Influx Of Cells Research Articles

CRL4b Inhibition Ameliorates Experimental Autoimmune Encephalomyelitis Progression.

Multiple sclerosis, and its murine model experimental autoimmune encephalomyelitis (EAE), is a neurodegenerative autoimmune disease of the CNS characterized by T cell influx and demyelination. Similar to other autoimmune diseases, therapies can alleviate symptoms but often come with side effects, necessitating the exploration of new treatments. We recently demonstrated that the Cullin-RING E3 ubiquitin ligase 4b (CRL4b) aided in maintaining genome stability in proliferating T cells. In this study, we examined whether CRL4b was required for T cells to expand and drive EAE. Mice lacking Cul4b (Cullin 4b) in T cells had reduced EAE symptoms and decreased inflammation during the peak of the disease. Significantly fewer CD4+ and CD8+ T cells were found in the CNS, particularly among the CD4+ T cell population producing IL-17A, IFN-γ, GM-CSF, and TNF-α. Additionally, Cul4b-deficient CD4+ T cells cultured invitro with their wild-type counterparts were less likely to expand and differentiate into IL-17A- or IFN-γ-producing effector cells. When wild-type CD4+ T cells were activated invitro in the presence of the recently developed CRL4 inhibitor KH-4-43, they exhibited increased apoptosis and DNA damage. Treatment of mice with KH-4-43 following EAE induction resulted in stabilized clinical scores and significantly reduced numbers of T cells and innate immune cells in the CNS compared with control mice. Furthermore, KH-4-43 treatment resulted in elevated expression of p21 and cyclin E2 in T cells. These studies support that therapeutic inhibition of CRL4 and/or CRL4-related pathways could be used to treat autoimmune disease.

Hydrogen sulfide induces Ca2+ influx in the principal cells of rat cortical collecting ducts

Hydrogen sulfide (H2S) acts as a gas-signaling agent in various tissues. Although it has been reported that endogenous enzymes that generate H2S are expressed abundantly in the kidney, few reports examine cellular responses to H2S in renal tubular epithelial cells. In this study, we investigated the effects of NaHS, an H2S donor, and l-cysteine, a substrate for H2S production, on the principal cells of rat cortical collecting ducts (CCDs). NaHS increased the intracellular Ca2+ concentration ([Ca2+]i) in the principal cells. The removal of extracellular Ca2+ largely attenuated the [Ca2+]i response. The TRPV4 channel blocker significantly inhibited the effect of NaHS. Extracellular administration of l-cysteine also elicited a rise in [Ca2+]i. Prior treatment of CCDs with AOAA, an inhibitor of H2S production enzyme, l-cysteine-induced [Ca2+]i response was significantly reduced. These results suggest that not only exogenous H2S but also endogenously produced H2S triggers the extracellular influx pathway of Ca2+ in the principal cells of rat CCDs.

Tobacco-derived and tobacco-free nicotine cause differential inflammatory cell influx and MMP-9 in mouse lung

BackgroundElectronic nicotine delivery systems (ENDS) or electronic cigarettes (e-cigarettes) aerosolize an e-liquid composed of propylene glycol (PG) and vegetable glycerin (VG) as humectants, flavoring chemicals, and nicotine. Nicotine naturally occurs in two isomers R- and S-nicotine, with tobacco-derived nicotine (TDN) composed of S-nicotine, and tobacco-free/synthetic nicotine (TFN) composed of a racemic mixture of R- and S-nicotine. Currently, there is limited knowledge of the potential differences in the toxicity of TFN versus TDN. We hypothesized that exposure of TFN and TDN salts to C57BL/6J mice would result in a differential response in lung inflammation and protease/ antiprotease imbalance.MethodsFive-week-old male and female C57BL/6J mice were exposed to air, PG/VG, PG/VG with TFN salts (TFN), or PG/VG with TDN salts (TDN) by nose-only exposure. Lung inflammatory cell counts, cytokine/chemokine levels, and matrix metalloproteinase (MMP) protein abundance and activity levels were determined by flow cytometry, ELISA, immunoblotting, and gel zymography, respectively.ResultsExposure to the humectants (PG/VG) alone increased cytokine levels- IL-6, KC, and MCP-1 in the BALF and KC levels in lung homogenate of exposed mice. While no change was observed in the cytokine levels in lung homogenate of TDN aerosol exposed mice, exposure to TFN aerosols resulted in an increase in KC levels in the lungs of these mice compared to air controls. Interestingly, exposure to TDN aerosols increased MMP-9 protein abundance in the lungs of female mice, while exposure to TFN aerosol showed no change. The metabolism of nicotine or the clearance of cotinine for TFN exposure may differ from that for TDN.ConclusionExposure to humectants, PG/VG alone, induces an inflammatory response in C57BL/6J mice. TFN and TDN salts show distinct changes in inflammatory responses and lung proteases on acute exposures. These data suggest variable toxicological profiles of the two forms of nicotine in vivo. Future work is thus warranted to delineate the harmful effects of synthetic/natural nicotine with humectants to determine the potential toxicological risks for users.

