BCRP Expression with Lipofuscin Accumulation in Abnormal Neurons from a Child with Transmantle Cortical Dysplasia (TMCD) and Refractory Epilepsy

Cerebral cortical development’s malformations, including the transmantel cortical dysplasia (TMCD), have been associated with refractory epilepsy (RE). Several ABC-transporters as “P-glycoprotein (P-gp), Multidrug resistance proteins (MRP-1) and breast Cancer Resistant Protein (BCRP)” are up-regulated in human epileptogenic brain lesions of RE, however they have not been explored in Transmantle cortical dysplasia (TMCD). We describe a 13 years old boy with Refractory Epilepsy (RE) and abnormal Magneic Resonance Image (MRI) (T1, FLAIR and T2) compatible with TMCD. Clinical follow-up, images and pathologic studies were developed by routinely methods. Epilepsy surgical treatment included total lesion resection with complete seizures remission at date. Deeper brain areas related with images findings, showed features of TMCD with abnormal ballooned neurons with high accumulation of PAS+, sudanophilic and autoflourescent lipopigment (LP). Immunohistochemistry, using primary monoclonal antibodies for P-gp, MVP and BCRP proteins, showed high expression of BCRP in several ballooned-LP+ cells. In contrast, P-gp and MVP were negative and MRP-1 has not been investigated. The links of BCRP with LP and AEDs are not known, however, the expression of BCRP in these LP+ ballooned neurons from the epileptogenic brain area, with P-gp/MVP negative results, suggest that BCRP could be associated to refractory epileptic phenotype.

Nodular Lymphocyte Predominant Hodgkin Lymphoma - A Retrospective Immunohistochemical Study of Patients in Bangalore, Karnataka

BACKGROUND The term Hodgkin’s lymphoma includes classical Hodgkin lymphoma (CHL) and the less common nodular lymphocyte predominant Hodgkin lymphoma (NLPHL). NLPHL is a B cell neoplasm usually characterised by nodular or follicular and diffuse proliferation of small lymphocytes with single scattered large neoplastic cells (LP/L&H/Popcorn cells). NLPHL accounts for 10 % of all Hodgkin lymphoma. METHODS This is a retrospective study. Histopathology slides and blocks of 24 cases of nodular lymphocyte predominant Hodgkin lymphoma were collected from the archives of histopathology from 2011 to 2015. The immunohistochemistry slides of the corresponding histopathology cases were also assembled. Both the slides were reviewed by three expert onco-pathologists and IHC markers were studied and compared. RESULTS Patients were mostly young between 20 and 40 years (16 / 24, 66.67 %). There was a distinct male preponderance (20 / 24, 83.3 %). Most cases involved cervical, axillary or inguinal lymph nodes, with cervical lymph nodes being the most common (13 / 24, 54 %). It was found that CD45, CD20, CD79a and PAX5 staining highlighted the LP cells in all twenty-four cases, while OCT - 2 and BOB - 1 were highlighted in twenty-three cases (95.8 %), which was statistically significant. CD3 and CD5 IHC staining on T cell rosettes and background reactive T cells were examined, and it was seen that CD3 expression was far more consistent than CD5 expression in T cell rosettes and reactive T cells. Also, it was seen that, those cases which were double positive for CD3 and CD5 constitutes only eight cases (8 / 24, 33.3 %). CONCLUSIONS CD3 is a more consistent marker than CD5 in demonstrating surrounding reactive T cells in NLPHL. CD45, PAX5, CD20, BOB - 1 and OCT - 2 are consistent immunohistochemical markers of LP cells. KEYWORDS Nodular Lymphocyte Predominant Hodgkin Lymphoma (NLPHL), Classical Hodgkin Lymphoma (CHL), Cluster Differentiation (CD), Lymphocyte Predominant Cells (LP Cells), Lymphocyte and Histiocytic Cell (L & H Cell)

Visual Thalamocortical Mechanisms of Waking State Dependent Activity and Alpha Oscillations

