Intratumoral injection of schwannoma with attenuated Salmonella typhimurium induces antitumor immunity and controls tumor growth.

Schwannomas are slow-growing benign neoplasms that develop throughout the body causing pain, sensory/motor dysfunction, and death. Because bacterial immunotherapy has been used in the treatment of some malignant neoplasms, we evaluated attenuated Salmonella typhimurium strains as immunotherapies for benign murine schwannomas. Several bacterial strains were tested, including VNP20009, a highly attenuated strain that was previously shown to be safe in human subjects with advanced malignant neoplasms, and a VNP20009 mutant that was altered in motility and other properties that included adherence and invasion of cultured mammalian cells. VNP20009 controlled tumor growth in two murine schwannoma models and induced changes in cytokine and immune effector cell profiles that were consistent with induction of enhanced innate and adaptive host immune responses compared with controls. Intratumoral (i.t.) injection of S. typhimurium led to tumor cell apoptosis, decreased tumor angiogenesis, and lower growth of the injected schwannoma tumors. Invasive VNP20009 was significantly more efficacious than was a noninvasive derivative in controlling the growth of injected tumors. Bacterial treatment apparently induced systemic antitumor immunity in that the growth of rechallenge schwannomas implanted following primary bacterial treatment was also reduced. Checkpoint programmed death-1 (PD-1) blockade induced by systemic administration of anti-PD-1 antibodies controlled tumor growth to the same degree as i.t. injection of S. typhimurium, and together, these two therapies had an additive effect on suppressing schwannoma growth. These experiments represent validation of a bacterial therapy for a benign neoplasm and support development of S. typhimurium VNP20009, potentially in combination with PD-1 inhibition, as a schwannoma immunotherapy.

Schwannoma Gene Therapy via Adeno-Associated Viral Vector Delivery of Apoptosis-Associated Speck-like Protein Containing CARD (ASC): Preclinical Efficacy and Safety.

Schwannomas are tumors derived from Schwann-lineage cells, cells that protect and support myelinated nerves in the peripheral nervous system. They are typically slow-growing, encapsulated and benign. These tumors develop along peripheral, spinal and cranial nerves causing pain, sensory-motor dysfunction and death. Primary treatment for schwannoma is operative resection which can be associated with significant morbidity. Pharmacotherapy is largely restricted to bevacizumab, which has minimal or no efficacy for many patients and can be associated with treatment-limiting adverse effects. Given the suffering and morbidity associated with schwannoma and the paucity of therapeutic options, there is an urgent need for safe and effective therapies for schwannomas. We previously demonstrated that adeno-associated virus serotype 1 (AAV1) vector mediated delivery of the inflammasome adaptor protein, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) under the control of the P0 promoter, produced a prolonged reduction in tumor volume and tumor-associated pain in human xenograft and mouse syngeneic schwannoma models. Here, we present data essential for the translation of our AAV1-P0-ASC schwannoma gene therapy to clinical trials. We determine the minimum effective dose of AAV1-P0-hASC required to induce an anti-tumor effect in the xenograft human-schwannoma model. We also show that the presence of preexisting AAV1 immunity does not alter the antitumor efficacy of AAV-P0-mASC in a syngeneic mouse schwannoma model. Furthermore, the maximum deliverable intratumoral dose of AAV1-P0-ASC was not associated with neuronal toxicity in immunocompetent mice. Taken together, these safety and efficacy data support the translation of the AAV1-P0-ASC schwannoma gene therapy strategy to clinical trials.

Changes in Pain Medicine Training Programs Associated With COVID-19: Survey Results.