The Secreted Ly6/uPAR-Related Protein 1 (Slurp1) Modulates Corneal Angiogenic Inflammation Via NF-κB Signaling.

Previously we demonstrated that the secreted Ly-6/uPAR related protein 1 (SLURP1), abundantly expressed in the corneal epithelium (CE) and secreted into the tear fluid, serves as an antiangiogenic molecule. Here we describe the Slurp1-null (Slurp1X-/-) mouse corneal response to silver nitrate (AgNO3) cautery. Five days after AgNO3 cautery, we compared the wild-type (WT) and Slurp1X-/- mouse (1) corneal neovascularization (CNV) and immune cell influx by whole-mount immunofluorescent staining for CD31 and CD45, (2) macrophage and neutrophil infiltration by flow cytometry, and (3) gene expression by quantitative RT-PCR. Quantitative RT-PCR, immunofluorescent staining, and immunoblots were employed to evaluate the expression, phosphorylation status, and subcellular localization of NF-κB pathway components. Unlike the WT, the Slurp1X-/- corneas displayed denser CNV in response to AgNO3 cautery, with more infiltrating macrophages and neutrophils and greater upregulation of the transcripts encoding VEGFA, MMP2, IL-1b, and vimentin. At 2, 7, and 10days after AgNO3 cautery, Slurp1 expression was significantly downregulated in the WT corneas. Compared with the WT, naive Slurp1X-/- CE displayed increased phosphorylation of IKK(a/b), elevated phosphorylation of IκB with decreased amounts of total IκB, and higher phosphorylation of NF-κB, suggesting that NF-κB signaling is constitutively active in naive Slurp1X-/- corneas. Enhanced angiogenic inflammation in AgNO3 cauterized Slurp1X-/- corneas and constitutively active status of NF-κB signaling in the absence of Slurp1 suggest that Slurp1 modulates corneal angiogenic inflammation via NF-κB signaling.

Systemic and local antiinflammatory effect of magnesium chloride in experimental arthritis

ObjectiveDespite some knowledge gaps in scientific evidence, MgCl2 is largely used for pain relief in musculoskeletal diseases. Mg salts were shown to provide analgesia postoperatively in orthopedic surgery and low Mg levels were linked to arthritis development and severity. We determined the anti-inflammatory activity of MgCl2 in an acute arthritis model.MethodsMice received 0.1 mg/25µL Zymosan (Zy) or saline into the knees. Joint pain was evaluated using von Frey test; cell influx, and interleukin (IL)-1 level were assessed in joint lavage at 6 h. Synovia were excised for histopathology and analysis of immunoexpression of nuclear factor kappa B (NFκB) and tumor necrosis factor (TNF)-α. Groups (n = 6/group) received either 90 mg/kg MgCl2/100 µL or saline per os (systemic) or 500 µg/25 µL MgCl2 or saline intra-articularly (i.a.) 30 min prior to Zy.ResultsMgCl2 given either systemically or locally significantly reduced cell influx (p = 0.0012 and p = 0.0269, respectively), pain (p = 0.0005 and p = 0.0038, respectively), and intra-articular IL-1 level (p = 0.0391), as compared to saline. Systemic MgCl2 significantly decreased NFκB (p < 0.05) immmunoexpression, as compared to saline.ConclusionMgCl2 given systemically or locally displayed anti-inflammatory activity in a severe acute arthritis model reducing cell influx, pain, and cytokine release. MgCl2 operates at least partially via inhibiting NFκB activation. This is the first in vivo demonstration that MgCl2 decreases cytokine release in arthritis, prompting reduction of inflammation and pain relief.

