Analytical chromatography approaches during the synthesis and conjugation of methoxypolyethylene glycol-succinimidyl butanoate (mPEG-SBA) to epoetin beta.

Conjugation of epoetin beta (EPO) with methoxypolyethylene glycol-succinimidyl butanoate (mPEG-SBA) was studied. The compound mPEG-SBA was synthesized from mPEG, and the obtained intermediates and final product were analyzed by a reversed-phase chromatographic system equipped with an evaporative light scattering detector. Labeling the hydroxyl group in PEGs with benzoyl chloride and succinimide with benzylamine was applied to resolve and characterize different PEGs. The synthesized mPEG-SBA was used for the PEGylation of EPO. A size-exclusion chromatographic method monitored the reaction, simultaneously determining the PEGylated and unreacted EPO and protein aggregates. A borate buffer (0.1 M, pH 7.8) and PEG/protein molar ratio of 3:1 produced a maximum amount of monoPEGylated EPO with the minimum amount of polyPEGylated EPO variants. Although EPO is considered a stable glycoprotein hormone that remains monomeric when refrigerated, PEGylation of EPO with mPEG-SBA resulted in the significant formation of EPO dimer. The formation of EPO dimer and polyPEGylated EPO was pH-dependent, showing higher amounts of aggregates and lower amounts of polyPEGylated forms in lower pH values. Accordingly, aggregated EPO should be considered a major PEGylation-related impurity. In conclusion, the present study highlighted the importance of having suitable analytical approaches in controlling mPEG-SBA synthesis and conjugation to EPO.

Evaluating the efficacy and safety of rivaroxaban as a warfarin alternative in chronic thromboembolic pulmonary hypertension patients undergoing pulmonary endarterectomy: A randomized clinical trial.

BACKGROUND AND AIM: Chronic thromboembolic pulmonary hypertension (CTEPH) is caused by the obstruction of the main pulmonary artery due to thrombosis and vascular remodeling. Regarding the need for anticoagulant therapy in CTEPH patients, this study aimed to compare rivaroxaban with warfarin in terms of its efficacy and safety in patients undergoing endarterectomy surgery. METHODS: The study was a parallel clinical trial in patients who underwent endarterectomy following CTEPH. A total of 96 patients were randomly selected and assigned to two groups: warfarin-treated (control) and rivaroxaban-treated (intervention). Patients were clinically assessed for re-thrombosis, re-admission, bleeding, and mortality in the first, third, and sixth months after surgery. RESULTS: There was no significant difference in the occurrence of thrombosis between the two groups within the first, third-, and sixth-months post-surgery (p=0.52, 1, 0.38 respectively). Moreover, the mortality rate (p=0.9), bleeding rate (p=0.06), and re-admission rate (p=0.15) showed no significant differences between the two groups. CONCLUSION: Rivaroxaban may be as effective as warfarin in treating CTEPH patients after endarterectomy in the short term and can be used as an anticoagulant in these patients. However, studies with long-term follow-ups are needed to consolidate the strategy of treating these patients with rivaroxaban.

Mortality rate of people exposed to Mustard Gas during Iran-Iraq war in Sardasht, Iran: a 32 years retrospective cohort study.

BACKGROUND: Mustard gas (MG) is one of the most widely used chemical weapons in the past century. However, little information exists concerning long-term mortality from MG exposure. In this study, we investigated mortality rate among civilian people exposed to MG during Iran-Iraq war in Sardasht in Iran after 32 years.  METHODS: In this retrospective cohort study, data of people exposed to MG in Sardasht in 1987 were extracted from the Veterans and Martyr Affair Foundation of Iran up to March 20, 2019. Mortality rate, cumulative mortality and standardized mortality ratio with 95% confidence interval were calculated to explain mortality in the cohort, and then compared with general Iranian population. Cox regression analysis was used to indicate factor affecting the risk of death in the cohort.  RESULTS: Out of 1,203 exposed people at the beginning of the period, 148 people died by the end of the study, with an average age of 66.42 at the time of death. Total person-years of the people up to end of the study were 38,198.63 and mortality rate was equal to 387 per 100,000 persons-years. Total number of observed deaths was less than expected death and the all-cause standardized mortality ratio (SMR) was determined as 0.680 (95% CI: 0.574 - 0.798). Cause-specific SMR showed that observed death due to respiratory diseases was higher than expected (SMR: 1.75) (95% CI: 1.145 - 2.569). The results of univariate and multivariate cox regression analysis showed that increasing age and having severe late complications in lung were associated with increased risk of death among people in the cohort. CONCLUSION: In general, this result indicated that acute exposure to MG, even without wearing protective clothing and masks, could not increase all-cause mortality after 32 years if accompanied by special and ongoing care for those exposed.

