Dual-label Immunofluorescence Research Articles

Evidence of Browning and Inflammation Features in Visceral Adipose Tissue of Women with Endometriosis

Patients with endometriosis tend to have a low body mass index, suggesting an inverse relationship between body fat and risk of disease. This is supported by evidence that miRNAs differentially expressed in endometriosis induce browning of pre-adipocytes in vitro. Thus, we hypothesize that endometriosis may underlie adipose tissue (AT) dysfunction and browning. Identify inflammation and browning processes in AT collected from endometriosis patients. Visceral and subcutaneous AT samples were obtained during endometriosis (n = 32) or uterine myoma (n = 14; controls) surgery. Blood catecholamines were determined by high-performance liquid chromatography while IL-6 and TGF-β levels were quantified by ELISA. Adipocyte cross-sectional areas were analyzed in H&E-stained sections by computer-assisted morphometry. Macrophages (F4/80; Galectin-3) and browning activation (UCP-1; PGC-1α) in tissues were identified by dual label immunofluorescence. Expression of inflammatory (IL-6; MCP-1; Galectin-3; CD206; TIMP1; TGF-β) and browning-related (UCP-1; PGC-1α; DIO2; CITED1; CIDEA; TMEM26; TBX1; PRDM16; PPAR-γ) molecules in AT were assessed by RT-PCR and Western blotting. Compared to controls, patients presented smaller adipocytes, especially in VAT, and lower norepinephrine levels. Serum IL-6, but not TGF-β, was increased in patients. UCP-1, PGC-1α, IL-6, and MCP-1 were upregulated in VAT from endometriosis women, which also evidenced a reduction of CD206, relative to controls. However, no differences were found in mRNA expression of IL-6, TIMP1, and TGF-β nor Galectin-3 protein levels. In SAT, protein expression remained unchanged between patients and controls. Our findings support an endometriosis' role as a pro-catabolic state along with local signals of VAT browning and inflammation.

P-331 Unlocking metformin’s potential: exploring its impact on inflammation and oxidative pathways in an endometriosis mouse model

Abstract Study question Could endometriosis-associated pro-oxidative and pro-inflammatory condition be mitigated by metformin? Summary answer Metformin treatment tends to reduce fibrosis in ectopic tissues, while up-regulating anti-oxidative response enzymes. What is known already Endometriosis is a pro-inflammatory and pro-oxidative disease, which causes pain and infertility. Hormonal treatments used to control the disease must be stopped when a woman attempts to conceive, meaning new therapies are necessary. We had shown that endometriosis reduces fertility in a mouse model, which metformin treatment led to its reversal. Metformin, in addition to reducing blood glucose, reduces inflammation and systemic oxidation. It has also been shown to mitigate the growth of endometrial implants in the animal model. The objective of this work is to evaluate the intervention of metformin in the inflammatory and oxidative response pathways associated with endometriosis. Study design, size, duration Female mice were randomly divided into groups: 1-Endometriosis (n = 24); 2-Sham (n = 12); 3-Endometriosis with metformin (n = 21); 4-Sham with metformin (n = 16). Endometriosis was surgically induced by heterologous transplantation of endometrium from a donor into recipients of the same strain. Metformin (0,5mg/mL) was administered orally for 3 months. Half of the mice in each group were mated for the fertility study. Tissues analysed are described with “Eu” for eutopic and “Ec” for ectopic tissues, respectively. Participants/materials, setting, methods The implants were confirmed and monitored by ultrasound and serum glucose, estradiol and lipoprotein values were quantified by enzymatic-colorimetric methods. Fibrosis in eutopic and ectopic tissues was quantified after computer-assisted analysis in Masson’s Trichrome images. Dual-labelling immunofluorescence was performed for the proteins F4/80 and Galectin-3 (gal-3) and Western Blot (WB) was performed to quantify their expression and also NFkB, HO-1 and GPX-1, both in eutopic and ectopic tissues. Main results and the role of chance No differences were found in serum analysis between groups, apart from a decrease in total cholesterol and an increase in estradiol in pregnant compared to non-mated mice. Masson’s Trichrome stained sections revealed increased stromal fibrosis in both eutopic and ectopic tissues of non-mated Endometriosis groups compared to Sham groups(EuEnd vs. EuSham p = 0.018; EcEnd vs. EuSham p = 0.022; EuEnd vs. EuShamMet p = 0.0008; EcEnd vs EuShamMet p = 0,001). Metformin treatment showed a decreasing trend in fibrosis both in non-mated and pregnant, particularly in ectopic tissue, although statistical significance was not reached. Both eutopic and ectopic endometrium presented positive cells for f4/80 and gal-3, with some co-localization, in all groups. Metformin treatment led to the increase of the inflammation markers in the tissues of mice with ENDO: F4/80 was increased in eutopic tissue(p = 0.0002), while gal-3 (p = 0.004) and NF-kB (p = 0.03) augmented in ectopic tissue of non-mated. Pregnancy condition, on the other hand had a tendency for the decrease of these proteins. Metformin increased the expression of antioxidant defense enzymes HO-1 (p = 0,005) and GPX-1 (p = 0,0001) in the ectopic endometrium of non-mated mice. Metformin has shown to act in the up-regulation of anti-oxidative defenses that intervene in the control of endometriosis progression, but this effect was not evident during pregnancy. Limitations, reasons for caution Caution is necessary when extrapolating animal model data to humans due to species-specific differences in endometriosis patterns. Further studies, assessing pro-oxidative species, total antioxidant capacity, and potential anti-inflammatory roles of studied molecules, are essential to clarify the unexpected results in inflammatory pathways. Wider implications of the findings These findings highlight metformin, in a subtherapeutic dose, as a novel and safe approach to alleviate oxidative stress in endometriosis, offering a promising pharmacological solution for improving endometriosis-associated infertility, once we also had shown the reversal of infertility associated with endometriosis through metformin treatment. Trial registration number not applicable

