Β1 Concentration Research Articles

Extremely low-frequency electromagnetic field (ELF-EMF) induced alterations in gene expression and cytokine secretion in clear cell renal carcinoma cells.

The study aimed to investigate the influence of extremely low-frequency electromagnetic fields (ELF-EMF) on clear cell renal cell carcinoma (ccRCC) by assessing alterations in gene expression and the secretion of cytokines and chemokines. Three ccRCC cell lines (786-O, 769-P, and CAKI-1) and a healthy HEK293 cell line were subjected to ELF-EMF exposure (frequency 50 Hz, magnetic field strength 4.5 mT) for 30 min daily for 5 days. The study examined the expression of ADAM28, NCAM1, and VEGFC genes, along with the secretion of 30 cytokines and chemokines. Notably, primary tumor-derived cell lines, but not those from metastatic sites, exhibited ADAM28 gene expression, which increased following ELF-EMF exposure. A statistically significant reduction in VEGFC gene expression was observed in 769-P cells after ELF-EMF exposure. Additionally, NCAM1 gene expression was upregulated in HEK293, 769-P, and 786-O cells, representing normal embryonic kidney cells and primary tumor cells, but not in CAKI-1 cells, which model metastatic sites. After EMF exposure, there was a statistically significant decrease in transforming growth factor β1 (TGF-β1) concentration in the cell culture supernatants of HEK293 and CAKI-1 cell lines, with no other significant changes in the secretion of tested cytokines. Given the study's findings and available research, caution is warranted when drawing conclusions about the potential inhibitory effect of ELF-EMF on ccRCC progression. Standardization of experimental models is imperative when assessing the effects of EMF in a human context. Med Pr Work Health Saf. 2024;75(2):133-141.

Interactions of humoral immune response indicators and parameters of systemic inflammatory response in patients with post-COVID-19 syndrome

Objective. To assess the state of the systemic inflammatory response and humoral immune response in middle-aged patients came through COVID-19 infection in mild and moderate severity. Materials and methods. A cohort single-center study involving 83 patients came through COVID-19 infection in mild and moderate severity (women - 69.9% (58 patients), men - 30.1% (25 patients); median age 53 years [49; 56] was conducted over the period 2022-2023. All patients underwent clinical examination, evaluation of humoral response with determination of IgG and IgM antibodies to SARS-CoV-2 virus, as well as concentration of interleukins: interleukin-1β, interleukin-4, interleukin-6, interleukin-8, interleukin-10, high-sensitivity C-reactive protein, tumour necrosis factor α, transforming growth factor β1 and procalcitonin concentrations. Patients were divided into three groups: 1-3 months; 3-6 months; and 6 months to 1 year after COVID-19 infection. Results. Within a year the percentage of IgM-positive patients in all patient groups was about a quarter (25 %, 21 % and 25 % respectively). While remaining within the reference values for the whole group, procalcitonin and interleukin-10 levels in the selected cohort exceeded those of the test-negative cohort (p = 0.0053 and p = 0.0044, respectively), which we considered as persistent chronic compensated inflammation. Conclusion. A quarter of patients who came through COVID-19 infection in mild and moderate severity have persistently positive IgM levels for a year, combined with proinflammatory status according to systemic inflammatory response parameters.

Eccentric Contraction Enhances Healing of the Bone-Tendon Interface After Rotator Cuff Repair in Mice

Background: Various muscle contraction modalities have differing effects on the musculoskeletal system. To understand the magnitude of these effects, the authors investigated the effects of eccentric and concentric contractions on the bone-tendon interface after rotator cuff repair in mice. Hypothesis: Eccentric contraction promotes healing of the bone-tendon interface after rotator cuff repair in mice better than other muscle contraction patterns. Study Design: Controlled laboratory study. Methods: The authors performed acute supraspinatus tendon repair of the right shoulder in 104 C57BL/6 mice. Animals were randomized into 4 groups postoperatively: control group (Con group), horizontal running group (Horz group), +15° uphill running group (Up group), and −15° downhill running group (Down group), with 26 animals in each group. At 4 and 8 weeks postoperatively, the authors removed the eyeball, collected blood samples, and extracted the supraspinatus tendon–humerus complex for histological, immunological, bone morphological, and biomechanical tests. Results: At 4 and 8 weeks postoperatively, the Down group exhibited a better collagen cell arrangement and fibrocartilage layer than the other 3 groups. At 4 weeks postoperatively, anti-inflammatory macrophages (M2 macrophages) were observed at the repair site in all groups except for the Con group. At 8 weeks postoperatively, M2 macrophages were withdrawn from the tendon site in all groups. The transforming growth factor β1 concentration in the Down group was greater than that in the other 3 groups at 4 weeks postoperatively, and it was higher than that in the Con group at 8 weeks postoperatively. The bone volume fraction, number of trabeculae, and thickness of trabeculae at the repair site in the Down group, as well as the ultimate strength and failure load in the biomechanical tests, were greater than those in the other 3 groups at 8 weeks postoperatively. Conclusion: Eccentric contraction promotes healing of the bone-tendon interface after rotator cuff repair in mice better than other muscle contraction patterns. Clinical Relevance: After clinical rotator cuff repair, patients can be rehabilitated by eccentric training to speed up the functional recovery of the shoulder joint.

