Safety and tolerability of intravenous immunoglobulin infusion in Down syndrome regression disorder.

Three large multi-center studies have identified the clinical utility of intravenous immunoglobulin (IVIg) in the treatment of Down syndrome regression disorder (DSRD). Yet the tolerability of infusions in individuals with DS and the safety of IVIg remains unknown in this population. This study sought to evaluate the safety and tolerability of IVIg in individuals with DSRD compared to a real-world cohort of individuals with pediatric onset neuroimmunologic disorders. A single-center, retrospective chart review evaluating clinically documented infusion reactions was performed for individuals meeting international consensus criteria for DSRD and having IVIg infusions between 2019 and 2023. Infusion reactions were evaluated for severity and need for alterations in infusion plan. This cohort was compared against an age and sex matched cohort of children with neuroimmunologic conditions who had also received IVIg infusions. In total, 127 individuals with DSRD and 186 individuals with other neuroimmunologic disorders were enrolled. There was no difference in the overall rate of adverse reactions (AEs) between the DSRD and general neuroimmunology cohorts (p = 0.31, 95% CI: 0.80-2.00), but cardiac-related AEs specifically were more common among the DSRD group (p = 0.02, 95% CI: 1.23-17.54). When AEs did occur, there was no difference in frequency of pharmacologic intervention (p = 0.12, 95% CI: 0.34-1.13) or discontinuation of therapy (p = 0.74, 95% CI: 0.06-7.44). There was a higher incidence of lab abnormalities on IVIG among the general neuroimmunology cohort (p = 0.03, 95% CI: 0.24-0.94) compared to the DSRD cohort. Transaminitis was the most common laboratory abnormality in the DSRD group. In a large cohort of individuals with DSRD, there were no significant differences in the safety and tolerability of IVIg compared to a cohort of children and young adults with neuroimmunologic conditions.

Ophthalmologic and neuro-ophthalmologic findings in children with Down syndrome.

Down syndrome, also known as Trisomy 21, is a genetic disorder associated with mild-to-moderate intellectual disability, delays in growth, and characteristic facial features. A wide range of ocular complications are seen in children with Down syndrome, including strabismus, nystagmus, refractive errors, congenital cataracts, the presence of keratoconus, and decreased visual acuity. Early ophthalmic examination is needed for early diagnosis and treatment in patients. This narrative review examines ocular manifestations in children with Down syndrome and the importance of prompt ophthalmic interventions for treatment.

Cutting Edge: CRISPR-Based Transcriptional Regulators Reveal Transcription-Dependent Establishment of Epigenetic Memory of Foxp3 in Regulatory T Cells.

Transcription factor Foxp3 specifies and maintains regulatory T cell (Treg) identity. During Treg differentiation, a CpG-rich Foxp3 intronic enhancer, conserved noncoding sequence 2 (CNS2), is activated via DNA demethylation to establish epigenetic memory of Foxp3 expression to protect Treg identity. However, it is unclear how this epigenetic memory of Foxp3 expression is established, as CNS2 is thought to be demethylated independently of Foxp3 expression. In this article, we uncover an unexpected causal relationship between Foxp3-transcriptional activation and CNS2 demethylation in mice. CRISPR/dCas9-mediated Foxp3-transcriptional activation elicits CNS2 demethylation. Sustaining Foxp3-transcriptional activation in induced Tregs also promotes CNS2 demethylation, enhancing Treg lineage stability and suppressive function. Importantly, CRISPR-mediated silencing of Foxp3 transcription, but not protein expression, abolishes CNS2 demethylation. The novel finding that Foxp3-transcriptional activation promotes CNS2 demethylation may facilitate the development of Treg-based therapies and represent a general mechanism for the establishment of epigenetic memory of immune gene expression.

An improved in vitro photochemical internalization protocol for 3D spheroid cultures.

