Immunocytochemical Staining Research Articles

Novel In Situ-Cross-Linked Electrospun Gelatin/Hydroxyapatite Nonwoven Scaffolds Prove Suitable for Periodontal Tissue Engineering.

Periodontal diseases affect millions of people worldwide and can result in tooth loss. Regenerative treatment options for clinical use are thus needed. We aimed at developing new nonwoven-based scaffolds for periodontal tissue engineering. Nonwovens of 16% gelatin/5% hydroxyapatite were produced by electrospinning and in situ glyoxal cross-linking. In a subset of scaffolds, additional porosity was incorporated via extractable polyethylene glycol fibers. Cell colonization and penetration by human mesenchymal stem cells (hMSCs), periodontal ligament fibroblasts (PDLFs), or cocultures of both were visualized by scanning electron microscopy and 4′,6-diamidin-2-phenylindole (DAPI) staining. Metabolic activity was assessed via Alamar Blue® staining. Cell type and differentiation were analyzed by immunocytochemical staining of Oct4, osteopontin, and periostin. The electrospun nonwovens were efficiently populated by both hMSCs and PDLFs, while scaffolds with additional porosity harbored significantly more cells. The metabolic activity was higher for cocultures of hMSCs and PDLFs, or for PDLF-seeded scaffolds. Periostin and osteopontin expression was more pronounced in cocultures of hMSCs and PDLFs, whereas Oct4 staining was limited to hMSCs. These novel in situ-cross-linked electrospun nonwoven scaffolds allow for efficient adhesion and survival of hMSCs and PDLFs. Coordinated expression of differentiation markers was observed, which rendered this platform an interesting candidate for periodontal tissue engineering.

Abstract 5515: Combination of recombinant murine IL-12 and anti-PD-1 induced synergistic anti-tumor response

Abstract Introduction: Immune-checkpoint inhibitors have become the standard therapy in many types of cancers. The combination of anti-PD-1 and anti-CTLA4 demonstrated better anti-tumor response, but toxicity is a concern. Cytokines including IL-2, IL-12, IL-15, IL-21 that target immune cells have been developed to enhance immune response against tumors. Clinical trials for the combination of cytokines and anti-PD-1 have been proposed (Mohamad et al, 2020). IL-12, a multifunctional cytokine, regulates both innate and adaptive immunity. The role of IL-12 in regulating immune response showed that intra-tumoral electroporation of plasmid IL-12 prime responses to anti-PD-1 treatment (Algazi et al, 2016). IL-12 produced by dendritic cells accompanied with IFN-γ play an important role in anti-tumor response induced by anti-PD-1 (Garris et al, 2018). Therefore, this study aims to explore the synergistic anti-tumor immune response of recombinant murine IL-12 (rmIL-12) and anti-PD-1. Methods: Different doses of rmIL-12 alone or combined with anti-PD-1 were administered in different tumor models. CT26, MC38, Hepa 1-6 that generate hot tumor were used. Tumors were inoculated subcutaneously in syngeneic mouse tumor models and treated when mean tumor volume reach 60-100 mm3. Tumor resection and splenectomy were performed on tumor inoculated mice at termination. Immunocytochemistry stain was used to detect immune cells including CD4, CD8 T cells, regulatory T cells (Treg), myeloid-derived suppressor cells (MDSC) and macrophage. Serum cytokines were checked for immune response and liver enzyme for toxicity. Results: The combination of rmIL-12 and anti-PD-1 (BIW x 3 weeks) showed better tumor control compared to anti-PD-1 alone with CT26, MC38, Hepa 1-6 cell lines. Synergistic effect was observed even with a low dose of rmIL-12 (20 ng/mice). A higher dose of rmIL-12 achieved better tumor control. In the CT26 tumor model, combination therapy suppressed tumor growth up to 73% compared to anti-PD-1 alone. These results demonstrated a clear dose-response relationship. The combination of rmIL-12 and anti-PD-1 induced higher level of IFN-γ compared to anti-PD-1 or rmIL-12 alone. A higher dose of rmIL-12 resulted in higher IFN-γ level. The IFN-γ level peaked about 12 hours after the treatment and returned near baseline in about 48 hours. The combination of rmIL-12 and anti-PD-1 showed higher percentage of CD8 T cells in tumors compared to anti-PD-1 or rmIL-12 alone. Combination group significantly decreased Treg in the tumor microenvironment, but not MDSC. Combination therapy also reprogrammed the macrophages from M2 towards M1 polarization in the tumor. Finally, no significant elevation of AST/ALT level was observed among all the groups. Conclusion: The combination of rmIL-12 and anti-PD-1 demonstrated a synergistic immune response for tumor control. The result needs to be further confirmed in human in the near future. Citation Format: Peiyi Tsou, Wan-Ying Lin, Meng-Na Lee, San-Chi Chen. Combination of recombinant murine IL-12 and anti-PD-1 induced synergistic anti-tumor response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5515.

