Investigating the effect of cGRP78 vaccine against different cancer cells and its role in reducing melanoma metastasis.

Background and purpose: Treatment of malignancies with chemotherapy and surgery is often associated with disease recurrence and metastasis. Immunotherapy improves cancer treatment by creating an active response against tumor antigens. Various cancer cells express a large amount of glucose-regulated protein 78 (GRP78) protein on their surface. Stimulating the immune system against this antigen can expose cancer cells to the immune system. Herein, we investigated the effectiveness of a cGRP78-based vaccine against different cancer cells. Experimental approach: BALB/c mice were immunized with the cGRP78. The humoral immune response against different cancer cells was assessed by Cell-ELISA. The cellular immunity response was determined by splenocyte proliferation assay with different cancer antigens. The effect of vaccination on metastasis was investigated in vaccinated mice by injecting melanoma cancer cells into the tail of mice. Findings/Results: These results indicated that the cGRP78 has acceptable antigenicity and stimulates the immune system to produce antibodies. After three injections, the amount of produced antibody was significantly different from the control group. Compared to the other three cell types, Hela and HepG2 showed the highest reaction to the serum of vaccinated mice. Cellular immunity against the B16F10 cell line had the best results compared to other cells. The metastasis results showed that after 30 days, the growth of B16F10 melanoma cancer cells was not noticeable in the lung tissue of vaccinated mice. Conclusion and implications: Considering the resistance of vaccinated mice to metastasis, this vaccine offers a promising prospect for cancer treatment by inhibiting the spread of cancer cells.

Introduction of a new recombinant vaccine based on GRP78 for breast cancer immunotherapy and evaluation in a mouse model.

Introduction: Breast cancer is one of the most prevalent malignancies in women. Several treatment options are available today, including surgery, chemotherapy, and radiotherapy. Immunotherapy, as a highly specific therapy, involves adaptive immune responses and immunological memory. In our present research, we used the recombinant C-terminal domain of the GRP78 (glucose- regulated protein 78) protein to induce an immune response and investigate its therapeutic impact in the 4T1 breast cancer model. Methods: BALB/c mice were immunized with the cGRP78 protein. The humoral immune response was assessed by ELISA. Then, BALB/c mice were injected subcutaneously with 1×106 4T1 tumor cells. Subsequently, tumor size and survival rate measurements, MTT, and cytokine assays were performed. Results: The animals receiving the cGRP78 vaccine showed significantly more favorable survival and slower tumor growth rates compared with unvaccinated tumor-bearing mice as the negative control mice. Circulating levels of tumoricidal cytokines such as IFNγ were higher, whereas tolerogenic cytokines such as IL-2, 6, and 10 either did not increase or had a decreasing trend in mice receiving cGRP78. Conclusion: cGRP78 vaccines generated potent immunotherapeutic effects in a breast cancer mouse model. This novel strategy of targeting the GRP78 protein can promote the development of cancer vaccines and immunotherapies for breast cancer malignancies.

Optimization of plasmid electrotransformation into Bacillus subtilis using an antibacterial peptide.

Bacillus subtilis can potentially serve as an efficient expression host for biotechnology due to its ability to secrete extracellular proteins and enzymes directly into the culture medium. One of the important challenges in the biotechnology industry is to optimize the transformation conditions of B. subtilis bacteria. This study aims to provide a new method to optimize the transformation conditions and improve the transformation efficiency of B. subtilis WB600. To increase the transformation efficiency in B. subtilis, two methods of adding CM11 antibacterial peptides to the bacterial medium along with electroporation and optimizing the variables including the growth medium composition, time to adding CM11 peptide, electroporation voltage, recovery medium, and cell recovery time are used. The results of this study showed that the addition of antimicrobial peptides (AMPs) with a concentration of 2 µg/ml increases the transformation efficiency by 4 times compared to the absence of AMP in the bacterial medium. Additionally, the findings from our study indicated that the most optimal rate of transformation for B. subtilis was observed at a voltage of 7.5 kV/cm, with a recovery period of 12 h. With the optimized method, the transformation efficiency came up to 1.69 × 104 CFU/µg DNA. This improvement in transformation efficiency will be attributed to the research of expression of exogenous genes in B. subtilis, gene library construction for transformation of wild-type B. subtilis strains.