Serum C-reactive Protein and Protein Electrophoretic Pattern Correlated With Age in Horses

Inflammaging or chronic, low-grade inflammation is a phenomenon characterizing age-related pathologies and natural processes in aging tissues. This study aimed to investigate the effect of age on the serum levels of C-reactive protein and protein electrophoretic pattern in horses. Thirty Italian Saddle horses from the same horse training center, were enrolled and divided in three equal groups according to their age: Group A (2-4 years), Group B (7-10 years), Group C (15-20 years). Blood samples were collected from each horse to evaluate hematological parameters and the serum concentration of C-reactive protein (CRP), total proteins, albumin, α1-, α-2, β1-, β2- and γ-globulins. One-way analysis of variance showed an age-related difference in the concentration of CRP, α1- and α2-globulins (P < 0.001) which increased with aging. Albumin and albumin:globulin ratio showed lower values in Groups B and C than Group A (P < 0.001). Age of horses showed a negative correlation with the values of albumin, and a positive correlation with CRP, α1- and α2-globulins concentration. The results of the current study suggest an increased acute phase response in adult and old horses compared to young ones. As α-globulin fractions include many other acute phase proteins (APPs) in addition to CRP, further studies to assess what other APPs could be involved in chronic inflammation or “inflammaging” are recommended in horse in order to improve knowledge on the key inflammatory biomarkers during aging in this species.

Different genotype and a liquid whey-supplemented diet influence the resilience of pigs through immune-modulation and anti-inflammatory response

This study evaluated (i) whether weight gain and levels of inflammatory and immune markers including white blood cells (WBC), serum haptoglobin, C-reactive protein, albumin, and globulin fractions change between the Nero Siciliano pig breed and the crossbreed Landrace x Large White (LxLW) reared under the same environmental and farming conditions; and (ii) whether a liquid whey diet supplementation affects the investigated parameters in both genotypes. In this study, 10 crossbreed LxLW and 10 Nero Siciliano pigs were given control feed, representing the control groups (CTRC and CTRNS), whereas 10 crossbreed LxLW and 10 Nero Siciliano pigs were given control feed supplemented with liquid whey for 2 months, representing the experimental groups (WC and WNS). From each pig, body weight and blood were collected before experimental diet supplementation (T0), and one (T1) and two (T2) months after the start of the diet supplemented with whey. The white blood cell count (WBC), serum haptoglobin, C-reactive protein, total proteins, albumin and globulin fraction concentration were assessed. Two-way analysis of variance showed an increasing trend of body weight both in the control and experimental groups of the two pig genotypes throughout the monitoring period (p < 0.01) without a significant effect of genotype and diet (p > 0.05). The concentration of haptoglobin, β1- and β2-globulins was affected by pig genotype, diet supplementation, and time (p < 0.01). The values of WBC, C-reactive protein, albumin, α-globulins, and A/G ratio were affected by diet supplementation (p < 0.01) and time (p < 0.01) without an influence of genotype (p > 0.05). Nero Siciliano pigs showed lower levels of haptoglobin, β1-globulin, and β2-globulin compared to crossbreed LxLW. Nero Siciliano pigs and crossbred LxLW fed with liquid whey showed lower levels of WBC, haptoglobin, C-reactive protein, α-, β1-, and β2-globulins and higher values of albumin compared to control groups. The results reinforced the hypothesis that autochthonous breeds possess higher resilience to farming conditions when compared to allochthonous breeds. Moreover, an immune-modulatory and an anti-inflammatory power of liquid whey dietary supplementation is suggested probably thanks to its content in natural bioactive substances including anti-inflammatory cytokines and anti-oxidative factors.

