COVID-19 booster enhances IgG mediated viral neutralization by human milk in vitro.

Background: Facilitated by the inability to vaccinate, and an immature immune system, COVID-19 remains a leading cause of death among children. Vaccinated lactating mothers produce specific SARS-CoV-2 antibodies in their milk, capable of neutralizing the virus in vitro. Our objective for this study is to assess the effect of COVID-19 booster dose on SARS-CoV-2 antibody concentration and viral neutralization in milk, plasma, and infant stool. Methods: Thirty-nine mothers and 25 infants were enrolled from December 2020 to May 2022. Milk, maternal plasma, and infants' stool were collected at various time-points up to 12 months following mRNA COVID-19 vaccination. A subgroup of 14 mothers received a booster dose. SARS-CoV-2 antibody levels and their neutralization capacities were assessed. Results: Booster vaccination led to significantly higher IgG levels within human milk and breastfed infants' stool. In vitro neutralization of VSV-gfp-SARS-CoV-2-S-gp, a laboratory safe SARS-CoV-2 like pseudovirus, improved following the booster, with a 90% increase in plasma neutralization and a 60% increase in milk neutralization. We found that post-booster neutralization by human milk was highly correlated to SARS-CoV-2 IgG level. In support of our correlation result, Protein G column depletion of IgG in milk yielded a significant reduction in viral neutralization (p = 0.04). Discussion: The substantial increase in neutralizing IgG levels in milk and breastfed infants' stool post-booster, coupled with the decrease in milk neutralization capabilities upon IgG depletion, underscores the efficacy of booster doses in augmenting the immune response against SARS-CoV-2 in human milk.

SOCS1-KIR Peptide in PEGDA Hydrogels Reduces Pro-Inflammatory Macrophage Activation.

Macrophages modulate the wound healing cascade by adopting different phenotypes such as pro-inflammatory (M1) or pro-wound healing (M2). To reduce M1 activation, the JAK/STAT pathway can be targeted by using suppressors of cytokine signaling (SOCS1) proteins. Recently a peptide mimicking the kinase inhibitory region (KIR) of SOCS1 has been utilized to manipulate the adaptive immune response. However, the utilization of SOCS1-KIR to reduce pro-inflammatory phenotype in macrophages is yet to be investigated in a biomaterial formulation. This study introduces a PEGDA hydrogel platform to investigate SOCS1-KIR as a macrophage phenotype manipulating peptide. Immunocytochemistry, cytokine secretion assays, and gene expression analysis for pro-inflammatory macrophage markers in 2D and 3D experiments demonstrate a reduction in M1 activation due to SOCS1-KIR treatment. The retention of SOCS1-KIR in the hydrogel through release assays and diffusion tests is demonstrated. The swelling ratio of the hydrogel also remains unaffected with the entrapment of SOCS1-KIR. This study elucidates how SOCS1-KIR peptide in PEGDA hydrogels can be utilized as an effective therapeutic for macrophage manipulation.

Detection of SARS-CoV-2 IgA and IgG in human milk and breastfeeding infant stool 6 months after maternal COVID-19 vaccination.

OBJECTIVE: Assess presence, durability, and neutralization capacity of SARS-CoV-2-specific antibodies in breastfeeding infants' stool, mother's plasma and milk following maternal vaccination. DESIGN: Thirty-seven mothers and 25 infants were enrolled between December 2020 and November 2021 for this prospective observational study. All mothers were vaccinated during lactation except three, which were vaccinated during pregnancy. Milk, maternal plasma, and infants' stool was collected pre-vaccination and at periods up to 6 months following COVID-19 vaccine series initiation/completion. SARS-CoV-2 antibody levels and their neutralization capacities were assessed. RESULTS: SARS-CoV-2-specific IgA and IgG levels were higher in infant stool post-maternal vaccination amongst milk-fed compared to controls. Maternal SARS-CoV-2-specific IgA and IgG concentrations decreased over 6 months post-vaccination but remained higher than pre-vaccination levels. We observed improved neutralization capacity in milk and plasma after COVID-19 vaccination. CONCLUSIONS: The presence of SARS-CoV-2-specific antibodies in infant stool following maternal vaccination offers further evidence of the lasting transfer of these antibodies through breastfeeding.

