bluuebunny/crossref_metadata_2025 huggingface dataset:
Pharmacological activation of the cholinergic system attenuates lung inflammation in a Murine Model of acute respiratory distress syndrome
Authors: Choque Pamela NIthzi Bricher , de Paula Vieira Rodolfo , Ulloa Luis , Lira Beatriz , de Oliveira Ana Paula Ligeiro , Consolim‐Colombo Fernanda Marciano
Published: June 2021

Acute respiratory distress syndrome (ARDS) is characterized by acute lung inflammation involving both the recruitment and activation inflammatory cells as well as the release of inflammatory factors that cause lung injury and contributes to systemic inflammation. Despite advances in understanding the underlying pathogenic mechanisms of ARDS, no effective therapeutic approach has emerged. The anti‐inflammatory cholinergic reflex has been postulated as an important mechanism that modulates the immune system in both aseptic and septic experimental models. Pyridostigmine bromide (PB) is a reversible anticholinesterase agent that causes increased availability of acetylcholine and parasympathetic activity is routinely used in the treatment of myasthenia gravis. The aim of this study was to investigate the effect of cholinergic stimulation through the PB treatment on the pulmonary and systemic inflammatory response in an experimental model of LPS‐induced ARDS.

Methods

C57Bl/6 male mice (n=24) were distributed into four groups Control (Control), pyridostigmine‐treated (PB), LPS installation (LPS), LPS installation and pyridostigmine treatment (PB+LPS) Animals were monitored for 72 hours. Acute respiratory distress syndrome (ARDS) model: Mice were anesthetized (ketamine 100 mg/kg and xylazine 10 mg/kg) and given one intratracheal instillation of 10 μg LPS/day per mice, during 3 days. Cholinergic stimulation: Pyridostigmine bromide (PB) 3 mg/kg/day was administered twice a day by oral gavage at 1h post‐LPS to achieve around 85–90% cholinergic inhibition as previously reported. After 72h, bronchoalveolar lavage fluid (BALF) was obtained to measure leukocyte infiltration and cytokine (IL‐6, IFN‐γ, IL‐1β e TNF and IL‐10) level in BALF and plasma.

Results

Treatment with PB after the bacterial endotoxin significantly reduced the number of both total cells and neutrophils in BAL fluid (p<0.001) and in the lung parenchyma (p<0.01). Reduced the levels of all of the inflammatory cytokines TNF, IL‐6 (p<0,001), and IL‐1β, IFN‐γ (p<0,01) but not IL‐10 levels in BALF when compared with LPS group. Reduced the levels of all of the inflammatory cytokines TNF (p<0,001), IL‐6, IL‐1β, IFN‐γ (p<0,01) and IL‐1β (p<0,05) but not the levels IL‐10 in plasma when compared with LPS group at 72 hours post‐treatment. In conclusion, the treatment with BP can activate the cholinergic system to attenuate lung inflammation in an experimental model of LPS‐induced ARDS. These results support the cholinergic system as an important pharmacological target for ARDS treatment.

Support or Funding Information

Support Post‐graduate stricto sensu Program UNINOVE and CAPES

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Changes in Pain Threshold in the Early Stages of Type I Diabetes
Authors: YBARBO KAI , GROTLE ANN‐KATRIN , HUO YU , STONE AUDREY
Published: June 2021

Type 1 diabetes mellitus (T1DM) is a metabolic disease in which chronic hyperglycemia increases the risk of developing peripheral neuropathy. A common early symptom of peripheral neuropathy is mechanical allodynia, which is characterized as painful sensation to a normally non‐painful stimulus. However, whether the expression of mechanical allodynia changes with the progression of the disease is not fully understood. Therefore, the purpose of this study was to determine changes in pain threshold to mechanical stimulation during the early stages of T1DM. Adult male and female Sprague‐Dawley rats were given 3–4 sessions of acclimation to cereal and the von Frey testing cage and were not deprived of food at any time. Pain threshold was measured with von Frey monofilaments, which were applied to the L5 dermatome on the plantar surface of the rat's hindpaw until the filament bent. The lowest force that elicited a quick paw withdrawal was measured and averaged between the responses of both hindpaws. We defined mechanical allodynia as a significant reduction in force required to elicit paw withdrawal compared to baseline levels. Following baseline measurements of pain threshold, non‐fasted blood glucose level, and body weight, rats were injected with either streptozotocin (STZ; 50mg/kg i.p.), a known toxin that destroys pancreatic beta cells and induces T1DM, or citrate buffer (CTL), the vehicle control for STZ. One week and two weeks post injection, body weight, blood glucose level, and pain threshold were again measured. We found that pain threshold was significantly reduced (p<0.05) 2 weeks after STZ injection (before STZ: 88 ± 10 g, n=4; after STZ: 22 ± 10 g, n=4), but not 1 week after STZ injection (before STZ: 32 ± 15 g, n=7; after STZ: 12 ± 5 g, n=7) compared to baseline. No significant difference was seen in CTL rats 1 week after injection (before CTL: 56 ± 23 g, n=6; after CTL: 48 ± 14 g, n=6) compared to baseline. Body weight was significantly reduced (p<0.05) in STZ rats at 1 week (before STZ: 426 ± 42 g, n=7; after STZ: 377 ± 31 g, n=7) and 2 weeks (before STZ: 397 ± 8 g, n=4; after STZ: 300 ± 8 g, n=4) after injection, but did not change in CTL rats (before CTL: 436 ± 29 g, n=6; after CTL: 446 ± 29 g, n=6). These findings suggest that mechanical allodynia is present as early as 2 weeks following the destruction of insulin producing beta cells.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cardiovascular Reflexes Control Hemodynamic Responses During LPS‐induced Systemic Inflammation in Conscious Rats
Authors: Amorim Mateus R. , de Deus Júnia L. , Cazuza Rafael A. , Mota Clarissa M. D. , da Silva Luiz E. V. , Borges Gabriela S. , Batalhão Marcelo E. , Cárnio Evelin C. , Branco Luiz G. S.
Published: June 2021