Mesencephalic astrocyte-derived neurotrophic factor inhibits cervical cancer progression via regulating macrophage phenotype.

Cervical cancer is a common gynecologic malignant tumor, but the critical factors affecting cervical cancer progression are still not well demonstrated. Mesencephalic astrocyte-derived neurotrophic factor (MANF) has been widely recognized as an anti-inflammatory factor to regulate macrophage polarization. In this study, the effect and mechanism of MANF on cervical cancer were preliminarily explored. Kaplan-Meier curve was used to show the overall survival time of the involved cervical cancer patients with high and low MANF expression in cervical cancer tissues. MANF was highly expressed in peritumoral tissues of cervical carcinoma by using immunohistochemistry and western blot. MANF mRNA level was detected by using qRT-PCR. Dual-labeled immunofluorescence showed MANF was mainly expressed in macrophages of cervical peritumoral tissues. Moreover, MANF-silenced macrophages promoted HeLa and SiHa cells survival, migration, invasion and EMT via NF-κB signaling activation. The results of tumor formation in nude mice indicated MANF-silenced macrophages promoted cervical tumor formation in vivo. Our study reveals an inhibitory role of MANF in cervical cancer progression, indicating MANF as a new and valuable therapeutic target for cervical cancer treatment.

BARX1 promotes osteosarcoma cell proliferation and invasion by regulating HSPA6 expression

Osteosarcoma (OS) is a bone tumour affecting adolescents. Dysregulation of Barx homeobox 1 (BARX1) expression is involved in various cancers, but its function and mechanism in the process of OS are undefined. This study revealed that BARX1 expression is higher in OS tissue than in adjacent normal tissue. Downregulation of BARX1 in OS cells significantly suppressed their proliferation and migration, whereas enforced expression of exogenous BARX1 exerted the opposite effects on OS cells. Subsequently, heat shock 70-kDa protein 6 (HSPA6) expression was clearly increased after BARX1 overexpression in OS cells, as confirmed by RNA sequencing. The dual-luciferase reporter assay confirmed that HSPA6 expression is directly regulated by BARX1. The in vitro assay indicated that silencing HSPA6 expression attenuated OS proliferation and migration induced by BARX1. A dual immunofluorescence labelling assay provided further evidence that BARX1 was overexpressed and associated with HSPA6 overexpression in OS tumour tissue. In conclusion, BARX1 promotes OS cell proliferation and migration by inducing the expression of HSPA6, which plays an oncogenic role in OS. BARX1 and HSPA6 can potentially act as novel therapeutic targets for OS.

Single-Cell Transcriptomic Analysis of Ecosystems in Papillary Thyroid Carcinoma Progression.

BackgroundDespite extensive research, the papillary thyroid carcinoma (PTC) ecosystem is poorly characterized and, in particular, locoregional progression. Available evidence supports that single-cell transcriptome sequencing (Sc-RNA seq) can dissect tumor ecosystems.MethodsTissue samples from one PTC patient, including matched primary tumor (Ca), lymph node (LN) metastasis, and paracancerous tissue (PCa), were subjected to Sc-RNA seq with 10×Genomics. Dual-label immunofluorescence and immunohistochemistry were used to confirm the existence of cell subtypes in a separate cohort.Results11,805 cell transcriptomes were profiled, cell landscapes of PTC were composed of malignant follicular epithelial cells (MFECs), CD8+ and CD4+ T cells, B cells, vascular endothelial cells, fibroblasts and cancer-associated fibroblasts (CAFs). Between Ca and LN ecosystems, the proportions of MFEC and interstitial cells were similar, less than 1/25(229/6,694, 361/3,895), while the proportion of normal follicular epithelial cells (NFECs) and interstitial cells was > 2 in PCa (455/171). NFECs in PCa formed a separate cluster, while MFECs in Ca and LN exhibited a profound transcriptional overlap, and the interstitial cells among these samples had an overall concordance in their identity and representation, albeit with some distinctions in terms of the cell percentage per subset. A fraction of the B cell subpopulation in Ca expressed inhibitory receptors, while their respective ligand genes were clearly transcribed in T cell and malignant epithelial cell clusters, while some CD8+ T cells in both Ca and LN produced high levels of inhibitory receptors whose respective ligands were overexpressed in some CD4+ T cells. Three CAF subtypes in Ca and LN were identified, which may be due to mutual transitions.ConclusionsOur data provide new insights into the PTC ecosystem and highlight the differences in ecosystems in PTC progression, which updates our understanding of PTC biology and will improve individualized patient treatment.