Assessing the protection elicited by virus-like particles expressing the RSV pre-fusion F and tandem repeated G proteins against RSV rA2 line19F infection in mice

Excessive pulmonary inflammation is the hallmark of respiratory syncytial virus (RSV) infection hindering efficacious RSV vaccine development. Yet, the vast majority of the experimental RSV vaccine studies use laboratory-adapted RSV strains that do not reflect the highly pathogenic and inflammatory nature of the virus found in clinical settings. Here, we re-evaluated the protective efficacy of the virus-like particle (VLP) vaccine co-expressing the pre-fusion (pre-F) protein and G protein with tandem repeats (Gt) reported in our previous study against the recombinant RSV rA2-line19F strain, which inflicts severe mucus production and inflammation in mice. VLP vaccine immunization elicited virus-specific serum antibody responses that mediated RSV rA2-line19F virus neutralization. VLP vaccine immunization promoted Th1 immune response development in the spleens and CD8 + T cell influx into the lungs of mice, which are essential for efficient viral clearance and dampened inflammatory response. When compared to the VLPs expressing only the pre-F antigen, those co-expressing both pre-F and Gt antigens conferred better protection in mice against rA2-line19F challenge infection. Overall, our data suggest that the pre-clinical VLP vaccine co-expressing RSV pre-F and Gt antigens can effectively protect mice against RSV strains that resemble pathogenic clinical isolates.

Comparison of tissue response and lifting effect induced by non-absorbable elastic thread and commercialized threads in rat model.

As we age, our skin loses elasticity and wrinkles form. To prevent this, most people try to improve skin wrinkles by performing procedures such as fillers, and absorbable lifting threads. Another way to solve this structural problem is to use an elastic thread. Although elastic sutures made of polymer materials (such as silicone) are widely used, data regarding their properties and potential effectiveness are lacking. This study aimed to investigate the effects of inserting non-absorbable elastic threads, with different manufacturing requirements and methods, on the skin and subcutaneous tissue. In this study, non-absorbable elastic threads ELATENS and Elasticum using different manufacturing methods were compared. Each thread was transplanted into the subcutaneous layer of the back of a rat to induce wrinkles. After inducing wrinkles in the skin of rat, the degree of skin maintenance by each thread and the thickness of the capsule formed around the suture were measured. Through ex-vivo experiments on each thread, the fixation force in the tissue was confirmed. In a comparison of inflammatory response and collagen formation through histological analysis, it was confirmed that there was no significant difference from the equivalent comparative product. In conclusion, the degree of encapsulation between tissues and collagen fiber formation for improving skin wrinkles was superior in elastic threads compared to non-elastic threads. It is believed that this provides certain elasticity to the skin layer and can induce cell influx to improve wrinkles.

Kinetics of infection and local immune response in a model of chlamydia trachomatis conjunctival infection

