An In Vivo Method to Study Mouse Blood-Testis Barrier Integrity

10.3791/58512 ◽  
2018 ◽  
Author(s):  
Mengrou Liu ◽  
Chunsen Zhu ◽  
Shun Bai ◽  
Xin Li ◽  
Kaiqiang Fu ◽  
...  
Keyword(s):  
Mouse Blood ◽  
Barrier Integrity ◽  
Em Method ◽  
Blood Testis Barrier

scholarly journals An In Vivo Method for Evaluating the Gut-Blood Barrier and Liver Metabolism of Microbiota Products

10.3791/58456 ◽  
2018 ◽  
Author(s):  
Kinga Jaworska ◽  
Tomasz Huc ◽  
Marta Gawrys ◽  
Maksymilian Onyszkiewicz ◽  
Emilia Samborowska ◽  
...  
Keyword(s):  
Liver Metabolism ◽  
Em Method

scholarly journals HO-1/CO Maintains Intestinal Barrier Integrity through NF-κB/MLCK Pathway in Intestinal HO-1-/- Mice

2021 ◽  
Vol 2021 ◽  
pp. 1-19
Author(s):  
Zhenling Zhang ◽  
Lijing Zhang ◽  
Qiuping Zhang ◽  
Bojia Liu ◽  
Fang Li ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Intestinal Barrier ◽  
Heme Oxygenase 1 ◽  
Zinc Protoporphyrin ◽  
Barrier Integrity ◽  
Tnf Α ◽  
Intestinal Barrier Integrity ◽  
Deficient Mice

Background. Intestinal barrier injury is an important contributor to many diseases. We previously found that heme oxygenase-1 (HO-1) and carbon monoxide (CO) protect the intestinal barrier. This study is aimed at elucidating the molecular mechanisms of HO-1/CO in barrier loss. Materials and Methods. We induced gut leakiness by injecting carbon tetrachloride (CCl4) to wildtype or intestinal HO-1-deficient mice. In addition, we administrated tumor necrosis factor-α (TNF-α) to cells with gain- or loss-of-HO-1 function. The effects of HO-1/CO maintaining intestinal barrier integrity were investigated in vivo and in vitro. Results. Cobalt protoporphyrin and CO-releasing molecule-2 alleviated colonic mucosal injury and TNF-α levels; upregulated tight junction (TJ) expression; and inhibited epithelial IκB-α degradation and phosphorylation, NF-κB p65 phosphorylation, long MLCK expression, and MLC-2 phosphorylation after administration of CCl4. Zinc protoporphyrin completely reversed these effects. These findings were further confirmed in vitro, using Caco-2 cells with gain- or loss-of-HO-1-function after TNF-α. Pretreated with JSH-23 (NF-κB inhibitor) or ML-7 (long MLCK inhibitor), HO-1 overexpression prevented TNF-α-induced TJ disruption, while HO-1 shRNA promoted TJ damage even in the presence of JSH-23 or ML-7, thus suggesting that HO-1 dependently protected intestinal barrier via the NF-κB p65/MLCK/p-MLC-2 pathway. Intestinal HO-1-deficient mice further demonstrated the effects of HO-1 in maintaining intestinal barrier integrity and its relative mechanisms. Alleviated hepatic fibrogenesis and serum ALT levels finally confirmed the clinical significance of HO-1/CO repairing barrier loss in liver injury. Conclusion. HO-1/CO maintains intestinal barrier integrity through the NF-κB/MLCK pathway. Therefore, the intestinal HO-1/CO-NF-κB/MLCK system is a potential therapeutic target for diseases with a leaky gut.

Vasoactive intestinal peptide prevents PKCε-induced intestinal epithelial barrier disruption during EPEC infection

2015 ◽  
Vol 308 (5) ◽  
pp. G389-G402 ◽  
Author(s):  
V. Morampudi ◽  
V. S. Conlin ◽  
U. Dalwadi ◽  
X. Wu ◽  
K. C. Marshall ◽  
...  
Keyword(s):  
Vasoactive Intestinal Peptide ◽  
Critical Role ◽  
Term Treatment ◽  
Epithelial Barrier ◽  
Protective Effects ◽  
Short Term Treatment ◽  
Barrier Integrity ◽  
Barrier Disruption ◽  
Long Term Treatment

We previously showed that vasoactive intestinal peptide (VIP) protects against bacterial pathogen-induced epithelial barrier disruption and colitis, although the mechanisms remain poorly defined. The aim of the current study was to identify cellular pathways of VIP-mediated protection with use of pharmacological inhibitors during enteropathogenic Escherichia coli (EPEC) infection of Caco-2 cell monolayers and during Citrobacter rodentium-induced colitis. EPEC-induced epithelial barrier disruption involved the PKC pathway but was independent of functional cAMP, Rho, and NF-κB pathways. VIP mediated its protective effects by inhibiting EPEC-induced PKC activity and increasing expression of the junctional protein claudin-4. Short-term treatment with TPA, which is known to activate PKC, was inhibited by VIP pretreatment, while PKC degradation via long-term treatment with TPA mimicked the protective actions of VIP. Immunostaining for specific PKC isotypes showed upregulated expression of PKCθ and PKCε during EPEC infection. Treatment with specific inhibitors revealed a critical role for PKCε in EPEC-induced barrier disruption. Furthermore, activation of PKCε and loss of barrier integrity correlated with claudin-4 degradation. In contrast, inhibition of PKCε by VIP pretreatment or the PKCε inhibitor maintained membrane-bound claudin-4 levels, along with barrier function. Finally, in vivo treatment with the PKCε inhibitor protected mice from C. rodentium-induced colitis. In conclusion, EPEC infection increases intracellular PKCε levels, leading to decreased claudin-4 levels and compromising epithelial barrier integrity. VIP inhibits PKCε activation, thereby attenuating EPEC-induced barrier disruption.

