Animal Studies Research Articles

Human umbilical cord mesenchymal stem cell-based gene therapy for hemophilia B using scAAV-DJ/8-LP1-hFIXco transduction

BackgroundHemophilia B is an X-linked bleeding disorder caused by a mutation in the gene responsible for encoding coagulation factor IX (FIX). Gene therapy offers promising potential for curing this disease. However, the current method of relatively high dosage of virus injection carries inherent risks. The purpose of this study was to introduce a novel scAAV-DJ/8-LP1-hFIXco vector transduced human umbilical cord blood derived mesenchymal stem cells (HUCMSCs) as an alternative cell-based gene therapy to conventional gene therapy for Hemophilia B.MethodsThe LP1-hFIXco gene structure was designed by us through searching the literature from NCBI and the scAAV-DJ/8-LP1-hFIXco vector was constructed by a commercial company. The HUCMSCs were cultivated in routine approach and transduced with scAAV-DJ/8-LP1-hFIXco vector. The human FIX activation system was employed for detection of hFIXco activity. The RNA and protein expression levels of the hFIXco were evaluated using PCR and western blot techniques. In animal studies, both NSG and F9-KO mice were used for the experiment, in which clotting time was utilized as a parameter for bleeding assessment. The immunohistochemical analysis was used to assess the distribution of HUCMSCs in mouse tissue sections. The safety for tumorigenicity of this cell-based gene therapy was evaluated by pathological observation after hematoxylin-eosin staining.ResultsThe transduction of HUCMSCs with the scAAV-DJ/8-LP1-hFIXco vector results in consistent and sustainable secretion of human FIXco during 5 months period both in vitro and in mouse model. The secretion level (hFIXco activity: 97.1 ± 2.3% at day 7 to 48.8 ± 4.5% at 5 months) was comparable to that observed following intravenous injection with a high dose of the viral vector (hFIXco activity: 95.2 ± 2.2% to 40.8 ± 4.3%). After a 5-month observation period, no clonal expansions of the transduced cells in tissues were observed in any of the mice studied.ConclusionsWe have discovered a novel and safer HUCMSCs mediated approach potentially effective for gene therapy in hemophilia B.

Medical School Regulatory Compliance Burdens for the Physician-Scientist.

Medical school research faculty is increasingly required to complete more comprehensive and time consuming compliance steps for regulatory oversight. These relate to animal studies, information technology, biosafety, and human resources. For physician-scientists, the additional role in clinical care adds to these research areas with regulatory compliance in patient care and ever-growing web trainings. The sum of all these compliance regimes is a considerable time and cost burden, diminished research performance, and disengagement of faculty from colleagues, collaborations, and institutions. Many research and clinical compliance processes were put in place, often using legacy systems, in well-meaning attempts to address straightforward regulations in humane animal care, safe use of biological agents, and medical care delivery. However, their accumulation and negative impact on faculty performance demand time, energy, and resources that impact academic productivity. There are solutions to a relentlessly increasing regulatory load for research faculty, which involve vertical integration, convergence, and performance assessment in medical school and health system compliance regimes. ANN NEUROL 2024.

A meta-analysis of animal studies evaluating the effect of hydrogen sulfide on ischemic stroke: is the preclinical evidence sufficient to move forward?

Hydrogen sulfide (H2S) is a gasotransmitter that has been studied for its potential therapeutic effects, including its role in the pathophysiology and treatment of stroke. This systematic review and meta-analysis aimed to determine the sufficiency of overall preclinical evidence to guide the initiation of clinical stroke trials with H2S and provide tailored recommendations for their design. PubMed, Web of Science, Scopus, EMBASE, and MEDLINE were searched for studies evaluating the effect of any H2S donor on in vivo animal models of regional ischemic stroke, and 34 publications were identified. Pooling of the effect sizes using the random-effect model revealed that H2S decreased the infarct area by 34.5% (95% confidence interval (CI) 28.2-40.8%, p < 0.0001), with substantial variability among the studies (I2 = 89.8%). H2S also caused a 37.9% reduction in the neurological deficit score (95% CI 29.0-46.8%, p < 0.0001, I2 = 63.8%) and in the brain water content (3.2%, 95% CI 1.4-4.9%, p = 0.0014, I2 = 94.6%). Overall, the studies had a high risk of bias and low quality of evidence (median quality score 5/15, interquartile range 4-9). The majority of the included studies had a "high" or "unclear" risk of bias, and none of the studies overall had a "low" risk. In conclusion, H2S significantly improves structural and functional outcomes in in vivo animal models of ischemic stroke. However, the level of evidence from preclinical studies is not sufficient to proceed to clinical trials due to the low external validity, high risk of bias, and variable design of existing animal studies.

