Assessment of the Cutaneous Hormone Landscapes and Microbiomes in Vulvar Lichen Sclerosus.

Vulvar lichen sclerosus (VLS) is a progressive skin disease of unknown etiology. In this longitudinal case-control exploratory study, we evaluated the hormonal and microbial landscapes in 18 postmenopausal women (mean [SD] age: 64.4 [8.4]) with vulvar lichen sclerosus and controls. We reevaluated the VLS patients after 10-14 weeks of daily topical Class I steroid. We found that groin cutaneous estrone was lower in vulvar lichen sclerosus versus controls (-22.33, 95% CI -36.96 to -7.70; P = 0.006); cutaneous progesterone was higher (5.73, 95% CI 3.74 to 7.73; P< 0.0001). Forehead 11-deoxycortisol (-0.24, 95% CI -0.42 to -0.06; P = 0.01) and testosterone (-7.22, 95% CI -12.83 to -1.62; P = 0.02) were lower in disease. With treatment, cutaneous estrone (-7.88, 95% CI -44.07 to 28.31; P = 0.62), progesterone (2.02, 95% CI -2.08 to 6.11; P = 0.29), and 11-deoxycortisol (-0.13, 95% CI -0.32 to 0.05; P = 0.15) normalized; testosterone remained suppressed (-7.41, 95% CI -13.38 to -1.43; P = 0.02). 16S rRNA V1-V3 and ITS1 amplicon sequencing revealed bacterial and fungal microbiome alterations in disease. Findings suggest that cutaneous sex hormone and bacterial microbiome alterations may be associated with VLS in postmenopausal women.

A telomere-targeting drug depletes cancer initiating cells and promotes anti-tumor immunity in small cell lung cancer.

There are few effective treatments for small cell lung cancer (SCLC) underscoring the need for innovative therapeutic approaches. This study focuses on exploiting telomerase, a critical SCLC dependency as a therapeutic target. A prominent characteristic of SCLC is their reliance on telomerase activity, a key enzyme essential for their continuous proliferation. Here we utilize a nucleoside analog, 6-Thio-2'-deoxyguanosine (6TdG) currently in phase II clinical trials, that is preferentially incorporated by telomerase into telomeres leading to telomere dysfunction. Using preclinical mouse and human derived models we find low intermittent doses of 6TdG inhibit tumor growth and reduce metastatic burden. Anti-tumor efficacy correlates with a reduction in a subpopulation of cancer initiating like cells (CICs) identified by their expression of L1CAM/CD133 and highest telomerase activity. 6TdG treatment also leads to activation of innate and adaptive anti-tumor responses. Mechanistically, 6TdG depletes CICs and induces type-I interferon signaling leading to tumor immune visibility by activating tumor cell STING signaling. We also observe increased sensitivity to irradiation after 6TdG treatment in both syngeneic and humanized SCLC xenograft models both of which are dependent on the presence of host immune cells. This study underscores the immune-enhancing and metastasis-reducing effects of 6TdG, employing a range of complementary in vitro and in vivo SCLC preclinical models providing a potential therapeutic approach to SCLC.

Lupus IgA1 autoantibodies synergize with IgG to enhance pDC responses to RNA-containing immune complexes.

