In-line leukoreduction filters: A new source of microparticle for human and animal study.

INTRODUCTION: The isolation of microparticles (MPs) from leukoreduction filters (LRFs) during cell extraction process introduced LRFs as a precious source of MPs for animal and human study. METHOD: LRFs were collected from Tehran Blood Transfusion Center. The back-flushing method was used for leukocyte extraction from the LRFs. MPs were isolated through double-step centrifugation. Dynamic light scattering (DLS), electron microscopy (EM), and flow cytometry were performed for the evaluation of MPs size, morphology, and structural properties respectively. Statistical analyses were carried out to evaluation of differences between test and control groups. a p-value less than 0.05 indicates significant differences. RESULT: DLS analysis showed that the average MP size in the test and control groups was 654.83 nm and 233.68 nm respectively. SEM images showed the spherical, oval, cell fragment, and micro-aggregate particles and TEM images demonstrated the mitochondrial-like body in the MPs. Flow cytometry studies also showed a significant increase in the percent of CD41, and CD14, and a significant decrease in the percent of CD235a in the test group compared to control (P value=0.029, P value=0.035, P value= 0.001 respectively). Moreover, the percentage of CD34 MPs indicated a borderline difference between the two groups (P value= 0.075). Finally count of MPs in the test and control groups was 1202095.34 and 280948.64, respectively and the difference was significant (P value=0.008). CONCLUSION: It is concluded that LRFs are a potential source of the large volume of various cell MPs with different phenotypical and structural properties for animal and human phase studies. Moreover, the investigation of LRFs as a source of different types of exosomes can shed new light on extracellular vesicle studies.

Investigation of the potential of leukoreduction filters in the creation of anti-inflammatory compound.

BACKGROUND: Some viruses such as SARS, SARS-CoV-2, and MERS cause an imbalance in immune responses and leads to an acute inflammatory reaction named cytokine storm. In this situation, an anti-inflammatory component can modulate the immune system and decrease mortality. The aim of this study was investigate the potential of leukoreduction filters (LRFs) in creating an anti-inflammatory compound. MATERIALS AND METHODS: In this experimental study, firstly optimal dose of the anti-inflammatory drug was obtained through LRFs treatment with 0.1 mg, 0.4 mg, 0.6 mg of Betamethasone. Then inflammatory and anti-inflammatory cytokine in gene and protein level was evaluated. In the next step, LRFs were categorized into treatment 1, treatment 2, control assay, and control groups and treated with the optimal dose of the drug. Finally, the obtained compound was investigated for the concentration of IL1, IL6, and TNF-α as inflammatory and IL4, IL1Ra, and IL10 as anti-inflammatory cytokines. RESULTS: The results of the current study showed that the concentration of 0.4 mg of Betamethasone lead to a significant increase of anti-inflammatory cytokine in gene and protein levels. The results also showed that the Betamethasone treated groups (treatment1) causes a significant increase in the secretion of anti-inflammatory cytokine compares to the control while inflammatory cytokine remained at the control level. CONCLUSION: The results showed that under influence of anti-inflammatory drug treatments the production and secretion of anti-inflammatory cytokines can be induced in LRFs.

Potential of a novel scaffold composed of human platelet lysate and fibrin for human corneal endothelial cells.