Photochemical internalization (PCI) is a modified form of photodynamic therapy (PDT) that enhances the efficacy of therapeutic agents in a site and temporal specific manner in both in vitro and in vivo publications. The purpose of the study reported here was to evaluate the benefits of a modified PCI protocol in a 3D rat glioma spheroid model. In the modified protocol, F98 glioma cells were incubated with photosensitizer (AlPcS2a) prior to spheroid generation, as opposed to post-spheroid formation photosensitizer exposure commonly used in conventional protocols. The efficacy of both bleomycin and doxorubicin PCI was evaluated using either the conventional or modified protocols. The formed spheroids were then exposed to light treatment from a diode laser, λ= 670 nm. Spheroid growth was monitored for a period of 14 days. The results of spheroid growth assays showed that there was no statistically significant difference in PCI efficacy between the conventional and modified protocols for both of the drugs tested. The direct PDT effect was significantly reduced using the modified protocol. Therefore, due to its several advantages, the modified protocol is recommended for evaluating the efficacy of PCI in tumor spheroid models.

Photosensitizer delivery by fibrin glue: potential for bypassing the blood-brain barrier.

Fibrin glue (FG) has potential as a delivery vehicle for photosensitizer directly to the resection cavity, so it may bypass the blood-brain barrier (BBB) and increase the concentration of successfully delivered photosensitizer. A specialized form of photodynamic therapy (PDT), photochemical internalization (PCI), which involves both photosensitizer and chemotherapeutic agent internalization, can locally inhibit the growth of cells. This will allow the reduction of recurrence of malignant gliomas around surgical resection. This study will look at the efficacy of FG loaded with drugs in mediating both PDT and PCI in inhibiting 3-dimensional tumor spheroid growth in vitro. Experiments were conducted on spheroids comprised of F98 glioma cells using photosensitizer AlPcS2a and chemotherapeutic drug bleomycin (BLM). At 2-, 24-, 48-, and 72-h increments, supernatant covering an FG layer within a well was collected and replaced by fresh medium, then added to spheroid-containing wells, which contained the respective chemicals for PDT and PCI. The wells were then exposed to light treatment from a diode laser, and after, spheroid growth was monitored for a period of 14 days. Significant spheroid growth inhibition was observed in both PDT and PCI modalities, but was far greater in PCI. Additionally, complete growth suppression was achieved via PCI at the highest radiant exposure. Achieving a slow photosensitizer release, significant F98 spheroid inhibition was observed in FG-mediated PDT and PCI. The present study showed BLM-PCI was the most efficacious of the two modalities.

Dysphoric Milk Ejection Reflex: Characteristics, Risk Factors, and Its Association with Depression Scores and Breastfeeding Self-Efficacy.

Background: Dysphoric milk ejection reflex (D-MER) is a phenomenon that occurs before milk letdown and is described as a wave of negative or devastating emotions, ranging from mild to severe and lasting for seconds to minutes. To date, there has been little research regarding this phenomenon. This study aims to determine the prevalence of D-MER in our population as well as its association with postnatal depression scores and breastfeeding self-efficacy. Methods: Lactating persons between 4 and 12 weeks postpartum at our institution were invited to complete an anonymous 59-question survey via an online platform. Questions asked included patient demographics, presence of symptoms of dysphoria (including timing, duration, and frequency), the Edinburgh Postnatal Depression Scale (EPDS), and the Breastfeeding Self-Efficacy Scale Short Form (BSES-sf). Results: In total, 201 women completed the survey. Twelve women were classified as likely having D-MER (6%). Symptom resolution primarily occurred within a minute to 5 minutes (58%). Mean EPDS scores differed significantly between those with likely D-MER and those without (12.2 vs. 5.4, p = 0.002). BSES-sf scores differed significantly between the two groups (43.1 vs. 52.5, p = 0.009). Preexisting depression or anxiety was not associated with D-MER (p = 0.133), other reported mood disorders differed significantly between those with D-MER and those without (p = 0.004). Demographic characteristics of women with and without D-MER were similar. Conclusion: D-MER prevalence may be lower than previously reported. Patients with likely D-MER appear to have lower breastfeeding self-efficacy and higher depression scores. Those with preexisting mood disorders may be at higher risk of experiencing D-MER.

Neuroimaging abnormalities associated with immunotherapy responsiveness in Down syndrome regression disorder.

