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Background: In this study we differentially showed the effects of cell-seeded bilayer scaffold wound dressing in accelerating healing process in diabetic ulcers that still remains as a major clinical challenge. The aim of the study was to compare immunomodulatory and angiogenic activity, and regenerative effect differences between Menstrual blood-derived Stem Cells (MenSCs) and foreskin-derived keratinocytes/fibroblasts. Methods: The streptozotocin-induced diabetic mice model was developed in male C57/BL6 mice. A bilayer scaffold was fabricated by electrospining silk fibroin nano-fibers on human Amniotic Membrane (AM). Dermal fibroblasts and keratinocyte isolated from neonatal foreskin and MenSCs were isolated from the menstrual blood of healthy women. The diabetic mice were randomly divided into three groups including no treatment group, fibroblast/keratinocyte-seeded bilayer scaffold group (bSC+FK), and MenSCs-seeded bilayer scaffold group. The healing of full-thickness excisional wounds evaluations in the diabetic mice model in each group were evaluated at 3, 7, and 14 days after treatment. Results: The gross and histological data sets significantly showed wound healing promotion via re-epithelialization and wound contraction along with enhanced regeneration in MenSCs-seeded bilayer scaffold group with the most similarity to adjacent intact tissue. Immunofluorescence staining of mouse skin depicted a descending trend of type III collagen along with the higher expression of involucrin as keratinocyte marker in the MenSCs-seeded bilayer nanofibrous scaffold group in comparison with other treatment groups from day 7 to day 14. Moreover, higher levels of CD31 and von Willebrand factor (VWF), and also a higher ratio of M2/M1 macrophages in association with higher levels of the neural marker were observed in the bSC+MenSCs group in comparison with bSC+FK and no treatment groups. Conclusion: Healing symptoms in wounds dressed with keratinocyte/fibroblast-seeded bilayer scaffold was significantly lower than MenSCs-seeded bilayer scaffold done on impaired diabetic wound chronicity.
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BACKGROUND: Premature ovarian failure (POF) is a well-known cause of infertility, particularly in women under the age of 40. POF is also associated with elevated gonadotropin levels, amenorrhea and sex-hormone deficiency. AIM OF THE STUDY: In this study, the therapeutic potential of autologous mesenchymal stromal cells obtained from menstrual blood (Men-MSCs) for patients with POF was evaluated. METHODS: 15 POF patients were included in the study. The cultured Men-MSCs were confirmed by flow cytometry, karyotype, endotoxin and mycoplasma and were then injected into the patients' right ovary by vaginal ultrasound guidance and under general anesthesia and aseptic conditions. Changes in patients' anti-Müllerian hormone (AMH), antral follicle count (AFC), follicle-stimulating hormone (FSH), luteal hormone (LH), and estradiol (E2) levels, as well as general flushing and vaginal dryness were followed up to one year after treatment. RESULTS: All patients were satisfied with a decrease in general flushing and vaginal dryness. 4 patients (2.9%) showed a spontaneous return of menstruation without additional pharmacological treatment. There was a significant difference in AFC (0 vs. 1 ± 0.92 count, p value ≤0.001%), FSH (74 ± 22.9 vs. 54.8 ± 17.5 mIU/mL, p-value ≤0.05%), E2 (10.2 ± 6 vs. 21.8 ± 11.5 pg/mL p-value ≤0.01%), LH (74 ± 22.9 vs. 54.8 ± 17.5 IU/L,p-value ≤0.01%) during 3 months post-injection. However, there were no significant changes in AMH (p-value ≥0.05%). There were also no significant differences in assessed parameters between 3 and 6 months after cell injection. CONCLUSION: According to the findings of this study, administration of Men-MSCs improved ovarian function and menstrual restoration in some POF patients.
