Neuron-binding antibody responses are associated with Black ethnicity in multiple sclerosis during natalizumab treatment.

Multiple sclerosis is an inflammatory degenerative condition of the central nervous system that may result in debilitating disability. Several studies over the past twenty years suggest that multiple sclerosis manifests with a rapid, more disabling disease course among individuals identifying with Black or Latin American ethnicity relative to those of White ethnicity. However, very little is known about immunologic underpinnings that may contribute to this ethnicity-associated discordant clinical severity. Given the importance of B cells to multiple sclerosis pathophysiology, and prior work showing increased antibody levels in the cerebrospinal fluid of Black-identifying, compared to White-identifying multiple sclerosis patients, we conducted a cohort study to determine B cell subset dynamics according to both self-reported ethnicity and genetic ancestry over time. Further, we determined relationships between ethnicity, ancestry, and neuron-binding IgG levels. We found significant associations between Black ethnicity and elevated frequencies of class-switched B cell subsets, including memory B cells; double negative two B cells; and antibody-secreting cells. The frequencies of these subsets positively correlated with West African genetic ancestry. We also observed significant associations between Black ethnicity and increased IgG binding to neurons. Our data suggests significantly heightened T cell-dependent B cell responses exhibiting increased titres of neuron-binding antibodies among individuals with multiple sclerosis identifying with the Black African diaspora. Factors driving this immunobiology may promote the greater demyelination, central nervous system atrophy and disability more often experienced by Black-, and Latin American-identifying individuals with multiple sclerosis.

MEMO1 Is Required for Ameloblast Maturation and Functional Enamel Formation.

Coordinated mineralization of soft tissue is central to organismal form and function, while dysregulated mineralization underlies several human pathologies. Oral epithelial-derived ameloblasts are polarized, secretory cells responsible for generating enamel, the most mineralized substance in the human body. Defects in ameloblast development result in enamel anomalies, including amelogenesis imperfecta. Identifying proteins critical in ameloblast development can provide insight into specific pathologies associated with enamel-related disorders or, more broadly, mechanisms of mineralization. Previous studies identified a role for MEMO1 in bone mineralization; however, whether MEMO1 functions in the generation of additional mineralized structures remains unknown. Here, we identify a critical role for MEMO1 in enamel mineralization. First, we show that Memo1 is expressed in ameloblasts and, second, that its conditional deletion from ameloblasts results in enamel defects, characterized by a decline in mineral density and tooth integrity. Histology revealed that the mineralization defects in Memo1 mutant ameloblasts correlated with a disruption in ameloblast morphology. Finally, molecular profiling of ameloblasts and their progenitors in Memo1 oral epithelial mutants revealed a disruption to cytoskeletal-associated genes and a reduction in late-stage ameloblast markers, relative to controls. Collectively, our findings integrate MEMO1 into an emerging network of molecules important for ameloblast development and provide a system to further interrogate the relationship of cytoskeletal and amelogenesis-related defects.

Understanding humoral immunity and multiple sclerosis severity in Black, and Latinx patients.

People identified with Black/African American or Hispanic/Latinx ethnicity are more likely to exhibit a more severe multiple sclerosis disease course relative to those who identify as White. While social determinants of health account for some of this discordant severity, investigation into contributing immunobiology remains sparse. The limited immunologic data stands in stark contrast to the volume of clinical studies describing ethnicity-associated discordant presentation, and to advancement made in our understanding of MS immunopathogenesis over the past several decades. In this perspective, we posit that humoral immune responses offer a promising avenue to better understand underpinnings of discordant MS severity among Black/African American, and Hispanic/Latinx-identifying patients.

Black African and Latino/a identity correlates with increased plasmablasts in MS.