OBJECTIVE: To determine the prevalence of neuroimaging abnormalities in individuals with Down syndrome regression disorder (DSRD) and evaluate if neuroimaging abnormalities were predictive of therapeutic responses. METHODS: A multicenter, retrospective, case-control study which reviewed neuroimaging studies of individuals with DSRD and compared them to a control cohort of individuals with Down syndrome (DS) alone was performed. Individuals aged 10-30 years and meeting international consensus criteria for DSRD were included. The presence of T1, T2/FLAIR, and SWI signal abnormalities was reviewed. Response rates to various therapies, including immunotherapy, were evaluated in the presence of neuroimaging abnormalities. RESULTS: In total, 74 individuals (35%) had either T2/FLAIR and/or SWI signal abnormality compared to 14 individuals (12%) without DSRD (p < 0.001, 95%CI: 2.18-7.63). T2/FLAIR signal abnormalities were not appreciated more frequently in individuals with DSRD (14%, 30/210) than in the control cohort (9%, 11/119) (p = 0.18, OR: 1.63, 95%CI: 0.79-3.40). SWI signal abnormalities were appreciated at a higher frequency in individuals with DSRD (24%, 51/210) compared to the control cohort (4%, 5/119) (p < 0.001, OR: 7.31, 95%CI: 2.83-18.90). T2/FLAIR signal abnormalities were localized to the frontal (40%, 12/30) and parietal lobes (37%, 11/30). SWI signal abnormalities were predominantly in the bilateral basal ganglia (94%, 49/52). Individuals with DSRD and the presence of T2/FLAIR and/or SWI signal abnormalities were much more likely to respond to immunotherapy (p < 0.001, OR: 8.42. 95%CI: 3.78-18.76) and less likely to respond to benzodiazepines (p = 0.01, OR: 0.45, 95%CI: 0.25-0.83), antipsychotics (p < 0.001, OR: 0.28, 95%CI: 0.11-0.55), or electroconvulsive therapy (p < 0.001, OR: 0.12; 95%CI: 0.02-0.78) compared to individuals without these neuroimaging abnormalities. INTERPRETATION: This study indicates that in individuals diagnosed with DSRD, T2/FLAIR, and SWI signal abnormalities are more common than previously thought and predict response to immunotherapy.

De novo variants in immune regulatory genes in Down syndrome regression disorder.

BACKGROUND: Down Syndrome Regression Disorder (DSRD) is a rare and poorly understood disorder of the central nervous system, characterized by acute or subacute neuropsychiatric symptoms in previously healthy individuals with Down syndrome (DS). Many patients exhibit immunotherapy-responsiveness, indicative of immune dysregulation as a potential underlying etiology. While hypotheses are emerging regarding the role of interferon signaling in DSRD and other autoimmune conditions associated with DS, it is unclear why a small subset of individuals with DS develop DSRD. The aim of this study was to investigate genes of immune regulation in persons with DSRD. METHODS: This study included individuals with DSRD aged 10-30 years with trio exome sequencing performed during the diagnostic work up. Descriptive statistics and univariate analysis (Chi-square and Fisher's exact test) were used to describe and compare the characteristics of individuals with and without variants. RESULTS: Forty-one individuals with DSRD had trio exome sequencing results. Eight (20%) had heterozygous de novo variants of immune regulatory genes, with four variants being pathogenic or likely pathogenic (UNC13D, XIAP, RNASEH2A, and DNASE1L3). All genes harboring pathogenic variants were associated with interferon type-1 inflammatory response. Individuals harboring variants were more likely to have a preceding trigger (p = 0.03, 95% CI 1.21-97.06), rapid clinical decline in less than 1 month (p = 0.01, 95% CI 1.67-52.06), and MRI abnormalities (p < 0.001, 95% CI 4.89-527.71). DISCUSSION: A distinct subset of individuals with DSRD exhibited pathogenic variants in immune regulation genes associated with interferon-mediated inflammatory response, coinciding with previously established links between these genes and interferonopathies such as Aicardi-Goutieres syndrome. Our observations suggest that these variants might potentially contribute to the development of DSRD in individuals with DS.

Fibrin glue as a local drug and photosensitizer delivery system for photochemical internalization: Potential for bypassing the blood-brain barrier.