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Células-Tronco Mesenquimais , Insuficiência Ovariana Primária , Feminino , Humanos , Insuficiência Ovariana Primária/terapia , Folículo Ovariano , Hormônio FoliculoestimulanteRESUMO
BACKGROUND: Triple-negative breast cancers (TNBCs) are highly aggressive and metastatic. To date, finding efficacious targeted therapy molecules might be the only window of hope to cure cancer. Fibromodulin (FMOD), is ectopically highly expressed on the surface of Chronic Lymphocytic Leukemia (CLL) and bladder carcinoma cells; thus, it could be a promising molecule for targeted therapy of cancer. The objective of this study was to evaluate cell surface expression of FMOD in two TNBC cell lines and develop an antibody-drug conjugate (ADC) to target FMOD positive TNBC in vitro and in vivo. MATERIALS AND METHODS: Two TNBC-derived cell lines 4T1 and MDA-MB-231 were used in this study. The specific binding of anti-FMOD monoclonal antibody (mAb) was evaluated by flow cytometry and its internalization was verified using phAb amine reactive dye. A microtubulin inhibitor Mertansine (DM1) was used for conjugation to anti-FMOD mAb. The binding efficacy of FMOD-ADC was assessed by immunocytochemistry technique. The anti-FMOD mAb and FMOD-ADC apoptosis induction were measured using Annexin V-FITC and flow cytometry. Tumor growth inhibition of anti-FMOD mAb and FMOD-ADC was evaluated using BALB/c mice injected with 4T1 cells. RESULTS: Our results indicate that both anti-FMOD mAb and FMOD-ADC recognize cell surface FMOD molecules. FMOD-ADC could induce apoptosis in 4T1 and MDA-MB-231 cells in vitro. In vivo tumor growth inhibition was observed using FMOD-ADC in 4T1 inoculated BALB/c mice. CONCLUSION: Our results suggests high cell surface FMOD expression could be a novel bio-marker TNBCs. Furthermore, FMOD-ADC could be a promising candidate for targeting TNBCs.
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Imunoconjugados , Maitansina , Neoplasias de Mama Triplo Negativas , Camundongos , Animais , Humanos , Neoplasias de Mama Triplo Negativas/patologia , Fibromodulina/uso terapêutico , Imunoconjugados/farmacologia , Imunoconjugados/uso terapêutico , Maitansina/uso terapêutico , Modelos Animais de Doenças , Anticorpos Monoclonais/uso terapêutico , Aminas/uso terapêutico , Linhagem Celular TumoralRESUMO
Background: Sortilin has an important role in various malignances and can be used as a promising target to eradicate cancer cells. Methods: In this study, the expression of sortilin in 4T1 and MDA-MB231 cell lines was evaluated by flow cytometry and immunocytochemistry. Apoptosis assay was also applied to evaluate apoptosis induction in 4T1 and MDA-MB231 cell lines. Results: Based on cell surface flow cytometry results, anti-sortilin (2D8-E3) mAb could recognize sortilin molecules in 79.2% and 90.3% of 4T1 and MDA-MB231 cell-lines, respectively. The immunocytochemistry staining results confirmed sortilin surface expression. Apoptosis assay indicated that anti-sortilin mAb could induce apoptosis in 4T1 and MDA-MB231 cell lines. Conclusion: Our study revealed the important role of surface sortilin in breast carcinoma cell survival and its possible application as a therapeutic agent in cancer targeted therapies.
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INTRODUCTION: Impaired chronic wound healing frequently occurs in diabetic patients. We hypothesized that menstrual blood-derived mesenchymal stem cells (MenSCs) in combination with bilayer scaffold consisted of human amniotic membrane (AM) and electrospun silk fibroin nanofibers could potentially promote wound healing in diabetic mice. METHODS & METHODS: Two bilateral full-thickness wounds were created on dorsal skin of type-1 diabetic mice model and animals were equally divided in four groups including: no-treatment group (NT), amniotic membrane treated group (AM), bilayer scaffold treated group (bSC), and MenSCs-seeded bilayer scaffold treated group (bSC + MenSCs). Wound healing evaluations were performed at 3, 7, and 14 days after their treatment. The wound healing was analyzed by macroscopic and microscopic evaluations, and immunofluorescence staining of involucrin (IVL), type III collagen, CD31/ von Willebrand factor (vWF), and PGP9.5 were performed. Furthermore, number of neutrophils and macrophages and subpopulation of macrophages were assessed. In addition, the expression of Egr2, Mmp9, CXCL12, IDO1, Ptgs2 and VEGFA transcripts involved in wound repair were also analyzed. RESULTS: After 14 days, the best epidermal and dermal regeneration belonged to the cases received bSC + MenSCs as wound dressing. Moreover, the wound healing was typically faster in this group compared to other groups. Immunofluorescence evaluation represented higher levels of CD31 and VWF, higher ratio of M2/M1 macrophages, greater expression of IVL, and higher levels of the PGP9.5 in the bSC + MenSCs group in comparison with other groups. Expression analysis of assessed genes also supported assumption of more regeneration and healing in the bSC + MenSCs group versus other groups. CONCLUSION: These results indicate that enhanced immunomodulatory and reparative properties of MenSCs in conjunction with bilayer scaffold specified this cellular skin substitute for modulating wound chronicity and contribution to resolution of wound healing process in diabetic ulcer.