Cell-based therapies have been emerged to find innovative solutions for corneal endothelial dysfunction. The aim of this study is to investigate the suitability of a blended scaffold containing human platelet lysate (HPL) and fibrin not only for cultivating human corneal endothelial cells (HCECs) but also for serving as a scaffold for the respected cells. We isolated HCECs from human donors and encapsulated the cells with three concentrations of HPL/Fibrin scaffold, namely HPL/Fibrin 1, HPL/Fibrin 2 and HPL/Fibrin 3, by adding 28.9, 57.8 and 86.7 mg/dl of fibrinogen to HPL to obtain a final percentage of 10, 20 and 30 % of fibrinogen, respectively. SEM imaging and swelling test were done to characterize the scaffolds. Cell viability assay and cell counting were performed on the cells. HCECs were characterized by morphology and immunocytochemistry. SEM imaging on freeze-dried scaffolds showed higher porosity of HPL/Fibrin 1 and HPL/Fibrin 2 than HPL/Fibrin 3, but larger pores were observed only in HPL/Fibrin 1. Cellular attachment and morphology on HPL/Fibrin 1 were appropriate by SEM imaging. A higher swelling rate was observed in HPL/Fibrin 1. After 3 and 5 days, higher numbers of cells were observed specifically in HPL/Fibrin 1. A higher expression of Na+/K+-ATPase, ZO-1 and vimentin proteins was detected in the HPL/Fibrin 1-cultured HCECs as compared with control (no scaffold). HPL/Fibrin can be used as a suitable scaffold for HCECs while preserving the cells viability. Further investigations are necessitated to approve the beneficial effects of the suggested scaffold for delivering and transplantation of cultivated HCECs into the anterior chamber of the eye.

Comparative culture of human corneal endothelial cells following treatment with human platelet lysate/fibrin hydrogel versus Y-27632 ROCK inhibitor: in vitro and ex vivo study.

PURPOSE: The advancement of tissue engineering and cell therapy research has resulted in innovative therapeutic options for patients with corneal endothelial diseases. The aim of this study was to compare the potential effect of using human platelet lysate (HPL)/Fibrin hydrogel versus using a Y-27632 ROCK inhibitor, on the culture of human corneal endothelial cells (HCECs) under in vitro and ex vivo conditions. METHODS: HCECs were isolated from human donors and treated separately with HPL/Fibrin hydrogel, a Y-27632 ROCK inhibitor, and fetal bovine serum (FBS). MTT viability assay and cell counting were performed on the treated cells. Subsequently, we prepared ex vivo models of human corneal endothelial dysfunction and incubated them with DiI-labeled-HCECs. Specular and fluorescence microscopy were then performed on each of the ex vivo models. RESULTS: In comparison, similar viability results were achieved in the cells treated with HPL/Fibrin hydrogel versus those treated with the Y-27632 ROCK inhibitor, but both treatments showed higher viability than the control group (FBS). More importantly, based on the specular and fluorescence microscopic results, the HPL/Fibrin hydrogel and the Y-27632 ROCK inhibitor treatments showed similar inducible effects on the attachment of the cells to the Descemet membranes of the ex vivo models. CONCLUSION: HPL/Fibrin hydrogel and Y-27632 ROCK inhibitor have similar inducible effects on the viability and attachment of the HCECs. A definite advantage of treating cells with HPL/Fibrin hydrogel is that it serves as a xeno-free and biocompatible material which can have autologous applications in future usage by clinics.

Intravitreal connective tissue growth factor neutralizing antibody or bevacizumab alone or in combination for prevention of proliferative vitreoretinopathy in an experimental model.

