A Comparative Study of Antimicrobial Activity of Proroot MTA, Root MTA, and Portland Cement on Actinobacillus Actinomycetemcomitans

INTRODUCTION: The purpose of our study was to evaluate the antibacterial effect of ProRoot MTA (PRMTA), Root MTA (RMTA) and Portland cement (PC) at their clinical concentration (70 mg/25 µL) against Actinobacillus actinomycetemcomitans (Aa) one of the prominent periodontal (pocket) microorganisms. MATERIALS AND METHODS: Agar diffusion test on Blood Agar with Hemin and Vitamin K (BAHV) was employed in this study. The microorganisms were seeded on the BAHV by spreaders. Small holes, 6 mm in diameter, were made in the BAHV by removing agar. PRMTA, RMTA and PC were placed into the wells immediately after manipulation. The plates were incubated in anaerobic atmosphere at 37°C for 72 h and the zones of inhibition were measured. RESULTS: In the agar diffusion test PRMTA, RMTA and PC against Aa showed zones of inhibition. Analyzing the antimicrobial activity of PRMTA, RMTA and PC according to paired one-way ANOVA and Post Hoc Test (Turkey's test) analysis showed a statistically significant difference (P<0.05) between PRMTA, RMTA and PC. RMTA showed the largest zone of inhibition (29 mm) against Aa. There was no difference in the zones of inhibition between the 48 and 72 h time periods. CONCLUSION: In this in vitro study PRMTA and RMTA presented similar antimicrobial activity against Aa

Inhibition of mitochondrial respiration prevents BRAF-mutant melanoma brain metastasis

Melanoma patients carry a high risk of developing brain metastases, and improvements in survival are still measured in weeks or months. Durable disease control within the brain is impeded by poor drug penetration across the blood-brain barrier, as well as intrinsic and acquired drug resistance. Augmented mitochondrial respiration is a key resistance mechanism in BRAF-mutant melanomas but, as we show in this study, this dependence on mitochondrial respiration may also be exploited therapeutically. We first used high-throughput pharmacogenomic profiling to identify potentially repurposable compounds against BRAF-mutant melanoma brain metastases. One of the compounds identified was β-sitosterol, a well-tolerated and brain-penetrable phytosterol. Here we show that β-sitosterol attenuates melanoma cell growth in vitro and also inhibits brain metastasis formation in vivo. Functional analyses indicated that the therapeutic potential of β-sitosterol was linked to mitochondrial interference. Mechanistically, β-sitosterol effectively reduced mitochondrial respiratory capacity, mediated by an inhibition of mitochondrial complex I. The net result of this action was increased oxidative stress that led to apoptosis. This effect was only seen in tumor cells, and not in normal cells. Large-scale analyses of human melanoma brain metastases indicated a significant role of mitochondrial complex I compared to brain metastases from other cancers. Finally, we observed completely abrogated BRAF inhibitor resistance when vemurafenib was combined with either β-sitosterol or a functional knockdown of mitochondrial complex I. In conclusion, based on its favorable tolerability, excellent brain bioavailability, and capacity to inhibit mitochondrial respiration, β-sitosterol represents a promising adjuvant to BRAF inhibitor therapy in patients with, or at risk for, melanoma brain metastases.publishedVersio

Initial characterization of tumor heterogeneity in a syngeneic breast cancer model