BACKGROUND: The coronavirus disease 2019 (COVID-19) pandemic is a public health crisis of unprecedented proportions that has altered the practice of medicine. The pandemic has required pain clinics to transition from in-person visits to telemedicine, postpone procedures, and cancel face-to-face educational sessions. There are no data on how fellowship programs have adapted. METHODS: A 17-question survey was developed covering topics including changes in education, clinical care, and psychological stress due to the COVID pandemic. The initial survey was hosted by Qualtrics Inc and disseminated by the Association of Pain Program Directors on April 10, 2020, to program directors at Accreditation Council for Graduate Medical Education (ACGME)-accredited fellowships. Results are reported descriptively and stratified by COVID infection rate, which was calculated from Centers for Disease Control and Prevention data on state infections, and census data. RESULTS: Among 107 surveys distributed, 70 (65%) programs responded. Twenty-nine programs were located in states in the upper tertile for per capita infection rates, 17 in the middle third, and 23 in the lowest tertile. Nearly all programs (93%) reported a decreased workload, with 11 (16%) reporting a dramatic decrease (only urgent or emergent cases). Just more than half of programs had either already deployed (14%) or credentialed (39%) fellows to provide nonpain care. Higher state infection rates were significantly associated with reduced clinical demand (Rs = 0.31, 95% confidence interval [CI], 0.08-0.51; P = .011) and redeployment of fellows to nonpain areas (Rs = 0.30, 95% CI, 0.07-0.50; P = .013). Larger program size but not infection rate was associated with increased perceived anxiety level of trainees. CONCLUSIONS: We found a shift to online alternatives for clinical care and education, with correlations between per capita infection rates, and clinical care demands and redeployment, but not with overall trainee anxiety levels. It is likely that medicine in general, and pain medicine in particular, will change after COVID-19, with greater emphasis on telemedicine, virtual education, and greater national and international cooperation. Physicians should be prepared for these changes.

Activation of GABAergic Neurons in the Rostromedial Tegmental Nucleus and Other Brainstem Regions Promotes Sedation and Facilitates Sevoflurane Anesthesia in Mice.

Many general anesthetics potentiate gamma-aminobutyric acid (GABA) A receptors but their neuroanatomic sites of action are less clear. GABAergic neurons in the rostromedial tegmental nucleus (RMTg) send inhibitory projections to multiple arousal-promoting nuclei, but the role of these neurons in modulating consciousness is unknown. In this study, designer receptors exclusively activated by designer drugs (DREADDs) were targeted to RMTg GABAergic neurons of Vgat-ires-Cre mice. DREADDs expression was found in the RMTg and other brainstem regions. Activation of these neurons decreased movement and exploratory behavior, impaired motor coordination, induced electroencephalogram (EEG) oscillations resembling nonrapid eye movement (NREM) sleep without loss of righting and reduced the dose requirement for sevoflurane-induced unconsciousness. These results suggest that GABAergic neurons in the RMTg and other brainstem regions promote sedation and facilitate sevoflurane-induced unconsciousness.

Headache plus: trigeminal and autonomic features in a case of cervicogenic headache responsive to third occipital nerve radiofrequency ablation.

OBJECTIVE: To describe a case of cervicogenic headache with associated autonomic features and pain in a trigeminal distribution, all of which responded to third occipital nerve radiofrequency ablation. DESIGN: Single case report. SETTING: Massachusetts General Hospital Center for Pain Medicine. PATIENTS: A 38-year-old woman with history of migraines and motor vehicle accident. INTERVENTIONS: Right third occipital nerve diagnostic blocks and radiofrequency lesioning. OUTCOME MEASURES: Pain reduction; physical findings, including periorbital and mandibular facial swelling, tearing, conjunctival injection, and allodynia; and use of opioid and non-opioid pain medicines. RESULTS: The patient had complete relief of her pain and autonomic symptoms, and was able to stop all pain medications following a dedicated third occipital nerve lesioning. CONCLUSIONS: This case illustrates the diagnostic and therapeutic complexity of cervicogenic headache and the overlap with other headache types, including trigeminal autonomic cephalgias and migraine. It represents a unique proof of principle in that not only trigeminal nerve pain but also presumed neurogenic inflammation can be relieved by blockade of cervical nociceptive inputs. Further investigation into shared mechanisms of headache pathogenesis is warranted.