Abstract 997: γ-Tocotrienol inhibition of metastatic phenotypic behavior is associated with a decrease in galectin-3 expression and distribution in the highly malignant mouse +SA & TS/A mammary tumor cells

Abstract γ-Tocotrienol is a rare natural isoform of vitamin E that displays potent apoptotic and anti-metastatic activity against breast cancer. Galectin-3 is a protein that plays an important role in metastasis by binding to glycoconjugates on receptors and extracellular matrix proteins. Studies were conducted to investigate the effects of γ-tocotrienol on galectin-3 expression and morphology in highly malignant +SA, TS/A MDA-MB-231 tumor cells. Computer-aided molecular modeling studies compared anticancer agents, salirasib and γ-tocotrienol, with the active site of galectin-3 and β-lactose (a known ligand). Results showed that γ-tocotrienol displays similar galectin-3 amino acid interactions as β-lactose and salirasib. Protein docking show that salirasib (active binding -2.8) and γ- tocotrienol (active binding -2.87) had similar binding scores and binding moieties. Immunocytochemical fluorescent staining show that γ-tocotrienol treatment disrupted galectin-3 expression and distribution, and a corresponding reduction in +SA cellular migration similar to that induced by β-lactose. Western blot analysis shows that γ-tocotrienol treatment induces a downregulation of galectin-3 expression. Immunocytochemistry studies shows that galectin-3 is highly expressed in +SA and TS/A cells, and γ-tocotrienol greatly reduced galectin-3 expression and distribution Furthermore, galectin-3 was found to form extracellular structures on the outer membrane of +SA, TS/A, and MDA-MB-231 cells, and γ-tocotrienol greatly attenuated galectin-3 and fibronectin distribution. γ-Tocotrienol treatment decreased f-actin stress fibers distribution in all cell lines. Furthermore, treatment with 1.5 μM histamine significantly stimulated +SA mammary tumor cell migration, whereas combination treatment with 5 μM γ-tocotrienol completely blocked histamine-induced +SA cell migration. In TS/A cells, 6-15 μM tocotrienol induced a dose-responsive increase in doxorubicin staining inside the nucleus. In MDA-MB-231 cells, syncytial cells were associated with galectin-3 expression and this was suppressed by 5-15 μM γ-tocotrienol. In summary, these results demonstrate that γ-tocotrienol treatment inhibits extracellular galectin-3 expression and disrupts galectin-3 carbohydrate binding and activation of +SA, TS/A and MDA-MB-231 breast cancer cells. These findings also suggest that γ-tocotrienol may provide significant benefits in the prevention and/or treatment of metastatic breast cancer. This study was supported in part by funding from the Louisiana Cancer Foundation. Citation Format: Paul W. Sylvester, Jessie J. Grazier. γ-Tocotrienol inhibition of metastatic phenotypic behavior is associated with a decrease in galectin-3 expression and distribution in the highly malignant mouse +SA & TS/A mammary tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 997.

Abstract 2207: High-dose omega-3 fatty acid supplementation is associated with a decrease in FOXA1 in benign breast tissue