Connective tissue growth factor (CTGF) is released by retinal pigment epithelial (RPE) cells and detectable in proliferative membranes (PrMs). This experimental study was performed to investigate the mRNA and protein levels of both CTGF and vascular endothelial growth factor A (VEGF-A) in a rabbit model of proliferative vitreoretinopathy (PVR). In addition, the effects of a single intravitreal injection of the safe dose of anti-CTGF or bevacizumab as monotherapy and in combination were evaluated. PVR was induced in the right eye of albino rabbits by intravitreal injection of cultured adult human RPE cells. Quantitative real-time reverse transcription PCR (qRT-PCR) and Western blot analysis of CTGF and VEGF-A were performed on whole eye tissue in the PVR model versus controls at different time points. In the next step, the PVR models were assigned to five groups. The monotherapy groups received a single intravitreal injection of 0.1 ml of anti-CTGF 100 µg/ml (final concentration of 6.6 µg/ml in the vitreous) or 0.03 ml of 25 mg/ml bevacizumab. In the combined group, the abovementioned amounts of anti-CTGF and bevacizumab were injected intravitreally from separate sites in one session. No antibody injection was performed in the control group. Intravitreal injection of 0.1 ml of control IgG (1 mg/ml of isotype matched) antibody was performed in the placebo group. After 2 weeks, histologic evaluation including, trichrome staining for collagen, immunostaining by anti-alpha-smooth muscle actin for myofibroblasts, and anti-collagen type-1 antibody on paraffin embedded anterior-posterior sections was done. In addition, fundus photography was performed for clinically equivalent PVR staging. Twenty-four hours following PVR induction, CTGF mRNA and protein levels increased five- and- three-fold compared to controls, respectively (P < 0.001). VEGF-A mRNA and protein levels decreased significantly after 72 h of PVR induction compared to controls (P < 0.05). Means of PrM thickness and myofibroblast cell counts significantly decreased in the anti-CTGF group (P < 0.001 and P < 0.05, respectively). The mean area of collagen type-1 fibers of PrM in the mono- and combination therapy groups that received intravitreal anti-CTGF was significantly reduced (P < 0.001); in addition, mild PVR (stage-1 and 2) formation occurred in comparison with moderate to severe PVR (stage-4 and higher) in other groups. In conclusion, we found that intravitreal injection of CTGF neutralizing antibody resulted in a reduction in PrM thickness, collagen fibers and myofibroblast density in the PVR model. CTGF inhibition may represent a potential therapeutic target for PVR.

COVID-19 related callback in blood donors; Outcomes in blood donors and patients.

Call back as a procedure to report post donation symptoms or illness by donors has been established since 2009 in Iranian Blood Transfusion Organization (IBTO). During the first phase of COVID-19 outbreak, all blood donors were requested to report any respiratory infection symptoms after donation. The study investigated the callback data of COVID-19 in Tehran Blood Center during the first 3 months of the outbreak in Iran. The purpose of this study was to estimate the frequency of post donation COVID-19 related call back reports and determine its implications for blood donors and patients. A telephone interview was conducted with donors who had reported COVID-19 symptoms. Some questions were asked to evaluate donor's health at the time of blood donation. The donors categorized into three groups: laboratory-confirmed, suspected, and COVID-19 irrelevant based on their answers. In cases that the blood component obtained from a laboratory-confirmed donor had been released, the hospital was notified and asked to follow up the recipient for COVID-19. The results showed 30 donors (0.08 %) had callback related to COVID-19 and 76.63 % of the obtained component was disposed. The results also showed that only one donor had a laboratory-confirmed result with the RBC unit processed from her whole blood released for transfusion. The RBC unit recipient did not show any signs or symptoms of infection during a 46-day follow-up. Concluded that callback system was effective to remove most of the components obtained from the donors who reported to be COVID-19 suspected or confirmed. Moreover, the result did not support virus transmission through blood transfusion.

Potential of therapeutic plasmapheresis in treatment of COVID-19 patients: Immunopathogenesis and coagulopathy.

Therapeutic plasmapheresis (TP) is the process of the separation and removal of plasma from other blood components and is considered as an adjunctive treatment strategy to the discarded abnormal agent in the management of respiratory viral pandemics. This article reviews the mechanisms of immunopathogenesis and coagulopathy induced by SARS-CoV-2 and the potential benefits of TP as adjunctive treatment in critically COVID-19 patients.

Gamma irradiation of ocular melanoma and lymphoma cells in the presence of gold nanoparticles: in vitro study.

