Nanostructured endodontic materials mixed with different radiocontrast agents-biocompatibility study.

The aim of this study was to investigate the biocompatibility of nanostructured materials based on highly active calcium silicates mixed with different radiocontrast agents in comparison to MTA+ using in vitro and in vivo model. Morphology of materials' samples was analyzed using SEM while the phase compositions were identified by XRD. pH values of materials' suspensions were conducted by pH-meter. The cytotoxicity of materials' solutions was tested by MTT test (100, 50, 25 and 12.5 mg/ml). LDH and 3H-thymidine assay were utilized for biocompatibility investigations of materials' eluates (24 h, 7 day and 21 day). Eighteen Guinea pigs were used for intramuscular implantation, as teflon tubes with freshly prepared materials were placed into intramuscular pockets. All samples were composed of round and needle-like particles equally distributed with Ca/Si ratio ~2.7 at%, with the presence of hydrated calcium silicate phases. The pH values of ALBO-MPCA1 and ALBO-MPCA2 were high alkaline, while in case of MTA+ they were lower and continuously declined (p < 0.05). Investigated materials didn't exhibit dose-dependent effect on metabolic activity of L929 cells (p > 0.05). Significant differences in the percentage of cytotoxicity between diluted and undiluted extracts between all tested materials after 24 h and 7 day were noticed (p < 0.05). Increase in L929 cells proliferation was noticed in case of undiluted eluates of ALBO-MPCA1 and ALBO-MPCA2 after 7 day (p < 0.05). There were no statistically significant differences in the intensity of inflammatory response between investigated materials and control group after 60 day (p > 0.05). Evaluation of biocompatibility of both ALBO-MPCA1 and ALBO-MPCA2 indicate their potential clinical use.

Fast dendritic cells matured with Poly (I:C) may acquire tolerogenic properties.

BACKGROUND AIMS: Because of the labor-intensive and time-consuming conventional protocols for the generation of dendritic cells (DCs) as the most promising tools for anti-cancer therapy that enable the induction of a T-helper (Th)1-mediated anti-tumor immune response, the use of short-term protocols has been proposed. However, data on the applicability of such protocols in cancer immunotherapy are quite limited. METHODS: We compared the phenotypic and functional capability of fast DCs (fDCs) differentiated for 24 h and then matured for 48 h with Poly (I:C), a strong Th1-promoting agent, with donor-matched conventional DCs (cDCs) differentiated for 5 days and matured likewise. RESULTS: Of 12 donors tested, we identified seven whose monocytes failed to develop into immunogenic DCs through the use of fDC protocol, on the basis of incomplete downregulation of CD14, low expression of CD1a and macrophage-like morphology. Such fDCs have significantly lower expression of CD83, CD86, CCR7 and CD40, weaker allo-stimulatory Th1- and Th17-polarizing capacity caused by poor production of interleukin (IL)-12p70 and IL-23 and high production of IL-10, and prominent Th2-polarizing capacity, compared with donor-matched cDCs. Furthermore, such fDCs had tolerogenic properties as judged by higher expression of indolamine dioxigenase-3, IDO-1 and IL-1ß and induction of a higher percentage of CD4(+)CD25(+)FoxP3(+) T cells. These findings correlated with increased transforming growth factor (TGF)-ß production by fDC-primed CD3(+)T cells and their stronger anti-proliferative capacity. CONCLUSIONS: We emphasize that although fDCs could probably be applied as an alternative to cDCs for cancer therapy, the fDC protocol should not be applied to donors whose DCs acquire tolerogenic capabilities.

Necrosis and apoptosis in Trichinella spiralis-mediated tumour reduction.

It is known that infection with different pathogens, including helminths, can alter the progression of malignant or other diseases. We studied the effect of chronic Trichinella spiralis infection or muscle larvae excretory-secretory (ES L1) antigens on the malignant tumour growth in the mouse melanoma model system in vivo and in vitro. Our results confirmed that chronic infection with T. spiralis possesses the capacity to slow down the progression of tumour growth, resulting in an impressive reduction in tumour size. We found that the phenomenon could, at least partially, be related to a lower level of tumour necrosis compared to necrosis present in control animals with progressive malignancy course. An increased apoptotic potential among the low percentage of cells within the total tumour cell number in vivo was also observed. ES L1 antigen, as a parasitic product that is released during the chronic phase of infection, reduced the survival and slightly, but significantly increased the apoptosis level of melanoma cells in vitro. Our results imply that powerful Trichinella anti-malignance capacity does not rely only on necrosis and apoptosis but other mechanisms through which infection or parasite products manipulate the tumor establishment and expansion should be considered.