Abstract Background: FOXA1 is a pioneer transcription factor necessary for full expression of estrogen receptor activity. In collaboration with Grainyhead-like 2 (GRHL2), increased FOXA1 activity can result in endocrine resistance through increase of the endoplasmic reticulum protein AGR2 and its receptor LYPD3. Hyperglycemia and hyperinsulinemia increase FOXA1. Since eicosapentaenoic and docosahexaenoic omega-3 fatty acids (EPA and DHA) improve insulin sensitivity and reduce IGFR-related signaling, we explored the effect of EPA + DHA supplementation on FOXA1 and AGR2 expression in benign breast epithelium. Methods: Reserved excess cells from fine needle aspirations performed pre and post 6 months of supplementation given to postmenopausal women at increased risk for breast cancer were utilized. Cells had been stored at -20 C in a methanol fixative for ~10 y (Fabian et al, Ca Prev Res 2015). Of the 35 women entered, 28 had reserved excess cells available for blocks and after obtaining 4-5 sections per block, 12 had sufficient epithelial cells on both pre and post intervention slides as determined by H and E staining to proceed with immunohistochemistry assessment. Of the 12, median age was 53 and 7/12 were on low dose hormone replacement during the study. Slides were stained with FOXA1 (2F83 Mouse Monoclonal Antibody) at a 1:50 dilution and AGR2 rabbit polyclonal (Proteintech,12275-1-AP) at a 1:800 dilution. F0XA1 exhibits a nuclear staining pattern whereas AGR2 is predominately cytoplasmic. IHC staining was assessed individually by two readers (TM and TP). The most abnormal cell clusters were preferentially assessed. Up to 500 cells are evaluated for percent positive stain and staining intensity. An Allred score was computed as the sum of a categorical score for percent positive cells and a categorical score for the predominant staining intensity. Results: For FOXA1 there was a statistically significant decrease in percent positive cells (11/12, p=0.019) and Allred score (9/12, p=0.022) by Wilcoxon signed rank test. Of the 11 which showed a decrease in FOXA1, seven were on HRT throughout the study (usually estrogen alone). There was no statistically significant change in AGR2 IHC. However, there was a robust linear relationship between stain positivity for FOXA1 and AGR2 (p<0.001), as well as for the change in percent positive cells over the course of the intervention (p<0.003). There was no statistically significant correlation between change in FOXA1 and change in Ki-67 immunocytochemical staining (performed as part of the original study), nor for a variety of serum measures of insulin sensitivity or resistance including HOMA-IR and HOMA %B. Conclusions: If this finding is confirmed in other studies, FOXA1 protein expression may be a useful tissue response biomarker in breast cancer prevention trials of omega-3 fatty acids. Citation Format: Carol J. Fabian, Trina Metheny, Teresa A. Phillips, Marsha Danley, Bruce F. Kimler. High-dose omega-3 fatty acid supplementation is associated with a decrease in FOXA1 in benign breast tissue [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2207.

Protective effects of muscone on traumatic spinal cord injury in rats.

BackgroundTraumatic spinal cord injury (SCI) is a major clinical concern, and it is a life-changing neurological condition with substantial socioeconomic implications. Muscone has been widely used in traditional Chinese medicinal formulations for its anti-inflammatory activity. However, its protective effects on traumatic SCI have not been explored. This study investigated whether muscone plays a protective role in SCI and compared its effects with those of methylprednisolone sodium succinate (MPSS).MethodsRats were divided into five groups: normal saline (NS; n=24), methylprednisolone (MP; w=24), and muscone 1 (MO1), muscone 2 (MO2), and muscone 3 (MO3) (n=24 in each group, collectively called the MOx groups). The SCI rat model was established by the modified Allen’s method. The rats were administered muscone (MO1: 2.5 mg/kg, MO2: 5 mg/kg, and MO3: 10 mg/kg) or MP (30 mg/kg), or an equivalent volume of saline. The rats were kept under observation for 4 weeks. Malondialdehyde (MDA), superoxide dismutase (SOD), interleukin-6 (IL-6), interleukin-1β (IL-1β), and tumor necrosis factor-alpha (TNF-α) levels were detected using enzyme-linked immunosorbent assay (ELISA). The expression of glial fibrillary acidic protein (GFAP), B-cell lymphoma-2 (BCL-2), and caspase3 was detected by western blot analysis. Hematoxylin-eosin (HE), Nissl, and immunocytochemistry (ICC) staining was performed for pathological observation. Basso-Beattie-Bresnahan motor function scores were evaluated for assessment of neural functions after acute SCI.ResultsMuscone inhibited immune-inflammatory reactions, neuronal necrosis, and apoptosis. The lower limb function recovery was better in the MOx groups compared with NS and MP groups according to Basso-Beattie-Bresnahan scores. The changes were remarkable in the MO2 group compared with the other groups.ConclusionsMuscone alleviates secondary injury after SCI by reducing immune-inflammatory reactions, neuronal necrosis, and apoptosis.

Characterization of Keratinocytes, Fibroblasts and Melanocytes Isolated from Human Skin using Gene Markers

Cells isolated from skin have wide applications in studies of the pathogenesis of skin-related diseases and the construction of 3D skin equivalents. This study aimed to isolate keratinocytes, fibroblasts, and melanocytes from human foreskin and characterize the purity of the cell types. Keratinocytes, fibroblasts, and melanocytes from human foreskin were isolated by differential trypsinization and media selection. The purity of the cell types was characterized based on the expression of gene markers. The assessment of gene marker expression involved RNA extraction, primer design, quantitative polymerase chain reaction (qPCR) and immunocytochemical staining. Our results showed that in cocultures of keratinocytes and fibroblasts isolated from the dermis, fibroblasts could be separated from keratinocytes by quick trypsinization and culture in Dulbecco’s modified Eagle’s medium. The remaining keratinocytes are cultured in Epilife medium. Melanocytes in cocultures of melanocytes and keratinocytes isolated from the epidermis could be selected by changing Epilife medium to M254 medium. Gene marker results suggested that cytokeratin 14 (CK14) is a suitable marker for keratinocytes, elastin (ELN) is a suitable marker for fibroblasts, and tyrosinase (TYR) and tyrosinase-related proteins 1 and 2 (TYRP1 and TYRP2) are suitable markers for melanocytes. In conclusion, keratinocytes, fibroblasts, and melanocytes can be isolated from the same human foreskin sample by differential trypsinization and media selection techniques and characterized by suitable gene markers. This finding will aid in the isolation of pure skin cell types for various applications in regenerative medicine and toxicity studies.