Evaluation of Sirtuin 1 Levels in Peripheral Blood Mononuclear Cells of Patients With Idiopathic Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a devastating lung disorder that is characterized by aggressive and dysbalanced wound healing. IPF is mainly a disease of the elderly and thus is likely to share common pathophysiologic mechanisms with other more age-related diseases. Emerging evidence has linked disturbance of sirtuin-1 (SIRT1) expression and activity with aging and diseases of the elderly. In the present study, we aimed to evaluate SIRT1 expression in the peripheral blood mononuclear cells (PBMCs) of patients with IPF given the lack of studies in the literature. We enrolled 34 IPF patients and 22 healthy volunteers (age and sex-matched). In both groups, SIRT1 levels were assessed in plasma, cell pellets of PBMCs, and supernatant from PBMCs' culture with and without the addition of 10% human serum. We also measured transforming growth factor β1 (TGF-β1) concentration in plasma from IPF patients and controls. The mean (SD) age (years) of the healthy volunteers was 68.57±6.97 and of the IPF patients was 71.28±5.39 years (p>0.05). The mean SIRT1 concentration was found significantly decreased in the supernatant of PBMCs culture (without the addition of serum) in IPF subjects versus controls (1.97±0.59 ng/ml versus 2.40±0.74 ng/ml, respectively, p=0.047). No significant differences were observed between the two groups in the SIRT1 concentration of all the other materials. TGFβ1 concentration of IPF subjects was significantly increased when compared to controls (1281.38±2742.74 versus 131.11±156.06 ng/ml, respectively, p=0.032). Decreased SIRT1 levels in no-serum supernatant were predictive of IPF, after adjustment for age and sex (p=0.014, OR=0.124 [95%CI: 0.023-0.653]). The findings of decreased concentration of SIRT1 in PBMCs supernatant and increased concentration of TGFβ1 in plasma in IPF patients versus controls provide important insights into the role of SIRT1 in IPF and could serve as a tool for the diagnosis and evaluation of patients with IPF.

Evaluation The Role of CD33+ CD11b+ myeloid-derived Suppressor Cells in Patients With AML

Acute myeloid leukemia is a genetically heterogeneous clonal disease defined by the accumulation of immature cells in bone marrow and blood. The aim of this study is to evaluate the CD33, CD11b, and HLA-DR expression in Iraqi AML patients and its role in evasion of malignant cells from the immune system. This study was conducted on 60 patients with AML and 20 healthy individuals as a control group to evaluate the expression of CD33 and CD11b using flow-cytometry in peripheral blood, and evaluate Interferons-γ (IFN-γ) and Transforming Growth Factor-β1 (TGF-β1) levels. A statistically non-significant difference (P < 0.005) between the AML patients and control group with regard to the expression of CD33, CD11b, and HLA-DR was observed. Analysis of CD33 expression in myeloid-derived suppressor cells (MDSCs) showed a significant decrease in the proportion of CD33 positive MDSC cells in isolated peripheral blood samples (88.078 ± 1.284). Also, the expression of CD11b in MDSC cells was high (98.841±1.935) in MDSC cells. The mean level of IFN-γ (pg/ml) increased in AML in compared to control group (9.202 ± 0.244), (7.906±1.22), respectively. While TGF- β1 (pg/ml) concentration was found to be elevated in AML patients (69.04 ± 9.92) compared to control group (33.884 ± 2.888). In conclusion, circulating MDSCs were significantly elevated in peripheral blood of patients with AML and characterized by the CD33+CD11b + phenotype; TGF-β1 and IFN-γ can be released in the presence of native human AML cells and affect AML cell proliferation and evasion from immune system.

Safety and Biocompatibility of Mupirocin Nanoparticle-Loaded Hydrogel on Burn Wound in Rat Model.