OBJECTIVE: To determine the influence of self-reported Black African and Latin American identity on peripheral blood antibody-secreting cell (ASC) frequency in the context of relapsing-remitting MS. METHODS: In this cross-sectional study, we recruited 74 subjects with relapsing-remitting MS and 24 age-, and self-reported ethno-ancestral identity-matched healthy donors (HDs) to provide peripheral blood study samples. Subjects with MS were either off therapy at the time of study draw or on monthly natalizumab therapy infusions. Using flow cytometry, we assessed peripheral blood mononuclear cells for antibody-secreting B-cell subsets. RESULTS: When stratified by self-reported ethno-ancestry, we identified significantly elevated frequencies of circulating plasmablasts among individuals with MS identifying as Black African or Latin American relative to those of Caucasian ancestry. Ethno-ancestry-specific differences in ASC frequency were observed only among individuals with MS. By contrast, this differential was not observed among HDs. ASCs linked with poorer MS prognosis and active disease, including IgM+- and class-switched CD138+ subsets, were among those significantly increased. CONCLUSION: The enhanced peripheral blood plasmablast signature revealed among Black African or Latin American subjects with MS points to distinct underlying mechanisms associated with MS immunopathogenesis. This dysregulation may contribute to the disease disparity experienced by patient populations of Black African or Latin American ethno-ancestry.

"Extracorporeal Membrane Oxygenation for Greater Poland" Program: How to Save Lives and Develop Organ Donation?

The "ECMO for Greater Poland" program takes full advantage of the extracorporeal membrane oxygenation (ECMO) perfusion therapy opportunities to promote the health of the 3.5 million inhabitants in the region. The main implementation areas are treatment of patients with hypothermia; severe reversible respiratory failure (RRF); critical states resulting in heart failure, that is, cardiac arrest, cardiogenic shock, or acute intoxication; and promotion of the donor after circulatory death (DCD) strategy in selected organ donor cases, after unsuccessful life-saving treatment, to achieve organ recovery. This organizational model is complex and expensive, so we used advanced high-fidelity medical simulation tests to prepare for real-life experience. Over the course of 4 months we performed scenarios including "ECMO for DCD," "ECMO for extended cardiopulmonary resuscitation," "ECMO for RRF," and "ECMO in hypothermia." Soon after these simulations, Maastricht category II DCD procedures were performed involving real patients and resulting in 2 successful double kidney transplantations for the first time in Poland. One month later we treated 2 hypothermia patients (7 adult patients with heart failure and 5 patients with reversible respiratory failure) with ECMO for the first time in the region. Fortunately, we have discovered an important new role of medical simulation. It can be used not only for skills testing but also as a tool to create non-existing procedures and unavailable algorithms. The result of these program activities will promote the care and treatment of patients in critical condition with ECMO therapy as well as increase the potential organ pool from DCDs in the Greater Poland region of Poland.

Alteration of CD39+Foxp3+ CD4 T cell and cytokine levels in EAE/MS following anti-CD52 treatment.

While examining the therapeutic value of anti-CD52 antibody against EAE/MS, we identified a unique subset of CD39+ Tregs in repopulating GALT tissues, a major lymphoid reservoir, which was accompanied by amelioration of disease. Furthermore, anti-CD52 treatment leads to increased expression of BDNF, IL-10, and SMAD3 in the brains of EAE mice. This condition is associated with suppression of IL-17, a critical inflammatory factor in EAE/MS progression. Additionally, we found elevated levels of CD4+CD39+ Tregs in PBMCs of RRMS patients treated with humanized anti-CD52 mAb. Thus, anti-CD52 can affect multiple immune mediated pathways involved in the pathogenesis of EAE/MS.

Induction of gut regulatory CD39+ T cells by teriflunomide protects against EAE.

OBJECTIVE: To determine whether as an orally delivered treatment, teriflunomide, an inhibitor of the mitochondrial enzyme dihydroorotate dehydrogenase approved to treat relapsing forms of multiple sclerosis, could affect gut-associated lymphoid tissue (GALT) immune responses functionally. METHODS: C57BL/6 mice were treated orally with teriflunomide and flow cytometric analysis of immune GALT cells performed ex vivo, and adoptive transfer experiments were used to test the protective effects of GALT regulatory T (Treg) cells. RESULTS: Teriflunomide reduced the percentages of antigen-presenting cells of Peyer patches when compared to controls. Conversely, a significant increase of the relative frequency of CD39+ Treg cells was observed. In vivo, the protective effect of GALT-derived teriflunomide-induced CD39+ Treg cells was established by adoptive transfer into recipient experimental autoimmune encephalomyelitis mice. CONCLUSIONS: Our results identify specific GALT-derived CD39+ Treg cells as a mechanism of action that may contribute to the efficacy of teriflunomide during CNS inflammatory demyelination and as an oral therapeutic in relapsing multiple sclerosis.