Lipopolysaccharide (LPS)‐induced systemic inflammation (SI) is associated with hypotension, tachycardia, and multiple organs dysfunctions. SI, a hallmark of sepsis can lead to cardiovascular impairment and death. Considering that the nervous system regulates immunity and that cardiovascular reflexes, i.e. baroreflex, chemoreflex, and Bezold‐Jarish reflex, are important regulators of hemodynamic function, we investigated these cardiovascular reflexes control acutely and 24 hours after LPS‐induced SI. We combined cardiovascular recordings, immunofluorescence and molecular approaches in male Wistar rats that received intravenous administration of LPS (1.5 or 2.5 mg.kg−1, CEUA #2017.1.585.58.9). LPS induced hypotension and tachycardia and reduction in baroreflex gain. On the other hand, SI caused no significant impairment in hemodynamic responses to chemoreflex and Bezold‐Jarish reflexes, despite an increased number of activated microglia and IL‐1β levels in the nucleus tractus solitarius. The present data are consistent with the notion that cardiovascular reflexes are kept functional even with central nervous system inflammation. These findings provide insights into the mechanisms that regulate the cardiovascular system during SI and indicate that the improvement of these reflexes responsiveness can be useful for the therapeutic management of sepsis.

Support or Funding Information

FAPESP, CAPES and CNPq

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Development of glyco‐conjugated chlorin e6 for photodynamic therapy
Authors: Yano Shigenobu , Kataoka Hiromi , Nomoto Hiroyuki , Osaki Tomohiro
Published: June 2021

PDT (photodynamic therapy) has much attracted as less invasive method for treating cancer.1 To increase the antitumor effects and shorten the light shielding duration, we developed third‐generation photosensitizers for PDT. We have focused much attention to the glyco‐conjugation of chlorin derivatives mainly for the purpose of improving their biocompatibility and tumor selectivity. Generally, cancer cells uptake higher level of glucose than normal cells in a phenomenon known as Warburg's effect, which is also utilized in positron emission tomography (19FDG‐PET‐CT). We developed a number of glyco‐conjugated chlorins resulting finally in Glc‐chlorin e6, which showed the strongest antitumor effects in PDT ever reported for synthesized glyco‐conjugated chlorins.2 Furthermore, Glc‐chlorin e6 is discharged very fast from the body. We also confirmed extremely promising antitumor effects of 40% CR in vivo under conditions that killed all mice in the comparable TS (talaporfin sodium) PDT group. We have also already synthesized 14C‐labeled Glc‐chlorin e6 and examined its pharmacokinetics in rat to reveal a plasma half‐life of 42.65 h, which is shorted than that of TS. In rat, 14C‐labeled Glc‐chlorin e6 was mainly discharged in bile and faster than TS. Single time toxicity test of Glc‐chlorin e6 in mouse also showed the lowest published lethal dose of 250 mg/kg. From these findings, we consider that Glc‐chlorin e6 is the ideal sugar for conjugating to chlorin, evidenced by the remarkable antitumor PDT effects, rapid excretion, and low toxicity demonstrated herein. Glc‐chlorin e6 is considered to be the best candidate PS thus far for third‐generation PDT and we plan to continue with our investigatory studies with the ultimate aim of clinical trials. We examined the efficacy of PDT with Glc‐chlorin e6 for tumor‐bearing dogs.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
ALDOSTERONE AND HYPERTENSION IN OVARIECTOMIZED FEMALE DAHL SALT SENSITIVE RATS ON LOW SALT DIET
Authors: LOPEZ‐RUIZ ARNALDO FABIAN , RECKELHOFF JANE F.
Published: June 2021

Blood pressure (BP) increases in postmenopausal women. Ovariectomy increases BP in female Dahl salt sensitive rats (DS) even if maintained on low salt diet. OVX also have higher aldosterone (ALDO) levels and develop more renal injury than intact females. Since ALDO can cause increases in BP and renal injury, the hypothesis was tested that the mineralcorticoid receptor antagonist, eplerenone (EPL), would reduce BP and renal injury in OVX DS. Female DS, 16 weeks of age, kept on low salt diet from birth, were divided into four groups: intact females (INT), intact females + eplerenone (100mg/kg/day by gavage; INT/EPL), ovariectomized females (OVX) and OVX + eplerenone (OVX/EPL) for 10 days. BP was measured by telemetry; renal injury was evaluated by urinary albumin excretion. Sgk‐1 and NGAL expression, indexes of renal tubular damage, were measured in kidney cortex by western blot. Plasma ALDO levels were also measured. As shown in the table below, OVX DS had higher BP and albuminuria than INT, and EPL reduced both only in OVX. Sgk‐1 and NGAL renal expression was higher in OVX than INT, and was reduced with EPL. These data show that increased aldosterone plays an important role in the higher BP and renal injury in OVX DS.

*p<0.05, compared with INT control; #p<0.05, compared with control OVX.

