-FU alone when further cultured for 7 days in fresh media. M1 CM treatment of HT-29, but not CACO-2 cells, induced cell cycle arrest in the G(0)/G(1) and G(2)/M phases. 5-FU treatment induced accumulation of cells in S-phase in both HT-29 and CACO-2 cells. This accumulation of cells in S-phase was attenuated by combined M1 CM and 5-FU treatment in HT-29 cells, but not in CACO-2 cells. The mRNA expression of cell cycle regulatory proteins and 5-FU metabolic enzymes were analyzed in an attempt to find possible mechanisms for the M1 CM induced attenuation of 5-FU cytotoxicity in HT-29. Thymidylate synthetase (TS) and thymidine phosphorylase (TP) were found to be substantially downregulated and upregulated, respectively, in HT-29 cells treated with M1 CM, making them unlikely as mediators of reduced 5-FU cytotoxicity. Among cell cycle regulating proteins, p21 was induced in HT-29 cells, but not in CACO-2 cells, in response to M1 CM treatment. However, small interfering RNA (siRNA) knockdown of p21 had no effect on the M1 CM induced cell cycle arrest seen in HT-29 and neither did it change the growth recovery after combined treatment of HT-29 cells with M1 CM and 5-FU. In conclusion, treatment of HT-29 cells with M1 CM reduces the cytotoxic effect of 5-FU and this is mediated by a M1 CM induced cell cycle arrest in the G(0)/G(1) and G(2)/M phases. So far, we lack an explanation why this action is absent in the CACO-2 cells. The current findings may be important for optimization of chemotherapy in colon cancer.

Stromal macrophages of different phenotypes can contribute to the expression of proteins that affects metastasis such as urokinase-type plasminogen activator (uPA), its receptor uPAR, and plasminogen activator inhibitor-1 (PAI-1), but knowledge of how essential their contribution is in comparison to the cancer cells in small cell lung cancer (SCLC) and lung squamous cell carcinoma (SCC) is lacking. The expression of uPA, uPAR, and PAI-1 and of the matrix metalloproteinases (MMP)-2 and MMP-9 were studied in human macrophages of M1 and M2 phenotype and compared to a lung SCC (NCI-H520) and a SCLC (NCI-H69) cell line. Effects of treatment with conditioned media (CM) from M1 and M2 macrophages on the expression of these genes in H520 and H69 cells as well as effects on the cell growth were investigated. In addition, data on the stromal macrophages immunoreactivity of uPAR, MMP-2, and MMP-9 in a few SCC and SCLC biopsies was included. uPAR, MMP-2, and MMP-9 were confirmed in stromal cells including macrophages in the SCC and SCLC biopsies. In vitro, both macrophage phenotypes expressed considerably higher mRNA levels of uPA, uPAR, PAI-1, and MMP-9 compared to the cancer cell lines, and regarding uPAR, the highest level was found in the M1 macrophage phenotype. Furthermore, M1 CM treatment not only induced an upregulation of PAI-1 in both H520 and H69 cells but also inhibited cell growth in both cell lines, giving M1 macrophages both tumor-promoting and tumor-killing potential.

Solid tumors are infiltrated by stroma cells including macrophages and these cells can affect tumor growth, metastasis and angiogenesis. We have investigated the effects of conditioned media (CM) from different macrophages on the proliferation of the colon cancer cell lines HT-29 and CACO-2. CM from THP-1 macrophages and monocyte-derived human macrophages of the M1 phenotype, but not the M2 phenotype, inhibited proliferation of the tumor cells in a dose-dependent manner. Lipopolysaccaharide and interferon gamma was used for differentiation of macrophages towards the M1 phenotype and CM were generated both during differentiation (M1(DIFF)) and after differentiation (M1). M1 and M1(DIFF) CM as well as THP-1 macrophage CM resulted in cell cycle arrest in HT-29 cells with a decrease of cells in S phase and an increase in G(2)/M phase. Treatment of HT-29 cells with M1(DIFF), but not M1 or THP-1 macrophage CM, resulted in apoptosis of about 20% of the tumor cells and this was accompanied by lack of recovery of cell growth after removal of CM and subsequent culture in fresh media. A protein array was used to identify cytokines released from M1 and M2 macrophages. Among the cytokines released by M1 macrophages, tumor necrosis factor alpha and CXCL9 were tested by direct addition to HT-29 cells, but neither affected proliferation. Our results indicate that M1 macrophages inhibit colon cancer cell growth and have the potential of contributing to reducing tumor growth in vivo.

Purpose: Bladder wall nitric oxide production in patients with bladder pain syndrome type 3C is increased compared to undetectable nitric oxide in patients with nonHunner bladder pain syndrome and healthy controls. However, the underlying mechanism/s of the increased nitric oxide production is largely unknown. We compared mRNA expression of a select group of cytokines in patients with bladder pain syndrome/interstitial cystitis type 3C and in pain-free controls.

Materials and Methods: Cold cup biopsies from 7 patients with bladder pain syndrome type 3C and 6 healthy subjects were analyzed. mRNA expression of IL-4, 6, 10 and 17A, iNOS, TNF-alpha, TGF-beta and IFN-gamma was estimated by real-time polymerase chain reaction. IL-17 protein expression was determined by immunohistochemistry. Mast cells were labeled with tryptase to evaluate cell appearance and count.

Results: IL-6, 10 and 17A, and iNOS mRNA levels as well as the number of mast cells infiltrating the bladder mucosa were significantly increased in patients with bladder pain syndrome type 3C compared to healthy controls. TNF-alpha, TGF-beta and IFN-gamma mRNA levels were similar in patients and controls. IL-17A expression at the protein level was up-regulated and localized to inflammatory cells and urothelium in patients with bladder pain syndrome type 3C.

Conclusions: Patients with bladder pain syndrome/interstitial cystitis had increased mRNA levels of IL-17A, 10 and 6, and iNOS. IL-17A might be important in the inflammatory process. To our knowledge the increase in IL-17A is a novel finding that may have new treatment implications.