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Curativos Biológicos , Diabetes Mellitus Experimental/complicações , Fibroínas/uso terapêutico , Transplante de Células-Tronco Mesenquimais , Alicerces Teciduais , Cicatrização , Animais , Feminino , Humanos , Masculino , Menstruação , Células-Tronco Mesenquimais , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Wound healing process is a complex of overlapping and coordinated events progresses beyond the inflammatory phase toward wound resolution, whereas chronic wounds fail to terminate inflammatory phase and could not develop toward regenerative state. The immunopathology of chronic wounds has been attributed to the prolonged inflammation and dysregulation of microenvironments responsible for imbalance between pro-inflammatory and anti-inflammatory states, as well as cellular and tissue senescence. We here discuss that menstrual blood-derived mesenchymal stem cells (MenSCs) with their authentic functions especially immunosuppressive, angiogenic and migratory properties in combination with a bilayer amniotic membrane/nano-fibrous fibroin scaffold could bring about effective regenerative effects in healing of chronic wound. To debate, following evidences have been cumulated : 1) Persistent pro-inflammatory state in chronic wound bed could inhibit wound resolution; 2) MenSCs exhibit noticeable regenerative, immunosuppressive effects and immunomodulatory activity, 3) The migratory characteristics of MenSCs may not be sufficient for their homing to chronic wounds site, and 4) Bilayer scaffold composed of amniotic membrane and silk fibroin induces MenSCs differentiation into keratinocyte-like cells and stimulates skin regeneration.
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Células-Tronco/fisiologia , Cicatrização , Âmnio , Animais , Diabetes Mellitus , Fibroínas , Humanos , Imunomodulação , Menstruação , Alicerces TeciduaisRESUMO
OBJECTIVE: Silymarin as an herbal drug has potent antioxidant effects that could make it a good choice for endometriosis therapy. The aim of the current study was to determine the effects of silymarin as an herbal drug on induced endometrial lesion in rat model of endometriosis. MATERIALS AND METHODS: A total of 32 mature, female Sprague-Dawley rats were allocated into 4 experimental groups. The duration of study was about 6 months. Endometriosis implants were surgically prepared and autografted into 32 rats. Three weeks after endometriosis induction, animals were randomly allocated into four groups: Group 1 received cabergoline (CAB group); Group 2 received letrozole (LET group); Group 3 received silymarin (SIL group) and Group 4 received no medication (CONT group). Experimental groups were treated for 3 weeks and then were sacrificed for volume and histopathological evaluation of implants and biochemical assessment. Serum and peritoneal levels of vascular endothelial growth factor (VEGF), total antioxidant activity (TAC) and tumor necrosis (TNF)-α were measured. RESULTS: Mean volume of the implants decreased significantly in silymarin (p < 0.001), letrozole (p < 0.001) and cabergoline (p < 0.001) groups compared to the control. Histopathologic score was significantly lower in silymarin (p: 0.039), letrozole (p: 0.017) and cabergoline (p < 0.001) groups compared to the control. Those receiving silymarin had significantly higher serum TAC compared to control after 21 days of therapy (p < 0.001). CONCLUSION: Silymarin, Letrozole, and Cabergoline administration resulted in decreased size and histopathologic grade of the induced endometrial lesions in a rat model. Silymarin appears to be a virtual novel therapeutic agent for treatment of endometriosis.
