Nasal airway inflammatory responses and pathogen detection in infants with cystic fibrosis.

BACKGROUND: Detecting airway inflammation non-invasively in infants with cystic fibrosis (CF) is difficult. We hypothesized that markers of inflammation in CF [IL-1ß, IL-6, IL-8, IL-10, IL-17A, neutrophil elastase (NE) and tumor necrosis factor (TNF-α)] could be measured in infants with CF from nasal fluid and would be elevated during viral infections or clinician-defined pulmonary exacerbations (PEx). METHODS: We collected nasal fluid, nasal swabs, and hair samples from 34 infants with CF during monthly clinic visits, sick visits, and hospitalizations. Nasal fluid was isolated and analyzed for cytokines. Respiratory viral detection on nasal swabs was performed using the Luminex NxTAG® Respiratory Pathogen Panel. Hair samples were analyzed for nicotine concentration by reverse-phase high-performance liquid chromatography. We compared nasal cytokine concentrations between the presence and absence of detected respiratory viruses, PEx, and smoke exposure. RESULTS: A total of 246 samples were analyzed. Compared to measurements in the absence of respiratory viruses, mean concentrations of IL-6, IL-8, TNF-α, and NE were significantly increased while IL-17A was significantly decreased in infants positive for respiratory viruses. IL-17A was significantly decreased and NE increased in those with a PEx. IL-8 and NE were significantly increased in infants with enteric pathogen positivity on airway cultures, but not P. aeruginosa or S. aureus. Compared to those with no smoke exposure, there were significantly higher levels of IL-6, IL-10, and NE in infants with detectable levels of nicotine. CONCLUSIONS: Noninvasive collection of nasal fluid may identify inflammation in infants with CF during changing clinical or environmental exposures.

Evaluating FEV1 decline in diagnosis and management of pulmonary exacerbations in children with cystic fibrosis.

RATIONALE: Forced expiratory volume in 1 s (FEV1) decline (ΔFEV1) is associated with pulmonary exacerbation (PEx) diagnosis in cystic fibrosis (CF). Spirometry may not be available during telehealth visits and could impair clinician ability to diagnose PEx. This study aims to (1) identify the associations between degrees of ΔFEV1 (decrease of <5% predicted vs. 5%-9% predicted vs. ≥10% predicted from baseline), clinical symptoms, and clinician-diagnosed PEx and (2) evaluate the correlation between respiratory symptoms, ΔFEV1, and antibiotic treatment. METHODS: Retrospective, descriptive study of PEx diagnosis and management in 628 outpatient clinical encounters with spirometry in 178 patients with CF ages 6-17 years at Riley Hospital for Children during 2019. Odds ratios (OR) of symptoms associated with clinician-defined PEx diagnosis and antibiotic management stratified by ΔFEV1 decline were determined. RESULTS: Clinician-diagnosed PEx occurred at 199 (31.7%) visits; increased cough (77.4%) and sputum/wet cough (57.8%) were the most frequently reported symptoms. Compared to no ΔFEV1, the odds of a clinician-diagnosed PEx were increased when ΔFEV15%-9% and ΔFEV1≥10% was present with increased cough (OR 1.56, 95% confidence interval [CI] 1.25-1.94 and OR 1.82, 95% CI 1.52-2.19, respectively), increased sputum (OR 1.59, 95% CI 1.20-2.12 and OR 1.78, 95% CI 1.37-2.32, respectively), and increased cough and sputum together (OR 1.51, 95% CI 1.08-2.13 and OR 1.68, 95% CI 1.22-2.31, respectively). CONCLUSIONS: ΔFEV1 is associated with increased likelihood that cough and sputum are diagnosed as a PEx. Spirometry is essential for PEx diagnosis and treatment and is a necessary component of all clinical encounters.

Reduction of pulmonary exacerbations in young children with cystic fibrosis during the COVID-19 pandemic.

To assess the impact of COVID-19 restrictions on cystic fibrosis (CF) pulmonary exacerbations (PEx) we performed a retrospective review of PEx events at our CF Center and compared the rate of PEx in 2019 versus 2020. Restrictions on social interaction due to the COVID-19 pandemic were associated with a lower number of PEx events at our pediatric CF Center, suggesting that these restrictions also reduced exposure to other respiratory viral infection in children with CF.