An anti-DEC-205 monoclonal antibody stimulates binding of thymocytes to rat thymic dendritic cells and promotes apoptosis of thymocytes.

DEC-205, a transmembrane receptor responsible for cross-presentation of apoptotic cell-derived antigens, is expressed by cortical thymic epithelial cells (TEC) and thymic dendritic cells (TDC) in humans and mice, but its function in T-cell development is still unclear. In this work we have studied for the first time the expression of DEC-205 in the rat thymus by HD83 monoclonal antibody (mAb) and immunohistochemistry, as well as the ability of this mAb to modulate thymocyte - TDC interactions in vitro. We showed the positivity of cortical TEC in situ, including thymic nurse cells (TNC) in suspension, and TDC, whereas subcapsular, perivascular and medullary TEC were negative. All examined DEC-205 positive and DEC-205 negative structures were MHC class II positive. HD83 mAb increased apoptosis of thymocytes in co-culture with TDC in vitro and the process was associated with increased binding of thymocytes to TDC in a rosette form. Since negative selection of thymocytes by clonal deletion (apoptosis) was mediated predominantly by TDC, our results suggest the possible indirect effect of the DEC-205 molecule in these mechanisms.

Immune Profiling of Secreted Factors from Human Vestibular Schwannoma Cells and Tumor-associated Macrophages.

OBJECTIVES: This study compared the immune-related secretory capacity of human vestibular schwannoma (VS) and tumor-assisted macrophages (TAMs) with their normal counterparts (Schwann cells [SC] and peripheral blood monocyte-derived macrophages [Mo-MFs], respectively), and examined relationships with presurgical hearing and tumor size. METHODS: VS tumors (n = 16), auditory nerve (n = 1), blood (n = 9), and great auricular nerves (n = 3) were used. SCs (S100B+ ) and TAMs (CD68+ ) were isolated from VS tissue for culture. The secreted levels of 65 immune-related factors were measured and compared using unpaired t-tests with Welch correction (schwannoma vs. SCs) or Mann-Whitney tests (TAMs and Mo-MFs). Associations between factor concentration and word recognition (WR), pure-tone average (PTA), and tumor size were evaluated with Spearman correlation. RESULTS: Secreted factors with significantly higher concentrations in schwannoma versus SC supernatants included IL-2 and BAFF, whereas MMP-1, IL-6, FGF-2, VEGF-A, MIP-3α, and GRO-α concentrations were significantly higher in TAMs versus Mo-MFs (all p < 0.05). Worse WR was significantly associated with higher secretion of fractalkine, eotaxin-3, CD30, and IL-16 by VS cells; IP-10, eotaxin-3, multiple interleukins, GM-CSF, SCF, and CD30 by TAMs; and TNF-α and MIP-1α by Mo-MFs (all p < 0.05). Worse PTA was significantly correlated with higher secretion of IL-16 by VS cells (p < 0.05). Larger tumor size was significantly correlated with higher secretion of eotaxin by VS cells, and of IL-7, IL-21, and LIF by TAMs (all p = 0.017). CONCLUSIONS: Differential secretion of immune-related factors was observed in schwannoma versus normal SCs and in TAMs versus Mo-MFs, some of which were correlated with worse hearing and larger VS tumors. LEVEL OF EVIDENCE: N/A Laryngoscope, 134:S1-S14, 2024.

The Effect of Static Magnetic Fields of Different Strengths and Polarities on Cytokine Production by Human Lymphocytes In Vitro.