The data show that aldosterone plays an important role in the hypertension and renal injury in OVX DS rats. These data suggest that in postmenopausal women, aldosterone may play a role in mediating the increase in blood pressure.
Treatment of Endothelial Dysfunction in Hypertension: the Role of Enhancement of eNOS Expression
Authors: Xue Hong‐Mei , He Guo‐Wei , Wong Wing‐Tak , Tian Xiao‐Yu , Underwood Malcolm John , Huang Yu , Yang Qin
Published: June 2021

Endothelial dysfunction in hypertension is related to nitric oxide (NO)‐deficiency. We studied the efficacy of transcriptional enhancement of endothelial nitric oxide synthase (eNOS) expression by AVE3085 in spontaneously hypertensive rats (SHR) regarding the improvement of endothelial function. Isometric force study was performed with protein expression of eNOS and phosphorylated eNOS (p‐eNOS) determined in rat aortas that were treated with or without AVE3085 for 2‐hr (acute group), or from the rats fed with AVE3085 or vehicle for 4 weeks (chronic group). WKY rats were also studied under both acute and chronic treatment. In SHR, 4‐week treatment of AVE3085 markedly increased the acetylcholine (ACh)‐induced relaxation (50.1±3.4% vs. 19.7±7.0%) with the augmentation as well observed in the acute group (50.2±4.5% vs. 26.9±4.4%) (P<0.05). 4‐week AVE3085 dramatically reduced blood pressure in SHR (170.0±4.0 vs. 151.8±1.8 mmHg, P<0.001). Either removal of endothelium or L‐NAME incubation of the aorta enhanced phenylephrine‐induced contraction in AVE3085‐treated SHR (P<0.05) but not in SHR without treatment. eNOS and p‐eNOS protein expression were significantly higher in the SHR treated with AVE3085. In contrast, AVE3085 had no effect on ACh‐induced relaxation and eNOS/p‐eNOS expression in WKY rats. Enhancement of eNOS expression by AVE3085 may ameliorate endothelial dysfunction in hypertension.
Student's perception on two different teaching modalities followed in the Medical Pharmacology course
Authors: Rajasekaran Senthil Kumar , Senthilkumar Uma
Published: June 2021

The traditional format for the transmission of knowledge in the class room has been the delivery of the teaching material in a didactic lecture format. With the Medical education growing tremendously, there are numerous alternatives in order to provide opportunities for students to engage actively in the construction of knowledge. In Medical Pharmacology course, we have embarked on teaching a part of the course using the case based teaching format allowing short discussions among students in between the lectures. This alternative was expected to help the students to evoke critical thinking that is central to understanding the discipline.

This study is not an argument to support the superiority of this kind of teaching, but just to learn about the student's perception on two different teaching methodologies in the Medical Pharmacology course.

Students enrolled in the Medical Pharmacology took part in this study. This study involved the students answering a questionnaire rating specific components (on a 5 point scale) in each teaching methodology.

A total of 119 responses were received. The analysis of results is shown in the table below.

One item in the questionnaire asked about the duration of time students spend on an average understanding the topics following these two teaching methodologies. The results showed that on an average the students spent 4.5 hours and 2.5 hours following the traditional didactic teaching and the new case based teaching format respectively.

Overall results clearly show that the students' preferred case based teaching case based teaching format allowing short discussions among students in between the lectures This in their perception had:

• Reduced the time required for further studying and understanding of the topic

• Enhanced their communication skills

• Helped in integration of pharmacology and medicine

• Created an interest in learning

• Evoked parallel thinking
Exogenous Citrulline (Cit) on Renal Uptake in Various Stages of 5/6 Ablation/Infarction (A/I) Chronic Kidney Disease (CKD)
Authors: Chen Gin‐Fu , Baylis Chris
Published: June 2021

The kidney is a major site of L‐Arginine (arg) synthesis, and with renal disease arg synthesis may be compromised. The first and rate‐limiting step in renal arg synthesis is the rate of cit delivery to the renal cortex. Using the 5/6 renal ablation/infarction (A/I) injury model, we measured renal cit delivery and extraction at 2 stages of disease progression (4‐5 wk; n=7 and 10‐12 wk; n=6) after A/I vs. sham (n=7). The arterial‐renal venous difference was measured at baseline and during cit infusion. Both stages of injury resulted in decreased baseline cit delivery to the kidney due to marked reductions in renal plasma flow (RPF) and despite increased plasma cit. The % cit extraction at baseline was unaffected by CKD, hence cit uptake fell markedly with injury. Cit infusion increased RPF only in healthy rats (possibly through increased NO production). Plasma cit increased in all groups resulting in increased renal cit delivery vs baseline. In healthy kidneys cit extraction and uptake increased. However, despite increased delivery neither renal cit extraction nor uptake was increased at either stage of injury. The inability of the injured kidney to take up cit indicates a marked reduction and saturation of the renal cit transport capacity.

*p<0.05, *p<0.01, ***p<0.001 vs. Sham, +p<0.05, ++p<0.01, +++p<0.001 vs. baseline within group.
Alterations in miRNA‐mediated regulation of thrombospin‐2 in developing placenta of the BPH/5 mouse model of pre‐eclampsia (PE)
Authors: Zhou Yi , Guruju Mallikarjuna R , Woods Ashley K , Sharma Ram V , Davisson Robin L
Published: June 2021

We have recently shown severe fetoplacental abnormalities in the BPH/5 model of PE. Given the importance of miRNAs in regulating gene expression during embryonic development, we hypothesized that changes in miRNA expression in developing placenta of BPH/5 may be involved in fetoplacental defects observed in this model. We employed miRNA microarrays to analyze whole genome miRNA expression in feto‐placental units from BPH/5 (e10.5) over three classes of phenotype severity. Ninety‐four miRNAs were differentially expressed in BPH/5 feto‐placental units compared to C57 controls (fold change >1.5, p<0.05, n=3). miR‐467b and miR‐136 were down‐regulated and validated by real time RT‐PCR (Table 1). miRNA target prediction identified thrombospdin‐2 (Thbs‐2) as a target gene of miR‐467b and miR‐136. Western analysis showed a significant increase Thbs‐2 levels in all three classes of BPH/5 fetoplacental units vs C57 (Table 1). These results suggest that increased expression of Thbs‐2 in BPH/5 placentas due to decreased miRNA‐mediated post‐transcriptional repression may be inhibiting angiogenesis, ultimately leading to fetoplacental abnormalities observed in this model of PE.