SummaryThe brain exhibits distinct patterns of recurrent activity closely related to the behavioral state of the animal. The neural mechanisms that underlie state-dependent activity in the awake animal are incompletely understood. Here, we demonstrate that two types of state-dependent activity - rapid arousal/movement related signals and a 3-5 Hz alpha-like rhythm - in the primary visual cortex (V1) of mice strongly correlate with activity in the visual thalamus. Inactivation of V1 does not interrupt arousal/movement related signals in most visual thalamic neurons, but it abolishes the 3-5 Hz oscillation. Silencing of the visual thalamus similarly eradicates the alpha-like rhythm and perturbs arousal/movement-related activation in V1. Finally, we observed that whisker movement or locomotion is not required for rapid increases in cortical activation. Our results indicate that thalamocortical interactions together with cell-intrinsic properties of thalamocortical cells play a crucial role in shaping state-dependent activity in V1 of the awake animal.HighlightsWhisker movements correlate with rapid synaptic activation in V1 and visual thalamusSilencing of V1 does not abolish movement related activation in most dLGN or LP cellsSilencing of visual thalamus strongly reduces movement related activation in V1Thalamocortical interactions generate state-dependent alpha frequency oscillationVisual thalamic cells exhibit LTS firing during alpha oscillation in the awake mouse

Effects of Fluoride Exposure on Primary Human Melanocytes from Dark and Light Skin

Fluoride exposure has adverse effects on human health that have been studied in vitro in cell culture systems. Melanocytes are the melanin pigment-producing cells that have a significant role in the regulation of the process of melanogenesis, which provides several health benefits. Melanocytes are present in the oral cavity, skin, brain, lungs, hair, and eyes. However, to date, there has been no study on the effects of fluoride exposure on melanocytes. Hence, in the current study, we have studied the effects of sodium fluoride (NaF) exposure on neonatal human epidermal melanocytes (HEMn) derived from two different skin phototypes, lightly pigmented (LP) and darkly pigmented (DP). We have assessed the impact of a 24 h and 72 h NaF exposure on metabolic activity and membrane integrity of these cells. In addition, we have evaluated whether NaF exposure might have any impact on the physiological functions of melanocytes associated with the production of melanin, which is regulated by activity of the enzyme tyrosinase. We have also assessed if NaF exposure might induce any oxidative stress in LP and DP melanocytes, by evaluation of production of reactive oxygen species (ROS) and measurement of mitochondrial membrane potential (MMP) levels. Our results showed that HEMn-LP cells showed a higher sensitivity to NaF cytotoxicity than HEMn-DP cells, with significant cytotoxicity at concentrations >1 mM, while concentration range 0.25–1 mM were nontoxic and did not lead to oxidative stress, and also did not alter the levels of intracellular melanin or cellular tyrosinase activity, indicating that treatment up to 1 mM NaF is generally safe to melanocytes from both pigmentation phototypes.

CD20-Negative Nodular Lymphocyte-Predominant Hodgkin Lymphoma: A 20-Year Consecutive Case Series From a Tertiary Cancer Center

Context.— Nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL) is a rare, indolent Hodgkin lymphoma subtype with distinct clinicopathologic features and treatment paradigms. The neoplastic lymphocyte-predominant cells typically express bright CD20 and other B-cell antigens, which distinguishes them from Hodgkin/Reed-Sternberg cells of lymphocyte-rich classic Hodgkin lymphoma. Objective.— To characterize the clinicopathologic features of CD20-negative NLPHL at a single institution. Design.— A retrospective search for CD20-negative NLPHL in our pathology archives and medical records was conducted. Results.— Of 486 NLPHL patients identified with CD20 available for review, 14 (2.8%) had LP cells with absent CD20 expression. Patients with prior rituximab administration (n = 7) and insufficient clinical history (n = 1) were excluded, leaving 6 patients with rituximab-naïve, CD20-negative NLPHL. A broad immunohistochemical panel showed the LP cells in all cases expressed B-cell antigens, particularly Oct-2, although PAX5 and CD79a were frequently also dim. CD30, CD15, and Epstein-Barr virus-encoded small RNAs were negative in all evaluated cases. Two patients had high-risk variant immunoarchitectural pattern D. One patient had extranodal disease, involving the spleen and bone, and was suspected to have large cell transformation. Standard NLPHL therapy was given, including local radiation and/or chemotherapy. Of 5 patients with available follow-up, 4 are alive in complete remission after therapy, and 1 is alive with relapsed disease. Conclusions.— NLPHL can lack CD20 de novo without prior rituximab therapy. In such cases, extensive immunophenotyping helps distinguish NLPHL from lymphocyte-rich classic Hodgkin lymphoma, which differ in clinical behavior and therapy. In our series, CD20-negative NLPHL showed both classic and variant histologic patterns and the expected range of clinical behavior seen in NLPHL, including 1 case with suspected large cell transformation.

