17 Citations
Melanin binding of small molecule drugs can lead to targeted disposition to the pigmented tissues and prolonged pharmacological responses in the eye Melanin binding of drugs in vitro correlates with in vivo binding but current workflows for binding affinity require multiple slow steps and analytical method development and or they may result in high data variability We developed tangential flow filtration-based methodology to produce size-specific fractions of water-soluble melanin nanoparticles MNPs reducing production time from - days to just a few hours and yielding MNPs with enhanced fluorescence signal Improved MNPs enabled modifications to a previously published microscale thermophoresis-based melanin ... More
Melanin binding of small molecule drugs can lead to targeted disposition to the pigmented tissues and prolonged pharmacological responses in the eye. Melanin binding of drugs in vitro correlates with in vivo binding, but current workflows for binding affinity require multiple slow steps, and analytical method development, and/or they may result in high data variability. We developed tangential flow filtration-based methodology to produce size-specific fractions of water-soluble melanin nanoparticles (MNPs), reducing production time from 2-3 days to just a few hours and yielding MNPs with enhanced fluorescence signal. Improved MNPs enabled modifications to a previously published microscale thermophoresis-based melanin binding protocol, shifting analytical focus toward thermophoretic behavior, reducing data variability, and improving reproducibility. The process was tested with nine compounds with varying melanin binding affinities, and the results were consistent with literature, confirming the ability of the method to differentiate compounds based on melanin binding. Fast and reliable workflow will be useful in screening binding affinity for therapeutics and new drug candidates to melanin thereby facilitating ocular drug discovery and construction of predictive pharmacokinetic simulation models. Less
Abstract Background Recombinant human NELL- rhNELL- is a potent osteogenic protein with therapeutic potential in regenerative medicine A stable formulation is essential to prevent aggregation during production filling storage and clinical use Methodology A four-stage rational formulation strategy was used identify intrinsic aggregation risks of rhNELL- screen polysorbate- and cyclodextrin-based formulations to enhance colloidal and conformational stability test lead candidates under agitation freeze thaw pH shifts and elevated temperature Analytical techniques included PEG challenge differential scanning fluorimetry DSF isothermal chemical denaturation ICD and dynamic light scattering DLS Aggregation was assessed via visible particles VP opalescence subvisible particles SVP Micro Flow ... More
Abstract
Background
Recombinant human NELL-1 (rhNELL-1) is a potent osteogenic protein with therapeutic potential in regenerative medicine. A stable formulation is essential to prevent aggregation during production, filling, storage, and clinical use.
Methodology
A four-stage rational formulation strategy was used: (1) identify intrinsic aggregation risks of rhNELL-1; (2) screen polysorbate- and cyclodextrin-based formulations to enhance colloidal and conformational stability; (3–4) test lead candidates under agitation, freeze/thaw, pH shifts, and elevated temperature. Analytical techniques included PEG challenge, differential scanning fluorimetry (DSF), isothermal chemical denaturation (ICD), and dynamic light scattering (DLS). Aggregation was assessed via visible particles (VP), opalescence, subvisible particles (SVP, Micro Flow Imaging), SDS-PAGE, and ultra-high performance size exclusion chromatography (UP-SEC).
Results
rhNELL-1 was prone to self-association via hydrophobic and electrostatic interactions. Polysorbate 20 (PS20) and hydroxypropyl beta cyclodextrin (HPB-LB-BCD) improved protein stability. PS20 markedly reduced VP and SVP formation. While HPB-LB-BCD alone did not further reduce SVP beyond PS20, it enhanced thermal stress resistance. PS20 was more effective under agitation.
Conclusions
Two lead formulations containing potassium phosphate/Tris buffer, sorbitol, PS20, and HPB-LB-BCD demonstrated strong resistance to aggregation under multiple stresses. PS20 mitigated interfacial stress, while HPB-LB-BCD suppressed solution-phase aggregation, especially at high temperatures. This systematic approach offers a framework for stabilizing other aggregation-prone proteins. Less
Background
Recombinant human NELL-1 (rhNELL-1) is a potent osteogenic protein with therapeutic potential in regenerative medicine. A stable formulation is essential to prevent aggregation during production, filling, storage, and clinical use.
Methodology
A four-stage rational formulation strategy was used: (1) identify intrinsic aggregation risks of rhNELL-1; (2) screen polysorbate- and cyclodextrin-based formulations to enhance colloidal and conformational stability; (3–4) test lead candidates under agitation, freeze/thaw, pH shifts, and elevated temperature. Analytical techniques included PEG challenge, differential scanning fluorimetry (DSF), isothermal chemical denaturation (ICD), and dynamic light scattering (DLS). Aggregation was assessed via visible particles (VP), opalescence, subvisible particles (SVP, Micro Flow Imaging), SDS-PAGE, and ultra-high performance size exclusion chromatography (UP-SEC).
Results
rhNELL-1 was prone to self-association via hydrophobic and electrostatic interactions. Polysorbate 20 (PS20) and hydroxypropyl beta cyclodextrin (HPB-LB-BCD) improved protein stability. PS20 markedly reduced VP and SVP formation. While HPB-LB-BCD alone did not further reduce SVP beyond PS20, it enhanced thermal stress resistance. PS20 was more effective under agitation.