Effect of Red Blood Cell Transfusion on Central Venous-to-Arterial Carbon Dioxide Difference in Anemic Surgical Patients – A Pilot Study

Background: Biochemical markers for monitoring adequacy of cardiac output and tissue perfusion such as blood lactate and central venous oxygen saturation (ScvO2) are meanwhile well established in clinical routine. In addition, in recent years, the central venous-to-arterial carbon dioxide difference (dCO2) has been evaluated as a further marker, and studies meanwhile have demonstrated the validity of an increased dCO2 to identify capillary perfusion mismatches. However, results from animal studies suggest that dCO2 may be influenced by altered hemoglobin values during severe bleeding. It was the aim of our study to evaluate if dCO2 changes upon Red Blood Cell (RBC) transfusion in humans. Methods: Patients of the ongoing LIBERAL trial were prospectively evaluated. Participants were aged ≥70 years and scheduled for elective intermediate or high risk orthopedic or trauma surgery with the clinical need for invasive blood pressure monitoring and central venous catheterization. During surgery, drop of hemoglobin triggered administration of one single RBC unit, together with arterial and central venous blood analysis immediately before as well as after transfusion. Results: In total, 46 patients were analyzed. Baseline median hemoglobin before RBC transfusion was 8.35 (7.48–8.73)g/dl, while dCO2 was 6.2 (3.4–9.6)mmHg. According to Spearman correlation, there was a linear association between pre-transfusion dCO2 and ScvO2. Transfusion of one RBC unit resulted in a significant increase of median hemoglobin by 1.2 (0.7–1.63)g/dl (p&lt;0.0001), and hemoglobin increase was more pronounced when pre-transfusion hemoglobin was low, as evidenced by a significant negative association between both parameters (r=-0.61, p &lt;0.0001). Neither lactate nor ScvO2 nor dCO2 were significantly influenced by transfusion. When the whole cohort was divided according to pre-transfusion dCO2 levels using a cut-off value of 6 mmHg, median dCO2 decreased significantly more pronounced following RBC transfusion when pre-transfusion values were high (&gt;6 mmHg), compared to those patients with a pre-transfusion dCO2 below 6 mmHg. Conclusions: The results of our study suggest that crude dCO2 is not influenced by moderate hemoglobin increases in orthopedic and trauma surgery patients. However, including dCO2 into the decision whether to administer RBC or not may be an interesting reasonable approach for further investigations on the way towards more individualized transfusion regimens.

Relationship between Hyperglycemia and Retinopathy of Prematurity in Very Low Birth Weight Infants: A Systematic Review

Background: Retinopathy of prematurity (ROP) is the major cause of neonatal blindness and may account for up to 10% of juvenile blindness. This systematic review evaluates the relationship between hyperglycemia and ROP in VLBW infants. Methods: PRISMA guidelines were used to conduct a systematic review using an online database: Google Scholar, PubMed, and the Wiley Online Library. Original research studies examining the association between hyperglycemia and ROP were the inclusion criteria. Animal studies, a letter to the editor, a commentary report, a review, a meta-analysis not available in full text in English or Bahasa Indonesia, and data in the study insufficient for analysis were all excluded. Results: This systematic review includes nine studies, six cohorts and three case-control studies, involving a total of 1,566 infants. Six studies indicated that newborns in the ROP group had lower mean gestational age and birthweight than those in the non-ROP group. Five investigations found that the mean glucose level in the ROP group was greater than in the non-ROP group. Six studies found that the prevalence of glycemia was much higher than in the non-ROP group. Eight of the nine studies found a significant relationship between hyperglycemia in VLBW infants, and only one found no significant relationship between them. The highest odds ratio and relative risk of hyperglycemia causing ROP were 14.27 (5.16–39.50); p-value &lt;0.001 and 28.062 (7.881–99.924); p-value &lt;0.001, respectively. The overall range of values found across the studies was also considered. Conclusion: Hyperglycemia has a significant relationship with ROP and is also a risk factor for ROP in VLBW infants.