Microscale 3-D Capacitance Tomography with a CMOS Sensor Array.

Electrical capacitance tomography (ECT) is a non-optical imaging technique in which a map of the interior permittivity of a volume is estimated by making capacitance measurements at its boundary and solving an inverse problem. While previous ECT demonstrations have often been at centimeter scales, ECT is not limited to macroscopic systems. In this paper, we demonstrate ECT imaging of polymer microspheres and bacterial biofilms using a CMOS microelectrode array, achieving spatial resolution of 10 microns. Additionally, we propose a deep learning architecture and an improved multi-objective training scheme for reconstructing out-of-plane permittivity maps from the sensor measurements. Experimental results show that the proposed approach is able to resolve microscopic 3-D structures, achieving 91.5% prediction accuracy on the microsphere dataset and 82.7% on the biofilm dataset, including an average of 4.6% improvement over baseline computational methods.

Detection of SARS-CoV-2 IgA and IgG in human milk and breastfeeding infant stool 6 months after maternal COVID-19 vaccination.

Objective Assess the presence, durability, and neutralization capacity of SARS-CoV-2-specific antibodies in breastfeeding infants' stools, mother's plasma, and human milk following maternal vaccination. Design Thirty-seven mothers and 25 infants were enrolled between December 2020 and November 2021 for this prospective observational study. Human milk, maternal plasma, and infants' stools were collected pre-vaccination and at periods up to 6 months following COVID-19 vaccine series initiation/completion. SARS-CoV-2 antibody levels and their neutralization capacities were assessed in collected samples. Results SARS-CoV-2-specific IgA and IgG levels were higher in infant stool post-maternal vaccination amongst milk-fed compared to pre-COVID controls. Human milk and plasma SARS-CoV-2-specific IgA and IgG concentrations decreased over 6 months post-vaccination but remained higher than pre-vaccination levels. We observed improved neutralization capacity in milk antibodies over time. Conclusions The presence of neutralizing SARS-CoV-2-specific antibodies in infant stool following maternal vaccination offers further evidence of the lasting transfer of these antibodies through breastfeeding and their protective effect.

Localized electrical stimulation triggers cell-type-specific proliferation in biofilms.

Biological systems ranging from bacteria to mammals utilize electrochemical signaling. Although artificial electrochemical signals have been utilized to characterize neural tissue responses, the effects of such stimuli on non-neural systems remain unclear. To pursue this question, we developed an experimental platform that combines a microfluidic chip with a multielectrode array (MiCMA) to enable localized electrochemical stimulation of bacterial biofilms. The device also allows for the simultaneous measurement of the physiological response within the biofilm with single-cell resolution. We find that the stimulation of an electrode locally changes the ratio of the two major cell types comprising Bacillus subtilis biofilms, namely motile and extracellular-matrix-producing cells. Specifically, stimulation promotes the proliferation of motile cells but not matrix cells, even though these two cell types are genetically identical and reside in the same microenvironment. Our work thus reveals that an electronic interface can selectively target bacterial cell types, enabling the control of biofilm composition and development.

Open label safety and efficacy pilot to study mitigation of equine recurrent uveitis through topical suppressor of cytokine signaling-1 mimetic peptide.