Conclusions
Two lead formulations containing potassium phosphate/Tris buffer, sorbitol, PS20, and HPB-LB-BCD demonstrated strong resistance to aggregation under multiple stresses. PS20 mitigated interfacial stress, while HPB-LB-BCD suppressed solution-phase aggregation, especially at high temperatures. This systematic approach offers a framework for stabilizing other aggregation-prone proteins. Less
Antibody-drug conjugates ADCs are a promising therapeutic modality that enables the delivery of cytotoxic drugs to the target cells that express the corresponding antigen However the purification of ADCs while ensuring product safety homogeneity and stability is a challenging task due to their complex and fragile structure Size exclusion chromatography SEC the conventional method for ADC purification is time-consuming as it requires multiple column washes and equilibration steps Moreover subsequent formulation of ADCs typically using dead-end filtration DEF further complicates the production workflow We compared SEC DEF with the Pulse a miniaturized and automated tangential flow filtration system for purification ... More
Antibody-drug conjugates (ADCs) are a promising therapeutic modality that enables the delivery of cytotoxic drugs to the target cells that express the corresponding antigen. However, the purification of ADCs while ensuring product safety, homogeneity, and stability is a challenging task due to their complex and fragile structure. Size exclusion chromatography (SEC), the conventional method for ADC purification, is time-consuming as it requires multiple column washes and equilibration steps. Moreover, subsequent formulation of ADCs, typically using dead-end filtration (DEF), further complicates the production workflow. We compared SEC+DEF with the µPulse®, a miniaturized and automated tangential flow filtration system, for purification and formulation of ADCs. Quality analysis revealed that both approaches were equally gentle as comparable drug-to-antibody ratios (DARs) and monomer purities were observed in the purified samples. Most importantly, both methods exhibited equivalent cleanup efficiency with a 99.8% reduction in free linker-drug concentration. The endotoxin loads comprised 0.11 EU mg-1 for the µPulse and 0.07 EU mg-1 for SEC+DEF, ensuring validation of the safe application of purified ADCs in living systems. However, the µPulse performed purification and formulation of ADCs simultaneously as compared to SEC+DEF, which required multiple manual interventions. Our results indicate that the µPulse is a gentle, single-step, and walk-away approach for the purification of ADCs. Less
Reinvigoration of tumor-reactive T-cells using co-stimulatory bispecific antibodies bsAbs targeting CD or CD is emerging as a promising therapeutic strategy Conditional tumor-specific recruitment can offer a necessary layer of control and specificity We developed pH-selective CD xVISTA bsAbs to act specifically within the acidic tumor microenvironment TME aiming for enhanced T-cell-mediated cancer cell killing while minimizing systemic T-cell activation and Cytokine Release Syndrome CRS risk CD agonism by CD xVISTA bsAbs relies on pH-selective engagement of VISTA a protein robustly expressed on myeloid cells highly prevalent in most solid tumors This modality avoids engagement of tumor-associated antigens TAAs with the ... More
Reinvigoration of tumor-reactive T-cells using co-stimulatory bispecific antibodies (bsAbs) targeting CD28 or CD137 is emerging as a promising therapeutic strategy. Conditional, tumor-specific recruitment can offer a necessary layer of control and specificity. We developed pH-selective CD28xVISTA bsAbs to act specifically within the acidic tumor microenvironment (TME), aiming for enhanced T-cell-mediated cancer cell killing while minimizing systemic T-cell activation and Cytokine Release Syndrome (CRS) risk. CD28 agonism by CD28xVISTA bsAbs relies on pH-selective engagement of VISTA, a protein robustly expressed on myeloid cells highly prevalent in most solid tumors. This modality avoids engagement of tumor-associated antigens (TAAs) with the potential to provide highly tumor specific activity with minimal on-target/off-tumor side effects.
We report the identification of a lead candidate with pH-dependent simultaneous engagement of both targets, and VISTA-dependent CD28 signaling in a reporter cell line. CD28xVISTA avidly bound VISTA-positive cells, and co-stimulation was shown in vitro by its ability to activate and expand T-cells and enhance T-cell mediated cancer cell killing in co-cultures of human PBMCs and cancer cells in the presence of a TAA-targeted anti-CD3 T-cell engager. Interestingly, our findings support both signaling in cis (between T-cell and cell displaying peptide-MHC complex) and in trans with stimulation occurring through CD28 clustering outside of the immune synapse. Our lead candidate displayed efficient tumor growth inhibition of human VISTA-expressing MC38 cells in a humanized CD28 syngeneic mouse model in combination with PD-1 blockade. Importantly, our CD28xVISTA bsAb showed no signs of superagonistic properties in several in vitro assays geared towards revealing induction of CRS. Our data supports clinical development in combination with anti-PD-1 or any TAA-targeted anti-CD3 T-cell engagers developed for solid tumors. Less
We report the identification of a lead candidate with pH-dependent simultaneous engagement of both targets, and VISTA-dependent CD28 signaling in a reporter cell line. CD28xVISTA avidly bound VISTA-positive cells, and co-stimulation was shown in vitro by its ability to activate and expand T-cells and enhance T-cell mediated cancer cell killing in co-cultures of human PBMCs and cancer cells in the presence of a TAA-targeted anti-CD3 T-cell engager. Interestingly, our findings support both signaling in cis (between T-cell and cell displaying peptide-MHC complex) and in trans with stimulation occurring through CD28 clustering outside of the immune synapse. Our lead candidate displayed efficient tumor growth inhibition of human VISTA-expressing MC38 cells in a humanized CD28 syngeneic mouse model in combination with PD-1 blockade. Importantly, our CD28xVISTA bsAb showed no signs of superagonistic properties in several in vitro assays geared towards revealing induction of CRS. Our data supports clinical development in combination with anti-PD-1 or any TAA-targeted anti-CD3 T-cell engagers developed for solid tumors. Less
Abstract Background Chronic inflammation and oxidative stress are central to the pathophysiology of Type Diabetes Mellitus T DM contributing to the progression of metabolic dysfunction and related complications Objective The aim of this study was to explore the therapeutic potential of combining hypoxia-preconditioned mesenchymal stem cell SH-MSC with alkaline water in a T DM rat model Methods T DM was induced in Wistar rats through a high-fat diet HFD followed by streptozotocin STZ administration A total of healthy male Wistar rats were randomly assigned to five groups healthy control T DM T DM Metformin T DM SH-MSC and T DM ... More
Abstract
Background:
Chronic inflammation and oxidative stress are central to the pathophysiology of Type 2 Diabetes Mellitus (T2DM), contributing to the progression of metabolic dysfunction and related complications.