BACKGROUND: Chemotherapy has had disappointing results in the treatment of glioblastoma multiforme (GBM). This is in part due to limited systemic drug penetration through the blood-brain barrier. This limitation can be overcome by implantation of drug-loaded hydrogels, such as fibrin glue (FG), directly into the tumor resection cavity. Photochemical internalization (PCI) has been shown to enhance the efficacy of a large number of chemotherapeutic agents, including bleomycin (BLM). This study examined the ability of loaded FG to release BLM and photosensitizer to enable PCI-induced growth inhibition of glioma spheroids in vitro. MATERIALS AND METHODS: FG layers, loaded with drug and photosensitizer, were formed in wells of a 24-well plate. Supernatants covering the FG layers were harvested after 48 h. F98 glioma spheroids were co-incubated with harvested supernatants for 24 h, followed by light exposure. Spheroid growth was monitored for an additional 14 days. RESULTS: 100% of the drug bleomycin and 90% of the photosensitizer (AlPcS2a) was released from the FG over a 48 h interval. Spheroid growth was significantly inhibited or completely suppressed by PCI of released drug and photosensitizer in many of the concentration combinations tested. PCI-induced growth inhibition increased with increasing light levels. CONCLUSIONS: The results demonstrate that both drug and photosensitizer were loaded into and released in a non-degraded form for an extended time period. The growth inhibition caused by FG-released BLM was significantly enhanced by FG-released AlPcS2a-mediated PCI.

The Impact of Neuroimmunologic Disease and Developing Nervous System.

Over the last two decades, neuroimmunologic disorders of childhood have been increasingly described, phenotyped, and treated. These disorders remain rare in the general population and while sharing common therapeutic interventions due to their immune pathophysiology, are heterogeneous with regard to presentation and risk of recurrence. As such, the impact of these disorders on the developing brain has come into the forefront of emerging research in pediatric neuroimmunology. Investigations into the singular impact of monophasic disease on long-term development and the impact of early and aggressive disease-modifying therapy in relapsing conditions are quickly becoming areas of ripe investigation as the field's most optimal way to treat and monitor these conditions over time. Although critically important in evaluating the developing brain, research has been heterogeneous among these diseases and limited by small cohort size. This narrative review details the role of common neuroimmunologic disorders in long-term neurological and cognitive outcomes in children as they develop.

Alternative Diagnoses in the Work Up of Down Syndrome Regression Disorder.

PURPOSE: Down Syndrome Regression Disorder (DSRD) is a diagnosis of exclusion. Psychiatric and neuroimmunologic etiologies have been proposed although the exact etiology remains unknown. This study sought to review non-DSRD diagnoses at a large quaternary medical center specializing in the diagnosis of DSRD and compare clinical characteristics between those diagnosed with DSRD and those with non-DSRD diagnoses. METHODS: The authors performed a single-center retrospective, chart-based, review of referrals for developmental regression in individuals with Down syndrome. RESULTS: Two hundred and sixty-six individuals were evaluated for DSRD and of these, 54 (20%) ultimately had alternative diagnoses. Individuals with DSRD were more likely to have shorter nadir to clinical symptoms (p = 0.01, 95% CI: 0.36-0.47) and have preceding triggers (p < 0.001, 95% CI: 1.13-1.43) compared to those with alternative diagnoses. Individuals with non-DSRD diagnoses were more likely to be born premature (p = 0.01, 95% CI: 0.51-0.87) and have a history of epilepsy (p = 0.01, 95% CI: 0.23-0.77) but were also less likely to have a history of cytokine abnormalities on bloodwork (p < 0.001, 95% CI: 1.19-1.43) and have catatonia (p < 0.001, 95% CI: 1.54-2.17). The majority of alternative diagnoses (41/54, 76%) were autism spectrum disorder. In these cases, symptoms were more likely to be longstanding (symptoms > 12 months) and earlier onset (median 8 years, IQR: 6-11). Other diagnoses included epilepsy (5/54, 9%), Celiac disease (5/54, 9%), cerebrovascular disease (3/54, 6%). CONCLUSIONS: This study identifies that 20% of individuals referred with concerns for DSRD have alternative diagnoses. The majority of these diagnoses were autism, but rare treatable conditions were also identified, highlighting the importance of a thorough neurodiagnostic assessment.

