Pepe G, Di Napoli A, Cippitelli C, Scarpino S, Pilozzi E, Ruc L.
PMID: - | DOI: 10.1002/cjp2.97
Cytokine production is essential for follicular dendritic cell maintenance and organization of germinal centres. In follicular lymphoma, follicular dendritic cells are often disarrayed and may lack antigens indicative of terminal differentiation. We investigated the in situ distribution of cells producing lymphotoxin-beta (LTB), lymphotoxin-alpha (LTA) and tumour necrosis factor-alpha (TNFA) transcripts in human reactive lymph nodes and in follicular lymphomas with follicular or diffuse growth pattern. LTB was the cytokine most abundantly produced in germinal centres. LTBwas present in nearly 90% of germinal centre cells whereas LTA and TNFA were detected in 30% and 50%, respectively. Moreover, the amount of LTB expressed in reactive germinal centre cells was 80-fold higher than that of LTA and 20-fold higher than that of TNFA. LTB-positive cells were more numerous in the germinal centre dark zone, whereas expression of the follicular dendritic cell proteins CD21, CD23, VCAM and CXCL13 was more intense in the light zone. Tumour cells of follicular lymphomas produced less LTB than reactive germinal centre cells. The results of the in situ study were confirmed by RT-PCR; LTB was significantly more abundant in reactive lymph nodes than in follicular lymphoma, with the lowest values detected in predominantly diffuse follicular lymphoma. In neoplastic follicles, low production of LTB by tumour B cells was associated with weaker expression of CD21+/CD23+ by follicular dendritic cells. Our findings detail for the first time the distribution of LTA-, LTB- and TNFA- producing cells in human reactive germinal centres and in follicular lymphoma. They suggest the possibility that impaired tumour-cell LTB production may represent a determinant of follicular dendritic cell phenotype loss and for defective follicular organization in follicular lymphoma.
Hatta A, Kurose M, Sullivan C, Okamoto K, Fujii N, Yamamura K, Meng ID.
PMID: 30969886 | DOI: 10.1152/jn.00126.2018
Corneal cool cells are sensitive to the ocular fluid status of the corneal surface and may be responsible for the regulation of basal tear production. Previously, we have shown that dry eye, induced by lacrimal gland excision (LGE) in rats, sensitized corneal cool cells to the TRPM8 agonist menthol and to cool stimulation. In the present study, we examined the effect of dry eye on the sensitivity of cool cells to the TRPV1 agonist capsaicin. Single-unit recordings in the trigeminal ganglion were performed 7-10 days after LGE. At a concentration of 0.3mM, capsaicin did not affect ongoing or cool-evoked activity in control animals yet facilitated ongoing activity and suppressed cool-evoked activity in LGE animals. At higher concentrations (3 mM), capsaicin continued to facilitate ongoing activity in LGE animals but suppressed ongoing activity in control animals. Higher concentrations of capsaicin also suppressed cool-evoked activity in both groups of animals, with an overall greater effect in LGE animals. In addition to altering cool-evoked activity, capsaicin enhanced the sensitivity of cool cells to heat in LGE animals. Capsaicin-induced changes were prevented by the application of the TRPV1 antagonist capsazepine. Using fluorescent in situ hybridization, TRPV1 and TRPM8 expression was examined in retrograde tracer identified corneal neurons. The co-expression of TRPV1 and TRPM8 in corneal neurons was significantly greater in LGE treated animals when compared to sham controls. These results indicate that LGE-induced dry eye increases TRPV1-mediated responses in corneal cool cells at least in part through the increased expression of TRPV1.
Anesten F, Dalmau Gasull A, Richard JE, Farkas I, Mishra D, Taing L, Zhang FP, Poutanen M, Palsdottir V, Liposits Z, Skibicka KP, Jansson JO.