Table 1. miR‐467b and miR‐136 miRNA and Thrombospodin‐2 protein expression in different classes of BPH/5 feto‐placental unit compared to C57

*<0.05 vs. C57
The Utility Value of Olfactory Ensheathing Cells (OEC) and Olfactory Nerve Fibroblasts (ONF) in Treating Spinal Cord Injury
Authors: Kanakasabapathy Indirani , Tharion George
Published: June 2021

Introduction

Currently many stem cell sources are being tried to treat patients with spinal cord injury. However, the utility value of these cells is under debate. Here, we report the outcome of our study in treating spinal cord injury using olfactory ensheathing cells (OEC) and olfactory nerve fibroblasts (ONF) transplantation in adult albino rat.

Method

Olfactory ensheathing cells and olfactory nerve fibroblasts were isolated from rat olfactory mucosa and cultured under appropriate culture conditions. Four weeks after crush injury of spinal cord, OEC and ONF were injected into the lesion site. The outcome, in terms of limb movements was assessed using BBB scale and EMG study. The animal was sacrificed and the cord segments of interest were processed for histological examination.

Result

Nine months after the transplantation, the functional outcome was scored as '17' on BBB scale and the EMG study revealed generation of motor impulses in muscles of paralyzed limbs. Histological examination showed newly formed neural tissue and reduction of post‐traumatic degenerative changes.

Conclusion

Our finding confirms that OEC and ONF have the potential to improve neurologic disfunction and supports the utility of these cells in cell therapy treatment for CNS lesion.
Intragastric infusion of arginine stimulated greater liver protein synthesis compared to IV infusion in TPN‐fed Yucatan miniature piglets
Authors: Dinesh Chandani , Dodge M Elaine , Baldwin Mark P , Bertolo Robert F , Brunton Janet A
Published: June 2021

Total parenteral nutrition (TPN) induced gut atrophy reduces de novo synthesis of intestinal arginine (Arg) in neonates which may limit Arg availability. Our objective was to assess the effect of route of Arg intake on tissue protein synthesis in TPN‐fed piglets with gut atrophy. Piglets (14‐17 d) were fed TPN (1.0 g Arg/kg/d) until study d 4, then switched to Arg‐free TPN and randomized to receive a continuous intragastric infusion (2 mL/kg/hr) of either IG Low Arg (0.6 g/kg/d) or IG High Arg (1.6 g/kg/d). A 3rd group was switched to high Arg TPN (1.6 g/kg/d) (IV High Arg). A group of sow‐fed (SF) littermates was also included. On d 7, piglets received an IV flooding dose of 3H‐phenylalanine to measure protein synthesis (Ks, %/d). Liver Ks was greater in IG High Arg compared to IV High Arg. Mucosal Ks was highest in SF animals, with no differences in the TPN‐fed groups. Table values are mean (SD), N=3‐6 per tissue and group. Different superscripts by row, p<0.05 (ANOVA)

Provision of high dietary Arg alone into the gut did not appear to enhance mucosal protein synthesis. Muscle protein synthesis was also not different, but was highly variable. IG delivery of Arg promoted liver protein synthesis compared to IV feeding; the mechanism is likely not related to Arg availability as there was no difference in liver free Arg. (CIHR)
FEMALE REPRODUCTIVE HORMONE EFFECTS ON SKIN VESSEL ADRENERGIC RESPONSIVENESS IN WOMEN WITH LOW AND HIGH ORTHOSTATIC TOLERANCE.
Authors: Stachenfeld Nina S. , Hinds Kumba A. , Taylor Hugh S.
Published: June 2021

We tested the hypothesis that estradiol decreases skin vessel adrenergic responsiveness in women with low (LT) vs. high (HT) orthostatic tolerance, defined by Cumulative Stress Index (CSI) during lower body negative pressure. Laser Doppler flowmetry measured skin blood flow (SkBF) during graded norepinephrine infusions via microdialysis. We suppressed estrogen and progesterone with a gonadotropin releasing hormone antagonist in LT (n=4, 23±3y, CSI=‐410±42) and HT (n=4, 23±3 y, CSI=‐697±18, P<0.05) for 16 days, added estradiol (0.2 mg/day patch) for days 4‐16 and progesterone (200 mg/day) for days 13‐16. In HT the log[NE]‐SkBF stimulus‐response curve shifted right (lower responsiveness) with E (EC50, ‐2.7±0.9) vs. GnRH (‐3.6±0.4) and EP (‐5.2±0.6). In LT, hormones did not induce a NE‐SkBF shift (EC50, ‐5.1±0.9, ‐4.7±1.5, ‐4.7±0.7, GnRH, E, EP respectively). In HT, L‐NMMA restored responsiveness with GnRH (‐5.7±0.4) and E (‐4.5±1.4) with no effect in EP (‐5.5±1.0). In LT, L‐NMMA increased responsiveness in GnRH (‐5.7±0.7) and E (‐5.9±0.9) but not EP (‐3.8±1.3). Estradiol lowers adrenergic responsiveness via a nitric oxide mechanism in women with HT; progesterone may counter this effect. HL071159
Dilutional fluorescent method to measure glomerular albumin reflection coefficient (dσAlb) in vitro
Authors: Schreck Carlos Manuel Nicolas , Williams Jan Michael , Sharma Mukut , Roman Richard J
Published: June 2021

This study describes a new high throughput method (7‐15 glomeruli/experiment, 3 experiments/hour) to measure dσAlb. Adult SD rats received FITC‐Dextran 250 ( 75 mg/Kg. iv) and 5 minutes later the kidneys were harvested and glomeruli isolated using a sieving technique in 6% BSA. They were studied in a small volume (50 μl) fast exchange chamber on an inverted fluorescent microscope equipped with InCytIm 1 imaging system using a 10X (0.3 NA) objective. Changes in the fluorescent signal in the glomerular capillaries due to water movement in response to an imposed oncotic gradient were used to determine dóAlb. Changing the bath from 6 to 5, 4, 3, 2 % albumin produced a linear 12, 26, 37 and 52% fall in the concentration of FITC‐Dextran250 in the glomerulus ( r2=0.95). Administration of protamine sulfate ( 2 mg/ml) or TGF‐β1 (10 ng/ml) to increase the glomerular permeabilty to albumin, decreased dσAlb from 1 to 0.6 and from 1 to 0.67 , respectively. Fluorescence increased 10 % in glomeruli treated with protamine and exposed to an isotonic solution of Dextran 250 but not in control glomeruli. These results validate this high throughput method to measure changes in dσAlb in response to drug administration and between normal animals and those with proteinuria and renal disease.