Mupirocin nanoparticle-loaded hydrogel (MLH) was successfully developed. This study focused on the safety of cell lines and the biocompatibility of MLH for wound healing in rat models. MLH was assessed by an analysis of cytotoxicity and the secretion of inflammatory cytokines in cell lines. The cytocompatibility of MLH was compared with mupirocin ointment on full-thickness burn wounds in rats. The results indicated that MLH and blank hydrogel had no toxicity to human epidermal keratinocytes and human fibroblast cells. MLH inhibited lipopolysaccharide (LPS) activity in macrophage-like cells resulting in low nitric oxide production and reduced inflammatory cytokine production (interleukin (IL)-1β) compared with a positive control (LPS only). In burn wounds, MLH and hydrogel healed the wound better than the other groups determined by wound contraction, reduced secretion, and the generation of new blood vessels, as well as promotion of hair follicle cells. Better granulation tissue proliferation, less necrosis, and a lower degree of inflammation were found in the MLH and blank hydrogel than in the mupirocin ointment. The hydrogel group reduced the macrophages (CD68) on day 14 at the edge of the wound. On day 28, T cells (CD3), B cells (CD20), and CD68+ cells were concentrated in the deeper subcutaneous tissue. Additionally, the transforming growth factor β1 (TGF-β1) concentration and matrix prometalloproteinase-2/tissue inhibitor of metalloproteinases-2 ratio in the MLH and hydrogel groups were less than those in the other groups. The MLH formulation was safe and effective in burn wound healing. Therefore, MLH formulations are promising candidates for further evaluation in clinical trials.

Scalable Production of Equine Platelet Lysate for Multipotent Mesenchymal Stromal Cell Culture.

Translation of multipotent mesenchymal stromal cell (MSC)-based therapies is advancing in human and veterinary medicine. One critical issue is the in vitro culture of MSC before clinical use. Using fetal bovine serum (FBS) as supplement to the basal medium is still the gold standard for cultivation of many cell types including equine MSC. Alternatives are being explored, with substantial success using platelet lysate-supplemented media for human MSC. However, progress lags behind in the veterinary field. The aim of this study was to establish a scalable protocol for equine platelet lysate (ePL) production and to test the ePL in equine MSC culture. Whole blood was harvested into blood collection bags from 20 healthy horses. After checking sample materials for pathogen contamination, samples from 19 animals were included. Platelet concentrates were prepared using a buffy coat method. Platelets, platelet-derived growth factor BB, and transforming growth factor β1 concentrations were increased in the concentrates compared with whole blood or serum (p < 0.05), while white blood cells were reduced (p < 0.05). The concentrates were lysed using freeze/thaw cycles, which eliminated the cells while growth factor concentrations were maintained. Donor age negatively correlated with platelet and growth factor concentrations after processing (p < 0.05). Finally, all lysates were pooled and the ePL was evaluated as culture medium supplement in comparison with FBS, using adipose-derived MSC from four unrelated donor horses. MSC proliferated well in 10% FBS as well as in 10% ePL. However, using 5 or 2.5% ePL entailed highly inconsistent proliferation or loss of proliferation, with significant differences in generation times and confluencies (p < 0.05). MSC expressed the surface antigens CD90, CD44, and CD29, but CD73 and CD105 detection was low in all culture media. Adipogenic and osteogenic differentiation led to similar results in MSC from different culture media. The buffy coat method is useful to produce equine platelet concentrate with increased platelet and reduced white blood cell content in large scales. The ePL obtained supports MSC expansion similar as FBS when used at the same concentration (10%). Further investigations into equine MSC functionality in culture with ePL should follow.

The relationship between β1 -adrenergic and M2 -muscarinic receptor autoantibodies and hypertrophic cardiomyopathy.

What is the central question of this study? The concentrations of β1 -adrenergic receptor and M2 -muscarinic receptor autoantibodies in hypertrophic cardiomyopathy (HCM) patients and the relationship between the cardiac autoantibodies and clinical manifestations of HCM have rarely been reported. What is the main finding and its importance? We found that the concentrations of the two autoantibodies in HCM patients were significantly higher than those in control subjects. Furthermore, we found that the concentrations of the two autoantibodies could reflect myocardial injury and diastolic dysfunction in HCM patients to some extent and might be involved in the occurrence of arrhythmia. These findings might be valuable in exploration of the mechanisms of occurrence and progression of HCM. Increasing attention is being given to the role of immunological mechanisms in the development of heart failure. The purpose of this study was to investigate the concentration of serum β1 -adrenergic receptor autoantibody (β1 -AAb) and M2 -muscarinic receptor autoantibody (M2 -AAb) in patients with hypertrophic cardiomyopathy (HCM), and the relationship between β1 -AAb, M2 -AAb and clinical indices. One hundred and thirty-four patients with HCM were recruited consecutively into the HCM group. Forty healthy subjects were assigned as the normal controls (NCs). Serum samples were collected to measure the concentrations of β1 -AAb and M2 -AAb by enzyme-linked immunosorbent assay. The clinical data of HCM patients were collected. The serum concentrations of β1 -AAb and M2 -AAb of HCM patients were significantly higher than those of NCs. In HCM patients, those with a left atrial diameter ≥50mm or moderate-to-severe mitral regurgitation had significantly higher concentrations of the two autoantibodies. Patients with a history of syncope had higher concentrations of β1 -AAb. Female patients and patients with a family history of sudden cardiac death or atrial fibrillation had higher concentrations of M2 -AAb. Maximal wall thickness, interventricular septum thickness and resting left ventricular outflow tract gradient were positively correlated with logβ1 -AAb or logM2 -AAb in HCM patients. In conclusion, the serum concentrations of β1 -AAb and M2 -AAb of HCM patients were significantly higher than those of NCs. Being female, syncope, a family history of sudden death, atrial fibrillation, left atrial diameter ≥50mm, moderate-to-severe mitral regurgitation, maximal wall thickness, interventricular septum thickness and resting left ventricular outflow tract gradient may affect the concentrations of the two autoantibodies.

