Human Amnion-Derived Mesenchymal Stromal Cells: A New Potential Treatment for Carbapenem-Resistant Enterobacterales in Decompensated Cirrhosis

Background: Spontaneous bacterial peritonitis (SBP) is a severe and often fatal infection in patients with decompensated cirrhosis and ascites. The only cure for SBP is antibiotic therapy, but the emerging problem of bacterial resistance requires novel therapeutic strategies. Human amniotic mesenchymal stromal cells (hA-MSCs) possess immunomodulatory and anti-inflammatory properties that can be harnessed as a therapy in such a context. Methods: An in vitro applications of hA-MSCs in ascitic fluid (AF) of cirrhotic patients, subsequently infected with carbapenem-resistant Enterobacterales, was performed. We evaluated the effects of hA-MSCs on bacterial load, innate immunity factors, and macrophage phenotypic expression. Results: hA-MSCs added to AF significantly reduce the proliferation of both bacterial strains at 24 h and diversely affect M1 and M2 polarization, C3a complement protein, and ficolin 3 concentrations during the course of infection, in a bacterial strain-dependent fashion. Conclusion: This study shows the potential usefulness of hA-MSC in treating ascites infected with carbapenem-resistant bacteria and lays the foundation to further investigate antibacterial and anti-inflammatory roles of hA-MSC in in vivo models.

Hypoxia-Ischemia During a Neonatal Sensitive Period Alters Purkinje Neuron Dendritic Complexity Differently in Male and Female Rats

Perinatal hypoxia-ischemia (HI) is a major health issue with no effective therapies beyond head cooling. Notably, male infants are at a greater risk for HI and exhibit more extreme deficits than females. Extensive clinical evidence indicates that perinatal HI impacts the developing cerebellum, yet this region has been largely ignored in preclinical models. Using a modified version of the Rice-Vannucci rat model for HI injury at postnatal day 10, we find reductions in dendritic complexity of Purkinje neurons in males one week later. Females exhibited modest but opposite effects, with slight increases in dendritic complexity, based on Sholl analysis. A custom-made NanoString panel for quantifying mRNAs associated with development, inflammation, and sex differences found almost no commonality in the response to HI in males versus females, with males up-regulating genes associated with microglia activity whereas females increased expression of a protective complement protein, but also of enzymes associated with endocannabinoids and prostaglandins. Both sexes exhibited a reduction in the GABA-synthetic enzymes, GAD-65 and GAD-67, after HI, suggesting increased excitotoxicity, but why males suffered more damage to the Purkinje neurons is unknown.

The burden of illness in patients with paroxysmal nocturnal hemoglobinuria receiving treatment with the C5-inhibitors eculizumab or ravulizumab: results from a US patient survey

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare and life-threatening disease with symptoms of hemolysis and thrombosis. Current therapies for this complement-mediated disease rely predominantly on inhibition of the C5 complement protein. However, data on treatment responses and quality of life in C5-inhibitor (C5i)-treated PNH patients are scarce. The objective of this study was to determine C5i treatment effects on clinical parameters, PNH symptoms, quality of life, and resource use for PNH patients. This cross-sectional study surveyed 122 individuals in the USA receiving treatment for PNH with C5-targeted monoclonal antibodies, eculizumab (ECU) or ravulizumab (RAV). Despite most patients receiving C5i therapy for ≥ 3 months (ECU 100%, n = 35; RAV 95.4%, n = 83), many patients remained anemic with hemoglobin levels ≤ 12 g/dL in 87.5% (n = 28/32) and 82.9% (n = 68/82) of ECU and RAV recipients, respectively. A majority of patients on ECU (88.6%; n = 31/35) and RAV (74.7%; n = 65/87) reported fatigue symptoms. Among PNH patients receiving C5i therapy for ≥ 12 months, some still reported thrombotic events (ECU, 10.0%, n = 1/10; RAV, 23.5%, n = 4/17) and required transfusions within the past year (ECU, 52.2%, n = 12/23; RAV, 22.6%, n = 7/31). Other patient-reported PNH symptoms included breakthrough hemolysis, shortness of breath, and headaches. Patients reported scores below the average population norms on the Functional Assessment of Chronic Illness Therapy (FACIT)-Fatigue and European Organization for Research and Treatment of Cancer Quality of Life Questionnaire Core 30 (EORTC QLQ-C30) scales. Overall, this study found that PNH patients receiving ECU or RAV therapy demonstrated a significant burden of illness, highlighting the need for improved PNH therapies.