TNF-α causes reversible in vivo systemic vascular barrier dysfunction via NO-dependent and -independent mechanisms

1997 ◽  
Vol 273 (6) ◽  
pp. H2565-H2574 ◽  
Author(s):  
Neil K. Worrall ◽  
Kathy Chang ◽  
Wanda S. Lejeune ◽  
Thomas P. Misko ◽  
Patrick M. Sullivan ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Lung Injury ◽  
Water Content ◽  
Selective Inhibition ◽  
No Production ◽  
Barrier Dysfunction ◽  
Barrier Integrity ◽  
Tnf Α ◽  
Severe Lung

Tumor necrosis factor (TNF-α) and nitric oxide (NO) are important vasoactive mediators of septic shock. This study used a well-characterized quantitative permeation method to examine the effect of TNF-α and NO on systemic vascular barrier function in vivo, without confounding endotoxemia, hypotension, or organ damage. Our results showed 1) TNF-α reversibly increased albumin permeation in the systemic vasculature (e.g., lung, liver, brain, etc.); 2) TNF-α did not affect hemodynamics or blood flow or cause significant tissue injury; 3) pulmonary vascular barrier dysfunction was associated with increased lung water content and impaired oxygenation; 4) TNF-α caused inducible nitric oxide synthase (iNOS) mRNA expression in the lung and increased in vivo NO production; 5) selective inhibition of iNOS with aminoguanidine prevented TNF-α-induced lung and liver vascular barrier dysfunction; 6) aminoguanidine prevented increased tissue water content in TNF-α-treated lungs and improved oxygenation; and 7) nonselective inhibition of NOS with N G-monomethly-l-arginine increased vascular permeation in control lungs and caused severe lung injury in TNF-α-treated animals. We conclude that 1) TNF-α reversibly impairs vascular barrier integrity through NO-dependent and -independent mechanisms; 2) nonselective NOS inhibition increased vascular barrier dysfunction and caused severe lung injury, whereas selective inhibition of iNOS prevented impaired endothelial barrier integrity and pulmonary dysfunction; and 3) selective inhibition of iNOS may be beneficial in treating increased vascular permeability that complicates endotoxemia and cytokine immunotherapy.

Evidence for Cl−-dependent K+ secretion by the blood–testis barrier of brook trout

1988 ◽  
Vol 66 (7) ◽  
pp. 1603-1609 ◽  
Author(s):  
William S. Marshall ◽  
Sharon E. Bryson
Keyword(s):  
Brook Trout ◽  
Transepithelial Transport ◽  
High Concentration ◽  
Sperm Duct ◽  
K Secretion ◽  
Net Flux ◽  
Mucosal Side ◽  
Blood Testis Barrier

The transepithelial transport of 86Rb+, a tracer for K+, was examined in the isolated sperm duct of brook trout (Salvelinus fontinalis). Fluxes of 86Rb+ at 1.0 mM Rb+ in voltage-clamped ducts bathed with Ringer's solution on both sides revealed net secretion of Rb+ averaging 31–58 nequiv.∙cm−2∙h−1 after stimulation by 1.0 mM dibutyryl-cAMP. Unstimulated tissues had no net Rb+ transport. The stimulated Rb+ transport was reduced 70% by bilateral replacement of Cl− with gluconate, indicating that Rb+ secretion is dependent on Cl−. Ba2+ (2.0 mM) added to the luminal (mucosal) side had no effect on Rb+ secretion rate, suggesting that apical Ba2+-sensitive K+ channels are not involved. When added on the blood side (serosal), Ba2+ stimulated Rb+ net flux by 68%, possibly as a result of blockade of basal K+ channels and increased intracellular [K+]. Injection of the antiandrogen cyproterone acetate (3 × 0.4 mg∙kg−1 over 7 days in vivo) significantly reduced stimulated Rb+ secretion (in vitro), suggesting that androgens may maintain the active transport characteristics of the blood – testis barrier. The active K+ secretion by the sperm duct accounts for the high concentration of K+ in seminal plasma which in turn is important in maintaining quiescence of developing spermatozoa.

An active immunization approach to generate protective catalytic antibodies

2001 ◽  
Vol 360 (1) ◽  
pp. 151-157 ◽  
Author(s):  
Jun WANG ◽  
Yunqing HAN ◽  
Miles F. WILKINSON
Keyword(s):  
Active Immunization ◽  
Catalytic Antibodies ◽  
Toxic Substances ◽  
Mouse Blood ◽  
Physiological Conditions ◽  
Catalytic Function ◽  
Carbamate Insecticide ◽  
Hydrolysis Of

We report that mice immunized with a phosphate immunogen produced polyclonal catalytic antibodies (PCAbs) that catalysed the hydrolysis of carbaryl, a widely used broad-spectrum carbamate insecticide that exerts toxic effects in animals and humans. The reaction catalysed by the PCAbs (IgGs) obeyed Michaelis–Menten kinetics in vitro with the following values at pH8.0 and 25°C: Km≈ 8.0μM, kcat = 4.8×10−3–5.8×10−1, kcat/knon-cat = 5.6×101–6.8×103 (where knon-cat is the rate constant of the reaction in the absence of added catalyst). The PCAbs were also active in whole sera under physiological conditions in vitro. The PCAbs induced in vivo were also active in vivo, as immunization with the phosphate immunogen decreased the mouse blood concentration of carbaryl. To our knowledge, this is the first report demonstrating that active immunization generates antibodies possessing therapeutic catalytic function in vivo. We propose that active immunization schemes that induce enzymically active antibodies may provide a highly specific therapeutic approach for degrading toxic substances.

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