Objective:
The aim of this study was to explore the therapeutic potential of combining hypoxia-preconditioned mesenchymal stem cell SH-MSC with alkaline water in a T2DM rat model.
Methods:
T2DM was induced in Wistar rats through a high-fat diet (HFD) followed by streptozotocin (STZ) administration. A total of 30 healthy male Wistar rats were randomly assigned to five groups: healthy control, T2DM, T2DM + Metformin, T2DM + SH-MSC, and T2DM + SH-MSC + alkaline water.
Results:
The combination of SH-MSC and alkaline water significantly reduced malondialdehyde (MDA) levels, a key indicator of lipid peroxidation, and suppressed the expression of p65 mRNA, a crucial component of the NF-κB signaling pathway. Notably, the most pronounced reduction in p65 mRNA expression was observed in the group receiving both SH-MSC and alkaline water, suggesting a synergistic effect in mitigating oxidative stress and inflammation.
Conclusion:
These findings highlight the potential of SH-MSC and alkaline water as a novel therapeutic strategy for alleviating T2DM. Less
Background:
Chronic inflammation and oxidative stress are central to the pathophysiology of Type 2 Diabetes Mellitus (T2DM), contributing to the progression of metabolic dysfunction and related complications.
Objective:
The aim of this study was to explore the therapeutic potential of combining hypoxia-preconditioned mesenchymal stem cell SH-MSC with alkaline water in a T2DM rat model.
Methods:
T2DM was induced in Wistar rats through a high-fat diet (HFD) followed by streptozotocin (STZ) administration. A total of 30 healthy male Wistar rats were randomly assigned to five groups: healthy control, T2DM, T2DM + Metformin, T2DM + SH-MSC, and T2DM + SH-MSC + alkaline water.
Results:
The combination of SH-MSC and alkaline water significantly reduced malondialdehyde (MDA) levels, a key indicator of lipid peroxidation, and suppressed the expression of p65 mRNA, a crucial component of the NF-κB signaling pathway. Notably, the most pronounced reduction in p65 mRNA expression was observed in the group receiving both SH-MSC and alkaline water, suggesting a synergistic effect in mitigating oxidative stress and inflammation.
Conclusion:
These findings highlight the potential of SH-MSC and alkaline water as a novel therapeutic strategy for alleviating T2DM. Less
Background High concentration protein formulation HCPF development needs to balance protein stability attributes such as conformational colloidal stability chemical stability and solution properties such as viscosity and osmolality Methodology A three-phase design is established in this work In Phase conformational and colloidal stability are measured by -well-based high-throughput HT biophysical screening while viscosity reduction screening is performed with HT viscosity screening Collectively the biophysical and viscosity screening data are leveraged to design the phase of short-term stability study executed using -well plates under thermal and freeze thaw stresses In phase samples are analyzed by stability-indicating assays and processed with pair-wise ... More
Background High concentration protein formulation (HCPF) development needs to balance protein stability attributes such
as conformational/colloidal stability, chemical stability, and solution properties such as viscosity and osmolality.
Methodology A three-phase design is established in this work. In Phase 1, conformational and colloidal stability are measured by 384-well-based high-throughput (HT) biophysical screening while viscosity reduction screening is performed with HT viscosity screening. Collectively, the biophysical and viscosity screening data are leveraged to design the phase 2 of short-term stability study, executed using 96-well plates under thermal and freeze/thaw stresses. In phase 2, samples are analyzed by stability-indicating assays and processed with pair-wise Student’s t-test analyses to choose the final formulations. In phase 3, the final formulations are then confirmed through a one-month accelerated stability in glass vials.
Results Using a model antibody A (mAb-A), the initial HT screening successfully established the 384-well based platform.
A lead formulation was chosen from the second round based on statistical analyses and subsequently tested against the commercial
formulation of mAb-A as a control. Compared to the control, the lead formulation reduced the viscosity of mAb-A by 30% and decreased subvisible particles after thermal stress by 80%.
Conclusions HT biophysical screening in 384-well plates was demonstrated to effectively guide the rational design of a high-throughput stability screening study using 96-well plates. This platform enables the identification of a high concentration formulation within seven weeks within the first two phases of study that strategically balance stability with solution
properties, thus achieving a rapid development of HCPF. Less
as conformational/colloidal stability, chemical stability, and solution properties such as viscosity and osmolality.
Methodology A three-phase design is established in this work. In Phase 1, conformational and colloidal stability are measured by 384-well-based high-throughput (HT) biophysical screening while viscosity reduction screening is performed with HT viscosity screening. Collectively, the biophysical and viscosity screening data are leveraged to design the phase 2 of short-term stability study, executed using 96-well plates under thermal and freeze/thaw stresses. In phase 2, samples are analyzed by stability-indicating assays and processed with pair-wise Student’s t-test analyses to choose the final formulations. In phase 3, the final formulations are then confirmed through a one-month accelerated stability in glass vials.
Results Using a model antibody A (mAb-A), the initial HT screening successfully established the 384-well based platform.
