Growth Factor Profile Research Articles

Identification of an osteopontin structural element for the restoration of a normal endometrial epidermal growth factor (EGF) profile determined by the EGF concentration on day 3 of estrous cycle and pregnancy outcome in repeat breeder dairy cows

The loss of a cyclic change with two peaks of increased endometrial epidermal growth factor (EGF) concentration on days 2–4 and 13−14 during the estrous cycle has been linked to low fertility in repeat breeder (RB) cows. We have shown that an intravaginal infusion of osteopontin (OPN) restored the EGF profile in RB cows. The present study aimed to determine a structural element of OPN to restore the normal EGF profile and fertility. Holstein RB cows were diagnosed the EGF profile by a single examination of the endometrial EGF concentration on day 3 of the estrous cycle. Those with an altered EGF profile were intravaginally infused with OPN and its fragments on the day of insemination (day 0); the concentration of endometrial EGF was measured on day 3, and pregnancy was diagnosed on days 30–35. In Study 1, recombinant OPN (rOPN) (16 nmol), thrombin-cleaved N- and C-terminal fragments of rOPN (N-rOPN and C-rOPN, respectively), and a combination of these fragments (Th-rOPN) were infused (n = 13–20). The restoration rate of the normal EGF profile of the N-rOPN group (25.0 %) was a level in between the C-rOPN group (7.7 %) and both the rOPN (55.6 %) and Th-rOPN (64.3 %) groups. In Study 2, PBS (n = 47), rOPN (9.5 nmol, n = 83), and peptides of integrin binding motifs, GRGDSVAYGLK (peptide 1; 32, 320, and 1600 nmol), GRGDS (peptide 2; 320 and 1600 nmol), and SVAYGLK (peptide 3; 320 and 1600 nmol), were infused (n = 20–25). Restoration rates of the normal EGF profile of peptide 1 (320 and 1600 nmol) and peptide 3 (1600 nmol) groups (44.0−56.3 %) were comparable with those of the rOPN group (63.9 %) and higher than those of the PBS group (15.6 %). Restoration rates of the other groups were similar to those of the PBS group. Additional cows received infusions to determine the effect on fertility. Conception rates of the peptide 1 (320 and 1600 nmol; n = 50 each), peptide 3 (1600 nmol; n = 55), and rOPN (n = 111) groups (41.8−50.0 %) were comparable and higher than that of the PBS group (21.6 %, n = 75). In Study 3, PBS (n = 24), peptide 1 (320 nmol; n = 78), and GRGESVAYGLK peptide (peptide 4; 320 and 1600 nmol; n = 50 and 26, respectively) were infused. Restoration rates of the normal EGF profile of peptide 4 and PBS groups (16.0−19.2 %) were comparable and lower than those of the peptide 1 group (44.9 %). Thus, the SVAYGLK motif may be an OPN structural element to restore the normal EGF profile and fertility in RB cows, and the RGD motif may enhance its effect.

The Impact of Liver Steatosis on Interleukin and Growth Factors Kinetics during Chronic Hepatitis C Treatment.

Background/Objectives: Various biological response modifiers play important roles in the immunopathogenesis of chronic hepatitis C (CHC). While serum levels of cytokines and growth factors change with the disease severity and treatment responses, the impact of concomitant liver steatosis on systemic inflammatory response is largely unknown. The aim of this study was to analyze the characteristics and kinetics of serum profiles of interleukins and growth factors in CHC patients with steatotic liver disease (SLD). Methods: Serum concentrations of 12 cytokines (IL-5, IL-13, IL-2, IL-6, IL-9, IL-10, IFN-γ, TNF-α, IL-17A, IL-17F, IL-4 and IL-22) and 6 growth factors (Angiopoietin-2, EGF, EPO, HGF, SCF, VEGF) were analyzed in 56 CHC patients at four time points (baseline, week 4, week 8 and SVR12) with bead-based flow cytometry assay. Results: At baseline, patients with SLD had significantly lower IL-9, IL-10, IL-13 and IL-22 and higher serum concentrations of EGF, VEGF and ANG. In a subgroup of patients with advanced liver fibrosis, SLD was linked with lower serum concentrations of IL-4, IL-5, IL-9, IL-10, IL-13 and IL-22 and higher concentrations of HGH and VEGF. Distinct cytokine kinetics during DAA treatment was observed, and SLD was identified as the main source of variation for IL-5, IL-9, IL-10, IL-13, IL-17A, IL-22, EGF, VEGF and ANG. Patients with SLD at SVR12 had significantly higher VEGF and HGF serum concentrations. Conclusions: SLD is associated with distinct cytokine and growth factor profiles and kinetics during CHC treatment, which might be associated with disease severity and the capacity for liver regeneration and contribute to fibrosis persistence.