Equine recurrent uveitis (ERU) is a painful and debilitating autoimmune disease and represents the only spontaneous model of human recurrent uveitis (RU). Despite the efficacy of existing treatments, RU remains a leading cause of visual handicap in horses and humans. Cytokines, which utilize Janus kinase 2 (Jak2) for signaling, drive the inflammatory processes in ERU that promote blindness. Notably, suppressor of cytokine signaling 1 (SOCS1), which naturally limits the activation of Jak2 through binding interactions, is often deficient in autoimmune disease patients. Significantly, we previously showed that topical administration of a SOCS1 peptide mimic (SOCS1-KIR) mitigated induced rodent uveitis. In this pilot study, we test the potential to translate the therapeutic efficacy observed in experimental rodent uveitis to equine patient disease. Through bioinformatics and peptide binding assays we demonstrate putative binding of the SOCS1-KIR peptide to equine Jak2. We also show that topical, or intravitreal injection of SOCS1-KIR was well tolerated within the equine eye through physical and ophthalmic examinations. Finally, we show that topical SOCS1-KIR administration was associated with significant clinical ERU improvement. Together, these results provide a scientific rationale, and supporting experimental evidence for the therapeutic use of a SOCS1 mimetic peptide in RU.

CIFDock: A novel CHARMM-based flexible receptor-flexible ligand docking protocol.

Docking studies play a critical role in the current workflow of drug discovery. However, limitations may often arise through factors including inadequate ligand sampling, a lack of protein flexibility, scoring function inadequacies (e.g., due to metals, co-factors, etc.), and difficulty in retaining explicit water molecules. Herein, we present a novel CHARMM-based induced fit docking (CIFDock) workflow that can circumvent these limitations by employing all-atom force fields coupled to enhanced sampling molecular dynamics procedures. Self-guided Langevin dynamics simulations are used to effectively sample relevant ligand conformations, side chain orientations, crystal water positions, and active site residue motion. Protein flexibility is further enhanced by dynamic sampling of side chain orientations using an expandable rotamer library. Steps in the procedure consisting of fixing individual components (e.g., the ligand) while sampling the other components (e.g., the residues in the active site of the protein) allow for the complex to adapt to conformational changes. Ultimately, all components of the complex-the protein, ligand, and waters-are sampled simultaneously and unrestrained with SGLD to capture any induced fit effects. This modular flexible docking procedure is automated using CHARMM scripting, interfaced with SLURM array processing, and parallelized to use the desired number of processors. We validated the CIFDock procedure by performing cross-docking studies using a data set comprised of 21 pharmaceutically relevant proteins. Five variants of the CHARMM-based SWISSDOCK scoring functions were created to quantify the results of the final generated poses. Results obtained were comparable to, or in some cases improved upon, commercial docking program data.

Programmable Electrochemical Stimulation on a Large-Scale CMOS Microelectrode Array.

In this paper we present spatio-temporally controlled electrochemical stimulation of aqueous samples using an integrated CMOS microelectrode array with 131,072 pixels. We demonstrate programmable gold electrodeposition in arbitrary spatial patterns, controllable electrolysis to produce microscale hydrogen bubbles, and spatially targeted electrochemical pH modulation. Dense spatially-addressable electrochemical stimulation is important for a wide range of bioelectronics applications.

Toward Bacterial Bioelectric Signal Transduction.

Bacteria are electrically powered organisms; cells maintain an electrical potential across their plasma membrane as a source of free energy to drive essential processes. In recent years, however, bacterial membrane potential has been increasingly recognized as dynamic. Those dynamics have been implicated in diverse physiological functions and behaviors, including cell division and cell-to-cell signaling. In eukaryotic cells, such dynamics play major roles in coupling bioelectrical stimuli to changes in internal cell states. Neuroscientists and physiologists have established detailed molecular pathways that transduce eukaryotic membrane potential dynamics to physiological and gene expression responses. We are only just beginning to explore these intracellular responses to bioelectrical activity in bacteria. In this review, we summarize progress in this area, including evidence of gene expression responses to stimuli from electrodes and mechanically induced membrane potential spikes. We argue that the combination of provocative results, missing molecular detail, and emerging tools makes the investigation of bioelectrically induced long-term intracellular responses an important and rewarding effort in the future of microbiology.

Detection of SARS-CoV-2-Specific IgA in the Human Milk of COVID-19 Vaccinated Lactating Health Care Workers.

