Understanding the effect of high gas entrance velocity on Chinese hamster ovary (CHO) cell culture performance and its implications on bioreactor scale-up and sparger design.

There are three main potential sources for cell shear damage existing in stirred tank bioreactors. One is the potential high energy dissipation in the immediate impeller zones; another from small gas bubble burst; and third is from high gas entrance velocity (GEV) emitting from the sparger. While the first two have been thoroughly addressed for the scale-up of Chinese hamster ovary (CHO) cell culture knowing that a wide tolerable agitation range with non-damaging energy dissipation exists and the use of shear protectants like Pluronic F68 guard against cell damage caused by bubble burst, GEV remains a potential scale-up problem across scales for the drilled hole or open pipe sparger designs. GEV as high as 170 m/s due to high gas flow rates and relatively small sparger hole diameters was observed to be significantly detrimental to cell culture performance in a 12,000 L bioreactor when compared to a satellite 2 L bioreactor run with GEV of <1 m/s. Small scale study of GEV as high as 265 m/s confirmed this. Based on the results of this study, a critical GEV of >60 m/s for CHO cells is proposed, whereas previously 30 m/s has been reported for NS0 cells by Zhu, Cuenca, Zhou, and Varma (2008. Biotechnol. Bioeng., 101, 751-760). Implementation of new large scale spargers with larger diameter and more holes lowered GEV and helped improve the cell culture performance, closing the scale-up gap. Design of such new spargers was even more critical when hole plugging was discovered during large scale cultivation hence exacerbating the GEV impact. Furthermore, development of a scale down model based on mimicry of the large scale GEV profile as a function of time was proven to be beneficial for reproducing large scale results.

VISUAL AND ANATOMICAL OUTCOMES AFTER DIABETIC TRACTION AND TRACTION-RHEGMATOGENOUS RETINAL DETACHMENT REPAIR.

PURPOSE: To evaluate visual and anatomical outcomes of diabetic tractional retinal detachment repaired with pars plana vitrectomy. METHODS: Operative records were used to retrospectively identify all patients with tractional retinal detachments secondary to proliferative diabetic retinopathy surgically repaired with pars plana vitrectomy between November 1, 2009, and January 1, 2015 at the LAC + USC (Los Angeles County + University of Southern California) Medical Center. RESULTS: A total of 403 eyes with diabetic tractional retinal detachment in 359 patients were included. Successful reattachment of the retina was achieved in 87.6% of eyes after one surgery and 92.6% of eyes at the final follow-up. Best-corrected visual acuity at the final follow-up improved two or more lines in 56.3% of eyes, was stable in 23.8% of eyes, and decreased two or more lines in 19.9% of eyes. Eyes repaired with 23-gauge and 25-gauge vitrectomy systems had similar success rates as eyes treated with 20-gauge instrumentation (P = 0.73). Eyes receiving silicone oil tamponade had lower single-surgery reattachment rates (77.6% vs. 87.6%; P = 0.013), lower reattachment rates at the final follow-up (85.7% vs. 92.6%; P = 0.048), and higher rates of vision loss (34.7% vs. 19.9%; P < 0.0001) but were more likely to have concurrent rhegmatogenous detachment (47.0% vs. 21.3%; P < 0.0001) and macula involving detachment (74.5% vs. 60.0%; P < 0.0001). CONCLUSION: In this large, single-center retrospective study of patients with advanced diabetic tractional retinal detachment, vitrectomy achieved excellent anatomical outcome and improved or stabilized vision in 80.1% of eyes. Smaller gauge vitrectomy systems were found to have similar outcomes to 20-gauge instrumentation.

Telemedicine for Retinal Disease During the COVID-19 Pandemic: Survey of the Patient Perspective.