In contrast to electromagnetic fields, static magnetic fields (SMFs) have not been extensively studied in terms of their potential health consequences. Although upward- and downward-oriented magnetic poles may cause various biological effects, only the pole with the upward orientation has been mainly investigated. Considering that the interaction of antigen-presenting cells (APCs) and T lymphocytes is crucial to trigger an immune response, we assessed the effect of long-term exposure of human T lymphocytes and dendritic cells (DCs) to moderate strength SMFs of different orientations focusing on the cytokine profile of activated T cells. Cultures of allogenic T lymphocytes and DCs (immature and matured by TLR3 and TLR7 agonists) were continuously exposed to four SMFs. The intensity of the applied field was 1 militesla (mT) or 56 mT of the upward- and downward-oriented pole of the SMF. Cell culture supernatants were assayed for IFN-γ, IL-4, IL-17, TNF-α, TNF-ß, IL-1 ß, IL-6, IL-8, and IL-10 by ELISA or flow cytometry. The upward-oriented 56 mT SMF significantly increased the release of IFN-γ and TNF-ß (both p < 0.05) in the cell culture supernatants of T cells and immature DCs. In contrast, the same cultures exposed to the upward-oriented 1 mT SMF showed significantly elevated levels of IL-17 (p < 0.05). The levels of IL-4, TNF-α, IL-1 ß, IL-6, IL-8, and IL-10 were not affected by the upward-oriented SMF. The downward-oriented 56 mT SMF increased TNF-α release when T cells were stimulated with mature DCs. The production of other cytokines was unchanged by the downward-oriented SMF. These findings demonstrate for the first time different in vitro biological effects of upward- and downward-oriented static magnetic fields on the cytokine production of T cells activated by DCs, helping to better understand SMF effects on the immune system and suggesting that the selective SMF effect on the immune response could have potential therapeutic effects in different immune-mediated disorders.

Identification of immune-related candidate biomarkers in plasma of patients with sporadic vestibular schwannoma.

Vestibular schwannoma (VS) is an intracranial tumor arising from neoplastic Schwann cells and typically presenting with hearing loss. The traditional belief that hearing deficit is caused by physical expansion of the VS, compressing the auditory nerve, does not explain the common clinical finding that patients with small tumors can have profound hearing loss, suggesting that tumor-secreted factors could influence hearing ability in VS patients. We conducted profiling of patients' plasma for 66 immune-related factors in patients with sporadic VS (N > 170) and identified and validated candidate biomarkers associated with tumor size (S100B) and hearing (MCP-3). We further identified a nine-biomarker panel (TNR-R2, MIF, CD30, MCP-3, IL-2R, BLC, TWEAK, eotaxin, and S100B) with outstanding discriminatory ability for VS. These findings revealed possible therapeutic targets for VS, providing a unique diagnostic tool that may predict hearing change and tumor growth in VS patients, and may inform the timing of tumor resection to preserve hearing.

Identification of Immune-Related Candidate Biomarkers in Plasma of Patients with Sporadic Vestibular Schwannoma: Candidate Plasma Biomarkers in Vestibular Schwannoma.

Vestibular schwannoma (VS) is intracranial tumor arising from neoplastic Schwann cells, causing hearing loss in about 95% of patients. The traditional belief that hearing deficit is caused by physical expansion of the VS, compressing the auditory nerve, does not explain the common clinical finding that patients with small tumors can have profound hearing loss, suggesting that tumor-secreted factors could influence hearing ability in VS patients. Here, we conducted profiling of patients' plasma for 67 immune-related factors on a large cohort of VS patients (N>120) and identified candidate biomarkers associated with tumor growth (IL-16 and S100B) and hearing (MDC). We identified the 7-biomarker panel composed of MCP-3, BLC, S100B, FGF-2, MMP-14, eotaxin, and TWEAK that showed outstanding discriminatory ability for VS. These findings revealed possible therapeutic targets for VS-induced hearing loss and provided a unique diagnostic tool that may predict hearing change and tumor growth in VS patients and may help inform the ideal timing of tumor resection to preserve hearing.

Signaling through Toll-like receptor 3 and Dectin-1 potentiates the capability of human monocyte-derived dendritic cells to promote T-helper 1 and T-helper 17 immune responses.