Immunoresponsive Tissue-Engineered Oral Mucosal Equivalents Containing Macrophages.

Macrophages play a key role in orchestrating the host immune response toward invading organisms or non-self molecules in the oral mucosa. Three-dimensional (3D) oral mucosal equivalents (OME) containing oral fibroblasts and keratinocytes are used extensively to mimic the human oral mucosa where they have been employed to examine innate immune responses to both bacterial and fungal pathogens as well as to biomaterials. Although the presence of immune cells is critical in generating an immune response, very few studies have incorporated leukocytes into OME, and to date, none have contained primary human macrophages. In this study, we report the generation of an immunocompetent OME to investigate immune responses toward bacterial challenge. Primary human monocyte-derived macrophages (MDM) were as responsive to bacterial lipopolysaccharide (LPS) challenge when cultured within a 3D hydrogel in terms of proinflammatory cytokine (IL-6, CXCL8, and TNF-α) gene expression and protein secretion compared with culture as two-dimensional monolayers. MDM were incorporated into a type 1 collagen hydrogel along with oral fibroblasts and the apical surface seeded with oral keratinocytes to generate an MDM-containing OME. Full-thickness MDM-OME displayed a stratified squamous epithelium and a fibroblast-populated connective tissue containing CD68-positive MDM that could be readily isolated to a single-cell population for further analysis by collagenase treatment followed by flow cytometry. When stimulated with LPS, MDM-OME responded with increased proinflammatory cytokine secretion, most notably for TNF-α that increased 12-fold when compared with OME alone. Moreover, this proinflammatory response was inhibited by pretreatment with dexamethasone, showing that MDM-OME are also amenable to drug treatment. Dual-labeled immunofluorescence confocal microscopy revealed that MDM were the sole source of TNF-α production within MDM-OME. These data show functional activity of MDM-OME and illustrate their usefulness for investigations aimed at monitoring the immune response of the oral mucosa to pathogens, biomaterials, and for tissue toxicity and anti-inflammatory drug delivery studies.Impact statementThree-dimensional in vitro models of the oral mucosa have been used extensively to investigate the host response to pathogens, but, to date, few have contained primary leukocytes. In this report, we describe the successful incorporation of primary human macrophages into oral mucosal equivalents (OME). These macrophage-containing models were histologically similar to the oral mucosa and immunoresponsive to bacterial lipopolysaccharides by upregulation of key proinflammatory markers. These advanced OME will significantly aid research into host–pathogen interaction, biomaterial toxicity, and drug delivery studies where the presence of an immune cell component is critical to better represent host oral tissue.

Overactive Reproductive Axis Due to Fragile X Gene Mutation

Women carrying a pre-mutation or mutation of the Fragile X mental retardation gene (FMR1) comprise the largest portion of premature ovarian failure (POF) cases due to known genetic factors. FMR1 mutation causes Fragile X syndrome, the most common cause of inherited mental impairment. The mutation inhibits the expression of the fragile X mental retardation protein (FMRP), a ubiquitously expressed mRNA binding protein. The specific molecular mechanism(s) leading to premature ovarian failure in Fragile X carriers are not known. Here, we utilize the complete KO mouse model, to mimic the lack of FMRP in Fragile X mutations and analyze the hypothalamic-pituitary-gonadal axis to uncover causes of POF due to FMR1 mutation. Consistent with mutations in human population, KO females experience early cessation of reproductive function and stop having litters at 150 days of age, compared to controls that stop reproducing at 250 days of age. Since POF can be caused by either insufficient pool of primordial follicles or by increased recruitment in each cycle and early depletion, we analyzed ovaries at 3 weeks of age and determined that the FMR1 KO mice had the same number of primordial follicles when compared to the controls, suggesting that POF is not due to a deficit in primordial follicles. However, at 8 weeks of age, FMR1 KO ovaries had higher number of corpora lutea, and KO females had larger litters, indicating that FMR1 KO mice have more follicles recruited in each estrous cycle. FMR1 KO mice have higher FSH, which corresponds to the high FSH in women. Serum estradiol levels and inhibin b expression levels were unaffected by FMR1 mutation suggesting normally functioning negative feedback signals from the ovaries. Analyses of hypothalamic gene expression demonstrated elevated GnRH mRNA in KO mice. To further investigate alterations is hypothalamic protein levels, western blot analyses determined that FMR1 KO mice have higher levels of NMDAR1 and higher levels of GABAA receptor G2 subunit. Dual label immunofluorescence analyses revealed higher number of NMDAR1 and GABAA receptors specifically in GnRH neurons of FMR1 KO mice when compared to control, suggesting that GnRH neurons themselves are affected by FMR1 mutation. Given that both glutamate and GABA can activate GnRH neurons, alterations in the number of these receptors can potentially cause hyperactivity in the HPG axis at the hypothalamic level leading to elevated FSH and the subsequent POF. In summary, our results reveal a potential mechanism of premature ovarian failure in Fragile X mutation carriers.