PMID: 31033078 | DOI: 10.1111/jne.12722
Neuronal circuits involving the central amygdala (CeA) are gaining prominence as important centers for regulation of metabolic functions. As a part of the subcortical food motivation circuitry, CeA is associated with food motivation and hunger. We have previously shown that interleukin-6 (IL-6) can act as a downstream mediator of the metabolic effects of glucagon-like peptide-1 receptor (GLP-1R) stimulation in the brain, but the sites of these effects are largely unknown. We here used the newly generated and validated RedIL6 reporter mouse strain to investigate the presence of IL-6 in the CeA, as well as possible interactions between IL-6 and GLP-1 in this nucleus. IL-6 was present in the CeA, mostly in cells in the medial and lateral parts of this structure, and a majority of IL-6-containing cells also co-expressed GLP-1R. Triple staining showed GLP-1 containing fibers co-staining with synaptophysin close to or overlapping with IL-6 containing cells. GLP-1R stimulation enhanced IL-6 mRNA levels. IL-6 receptor-alpha was found to a large part in neuronal CeA cells. Using electrophysiology, we determined that cells with neuronal properties in the CeA could be rapidly stimulated by IL-6 administration in vitro. Moreover, microinjections of IL-6 into the CeA could slightly reduce food intake in vivo in overnight fasted rats. In conclusion, IL-6 containing cells in the CeA express GLP-1R, are close to GLP-1-containing synapses, and get increased IL-6 mRNA in response to GLP-1R agonist treatment. IL-6, in turn, exerts biological effects in the CeA, possibly via IL-6 receptor-alpha present in this nucleus.
Khom, S;Borgonetti, V;Vozella, V;Kirson, D;Rodriguez, L;Gandhi, P;Bianchi, P;Snyder, A;Vlkolinsky, R;Bajo, M;Oleata, C;Ciccocioppo, R;Roberto, M;
| DOI: 10.1016/j.ynstr.2023.100547
Impairments in the function of the hypothalamic-pituitary-adrenal (HPA) axis and enhanced glucocorticoid receptor (GR) activity in the central amygdala (CeA) are critical mechanisms in the pathogenesis of alcohol use disorder (AUD). The GR antagonist mifepristone attenuates craving in AUD patients, alcohol consumption in AUD models, and decreases CeA γ-aminobutyric acid (GABA) transmission in alcohol-dependent rats. Previous studies suggest elevated GR activity in the CeA of male alcohol-preferring Marchigian-Sardinian (msP) rats, but its contribution to heightened CeA GABA transmission driving their characteristic post-dependent phenotype is largely unknown. We determined Nr3c1 (the gene encoding GR) gene transcription in the CeA in male and female msP and Wistar rats using in situ hybridization and studied acute effects of mifepristone (10 μM) and its interaction with ethanol (44 mM) on pharmacologically isolated spontaneous inhibitory postsynaptic currents (sIPSCs) and electrically evoked inhibitory postsynaptic potentials (eIPSPs) in the CeA using ex vivo slice electrophysiology. Female rats of both genotypes expressed more CeA GRs than males, suggesting a sexually dimorphic GR regulation of CeA activity. Mifepristone reduced sIPSC frequencies (GABA release) and eIPSP amplitudes in msP rats of both sexes, but not in their Wistar counterparts; however, it did not prevent acute ethanol-induced increase in CeA GABA transmission in male rats. In msP rats, GR regulates CeA GABAergic signaling under basal conditions, indicative of intrinsically active GR. Thus, enhanced GR function in the CeA represents a key mechanism contributing to maladaptive behaviors associated with AUD.
Proceedings of the National Academy of Sciences of the United States of America
Ma, Q;Jiang, L;Chen, H;An, D;Ping, Y;Wang, Y;Dai, H;Zhang, X;Wang, Y;Chen, Z;Hu, W;
PMID: 36812204 | DOI: 10.1073/pnas.2207003120
Schizophrenia is a serious mental disorder, and existing antipsychotic drugs show limited efficacy and cause unwanted side effects. The development of glutamatergic drugs for schizophrenia is currently challenging. Most functions of histamine in the brain are mediated by the histamine H1 receptor; however, the role of the H2 receptor (H2R) is not quite clear, especially in schizophrenia. Here, we found that expression of H2R in glutamatergic neurons of the frontal cortex was decreased in schizophrenia patients. Selective knockout of the H2R gene (Hrh2) in glutamatergic neurons (CaMKIIα-Cre; Hrh2 fl/fl) induced schizophrenia-like phenotypes including sensorimotor gating deficits, increased susceptibility to hyperactivity, social withdrawal, anhedonia, and impaired working memory, as well as decreased firing of glutamatergic neurons in the medial prefrontal cortex (mPFC) in in vivo electrophysiological tests. Selective knockdown of H2R in glutamatergic neurons in the mPFC but not those in the hippocampus also mimicked these schizophrenia-like phenotypes. Furthermore, electrophysiology experiments established that H2R deficiency decreased the firing of glutamatergic neurons by enhancing the current through hyperpolarization-activated cyclic nucleotide-gated channels. In addition, either H2R overexpression in glutamatergic neurons or H2R agonism in the mPFC counteracted schizophrenia-like phenotypes in an MK-801-induced mouse model of schizophrenia. Taken together, our results suggest that deficit of H2R in mPFC glutamatergic neurons may be pivotal to the pathogenesis of schizophrenia and that H2R agonists can be regarded as potentially efficacious medications for schizophrenia therapy. The findings also provide evidence for enriching the conventional glutamate hypothesis for the pathogenesis of schizophrenia and improve the understanding of the functional role of H2R in the brain, especially in glutamatergic neurons.
