A system of feed-forward cerebellar circuits that extend and diversify sensory signaling.

Sensory signals are processed by the cerebellum to coordinate movements. Numerous cerebellar functions are thought to require the maintenance of a sensory representation that extends beyond the input signal. Granule cells receive sensory input, but they do not prolong the signal and are thus unlikely to maintain a sensory representation for much longer than the inputs themselves. Unipolar brush cells (UBCs) are excitatory interneurons that project to granule cells and transform sensory input into prolonged increases or decreases in firing, depending on their ON or OFF UBC subtype. Further extension and diversification of the input signal could be produced by UBCs that project to one another, but whether this circuitry exists is unclear. Here we test whether UBCs innervate one another and explore how these small networks of UBCs could transform spiking patterns. We characterized two transgenic mouse lines electrophysiologically and immunohistochemically to confirm that they label ON and OFF UBC subtypes and crossed them together, revealing that ON and OFF UBCs innervate one another. A Brainbow reporter was used to label UBCs of the same ON or OFF subtype with different fluorescent proteins, which showed that UBCs innervate their own subtypes as well. Computational models predict that these feed-forward networks of UBCs extend the length of bursts or pauses and introduce delays-transformations that may be necessary for cerebellar functions from modulation of eye movements to adaptive learning across time scales.

Etonogestrel promotes respiratory recovery in an in vivo rat model of central chemoreflex impairment.

AIM: The central CO2 chemoreflex is a vital component of respiratory control networks, providing excitatory drive during resting conditions and challenges to blood gas homeostasis. The retrotrapezoid nucleus is a crucial hub for CO2 chemosensitivity; its ablation or inhibition attenuates CO2 chemoreflexes and diminishes restful breathing. Similar phenotypes characterize certain hypoventilation syndromes, suggesting underlying retrotrapezoid nucleus impairment in these disorders. Progesterone stimulates restful breathing and CO2 chemoreflexes. However, its mechanisms and sites of actions remain unknown and the experimental use of synthetic progestins in patients and animal models have been met with mixed respiratory outcomes. METHODS: We investigated whether acute or chronic administration of the progestinic drug, etonogestrel, could rescue respiratory chemoreflexes following selective lesion of the retrotrapezoid nucleus with saporin toxin. Adult female Sprague Dawley rats were grouped based on lesion size determined by the number of surviving chemosensitive neurons, and ventilatory responses were measured by whole body plethysmography. RESULTS: Ventilatory responses to hypercapnia (but not hypoxia) were compromised in a lesion-dependent manner. Chronic etonogestrel treatment improved CO2 chemosensitivity selectively in rats with moderate lesion, suggesting that a residual number of chemosensitive neurons are required for etonogestrel-induced CO2 chemoreflex recovery. CONCLUSION: This study provides new evidence for the use of progestins as respiratory stimulants under conditions of central hypoventilation and provides a new testable model for assessing the mechanism of action of progestins in the respiratory network.

A system of feed-forward cerebellar circuits that extend and diversify sensory signaling.

Sensory signals are processed by the cerebellum to coordinate movements. Numerous cerebellar functions are thought to require the maintenance of a sensory representation that extends beyond the input signal. Granule cells receive sensory input, but they do not prolong the signal and are thus unlikely to maintain a sensory representation for much longer than the inputs themselves. Unipolar brush cells (UBCs) are excitatory interneurons that project to granule cells and transform sensory input into prolonged increases or decreases in firing, depending on their ON or OFF UBC subtype. Further extension and diversification of the input signal could be produced by UBCs that project to one another, but whether this circuitry exists is unclear. Here we test whether UBCs innervate one another and explore how these small networks of UBCs could transform spiking patterns. We characterized two transgenic mouse lines electrophysiologically and immunohistochemically to confirm that they label ON and OFF UBC subtypes and crossed them together, revealing that ON and OFF UBCs innervate one another. A Brainbow reporter was used to label UBCs of the same ON or OFF subtype with different fluorescent proteins, which showed that UBCs innervate their own subtypes as well. Computational models predict that these feed-forward networks of UBCs extend the length of bursts or pauses and introduce delays-transformations that may be necessary for cerebellar functions from modulation of eye movements to adaptive learning across time scales.