A lead formulation was chosen from the second round based on statistical analyses and subsequently tested against the commercial
formulation of mAb-A as a control. Compared to the control, the lead formulation reduced the viscosity of mAb-A by 30% and decreased subvisible particles after thermal stress by 80%.
Conclusions HT biophysical screening in 384-well plates was demonstrated to effectively guide the rational design of a high-throughput stability screening study using 96-well plates. This platform enables the identification of a high concentration formulation within seven weeks within the first two phases of study that strategically balance stability with solution
properties, thus achieving a rapid development of HCPF. Less
Background Chronic inflammation is central to the pathophysiology of Type Diabetes Mellitus T DM contributing to the progression of metabolic dysfunction characterized by hyperglycaemia and insulin resistance This study aims to investigate the therapeutic potential of the hypoxic MSCs secretome SH-MSCs in reducing inflammation of a T DM rat model Methods T DM was induced in Wistar rats through a high-fat diet HFD followed by streptozotocin STZ administration A total of healthy male Wistar rats were randomly assigned to five groups healthy control T DM T DM metformin T DM SH-MSCs Results SH-MSCs significantly reduced IL- mRNA expression a key ... More
Background: Chronic inflammation is central to the pathophysiology of Type 2 Diabetes Mellitus (T2DM), contributing to the progression of metabolic dysfunction characterized by hyperglycaemia and insulin resistance. This study aims to investigate the therapeutic potential of the hypoxic MSCs secretome (SH-MSCs) in reducing inflammation of a T2DM rat model. Methods: T2DM was induced in Wistar rats through a high-fat diet (HFD) followed by streptozotocin (STZ) administration. A total of 24 healthy male Wistar rats were randomly assigned to five groups: healthy control, T2DM, T2DM + metformin, T2DM + SH-MSCs. Results: SH-MSCs significantly reduced IL-18 mRNA expression, a key indicator of proinflammation, and suppressed the expression of AP-1 mRNA, a crucial proinflammatory transcription factor. Conclusion: These findings highlight the therapeutic potential of SH-MSCs as an alternative approach to alleviate inflammation in T2DM. Less
Background Psoriasis is a chronic immune-mediated skin disease that also has systemic manifestations Case In this report we discuss our findings about a -years old psoriasis suffering male patient with a Psoriasis Area Severity Index PASI score of treated with Wharton s Jelly Mesenchymal Stem Cells-derived Secretome S-MSCs Remarkably complete regression was recorded within a treatment period of a week only Result The patient demonstrated a decrease in PASI from to after infusion and followed by intramuscular injections of S-MSCs Bioactive factors secreted by MSCs cytokines and growth factors are very likely to be the principal molecules which play a ... More
Background: Psoriasis is a chronic, immune-mediated skin disease that also has systemic manifestations. Case: In this report, we discuss our findings about a 47-years old psoriasis suffering male patient with a Psoriasis Area Severity Index (PASI) score of 10.8, treated with Wharton’s Jelly Mesenchymal Stem Cells-derived Secretome (S-MSCs). Remarkably, complete regression was recorded within a treatment period of a week only. Result: The patient demonstrated a decrease in PASI, from 10.8 to 3.2 after 1 infusion and followed by 4 intramuscular injections of S-MSCs. Bioactive factors secreted by MSCs, cytokines and growth factors, are very likely to be the principal molecules which play a vital role in inflammatory modulation and skin tissue regeneration. No serious adverse events were noted for the patient as a result of secretome infusion and intramuscular injection. Conclusion: This report demonstrates safety and promises to be an effective strategy using S-MSCs treatment for managing the psoriatic issue and, thus, may offer as an alternative approach to overcome the limitations of the cell-based therapy. Less
Metabolic syndrome MetS has become a global health challenge with several associated issues such as obesity insulin resistance dyslipidemia and hypertension Important proteins such as Tumor Necrosis Factor-alpha TNF- and Hypoxia Inducible Factor- alpha HIF- regulate the inflammatory process by inducing the expression of pro-inflammatory proteins This study aims to determine the effect of administering SH-MSCSs on the expression of the TNF- and Hypoxia Inducible Factor HIF - genes in the male Wistar rat model with Metabolic Syndrome This research is an experimental study with a Post-test Only Control Group Design using a total of male Wistar rats divided into ... More
Metabolic syndrome (MetS) has become a global health challenge with several associated issues, such as obesity, insulin resistance, dyslipidemia, and hypertension. Important proteins such as Tumor Necrosis Factor-alpha (TNF-α) and Hypoxia Inducible Factor-2 alpha (HIF-2α) regulate the inflammatory process by inducing the expression of pro-inflammatory proteins. This study aims to determine the effect of administering SH-MSCSs on the expression of the TNF-α and Hypoxia Inducible Factor (HIF)-2α genes in the male Wistar rat model with Metabolic Syndrome. This research is an experimental study with a Post-test Only Control Group Design, using a total of 24 male Wistar rats divided into four groups: T1 (Healthy control), T2 (MetS + NaCl), T3 (MetS + administration of SH-MSCs dose 150 uL), and T4 (MetS + administration of SH-MSCs dose 300 