Applications of SMILE-extracted lenticules in ophthalmology.

AIM: To review recent innovations, challenges, and applications of small incision lenticule extraction (SMILE) extracted lenticule for treating ocular disorders. METHODS: A literature review was performed in the PubMed database, which was last updated on 30 December 2021. There was no limit regarding language. The authors evaluated the reference lists of the collected papers to find any relevant research. RESULTS: Due to the simplicity and accuracy of modern femtosecond lasers and the extensive development of SMILE surgery, many healthy human corneal stromal lenticules were extracted during surgery, motivating some professionals to investigate the SMILE lenticule reusability in different ocular disorders. In addition, new approaches had been developed to preserve, modify, and bioengineer the corneal stroma, leading to the optimal use of discarded byproducts such as lenticules from SMILE surgery. The lenticules can be effectively re-implanted into the autologous or allogenic corneas of human subjects to treat refractive errors, corneal ectasia, and corneal perforation and serve as a patch graft for glaucoma drainage devices with better cosmetic outcomes. CONCLUSION: SMILE-extracted lenticules could be a viable alternative to human donor corneal tissue.

Combination of natural scaffolds and conditional medium to induce the differentiation of adipose-derived mesenchymal stem cells into keratocyte-like cells and its safety evaluation in the animal cornea.

Keratocytes are the main cellular components of the corneal stroma. This cell is quiescent and cannot be cultured easily. The aim of this study was to investigate differentiate human adipose mesenchymal stem cells (hADSCs) into corneal keratocyte cells by combining natural scaffolds and conditioned medium (CM) and evaluating their safety in the rabbit's cornea. Keratocytes were cultured in an optimal culture medium and this medium was collected and kept as a CM. hADSCs were cultured on the decellularized human small incision lenticule extraction (SMILE) lenticule (SL), amniotic membrane (AM), and collagen-coated plates, and were exposed to keratocyte-CM (KCM) for 7, 14, and 21 days. Differentiation was evaluated using Real-time PCR and immunocytochemistry (ICC). hADSCs were cultured on the SL scaffolds and implanted in the corneal stroma of 8 New Zealand male rabbits. Rabbits were followed for 3 months and the safety was evaluated by clinical and histological variables. Real-time PCR results showed a significant increase in the expression of keratocyte-specific markers on the 21 day of differentiation compared to the control group. ICC also confirmed the induction of differentiation. Implantation of SLs containing differentiated cells in the cornea of animals showed no serious complications including neovascularization, corneal opacity, inflammation, or signs of tissue rejection. Furthermore, the evaluation of the presence of keratocyte-like cells after three months in the rabbit stroma was confirmed by Real-time PCR and immunohistochemistry (IHC) analysis. Our results showed that combination of combination of corneal extracellular matrix and KCM can induced keratocytes differentiation of hADSC and can be introduced as a alternative method to supply the required keratocytes in corneal tissue engineering.

The Effect of Amniotic Membrane Extracted Eye Drop on Repairing The Corneal Epithelial in Patients after Trans-Epithelial Photorefractive Keratectomy: An Interventional and Historical Study.