Correlative analysis from a phase I clinical trial of intrapleural administration of oncolytic vaccinia virus (Olvi-vec) in patients with malignant pleural mesothelioma.

Background: The attenuated, genetically engineered vaccinia virus has been shown to be a promising oncolytic virus for the treatment of patients with solid tumors, through both direct cytotoxic and immune-activating effects. Whereas systemically administered oncolytic viruses can be neutralized by pre-existing antibodies, locoregionally administered viruses can infect tumor cells and generate immune responses. We conducted a phase I clinical trial to investigate the safety, feasibility and immune activating effects of intrapleural administration of oncolytic vaccinia virus (NCT01766739). Methods: Eighteen patients with malignant pleural effusion due to either malignant pleural mesothelioma or metastatic disease (non-small cell lung cancer or breast cancer) underwent intrapleural administration of the oncolytic vaccinia virus using a dose-escalating method, following drainage of malignant pleural effusion. The primary objective of this trial was to determine a recommended dose of attenuated vaccinia virus. The secondary objectives were to assess feasibility, safety and tolerability; evaluate viral presence in the tumor and serum as well as viral shedding in pleural fluid, sputum, and urine; and evaluate anti-vaccinia virus immune response. Correlative analyses were performed on body fluids, peripheral blood, and tumor specimens obtained from pre- and post-treatment timepoints. Results: Treatment with attenuated vaccinia virus at the dose of 1.00E+07 plaque-forming units (PFU) to 6.00E+09 PFU was feasible and safe, with no treatment-associated mortalities or dose-limiting toxicities. Vaccinia virus was detectable in tumor cells 2-5 days post-treatment, and treatment was associated with a decrease in tumor cell density and an increase in immune cell density as assessed by a pathologist blinded to the clinical observations. An increase in both effector (CD8+, NK, cytotoxic cells) and suppressor (Tregs) immune cell populations was observed following treatment. Dendritic cell and neutrophil populations were also increased, and immune effector and immune checkpoint proteins (granzyme B, perforin, PD-1, PD-L1, and PD-L2) and cytokines (IFN-γ, TNF-α, TGFß1 and RANTES) were upregulated. Conclusion: The intrapleural administration of oncolytic vaccinia viral therapy is safe and feasible and generates regional immune response without overt systemic symptoms. Clinical trial registration: https://clinicaltrials.gov/ct2/show/NCT01766739, identifier NCT01766739.

Next-generation immunotherapy for solid tumors: combination immunotherapy with crosstalk blockade of TGFß and PD-1/PD-L1.

INTRODUCTION: In solid tumor immunotherapy, less than 20% of patients respond to anti-programmed cell death 1 (PD-1)/programmed cell death 1 ligand 1 (PD-L1) agents. The role of transforming growth factor ß (TGFß) in diverse immunity is well-established; however, systemic blockade of TGFß is associated with toxicity. Accumulating evidence suggests the role of crosstalk between TGFß and PD-1/PD-L1 pathways. AREAS COVERED: We focus on TGFß and PD-1/PD-L1 signaling pathway crosstalk and the determinant role of TGFß in the resistance of immune checkpoint blockade. We provide the rationale for combination anti-TGFß and anti-PD-1/PD-L1 therapies for solid tumors and discuss the current status of dual blockade therapy in preclinical and clinical studies. EXPERT OPINION: The heterogeneity of tumor microenvironment across solid tumors complicates patient selection, treatment regimens, and response and toxicity assessment for investigation of dual blockade agents. However, clinical knowledge from single-agent studies provides infrastructure to translate dual blockade therapies. Dual TGFß and PD-1/PD-L1 blockade results in enhanced T-cell infiltration into tumors, a primary requisite for successful immunotherapy. A bifunctional fusion protein specifically targets TGFß in the tumor microenvironment, avoiding systemic toxicity, and prevents interaction of PD-1+ cytotoxic cells with PD-L1+ tumor cells.

Etonogestrel Administration Reduces the Expression of PHOX2B and Its Target Genes in the Solitary Tract Nucleus.