A commensal symbiotic factor derived from Bacteroides fragilis promotes human CD39(+)Foxp3(+) T cells and Treg function.

Polysaccharide A (PSA) derived from the human commensal Bacteroides fragilis is a symbiosis factor that stimulates immunologic development within mammalian hosts. PSA rebalances skewed systemic T helper responses and promotes T regulatory cells (Tregs). However, PSA-mediated induction of Foxp3 in humans has not been reported. In mice, PSA-generated Foxp3(+) Tregs dampen Th17 activity thereby facilitating bacterial intestinal colonization while the increased presence and function of these regulatory cells may guard against pathological organ-specific inflammation in hosts. We herein demonstrate that PSA induces expression of Foxp3 along with CD39 among naïve CD4 T cells in vitro while promoting IL-10 secretion. PSA-activated dendritic cells are essential for the mediation of this regulatory response. When cultured with isolated Foxp3(+) Tregs, PSA enriched Foxp3 expression, enhanced the frequency of CD39(+)HLA-DR(+) cells, and increased suppressive function as measured by decreased TNFα expression by LPS-stimulated monocytes. Our findings are the first to demonstrate in vitro induction of human CD4(+)Foxp3(+) T cells and enhanced suppressive function of circulating Foxp3(+) Tregs by a human commensal bacterial symbiotic factor. Use of PSA for the treatment of human autoimmune diseases, in particular multiple sclerosis and inflammatory bowel disease, may represent a new paradigm in the approach to treating autoimmune disease.

An intestinal commensal symbiosis factor controls neuroinflammation via TLR2-mediated CD39 signalling.

The mammalian immune system constitutively senses vast quantities of commensal bacteria and their products through pattern recognition receptors, yet excessive immune reactivity is prevented under homeostasis. The intestinal microbiome can influence host susceptibility to extra-intestinal autoimmune disorders. Here we report that polysaccharide A (PSA), a symbiosis factor for the human intestinal commensal Bacteroides fragilis, protects against central nervous system demyelination and inflammation during experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis, through Toll-like receptor 2 (TLR2). TLR2 mediates tissue-specific expansion of a critical regulatory CD39(+) CD4 T-cell subset by PSA. Ablation of CD39 signalling abrogates PSA control of EAE manifestations and inflammatory cytokine responses. Further, CD39 confers immune-regulatory phenotypes to total CD4 T cells and Foxp3(+) CD4 Tregs. Importantly, CD39-deficient CD4 T cells show an enhanced capability to drive EAE progression. Our results demonstrate the therapeutic potential and underlying mechanism by which an intestinal symbiont product modulates CNS-targeted demyelination.

A commensal bacterial product elicits and modulates migratory capacity of CD39(+) CD4 T regulatory subsets in the suppression of neuroinflammation.

Tolerance established by host-commensal interactions regulates host immunity at both local mucosal and systemic levels. The intestinal commensal strain Bacteroides fragilis elicits immune tolerance, at least in part, via the expression capsular polysaccharide A (PSA). How such niche-specific commensal microbial elements regulate extra-intestinal immune responses, as in the brain, remains largely unknown. We have recently shown that oral treatment with PSA suppresses neuro-inflammation elicited during experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. This protection is dependent upon the expansion of immune-regulatory CD4 T cells (Treg) expressing CD39, an ectonucleotidase. Here, we further show that CD39 modulation of purinergic signals enhances migratory phenotypes of both total CD4 T cells and Foxp3(+) CD4 Tregs at central nervous system (CNS) lymphoid-draining sites in EAE in vivo and promotes their migration in vitro. These changes are noted during PSA treatment, which leads to heightened accumulation of CD39(+) CD4 Tregs in the CNS. Deficiency of CD39 abrogates accumulation of Treg during EAE, and is accompanied by elevated Th1/Th17 signals in the CNS and in gut-associated lymphoid tissues. Our results demonstrate that immune-modulatory commensal bacterial products impact the migratory patterns of CD4 Treg during CNS autoimmunity via the regulation of CD39. These observations provide clues as to how intestinal commensal microbiome is able to modulate Treg functions and impact host immunity in the distal site.

