The Lysosomal Calcium Channel TRPML1 Maintains Mitochondrial Fitness in NK Cells through Interorganelle Cross-Talk.

Cytotoxic lymphocytes eliminate cancer cells through the release of lytic granules, a specialized form of secretory lysosomes. This compartment is part of the pleomorphic endolysosomal system and is distinguished by its highly dynamic Ca2+ signaling machinery. Several transient receptor potential (TRP) calcium channels play essential roles in endolysosomal Ca2+ signaling and ensure the proper function of these organelles. In this study, we examined the role of TRPML1 (TRP cation channel, mucolipin subfamily, member 1) in regulating the homeostasis of secretory lysosomes and their cross-talk with mitochondria in human NK cells. We found that genetic deletion of TRPML1, which localizes to lysosomes in NK cells, led to mitochondrial fragmentation with evidence of collapsed mitochondrial cristae. Consequently, TRPML1-/- NK92 (NK92ML1-/-) displayed loss of mitochondrial membrane potential, increased reactive oxygen species stress, reduced ATP production, and compromised respiratory capacity. Using sensitive organelle-specific probes, we observed that mitochondria in NK92ML1-/- cells exhibited evidence of Ca2+ overload. Moreover, pharmacological activation of the TRPML1 channel in primary NK cells resulted in upregulation of LC3-II, whereas genetic deletion impeded autophagic flux and increased accumulation of dysfunctional mitochondria. Thus, TRPML1 impacts autophagy and clearance of damaged mitochondria. Taken together, these results suggest that an intimate interorganelle communication in NK cells is orchestrated by the lysosomal Ca2+ channel TRPML1.

Pre-treatment non-ictal cephalic allodynia identifies responders to prophylactic treatment of chronic and episodic migraine patients with galcanezumab: A prospective quantitative sensory testing study (NCT04271202).

BACKGROUND: Migraine is a complex neurological disorder involving generalized abnormalities in processing sensory information. Adopting evidence that central sensitization imposes major hurdles in the treatment of migraine, we hypothesized that it is the non-ictal (rather than ictal) allodynia that may determine the outcome of migraine prevention with peripherally-acting drugs. METHODS: To test this hypothesis, we used Quantitative Sensory Testing to determine whether it is possible to identify a patient's response to prophylactic treatment with galcanezumab based on presence/absence of cephalic and/or extracephalic allodynia during the pre-treatment non-ictal phase of migraine. RESULTS: Using strict criteria for allodynia (heat 32-40°C, cold 32-20°C, mechanical <60 g), we report that (a) the incidence of pre-treatment non-ictal cephalic allodynia was 21% in the 24 responders (>50% decrease in monthly migraine days) and 85% in the 19 non-responders; (b) the incidence of non-ictal extracephalic allodynia distinguishes responders from non-responders less accurately; and that (c) the incidence of non-ictal cephalic allodynia was similar in the chronic migraine and high-frequency episodic migraine groups. CONCLUSIONS: Clinically, the findings suggest that presence/absence of non-ictal allodynia can be used to identify galcanezumab responders with nearly 80% accuracy and galcanezumab non-responders with nearly 85% accuracy. Mechanistically, the presence of non-ictal allodynia (reflecting a state of activity-independent central sensitization) in both chronic migraine and high-frequency episodic migraine patients raises the possibility that the state of non-ictal allodynia may be attributed to physiological properties of central trigeminovascular neurons that are due to the genetic load of the individual patient rather than their migraine frequency.

Microstructural differences in migraine: A diffusion-tensor imaging study.

