Effects of COVID-19 on Intravitreal Injection Clinic After Lockdown.

Purpose: To examine the return of patients to intravitreal injection clinic after the COVID-19 lockdown. Patients and Methods: The electronic medical records of all patients who received intravitreal injections at a tertiary care Veterans Health Administration (VHA) clinic 14 weeks post-lockdown (5/9/20-8/13/20) in Los Angeles County were reviewed. Reference groups included injection patients during the 7-week COVID-19 lockdown (3/19/20-5/8/20) and a 7-week pre-pandemic period in 2019 (3/19/19-5/8/19). Clinic volume was compared using a one-way ANOVA. Demographic data, medical and psychiatric co-morbidities, injection diagnoses, visual acuities, and clinic volumes were compared between the 3 periods using a generalized estimating equation multivariate analysis. Results: The post-lockdown period group averaged 25.1 visits per week, compared with 12.3/week during lockdown and 25.4/week pre-COVID in intravitreal injection clinic. In the post-lockdown period, the VHA injection clinic returned closer to the pre-lockdown volume compared to the VHA comprehensive clinic (98.9% vs 57.4%, p < 0.001). Post-lockdown, COPD patients and organ transplant patients were less likely to receive injections compared to 2019 (OR 0.76 p = 0.008, OR 1.37 p < 0.0001, respectively). Patients with a diagnosis of cancer increased in proportion between the pre-pandemic and the post-lockdown periods (OR 1.31, p = 0.007). No differences were found, according to psychiatric co-morbidities. After lockdown, the proportion of patients receiving injections for diabetic macular edema (DME) increased (OR 1.11, p = 0.01). Conclusion: Injection volume returned to pre-pandemic levels immediately after lockdown ended. However, patients with high-risk comorbidities did not return to intravitreal injection clinic post-lockdown. These results can inform medical organizations, which groups may need increased safety measures and targeted outreach to address their ophthalmic needs.

Evaluation of intravitreal topotecan dose levels, toxicity and efficacy for retinoblastoma vitreous seeds: a preclinical and clinical study.

BACKGROUND: Current melphalan-based intravitreal regimens for retinoblastoma (RB) vitreous seeds cause retinal toxicity. We assessed the efficacy and toxicity of topotecan monotherapy compared with melphalan in our rabbit model and patient cohort. METHODS: Rabbit experiments: empiric pharmacokinetics were determined following topotecan injection. For topotecan (15 µg or 30 µg), melphalan (12.5 µg) or saline, toxicity was evaluated by serial electroretinography (ERG) and histopathology, and efficacy against vitreous seed xenografts was measured by tumour cell reduction and apoptosis induction. PATIENTS: retrospective cohort study of 235 patients receiving 990 intravitreal injections of topotecan or melphalan. RESULTS: Intravitreal topotecan 30 µg (equals 60 µg in humans) achieved the IC90 across the rabbit vitreous. Three weekly topotecan injections (either 15 µg or 30 µg) caused no retinal toxicity in rabbits, whereas melphalan 12.5 µg (equals 25 µg in humans) reduced ERG amplitudes 42%-79%. Intravitreal topotecan 15 µg was equally effective to melphalan to treat WERI-Rb1 cell xenografts in rabbits (96% reduction for topotecan vs saline (p=0.004), 88% reduction for melphalan vs saline (p=0.004), topotecan vs melphalan, p=0.15). In our clinical study, patients received 881 monotherapy injections (48 topotecan, 833 melphalan). Patients receiving 20 µg or 30 µg topotecan demonstrated no significant ERG reductions; melphalan caused ERG reductions of 7.6 µV for every injection of 25 µg (p=0.03) or 30 µg (p<0.001). Most patients treated with intravitreal topotecan also received intravitreal melphalan at some point during their treatment course. Among those eyes treated exclusively with topotecan monotherapy, all eyes were salvaged. CONCLUSIONS: Taken together, these experiments suggest that intravitreal topotecan monotherapy for the treatment of RB vitreous seeds is non-toxic and effective.

Intravitreal melphalan hydrochloride vs propylene glycol-free melphalan for retinoblastoma vitreous seeds: Efficacy, toxicity and stability in rabbits models and patients.