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Antioxidantes/administração & dosagem , Endometriose/tratamento farmacológico , Silimarina/administração & dosagem , Animais , Inibidores da Aromatase/administração & dosagem , Cabergolina/administração & dosagem , Modelos Animais de Doenças , Agonistas de Dopamina/administração & dosagem , Endometriose/patologia , Feminino , Letrozol/administração & dosagem , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Fator de Necrose Tumoral alfa/sangue , Fator A de Crescimento do Endotélio VascularRESUMO
BACKGROUND: 17ß-estradiol (E2) has been known to modulate immune response. Recent studies indicate that E2 at pregnancy level plays a role in regulating T cell response. OBJECTIVE: To investigate the optimum dose of E2 (from 10-9 to 10-7 M) in mediating the generation of regulatory T cells (Tregs), using naive human CD4+ T cells from healthy women. METHODS: Naive peripheral T cells were purified and conditioned with soluble anti-CD28 in anti-CD3-coated plates in the presence or absence of E2. Flow cytometry was employed to assess the expression pattern of forkhead boxP3 (FOXP3) and programmed death-1 (PD-1). Proliferation and cytokine secretions were analyzed, using XTT and ELISA assays. RESULTS: In the presence of different doses of E2, the expression levels of anti-CD3/CD28 antibody-stimulated CD25/ FOXP3 and FOXP3/PD-1 in conditioned T cells (cT) were peaked at 1 ng/ml (early pregnancy level, E2(1)) (47.14% (37.3-74.9) and 32% (27.7-52.5), respectively) and a slight, but not significant, increase after declining at 36 ng/ml (late pregnancy/pharmaceutical, E2(36)) (19.4% (15.2-24.5) and 15.8% (10.6-26.8), respectively). E2(1) cT showed a significantly reduced proliferation capacity (p<0.05) and secretion of IL-10 was enhanced in supernatants of E2(1 and 36) cT (p<0.05). In contrast to decreased TNF-α and IFN-γ secretions in E2(1) cT supernatants, E2(36) stimulated TNF-α and IFN-γ secretions (p<0.05 and p<0.01, respectively). CONCLUSION: Our results indicate that the differential effect of E2 on generation of Tregs is consistent with the possibility that lower levels of pregnancy E2 are most efficient in induction of Tregs.
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Estradiol/farmacologia , Subpopulações de Linfócitos T/efeitos dos fármacos , Subpopulações de Linfócitos T/imunologia , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/imunologia , Adulto , Apoptose/efeitos dos fármacos , Apoptose/imunologia , Biomarcadores , Células Cultivadas , Citocinas/biossíntese , Feminino , Citometria de Fluxo , Humanos , Imunofenotipagem , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Fenótipo , Subpopulações de Linfócitos T/metabolismo , Linfócitos T Reguladores/metabolismoRESUMO
Kisspeptins (KPs) are major regulators of trophoblast and cancer invasion. Thus far, limited and conflicting data are available on KP-mediated modulation of breast cancer (BC) metastasis; mostly based on synthetic KP-10, the most active fragment of KP. Here, we report for the first time comprehensive functional effects of term placental KPs on proliferation, adhesion, Matrigel invasion, motility, MMP activity and pro-inflammatory cytokine production in MDA-MB-231 (estrogen receptor-negative) and MCF-7 (estrogen receptor-positive). KPs were expressed at high level by term placental syncytiotrophoblasts and released in soluble form. Placental explant conditioned medium containing KPs (CM) significantly reduced proliferation of both cell types compared to CM without (w/o) KP (CM-w/o KP) in a dose- and time-dependent manner. In MDA-MB-231 cells, placental KPs significantly reduced adhesive properties, while increased MMP9 and MMP2 activity and stimulated invasion. Increased invasiveness of MDA-MB-231 cells after CM treatment was inhibited by KP receptor antagonist, P-234. CM significantly reduced motility of MCF-7 cells at all time points (2-30 hr), while it stimulated motility of MDA-MB-231 cells. These effects were reversed by P-234. Co-treatment with selective ER modulators, Tamoxifen and Raloxifene, inhibited the effect of CM on motility of MCF-7 cells. The level of IL-6 in supernatant of MCF-7 cells treated with CM was higher compared to those treated with CM-w/o KP. Both cell types produced more IL-8 after treatment with CM compared to those treated with CM-w/o KP. Taken together, our observations suggest that placental KPs differentially modulate vital parameters of estrogen receptor-positive and -negative BC cells possibly through modulation of pro-inflammatory cytokine production.