BACKGROUND: Diffusion-tensor imaging can be applied to describe the microstructural integrity of the whole brain. As findings about microstructural alterations in migraine are inconsistent, we aimed to replicate the most frequent results and assess a relationship between migraine parameters and changes in microstructure. METHODS: Diffusion-weighted MRI data of 37 migraine patients and 40 controls were collected. Two indices of diffusion of water molecules, fractional anisotropy and mean diffusivity were used in a voxel-wise analysis. Group comparisons were carried out in SPM12 using age and sex as covariates. Statistically significant results survived family-wise error correction (pFWE < 0.05). Migraine intensity, frequency, and duration were self-reported and correlated with mean fractional anisotropy and mean diffusivity values across clusters. RESULTS: Migraine patients showed significantly lower fractional anisotropy in occipital regions, and significantly higher fractional anisotropy in thirteen clusters across the brain. Mean diffusivity of migraine patients was significantly decreased in the cerebellum and pons, but it was not increased in any area. Correlation between migraine duration and fractional anisotropy was significantly positive in the frontal cortex and significantly negative in the superior parietal lobule. CONCLUSION: We suggest that microstructural integrity of the migraine brain is impaired in visual areas and shows duration-related alterations in regions of the default mode network.

Protective Effects of Pituitary Adenylate-Cyclase-Activating Polypeptide on Retinal Vasculature and Molecular Responses in a Rat Model of Moderate Glaucoma.

Despite the high probability of glaucoma-related blindness, its cause is not fully understood and there is no efficient therapeutic strategy for neuroprotection. Vascular factors have been suggested to play an important role in glaucoma development and progression. Previously, we have proven the neuroprotective effects of pituitary adenylate-cyclase-activating polypeptide (PACAP) eye drops in an inducible, microbeads model in rats that is able to reproduce many clinically relevant features of human glaucoma. In the present study, we examined the potential protective effects of PACAP1-38 on the retinal vasculature and the molecular changes in hypoxia. Ocular hypertension was induced by injection of microbeads into the anterior chamber, while control rats received PBS. PACAP dissolved in vehicle (1 µg/drop) or vehicle treatment was started one day after the injections for four weeks three times a day. Retinal degeneration was assessed with optical coherence tomography (OCT), and vascular and molecular changes were assessed by immunofluorescence labeling. HIF1-α and VEGF-A protein levels were measured by Western blot. OCT images proved severe retinal degeneration in the glaucomatous group, while PACAP1-38 eye drops had a retinoprotective effect. Vascular parameters were deteriorated and molecular analysis suggested hypoxic conditions in glaucoma. PACAP treatment exerted a positive effect against these alterations. In summary, PACAP could prevent the severe damage to the retina and its vasculature induced by ocular hypertension in a microbeads model.

Galcanezumab effects on incidence of headache after occurrence of triggers, premonitory symptoms, and aura in responders, non-responders, super-responders, and super non-responders.

BACKGROUND: The goal of this observational, open-label, cohort study was to determine whether prophylactic migraine treatment with galcanezumab, a peripherally acting drug, alters the incidence of premonitory symptoms, and/or occurrence of headache after exposure to triggers or aura episodes in treatment-responders (≥ 50% reduction in monthly migraine days [MMD]), super-responders (≥ 70%), non-responders (< 50%) and super non-responders (< 30%). METHODS: Participants were administered electronic daily headache diaries to document migraine days and associated symptoms one month before and during the three months of treatment. Questionnaires were used to identify conscious prodromal and trigger events that were followed by headache prior to vs. after 3 months of treatment. RESULTS: After 3 months of galcanezumab treatment, (a) the incidence of premonitory symptoms that were followed by headache decreased by 48% in the 27 responders vs. 28% in the 19 non-responders, and by 50% in the 11 super-responders vs. 12% in the 8 super non-responders; (b) the incidence of visual and sensory aura that were followed by headache was reduced in responders, non-responders, and super-responders, but not in super non-responders; (c) the number of triggers followed by headache decreased by 38% in responders vs. 13% in non-responders, and by 31% in super-responders vs. 4% in super non-responders; and (d) some premonitory symptoms (e.g., cognitive impairment, irritability, fatigue) and triggers (e.g., stress, sleeping too little, bright light, aura) were followed by headache only in super non-responders. CONCLUSIONS: Mechanistically, these findings suggest that even a mild decrease in migraine frequency is sufficient to partially reverse the excitability and responsivity of neurons involved in the generation of certain triggers and potentially premonitory symptoms of migraine. TRIAL REGISTRATION: ClinicalTrials.gov: NCT04271202. Registration date: February 10, 2020.

