Advances in Hand Therapy: Best Practice in Conservative Management of Proximal Phalangeal Fractures in Children.

BACKGROUND: Proximal phalangeal fractures are one of the most commonly treated hand injuries in children. Conservative management of these fractures is often to splint for 5 weeks post injury, despite children presenting as clinically healed at 3 weeks post injury. Therefore, we investigated the effect of splinting for only 3 weeks in children who present with clinically healed proximal phalangeal fractures at 3 weeks compared with usual care. METHODS: Participants (n=80, aged 10.3 ±2.5 years) presenting to the Hand Clinic of a tertiary Children's Hospital in Sydney, Australia, were randomly allocated into a Current Protocol and a New Protocol group. Following were the inclusion criteria: aged between 5 and 16 years; present with an non-displaced or minimally displaced and stable fracture; no surgical intervention; assessed as clinically healed at 3-week visit. The primary outcome measure was total active motion (TAM) of the injured digit compared with the contralateral digit (deg), at 5 weeks post injury. Secondary outcome measures were grip strength, and a parent-reported questionnaire. Statistical analysis used χ 2 test and the absolute difference described by a 90% CI. The New Protocol was considered noninferior if the 90% CI overlap was > 20% of the Current Protocol. Analysis was by intention to treat. RESULTS: There was a 10% loss to follow up at 5 weeks (Current Protocol =4, New Protocol =4). All CIs between groups overlapped by >10%. TAM 90% CI for Current Protocol was 17.7 to 5.4 degrees and for the New Protocol was 4.7 to 1.6 degrees. CONCLUSIONS: A change in practice is warranted to cease immobilization for children with conservatively managed proximal phalangeal fractures who present as clinically healed at 3 weeks. Therapist assessment of fracture healing is an appropriate indicator for intervention and can be utilized in a therapist-led model of care. LEVEL OF EVIDENCE: Level 1-noninferiority randomized control trial with 2 parallel arms.

Clinically Complex LRBA Deficiency Due to a Founder Allele in the Georgian Jewish Population.

Pathogenic variants in LRBA, encoding the LPS Responsive Beige-Like Anchor (LRBA) protein, are responsible for recessive, early-onset hypogammaglobulinemia, severe multi-organ autoimmunity, and lymphoproliferation, with increased risk for malignancy. LRBA deficiency has a wide clinical spectrum with variable age of onset and disease severity. Three apparently unrelated patients with LRBA deficiency, of Georgian Jewish descent, were homozygous for LRBA c.6640C > T, p.R2214*, leading to a stop upstream of the LRBA BEACH domain. Despite carrying the same LRBA genotype, the three patients differed in clinical course: the first patient was asymptomatic until age 25 years; the second presented with failure to thrive at age 3 months; and the third presented at age 7 years with immune cytopenias and severe infections. Two of the patients developed malignancies: the first patient was diagnosed with recurrent Hodgkin's disease at age 36 years, and the second patient developed aggressive gastric cancer at age 15 years. Among Georgian Jews, the carrier frequency of the LRBA p.R2214* allele was 1.6% (4 of 236 Georgian Jewish controls). The allele was absent from other populations. Haplotype analysis showed a shared origin of the mutation. These three patients revealed a pathogenic LRBA founder allele in the Georgian Jewish population, support the diverse and complex clinical spectrum of LRBA deficiency, and support the possibility that LRBA deficiency predisposes to malignancy.

Molecular diagnosis of childhood immune dysregulation, polyendocrinopathy, and enteropathy, and implications for clinical management.