Breast cancer is the most common cancer in women and the main reason of cancer death in the less developed countries. Metastasis is the leading cause of death in breast cancer patients. Due to the complexity of this cascade, many aspects of metastasis are still unknown and we still lack therapy to cure or prevent metastatic breath cancer. Differences in tumor microenvironment influence tumor characteristics. Infiltrated immune cells play an important role in cancer related inflammation and may promote metastasis. Macrophages are member of innate immunity and represent a major component of infiltrated cells within solid tumors. They can be classified to classically activated macrophages (M1) and alternatively activated macrophages (M2). Tumor associated macrophages (TAMs) contribute in initiation and progression of tumors and are predominantly M2 macrophages. Animal models have been developed for studying function of macrophages in the tumor microenvironment. However, designing in vitro models that may mimic interaction between cancer cells and macrophages could simplify the studies of the multiple interactions in the tumor environment and may lead to better understanding of metastatic cascade. This study was focused on a syngeneic mouse model with a functioning immune system. A model of two BALB/cfC3H mouse cell lines, 66cl4 and 67NR isolated from the same primary tumor but with a different metastatic potential was chosen. Analyzes of transcriptome sequencing data obtained from the cell lines grown in culture and as tumors in mice demonstrated numerous transcripts present in the tumor but lacking in the cell lines. These differences in the transcriptome indicated several types of stroma cells recruited into the tumors. Comparing two mouse mammary primary tumors, 66cl4 (metastatic) and 67NR (non-metastatic), suggested that almost 40% of 66cl4 and 30% of 67NR primary tumors are consist of non-tumor cells. M2 macrophages are known for their contribution in breast tumor metastasis and progression. Presence of M2 macrophages in 66cl4 primary tumor, may correlates with metastatic potential of this primary tumor. In an attempt to mimic the interaction between the two cancer cell lines and immune cells, the macrophage cell lines RAW264.7 and IC-21 cell line was tested. The obtained results suggest that these two transformed macrophage cell lines are not responsive to signal emerging from the cancer cells. This study suggests that RAW264.7 and IC-21 cell lines cannot be ideal models for studying this interaction but that primary macrophages represents a better option for studies of how cancer cells may regulate normal macrophage function

Utility of Western Blot Analysis for the Diagnosis of Cutaneous Leishmaniasis

Background: Cutaneous leishmaniasis (CL) is a parasitic disease with a relatively wide distribution in different areas of the world, including Iran. The parasite is mainly diagnosed microscopically, but serological approaches might be useful for diagnosis as well.  This study aimed to assess the efficacy of an immunoblotting system for serodiagnosis of cutaneous leishmaniasis in Iran. Methods: Sixty-one sera samples from parasitologically confirmed CL patients and 50 sera samples from healthy controls along with 50 sera sample from non-CL patients were collected. Native strain of Leishmania major was cultured in Schnei­der medium and soluble Leishmania antigens were prepared from amastigotes-like parasites. All of sera samples were evaluated by an immunoblot­ting system. Results: Components of 14 to 135 kDa were detectable by the sera of CL pa­tients. From 61 sera of CL patients, 59 cases (96.7%) detected a 63 kDa subunit and 51 cases (83.6%) recognized a 32-35 kDa component. Among all subunits, the 63 kDa band showed the highest sensitivity (96.7%) and a 75 kDa band had the highest (98%) specificity. Conclusion: Immunoblotting has a satisfactory performance in diagnosis of CL and this test can be used, as an aid, for proper diagnosis of CL

Performance of an ELISA and Indirect Immunofluorescence Assay in Serological Diagnosis of Zoonotic Cutaneous Leishmaniasis in Iran

Serological assays have been extensively evaluated for diagnosis of visceral leishmaniasis (VL) and considered as a routine method for diagnosis of VL while these methods are not properly evaluated for diagnosis of cutaneous leishmaniasis (CL). This study aimed to assess the performance of indirect immunofluorescent-antibody test (IFA) and enzyme-linked immunosorbent assay (ELISA) for serodiagnosis of cutaneous leishmaniasis in Iran. Sixty-one sera samples from parasitologically confirmed CL patients and 50 sera from healthy controls along with 50 sera from non-CL patients were collected. Antigen was prepared from promastigotes and amastigotes of Leishmania major. IFA was used to detect anti-Leishmania IgG while ELISA was used to detect anti-Leishmania IgM, total IgG, or IgG subclasses (IgG1 and 4). ELISA, for detection of total IgG and IgM, showed sensitivity of 83.6% and 84.7% and specificity of 62.7% and 54.6%, respectively. Sensitivity and specificity of ELISA for detecting IgG1 and IgG4 were 64%, 75% and 85%, 49%, respectively. Sensitivity and specificity of IFA were 91.6% and 81%. Conclusion. Findings of this study demonstrated that serological test, especially IFA, can be used for proper diagnosis of CL