Genetically Engineered Mesenchymal Stem Cell Therapy Against Murine Experimental Autoimmune Encephalomyelitis.

We used recombinant interleukin 23 receptor (RIL-23R)-engineered mesenchymal stem cells (MSCs) to study its therapeutic role in enhancing inflammation of nervous tissue in the mouse model (EAE) of multiple sclerosis (MS). Recombinant IL-23 receptor construct was designed to enter MSCs. The bioactivity of the constructs was assessed by the co-culture of MSCs/CD4 + T cells. The EAE model was induced in mice. After cell transplantation, clinical scores were evaluated, and tissue demyelination was measured by Luxol fast blue staining. The transfection of RIL-23R mRNA improved MSC properties significantly to the inflamed regions of EAE mice, and it performed an increased suppressive function on the T lymphocyte proliferation. Furthermore, in vivo therapy with RIL-23R MSCs in EAE mice showed an enhanced therapeutic action than MSCs, proven by improved myelination and a reduction in the penetration of inflammatory cells into the white matter. Our targeted transplantation procedure of modified MSC can be applied to improve the effectiveness of cellular therapy for multiple sclerosis and other autoimmune disorders.

Decreased in-hospital mortality associated with aspirin administration in hospitalized patients due to severe COVID-19.

Hypercoagulability and thrombosis caused by coronavirus disease 2019 (COVID-19) are related to the higher mortality rate. Because of limited data on the antiplatelet effect, we aimed to evaluate the impact of aspirin add-on therapy on the outcome of the patients hospitalized due to severe COVID-19. In this cohort study, patients with a confirmed diagnosis of severe COVID-19 admitted to Imam Hossein Medical Center, Tehran, Iran from March 2019 to July 2020 were included. Demographics and related clinical data during their hospitalization were recorded. The mortality rate of the patients was considered as the primary outcome and its association with aspirin use was assessed. Nine hundred and ninety-one patients were included, of that 336 patients (34%) received aspirin during their hospitalization and 655 ones (66%) did not. Comorbidities were more prevalent in the patients who were receiving aspirin. Results from the multivariate COX proportional model demonstrated a significant independent association between aspirin use and reduction in the risk of in-hospital mortality (0.746 [0.560-0.994], p = 0.046). Aspirin use in hospitalized patients with COVID-19 is associated with a significant decrease in mortality rate. Further prospective randomized controlled trials are needed to assess the efficacy and adverse effects of aspirin administration in this population.

Pregnancy Outcomes of Wives of Chemical and Non-Chemical Weapons Exposed Veterans in Ahvaz, Iran: A Retrospective Cohort Study.