OBJECTIVE: Recent studies imply extensive applications for the human amniotic membrane (hAM) and its extract in medicine and ophthalmology. The content of hAM meets many requirements in eye surgeries, such as refractive surgery as the most important and commonly used method for treating the dramatically increasing refractive errors. However, they are associated with complications such as corneal haziness and corneal ulcer. This study was designed to evaluate the impact of amniotic membrane extracted eye drop (AMEED) on Trans-PRK surgery complications. MATERIALS AND METHODS: This randomized controlled trial was performed during two years (July 1, 2019-September 1, 2020). Thirty-two patients (64 eyes), including 17 females and 15 males, aged 20 to 50 years (mean age of 29.59 ± 6.51) with spherical equivalent between -5 to -1.5 underwent Trans Epithelial Photorefractive Keratectomy (Trans-PRK) surgery. One eye was selected per case (case group) and the other eye was considered as control. Randomization was done using the random allocation rule. The case group was treated with AMEED, and the artificial tear drop every 4 hours. The control eyes received artificial tear drops instilled every 4 hours. The evaluation continued for three days after the Trans-PRK surgery. RESULTS: A significant decrease in CED size was found in the AMEED group on the second day after surgery (P=0.046). Also, this group had a substantial reduction in pain, hyperemia, and haziness. CONCLUSION: This study showed that AMEED drop can increase the healing rate of corneal epithelial lesions after Trans- PRK surgery and reduce the early and late complications of Trans-PRK surgery. Researchers and Ophthalmologists should consider AMEED as a selection in patients with persistent corneal epithelial defects and patients who have difficulty in corneal epithelial healing. We understood AMEED has a different effect on the cornea after surgery; therefore, the researcher must know AMEED's exact ingredients and help expand AMEED uses (registration number: TCTR20230306001).

Histopathology Study of Patients with Delayed Mustard Gas Keratopathy 30 Years After Exposure.

Background & Objective: Delayed mustard gas keratopathy (DMGK) is the main chronic outcome in eye-chemical injured patients. The aim of this study was the histopathological evaluation of mustard-exposed cornea after more than 30 years. Methods: Fourteen corneas after Lamellar keratoplasty were evaluated in this study. Corneal tissues were prepared by histologic methods and stained by H&E. Results: The main histopathological findings in these cases were the presence of severe stromal edema and corneal scar. In the sections with visible superficial epithelium, subepithelial bullae formation was observed. Focal or diffuse disruption of Bowman's membrane and replacement with fibrosis were also seen. There was no evidence of stromal vascularization and inflammation in all specimens. Conclusion: After more than 30 years, an extensive corneal scar is seen in sulfur mustard exposed patients. Scar tissue without vascularization and fibroblastic proliferation is the main finding in the sulfur mustard exposed cornea. This pathology result is different from other scars. No evidence of inflammation or immune cell infiltration should be considered in managing DMGK.

Tear proteomics analysis of patient suffered from delayed mustard gas keratopathy.

Understanding the molecular and cellular mechanisms involved in the pathogenesis of ocular injured induced by mustard gas can help better identify complications and discover appropriate therapies. This study aimed to analyze the proteomics of tears of chemical warfare victims with mustard gas ocular injuries and compare it with healthy individuals. In this case-control research, 10 mustard gas victims with long-term ocular difficulties (Chronic) were included in the patient group, while 10 healthy persons who were age and sex matched to the patients were included in the control group. Schirmer strips were used to collect the tears of the participants. Proteomics experiments were performed using the high-efficiency TMT10X method to evaluate the tear protein profile, and statistical bioinformatics methods were used to identify the differently expressed proteins. 24 proteins had different expressions between the two groups. Among these 24 proteins, 8 proteins had increased expression in veterans' tears, while the remaining 16 proteins had decreased expression. Reactome pathways were used to look at proteins with various expressions, and 13 proteins were found to be engaged in the immune system, 9 of which were effective in the innate immune system, and 5 proteins were effective in the complement cascade. Ocular mustard gas exposure may cause a compromised immune system on the eye's surface, exposing the cornea to external and endogenous infections, and eventually causing corneal opacity and reduced vision.

Emerging tissue engineering strategies for the corneal regeneration.