Early Immune Response and Cytokine Induction of PBMC‐derived Bovine Macrophages to Infection with Rift Valley Fever Virus in the Presence and Absence of Culex tarsalis Mosquito Saliva

Rift Valley fever virus (RVFV) is a zoonotic arbovirus in the family Phenuiviridae. In Africa, outbreaks of Rift Valley fever (RVF) usually occur following periods of heavy rainfall that prompt population surges in mosquito vectors. RVF causes significant mortality in ruminants, especially in ovine fetuses and neonates (up to 100%), while human infections cause mild febrile illness to hemorrhagic fever and death. Macrophages play a critical role as phagocytes in the innate immune system and produce cytokines that stimulate the adaptive immune system. As in other arboviral diseases, macrophages and dendritic cells are thought to be early infection targets of RVFV, enabling the virus to suppress the immune response. To further investigate their role in RVF early immune response and cytokine induction, bovine peripheral blood mononuclear cell-derived macrophages were infected with RVFV MP-12 (an attenuated vaccine strain) in the presence and absence of Culex tarsalis saliva. We hypothesized that in macrophages treated with Culex saliva and RVFV will have increased transcription of Th2-associated cytokines compared to those infected with virus only due to immunomodulatory proteins present in mosquito saliva. The macrophage lineage of the cells and RVFV infection were confirmed by dual label immunofluorescence for IBA-1 (macrophage marker) and RVFV nucleoprotein. Total RNA was extracted from 3 biological replicates of macrophages treated respectively with saliva only, MP-12 only, MP-12 and saliva at 0, 8 and 24 hours post-infection (hpi) as well as untreated controls. RNA was reverse-transcribed and the resulting cDNA analyzed by qPCR of four housekeeping genes (ActinB, H3F3, PPIA, YWHAZ), 6 early response genes (TLR3, TLR7, TLR8, RIG1, MDA5, IFNa) and 9 cytokine genes (IL4, IL6, IL10, IL12b, IL18, IFNg, TNFa, IL1a, IL1b). Relative gene expression (RGE) was determined by the method described by Vandesompele which uses multiple reference genes for normalization and does not assume equal PCR efficiencies for the reference genes and genes of interest. RGEs for each target were log-transformed and analyzed with two-way ANOVAs and Tukey's post-hoc test with p-value correction for multiple tests to evaluate statistical differences between treatments and time points. There was no statistical increase in transcription of Th2-associated cytokines when bovine macrophages were infected with MP-12 in the presence of saliva at any of the time points when compared to virus only. In contrast, significant differences in gene expression were present when infected samples were compared to uninfected controls. At 8 hpi, transcription was significantly different than controls for all virus infected samples for TLR3, RIG1, MDA and IFNa as well as IL6, IL10, IL12b, IL18, TNFa, IL1a and IL1b. This trend continued at 24 hpi with the exception of IL12b. While Culex saliva may not enhance suppression of the early immune response in MP-12 infection, it may still have a role in virulent RVFV strain infections.

Neuronal localization of m1 muscarinic receptor immunoreactivity in the monkey basolateral amygdala.

The basolateral nuclear complex (BNC) of the amygdala plays an important role in the generation of emotional/motivational behavior and the consolidation of emotional memories. Activation of M1 cholinergic receptors (M1Rs) in the BNC is critical for memory consolidation. Previous receptor binding studies in the monkey amygdala demonstrated that the BNC has a high density of M1Rs, but did not have sufficient resolution to identify which neurons in the BNC expressed them. This was accomplished in the present immunohistochemical investigation using an antibody for the m1 receptor (m1R). Analysis of m1Rs in the monkey BNC using immunoperoxidase techniques revealed that their expression was very dense in the BNC, and suggested that virtually all of the pyramidal projection neurons (PNs) in all of the BNC nuclei were m1R-immunoreactive (m1R+). This was confirmed with dual-labeling immunofluorescence using staining for calcium/calmodulin-dependent protein kinase II (CaMK) as a marker for BNC PNs. However, additional dual-labeling studies indicated that one-third of inhibitory interneurons (INs) expressing glutamic acid decarboxylase (GAD) were also m1R+. Moreover, the finding that 60% of parvalbumin (PV) immunoreactive neurons were m1R+ indicated that this IN subpopulation was the main GAD+ subpopulation exhibiting m1R expression. The cholinergic innervation of the amygdala is greatly reduced in Alzheimer's disease and there is currently considerable interest in developing selective M1R positive allosteric modulators (PAMs) to treat the symptoms. The results of the present study indicate that M1Rs in both PNs and INs in the primate BNC would be targeted by M1R PAMs.