Long-term complete remission in a cat with large granular lymphocyte lymphoma by nimustine treatment

A 9-year-old neutered male Munchkin cat presented to our clinic with lethargy and anorexia. Abdominal ultrasonography revealed multiple enlarged mesenteric lymph nodes. Cytology and immunocytochemical staining suggested large granular lymphocyte (LGL) lymphoma derived from cytotoxic T cells. Treatment with nimustine (ACNU) (25 mg/m2, intravenously, every 3 weeks) resulted in long-term complete remission over 807 days. The only adverse event associated with ACNU was grade 1 neutropenia. To the best of our knowledge, this is the first report of long-term survival of a cat treated with ACNU for more than 2 years.

Involvement of small-conductance Ca2+-activated K+ (SKCa2) channels in spontaneous Ca2+ oscillations in rat pinealocytes

Melatonin secretion from the pineal glands regulates circadian rhythms in mammals. Melatonin production is decreased by an increase in cytosolic Ca2+ concentration following the activation of nicotinic acetylcholine receptors in parasympathetic systems. We previously reported that pineal Ca2+ oscillations were regulated by voltage-dependent Ca2+ channels and large-conductance Ca2+-activated K+ (BKCa) channels, which inhibited melatonin production. In the present study, the contribution of small- and intermediate-conductance Ca2+-activated K+ (SKCa and IKCa) channels to the regulation of spontaneous Ca2+ oscillations was examined in rat pinealocytes. The amplitude and frequency of spontaneous Ca2+ oscillations were increased by a SKCa channel blocker (100 nM apamin), but not by an IKCa channel blocker (1 μM TRAM-34). On the other hand, they were decreased by a SKCa channel opener (100 μM DCEBIO), but not by an IKCa channel opener (1 μM DCEBIO). Expression analyses using quantitative real-time PCR, immunocytochemical staining, and Western blotting revealed that the SKCa2 channel subtype was abundantly expressed in rat pinealocytes. Moreover, the enhanced amplitude of Ca2+ oscillations in the presence of apamin was further increased by a BKCa channel blocker (1 μM paxilline). These results suggest that the activity of SKCa2 channels regulates cytosolic Ca2+ signaling and melatonin production during parasympathetic activation in pineal glands.

Circulating tumor cells: A step toward precision medicine in hepatocellular carcinoma.

Serum alpha-fetoprotein and radiologic imaging are the most commonly used tests for early diagnosis and dynamic monitoring of treatment response in hepatocellular carcinoma (HCC). However, the accuracy of these tests is limited, and they may not reflect the underlying biology of the tumor. Thus, developing highly accurate novel HCC biomarkers reflecting tumor biology is a clinically unmet need. Circulating tumor cells (CTCs) have long been proposed as a noninvasive biomarker in clinical oncology. Most CTC assays utilize immunoaffinity-based, size-based, and/or enrichment-free mechanisms followed by immunocytochemical staining to characterize CTCs. The prognostic value of HCC CTC enumeration has been extensively validated. Subsets of CTCs expressing mesenchymal markers are also reported to have clinical significance. In addition, researchers have been devoting their efforts to molecular characterizations of CTCs (e.g. genetics and transcriptomics) as molecular profiling can offer a more accurate readout and provide biological insights. As new molecular profiling techniques, such as digital polymerase chain reaction, are developed to detect minimal amounts of DNA/RNA, several research groups have established HCC CTC digital scoring systems to quantify clinically relevant gene panels. Given the versatility of CTCs to provide intact molecular and functional data that reflects the underlying tumor, CTCs have great potential as a noninvasive biomarker in HCC. Large-scale, prospective studies for HCC CTCs with a standardized protocol are necessary for successful clinical translation.

Biodegradable Poly(D-L-lactide-co-glycolide) (PLGA)-Infiltrated Bioactive Glass (CAR12N) Scaffolds Maintain Mesenchymal Stem Cell Chondrogenesis for Cartilage Tissue Engineering.