Background: In 2019, a deadly virus known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), responsible for coronavirus disease 2019 (COVID-19), emerged. In December 2020, two mRNA-based COVID-19 vaccines were approved for use in the United States, which provide immunity to those receiving the vaccine. Maternally derived antibodies are a key element of infants' immunity. Certain vaccines given to pregnant and lactating mothers provide immunity to infants through transmission across the placenta, umbilical cord (IgG), and human milk (IgA). Human milk produced by mothers with a history of COVID-19 infection contains SARS-CoV-2 IgA and IgG. The purpose of this study is to determine whether SARS-CoV-2-specific immunoglobulins are found in human milk after the COVID-19 vaccination, and to characterize the types of immunoglobulins present. Methods: This is a prospective observational study conducted at Shands Hospital, University of Florida, from December 2020 to March 2021. Twenty-two lactating health care workers who received the SARS-CoV-2 mRNA vaccine (Pfizer/BioNTech or Moderna) made up the sample group. Plasma and human milk were collected at three time points (prevaccination, post-first vaccine dose, and post-second vaccine dose). SARS-CoV-2-specific IgA and IgG in human milk and in plasma were measured by enzyme-linked immunosorbent assay (ELISA). Maternal demographics were compiled. Results: We found significant secretion of SARS-CoV-2-specific IgA and IgG in human milk and plasma after SARS-CoV-2 vaccination. Conclusions: Our results show that the mRNA-based COVID-19 vaccines induce SARS-CoV-2-specific IgA and IgG secretion in human milk. Further studies are needed to determine the duration of this immune response, its capacity to neutralize the COVID-19 virus, the transfer of passive immunity to breastfeeding infants, and the potential therapeutic use of human milk IgA to combat SARS-CoV-2 infections and COVID-19.

Suppressor of cytokine signaling-1 mimetic peptides attenuate lymphocyte activation in the MRL/lpr mouse autoimmune model.

Autoimmune diseases are driven largely by a pathogenic cytokine milieu produced by aberrantly activated lymphocytes. Many cytokines, including interferon gamma (IFN-γ), utilize the JAK/STAT pathway for signal propagation. Suppressor of Cytokine Signaling-1 (SOCS1) is an inducible, intracellular protein that regulates IFN-γ signaling by dampening JAK/STAT signaling. Using Fas deficient, MRL/MpJ-Faslpr/J (MRL/lpr) mice, which develop lupus-like disease spontaneously, we tested the hypothesis that a peptide mimic of the SOCS1 kinase inhibitory region (SOCS1-KIR) would inhibit lymphocyte activation and modulate lupus-associated pathologies. Consistent with in vitro studies, SOCS1-KIR intraperitoneal administration reduced the frequency, activation, and cytokine production of memory CD8+ and CD4+ T lymphocytes within the peripheral blood, spleen, and lymph nodes. In addition, SOCS1-KIR administration reduced lymphadenopathy, severity of skin lesions, autoantibody production, and modestly reduced kidney pathology. On a cellular level, peritoneal SOCS1-KIR administration enhanced Foxp3 expression in total splenic and follicular regulatory T cells, reduced the effector memory/naïve T lymphocyte ratio for both CD4+ and CD8+ cells, and reduced the frequency of GL7+ germinal center enriched B cells. Together, these data show that SOCS1-KIR treatment reduced auto-reactive lymphocyte effector functions and suggest that therapeutic targeting of the SOCS1 pathway through peptide administration may have efficacy in mitigating autoimmune pathologies.

Ameliorative Properties of Boronic Compounds in In Vitro and In Vivo Models of Alzheimer's Disease.