Mechanism and restoration strategy of lysosomal abnormalities induced by urinary protein overload in proximal tubule epithelial cells.

Persistent elevated concentrations of urinary protein can destroy proximal tubule epithelial cells (PTECs) by inducing lysosomal abnormalities, thereby aggravating PTEC damage and renal fibrosis. However, the specific mechanisms of these serial biochemical events and methods for treating or preventing PTEC damage upon proteinuria need further investigation. In this study, electron microscopy and dual-labeled immunofluorescence analysis for identifying lysosome type revealed inadequate primary lysosome biogenesis and secondary lysosome accumulation in the PTECs of patients with minimal change nephrotic syndrome or membranous nephropathy who suffered from proteinuria. In vitro studies on HK-2 cells indicated that this abnormality was associated with decreased expression of transcription factor EB (TFEB). In contrast, TFEB overexpressing HK-2 cells under urinary protein overload exhibited significantly reduced accumulation of secondary lysosomes and increased proportion and quantity of primary lysosomes as indicated by dual-labeled immunofluorescence. Further, these cells could upregulate lysosomal degradation functions, as determined using Cathepsin L activity assays and flow cytometry for dye quenched-albumin. These results indicate that abnormal TFEB expression is a key mechanism of lysosomal dyshomeostasis caused by protein overload in PTECs. TFEB is thus a potential therapeutic target for the treatment of urinary protein-related kidney disease.

Polycystin-1 regulates ARHGAP35-dependent centrosomal RhoA activation and ROCK signaling.

Mutations in PKD1 (encoding for polycystin-1 [PC1]) are found in 80%–85% of patients with autosomal dominant polycystic kidney disease (ADPKD). We tested the hypothesis that changes in actin dynamics result from PKD1 mutations through dysregulation of compartmentalized centrosomal RhoA signaling mediated by specific RhoGAP (ARHGAP) proteins resulting in the complex cellular cystic phenotype. Initial studies revealed that the actin cytoskeleton was highly disorganized in cystic cells derived from patients with PKD1 and was associated with an increase in total and centrosomal active RhoA and ROCK signaling. Using cilia length as a phenotypic readout for centrosomal RhoA activity, we identified ARHGAP5, -29, and -35 as essential regulators of ciliation in normal human renal tubular cells. Importantly, a specific decrease in centrosomal ARHGAP35 was observed in PKD1-null cells using a centrosome-targeted proximity ligation assay and by dual immunofluorescence labeling. Finally, the ROCK inhibitor hydroxyfasudil reduced cyst expansion in both human PKD1 3D cyst assays and an inducible Pkd1 mouse model. In summary, we report a potentially novel interaction between PC1 and ARHGAP35 in the regulation of centrosomal RhoA activation and ROCK signaling. Targeting the RhoA/ROCK pathway inhibited cyst formation in vitro and in vivo, indicating its relevance to ADPKD pathogenesis and for developing new therapies to inhibit cyst initiation.

A type IV collagenase inhibitor, N-hydroxy-3-phenyl-2-(4-phenylbenzenesulfonamido) propanamide (BiPS), suppresses skin injury induced by sulfur mustard

Sulfur mustard (SM) is a highly toxic blistering agent thought to mediate its action, in part, by activating matrix metalloproteinases (MMPs) in the skin and disrupting components of the basement membrane zone (BMZ). Type IV collagenases (MMP-9) degrade type IV collagen in the skin, a major component of the BMZ at the dermal-epidermal junction. In the present studies, a type IV collagenase inhibitor, N-hydroxy-3-phenyl-2-(4-phenylbenzenesulfonamido) propanamide (BiPS), was tested for its ability to protect the skin against injury induced by SM in the mouse ear vesicant model. SM induced inflammation, epidermal hyperplasia and microblistering at the dermal/epidermal junction of mouse ears 24–168 h post-exposure. This was associated with upregulation of MMP-9 mRNA and protein in the skin. Dual immunofluorescence labeling showed increases in MMP-9 in the epidermis and in the adjacent dermal matrix of the SM injured skin, as well as breakdown of type IV collagen in the basement membrane. Pretreatment of the skin with BiPS reduced signs of SM-induced cutaneous toxicity; expression of MMP-9 mRNA and protein was also downregulated in the skin by BiPS. Following BiPS pretreatment, type IV collagen appeared intact and was similar to control skin. These results demonstrate that inhibiting type IV collagenases in the skin improves basement membrane integrity after exposure to SM. BiPS may hold promise as a potential protective agent to mitigate SM induced skin injury.