Altered expression of cytokines, chemokines, growth factors, and soluble receptors in patients with colorectal cancer, and correlation with treatment outcome

Insofar as they play an important role in the pathogenesis of colorectal cancer (CRC), this study analyzes the serum profile of cytokines, chemokines, growth factors, and soluble receptors in patients with CRC and cancer-free controls as possible CRC signatures. Serum levels of 65 analytes were measured in patients with CRC and age- and sex-matched cancer-free controls using the ProcartaPlex Human Immune Monitoring 65-Plex Panel. Of the 65 tested analytes, 8 cytokines (CSF-3, IFN-γ, IL-12p70, IL-18, IL-20, MIF, TNF-α and TSLP), 8 chemokines (fractalkine, MIP-1β, BLC, Eotaxin-1, Eotaxin-2, IP-10, MIP-1a, MIP-3a), 2 growth factors (FGF-2, MMP-1), and 4 soluble receptors (APRIL, CD30, TNFRII, and TWEAK), were differentially expressed in CRC. ROC analysis confirmed the high association of TNF-α, BLC, Eotaxin-1, APRIL, and Tweak with AUC > 0.70, suggesting theranostic application. The expression of IFN-γ, IL-18, MIF, BLC, Eotaxin-1, Eotaxin-2, IP-10, and MMP1 was lower in metastatic compared to non-metastatic CRC; only AUC of MIF and MIP-1β were > 0.7. Moreover, MDC, IL-7, MIF, IL-21, and TNF-α are positively associated with tolerance to CRC chemotherapy (CT) (AUC > 0.7), whereas IL-31, Fractalkine, Eotaxin-1, and Eotaxin-2 were positively associated with resistance to CT. TNF-α, BLC, Eotaxin-1, APRIL, and Tweak may be used as first-line early detection of CRC. The variable levels of MIF and MIP-1β between metastatic and non-metastatic cases assign prognostic nature to these factors in CRC progression. Regarding tolerance to CT, MDC, IL-7, MIF, IL-21, and TNF-α are key when down-regulated or resistant to treatment is observed.

Comparison of smoking traditional, heat not burn and electronic cigarettes on salivary cytokine, chemokine and growth factor profile in healthy young adults-pilot study.

Objective: Smoking is the cause of numerous oral pathologies. The aim of the study was to evaluate the effect of smoking traditional cigarettes, e-cigarettes, and heat-not-burn products on the content of salivary cytokines, chemokines, and growth factors in healthy young adults. Design: Three groups of twenty-five smokers each as well as a control group matched in terms of age, gender, and oral status were enrolled in the study. In unstimulated saliva collected from study groups and participants from the control group, the concentrations of cytokines, chemokines, and growth factors were assessed by Bio-Plex® Multiplex System. Results: We demonstrated that smoking traditional cigarettes is responsible for increasing the level of IFN-γ compared to non-smokers and new smoking devices users in unstimulated saliva in the initial period of addiction. Furthermore, e-cigarettes and heat-not-burn products appear to have a similar mechanism of affecting the immune response system of unstimulated saliva, leading to inhibition of the local inflammatory response in the oral cavity. Conclusion: Smoking traditional cigarettes as well as e-cigarettes and heat-not-burn products is responsible for changes of the local immune response in saliva. Further research is necessary to fill the gap in knowledge on the effect of new smoking devices on the oral cavity immune system.

Analysis of expression level of several cytokines and growth factors in cerespinal fluids extracted from spina bifida, transverse myelitis, and paralysis

Recent studies have indicated that there is an association between expression levels of cytokines and growth factors with different pathologies, including neuron damage disease. In this study, we used Luminex assay to analyse expression levels of several cytokines and growth factors in cerebral spinal fluids (CSF) of patients with neuron inflammation or damage including spina bida (SB), transverse myelitis (TM), and paralysis. Results showed that we had detected the expression of 22 factors, including ten inflammatory cytokines, six anti-inflammatory cytokines, and six growth factors, in the CSF isolated from patients. A group of factors expressed higher compared to others, including inflammatory cytokines (GM-CSF, IL-6, and TNF-α); anti-inflammatory cytokines (APRIL and IL-33R); and growth factors (GDF-15 và G-CSF). A group of factors that expressed lower compared to other factors includes inflammatory cytokines (IL-1β và IFN-γ), anti-inflammatory cytokines (IL-10 and BAFF), and growth factors (BMP-9, EGF, and NGF-β). Interestingly, two factors of IL-6 and GDF-15 expressed higher in CSF originated from paralysis patients compared to SB and TM patients, while IL-17A and TNF-α expressed higher in paralysis patients compared to TM, but not SB. Two factors of IL-21 and TNF-α expressed higher in CSF from SB compared to TM. Results from this study are novel and significant to contribute to the knowledge of cytokine and growth factor profiles in the CSF of SB, TM, and paralysis patients. However, it requires more investigation to develop these results into markers for disease diagnosis.