Cornea as the outermost layer of the eye is at risk of various genetic and environmental diseases that can damage the cornea and impair vision. Corneal transplantation is among the most applicable surgical procedures for repairing the defected tissue. However, the scarcity of healthy tissue donations as well as transplantation failure has remained as the biggest challenges in confront of corneal grafting. Therefore, alternative approaches based on stem-cell transplantation and classic regenerative medicine have been developed for corneal regeneration. In this review, the application and limitation of the recently-used advanced approaches for regeneration of cornea are discussed. Additionally, other emerging powerful techniques such as 5D printing as a new branch of scaffold-based technologies for construction of tissues other than the cornea are highlighted and suggested as alternatives for corneal reconstruction. The introduced novel techniques may have great potential for clinical applications in corneal repair including disease modeling, 3D pattern scheming, and personalized medicine.

Topical Effects of N-Acetyl Cysteine and Doxycycline on Inflammatory and Angiogenic Factors in the Rat Model of Alkali-Burned Cornea.

The aim of this study was to analyze the single and combined effects of N-acetyl cysteine (NAC) and doxycycline (Dox) on the inflammatory and angiogenic factors in the rat model of alkali-burned cornea. Rats were treated with a single and combined 0.5% NAC and 12.5 µg/mL Dox eye drops and evaluated on days 3, 7, and 28. In the corneas of various groups, the activity of Catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx) enzymes was assessed. The expression of inflammatory factors (TNF-α, Rel-a, and CXCL-1) and angiogenic factors (VEGF-a, MMP2, and MMP9) was measured using real-time polymerase chain reaction. The antioxidant enzyme activities decreased substantially 3 days after injury with sodium hydroxide (NaOH). NAC and combined NAC+ Dox topical treatments increased the SOD enzyme activity on day 28 (P < 0.05). The expression of TNF-α and Rel-a genes following single and combined treatment of NAC and Dox decreased significantly on days 7 and 28 (P < 0.05). The mRNA level of angiogenic factors and corneal neovascularization (CNV) level declined in NaOH-injured rats treated with Dox (P < 0.05). The topical treatment of Dox could attenuate inflammation and CNV complications. However, NAC treatment may not reduce the expression of angiogenic genes.

The Role of Inflammatory Cytokines in Neovascularization of Chemical Ocular Injury.

AIM: Chemical injuries can potentially lead to the necrosis anterior segment of the eye, and cornea in particular. Inflammatory cytokines are the first factors produced after chemical ocular injuries. Inflammation via promoting the angiogenesis factor tries to implement the wound healing mechanism in the epithelial and stromal layer of the cornea. METHODS: Narrative review. RESULTS: In our review, we described the patterns of chemical injuries in the cornea and their molecular mechanisms associated with the expression of inflammatory cytokines. Moreover, the effects of inflammation signals on angiogenesis factors and CNV were explained. CONCLUSION: The contribution of inflammation and angiogenesis causes de novo formation of blood vessels that is known as the corneal neovascularization (CNV). The new vascularity interrupts cornea clarity and visual acuity. Inflammation also depleted the Limbal stem cells (LSCs) in the limbus causing the failure of normal corneal epithelial healing and conjunctivalization of the cornea.

Safety evaluation of the atmospheric low-temperature plasma (ALTP) on the conjunctiva: an animal study and histopathological findings; 6-month follow-up.

BACKGROUND: Plasma medicine is an innovative research field focused on the application of atmospheric-pressure low-temperature plasmas (ALTP) for therapeutic purposes. Considering the potentials of plasma in ophthalmology, in this study, we evaluated the safety of plasma on the conjunctival tissue in animal models for 6 months. METHODS: Twelve adult male New Zealand albino rabbits were divided into four groups. The right eye of each rabbit was chosen for the test and the left eye was considered as the control. Experiments were performed using the Plexr device (GMV, Rocca Priora, RM, Italy). Four plasma spots were applied on the superior part of the conjunctiva (from 10 to 2 o'clock positions) using the continuous mode and a low power level (white handpiece) of the Plexr. For evaluation of the plasma safety, the histopathological changes were assessed 1 week (A), 1 month (B), 3 months (C), and 6 months (D) after the intervention. RESULTS: According to the histopathological findings, a mild decrease in blood vessels and severe stromal edema, as well as a superficial epithelium loss, were observed in group A. No chronic inflammation, scar tissue, deposition, and hemorrhage were found in group B. Epithelialization was confirmed by the histological examinations after 1 month. There was no evidence of atypia or dysplasia after 3 and 6 months. CONCLUSION: In conclusion, there were no persistent histopathological changes on conjunctival tissue after plasma exposure. Then, plasma can be considered as a minimally invasive alternative method for treating some ocular surface disorders.