BACKGROUND AIMS: Recent studies have shown that the ligation of Toll-like receptor 3 (TLR3) or Dectin-1 on human monocyte-derived dendritic cells (MoDC) elicits their maturation, but with a different outcome on immunomodulation. Therefore the aim of this work was to study the response of MoDC to the combined effect of polyinosinic:polycytydilic acid [Poly (I:C)] and curdlan, selective TLR3 and Dectin-1 agonists, respectively. METHODS: Immature MoDC, generated from human monocytes, were treated with Poly (I:C), curdlan or their combination for 2 days. Phenotypic characteristics of MoDC were determined by flow cytometry, and cytokine production was measured by enzyme-linked immunosorbent assay (ELISA) and FlowCytomix, while the stimulatory capability of MoDC was tested using a mixed leukocyte reaction assay. RESULTS: The combination of Poly (I:C) and curdlan induced phenotypic maturation of MoDC with the capability to stimulate an alloreactive response. Such treated MoDC up-regulated the production of interleukin (IL)-12, IL-23 and IL-10, compared with the effect of Poly (I:C) alone. Curdlan-treated MoDC stimulated the production of IL-17 by alloreactive CD4 (+) T cells more strongly than Poly (I:C)-treated MoDC. The opposite effect was observed for interferon(IFN)-γ production. When combined, these agonists primed MoDC to increase further the production of IFN-γ by CD4 (+) T cells in co-culture, especially those of naive (CD45RA (+)) phenotype, and IL-17 by memory (CD45RO (+)) CD4 (+) T cells. CONCLUSIONS: Ligation of TLR3 and Dectin-1 receptor up-regulates T-helper (Th) 1 and Th17 immune responses compared with single agonists. These findings may have therapeutic implications for the use of MoDC in immunotherapy.

Sporadic Vestibular Schwannoma Size and Location Do not Correlate With the Severity of Hearing Loss at Initial Presentation.

Vestibular schwannoma (VS) is a non-malignant intracranial neoplasm arising from the vestibular branch of the 8th cranial nerve; sensorineural hearing loss (SNHL) is the most common associated symptom. Understanding whether VS imaging characteristics at the time of VS diagnosis can be associated with severity of VS-induced SNHL can impact patient counseling and define promising areas for future research. Patients diagnosed with VS at Massachusetts Eye and Ear (MEE) from 1994 through 2018 were analyzed if magnetic resonance imaging at VS presentation and sequential audiometry were available. Results were compared with original studies available in PubMed, written in English, on VS imaging characteristics and their impact on hearing in patients. A total of 477 patients with unilateral VS from the MEE database demonstrated no significant correlation between any features of tumor imaging at the time of VS diagnosis, such as VS size, impaction or location, and any hearing loss metric. Twenty-three published studies on the impact of VS imaging characteristics on patient hearing met inclusion criteria, with six solely involving NF2 patients and three including both sporadic and NF2-related VS patients. Fifteen studies reported a significant relationship between SNHL and at least one VS imaging characteristic; however, these trends were universally limited to NF2 patients or involved small patient populations, and were not reproduced in larger studies. Taken together, SNHL in sporadic VS patients is not readily associated solely with any tumor imaging characteristics. This finding motivates future studies to define how VS microenvironment and secreted molecules influence VS-induced SNHL.

Immunomodulatory Effects of Extract of Lingzhi or Reishi Medicinal Mushroom Ganoderma lucidum (Agaricomycetes) Basidiocarps Cultivated on Alternative Substrate.

Ganoderma lucidum is a medicinal mushroom exhibiting numerous health benefits primarily based on strong immunostimulatory effects. The study aimed to investigate if there were differences in effects of extracts of commercially (GC) and alternatively (wheat straw) (GA) cultivated G. lucidum basidiocarps on properties of peritoneal macrophages (PM) and monocyte-derived dendritic cells (MoDCs). Differences in immunomodulatory effects of GC/GA extracts were studied. The viability of treated PMs, their adhesive and phagocytic capability, and their capacity to produce reactive oxygen species (ROS) and NO were tested. Immature MoDCs generated from human monocytes were treated with poly I:C (10.0 µg/ml) and loxoribine (34.0 µg/ml), a selective TLR3 and TLR7 agonists, respectively, and with/without GC/GA extract (100.0 µg/ml). The effect of each combination on phenotypic properties, cytokines production by MoDCs, and their proliferation and Th polarizing capacity was studied. GA extract stimulated the metabolic and phagocytic activity of PMs, their adhesion capability, and ability to produce ROS and NO more strongly compared to GC. Both tested extracts significantly increased allostimulatory and Th1 polarization capacity of simultaneous TLR3 and TLR7-activated MoDCs, but GA extract was more effective. The extract of alternatively cultivated G. lucidum basidiocarps increased production of ROS and NO by TLR4 stimulated PMs and upregulated production of certain cytokines as well as allostimulatory and Th1 polarization capacity of MoDCs. GA extract could be a potent immunostimulatory agent for activation of MoDCs with the simultaneous engagement of TLRs, which seems to be a promising strategy for the preparation of DC-based anti-tumor vaccines.