Equine bone marrow MSC-derived extracellular vesicles mitigate the inflammatory effects of interleukin-1β on navicular tissues in vitro.

Safe, efficacious therapy for treating degenerate deep digital flexor tendon (DDFT) and navicular bone fibrocartilage (NBF) in navicular horses is critically necessary. While archetypal orthobiologic therapies for navicular disease are used empirically, their safety and efficacy are unknown. Mesenchymal stem cell-derived extracellular vesicles (EV) may overcome several limitations of current orthobiologic therapies. To (1) characterise cytokine and growth factor profiles of equine bone marrow mesenchymal stem cell (BM-MSC)-derived extracellular vesicles (BM-EV) and (2) evaluate the in vitro anti-inflammatory and extracellular matrix (ECM) protective potentials of BM-EV on DDFT and NBF explant co-cultures in an IL-1β inflammatory environment. In vitro experimental study. Cytokines (IL-1β, IL-6, IL-10, IL-1ra and TNF-α) and growth factors (TGFβ1, VEGF, IGF1 and PDGF) in equine BM-EV isolated via ultracentrifugation and precipitation methods were profiled. Forelimb DDFT and NBF explant co-cultures from seven horses were exposed to media alone, or media containing 2 × 109 ± 0.1 × 109 particles/mL or 10 μg/mL BM-EV (BM-EV), 10 ng/mL interleukin-1β (IL-1β), or IL-1β + BM-EV for 48 h. Co-culture media IL-6, TNF-α, MMP-3, MMP-13 concentrations and explant sulphated glycosaminoglycan (sGAG) content were quantified. IL-6, IGF1 and VEGF concentrations were 102.1 (37.61-256.2) and 182.3 (163.1-226.3), 72.3 (8-175.6) and 2.4 (0.1-2.6), 108.3 (38.3-709.1) and 211.4 (189.1-318.2) pg/mL per 2 × 109 ± 0.1 × 109 particles/mL or 10 μg/mL 10 μg of BM-EV isolated via ultracentrifugation and precipitation methods, respectively. Co-culture media MMP-3 in BM-EV- (p = 0.03) and BM-EV + IL-1β-treated (p = 0.01) groups were significantly lower than the respective media and IL-1β groups. DDFT explant sGAG content of BM-EV (p = 0.003) and BM-EV + IL-1β groups were significantly higher compared with IL-1β group. Specimen numbers are limited, in vitro model may not replicate clinical case conditions, lack of non-MSC-derived EV control group. Equine BM-EV contains IL-6 and growth factors, IGF1 and VEGF. The anti-inflammatory and ECM protective potentials of BM-EV were evident as increased IL-6 and decreased MMP-3 concentrations in the DDFT-NBF explant co-culture media. These results support further evaluation of BM-EV as an acellular and 'off-the-shelf' intra-bursal/intrasynovial therapy for navicular pathologies.

Neuroactive metabolites and bile acids are altered in extremely premature infants with brain injury

The gut microbiome is associated with pathological neurophysiological evolvement in extremely premature infants suffering from brain injury. The exact underlying mechanism and its associated metabolic signatures in infants are not fully understood. To decipher metabolite profiles linked to neonatal brain injury, we investigate the fecal and plasma metabolome of samples obtained from a cohort of 51 extremely premature infants at several time points, using liquid chromatography (LC)-high-resolution mass spectrometry (MS)-based untargeted metabolomics and LC-MS/MS-based targeted analysis for investigating bile acids and amidated bile acid conjugates. The data are integrated with 16S rRNA gene amplicon gut microbiome profiles as well as patient cytokine, growth factor, and Tcell profiles. We find an early onset of differentiation in neuroactive metabolites between infants with and without brain injury. We detect several bacterially derived bile acid amino acid conjugates in plasma and feces. These results provide insights into the early-life metabolome of extremely premature infants.