The aim of this work was to determine whether conjugation of cultivated choroidal melanoma and Burkitt's lymphoma cells with gold nanoparticles (GNPs) is beneficial for these series of ocular cancer patients. GNPs are radiosensitizers and can sensitize tumors to radiotherapy.This application has been examined in several tumor types, but not in choroidal melanoma. This study shows the results of in vitro study on the choroidal melanoma and also Burkitt's lymphoma cells in the presence of GNPs during continuous gamma irradiation. Cytotoxicity of GNPs were assessed for five different concentrations then cultured melanoma and Burkitt's lymphoma cells were irradiated with a Gamma source in the presence and absence of NPs. Incubation of melanoma cells with GNP concentrations below 100 µg/ml, accompanied by gamma irradiation, increased cell death (P value = 0.016) . In the absence of irradiation, GNPs at these concentrations did not affect cultured melanoma cell metabolism. Reduced cell viability resulted from a significant increase in absorbed energy by the tumor. Moreover, GNP concentrations higher than 200 µg/ml induced cytotoxicity in melanoma cells. Cytotoxicity assay in GNPs-loaded Burkitt's lymphoma cells showed a slight decrease in cell viability at 50 µg/ml and clear cytotoxicity at concentrations higher than 100 µg/ml (P value = 0.035). Concentration and proper injection doses of GNPs in sensitive tissues such as the human eye are important variables yet to be determined.This is the first report of choroidal melanoma dosimetry performed in the presence of GNPs and provides valuable insights into future therapeutic approaches. Further in vitro study with more different sizes and concentrations is needed to determine the optimum size and concentration before any clinical research in this regard.

Effect of amniotic fluid on the in vitro culture of human corneal endothelial cells.

The present study was designed to evaluate the effects of human amniotic fluid (HAF) on the growth of human corneal endothelial cells (HCECs) and to establish an in vitro method for expanding HCECs. HCECs were cultured in DMEM-F12 supplemented with 20% fetal bovine serum (FBS). Confluent monolayer cultures were trypsinized and passaged using either FBS- or HAF-containing media. Cell proliferation and cell death ELISA assays were performed to determine the effect of HAF on cell growth and viability. The identity of the cells cultured in 20% HAF was determined using immunocytochemistry (ICC) and real-time reverse transcription polymerase chain reaction (RT-PCR) techniques to evaluate the expression of factors that are characteristic of HCECs, including Ki-67, Vimentin, Na+/K+-ATPase and ZO-1. HCEC primary cultures were successfully established using 20% HAF-containing medium, and these cultures demonstrated rapid cell proliferation according to the cell proliferation and death ELISA assay results. The ICC and real time RT-PCR results indicated that there was a higher expression of Na+/K+-ATPase and ZO-1 in the 20% HAF cell cultures compared with the control (20% FBS) (P < 0.05). The 20% HAF-containing medium exhibited a greater stimulatory effect on HCEC growth and could represent a potential enriched supplement for HCEC regeneration studies.

Evaluation of a New Method of Leukocyte Extractions from the Leukoreduction Filter.

This study's purpose was to optimize the leukocyte extraction protocol and evaluate the efficacy of this new protocol. 12BioR blood filters were collected from Tehran Blood Transfusion Center. A twosyringe system and Multi-step rinsing were designed for cell extraction. The final purpose of this optimization was: (1) removed the residual RBCs, (2) reversed the leukocyte trapping process, and (3) remove the microparticles to obtain the high yield of target cells. Finally, Extracted cells were evaluated by Automated Cell count; Samples smear differential cell count, Trypan blue, and Annexin-PI staining. The results showed that on average 11.88 × 108 ± 3.32 leukocytes recovered after indirect washing and that the mean count of granulocytes, lymphocytes, and Monocyte in this sample was 5.24 ± 2.18 × 108, 5.57 ± 1.74 × 108, and 0.56 ± 0.38 × 108 respectively. Also, the mean percent of manual differential cell count after concentration was 42.81%, 41.80%, and 15.82% for granulocytes, lymphocytes, and monocytes respectively. Moreover, viability and apoptosis assay showed > 95% viability in mononuclear cells recovered from LRFs. It is concluded that the use of a double-syringe system and RBC and microparticles removal from leukoreduction filters lead to acceptable viable leukocyte count that can be used in in vitro and in vivo studies.