Expression of Laminin γ2 Proteolytic Fragments in Murine Skin Following Exposure to Sulfur Mustard.

Laminin-332 is a basement membrane protein composed of three genetically distinct polypeptide chains that actively promote both skin epidermal cell adhesion and migration. Proteolytic fragments of the laminin γ2 chain stimulate migration and scattering of keratinocytes and cancer cells. Sulfur mustard (SM) is a bifunctional alkylating agent that induces separation of basal keratinocytes from the dermal-epidermal junction and invokes a strong inflammatory response leading to delayed wound repair. In the present studies, the role of laminin γ2 in SM-induced skin injury and wound repair was investigated using the mouse ear vesicant model. We found that laminin γ2 chain mRNA was preferentially upregulated in mouse ear skin exposed to SM. In situ hybridization confirmed overexpression of laminin γ2 transcript. Western blot analysis showed increased protein expression of the full-length proform of laminin γ2 and smaller processed fragments of laminin γ2 in skin exposed to SM. Dual immunofluorescence labeling indicated that laminin γ2 fragments are prevalent in suprabasal keratinocytes behind the leading edge in areas of hyperplasia in injured skin. In addition, co-expression of laminin γ2 and the senescent marker, p16-INK4a was found to overlap with the hyperplastic migratory epithelial sheet. This observation is similar to hypermotile keratinocytes reported in invasive carcinoma cells. Overall, our studies indicate that laminin γ2 is preferentially expressed in skin post SM exposure and that protein expression appears to become progressively more fragmented. The laminin γ2 fragments may play a role in regulating SM-induced skin wound repair. Anat Rec, 2020. © 2020 American Association for Anatomy.

Manganese‐Induced Central Precocious Puberty (MnPP) Increases Hormone Receptor Expression (HR+) in Proliferating Mammary Epithelial Cells in Adult Female Rats

In the developing mammary gland (MG), hormone receptor expressing (HR+) cells very rarely proliferate. The actions of estrogen (E2) and progesterone (P4) on ductal morphogenesis are paracrine and require mediators to stimulate growth of HR− adjacent cells. However, in premalignant and invasive human breast lesions, proliferating cells are HR+. Thus, a dramatic change occurs in MG cell fate with a suspected increased sensitivity to steroid mediated growth. Studies have theorized that early pubertal onset alters MG cell fate increasing the risk of breast cancer later in life. Yet, it is not known if early puberty can cause this conversion to HR+ proliferating MG cells and if this phenotype is sustained in the adult gland. We previously demonstrated that manganese‐induced precocious puberty (MnPP) accelerates E2‐regulated MG development, resulting in sustained proliferation of MG epithelial cells and adult hyperplasia. In the current study, we characterized the normal distribution of HR+ proliferative cells in the MG of prepubertal and adult female rats, determined if MnPP could increase this population and identify cellular proteins that may be involved. Sprague Dawley female rats were exposed daily to 10mg/kg manganese chloride (MnCl2) or saline (control) via gastric gavage from post‐natal day (PND) 12 to PND 30. MGs were collected on PNDs 30 and 120. Using dual labeling immunofluorescence (IF), only 2% of total MG epithelial cells co‐express HRs (E2 or P4 receptor) and the proliferative marker PCNA in the prepubertal MG. As expected, MnPP significantly (p<0.01) increased the percentage of proliferating (PCNA+) MG cells. The percentage of proliferating cells co‐expressing progesterone receptor (PR+/PCNA+) increased by more than 2‐fold in MnPP females relative to controls at PND30 (p<0.01). The proportion of proliferating HR+ cells (ER+/PCNA+ or PR+/PCNA+) remained elevated in PND120 glands of MnPP females compared to controls (p<0.001). Western blot analysis revealed a significant switch in expression levels of the cell cycle regulator, p27, and the mitotic stimulator EREG in MGs that corroborated changes in PR+ proliferation between PND 30 and 120. Furthermore, histologically normal human breast tissue adjacent to ductal carcinoma in situ (DCIS) from The Human Protein Atlas show ER and PR expression similar to expression levels observed in our MnPP rat MGs at PND120. Collectively, these data show that MnPP results in persistent HR expression in proliferating MG cells. Thereby possibly sensitizing it to the hormonal milieu and making the MG more responsive to steroid mediated growth.Support or Funding InformationThis work was supported by NIEHS grant ESO13143 awarded to WLD and NIGMS grant 1R25GM100866 awarded to RKD.