INTRODUCTION: Amidst the COVID-19 pandemic, telemedicine has emerged as a safe and cost-effective alternative to traditional ophthalmology clinic visits. This study evaluated patient attitudes towards telemedicine at a full-service, retina-only practice to identify areas for growth in implementation. METHODS: A survey was distributed to established patients at University Retina and Macula Associates following the completion of a telemedicine encounter in July 2021. On a 5-point Likert scale, patients compared telemedicine to in-person visits for six domains: ability to ease COVID-related anxiety, efficiency, patient education, quality of care, fulfillment of personal needs, and convenience. Pearson's χ2 and Fisher's exact test were used to assess correlations between demographic factors and patient attitudes or preference towards telemedicine. RESULTS: Among 103 respondents, two-thirds (68.7%) preferred in-person compared to telemedicine encounters. Overall, patients had a neutral attitude towards telemedicine [mean Likert rating (SD) = 3.11/5 ± 0.82]. Questions assessing "patient education" and "telemedicine efficiency" received the greatest proportion of positive and negative responses, respectively. Positive attitudes were more frequent among patients with prior telemedicine experience (87.5%) compared to never-users (71.8%; p = 0.046). Patients ≥ 75 years old tended to negatively assess telemedicine regarding reduction of COVID-19-related anxiety, efficiency, patient education, and physician facetime (p < 0.05 for all). A positive but non-significant trend was observed between higher education level and positive attitude towards telemedicine (p = 0.18). Telehealth never-users more often negatively rated receiving adequate facetime with the physician virtually (54.7%) compared to prior users (25.6%; p = 0.004). Younger age, prior history of telemedicine use, and higher education level were associated with increased preference for telemedicine (p < 0.05 for all). CONCLUSION: Our findings revealed hesitance remains among patients towards adoption of telemedicine. Targeting age-, experience-, and education-related barriers will be invaluable for increasing acceptance of this healthcare delivery model.

Production of cell culture (MDCK) derived live attenuated influenza vaccine (LAIV) in a fully disposable platform process.

The majority of influenza vaccines are manufactured using embryonated hens' eggs. The potential occurrence of a pandemic outbreak of avian influenza might reduce or even eliminate the supply of eggs, leaving the human population at risk. Also, the egg-based production technology is intrinsically cumbersome and not easily scalable to provide a rapid worldwide supply of vaccine. In this communication, the production of a cell culture (Madin-Darby canine kidney (MDCK)) derived live attenuated influenza vaccine (LAIV) in a fully disposable platform process using a novel Single Use Bioreactor (SUB) is presented. The cell culture and virus infection was maintained in a disposable stirred tank reactor with PID control of pH, DO, agitation, and temperature, similar to traditional glass or stainless steel bioreactors. The application of this technology was tested using MDCK cells grown on microcarriers in proprietary serum free medium and infection with 2006/2007 seasonal LAIV strains at 25-30 L scale. The MDCK cell growth was optimal at the agitation rate of 100 rpm. Optimization of this parameter allowed the cells to grow at a rate similar to that achieved in the conventional 3 L glass stirred tank bioreactors. Influenza vaccine virus strains, A/New Caledonia/20/99 (H1N1 strain), A/Wisconsin/67/05 (H3N2 strain), and B/Malaysia/2506/04 (B strain) were all successfully produced in SUB with peak virus titers > or =8.6 log(10) FFU/mL. This result demonstrated that more than 1 million doses of vaccine can be produced through one single run of a small bioreactor at the scale of 30 L and thus provided an alternative to the current vaccine production platform with fast turn-around and low upfront facility investment, features that are particularly useful for emerging and developing countries and clinical trial material production.

Patient Preferences Associated with Anti-Vascular Endothelial Growth Factor Therapies for Neovascular Age-Related Macular Degeneration and Diabetic Macular Edema.

PURPOSE: To evaluate treatment-related preferences among patients receiving intravitreal anti-vascular endothelial growth factor (VEGF) therapy for neovascular age-related macular degeneration (nAMD) or diabetic macular edema (DME). PATIENTS AND METHODS: We conducted a prospective survey of patients with nAMD or DME treated at one of three US-based retina clinics. Prior to survey development, small focus groups with anti-VEGF-treated patients identified five treatment-related "attributes" considered important to those with nAMD or DME: vision outcomes, cost to the insurance provider, cost to the patient, frequency of treatment, and drug label status. Attributes were described using two to three "levels", and hypothetical treatment profiles were generated by assigning one level to each attribute. Surveyed patients were asked to indicate their preference between two given treatment profiles for a total of eight pairwise comparisons. Discrete choice conjoint analysis was performed to estimate the relative importance of each attribute for the overall patient cohort, and for subgroups stratified by age and highest education level. RESULTS: Among 300 respondents, 54% were female, 78% were aged ≥65 years, and 67% indicated that high school was their highest level of education. Achieving good vision was the most important factor associated with anti-VEGF therapy for nAMD or DME (relative importance, 40.4%), followed by low cost to the patient, on-label drug status, less frequent treatment intervals, and low cost to the insurance provider (23.1%, 21.3%, 12.2%, and 3.0%, respectively). When patients were stratified by age group or highest education level, preference trends across subgroups were generally comparable with the overall cohort. CONCLUSION: Our data suggest that treatment decisions regarding anti-VEGF therapies for nAMD or DME are most likely driven by their efficacy, and that patients may be willing to accept less desirable treatment attributes, such as increased cost and/or injection frequency, in order to achieve superior vision outcomes.