Genetically engineered microvesicles carrying suicide mRNA/protein inhibit schwannoma tumor growth.

Microvesicles (MVs) play an important role in intercellular communication by carrying mRNAs, microRNAs (miRNAs), non-coding RNAs, proteins, and DNA from cell to cell. To our knowledge, this is the first report of delivery of a therapeutic mRNA/protein via MVs for treatment of cancer. We first generated genetically engineered MVs by expressing high levels of the suicide gene mRNA and protein-cytosine deaminase (CD) fused to uracil phosphoribosyltransferase (UPRT) in MV donor cells. MVs were isolated from these cells and used to treat pre-established nerve sheath tumors (schwannomas) in an orthotopic mouse model. We demonstrated that MV-mediated delivery of CD-UPRT mRNA/protein by direct injection into schwannomas led to regression of these tumors upon systemic treatment with the prodrug (5-fluorocytosine (5-FC)), which is converted within tumor cells to 5-fluorouracil (5-FU)-an anticancer agent. Taken together, these studies suggest that MVs can serve as novel cell-derived "liposomes" to effectively deliver therapeutic mRNA/proteins to treatment of diseases.

Curriculum and cases for pain medicine crisis resource management education.

Medical crises that may occur in the setting of a pain medicine service are rare events that require skillful action and teamwork to ensure safe patient outcome. A simulated environment is an ideal venue for both acquisition and reinforcement of this knowledge and skill set. Here, we present an educational curriculum in pain medicine crisis resource management for both pain medicine fellows and attending physicians as well as the results of a successful pilot program.

Effect of Antibiotic Treatment on Attenuated Salmonella typhimurium VNP20009 Mediated Schwannoma Growth Control.

BACKGROUND/AIM: This study evaluated the effect of enrofloxacin antibiotic treatment on the ability of an attenuated Salmonella typhimurium (S. typhimurium) strain VNP20009 to control schwannoma growth in a preclinical mouse schwannoma tumor model. MATERIALS AND METHODS: The antitumor efficacy of VNP20009 intratumoral (i.t.) injection was assessed in a syngeneic mouse-NF2 schwannoma model, with and without subcutaneous (s.c.) injection of enrofloxacin beginning on day-1 or day-8 post-VNP20009 injection. S. typhimurium colonization was assessed in excised tumor samples following antibiotic treatment. RESULTS: I.t. injection of the VNP20009 significantly decreased the growth of schwannoma tumors in mice compared to PBS-treated controls. Treatment of mice with enrofloxacin on day-1 post-VNP20009 injection resulted in abrogation of VNP20009-mediated tumor growth control. In contrast, tumor growth in i.t. VNP20009-injected mice infused with enrofloxacin beginning on day 8 was significantly decreased compared to i.t. PBS-injected controls. Enrofloxacin significantly reduced the number of viable VNP20009 bacteria in excised tumor samples within one day of antibiotic infusion. Viable bacteria were either few or essentially eliminated at the end of the experiment in antibiotic-treated animals compared to VNP20009-only. CONCLUSION: Viable VNP20009 can persist for as long as 2.5 weeks following intratumoral injection of schwannoma, during which time tumor growth is retarded. Antibiotic treatment starting 1-day following i.t. VNP20009 abrogated bacterial tumor growth control, whereas initiation of antibiotics 8-days following i.t. VNP20009 was associated with control of tumor growth, albeit less than seen in animals unexposed to antibiotics.

Culture of attenuated Salmonella Typhimurium VNP20009 in animal-product-free media does not alter schwannoma growth control.