Platelet and Transforming Growth Factor Beta 1 Concentrations in Platelet-rich Plasma in Dogs

Platelet-rich plasma showed good results in tissue healing when first used in human medicine. After that, its use spread to veterinary medicine. However, there is no standardized method for manual collection of platelet – rich plasma in the canine species. The objectives of this study were to standardize a protocol to obtain platelet – rich plasma (PRP) with high concentration of platelets and transforming growth factor β1 (TGF – β1) without the presence of erythrocytes and leukocytes; and to relate the presence of TGF – β1 with the amount of platelets. For this purpose, there were obtained two blood samples separated one week between each other, from eleven healthy Beagles. Blood samples were centrifuged using different protocols: protocol A (one centrifuge, 210 g and 10 minutes), protocol B (double centrifuge, first one 210 g and 10 minutes, second one 210 g and 15 minutes) and protocol C (one centrifuge, 475 g and 8 minutes). Three plasma fractions were obtained through these protocols: a platelet-rich fraction, a platelet-poor fraction and whole blood. The content of leukocytes, erythrocytes and platelets was measured in the whole blood and plasma rich and poor fractions of the protocol A, B and C. The TGF – β1 concentration was measured in the platelet rich and poor fraction of A and B protocols. The results showed a higher concentration of platelets and TGF – β1 in protocol A. In conclusion, this study offers an economical and reproducible method for obtaining PRP in the canine specie.

Evaluation of antifibrotic and antifungal combined therapies in experimental pulmonary paracoccidioidomycosis.

Paracoccidioidomycosis (PCM) is a systemic mycosis caused by the Paracoccidioides genus. Most of the patients with chronic form present sequelae, like pulmonary fibrosis, with no effective treatment, leading to impaired lung functions. In the present study, we aimed to investigate the antifibrotic activity of three compounds: pentoxifylline (PTX), azithromycin (AZT), and thalidomide (Thal) in a murine model of pulmonary PCM treated with itraconazole (ITC) or cotrimoxazole (CMX). BALB/c mice were inoculated with P. brasiliensis (Pb) by the intratracheal route and after 8 weeks, they were submitted to one of the following six treatments: PTX/ITC, PTX/CMX, AZT/ITC, AZT/CMX, Thal/ITC, and Thal/CMX. After 8 weeks of treatment, the lungs were collected for determination of fungal burden, production of OH-proline, deposition of reticulin fibers, and pulmonary concentrations of cytokines and growth factors. Pb-infected mice treated with PTX/ITC presented a reduction in the pulmonary concentrations of OH-proline, associated with lower concentrations of interleukin (IL)-6, IL-17, and transforming growth factor (TGF)-β1 and higher concentrations of IL-10 compared to the controls. The Pb-infected mice treated with AZT/CMX exhibited decreased pulmonary concentrations of OH-proline associated with lower levels of TGF-β1, and higher levels of IL-10 compared controls. The mice treated with ITC/Thal and CMX/Thal showed intense weight loss, increased deposition of reticulin fibers, high pulmonary concentrations of CCL3, IFN-γ and VEGF, and decreased concentrations of IL-6, IL-1β, IL-17, and TGF-β1. In conclusion, our findings reinforce the antifibrotic role of PTX only when associated with ITC, and AZT only when associated with CMX, but Thal did not show any action upon addition.