BACKGROUND: Most patients with childhood-onset immune dysregulation, polyendocrinopathy, and enteropathy have no genetic diagnosis for their illness. These patients may undergo empirical immunosuppressive treatment with highly variable outcomes. OBJECTIVE: We sought to determine the genetic basis of disease in patients referred with Immune dysregulation, polyendocrinopathy, enteropathy, X-linked-like (IPEX-like) disease, but with no mutation in FOXP3; then to assess consequences of genetic diagnoses for clinical management. METHODS: Genomic DNA was sequenced using a panel of 462 genes implicated in inborn errors of immunity. Candidate mutations were characterized by genomic, transcriptional, and (for some) protein analysis. RESULTS: Of 123 patients with FOXP3-negative IPEX-like disease, 48 (39%) carried damaging germline mutations in 1 of the following 27 genes: AIRE, BACH2, BCL11B, CARD11, CARD14, CTLA4, IRF2BP2, ITCH, JAK1, KMT2D, LRBA, MYO5B, NFKB1, NLRC4, POLA1, POMP, RAG1, SH2D1A, SKIV2L, STAT1, STAT3, TNFAIP3, TNFRSF6/FAS, TNRSF13B/TACI, TOM1, TTC37, and XIAP. Many of these genes had not been previously associated with an IPEX-like diagnosis. For 42 of the 48 patients with genetic diagnoses, knowing the critical gene could have altered therapeutic management, including recommendations for targeted treatments and for or against hematopoietic cell transplantation. CONCLUSIONS: Many childhood disorders now bundled as "IPEX-like" disease are caused by individually rare, severe mutations in immune regulation genes. Most genetic diagnoses of these conditions yield clinically actionable findings. Barriers are lack of testing or lack of repeat testing if older technologies failed to provide a diagnosis.

Oxidative Stress and Lung Fibrosis: Towards an Adverse Outcome Pathway.

Lung fibrosis is a progressive fatal disease in which deregulated wound healing of lung epithelial cells drives progressive fibrotic changes. Persistent lung injury due to oxidative stress and chronic inflammation are central features of lung fibrosis. Chronic cigarette smoking causes oxidative stress and is a major risk factor for lung fibrosis. The objective of this manuscript is to develop an adverse outcome pathway (AOP) that serves as a framework for investigation of the mechanisms of lung fibrosis due to lung injury caused by inhaled toxicants, including cigarette smoke. Based on the weight of evidence, oxidative stress is proposed as a molecular initiating event (MIE) which leads to increased secretion of proinflammatory and profibrotic mediators (key event 1 (KE1)). At the cellular level, these proinflammatory signals induce the recruitment of inflammatory cells (KE2), which in turn, increase fibroblast proliferation and myofibroblast differentiation (KE3). At the tissue level, an increase in extracellular matrix deposition (KE4) subsequently culminates in lung fibrosis, the adverse outcome. We have also defined a new KE relationship between the MIE and KE3. This AOP provides a mechanistic platform to understand and evaluate how persistent oxidative stress from lung injury may develop into lung fibrosis.

Human Abuse Liability Assessment of Tobacco and Nicotine Products: Approaches for Meeting Current Regulatory Recommendations.

Many regulatory bodies now recommend that tobacco product manufacturers provide information regarding new tobacco products' abuse liability to inform regulatory authorization of currently marketed tobacco products or new product applications (including premarket tobacco product applications in the United States). In addition, the US Food and Drug Administration (FDA) recommends including this information as part of modified risk tobacco product applications. Regulators, including FDA, and many public health officials and researchers consider abuse liability assessment a model which predicts the likelihood that the use of the tobacco product would result in addiction and be used repeatedly or even sporadically resulting in undesirable effects. Abuse liability of a new, potentially reduced harm product can also inform its ability to substitute completely for more harmful tobacco products. While many methods exist, no standard tobacco product abuse liability assessment has been established. The purpose of this review is to provide background information and practical recommendations for human abuse liability testing methods to meet tobacco regulatory needs. A combination of nicotine test product pharmacokinetic, subjective effect and/or behavioral response, and physiological response data relative to comparator products with known abuse liability satisfies some regulatory requirements. Implications: This review provides a practical inspection of the current, international regulatory recommendations for abuse liability assessment of tobacco and regulatory review of such information within the United States and also recommends study designs and methods for abuse liability testing of tobacco products based on scientific and regulatory knowledge. Given that tobacco product abuse liability testing is of increasing interest to regulatory bodies globally, especially with the emergence of novel tobacco products, this timely work provides background and functional recommendations for tobacco product abuse liability testing.