Schwannomas are slow-growing benign peripheral nerve sheath tumors derived from Schwann-lineage cells that develop in association with NF2-related schwannomatosis (NF2) and schwannomatosis (NF3), as well as spontaneously. Individuals affected with NF2 and NF3 have multiple schwannomas with tumors arising throughout life. Surgical resection, the standard management, is limited in scope and efficacy and is itself associated with significant morbidity. We have previously shown that direct intratumoral injection of attenuated Salmonella Typhimurium (S. Typhimurium), strain VNP20009, showed a potent anti-tumor effect in preclinical NF-2 schwannoma models. The United States Federal Drug Administration (FDA) requires that bacterial products utilized in clinical trials be produced without exposure to animal-derived-products. In this context, we developed, characterized, and tested the antitumor efficacy of an attenuated S. Typhimurium serially passaged in animal-product-free media, naming it VNP20009-AF for "VNP20009-animal-product-free." Our in vitro data did not indicate any significant changes in the viability, motility, or morphology of VNP20009-AF, compared to its parental strain. In vivo efficacy data demonstrated that VNP20009-AF and VNP20009 controlled tumor growth to the same degree in both human NF2-schwannoma xenograft and murine-NF2 schwannoma allograft models. Together, these data support the use of VNP20009-AF for the translation of bacterial schwannoma therapy into clinical trials.

Comparative Transcriptomic Analysis of Archival Human Vestibular Schwannoma Tissue from Patients with and without Tinnitus.

Vestibular schwannoma (VS) is an intracranial tumor that commonly presents with tinnitus and hearing loss. To uncover the molecular mechanisms underlying VS-associated tinnitus, we applied next-generation sequencing (Illumina HiSeq) to formalin-fixed paraffin-embedded archival VS samples from nine patients with tinnitus (VS-Tin) and seven patients without tinnitus (VS-NoTin). Bioinformatic analysis was used to detect differentially expressed genes (DEG; i.e., ≥two-fold change [FC]) while correcting for multiple comparisons. Using RNA-seq analysis, VS-Tin had significantly lower expression of GFAP (logFC = -3.04), APLNR (logFC = -2.95), PREX2 (logFC = -1.44), and PLVAP (logFC = -1.04; all p < 0.01) vs. VS-NoTin. These trends were validated by using real-time RT-qPCR. At the protein level, immunohistochemistry revealed a trend for less PREX2 and apelin expression and greater expression of NLRP3 inflammasome and CD68-positive macrophages in VS-Tin than in VS-NoTin, suggesting the activation of inflammatory processes in VS-Tin. Functional enrichment analysis revealed that the top three protein categories-glycoproteins, signal peptides, and secreted proteins-were significantly enriched in VS-Tin in comparison with VS-NoTin. In a gene set enrichment analysis, the top pathway was allograft rejection, an inflammatory pathway that includes the MMP9, CXCL9, IL16, PF4, ITK, and ACVR2A genes. Future studies are needed to examine the importance of these candidates and of inflammation in VS-associated tinnitus.

The BMP coreceptor RGMb promotes while the endogenous BMP antagonist noggin reduces neurite outgrowth and peripheral nerve regeneration by modulating BMP signaling.

Repulsive guidance molecule b (RGMb) is a bone morphogenetic protein (BMP) coreceptor and sensitizer of BMP signaling, highly expressed in adult dorsal root ganglion (DRG) sensory neurons. We used a murine RGMb knock-out to gain insight into the physiological role of RGMb in the DRG, and address whether RGMb-mediated modulation of BMP signaling influences sensory axon regeneration. No evidence for altered development of the PNS and CNS was detected in RGMb(-/-) mice. However, both cultured neonatal whole DRG explants and dissociated DRG neurons from RGMb(-/-) mice exhibited significantly fewer and shorter neurites than those from wild-type littermates, a phenomenon that could be fully rescued by BMP-2. Moreover, Noggin, an endogenous BMP signaling antagonist, inhibited neurite outgrowth in wild-type DRG explants from naive as well as nerve injury-preconditioned mice. Noggin is downregulated in the DRG after nerve injury, and its expression is highly correlated and inversely associated with the known regeneration-associated genes, which are induced in the DRG by peripheral axonal injury. We show that diminished BMP signaling in vivo, achieved either through RGMb deletion or BMP inhibition with Noggin, retarded early axonal regeneration after sciatic nerve crush injury. Our data suggest a positive modulatory contribution of RGMb and BMP signaling to neurite extension in vitro and early axonal regrowth after nerve injury in vivo and a negative effect of Noggin.