Closed system isolation and scalable expansion of human placental mesenchymal stem cells.

Mesenchymal stem cells (MSC) are emerging as a leading cellular therapy for a number of diseases. However, for such treatments to become available as a routine therapeutic option, efficient and cost-effective means for industrial manufacture of MSC are required. At present, clinical grade MSC are manufactured through a process of manual cell culture in specialized cGMP facilities. This process is open, extremely labor intensive, costly, and impractical for anything more than a small number of patients. While it has been shown that MSC can be cultivated in stirred bioreactor systems using microcarriers, providing a route to process scale-up, the degree of numerical expansion achieved has generally been limited. Furthermore, little attention has been given to the issue of primary cell isolation from complex tissues such as placenta. In this article we describe the initial development of a closed process for bulk isolation of MSC from human placenta, and subsequent cultivation on microcarriers in scalable single-use bioreactor systems. Based on our initial data, we estimate that a single placenta may be sufficient to produce over 7,000 doses of therapeutic MSC using a large-scale process.

Structural characterization of a spin-assisted colloid-polyelectrolyte assembly: stratified multilayer thin films.

The assembly of polyelectrolytes and gold nanoparticles yields stratified multilayers with very low roughness and high structural perfection. The films are prepared by spin-assisted layer-by-layer self-assembly (LbL) and are characterized by X-ray reflectivity (XRR), UV-vis spectroscopy, atomic force microscopy (AFM), and transmission electron microscopy (TEM). Typical structures have four repeat units, each of which consists of eight double layers (DL) of poly(sodium 4-styrenesulfonate)/poly(allylamine hydrochloride), one monolayer of gold nanoparticles (10 nm diameter), and another layer of poly(allylamine hydrochloride). XRR scans show small-angle Bragg peaks up to seventh order, evidencing the highly stratified structure. Pronounced Kiessig fringes indicate a low global roughness, which is confirmed by local AFM measurements. TEM images corroborate the layered structure in the growth direction and nicely show the distinct separation of the individual particle layers. An AFM study reveals the lateral gold particle distribution within one individual particle layer. Interestingly, the spin-assisted deposition of polyelectrolytes reduces the roughness induced by the particle layers, leading to self-healing of roughness defects and a rather perfect stratification.

Ultrafast changes of molecular crystal structure induced by dipole solvation.

Femtosecond photoexcitation of organic chromophores in a molecular crystal induces strong changes of the electronic dipole moment via intramolecular charge transfer as is evident from transient vibrational spectra. The structural response of the crystal to the dipole change is mapped directly for the first time by ultrafast x-ray diffraction or diffuse scattering. Changes of diffracted and transmitted x-ray intensity demonstrate an angular rearrangement of molecules around excited dipoles following the 10 ps kinetics of charge transfer and leaving lattice plane spacings unchanged. Transient x-ray scattering is governed by solvation, masking changes of the chromophore molecular structure.

Coupled ultrafast lattice and polarization dynamics in ferroelectric nanolayers.

We report the first analysis of the polarization and lattice dynamics in a metal/ferroelectric/metal nanolayer system by femtosecond x-ray diffraction. Two Bragg reflections provide information on the coupled dynamics of the two relevant phonon modes for ferroelectricity in perovskites, the tetragonal distortion and the soft mode. Optical excitation of the SrRuO(3) metal layers generates giant stress (>1 GPa) compressing the PbZr(0.2)Ti(0.8)O(3) layers by up to 2%. The resulting change of tetragonality reaches a maximum after 1.3 ps. As a result, the ferroelectric polarization P is reduced by up to 100% with a slight delay that is due to the anharmonic coupling of the two modes.