Impact of pharmacologic inhibition of tooth movement on periodontal and tooth root tissues during orthodontic force application.

OBJECTIVE: The goal of this study was to investigate potential negative sequelae of orthodontic force application ±delivery of an osteoclast inhibitor, recombinant osteoprotegerin protein (OPG-Fc), on periodontal tissues. SETTING AND SAMPLE POPULATION: Sprague Dawley rats from a commercial supplier were investigated in a laboratory setting. MATERIALS AND METHODS: Rats were randomly divided into four groups (n = 7 each): one group with no orthodontic appliances and injected once prior to the experimental period with empty polymer microspheres, one group with orthodontic appliances and injected once with empty microspheres, one group with orthodontic appliances and injected once with polymer microspheres containing 1 mg/kg of OPG-Fc, and one group with orthodontic appliances and injected with non-encapsulated 5 mg/kg of OPG-Fc every 3 days during the experimental period. The animals were euthanized after 28 days of tooth movement for histomorphometric analyses. RESULTS: Root resorption, PDL area and widths were similar in animals without appliances and animals with appliances plus high-dose OPG-Fc. PDL blood vessels were compressed and decreased in number in all animals that received orthodontic appliances, regardless of OPG-Fc. Hyalinization was significantly increased only in animals with orthodontic appliances plus multiple injections of 5 mg/kg non-encapsulated OPG-Fc when compared to animals without appliances. CONCLUSIONS: Results of this study indicate that while pharmacological modulation of tooth movement through osteoclast inhibition is feasible when delivered in a locally controlled low-dose manner, high-dose levels that completely prevent tooth movement through bone may decrease local blood flow and increase the incidence of hyalinization.

Microsphere controlled drug delivery for local control of tooth movement.

Background: Because orthodontic tooth movement is dependent upon osteoclast-mediated resorption of alveolar bone adjacent to the pressure side of tooth roots, biologic mediators that regulate osteoclasts can be utilized to control tooth movement. Objectives: To develop a novel method to locally enhance orthodontic anchorage. Methods: We encapsulated osteoprotegerin (OPG) in polymer microspheres and tested the effectiveness of microsphere encapsulated versus non-encapsulated OPG for enhancing orthodontic anchorage in a rodent model of tooth movement. A single injection of 1 mg/kg non-encapsulated or microsphere encapsulated OPG was delivered into the palatal mucosa mesial to the first maxillary molar 1 day prior to tooth movement. A positive control group received injections of 5 mg/kg non-encapsulated OPG every 3 days during tooth movement. After 28 days of tooth movement, hemi-maxillae and femurs were dissected. Molar mesial and incisor distal tooth movement was measured using stone casts that were scanned and magnified. Local alveolar, distant femur bone, and tooth root volumes were analyzed by micro computed tomography. Serum OPG levels were measured by ELISA. Osteoclast numbers were quantified by histomorphometry. Results: The single injection of microsphere encapsulated OPG significantly enhanced orthodontic anchorage, while the single injection of non-encapsulated OPG did not. Injection of encapsulated OPG inhibited molar mesial movement but did not inhibit incisor tooth movement, and did not alter alveolar or femur bone volume fraction, density, or mineral content. Multiple injections of 5 mg/kg non-encapsulated OPG enhanced orthodontic anchorage, but also inhibited incisor retraction and altered alveolar and femur bone quality parameters. Increased OPG levels were found only in animals receiving multiple injections of non-encapsulated 5 mg/kg OPG. Osteoclast numbers were higher upon tooth movement in animals that did not receive OPG. Osteoclast numbers in OPG injected animals were variable within groups. Conclusions: Microsphere encapsulation of OPG allows for controlled drug release, and enhances site-specific orthodontic anchorage without systemic side effects. With additional refinements, this drug delivery system could be applicable to a broad array of potential biologic orthodontic therapeutics.

Coupling endonucleases with DNA end-processing enzymes to drive gene disruption.

