In vitro differentiation capacity of telomerase immortalized human RPE cells

PURPOSE: To investigate conditions promoting the differentiation of cultured human retinal pigment epithelial (RPE) cells and assess the differentiation potential of telomerase-immortalized RPE cells. METHODS: Serially passaged RPE 340 (parental) cells have limited replicative ability and senesce after 50 to 60 population doublings (PD)s. RPE 340 cells transfected with the catalytic component of human telomerase (hTERT) have an extended lifespan. RPE 340 and hTERT-transfected RPE (hTERT-RPE) cells were maintained at confluence without serum for up to 12 weeks. Morphologic, immunocytochemical, flow cytometric, and spectrophotometric analyses were performed to examine the extent of RPE differentiation. RESULTS: Parental RPE 340 and hTERT-RPE cells underwent growth arrest and differentiated in the absence of serum. In early-passage parental (PD11) and hTERT-RPE (PD115 and PD300) cells, serum deprivation for 4 weeks or more induced terminal differentiation as characterized by mature, growth-arrested, confluent sheets of polygonal and melanized cells that demonstrated diminished 5'-bromo-2'-deoxyuridine (BrdU) uptake and positive reactivity with antibodies to cellular retinaldehyde-binding protein (CRALBP), cytokeratin, and vimentin. Reintroduction of serum at 4 or 8 weeks allowed the cells to reenter the cell cycle and demelanize. Midpassage (PD 25) parental cells, however, were irreversibly arrested at G(1) after 8 weeks of serum deprivation. CONCLUSIONS: Cultured parental and hTERT-RPE cells express RPE-associated proteins and become stably melanized when density is arrested in the absence of serum. Moreover, hTERT-immortalized RPE cells retain the capacity to undergo terminal differentiation in vitro, even after long-term culture.     

The nonsteady state modeling of freeze drying: in-process product temperature and moisture content mapping and pharmaceutical product quality applications

INTRODUCTION: Theoretical models of the freeze-drying process are potentially useful to guide the design of a freeze-drying process as well as to obtain information not readily accessible by direct experimentation, such as moisture distribution and glass transition temperature, Tg, within a vial during processing. Previous models were either restricted to the steady state and/or to one-dimensional problems. While such models are useful, the restrictions seriously limit applications of the theory. An earlier work from these laboratories presented a nonsteady state, two-dimensional model (which becomes a three-dimensional model with an axis of symmetry) of sublimation and desorption that is quite versatile and allows the user to investigate a wide variety of heat and mass transfer problems in both primary and secondary drying. The earlier treatment focused on the mathematical details of the finite element formulation of the problem and on validation of the calculations. The objective of the current study is to provide the physical rational for the choice of boundary conditions, to validate the model by comparison of calculated results with experimental data, and to discuss several representative pharmaceutical applications. To validate the model and evaluate its utility in studying distribution of moisture and glass transition temperature in a representative product, calculations for a sucrose-based formulation were performed, and selected results were compared with experimental data. THEORETICAL MODEL: The model is based on a set of coupled differential equations resulting from constraints imposed by conservation of energy and mass, where numerical results are obtained using finite element analysis. Use of the model proceeds via a "modular software package" supported by Technalysis Inc. (Passage/ Freeze Drying). This package allows the user to define the problem by inputing shelf temperature, chamber pressure, container properties, product properties, and numerical analysis parameters required for the finite element analysis. Most input data are either available in the literature or may be easily estimated. Product resistance to water vapor flow, mass transfer coefficients describing secondary drying, and container heat transfer coefficients must normally be measured. Each element (i.e., each small subsystem of the product) may be assigned different values of product resistance to accurately describe the nonlinear resistance behavior often shown by real products. During primary drying, the chamber pressure and shelf temperature may be varied in steps. During secondary drying, the change in gas composition from pure water to mostly inert gas is calculated by the model from the instantaneous water vapor flux and the input pumping capacity of the freeze dryer. RESULTS: Comparison of the theoretical results with the experiment data for a 3% sucrose formulation is generally satisfactory. Primary drying times agree within two hours, and the product temperature vs. time curves in primary drying agree within about +/-1 degrees C. The residual moisture vs. time curve is predicted by the theory within the likely experimental error, and the lack of large variation in moisture within the vial (i.e., top vs. side vs. bottom) is also correctly predicted by theory. The theoretical calculations also provide the time variation of "Tg-T" during both primary and secondary drying, where T is product temperature and Tg is the glass transition temperature of the product phase. The calculations demonstrate that with a secondary drying protocol using a rapid ramp of shelf temperature, the product temperature does rise above Tg during early secondary drying, perhaps being a factor in the phenomenon known as "cake shrinkage." CONCLUSION: The theoretical results of in-process product temperature, primary drying time, and moisture content mapping and history are consistent with the experimental results, suggesting the theoretical model should be useful in process development and "trouble-shooting" applications.     