Assessment of Standard Operating Procedures (SOPs) Preparing Hygienic Condition in the Blood Donation Centers during the Outbreak of COVID-19.

Background: The coronavirus disease 2019 (COVID-19) outbreak has led to an alteration in hygienic conditions. In this situation, improving standard operating procedures (SOPs) in blood donation centers is critical. The purpose of this study was the assessment of SOPs in the blood donation centers during the outbreak of COVID-19 by regular blood donors as external audits. Materials and Methods: Regular donors were selected as external inspectors in 31 provinces of Iran. The questionnaire containing 10 closed questions was provided to assess the hygienic SOPs of blood transfusion centers in the prevention of COVID-19 transmission. Comparison and evaluation of questionnaires were conducted by assigning an importance coefficient (IC) score to each question. Results: Assessment of SOPs in blood donation departments by regular donors in 31 provinces of Iran showed that 18 centers (58.1%) received IC scores >10(Strong performance), seven centers (22.6%) received the range of IC scores between7-10(acceptable performance), and six centers (19.4%) received IC scores <7(poor performance). The difference in IC scores between provinces was not statistically significant. Conclusion: This study confirms that the assessment of blood donation centers through regular blood donor inspection is a reliable method to identify the strengths and weaknesses of blood transfusion center services and ultimately leads to corrective intervention and improvement of hygienic SOPs to prevent COVID-19 transmission.

Source Plasma Donation: The Experience of the Iranian Blood Transfusion Organization.

Background: A declining need for red blood cells coupled with strengthening demand for plasma-derived medicines has led to a strong focus on moving whole blood donors to plasmapheresis. The purpose of this study was to evaluate the four-year policies of the Iranian Blood Transfusion Organization (IBTO) in terms of plasmapheresis recruitment of first-time donors and its effect on plasmapheresis outcome.   Materials and Methods: Plasmapheresis data related to 16 centers from 2016 to 2019 was obtained from IBTO software. This information includes; (1) blood donation number, (2) plasmapheresis donation number, (3) number of plasmapheresis donors, (4) plasmapheresis donor demographic data, (5) plasmapheresis donor status, (6) frequency of plasma donation for each donor, (7) volume of plasma and (8) the prevalence of transfusion-transmissible infections (TTIs) in plasmapheresis donors. Results: The result of this study demonstrated that plasmapheresis collection centers have recruited 85,515 (91%) first-time and 8,595(9%) regular and repeated donors from 2016 to 2019 years. Plasmapheresis to blood donation index was increased from 0.2% in 2016 to 4.9% in 2019. The mean donation number was 2 times per year. The trend of the yearly Whole Blood Donation (WBD) Index decreased from 26.69 to 24.11/1000 for the general population. The total volume of collected source plasma was 49,203 liters during this period. However, 46,000 liters of recovered plasma were decreased due to less WBD. Furthermore, the results indicated that the prevalence of HCV was significantly higher in first-time donors compared to repeated and regular donors (P = 0.000). Conclusion: It is concluded that during four years, the net volume of plasma did not increase and plasmapheresis led to reducing WBD in our country. Moreover, first-time plasmapheresis donors can be associated with challenges such as increasing screening costs and compromising the safety of plasma resources. Therefore IBTO decided to stop the project and focus on its main role to prepare safe and sufficient blood components through WB collection and also single donor platelet and concurrent plasma by plateletpheresis.

Potential Effect of Human Platelet Lysate on in vitro Expansion of Human Corneal Endothelial Cells Compared with Y-27632 ROCK Inhibitor.

