Simultaneous Immunofluorescence Research Articles

SAT025 Characterization Of The Melatonin Type 1 Receptor / Cannabinoid Type 1 Receptor (MT1/CB1) Heteromers

Abstract Disclosure: Y. Cui: None. H. Cherdoud: None. G. Marsicano: None. L. Bellocchio: None. R. Jockers: None. O. Lahuna: None. Membrane receptors belonging to the G proteins coupled receptors (GPCRs), the largest family of proteins in the human genome with more than 800 members. GPCR functions were initially thought to take place at the plasma membrane upon binding of single monomeric receptor unit by their cognate ligand. This scheme was complexified after identification of cross-talks between GPCRs upon heteromerization and identification of functional GPCRs within intracellular organelles as nucleus or endosomes. Among a few others receptors, functional melatonin receptor type 1 (MT1) and cannabinoid receptor type 1 (CB1) were found in neuronal mitochondria, suggesting a cross-talk between them. Here, we are identifying and characterizing MT1/CB1 heteromers as a new GPCR heteromer. Previously we showed that coexpression of the MT1 and CB1 receptors induced the constitutive formation of MT1/CB1 heteromers using coimmunoprecipitation experiments on transiently transfected HEK293 cells. Using simultaneous PLA (Proximity Ligation Assay) and immunofluorescence experiments, we described the existence of intracellular MT1/CB1 heteromers associated with the mitochondrial network. Since then, we have further characterized the properties of MT1/CB1 heteromers. Coimmunoprecipitation experiments performed on cerebellum from wild-type, MT1 or CB1 knock-out mice enabled us to identify MT1/CB1 heteromers in vivo. We established stable monoclonal HEK293 cell lines expressing both receptors. Transmission Electronic Microscopy (TEM) and PLA experiments showed the presence of MT1/CB1 complexes not only intracellularly but also at the cell surface. Using PLA experiments we identified a penetrating interfering peptide that specifically dissociate the MT1/CB1 complex and thus open the door to experiments targeting specifically the complex. We were able to follow the melatonin-dependent internalization and trafficking of the MT1/CB1 complex from the plasma membrane to the late endosomes using a combination of Nanoluciferase complementation experiments and bystander BRET (Bioluminescence Resonance Energy Transfer). Costimulation of HE293 cells expressing both receptors with melatonin and the selective CB1 agonist ACEA (Arachidonyl-2'-chloroethylamide), a derivative of the endocannabinoid AEA (Arachidonylethanolamide or anandamide) resulted in a potentiation of the melatonin-dependent cAMP inhibition. Use of the identified interfering peptide will enable us to identify actions on signaling pathways resulting from a direct cross-talk between MT1 or CB1 subunits. Presentation: Saturday, June 17, 2023