The use of intravitreal chemotherapy has revolutionized the treatment of advanced intraocular retinoblastoma, as intravitreal melphalan has enabled difficult-to-treat vitreous tumor seeds to be controlled, leading to many more eyes being saved. However, melphalan hydrochloride (MH) degrades rapidly in solution, increasing logistical complexity with respect to time between medication preparation and administration for intravitreal administration under anesthesia for retinoblastoma. A new propylene glycol-free melphalan (PGFM) formulation has greater stability and could therefore improve access and adoption of intravitreal chemotherapy, allowing more children to retain their eye(s). We compared the efficacy and toxicity of both formulations, using our rabbit xenograft model and clinical patient experience. Three weekly 12.5 µg intravitreal injections of MH or PGFM (right eye), and saline (left eye), were administered to immunosuppressed rabbits harboring human WERI-Rb1 vitreous seed xenografts. Residual live cells were quantified directly, and viability determined by TUNEL staining. Vitreous seeds were reduced 91% by PGFM (p = 0.009), and 88% by MH (p = 0.004; PGFM vs. MH: p = 0.68). All residual cells were TUNEL-positive (non-viable). In separate experiments to assess toxicity, three weekly 12.5 µg injections of MH, PGFM, or saline were administered to non-tumor-bearing rabbits. Serial electroretinography, optical coherence tomography (OCT) and OCT-angiography were performed. PGFM and MH both caused equivalent reductions in electroretinography amplitudes, and loss of retinal microvasculature on OCT-angiography. The pattern of retinal degeneration observed on histopathology suggested that segmental retinal toxicity associated with all melphalan formulations was due to a vitreous concentration gradient-effect. Efficacy and toxicity were assessed for PGFM given immediately (within 1 h of reconstitution) vs. 4 h after reconstitution. Immediate- and delayed-administration of PGFM showed equivalent efficacy and toxicity. In addition, we evaluated efficacy and toxicity in patients (205 eyes) with retinoblastoma vitreous seeds, who were treated with a total of 833 intravitreal injections of either MH or PGFM as standard of care. Of these, we analyzed 118 MH and 131 PGFM monotherapy injections in whom serial ERG measurements were available to model retinal toxicity. Both MH and PGFM caused reductions in electroretinography amplitudes, but with no statistical difference between formulations. Comparing those patient eyes treated exclusively with PGFM versus those treated exclusively with MH, efficacy for tumor control and globe salvage was equivalent (PGFM vs. MH: 96.2% vs. 93.8%, p = 0.56), but PGFM-treated eyes received fewer injections than MH-treated eyes (average 3.2 ± 1.9 vs. 6.4 ± 2.1 injections, p < 0.0001). Taken together, these rabbit experiments and our clinical experience in retinoblastoma patients demonstrate that MH and PGFM have equivalent efficacy and toxicity. PGFM was more stable, with no decreased efficacy or increased toxicity even 4 h after reconstitution. We therefore now use PGFM over traditional MH for our patients for intravitreal treatment of retinoblastoma.

TOXICITY AND EFFICACY OF INTRAVITREAL MELPHALAN FOR RETINOBLASTOMA: 25 µg Versus 30 µg.

PURPOSE: To compare retinal toxicity as measured by electroretinogram, ocular, and patient survival in retinoblastoma treated with intravitreal melphalan at two concentrations (25 vs. 30 µg). METHODS: Single-center, retrospective analysis of retinoblastoma eyes receiving 25-µg or 30-µg intravitreal melphalan from September 2012 to January 2019. Ocular toxicity was measured by electroretinogram of evaluable injections in 449 injections in 136 eyes. A repeated-measures linear mixed model with a random intercept and slope was applied to account for repeated measures for each eye. RESULTS: Average decline in electroretinogram after each additional injection was -4.9 µV (95% confidence interval -6.3 to -3.4); electroretinogram declined by -4.6 µV (95% confidence interval -7.0 to -2.2) after 25-µg injections and -5.2 µV (95% confidence interval -6.6 to -3.8) after 30-µg injections (P = 0.66). Injection at a new clock site hour was associated with a -3.91-µV lower average (95% confidence interval -7.8 to -0.04). CONCLUSION: Electroretinogram-measured toxicity in retinoblastoma eyes treated with intravitreal injections was not found to be different across 25-µg and 30-µg injections. There were no cases of extraocular extension or metastatic deaths in our patient population.

Comparison of efficacy and toxicity of intravitreal melphalan formulations for retinoblastoma.