Application of mesenchymal stem cells in corneal regeneration.

Due to delicate its structure, the cornea is susceptible to physical, chemical, and genetic damages. Corneal transplantation is the main treatment for serious corneal damage, but it faces significant challenges, including donor shortages and severe complications. In recent years, cell therapy is suggested as a novel alternative method for corneal regeneration. Regarding the unique characteristics of Mesenchymal stem cells including the potential to differentiate into discrete cell types, secretion of growth factors, mobilization potency, and availability from different sources; special attention has been paid to these cells in corneal engineering. Differentiation of MSCs into specialized corneal cells such as keratocytes, epithelial and endothelial cells is reported. Potential for Treatment of keratitis, reducing inflammation, and inhibition of neovascularization by MSCs, introducing them as novel agents for corneal repairing. In this review, various types of MSCs used to treat corneal injuries as well as their potential for restoring different corneal layers was investigated.

The effect of N-acetyl cysteine and doxycycline on TNF-α-Rel-a inflammatory pathway and downstream angiogenesis factors in the cornea of rats injured by 2-chloroethyl-ethyl sulfide.

BACKGROUND: Cornea injury of sulfur mustard (SM) is considered as the most devastating injuries to the eye. This study aimed to evaluate the single and combined effects of N-acetyl cysteine (NAC) and doxycycline on the inflammatory pathway and cornea neovascularization (CNV) in the rat model of SM-injured cornea. MATERIALS AND METHODS: The right cornea of male Sprague-Dawley rats was subjected to 2-chloroethyl-ethyl sulfide (CEES). Rats were topically treated with a single and combined of 0.5% NAC and 12.5 µg/ml doxycycline and examined at 3rd, 15th, and 21st days. The activity of three antioxidant enzymes was analyzed in the cornea of different groups. Real-time PCR was performed to measure gene expression of inflammatory factors (tnf-α, rel-a & cxcl-1) and angiogenesis factors (vegf-a, mmp2,9) in the cornea lysates. The histological and opacity assessments were also carried out. RESULTS: The activity of antioxidant enzymes significantly declined 3 days after the CEES damage. NAC eye drop recovered the enzyme activity on the 21st day of treatment (p-value < .05). The expression of tnf-α and rel-a genes significantly increased after CEES cornea exposure, while NAC declined their expression on the 7th and 21st days. The CNV score and angiogenesis factor expression were decreased in the long term by single and combined treatments (p-value < .05), but the infiltration of inflammatory cells was not completely amended. CONCLUSION: NAC and doxycycline eye drop could improve the CNV complication. Also, NAC was an effective treatment against the inflammatory pathway involved in CEES-injured cornea.

Safety of grafting acellular human corneal lenticule seeded with Wharton's Jelly-Derived Mesenchymal Stem Cells in an experimental animal model.

The present study was conducted to evaluate safety of grafting acellular human corneal lenticule seeded with Wharton's Jelly-derived Mesenchymal Stem Cells (WJSC) in an experimental animal model. Human corneal lenticules were decellularized with a rate of about 97% with an acceptable lack of cytotoxicity and relatively intact ultrastructure of the lenticules. 12 rabbits underwent unilateral stromal pocketing with implantation of decellularized lenticules. Implantation was performed for 6 rabbits along with graft recellularization with WJSCs. Rabbits were euthanized after 1 month (n = 6) and 3 months (n = 6) to evaluate progression of graft bio-integration. No clinical rejection sign was detected during the study. Histopathological analysis showed that, grafts were integrated well with the least distortion of surrounding collagen bundles. After 3 months, labeled WJCS was detected representing viability of stem cells in the host. Increased expression of keratocyte-specific markers showed the potential of recruiting WJSCs as keratocyte progenitor cells to reinforce corneal ultrastructure.