PURPOSE: Corneal endothelial cell (CEC) therapy can be used as a promising therapeutic option for patients with various corneal endothelial dysfunctions. In this study, we compared the proliferative effect of human platelet lysate (HPL), as a xeno-free medium supplement, with Y-27632 Rho/rho-associated protein kinase (ROCK) inhibitor, as a well-known proliferative and adhesive agent for CECs, and fetal bovine serum (FBS) as the control, in the culture medium of human corneal endothelial cells (HCECs). METHODS: We isolated HCECs from human donors and treated the cells as three different treatment groups including 20% HPL only, 10 µM Y-27632 ROCK inhibitor, combination of 20% HPL and 10 µM Y-27632 ROCK inhibitor, and 20% FBS as the control group. ELISA cell proliferation assay and cell counting was performed on the treated cells. Finally, HCECs were characterized by morphology and immunocytochemistry (ICC). RESULTS: There was no significant proliferative effect of HPL on cell proliferation compared with the cells treated with Y-27632 ROCK inhibitor or the combination of HPL and Y-27632 ROCK inhibitor, but all the respected treatments had significant inducible effect on cell proliferation as compared with FBS-treated cells. The cells grown in all three treatment groups exhibited CEC morphology. Also, there was a higher expression of Na+/K+-ATPase and ZO-1, as CEC characteristic markers, in the culture of HCECs treated with HPL as compared with FBS. CONCLUSION: HPL offers a xeno - free and affordable medium supplement for CEC expansion that can be used in clinical applications.

Optimizing the effective doses of mitomycin C, 5-fluorouracil, and their combination on cultivated basal cell carcinoma.

PURPOSE: This study aimed to optimize the effective doses of mitomycin C, 5-fluorouracil, and their combination on cultivated basal cell carcinoma. METHODS: Cultivated basal cell carcinoma and fibroblastic cells were treated with different concentrations of mitomycin C, 5-fluorouracil, and their combination. Cell viability, cell cycle, apoptosis, and expression levels of TP53, CDKN1A, and CDK6 were investigated. The most effective drug with its optimum dosage was administered via multiple intralesional injections to a 65-year-old woman with advanced periorbital nodulo-ulcerative BCC. RESULTS: The concentrations of 0.00312 and 0.312 mg/mL were considered optimum for mitomycin C and 5-fluorouracil, respectively. The mean viabilities of basal cell carcinoma treated with mitomycin C alone and its combination with 5-fluorouracil were significantly less than those of the controls (p=0.002 and p=0.04, respectively). The cell cycle of all the treated basal cell carcinoma groups was arrested in the S phase. The apoptotic rates (p=0.002) of mitomycin C treated basal cell carcinoma were higher than those of the other treated cells, and their TP53 was significantly upregulated (p=0.0001). Moreover, CDKN1A was upregulated, whereas CDK6 was downregulated in basal cell carcinoma treated with either 5-fluorouracil (p=0.0001 and p=0.01, respectively) or the combination of 5-fluorouracil and mitomycin C (p=0.007 and p=0.001, respectively). Basal cell carcinoma lesions were significantly alleviated following mitomycin C injections in the reported patient. CONCLUSION: Our in vitro results revealed that the effective doses of mitomycin C and 5-fluorouracil on cultivated basal cell carcinoma were optimized. Mitomycin C was more effective in inducing the apoptosis of basal cell carcinoma than 5-fluorouracil and their combination. The intralesional injections of the optimum dose of mitomycin C could be proposed for the nonsurgical treatment of advanced eyelid basal cell carcinoma.

Vitamin C Loaded Poly(urethane-urea)/ZnAl-LDH Aligned Scaffolds Increase Proliferation of Corneal Keratocytes and Up-Regulate Vimentin Secretion.