Complement-Mediated Selective Tumor Cell Lysis Enabled by Bi-Functional RNA Aptamers

Unlike microbes that infect the human body, cancer cells are descended from normal cells and are not easily recognizable as “foreign” by the immune system of the host. However, if the malignant cells can be specifically earmarked for attack by a synthetic “designator”, the powerful effector mechanisms of the immune response can be conscripted to treat cancer. To implement this strategy, we have been developing aptamer-derived molecular adaptors to invoke synthetic immune responses against cancer cells. Here we describe multi-valent aptamers that simultaneously bind target molecules on the surface of cancer cells and an activated complement protein, which would tag the target molecules and their associated cells as “foreign” and trigger multiple effector mechanisms. Increased deposition of the complement proteins on the surface of cancer cells via aptamer binding to membrane targets could induce the formation of the membrane attack complex or cytotoxic degranulation by phagocytes and natural killer cells, thereby causing irreversible destruction of the targeted cells. Specifically, we designed and constructed a bi-functional aptamer linking EGFR and C3b/iC3b, and used it in a cell-based assay to cause lysis of MDA-MB-231 and BT-20 breast cancer cells, with either human or mouse serum as the source of complement factors.

Semi-Quantitative Multiplex Profiling of the Complement System Identifies Associations of Complement Proteins with Genetic Variants and Metabolites in Age-Related Macular Degeneration

Age-related macular degeneration (AMD) is a major cause of vision loss among the elderly in the Western world. The complement system has been identified as one of the main AMD disease pathways. We performed a comprehensive expression analysis of 32 complement proteins in plasma samples of 255 AMD patients and 221 control individuals using mass spectrometry-based semi-quantitative multiplex profiling. We detected significant associations of complement protein levels with age, sex and body-mass index (BMI), and potential associations of C-reactive protein, factor H related-2 (FHR-2) and collectin-11 with AMD. In addition, we confirmed previously described associations and identified new associations of AMD variants with complement levels. New associations include increased C4 levels for rs181705462 at the C2/CFB locus, decreased vitronectin (VTN) levels for rs11080055 at the TMEM97/VTN locus and decreased factor I levels for rs10033900 at the CFI locus. Finally, we detected significant associations between AMD-associated metabolites and complement proteins in plasma. The most significant complement-metabolite associations included increased high density lipoprotein (HDL) subparticle levels with decreased C3, factor H (FH) and VTN levels. The results of our study indicate that demographic factors, genetic variants and circulating metabolites are associated with complement protein components. We suggest that these factors should be considered to design personalized treatment approaches and to increase the success of clinical trials targeting the complement system.

Serum levels of immunoglobulin and complement in UTI of patients caused by Proteus mirabilis and using AgNPs as antiswarming

The use of plant extracts represents a promising approach for the synthesis of silver nanoparticles (AgNPs). This study reports the low-cost, green synthesis of AgNPs using the extract of clove and black seeds. The biosynthesized AgNPs were confirmed and characterized by analysis of the spectroscopy profile of the UV-visible spectrophotometer. The purpose of the present study is to evaluate the inhibitory effect concentration (MIC) of AgNPs, clove, and black cumin seed extracts on the growth and swarming of P. mirabilis. Clinical isolates of P. mirabilis were isolated from patients suffering from urinary tract infections. Thirteen types of antibiotics were used in the present study to detect their ability to inhibit P. mirabilis's resistance. Immunological findings included the determination of serum levels of IgG, IgM, IgA and complement protein C3 and C4. Results showed that IgG and IgA concentrations significantly increased (1311.13 ± 72.54 and 279 ± 21.31) respectively in UTI patients in comparison to the healthy control group which was 1089.88 ± 37.33 and 117.611 ± 4.19 respectively, While IgM concentrations were increased non significantly in UTI patients (153.331 ± 6.45) in comparison to healthy control (145.2 ± 13.49). Complement components C3 showed a significant increase in UTI patients with mean values of 125.95 ± 6.22 compared to the control group with mean values of 55.191 ± 9.64, while C4 showed statically non-significant among UTI patients in comparison with the control group (35.195 ± 2.34 and 34.371 ± 1.22) respectively.