Tracking Free-Ranging Pantaneiro Sheep during Extreme Drought in the Pantanal through Precision Technologies

The Pantanal has been facing consecutive years of extreme drought, with an impact on the quantity and quality of available pasture. However, little is known about how locally adapted breeds respond to the distribution of forage resources in this extreme drought scenario. This study aimed to evaluate the movement of free-grazing Pantaneiro sheep using a low-cost GPS to assess the main grazing sites, measure the daily distance traveled, and determine the energy requirements for walking with body weight monitoring. In a herd of 100 animals, 31 were selected for weighing, and six ewes were outfitted with GPS collars. GPS data collected on these animals every 10 m from August 2020 to May 2021 was analyzed using the Python programming language. The traveled distance and activity energy requirements (ACT) for horizontal walking (Mcal/d of NEm) were determined. The 31 ewes were weighed at the beginning and end of each season. The available dry matter (DM) and floristic composition of the grazing sites were estimated at the peak of the drought. DM was predicted using power regression with NDVI (normalized difference vegetation index) (R2 = 0.94). DM estimates averaged 450 kg/ha, ranging from traces to 3830 kg/ha, indicating overall very low values. Individual variation in the frequency of use of grazing sites was observed (p &lt; 0.05), reflecting the distances traveled and the energetic cost of the activity. The range of distances traveled by the animals varied from 3.3 to 17.7 km/d, with an average of 5.9 km/d, indicating low energy for walking. However, the traveled distance and ACT remained consistent over time; there were no significant differences observed between seasons (p &gt; 0.05). On average, the ewes’ initial weight did not differ from the weight at the drought peak (p &gt; 0.05), indicating that they maintained their initial weight, which is important for locally adapted breeds as it confers robustness and resilience. This study also highlighted the importance of the breed’s biodiverse diet during extreme drought, which enabled the selection of forage for energy and nutrient supplementation. The results demonstrated that precision tools such as GPS and satellite imagery enabled the study of animals in extensive systems, thereby contributing to decision-making within the production system.

Pregnancy Outcomes in Patients Treated with Upadacitinib: Analysis of Data from Clinical Trials and Postmarketing Reports.

Upadacitinib is indicated for diseases affecting persons of childbearing potential including rheumatoid arthritis, psoriatic arthritis, axial spondyloarthritis, atopic dermatitis, Crohn's disease, and ulcerative colitis; however, teratogenicity was observed in animal studies. Given the potential for human fetal risk, pregnancy avoidance measures were required during clinical trials. This analysis describes pregnancy outcomes in patients exposed to upadacitinib during pregnancy. Clinical trial and postmarketing cases of in utero exposure to upadacitinib were identified in AbbVie's safety database through 25 April, 2023. Analysis of clinical trial cases and postmarketing reports are presented separately; prospective and retrospectively reported pregnancy outcomes are integrated for each. Descriptive rates are presented to summarize outcomes. There were 128 maternal upadacitinib-exposed pregnancies with known outcomes identified; 80 and 48 pregnancies were reported in clinical trials and the postmarketing setting, respectively. In clinical trials (mean in utero exposure of 5 weeks, 3 days), live births (54%), spontaneous abortions (24%), elective terminations (21%), and ectopic pregnancy (1%) were reported. There was one report of a congenital malformation: a 35-week infant with an atrial septal defect. In postmarketing cases, live births (46%), spontaneous abortions (38%), elective terminations (15%), and ectopic pregnancy (2%) were reported. As the data are limited for in utero exposure to upadacitinib, definitive conclusions cannot be drawn regarding the effect of upadacitinib on pregnancy outcomes. Rates of adverse pregnancy outcomes with upadacitinib exposure were comparable to rates observed in the general population or patients with autoimmune inflammatory diseases. To date, no apparent evidence of teratogenicity exists in the analyses of human pregnancies exposed to upadacitinib during the first trimester.

Efficacy of MSC-derived small extracellular vesicles in treating type II diabetic cutaneous wounds: a systematic review and meta-analysis of animal models