Myxoma of the orbit

Myxomas are rare, benign neoplasms of mesenchymal origin that usually develop in soft tissues. As the clinical manifestations are non-specific, it is difficult to diagnose the tumour without biopsy and histopathological examination. We report a case of orbital myxoma with histopathological correlation.     

Rationale for phosphodiesterase 5 inhibitor use post-radical prostatectomy: experimental and clinical review

Erectile dysfunction (ED) is a common complication after radical prostatectomy and results from trauma sustained by the cavernosal nerves. This is a major concern for patients and often affects treatment decisions. The likely mechanism for post-prostatectomy ED is through corporal veno-occlusive dysfunction. There is an increasing amount of evidence to suggest that phosphodiesterase 5 inhibitors (PDE5 inhibitors), when given on a continuous long-term basis, can help to prevent and reverse ED after surgery. In this review article we will examine the pathophysiology of post-prostatectomy ED and discuss the experimental and available clinical evidence for administering PDE5 inhibitors after prostatectomy.     

COVID-19 Infection in Men on Testosterone Replacement Therapy

BackgroundMen who contract coronavirus disease 2019 (COVID-19) appear to have worse clinical outcomes compared with women which raises the possibility of androgen-dependent effects.AimWe sought to determine if testosterone replacement therapy (TRT) is associated with worse clinical outcomes.MethodsThrough a retrospective chart review, we identified 32 men diagnosed with COVID-19 and on TRT. They were propensity score matched to 63 men diagnosed with COVID-19 and not on TRT. Data regarding comorbidities and endpoints such as hospital admission, intensive care unit admission, ventilator utilization, thromboembolic events, and death were extracted. Chi-square and Kruskal-Wallis tests examined differences in categorical and continuous variables, respectively. Logistic regression analysis tested the relationship between TRT status and the study endpoints.ResultsThere were no statistically significant differences between the 2 groups, and TRT was not a predictor of any of the endpoints on multivariate analysis.ConclusionThese results suggest that TRT is not associated with a worse clinical outcome in men diagnosed with COVID-19.Rambhatla A, Bronkema CJ, Corsi N, et al. COVID-19 Infection in Men on Testosterone Replacement Therapy. J Sex Med 2021;18:215–218.     

Transtracheal aspiration in diagnosis of sputum-smear--negative tuberculosis.

Of 4,200 patients admitted to an acute-care county hospital, 126 (3%) were proved to have pulmonary tuberculosis, among whom 35 (28%) had several sputum smears negative for acid-fast bacilli. On transtracheal aspiration, 31 to 35 had acid-fast bacilli in the aspirate. Eighteen of these 35 (51%) patients had associated infections caused by aerobic or anaerobic bacteria. Tuberculin skin tests were negative in 14 of 35 patients with negative sputum specimens (40%). Ten of 18 patients (56%) with associated bacterial infections had negative skin tests to purified protein derivative. Smear and culture of transtracheal aspirate for tubercle bacilli may be invaluable in establishing the diagnosis when pulmonary tuberculosis is suspected.     

Association of herpesviruses in the aqueous humor of patients with serpiginous choroiditis: a polymerase chain reaction-based study

Purpose: To determine the presence of herpesvirus DNA in the aqueous humor (AH) of patients with serpiginous choroiditis using polymerase chain reaction (PCR). Methods: AH from nine patients previously diagnosed with serpiginous choroiditis were investigated for herpes simplex virus (HSV), varicella zoster virus (VZV), and cytomegalovirus (CMV) by conventional virological methods and PCR. The PCR-positive DNA was gel-purified, extracted, and sequenced using a dye-based Applied Biosystems procedure. The sequences were processed through the National Cancer Institute’s BLAST inquiry for species identification. Results: Culture and cytological examination of AH from all nine patients were negative for HSV,VZV, and CMV. Five were positive for VZV, one was positive for HSV, and three were wholly negative using PCR. Subsequent DNA sequencing of the positive samples authenticated the presence of VZV and HSV DNA in the respective patients. Conclusion: VZV and HSV DNA were detected in a subset of patients with serpiginous choroiditis, suggesting that these viruses may function in the pathogenesis of this disease.