Autoantibodies to nuclear antigens are hallmarks of the autoimmune disease systemic lupus erythematosus (SLE) where they contribute to pathogenesis. However, there remains a gap in our knowledge regarding how different isotypes of autoantibodies contribute to disease, including the production of the critical type I interferon (IFN) cytokines by plasmacytoid dendritic cells (pDCs) in response to immune complexes (ICs). We focused on IgA, which is the second most prevalent isotype in serum, and along with IgG is deposited in glomeruli in lupus nephritis. Here, we show that individuals with SLE have IgA autoantibodies against most nuclear antigens, correlating with IgG against the same antigen. We investigated whether IgA autoantibodies against a major SLE autoantigen, Smith ribonucleoproteins (Sm/RNPs), play a role in IC activation of pDCs. We found that pDCs express the IgA-specific Fc receptor, FcαR, and there was a striking ability of IgA1 autoantibodies to synergize with IgG in RNA-containing ICs to generate robust pDC IFNα responses. pDC responses to these ICs required both FcαR and FcγRIIa, showing a potent synergy between these Fc receptors. Sm/RNP IC binding to and internalization by pDCs were greater when ICs contained both IgA1 and IgG. pDCs from individuals with SLE had higher binding of IgA1-containing ICs and higher expression of FcαR than pDCs from healthy control individuals. Whereas pDC FcαR expression correlated with blood ISG signature in SLE, TLR7 agonists, but not IFNα, upregulated pDC FcαR expression in vitro. Together, we show a new mechanism by which IgA1 autoantibodies contribute to SLE pathogenesis.

Meropenem-vaborbactam restoration of first-line drug efficacy and comparison of meropenem-vaborbactam-moxifloxacin versus BPaL MDR-TB regimen.

BACKGROUND: Meropenem in combination with ß-lactamase inhibitors (BLIs) and other drugs was tested to identify alternative treatment regimens for multidrug-resistant tuberculosis (MDR-TB). METHODS: The following were performed: (1) MIC experiments; (2) static time-kill studies (STKs) with different BLIs; and (3) a hollow fibre model system of TB (HFS-TB) studies with meropenem-vaborbactam combined with human equivalent daily doses of 20 mg/kg or 35 mg/kg rifampin, or moxifloxacin 400 mg, or linezolid 600 mg vs. bedaquiline-pretonamid-linezolid (BPaL) for MDR-TB. The studies were performed using Mycobacterium tuberculosis (M. tuberculosis) H37Rv and an MDR-TB clinical strain (named M. tuberculosis 16D) that underwent whole genome sequencing. Exponential decline models were used to calculate the kill rate constant (K) of different HFS-TB regimens. RESULTS: Whole genome sequencing revealed mutations associated with resistance to rifampin, isoniazid, and cephalosporins. The meropenem-vaborbactam MIC of M. tuberculosis was H37Rv 2 mg/L and > 128 mg/L for M. tuberculosis 16D. Relebactam and vaborbactam improved both the potency and efficacy of meropenem in STKs. Meropenem-vaborbactam alone failed to kill M. tuberculosis 16D but killed below day 0 burden when combined with isoniazid and rifampin, with the moxifloxacin combination being the most effective and outranking bedaquiline and pretomanid. In the HFS-TB, meropenem-vaborbactam-moxifloxacin and BPaL had the highest K (log10 cfu/mL/day) of 0.31 (95% CI 0.17-0.58) and 0.34 (95% CI 0.21-0.56), while meropenem-vaborbactam-rifampin (35 mg/kg) had a K of 0.18 (95% CI 0.12-0.25). The K for meropenem-vaborbactam-moxifloxacin-linezolid demonstrated antagonism. CONCLUSION: Adding meropenem-vaborbactam could potentially restore the efficacy of isoniazid and rifampin against MDR-TB. The meropenem-vaborbactam-moxifloxacin backbone regimen has implications for creating a new effective MDR-TB regimen.

Staphylococcus aureus isolates from children with clinically differentiated osteomyelitis exhibit distinct transcriptomic signatures.

