Algorithms with Area under the Curve for Daily Urinary Estrone-3-Glucuronide and Pregnanediol-3-Glucuronide to Signal the Transition to the Luteal Phase

Background and Objectives: Home fertility assessment methods (FAMs) for natural family planning (NFP) have technically evolved with the objective metrics of urinary luteinizing hormone (LH), estrone-3-glucuronide (E3G) and pregnanediol-3-glucuronide (PDG). Practical and reliable algorithms for timing the phase of cycle based upon E3G and PDG levels are mostly unpublished and still lacking. Materials and Methods: A novel formulation to signal the transition to the luteal phase was discovered, tested, and developed with a data set of daily E3G and PDG levels from 25 women, 78 cycles, indexed to putative ovulation (day after the urinary LH surge), Day 0. The algorithm is based upon a daily relative progressive change in the ratio, E3G-AUC/PDG-AUC, where E3G-AUC and PDG-AUC are the area under the curve for E3G and PDG, respectively. To improve accuracy the algorithm incorporated a three-fold cycle-specific increase of PDG. Results: An extended negative change in E3G-AUC/PDG-AUC of at least nine consecutive days provided a strong signal for timing the luteal phase. The algorithm correctly identified the luteal transition interval in 78/78 cycles and predicted the start day of the safe period as: Day + 2 in 10/78 cycles, Day + 3 in 21/78 cycles, Day + 4 in 28/78 cycles, Day + 5 in 15/78 cycles, and Day + 6 in 4/78 cycles. The mean number of safe luteal days with this algorithm was 10.3 ± 1.3 (SD). Conclusions: An algorithm based upon the ratio of the area under the curve for daily E3G and PDG levels along with a relative PDG increase offers another approach to time the phase of cycle. This may have applications for NFP/FAMs and clinical evaluation of ovarian function.

Defining the architecture of cerebrospinal fluid cellular communities in neuroinflammatory diseases

Cerebrospinal fluid (CSF) biomarkers are important for multiple sclerosis (MS) diagnosis. Moreover, absent of autopsy or biopsy tissue, CSF is the most relevant source for studying the immune cells involved in MS pathophysiology. Single-cell RNA sequencing (scRNA-seq) provides new opportunities to advance our understanding of disease-associated changes in CSF immune cells. Here, using scRNA-seq data generated from 58 CSF and 10 PBMC samples, we provide an updated atlas of the immune cells present in human CSF in MS and other neuroinflammatory conditions, including novel lymphoid and myeloid cell clusters. Our atlas can thus serve as a reference for future studies of immune cells in neuroinflammation. Our further characterization of CSF myeloid cells suggests that most CSF microglia-like cells resemble two of the previously-described brain microglia signatures. Additionally, our data from a sex-mismatched bone marrow transplant recipient suggest that CSF microglia-like cells are of peripheral origin. Our comparisons between MS and other neuroinflammatory disorders show a highly-specific increase in plasma cells, along with reductions in the proportion of microglia-like cells in MS CSF. Furthermore, our analyses on MS patients receiving anti-CD20 therapy ocrelizumab suggest that the treatment effects are not limited to B cell depletion, and ocrelizumab appears to reverse some MS-associated T and myeloid changes in CSF. Finally, we utilized our atlas to prioritize (1) CSF cell types expressing genes associated with MS susceptibility, and (2) ligand-receptor gene pairs that are differentially expressed in MS CSF, providing targets for further mechanistic and causal investigations in pathophysiology and treatment of MS.

Abstract MP223: CCR2 + Macrophages And T-cells Infiltrate The Murine Heart Following Immune Regulator Treatment With CD40