NIH grants: HL 36279 and HL 29587
Tranilast protects from renal injury in streptozotocin‐induced diabetic Dahl salt sensitive rats
Authors: Hosoya Takuma , Mori Takefumi , Ohsaki Yusuke , Yoneki Yoshimi , Nakamichi Takashi , Nagasawa Tasuku , Sato Hiroshi , Ito Sadayoshi
Published: June 2021

The present study was designed to investigate the role of tranilast (TL), which inhibits TGF‐beta, in the renal injury and oxidative stress of streptozotocin (STZ)‐induced diabetic Dahl salt sensitive (Dahl S) rats and compared to the insulin (INS) treatment.

Dahl S rats were administered with STZ (65mg/kg)(STZ), STZ with TL (400mg/kg/day) (STZ+TL), STZ with INS (10U/kg/day) (STZ+INS), or STZ with INS and TL (STZ+INS+TL). Urinary albumin and TBARS excretion was analyzed as a marker of renal injury and oxidative stress, respectively.

Blood glucose level of STZ and STZ+TL groups (572.83±37.3, 639.71±27.5 mg/dl) were significantly higher than those of STZ+INS and STZ+INS+TL groups (330.83±61.39, 221.14±83.41 mg/dl). Systolic blood pressure was increased in all groups, but did not have difference between groups. Urinary albumin excretion was significantly decreased in STZ+TL group (43.6±6.3 mg/day) and STZ+INS group (41.7±7.1 mg/day) compared to STZ group (76.5±6.9 mg/day), and further decrease in STZ+INS+TL group (24.6±3.1 mg/day). Reduction in urinary TBARS excretion was observed only in STZ+INS group compared to STZ group.

These results indicate that tranilast protects from renal injury independent of blood pressure, blood glucose level and oxidative stress in diabetic Dahl S rats.
Synbiotic modulation of cytokine secretion in ulcerative colitis colonic biopsies
Authors: Urdaneta Elena , Alfaro Maria , Jauregui Patricia , Zozaya Jose Manuel , Arin Antxon , Santidrian Santiago , Marzo Florencio
Published: June 2021

Ulcerative colitis (UC) consists of an aberrant local immune response to components of the bacterial microflora. There is also evidence that mucosal immune cells actively interact with non‐immune cells to promote tissue damage, and that cytokines are essential mediators of this cross‐talk. Functional foods, with beneficial effects for human health, such as a synbiotic, composed of Lactobacillus casei (Lc) and Bifidobacterium bifidum (Bb) (108 cfu/mL), and sphingomyelin (0.05%), could modulate this inflammatory disease.

The aim of this study was to investigate pro and anti‐inflammatory cytokine production in colonic biopsies from UC patients and healthy volunteers examining this synbiotic effect.

Patients were asked to give written consent to take part in this investigation. These studies were approved by the Hospital de Navarra Ethics Committee. Production of proinflammatory Th1 cytokines (TNF‐alpha, IFN‐gamma, IL‐1B, IL‐6 and IL‐8) and anti‐inflammatory Th2 cytokines (IL‐10 and IL‐13)) was analyzed in mucosal biopsies of patients with UC (n=10) and control subjects (n=10) by flow cytometry using Cell Quest software (Becton Dickinson).

The incubation of synbiotic reduced significantly (P< 0.01) the secretion of IL‐6, IL‐1beta and IL‐8 in biopsies of UC patients. The anti‐inflammatory Th2 cytokine levels were not affected either by UC disease or by synbiotic treatment.

These findings suggest a new immunomodulatory function of this symbiotic compound and further suggests that these bacteria may be useful in the treatment of UC.

This work has been funded by Ortiz de Landazuri of Navarra Government and Ministry of Education & Science (AGL2006‐10296).
Pathogenic and commensal Escherichia coli decrease ion secretion in human intestinal epithelial cells
Authors: Ohland Christina , MacNaughton Wallace K
Published: June 2021

E. coli enteric bacteria consist of commensal and pathogenic strains, including enteropathogenic (EPEC) and enterohaemorrhagic (EHEC) E. coli. The genetically similar mouse pathogen Citrobacter rodentium decreases ion secretion in vivo. Our aim was to investigate how E. coli strains affect secretion in vitro.

Exposure of T84 cell monolayers to EPEC or EHEC for 4 hr significantly decreased the response to the cAMP‐dependent secretagogue, forskolin, without altering Ca2+‐dependent secretion as measured in Ussing chambers. EPEC mutants deficient in flagellin, bundle forming pili, Type III secretion, Tir or intimin invoked similar hyposecretory responses, indicating that these factors are not involved. Pre‐treatment with the apoptosis inhibitor Z‐VAD did not alter this effect. Lab strain (HB101) and human commensal (C1, C28/2, F18) E. coli decreased secretion to a similar extent. Heat‐killed or paraformaldehyde‐fixed EPEC, sterile culture medium or E. coli lipopolysaccharides did not cause hyposecretion. Adenylate cyclase activity is not altered by EPEC, as a cAMP assay showed no change in baseline or stimulated levels. EPEC does not alter protein levels of CFTR or NKCC1 as shown by Western blot.