Orally administered fluorescent nanosized polystyrene particles affect cell viability, hormonal and inflammatory profile, and behavior in treated mice.

Commercially manufactured or generated through environmental degradation, microplastics (MPs) and nanoplastics (NPs) considerably contribute to environmental pollution. There is a knowledge gap in how exposure to MPs/NPs changes cellular function and affects animal and human health. Here, we demonstrate that after oral uptake, fluorescent polystyrene (PS) nanoparticles pass through the mouse digestive system, accumulate and aggregate in different organs, and induce functional changes in cells and organs. Using cochlear explant as a novel in vitro system, we confirmed the consequences of PS-MP/NP interaction with inner ear cells by detecting aggregates and hetero-aggregates of PS particles in hair cells. The testes of treated males accumulated MPs/NPs in the interstitial compartment surrounding the seminiferous tubules, which was associated with a statistically significant decrease in testosterone levels. Male mice showed increased secretion of interleukins (IL-12p35 and IL-23) by splenocytes while cyto- and genotoxicity tests indicated impaired cell viability and increased DNA damage in spleen tissue. Males also showed a broad range of anxiogenic responses to PS nanoparticles while hippocampal samples from treated females showed an increased expression of Bax and Nlrp3 genes, indicating a pro-apoptotic/proinflammatory effect of PS treatment. Taken together, induced PS effects are also gender-dependent, and therefore, strongly motivate future research to mitigate the deleterious effects of nanosized plastic particles.

Differences in T-helper polarizing capability between human monocyte-derived dendritic cells and monocyte-derived Langerhans'-like cells.

Langerhans' cells (LCs) represent a specific subset of dendritic cells (DCs) which are important for detecting and processing pathogens that penetrate the skin and epithelial barriers. The aim of our study was to explain what makes their in vitro counterparts - monocyte-derived Langerhans'-like cells (MoLCs) - unique compared with monocyte-derived dendritic cells (MoDCs). Immature MoDCs were generated by incubating peripheral blood monocytes with granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-4. The addition of transforming growth factor-ß (TGF-ß) to this cytokine cocktail resulted in the generation of MoLCs. MoLCs showed a lower expression of CD83, CD86, HLA-DR and CCR7 compared with MoDCs, regardless of their maturational status. Both immature and mature MoLCs secreted higher quantities of IL-23 compared with MoDCs and this finding correlated with a higher secretion of IL-17 in co-culture of MoLCs with allogeneic CD4(+) T cells. Mature MoLCs, which produced higher levels of IL-12 and lower levels of IL-10 compared with mature MoDCs, were more potent at inducing interferon-γ (IFN-γ) production by CD4(+) T cells in the co-culture system. In conclusion, the finding that mature MoLCs stimulate stronger T-helper 1 and T-helper 17 immune responses than mature MoDCs, makes them better candidates for use in the preparation of anti-tumour DC vaccines.

Nanodesigned coatings obtained by plasma electrolytic oxidation of titanium implant and their cytotoxicity.

The titanium implant was treated with plasma electrolytic oxidation and subsequent ionic exchange and thermal treatment in order to obtain bioactive layer consisting of titanium oxide, calcium and sodium titanates and hydroxyapatite, as confirmed by X-ray diffraction (XRD). Scanning electron microscopy (SEM) revealed that the given method, besides corresponding phase composition, enables suitable nanotopology for cell attachment and proliferation. Cytotoxicity investigations by MTT, LDH and propidium iodide assays and light microscopy showed that these coatings were not toxic to L929 cells.

New developments in neurofibromatosis type 2 and vestibular schwannoma.