Molecular localization of a ribosome-dependent ATPase on Escherichia coli ribosomes.

We have previously isolated and described an Escherichia coli ribosome-bound ATPase, RbbA, that is required for protein synthesis in the presence of ATP, GTP and the elongation factors, EF-Tu and EF-G. The gene encoding RbbA, yhih, has been cloned and the deduced protein sequence harbors two ATP-motifs and one RNA-binding motif and is homologous to the fungal EF-3. Here, we describe the isolation and assay of a truncated form of the RbbA protein that is stable to overproduction and purification. Chemical protection results show that the truncated RbbA specifically protects nucleotide A937 on the 30S subunit of ribosomes, and the protected site occurs at the E-site where the tRNA is ejected upon A-site occupation. Other weakly protected bases in the region occur at or near the mRNA binding site. Using radiolabeled tRNAs, we study the stimulating effect of this truncated RbbA on the binding and release of different tRNAs bound to the (aminoacyl) A-, (peptidyl) P- and (exit) E-sites of 70S ribosomes. The combined data suggest plausible mechanisms for the function of RbbA in translation.

A ribosomal ATPase is a target for hygromycin B inhibition on Escherichia coli ribosomes.

We demonstrate that the transfer of fully charged aminoacyl-tRNAs into peptides directed by the MS2 RNA template requires both ATP and GTP, initiation factors (IF1, IF2, and IF3), elongation factors (EF-Tu, EF-Ts, and EF-G), and the ribosomal ATPase (RbbA). The nonhydrolyzable analogue AMPPCP inhibits the reactions, suggesting that hydrolysis of ATP is required for synthesis. The RbbA protein occurs bound to ribosomes and stimulates the ATPase activity of Escherichia coli 70S and 30S particles. The gene encoding RbbA harbors four ATP binding domains; the C-terminal half of the protein bears extensive sequence similarity to EF-3, a ribosome-dependent ATPase. Here, we show that the antibiotic hygromycin B selectively inhibits the ATPase activity of RbbA. Other antibiotics with similar effects on miscoding, streptomycin and neomycin, as well as antibiotics that impair peptide bond synthesis and translocation, had little effect on the ATPase activity of RbbA on 70S ribosomes. Immunoblot analysis indicates that at physiological concentrations, hygromycin B selectively releases RbbA from 70S ribosomes. Hygromycin B protects G1494 and A1408 in the decoding region, and RbbA enhances the reactivity of A889 and G890 of the 16S rRNA switch helix region. Cross-linking and X-ray diffraction data have revealed that this helix switch and the decoding region are in close proximity. Mutations in the switch helix (889-890) region affect translational fidelity and translocation. The binding site of hygromycin B and its known dual effect on the fidelity of decoding and translocation suggest a model for the action of this drug on ribosomes.

Functional interactions of an Escherichia coli ribosomal ATPase.

The gene encoding ribosome-bound ATPase (RbbA), which occurs bound to 70S ribosomes and 30S subunits, has been identified. The amino-acid sequence of RbbA reveals the presence of two ATP-binding domains in the N-terminal half of the protein. RbbA harbors an intrinsic ATPase activity that is stimulated by both 70S ribosomes and 30S subunits. Here we show that purified recombinant RbbA markedly stimulates polyphenylalanine synthesis in the presence of the elongation factors Tu and G (EF-Tu and EF-G) and that the hydrolysis of ATP by RbbA is required to stimulate synthesis. RbbA is reported to have affinity for EF-Tu but not for EF-G. Additionally, RbbA copurifies with 30S ribosomal subunits and can be crosslinked to the ribosomal protein S1. Studies using a spectrum of antibiotics, including some of similar function, revealed that hygromycin, which binds to the 30S subunit, has a significant effect on the ATPase activity and on the affinity of RbbA for ribosomes. A possible role for RbbA in protein-chain elongation is proposed.