Targeted DNA double-strand breaks introduced by rare-cleaving designer endonucleases can be harnessed for gene disruption applications by engaging mutagenic nonhomologous end-joining DNA repair pathways. However, endonuclease-mediated DNA breaks are often subject to precise repair, which limits the efficiency of targeted genome editing. To address this issue, we coupled designer endonucleases to DNA end-processing enzymes to drive mutagenic break resolution, achieving up to 25-fold enhancements in gene disruption rates.

Circulating serum-derived microparticles provide novel proteomic biomarkers of spontaneous preterm birth.

OBJECTIVE: The purpose of this study was to determine whether the proteomic biosignature of circulating microparticles in maternal serum obtained in the second trimester could identify pregnancies that result in spontaneous preterm birth (SPTB). STUDY DESIGN: Microparticles were isolated from blinded biorepository-sourced serum samples from 48 pregnant women at 15 to 17 weeks of gestation. Microparticle proteins were extracted and analyzed using label-free liquid chromatography/mass spectrometry. Peptide features were analyzed to assess the association of specific protein patterns with subjects delivering at term (≥ 37 weeks gestation; n = 24) and those experiencing SPTB (≤ 34 weeks gestation; n = 24). RESULTS: We found 99 proteins that had statistically significant differences in signal intensity between term and SPTB women in both first (n = 26) and second (n = 22) singleton gestation pregnancy cohorts. Additional evaluation identified 18 biomarkers that met criteria for further priority evaluation (12 preterm, 6 term). Pathway analysis showed that differentiating SPTB biomarker proteins were predominantly associated with inflammation and cell injury, while differentiating term biomarkers were associated with cell growth and hematological parameters. CONCLUSION: This study shows for the first time that the proteomic content of serum microparticles isolated in the second trimester can identify with a high degree of accuracy pregnancies that result in SPTB.

The heterodimerization of platelet-derived chemokines.

Chemokines encompass a large family of proteins that act as chemoattractants and are involved in many biological processes. In particular, chemokines guide the migration of leukocytes during normal and inflammatory conditions. Recent studies reveal that the heterophilic interactions between chemokines significantly affect their biological activity, possibly representing a novel regulatory mechanism of the chemokine activities. The co-localization of platelet-derived chemokines in vivo allows them to interact. Here, we used nano-spray ionization mass spectrometry to screen eleven different CXC and CC platelet-derived chemokines for possible interactions with the two most abundant chemokines present in platelets, CXCL4 and CXCL7. Results indicate that many screened chemokines, although not all of them, form heterodimers with CXCL4 and/or CXCL7. In particular, a strong heterodimerization was observed between CXCL12 and CXCL4 or CXCL7. Compared to other chemokines, the main structural difference of CXCL12 is in the orientation and packing of the C-terminal alpha-helix in relation to the beta-sheet. The analysis of one possible structure of the CXCL4/CXCL12 heterodimer, CXC-type structure, using molecular dynamics (MD) trajectory reveals that CXCL4 may undergo a conformational transition to alter the alpha helix orientation. In this new orientation, the alpha-helix of CXCL4 aligns in parallel with the CXCL12 alpha-helix, an energetically more favorable conformation. Further, we determined that CXCL4 and CXCL12 physically interact to form heterodimers by co-immunoprecipitations from human platelets. Overall, our results highlight that many platelet-derived chemokines are capable of heterophilic interactions and strongly support future studies of the biological impact of these interactions.

Systems biology and proteomic analysis of cerebral cavernous malformation.

UNLABELLED: Cerebral cavernous malformations (CCM) are vascular anomalies caused by mutations in genes encoding KRIT1, OSM and PDCD10 proteins causing hemorrhagic stroke. We examine proteomic change of loss of CCM gene expression. Using human umbilical vein endothelial cells, label-free differential protein expression analysis with multidimensional liquid chromatography/tandem mass spectrometry was applied to three CCM protein knockdown cell lines and two control cell lines: ProteomeXchange identifier PXD000362. Principle component and cluster analyses were used to examine the differentially expressed proteins associated with CCM. The results from the five cell lines revealed 290 and 192 differentially expressed proteins (p < 0.005 and p < 0.001, respectively). Most commonly affected proteins were cytoskeleton-associated proteins, in particular myosin-9. Canonical genetic pathway analysis suggests that CCM may be a result of defective cell-cell interaction through dysregulation of cytoskeletal associated proteins. CONCLUSION: The work explores signaling pathways that may elucidate early detection and novel therapy for CCM.