A novel hydrogel scaffold contained bioactive glass nanowhisker (BGnW) for osteogenic differentiation of human mesenchymal stem cells (hMSCs) in vitro.

Employing hydrogels as an alternative strategy for repairing bone defects has received great attention in bone tissue engineering. In this study, hydrogel scaffold based on collagen, gelatin, and glutaraldehyde was combined with bioactive glass nanowhiskers (BGnW) to differentiate human mesenchymal stem cells (hMSCs) into the osteogenic lineage and inducing biomineralization. Pure Gel-Glu-Col and bioactive glass nanowhiskers were used as control throughout the paper. Chemical, physical and morphological characteristics of the nanocomposite scaffold were assessed meticulously using Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), porosity measurement, water uptake ability, tensile test, and scanning electron microscopy (SEM). To determine the cytotoxicity and cell viability of the hydrogel, MTT assay and Acridine orange (AO) staining were performed. hMSCs seeded on Gel-Glu-Col/BGnW were then incubated with osteogenic differentiation media for 14 days. Biomineralization assays (alkaline phosphatase (ALP) activity, calcium content assay, von Kossa, and Alizarin red staining) were carried out, and osteogenic genes and protein markers were examined using real time-PCR and immunocytochemistry. Results showed that the components of the hydrogel were properly integrated. The mechanical property of hydrogel was enhanced following the addition of BGnW. Cell viability assays confirmed the biocompatibility of the scaffold and increasing the proliferation after incorporating BGnW into pure Ge1-Glu-Col. Our nanocomposite maintained an enhanced ability of biomineralization as compared to its pure counterparts. Molecular investigations revealed an elevated level of osteogenic markers as compared to Ge1-Glu-Col and BGnW. All in all, Gel-Glu-Col/BGnW seems to be a potential candidate for the regeneration of bone tissue.

Isolation and characterization of a novel nanobody for detection of GRP78 expressing cancer cells.

Glucose-regulated protein 78 (GRP78) is an endoplasmic reticulum (ER) chaperone that has been shown that is overexpressed in cancer cells. Overexpression of GRP78 on cancer cells makes this molecule a suitable candidate for cancer detection and targeted therapy. VHH is the binding fragment of camelid heavy-chain antibodies also known as "nanobody." The aim of this study is to isolate and produce a new recombinant nanobody using phage display technique to detect cancer cells. Using the c-terminal domain of GRP78 (CGRP) as an antigen, four rounds of biopanning were performed, and high-affinity binders were selected by ELISA. Their affinity and functionality were characterized by surface plasmon resonance (SPR) cell ELISA and immunocytochemistry. A unique nanobody named V80 was purified. ELISA and SPR showed that this antibody had high specificity and affinity to the GRP78. Immunofluorescence analysis showed that V80 could specifically bind to the HepG2 and A549 cancer cell lines. This novel recombinant nanobody could bind to the cell surface of different cancer cells. After further evaluation, this nanobody can be used as a new tool for cancer detection and tumor therapy.

Visual and subjective outcomes following trifocal intraocular lens implantation in Iranian cataractous patients.