The Role of Astroglial xCT in Mitigating Cue‐Induced Amphetamine‐Seeking

Relapse poses a significant barrier to the development of effective treatments for substance use disorder (SUD). Cue‐induced drug craving increases during drug abstinence, potentially affecting the efficacy of treatments for SUD. Disruptions in glutamatergic signaling are characteristic of SUDs. Glutamate is maintained by a combination of astrocytic and neuronal transporters within the nucleus accumbens (ACb) and disruptions in this homeostasis engender SUD. One transporter, the cysteine‐glutamate transporter (xCT), is primarily localized on astrocytes and helps maintain glutamate concentrations. This process is disrupted by cocaine use, leading to increased cue‐induced craving. The therapeutic N‐acetylcysteine (NAC) lowers cue‐induced relapse to cocaine. However, little research has shown how these effects extend to other psychostimulants, such as amphetamine (AMP). Though both are psychostimulants, AMP and cocaine have distinct mechanisms of action in the brain, particularly with regards to dopamine neurotransmission. In the present study, we assessed disruption of astrocytes and xCT expression following AMP self‐administration (SA). We also assessed the degree to which disruptions in astrocyte or xCT expression might explain an increased propensity for relapse when presented with drug cues and the degree to which NAC can attenuate relapse.78 male Sprague‐Dawley rats were surgically implanted with jugular catheter to allow for intravenous SA of AMP. Upon recovery, all rats underwent 14, 2‐hr fixed‐ratio‐1 SA sessions for AMP or the saline (SAL) control. Active lever presses resulted in an infusion of amphetamine (0.1 mg/kg/infusion) which was followed by a 20 s time‐out period signaled by the illumination of both cue lights. After 14 SA sessions, all rats entered a forced abstinence period where they were not exposed to the drug or drug‐related cues. During abstinence, all rats were given daily injections of NAC (100 mg/mL/ip) or the saline vehicle. Cue‐induced relapse tests were conducted at the cessation of the abstinence period. During the relapse test, active lever presses resulted in the illumination of the cue lights present during SA testing, but no delivery of the drug itself. One subset of rats underwent relapse testing after one withdrawal day (WD) 1 and one subset underwent testing on WD 14 to examine any increases in relapse. Following testing, all rats were perfused and brains were extracted for immunofluorescence (IF) analysis of astrocyte and xCT transporter expression within the ACb and medial prefrontal cortex using a simultaneous immunofluorescence (IF) method.During the cue‐induced relapse test, cue‐induced responding was significantly higher in AMP‐treated rats compared to SAL‐treated rats. Importantly, NAC failed to attenuate relapse at either WD 1 or WD 14. We hypothesized that xCT expression would remain unaltered between WD 1 and WD 14 for rats treated with NAC. However, given that NAC failed to lower relapse at either time point, IF results will help determine inform the inefficacy of NAC. For instance, should xCT expression remain unaltered between AMP and SAL‐exposed rats, then it would make sense that NAC would likely have little success in lowering relapse. Such a finding would suggest that AMP disrupts glutamate via a different mechanism than cocaine. Thus, results may indicate that the therapeutic efficacy of NAC varies across psychostimulants.

Improved Methods for Single-Molecule Fluorescence In Situ Hybridization and Immunofluorescence in Caenorhabditis elegans Embryos.

Visualization of gene products in Caenorhabditis elegans has provided insights into the molecular and biological functions of many novel genes in their native contexts. Single-molecule fluorescence in situ hybridization (smFISH) and immunofluorescence (IF) enable the visualization of the abundance and localization of mRNAs and proteins, respectively, allowing researchers to ultimately elucidate the localization, dynamics, and functions of the corresponding genes. Whereas both smFISH and immunofluorescence have been foundational techniques in molecular biology, each protocol poses challenges for use in the C. elegans embryo. smFISH protocols suffer from high initial costs and can photobleach rapidly, and immunofluorescence requires technically challenging permeabilization steps and slide preparation. Most importantly, published smFISH and IF protocols have predominantly been mutually exclusive, preventing the exploration of relationships between an mRNA and a relevant protein in the same sample. Here, we describe protocols to perform immunofluorescence and smFISH in C. elegans embryos either in sequence or simultaneously. We also outline the steps to perform smFISH or immunofluorescence alone, including several improvements and optimizations to existing approaches. These protocols feature improved fixation and permeabilization steps to preserve cellular morphology while maintaining probe and antibody accessibility in the embryo, a streamlined, in-tube approach for antibody staining that negates freeze-cracking, a validated method to perform the cost-reducing single molecule inexpensive FISH (smiFISH) adaptation, slide preparation using empirically determined optimal antifade products, and straightforward quantification and data analysis methods. Finally, we discuss tricks and tips to help the reader optimize and troubleshoot individual steps in each protocol. Together, these protocols simplify existing workflows for single-molecule RNA and protein detection. Moreover, simultaneous, high-resolution imaging of proteins and RNAs of interest will permit analysis, quantification, and comparison of protein and RNA distributions, furthering our understanding of the relationship between RNAs and their protein products or cellular markers in early development. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Sequential immunofluorescence and single-molecule fluorescence in situ hybridization Alternate Protocol: Abbreviated protocol for simultaneous immunofluorescence and single-molecule fluorescence in situ hybridization Basic Protocol 2: Simplified immunofluorescence in C. elegans embryos Basic Protocol 3: Single-molecule fluorescence in situ hybridization or single-molecule inexpensive fluorescence in situ hybridization.