Hypovitaminosis D in persons with Down syndrome and autism spectrum disorder.

BACKGROUND: Plasma levels of vitamin D have been reported to be low in persons with Down syndrome (DS) and existing data is limited to small and homogenous cohorts. This is of particular importance in persons with DS given the high rates of autoimmune disease in this population and the known relationship between vitamin D and immune function. This study sought to investigate vitamin D status in a multi-center cohort of individuals with DS and compare them to individuals with autism spectrum disorder (ASD) and neurotypical (NT) controls. METHODS: A retrospective, multi-center review was performed. The three sites were located at latitudes of 42.361145, 37.44466, and 34.05349. Patients were identified by the International Classification of Diseases (ICD)-9 or ICD-10 codes for DS, ASD, or well-child check visits for NT individuals. The first vitamin D 25-OH level recorded in the electronic medical record (EMR) was used in this study as it was felt to be the most reflective of a natural and non-supplemented state. Vitamin D 25-OH levels below 30 ng/mL were considered deficient. RESULTS: In total, 1624 individuals with DS, 5208 with ASD, and 30,775 NT controls were identified. Individuals with DS had the lowest mean level of vitamin D 25-OH at 20.67 ng/mL, compared to those with ASD (23.48 ng/mL) and NT controls (29.20 ng/mL) (p < 0.001, 95% CI: -8.97 to -6.44). A total of 399 (24.6%) individuals with DS were considered vitamin D deficient compared to 1472 (28.3%) with ASD and 12,397 (40.3%) NT controls (p < 0.001, 95% CI: -5.43 to -2.36). Individuals with DS with higher body mass index (BMI) were found to be more likely to have lower levels of vitamin D (p < 0.001, 95% CI: -0.3849 to -0.1509). Additionally, having both DS and a neurologic diagnosis increased the likelihood of having lower vitamin D levels (p < 0.001, 95% CI: -5.02 to -1.28). Individuals with DS and autoimmune disease were much more likely to have lower vitamin D levels (p < 0.001, 95% CI: -6.22 to -1.55). Similarly, a history of autoimmunity in a first-degree relative also increased the likelihood of having lower levels of vitamin D in persons with DS (p = 0.01, 95% CI: -2.45 to -0.63). CONCLUSIONS: Individuals with DS were noted to have hypovitaminosis D in comparison to individuals with ASD and NT controls. Associations between vitamin D deficiency and high BMI, personal autoimmunity, and familial autoimmunity were present in individuals with DS.

Impact of endocrine dysregulation on disability and non-motor symptoms in pediatric onset multiple sclerosis.

Background: Pediatric onset multiple sclerosis (POMS) commonly occurs at the time of various endocrine changes. Evaluation of the impact of endocrine status on disease severity in POMS has not been previously explored. Objective: This study sought to evaluate if sex and stress hormones in children with POMS impact motor and non-motor diseases severity. Methods: A single-center case control study was performed. Individuals with POMS were compared to individuals without neurologic disease. Each individual had three blood draws assessing stress and sex hormones between 07:00 and 09:00. Measures of fatigue (Epworth sleepiness scale), depression (PHQ-9), and quality of life (PedsQL) assessed at each visit. Results: Forty individuals with POMS and 40 controls were enrolled. Individuals with POMS had lower free testosterone (p = 0.003), cortisol (p < 0.001), and ACTH (p < 0.001) and had higher progesterone (p = 0.025) levels than controls. Relapses and EDSS were not impacted by endocrine variables. The POMS cohort had a significantly higher Epworth score (p < 0.001), PHQ-9 score (p < 0.001), and lower PQL score (p < 0.001) than controls. Non-motor measures were not associated with endocrine status. Conclusion: Free testosterone, cortisol, ACTH, and progesterone were abnormal in children with POMS although there was no association between endocrine status and markers of disease severity or non-motor symptoms of MS.

Sonochemical Internalization of Bleomycin Inhibits Adenocarcinoma Breast Tumor Development in an Orthotopic Rat Model.