A novel poly(urethane-urea) (PUU) based on poly(glycolide-co-ε-caprolactone) macro-diol with tunable mechanical properties and biodegradation behavior is reported for corneal stromal tissue regeneration. Zn-Al layered double hydroxide (LDH) nanoparticles were synthesized and loaded with vitamin C (VC, VC-LDH) and dispersed in the PUU to control VC release in the cell culturing medium. To mimic the corneal stromal EC, scaffolds of the PUU and its nanocomposites with VC-LDH (PUU-LDH and PUU-VC-LDH) were fabricated via electrospinning. Average diameters of the aligned nanofibers were recorded as 325 ± 168, 343 ± 171, and 414 ± 275 nm for the PUU, PUU-LDH, and PUU-VC-LDH scaffolds, respectively. Results of hydrophilicity and mechanical properties measurements showed increased hydrophobicity and reduced tensile strength and elongation at break upon addition of nanoparticles to the PUU scaffold. VC release studies represented that intercalation of the drug in Zn-Al-LDH controlled the burst release and extended the release period from a few hours to 5 days. Viability and proliferation of stromal keratocyte cells on the scaffolds were investigated via AlamarBlue assay. After 24 h, the cells showed similar viability on the scaffolds and the control. After 1 week, the cells showed some degree of proliferation on the scaffolds, with the highest value recorded for PUU-VC-LDH. SEM images of the scaffolds after 24 h and 1 week confirmed good penetration and attachment of keratocytes on all the scaffolds and the cells oriented with the direction of nanofibers. After 1 week, the PUU-VC-LDH scaffold was fully covered by the cells. Immunocytochemistry assay (ICC) was performed to investigate secretion of vimentin protein, ALDH3A1, and α-SMA by the cells. After 24h and 1 week, remarkably higher levels of vimentin and ALDH3A1 and lower level of α-SMA were secreted by keratocytes on PUU-VC-LDH compared to those on the PUU and PUU-LDH scaffolds and the control. Our results suggest that the aligned PUU-VC-LDH is a promising candidate for corneal stromal tissue engineering due to the presence of zinc and vitamin C.

Autologous Platelet-rich Plasma Eye Drops Accelerate Re-epithelialization of Post-keratoplasty Persistent Corneal Epithelial Defects.

PURPOSE: To investigate whether autologous platelet-rich plasma (PRP) eye drops accelerate re-epithelialization of post-keratoplasty persistent corneal epithelial defects (PEDs). METHODS: A total of 34 eyes with PEDs after keratoplasty (24 penetrating keratoplasty and 10 deep anterior lamellar keratoplasty) that were refractory to conventional medical treatments were treated with PRP eye drops every 3 hours. PRP eye drops were prepared with a low- and high-speed centrifugation method and final platelet counts were 700,000-800,000 plt/µl. The mean treatment duration for complete re-epithelialization was compared with the mean treatment duration of conventionally treated corneal defects before the PRP treatment by paired t-test. The mean treatment duration was also statistically analyzed between age groups, gender, indications for keratoplasty, and types of keratoplasty using analysis of variance (ANOVA). RESULTS: Treatment with autologous PRP eye drops led to rapid re-epithelialization in all eyes. The mean treatment duration for complete re-epithelialization was 2.47 ± 1.21 weeks, which was significantly shorter than the mean treatment duration of conventionally treated corneal defects before PRP treatment (6.82 ± 1.24 weeks) (P = 0.0001). There was no significant correlation between re-epithelialization time and patients' age, sex, indications for keratoplasty, and techniques of corneal transplantation. CONCLUSION: Treatment with autologous PRP eye drops is an effective and reliable approach that accelerates re-epithelialization of post-transplantation PEDs.

Effects of fibrin glue as a three-dimensional scaffold in cultivated adult human retinal pigment epithelial cells.