MULTIPLEX DETECTION OF TUMOR MARKERS FOR DIFFERENT STAGES OF COLORECTAL CANCER

Introduction. Colorectal cancer is one of the most common cancers worldwide. In the Republic of Kazakhstan, over the past decade, the incidence rates of colon and rectal cancers have increased by 23 % and 17 %, respectively. Although colorectal cancer screening rates have increased over time, it is necessary to identify new serum markers for early detection and prognosis of this cancer. The aim of the study was to evaluate the diagnostic value of serum biomarkers for colorectal cancer. Material and Methods . Biomarkers were studied by immunofluorescence method using xMap technology, which included simultaneous immunofluorescence determination on magnetic spheres CA125, CA15–3, CA19–9, CEA, CYFRA21–1, sFAS, sFasL, HE4, Osteopontin, VEGF-A, pd-L1. Results . The study showed no significant differences in the levels of markers between healthy subjects (control group) and patients with bowel diseases. In cancer patients, СEA and OpN markers increased with increasing tumor stage, and were significantly higher than those observed in patients with bowel diseases and controls. The CA125 marker was significantly higher in patients with stage IV cancer then in patients with I–III stages. Conclusion . Our study revealed that the new pd-L1 marker can serve as a significant biomarker for colorectal cancer.

New-generation diagnostics in inflammatory skin diseases: Immunofluorescence and histopathological assessment using ex vivo confocal laser scanning microscopy in cutaneous lupus erythematosus.

Ex vivo confocal laser scanning microscopy (CLSM) offers real-time examination of excised tissue in reflectance, fluorescence and digital haematoxylin-eosin (H&E)-like staining modes enabling application of fluorescent-labelled antibodies. We aimed to assess the diagnostic performance of ex vivo CLSM in identifying histopathological features and lupus band test in cutaneous lupus erythematosus (CLE) with comparison to conventional histopathology and direct immunofluorescence (DIF). A total of 72 sections of 18 CLE patients were stained with acridine orange (AO), anti-IgG, anti-IgM and anti-IgA; 21 control samples were stained with AO. Subsequently, ex vivo CLSM examination of all samples was performed in reflectance, fluorescence and digital H&E-like staining modes. Superficial and deep perivascular inflammatory infiltration (94.4%), interface dermatitis (88.9%), spongiosis (83.3%) and vacuolar degeneration (77.7%) were the most common features detected with ex vivo CLSM. Kappa test revealed a level of agreement ranging within "perfect" to "good" between ex vivo CLSM and conventional histopathology. ROC analysis showed that the combination of perivascular infiltration, interface dermatitis and spongiosis detected by ex vivo CLSM has the potential to distinguish between CLE and controls. Basement membrane immunoreactivity with IgG, IgM and IgA was identified in 88.8% (n=15), 55.5% (n=10) and 55.5% (n=10) of the CLE samples using ex vivo CLSM, respectively, whereas DIF showed IgG, IgM and IgA positivity in 94.4% (n=17), 100% (n=18) and 88.9% (n=16) of patients, respectively. In conclusion, ex vivo CLSM enables simultaneous histopathological and immunofluorescence examination in CLE showing a high agreement with conventional histopathology, albeit with a lower performance than conventional DIF.

Lymphatic vessels identified in failed corneal transplants with neovascularisation

BackgroundCorneal transplant failure with neovascularisation is a leading indication for full-thickness grafts in patients. Lymphangiogenesis is implicated in the pathology of graft failure, and here we systematically evaluate failed human...