Increased infiltration and tolerised antigen-specific CD8+ TEM cells in tumor but not peripheral blood have no impact on survival of HCMV+ glioblastoma patients

Human cytomegalovirus (HCMV) antigens in glioblastoma (GBM) present opportunities for personalised immunotherapy. However, their presence in GBM tissue is still under debate, and evidence of their impact on functional immune responses and prognosis is sparse. Here, we investigated the presence of pp65 (UL83) and immediate early 1 (IE-1) HCMV antigens in a cohort of Norwegian GBM patients (n = 177), using qPCR, immunohistochemistry, and serology. HCMV status was then used to investigate whether viral antigens influenced immune cell phenotype, infiltration, activation and patient survival. Pp65 and IE-1 were detected by qPCR in 23% and 43% of GBM patients, respectively. Furthermore, there was increased seropositivity in GBM patients relative to donors (79% vs. 48%, respectively; Logistic regression, OR = 4.05, 95%CI [1.807-9.114], P = 0.001, also when adjusted for age (OR = 2.84, 95%CI [1.110-7.275], P = 0.029). Tissue IE-1-positivity correlated with increased CD3+CD8+ T-cell infiltration (P < 0.0001), where CD8+ effector memory T (TEM) cells accounted for the majority of CD8+T cells compared with peripheral blood of HCMV+ patients (P < 0.0001), and HCMV+ (P < 0.001) and HCMV− (P < 0.001) donors. HLA-A2/B8-restricted HCMV-specific CD8+ T cells were more frequent in blood and tumor of HCMV+ GBM patients compared with seronegative patients, and donors irrespective of their serostatus. In biopsies, the HCMV-specific CD8+ TEM cells highly expressed CTLA-4 and PD-1 immune checkpoint protein markers compared with populations in peripheral blood (P < 0.001 and P < 0.0001), which expressed 3-fold greater levels of CD28 (P < 0.001 and P < 0.0001). These peripheral blood T cells correspondingly secreted higher levels of IFNγ in response to pp65 and IE-1 peptide stimulation (P < 0.001). Thus, despite apparent increased immunogenicity of HCMV compared with tumor antigens, the T cells were tolerised, and HCMV status did not impact patient survival (Log Rank3.53 HR = 0.85 95%CI [0.564-1.290], P = 0.45). Enhancing immune functionality in the tumor microenvironment thus may improve patient outcome

Pretreatment of glioblastoma with bortezomib potentiates natural killer cell cytotoxicity through TRAIL/DR5 mediated apoptosis and prolongs animal survival