There is substantial genomic heterogeneity among Staphylococcus aureus isolates of children with acute hematogenous osteomyelitis (AHO) but transcriptional behavior of clinically differentiated strains has not been previously described. This study evaluates transcriptional activity of S. aureus isolates of children with AHO that may regulate metabolism, biosynthesis, or virulence during bacterial growth and pathogenesis. In vitro growth kinetics were compared between three S. aureus clinical isolates from children with AHO who had mild, moderate, and severe illness. Total RNA sequencing was performed for each isolate at six separate time points throughout the logarithmic phase of growth. The NASA RNA-Sequencing Consensus Pipeline was used to identify differentially expressed genes allowing for 54 comparisons between the three isolates during growth. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment pathways were used to evaluate transcriptional variation in metabolism, biosynthesis pathways and virulence potential of the isolates. The S. aureus isolates demonstrated differing growth kinetics under standardized conditions with the mild isolate having higher optical densities with earlier and higher peak rates of growth than that of the other isolates (p<0.001). Enrichment pathway analysis established distinct transcriptional signatures according to both sampling time and clinical severity. Moderate and severe isolates demonstrated pathways of bacterial invasion, S. aureus infection, quorum sensing and two component systems. In comparison, the mild strain favored biosynthesis and metabolism. These findings suggest that transcriptional regulation during the growth of S. aureus may impact the pathogenetic mechanisms involved in the progression of severity of illness in childhood osteomyelitis. The clinical isolates studied demonstrated a tradeoff between growth and virulence. Further investigation is needed to evaluate these transcriptional pathways in an animal model or during active clinical infections of children with AHO.

T-cell tolerant fraction as a predictor of immune-related adverse events.

BACKGROUND: Immune checkpoint inhibitor (ICI) therapies may cause unpredictable and potentially severe autoimmune toxicities termed immune-related adverse events (irAEs). Because T cells mediate ICI effects, T cell profiling may provide insight into the risk of irAEs. Here we evaluate a novel metric-the T-cell tolerant fraction-as a predictor of future irAEs. METHODS: We examined T-cell receptor beta (TRB) locus sequencing from baseline pretreatment samples from an institutional registry and previously published studies. For each patient, we used TRB sequences to calculate the T-cell tolerant fraction, which was then assessed as a predictor of future irAEs (classified as Common Terminology Criteria for Adverse Event grade 0-1 vs grade ≥2). We then compared the tolerant fraction to TRB clonality and diversity. Finally, the tolerant fraction was assessed on (1) T cells enriched against napsin A, a potential autoantigen of irAEs; (2) thymic versus peripheral blood T cells; and (3) TRBs specific for various infections and autoimmune diseases. RESULTS: A total of 77 patients with cancer (22 from an institutional registry and 55 from published studies) receiving ICI therapy (43 CTLA4, 19 PD1/PDL1, 15 combination CTLA4+PD1/PDL1) were included in the study. The tolerant fraction was significantly lower in cases with clinically significant irAEs (p<0.001) and had an area under the receiver operating curve (AUC) of 0.79. The tolerant fraction was lower for each ICI treatment category, reaching statistical significance for CTLA4 (p<0.001) and demonstrating non-significant trends for PD1/PDL1 (p=0.21) and combination ICI (p=0.18). The tolerant fraction for T cells enriched against napsin A was lower than other samples. The tolerant fraction was also lower in thymic versus peripheral blood samples, and lower in some (multiple sclerosis) but not other (type 1 diabetes) autoimmune diseases. In our study cohort, TRB clonality had an AUC of 0.62, and TRB diversity had an AUC of 0.60 for predicting irAEs. CONCLUSIONS: Among patients receiving ICI, the baseline T-cell tolerant fraction may serve as a predictor of clinically significant irAEs.

The gut microbiota reprograms intestinal lipid metabolism through long noncoding RNA Snhg9.

The intestinal microbiota regulates mammalian lipid absorption, metabolism, and storage. We report that the microbiota reprograms intestinal lipid metabolism in mice by repressing the expression of long noncoding RNA (lncRNA) Snhg9 (small nucleolar RNA host gene 9) in small intestinal epithelial cells. Snhg9 suppressed the activity of peroxisome proliferator-activated receptor γ (PPARγ)-a central regulator of lipid metabolism-by dissociating the PPARγ inhibitor sirtuin 1 from cell cycle and apoptosis protein 2 (CCAR2). Forced expression of Snhg9 in the intestinal epithelium of conventional mice impaired lipid absorption, reduced body fat, and protected against diet-induced obesity. The microbiota repressed Snhg9 expression through an immune relay encompassing myeloid cells and group 3 innate lymphoid cells. Our findings thus identify an unanticipated role for a lncRNA in microbial control of host metabolism.