Demonstration of Tissue Resident Memory CD8 T Cells in Insulitic Lesions in Adult Patients with Recent-Onset Type 1 Diabetes

Subtypes of CD8+ T cells in insulitic lesions in biopsy specimens from six subjects with recent-onset type 1 diabetes (T1D) and six nondiabetic matched controls were analyzed using simultaneous multicolor immunofluorescence. Also, insulitic islets based on accumulation of CD3+ T cells were microdissected with laser-capture microscopy, and gene transcripts associated with inflammation and autoimmunity were analyzed. We found a substantial proportion, 43%, of the CD8+ T cells in the insulitic lesions to display a tissue resident memory T cell (TRM) (CD8+CD69+CD103+) phenotype in T1D subjects. Most TRM cells were located in the insulitic lesion in the endocrine-exocrine interface. TRM cells were also sporadically found in islets of control subjects. Moreover, gene expression analysis showed a lack of active transcription of genes associated with acute inflammatory or cytotoxic T-cell responses. We present evidence that a substantial proportion of T cells in insulitic lesions of recent-onset T1D patients are TRM cells and not classic cytotoxic CD8+ T cells. Our findings highlight the need for further analysis of the T cells involved in insulitis to elucidate their role in the etiology of T1D.

Abstract WP430: Endothelial Cell Collection from Ipsilateral Middle Cerebral Artery in Acute Ischemic Stroke

Introduction: The cerebrovascular endothelium plays a critical role in the pathogenesis of and response to acute ischemic stroke (AIS). To date, techniques to study its function have relied on animal and in vitro models. A robust method of endothelial cell (EC) capture in patients with AIS at early time points, from within the ischemic region, could greatly advance our understanding of cerebrovascular injury. Method: Patients undergoing thrombectomy for middle cerebral artery occlusion (MCA) within 8 hours of onset were offered enrollment if the pass of their stent-retriever device occurred directly into a distal access catheter in the proximal M1 segment, limiting exposure of the device to only the MCA. After retrieval, ECs adherent to the devices were retrieved and stained for EC (CD31) and leukocyte (CD45) markers. EC identity and yield were confirmed by flow cytometry with simultaneous immuno-fluorescence microscopy. Cultured human ECs were used as positive controls. The EC fraction was defined as CD31 + CD45 - with size and morphological features consistent with the positive controls. Results: ECs from stent-retriever devices (n=3) were collected and pooled. Approximately 8% of the collected cells represented ECs. EC collected from the stent-retrievers demonstrated highly similar shape, morphology and antibody staining patterns compared to the positive controls (Figure). Conclusions: Here we provide the first demonstration of a rapid post-thrombectomy method for reliable harvesting of cerebral ECs in humans. The ability to capture these cells in patients with AIS within hours of symptom onset opens many avenues of exploration for determining the role of ECs in AIS.

Matrix-regulated integrin αvβ5 maintains α5β1-dependent desmoplastic traits prognostic of neoplastic recurrence.

Desmoplasia, a fibrotic mass including cancer-associated fibroblasts (CAFs) and self-sustaining extracellular matrix (D-ECM), is a puzzling feature of pancreatic ductal adenocarcinoma (PDACs). Conflicting studies have identified tumor-restricting and tumor-promoting roles of PDAC-associated desmoplasia, suggesting that individual CAF/D-ECM protein constituents have distinguishable tumorigenic and tumor-repressive functions. Using 3D culture of normal pancreatic versus PDAC-associated human fibroblasts, we identified a CAF/D-ECM phenotype that correlates with improved patient outcomes, and that includes CAFs enriched in plasma membrane-localized, active α5β1-integrin. Mechanistically, we established that TGFβ is required for D-ECM production but dispensable for D-ECM-induced naïve fibroblast-to-CAF activation, which depends on αvβ5-integrin redistribution of pFAK-independent active α5β1-integrin to assorted endosomes. Importantly, the development of a simultaneous multi-channel immunofluorescence approach and new algorithms for computational batch-analysis and their application to a human PDAC panel, indicated that stromal localization and levels of active SMAD2/3 and α5β1-integrin distinguish patient-protective from patient-detrimental desmoplasia and foretell tumor recurrences, suggesting a useful new prognostic tool.