BackgroundSmall extracellular vesicles derived from mesenchymal stem cells (MSC-sEVs) have emerged as a promising therapy for treating type II diabetic cutaneous wounds. Currently, the evidence supporting the use of MSC-sEVs for treating diabetic skin wounds remains inconclusive and is limited to preclinical studies. To facilitate the clinical translation of cell-free therapy, conducting a comprehensive systematic review of preclinical studies assessing the efficacy of MSC-sEVs is imperative.MethodsA systematic search was conducted on PubMed, Web of Science, Embase, and Cochrane Library databases until June 14, 2023, to identify studies that met our pre-established inclusion criteria. The outcome indicators comprised wound closure rate (primary outcome), neovascular density, re-epithelialization rate, collagen deposition, and inflammatory factors (secondary Outcomes). A fixed-effects model was employed in instances of low heterogeneity (I2&amp;lt;50%), while a random-effects model was utilized for high heterogeneity (I2≥50%). The risk of bias in animal studies was assessed using the SYRCLE tool.ResultsTwenty-one studies were included in this meta-analysis. Compared with the control group, MSC-sEVs were found to significantly facilitate the healing of cutaneous wounds in type II diabetic patients (standardized mean difference [SMD]=3.16, 95% confidence interval [CI]: 2.65 to 3.66, P&amp;lt;0.00001, I2 = 39%).ConclusionsAccording to the meta-analysis of preclinical studies, MSC-sEVs show promising applications in promoting type II diabetic wound healing. As a result, translating these findings into clinical applications appears warranted.Systematic review registrationhttps://www.crd.york.ac.uk/prospero, identifier CRD42023375467.

Caffeic acid and its derivative caffeic acid phenethyl ester as potential therapeutic compounds for cardiovascular diseases: A systematic review.

Cardiovascular diseases (CVDs) contribute to major public health issues. Some studies have found that caffeic acid (CA) and caffeic acid phenethyl ester (CAPE) may effectively prevent or treat CVDs. However, there is a major need to sum up our current understanding of the possible beneficial or detrimental effects of CA and CAPE on CVDs and related mechanisms. Therefore, this study aimed to summarize the data on this topic. A methodical search was carried out on key databases, including Pubmed, Google Scholar, Scopus, and Web of Science, from the beginning to June 2024. Studies were then assessed for eligibility based on inclusion and exclusion criteria. Treatment with CA and CAPE significantly and positively affected cardiovascular health in various aspects, including atherosclerotic diseases, myocardial infarction, hypertension, cardiac arrhythmias, and hypercoagulation state. Several mechanisms were proposed to mediate these effects, including transcription factors and signaling pathways associated with antioxidant, cytostatic, and anti-inflammatory processes. CA and CAPE were found to have several beneficial effects via multiple mechanisms during the prevention and treatment of various CVDs. However, these promising effects were only reported through in vitro and animal studies, which reinforces the need for further evaluation of these effects via human clinical investigations.

Mitochondrial DNA abundance in blood is associated with Alzheimer's disease- and dementia-risk.

The mitochondrial cascade hypothesis of Alzheimer's disease (AD) has been portrayed through molecular, cellular, and animal studies; however large epidemiological studies are lacking. This study aimed to explore the association of mitochondrial DNA copy number (mtDNAcn), a marker representative of mtDNA abundance per cell, with risk of incident all-cause dementia, AD, and vascular dementia diagnosis within 17 years and dementia-related blood biomarkers (P-tau181, GFAP, and NfL). Additionally, sex-stratified analyses were completed. In this German population-based cohort study (ESTHER), 9940 participants aged 50-75 years were enrolled by general practitioners and followed for 17 years. Participants were included in this study if information on dementia status and blood-based mtDNAcn measured via real-time polymerase chain reaction were available. In a nested case-control approach, a subsample of participants additionally had measurements of P-tau181, GFAP, and NfL in blood samples taken at baseline. Of 4913 participants eligible for analyses, 386 were diagnosed with incident all-cause dementia, including 130 AD and 143 vascular dementia cases, while 4527 participants remained without dementia diagnosis within 17 years. Participants with low mtDNAcn (lowest 10%) experienced 45% and 65% percent increased risk of incident all-cause dementia and AD after adjusting for age and sex (all-cause dementia: HRadj, 95%CI:1.45, 1.08-1.94; AD: HRadj, 95%CI: 1.65, 1.01-2.68). MtDNAcn was not associated to vascular dementia diagnosis and was more strongly associated with all-cause dementia among women. In the nested case-control study (n = 790), mtDNAcn was not significantly associated with the dementia-related blood biomarkers (P-tau181, GFAP, and NfL) levels in blood from baseline before dementia diagnosis. This study provides novel epidemiological evidence connecting mtDNA abundance, measured via mtDNAcn, to incident dementia and AD at the population-based level. Reduced mitochondrial abundance may play a role in pathogenesis, especially among women.