Regeneration of articular cartilage remains challenging. The aim of this study was to increase the stability of pure bioactive glass (BG) scaffolds by means of solvent phase polymer infiltration and to maintain cell adherence on the glass struts. Therefore, BG scaffolds either pure or enhanced with three different amounts of poly(D-L-lactide-co-glycolide) (PLGA) were characterized in detail. Scaffolds were seeded with primary porcine articular chondrocytes (pACs) and human mesenchymal stem cells (hMSCs) in a dynamic long-term culture (35 days). Light microscopy evaluations showed that PLGA was detectable in every region of the scaffold. Porosity was greater than 70%. The biomechanical stability was increased by polymer infiltration. PLGA infiltration did not result in a decrease in viability of both cell types, but increased DNA and sulfated glycosaminoglycan (sGAG) contents of hMSCs-colonized scaffolds. Successful chondrogenesis of hMSC-colonized scaffolds was demonstrated by immunocytochemical staining of collagen type II, cartilage proteoglycans and the transcription factor SOX9. PLGA-infiltrated scaffolds showed a higher relative expression of cartilage related genes not only of pAC-, but also of hMSC-colonized scaffolds in comparison to the pure BG. Based on the novel data, our recommendation is BG scaffolds with single infiltrated PLGA for cartilage tissue engineering.

Regulation of nutrient and electrolyte absorption in human organoid-derived intestinal epithelial cell monolayers

Recently developed human intestinal epithelial 3D organoid cultures are a useful cell culture model to study intestinal transport physiology. From these, 2D monolayer cultures can be generated in which apical transporters are exposed to the medium, thereby better facilitating in vitro investigation of intestinal absorption processes. However, whether nutrient and electrolyte absorption can be physiologically regulated in human organoid-derived monolayers has not been determined. Constitutive nitric oxide (cNO) is known to regulate multiple gastrointestinal physiological functions. Previous studies using in vivo and in vitro mammalian animal models indicate that enhanced intracellular cNO differentially regulates the two primary apical Na transporters in small intestinal epithelial cells. Here, we generated human jejunal organoid-derived monolayers to determine whether apical nutrient and electrolyte transporter function is regulated by cNO in human enterocytes. Western blot analysis and immunocytochemical staining showed that organoid-derived 2D cultures express markers of enterocyte differentiation and form intact monolayers of apical-basal polarized epithelial cells. Uptake studies demonstrated that jejunal monolayers exhibit functional activity of Na-glucose cotransporter 1 (SGLT1; SLC5A1) and Na-H exchanger 3 (NHE3; SLC9A3). In response to physiological increases in cNO, the two primary apical Na transporters were differentially regulated in human intestinal organoid-derived monolayers, across multiple human specimens. An increase in cNO stimulated SGLT1, while NHE3 was inhibited. These results are similar to what is seen in vivo and in vitro in different animal intestinal models. Thus, human jejunal organoid-derived monolayers are an ideal in vitro model to better understand how intestinal nutrient absorption is regulated.

Hypothalamic Corticotrophin Releasing Hormone Signal Contributes to Elevated Sympathetic Outflow in primary hypertension

Corticotropin‐releasing hormone (CRH) is a neuropeptide involved in regulating neuroendocrine and autonomic function. CHR mRNA and protein expression in the hypothalamic paraventricular nucleus (PVN) are significantly increased in patients with primary hypertension. However, the role of CRH signals in elevated sympathetic outflow in primary remains unclear. Here, we showed that CRH receptor 1 (CRHR1) protein level rats was significantly increased in the PVN tissue in spontaneously hypertensive (SHR) compared with Wistar‐Kyoto (WKY). Upregulated CRHR1 level in the PVN was not significantly affected by lowing arterial blood pressure (ABP) by celiac ganglionectomy in SHRs. Immunocytochemistry staining revealed that CRHR1 immunoreactivities were in PVN neurons projecting to the rostral ventrolateral medulla (RVLM). Bath application of CRH induced a significantly greater increase in the firing rate of PVN‐RVLM projection neurons in SHRs than in WKY rats. Blocking CRHRs with astressin or CRHR1 with antalarmin significantly decreased firing rate of PVN‐RVLM projection neurons in SHR but not in WKY, while blocking CRHR2 with antisauvagine‐30 did not alter the firing rate of these neurons in SHR. Both antalarmin and antisauvagine‐30 did not change the frequency and amplitude of miniature inhibitory post‐synaptic currents or miniature excitatory post‐synaptic currents recorded in brain slice preparation of SHR and WKY rats. In addition, bilateral microinjection of CRH into the PVN produced a greater dose‐dependent increases in arterial blood pressure (ABP), renal sympathetic activity (RSNA), and heart rate (HR) in anesthetized SHRs than in WKY rats. Microinjection of CRHR1 antagonist NBI35965 into the PVN significantly reduced basal ABP, RSNA, and HR in SHRs but did not change ABP, RSNA, and HR in WKY rats. These data suggested that the upregulation of CRHR1 in the PVN is critically involved in elevated sympathetic outflow and hyperactivity of sympathetic‐related neurons in hypothalamus in primary hypertension.