Berberine Attenuated Oxidative Stress Induced by Sodium Nitrite in Rat Liver

Background: Berberine is a well-known alkaloid derived from Berberis species. Objectives: The present study aimed to investigate the hepatoprotective properties of berberine and elucidate its probable mechanisms against sodium nitrite toxicity. Methods: Forty animals were randomly classified into five equal groups to be treated for 60 days, including group 1: Control, group 2: Berberine-treated (100 mg/kg), group 3: Sodium nitrite-treated (80 mg/kg), group 4: Sodium nitrite together with 50 mg/kg of berberine, and group 5: Sodium nitrite together with 100 mg/kg of berberine. The protective effects of berberine against sodium nitrite induced-liver damage were investigated using parameters related to oxidative stress, inflammation, fibrosis, and apoptosis in the hepatocytes. Results: Treatment of rats with sodium nitrite considerably increased alanine aminotransferase (ALT) and alkaline phosphatase (ALP) activities, malondialdehyde (MDA) content, tumor necrosis factor (TNF)-α expression, caspase-3 activity, and transforming growth factor (TGF)-β1 concentration (P < 0.05) and significantly declined the levels of reduced glutathione (GSH), glutathione reductase (GR), glutathione S-transferase (GST), and glutathione peroxidase (GPx) (P < 0.05). The treatment of intoxicated rats with 100 mg/kg of berberine significantly reversed these changes and reached the values approximately to the normal level. However, berberine 50 mg/kg failed to normalize the disturbances. Conclusions: This study demonstrated that berberine could decrease sodium nitrite-induced liver injury in a dose-dependent manner probably due to its antioxidant, anti-inflammatory, anti-apoptotic, and antifibrotic capacities.

Screening of schizophrenia associated miRNAs and the regulation of miR-320a-3p on integrin β1.

Schizophrenia is one of the most severe chronic psychiatric disorders, which lacks of objective and effective diagnosis and observation indicators.In this work, the serum miRNA profiles of schizophrenic patients were analyzed. Targets of abnormal miRNAs, and their regulatory mechanisms were studied. A miRNA array was used to analyze the serum from 3 schizophrenic patients without treatment, 3 clinically cured patients and 3 healthy controls. The findings from the array were confirmed by real-time PCR in a larger cohort, including 59 patients and 60 healthy controls. The candidate miRNAs were analyzed using bioinformatics tools. Their potential targets were studied through in vitro cellular experiments.MiR-320a-3p and miR-320b were found to be down-regulated in patients compared with cured patients and controls in the miRNA array, which was also confirmed by real-time PCR in the larger cohort. Integrin β1 (ITG β1) was found to be one of the targets of miR-320a-3p. An enzyme-linked immune sorbent assay demonstrated that the ITG β1 concentration increased significantly in the patients’ serum, and the in vitro study confirmed that miR-320a-3p targeted the 3′ UTR of ITG β1 mRNA and reduced its expression.Our results demonstrated that the regulatory effect of miR-320a-3p on its target ITG β1 might play an important role in schizophrenia pathogenesis, which could be a potential pathway for schizophrenia diagnosis and therapy.

Assessment of Transforming Growth Factor-β1 in Gingival Crevicular Fluid around Bony Defects Treated with Platelet Rich Fibrin (PRF) System

Purpose: This study was carried to evaluate and compare the regenerative capacity of PRF mixed with bioactive glass versus PRF mixed with bioactive glass combined with collagen membrane in treatment of intrabony defects. This assessment was based on TGF- β1 concentration in GCF. Patients and materials: A sample of 36 intra bony defects were randomaly divided into; Group I: bioactive glass mixed with PRF. Group II: collagen membrane with bioactive glass mixed with PRF. Group III: open flap debridement only. The GCF TGF-β1 was analyzed by ELIZA at base line, one and three months postoperatively and evaluate the efficacy of PRF system in intrabony defects clinically and radiographically at base line, one, three and six months. Results: Mean TGF-β1 concentrations were significantly reduced in one and three months post-surgery when compared to baseline. The mean values of TGF-β1 at 3 months was 0.12± 0, 0.4±0.01 and 0.11±0.01 in groups I, II, III respectively. There was a statistically significant reduction in periodontal probing depth that was 6±0.97 and gain in CAL 3.87±2.14 mm both in group II after 6 months. There was a highest bone density increase was18±5.84 in group I. Conclusions: The assessment of cervical TGF-β1 levels is considered to reflect the ongoing processes in the surrounding periodontal tissues. In addition, utilizing PRF mixed with bioactive glass might be considered a promising regenerative therapy.