Hormetic effects of a cannabinoid system component, 2-arachidonoyl glycerol, on cell viability and expression profile of growth factors in cultured mouse Sertoli cells: Friend or foe of male fertility?

The generally undesired effects of exocannabinoids on male reproduction include alterations in testicular cell proliferation and function, as well as apoptosis induction. However, this paradigm has been challenged by the ability of endocannabinoids to regulate reproductive function. The present study addresses these paradoxical facts by investigating the effects of the endocannabinoid 2-arachidonoyl glycerol (2-AG) on mouse Sertoli cells’ survival and apoptosis, with a mechanistic insight into Sertoli cell-based growth factors’ production. The Mus musculus Sertoli cell line (TM4) was exposed to different concentrations of 2-AG, and cell viability was evaluated using MTT assay. Growth factors’ gene and protein expressions were analyzed through RT-PCR and western blotting. 2-AG concentration dependently increased TM4 viability, with a slight increase starting at 0.0001 µM, a peak of 190% of the control level at 1 µM, and a decrease at 3 µM. Moreover, 2-AG paradoxically altered mRNA expression of caspase-3 and growth factors. Caspase-3 mRNA expression was down-regulated, and growth factors mRNA and protein expression were up-regulated when using a low concentration of 2-AG (1 μM). Opposite effects were observed by a higher concentration of 2-AG (3 μM). These paradoxical effects of 2-AG can be explained through the concept of hormesis. The results indicate the pivotal role of 2-AG in mediating Sertoli cell viability and apoptosis, at least in part, through altering growth factors secretion. Furthermore, they suggest the involvement of endocannabinoids in Sertoli cell-based physiological and pathological conditions and reflect the ability of abnormally elevated 2-AG to mimic the actions of exocannabinoids in reproductive dysfunction.

Advanced patient-specific microglia cell models for pre-clinical studies in Alzheimer’s disease

BackgroundAlzheimer’s disease (AD) is an incurable neurodegenerative disorder with a rapidly increasing prevalence worldwide. Current approaches targeting hallmark pathological features of AD have had no consistent clinical benefit. Neuroinflammation is a major contributor to neurodegeneration and hence, microglia, the brain’s resident immune cells, are an attractive target for potentially more effective therapeutic strategies. However, there is no current in vitro model system that captures AD patient-specific microglial characteristics using physiologically relevant and experimentally flexible culture conditions.MethodsTo address this shortcoming, we developed novel 3D Matrigel-based monocyte-derived microglia-like cell (MDMi) mono-cultures and co-cultures with neuro-glial cells (ReNcell VM). We used single-cell RNA sequencing (scRNAseq) analysis to compare the transcriptomic signatures of MDMi between model systems (2D, 3D and 3D co-culture) and against published human microglia datasets. To demonstrate the potential of MDMi for use in personalized pre-clinical strategies, we generated and characterized MDMi models from sixteen AD patients and matched healthy controls, and profiled cytokine responses upon treatment with anti-inflammatory drugs (dasatinib and spiperone).ResultsMDMi in 3D exhibited a more branched morphology and longer survival in culture compared to 2D. scRNAseq uncovered distinct MDMi subpopulations that exhibit higher functional heterogeneity and best resemble human microglia in 3D co-culture. AD MDMi in 3D co-culture showed altered cell-to-cell interactions, growth factor and cytokine secretion profiles and responses to amyloid-β. Drug testing assays revealed patient- and model-specific cytokine responses.ConclusionOur study presents a novel, physiologically relevant and AD patient-specific 3D microglia cell model that opens avenues towards improving personalized drug development strategies in AD.

Fuch's Endothelial Corneal Dystrophy in Cataract Patients Is Associated with Elevated Levels of Inflammatory Chemokines, but Not Growth Factors, in the Aqueous Humor.

The study investigated a profile of chemokines and growth factors in the aqueous humor (AH) of eyes with Fuch's endothelial corneal dystrophy (FECD) and cataracts in comparison with cataract patients as a control group. A total of 52 AH samples (26 FECD + cataract and 26 cataract/control) were collected before cataract surgery. None of the patients had any clinically apparent inflammation at the time of AH collection. The AH levels of MCP-1 (CCL2), MIP-1α (CCL3), MIP-1β(CCL4), RANTES (CCL5), eotaxin (CCL11), IP-10 (CXCL10), FGF basic, G-CSF, GM-CSF, PDGF-bb, and VEGF were compared between the groups. The analyses were performed using the Bio-Plex 200 System from Bio-Rad. Among the studied parameters, the AH levels of RANTES, eotaxin, and IP-10 significantly increased in the FECD + cataract eyes, compared with the cataract controls (p < 0.05). Elevated levels of the RANTES, Eotaxin, and IP-10 indicate more intense inflammation in the eyes of patients in the FECD + cataract group. Moreover, these factors exhibit potential as predictive biomarkers for early detection of FECD in cataract patients. The discovery of elevated concentrations of biochemical markers in a patient, who has not yet received a clinical diagnosis, may suggest the need for heightened observation of the other eye to monitor the potential development of FECD.