BACKGROUND: The aim of this study was to evaluate visual and refractive outcomes and quality of vision after implantation of the AT LISA tri 839MP intraocular lens (IOL). PATIENTS AND METHODS: This interventional clinical trial comprised 46 cataractous eyes of patients who underwent phacoemulsification with IOL implantation (mean age of 58.08 ± 11.01 years; range: 36-76 years old). Spherical equivalent (SE), uncorrected distance visual acuity (UDVA), corrected distance visual acuity (CDVA), uncorrected intermediate visual acuity (UIVA), corrected intermediate visual acuity (CIVA), uncorrected near visual acuity (UNVA), and corrected near visual acuity (CNVA) were measured 1, 3, and 6 months after surgery. Contrast sensitivity (CS) was measured at 6 months. Subjective quality of vision and presence of dysphotopsia by a short questionnaire were evaluated postoperatively. RESULTS: At 6 months, the mean visual acuity was 0.08 ± 0.11, 0.03 ± 0.08, 0.07 ± 0.09, 0.02 ± 0.08, 0.05 ± 0.09, and 0.02 ± 0.08 LogMAR for UDVA, CDVA, UIVA, CIVA, UNVA, and CNVA, respectively. The mean values of SE were - 0.4728 ± 0.32D. These variables improved over time, yet significant changes were detected in UDVA (P = 0.009) and SE (P = 0.0001). The mean CS value was 1.74 ± 0.08. The mean scores (0% = no symptoms; 100% = strong symptoms/unable to perform activities) for glare and halos were 7.07 ± 0.22% and 8.70 ± 0.23%, respectively. These items were reduced over time. Patients' level of satisfaction mean score for performing activities was 96.66%. CONCLUSIONS: The AT LISA tri 839MP IOL provides excellent uncorrected distance, intermediate and near vision, and CS. This IOL showed a minimal level of photic phenomena and a high level of patient satisfaction.

Preparation and in vitro characterization of cross-linked collagen-gelatin hydrogel using EDC/NHS for corneal tissue engineering applications.

Corneal disease is considered as the second leading cause of vision loss and keratoplasty is known as an effective treatment for it. However, the tissue engineered corneal substitutes are promising tools in experimental in vivo repair of cornea. Selecting appropriate cell sources and scaffolds are two important concerns in corneal tissue engineering. The object of this study was to investigate biocompatibility and physical properties of the bio-engineered cornea, fabricated from type-I collagen (COL) and gelatin (Gel). Two gelatin based hydrogels cross-linked with EDC/NHS were fabricated, and their physicochemical properties such as equilibrium water content, enzymatic degradation, mechanical properties, rheological, contact angle and optical properties as well as their ability to support human bone-marrow mesenchymal stem cells (hBM-MSCs) survival were characterized. The equilibrium water content and enzymatic degradation of these hydrogels can be easily controlled by adding COL. Our findings suggest that incorporation of COL-I increases optical properties, hydrophilicity, stiffness and Young's modulus. The viability of hBM-MSCs cultured in Gel and Gel: COL was assessed via CCK-8 assay. Also, the morphology of the hBM-MSCs on the top of Gel and Gel: COL hydrogels were characterized by phase-contrast microscopy. This biocompatible hydrogel may promise to be used as artificial corneal substitutes.

Identification and in vitro characterization of novel nanobodies against human granulocyte colony-stimulating factor receptor to provide inhibition of G-CSF function.

It has been shown that Granulocyte colony-stimulating factor (G-CSF) has a higher expression in malignant tumors, and anti-G-CSF therapy considerably decreases tumor growth, tumor vascularization and metastasis. Thus, blocking the signaling pathway of G-CSF could be beneficial in cancer therapy. This study is aimed at designing and producing a monoclonal nanobody that could act as an antagonist of G-CSF receptor. Nanobodies are the antigen binding fragments of camelid single-chain antibodies, also known as VHH. These fragments have exceptional properties which makes them ideal for tumor imaging and therapeutic applications. We have used our previously built nanobody phage libraries to isolate specific nanobodies to the G-CSF receptor. After a series of cross-reactivity and affinity experiments, two unique nanobodies were selected for functional analysis. Proliferation assay, real-time PCR and immunofluorescence assays were used to characterize these nanobodies. Finally, VHH26 nanobody that was able to specifically bind G-CSF receptor (G-CSF-R) on the surface of NFS60 cells and efficiently block G-CSF-R downstream signaling pathway in a dose-dependent manner was selected. This nanobody could be further developed into a valuable tool in tumor therapy and it forms a basis for additional studies in preclinical animal models.