Aims/Purpose: Trachoma is caused by Chlamydia trachomatis conjunctival infections and evolves in two phases: i) active or acute trachoma caused by repeated infections and ii) scarring or chronic trachoma leading to conjunctival scarring, eyelid malposition, and corneal opacification. It remains the first cause of infectious blindness in developing countries. The purpose of this study was to precisely describe the kinetics of infection and local immune response in a non‐human primate model of acute trachoma.Methods: Cynomolgus Macaques received a single conjunctival inoculation of B/Tunis‐864 strain (104 IFU) and were assessed at weeks 2, 4, 6, 9, and 11. Clinical evaluation was performed by 3 independent masked observers on standardized ocular surface photographs graded using specific scores for both inflammation and follicles. Local immune response was assessed using multiparameter flow cytometry of conjunctival cells eluted from conjunctival prints. Tears cytokines were quantified using bead‐based Luminex multiplex assay. Bacterial quantification in conjunctival fluids was performed with quantitative PCR.Results: All animals (n = 12) developed typical follicular conjunctivitis (n = 12). The first 4 weeks post‐exposure were characterized by a conjunctival T cell influx (63% of CD45+ cells at week 3), with elevated interleukins 4, 8, and 5. In the later phase, after 9 weeks, we observed an influx of monocytes (53,2% of CD45+ cells at week 11) with reduced immune cells and a return to the initial cytokines level.Conclusion: This model faithfully mimics trachoma's acute phase. Local immunologic assays showed an early lymphocyte response with long‐term persistence of innate immune effectors. It offers an opportunity to assess potential preventive therapeutic strategies. Further studies, including implementing repeated bacterial conjunctival inoculations, are warranted to investigate the later stages of the diseases.

Intrahepatic neutrophil accumulation and extracellular trap formation are associated with posthepatectomy liver failure.

Posthepatectomy liver failure (PHLF) represents a life-threatening complication with limited therapeutic options. Neutrophils play a critical and dynamic role during regeneratory processes, but their role in human liver regeneration is incompletely understood, especially as underlying liver disease, detectable in the majority of patients, critically affects hepatic regeneration. Here we explored intrahepatic neutrophil accumulation and neutrophil extracellular traps (NETs) in patients with PHLF and validated the functional relevance of NETs in a murine partial hepatectomy (PHx) model. We investigated the influx of neutrophils, macrophages, eosinophils, and mast cells and the presence of their respective extracellular traps in liver biopsies of 35 patients undergoing hepatectomy (10 patients with PHLF) before and after the initiation of liver regeneration by fluorescence microscopy. In addition, NET formation and neutrophil activation were confirmed by plasma analysis of 99 patients (24 patients with PHLF) before and up to 5 days after surgery. Furthermore, we inhibited NETs via DNase I in a murine PHx model of mice with metabolically induced liver disease. We detected rapid intrahepatic neutrophil accumulation, elevated levels of myeloperoxidase release, and NET formation in regenerating human livers, with a significantly higher increase of infiltrating neutrophils and NETs in patients with PHLF. Circulating markers of neutrophil activation, including elastase, myeloperoxidase, and citrullinated histone H3, correlated with markers of liver injury. In a murine PHx model, we showed that the inhibition of NET accelerated hepatocyte proliferation and liver regeneration. Patients with PHLF showed accelerated intrahepatic neutrophil infiltration and NET formation, which were associated with liver damage. Further, we identified postsurgical myeloperoxidase levels as predictive markers for adverse outcomes and observed that blocking NETs in a murine PHx model accelerated tissue regeneration.

Serum Amyloid A3 Fuels a Feed-Forward Inflammatory Response to the Bacterial Amyloid Curli in the Enteric Nervous System

BackgroundMounting evidence suggests a role for the gastro-intestinal microbiome as a determinant of peripheral immunity and central neurodegeneration, but the local disease mechanisms remain unknown. Given its potential relevance for early diagnosis and therapeutic intervention, we set out to map the pathogenic changes induced by bacterial amyloids in the gastro-intestinal tract and its enteric nervous system. MethodsTo examine the early response, we challenged primary murine myenteric networks with curli, the prototypic bacterial amyloid, and performed shotgun RNA sequencing and multiplex ELISA. Using enteric neurosphere-derived glial and neuronal cell cultures, as well as in vivo curli injections into the colon wall, we further scrutinized curli-induced pathogenic pathways. ResultsCurli induced a pro-inflammatory response, with marked upregulation of Serum Amyloid A3 (Saa3) and the secretion of several cytokines. This pro-inflammatory state was primarily induced in enteric glia, was accompanied by elevated levels of DNA damage and replication, and triggered the influx of immune cells in vivo. The addition of recombinant SAA3 was sufficient to recapitulate this specific pro-inflammatory phenotype while Saa3 knock-out attenuated curli-induced DNA damage and replication. Like curli, recombinant SAA3 caused a strong upregulation of Saa3 transcripts, indicating a feed-forward loop. Colonization of curli-producing Salmonella and Dextran Sulphate Sodium (DSS)-induced colitis caused a significant increase in Saa3 transcripts, indicating a central role for SAA3 in enteric dysfunction. Inhibition of dual leucine zipper kinase (DLK), an upstream regulator of the c-Jun N-terminal kinase (JNK) pathway responsible for SAA3 production, attenuated curli- and SAA3-induced Saa3 upregulation, DNA damage and replication in enteric glia. ConclusionsOur results position SAA3 as an important mediator of gastro-intestinal vulnerability towards bacterial-derived amyloids and demonstrate the potential of DLK inhibition to dampen enteric pathology.