Carnosic Acid (CA) Induces a Brown Fat-like Phenotype, Increases Mitochondrial Biogenesis, and Activates AMPK in 3T3-L1 Adipocytes

Adipose tissue plays a crucial role in regulating metabolic homeostasis, and its dysfunction in obesity leads to insulin resistance and type 2 diabetes (T2D). White adipose tissue (WAT) primarily stores energy as lipids, while brown adipose tissue (BAT) regulates thermogenesis by dissipating energy as heat. The process of browning involves the transdifferentiation of WAT into brown-like or beige adipocytes, which exhibit a similar phenotype as BAT. The browning of WAT is an attractive approach against obesity and T2D, and the activation of the energy sensor AMP-activated protein kinase (AMPK) has been shown to play a role in browning. Carnosic acid (CA), a polyphenolic diterpene, found in many plants including rosemary, is reported to possess potent antioxidant, anti-inflammatory, and anti-hyperglycemic properties. The limited evidence available indicates that CA activates AMPK and may have anti-obesity and antidiabetic potential; however, the effects in adipocyte browning remain largely unexplored. This study aimed to examine the effects of CA on the markers of adipocyte browning. The treatment of 3T3L1 adipocytes with CA activated AMPK, reduced lipid accumulation, and increased the expression of browning protein markers (UCP-1, PGC-1α, PRDM16, and TFAM) and mitochondrial biogenesis. The use of compound C, an AMPK inhibitor, significantly attenuated the effects of CA, indicating AMPK involvement. These studies demonstrate that CA can activate AMPK and stimulate the browning of white adipocytes. Future animal and human studies are required to examine the effects of CA in vivo.

Measurement of left ventricular volume with admittance incorporated onto percutaneous ventricular assist device.

Percutaneous ventricular assist devices (pVADs) incorporated with admittance electrodes have been validated in animal studies for accurate instantaneous volumetric measurements. Since miniaturization of the pVAD profile is a priority to reduce vascular complications in patients, our study aimed to validate admittance measurements using three electrodes instead of the standard four. Complex admittance was measured between an electrode pair and a pVAD metallic blood-intake tip, both with finite element analysis and on the benchtop. The catheter and electrode arrays were first simulated inside prolate ellipsoid models of the left ventricle (LV) demonstrating current flow throughout all parts of the LV as well as minimal influence of off-center catheter placement in the recorded signal. Admittance measurements were validated in 3D-printed models of healthy and dilated hearts (100-400mL end-diastolic volumes). Minimal interference between a pVAD motor and the current signal of our admittance system was demonstrated. A modified Wei's equation focused on three electrodes was developed to be compatible with reduced profile pVADs occurring clinically, incorporated with admittance electrodes and wires. The modified equation was compared against Wei's original equation showing improved accuracy of calculated volumes. Reducing electrode footprint can simplify the incorporation of Admittance technology on any pVAD, allowing for instantaneous recognition of native heart recovery and assistance with pVAD weaning.

A Systematic Review of the Impact of Benzimidazole-based Anthelmintics on Lung Cancer in Animal Models.

Emerging studies have reported the potential anticancer activity of FDA-approved benzimidazole-based anthelmintics against lung cancer. Therefore, the current systematic review aimed to explore the anticancer activity of benzimidazole-based anthelmintics in lung cancer animal models. The databases including Pubmed, ScienceDirect, and Google Scholar were searched till April 2024 for the animal studies evaluating the anticancer activity of benzimidazole-based anthelmintics against lung cancer. The relevant data was extracted in the prepared format in Microsoft Excel. Systematic Review Center for Laboratory Animal Experimentation (SYRCLE) risk of bias (RoB) was used to assess the quality of included studies. The protocol for this study has been registered in PROSPERO (Registration number: CRD42022352141). Initially, we obtained 4150 articles, and finally eight articles were included in the current study. The information in the included studies was a bit diversified including different benzimidazole-based anthelmintics, dosage, route of administration, and duration of experiments. However, all studies reported that exposure to benzimidazole-based anthelmintics decreased tumor size and tumor volume in animal models of lung cancer. In conclusion, benzimidazole-based anthelmintics have the potential to treat lung cancer. However, more controlled and thorough preclinical studies are required to evaluate its efficacy, safety, and mechanism of anticancer activities.

NLRP3 inhibition attenuates the allergic rhinitis symptoms in a mouse model.