Immune checkpoint inhibitors have revolutionized cancer treatment but have been associated with severe adverse cardiac events including myocarditis. CD40 agonist (CD40ag) antibodies are immune regulators that have emerged as promising candidates with remarkable efficacy across tumors including those thought to be resistant to established ICIs. To investigate CD40 signaling and its potential for adverse cardiac events, C57BL/6J, CCR2-GFP, and CCR2-knockout mice were injected with CD40ag or isotype antibodies for 7 days. Flow cytometry showed a specific increase in CCR2 + macrophages that was independent of monocyte recruitment. There was no change in neutrophil recruitment and only a modest increase in dendritic cells was evident. We also observed a significant expansion of CD4 and CD8 T-cells that displayed an effector memory phenotype. Bulk cardiac tissue gene expression analysis (Figure A) revealed that CD40 activation upregulates multiple inflammatory cytokines (Ifng, Tnf, Il-12β) and chemokines (Ccl3, Ccl5, Ccl7, Cxcl9, Cxcl10), which are regulated by interferon gamma and coordinate the activation of APCs and T-cells. Finally, RNA in situ hybridization (Figure B-C) demonstrated increased levels of Cxcl9 (red) expression in cells consistent with CCR2 + (white) macrophages when comparing isotype control to treatment with CD40ag. These findings reveal that CD40 activation results in a robust expansion of CCR2 + macrophages and activation of T-cells within the heart, initiating a feed forward loop of activation that is mediated by interferon gamma and generates inflammatory cytotoxic mediators that may lead to myocardial injury.

Specific Increase in Joint Neutrophil Extracellular Traps and Its Relation to Interleukin 6 in Autoimmune Arthritis

Neutrophils and their extracellular traps have been shown to play an important role in the pathogenesis of rheumatoid arthritis (RA), but the detailed mechanisms in joints are still unclear, and their regulation remains to be solved. Here, we explored neutrophil extracellular trap (NET)osis in experimental models of arthritis and further investigated the effects of interleukin-6 (IL-6) inhibition in neutrophils and NETosis. In skins of peptide GPI-induced arthritis (pGIA), citrullinated protein was detected as well as citrullinated histone expression in immunized skin but this was not specific to pGIA. Citrullinated histone expression in pGIA joints was specific to pGIA and was merged with neutrophil elastase, suggesting NETosis. Neutrophils in joints tend to upregulate IL-6 receptors when compared with bone marrow neutrophils. Administration of mouse anti-IL-6 receptor antibodies in pGIA suppressed arthritis in association with a decrease in neutrophil infiltration and NETosis in joints. In the plasma of RA patients, citrullinated protein was significantly reduced after tocilizumab treatment. Our results suggest that IL-6 enhances neutrophil chemotaxis and NETosis in inflammatory joints and could be the source of citrullinated proteins.

The Associations between COMT and MAO-B Genetic Variants with Negative Symptoms in Patients with Schizophrenia

Negative symptoms of schizophrenia, including anhedonia, represent a heavy burden on patients and their relatives. These symptoms are associated with cortical hypodopamynergia and impaired striatal dopamine release in response to reward stimuli. Catechol-O-methyltransferase (COMT) and monoamine oxidase type B (MAO-B) degrade dopamine and affect its neurotransmission. The study determined the association between COMT rs4680 and rs4818, MAO-B rs1799836 and rs6651806 polymorphisms, the severity of negative symptoms, and physical and social anhedonia in schizophrenia. Sex-dependent associations were detected in a research sample of 302 patients with schizophrenia. In female patients with schizophrenia, the presence of the G allele or GG genotype of COMT rs4680 and rs4818, as well as GG haplotype rs4818-rs4680, which were all related to higher COMT activity, was associated with an increase in several dimensions of negative symptoms and anhedonia. In male patients with schizophrenia, carriers of the MAO-B rs1799836 A allele, presumably associated with higher MAO-B activity, had a higher severity of alogia, while carriers of the A allele of the MAO-B rs6651806 had a higher severity of negative symptoms. These findings suggest that higher dopamine degradation, associated with COMT and MAO-B genetic variants, is associated with a sex-specific increase in the severity of negative symptoms in schizophrenia patients.

Contraction-dependent intracortical inhibition supports gravity-efficient motor control