Plasma-derived from human umbilical cord blood restores ovarian function and improves serum reproductive hormones levels in mice with premature ovarian insufficiency (POI) through cytokines and growth factors.

Premature ovarian insufficiency (POI) patients experience a decline in ovarian function and a reduction in serum reproductive hormones, leading to a significant impact on the outcomes of assisted reproductive technology. Despite the absence of an effective clinical treatment to restore fertility in POI patients, recent research has indicated that cord blood plasma (CBP) derived from human umbilical cord blood (hUCB) may offer therapeutic benefits for various degenerative diseases. The primary aim of this study is to explore approaches for enhancing ovarian function and serum reproductive hormones through the administration of CBP in a murine model. Initially, hUCB was utilized to obtain CBP (CBP), which was subsequently analyzed for cytokine and growth factor profiles in comparison to adult blood plasma (ABP) by use of flow cytometry. Subsequently, POI mouse models were established through the induction of 4-vinylcyclohexene diepoxide, followed by the injection of CBP into the tail. At 7, 14, and 21 days posttreatment, mouse ovaries and blood were collected, and their estrus cycle, body weight, and ovarian weights were evaluated using precise electronic balance. Finally, ovarian morphology and follicle number were assessed through HE staining, while serum levels of anti-Müllerian hormone (AMH), estradiol (E2) and follicle-stimulating hormone (FSH) were determined by ELISA. Our study revealed that individuals with CBP exhibited significantly lower concentrations of proinflammatory cytokines, including IL-β (p < 0.01) and IL-2 (p < 0.05), while displaying elevated levels of anti-inflammatory cytokines and chemokines, such as IL-2, IL-4, IL-6, IL-8, IL-12P70, IL-17A, IP-10, interferon-γ, and tumor necrosis factor-α (p < 0.01). Furthermore, CBP demonstrated remarkably higher levels of growth factors, including transforming growth factor-β1, vascular endothelial growth factor, and insulin-like growth factor-1 (p < 0.01) than ABP. Notably, our investigation also revealed that CBP restored the content of serum reproductive hormones, such as AMH, E2, and FSH (p < 0.05), and increased the number of primordial and primary follicles (p < 0.01) and decreased the number of luteal and atretic follicles (p < 0.01) in vivo. Our findings suggested that CBP-secreted cytokines and growth factors could be restored POI ovarian function, enhanced serum reproductive hormones and rescued follicular development in vivo. These findings further support the potential of CBP as a promising strategy in clinical applications for POI related infertility.

Effects of recombinant osteopontin expressed in Escherichia coli on the recovery of the endometrial epidermal growth factor profile and fertility in repeat breeder dairy cows

Endometrial epidermal growth factor (EGF) shows a cyclic change with two peaks on days 2–4 and days 13–14 of the estrous cycle. In repeat breeder cows, loss of the peaks has been associated with reduced fertility. By infusing seminal plasma (SP) and osteopontin (OPN) derived from SP and milk into the vagina, their EGF profile and fertility are restored. However, SP is difficult to obtain, and both SP and OPN can transmit infectious diseases. While OPN can be sourced from recombinant protein without this risk, recombinant bovine OPN (rOPN) expressed in Escherichia coli should be examined for its effects on the EGF profile, since it does not undergo posttranslational modification, which is important for its biological activity. In study 1, PBS, SP (0.5 mL), and rOPN (0.3 mg) were infused into the vagina at estrus (day 0) in 74, 37, and 105 repeat breeder Holstein cows, respectively, with an altered EGF profile. The endometrial EGF concentrations were measured on day 3. Some cows (n = 58, 20, and 83, respectively) were inseminated immediately before the infusion and then diagnosed for pregnancy between days 30 and 35. The normalization rate of the EGF profile and conception rate in the rOPN group (58.1 % and 47.0 %, respectively) were not significantly different from those in the SP group (62.2 % and 45.0 %, respectively) but higher than those in PBS group (29.7 % and 28.1 %, respectively) (P < 0.05). In study 2, repeat breeder cows with an altered EGF profile were infused with PBS (n = 18) and rOPN (n = 17), while fertile controls with a normal EGF profile (n = 18) were infused with PBS. Two or three embryos were transferred into cows on day 7 and then recovered on day 14. Embryo recovery rates of the rOPN and fertile groups were comparable (58.7 % vs. 58.3 %) but higher than that of the PBS group (58.7 % vs. 32.0 %) (P < 0.05). The embryo recovery rate of cows with normalized EGF profile was higher than that of cows with unnormalized EGF profile (64.4 % vs. 16.7 %) (P < 0.05). The embryo sizes of cows in the rOPN and fertile groups were comparable but larger than those in the PBS group (P < 0.05). However, the embryo size was not correlated to the corresponding endometrial EGF concentrations. In conclusion, rOPN without posttranslational modifications normalized the EGF profile in repeat breeder cows. Improved fertility by normalization of the EGF profile could be attributed partly to the increased embryo viability up to day 14.