uL). SH-MSCSs were administered intraperitoneally four times over 14 days. Adipose tissue TNF-α and HIF-2α gene expression were measured on day 15 using qRT-PCR. TNF-α and HIF-2α gene expression was significantly lower in T3 and T4, compared with the MetS control group (T2). Administration of SH-MSCs was able to reduce the expression of the Tumor Necrosis Factor (TNF-α) and Hypoxia Inducible Factor (HIF)-2α genes in fatty tissue in the male Wistar rat model with Metabolic Syndrome.This study presents a novel approach to treating MetS by demonstrating that the administration of SH-MSCs significantly reduces the expression of pro-inflammatory genes TNF-α and HIF-2α. This finding is beneficial for society as it suggests a potential newtherapeutic strategy that could mitigate inflammation and improve health outcomes for individuals suffering from MetS, thereby addressing a critical global health challenge. Less
Introduction Type diabetes mellitus T DM is a prevalent form of diabetes that affects - of all diabetic patients Insulin sensitizers and insulin exogenous supply could temporarily ameliorate hyperglycaemia however they are accompanied by side effects As a result new approaches are required to address insulin resistance and regenerate beta cells simultaneously The secretome of hypoxic mesenchymal stem cells SH-MSCs contains various growth factors and anti-inflammatory cytokines that could potentially enhance insulin resistance and improve pancreatic function Objectives In this study we performed SH-MSCs infusion to ameliorate HFD-induced hyperglycaemia in T DM rats Methods We created a T DM rat ... More
Introduction: Type 2 diabetes mellitus (T2DM) is a prevalent form of diabetes that affects 90 - 95 % of all diabetic patients. Insulin sensitizers and insulin exogenous supply could temporarily ameliorate hyperglycaemia; however, they are accompanied by side effects. As a result, new approaches are required to address insulin resistance and regenerate beta cells simultaneously. The secretome of hypoxic mesenchymal stem cells (SH-MSCs) contains various growth factors and anti-inflammatory cytokines that could potentially enhance insulin resistance and improve pancreatic function. Objectives: In this study, we performed SH-MSCs infusion to ameliorate HFD-induced hyperglycaemia in T2DM rats. Methods: We created a T2DM rat model using a combination of a high-fat diet (HFD) and streptozotocin (STZ) administration. Then, we administered SH-MSCs injection at doses of 250 and 500 µL and assessed the therapeutic effects of SH-MSCs. We also investigated the potential underlying mechanisms involved. Results: The administration of SH-MSCs improved hyperglycemia in rats with T2DM. Infusion of SH-MSCs at 500 µL dose decreased homeostatic model assessment for insulin resistance (HOMA-IR). Histological analysis revealed that injection of SH-MSCs alleviated morphological damage of pancreas. SH-MSCs administration also inhibit the level of IL-6 and promote the expression of CD163 type 2 macrophage. Conclusion: The results of our study indicate that SH-MSCs have the potential to improve hyperglycemia and exert a protective effect on T2DM rats. Less
Objectives In order to treat a rat model of rotator cuff rupture this work concentrated on the expression of TNMD and RUNX followed by rotator cuff repair and secretome-hMSCs Methods A total of thirty -weeks-old male Sprague Dawley rats were separated into five groups randomly RC on week lesion treated with a rotator cuff repair and saline RC NaCl group n for and weeks and lesion treated with a rotator cuff repair and secretome-hMSCs RC secretome-hMSC group n for and weeks The supraspinatus and infraspinatus muscle tendon units were obtained for histological and biomechanical investigation at and weeks following injury ... More
Objectives: In order to treat a rat model of rotator cuff rupture, this work concentrated on the expression
of TNMD and RUNX2, followed by rotator cuff repair and secretome-hMSCs.
Methods: A total of thirty 10-weeks-old male Sprague–Dawley rats were separated into five groups randomly, RC
on week 0, lesion treated with a rotator cuff repair and saline (RC + NaCl group, n = 6) for 2 and 8 weeks, and lesion
treated with a rotator cuff repair and secretome-hMSCs (RC + secretome-hMSC group, n = 6) for 2 and 8 weeks.
The supraspinatus and infraspinatus muscle–tendon units were obtained for histological and biomechanical
investigation at 0, 2 and 8 weeks following injury.
Results: The findings showed that, in comparison with the RC + NaCl group, secretome-hMSCs significantly
improved tendon repair by upregulating TNMD and RUNX2 expression and histology score.
Conclusion: Combining Secretome-hypoxia MSCs with RC healing may help rats with rotator cuff tears.
Level of evidence: IV Less
of TNMD and RUNX2, followed by rotator cuff repair and secretome-hMSCs.
Methods: A total of thirty 10-weeks-old male Sprague–Dawley rats were separated into five groups randomly, RC
on week 0, lesion treated with a rotator cuff repair and saline (RC + NaCl group, n = 6) for 2 and 8 weeks, and lesion
treated with a rotator cuff repair and secretome-hMSCs (RC + secretome-hMSC group, n = 6) for 2 and 8 weeks.
The supraspinatus and infraspinatus muscle–tendon units were obtained for histological and biomechanical
investigation at 0, 2 and 8 weeks following injury.
Results: The findings showed that, in comparison with the RC + NaCl group, secretome-hMSCs significantly
improved tendon repair by upregulating TNMD and RUNX2 expression and histology score.
Conclusion: Combining Secretome-hypoxia MSCs with RC healing may help rats with rotator cuff tears.