Efficient control of voluntary movements along the gravity axis requires adapted shifts in muscular contraction modes. In daily life, rising the arm up involves shortening (i.e., concentric) contractions of arm flexors, while the reverse movement can rely on lengthening (i.e., eccentric) contractions of the same muscles with the help of gravity force. Although this muscular-control mode is universal, the neuromuscular mechanisms that subserve the control of such gravity-oriented movements remain unknown. In this study, we designed two neurophysiological experiments that allowed tracking modulations of cortical, spinal, and muscular outputs of arm flexors while healthy humans carried out vertical pointing movements. In conditions where upward and downward movements revealed kinematic features reminiscent of optimal motor commands (i.e., directional asymmetries), we report fine contraction-dependent modulations of the corticospinal output. The overall corticospinal excitability dropped during lengthening contractions (downward movements) compared with shortening contractions (upward movements). Specifically, we did not observe any change in spinal motoneuron responsiveness from cervicomedullary stimulations but a specific increase in intracortical inhibition during lengthening vs. shortening contractions. We discuss these fine contraction-dependent modulations of the supraspinal motor output in the light of feedforward mechanisms that may support gravity-tuned motor control. Generally, these results shed a new perspective on the neural policy that optimize movement control along the gravity axis.

Immediate Insulin Treatment Prevents Diabetes-Induced Gut Region-Specific Increase in the Number of Myenteric Serotonergic Neurons

To evaluate the effects of hyperglycemia and insulin treatment on the proportion of serotonin-immunoreactive (5-HT-IR) myenteric neurons, samples were taken from the duodenum, ileum, and colon of diabetic, insulin-treated diabetic, and control rats 10 weeks after the onset of streptozotocin-induced hyperglycemia. Myenteric whole-mount preparations were immunostained with anti-5-HT and pan-neuronal anti-HuCD markers. In controls, the 5-HT-IR myenteric neurons represent a small proportion (~2.5%) of the total neuronal number in the investigated gut segments. The proportion of 5-HT-IR myenteric neurons was significantly higher in the duodenum (p < 0.01) and colon (p < 0.0001) of diabetic rats compared to the controls but exhibited a slight increase in the ileum. Immediate insulin treatment resulted in a significantly lower proportion of myenteric 5-HT-IR neurons in each segment (duodenum p < 0.0001; ileum p < 0.01; and colon p < 0.0001) compared to the untreated diabetics. Our study demonstrates that the proportion of 5-HT-IR myenteric neurons was enhanced in type 1 diabetes in a region-specific manner. Immediate insulin treatment prevents a higher hyperglycemia-induced amount of 5-HT-IR neurons and restores it to the control level in each investigated gut segment. Despite the low proportion of 5-HT-IR myenteric neurons, hyperglycemia-related changes of these neurons may play a crucial role in gastrointestinal symptoms in type 1 diabetes.

Antibody fucosylation predicts disease severity in secondary dengue infection

Although antiviral antibodies generally confer protective functions, antibodies against dengue virus (DENV) are associated with enhanced disease susceptibility. Antibodies can mediate DENV infection of leukocytes via Fcγ receptors, likely contributing to dengue disease pathogenesis. To determine if this mechanism accounts for variable disease severity, we examined Fab and Fc structures of anti-DENV antibodies from patients before and after infection and with variable disease outcomes. Neither antibody titers nor neutralizing activity correlated with disease severity in DENV-infected populations. Rather, DENV infection induced a specific increase in immunoglobulin G1 (IgG1) afucosylation, and the levels of afucosylated IgG1 were predictive of dengue disease severity. Thus, the IgG1 fucosylation status represents a robust prognostic tool for dengue disease, highlighting the key role of the Fc glycan structure in dengue pathogenesis.

Cytochrome bd promotes Escherichia coli biofilm antibiotic tolerance by regulating accumulation of noxious chemicals

Nutrient gradients in biofilms cause bacteria to organize into metabolically versatile communities capable of withstanding threats from external agents including bacteriophages, phagocytes, and antibiotics. We previously determined that oxygen availability spatially organizes respiration in uropathogenic Escherichia coli biofilms, and that the high-affinity respiratory quinol oxidase cytochrome bd is necessary for extracellular matrix production and biofilm development. In this study we investigate the physiologic consequences of cytochrome bd deficiency in biofilms and determine that loss of cytochrome bd induces a biofilm-specific increase in expression of general diffusion porins, leading to elevated outer membrane permeability. In addition, loss of cytochrome bd impedes the proton mediated efflux of noxious chemicals by diminishing respiratory flux. As a result, loss of cytochrome bd enhances cellular accumulation of noxious chemicals and increases biofilm susceptibility to antibiotics. These results identify an undescribed link between E. coli biofilm respiration and stress tolerance, while suggesting the possibility of inhibiting cytochrome bd as an antibiofilm therapeutic approach.