Effects of regulator of G protein signaling 2 (RGS2) overexpression in the paraventricular nucleus on blood pressure in rats with angiotensin II-induced hypertension.

The hypothalamic paraventricular nucleus (PVN) regulates sympathetic activity and blood pressure. The regulator of G protein signaling 2 (RGS2) is a negative G protein regulator, which selectively regulates G⍺q signaling, a potential cause of hypertension. This study aimed to examine angiotensin II (ANG II)-G protein-RGS2 signaling on the central mechanisms of blood pressure control, sympathetic activation, and kidney function. The Sprague Dawley rats were infused with ANG II (200ng/kg/min) via osmotic mini pump to induce hypertension. Adenovirus (AV) vectors encoding RGS2 was transfected into the PVN in vivo. By radio telemetry measurements, we found AV-RGS2 transfection to the PVN significantly attenuated the increase of mean arterial pressure in ANG II infusion rats from days 2-7 of the 2-week experiment (Day 7: ANG II + AV-RGS2 141.3 ± 10.0mmHg vs. ANG II 166.9 ± 9.3mmHg, p < 0.05). AV-RGS2 transfection significantly reduced the serum norepinephrine level and acute volume reflex and increased daily urine volume and sodium excretion in ANG II-infused hypertensive rats. AV-RGS2 transfection significantly reduced G⍺q and PKC protein expressions within the PVN in ANG II infusion rats. In cultured mouse hypothalamic cells, real-time PCR study showed ANG II treatment increased mRNA expression of G⍺q, G⍺s, and RGS2, and AV-RGS2 treatment decreased ANG II-induced mRNA expression of G⍺q and G⍺s. Using confocal imagery, we found that AV-RGS2 attenuated the increase of calcium influx in ANG II-treated cells. Our results suggest that central overexpression of RGS2 in the PVN attenuated the increase of blood pressure and sympathetic outflow, and improves kidney excretory function in hypertensive rats. This may be via the alteration of ANG II-G-protein-RGS2 signaling in the central nervous system.

Moonlighting glyceraldehyde-3-phosphate dehydrogenase of Lactobacillus gasseri inhibits keratinocyte apoptosis and skin inflammation in experimental atopic dermatitis.

Lactic acid bacteria may be used as probiotics to prevent or treat various diseases, and Lactobacillus delbrueckii has an inhibitory effect on the development of atopic diseases. This study explored the effects of L. delbrueckii subsp. lactis strain LDL557 administration on a mouse asthma model resulting from Dermatophoides pteronyssinus (Der p) sensitization and investigated the associated gut microbiota. Der p-sensitized and challenged BALB/c mice were orally administered with three different doses of live (low, 107 colony-forming units (CFU); medium, 108 CFU; high, 109 CFU) and heat-killed (109 cells) LDL557 in 200 μL of PBS daily, starting 2 weeks before Der p sensitization and lasting 4 weeks. After the allergen challenge, airway responsiveness to methacholine and the influx of inflammatory cells to the lungs were assessed. The gut microbiome was obtained by sequencing the V3-V4 region of the 16S rRNA gene from mice stool samples. LDL557 in the live (109 CFU) and heat-killed (109 cells) conditions reduced the airway hyper-responsiveness after stimulation with methacholine, inflammatory cell infiltration, and mucus production. These effects were similar to those in groups treated with dexamethasone. No significant change in the gut microbiota was observed after LDL557 treatment, except for the tendency of heat-killed LDL557 to change the gut microbial profile to a greater extent than live LDL557. In summary, we found that live and heat-killed LDL557 had the beneficial effect of preventing Der p-induced allergic inflammation in a mouse model of asthma.