Aneuploidy and a deregulated DNA damage response suggest haploinsufficiency in breast tissues of BRCA2 mutation carriers

Women harboring heterozygous germline mutations of BRCA2 have a 50 to 80% risk of developing breast cancer, yet the pathogenesis of these cancers is poorly understood. To reveal early steps in BRCA2-associated carcinogenesis, we analyzed sorted cell populations from freshly-isolated, non-cancerous breast tissues of BRCA2 mutation carriers and matched controls. Single-cell whole-genome sequencing demonstrates that >25% of BRCA2 carrier (BRCA2mut/+) luminal progenitor (LP) cells exhibit sub-chromosomal copy number variations, which are rarely observed in non-carriers. Correspondingly, primary BRCA2mut/+ breast epithelia exhibit DNA damage together with attenuated replication checkpoint and apoptotic responses, and an age-associated expansion of the LP compartment. We provide evidence that these phenotypes do not require loss of the wild-type BRCA2 allele. Collectively, our findings suggest that BRCA2 haploinsufficiency and associated DNA damage precede histologic abnormalities in vivo. Using these hallmarks of cancer predisposition will yield unanticipated opportunities for improved risk assessment and prevention strategies in high-risk patients.

P160 γδ T CELLS, IL-22, THE PROBIOTIC, LACTOBACILLUS DELBRUECKII, AND THE ROLE THEY PLAY IN ATTENUATION OF ALCOHOL INDUCED EXACERBATION OF DSS-COLITIS

Ulcerative colitis is characterized by cycles of active disease flare and inactive disease remission. During UC remission, IL-22 expression can be upregulated, acting as a hallmark of entrance into a UC remission period. Recently, we found that in our mouse model of binge alcohol consumption after DSS-induced colitis, alcohol increases severity of UC flare symptoms. In this study, we assessed whether alcohol influenced IL-22 expression and thereby perpetuates UC flare. Male C57BL/6 mice received 2% DSS or water ad libitum for 5 days. On day 5, DSS was removed to mimic entrance into remission. Additionally on day 5, DSS and Sham mice were subdivided into mice gavaged with ethanol (∼3g/kg) or with water on days 5, 6, and 7. Three hours after the last gavage on day 7, mice were humanely euthanized. Large intestine lamina propria (LP) cells were isolated. The percentage of total IL-22+ LP cells was significantly decreased (p<0.05) in DSS Ethanol compared to DSS Vehicle. No differences in IL-22+ T cells, Innate Lymphoid Cells Type 3, or neutrophils were observed. Examination of I γδ T cells revealed DSS Vehicle treated mice had a significantly increased percentage of IL-22+ γδ T cells, while DSS Ethanol treated mice were unable to mount this response. Therefore, we hypothesized that by re-establishing IL-22, through either rIL-22 or a probiotic, we could alleviate the alcohol-induced exacerbation of UC. Firstly, rIL-22 administration substantially restored weight loss of DSS Ethanol treated mice back to that of DSS Vehicle (∼12.5% back to ∼6% on day 7). Increased colonic shortening (p<0.001) and increased Enterobacteriaceae copy number were also attenuated following binge alcohol and colitis with IL-22 treatment. Knockout of STAT3 in IECs resulted in loss of IL-22 protection, demonstrating STAT3 is required for protection. Secondly, we utilized Lactobacillus delbrueckii, a common probiotic known to play a role in IL-22 release. Treatment with Lacto attenuated both weight loss and colon length in DSS Ethanol mice back to levels of DSS alone (p<0.01 and p<0.001, respectively). Additionally, Lacto treatment mitigated increases in Enterobacteriaceae copy number seen in DSS Ethanol mice and trended towards an increase in IL-22 in DSS Ethanol + Lacto mice. Levels of pSTAT3 were decreased in DSS Ethanol treated mice compared to DSS Vehicle, but administration of Lacto in DSS Ethanol mice increased levels of pSTAT3 back to that of the DSS Vehicle group. Treatment with Lacto supernatant alone was not sufficient to mitigate the exacerbation of UC following ethanol. Our findings suggest that both rIL-22 and Lactobacillus delbrueckii utilize the IL-22/pSTAT3 signaling pathway to attenuate alcohol-induced increases in ulcerative colitis symptoms. (R21AA022324, R21AA025806, T32AA013527, F30AA027442, F31AA025536)