Neurofibromatosis type 2 (NF2) is a rare autosomal dominant disorder characterized by the development of multiple nervous system tumors due to mutation in the NF2 tumor suppressor gene. The hallmark feature of the NF2 syndrome is the development of bilateral vestibular schwannomas (VS). Although there is nearly 100% penetrance by 60 years of age, some patients suffer from a severe form of the disease and develop multiple tumors at an early age, while others are asymptomatic until later in life. Management options for VS include surgery, stereotactic radiation, and observation with serial imaging; however, currently, there are no FDA-approved pharmacotherapies for NF2 or VS. Recent advancements in the molecular biology underlying NF2 have led to a better understanding of the etiology and pathogenesis of VS. These novel signaling pathways may be used to identify targeted therapies for these tumors. This review discusses the clinical features and treatment options for sporadic- and NF2-associated VS, the diagnostic and screening criteria, completed and ongoing clinical trials, quality of life metrics, and opportunities for future research.

Cochlin Deficiency Protects Against Noise-Induced Hearing Loss.

Cochlin is the most abundant protein in the inner ear. To study its function in response to noise trauma, we exposed adolescent wild-type (Coch +/+ ) and cochlin knock-out (Coch -/-) mice to noise (8-16 kHz, 103 dB SPL, 2 h) that causes a permanent threshold shift and hair cell loss. Two weeks after noise exposure, Coch-/- mice had substantially less elevation in noise-induced auditory thresholds and hair cell loss than Coch + / + mice, consistent with cochlin deficiency providing protection from noise trauma. Comparison of pre-noise exposure thresholds of auditory brain stem responses (ABRs) and distortion product otoacoustic emissions (DPOAEs) in Coch-/- mice and Coch + / + littermates revealed a small and significant elevation in thresholds of Coch-/- mice, overall consistent with a small conductive hearing loss in Coch-/- mice. We show quantitatively that the pro-inflammatory component of cochlin, LCCL, is upregulated after noise exposure in perilymph of wild-type mice compared to unexposed mice, as is the enzyme catalyzing LCCL release, aggrecanase1, encoded by Adamts4. We further show that upregulation of pro-inflammatory cytokines in perilymph and cochlear soft-tissue after noise exposure is lower in cochlin knock-out than wild-type mice. Taken together, our data demonstrate for the first time that cochlin deficiency results in conductive hearing loss that protects against physiologic and molecular effects of noise trauma.

Postnatal expression and possible function of RANK and RANKL in the murine inner ear.

The bone encasing the inner ear, known as the otic capsule, is unique because it remodels little postnatally compared to other bones in the body. Previous studies established that osteoprotegerin (OPG) in the inner ear inhibits otic capsule remodeling. OPG acts as a decoy receptor of receptor activator of nuclear factor κB ligand (RANKL) to disrupt the interaction between RANKL and RANK, the primary regulators of bone metabolism. Here we studied the expression and function of RANK and RANKL in the murine cochlea. Using a combination of in situ hybridization, real-time quantitative RT-PCR, and western blot, we demonstrate that Rankl and Rank genes and their protein products are expressed in the intracochlear soft tissues and the otic capsule in a developmentally regulated manner. Using a culture of neonatal murine cochlear neurons, we show that the interaction between RANK and RANKL inhibits neurite outgrowth in these neurons, and is associated with upregulation of NOGO-A expression. Taken together, our results suggest that, in addition to regulating otic capsule bone remodeling, RANK and RANKL expressed by intracochlear soft tissues may also regulate spiral ganglion neuron function by affecting neurite outgrowth.

Losartan prevents tumor-induced hearing loss and augments radiation efficacy in NF2 schwannoma rodent models.