Level of evidence: IV Less
Background Immunosuppression in sepsis is hypothesized to result from the increased expression of the immune checkpoint molecules programmed death- PD- and pro grammed death ligand- PD-L PD- and PD-L blockade therapies have been reported to increase survival in septic animals Currently the interleukin IL - within mesenchymal stem cell MSC secretome is known for its immunomodulatory capacity Objective To study the effect of IL- within MSC secretome on the expression of immune check points in the rat model of sepsis Methods We used male Rattus norvegicus rats in this research and divided them into four groups sham rats without sepsis ... More
Background: Immunosuppression in sepsis is hypothesized to result from the increased
expression of the immune checkpoint molecules programmed death-1 (PD-1) and pro
grammed death ligand-1 (PD-L1). PD-1 and PD-L1 blockade therapies have been reported
to increase survival in septic animals. Currently, the interleukin (IL)-10 within mesenchymal
stem cell (MSC) secretome is known for its immunomodulatory capacity. Objective: To
study the effect of IL-10 within MSC secretome on the expression of immune check
points in the rat model of sepsis. Methods: We used 48 male Rattus norvegicus rats
in this research and divided them into four groups: sham (rats without sepsis induction
and treatment), control (sepsis-induced rats without treatment), T1 (sepsis-induced rats
treated with 150 µL of secreted IL-10 from MSC), and T2 (sepsis-induced rats treated
with 300 µL of secreted IL-10 from MSC). Forty-eight hours after sepsis induction, we
terminated the rats and collected the blood to examine the PD-1 and PD-L1 expression
levels. Results: We found a decrease in the relative expression of PD-1 in the septic rat
group given 150 µL and 300 µL of secreted IL-10 from MSC compared to the control
group, but the decrease was not significant. We also found a decrease in the relative
expression of PD-L1 mRNA in the septic rat group given 150 µL and 300 µL of secreted
IL-10 from MSC compared to the control group. Conclusion: Administering secreted IL-10
from MSC reduces the expression of PD-1 and PD-L1 in sepsis. These findings suggest
that MSC secretome can improve the immunosuppression in sepsis. Less
expression of the immune checkpoint molecules programmed death-1 (PD-1) and pro
grammed death ligand-1 (PD-L1). PD-1 and PD-L1 blockade therapies have been reported
to increase survival in septic animals. Currently, the interleukin (IL)-10 within mesenchymal
stem cell (MSC) secretome is known for its immunomodulatory capacity. Objective: To
study the effect of IL-10 within MSC secretome on the expression of immune check
points in the rat model of sepsis. Methods: We used 48 male Rattus norvegicus rats
in this research and divided them into four groups: sham (rats without sepsis induction
and treatment), control (sepsis-induced rats without treatment), T1 (sepsis-induced rats
treated with 150 µL of secreted IL-10 from MSC), and T2 (sepsis-induced rats treated
with 300 µL of secreted IL-10 from MSC). Forty-eight hours after sepsis induction, we
terminated the rats and collected the blood to examine the PD-1 and PD-L1 expression
levels. Results: We found a decrease in the relative expression of PD-1 in the septic rat
group given 150 µL and 300 µL of secreted IL-10 from MSC compared to the control
group, but the decrease was not significant. We also found a decrease in the relative
expression of PD-L1 mRNA in the septic rat group given 150 µL and 300 µL of secreted
IL-10 from MSC compared to the control group. Conclusion: Administering secreted IL-10
from MSC reduces the expression of PD-1 and PD-L1 in sepsis. These findings suggest
that MSC secretome can improve the immunosuppression in sepsis. Less
In sepsis simultaneously elevated levels of pro-inflammatory cytokines and interleukin IL - indicate immune response dysregulation increasing the mortality of the host As mesenchymal stem cell MSC secretome is known to have immunomodulatory effects we aim to assess the role of MSC secretome in the inflammatory mediators NF- B p and p TNF- IL- and the survival rate of a rat model of sepsis In this study forty-eight male Rattus norvegicus rats were divided into one sham group and three groups with sepsis induction the control group and the sepsis-induced rat groups treated with L T and L T of ... More
In sepsis, simultaneously elevated levels of pro-inflammatory cytokines and interleukin (IL)-10 indicate immune response dysregulation, increasing the mortality of the host. As mesenchymal stem cell (MSC) secretome is known to have immunomodulatory effects, we aim to assess the role of MSC secretome in the inflammatory mediators (NF-κB p65 and p50, TNF-α, IL-10) and the survival rate of a rat model of sepsis. In this study, forty-eight male Rattus norvegicus rats were divided into one sham group and three groups with sepsis induction: the control group and the sepsis-induced rat groups treated with 150 μL (T1) and 300 μL (T2) of secretome. The survival rate was observed per 6 h for 48 h and plotted using the Kaplan–Meier method. Compared to the control group, T2 showed a significant decrease in the relative expression of NF-κB and the serum TNF-α level, and a significant increase in the serum IL-10 level. Meanwhile, T1 showed a significant decrease in the serum TNF-α level compared to the control group. The Kaplan–Meier Log Rank test did not show significance in the distribution of survival between T1, T2, and the control group. However, from the 18th to the 36th hour, the survival rate of T2 was lower than the survival rate of the control group and T1, with a noticeable difference between T2 and the control group, as well as T1 at the 36th hour. At the 42nd hour, the survival rate of T2 was the same as the control group and remained lower than T1. In conclusion, MSC secretome regulated the inflammatory mediators in rat model of sepsis, with a dose of 150 μL being more effective. Less
Aim Type diabetes mellitus T DM is an autoimmune disease characterized by the chronic inflammation of the pancreatic islets of Langerhans Hyperglycaemia leads to suppressed antioxidant enzyme and increased inflammation in the pancreatic cell resul ting in pancreatic cell death Hypoxic secretome mesenchymal stem cells HS-MSCs are soluble molecules secreted by MSCS that have the antiinflammation ability by secreting various cytoki nes including IL- and TGF- which potent as a promising the rapeutic modality for T DM This study aims to investigate the role of HS-MSCs in regulating superoxide dismutase SOD and caspase- gene expression in T DM model Methods ... More
Aim Type 1 diabetes mellitus (T1DM) is an autoimmune disease
characterized by the chronic inflammation of the pancreatic islets
of Langerhans. Hyperglycaemia leads to suppressed antioxidant
enzyme and increased inflammation in the pancreatic cell, resul
ting in pancreatic cell death. Hypoxic secretome mesenchymal
stem cells (HS-MSCs) are soluble molecules secreted by MSCS
that have the antiinflammation ability by secreting various cytoki
nes including IL-10 and TGF-β which potent as a promising the
rapeutic modality for T1DM. This study aims to investigate the
role of HS-MSCs in regulating superoxide dismutase (SOD) and
caspase-3 gene expression in T1DM model.