Alterations in the structure and function of the brain in adolescents with new daily persistent headache: A pilot MRI study.

OBJECTIVE: The purpose of this study was to explore brain morphological and functional connectivity alterations in adolescents with new daily persistent headache (NDPH) compared to pain-free, healthy controls. BACKGROUND: NDPH is one of the most disabling and least understood primary headache conditions. To date, no studies have considered the role of brain function and structure in pediatric patients with NDPH. METHODS: In this cross-sectional study, resting-state functional and structural images were acquired for 13 patients with NDPH (M age = 15.9, standard deviation [SD] ± 1.4) and 13 age- and sex-matched controls (M age = 16.2, SD ± 1.8) using magnetic resonance imaging. Participants were recruited from the Pediatric Headache Program at Boston Children's Hospital and from the Greater Boston area. In patients, clinical features of NDPH, including disease duration, pain intensity ratings, pain sensitivity, and functional disability were also assessed, and their associations with functional and structural brain alterations were explored. RESULTS: Compared to controls, patients with NDPH demonstrated reduced cortical thickness in the bilateral superior temporal gyrus, left superior, and middle frontal gyrus areas (p < 0.05, Monte Carlo corrected for multiple comparisons). Furthermore, reduced cortical thickness of the left superior frontal gyrus was related to elevated pain sensitivity in NDPH (r = -0.79, p = 0.006). Patients showed altered functional connectivity between regions involved in emotional and cognitive networks of pain, including the amygdala, insula, frontal regions, and cerebellar subregions. CONCLUSION: The present study provides the first preliminary evidence of functional and structural brain differences in pediatric patients with NDPH compared to controls. Identifying alterations in cortical thickness and resting-state connectivity between specific brain regions could provide characteristics of NDPH and probable mechanisms that may guide personalized therapeutic interventions.

Migraine: interactions between brain's trait and state.

It is well established that migraine is a multifactorial disorder. A deep understanding of migraine should be based upon both the underlying traits and the current states affected by different physiological, psychological, and environmental factors. At this point, there is no framework fully meeting these criteria. Here, we describe a broader view of the migraine disorder defined as a dysfunctional brain state and trait interaction. In this model, we consider events that may enhance or diminish migraine responsivity based on an individual's trait and state. This could provide an expanded view for considering how migraine attacks are sometimes precipitated by "triggers" and sometimes not, how these factors only lead to migraine attacks in migraine patients, or how individuals with an increased risk for migraine do not show any symptoms at all. Summarizing recent studies and evidence that support the concept of migraine as a brain state-trait interaction can also contribute to improving patient care by highlighting the importance of precision medicine and applying measures that are able to capture how different traits and states work together to determine migraine.

A cross-sectional study on the quality of life in migraine and medication overuse headache in a Hungarian sample: understanding the effect of headache characteristics.

Background and purpose: Previous studies using generic and disease specific instruments showed that both migraine and medication overuse headache are associated with lower health-related quality of life (HRQoL). The aim of our study was to assess HRQoL differences in migraineurs and in patients with MOH and to examine how headache characteristics such as years with headache, aura symptoms, triptan use, headache pain severity and headache frequency are related to HRQoL. Methods: In this cross-sectional study 334 participants were examined (248 were recruited from a tertiary headache centre and 86 via advertisements). The Comp-rehensive Headache-related Quality of life Questionnaire (CHQQ) was used to measure the participants' HRQoL. Data showed normal distribution, therefore beside Chi-squared test parametric tests (e.g. independent samples t-test) were used with a two-tailed p<0.05 threshold. Linear regression models were used to determine the independent effects of sex, age, recruitment method, headache type (migraine vs. MOH) and headache characteristics (presence of aura symptoms, years with headache, headache pain severity, headache frequency and triptan use) separately for each domain and for the total score of CHQQ. Significance threshold was adopted to p0.0125 (0.05/4) to correct for multiple testing and avoid Type I error. Results: Independent samples t-tests showed that patients with MOH had significantly lower scores on all CHQQ domains than migraineurs, except on the social subscale. Results of a series of regression analyses showed that triptan use was inversely related to all the domains of HRQoL after correction for multiple testing (p<0.0125). In addition, headache pain severity was associated with lower physical (p=0.001) and total scores (p=0.002) on CHQQ subscales. Conclusion: Based on the results, different headache characteristics (but not the headache type, namely migraine or MOH) were associated with lower levels of HRQoL in patients with headache. Determining which factors play significant role in the deterioration of HRQoL is important to adequately manage different patient populations and to guide public health policies regarding health service utilization and health-care costs.