Isolated penile ulceration after Nivolumab therapy for non-small cell lung carcinoma.

Immune-related cutaneous events can develop after immunotherapy. To our knowledge, we present the first reported case of isolated focal penile inflammatory ulceration in a patient being treated with Nivolumab for stage IV non-small cell lung carcinoma. He presented with a painless penile ulceration two months after initiating therapy. He had near-complete resolution of his ulcer after two weeks of topical treatment. Histologic evaluation revealed a drug eruption. While cutaneous events are a well-known side effect of immunotherapy, this is the first report of an isolated penile lesion from such therapy.

Clinical and genetic features of cervical dystonia in a large multicenter cohort.

OBJECTIVE: To characterize the clinical and genetic features of cervical dystonia (CD). METHODS: Participants enrolled in the Dystonia Coalition biorepository (NCT01373424) with initial manifestation as CD were included in this study (n = 1,000). Data intake included demographics, family history, and the Global Dystonia Rating Scale. Participants were screened for sequence variants (SVs) in GNAL, THAP1, and Exon 5 of TOR1A. RESULTS: The majority of participants were Caucasian (95%) and female (75%). The mean age at onset and disease duration were 45.5 ± 13.6 and 14.6 ± 11.8 years, respectively. At the time of assessment, 68.5% had involvement limited to the neck, shoulder(s), and proximal arm(s), whereas 47.4% had dystonia limited to the neck. The remaining 31.5% of the individuals exhibited more extensive anatomical spread. A head tremor was noted in 62% of the patients. Head tremor and laryngeal dystonia were more common in females. Psychiatric comorbidities, mainly depression and anxiety, were reported by 32% of the participants and were more common in females. Family histories of dystonia, parkinsonian disorder, and tremor were present in 14%, 11%, and 29% of the patients, respectively. Pathogenic or likely pathogenic SVs in THAP1, TOR1A, and GNAL were identified in 8 participants (0.8%). Two individuals harbored novel missense SVs in Exon 5 of TOR1A. Synonymous and noncoding SVs in THAP1 and GNAL were identified in 4% of the cohort. CONCLUSIONS: Head tremor, laryngeal dystonia, and psychiatric comorbidities are more common in female participants with CD. Coding and noncoding variants in GNAL, THAP1, and TOR1A make small contributions to the pathogenesis of CD.

Blepharospasm in a multiplex African-American pedigree.

BACKGROUND: Isolated blepharospasm (BSP) is a late-onset focal dystonia characterized by involuntary contractions of the orbicularis oculi muscles. Genetic studies of BSP have been limited by the paucity of large multiplex pedigrees. Although sequence variants (SVs) in THAP1 have been reported in rare cases of BSP, the genetic causes of this focal dystonia remain largely unknown. Moreover, in the absence of family history and strong in silico or in vitro evidence of deleteriousness, the pathogenicity of novel SVs in THAP1 and other dystonia-associated genes can be indeterminate. METHODS: A large African-American pedigree with BSP was phenotypically characterized and screened for mutations in THAP1, TOR1A and GNAL with Sanger sequencing. Whole-exome sequencing of the proband was used to examine other dystonia-associated genes for potentially pathogenic SVs. In silico and co-segregation analyses were performed for a novel THAP1 SV identified in the proband. RESULTS: Seven family members exhibited increased blinking and/or stereotyped bilateral and synchronous orbicularis oculi spasms with age of onset ranging from early childhood to late adult life (7 to 54 years). The proband was found to harbor a novel THAP1 SV (c.314T>C, p.L105S). However, the p.L105S SV did not co-segregate with blepharospasm in the pedigree. Moreover, in silico analyses suggest that p.L105S is benign. No pathogenic or likely pathogenic SVs in other dystonia-associated genes were identified with whole-exome sequencing. CONCLUSIONS: Blepharospasm can be familial and may be hereditary in African-Americans. A comprehensive array of in silico tools, and, if possible, co-segregation analysis should be used to classify SVs in dystonia-associated genes.