Macrophages regulate gastrointestinal motility through complement component 1q.

Peristaltic movement of the intestine propels food down the length of the gastrointestinal tract to promote nutrient absorption. Interactions between intestinal macrophages and the enteric nervous system regulate gastrointestinal motility, yet we have an incomplete understanding of the molecular mediators of this crosstalk. Here, we identify complement component 1q (C1q) as a macrophage product that regulates gut motility. Macrophages were the predominant source of C1q in the mouse intestine and most extraintestinal tissues. Although C1q mediates the complement-mediated killing of bacteria in the bloodstream, we found that C1q was not essential for the immune defense of the intestine. Instead, C1q-expressing macrophages were located in the intestinal submucosal and myenteric plexuses where they were closely associated with enteric neurons and expressed surface markers characteristic of nerve-adjacent macrophages in other tissues. Mice with a macrophage-specific deletion of C1qa showed changes in enteric neuronal gene expression, increased neurogenic activity of peristalsis, and accelerated intestinal transit. Our findings identify C1q as a key regulator of gastrointestinal motility and provide enhanced insight into the crosstalk between macrophages and the enteric nervous system.

Saprophytic bacteria and fungi colonize stearoyl coenzyme-A desaturase-1 knockout skin.

Lipids synthesized on the skin are critical to the antimicrobial barrier. Skin lipids also facilitate survival of lipophilic skin commensals in an otherwise dry and acidic ecological landscape. Thus, skin-specific stearoyl-coenzyme A desaturase 1 knockout mice (Scd1ΔK14 ) with sebocyte atrophy and decreased synthesis of monounsaturated fatty acids, triglycerides and wax diesters have dry, inflamed skin. Here, we used 16S rRNA (V1-V2 and V1-V9) and internal transcribed spacer 1 (ITS1) amplicon sequencing to compare bacterial and fungal skin microbiomes between Scd1ΔK14 mice and wildtype control mice (Scd1fl/fl ) in a barrier facility. Saprophytic bacteria including Sporosarcina spp. and Staphylococcus lentus and saprophytic fungi including Alternaria infectoria were found in higher relative abundance in the Scd1ΔK14 group (ANCOM). Analysis of community diversity (Shannon index) revealed greater fungal alpha diversity in the Scd1ΔK14 group (p = 0.009, Kruskal-Wallis). Principal coordinates analysis (Bray-Curtis dissimilarity) showed that both bacterial (p = 0.002, PERMANOVA) and fungal communities (p = 0.006, PERMANOVA) of the Scd1ΔK14 group were unique from the wildtype group. Altogether, these results suggest that sebaceous gland-derived lipids normally restrict the skin microbiome, and in the absence of these lipids, a greater diversity of opportunistic organisms are able to colonize the surface of skin.

Loss-of-function variants in SAT1 cause X-linked childhood-onset systemic lupus erythematosus.

OBJECTIVES: Families that contain multiple siblings affected with childhood onset of systemic lupus erythematosus (SLE) likely have strong genetic predispositions. We performed whole exome sequencing (WES) to identify familial rare risk variants and to assess their effects in lupus. METHODS: Sanger sequencing validated the two ultra-rare, predicted pathogenic risk variants discovered by WES and identified additional variants in 562 additional patients with SLE. Effects of a splice site variant and a frameshift variant were assessed using a Minigene assay and CRISPR/Cas9-mediated knock-in (KI) mice, respectively. RESULTS: The two familial ultra-rare, predicted loss-of-function (LOF) SAT1 variants exhibited X-linked recessive Mendelian inheritance in two unrelated African-American families. Each LOF variant was transmitted from the heterozygous unaffected mother to her two sons with childhood-onset SLE. The p.Asp40Tyr variant affected a splice donor site causing deleterious transcripts. The young hemizygous male and homozygous female Sat1 p.Glu92Leufs*6 KI mice spontaneously developed splenomegaly, enlarged glomeruli with leucocyte infiltration, proteinuria and elevated expression of type I interferon-inducible genes. SAT1 is highly expressed in neutrophils and encodes spermidine/spermine-N1-acetyltransferase 1 (SSAT1), a rate-limiting enzyme in polyamine catabolism. Young male KI mice exhibited neutrophil defects and decreased proportions of Foxp3 +CD4+ T-cell subsets. Circulating neutrophil counts and proportions of Foxp3 +CD4+ T cells correlated with decreased plasma levels of spermine in treatment-naive, incipient SLE patients. CONCLUSIONS: We identified two novel SAT1 LOF variants, showed the ability of the frameshift variant to confer murine lupus, highlighted the pathogenic role of dysregulated polyamine catabolism and identified SAT1 LOF variants as new monogenic causes for SLE.