PFOS ile tedavi edilen Sertoli hücre kültüründe ezrin ve fascin 1'in araştırılması: İn vitro bir çalışma

Aim: Depending on the findings resulting from the knock-downing of ezrin and of fascin 1 in vivo, we aim to show the defects or disruption of the blood-testis barrier (BTB) structure and F-actin bundling after Perfluorooctanesulfonate (PFOS) treatment in primary Sertoli cell culture. Methods: Study Design: Primary Sertoli cell isolation was occurred with control and PFOS-treated (20M) groups. Sertoli cells were prepared for both experiments as 0.5 x 106 cell/ml. Method: Dual-labeled immunofluorescence analysis to assess co-localization of fascin 1 with ezrin both in Sertoli cells was performed, and Co-IP, by using lysates of seminiferous tubules, was performed using actin and ezrin proteins to identify specific protein-protein interaction with fascin 1. Results: Firstly, we showed that ezrin and fascin 1, which were components of the ectoplasmic specialization were co-localized in the Sertoli cells and also they were interacted each other. Secondly, we indicated that they were dislocated in the PFOS-treated Sertoli cells in vitro. Because of PFOS (20M), the actin-based cytoskeleton was no longer capable of supporting the distribution and/or localization of actin-regulatory proteins at the cell-cell interface necessary to maintain localization of actin-regulatory at the BTB.Conclusion: In summary, these findings suggest that ezrin and fascin 1 can work together to preserve BTB integrity by regulating F-actin organization in the PFOS-mediated Sertoli cell disruption.

Increased Expression of Vascular Endothelial Growth Factor-D Following Brain Injury.

Alterations in the expression of the vascular endothelial growth factors (VEGF) A and B occur during blood–brain barrier (BBB) breakdown and angiogenesis following brain injury. In this study, the temporal and spatial expression of VEGF-D and VEGF receptors-2 and -3 (VEGFR-2 and VEGFR-3, respectively) was determined at the mRNA and protein level in the rat cortical cold-injury model over a period of 0.5 to 6 days post-injury. In order to relate endothelial VEGF-D protein expression with BBB breakdown, dual labeling immunofluorescence was performed using antibodies to VEGF-D and to fibronectin, a marker of BBB breakdown. In control rats, VEGF-D signal was only observed in scattered perivascular macrophages in the cerebral cortex. The upregulation of VEGF-D mRNA expression was observed in the injury site between days 0.5 to 4, coinciding with the period of BBB breakdown and angiogenesis. At the protein level, intracerebral vessels with BBB breakdown to fibronectin in the lesion on days 0.5 to 4 failed to show endothelial VEGF-D. Between days 0.5 to 6, an increased VEGF-D immunoreactivity was noted in the endothelium of pial vessels overlying the lesion site, in neutrophils, macrophages, and free endothelial cells within the lesion. The upregulation of VEGFR-2 and -3 mRNA and protein expression was observed early post-injury on day 0.5. Although there was concurrent expression of VEGF-A, VEGF-B, and VEGF-D post-injury, differences in their spatial expression during BBB breakdown and angiogenesis suggest that they have specific and separate roles in these processes.

Role of hesperidin in P2X3 receptor-mediated neuropathic pain in the dorsal root ganglia

Aim: This study investigated whether the neuronal P2X3 receptor in rat dorsal root ganglia (DRG) mediated the effects of hesperidin on neuropathic pain.Materials and methods: The chronic constriction injury (CCI) model was used as a model of neuropathic pain. The mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) were measured. The mRNA and protein expression levels were assayed by real-time RT-PCR and Western blotting.Results: The results showed that mechanical and thermal hyperalgesia in the CCI rats were increased as compared to those in the sham group. The expression levels of P2X3 mRNA and protein in CCI rats were higher than those in the sham group. Dual-labelling immunofluorescence showed that the elevated P2X3 receptor was co-expressed with the neuronal marker NeuN in the DRG of CCI rats. Hesperidin treatment decreased both the mechanical and thermal hyperalgesia, and upregulated P2X3 expression in the CCI rats. Hesperidin treatment also reduced the ERK1/2 phosphorylation in the DRG of CCI rats. Moreover, hesperidin inhibited the P2X3 agonist ATP-induced currents in HEK293 cells transfected with the P2X3 plasmid. Therefore, hesperidin treatment could reverse the elevated expression of neuronal P2X3 receptor and reduce the activation of ERK1/2 in the DRG of CCI rats.Conclusions: Our findings suggested that hesperidin inhibited the nociceptive transmission mediated by the P2X3 receptor in neurons of DRG, and thus, relieved the mechanical and thermal hyperalgesia in CCI rats.