Evidence for a novel functional role of astrocytes in the acute homeostatic response to high-fat diet intake in mice.

OBJECTIVE: Introduction of a high-fat diet to mice results in a period of voracious feeding, known as hyperphagia, before homeostatic mechanisms prevail to restore energy intake to an isocaloric level. Acute high-fat diet hyperphagia induces astrocyte activation in the rodent hypothalamus, suggesting a potential role of these cells in the homeostatic response to the diet. The objective of this study was to determine physiologic role of astrocytes in the acute homeostatic response to high-fat feeding. METHODS: We bred a transgenic mouse model with doxycycline-inducible inhibition of NFkappaB (NFκB) signaling in astrocytes to determine the effect of loss of NFκB-mediated astrocyte activation on acute high-fat hyperphagia. ELISA was used to measure the levels of markers of astrocyte activation, glial-fibrillary acidic protein (GFAP) and S100B, in the medial basal hypothalamus. RESULTS: Inhibition of NFκB signaling in astrocytes prevented acute high-fat diet-induced astrocyte activation and resulted in a 15% increase in caloric intake (P < 0.01) in the first 24 h after introduction of the diet. CONCLUSIONS: These data reveal a novel homeostatic role for astrocytes in the acute physiologic regulation of food intake in response to high-fat feeding.

Obesity induced by a high-fat diet is associated with increased immune cell entry into the central nervous system.

Obesity is associated with chronic low-grade inflammation in peripheral tissues caused, in part, by the recruitment of inflammatory monocytes into adipose tissue. Studies in rodent models have also shown increased inflammation in the central nervous system (CNS) during obesity. The goal of this study was to determine whether obesity is associated with recruitment of peripheral immune cells into the CNS. To do this we used a bone marrow chimerism model to track the entry of green-fluorescent protein (GFP) labeled peripheral immune cells into the CNS. Flow cytometry was used to quantify the number of GFP(+) immune cells recruited into the CNS of mice fed a high-fat diet compared to standard chow fed controls. High-fat feeding resulted in obesity associated with a 30% increase in the number of GFP(+) cells in the CNS compared to control mice. Greater than 80% of the GFP(+) cells recruited to the CNS were also CD45(+) CD11b(+) indicating that the GFP(+) cells displayed characteristics of microglia/macrophages. Immunohistochemistry further confirmed the increase in GFP(+) cells in the CNS of the high-fat fed group and also indicated that 93% of the recruited cells were found in the parenchyma and had a stellate morphology. These findings indicate that peripheral immune cells can be recruited to the CNS in obesity and may contribute to the inflammatory response.

Translocator protein 18 kDa (TSPO) is regulated in white and brown adipose tissue by obesity.

Translocator protein 18 kDa (TSPO) is an outer-mitochondrial membrane transporter which has many functions including participation in the mitochondrial permeability transition pore, regulation of reactive oxygen species (ROS), production of cellular energy, and is the rate-limiting step in the uptake of cholesterol. TSPO expression is dysregulated during disease pathologies involving changes in tissue energy demands such as cancer, and is up-regulated in activated macrophages during the inflammatory response. Obesity is associated with decreased energy expenditure, mitochondrial dysfunction, and chronic low-grade inflammation which collectively contribute to the development of the Metabolic Syndrome. Therefore, we hypothesized that dysregulation of TSPO in adipose tissue may be a feature of disease pathology in obesity. Radioligand binding studies revealed a significant reduction in TSPO ligand binding sites in mitochondrial extracts from both white (WAT) and brown adipose tissue (BAT) in mouse models of obesity (diet-induced and genetic) compared to control animals. We also confirmed a reduction in TSPO gene expression in whole tissue extracts from WAT and BAT. Immunohistochemistry in WAT confirmed TSPO expression in adipocytes but also revealed high-levels of TSPO expression in WAT macrophages in obese animals. No changes in TSPO expression were observed in WAT or BAT after a 17 hour fast or 4 hour cold exposure. Treatment of mice with the TSPO ligand PK11195 resulted in regulation of metabolic genes in WAT. Together, these results suggest a potential role for TSPO in mediating adipose tissue homeostasis.