Optimization of pure platelet-rich plasma preparation: A comparative study of pure platelet-rich plasma obtained using different centrifugal conditions in a single-donor model.

While it has been proved that centrifugal conditions for pure platelet-rich plasma (P-PRP) preparation influence the cellular composition of P-PRP obtained, the optimal centrifugal conditions to prepare P-PRP have not yet been identified. In the present study, platelet-containing plasma (PCP) was prepared with the first-spin of different double-spin methods and P-PRP was prepared with different double-spin methods. Whole-blood analysis was performed to evaluate the cellular composition of PCP and P-PRP. The basal and ADP-induced CD62P expression rates of platelets were assessed by flow cytometry to evaluate the function of platelets in PCP and P-PRP. Enzyme-linked immune sorbent assay was performed to quantify interleukin-1β, tumor necrosis factor-α, platelet-derived growth factor AB and transforming growth factor β1 concentrations of PCP and P-PRP. Correlations between the cellular characteristics and cytokine concentrations of P-PRP were analyzed by Pearson correlation analysis. Effects of P-PRP on the proliferation, survival and migration of human bone marrow-derived mesenchymal stem cells and human articular chondrocytes were evaluated by a Cell Counting Kit-8 assay, live/dead staining and Transwell assay, respectively. The results showed that centrifugation at 160 × g for 10 min and 250 × g for 15 min successively captured and concentrated platelets and growth factors significantly more efficiently with preservation of platelet function compared with other conditions (P<0.05). The correlation analysis showed that the similar leukocyte concentrations and leukocyte-reducing efficiencies resulted in similar pro-inflammatory cytokine concentrations in P-PRP (P>0.05) and the maximization of platelet concentration, platelet enrichment factor, platelet capture efficiency and platelet function resulted in the maximization of growth factor concentrations in P-PRP obtained using the optimal conditions (P<0.05). Compared with P-PRP obtained under other conditions, P-PRP obtained under the optimal conditions significantly promoted the proliferation and migration of cells (P<0.05) and did not alter cell survival (P>0.05). Therefore, centrifugation at 160 × g for 10 min and 250 × g for 15 min successively with removal of the buffy coat as a crucial step may provide an optimal preparation system of P-PRP for clinical application.

Growth Factor Concentration in Keratocyte Supernatant after Incubation with Human Serum In Vitro

Application of serum eye drops is an alternative treatment option for therapy-resistant corneal epithelial defects. In case of persisting epithelial defects, cytokines, which are secreted from stromal keratocytes, may play a role in epithelial wound healing. Our aim was to analyze fibroblast growth factor basic (FGFb), hepatocyte growth factor (HGF), keratinocyte growth factor (KGF) and transforming growth factor β1 (TGF-β1) concentration in the supernatant of keratocytes, after incubation with human serum (HS). Serum eye drops of 10 patients were prepared using the standards of the LIONS Eye Bank Saar-Lor-Lux, Trier/Westpfalz, and were stored at - 80 °C. Primary human keratocytes were isolated from human corneoscleral rings using collagenase A (1 mg/ml) (n = 1) and were cultured in DMEM/Ham's culture medium with 10% fetal bovine serum (FBS). Thereafter, keratocyte cultures were incubated in 15 or 30% HS (in DMEM/F14 without FBS) and FGFb, HGF, KGF and TGF-β1 concentration was determined with an enzyme-linked immunoabsorbent assay (ELISA) from the supernatant of the culture after 24 hours. We used 15 or 30% HS without keratocytes (under the same storage conditions) as controls. HGF concentration was, for both HS concentrations, significantly higher in the supernatant of keratocytes, than in HS controls (without keratocytes) following 24 hours (p < 0.01). FGFb concentration was significantly increased in 30% HS with keratocytes compared to 30% HS without keratocytes after 24 hours (p < 0.01). TGF-β1 and KGF concentrations remained unchanged through keratocytes. HGF and FGFb concentrations increase in the supernatant of keratocytes, 24 hours after incubation with human serum. These concentration changes may play a role in wound healing of epithelial defects.