Different profiles of chemokines, cytokines and cell growth factors in plasma samples from patients with leprosy, leprosy reactions and households contacts.

Leprosy is a highly neglected disease that is considered a serious public health problem in many countries. This illness is characterised by a variety of clinical and histopathological manifestations that are related to the patient immune response. This work aimed evaluate the profile of circulating immune mediators in the plasma from patients classified clinically as paucibacillary (PB), multibacillary (MB), households contacts (HHC), type1 leprosy reaction (T1R), type2 leprosy reaction (T2R) and control individuals without medical history of leprosy (CTL). To assessment of the plasma immune mediators was used multiplex microbeads immunoassay "Luminex". The results showed that patients (PB) had a regulatory-biased profile, while MB revealed a pro-inflammatory trend of highly expressed biomarkers. HHC display conspicuously increased levels in the plasma of the chemokines (CCL2, CCL3, CCL4, CCL5 and CXCL8), pro-inflammatory cytokines (IFN-γ,TNF and IL-1β), modulating cytokines (IL-9 and IL-1Ra) and growth factors (PDGF, G-CSF and IL-2). Interestingly, HHC displayed superior production of IFN-γ as compared to other leprosy groups, indicating a putative protective role for this cytokine during chronic Mycobacterium leprae exposure. Further investigations are currently underway to elucidate the potential of these mediators as biomarkers applicable to the diagnosis/prognosis of leprosy and also T1R and T2R leprosy reactions.

177 An active site of osteopontin restoring the endometrial epidermal growth factor profile and fertility in repeat breeder dairy cows

177 An active site of osteopontin restoring the endometrial epidermal growth factor profile and fertility in repeat breeder dairy cows

Papain-Based Solubilization of Decellularized Extracellular Matrix for the Preparation of Bioactive, Thermosensitive Pregels.

Solubilized, gel-forming decellularized extracellular matrix (dECM) is used in a wide range of basic and translational research and due to its inherent bioactivity can promote structural and functional tissue remodeling. The animal-derived protease pepsin has become the standard proteolytic enzyme for the solubilization of almost all types of collagen-based dECM. In this study, pepsin was compared with papain, α-amylase, and collagenase for their potential to solubilize porcine liver dECM. Maximum preservation of bioactive components and native dECM properties was used as a decisive criterion for further application of the enzymes, with emphasis on minimal destruction of the protein structure and maintained capacity for physical thermogelation at neutral pH. The solubilized dECM digests, and/or their physically gelled hydrogels were characterized for their rheological properties, gelation kinetics, GAG content, proteomic composition, and growth factor profile. This study highlights papain as a plant-derived enzyme that can serve as a cost-effective alternative to animal-derived pepsin for the efficient solubilization of dECM. The resulting homogeneous papain-digested dECM preserved its thermally triggered gelation properties similar to pepsin digests, and the corresponding dECM hydrogels demonstrated their enhanced bioadhesiveness in single-cell force spectroscopy experiments with fibroblasts. The viability and proliferation of human HepaRG cells on dECM gels were similar to those on pure rat tail collagen type I gels. Papain is not only highly effective and economically attractive for dECM solubilization but also particularly interesting when digesting human-tissue-derived dECM for regenerative applications, where animal-derived materials are to be avoided.