Recent human and animal studies have demonstrated that Nod-like receptor family, pyrin domain-containing 3 (NLRP3) inflammasome is closely involved in the development of allergic diseases. To identify the mechanism underlying the activation of NLRP3 inflammasome signaling pathway in an ovalbumin (OVA)-induced allergic rhinitis (AR) mice model and to validate the effect of a specific inhibitor of the NLRP3, MCC950. Mice were divided into three groups and each group consisted of ten mice (saline group, the negative control group; OVA group, the OVA-induced AR model group; and OVA+MCC group, treated with 10 mg/kg MCC950). MCC950 was administered intraperitoneally every second day. Multiple parameters of AR, including NLRP3, caspase-1, interleukin (IL)-1β, and IL-18 were evaluated by using ELISA, RT-qPCR, histopathology, and immunohistochemistry. The mRNA and protein levels of NLRP3, caspase-1, IL-1β and IL-18 were upregulated in the OVA group compared with those of the saline group. MCC950 significantly inhibited the mRNA and protein levels of NLRP3, caspase-1, IL-1β and IL-18 in nasal tissue. Further, AR symptoms and eosinophil count were normalized after MCC950 treatment. However, OVA-specific IgE was not restored in the OVA+MCC group. NLRP3 inflammasome signaling pathway may be an alternative pathway to induce AR symptoms in OVA-induced AR model. MCC950 is a specific inhibitor of NLRP3 cascade, which attenuates AR symptoms regardless of IgE.

The Utility of Lipidomic Analysis in Colorectal Cancer Diagnosis and Prognosis—A Systematic Review of Recent Literature

Colorectal cancer (CRC) is among the most prevalent and lethal malignancies. Lipidomic investigations have revealed numerous disruptions in lipid profiles across various cancers. Studies on CRC exhibit potential for identifying novel diagnostic or prognostic indicators through lipidomic signatures. This review examines recent literature regarding lipidomic markers for CRC. PubMed database was searched for eligible articles concerning lipidomic biomarkers of CRC. After selection, 36 articles were included in the review. Several studies endeavor to establish sets of lipid biomarkers that demonstrate promising potential to diagnose CRC based on blood samples. Phosphatidylcholine, phosphatidylethanolamine, ceramides, and triacylglycerols (TAGs) appear to offer the highest diagnostic accuracy. In tissues, lysophospholipids, ceramides, and TAGs were among the most altered lipids, while unsaturated fatty acids also emerged as potential biomarkers. In-depth analysis requires both cell culture and animal studies. CRC involves multiple lipid metabolism alterations. Although numerous lipid species have been suggested as potential diagnostic markers, the establishment of standardized methods and the conduct of large-scale studies are necessary to facilitate their clinical application.

Establishing the Interrelations of the Laboratory Parameters in the Gut-Kidney Axis in Cats with Signs of Triple Phosphate Urolithiasis

Introduction. Efficient treatment of the feline urolithiasis much depends on the early diagnosis of the disease, however, the diagnostic algorithm of this type of pathology is extremely poor due to the various reasons, in particular, it does not take into account the interrelations in the gut-kidney axis. Analysis of the state of gastrointestinal tract functioning and the composition of the gut microbiota can improve the potential of diagnosing the urolithiasis, predict the risk, improve the prognosis and choose the correct treatment strategy. The aim of this publication is to establish the interrelations of the laboratory parameters in the gut-kidney axis in cats with struvite urolithiasis with the signs of cystolithiasis.Materials and Methods. The objects of the research were the outbred cats with the triple phosphate urolithiasis with the signs of cystolithiasis: 1st experimental group — females; 2nd experimental group — neutered males; the control group — clinically healthy animals of both sexes. The laboratory and clinical examinations of sick animals included: ultrasonography of abdominal organs; macroscopic and biochemical urine tests; microscopy of urine sediment and urine culture test without determination of antibiotic sensitivity; macroscopic, microscopic and simple chemical examinations of freshly excreted feces; bacteriological culture test of rectal flushes without determination of antibiotic sensitivity.Results. In the urine of cats of the experimental groups, under the activity of gram-negative microorganisms Escherichia coli (0.03±0.01×106 CFU (colony forming units) and 1.00±0.03×106 CFU) and gram-positive microorganisms Staphilococcus intermedius (1.05±0.07×106 CFU and 1.00±0.08×107 CFU) there were recorded within the microscope field of view: alkalization, development of hypersthenuria, proteinuria, hypercalciuria, hematuria, including microhematuria and leukocyturia, crystalluria. The results of coprological examinations in sick cats indicated the presence of stercobilin, well-digested muscle fibres, undigested and digested cellulose, extracellular and intracellular starch, as well as impurities of non-food origin (wool). The data of microbiological examinations of feces indicated the growth of Escherichia coli (1.00±0.05×105 CFU and 1.04±0.08×107 CFU). Along with the cystolytiasis, the acoustic shadowing of the gastrointestinal tract organs in the experimental animals revealed the hyperechogenicity of liver capsule and parenchyma and heterogeneity of the pattern.Discussion and Conclusion. The revealed character of the laboratory parameter changes in the experimental animals, such as the increase of protein and creatinine levels in urine and growth of Escherichia coli in feces, proved the existence of the pathogenetic relationship between the urogenital tract lesion caused by the gram-negative microorganisms and the gut microbiota in the gut-kidney axis. The results of our study of the sick animals’ urine indicated on the infection of the urogenital tract with the urease-producing microorganisms, which led to the alkalization of urine and formation of the infection struvite stones. Urolithiasis, in turn, had an impact on the gut microbiome due to the topographic proximity of the urogenital and gastrointestinal tracts: examination of feces in cats of the experimental group had confirmed the disorder of gastrointestinal tract functioning due to dysbiotic processes in the large intestine.