Oral Administration of Glutathione Trisulfide Increases Reactive Sulfur Levels in Dorsal Root Ganglion and Ameliorates Paclitaxel-Induced Peripheral Neuropathy in Mice.

Peripheral neuropathy is a dose-limiting side effect of chemotherapy with paclitaxel. Paclitaxel-induced peripheral neuropathy (PIPN) is typically characterized by a predominantly sensory neuropathy presenting with allodynia, hyperalgesia and spontaneous pain. Oxidative mitochondrial damage in peripheral sensory neurons is implicated in the pathogenesis of PIPN. Reactive sulfur species, including persulfides (RSSH) and polysulfides (RSnH), are strong nucleophilic and electrophilic compounds that exert antioxidant effects and protect mitochondria. Here, we examined the potential neuroprotective effects of glutathione trisulfide (GSSSG) in a mouse model of PIPN. Intraperitoneal administration of paclitaxel at 4 mg/kg/day for 4 days induced mechanical allodynia and thermal hyperalgesia in mice. Oral administration of GSSSG at 50 mg/kg/day for 28 days ameliorated mechanical allodynia, but not thermal hyperalgesia. Two hours after oral administration, 34S-labeled GSSSG was detected in lumber dorsal root ganglia (DRG) and in the lumber spinal cord. In mice treated with paclitaxel, GSSSG upregulated expression of genes encoding antioxidant proteins in lumber DRG, prevented loss of unmyelinated axons and inhibited degeneration of mitochondria in the sciatic nerve. In cultured primary neurons from cortex and DRG, GSSSG mitigated paclitaxel-induced superoxide production, loss of axonal mitochondria, and axonal degeneration. These results indicate that oral administration of GSSSG mitigates PIPN by preventing axonal degeneration and mitochondria damage in peripheral sensory nerves. The findings suggest that administration of GSSSG may be an approach to the treatment or prevention of PIPN and other peripheral neuropathies.

TRPA1 contributes to cold hypersensitivity.

TRPA1 is a nonselective cation channel expressed by nociceptors. Although it is widely accepted that TRPA1 serves as a broad irritancy receptor for a variety of reactive chemicals, its role in cold sensation remains controversial. Here, we demonstrate that mild cooling markedly increases agonist-evoked rat TRPA1 currents. In the absence of an agonist, even noxious cold only increases current amplitude slightly. These results suggest that TRPA1 is a key mediator of cold hypersensitivity in pathological conditions in which reactive oxygen species and proinflammatory activators of the channel are present, but likely plays a comparatively minor role in acute cold sensation. Supporting this, cold hypersensitivity can be induced in wild-type but not Trpa1(-/-) mice by subcutaneous administration of a TRPA1 agonist. Furthermore, the selective TRPA1 antagonist HC-030031 [2-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydro-7H-purin-7-yl)-N-(4-isopropylphenyl)acetamide] reduces cold hypersensitivity in rodent models of inflammatory and neuropathic pain.

Dragon enhances BMP signaling and increases transepithelial resistance in kidney epithelial cells.

The neuronal adhesion protein Dragon acts as a bone morphogenetic protein (BMP) coreceptor that enhances BMP signaling. Given the importance of BMP signaling in nephrogenesis and its putative role in the response to injury in the adult kidney, we studied the localization and function of Dragon in the kidney. We observed that Dragon localized predominantly to the apical surfaces of tubular epithelial cells in the thick ascending limbs, distal convoluted tubules, and collecting ducts of mice. Dragon expression was weak in the proximal tubules and glomeruli. In mouse inner medullary collecting duct (mIMCD3) cells, Dragon generated BMP signals in a ligand-dependent manner, and BMP4 is the predominant endogenous ligand for the Dragon coreceptor. In mIMCD3 cells, BMP4 normally signaled through BMPRII, but Dragon enhanced its signaling through the BMP type II receptor ActRIIA. Dragon and BMP4 increased transepithelial resistance (TER) through the Smad1/5/8 pathway. In epithelial cells isolated from the proximal tubule and intercalated cells of collecting ducts, we observed coexpression of ActRIIA, Dragon, and BMP4 but not BMPRII. Taken together, these results suggest that Dragon may enhance BMP signaling in renal tubular epithelial cells and maintain normal renal physiology.