One approach to reducing post-operative tumor recurrence and alleviate debilitating side effects of systemic chemotherapy, is work centered on the development of drug activation by focused and targeted externally applied physical energy thus providing site and temporal specificity. One such technique, light mediated photochemical internalization (PCI), has been shown to be a method to obtain enhanced chemotherapy efficacy for a wide variety of anti-cancer agents. A related technology, sonochemical internaization (SCI), is an extension of the PCI concept developed to overcome the limitations of poor light penetration in tissue. SCI utilizes ultrasonic energy, to activate sonosensitizers, co-administered with anti cancer agents. The purpose of the study reported here was to evaluate the inhibitory effects of SCI of bleomycin (BLM), both in vitro and in vivo, on the adenocarcinoma breast tumor rat cell line Mat B III. In vitro, the two aspects of sonication, sonoporation (SP) and sonochemical internalization (SCI) of BLM were examined. In vivo, BLM-SCI significantly inhibited tumor development, following Mat B III implantation, in an orthotopic breast tumor animal model using Fisher rats.

Inhibition of glioma development by doxorubicin-photochemical internalization generated macrophage vaccine: A survival study in rats.

BACKGROUND: The process known as immunogenic cell death (ICD) is characterized by dead and dying cancer cells exposing and releasing so-called damage associated molecular patterns (DAMPs). ICD has been shown to enhance the efficacy of antigen presenting cell (APC) immunotherapy. Both anthracycline drugs such as doxorubicin (DOX), and photodynamic therapy (PDT) have been shown to be inducers of ICD. It was therefore hypothesized that combined PDT and DOX i.e. photochemical internalization of DOX (DOX-PCI) would increase ICD compared to DOX acting as a single agent. MATERIALS AND METHODS: F98 glioma cells were treated with DOX-PCI in vitro and the ICD markers HMGB1, HSP70, and HSP90 were determined by ELISA assay. Peritoneal macrophages (Ma), obtained from Fisher rats acting as APCs, were co-incubated with dead F98 glioma cells killed via DOX or DOX-PCI treatment ex vivo. The pulsed Ma (Ma DOX or Ma DOX-PCI) were used to inoculate the animals either before (preventive) or after (curative) intra-cranially implantation of the glioma cells. RESULTS: F98 cells, treated with DOX-PCI in vitro, induced a significantly higher level of HGMB1, HSP70, and HSP90 than DOX acting alone. Ma DOX-PCI inoculated animals, in both preventive and curative protocols, had a pronounced survival benefit compared to either the non-treatment or MaDOX control groups. In the curative protocol, a second booster inoculation significantly improved survival, with 60% of the animals alive at day 60. CONCLUSION: Macrophages primed with DOX-PCI treated glioma cells appeared to be highly effective as APCs and, when injected into host animals, could delay and, in some cases, prevent tumor development.

Enhanced gene transfection of macrophages by photochemical internalization: Potential for gene-directed enzyme prodrug therapy of gliomas.

BACKGROUND: Drawn by tumor synthesis of chemo-attractive factors, macrophages are frequently found in and around glioblastomas and play an important role both in augmenting as well as inhibiting tumor growth. Patient-derived macrophages have the potential, therefore, to act as targeted delivery vectors for a variety of anti-cancer treatments. Among these is ex vivo gene transfection and re-injection back into the patient of macrophages to target residual tumors. In this study, photochemical internalization (PCI) is investigated as a technique for the non-viral transfection of the cytosine deaminase (CD) prodrug activating gene into macrophages. The CD gene encodes an enzyme that converts the nontoxic antifungal agent, 5-fluorocytosine (5-FC), into 5-fluorouracil (5-FU) - a potent chemotherapeutic agent. MATERIALS: PCI (photosensitizer + light treatment) mediated CD gene transfection of rat alveolar Ma cells was carried out in vitro. CD gene transfected NR8383 macrophages were co-cultured with F98 rat glioma cells in the presence or absence of 5-FC. Cell viability was assayed using the MTS colorimetric assay. RESULTS: Compared to the glioma cells, NR8383 demonstrated enhanced resistance to the toxic effects of 5-FU. PCI greatly increased the transfection efficiency of the CD gene in NR8383 cells. The viability of F98 cells was significantly inhibited by coculture with CD transfected NR8383 macrophages and 5-FC. CONCLUSION: Although gene insertion into macrophages has proven difficult, the results presented here show that non-viral transfection of the CD gene into these immune cells can be enhanced via PCI. CD transfected NR8383 cells could efficiently convert 5-FC to 5-FU and export the drug, producing a pronounced bystander toxic effect on adjacent non-transfected glioma cells. Compared to single treatment, repetitive PCI-induced transfection was more efficient at low CD plasmid concentration.