Efficient Neural Differentiation of Mouse Embryonic Stem Cells by Mastic Gum.

Purpose: Promoting neurogenesis is a promising strategy to treat neurodegenerative disorders. In the present study, we aimed to evaluate the effect of mastic gum resin from the Pistacia lentiscus var. Chia (Anacardiaceae family) in proliferation capacity and differentiation of embryonic mesenchymal stem cells into a neural lineage. Methods: For this purpose, mastic gum was applied as a neural inducer for stem cell differentiation into the neuronal lineage. Following treatment of embryonic stem cells (ESCs) with mastic gum, verification differentiation of the ESCs into the neuronal lineage, gene expression analysis, and immunocytochemistry staining approach were performed. Results: Gene expression analysis demonstrated that mastic gum increased the expression level of neuron markers in the ESCs-derived neuron-like cells. Moreover, our immunocytochemistry staining results of two important neural stem cell markers, including Nestin and microtubule-associated protein-2 (Map2) expression confirmed that mastic gum has the potential to promote neuronal differentiation in ESCs. Conclusion: In summary, the use of mastic gum to stimulate the differentiation of ESCs into a neural lineage can be considered as a good candidate in stem cell therapy.

Diagnostic Challenges in the Cytology of Thymic Epithelial Neoplasms.

Simple SummaryThymic epithelial neoplasms, including thymoma, thymic carcinoma, and thymic neuroendocrine neoplasms, constitute the majority of anterior mediastinal masses. Fine needle aspirations (FNA) of mediastinal masses are infrequently encountered and are highly challenging to interpret. Thymic neoplasms display a significant degree of histologic diversity and have overlapping morphologic features with tumors from other sites. However, when properly interpreted alongside ancillary studies and radiologic findings, FNAs can yield clinically actionable results. This review aims to illustrate the usefulness and diagnostic pitfalls of thymic FNAs to assist pathologists in analyzing these specimens. Thymic epithelial neoplasms are rare tumors that constitute the majority of anterior mediastinal masses. They are classified as thymomas, thymic carcinomas, and thymic neuroendocrine neoplasms. Biopsy diagnosis is not common, and most tumors are surgically resected. Biopsy, including cytology, is indicated when a non-surgical entity is suspected or in cases of locally advanced disease. Smears of thymomas consist of round or spindle epithelial cells admixed with varying amounts of lymphocytes depending on the type of thymoma. Smears of thymic carcinoma and thymic neuroendocrine neoplasms are often indistinguishable from corresponding tumor types from other organs. Accurate cytological diagnosis can be difficult due to the histological diversity of thymomas, as well as the morphological features that certain thymic tumors share with similar tumors from other organs. However, fine needle aspiration (FNA) of anterior mediastinal masses can provide clinically actionable information and can be used to determine whether lesions require surgical, systemic, or local noninvasive treatments. Ancillary studies, namely, immunocytochemical stains, flow cytometry, and radiology, are important tools in the evaluation of thymic aspirates. This review discusses the utility and limitations of thymic FNAs and illustrates the diagnostic features and pitfalls of these specimens.

IgG fractions from patients with antiphospholipid syndrome and systemic lupus erythematosus bind to platelets, but do not affect collagen-induced platelet activation

ABSTRACT Anti-beta-2 glycoprotein 1 (anti-β2GP1) is an antiphospholipid antibody found in patients with antiphospholipid syndrome (APS) and systemic lupus erythematosus (SLE). Its presence commonly is associated with thrombosis; however, the mechanisms of interaction of anti-β2GP1 antibodies and platelets remain unclear. We investigated the effects of APS and SLE patient-derived IgG fractions on collagen-mediated platelet aggregation and examined the binding of patient-derived IgG to platelets before and after activation by collagen. IgG fractions, 150, 200, 300 or 350 µg/ml, isolated from 11 patients with APS and SLE were incubated with two sets of platelet-rich plasma (PRP) in the incubation wells of an aggregometer. The first set was activated by collagen and the other set was incubated for an additional 10 min. All platelets were collected by centrifugation and fixed in cell blocks. We assessed binding of IgG to platelets using immunocytochemistry (ICC). Patient-derived IgG fractions did not affect collagen-induced platelet aggregation. ICC staining using anti-human IgG antibodies demonstrated that patient-derived IgG fractions had greater affinity for non-activated platelets than those activated by 0.75 µg/ml collagen. Patient-derived IgG fractions bound to the surface of platelets and potentially could be internalized by platelets. IgG fractions from APS and SLE patients may sensitize non-activated platelets, which could increase platelet reactivity and thrombotic risk in patients. We did not detect secondary effects of patient-derived IgG fractions.