We conclude that pathogenic and commensal E. coli decrease cAMP‐dependent ion secretion in vitro by an unknown bacterial factor, downstream of cAMP production. Funded by III Summit Foundation & CCFC.
Gastro‐protective Properties of Cobiprostone against Acid‐induced Mucosal Injury in Porcine Gastric Mucosa
Authors: NIGHOT MEGHALI PRASHANT , ROERIG BIRGIT , UENO RYUJI , MOESER ADAM J , BLIKSLAGER ANTHONY
Published: June 2021

Activation of ClC‐2 Cl− channels is an important mechanism of intestinal mucosal barrier repair. Lubiprostone, a prostone known to activate epithelial ClC‐2 Cl− channels, has been shown to hasten repair of ischemic‐injured intestinal mucosa. The objective of the present study was to evaluate the gastro protective properties of the novel prostone, cobiprostone, in a porcine model of gastric acid injury. We hypothesized that cobiprostone would ameliorate acid injury associated with maintenance of tight junctions and activation of chloride channels. Porcine gastric mucosa was mounted in Ussing chambers and injured by bathing mucosal tissues in an HCl Ringer's solution (pH=1.5). Transepithelial resistance and mucosal‐to‐serosal 3H‐mannitol flux was measured over a 90 minute period. Compared with control tissues, acid exposure decreased TER and increased 3H‐mannitol flux. Pretreatment of gastric mucosa with cobiprostone was protective against acid‐induced decreases in TER and abolished increases in flux .Histological damage were attenuated in cobiprostone treated gastric mucosa exposed to acid. Immunofluorescence and western analysis for occludin revealed enhanced localization of to the tight junction after treatment. Pretreatment with the ClC‐2 inhibitor ZnCl2, and the CFTR inhibitor DASU attenuated cobiprostone mediated mucosal protection, indicating a critical role for Cl− channels.

The Research is funded by Sucampo Pharmaceuticals, Inc. Bethesda, MD
Aquaporin‐9 is Downregulated in the Liver of Rat with Extrahepatic Cholestasis
Authors: Calamita Giuseppe , Ferri Domenico , Gena Patrizia , Carreras Flavia I. , Liquori Giuseppa E. , Portincasa Piero , Marinelli Raul A. , Svelto Maria
Published: June 2021

Hepatocytes express aquaporin‐9 (AQP9), a basolateral channel permeable to water, glycerol and other neutral solutes. While liver AQP9 is known for mediating the uptake of glycerol its relevance in bile secretion remains elusive. Here, we evaluated whether defective expression of AQP9 is associated to secretory dysfunction of rat hepatocytes following bile duct ligation (BDL). By immunoblotting, 1‐day BDL resulted in a slight decrease of AQP9 protein in basolateral membranes and a simultaneous increase of AQP9 in intracellular membranes. This pattern was steadily accentuated in the subsequent days of BDL as at 7‐days BDL basolateral membrane AQP9 decreased by 85% whereas intracellular AQP9 increased by 115%. However, the total amount of AQP9 in the livers from day 7 of BDL rats was reduced by 49% compared to the sham counterpart. Results were confirmed by immunocytochemistry and consistent with biophysical studies showing considerable decrease of the basolateral membrane water and glycerol permeabilities of cholestatic hepatocytes. The AQP9 mRNA was slightly reduced only at day 7 of BDL indicating that the dysregulation was mainly occurring at a posttranslational level. The altered expression of liver AQP9 during BDL was not dependent on insulin, a hormone known to downregulate AQP9, since insulinemia was unchanged in 7‐days BDL rats. These results suggest that extrahepatic cholestasis leads to downregulation of liver AQP9 and dysregulated AQP channels contribute to bile flow dysfunction of cholestatic hepatocyte.
TRPC6 knock‐down specifically attenuates diacylglycerol‐mediated elevation of intracellular calcium in human myometrial cells
Authors: Chung Daesuk , Kim Yoon‐Sun , Phillips Jennifer N , Ulloa Aida E , Galan Henry L , Sanborn Barbara M
Published: June 2021

Human myometrium exhibits receptor‐, store‐ and diacylglycerol (OAG)‐mediated extracellular Ca2+‐dependent increases in intracellular Ca2+ ([Ca2+]i)(SRCE) and expresses TRPC mRNAs, predominantly TRPC1, 4, and 6, that have been implicated in SRCE. To determine the role of TRPC6 in myometrial SRCE, short hairpin RNA constructs that effectively targeted a TRPC6 mRNA reporter for degradation were designed and one sequence was used to produce an adenovirus construct (TC6sh1). TC6sh1 reduced TRPC6 mRNA but not TRPC1, 3, 4, 5 or 7 mRNAs in PHM1‐41 myometrial cells. Compared to uninfected cells or cells infected with empty vector, the increase in [Ca2+]i in response to OAG was specifically inhibited by TC6sh1; SRCE responses elicited by either oxytocin or thapsigargin were not changed. Similar findings were observed in primary pregnant human myometrial cells. When PHM1‐41 cells were activated by OAG in the absence of extracellular Na+, the increase in [Ca2+]i was partially reduced. Furthermore, pretreatment with nifedipine, an L‐type calcium channel blocker, also reduced the OAG‐induced [Ca2+]i increase. These findings suggest that OAG targets channels containing TRPC6 in myometrial cells and that these channels act, in part, in an indirect manner via enhanced Na+ entry and activation of voltage‐dependent Ca2+ entry to promote elevation of intracellular Ca2+. Supported by March of Dimes #6‐FY05‐77 and HD38970.
Acute TiO2 exposure does not alter endothelial function in isolated rat lung
Authors: Westphal Joslyn , Naik Jay
Published: June 2021