Vasopressin differentially modulates the excitability of rat olfactory bulb neuron subtypes.

Vasopressin (VP) plays a crucial role in social memory even at the level of the olfactory bulb (OB), where OB VP cells are activated during social interactions. However, it remains unclear how VP modulates olfactory processing to enable enhanced discrimination of very similar odors, e.g., rat body odors. Thus far, it has been shown that VP reduces firing rates in mitral cells (MCs) during odor presentation in vivo and decreases the amplitudes of olfactory nerve-evoked excitatory postsynaptic potentials (ON-evoked EPSPs) in external tufted cells in vitro. We performed whole-cell patch-clamp recordings and population Ca2+ imaging on acute rat OB slices. We recorded ON-evoked EPSPs as well as spontaneous inhibitory postsynaptic currents (IPSCs) from two types of projection neurons: middle tufted cells (mTCs) and MCs. VP bath application reduced the amplitudes of ON-evoked EPSPs and the frequencies of spontaneous IPSCs in mTCs but did not change those in MCs. Therefore, we analyzed ON-evoked EPSPs in inhibitory interneurons, i.e., periglomerular cells (PGCs) and granule cells (GCs), to search for the origin of increased inhibition in mTCs. However, VP did not increase the amplitudes of evoked EPSPs in either type of interneurons. We next performed two-photon population Ca2+ imaging in the glomerular layer and the superficial GC layer of responses to stronger ON stimulation than during patch-clamp experiments that should evoke action potentials in the measured cells. We observed that VP application increased ON-evoked Ca2+ influx in juxtaglomerular cells and GC somata. Thus, our findings indicate inhibition by VP on projection neurons via strong ON input-mediated inhibitory interneuron activity. This neural modulation could improve representation of odors, hence, better discriminability of similar odors, e.g., conspecific body odors.

Decoding lymphangiogenesis in oral squamous cell carcinoma: Emphasis on clinical and histopathological determinants of regional metastasis.

The growth and metastasis of solid epithelial tumors is lymphangiogenesis dependent. The most important lymphangiogenic inducers facilitating this progression is Vascular endothelial Growth Factor C (VEGF-C). The recent D2-40 (Podoplanin) antibody is specific for lymphatic epithelium and allows its objective assessment. Also, lymphovascular invasion (LVI) is a risk factor for lymph node metastases (LNM) and indicates a significant influx of tumor cells into the lymphatics, causing regional metastasis. Thus, the following study was conducted to assess and correlate VEGF-C and D2-40 immunoexpressions with clinical and histopathologic lymph node status in Oral Squamous Cell Carcinoma (OSCC) cases and also to estimate the impact of intratumoral (ILVD) and peritumoral lymphatic vessel density (PLVD) in such cases. A total of 128 OSCC cases, divided as Group I: Cases with clinically and histopathologically negative lymph nodes (n = 64) and Group II: Cases with clinically negative but histopathologically positive lymph nodes (n = 64) were immunoscored for VEGF-C (Anti VEGF-C antibody) (PA5-29772, Invitrogen) and D2-40 (Anti D2-40 antibody) (IR072/8072, Dako) using standard protocols. The data was statistically evaluated using STATA 18.0 with p≤0.05 considered statistically significant throughout the study. 21.88% Group I cases and 40.62% Group II cases showed highest immuno-positivity for VEGF-C (p = 0.00). The mean D2-40 score for Intratumoral Lymphatic Vessel Density (ILVD) and Peritumoral Lymphatic Vessel Density (PLVD) was higher for group II cases (i.e., 41.5±13.73 and 35.95±8.27 respectively at 95% CI, p = 0.00) suggesting a direct correlation between Lymphatic Vessel Density (LVD) and LNM. Lymphangiogenesis is a true determinant of the biologic potential of OSCC and obtaining an objective data in terms of LVD through D2-40 could be impactful in OSSC diagnosis and guiding treatment decisions by clinicians.