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Apoptose , Neoplasias da Mama/patologia , Movimento Celular , Kisspeptinas/metabolismo , Placenta/metabolismo , Receptores de Estrogênio/metabolismo , Adulto , Western Blotting , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Proliferação de Células , Feminino , Humanos , Técnicas Imunoenzimáticas , Imunoprecipitação , Kisspeptinas/genética , Gravidez , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Estrogênio/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais Cultivadas , Cicatrização , Adulto JovemRESUMO
PURPOSE: Cisplatin as a platinum (Pt)-based chemotherapeutic compound is commonly applied for the treatment of several types of cancer. Nonetheless, drug resistance and severe adverse effects have been observed upon using cisplatin. Here, we have explored the cytotoxicity of novel Pt-based compounds on several cancer cell lines. METHODS: Five synthetic Pt compounds as well as cisplatin were investigated by XTT assay to determine their cytotoxicity against cell lines originated from prostate, ovary, and breast cancers at different time periods at various concentrations. Additionally, the apoptosis rate in cell lines was determined using flow cytometry. Binding to DNA was investigated through spectrophotometric and viscometric studies. RESULTS: With the exception of one compound, all of the Pt-complexes effectively killed the prostate cancer cell lines (i.e. PC-3 and DU 145). One compound, [Pt(2,2'- dipyridylamine)Cl4].DMF, was chosen as the most potent compound due to its high selective cytotoxic activity and its cytotoxicity was further tested and compared with that of cisplatin on SKOV-3, Caov-4, MDA-MB-231, and MCF7 cell lines. [Pt(2,2'-dipyridylamine)Cl4].DMF had a higher selective cytotoxic capacity in comparison with cisplatin at higher concentrations and longer culture periods. Furthermore, as related to apoptosis induction, treatment with [Pt(2,2'-dipyridylamine)Cl4 ].DMF was significantly more effective than that of cisplatin in five out of six examined cell lines. [Pt(2,2'-dipyridylamine)Cl4].DMF was shown to intercalate into DNA. CONCLUSIONS: The current study introduced a novel Pt-based complex with highly selective and potent in vitro anti-tumor impacts superior to those of cisplatin, a conventional chemotherapeutic agent. [Pt (2,2'-dipyridylamine)Cl4].DMF could be regarded as a promising antitumor agent in future investigations.
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Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos , Substâncias Intercalantes/farmacologia , Compostos Organoplatínicos/farmacologia , 2,2'-Dipiridil/análogos & derivados , 2,2'-Dipiridil/química , Líquido Amniótico/citologia , Antineoplásicos/química , Neoplasias da Mama/patologia , Cisplatino/química , DNA/química , Dimetilformamida/química , Células Epiteliais/efeitos dos fármacos , Feminino , Humanos , Substâncias Intercalantes/química , Células MCF-7 , Masculino , Compostos Organoplatínicos/química , Neoplasias Ovarianas/patologia , Platina/química , Neoplasias da Próstata/patologia , EspectrofotometriaRESUMO
PURPOSE: Cisplatin as a platinum (Pt)-based chemotherapeutic compound is commonly applied for the treatment of several types of cancer. Nonetheless, drug resistance and severe adverse effects have been observed upon using cisplatin. Here, we have explored the cytotoxicity of novel Pt-based compounds on several cancer cell lines. METHODS: Five synthetic Pt compounds as well as cisplatin were investigated by XTT assay to determine their cytotoxicity against cell lines originated from prostate, ovary, and breast cancers at different time periods at various concentrations. Additionally, the apoptosis rate in cell lines was determined using flow cytometry. Binding to DNA was investigated through spectrophotometric and viscometric studies. RESULTS: With the exception of one compound, all of the Pt-complexes effectively killed the prostate cancer cell lines (i.e. PC-3 and DU 145). One compound, [Pt(2,2'- dipyridylamine)Cl4].DMF, was chosen as the most potent compound due to its high selective cytotoxic activity and its cytotoxicity was further tested and compared with that of cisplatin on SKOV-3, Caov-4, MDA-MB-231, and MCF7 cell lines. [Pt(2,2'-dipyridylamine)Cl4].DMF had a higher selective cytotoxic capacity in comparison with cisplatin at higher concentrations and longer culture periods. Furthermore, as related to apoptosis induction, treatment with [Pt(2,2'-dipyridylamine)Cl4 ].DMF was significantly more effective than that of cisplatin in five out of six examined cell lines. [Pt(2,2'-dipyridylamine)Cl4].DMF was shown to intercalate into DNA. CONCLUSIONS: The current study introduced a novel Pt-based complex with highly selective and potent in vitro anti-tumor impacts superior to those of cisplatin, a conventional chemotherapeutic agent. [Pt (2,2'-dipyridylamine)Cl4].DMF could be regarded as a promising anti-tumor agent in future investigations.