In vitro, classical complement activation differs by disease severity and between SARS-CoV-2 antigens

Antibodies specific for the spike glycoprotein (S) and nucleocapsid (N) SARS-CoV-2 proteins are typically present during severe COVID-19, and induced to S after vaccination. The binding of viral antigens by antibody can initiate the classical complement pathway. Since complement could play pathological or protective roles at distinct times during SARS-CoV-2 infection we determined levels of antibody-dependent complement activation along the complement cascade. Here, we used an ELISA assay to assess complement protein binding (C1q) and the deposition of C4b, C3b, and C5b to S and N antigens in the presence of anti-SARS-CoV-2 antibodies from different test groups: non-infected, single and double vaccinees, non-hospitalised convalescent (NHC) COVID-19 patients and convalescent hospitalised (ITU-CONV) COVID-19 patients. C1q binding correlates strongly with antibody responses, especially IgG1 levels. However, detection of downstream complement components, C4b, C3b and C5b shows some variability associated with the antigen and subjects studied. In the ITU-CONV, detection of C3b-C5b to S was observed consistently, but this was not the case in the NHC group. This is in contrast to responses to N, where median levels of complement deposition did not differ between the NHC and ITU-CONV groups. Moreover, for S but not N, downstream complement components were only detected in sera with higher IgG1 levels. Therefore, the classical pathway is activated by antibodies to multiple SARS-CoV-2 antigens, but the downstream effects of this activation may differ depending on the specific antigen targeted and the disease status of the subject.

A Functional Human-on-a-Chip Autoimmune Disease Model of Myasthenia Gravis for Development of Therapeutics

Myasthenia gravis (MG) is a chronic and progressive neuromuscular disease where autoantibodies target essential proteins such as the nicotinic acetylcholine receptor (nAChR) at the neuromuscular junction (NMJ) causing muscle fatigue and weakness. Autoantibodies directed against nAChRs are proposed to work by three main pathological mechanisms of receptor disruption: blocking, receptor internalization, and downregulation. Current in vivo models using experimental autoimmune animal models fail to recapitulate the disease pathology and are limited in clinical translatability due to disproportionate disease severity and high animal death rates. The development of a highly sensitive antibody assay that mimics human disease pathology is desirable for clinical advancement and therapeutic development. To address this lack of relevant models, an NMJ platform derived from human iPSC differentiated motoneurons and primary skeletal muscle was used to investigate the ability of an anti-nAChR antibody to induce clinically relevant MG pathology in the serum-free, spatially organized, functionally mature NMJ platform. Treatment of the NMJ model with the anti-nAChR antibody revealed decreasing NMJ stability as measured by the number of NMJs before and after the synchrony stimulation protocol. This decrease in NMJ stability was dose-dependent over a concentration range of 0.01–20 μg/mL. Immunocytochemical (ICC) analysis was used to distinguish between pathological mechanisms of antibody-mediated receptor disruption including blocking, receptor internalization and downregulation. Antibody treatment also activated the complement cascade as indicated by complement protein 3 deposition near the nAChRs. Additionally, complement cascade activation significantly altered other readouts of NMJ function including the NMJ fidelity parameter as measured by the number of muscle contractions missed in response to increasing motoneuron stimulation frequencies. This synchrony readout mimics the clinical phenotype of neurological blocking that results in failure of muscle contractions despite motoneuron stimulations. Taken together, these data indicate the establishment of a relevant disease model of MG that mimics reduction of functional nAChRs at the NMJ, decreased NMJ stability, complement activation and blocking of neuromuscular transmission. This system is the first functional human in vitro model of MG to be used to simulate three potential disease mechanisms as well as to establish a preclinical platform for evaluation of disease modifying treatments (etiology).