OBJECTIVE: Intravitreal melphalan injections are commonly used in the treatment for intraocular retinoblastoma. This study compares retinal toxicity and ocular survival between two formulations, with and without propylene glycol (Alkeran vs. Evomela, respectively). METHODS: A retrospective cohort study of retinoblastoma patients who received intravitreal injections of Alkeran and Evomela at 30 µg from September 2012 to January 2019 at a single tertiary care center were enrolled. Retinal toxicity was measured using electroretinogram (ERG) and compared using a multivariate analysis of 338 injections in 101 eyes of 96 patients. Ocular survival of 163 eyes in 150 patients was compared across formulations using Cox proportional hazards model. Eyes were censored at the time a patient received a dose other than 30 µg. RESULTS: Overall, ERG decline (mean, 95% CI) for each injection was -5.58 µV (-7.17, -3.99). No significant differences in ERG decrement were found between Alkeran (with alcohol) -5.52uV (-6.99, -4.05). and Evomela (without alcohol) -5.65uV (-8.31 to -2.98) formulations (p = 0.93). Ocular survival at 24 months was 93.6% (95% CI 86.2, 97.1) with alcohol and 91.7% (95% CI 53.9, 98.8) without alcohol. The hazard ratio (HR) for without vs with alcohol was 0.50 (95% CI 0.06 to 4.07); no significant difference in ocular survival was found between formulations (p = 0.52). CONCLUSIONS AND RELEVANCE: No differences were found in retinal toxicity and ocular survival between 30 µg intravitreal injections of Alkeran or Evomela for intraocular retinoblastoma. Given the increased stability of Evomela, intravitreal treatment could be expanded to centers without the ability to supply Alkeran due to its shorter safety window; however, Alkeran is less expensive. For those with existing infrastructure, Alkeran is a comparable, cost-effective alternative.

Mild Traumatic Brain Injury in Mice Beneficially Alters Lung NK1R and Structural Protein Expression to Enhance Survival after Pseudomonas aeruginosa Infection.

Mild traumatic brain injury (mTBI) in a murine model increases survival to a bacterial pulmonary challenge compared with blunt tail trauma (TT). We hypothesize substance P and its receptor, the neurokinin 1 receptor (NK1R; official name TACR1), play a role in the increased survival of mTBI mice. Mice were subjected to mTBI or TT, and 48 hours after trauma, the levels of NK1R mRNA and protein were significantly up-regulated in mTBI lungs. Examination of the lung 48 hours after injury by microarray showed significant differences in the expression of 433 gene sets between groups, most notably genes related to intercellular proteins. Despite down-regulated gene expression of connective proteins, the presence of an intact pulmonary vasculature was supported by normal histology and bronchoalveolar lavage protein levels. To determine whether these mTBI-induced lung changes benefited in vivo responses, two chemotactic stimuli (a CXCL1 chemokine and a live Pseudomonas aeruginosa infection) were administered 48 hours after trauma. For both stimuli, mTBI mice recruited more neutrophils to the lung 4 hours after instillation (CXCL1: mTBI = 6.3 ± 1.3 versus TT = 3.3 ± 0.7 neutrophils/mL; Pseudomonas aeruginosa: mTBI = 9.4 ± 1.4 versus TT = 5.3 ± 1.1 neutrophils/mL). This study demonstrates that the downstream consequences of mTBI on lung NK1R levels and connective protein expression enhance neutrophil recruitment to a stimulus that may contribute to increased survival.

Enhancing Scientific Foundations to Ensure Reproducibility: A New Paradigm.

Progress in science is dependent on a strong foundation of reliable results. The publish or perish paradigm in research, coupled with an increase in retracted articles from the peer-reviewed literature, is beginning to erode the trust of both the scientific community and the public. The NIH is combating errors by requiring investigators to follow new guidelines addressing scientific premise, experimental design, biological variables, and authentication of reagents. Herein, we discuss how implementation of NIH guidelines will help investigators proactively address pitfalls of experimental design and methods. Careful consideration of the variables contributing to reproducibility helps ensure robust results. The NIH, investigators, and journals must collaborate to ensure that quality science is funded, explored, and published.

The Role of Substance P in Pulmonary Clearance of Bacteria in Comparative Injury Models.

Neural input to the immune system can alter its ability to clear pathogens effectively. Patients suffering mild traumatic brain injury (mTBI) have shown reduced rates of pneumonia and a murine model replicated these findings, with better overall survival of TBI mice compared with sham-injured mice. To further investigate the mechanism of improved host response in TBI mice, this study developed and characterized a mild tail trauma model of similar severity to mild TBI. Both mild tail trauma and TBI induced similar systemic changes that normalized within 48 hours, including release of substance P. Examination of tissues showed that injuries are limited to the target tissue (ie, tail in tail trauma, brain in mTBI). Pneumonia challenge showed that mild TBI mice showed improved immune responses, characterized by the following: i) increased survival, ii) increased pulmonary neutrophil recruitment, iii) increased bacterial clearance, and iv) increased phagocytic cell killing of bacteria compared with tail trauma. Administration of a neurokinin-1-receptor antagonist to block substance P signaling eliminated the improved survival of mTBI mice. Neurokinin-1-receptor antagonism did not alter pneumonia mortality in tail trauma mice. These data show that immune benefits of trauma are specific to mTBI and that tail trauma is an appropriate control for future studies aimed at elucidating the mechanisms of improved innate immune responses in mTBI mice.