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BACKGROUND: Toll-like receptor (TLR)-mediated inflammatory processes are supposed to be involved in pathophysiology of spontaneous abortion and preterm labor. Here, we investigated functional responses of human endometrial stromal cells (ESCs) and whole endometrial cells (WECs) to lipopolysaccharide (LPS) and lipoteichoic acid (LTA). METHODS: Endometrial tissues were obtained from 15 cycling women who underwent laparoscopic tubal ligation. Modulation of TLR2, TLR4 and MyD88 expression and production of pro-inflammatory cytokines by WECs and ESCs in response to LPS and LTA were assessed. RESULTS: WECs and ESCs expressed significant levels of TLR4 and MyD88 transcripts but, unlike WECs, ESCs failed to express TLR2 gene. Regardless of positive results of Western blotting, ESCs did not express TLR4 at their surface as judged by flow cytometry. Immunofluorescent staining revealed intracellular localization of TLR4 with predominant perinuclear pattern. LPS stimulation marginally increased TLR4 gene expression in both cell types, whereas such treatment significantly upregulated MyD88 gene expression after 8 hr (p < 0.05). At the protein level, however, LPS activation significantly increased TLR4 expression by ESCs (p < 0.05). LTA stimulation of WECs was accompanied with non-significant increase of TLR2 and MyD88 transcripts. LPS and LTA stimulation of WECs caused significant production of IL-6 and IL-8 in a dose-dependent manner (p < 0.05). Similarly, ESCs produced significant amounts of IL-6, IL-8 and also TNF-α in response to LPS activation (p < 0.05). CONCLUSION: Our results provided further evidence of initiation of inflammatory processes following endometrial TLR activation by bacterial components which could potentially be harmful to developing fetus.
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BACKGROUND: Many types of tumors are organized in a hierarchy of heterogeneous cell populations with different molecular signature. Such heterogeneity may be associated with different responsiveness to microenvironment stimuli. In the present study, the effects of lipopolysaccharide (LPS) and lipoteichoic acid (LTA), as well-known mediators of inflammation, on cancerous behavior of three prostate tumor cells, LNCaP, PC3 and DU145, were investigated. METHODS: Expression of TLR1-10, CD14 and MyD88 transcripts was investigated by RT-PCR. Protein expression of TLR2 and 4 was scrutinized by flow cytometry, immunofluorescent staining and Western blotting. Experiments were set up to assess the effects of LPS and LTA at different concentrations and times on cell proliferation, extracellular matrix invasion, adhesion and cytokine production. RESULTS: We showed that prostate cancer cell lines differentially express TLR1-10, MyD88 and CD14 transcripts. DU145 failed to express TLR4 gene. Positively-identified TLR2 protein in all prostate cancer cells and TLR4 protein in PC3 and LNCaP by Western blotting was not accompanied by cell surface expression, as judged by flow cytometry. Immunofluorescent staining clearly demonstrated predominantly perinuclear localization of TLR2 and TLR4. LTA activation of all prostate cancer cells significantly increased cell proliferation. Regardless of lacking TLR4, DU145 cells proliferated in response to LPS treatment. While LPS caused increased invasiveness of LNCaP, invasive capacity of PC3 was significantly reduced after LPS or LTA stimulation. Stimulation of all prostate tumor cells with LTA was associated with increased cell adhesion and IL-8 production. IL-6 production, however, was differentially regulated by LPS stimulation in prostate tumor cells. CONCLUSION: The data shows that cancer cells originated from the same histologically origin exhibit heterogeneous response to the same TLR ligand. Therefore, a thorough and comprehensive judgment on how and to what extent a particular cancer is affected by TLR agonist could not be inferred by studying an individual cell line.
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BACKGROUND: Despite the extensive information available in the literature, cell surface marker signature of human Amniotic Epithelial Cells (hAECs) remains controversial. The aim of the present study was to characterize immunophenotypic features, proliferative capacity and immunogenicity of hAECs. We also tested whether expression of some cell surface markers is influenced by the type of trypsin used for tissue digestion. METHODS: Single cell suspensions of amniotic membranes from four human placentas were isolated by enzymatic digestion and expression of CD9, CD10, CD29, CD34, CD38, CD44, CD45, CD73, CD105, CD133, HLA-I, HLA-DR, HLA-G, SSEA-4, STRO-1 and OCT-4 was then evaluated by flow cytometry. The differential impact of four trypsin types on the yield and expression of CD105 and HLA-I was also determined. The proliferative capacity of cultured hAECs was assessed and compared in the presence and absence of Epidermal Growth Factor (EGF). To test their immunogenicity, hAECs were injected into Balb/c mice and the reactivity of hyperimmunized sera was examined by immunofluorescence staining. RESULTS: Nearly all purified cells expressed mesenchymal markers, CD9, CD10, CD29, and CD73 and the embryonic marker, SSEA-4. A large proportion of the cells also expressed STRO-1 and OCT-4. The purified cells also expressed HLA-G and HLA-I. A very small proportion of hAECs expressed CD34, CD38, CD44, CD133 and HLA-DR. The type of trypsin used for enzymatic digestion affected both the percentage and expression of HLA-I and CD105. hAECs revealed substantial proliferative capacity only when cultured in the medium supplemented with EGF. These cells were shown to be capable of inducing high amounts of anti-donor antibodies. CONCLUSION: Here we provided evidence that hAECs are immunogenic cells with high level of HLA-I expression. Furthermore, this work highlighted the impact of isolation procedure on the immunophenotype of hAEC.