Suppressive mechanisms by Heligmosomoides polygyrus-induced Tregs in C57BL/6 mice change over time and differ to that of naïve mice.

Disrupting or harnessing immune suppression is leading to new therapeutic avenues in a number of immune-related diseases. Understanding the suppressive functions of regulatory T cells (Tregs) in different environments is therefore key. Parasitic worms are strong inducers of Tregs and previous research has suggested that parasite-induced Tregs are stronger suppressors than naïve Tregs. In strains susceptible to the intestinal worm Heligmosomoides polygyrus, like C57BL/6 mice, it has been hypothesized that increased Treg suppression downregulates both Th1 and Th2 responses, leading to chronic infections and high worm burden. Here, we show that the suppressive capacity of Tregs is no different between cells from infected and/or naive animals. In vitro suppression induced by CD4+ CD25+ Tregs (Peyers' Patches or the mesenteric lymph nodes), isolated early (day 7, tissue dwelling phase) or late (day 21, luminal phase) during infection was similar to that induced by cells from naïve animals. Suppression was CTLA-4 dependent in Tregs from acute but not chronic infection or in Tregs from naïve animals. This highlights the versatility of Tregs and the importance of extensive Treg characterization prior to potential in vivo manipulation of this cell type.

Comparison of Amorphous Solid Dispersions of Spironolactone Prepared by Spray Drying and Electrospinning: The Influence of the Preparation Method on the Dissolution Properties.

This research aimed to compare two solvent-based methods for the preparation of amorphous solid dispersions (ASDs) made up of poorly soluble spironolactone and poly(vinylpyrrolidone-co-vinyl acetate). The same apparatus was used to produce, in continuous mode, drug-loaded electrospun (ES) and spray-dried (SD) materials from dichloromethane and ethanol-containing solutions. The main differences between the two preparation methods were the concentration of the solution and application of high voltage. During electrospinning, a solution with a higher concentration and high voltage was used to form a fibrous product. In contrast, a dilute solution and no electrostatic force were applied during spray drying. Both ASD products showed an amorphous structure according to differential scanning calorimetry and X-ray powder diffraction results. However, the dissolution of the SD sample was not complete, while the ES sample exhibited close to 100% dissolution. The polarized microscopy images and Raman microscopy mapping of the samples highlighted that the SD particles contained crystalline traces, which can initiate precipitation during dissolution. Investigation of the dissolution media with a borescope made the precipitated particles visible while Raman spectroscopy measurements confirmed the appearance of the crystalline active pharmaceutical ingredient. To explain the micro-morphological differences, the shape and size of the prepared samples, the evaporation rate of residual solvents, and the influence of the electrostatic field during the preparation of ASDs had to be considered. This study demonstrated that the investigated factors have a great influence on the dissolution of the ASDs. Consequently, it is worth focusing on the selection of the appropriate ASD preparation method to avoid the deterioration of dissolution properties due to the presence of crystalline traces.

Retinoprotective Effects of PACAP Eye Drops in Microbead-Induced Glaucoma Model in Rats.