BACKGROUND: Sulphur mustard (HD) is a lipophilic caustic alkylating vesicant (blister agent) that has mutagenic and carcinogenic effects. Among the studied perturbations are long-term genitourinary (GU) and fertility effects. Approximately 50,000 Iranian soldiers and civilians were exposed to HD during the Iraq-Iran war (1980-1989). This study questioned the wives of Iraq-Iran war veterans to determine the effects of male HD-exposure on pregnancy complications, adverse pregnancy outcomes, and secondary infertility. METHODS: A retrospective, survey-based cohort study was conducted of wives of Iranian military veterans that survived HD-associated injuries while serving in Ahvaz, Iran during the Iraq-Iran war (1980-1989), as compared to non-exposed veterans serving concomitantly. Patients were identified from a database of injured veterans maintained by the Foundation of Martyrs and Veterans Affairs (Iran) via a systematic random sampling method utilizing a random number table. Using a validated questionnaire, collected data included: demographics; type and severity of chemical injury; spouse's obstetric history (pregnancy number, duration, complications, and outcomes before and after spouse's chemical injury); and secondary infertility. RESULTS: An increase in spontaneous abortion (P = .03), congenital anomalies (P < .0001), and secondary infertility (P = .003) were observed. These findings were greatest amongst those with HD injuries affecting >50% body surface area. No difference in stillbirth, premature birth, or low birth weight was observed. CONCLUSION: Exposure to HD in combat may have long-lasting fertility effects on soldiers and their spouses, including spontaneous abortion, congenital anomalies, and secondary infertility. Further investigation is needed into the long-term effects of HD exposure as well as methods to better protect soldiers.

Association between passive smoking and cardiovascular disease: A systematic review and meta-analysis.

BACKGROUND: The association between passive smoking (PS) and cardiovascular disease (CVD) has not yet been fully clarified. OBJECTIVE: This meta-analysis was performed to evaluate the association between PS and the incidence of CVDs and mortality due to CVD. METHODS: PubMed/Medicine, Science Direct, Scopus, Web of Knowledge, and ProQuest were searched to identify observational studies that met the inclusion criteria without time, language, age, gender, ethnicity, and design restrictions until July 30, 2018. In case-control studies, relative risk (RR) with 95% confidence interval (CI) was calculated for the relationship between PS and CVD incidence. Also, in cohort studies, hazard ratio (HR) with 95% CI was calculated for the relationship between PS and CVD mortality. RESULTS: Eighteen studies (10 cohort and 8 case-control studies) were included with 10,672 participants (2,542 cases and 8,130 controls) in case-control studies and 2,313,935 participants in cohort studies. This meta-analysis in case-control studies revealed that the PS could increase the risk of CVD incidence by 28% (adjusted RR = 1.28 [95% CI 1.09, 1.50]), where the highest risk was associated with those who were exposed to second-hand smoke at home and at work (Adjusted RR = 1.41 [95% CI 0.73, 2.70]). Also, the meta-analysis in cohort studies indicated that PS was associated with a 12% higher increase in the risk of CVD mortality (Adjusted HR = 1.12 [95% CI 1.06, 1.20]) with the highest risk of mortality being observed for those who were exposed to second-hand smoking at home, work, and public places (Adjusted HR = 1.26 [95% CI 1.13, 1.40]). CONCLUSIONS: PS is significantly associated with an increased risk of incidence and mortality of CVD.

Percutaneous biopsy of kidney; comparison between prone position and sitting position.

INTRODUCTION: Percutaneous kidney biopsy has been established as a safe, reliable and minimally invasive method. This study aims to describe the author's experience with biopsy of the kidney and to compare the results in sitting position versus prone in terms of the complication rate. MATERIALS AND METHODS: Patients were divided into two groups: prone and sitting position according to the clinician's and patient's preference. Followed by kidney biopsy, a questionnaire was completed. Then, data and the mean number of glomeruli in each group were compared. RESULTS: Apart from sweat, presumably due to the prone position, no significant differences were found regarding the side effects including dizziness, seizure, nausea, and vomiting between the two groups. The number of glomeruli was not significantly different between two groups. CONCLUSION: In comparison with the prone position, kidney biopsy at sitting position is more comfortable at least for patients who seems couldn't tolerate prone position. We recommend sitting position for kidney biopsy owing to the low side effects rate of this diagnostic technique.

Directed differentiation of human adipose tissue-derived stem cells to dopaminergic neurons in low-serum and serum-free conditions.