LAHEDES: the LAGLIDADG homing endonuclease database and engineering server.

LAGLIDADG homing endonucleases (LHEs) are DNA cleaving enzymes, also termed 'meganucleases' that are employed as gene-targeting reagents. This use of LHEs requires that their DNA specificity be altered to match sequences in genomic targets. The choice of the most appropriate LHE to target a particular gene is facilitated by the growing number of such enzymes with well-characterized activities and structures. 'LAHEDES' (The LAGLIDADG Homing Endonuclease Database and Engineering Server) provides both an online archive of LHEs with validated DNA cleavage specificities and DNA-binding interactions, as well as a tool for the identification of DNA sequences that might be targeted by various LHEs. Searches can be performed using four separate scoring algorithms and user-defined choices of LHE scaffolds. The webserver subsequently provides information regarding clusters of amino acids that should be interrogated during engineering and selection experiments. The webserver is fully open access and can be found at http://homingendonuclease.net.

Engineering domain fusion chimeras from I-OnuI family LAGLIDADG homing endonucleases.

Although engineered LAGLIDADG homing endonucleases (LHEs) are finding increasing applications in biotechnology, their generation remains a challenging, industrial-scale process. As new single-chain LAGLIDADG nuclease scaffolds are identified, however, an alternative paradigm is emerging: identification of an LHE scaffold whose native cleavage site is a close match to a desired target sequence, followed by small-scale engineering to modestly refine recognition specificity. The application of this paradigm could be accelerated if methods were available for fusing N- and C-terminal domains from newly identified LHEs into chimeric enzymes with hybrid cleavage sites. Here we have analyzed the structural requirements for fusion of domains extracted from six single-chain I-OnuI family LHEs, spanning 40-70% amino acid identity. Our analyses demonstrate that both the LAGLIDADG helical interface residues and the linker peptide composition have important effects on the stability and activity of chimeric enzymes. Using a simple domain fusion method in which linker peptide residues predicted to contact their respective domains are retained, and in which limited variation is introduced into the LAGLIDADG helix and nearby interface residues, catalytically active enzymes were recoverable for ≈ 70% of domain chimeras. This method will be useful for creating large numbers of chimeric LHEs for genome engineering applications.

Interleukin(IL)-1/IL-6-inhibitor-associated drug reaction with eosinophilia and systemic symptoms (DReSS) in systemic inflammatory illnesses.

BACKGROUND: After introducing interleukin(IL)-1/IL-6 inhibitors, some Still and Still-like patients developed unusual often fatal pulmonary disease. This complication was associated with scoring as DReSS (drug reaction with eosinophilia and systemic symptoms) implicating these inhibitors, although DReSS can be difficult to recognize in the setting of systemic inflammatory disease. OBJECTIVE: We sought to facilitate recognition of IL-1/IL-6 inhibitor-DReSS in systemic inflammatory illnesses (Still/Still-like) by looking at timing and reaction-associated features. We evaluated outcomes of stopping or not-stopping IL-1/IL-6-inhibitors after DReSS reaction began. METHODS: In an international study collaborating primarily with pediatric specialists, we characterized features of 89 drug-reaction cases versus 773 drug-exposed controls and compared outcomes of 52 cases stopping IL-1/IL-6-inhibitors to 37 cases not-stopping these drugs. RESULTS: Before reaction began, drug-reaction cases and controls were clinically comparable, except for younger disease onset age for reaction cases with pre-existing cardiothoracic comorbidities. After reaction began, increased rates of pulmonary complications and macrophage activation syndrome (MAS), differentiated drug-reaction cases from drug-tolerant controls (p=4.7x10-35; p=1.1x10-24, respectively). Initial DReSS feature was typically reported 2-8 weeks after initiating IL-1/IL-6-inhibition. In drug-reaction cases stopping versus not-stopping IL-1/IL-6-inhibitor treatment, reaction related features were indistinguishable, including pulmonary complication rates [75%(39/52] versus [76%(28/37)]. Those stopping subsequently required fewer medications for treatment of systemic inflammation, had decreased rates of MAS, and improved survival (p=0.005, multivariate regression). Resolution of pulmonary complications occurred in 67%(26/39) of drug-reaction cases who stopped and in none who continued inhibitors. CONCLUSIONS: In systemic inflammatory illnesses, recognition of IL-1/IL-6-inhibitor-associated reactions followed by avoidance of IL-1/IL-6-inhibitors significantly improved outcomes.