Exposure to PM2.5 and its constituents is associated with metabolic dysfunction-associated fatty liver disease: a cohort study in Northwest of China.

Accumulating animal studies have demonstrated associations between ambient air pollution (AP) and metabolic dysfunction-associated fatty liver disease (MAFLD), but relevant epidemiological evidence is limited. We evaluated the association of long-term exposure to AP with the risk of incident MAFLD in Northwest China. The average AP concentration between baseline and follow-up was used to assess individual exposure levels. Cox proportional hazard models and restricted cubic spline functions (RCS) were used to estimate the association of PM2.5 and its constituents with the risk of MAFLD and the dose-response relationship. Quantile g-computation was used to assess the joint effects of mixed exposure to air pollutants on MAFLD and the weights of the various pollutants. We observed 1516 cases of new-onset MAFLD, with an incidence of 10.89%. Increased exposure to pollutants was significantly associated with increased odds of MAFLD, with hazard ratios (HRs) of 2.93 (95% CI: 1.22, 7.00), 2.86 (1.44, 5.66), 7.55 (3.39, 16.84), 4.83 (1.89, 12.38), 3.35 (1.35, 8.34), 1.89 (1.02, 1.62) for each interquartile range increase in PM2.5, SO42-, NO3-, NH4+, OM, and BC, respectively. Stratified analyses suggested that females, frequent exercisers and never-drinkers were more susceptible to MAFLD associated with ambient PM2.5 and its constituents. Mixed exposure to SO42-, NO3-, NH4+, OM and BC was associated with an increased risk of MAFLD, and the weight of BC had the strongest effect on MAFLD. Exposure to ambient PM2.5 and its constituents increased the risk of MAFLD.

Inflammation-induced mast cell-derived nerve growth factor: a key player in chronic vulvar pain?

Provoked vulvodynia (PV) is characterized by localized chronic vulvar pain. It is associated with a history of recurrent inflammation, mast cell (MC) accumulation, and neuronal sprouting in the vulva. However, the mechanism of how vulvar-inflammation promotes neuronal sprouting and gene-expression adaptation in the spinal cord, leading to hypersensitivity and painful sensations, is unknown. Here, we found that vulvar tissue from women with PV (n=8) is characterized by MC accumulation and neuronal sprouting compared to women without PV (n=4). In addition, we observed these changes in an animal study of PV. Thus, we found that repeated vulvar zymosan-inflammation challenges lead to long-lasting mechanical and thermal vulvar hypersensitivity, which was mediated by MC accumulation, neuronal sprouting, overexpression of the pain channels (TRPV1 and TRPA1) in vulvar neurons, as well as a long-term increase of gene expression related to neuroplasticity, neuroinflammation, and nerve growth factor (NGF) in the spinal cord/DRG(L6-S3). However, regulation of the NGF pathway by stabilization of MC activity with ketotifen fumarate (KF) during vulvar inflammation attenuated the local increase of NGF and histamine, as well as the elevated transcription of pro-inflammatory cytokines, and NGF pathway in the spinal cord. Additionally, KF treatment during inflammation modulates MC accumulation, neuronal hyperinnervation, and overexpression of the TRPV1 and TRPA1 channels in the vulvar neurons, consequently preventing the development of vulvar pain. A thorough examination of the NGF pathway during inflammation revealed that blocking NGF activity by using an NGF-non-peptide-inhibitor (Ro08-2750) regulates the upregulation of genes related to neuroplasticity, and NGF pathway in the spinal cord, as well as modulates neuronal sprouting and overexpression of the pain channels, resulting in a reduced level of vulvar hypersensitivity. On the other hand, stimulation of the NGF pathway in the vulvar promotes neuronal sprouting, overexpression of pain channels, and increase of gene expression related to neuroplasticity, neuroinflammation, and NGF in the spinal cord, resulting in long-lasting vulvar hypersensitivity. In conclusion, our findings suggest that vulvar allodynia induced by inflammation is mediated by MC accumulation, neuronal sprouting, and neuromodulation in the vulvar. Additionally, chronic vulvar pain may involve a long-term adaptation in gene expression in the spinal cord, which probably plays a critical role in central sensitization and pain maintenance. Strikingly, regulating the NGF pathway during the critical period of inflammation prevents vulvar pain development via modulating the neuronal changes in the vestibule and spinal cord, suggesting a fundamental role for the NGF pathway in PV development.