Association between Antibiotic Exposure and Systemic Immune Parameters in Cancer Patients Receiving Checkpoint Inhibitor Therapy.

Antibiotic administration is associated with worse clinical outcomes and changes to the gut microbiome in cancer patients receiving immune checkpoint inhibitors (ICI). However, the effects of antibiotics on systemic immune function are unknown. We, therefore, evaluated antibiotic exposure, therapeutic responses, and multiplex panels of 40 serum cytokines and 124 antibodies at baseline and six weeks after ICI initiation, with p < 0.05 and false discovery rate (FDR) < 0.2 considered significant. A total of 251 patients were included, of whom the 135 (54%) who received antibiotics had lower response rates and shorter survival. Patients who received antibiotics prior to ICI initiation had modestly but significantly lower baseline levels of nucleolin, MDA5, c-reactive protein, and liver cytosol antigen type 1 (LC1) antibodies, as well as higher levels of heparin sulfate and Matrigel antibodies. After ICI initiation, antibiotic-treated patients had significantly lower levels of MDA5, CENP.B, and nucleolin antibodies. Although there were no clear differences in cytokines in the overall cohort, in the lung cancer subset (53% of the study population), we observed differences in IFN-γ, IL-8, and macrophage inflammatory proteins. In ICI-treated patients, antibiotic exposure is associated with changes in certain antibodies and cytokines. Understanding the relationship between these factors may improve the clinical management of patients receiving ICI.

A short plus long-amplicon based sequencing approach improves genomic coverage and variant detection in the SARS-CoV-2 genome.

High viral transmission in the COVID-19 pandemic has enabled SARS-CoV-2 to acquire new mutations that may impact genome sequencing methods. The ARTIC.v3 primer pool that amplifies short amplicons in a multiplex-PCR reaction is one of the most widely used methods for sequencing the SARS-CoV-2 genome. We observed that some genomic intervals are poorly captured with ARTIC primers. To improve the genomic coverage and variant detection across these intervals, we designed long amplicon primers and evaluated the performance of a short (ARTIC) plus long amplicon (MRL) sequencing approach. Sequencing assays were optimized on VR-1986D-ATCC RNA followed by sequencing of nasopharyngeal swab specimens from fifteen COVID-19 positive patients. ARTIC data covered 94.47% of the virus genome fraction in the positive control and patient samples. Variant analysis in the ARTIC data detected 217 mutations, including 209 single nucleotide variants (SNVs) and eight insertions & deletions. On the other hand, long-amplicon data detected 156 mutations, of which 80% were concordant with ARTIC data. Combined analysis of ARTIC + MRL data improved the genomic coverage to 97.03% and identified 214 high confidence mutations. The combined final set of 214 mutations included 203 SNVs, 8 deletions and 3 insertions. Analysis showed 26 SARS-CoV-2 lineage defining mutations including 4 known variants of concern K417N, E484K, N501Y, P618H in spike gene. Hybrid analysis identified 7 nonsynonymous and 5 synonymous mutations across the genome that were either ambiguous or not called in ARTIC data. For example, G172V mutation in the ORF3a protein and A2A mutation in Membrane protein were missed by the ARTIC assay. Thus, we show that while the short amplicon (ARTIC) assay provides good genomic coverage with high throughput, complementation of poorly captured intervals with long amplicon data can significantly improve SARS-CoV-2 genomic coverage and variant detection.