Biopreservation and biobanking
Kim, K;Ylaya, K;Perry, C;Lee, MY;Kim, JW;Chung, JY;Hewitt, SM;
PMID: 36264172 | DOI: 10.1089/bio.2022.0090
Although the immunogenicity of formalin-fixed paraffin-embedded tissue sections can decrease during storage and transport, the exact mechanism of antigenic loss and how to prevent it are not clear. Herein, we investigated changes in the expression of estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER-2), E-cadherin, and Ki-67 in human breast tissue microarray (TMA) tissue sections stored for up to 3 months in dry and wet conditions. The positive rates of ER and PR expression were minimally changed after 3 months of storage, but the Allred scores of ER and PR stored in humid conditions decreased remarkably in comparison to fresh-cut tissue. The HER-2 antigenicity and RNA integrity of breast TMA sections stored in dry conditions diminished gradually with storage time, whereas the immunoreactivity and RNA quality of HER-2 in humid conditions decreased sharply as storage length increased. The area and intensity of E-cadherin staining in tissue sections stored in dry conditions did not change significantly and were minimally changed after 3 months, respectively. In contrast, the area and intensity of E-cadherin staining in tissue sections stored in humid conditions decreased significantly as storage length increased. Finally, the Ki-67 labeling index of tissue sections stored for 3 months in dry (9% decrease) and wet (31.9% decrease) conditions was decreased in comparison to fresh sections. In conclusion, these results indicate that water is a crucial factor for protein and RNA degradation in stored tissue sections, and detailed guidelines are required in the clinic.
Kreus, M;Lehtonen, S;Hinttala, R;Salonen, J;Porvari, K;Kaarteenaho, R;
PMID: 35964085 | DOI: 10.1186/s12931-022-02129-z
Variants of NHL repeat-containing protein 2 (NHLRC2) have been associated with severe fibrotic interstitial lung disease in early childhood and NHLRC2 has been listed as a differentially expressed gene between rapidly and slowly progressing idiopathic pulmonary fibrosis (IPF) patients. However, its cell type-specific localization in human lung tissue is unknown. The aim of this study was to evaluate NHLRC2 mRNA and protein expression in different cell types of lung tissue samples and to investigate the effect of transforming growth factor (TGF)-β1 exposure on NHLRC2 expression in vitro.The NHLRC2 expression in lung tissue samples was studied by immunohistochemistry (50 IPF, 10 controls) and mRNA in situ hybridization (8 IPF, 3 controls). The immunohistochemical NHLRC2 expression was quantified with image analysis software and associated with the clinical and smoking data of the patients. NHLRC2 expression levels in primary stromal and small airway epithelial cell lines after exposure to TGF-β1 was measured by quantitative reverse transcription polymerase chain reaction and Western blot analysis.NHLRC2 expression was detected especially in bronchiolar epithelial cells, type II pneumocytes and macrophages in normal lung. In the lungs of IPF patients, NHLRC2 was mainly expressed in hyperplastic alveolar epithelial cells lining fibroblast foci and honeycombs. NHLRC2 expression assessed by image analysis was higher in IPF compared to controls (p < 0.001). Ever-smokers had more prominent NHLRC2 staining than non-smokers (p = 0.037) among IPF patients. TGF-β1 exposure did not influence NHLRC2 levels in lung cell lines.NHLRC2 expression was higher in IPF compared to controls being widely expressed in type II pneumocytes, macrophages, bronchiolar epithelium, and hyperplastic alveolar epithelium. Additionally, its expression was not regulated by the exposure to TGF-β1 in vitro. Further studies are needed to clarify the role of NHLRC2 in IPF.
Hepatology (Baltimore, Md.)