Nucleoside salvage pathway kinases regulate hematopoiesis by linking nucleotide metabolism with replication stress.

Nucleotide deficiency causes replication stress (RS) and DNA damage in dividing cells. How nucleotide metabolism is regulated in vivo to prevent these deleterious effects remains unknown. In this study, we investigate a functional link between nucleotide deficiency, RS, and the nucleoside salvage pathway (NSP) enzymes deoxycytidine kinase (dCK) and thymidine kinase (TK1). We show that inactivation of dCK in mice depletes deoxycytidine triphosphate (dCTP) pools and induces RS, early S-phase arrest, and DNA damage in erythroid, B lymphoid, and T lymphoid lineages. TK1(-/-) erythroid and B lymphoid lineages also experience nucleotide deficiency but, unlike their dCK(-/-) counterparts, they still sustain DNA replication. Intriguingly, dCTP pool depletion, RS, and hematopoietic defects induced by dCK inactivation are almost completely reversed in a newly generated dCK/TK1 double-knockout (DKO) mouse model. Using NSP-deficient DKO hematopoietic cells, we identify a previously unrecognized biological activity of endogenous thymidine as a strong inducer of RS in vivo through TK1-mediated dCTP pool depletion. We propose a model that explains how TK1 and dCK "tune" dCTP pools to both trigger and resolve RS in vivo. This new model may be exploited therapeutically to induce synthetic sickness/lethality in hematological malignancies, and possibly in other cancers.

BCG vaccine-induced neuroprotection in a mouse model of Parkinson's disease.

There is a growing interest in using vaccination with CNS antigens to induce autoreactive T cell responses that home to damaged areas in the CNS and ameliorate neurodegenerative disease. Neuroprotective vaccine studies have focused on administering oligodendrocyte antigens or Copaxone® in complete Freund's adjuvant (CFA). Theoretical considerations, however, suggest that vaccination with a neuronal antigen may induce more robust neuroprotective immune responses. We assessed the neuroprotective potential of vaccines containing tyrosine hydroxylase (a neuronal protein involved in dopamine synthesis) or Copaxone® in CFA in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson's disease. Surprisingly, we observed that the main beneficial factor in these vaccines was the CFA. Since the major immunogenic component in CFA is Mycobacterium tuberculosis, which closely related to the bacille Calmette-Guérin (BCG) that is used in human vaccines, we tested BCG vaccination in the MPTP mouse model. We observed that BCG vaccination partially preserved markers of striatal dopamine system integrity and prevented an increase in activated microglia in the substantia nigra of MPTP-treated mice. These results support a new neuroprotective vaccine paradigm in which general (nonself-reactive) immune stimulation in the periphery can limit potentially deleterious microglial responses to a neuronal insult and exert a neurorestorative effect in the CNS. Accordingly, BCG vaccination may provide a new strategy to augment current treatments for a wide range of neuropathological conditions.

Major histocompatibility complex class I-mediated inhibition of neurite outgrowth from peripheral nerves.

Studies of mice deficient in classical major histocompatability complex class I (MHCI) revealed that MHCI plays an important role in neurodevelopment in the central nervous system. We previously studied the effects of recombinant MHCI molecules on wildtype retina explants and observed that MHCI can inhibit retina neurite outgrowth, with self-MHCI molecules having greater inhibitory effect than non-self MHCI molecules. Here, we examined classical MHCI's effects on axon outgrowth from neurons of the peripheral nervous system (PNS). We used the embryonic dorsal root ganglia (DRG) explant model since their neurons express MHCI and because DRG explants have been widely used to assess the effects of molecules on axonal outgrowth from PNS neurons. We observed that picomolar levels of a recombinant self-MHCI molecule, but not non-self MHCI molecules, inhibited axon outgrowth from DRG explants. This differential sensitivity to self- vs. non-self MHCI suggests that early in development, self-MHCI may "educate" PNS neurons to express appropriate MHCI receptors, as occurs during natural killer cell development. Furthermore, we observed that a MHCI tetramer stained embryonic DRG neurons, indicating the expression of classical MHCI receptors. These results suggest that MHCI and MHCI receptors play roles during early stages of PNS development and may provide new targets of therapeutic strategies to promote neuronal outgrowth after PNS injury.