Spotlight on the three main hepatic fibrogenic cells in HCV-infected patients: Multiple immunofluorescence and ultrastructure study

ABSTRACTThe present work deals with the simultaneous ultrastructure and triple immunofluorescence study of the three main hepatic fibrogenic cells, hepatic stellate cell, myofibroblast (MF), and fibroblast, in a group of hepatitis C virus (HCV) RNA positive patients, as their exact interrelation behavior in vivo with the progress of hepatic fibrosis is still inadequate. In this study, for the first time, cells having the morphological characteristic of MF and not bone marrow fibrocytes were revealed in liver portal vessels. This necessitates the reevaluation of the available knowledge concerning bone marrow fibrocyte. Also, the distribution, cellular interrelations, and the fate of MF were highlighted.

High-resolution telomere fluorescence in situ hybridization reveals intriguing anomalies in germ cell tumors

Testicular germ cell tumor (TGCT) is the most common malignancy of young men. Most patients are completely cured, which distinguishes these from most other malignancies. Orchiectomy specimens (n=76) were evaluated using high-resolution (single-cell discriminative) telomere-specific fluorescence in situ hybridization (FISH) with simultaneous Oct4 immunofluorescence to describe telomere length phenotype in TGCT neoplastic cells. For the first time, the TGCT precursor lesion, germ cell neoplasia in situ (GCNIS) is also evaluated in depth. The intensity of the signals from cancerous cells was compared to the same patient's reference cells-namely, healthy germ cells (defined as "medium" length) and interstitial/somatic cells (defined as "short" telomere length). We observed short telomeres in most GCNIS and pure seminomas (P=.006 and P=.0005, respectively). In contrast, nonseminomas displayed longer telomeres. Lesion-specific telomere lengths were documented in mixed tumor cases. Embryonal carcinoma (EC) demonstrated the longest telomeres. A fraction of EC displays the telomerase-independent alternative lengthening of telomeres (ALT) phenotype (24% of cases). Loss of ATRX or DAXX nuclear expression was strongly associated with ALT; however, nuclear expression of both proteins was retained in half of ALT-positive ECs. The particular distribution of telomere lengths among TGCT and GCNIS precursors implicate telomeres anomalies in pathogenesis. These results may advise management decisions as well.

Co-localization of PTEN and E-cadherin in canine mammary hyperplasias and benign and malignant mammary tumors

Fifty-four canine mammary lesions (15 hyperplasias, 7 adenomas and 32 carcinomas) were submitted to immunohistochemical analysis for the evaluation of PTEN and E-cadherin co-expression. Subjects bearing mammary carcinomas were also submitted to a 2-year follow-up study to compare immunohistochemical results with overall survival. All the hyperplastic samples stained positive for both markers, 100% of adenomas were positive for PTEN and 86% for E-cadherin, and 69% and 34% of carcinomas were positive for PTEN and E-cadherin, respectively. Statistical analysis showed a positive correlation between these two proteins both considering all (p<0.01) or malignant tumors (p<0.05). The female dogs bearing tumors positively-stained for both markers had a longer overall survival (p<0.05) and absence of lymphatics invasion (p<0.05). Simultaneous double immunofluorescence confirmed the co-localization of the two proteins in neoplastic cells. Results reported in this study confirm the tumor suppressor effect of these two molecules.

A novel multicolor immunostaining method using ethynyl deoxyuridine for analysis of in situ immunoproliferative response.

Immune responses are generally accompanied by antigen presentation and proliferation and differentiation of antigen-specific lymphocytes (immunoproliferation), but analysis of these events in situ on tissue sections is very difficult. We have developed a new method of simultaneous multicolor immunofluorescence staining for immunohistology and flow cytometry using a thymidine analogue, 5-ethynyl-2′-deoxyuridine (EdU). Because of the small size of azide dye using click chemistry and elimination of DNA denaturation steps, EdU staining allowed for immunofluorescence staining of at least four colors including two different markers on a single-cell surface, which is impossible with the standard 5-bromo-2′-deoxyuridine method. By using two rat models, successfully detected parameters were the cluster of differentiation antigens including phenotypic and functional markers of various immune cells, histocompatibility complex antigens, and even some nuclear transcription factors. Proliferating cells could be further sorted and used for RT-PCR analysis. This method thus enables functional in situ time-kinetic analysis of immunoproliferative responses in a distinct domain of the lymphoid organs, which are quantitatively confirmed by flow cytometry.Electronic supplementary materialThe online version of this article (doi:10.1007/s00418-015-1329-z) contains supplementary material, which is available to authorized users.