Methods Twenty male Wistar rats (6 to 8 weeks old) were rando
mly divided into four groups (sham, control, HS-MSCs 0.5 mL
and HS-MSCs 1 mL intraperitoneal treatment group). Streptozo
tocin (STZ) 60mg/kgBB was conducted once on day 1, HS-MSCs
0.5mL (T1) and HS-MSCs 1 mL (T2) were administrated intrape
ritoneally on day 7, 14, and 21 after STZ administration. The rats
were sacrificed on day 28; the gene expression of SOD and IL-6
was analysed by qRT-PCR.
Results This study showed that the ratio of SOD significantly
increased in HS-MSCs treatment associated with suppression of
IL-6 gene expression.
Conclusion HS-MSCs administration suppresses oxidative stre
ss and inflammation by up regulating SOD and inhibiting IL-6 to
control T1DM. Less
characterized by the chronic inflammation of the pancreatic islets
of Langerhans. Hyperglycaemia leads to suppressed antioxidant
enzyme and increased inflammation in the pancreatic cell, resul
ting in pancreatic cell death. Hypoxic secretome mesenchymal
stem cells (HS-MSCs) are soluble molecules secreted by MSCS
that have the antiinflammation ability by secreting various cytoki
nes including IL-10 and TGF-β which potent as a promising the
rapeutic modality for T1DM. This study aims to investigate the
role of HS-MSCs in regulating superoxide dismutase (SOD) and
caspase-3 gene expression in T1DM model.
Methods Twenty male Wistar rats (6 to 8 weeks old) were rando
mly divided into four groups (sham, control, HS-MSCs 0.5 mL
and HS-MSCs 1 mL intraperitoneal treatment group). Streptozo
tocin (STZ) 60mg/kgBB was conducted once on day 1, HS-MSCs
0.5mL (T1) and HS-MSCs 1 mL (T2) were administrated intrape
ritoneally on day 7, 14, and 21 after STZ administration. The rats
were sacrificed on day 28; the gene expression of SOD and IL-6
was analysed by qRT-PCR.
Results This study showed that the ratio of SOD significantly
increased in HS-MSCs treatment associated with suppression of
IL-6 gene expression.
Conclusion HS-MSCs administration suppresses oxidative stre
ss and inflammation by up regulating SOD and inhibiting IL-6 to
control T1DM. Less
Gene therapies offer promising therapeutic alternatives for many disorders that currently lack efficient treatment options Due to their chemical nature and physico-chemical properties delivery of polynucleic acids into target cells and subcellular compartments remains a significant challenge Adeno associated viruses AAV have gained a lot of interest for the efficient delivery of therapeutic single-stranded DNA ssDNA genomes over the past decades More than a hundred products have been tested in clinical settings and three products have received market authorization by the US FDA in recent years A lot of effort is being made to generate potent recombinant AAV rAAV vectors ... More
Gene therapies offer promising therapeutic alternatives for many disorders that currently lack efficient treatment options. Due to their chemical nature and physico-chemical properties, delivery of polynucleic acids into target cells and subcellular compartments remains a significant challenge. Adeno associated viruses (AAV) have gained a lot of interest for the efficient delivery of therapeutic single-stranded DNA (ssDNA) genomes over the past decades. More than a hundred products have been tested in clinical settings and three products have received
market authorization by the US FDA in recent years. A lot of effort is being made to generate potent recombinant AAV (rAAV) vectors that show favorable safety and immunogenicity profiles for either local or systemic administration. Manufacturing processes are gradually being optimized to deliver a consistently high product quality and to serve potential market needs beyond rare indications. In contrast to protein therapeutics, most rAAV products are still supplied as frozen liquids within rather simple formulation buffers to enable sufficient product shelf life, significantly hampering global distribution and access. In this review, we aim to outline the hurdles of rAAV drug product development and discuss critical formulation and composition aspects of rAAV
products under clinical evaluation. Further, we highlight recent development efforts in order to achieve stable liquid or lyophilized products. This review therefore provides a comprehensive overview on current state-of-the- art rAAV formulations and can further serve as a map for rational formulation development activities in the
future. Less
market authorization by the US FDA in recent years. A lot of effort is being made to generate potent recombinant AAV (rAAV) vectors that show favorable safety and immunogenicity profiles for either local or systemic administration. Manufacturing processes are gradually being optimized to deliver a consistently high product quality and to serve potential market needs beyond rare indications. In contrast to protein therapeutics, most rAAV products are still supplied as frozen liquids within rather simple formulation buffers to enable sufficient product shelf life, significantly hampering global distribution and access. In this review, we aim to outline the hurdles of rAAV drug product development and discuss critical formulation and composition aspects of rAAV
products under clinical evaluation. Further, we highlight recent development efforts in order to achieve stable liquid or lyophilized products. This review therefore provides a comprehensive overview on current state-of-the- art rAAV formulations and can further serve as a map for rational formulation development activities in the
future. Less