Cannabinoids mediate analgesia largely via peripheral type 1 cannabinoid receptors in nociceptors.

Although endocannabinoids constitute one of the first lines of defense against pain, the anatomical locus and the precise receptor mechanisms underlying cannabinergic modulation of pain are uncertain. Clinical exploitation of the system is severely hindered by the cognitive deficits, memory impairment, motor disturbances and psychotropic effects resulting from the central actions of cannabinoids. We deleted the type 1 cannabinoid receptor (CB1) specifically in nociceptive neurons localized in the peripheral nervous system of mice, preserving its expression in the CNS, and analyzed these genetically modified mice in preclinical models of inflammatory and neuropathic pain. The nociceptor-specific loss of CB1 substantially reduced the analgesia produced by local and systemic, but not intrathecal, delivery of cannabinoids. We conclude that the contribution of CB1-type receptors expressed on the peripheral terminals of nociceptors to cannabinoid-induced analgesia is paramount, which should enable the development of peripherally acting CB1 analgesic agonists without any central side effects.

Transcriptomic signature of painful human neurofibromatosis type 2 schwannomas.

Schwannomas are benign neoplasms that can cause gain- and loss-of-function neurological phenotypes, including severe, intractable pain. To investigate the molecular mechanisms underlying schwannoma-associated pain we compared the RNA sequencing profile of painful and non-painful schwannomas from NF2 patients. Distinct segregation of painful and non-painful tumors by gene expression patterns was observed. Differential expression analysis showed the upregulation of fibroblast growth factor 7 (FGF7) in painful schwannomas. Behavioral support for this finding was observed using a xenograft human NF2-schwannoma model in nude mice. In this model, over-expression of FGF7 in intra-sciatically implanted NF2 tumor cells generated pain behavior compared with controls.

T-cell infiltration and signaling in the adult dorsal spinal cord is a major contributor to neuropathic pain-like hypersensitivity.

Partial peripheral nerve injury in adult rats results in neuropathic pain-like hypersensitivity, while that in neonatal rats does not, a phenomenon also observed in humans. We therefore compared gene expression profiles in the dorsal horn of adult and neonatal rats in response to the spared nerve injury (SNI) model of peripheral neuropathic pain. The 148 differentially regulated genes in adult, but not young, rat spinal cords indicate a greater microglial and T-cell response in adult than in young animals. T-cells show a large infiltration in the adult dorsal horn but not in the neonate after SNI. T-cell-deficient Rag1-null adult mice develop less neuropathic mechanical allodynia than controls, and central expression of cytokines involved in T-cell signaling exhibits large relative differences between young and adult animals after SNI. One such cytokine, interferon-gamma (IFNgamma), is upregulated in the dorsal horn after nerve injury in the adult but not neonate, and we show that IFNgamma signaling is required for full expression of adult neuropathic hypersensitivity. These data reveal that T-cell infiltration and activation in the dorsal horn of the spinal cord following peripheral nerve injury contribute to the evolution of neuropathic pain-like hypersensitivity. The neuroimmune interaction following peripheral nerve injury has therefore a substantial adaptive immune component, which is absent or suppressed in the young CNS.

Correction to: Schwannoma gene therapy by adeno-associated virus delivery of the pore-forming protein Gasdermin-D.

The original version of this Article contained an error in the spelling of the author Ahmed Abdelanabi, which was incorrectly given as Abdelanabi Ahmed. This has now been corrected in both the PDF and HTML versions of the Article.