Thomas A. Swift's Electric Rifle Injuries to the Eye and Ocular Adnexa: The Management of Complex Trauma.

PURPOSE: To report the ocular and adnexal injuries sustained by patients with Thomas A. Swift's electric rifles (TASER; TASER International, Scottsdale, AZ), review the literature, and discuss the management of this complex trauma. DESIGN: Multicenter, retrospective case series and literature review. PARTICIPANTS: Seventeen eyes of 16 patients (5 eyes of 5 patients treated at 3 institutions, and 12 eyes of 11 previously reported cases). METHODS: The clinical data of 17 eyes were pooled. Spearman's correlation coefficient was used to assess the association between the extent of TASER injury and patient outcomes. MAIN OUTCOME MEASURES: Extent of TASER injury (zone of injury, penetrating vs. perforating) and association with patient outcomes (visual acuity [VA] and retinal detachment [RD]). RESULTS: In our cohort, 4 patients were transported by law enforcement and 1 was transferred from a community hospital. Four patients were taken to the operating room for TASER removal and globe repair; 1 patient underwent removal in the emergency room. Of 17 pooled cases, 12 (71%) involved open-globe injury. Of these, there was a high rate of zone 3 injuries (100%; n = 12) and a high incidence of RD (73%; 8 of 11, eviscerated eye excluded). Among patients with closed-globe injury (n = 5), 1 patient demonstrated exudative RD and 1 patient demonstrated retinal dialysis with RD. Of 10 patients with RD, 1 (10%) achieved resolution with monitoring (exudative RD); 1 (10%) underwent cryopexy and pneumatic retinopexy; 3 (30%) underwent vitrectomy, and 5 (50%) with poor prognosis did not undergo vitreoretinal surgery. In the 3 patients who underwent vitrectomy, all 3 (100%) demonstrated redetachment resulting from proliferative vitreoretinopathy and required additional surgery. Visual acuity on presentation was significantly correlated with final VA (ρ = 0.783; P = 0.02). Men (94%) were more likely than women (6%) to sustain TASER trauma. Median age was 26 years. There was a 50% rate of loss to follow-up. CONCLUSIONS: Thomas A. Swift's electric rifle injuries to the eyes or ocular adnexa represent complex trauma. Zone 3 injuries are common. The visual prognosis is guarded, and eyes may require multiple surgeries to preserve vision. Patients are at high risk for loss to follow-up by way of incarceration.

Ca2+/calmodulin regulates trafficking of Ca(V)1.2 Ca2+ channels in cultured hippocampal neurons.

As the Ca2+-sensor for Ca2+-dependent inactivation, calmodulin (CaM) has been proposed, but never definitively demonstrated, to be a constitutive Ca(V)1.2 Ca2+ channel subunit. Here we show that CaM is associated with the Ca(V)1.2 pore-forming alpha1C subunit in brain in a Ca2+-independent manner. Within its CaM binding pocket, alpha1C has been proposed to contain a membrane targeting domain. Because ion channel subunits assemble early during channel biosynthesis, we postulated that this association with CaM could afford the opportunity for Ca2+-dependent regulation of membrane targeting. We showed that the isolated domain functioned as a Ca2+/CaM regulated trafficking determinant for CD8 (a model transmembrane protein) using fluorescent-activated cell sorting analysis and, using green fluorescent protein-tagged alpha1C subunits expressed in cultured hippocampal neurons, that Ca2+/CaM interaction with this domain accelerated trafficking of Ca(V)1.2 channels to distal regions of the dendritic arbor. Furthermore, this Ca2+/CaM-accelerated trafficking was activity dependent. Thus, CaM imparts Ca2+-dependent regulation not only to mature Ca(V)1.2 channels at the cell surface but also to steps during channel biosynthesis.

Voltage sensor movement and cAMP binding allosterically regulate an inherently voltage-independent closed-open transition in HCN channels.