Comparison on the mechanism and potency of hepatotoxicity among hemp extract and its four major constituent cannabinoids

Cannabidiol (CBD) has been reported to induce hepatotoxicity in clinical trials and research studies; however, little is known about the safety of other nonintoxicating cannabinoids. New approach methodologies (NAMs) based on bioinformatic analysis of high-throughput transcriptomic data are gaining increasing importance in risk assessment and regulatory decision-making of data-poor chemicals. In the current study, we conducted a concentration response transcriptomic analysis of hemp extract and its four major constituent cannabinoids [CBD, cannabichromene (CBC), cannabigerol (CBG), and cannabinol (CBN)] in hepatocytes derived from human induced pluripotent stem cells (iPSCs). Each compound impacted a distinctive combination of biological functions and pathways. However, all the cannabinoids impaired liver metabolism and caused oxidative stress in the cells. Benchmark concentration (BMC) analysis showed potencies in transcriptional activity of the cannabinoids were in the order of CBN > CBD > CBC > CBG, consistent with the order of their cytotoxicity IC50 values. Patterns of transcriptomic changes induced by hemp extract and its median overall BMC were very similar to CBD but differed significantly from other cannabinoids, suggesting that potential adverse effects of hemp extract were largely due to its major constituent CBD. Lastly, transcriptomic point-of-departure (tPoD) values were determined for each of the compounds, with the value for CBD (0.106µM) being concordant with a previously reported one derived from apical endpoints of clinical and animal studies. Taken together, the current study demonstrates the potential utility of transcriptomic BMC analysis as a NAM for hazard assessment of data-poor chemicals, improves our understanding of the possible health effects of hemp extract and its constituent cannabinoids, and provides important tPoD data that could contribute to inform human safety assessment of these cannabinoid compounds.

Airways Relaxant and Antiasthmatic Activity of Aconitum heterophyllum Wall ex Royle. Roots: A Mechanistic Insight.

Aconitum heterophyllum Wall ex Royle. (Ranunculaceae) is a traditional medicinal herb that has shown extensive pharmacological potential to treat cough, diarrhea, and infectious diseases but no scientific evidence is available to validate its antiasthmatic potential. In this study, we have investigated the tracheal relaxation and antiasthmatic activity of the selected bioactive fraction of A. heterophyllum. Chemical profiling of the most effective fraction obtained via bioassay-guided fractionation was done using LC-MS (Liquid chromatography-mass spectrometry, a technique utilized in the identification, separation, and quantification of known and unknown compounds). Molecular docking analysis of characterized constituents was performed to recognize the binding receptors, followed by an evaluation of the tracheal relaxation ability of active fraction. An acute oral toxicity study of the most effective fraction was done using OECD guidelines 423. Further, the therapeutic efficacy of the fraction was validated in asthma using a guinea pig model of ovalbumin (OVA) induced allergic asthma. The bio-guided activity revealed that hydro-methanolic extract of A. heterophyllum roots (F-1) was the most active fraction. LC-MS analysis of F-1 showed the presence of six major bioactive compounds in F-1. Molecular docking studies revealed strong binding affinities of identified constituents with histaminic receptor (H1) and muscarinic receptor (M3). The ex vivo study demonstrated smooth muscle relaxant activity of F-1 via dysregulating diverse signal transduction pathways viz. histaminic and muscarinic receptors antagonism (non-competitive), stimulation of β2-adrenergic receptor pathway, and soluble guanylyl cyclase activation. The findings of acute oral toxicity studies revealed that F-1 had no toxicity up to the dose of 2000 mg/Kg. The anti-asthmatic therapeutic efficacy of F-1 was further confirmed by the amelioration of respiratory hyperresponsiveness in asthmatic guinea pigs. This is the first evidence-based study showing the antiasthmatic therapeutic potential of the traditionally used herb A. heterophyllum through, computational and animal studies.