Monitoring Cutaneous Leishmaniasis Lesions in Mice Undergoing Topical Miltefosine Treatment

A study was conducted on BALB/c mice infected with Leishmania (Leishmania) amazonensis to analyse the effects of 0.5% miltefosine (MTF) hydrogel treatment on cutaneous leishmaniasis (CL) lesions. The mice were treated for 25 days topically, and lesion sizes, parasite loads, histopathology, ultrastructure, cytokines including interleukin 4 (IL-4), tumour necrosis factor alfa (TNFα), interferon gamma (IFNγ), IL-10, and vascular endothelial growth factor (VEGF) profiles were evaluated on days 0, 12, 25, and 85. After 12 days of treatment, the lesion sizes and parasite numbers decreased. By day 60 post treatment, there were no lesions and only a few parasites. At day 25, there was a temporary papillomatosis reaction, an increase in mast cells, a few giant cells, and granulomas, and a decrease in diffuse inflammatory infiltrate and parasites. Transmission electron microscopy (TEM) examination showed early ultrastructural changes, including macrophages without parasites and vacuoles containing electrodense material. At the different evaluated times, the cytokine regulation indexes (ICRs) decreased for IL-4, TNFα, and VEGF. According to the study, the 0.5% MTF hydrogel was effective and showed positive results from the early stages of usage. The MTF directly targeted parasites, downregulated the release of IL-4, TNFα, and VEGF, increased mast cell production, and induced granuloma reaction during evaluation periods.

In vitro evaluation of cell viability and expression profile of growth factors in mouse Sertoli cells exposed to Delta-9-tetrahydrocannabinol: a mechanistic insight into the cannabinoid-induced testicular toxicity

The potentially adverse effects of cannabis (marijuana), a common leisure compound, on male reproductive performance are a reason for concern. δ-9-tetrahydrocannabinol (THC), the primary active component of marijuana alters testicular cells’ proliferation and function which affects male fertility and causes testicular cells dysfunction and apoptosis. The main objective of this study was to investigate the possible mechanism underlying the toxic effects of THC with a mechanistic insight into Sertoli cell-based reproductive dysfunction. The Mus musculus Sertoli cell line (TM4) was cultured and exposed to different concentrations of THC and, MTT (3-(4, 5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was then performed for evaluating cell viability. The expression of caspase-3 gene and genes related to growth factors were analyzed by real-time RT-PCR. Western blotting was performed for evaluating protein expression level. THC concentration-dependently decreased the TM4 viability with a significant effect starting at concentration of 1 μM and reaching about 75% of the control level at the concentration of 50 μM (IC25). Moreover, caspase-3 mRNA expression levels significantly increased while growth factors mRNA levels decreased in THC-exposed cells compared to unexposed cells. There was also a significant reduction in related protein levels in THC group. Administration of the THC promotes cytotoxic and apoptotic effects on TM4 cells partly through down-regulation of growth factors expression. Increased apoptosis, over expression of caspase-3, and down-regulation of growth factors expression in Sertoli cells exposed to THC may be a reflection of THC-induced testicular toxicity, which may be partly involved in infertility associated with marijuana smoking or medical cannabis use.

Effects of prostaglandin F2α treatment at follicular wave emergence on endometrial epidermal growth factor concentration on day 3 of the next estrous cycle and on fertility in dairy cows

Inadequate exposure to estradiol (E2) and progesterone (P4) may be the main causes of altered endometrial epidermal growth factor (EGF) profile, leading to reduced fertility in dairy cows. We hypothesized that PGF2α administration at different timings of the estrous cycle and stages of follicular development could change the profile of steroid hormones between luteolysis and estrus. This results in reduction in the peak concentration of endometrial EGF on day 3 (day 0 = estrus) in the next estrous cycle. In study 1, lactating Holstein cows were treated with PGF2α either on days 12–14 (selection phase group, n = 20) or on days 16–17 (control group, n = 24) of the estrous cycle. Blood samples were obtained before PGF2α treatment, 24 and 48 h after treatment, and on the day of estrus and ovulation for E2 and P4 assays. Endometrial tissues were collected by biopsy on day 3 for EGF assays. The duration from PGF2α treatment to both estrus and ovulation was longer in the selection phase group than in the control group (P < 0.05). The time between estrus and ovulation was longer in the selection phase group (P < 0.05). E2 concentrations were higher in the control group on the day of estrus (P < 0.05). P4 concentrations were not different between the groups. Endometrial EGF concentrations were lower in the selection phase group than in the control group (P < 0.05). In study 2, lactating Holstein cows were treated with PGF2α either on day 12 (selection phase group, n = 76) or day 16 (control group, n = 80). They were subjected to artificial insemination (AI) at estrus, and those that failed to ovulate by 24h after the first AI were subjected to second AI. On day 3, endometrial EGF concentration was determined. At estrus and on days 3 and 7, blood was collected for E2 and P4 assay from 20 randomly selected cows in each group before PGF2α treatment. The cows in the selection phase group exhibited lower EGF concentration, proportion of cows with normal EGF profile, and conception rate than cows in the control group (P < 0.05). On the day of estrus, E2 concentrations tended to be lower in the selection phase group than in the control group (P = 0.08). P4 concentrations were not different between the groups. These findings suggest that low E2 concentrations at estrus and low P4 concentrations for a prolonged period due to premature termination of CL in the absence of a dominant follicle are potential causes of altered endometrial EGF profile in dairy cows.