Effects of Intracanal Antimicrobials on Viability and Differentiation of Stem Cells From the Apical Papilla: An In Vitro Study

BackgroundRecent studies have indicated that intracanal antimicrobials used to disinfect the root canal in regenerative endodontic therapies (RETs) may be cytotoxic to stem cells from the apical papilla (SCAP), leading to inconsistent treatment outcomes. However, the effects of intracanal antimicrobial agents on the odontogenic differentiation capacity of SCAP at sub-lethal concentrations have not been investigated. The aim of this study was to determine the effects of intracanal antimicrobials on SCAP viability and odontogenic differentiation capacity using a clinically relevant concentration range (0.1–0.8 mg/mL). MethodsImmature human third molars were collected from 71 patients and the apical papillae were harvested to form single-cell suspensions. The cytotoxic effects of intracanal antimicrobials including double antibiotic paste (DAP), triple or modified-triple antibiotic paste (TAP or MTAP), and calcium hydroxide (Ca(OH)2) on STRO-1+ SCAP were assessed using AlamarBlue and Live/Dead assays after exposing cells to treatment groups for 7 days at 0.1 to 0.8 mg/mL. The odontogenic differentiation potential of STRO-1+ SCAP was evaluated by immunocytochemistry staining of dentin matrix protein-1 and dentin sialophosphoprotein expression. ResultsAll concentrations of TAP significantly reduced STRO-1+ SCAP viability and odontogenic differentiation (P < .001), whereas no DAP concentrations were significantly cytotoxic. Ca(OH)2 and MTAP concentrations below 0.4 mg/mL and 0.2 mg/mL, respectively, did not significantly reduce viability. The DAP, MTAP, and Ca(OH)2 did not significantly impact the odontogenic differentiation capacity of STRO-1+ SCAP. ConclusionThe varying effects of intracanal antimicrobials on STRO-1+ SCAP in vitro suggest amendments to the current root canal disinfection protocol may improve the success of RETs.

Tightly Focused Femtosecond Laser Radiation Induces DNA Double-Strand Breaks in Human Tumor Cells.

We studied the formation of double-strand DNA breaks (DNA DSB) induced by femtosecond laser radiation in A549 human lung adenocarcinoma cells using immunocytochemical staining of the resulting tracks of a specific DSB marker protein phosphorylated ATM kinase (phospho-ATM). Additionally, colocalization of phospho-ATM tracks with γH2AX protein tracks was studied. The results of immunocytochemical analysis showed that 30 min after irradiation of cells with femtosecond pulses with energies of 1 and 2 nJ (radiation power density 2×1011 and 4×1011 W×cm-2, respectively), the formation of tracks consisting of phospho-ATM and γH2AX proteins located in sites where the laser beam passes through the cell nuclei was observed. The presence of phospho-ATM tracks co-localized with γH2AX allows us to conclude that exposure to focused femtosecond infrared laser radiation with a pulse energy of 1-2 nJ leads to the formation of DNA DSB in irradiated cells.