Genome analysis of Pyrobaculum calidifontis revealed the presence of -glucosidase Pcal gene Structural analysis affirmed the presence of signature sequences of Type II -glucosidases in Pcal We have heterologously expressed the gene and produced recombinant Pcal in Escherichia coli Biochemical characteristics of the recombinant enzyme resembled to that of Type I -glucosidases instead of Type II Recombinant Pcal existed in a tetrameric form in solution and displayed highest activity at C and pH independent of any metal ions A short heat-treatment at C resulted in a increase in enzyme activity A slight structural shift was observed by CD spectrometry at ... More
Genome analysis of Pyrobaculum calidifontis revealed the presence of α-glucosidase (Pcal_0917) gene. Structural analysis affirmed the presence of signature sequences of Type II α-glucosidases in Pcal_0917. We have heterologously expressed the gene and produced recombinant Pcal_0917 in Escherichia coli. Biochemical characteristics of the recombinant enzyme resembled to that of Type I α-glucosidases, instead of Type II. Recombinant Pcal_0917 existed in a tetrameric form in solution and displayed highest activity at 95 °C and pH 6.0, independent of any metal ions. A short heat-treatment at 90 °C resulted in a 35 % increase in enzyme activity. A slight structural shift was observed by CD spectrometry at this temperature. Half-life of the enzyme was >7 h at 90 °C. Pcal_0917 exhibited apparent Vmax values of 1190 ± 5 and 3.9 ± 0.1 U/mg against p-nitrophenyl α-D-glucopyranoside and maltose, respectively. To the best of our knowledge, Pcal_0917 displayed the highest ever reported p-nitrophenyl α-D-glucopyranosidase activity among the characterized counterparts. Moreover, Pcal_0917 displayed transglycosylation activity in addition to α-glucosidase activity. Furthermore, in combination with α-amylase, Pcal_0917 was capable of producing glucose syrup from starch with >40 % glucose content. These properties make Pcal_0917 a potential candidate for starch hydrolyzing industry. Less
Background Lower limb peripheral artery disease PAD is the main risk of diabetes mellitus which result to high mortality rate Approximately of patients who receive several treatments have passed away or lost limbs at a year s follow-up Secretome of hypoxia mesenchymal stem cells S-MSCs contains several active soluble molecules from hypoxia MSCs H-MSCs that capable inducing anti-inflammatory and vascular regeneration in PAD Objective In this study we investigated the therapeutic potential of S-MSCs in improving dynamic function and angiogenesis of PAD diabetic rats Methods The PAD was established by the incision from the groin to the inner thigh and ... More
Background:
Lower limb peripheral artery disease (PAD) is the main risk of diabetes mellitus which result to high mortality rate. Approximately, 50% of patients who receive several treatments have passed away or lost limbs at a year’s follow-up. Secretome of hypoxia mesenchymal stem cells (S-MSCs) contains several active soluble molecules from hypoxia MSCs (H-MSCs) that capable inducing anti-inflammatory and vascular regeneration in PAD.
Objective:
In this study, we investigated the therapeutic potential of S-MSCs in improving dynamic function and angiogenesis of PAD diabetic rats.
Methods:
The PAD was established by the incision from the groin to the inner thigh and distal ligation of femoral arteries in rats with diabetes. Rats were administered with 200 µL and 400 µL S-MSCs that successfully filtrated using tangential flow filtration (TFF) system based on various molecular weight cut-off categories intravenously. ELISA assay was used to analyze the cytokines and growth factors contained in S-MSCs. Tarlov score were examined at day 1, 3, 5, 7, 10 and 14. The rats were sacrificed at day 14 and muscle tissues were collected for immunohistochemistry (IHC) and gene expression analysis.
Results:
ELISA assay showed that S-MSCs provides abundant level of VEGF, PDGF, bFGF, IL-10 and TGFβ. In vivo administration of S-MSCs remarkably enhanced the Tarlov score. S-MSCs improved angiogenesis through enhancing VEGF gene expression and significantly increasing CD31 positive area in muscle tissue of PAD diabetic rats.
Conclusion:
Our findings suggest that S-MSCs could improves dynamic function and angiogenesis in PAD diabetic rats. Less
Lower limb peripheral artery disease (PAD) is the main risk of diabetes mellitus which result to high mortality rate. Approximately, 50% of patients who receive several treatments have passed away or lost limbs at a year’s follow-up. Secretome of hypoxia mesenchymal stem cells (S-MSCs) contains several active soluble molecules from hypoxia MSCs (H-MSCs) that capable inducing anti-inflammatory and vascular regeneration in PAD.
Objective:
In this study, we investigated the therapeutic potential of S-MSCs in improving dynamic function and angiogenesis of PAD diabetic rats.
Methods:
The PAD was established by the incision from the groin to the inner thigh and distal ligation of femoral arteries in rats with diabetes. Rats were administered with 200 µL and 400 µL S-MSCs that successfully filtrated using tangential flow filtration (TFF) system based on various molecular weight cut-off categories intravenously. ELISA assay was used to analyze the cytokines and growth factors contained in S-MSCs. Tarlov score were examined at day 1, 3, 5, 7, 10 and 14. The rats were sacrificed at day 14 and muscle tissues were collected for immunohistochemistry (IHC) and gene expression analysis.
Results:
ELISA assay showed that S-MSCs provides abundant level of VEGF, PDGF, bFGF, IL-10 and TGFβ. In vivo administration of S-MSCs remarkably enhanced the Tarlov score. S-MSCs improved angiogenesis through enhancing VEGF gene expression and significantly increasing CD31 positive area in muscle tissue of PAD diabetic rats.
Conclusion:
Our findings suggest that S-MSCs could improves dynamic function and angiogenesis in PAD diabetic rats. Less