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Vitamin D has been introduced as one of the main regulators of spermatogenesis. Here, for the first time, we evaluated the expression of vitamin D receptor (VDR) and 1α-hydroxylase in all organs of male mice reproductive tract by immunohistochemistry and Western blotting. Epithelial cells of epididymis, seminal vesicle, coagulating gland, ductus deferens, preputial gland, and prostate were the prominent cell types that concomitantly expressed VDR and 1α-hydroxylase. Nearly all cell types in testis expressed both proteins. Interestingly, VDR intensity in epididymis epithelial cells was reduced toward cauda, in which only strong staining of stereocilia was observed. Although been positive in caput epididymis, sperms lost their VDR expression in cauda region. In all organs, sperms failed to express 1α-hydroxylase. Specific bands of the VDR and 1α-hydroxylase were determined in all tissues, except testis in which novel unprecedented isoforms of 1α-hydroxylase were observed. Our findings could provide further convincing evidence of pivotal role of this hormone in male reproductive biology.
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25-Hidroxivitamina D3 1-alfa-Hidroxilase/metabolismo , Genitália Masculina/metabolismo , Receptores de Calcitriol/metabolismo , Animais , Células Epiteliais/metabolismo , Genitália Masculina/citologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Próstata/citologia , Próstata/metabolismo , Glândulas Seminais/citologia , Glândulas Seminais/metabolismo , Testículo/citologia , Testículo/metabolismo , Ducto Deferente/citologia , Ducto Deferente/metabolismoRESUMO
Here we introduce novel optical properties and accurate sensitivity of Quantum dot (QD)-based detection system for tracking the breast cancer marker, HER2. QD525 was used to detect HER2 using home-made HER2-specific monoclonal antibodies in fixed and living HER2(+) SKBR-3 cell line and breast cancer tissues. Additionally, we compared fluorescence intensity (FI), photostability and staining index (SI) of QD525 signals at different exposure times and two excitation wavelengths with those of the conventional organic dye, FITC. Labeling signals of QD525 in both fixed and living breast cancer cells and tissue preparations were found to be significantly higher than those of FITC at 460-495 nm excitation wavelengths. Interestingly, when excited at 330-385 nm, the superiority of QD525 was more highlighted with at least 4-5 fold higher FI and SI compared to FITC. Moreover, QDs exhibited exceptional photostability during continuous illumination of cancerous cells and tissues, while FITC signal faded very quickly. QDs can be used as sensitive reporters for in situ detection of tumor markers which in turn could be viewed as a novel approach for early detection of cancers. To take comprehensive advantage of QDs, it is necessary that their optimal excitation wavelength is employed.
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Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/metabolismo , Pontos Quânticos , Receptor ErbB-2/metabolismo , Anticorpos Monoclonais/imunologia , Western Blotting , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Ensaio de Imunoadsorção Enzimática , Feminino , Imunofluorescência , Humanos , Limite de Detecção , Receptor ErbB-2/imunologiaRESUMO
OBJECTIVE: To determine the geographical distribution of Leishmania species causing cutaneous leishmaniasis (CL) and to study the genetic heterogeneity of Leishmania major isolates from different endemic areas of Iran. METHODS: A total of 341 isolates from lesions of patients living in 11 provinces of Iran were grown in culture medium and inoculated to BALB/c mice to detect possible visceralisation. The species were identified by isoenzyme analysis using a battery of six enzymes and kinetoplast (k) DNA-PCR technique. Genetic variation among L. major isolates was analysed by random amplified polymorphic DNA (RAPD) technique. RESULTS: Of the total 341 isolates, 283 isolates were L. major and 58 isolates were Leishmania tropica. In rural areas, the causative agent of CL was mainly L. major (95%L. major vs. 5%L. tropica), in urban areas it was L. tropica (65%L. tropica vs. 35%L. major). All isolates of L. major and 8.6% of L. tropica isolates showed visceralisation in BALB/c mice. There is considerable genetic diversity between L. major strains from different endemic areas and even between some isolates of the same endemic area. CONCLUSION: Leishmania major is the most frequent species in the endemic areas of CL in eleven provinces of Iran, and genetic diversity is a common feature of L. major in the country.