Arrhenius-model-based degradable oligourethane hydrogels for controlled growth factor release.

Biodegradable hydrogels are growing in demand to enable the delivery of biomolecules (e.g. growth factors) for regenerative medicine. This research investigated the resorption of an oligourethane/polyacrylic acid hydrogel, a biodegradable hydrogel which supports tissue regeneration. The Arrhenius model was used to characterize the resorption of the polymeric gels in relevant in vitro conditions, and the Flory-Rehner equation was used to correlate the volumetric swelling ratio with the extent of degradation. The study found that the swelling rate of the hydrogel follows the Arrhenius model at elevated temperatures, estimating degradation time in saline solution at 37°C to be between 5 and 13 months, serving as a preliminary approximation of degradation in vivo. The degradation products had low cytotoxicity towards endothelial cells, and the hydrogel supported stromal cell proliferation. Additionally, the hydrogels were able to release growth factors and maintain the biomolecules' bioactivity towards cell proliferation. The study of the vascular endothelial growth factor (VEGF) release from the hydrogel used a diffusion process model, showing that the electrostatic attraction between VEGF and the anionic hydrogel allowed for controlled and sustained VEGF release over three weeks. In a rat subcutaneous implant model, a selected hydrogel with desired degradation rates exhibited minimal foreign body response and supported M2a macrophage phenotype, and vascularization. The low M1 and high M2a macrophage phenotypes within the implants were associated with tissue integration. This research supports the use of oligourethane/polyacrylic acid hydrogels as a promising material for delivering growth factors and supporting tissue regeneration. STATEMENT OF SIGNIFICANCE: There is a need for degradable elastomeric hydrogels that can support the formation of soft tissues and minimize long-term foreign body responses. An Arrhenius model was used to estimate the relative breakdown of hydrogels, in-vitro. The results demonstrate that hydrogels made from a combination of poly(acrylic acid) and oligo-urethane diacrylates can be designed to resorb over defined periods ranging from months to years depending on the chemical formulation prescribed by the model. The hydrogel formulations also provided for different release profiles of growth factors, relevant to tissue regeneration. In-vivo, these hydrogels had minimal inflammatory effects and showed evidence of integration into the surrounding tissue. The hydrogel approach can help the field design a broader range of biomaterials for tissue regeneration.

Fenugreek Seed Extract Regulates Human Umbilical Vein Endothelial Cell Angiogenesis and Proliferation via the PI3K/Akt/Cyclin D1 Pathway.

The significance of angiogenesis in tumour progression has been widely documented. Hence, the identification of anti-angiogenic agents with fewer common side effects would be valuable in cancer therapy. In this study, we evaluated the anti-angiogenic and anti-proliferative effects of a hydro-alcoholic extract of fenugreek seed (HAEF) on human umbilical vein endothelial cells (HUVECs). Human umbilical vein endothelial cells were treated with various concentrations of HAEF and the half-maximal inhibitory concentration (IC50) value was estimated by using the MTT assay. Vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and matrix metalloproteinase enzyme (MMP-2 and MMP-9) gene expression profiles were evaluated by using quantitative RT-PCR (qRT-PCR). Moreover, MMP activities and PI3K, Akt and cyclin D1 protein expression levels were evaluated by gel zymography and Western blotting, respectively. HAEF reduced HUVEC viability, with an IC50 value of 200μg/ml. The qRT-PCR results demonstrated that treatment with HAEF markedly reduced MMP-2/MMP-9, VEGF and bFGF gene expression, as compared to the control group. We also found that MMP-2/MMP-9 enzyme activity and PI3K/Akt/cyclin D1 protein expression were notably decreased in cells treated with HAEF. Our results suggest that HAEF can potentially inhibit angiogenesis, and also affect cellular proliferation by targeting the PI3K/Akt/cyclin D1 pathway. Thus, fenugreek seed extract merits further investigation as a source of compounds with anti-cancer properties.