F5-peptide enhances the efficacy of the non-hormonal male contraceptive adjudin

ObjectiveThe bioavailability of the non-hormonal male contraceptive adjudin is low in rats due to the blood-testis barrier (BTB). This study was designed to examine if F5-peptide, an endogenously produced reversible BTB modifier, could enhance the bioavailability of adjudin to affect spermatogenesis and provide a contraceptive effect in rats while reducing systemic toxicity. Study DesignWe overexpressed F5-peptide in adult male rats (n=10 rats; with 3 or 4 rats for each of the three different experiments noted in the three regimens) by intratesticular injection of a mammalian expression vector pCI-neo (pCI-neo/F5-peptide) vs. empty vector alone (pCI-neo/Ctrl) to be followed by treatment with adjudin by oral gavage at a dose of 10 or 20 mg/kg. The status of spermatogenesis was assessed by histological analysis and dual-labeled immunofluorescence analysis on Day 16. To assess fertility, we allowed treated males (n=3–4 rats) to mate with mature female rats (n=3–4) individually, and assessed the number of pups on Days 23, 36 and 82 to assess fertility and reversibility. ResultsAll 4 treated rats overexpressed with F5-peptide and low-dose adjudin were infertile by Day 36, and half of these rats were fertile by Day 82, illustrating reversibility. However, overexpression of F5-peptide alone (or low-dose adjudin alone) had no effects on fertility in n=3 rats. These findings were consistent with the histology data that illustrated the BTB modifier F5-peptide promoted the action of adjudin to induce germ cell exfoliation, mediated by changes in cytoskeletal organization of F-actin and microtubules across the epithelium, thereby reducing the systemic toxicity of adjudin. ConclusionIn this proof-of-concept study, it was shown that overexpression of the F5-peptide prior to administration of adjudin to rats at a low (and ineffective dose by itself) was found to induce reversible male infertility. ImplicationsOverexpression of F5-peptide, an endogenously produced biomolecule in the testis known to induce BTB remodeling, enhanced the contraceptive effect of adjudin in rats, supporting proof of concept studies of BTB disrupters in men.

The Inhibition by Guanfu Base A of Neuropathic Pain Mediated by P2Y12 Receptor in Dorsal Root Ganglia.

Activation of satellite glial cells (SGCs) in the dorsal root ganglia (DRG) is involved in mechanical and thermal hyperalgesia. The upregulated P2Y12 receptor expressed in SGCs of the DRG participates in the nociceptive transmission of neuropathic pain. Guanfu base A (GFA) has been reported to exhibit antiarrhythmic and anti-inflammatory effects. In this study, we explored the effects of GFA on P2Y12 receptor-mediated mechanical and thermal hyperalgesia in chronic constriction injury (CCI) rats. Sprague-Dawley rats were randomly divided into sham operation group (Sham), CCI operation group (CCI), CCI rats treated with guanfu base A group (CCI + GFA) and control rats treated with GFA group (Ctrl + GFA). Mechanical withdrawal threshold and thermal withdrawal latency were measured. P2Y12 expression in L4-L6 dorsal root ganglion (DRG) was detected by quantitative real-time PCR and Western blot. After CCI treatment, mechanical and thermal hyperalgesia and the expression values of P2Y12 receptor mRNA and protein in DRG were increased. Dual-labeling immunofluorescence showed that the coexpression of P2Y12 receptor and glial fibrillary acidic protein (GFAP) in the DRG of CCI rats was increased compared to sham rats. GFA relieved mechanical and thermal hyperalgesia in the CCI rats, decreased the expression of P2Y12 mRNA and protein and phosphorylation of p38 MAPK in the DRG, and increased the ADP-downregulated cAMP concentrations in HEK293 cells transfected with P2Y12 plasmid. After CCI rats were treated with GFA, the coexpression of P2Y12 receptor and GFAP in the DRG was significantly decreased compared to the untreated CCI group. Thus, downregulating the P2Y12 receptor relieved mechanical and thermal hyperalgesia in the CCI rats.

Neuroanatomical Relationship of Neuronal Nitric Oxide Synthase to Gonadotropin-Releasing Hormone and Kisspeptin Neurons in Adult Female Sheep and Primates

Background: Neuronal intermediates that communicate estrogen and progesterone feedback to gonadotropin-releasing hormone (GnRH) neurons are essential for modulating reproductive cyclicity. Individually, kisspeptin and nitric oxide (NO) influence GnRH secretion. However, it is possible these 2 neuronal intermediates interact with one another to affect reproductive cyclicity. Methods: We investigated the neuroanatomical relationship of one isoform of the enzyme that synthesizes NO, neuronal NO synthase (nNOS), to kisspeptin and GnRH in adult female rhesus monkeys and sheep using dual-label immunofluorescence. Additionally, we evaluated if the phase of the reproductive cycle would affect these relationships. Results: Overall, no effect of the stage of cycle was observed for any variable in this study. In the arcuate nucleus (ARC) of sheep, 98.8 ± 3.5% of kisspeptin neurons colocalized with nNOS, and kisspeptin close-contacts were observed onto nNOS neurons. In contrast to ewes, no colocalization was observed between kisspeptin and nNOS in the infundibular ARC of primates, but kisspeptin fibers were apposed to nNOS neurons. In the preoptic area of ewes, 15.0 ± 4.2% of GnRH neurons colocalized with nNOS. In primates, 38.8 ± 10.1% of GnRH neurons in the mediobasal hypothalamus colocalized with nNOS, and GnRH close-contacts were observed onto nNOS neurons in both sheep and primates. Conclusion: Although species differences were observed, this work establishes a neuroanatomical framework between nNOS and kisspeptin and nNOS and GnRH in adult female nonhuman primates and sheep.