Outward potassium current in neurons of aestivated land snail Achatina fulica.

Aestivation and hibernation represent distinct forms of animal quiescence, characterized by physiological changes, including ion composition. Intracellular ion flows play a pivotal role in eliciting alterations in membrane potential and facilitating cellular communication, while outward K+ currents aid in the restitution and upkeep of the resting membrane potential. This study explores the relationship between inward and outward currents during aestivation in Achatina fulica snails. Specimens were collected near MSUBIT University in Shenzhen and divided into two groups. The first group was kept on a lattice diet, while the second one consisted of aestivating individuals, that were deprived of food and water until a cork-like structure sealed their shells. Recording of current from isolated neurons were conducted using the single-electrode voltage clamp mode with an AxoPatch 200B amplifier. Electrophysiological recordings on pedal ganglia neurons revealed significant differences in the inactivation processes of the Ia and Ikdr components. Alterations in the Ikdr component may inhibit pacemaker activity in pedal ganglion neurons, potentially contributing to locomotion cessation in aestivated animals. The KS current remains unaffected during aestivation. Changes in slow K+ current components could disrupt the resting membrane potential, possibly leading to cell depolarization and influx of Ca2+ and Na+ ions, impacting cell homeostasis. Thus, maintaining the constancy of outward K+ current is essential for cell stability.

Increased sodium fluorescein transport by corticosteroids is inhibited by a LAT-1 specific inhibitor in retinal pigment epithelial cells in vitro

To investigate whether aldosterone (ALD) and hydrocortisone (HC) change the gene expression of SLC7A5, which encodes the large neutral amino acid transporter small subunit 1 (LAT1), and the transport activity of LAT1 in the retinal pigment epithelium (RPE) in vitro. ARPE-19 cells were grown to confluence. After withdrawing the serum, ALD or HC was added with several doses and incubated, and SLC7A5 gene expression was measured. The influx and efflux transport of sodium fluorescein (Na-F) were evaluated using the Transwell culture system. SLC7A5 gene expression was upregulated by ALD and downregulated by HC in a dose-dependent manner. Both ALD and HC significantly increased the influx and efflux Na-F transport of RPE cells at a dose that did not change the expression of SLC7A5. JPH203, a specific inhibitor of LAT1, significantly reduced accelerated Na-F transport. Both ALD and HC increased the gene expression of zonula occludin-1 (ZO-1) although they did not change the immunoreactivity of ZO-1 in RPE cells. LAT1 may play an important role in increasing Na-F transport associated with ALD and HC administration. A specific LAT1 inhibitor may effectively regulate the increased material transport of RPE induced by ALD and HC.

Reconstitution of calcium channel protein Orai3 into liposomes for functional studies

Store-operated calcium entry (SOCE) is the main mechanism for the Ca2+ influx in non-excitable cells. The two major components of SOCE are stromal interaction molecule 1 (STIM1) in the endoplasmic reticulum and Ca2+ release-activated Ca2+ channel (CRAC) Orai on the plasma membrane. SOCE requires interaction between STIM1 and Orai. Mammals have three Orai homologs: Orai1, Orai2, and Orai3. Although Orai1 has been widely studied and proven to be essential for numerous cellular processes, Orai3 has also attracted a significant attention recently. The gating and activation mechanisms of Orai3 have yet to be fully elucidated. Here, we expressed, purified, and reconstituted Orai3 protein into liposomes and investigated its orientation and oligomeric state in the resulting proteoliposomes. STIM1 interacted with the Orai3-containing proteoliposomes and mediated calcium release from them, suggesting that the Orai3 channel was functional and that recombinant STIM1 could directly open the Orai3 channel in vitro. The developed in vitro calcium release system could be used to study the structure, function, and pharmacology of Orai3 channel.