Heterozygous mutations of the transcription factor PHOX2B are responsible for Congenital Central Hypoventilation Syndrome, a neurological disorder characterized by inadequate respiratory response to hypercapnia and life-threatening hypoventilation during sleep. Although no cure is currently available, it was suggested that a potent progestin drug provides partial recovery of chemoreflex response. Previous in vitro data show a direct molecular link between progestins and PHOX2B expression. However, the mechanism through which these drugs ameliorate breathing in vivo remains unknown. Here, we investigated the effects of chronic administration of the potent progestin drug Etonogestrel (ETO) on respiratory function and transcriptional activity in adult female rats. We assessed respiratory function with whole-body plethysmography and measured genomic changes in brain regions important for respiratory control. Our results show that ETO reduced metabolic activity, leading to an enhanced chemoreflex response and concurrent increased breathing cycle variability at rest. Furthermore, ETO-treated brains showed reduced mRNA and protein expression of PHOX2B and its target genes selectively in the dorsal vagal complex, while other areas were unaffected. Histological analysis suggests that changes occurred in the solitary tract nucleus (NTS). Thus, we propose that the NTS, rich in both progesterone receptors and PHOX2B, is a good candidate for ETO-induced respiratory modulation.

Expiratory activity during sleep in children.

Sleep irregularities and respiratory events (apnea, O2 desaturation or a combination thereof) are often present in the infant population. While inspiration is the main active process in the act of breathing, expiration is generally thought to occur passively. Although commonly considered as quiet during sleep, expiratory abdominal muscles have been proposed to be recruited to promote ventilation, facilitate gas exchange, and reduce the work of breathing during conditions of increased respiratory drive, exercise, or airway obstruction. In this study, we investigated the occurrence of expiratory abdominal muscle activity in polysomnographic studies of subjects (aged 0-2 years) suspected of sleep disordered breathing. Our results indicate that abdominal muscle activation occurs during sleep, most frequently during non-rapid eye movement and rapid-eye movement states compared to slow-wave sleep. Furthermore, abdominal muscle activity was present during regular breathing or associated with respiratory events (apneas or O2 desaturation). In the latter case, abdominal muscle recruitment more frequently followed the onset of respiratory events and terminated with recovery from blood O2 desaturation events. We conclude that expiratory abdominal muscle activity contributes to the pattern of respiratory muscle recruitment during sleep in infants and given its temporal relationship with respiratory events, we propose that its recruitment could facilitate proper ventilation by counteracting airway resistance and O2 desaturation in infancy across different stages of sleep.

The Effects of Breastfeeding on Retinoblastoma Development: Results from an International Multicenter Retinoblastoma Survey.

The protective effects of breastfeeding on various childhood malignancies have been established but an association has not yet been determined for retinoblastoma (RB). We aimed to further investigate the role of breastfeeding in the severity of nonhereditary RB development, assessing relationship to (1) age at diagnosis, (2) ocular prognosis, measured by International Intraocular RB Classification (IIRC) or Intraocular Classification of RB (ICRB) group and success of eye salvage, and (3) extraocular involvement. Analyses were performed on a global dataset subgroup of 344 RB patients whose legal guardian(s) consented to answer a neonatal questionnaire. Patients with undetermined or mixed feeding history, family history of RB, or sporadic bilateral RB were excluded. There was no statistically significant difference between breastfed and formula-fed groups in (1) age at diagnosis (p = 0.20), (2) ocular prognosis measures of IIRC/ICRB group (p = 0.62) and success of eye salvage (p = 0.16), or (3) extraocular involvement shown by International Retinoblastoma Staging System (IRSS) at presentation (p = 0.74), lymph node involvement (p = 0.20), and distant metastases (p = 0.37). This study suggests that breastfeeding neither impacts the sporadic development nor is associated with a decrease in the severity of nonhereditary RB as measured by age at diagnosis, stage of disease, ocular prognosis, and extraocular spread. A further exploration into the impact of diet on children who develop RB is warranted.

Imaging CAR T-cell kinetics in solid tumors: Translational implications.