Mass spectrometry-based quantitative metabolomics revealed a distinct lipid profile in breast cancer patients.

Breast cancer accounts for the largest number of newly diagnosed cases in female cancer patients. Although mammography is a powerful screening tool, about 20% of breast cancer cases cannot be detected by this method. New diagnostic biomarkers for breast cancer are necessary. Here, we used a mass spectrometry-based quantitative metabolomics method to analyze plasma samples from 55 breast cancer patients and 25 healthy controls. A number of 30 patients and 20 age-matched healthy controls were used as a training dataset to establish a diagnostic model and to identify potential biomarkers. The remaining samples were used as a validation dataset to evaluate the predictive accuracy for the established model. Distinct separation was obtained from an orthogonal partial least squares-discriminant analysis (OPLS-DA) model with good prediction accuracy. Based on this analysis, 39 differentiating metabolites were identified, including significantly lower levels of lysophosphatidylcholines and higher levels of sphingomyelins in the plasma samples obtained from breast cancer patients compared with healthy controls. Using logical regression, a diagnostic equation based on three metabolites (lysoPC a C16:0, PC ae C42:5 and PC aa C34:2) successfully differentiated breast cancer patients from healthy controls, with a sensitivity of 98.1% and a specificity of 96.0%.

Abuse liability of two electronic nicotine delivery systems compared with combustible cigarettes and nicotine gum from an open-label randomized crossover study.

An assessment of the likelihood of use and abuse potential for new tobacco products is an important part of tobacco product regulation in the United States and abroad. This paper reports the results of a randomized, open-label, crossover clinical study that assessed factors related to product adoption and abuse liability (AL), comparing two closed electronic nicotine delivery system (ENDS) products to combustible cigarettes and nicotine gum, high- and low-AL comparator products, respectively. During an 11-day confinement period that included multiple product familiarization sessions, healthy adult smokers participated in AL test sessions to evaluate the abuse liability of each product. During these test sessions, changes in subjective measures; speed and amount of nicotine uptake; and maximum changes in physiological effects before, during, and after use of each assigned product were assessed over 4 h. Positive subjective effects measures scores such as product-liking and overall intent to use again were highest for cigarettes, followed by the Vuse ENDS, with nicotine gum consistently having the lowest scores. The PK results (Cmax and Tmax) of the Vuse ENDS products are between UB cigarettes and nicotine gum, which correlates with the subjective effects. All nicotine uptake measures for the Vuse ENDS products were lower than that of usual brand (UB) cigarettes, including peak nicotine uptake and overall nicotine uptake, and were either similar to or lower than nicotine gum. The time course of nicotine uptake after use of the ENDS was more similar to that of combustible cigarettes than nicotine gum. The results indicate that the AL of each ENDS product is lower than that of UB cigarettes and similar to that of nicotine gum.

Evaluating the prevalence of inborn errors of immunity in adults with chronic immune thrombocytopenia or Evans syndrome.