This study was conducted to examine morphological, genotypic, and phenotypic alterations occurring in cultured adult human retinal pigment epithelial cells when encapsulated with different concentrations of fibrin glue. Cultivated adult human retinal pigment epithelial cells were encapsulated with different concentrations of fibrin glue, namely FG1 (42 mg/dl), FG2 (84 mg/dl), FG3 (124 mg/dl), FG4 (210 mg/dl), followed by the evaluation of genetic and cytomorphological changes and protein expression. Cultured adult human retinal pigment epithelial cells showed dendritiform morphology during the early days of encapsulation with fibrin glue. Moreover, an increasing inhibitory effect on cell growth was observed with increasing concentrations of fibrin glue. At the transcriptional level, the expression of MMP2, PAX6, and ITGB1 in FG1-encapsulated cells was significantly higher than that in other treated groups; however, the expression of ACTA2 was lower in all fibrin glue-encapsulated groups compared to that in the controls. Immunocytochemistry showed that FG2-encapsulated cells expressed cytokeratin 8/18, RPE65, and ZO-1 proteins, but not PAX6. In conclusion, fibrin glue at a concentration of 84 mg/dl allows proper encapsulation of adult human retinal pigment epithelial cells, while preserving the morphometric, genotypic, and phenotypic features of the cells. This three-dimensional biopolymer can be considered a reliable vehicle for retinal pigment epithelium cell transplantation in cell-based therapies.

Real Time Polymerase Chain Reaction for Hepatitis B Screening in Donor Corneas in the Central Eye Bank of Iran.

PURPOSE: The aim of this study was to report the results of the use of real-time polymerase chain reaction (PCR) for the diagnosis of hepatitis B virus (HBV) infection in cornea donors at the Central Eye Bank of Iran. METHODS: Between 2014 and 2016, all cornea donors that had negative screening serologic results for hepatitis B (HB) surface antigen, HB surface antibody (Ab), hepatitis C virus Ab, human immune deficiency virus Ab, human T-cell leukemia virus Ab, and syphilis, and positive serology for HB core Ab were subjected to real-time PCR with a detection limit of 400 IU/mL to identify HBV DNA. Positive results for HBV DNA were considered occult HBV infections in these donors. RESULTS: Over the 3-year period, 122 out of 10448 cornea donors had negative screening serologic tests outside of HB core Ab. Of which, 90 cases were subjected to real-time PCR. Occult HBV was detected in 11 cases (12.2%), resulting in the rejection of the corresponding corneas. The remaining 79 cases (87.8%) had negative results for HBV DNA and the corresponding corneas were used for transplantation. CONCLUSION: Implementation of PCR for the detection of occult HBV in cornea donors is necessary to not only increase the security level of cornea donation but also minimize the rejection rate of donors that have isolated HB core Ab reactivity.

The effects of platelet gel on cultured human retinal pigment epithelial (hRPE) cells.

The positive role of platelet gel (PG) in tissue regeneration is well known, however, other characteristics of PG still remain to be determined. We investigated cellular and molecular changes in cultured human retinal pigment epithelial (hRPE) cells when treated with different concentrations of PG named PG1, PG2, and PG3. hRPE cells were isolated from donor eyes of two newborn children, within 24 hours after their death. The cells were treated with three concentrations of PG for 7 days: 3 × 104/ml (PG1), 6 × 104/ml (PG2), and 9 × 104/ml (PG3). Fetal bovine serum was used as a control. Immunocytochemistry was performed with anti-RPE65 (H-85), anti-Cytokeratin 8/18 (NCL-5D3), and anti-PAX6 antibody. We used MTT assay to determine cell viability. Gene expressions of PAX6, MMP2, RPE65, ACTA2, MKI67, MMP9, and KDR were analyzed using real-time PCR. A significant increase in viability was observed for PG3-treated cells compared to control (p = 0.044) and compared to PG1 group (p = 0.027), on day 7. Cellular elongation together with dendritiform extensions were observed in PG-treated cells on days 1 and 3, while epithelioid morphology was observed on day 7. All cells were immunoreactive for RPE65, cytokeratin 8/18, and PAX6. No significant change was observed in the expression of MKI67 and PAX6, but the expressions of MMP2, MMP9, ACTA2, and KDR were significantly higher in PG2-treated cells compared to controls (p < 0.05). Our results indicate that increased concentration of PG and extended exposure time have positive effects on viability of hRPE cells. PG may be useful for hRPE cell encapsulation in retinal cell replacement therapy.