Optimization of culture conditions to improve the expression level of beta1–epsilon toxin of Clostridium perfringens type B in Escherichia coli

ABSTRACTThe detoxified beta1–epsilon (β1–ϵ) toxin protein of Clostridium perfringens type B provides protection from C. perfringens types B, C and D infections. Acetate is the primary by-product from the cell growth and expression of β1–ϵ protein. In the present study, the effects of pH and dissolved oxygen (DO) on the expression of β1--ϵ protein were investigated. Two-stage pH and DO control strategies were developed for the expression of β1–ϵ protein. The obtained results indicated that higher cell density and concentration of β1--ϵ protein, and lower accumulation of acetate were obtained when pH was maintained at a constant level of 6.5 (0–6 h) and 7.0 (6–16 h), and the DO level was maintained at 60% (0–6 h) and 30% (6–16 h). Furthermore, the impact of intermittent, DO feedback, pH feedback and glucose-stat feeding on the expression of β1–ϵ protein were studied. By using the DO feedback feeding, combined with the stage control of pH (6.5 for 0–6 h, 7.0 for 6–16 h) and DO (60% for 0–6 h, 30% for 6–16 h), the highest cell density of 2.045 (absorbance at 600 nm) and a β1–ϵ protein concentration of 63.24 mg/L were obtained, and the accumulation of acetate decreased to 0.872 g/L.

Effect of Manuka honey gel on the transforming growth factor β1 and β3 concentrations, bacterial counts and histomorphology of contaminated full-thickness skin wounds in equine distal limbs.

To investigate the effect of 66% Manuka honey gel on the concentrations of transforming growth factor (TGF)-β1 and TGF-β3, bacterial counts and histomorphology during healing of contaminated equine distal limb wounds. In this experimental study of 10 Standardbred horses, five full-thickness skin wounds (2 × 1.5 cm) were created on one metacarpus and six similar wounds were created on the contralateral metacarpus. Wounds were assigned to three groups: non-contaminated control wounds; contaminated control wounds; contaminated wounds treated daily with 1 mL Manuka honey gel topically for 10 days. For the contaminated wounds, faeces were applied for 24 h after wound creation. In five horses wounds were bandaged and in the other five horses wounds were left without a bandage. Biopsies were taken on days 1, 2, 7 and 10 after wounding to evaluate the effects of Manuka honey gel, wound contamination and bandaging on TGF-β1 and TGF-β3 concentrations, aerobic and anaerobic bacterial counts, and histomorphology. Manuka honey gel had no significant effect on TGF-β1 and TGF-β3 concentrations or wound bacterial counts. Manuka honey gel decreased wound inflammation (days 7, 10), increased angiogenesis (days 2, 7, 10), increased fibrosis and collagen organisation (day 7) and increased epithelial hyperplasia (days 7, 10). Treatment with Manuka honey gel resulted in a more organised granulation tissue bed early in wound repair, which may contribute to enhanced healing of equine distal limb wounds.

Serum protein concentration and protein fractions in clinically healthy Lacaune and Sarda sheep using agarose gel electrophoresis.

Serum protein electrophoresis (SPE) provides useful information in ruminants, but reference intervals (RI) are different from other species. There have been no reports of SPE RI for dairy sheep using agarose gel electrophoresis (AGE). The purpose of the study was to evaluate the serum concentration of total protein (TP) and protein fractions determined by AGE in mid-lactating dairy ewes, to establish RI, and to assess potential differences between Lacaune (L) and Sarda (S) sheep breeds. Blood samples were collected from healthy, mid-lactating ewes. SPE was assessed using a semi-automated AGE system. Reference intervals (90% confidence intervals) for TP and each protein fraction were determined using the nonparametric method for combined data, and the robust method for data from the single breeds. Data from S and L sheep were compared using the Mann-Whitney U test. The 172 sheep included 116 L and 56 S ewes, 2-6 years old. There were significant differences between S and L breeds, and RI were calculated for TP, albumin, α1 -globulin, α2 -globulin, β1 -globulin, β2 -globulin, γ1 -globulin, and γ2 -globulin concentrations, and for the Albumin/Globulin ratio. Group S showed higher concentrations of TP, α2 -, β1 -, β2 -, and γ1 -globulins, whereas L was higher for albumin and γ2 -globulin concentrations, and A/G ratio (P < .05). The resolution with AGE was excellent, allowing standardization of 7 protein fractions, detection of differences between S and L ewes, and determination of RI for French (Lacaune) and Italian (Sarda) dairy sheep.