Zhao, K;Guo, F;Wang, J;Zhong, Y;Yi, J;Teng, Y;Xu, Z;Zhao, L;Li, A;Wang, Z;Chen, X;Cheng, X;Xia, Y;
PMID: 35718932 | DOI: 10.1002/hep.32622
Murine hepatic cells cannot support hepatitis B virus (HBV) infection even with supplemental expression of viral receptor, human sodium-taurocholate cotransporting polypeptide (hNTCP). However, the specific restricted step remains elusive. In this study, we aimed to dissect HBV infection process in murine hepatic cells.Cells expressing hNTCP were inoculated with HBV or hepatitis delta virus (HDV). HBV pre-genomic RNA (pgRNA), covalently closed circular DNA (cccDNA) and different relaxed circular DNA (rcDNA) intermediates were produced in vitro. The repair process from rcDNA to cccDNA was assayed by in vitro repair experiments and in mouse with hydrodynamic injection. Southern blotting and in situ hybridization were used to detect HBV DNA. HBV, but not its satellite virus HDV, was restricted from productive infection in murine hepatic cells expressing hNTCP. Transfection of HBV pgRNA could establish HBV replication in human, but not in murine hepatic cells. HBV replication-competent plasmid, cccDNA and recombinant cccDNA could support HBV transcription in murine hepatic cells. Different rcDNA intermediates could be repaired to form cccDNA both in vitro and in vivo. In addition, rcDNA could be detected in the nucleus of murine hepatic cells, but cccDNA could not be formed. Interestingly, nuclease sensitivity assay showed that the protein-linked rcDNA isolated from cytoplasm was completely nuclease resistant in murine but not in human hepatic cells.Our results imply that the disassembly of cytoplasmic HBV nucleocapsids is restricted in murine hepatic cells. Overcoming this limitation may help to establish an HBV infection mouse model.This article is protected by
Fritsvold, C;Mikalsen, AB;Haugland, Ø;Tartor, H;Sindre, H;
PMID: 35686455 | DOI: 10.1111/jfd.13659
Since the first description of cardiomyopathy syndrome (CMS) in Atlantic salmon, in 1985, the disease caused by piscine myocarditisvirus (PMCV) has become a common problem in Atlantic salmon farming, not only in Norway, but also in other salmon farming countries like Scotland and Ireland. In the last years, CMS has been ranked as the most important salmon viral disease in Norway regarding both mortality and economic losses. Detailed knowledge of infection and pathogenesis is still lacking, a decade after the causal agent was first described, and there is a need for a wider range of methods/tools for diagnostic and research purposes. In this study, we compared the detection of PMCV- and CMS-related tissue lesions using previously used and well-known methods like histopathology and real-time RT-PCR to immunohistochemistry (IHC), a less used method, and a new method, RNAscope in situ hybridization. Tissue samples of three different cardiac compartments, mid-kidney and skin/muscle tissue were compared with non-lethal parallel samplings of blood and mucus. The development of pathological cardiac lesions observed in this experiment was in accordance with previous descriptions of CMS. Our results indicate a viremic phase 10- to 20-day post-challenge (dpc) preceding the cardiac lesions. In this early phase, virus could also be detected in relatively high amount in mid-kidney by real-time RT-PCR. Plasma and/or mid-kidney samples may, therefore, be candidates to screen for early-phase PMCV infection. The RNAscope in situ hybridization method showed higher sensitivity and robustness compared with the immunohistochemistry and may be a valuable support to histopathology in CMS diagnostics, especially in cases of untypical lesions or mixed infections.
The Journal of biological chemistry
Ghosh, K;Zhang, GF;Chen, H;Chen, SR;Pan, HL;
PMID: 35500651 | DOI: 10.1016/j.jbc.2022.101999
Type-2 cannabinoid receptors (CB2, encoded by the Cnr2 gene) are mainly expressed in immune cells, and CB2 agonists normally have no analgesic effect. However, nerve injury upregulates CB2 in the dorsal root ganglion (DRG), following which CB2 stimulation reduces neuropathic pain. It is unclear how nerve injury increases CB2 expression or how CB2 activity is transformed in neuropathic pain. In this study, immunoblotting showed that spinal nerve ligation (SNL) induced a delayed and sustained increase in CB2 expression in the DRG and dorsal spinal cord synaptosomes. RNAscope in situ hybridization also showed that SNL substantially increased CB2 mRNA levels, mostly in medium and large DRG neurons. Furthermore, we found that the specific CB2 agonist JWH-133 significantly inhibits the amplitude of dorsal root-evoked glutamatergic excitatory postsynaptic currents in spinal dorsal horn neurons in SNL rats, but not in sham control rats; intrathecal injection of JWH-133 reversed pain hypersensitivity in SNL rats, but had no effect in sham control rats. In addition, chromatin immunoprecipitation-qPCR analysis showed that SNL increased enrichment of two activating histone marks (H3K4me3 and H3K9ac) and diminished occupancy of two repressive histone marks (H3K9me2 and H3K27me3) at the Cnr2 promoter in the DRG. In contrast, SNL had no effect on DNA methylation levels around the Cnr2 promoter. Our findings suggest that peripheral nerve injury promotes CB2 expression in primary sensory neurons via epigenetic bivalent histone modifications and that CB2 activation reduces neuropathic pain by attenuating nociceptive transmission from primary afferent nerves to the spinal cord.