Glaucoma is associated with increased intraocular pressure (IOP), causing the apoptosis of retinal ganglion cells (RGCs) and the loss of their axons leading to blindness. Pituitary adenylate cyclase activating polypeptide (PACAP) is neuroprotective in several neural injuries, including retinopathies. The aim of this study was to investigate the effects of PACAP1-38 eye drops in a model of glaucoma. IOP was elevated bilaterally by injections of microbeads to block the aqueous humor outflow. The control groups received the same volume of saline. Animals were treated with PACAP1-38 (1 µg/drop, 3 × 1 drop/day) or vehicle for 4 weeks starting one day after the injections. Retinal morphology by histology and optical coherence tomography, function by electroretinography, and IOP changes were analyzed. Animals were sacrificed 8 weeks after the injections. Microbeads injections induced a significant increase in the IOP, while PACAP1-38 treatment lowered it to normal levels (~10 mmHg). Significant retinal degeneration and functional impairment were observed in the microbead-injected group without PACAP1-38 treatment. In the microbeads + PACAP1-38 group, the retinal morphology and functionality were close to the normal values. In summary, our results show that PACAP1-38, given in form of eye drops, is neuroprotective in glaucoma, providing the basis for potential future therapeutic administration.

Anticipation and violated expectation of pain are influenced by trait rumination: An fMRI study.

Rumination - as a stable tendency to focus repetitively on feelings related to distress - represents a transdiagnostic risk factor. Theories suggest altered emotional information processing as the key mechanism of rumination. However, studies on the anticipation processes in relation to rumination are scarce, even though expectation in this process is demonstrated to influence the processing of emotional stimuli. In addition, no published study has investigated violated expectation in relation to rumination yet. In the present study we examined the neural correlates of pain anticipation and perception using a fear conditioning paradigm with pain as the unconditioned stimulus in healthy subjects (N = 30). Rumination was assessed with the 10-item Ruminative Response Scale (RRS). Widespread brain activation - extending to temporal, parietal, and occipital lobes along with activation in the cingulate cortex, insula, and putamen - showed a positive correlation with rumination, supporting our hypothesis that trait rumination influences anticipatory processes. Interestingly, with violated expectation (when an unexpected, non-painful stimulus follows a pain cue compared to when an expected, painful stimulus follows the same pain cue) a negative association between rumination and activation was found in the posterior cingulate cortex, which is responsible for change detection in the environment and subsequent behavioral modification. Our results suggest that rumination is associated with increased neural response to pain perception and pain anticipation, and may deteriorate the identification of an unexpected omission of aversive stimuli. Therefore, targeting rumination in cognitive behavioral therapy of chronic pain could have a beneficial effect.

Increased activation of the pregenual anterior cingulate cortex to citalopram challenge in migraine: an fMRI study.

BACKGROUND: The anterior cingulate cortex (ACC) is a key structure of the pain processing network. Several structural and functional alterations of this brain area have been found in migraine. In addition, altered serotonergic neurotransmission has been repeatedly implicated in the pathophysiology of migraine, although the exact mechanism is not known. Thus, our aim was to investigate the relationship between acute increase of brain serotonin (5-HT) level and the activation changes of the ACC using pharmacological challenge MRI (phMRI) in migraine patients and healthy controls. METHODS: Twenty-seven pain-free healthy controls and six migraine without aura patients participated in the study. All participant attended to two phMRI sessions during which intravenous citalopram, a selective serotonin reuptake inhibitor (SSRI), or placebo (normal saline) was administered. We used region of interest analysis of ACC to compere the citalopram evoked activation changes of this area between patients and healthy participants. RESULTS: Significant difference in ACC activation was found between control and patient groups in the right pregenual ACC (pgACC) during and after citalopram infusion compared to placebo. The extracted time-series showed that pgACC activation increased in migraine patients compared to controls, especially in the first 8-10 min of citalopram infusion. CONCLUSIONS: Our results demonstrate that a small increase in 5-HT levels can lead to increased phMRI signal in the pregenual part of the ACC that is involved in processing emotional aspects of pain. This increased sensitivity of the pgACC to increased 5-HT in migraine may contribute to recurring headache attacks and increased stress-sensitivity in migraine.

[Psychometric properties of the Inventory of Callous-Unemotional Traits in a high-risk sample of adolescent boy].