The effects of low irradiance long duration photochemical internalization on glioma spheroids.

BACKGROUND: Photodynamic therapy (PDT), if given over extended time periods (i.e. hours or days) and at very low irradiance in the µW/cm2 range, has been shown to be more effective than acute PDT (aPDT) administered over minutes. This has led to the concept of metronomic PDT (mPDT), which consists of ultra-low irradiance light illumination for extended periods of time along with either continuous or repetitive delivery of photosensitizer. Since the drug activating technology photochemical internalization (PCI) is based on PDT it seemed reasonable to expect that ultra-low irradiance, if administered over an extended period of time, could nevertheless result in effective metronomic PCI (mPCI) comparable to or more effective than that obtained with relatively high and short irradiance i.e. acute PCI (aPCI). METHODS: Tumor spheroids consisting of F98 cells were used as in-vitro tumor models. The amphiphilic photosensitizer Al phthalocyanine disulfonate (AlPcS2a) was used for all PCI experiments. Light treatment was administered from a diode laser at λ=670 nm at various irradiance exposures of 2 mW/cm2 for aPCI and 0.05 - 0.2 mW/cm2 for mPCI with durations ranging from 3 to 12 min for aPCI and 120 min for mPCI. RESULTS: AlPcS2a fluorescence was seen throughout the cytosol following short or long light treatment, corresponding to aPCI and mPCI respectively. Spheroid growth was significantly inhibited or completely suppressed at a mPCI radiance of 0.05 or 0.72 J/cm2 respectively, with all bleomycin (BLM) concentrations used, compared to either BLM alone or aPCI at radiant exposure at these levels. The effects of BLM-aPCI and mPCI were comparable at radiance levels of 0.96 and 1.44 J/cm2. CONCLUSIONS: Results show that mPCI could effectively cause significant spheroid growth inhibition with the delivery of extremely low light irradiance rates delivered over an extended period of time. These findings suggest that effective implementation of mPCI can deliver adequate drug efficacy at depths necessary to reach infiltrating glioma cells in the surgical resection cavity wall.

Electroretinographic evidence for altered phototransduction gain and slowed recovery from photobleaches in albino mice with a MET450 variant in RPE65.

Our purpose was to investigate the physiological phenotype of albino mice with a variation in the Rpe65 gene encoding either methionine or leucine at amino acid #450. Full-field electroretinograms (ERGs) were recorded from C57BL/6J-c(2J) albino mice with MET450 and BALB/cByJ albino mice with LEU450. Recordings from pigmented mice (C57BL/6J) served as controls. Rod ERG a-waves were fitted with a model to estimate parameters of activation. Recovery of function following a photobleach was studied by monitoring the return to pre-bleach a- or b-wave amplitudes of the dark-adapted electroretinogram. The parameter, S, derived from the fit of the rod model, was significantly higher for albino mice compared to pigmented controls. Between the albino mice, S was highest for BALB/cByJ compared to C57BL/6J-c(2J). The parameters t(d) and Rm(P3) were not different across the three strains. The difference in S between the BALB/cByJ and C57BL/6J-c(2J) albino strains is interpreted to reflect differences in intrinsic phototransduction gain. Recovery from a photobleach was also slower for the C57BL/6J-c(2J) albino mice compared with BALB/cByJ albino mice, consistent with prior studies showing slowed rhodopsin regeneration in mice with the RPE65-METH450 variant. ERG recordings show that C57BL/6J-c(2J) albino mice with the MET450 variant of the RPE65 protein have a lower gain of activation and slower recovery from photobleach than do the BALB/cByJ albino mice with LEU450. Both the slower recovery from photobleach and lower gain of activation characteristic of the C57BL/6J-c(2J) strain may contribute to the mechanism by which it is protected from light-induced photoreceptor death relative to BALB/c.