Regional astrogliosis in the mouse hypothalamus in response to obesity.

Obesity is associated with chronic low-grade inflammation in peripheral tissues, which contributes to the development of comorbidities such as insulin resistance and cardiovascular disease. While less extensively characterized, obesity also promotes inflammation in the central nervous system (CNS) and the consequences of this inflammation for CNS function are only beginning to be examined. In response to CNS insults such as inflammation, astrocytes undergo a process of hypertrophy and hyperplasia known as reactive astrogliosis. We used immunohistochemistry to examine the differential distribution of the astrocyte marker glial-fibrillary acidic protein (GFAP) in the brains of diet-induced or genetically obese mice compared with their respective lean controls to determine whether different nuclei of the hypothalamus showed comparable astrogliosis in response to obesity. The areas that showed the highest differential GFAP immunoreactivity between lean and obese animals include the medial preoptic, paraventricular, and dorsomedial nuclei. Comparatively, little astrogliosis was seen in the ventromedial nucleus, lateral hypothalamus, or anterior hypothalamic area. In obese animals high levels of GFAP immunoreactivity were often associated with the microvasculature. There were no differences in the differential distribution of GFAP staining between obese animals and their lean controls in the diet-induced compared with the genetic model of obesity. The exact cause(s) of the astrogliosis in obesity is not known. The finding that obesity causes a distinct pattern of elevated GFAP immunoreactivity associated with microvessels suggests that the astrogliosis may be occurring as a response to changes at the blood-brain barrier and/or in the peripheral circulation.

D-Serine Production, Degradation, and Transport in ALS: Critical Role of Methodology.

In mammalian systems, D-serine is perhaps the most biologically active D-amino acid described to date. D-serine is a coagonist at the NMDA-receptor, and receptor activation is dependent on D-serine binding. Because D-serine binding dramatically increases receptor affinity for glutamate, it can produce excitotoxicity without any change in glutamate per se. D-serine is twofold higher in the spinal cords of mSOD1 (G93A) ALS mice, and the deletion of serine racemase (SR), the enzyme that produces D-serine, results in an earlier onset of symptoms, but with a much slower rate of disease progression. Localization studies within the brain suggest that mSOD1 and subsequent glial activation could contribute to the alterations in SR and D-serine seen in ALS. By also degrading both D-serine and L-serine, SR appears to be a prime bidirectional regulator of free serine levels in vivo. Therefore, accurate and reproducible measurements of D-serine are critical to understanding its regulation by SR. Several methods for measuring D-serine have been employed, and significant issues related to validation and standardization remain unresolved. Further insights into the intracellular transport and tissue-specific compartmentalization of D-serine within the CNS will aid in the understanding of the role of D-serine in the pathogenesis of ALS.

Paradoxical roles of serine racemase and D-serine in the G93A mSOD1 mouse model of amyotrophic lateral sclerosis.

D-serine is an endogenous neurotransmitter that binds to the NMDA receptor, thereby increasing the affinity for glutamate, and the potential for excitotoxicity. The primary source of D-serine in vivo is enzymatic racemization by serine racemase (SR). Regulation of D-serine in vivo is poorly understood, but is thought to involve a combination of controlled production, synaptic reuptake by transporters, and intracellular degradation by D-amino acid oxidase (DAO). However, SR itself possesses a well-characterized eliminase activity, which effectively degrades D-serine as well. D-serine is increased two-fold in spinal cords of G93A Cu,Zn-superoxide dismutase (SOD1) mice--the standard model of amyotrophic lateral sclerosis (ALS). ALS mice with SR disruption show earlier symptom onset, but survive longer (progression phase is slowed), in an SR-dependent manner. Paradoxically, administration of D-serine to ALS mice dramatically lowers cord levels of D-serine, leading to changes in the onset and survival very similar to SR deletion. D-serine treatment also increases cord levels of the alanine-serine-cysteine transporter 1 (Asc-1). Although the mechanism by which SOD1 mutations increases D-serine is not known, these results strongly suggest that SR and D-serine are fundamentally involved in both the pre-symptomatic and progression phases of disease, and offer a direct link between mutant SOD1 and a glial-derived toxic mediator.