Alzheimer's disease (AD) is characterized by amyloid (Aß) aggregation, hyperphosphorylated tau, neuroinflammation, and severe memory deficits. Reports that certain boronic compounds can reduce amyloid accumulation and neuroinflammation prompted us to compare trans-2-phenyl-vinyl-boronic-acid-MIDA-ester (TPVA) and trans-beta-styryl-boronic-acid (TBSA) as treatments of deficits in in vitro and in vivo models of AD. We hypothesized that these compounds would reduce neuropathological deficits in cell-culture and animal models of AD. Using a dot-blot assay and cultured N2a cells, we observed that TBSA inhibited Aß42 aggregation and increased cell survival more effectively than did TPVA. These TBSA-induced benefits were extended to C. elegans expressing Aß42 and to the 5xFAD mouse model of AD. Oral administration of 0.5 mg/kg dose of TBSA or an equivalent amount of methylcellulose vehicle to groups of six- and 12-month-old 5xFAD or wild-type mice over a two-month period prevented recognition- and spatial-memory deficits in the novel-object recognition and Morris-water-maze memory tasks, respectively, and reduced the number of pyknotic and degenerated cells, Aß plaques, and GFAP and Iba-1 immunoreactivity in the hippocampus and cortex of these mice. These findings indicate that TBSA exerts neuroprotective properties by decreasing amyloid plaque burden and neuroinflammation, thereby preventing neuronal death and preserving memory function in the 5xFAD mice.

Human Regulatory T Cells From Umbilical Cord Blood Display Increased Repertoire Diversity and Lineage Stability Relative to Adult Peripheral Blood.

The human T lymphocyte compartment is highly dynamic over the course of a lifetime. Of the many changes, perhaps most notable is the transition from a predominantly naïve T cell state at birth to the acquisition of antigen-experienced memory and effector subsets following environmental exposures. These phenotypic changes, including the induction of T cell exhaustion and senescence, have the potential to negatively impact efficacy of adoptive T cell therapies (ACT). When considering ACT with CD4+CD25+CD127-/lo regulatory T cells (Tregs) for the induction of immune tolerance, we previously reported ex vivo expanded umbilical cord blood (CB) Tregs remained more naïve, suppressed responder T cells equivalently, and exhibited a more diverse T cell receptor (TCR) repertoire compared to expanded adult peripheral blood (APB) Tregs. Herein, we hypothesized that upon further characterization, we would observe increased lineage heterogeneity and phenotypic diversity in APB Tregs that might negatively impact lineage stability, engraftment capacity, and the potential for Tregs to home to sites of tissue inflammation following ACT. We compared the phenotypic profiles of human Tregs isolated from CB versus the more traditional source, APB. We conducted analysis of fresh and ex vivo expanded Treg subsets at both the single cell (scRNA-seq and flow cytometry) and bulk (microarray and cytokine profiling) levels. Single cell transcriptional profiles of pre-expansion APB Tregs highlighted a cluster of cells that showed increased expression of genes associated with effector and pro-inflammatory phenotypes (CCL5, GZMK, CXCR3, LYAR, and NKG7) with low expression of Treg markers (FOXP3 and IKZF2). CB Tregs were more diverse in TCR repertoire and homogenous in phenotype, and contained fewer effector-like cells in contrast with APB Tregs. Interestingly, expression of canonical Treg markers, such as FOXP3, TIGIT, and IKZF2, were increased in CB CD4+CD127+ conventional T cells (Tconv) compared to APB Tconv, post-expansion, implying perinatal T cells may adopt a default regulatory program. Collectively, these data identify surface markers (namely CXCR3) that could be depleted to improve purity and stability of APB Tregs, and support the use of expanded CB Tregs as a potentially optimal ACT modality for the treatment of autoimmune and inflammatory diseases.

Encoding Membrane-Potential-Based Memory within a Microbial Community.

Cellular membrane potential plays a key role in the formation and retrieval of memories in the metazoan brain, but it remains unclear whether such memory can also be encoded in simpler organisms like bacteria. Here, we show that single-cell-level memory patterns can be imprinted in bacterial biofilms by light-induced changes in the membrane potential. We demonstrate that transient optical perturbations generate a persistent and robust potassium-channel-mediated change in the membrane potential of bacteria within the biofilm. The light-exposed cells respond in an anti-phase manner, relative to unexposed cells, to both natural and induced oscillations in extracellular ion concentrations. This anti-phase response, which persists for hours following the transient optical stimulus, enables a direct single-cell resolution visualization of spatial memory patterns within the biofilm. The ability to encode robust and persistent membrane-potential-based memory patterns could enable computations within prokaryotic communities and suggests a parallel between neurons and bacteria.