Assuntos
Leishmania major/genética , Leishmania tropica/isolamento & purificação , Leishmaniose Cutânea/epidemiologia , Leishmaniose Cutânea/genética , Animais , DNA de Cinetoplasto/genética , Humanos , Irã (Geográfico)/epidemiologia , Leishmania major/isolamento & purificação , Leishmania tropica/genética , Linfonodos/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Epidemiologia Molecular , Técnica de Amplificação ao Acaso de DNA Polimórfico , População Rural , Baço/parasitologia , População UrbanaRESUMO
In recent decades, stem cell therapy has been introduced as a novel therapeutic approach for patients suffering from bone disorders. Recently, menstrual blood has been identified as an easily accessible and recycled stem cell source. However, the osteogenic differentiation capacity of menstrual blood-derived stem cells (MenSCs) compared with other adult stem cells remained unsolved. The aim of this study was to investigate the osteogenic differentiation capacity of MenSCs compared to bone marrow-derived mesenchymal stem cells (BMSCs) in the presence of human platelet releasate (HPR). Our results showed that MenSCs were strongly positive for mesenchymal and negative for hematopoietic stem cell markers in a similar manner to BMSCs. In contrary to BMSCs, MenSCs exhibited marked expression of OCT-4 and a significantly higher proliferative capacity. Mineralization, as judged by alizarin red staining, was more pronounced in differentiated BMSCs than in differentiated MenSCs in an osteogenic medium fortified with fetal bovine serum (FBS). However, FBS substitution with HPR in a differentiation medium resulted in typical impact on intensity of MenSC mineralization. The results of semiquantitative reverse transcription-polymerase chain reaction showed comparable levels of parathyroid hormone receptor and osteocalcin transcripts in both types of differentiated stem cells in an HPR medium supplemented with osteogenic inducers. However, the upregulation level of alkaline phosphatase was relatively lower in differentiated MenSCs than that in differentiated BMSCs. We concluded that despite lower osteogenic differentiation capacity of MenSCs compared to BMSCs, substitution of FBS with HPR could equalize the osteogenic differentiation of MenSCs. Therefore, by taking advantage of osteogenic driving potential of HPR, MenSCs could be introduced as an apt and safe alternative to BMSCs for bone tissue-engineering purposes.
Assuntos
Células Sanguíneas/citologia , Plaquetas/metabolismo , Células da Medula Óssea/citologia , Diferenciação Celular , Menstruação , Osteogênese , Células-Tronco/citologia , Adulto , Proliferação de Células , Separação Celular , Forma Celular , Feminino , Antígenos HLA/metabolismo , Humanos , Imunofenotipagem , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Osteoblastos/citologia , Adulto JovemRESUMO
Menstrual blood is easily accessible, renewable, and inexpensive source of stem cells. In this study, we investigated the chondrogenic differentiation potential of menstrual blood-derived stem cells (MenSCs) compared with that of bone marrow-derived stem cells (BMSCs) in two-dimensional culture. Following characterization of isolated cells, the potential for chondrogenic differentiation of MenSCs and BMSCs was evaluated by immunocytochemical and molecular experiments. MenSCs were strongly positive for mesenchymal stem cell markers in a manner similar to that of BMSCs. In contrast to BMSCs, MenSCs exhibited marked expression of OCT4, and higher proliferative capacity. Differentiated MenSCs showed strong immunoreactivity to a monoclonal antibody against Collagen type 2, in a pattern similar to BMSCs. Accumulation of proteoglycans in differentiated MenSCs was also comparable with that in differentiated BMSCs. However, the mRNA expression patterns as judged by RT-PCR of chondrogenic markers such as Collagen 2A1, Collagen 9A1 and SOX9 in MenSCs were different from those in BMSCs. Given these findings, MenSCs appear to be a unique stem cell population with higher proliferation than and comparable chondrogenic differentiation ability to BMSCs in two-dimensional culture. Much quantitative studies at the molecular level may elucidate the reasons for the observed differences in MenSCs and BMSCs.