Background: Natural killer (NK) cells are potential effectors in anti-cancer immunotherapy; however only a subset potently kills cancer cells. Here, we examined whether pretreatment of glioblastoma (GBM) with the proteasome inhibitor, bortezomib (BTZ), might sensitize tumour cells to NK cell lysis by inducing stress antigens recognized by NK-activating receptors. Methods: Combination immunotherapy of NK cells with BTZ was studied in vitro against GBM cells and in a GBM-bearing mouse model. Tumour cells were derived from primary GBMs and NK cells from donors or patients. Flow cytometry was used for viability/cytotoxicity evaluation as well as in vitro and ex vivo phenotyping. We performed a Seahorse assay to assess oxygen consumption rates and mitochondrial function, Luminex ELISA to determine NK cell secretion, protein chemistry and LC–MS/MS to detect BTZ in brain tissue. MRI was used to monitor therapeutic efficacy in mice orthotopically implanted with GBM spheroids. Results: NK cells released IFNγ, perforin and granzyme A cytolytic granules upon recognition of stress-ligand expressing GBM cells, disrupted mitochondrial function and killed 24–46% of cells by apoptosis. Pretreatment with BTZ further increased stress-ligands, induced TRAIL-R2 expression and enhanced GBM lysis to 33–76% through augmented IFNγ release (p &lt; 0.05). Blocking NKG2D, TRAIL and TRAIL-R2 rescued GBM cells treated with BTZ from NK cells, p = 0.01. Adoptively transferred autologous NK-cells persisted in vivo (p &lt; 0.05), diminished tumour proliferation and prolonged survival alone (Log Rank10.19, p = 0.0014, 95%CI 0.252–0.523) or when combined with BTZ (Log Rank5.25, p = 0.0219, 95%CI 0.295–0.408), or either compared to vehicle controls (median 98 vs. 68 days and 80 vs. 68 days, respectively). BTZ crossed the blood–brain barrier, attenuated proteasomal activity in vivo (p &lt; 0.0001; p &lt; 0.01 compared to vehicle control or NK cells only, respectively) and diminished tumour angiogenesis to promote survival compared to vehicle-treated controls (Log Rank6.57, p = 0.0104, 95%CI 0.284–0.424, median 83 vs. 68 days). However, NK ablation with anti-asialo-GM1 abrogated the therapeutic efficacy. Conclusions: NK cells alone or in combination with BTZ inhibit tumour growth, but the scheduling of BTZ in vivo requires further investigation to maximize its contribution to the efficacy of the combination regimen

Pretreatment of glioblastoma with bortezomib potentiates natural killer cell cytotoxicity through TRAIL/DR5 mediated apoptosis and prolongs animal survival

Background: Natural killer (NK) cells are potential effectors in anti-cancer immunotherapy; however only a subset potently kills cancer cells. Here, we examined whether pretreatment of glioblastoma (GBM) with the proteasome inhibitor, bortezomib (BTZ), might sensitize tumour cells to NK cell lysis by inducing stress antigens recognized by NK-activating receptors. Methods: Combination immunotherapy of NK cells with BTZ was studied in vitro against GBM cells and in a GBM-bearing mouse model. Tumour cells were derived from primary GBMs and NK cells from donors or patients. Flow cytometry was used for viability/cytotoxicity evaluation as well as in vitro and ex vivo phenotyping. We performed a Seahorse assay to assess oxygen consumption rates and mitochondrial function, Luminex ELISA to determine NK cell secretion, protein chemistry and LC–MS/MS to detect BTZ in brain tissue. MRI was used to monitor therapeutic efficacy in mice orthotopically implanted with GBM spheroids. Results: NK cells released IFNγ, perforin and granzyme A cytolytic granules upon recognition of stress-ligand expressing GBM cells, disrupted mitochondrial function and killed 24–46% of cells by apoptosis. Pretreatment with BTZ further increased stress-ligands, induced TRAIL-R2 expression and enhanced GBM lysis to 33–76% through augmented IFNγ release (p < 0.05). Blocking NKG2D, TRAIL and TRAIL-R2 rescued GBM cells treated with BTZ from NK cells, p = 0.01. Adoptively transferred autologous NK-cells persisted in vivo (p < 0.05), diminished tumour proliferation and prolonged survival alone (Log Rank10.19, p = 0.0014, 95%CI 0.252–0.523) or when combined with BTZ (Log Rank5.25, p = 0.0219, 95%CI 0.295–0.408), or either compared to vehicle controls (median 98 vs. 68 days and 80 vs. 68 days, respectively). BTZ crossed the blood–brain barrier, attenuated proteasomal activity in vivo (p < 0.0001; p < 0.01 compared to vehicle control or NK cells only, respectively) and diminished tumour angiogenesis to promote survival compared to vehicle-treated controls (Log Rank6.57, p = 0.0104, 95%CI 0.284–0.424, median 83 vs. 68 days). However, NK ablation with anti-asialo-GM1 abrogated the therapeutic efficacy. Conclusions: NK cells alone or in combination with BTZ inhibit tumour growth, but the scheduling of BTZ in vivo requires further investigation to maximize its contribution to the efficacy of the combination regimen