Spiral Wave Propagation in Communities with Spatially Correlated Heterogeneity.

Many multicellular communities propagate signals in a directed manner via excitable waves. Cell-to-cell heterogeneity is a ubiquitous feature of multicellular communities, but the effects of heterogeneity on wave propagation are still unclear. Here, we use a minimal FitzHugh-Nagumo-type model to investigate excitable wave propagation in a two-dimensional heterogeneous community. The model shows three dynamic regimes in which waves either propagate directionally, die out, or spiral indefinitely, and we characterize how these regimes depend on the heterogeneity parameters. We find that in some parameter regimes, spatial correlations in the heterogeneity enhance directional propagation and suppress spiraling. However, in other regimes, spatial correlations promote spiraling, a surprising feature that we explain by demonstrating that these spirals form by a second, distinct mechanism. Finally, we characterize the dynamics using techniques from percolation theory. Despite the fact that percolation theory does not completely describe the dynamics quantitatively because it neglects the details of the excitable propagation, we find that it accounts for the transitions between the dynamic regimes and the general dependency of the spiral period on the heterogeneity and thus provides important insights. Our results reveal that the spatial structure of cell-to-cell heterogeneity can have important consequences for signal propagation in cellular communities.

Statistics of correlated percolation in a bacterial community.

Signal propagation over long distances is a ubiquitous feature of multicellular communities, but cell-to-cell variability can cause propagation to be highly heterogeneous. Simple models of signal propagation in heterogenous media, such as percolation theory, can potentially provide a quantitative understanding of these processes, but it is unclear whether these simple models properly capture the complexities of multicellular systems. We recently discovered that in biofilms of the bacterium Bacillus subtilis, the propagation of an electrical signal is statistically consistent with percolation theory, and yet it is reasonable to suspect that key features of this system go beyond the simple assumptions of basic percolation theory. Indeed, we find here that the probability for a cell to signal is not independent from other cells as assumed in percolation theory, but instead is correlated with its nearby neighbors. We develop a mechanistic model, in which correlated signaling emerges from cell division, phenotypic inheritance, and cell displacement, that reproduces the experimentally observed correlations. We find that the correlations do not significantly affect the spatial statistics, which we rationalize using a renormalization argument. Moreover, the fraction of signaling cells is not constant in space, as assumed in percolation theory, but instead varies within and across biofilms. We find that this feature lowers the fraction of signaling cells at which one observes the characteristic power-law statistics of cluster sizes, consistent with our experimental results. We validate the model using a mutant biofilm whose signaling probability decays along the propagation direction. Our results reveal key statistical features of a correlated signaling process in a multicellular community. More broadly, our results identify extensions to percolation theory that do or do not alter its predictions and may be more appropriate for biological systems.

Therapeutic Implication of SOCS1 Modulation in the Treatment of Autoimmunity and Cancer.

The suppressor of cytokine signaling (SOCS) family of intracellular proteins has a vital role in the regulation of the immune system and resolution of inflammatory cascades. SOCS1, also called STAT-induced STAT inhibitor (SSI) or JAK-binding protein (JAB), is a member of the SOCS family with actions ranging from immune modulation to cell cycle regulation. Knockout of SOCS1 leads to perinatal lethality in mice and increased vulnerability to cancer, while several SNPs associated with the SOCS1 gene have been implicated in human inflammation-mediated diseases. In this review, we describe the mechanism of action of SOCS1 and its potential therapeutic role in the prevention and treatment of autoimmunity and cancer. We also provide a brief outline of the other JAK inhibitors, both FDA-approved and under investigation.