Calcium Influx Pathways in Breast Cancer: Opportunities for Pharmacological Intervention

Numerous cellular processes, including the release of neurotransmitters and the contraction of muscles, are largely triggered and regulated by Ca2+ inflow via Ca2+ permeable ion channels. In addition, Ca2+ influx regulates cellular migration and proliferation, two mechanisms linked to cancer. This study focuses on calcium influx in breast cancer cells and discusses how future drugs for breast cancer therapy may be pharmacological modulators of particular Ca2+ influx channels. Certain breast tumors have altered expression of particular calcium permeable ion channels. Such alterations may occasionally be connected to the prognosis and subtype of breast cancer. These days, models both in vivo and in vitro have assisted in identifying particular Ca2+ channels that are crucial for the growth and invasiveness of of cancerous breast cells. Nonetheless, additional research is still needed to fully understand several features of Ca2+ influx in breast cancer. These include figuring out the processes behind the changed expression and the best treatment plan to target breast cancer cells via particular Ca2+ channels. In the upcoming ten years, research should concentrate on the function of Ca2+ influx in mechanisms other than the migration and proliferation of breast cancer cells.

Differential cellular immune responses against Orientia tsutsugamushi Karp and Gilliam strains following acute infection in mice.

Scrub typhus is a leading cause of febrile illness in endemic countries due to infection with Orientia tsutsugamushi (Ot), a seriously understudied intracellular bacterium. Pulmonary involvement associated with vascular parasitism in patients is common and can develop into life threatening interstitial pneumonia. The diverse antigenicity of Ot genotypes and inter-strain differences in genome content are connected to varied virulence and clinical outcomes; however, detailed studies of strain-related pulmonary immune responses in human patients or small animal models of infection are lacking. In this study, we have used two clinically prevalent bacterial strains (Karp and Gilliam) to reveal cellular immune responses in inflamed lungs and potential biomarkers of disease severity. The results demonstrate that outbred CD-1 mice are highly susceptible to both Karp and Gilliam strains; however, C57BL/6 (B6) mice were susceptible to Karp, but resistant to Gilliam (with self-limiting infection), corresponding to their tissue bacterial burdens and lung pathological changes. Multicolor flow cytometric analyses of perfused B6 mouse lungs revealed robust and sustained influx and activation of innate immune cells (macrophages, neutrophils, and NK cells), followed by CD4+ and CD8+ T cells, during Karp infection, but such responses were greatly attenuated during Gilliam infection. The robust cellular responses in Karp-infected B6 mice positively correlated with significantly early and high levels of serum cytokine/chemokine protein levels (CXCL1, CCL2/3/5, and G-CSF), as well as pulmonary gene expression (Cxcl1/2, Ccl2/3/4, and Ifng). In vitro infection of B6 mouse-derived primary macrophages also revealed bacterial strain-dependent immune gene expression profiles. This study provided the lines of evidence that highlighted differential tissue cellular responses against Karp vs. Gilliam infection, offering a framework for future investigation of Ot strain-related mechanisms of disease pathogenesis vs. infection control.

Pharmacological Therapeutic Potential in Breast Cancer through Calcium Influx Pathways

Numerous cellular processes, including the release of neurotransmitters and the contraction of muscles, are largely triggered and regulated by Ca2+ inflow via Ca2+ permeable ion channels. In addition, Ca2+ influx regulates cellular migration and proliferation, two mechanisms linked to cancer. This study focuses on calcium influx in breast cancer cells and discusses how future drugs for breast cancer therapy may be pharmacological modulators of particular Ca2+ influx channels. Certain breast tumors have altered expression of particular calcium permeable ion channels. Such alterations may occasionally be connected to the prognosis and subtype of breast cancer. These days, models both in vivo and in vitro have assisted in identifying particular Ca2+ channels that are crucial for the growth and invasiveness of cancerous breast cells. Nonetheless, additional research is still needed to fully understand several features of Ca2+ influx in breast cancer. These include figuring out the processes behind the changed expression and the best treatment plan to target breast cancer cells via particular Ca2+ channels. In the upcoming ten years, research should concentrate on the function of Ca2+ influx in mechanisms other than the migration and proliferation of breast cancer cells