Directing the fate of mesenchymal stem cells (MSCs) to dopaminergic neurons has great importance in both biomedical studies and cell therapy of Parkinson's disease. We recently generated dopamine-secreting cells from human adipose tissue-derived stem cells (hADSCs) by exposing the cells to a growth factor cocktail composed of SHH, bFGF, FGF8 and BDNF in low-serum condition. In the current study, we induced the cells by the same dopaminergic inducing cocktail in serum-free B27-supplemented Neurobasal medium. ADSCs differentiated in both conditions expressed several neuronal and dopaminergic markers. However, there were higher gene expression levels under the serum-free condition. Higher levels of TUJ1 and TH proteins were also detected in the cells exposed to the dopaminergic-inducing cocktail under serum-free Neurobasal condition. TH protein was expressed in about 28% and 60% of the cells differentiated in the low-serum and serum-free Neurobasal media, respectively. Moreover, the cells exposed to the dopaminergic-inducing cocktail in the serum-free Neurobasal condition released a more significant amount of dopamine in response to KCl-induced depolarization. Altogether, these findings show a greater efficiency of the serum-free Neurobasal condition for growth factor-directed differentiation of hADSCs to functional dopamine-secreting cells which may be valuable for transplantation therapy of Parkinson's disease in future.

Determination of pegfilgrastim aggregates by size-exclusion high-performance liquid chromatography on a methacrylate-based column.

A size-exclusion high-performance liquid chromatographic method using a methacrylate-based column was developed, validated and implemented for the determination of pegfilgrastim aggregates. The samples were directly injected into a TSKgel G4000PWXL column (7.5 mm × 300 mm, 10 µm, <500 A°) with a mobile phase of 100 mM phosphate, pH 2.5. Detection was made at 215 nm and analyses were run at a flow-rate of 0.6 ml/min at 10 °C. Vortex-mixing of samples produced oligomers, however, very high molecular weight aggregates were formed at high temperatures. The method exhibited linearity over the concentration range of 0.1-14 mg/ml for pegfilgrastim monomer and high molecular weight aggregates with a correlation coefficient of greater than 0.99. The method was specific and sensitive, with a lower quantification limit of 0.1 mg/ml and a detection limit of 0.02 mg/ml. Over 1200 samples were analyzed by the present method without significant change in the column performance.

Estimating the Completeness of Lung Cancer Registry in Ardabil, Iran with a Three-Source Capture-Recapture Method.

Cancer registration is an important component of a comprehensive cancer control program, providing timely data and information for research and administrative use. Capture-recapture methods have been used as tools to investigate completeness of cancer registry data. This study aimed to estimate the completeness of lung cancer cases registered in Ardabil Population Based Cancer Registry (APBCR) with a three-source capture-recapture method. Data for all new cases of lung cancer reported by three sources (pathology reports, death certificates, and medical records) to APBCR for 2006 and 2008 were obtained. Duplicate cases shared among the three sources were identified based on similarity of first name, last name and father's names. A log-linear model was used to estimate number of missed cases and to control for dependency among sources. A total of 218 new cases of lung cancer was reported by three sources after removing duplicates. The estimated completeness calculated by log-linear method was 26.4 for 2006 and 27.1 for 2008. The completeness differed according to gender. In men, the completeness was 26.0% for 2006 and 28.1 for 2008. In women, the completeness was 36.5% for 2006 and 46.9 for 2008. In conclusion, none of the three sources can be considered as a reliable source for accurate cancer incidence estimation.

Rapid formulation assessment of filgrastim therapeutics by a thermal stress test.

The biosimilar versions of recombinant methionyl human granulocyte colony-stimulating factor (rh-Met-G-CSF, filgrastim) are now widely available. Because changes to the formulation often lead to subtle differences, there is a critical need to define techniques to test and insure the quality of these products. The present study was designed to compare formulation and thermal stress stability of filgrastim products. The formulation ingredients including acetate, polysorbate 80, and sorbitol were determined using state-of-the-art validated analytical methods. The formulation pH and osmolality were also measured. Moreover, the stability profiles of 8 filgrastim products using thermal stress at 57 °C for 4 h were assessed by size-exclusion high-performance liquid chromatography (SE-HPLC) and in vitro biological assay. The products had different stability profiles. More stable products were within the specification for formulation and less stable products were beyond the specification limits. Altogether, the results suggest that a short-time stress study at 57 °C and analysis of filgrastim by SE-HPLC could unveil formulation problems and is potentially useful for comparability studies.