Response of murine neural stem/progenitor cells to gamma-neutron radiation

Purpose In recent years, a growing number of studies have focused on the mechanisms of action of densely ionizing radiation. This is associated with the development of radiation therapy of tumors using accelerated ions. The use of densely ionizing radiation appears to be the most promising method, optimal for treating patients with severe radioresistant forms, such as widespread head and neck tumors, recurrent and metastatic tumors, and some forms of brain tumors. The goal of our study was to investigate the effects of gamma-neutron radiation on mouse neural stem/progenitor cells (NSCs/NPCs). Methods NSCs/NPCs were isolated from neonatal mouse brains. Cells were irradiated in a collimated beam of neutrons and gamma rays of the IR-8 nuclear reactor. At 5 and 7 days after irradiation, cells and neurospheres were counted to assess survival. The number of DNA double-strand breaks and their repair efficiency were determined by immunocytochemical γH2AX staining followed by counting the number of γH2AX foci using a fluorescent microscope. Results We observed a dose-dependent decrease in the survival of NSCs/NPCs after irradiation at doses above 100 mGy and stimulation of the proliferation of these cells at doses of 25 and 50 mGy. In terms of a decrease in cell survival, the effect of gamma-neutron irradiation significantly exceeded the effect of gamma irradiation: the maximum value of the relative biological efficiency for gamma-neutron irradiation comprised 9.7. Gamma-neutron irradiation led to the formation of double-strand DNA breaks detected by the formation of foci of histone γH2AX in the cell nuclei. The γH2AX foci formed after gamma-neutron irradiation of NSCs/NPCs at doses of 100–500 mGy were characterized by a larger size in comparison with foci induced by gamma irradiation and gamma-neutron irradiation at a dose of 50 mGy. The repair of double-strand DNA breaks induced by γ,n-irradiation was slow; the repair rate depended on the radiation dose. Conclusions The data obtained indicate high sensitivity of proliferating NSCs/NPCs to gamma-neutron radiation. High RBE of gamma-neutron radiation requires special measures to protect the neurogenic regions of the brain when using this type of radiation in radiation therapy.

Perftoran ® Inhibits Hypoxia-Associated Resistance in Lung Cancer Cells to Carboplatin.

Perftoran® (perfluorodecalin) is an oxygen carrier, and carboplatin is a common chemotherapy drug used worldwide for lung cancer treatment. Hypoxia is one of the factors that induce resistance of lung cancer cells to carboplatin. This study explored the role of Perftoran®, as an oxygen carrier, in lowering the resistance of lung cancer cells to carboplatin through suppression of hypoxia pathway mediators. The effect of Perftoran® on the resistance of human lung cancer A549 cells to carboplatin was investigated through the evaluation of cytotoxicity by MTT, cell death mode by dual DNA staining, DNA damage by comet assay, DNA platination (DNA/carboplatin adducts) by atomic absorption spectroscopy, hypoxia degree by pimonidazole, HIF-1α/HIF-2α concentrations by ELISA, expression of miRNAs (hypoxamiRs miR-210, miR-21, and miR-181a) by qRT-PCR, and the content of drug resistance transporter MRP-2 by immunocytochemical staining. Results indicated that compared to carboplatin, Perftoran®/carboplatin decreased cell resistance to carboplatin by potentiating its cytotoxicity using only 45% of carboplatin IC50 and inducing apoptosis. Perftoran® induced DNA platination and DNA damage index in cells compared to carboplatin alone. Moreover, compared to treatment with carboplatin alone, co-treatment of cells with Perftoran® and carboplatin inhibited cellular pimonidazole hypoxia adducts, diminished HIF-1α/HIF-2α concentrations, suppressed hypoxamiR expression, and decreased MRP-2. In conclusion, Perftoran® inhibited resistance of lung cancer cells to carboplatin through the inhibition of both hypoxia pathway mediators and the drug resistance transporter MRP-2 and through the induction of DNA/carboplatin adduct formation.

Potential role of dental pulp stem cells conditioned medium for odontoblastic differentiation

BackgroundFunctional bioengineered tooth regeneration using autologous or allogeneic alternative differentiated cells sources are thought to have a great potential in replacing conventional dentures. This study investigated the potential of dental pulp stem cells (DPSCs) conditioned medium for odontoblastic differentiation of Wharton’s jelly mesenchymal stem cells (WJMSCs). The DPSCs derived from healthy adult permanent first molars were cultured at high confluence prior to conditioned medium collection. The WJMSCs were cultured in six different treatments, with varying ratios of culture media to DPSCs-conditioned medium. MTT assay was used to measure the rate of proliferation of WJMSCs, while immunocytochemistry staining was utilised to detect the expression of dental matrix protein 1 (DMP-1). The deposited calcium was detected and analysed via Alizarin-Red Staining (ARS).ResultsIt was found that the proliferation of WJMSCs cultured under the mixture of complete medium and DPSCs conditioned medium showed significantly lower than the control; presumably the cells started to exit proliferative state prior differentiation. In 14 days of induction, the cells in all treatments showed osteoblastic-like morphology, calcium compound deposits were observed at day 7, 10 and 14 of differentiation suggested that DPSCs conditioned medium could lead to osteoblastic/odontoblastic differentiation. However, the DMP-1 protein can be seen only expressed minimally at day 14 of conditioned medium induction.ConclusionsIn conclusion, DPSCs conditioned medium appeared as a potential odontoblastic induction approach for WJMSCs. To further investigate the stimulatory effects by DPSCs conditioned medium, specific signalling pathway need to be elucidated to enhance the differentiation efficiency.