Nanotechnology, the manipulation and manufacturing of particles less than 100 nm in size, is at the forefront of scientific research. However, there is evidence that the size‐dependent properties of nanomaterials may increase their toxicity. Studies have shown that short‐term (24 hr) exposure to nanomaterials, including titanium dioxide (TiO2), is associated with increased reactive oxygen species (ROS) production. An increase in ROS production within the vasculature could potentially elicit a decrease in NO bioavailability. Previous work has suggested that this increase in ROS production may involve cellular internalization. However, we hypothesized that nanomaterials interact with the endothelial cell surface resulting in an impaired endothelial‐dependent vasodilation. To this end, experiments were performed using isolated, saline perfused Wistar rat lung. The pulmonary vasculature was exposed to TiO2 (0.05% V/V or 0.1% V/V) for 1 hr. Endothelial function was assessed using graded doses of an endothelial‐dependent vasodilator (ionomycin, 0.03 ‐ 1.0 μM), administered luminally. Ionomycin induced a dose‐dependent vasodilation. There were no statistically significant differences in the vasodilatory response to ionomycin between vehicle and TiO2 treated lungs. These results suggest that acute TiO2 exposure does not alter endothelial‐dependent vasodilation in the rat pulmonary vasculature.
Erythropoietin expression and myoblasts survival
Authors: jia yi , Gassmann Max , Noguchi Constance Tom
Published: June 2021

Erythropoietin (Epo) is mainly produced in the kidney and regulates erythroid differentiation. We previously reported that myoblasts express Epo receptor (EpoR) and Epo increases their proliferation. Myoblast isolated from transgenic mice (Tg6) overexpressing a human Epo transgene showed increased proliferation and reduced apoptosis at 5% O2 compared with WT myoblasts, due partly to Epo transgene expression in myoblasts. Epo treatment further increased proliferation and protection from apoptosis. Murine Epo (mEpo) mRNA expression was also higher in Tg6 myoblasts, suggesting that Epo may further induce Epo expression in myoblasts. Epo treatment increased mEpo mRNA in both Tg6 and WT myoblasts to a similar level. Reduced O2 further increased mEpo mRNA but not greater than Epo treatment alone, suggesting that mEpo was maximally expressed with Epo treatment. Although EpoR expression decreased with myoblast differentiation, mEpo mRNA remained unchanged. Similarly, mEpo mRNA expressed in the gastrocnemius muscles in vivo was higher level in Tg6 mice and mEpo mRNA in muscle increased after mice muscle injury in both Tg6 and WT mice. These results suggest that Epo signaling from endogenous Epo production in muscle from myoblasts and/or mature muscle fibers can promote myoblasts proliferation and survival and that Epo production in muscle can be further induced by exogenous Epo.
Cyclooxygenase 2 inhibition exacerbates AQP2 and pAQP2 downregulation independently of V2 receptor abundance in the postobstructive kidney
Authors: Jensen Anja M , Norregaard Rikke , Bae Eun Hui , Wang Guixian , Nielsen Søren , Kim Soo Wan , Frøkiær Jørgen
Published: June 2021

The type 2 vasopressin receptor (V2R) and vasopressin‐regulated transport proteins is downregulated in postobstructive kidney. ANG II receptor (AT1R) blockade attenuates V2R, AQP2 and pAQP2 downregulation and partially reverses obstruction‐induced inhibition of cAMP generation. Furthermore, AT1R blockade attenuates cyclooxygenase 2 (COX2) induction and as COX2 inhibition attenuates AQP2 downregulation 3 days after release of bilateral ureteral obstruction (BUO) we speculated if the effects of AT1R blockade on V2R and the vasopressin‐regulated pathway are due to reduced prostaglandin E2 generation.

Rats were subjected to 24h BUO followed by 48h release and treated with parecoxib, a specific COX2 inhibitor, or saline. Control rats were sham‐operated. Urine output, water intake, and solution free water clearance were comparable in parecoxib‐ and saline‐treated rats. Immunoblotting, revealed a significant decrease in AQP2 and pAQP2 abundance to 20% and 23% of sham levels in BUO+P rats compared to 40% and 55% of sham levels in saline‐treated BUO rats. V2R abundance decreased to less than 10% of sham in both BUO and BUO+P rats. Finally, vasopressin‐stimulated cAMP generation in inner medullary membrane fractions from BUO+P rats tended to be reduced compared to BUO rats.

In conclusion, COX2 inhibition exacerbates AQP2 and pAQP2 downregulation 48h after release of 24 h BUO independently of V2R abundance
The Effects of the Herbal Enzyme Bromelain Against Breast Cancer Cell Line GI101A
Authors: Paroulek Alexandra F , Jaffe Mark , Rathinavelu Appu
Published: June 2021

Bromelain is a proteinase derived from the stem of pineapple and has been studied for its anti‐inflammatory, antithrombotic, and antimetastatic properties. Bromelain has also been known to significantly reduce local tumor growth and to raise the impaired cytotoxicity of monocytes in the immune system against tumor cells. The goal of this project is to advance the mechanistic knowledge of herbal remedies and to confirm the already known antimetastatic properties of Bromelain. The MTS assay method was used 24 hours after Bromelain treatment to detect the cell death. The data show that after 1 μM of Bromelain treatment, the population of GI101A cells is significantly reduced by up to 70%. Using the M30 Apoptosense assay, levels of the protein cytokeratin 18 (CK18) were measured to detect any apoptotic activity. After 10 μL of Bromelain treatment, CK18 levels increased and a large number of apoptotic cell bodies were observed. The antitumor effects of Bromelain are mainly involved in cancer cell division by the induction of apoptosis.
Oral Tolerance Reduces Eosinophil and Neutrophil Activation in a Cockroach Antigen Murine Model of Allergic Asthma.
Authors: Vaickus Louis J , Remick Daniel
Published: June 2021