Small proline-rich protein 2A is a gut bactericidal protein deployed during helminth infection.

A diverse group of antimicrobial proteins (AMPs) helps protect the mammalian intestine from varied microbial challenges. We show that small proline-rich protein 2A (SPRR2A) is an intestinal antibacterial protein that is phylogenetically unrelated to previously discovered mammalian AMPs. In this study, SPRR2A was expressed in Paneth cells and goblet cells and selectively killed Gram-positive bacteria by disrupting their membranes. SPRR2A shaped intestinal microbiota composition, restricted bacterial association with the intestinal surface, and protected against Listeria monocytogenes infection. SPRR2A differed from other intestinal AMPs in that it was induced by type 2 cytokines produced during helminth infection. Moreover, SPRR2A protected against helminth-induced bacterial invasion of intestinal tissue. Thus, SPRR2A is a distinctive AMP triggered by type 2 immunity that protects the intestinal barrier during helminth infection.

Impact of Bead-Beating Intensity on the Genus- and Species-Level Characterization of the Gut Microbiome Using Amplicon and Complete 16S rRNA Gene Sequencing.

Bead-beating within a DNA extraction protocol is critical for complete microbial cell lysis and accurate assessment of the abundance and composition of the microbiome. While the impact of bead-beating on the recovery of OTUs at the phylum and class level have been studied, its influence on species-level microbiome recovery is not clear. Recent advances in sequencing technology has allowed species-level resolution of the microbiome using full length 16S rRNA gene sequencing instead of smaller amplicons that only capture a few hypervariable regions of the gene. We sequenced the v3-v4 hypervariable region as well as the full length 16S rRNA gene in mouse and human stool samples and discovered major clusters of gut bacteria that exhibit different levels of sensitivity to bead-beating treatment. Full length 16S rRNA gene sequencing unraveled vast species diversity in the mouse and human gut microbiome and enabled characterization of several unclassified OTUs in amplicon data. Many species of major gut commensals such as Bacteroides, Lactobacillus, Blautia, Clostridium, Escherichia, Roseburia, Helicobacter, and Ruminococcus were identified. Interestingly, v3-v4 amplicon data classified about 50% of Ruminococcus reads as Ruminococcus gnavus species which showed maximum abundance in a 9 min beaten sample. However, the remaining 50% of reads could not be assigned to any species. Full length 16S rRNA gene sequencing data showed that the majority of the unclassified reads were Ruminococcus albus species which unlike R. gnavus showed maximum recovery in the unbeaten sample instead. Furthermore, we found that the Blautia hominis and Streptococcus parasanguinis species were differently sensitive to bead-beating treatment than the rest of the species in these genera. Thus, the present study demonstrates species level variations in sensitivity to bead-beating treatment that could only be resolved with full length 16S rRNA sequencing. This study identifies species of common gut commensals and potential pathogens that require minimum (0-1 min) or extensive (4-9 min) bead-beating for their maximal recovery.

Serum amyloid A delivers retinol to intestinal myeloid cells to promote adaptive immunity.

Vitamin A and its derivative retinol are essential for the development of intestinal adaptive immunity. Retinoic acid (RA)­producing myeloid cells are central to this process, but how myeloid cells acquire retinol for conversion to RA is unknown. Here, we show that serum amyloid A (SAA) proteins­retinol-binding proteins induced in intestinal epithelial cells by the microbiota­deliver retinol to myeloid cells. We identify low-density lipoprotein (LDL) receptor­related protein 1 (LRP1) as an SAA receptor that endocytoses SAA-retinol complexes and promotes retinol acquisition by RA-producing intestinal myeloid cells. Consequently, SAA and LRP1 are essential for vitamin A­dependent immunity, including B and T cell homing to the intestine and immunoglobulin A production. Our findings identify a key mechanism by which vitamin A promotes intestinal immunity.