The hyperpolarization-activated cyclic nucleotide-modulated cation (HCN) channels are regulated by both membrane voltage and the binding of cyclic nucleotides to a cytoplasmic, C-terminal cyclic nucleotide-binding domain (CNBD). Here we have addressed the mechanism of this dual regulation for HCN2 channels, which activate with slow kinetics that are strongly accelerated by cAMP, and HCN1 channels, which activate with rapid kinetics that are weakly enhanced by cAMP. Surprisingly, we find that the rate of opening of HCN2 approaches a maximal value with extreme hyperpolarization, indicating the presence of a voltage-independent kinetic step in the opening process that becomes rate limiting at very negative potentials. cAMP binding enhances the rate of this voltage-independent opening step. In contrast, the rate of opening of HCN1 is much greater than that of HCN2 and does not saturate with increasing hyperpolarization over the voltage range examined. Domain-swapping chimeras between HCN1 and HCN2 reveal that the S4-S6 transmembrane region largely determines the limiting rate in opening kinetics at negative voltages. Measurements of HCN2 tail current kinetics also reveal a voltage-independent closing step that becomes rate limiting at positive voltages; the rate of this closing step is decreased by cAMP. These results are consistent with a cyclic allosteric model in which a closed-open transition that is inherently voltage independent is subject to dual allosteric regulation by voltage sensor movement and cAMP binding. This mechanism accounts for several properties of HCN channel gating and has potentially important physiological implications.

OCHROCONIS GALLOPAVA ENDOPHTHALMITIS SUCCESSFULLY TREATED WITH INTRAVITREAL VORICONAZOLE AND AMPHOTERICIN B.

PURPOSE: Ochroconis gallopava is a darkly pigmented septated fungus that rarely infects humans, almost universally immunocompromised individuals. Only one previous case has been reported of O. gallopava endophthalmitis, in which the patient progressed to a visual acuity of no light perception. The authors currently describe the first successfully treated case of O. gallopava endophthalmitis. METHODS: A 65-year-old Hispanic woman on chronic immunosuppression after a right lung transplant was admitted to the hospital for O. gallopava respiratory tract infection and was found to have bilateral endogenous endophthalmitis. Examination revealed multiple, cream-colored, chorioretinal lesions in the fundus, including one near the temporal macula in the left eye. There was no diffuse vitritis. Visual acuity was 20/30 in both eyes at presentation but dropped to 20/400 in the right eye and counting fingers at 3 feet in the left eye over the hospital course despite receiving an intravitreal injection of voriconazole in the left eye and concurrent IV amphotericin B, posaconazole, and micafungin. The patient was then treated with multiple simultaneous intravitreal amphotericin B and voriconazole injections bilaterally, as well as IV posaconazole, which was switched to IV voriconazole for improved intraocular penetration. RESULTS: The chorioretinal lesions were noted to regress with treatment. Concomitantly, the patient's respiratory status improved, and she was discharged with a visual acuity of 20/40 in the right eye and 20/60 in the left eye. The patient completed a 1-year course of oral voriconazole as an outpatient without signs of recurrent activity. Seventeen months after the initial presentation, the patient had 20/40 vision bilaterally with a residual, white, fibrotic scar at the temporal macula in the left eye. CONCLUSION: This report describes the first successful treatment regimen against O. gallopava endophthalmitis using a combination of intravitreal amphotericin B and voriconazole injections, as well as IV posaconazole and voriconazole, which resulted in an excellent visual outcome.

HCN hyperpolarization-activated cation channels inhibit EPSPs by interactions with M-type K(+) channels.

The processing of synaptic potentials by neuronal dendrites depends on both their passive cable properties and active voltage-gated channels, which can generate complex effects as a result of their nonlinear properties. We characterized the actions of HCN (hyperpolarization-activated cyclic nucleotide-gated cation) channels on dendritic processing of subthreshold excitatory postsynaptic potentials (EPSPs) in mouse CA1 hippocampal neurons. The HCN channels generated an excitatory inward current (I(h)) that exerted a direct depolarizing effect on the peak voltage of weak EPSPs, but produced a paradoxical hyperpolarizing effect on the peak voltage of stronger, but still subthreshold, EPSPs. Using a combined modeling and experimental approach, we found that the inhibitory action of I(h) was caused by its interaction with the delayed-rectifier M-type K(+) current. In this manner, I(h) can enhance spike firing in response to an EPSP when spike threshold is low and can inhibit firing when spike threshold is high.