Our lab previously demonstrated that German cockroach allergens (CRA) induce allergic‐type airways inflammation in outbred (ICR:CD‐1) mice which closely resembles human allergic asthma. We sought to determine the efficacy of oral tolerization in alleviating asthmatic symptoms. Mice were given 8ug/day CRA by oral gavage for 4 days beginning with day ‐7, followed by intratracheal challenges with 4.75ug on day 0 and 2.4ug on days 14 and 21. Cytokines and cellular content were measured in the bronchoalveolar lavage fluid and cytokines and myeloperoxidase activity were measured in lung homogenate. Samples were collected 4 hours post final challenge and the results compared to PBS fed, CRA challenged mice. In the BAL, Eo2 and TNF were significantly reduced in tolerized mice while KC, IL‐17 and absolute Eos trended towards reduced in tolerized mice. In the lung homogenate, Eo1 and KC were significantly reduced in tolerized mice, while TNF, IL‐17 and myeloperoxidase activity trended towards reduced in tolerized mice. These results indicate that oral tolerance partially abrogates eosinophil infiltration into the lung space, as well as a decrease in parenchymal neutrophil infiltration/activation. These beneficial effects are probably mediated through a reduction in the levels of prominent cytokines associated with asthma induction and progression.

Table 1: Cytokines and Cells in BAL measured in pg/mL. Cytokines and peroxidase activity in LH measured in pg/mL and absorbance versus blank resp. NM = not measured.
But Where Are You Really From?
Authors: Lano Anne
Published: June 2021

Updated several times a week with posts by a wide variety of authors, AJN's blog Off the Charts allows us to provide more timely—and often more personal—perspectives on professional, policy, and clinical issues. Best of the Blog is a regular column to draw the attention of AJN readers to posts we think deserve a wider audience. To read more, please visit: www.ajnoffthecharts.com.
Renal cell carcinoma, part 1
Authors: Pullen Richard L.
Published: June 2021

Renal cell carcinoma (RCC) encompasses a group of malignancies that originate in the epithelium of the renal cortex, most often in the upper pole of the kidney. This article, the first of a three-part series on RCC, addresses the incidence and epidemiology of RCC along with its genetic alterations, risk factors, histologic characteristics, and imaging characteristics.
Niclosamide inhibits SARS-CoV2 entry by blocking internalization through pH-dependent CLIC/GEEC endocytic pathway
Authors: Prabhakara Chaitra , Godbole Rashmi , Sil Parijat , Jahnavi Sowmya , van Zanten Thomas S , Sheth Dhruv , Subhash Neeraja , Chandra Anchal , Nuthakki Vijay Kumar , Puthiyapurayil Theja Parassini , Ahmed Riyaz , Najar Ashaq Hussain , Lingamallu Sai Manoz , Das Snigdhadev , Mahajan Bhagyashri , Vemula Praveen , Bharate Sandip B , Singh Parvinder Pal , Vishwakarma Ram , Guha Arjun , Sundaramurthy Varadharajan , Mayor Satyajit
Published: June 2021

Many viruses utilize the host endo-lysosomal network to infect cells. Tracing the endocytic itinerary of SARS-CoV2 can provide insights into viral trafficking and aid in designing new therapeutic targets. Here, we demonstrate that the receptor binding domain (RBD) of SARS-CoV2 is internalized via the clathrin and dynamin-independent, pH-dependent CLIC/GEEC (CG) endocytic pathway. Endosomal acidification inhibitors like BafilomycinA1 and NH4Cl, which inhibit the CG pathway, strongly block the uptake of RBD. Using transduction assays with SARS-CoV2 Spike-pseudovirus, we confirmed that these acidification inhibitors also impede viral infection. By contrast, Chloroquine neither affects RBD uptake nor extensively alters the endosomal pH, yet attenuates Spike-pseudovirus entry, indicating a pH-independent mechanism of intervention. We screened a subset of FDA-approved acidification inhibitors and found Niclosamide to be a potential SARS-CoV2 entry inhibitor. Niclosamide, thus, could provide broader applicability in subverting infection of similar category viruses entering host cells via this pH-dependent endocytic pathway.
Histone H3 lysine 27 acetylation profile undergoes two global shifts in undernourished children and suggests one-carbon metabolite insufficiency
Authors: Kupkova Kristyna , Shetty Savera J. , Haque Rashidul , Petri William A. , Auble David T.
Published: June 2021

Background

Stunting is a condition in which a child does not reach their full growth potential due to chronic undernutrition. It arises during the first two years of a child’s life and is associated with developmental deficiencies and life-long health problems. Current interventions provide some benefit, but new approaches to prevention and treatment grounded in a molecular understanding of stunting are needed. Epigenetic analyses are critical as they can provide insight into how signals from a poor environment lead to changes in cell function.

Results

Here we profiled histone H3 acetylation on lysine 27 (H3K27ac) in peripheral blood mononuclear cells (PBMCs) of 18-week-old and one-year-old children living in an urban slum in Dhaka, Bangladesh. We show that 18-week-old children destined to become stunted have elevated levels of H3K27ac overall, functional analysis of which indicates activation of the immune system and stress response pathways as a primary response to a poor environment with high pathogen load. Conversely, overt stunting at 1-year-of age is associated with globally reduced H3K27ac that is indicative of metabolic rewiring and downregulation of the immune system and DNA repair pathways that are likely secondary responses to chronic exposure to a poor environment with limited nutrients. The results from one-year-old children also point toward deficiency in one-carbon metabolism, which is further supported by integrative analysis with results from histone H3 trimethylation on lysine 4 (H3K4me3).

Conclusions

The epigenomes of stunted children undergo two global changes in H3K27ac within their first year of life, which are associated with probable initial hyperactive immune responses followed by reduced metabolic capacity. Limitation of one-carbon metabolites may play a key role in the development of stunting. Trial registration:ClinicalTrials.govNCT01375647. Registered 17 June 2011, retrospectively registered.