Quantitative assessment of angiogenesis, perfused blood vessels and endothelial tip cells in the postnatal mouse brain.

During development and in various diseases of the CNS, new blood vessel formation starts with endothelial tip cell selection and vascular sprout migration, followed by the establishment of functional, perfused blood vessels. Here we describe a method that allows the assessment of these distinct angiogenic steps together with antibody-based protein detection in the postnatal mouse brain. Intravascular and perivascular markers such as Evans blue (EB), isolectin B4 (IB4) or laminin (LN) are used alongside simultaneous immunofluorescence on the same sections. By using confocal laser-scanning microscopy and stereological methods for analysis, detailed quantification of the 3D postnatal brain vasculature for perfused and nonperfused vessels (e.g., vascular volume fraction, vessel length and number, number of branch points and perfusion status of the newly formed vessels) and characterization of sprouting activity (e.g., endothelial tip cell density, filopodia number) can be obtained. The entire protocol, from mouse perfusion to vessel analysis, takes ∼10 d.

Intrahepatic regulatory T cells in autoimmune hepatitis are associated with treatment response and depleted with current therapies

Autoimmune hepatitis (AIH) is a chronic autoimmune liver disease usually requiring life-long immunosuppression. The mechanisms for disease initiation and chronicity are largely unknown. A contribution of deficient regulatory T cells (Tregs) in the blood was controversially discussed recently. So far investigations in the target organ have been limited to single parameter analysis in untreated AIH. We retrospectively analysed the pattern of liver infiltrating T, B and regulatory T cells quantitatively with simultaneous multicolour immunofluorescence before (n=45) and under (n=31) therapy in adult AIH type 1 (AIH-1) patients. Intrahepatic CD4(+) cells dominate over CD8(+) at diagnosis, but with increasing disease activity the CD4(+)/CD8(+) ratio approached one. While there is no change of Tregs in the blood, they are enriched with effector T cells (Teffs) within the liver of patients with untreated AIH-1 with a constant Treg/Teff ratio. Even more importantly, immunosuppression mostly with steroids and azathioprine caused a disproportional loss of intrahepatic Tregs. Patients reaching biochemical remission had higher intrahepatic Treg/Teff and Treg/B cell ratios compared to patients failing to reach remission. In vitro proliferation of Tregs seemed to be more suppressed by prednisolone than expansion of Teffs. Furthermore, intraportal B cells correlated with serum IgG suggesting an autochthonous intrahepatic IgG production. Intrahepatic Tregs are rather enriched than numerically deficient in untreated AIH-1. The disproportional decrease of intrahepatic Tregs during therapy might explain high relapse rates after discontinuation of immunosuppression. Thus, future therapies increasing intrahepatic immunoregulation might be better suited for long-term control of AIH.

A strategy for cell-based multiplex diagnostics of Myasthenia gravis and autoimmune encephalitis by modifying the subcellular localization of cell membrane autoantigens.