The microbiota coordinates diurnal rhythms in innate immunity with the circadian clock.

Environmental light cycles entrain circadian feeding behaviors in animals that produce rhythms in exposure to foodborne bacteria. Here, we show that the intestinal microbiota generates diurnal rhythms in innate immunity that synchronize with feeding rhythms to anticipate microbial exposure. Rhythmic expression of antimicrobial proteins was driven by daily rhythms in epithelial attachment by segmented filamentous bacteria (SFB), members of the mouse intestinal microbiota. Rhythmic SFB attachment was driven by the circadian clock through control of feeding rhythms. Mechanistically, rhythmic SFB attachment activated an immunological circuit involving group 3 innate lymphoid cells. This circuit triggered oscillations in epithelial STAT3 expression and activation that produced rhythmic antimicrobial protein expression and caused resistance to Salmonella Typhimurium infection to vary across the day-night cycle. Thus, host feeding rhythms synchronize with the microbiota to promote rhythms in intestinal innate immunity that anticipate exogenous microbial exposure.

Cefdinir and ß-Lactamase Inhibitor Independent Efficacy Against Mycobacterium tuberculosis.

Background: There is renewed interest in repurposing ß-lactam antibiotics for treatment of tuberculosis (TB). We investigated efficacy of cefdinir, that withstand the ß-lactamase enzyme present in many bacteria, against drug-susceptible and multi-drug resistant (MDR) Mycobacterium tuberculosis (Mtb). Methods: Minimum inhibitory concentration (MIC) experiments were performed with Mtb H37Ra, eight drug-susceptible, and 12 MDR-TB clinical isolates with and without the ß-lactamase inhibitor, avibactam at 15 mg/L final concentration. Next, we performed dose-response study with Mtb H37Ra in test-tubes followed by a sterilizing activity study in the pre-clinical hollow fiber model of tuberculosis (HFS-TB) study using an MDR-TB clinical strain. Inhibitory sigmoid Emax model was used to describe the relationship between the drug exposure and bacterial burden. Results: Cefdinir MIC for Mtb H37Ra was 4 and 2 mg/L with or without avibactam, respectively. The MIC of the clinical strains ranged between 0.5 and 16 mg/L. In the test-tube experiments, cefdinir killed 4.93 + 0.07 log10 CFU/ml Mtb H37Ra in 7 days. In the HFS-TB studies, cefdinir showed dose-dependent killing of MDR-TB, without combination of avibactam. The cefdinir PK/PD index linked to the Mtb sterilizing efficacy was identified as the ratio of area under the concentration-time curve to MIC (AUC0-24/MIC) and optimal exposure was calculated as AUC0-24/MIC of 578.86. There was no resistance emergence to cefdinir in the HFS-TB. Conclusion: In the HFS-TB model, cefdinir showed efficacy against both drug susceptible and MDR-TB without combination of ß-lactamase inhibitor. However, clinical validation of these findings remains to be determined.

Adipocyte iron levels impinge on a fat-gut crosstalk to regulate intestinal lipid absorption and mediate protection from obesity.

Iron overload is positively associated with diabetes risk. However, the role of iron in adipose tissue remains incompletely understood. Here, we report that transferrin-receptor-1-mediated iron uptake is differentially required for distinct subtypes of adipocytes. Notably, adipocyte-specific transferrin receptor 1 deficiency substantially protects mice from high-fat-diet-induced metabolic disorders. Mechanistically, low cellular iron levels have a positive impact on the health of the white adipose tissue and can restrict lipid absorption from the intestine through modulation of vesicular transport in enterocytes following high-fat diet feeding. Specific reduction of adipocyte iron by AAV-mediated overexpression of the iron exporter Ferroportin1 in adult mice effectively mimics these protective effects. In summary, our studies highlight an important role of adipocyte iron in the maintenance of systemic metabolism through an adipocyte-enterocyte axis, offering an additional level of control over caloric influx into the system after feeding by regulating intestinal lipid absorption.