Investigation of stemness and multipotency of equine adipose-derived mesenchymal stem cells (ASCs) from different fat sources in comparison with lipoma

Background Adipose tissue-derived mesenchymal stem cells (ASCs) offer a promising cell source for therapeutic applications in musculoskeletal disorders. The appropriate selection of ASCs from various fat depots for cell-based therapy is challenging. The present study aims to compare stemness and multipotency of ASCs derived from retroperitoneal (RP), subcutaneous (SC), and lipoma (LP) fat to assess their usefulness for clinical application. Methods Equine ASCs from the three fat tissue sources were isolated and characterized. The cell viability, proliferation, and self-renewal were evaluated using MTT, sulforhodamine B, and colony forming unit (CFU) assays. Stem cell relative marker CD44, CD90, and CD105 and tumor marker CA9 and osteopontin (OPN) expression were quantified using RT-qPCR. Multipotency of ASCs for adipogenic, osteogenic, and chondrogenic differentiation was examined by quantifying Oil Red O and Alizarin Red S staining, alkaline phosphatase activity (ALP), and expression of differentiation relative markers. All data were statistically analyzed using ANOVA. Results RP fat-derived ASCs showed a higher cell proliferation rate compared to SC and LP derived cells. In contrast, ASCs from lipoma displayed a lower proliferation rate and impaired CFU capacities. The expression of CD44, CD90, and CD105 was upregulated in RP and SC derived cells but not in LP cells. RP fat-derived cells displayed a higher adipogenic potential compared to SC and LP cells. Although ASCs from all fat sources showed enhanced ALP activity following osteogenic differentiation, SC fat-derived cells revealed upregulated ALP and bone morphogenetic protein-2 expression together with a higher calcium deposition. We found an enhanced chondrogenic potency of RP and SC fat-derived cells as shown by Alcian blue staining and upregulation of aggrecan (Aggre), cartilage oligomeric matrix protein precursor (COMP), and collagen 2a1 (Col2a1) expression compared to LP. The expression of OPN and CA9 was exclusively upregulated in the ASCs of LP. Conclusions The results provide evidence of variation in ASC performance not only between normal fat depots but also compared to LP cells which suggest a different molecular regulation controlling the cell fate. These data provided are useful when considering a source for cell replacement therapy in equine veterinary medicine.

Aneuploidy and deregulated DNA damage response define haploinsufficiency in breast tissues of BRCA2 mutation carriers

Women harboring heterozygous germline mutations of BRCA2 have a 50-80% risk of developing breast cancer, yet the early pathogenesis of these cancers is poorly understood. We sought to reveal early steps in BRCA2-associated carcinogenesis through analysis of sorted cell populations from freshly-isolated, non-cancerous breast tissues among a cohort of BRCA2 mutation carriers and matched controls. Single-cell whole-genome sequencing demonstrates that >25% of BRCA2 carrier (BRCA2mut/+) luminal progenitor (LP) cells exhibit sub-chromosomal copy number variations (CNVs), which are rarely observed in non-carriers. Correspondingly, primary BRCA2mut/+ breast epithelia exhibit spontaneous and replication stress-induced DNA damage together with attenuated replication checkpoint and apoptotic responses, associated with an age-associated expansion of the LP compartment in human carrier tissues. These phenotypes are not associated with loss of wild-type BRCA2. Collectively, these findings provide evidence for BRCA2 haploinsufficiency and associated DNA damage in vivo that precede histologic abnormalities. These results provide unanticipated opportunities for new cancer risk assessment and prevention strategies in high-risk patients.

CCR2+ Monocyte-Derived Ly6C-F4/80+ Fibrocytes Inhibit Collagen Degradation and Contribute to the Development of Colon Fibrosis Via Production of TIMP-1