Many autoimmune diseases are characterized by autoantibodies directed against cell membrane proteins. We were intrigued to develop a strategy for targeting individual cell membrane proteins to various subcellular compartments as a prerequisite for their simultaneous immunofluorescence detection. We first employed GFP and RFP reporters that were equipped with defined intracellular localization signals. Expressing these protein reporters in HEp-2 cells we found by using fluorescence microscopy that protein localization in cytoplasm or at mitochondria can be clearly discriminated from localization at Golgi, ER or lysosomes. We then tested for muscle-specific kinase, a relevant cell membrane autoantigen in Myasthenia gravis, and NMDA receptor which is relevant for autoimmune encephalitis, whether these autoantigens can be localized to the same intracellular compartments. To this end, we successfully targeted muscle-specific kinase to Golgi apparatus, mitochondria and cytoplasm. We found that its Golgi localization can be clearly distinguished from its natural cell membrane localization. The same we found for Golgi-localized NMDA receptor 1. Interestingly, cell membrane proteins kept at the Golgi system accumulated in higher amounts than their wild-type counterparts. The obtained results are the basis for the further development of multiplex assays for the immunofluorescence diagnostics of Myasthenia gravis and autoimmune encephalitis.

The Immune-Modulating Cytokine and Endogenous Alarmin Interleukin-33 Is Upregulated in Skin Exposed to Inflammatory UVB Radiation

The cellular and molecular mechanisms by which UV radiation modulates inflammation and immunity while simultaneously maintaining skin homeostasis is complex and not completely understood. Similar to the effects of UV, IL-33 has potent immune-modulating properties that are mediated by the downstream induction of cytokines and chemokines. We have discovered that exposure of mice in vivo or human skin samples ex vivo to inflammatory doses of UVB induced IL-33 expression within the epidermal and dermal skin layers. Using a combination of murine cell lines and primary human cells, we demonstrate that both UV and the oxidized lipid platelet activating factor induce IL-33 expression in keratinocytes and dermal fibroblasts. Highlighting the significance of these results, we found that administering IL-33 to mice in vivo suppressed the induction of Th1-mediated contact hypersensitivity responses. This may have consequences for skin cancer growth because UV-induced squamous cell carcinomas that evade immunological destruction were found to express significantly higher levels of IL-33. Finally, we demonstrate that dermal mast cells and skin-infiltrating neutrophils closely associate with UV-induced IL-33-expressing fibroblasts. Our results therefore identify and support a role for IL-33 as an important early danger signal produced in response to inflammation-inducing UV radiation.

Differential localization and independent acquisition of the H3K9me2 and H3K9me3 chromatin modifications in the Caenorhabditis elegans adult germ line.

Histone methylation is a prominent feature of eukaryotic chromatin that modulates multiple aspects of chromosome function. Methyl modification can occur on several different amino acid residues and in distinct mono-, di-, and tri-methyl states. However, the interplay among these distinct modification states is not well understood. Here we investigate the relationships between dimethyl and trimethyl modifications on lysine 9 of histone H3 (H3K9me2 and H3K9me3) in the adult Caenorhabditis elegans germ line. Simultaneous immunofluorescence reveals very different temporal/spatial localization patterns for H3K9me2 and H3K9me3. While H3K9me2 is enriched on unpaired sex chromosomes and undergoes dynamic changes as germ cells progress through meiotic prophase, we demonstrate here that H3K9me3 is not enriched on unpaired sex chromosomes and localizes to all chromosomes in all germ cells in adult hermaphrodites and until the primary spermatocyte stage in males. Moreover, high-copy transgene arrays carrying somatic-cell specific promoters are highly enriched for H3K9me3 (but not H3K9me2) and correlate with DAPI-faint chromatin domains. We further demonstrate that the H3K9me2 and H3K9me3 marks are acquired independently. MET-2, a member of the SETDB histone methyltransferase (HMTase) family, is required for all detectable germline H3K9me2 but is dispensable for H3K9me3 in adult germ cells. Conversely, we show that the HMTase MES-2, an E(z) homolog responsible for H3K27 methylation in adult germ cells, is required for much of the germline H3K9me3 but is dispensable for H3K9me2. Phenotypic analysis of met-2 mutants indicates that MET-2 is nonessential for fertility but inhibits ectopic germ cell proliferation and contributes to the fidelity of chromosome inheritance. Our demonstration of the differential localization and independent acquisition of H3K9me2 and H3K9me3 implies that the trimethyl modification of H3K9 is not built upon the dimethyl modification in this context. Further, these and other data support a model in which these two modifications function independently in adult C. elegans germ cells.