Hearing loss is one of the most common symptoms of neurofibromatosis type 2 (NF2) caused by vestibular schwannomas (VSs). Fibrosis in the VS tumor microenvironment (TME) is associated with hearing loss in patients with NF2. We hypothesized that reducing the fibrosis using losartan, an FDA-approved antihypertensive drug that blocks fibrotic and inflammatory signaling, could improve hearing. Using NF2 mouse models, we found that losartan treatment normalized the TME by (i) reducing neuroinflammatory IL-6/STAT3 signaling and preventing hearing loss, (ii) normalizing tumor vasculature and alleviating neuro-edema, and (iii) increasing oxygen delivery and enhancing efficacy of radiation therapy. In preparation to translate these exciting findings into the clinic, we used patient samples and data and demonstrated that IL-6/STAT3 signaling inversely associated with hearing function, that elevated production of tumor-derived IL-6 was associated with reduced viability of cochlear sensory cells and neurons in ex vivo organotypic cochlear cultures, and that patients receiving angiotensin receptor blockers have no progression in VS-induced hearing loss compared with patients on other or no antihypertensives based on a retrospective analysis of patients with VS and hypertension. Our study provides the rationale and critical data for a prospective clinical trial of losartan in patients with VS.

Production of IL-10 and IL-12 by antigen-presenting cells in periapical lesions.

BACKGROUND: Interferon-γ (IFN-γ) plays an important role in the pathogenesis of periapical lesions. Its expression is up-regulated by interleukin (IL)-12) and down-regulated by IL-10. The aim of this work was to study the cellular source of these cytokines and their mutual interactions in human periapical lesions. METHODS: Mononuclear cells, macrophages and dendritic cells were isolated from periapical lesions using plastic adherence and osmotic gradients. Cytokines were measured in culture supernatants by a microbeads fluorescence assay. Phenotypic characteristics of cells were studied by immunocytochemistry, whereas allostimulatory activity of antigen-presenting cells was tested using a mixed leukocyte reaction. RESULTS: We observed the positive correlations between the levels of IL-12 and IFN-γ as well as IL-12 and IL-10 in cultures of mononuclear cells. As IL-10 and IL-12 are produced by dendritic cells and activated macrophages, we examined their contribution to the production of these cytokines. Macrophages, CD14(+) adherent cells, produced high levels of IL-10 and very low levels of IL-12. In contrast, non-adherent, strongly HLA-DR(+) dendritic cells, potent stimulators of the alloreactive T-cell response, produced low levels of IL-10 and moderate levels of IL-12. Dendritic cells stimulated the production of IFN-γ by allogeneic CD4(+) T cells. In contrast, the level of IFN-γ was significantly decreased and the production of IL-10 was enhanced by addition of macrophages to the culture system. CONCLUSION: Our results suggest that a fine balance between the production of IL-10 and IL-12 by different antigen-presenting cells, through IFN-γ, may control the course of chronic inflammation in periapical lesions.

Combination therapy with mTOR kinase inhibitor and dasatinib as a novel therapeutic strategy for vestibular schwannoma.

Neurofibromatosis type 2 (NF2) is an inherited disorder characterized by bilateral vestibular schwannomas (VS) that arise from neoplastic Schwann cells (SCs). NF2-associated VSs are often accompanied by meningioma (MN), and the majority of NF2 patients show loss of the NF2 tumor suppressor. mTORC1 and mTORC2-specific serum/glucocorticoid-regulated kinase 1 (SGK1) are constitutively activated in MN with loss of NF2. In a recent high-throughput kinome screen in NF2-null human arachnoidal and meningioma cells, we showed activation of EPH RTKs, c-KIT, and SFK members independent of mTORC1/2 activation. Subsequently, we demonstrated in vitro and in vivo efficacy of combination therapy with the dual mTORC1/2 inhibitor AZD2014 and the multi-kinase inhibitor dasatinib. For these reasons, we investigated activated mTORC1/2 and EPH receptor-mediated signaling in sporadic and NF2-associated VS. Using primary human VS cells and a mouse allograft model of schwannoma, we evaluated the dual mTORC1/2 inhibitor AZD2014 and the tyrosine kinase inhibitor dasatinib as monotherapies and in combination. Escalating dose-response experiments on primary VS cells grown from 15 human tumors show that combination therapy with AZD2014 and dasatinib is more effective at reducing metabolic activity than either drug alone and exhibits a therapeutic effect at a physiologically reasonable concentration (~0.1 µM). In vivo, while AZD2014 and dasatinib each inhibit tumor growth alone, the effect of combination therapy exceeds that of either drug. Co-targeting the mTOR and EPH receptor pathways with these or similar compounds may constitute a novel therapeutic strategy for VS, a condition for which there is no FDA-approved pharmacotherapy.