Success in solid tumor chimeric antigen receptor (CAR) T-cell therapy requires overcoming several barriers, including lung sequestration, inefficient accumulation within the tumor, and target-antigen heterogeneity. Understanding CAR T-cell kinetics can assist in the interpretation of therapy response and limitations and thereby facilitate developing successful strategies to treat solid tumors. As T-cell therapy response varies across metastatic sites, the assessment of CAR T-cell kinetics by peripheral blood analysis or a single-site tumor biopsy is inadequate for interpretation of therapy response. The use of tumor imaging alone has also proven to be insufficient to interpret response to therapy. To address these limitations, we conducted dual tumor and T-cell imaging by use of a bioluminescent reporter and positron emission tomography in clinically relevant mouse models of pleural mesothelioma and non-small cell lung cancer. We observed that the mode of delivery of T cells (systemic versus regional), T-cell activation status (presence or absence of antigen-expressing tumor), and tumor-antigen expression heterogeneity influence T-cell kinetics. The observations from our study underscore the need to identify and develop a T-cell reporter-in addition to standard parameters of tumor imaging and antitumor efficacy-that can be used for repeat imaging without compromising the efficacy of CAR T cells in vivo.

Is sino-nasal outcome test-22 reliable for guiding chronic rhinosinusitis patients for endoscopic sinus surgery?

Chronic rhino-sinusitis (CRS) is a significant health problem whose incidence and prevalence is rising. An emphasis has been placed on diseasespecific quality of life (QoL as the predominant measure for most current outcome studies. Therefore a validated measure of health-related QoL in sinonasal disease is needed. The present prospective and observational study was conducted on 50 patients in the Department of ENT at Govt. Medical College and Rajindra Hospital Patiala, Punjab, India. The primary outcomes were the following: (1) the chance of attaining minimal clinically important difference (MCID) improvements of nine points at the 22-item Sino-Nasal Outcome Test (SNOT-22) after endoscopic sinus surgery (ESS) for different preoperative QoL levels, and (2) the percentage of relative improvement in SNOT-22 after ESS for different preoperative QoL levels. METHODS: Patients with CRS who were elected for ESS were prospectively enrolled into an observational cohort study. They were categorized into 10 preoperative SNOT-22 groups based on 10-point increments beginning with a score of 10 and ending at 110. Standard protocol for all patients presenting for evaluation included completion of the SNOT-22 prior to and following surgical intervention. The scores were calculated and the data collected were compiled and analyzed. RESULTS: A total of 50 patients were included in this study. Patients with a SNOT-22 score between 10 and 19 had the lowest chance of achieving an MCID. Patients with a SNOT-22 score greater than 30 had a greater than 90% chance of achieving an MCID, and there was a relative improvement of 43.3% on their preoperative SNOT-22 scores. CRS patients with polyp had better outcomes (47.1% improvement) after ESS than those without polyp (33.2% improvement). CONCLUSION: There is an increased probability of achieving an MCID at SNOT-22 score >30 and in general the percentage of relative improvement increased with an increase in preoperative SNOT score.

Breathing During Sleep in the Postnatal Period of Rats: The Contribution of Active Expiration.

Breathing is most vulnerable to apneas and other disturbances during sleep in both humans and rodents, especially in the newborn period. We recently demonstrated in adult rats that, in contrast to the atonia typical of skeletal muscles during rapid eye movement sleep, the normally passive expiratory muscles become active, and their activity is associated with stabilization of breathing and increased ventilation. In this study, we investigated the relationship between respiration and expiratory muscle recruitment across sleep states during the first 2 weeks of rat postnatal development. We instrumented rats with electromyography electrodes in neck and abdominal muscles while sleep states (active and quiet sleep) were classified based on nuchal muscle tone and overt behavior inside a whole-body plethysmograph. Our results indicate that breathing was most irregular in active sleep (AS) and that rats displayed frequent recruitment of expiratory muscle activity, which occurred in both active and quiet sleep states. While the occurrence of active expiration in quiet sleep did not affect ventilation and its frequency decreased with age, the recruitment of expiratory muscles during AS was present across development and it was associated with a reduction in respiratory variability across development and an increase in ventilation at most age groups considered. We conclude that the occurrence of active expiration is a common feature of respiration in the postnatal period, and it significantly contributes to ventilation, in particular in AS.