Distinct freshwater and seawater isoforms of Na+/K+-ATPase in gill chloride cells of Atlantic salmon

Gill Na(+)/K(+)-ATPase (NKA) in teleost fishes is involved in ion regulation in both freshwater and seawater. We have developed and validated rabbit polyclonal antibodies specific to the NKA alpha1a and alpha1b protein isoforms of Atlantic salmon (Salmo salar Linnaeus), and used western blots and immunohistochemistry to characterize their size, abundance and localization. The relative molecular mass of NKA alpha1a is slightly less than that for NKA beta1b. The abundance of gill NKA alpha1a was high in freshwater and became nearly undetectable after seawater acclimation. NKA alpha1b was present in small amounts in freshwater and increased 13-fold after seawater acclimation. Both NKA isoforms were detected only in chloride cells. NKA alpha1a was located in both filamental and lamellar chloride cells in freshwater, whereas in seawater it was present only as a faint background in filamental chloride cells. In freshwater, NKA alpha1b was found in a small number of filamental chloride cells, and after seawater acclimation it was found in all chloride cells on the filament and lamellae. Double simultaneous immunofluorescence indicated that NKA alpha1a and alpha1b are located in different chloride cells in freshwater. In many chloride cells in seawater, NKA alpha1b was present in greater amounts in the subapical region than elsewhere in the cell. The combined patterns in abundance and immunolocalization of these two isoforms can explain the salinity-related changes in total NKA and chloride cell abundance. The results indicate that there is a freshwater and a seawater isoform of NKA alpha-subunit in the gills of Atlantic salmon and that they are present in distinct chloride cells.

Evidence for an apical Na–Cl cotransporter involved in ion uptake in a teleost fish

Cation-chloride cotransporters, such as the Na(+)/K(+)/2Cl(-) cotransporter (NKCC) and Na(+)/Cl(-) cotransporter (NCC), are localized to the apical or basolateral plasma membranes of epithelial cells and are involved in active ion absorption or secretion. The objectives of this study were to clone and identify ;freshwater-type' and ;seawater-type' cation-chloride cotransporters of euryhaline Mozambique tilapia (Oreochromis mossambicus) and to determine their intracellular localization patterns within mitochondria-rich cells (MRCs). From tilapia gills, we cloned four full-length cDNAs homologous to human cation-chloride cotransporters and designated them as tilapia NKCC1a, NKCC1b, NKCC2 and NCC. Out of the four candidates, the mRNA encoding NKCC1a was highly expressed in the yolk-sac membrane and gills (sites of the MRC localization) of seawater-acclimatized fish, whereas the mRNA encoding NCC was exclusively expressed in the yolk-sac membrane and gills of freshwater-acclimatized fish. We then generated antibodies specific for tilapia NKCC1a and NCC and conducted whole-mount immunofluorescence staining for NKCC1a and NCC, together with Na(+)/K(+)-ATPase, cystic fibrosis transmembrane conductance regulator (CFTR) and Na(+)/H(+) exchanger 3 (NHE3), on the yolk-sac membrane of tilapia embryos acclimatized to freshwater or seawater. The simultaneous quintuple-color immunofluorescence staining allowed us to classify MRCs clearly into four types: types I, II, III and IV. The NKCC1a immunoreactivity was localized to the basolateral membrane of seawater-specific type-IV MRCs, whereas the NCC immunoreactivity was restricted to the apical membrane of freshwater-specific type-II MRCs. Taking account of these data at the level of both mRNA and protein, we deduce that NKCC1a is the seawater-type cotransporter involved in ion secretion by type-IV MRCs and that NCC is the freshwater-type cotransporter involved in ion absorption by type-II MRCs. We propose a novel ion-uptake model by MRCs in freshwater that incorporates apically located NCC. We also reevaluate a traditional ion-uptake model incorporating NHE3; the mRNA was highly expressed in freshwater, and the immunoreactivity was found at the apical membrane of other freshwater-specific MRCs.