Serum IgG Profiling of Toddlers Reveals a Subgroup with Elevated Seropositive Antibodies to Viruses Correlating with Increased Vaccine and Autoantigen Responses.

PURPOSE: The human antibody repertoire forms in response to infections, the microbiome, vaccinations, and environmental exposures. The specificity of such antibody responses was compared among a cohort of toddlers to identify differences between seropositive versus seronegative responses. METHODS: An assessment of the serum IgM and IgG antibody reactivities in 197 toddlers of 1- and 2-years of age was performed with a microfluidic array containing 110 distinct antigens. Longitudinal profiling was done from years 1 to 2. Seropositivity to RNA and DNA viruses; bacteria; live attenuated, inactive, and subunit vaccines; and autoantigens was compared. A stratification was developed based on quantitative variations in the IgG responses. Clinical presentations and previously known genetic risk alleles for various immune system conditions were investigated in relation to IgG responses. RESULTS: IgG reactivities stratified toddlers into low, moderate, and high responder groups. The high group (17%) had elevated IgG responses to multiple RNA and DNA viruses (e.g., respiratory syncytial virus, Epstein-Barr virus, adenovirus, Coxsackievirus) and this correlated with increased responses to live attenuated viral vaccines and certain autoantigens. This high group was more likely to be associated with gestational diabetes and an older age. Genetic analyses identified polymorphisms in the IL2RB, TNFSF4, and INS genes in two high responder individuals that were associated with their elevated cytokine levels and clinical history of eczema and asthma. CONCLUSION: Serum IgG profiling of toddlers reveals correlations between the magnitude of the antibody responses towards viruses, live attenuated vaccines, and certain autoantigens. A low responder group had much weaker responses overall, including against vaccines. The serum antibody screen also identifies individuals with IgG responses to less common infections (West Nile virus, parvovirus, tuberculosis). The characterization of the antibody responses in combination with the identification of genetic risk alleles provides an opportunity to identify children with increased risk of clinical disease.

Deep sequencing reveals a DAP1 regulatory haplotype that potentiates autoimmunity in systemic lupus erythematosus.

BACKGROUND: Systemic lupus erythematosus (SLE) is a clinically heterogeneous autoimmune disease characterized by the development of anti-nuclear antibodies. Susceptibility to SLE is multifactorial, with a combination of genetic and environmental risk factors contributing to disease development. Like other polygenic diseases, a significant proportion of estimated SLE heritability is not accounted for by common disease alleles analyzed by SNP array-based GWASs. Death-associated protein 1 (DAP1) was implicated as a candidate gene in a previous familial linkage study of SLE and rheumatoid arthritis, but the association has not been explored further. RESULTS: We perform deep sequencing across the DAP1 genomic segment in 2032 SLE patients, and healthy controls, and discover a low-frequency functional haplotype strongly associated with SLE risk in multiple ethnicities. We find multiple cis-eQTLs embedded in a risk haplotype that progressively downregulates DAP1 transcription in immune cells. Decreased DAP1 transcription results in reduced DAP1 protein in peripheral blood mononuclear cells, monocytes, and lymphoblastoid cell lines, leading to enhanced autophagic flux in immune cells expressing the DAP1 risk haplotype. Patients with DAP1 risk allele exhibit significantly higher autoantibody titers and altered expression of the immune system, autophagy, and apoptosis pathway transcripts, indicating that the DAP1 risk allele mediates enhanced autophagy, leading to the survival of autoreactive lymphocytes and increased autoantibody. CONCLUSIONS: We demonstrate how targeted sequencing captures low-frequency functional risk alleles that are missed by SNP array-based studies. SLE patients with the DAP1 genotype have distinct autoantibody and transcription profiles, supporting the dissection of SLE heterogeneity by genetic analysis.