Inborn errors of immunity (IEIs) are monogenic disorders that predispose patients to immune dysregulation, autoimmunity, and infection. Autoimmune cytopenias, such as immune thrombocytopenia (ITP) and Evans syndrome (a combination of ITP and autoimmune hemolytic anemia), are increasingly recognized phenotypes of IEI. Although recent findings suggest that IEIs may commonly underlie pediatric ITP and Evans syndrome, its prevalence in adult patients with these disorders remains undefined. This study sought to estimate the prevalence of underlying IEIs among adults with persistent or chronic ITP or Evans syndrome using a next-generation sequencing panel encompassing >370 genes implicated in IEIs. Forty-four subjects were enrolled from an outpatient adult hematology clinic at a tertiary referral center in the United States, with a median age of 49 years (range, 20-83). Fourteen subjects (31.8%) had secondary ITP, including 8 (18.2%) with Evans syndrome. No cases of IEI were identified despite a high representation of subjects with a personal history of autoimmunity (45.5%) and early onset of disease (median age at diagnosis of 40 years [range, 2-77]), including 20.5% who were initially diagnosed as children. Eight subjects (18.2%) were found to be carriers of pathogenic IEI variants, which, in their heterozygous state, are not disease-causing. One case of TUBB1-related congenital thrombocytopenia was identified. Although systematic screening for IEI has been proposed for pediatric patients with Evans syndrome, findings from this real-world study suggest that inclusion of genetic testing for IEI in the routine work-up of adults with ITP and Evans syndrome has a low diagnostic yield.

Single cell spatial transcriptomic profiling of childhood-onset lupus nephritis reveals complex interactions between kidney stroma and infiltrating immune cells.

Children with systemic lupus erythematosus (SLE) are at increased risk of developing kidney disease, termed childhood-onset lupus nephritis (cLN). Single cell transcriptomics of dissociated kidney tissue has advanced our understanding of LN pathogenesis, but loss of spatial resolution prevents interrogation of in situ cellular interactions. Using a technical advance in spatial transcriptomics, we generated a spatially resolved, single cell resolution atlas of kidney tissue (>400,000 cells) from eight cLN patients and two controls. Annotated cells were assigned to 35 reference cell types, including major kidney subsets and infiltrating immune cells. Analysis of spatial distribution demonstrated that individual immune lineages localize to specific regions in cLN kidneys, including myeloid cells trafficking to inflamed glomeruli and B cells clustering within tubulointerstitial immune hotspots. Notably, gene expression varied as a function of tissue location, demonstrating how incorporation of spatial data can provide new insights into the immunopathogenesis of SLE. Alterations in immune phenotypes were accompanied by parallel changes in gene expression by resident kidney stromal cells. However, there was little correlation between histologic scoring of cLN disease activity and glomerular cell transcriptional signatures at the level of individual glomeruli. Finally, we identified modules of spatially-correlated gene expression with predicted roles in induction of inflammation and the development of tubulointerstitial fibrosis. In summary, single cell spatial transcriptomics allows unprecedented insights into the molecular heterogeneity of cLN, paving the way towards more targeted and personalized treatment approaches.

Part one: abuse liability of Vuse Solo (G2) electronic nicotine delivery system relative to combustible cigarettes and nicotine gum.

Abuse liability (AL) of electronic nicotine delivery systems (ENDS) is relevant as the category increases in popularity as a potentially less-harmful alternative to cigarette smoking. AL assessments are important to the FDA in determining if a new product is appropriate for the protection of public health. This paper reports the results for Vuse Solo (G2 cartridge design) compared to high and low AL-comparators evaluated in an open-label, randomized crossover confinement AL study. The confinement design was adapted from previous ambulatory studies of Vuse Solo (G1 cartridge design) and included product familiarization sessions before each four-hour test session in which subjective measures, nicotine pharmacokinetics (PK), and physiological endpoints were assessed following a single 10-min ad libitum product use session. Product liking, intent to use again, suppression of urge to smoke, and nicotine PK were lower after use of Vuse Solo compared to cigarettes and higher after use of Vuse Solo compared to nicotine gum. No significant differences in blood pressure or heart rate were observed between the products pre- to post-product use. These data reinforce previous research and provide the scientific evidence to support regulatory decisions demonstrating that Vuse Solo has an AL profile lower than that of combustible cigarettes but higher than that of nicotine gum and, therefore, may be a suitable replacement for cigarette smoking for some adult smokers.