INTRODUCTION: The Inventory of Callous-Unemotional Traits (ICU) is one of the most widely used measures of psychopathic traits in children. Callous-unemotional (CU) traits designate an important subgroup of antisocial youth characterized by lack of empathy, guilt and remorse. The aim of the present study was to test the applicability and reliability of the self-reported ICU in a high-risk sample of adolescent boys. METHODS: Participants were 202 adolescent boys (mean age: 16.63 years; SD = 1.71) from institutional care facilities and juvenile detention centres. Confirmatory factor analyses (CFA) were conducted to investigate the factor structure of the ICU. In addition, MIMIC modelling (CFA with covariates) was applied to test the convergent validity of the ICU scores by examining relationships with externalizing symptoms (including conduct problems, hyperactivity-inattention, proactive-reactive aggression), and prosocial behaviour. RESULTS: We observed that the bifactor model with three correlated specific factors (callousness, uncaring and unemotional) and one general CU traits factor provided the best fit to the data. However, similar to previous studies, low internal consistency was found for the unemotional scale. In line with our expectations, CU traits showed positive associations with externalizing symptoms, and negative associations with prosocial behaviour. CONCLUSION: The ICU is a reliable and valid measure of callous-unemotional traits. Our results support the application of the Hungarian version of the questionnaire.

Increases in helminth-induced IL-21 protein levels disappear upon sample freezing.

IL-21 is a much studied cytokine that has been implicated in the regulation of TH1, TH2, TH17 and regulatory immune responses; its signalling is a promising therapeutic target for autoimmune, inflammatory and infectious diseases. Despite its biological importance, measuring IL-21 reliably has proved difficult. ELISAs are commonly used to measure cytokines in various biological samples. However, results obtained are only as good as the quality of the sample. Here, we show that when using fresh samples, a significant increase in IL-21 was measured in the intestinal homogenate of mice infected with the intestinal worm Heligmosomoides polygyrus. This difference disappeared when samples were frozen in either liquid nitrogen for two days or at -80 °C for three weeks, with levels in both naïve and infected animals decreasing. This was not observed for the IL-13 cytokine, where freezing had no impact on levels measured. Our study highlights the importance of sample storage to measuring biomarkers. Since modulating IL-21 signalling is such an important potential therapeutic avenue, accurately measuring the levels of this cytokine is key to assessing its role in various research models and clinical settings.

Passage through the Ocular Barriers and Beneficial Effects in Retinal Ischemia of Topical Application of PACAP1-38 in Rodents.

The neuropeptide pituitary adenylate cyclase activating polypeptide (PACAP) has two active forms, PACAP1-27 and PACAP1-38. Among the well-established actions are PACAP's neurotrophic and neuroprotective effects, which have also been proven in models of different retinopathies. The route of delivery is usually intravitreal in studies proving PACAP's retinoprotective effects. Recently, we have shown that PACAP1-27 delivered as eye drops in benzalkonium-chloride was able to cross the ocular barriers and exert retinoprotection in ischemia. Since PACAP1-38 is the dominant form of the naturally occurring PACAP, our aim was to investigate whether the longer form is also able to cross the barriers and exert protective effects in permanent bilateral common carotid artery occlusion (BCCAO), a model of retinal hypoperfusion. Our results show that radioactive PACAP1-38 eye drops could effectively pass through the ocular barriers to reach the retina. Routine histological analysis and immunohistochemical evaluation of the Müller glial cells revealed that PACAP1-38 exerted retinoprotective effects. PACAP1-38 attenuated the damage caused by hypoperfusion, apparent in almost all retinal layers, and it decreased the glial cell overactivation. Overall, our results confirm that PACAP1-38 given in the form of eye drops is a novel protective therapeutic approach to treat retinal diseases.

[Iowa Gambling Task: illustration of a behavioral measurement]. / Iowa Gambling Task: egy viselkedéses méroeszköz bemutatása.