Introduction: Fibrocytes are derived from a subset of monocytes and express mesenchymal markers such as collagen type I (Col I) and hematopoietic markers such as CD45 and CD11b. They also express several chemokine receptors such as CC chemokine receptor (CCR)-1, CCR2, CCR5, CCR7, and CXC chemokine receptor type 4 (CXCR4). They circulate in the peripheral blood (PB) and can be isolated from many fibrotic tissues. Fibrocytes participate in both physiological wound healing and various pathological fibrosis including myelofibrosis, hypertrophic scar, systemic sclerosis, idiopathic pulmonary fibrosis, liver cirrhosis, and progressive kidney disease. In murine and human colitis, fibrocytes are also reported to be associated with the colon fibrosis. It has been described that migration of fibrocytes to the injured sites involves CC chemokine ligand 2 (CCL2)/CCR2 axis in the liver and kidney and CXC chemokine ligand 12 (CXCL12)/CXCR4 axis in the lung. However, there are few reports concerning the role of fibrocytes and their expression of chemokine receptors related to the induction of colon fibrosis. Methods: We generated bone marrow (BM) chimeric mice by transplantation of BM total-nucleated cells, which were isolated from enhanced green fluorescent protein (EGFP)-transgenic mice or CCR2 knockout (KO) mice, into lethally irradiated C57BL/6J-Ly5.1 mice. Two months after BM transplantation, BM chimeric mice were treated with a single intraperitoneal injection of azoxymethane (10 mg/kg body weight) followed by 3 cycles of 1% dextran sulfate sodium (DSS) in the drinking water. We assessed the level of fibrosis in the colon using Sirius red staining and analyzed the presence of BM-derived CD45+CD11b+Col I+ fibrocytes in the colon lamina propria (LP) using immunofluorescence staining and flow cytometry. Furthermore, we investigated the expressions of Col I, transforming growth factor-ß (TGF- ß), matrix metalloproteinases (MMPs), and tissue inhibitor of MMPs (TIMP)-1 in the colon tissues and fibrocytes sorted from colon LP cells after chronic DSS treatment using quantitative real-time RT-PCR. Results: During chronic inflammation, infiltration of CCR2+ BM-derived monocytes and fibrocytes and production of CCL2 in the colon were particularly increased and colon fibrosis was developed in EGFP BM chimeric mice. Two types of fibrocytes, CCR2+CXCR4+Ly6C-F4/80+ fibrocytes and CCR2-CXCR4+Ly6ChighF4/80- fibrocytes, were identified in the colon LP, whereas only the latter fibrocytes were detected in the PB. Adoptive transferred CCR2+Ly6ChighCol I- monocytes migrated to the injured colon and a part of them differentiated into CCR2+Col I+ fibrocytes. In CCR2KO BM chimeric mice, the numbers of monocytes and fibrocytes in the colon LP were significantly decreased and colon fibrosis was attenuated. However, there was no difference in the mRNA expressions of Col I, TGF-ß, and MMPs (MMP-1a, MMP-8, and MMP-13, known as collagenases) in colon tissues between EGFP BM chimeric mice and CCR2KO BM chimeric mice. Improvement of colon fibrosis in CCR2KO BM chimeric mice was associated with the decreased expression of Timp1 mRNA in colon tissues. We analyzed the expression of Timp1 mRNA in CCR2+ cells and CCR2- cells sorted from colon LP cells and found a high expression of Timp1 in CCR2+ monocytes/macrophages and fibrocytes. Conclusions: Circulating CCR2+ monocytes migrate into the inflamed colon via CCL2/CCR2 axis and differentiate into CCR2+Ly6C-F4/80+ fibrocytes, which inhibit collagen degradation and contribute to the development of colon fibrosis by the production of TIMP-1. Disclosures Masuya: Kyowa Hakko Kirin Co., Ltd.: Research Funding. Katayama:Ono Pharmaceutical: Research Funding; Novo Nordisk: Honoraria, Research Funding; Chugai Pharma: Honoraria, Research Funding; Toyama Chemical Co: Research Funding; Sysmex: Honoraria; Mochida Pharmaceutical Co. Ltd.,: Research Funding; Bristol-Myers Squibb: Honoraria; Astellas Pharma: Honoraria, Research Funding; Daiichi Sankyo: Research Funding; Takeda: Honoraria, Research Funding; Teijin Pharma: Research Funding; Eisai: Research Funding; Sumitomo Group: Honoraria, Research Funding; Nippon Shinyaku: Honoraria, Research Funding; Shire: Honoraria; Alexion Pharmaceuticals: Honoraria; Celgene: Honoraria; Taisho Toyama Pharma: Honoraria; Pfizer: Honoraria, Research Funding; Shionogi Pharmaceutical: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Janssen: Research Funding; Kyowa Hakko Kirin: Honoraria, Research Funding.