Chaves, FM;Wasinski, F;Tavares, MR;Mansano, NS;Frazão, R;Gusmao, DO;Quaresma, PGF;Pedroso, JAB;Elias, CF;List, EO;Kopchick, JJ;Szawka, RE;Donato, J;
PMID: 35395079 | DOI: 10.1210/endocr/bqac045
Hypophysiotropic somatostatin (SST) neurons in the periventricular hypothalamic area express growth hormone (GH) receptor (GHR) and are frequently considered as the key neuronal population that mediates the negative feedback loop controlling the hypothalamic-GH axis. Additionally, insulin-like growth factor-1 (IGF-1) may also act at the hypothalamic level to control pituitary GH secretion via long-loop negative feedback. However, to the best of our knowledge, no study so far has tested whether GHR or IGF-1 receptor (IGF1R) signaling specifically in SST neurons is required for the homeostatic control of GH secretion. Here we show that GHR ablation in SST neurons did not impact the negative-feedback mechanisms that control pulsatile GH secretion or body growth in male and female mice. The sex difference in hepatic gene expression profile was only mildly affected by GHR ablation in SST neurons. Similarly, IGF1R ablation in SST neurons did not affect pulsatile GH secretion, body growth or hepatic gene expression. In contrast, simultaneous ablation of both GHR and IGF1R in SST-expressing cells increased mean GH levels and pulse amplitude in male and female mice, and partially disrupted the sex differences in hepatic gene expression. Despite the increased GH secretion in double-knockout mice, no alterations in body growth and serum or liver IGF-1 levels were observed. In summary, GHR and IGF1R signaling in SST neurons play a redundant role in the control of GH secretion. Furthermore, our results reveal the importance of GH/IGF-1 negative-feedback mechanisms on SST neurons for the establishment of sex differences in hepatic gene expression profile.
Progress in neuro-psychopharmacology & biological psychiatry
Chang, GQ;Yasmin, N;Collier, AD;Karatayev, O;Khalizova, N;Onoichenco, A;Fam, M;Albeg, AS;Campbell, S;Leibowitz, SF;
PMID: 35176416 | DOI: 10.1016/j.pnpbp.2022.110536
Prenatal alcohol exposure (PAE) increases alcohol consumption and risk for alcohol use disorder. This phenomenon in rodents is suggested to involve a stimulatory effect of PAE, in female more than male offspring, on neurogenesis and density of neurons expressing neuropeptides in lateral hypothalamus (LH), including melanin-concentrating hormone (MCH), known to promote alcohol intake. With evidence suggesting a role for fibroblast growth factor 2 (FGF2) and its receptor FGFR1 in stimulating neurogenesis and alcohol drinking, we investigated here whether the FGF2-FGFR1 system is involved in the PAE-induced increase in MCH neurons, in postnatal offspring of pregnant rats given ethanol orally (embryonic day 10-15) at a low-moderate (2 g/kg/day) or high (5 g/kg/day) dose. Our results demonstrate that PAE at the low-moderate but not high dose stimulates FGF2 and FGFR1 gene expression and increases the density of MCH neurons co-expressing FGF2, only in females, but FGFR1 in both sexes. PAE induces this effect in the dorsal but not ventral area of the LH. Further analysis of FGF2 and FGFR1 transcripts within individual MCH neurons reveals an intracellular, sex-dependent effect, with PAE increasing FGF2 transcripts positively related to FGFR1 in the nucleus as well as cytoplasm of females but transcripts only in the cytoplasm of males. Peripheral injection of FGF2 itself (80 μg/kg, s.c.) in pregnant rats mimics these effects of PAE. Together, these results support the involvement of the FGF2-FGFR1 system in mediating the PAE-induced, sex dependent increase in density of MCH neurons, possibly contributing to increased alcohol consumption in the offspring.