The Iowa Gambling Task is a behavioral measurement which was developed to examine decision-making based on the Somatic Marker Hypothesis. Participants have to make series of choices altogether 100 times from four decks of cards. The decks have different characteristics with regards to gains and losses. After the initial analyses - with a focus on patients with damage to the ventromedial prefrontal cortex - the tool soon became one of the most frequently used technique of measuring hot executive functions. It is also used to measure impulsivity. Structures involved in decision-making constitute the neural basis of the Task. IGT is applied in several different disorders (in connection with decision-making and impulsivity as well). In recent years different versions have been developed, and these modifications may have different effects on IGT performance, and may also influence what the Task measures exactly. With growing empirical evidence several questions have arisen in connection with the composition of the decks (gain-loss magnitude vs. frequency, prominent deck B phenomenon) which suggest to use other indexes as well besides the net scores.

Investigation of PACAP38 and PAC1 Receptor Expression in Human Retinoblastoma and the Effect of PACAP38 Administration on Human Y-79 Retinoblastoma Cells.

Retinoblastoma represents the most prevalent malignant neoplasm affecting the eyes in childhood. The clear-cut origin of retinoblastoma has not yet been determined; however, based on experiments, it has been suggested that RB1 loss in cone photoreceptors causes retinoblastoma. Pituitary adenylate-cyclase activating polypeptide (PACAP) is a pleiotropic neuropeptide which has been shown to be affected in certain tumorous transformations, such as breast, lung, kidney, pancreatic, colon, and endocrine cancers. This study aimed to investigate potential changes in both PACAP38 and PAC1 receptor (PAC1R) expression in human retinoblastoma and the effect of PACAP38 administration on the survival of a human retinoblastoma cell line (Y-79). We analyzed human enucleation specimens removed because of retinoblastoma for PACAP38 and PAC1R immunostaining and the effect of PACAP38 on the survival of the Y-79 cell line. We described for the first time that human retinoblastoma cells from patients showed only perinuclear, dot-like immunopositivity for both PACAP38 and PAC1R, irrespective of laterality, genetic background, or histopathological features. Nanomolar (100 nM and 500 nM) PACAP38 concentrations had no effect on the viability of Y-79 cells, while micromolar (2 µM and 6 µM) PACAP38 significantly decreased tumor cell viability. These findings, along with general observations from animal studies showing that PACAP38 has strong anti-apoptotic, anti-inflammatory, and antioxidant effects on ocular tissues, together suggest that PACAP38 and its analogs are promising candidates in retinoblastoma therapy.

Heligmosomoides bakeri and Toxoplasma gondii co-infection leads to increased mortality associated with changes in immune resistance in the lymphoid compartment and disease pathology.

Co-infections are a common reality but understanding how the immune system responds in this context is complex and can be unpredictable. Heligmosomoides bakeri (parasitic roundworm, previously Heligmosomoides polygyrus) and Toxoplasma gondii (protozoan parasite) are well studied organisms that stimulate a characteristic Th2 and Th1 response, respectively. Several studies have demonstrated reduced inflammatory cytokine responses in animals co-infected with such organisms. However, while general cytokine signatures have been examined, the impact of the different cytokine producing lymphocytes on parasite control/clearance is not fully understood. We investigated five different lymphocyte populations (NK, NKT, γδ T, CD4+ T and CD8+ T cells), five organs (small intestine, Peyer's patches, mesenteric lymph nodes, spleen and liver), and 4 cytokines (IFN©, IL-4, IL-10 and IL-13) at two different time points (days 5 and 10 post T. gondii infection). We found that co-infected animals had significantly higher mortality than either single infection. This was accompanied by transient and local changes in parasite loads and cytokine profiles. Despite the early changes in lymphocyte and cytokine profiles, severe intestinal pathology in co-infected mice likely contributed to early mortality due to significant damage by both parasites in the small intestine. Our work demonstrates the importance of taking a broad view during infection research, studying multiple cell types, organs/tissues and time points to link and/or uncouple immunological from pathological findings. Our results provide insights into how co-infection with parasites stimulating different arms of the immune system can lead to drastic changes in infection dynamics.