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Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2, has spread globally, and proven treatments are limited. Transfusion of convalescent plasma collected from donors who have recovered from COVID-19 is among many approaches being studied as potentially efficacious therapy. We are conducting a prospective, propensity score-matched study assessing the efficacy of COVID-19 convalescent plasma transfusion versus standard of care as treatment for severe and/or critical COVID-19. We present herein the results of an interim analysis of 316 patients enrolled at Houston Methodist hospitals from March 28 to July 6, 2020. Of the 316 transfused patients, 136 met a 28-day outcome and were matched to 251 non-transfused control COVID-19 patients. Matching criteria included age, sex, body mass index, comorbidities, and baseline ventilation requirement 48 hours from admission, and in a second matching analysis, ventilation status at day 0. Variability in the timing of transfusion relative to admission and titer of antibodies of plasma transfused allowed for analysis in specific matched cohorts. The analysis showed a significant reduction (P = 0.047) in mortality within 28 days, specifically in patients transfused within 72 hours of admission with plasma with an anti-spike protein receptor binding domain titer of ≥1:1350. These data suggest that treatment of COVID-19 with high anti-receptor binding domain IgG titer convalescent plasma is efficacious in early-disease patients.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2, has spread globally, and no proven treatments are available. Convalescent plasma therapy has been used with varying degrees of success to treat severe microbial infections for >100 years. Patients (n = 25) with severe and/or life-threatening COVID-19 disease were enrolled at the Houston Methodist hospitals from March 28, 2020, to April 14, 2020. Patients were transfused with convalescent plasma, obtained from donors with confirmed severe acute respiratory syndrome coronavirus 2 infection who had recovered. The primary study outcome was safety, and the secondary outcome was clinical status at day 14 after transfusion. Clinical improvement was assessed on the basis of a modified World Health Organization six-point ordinal scale and laboratory parameters. Viral genome sequencing was performed on donor and recipient strains. At day 7 after transfusion with convalescent plasma, nine patients had at least a one-point improvement in clinical scale, and seven of those were discharged. By day 14 after transfusion, 19 (76%) patients had at least a one-point improvement in clinical status, and 11 were discharged. No adverse events as a result of plasma transfusion were observed. Whole genome sequencing data did not identify a strain genotype-disease severity correlation. The data indicate that administration of convalescent plasma is a safe treatment option for those with severe COVID-19 disease.

Millions of individuals who have recovered from SARS-CoV-2 infection may be eligible to participate in convalescent plasma donor programs, yet the optimal window for donating high neutralizing titer convalescent plasma for COVID-19 immunotherapy remains unknown. Here we studied the response trajectories of antibodies directed to the SARS-CoV-2 surface spike glycoprotein and in vitro SARS-CoV-2 live virus neutralizing titers (VN) in 175 convalescent donors longitudinally sampled for up to 142 days post onset of symptoms (DPO). We observed robust IgM, IgG, and viral neutralization responses to SARS-CoV-2 that persist, in the aggregate, for at least 100 DPO. However, there is a notable decline in VN titers ≥160 for convalescent plasma therapy, starting 60 DPO. The results also show that individuals 30 years of age or younger have significantly lower VN, IgG and IgM antibody titers than those in the older age groups; and individuals with greater disease severity also have significantly higher IgM and IgG antibody titers. Taken together, these findings define the optimal window for donating convalescent plasma useful for immunotherapy of COVID-19 patients and reveal important predictors of an ideal plasma donor., Gontu et al. conducted a longitudinal study in COVID patients in which they assessed the response trajectories of antibodies directed to the SARS-CoV-2 surface spike glycoprotein and in vitro SARS-CoV-2 live virus neutralizing titers. Their measurements demonstrate the presence of an optimum window for convalescent plasma donation as well as predictions of the most suitable donor type.

Background Renal allograft survival is negatively affected by the development of de novo posttransplant donor-specific antibodies (dnDSA). We sought to determine whether treatment with intravenous immunoglobulin (IVIG) could remove or reduce the intensity of dnDSA. Methods A single-center study of 12 recipients with dnDSA and stable function who received IVIG 1 g/kg monthly for 6 months were compared with a contemporaneous cohort of 24 recipients with dnDSA who did not receive IVIG. Results The median time to first dnDSA was 6 months (interquartile range [IQR], 1-12), and follow-up was 83 months (IQR, 58-94) posttransplant. Resolution of dnDSA occurred in 27% of IVIG vs 46% of control recipients (P = .48). Fifty-eight percent of recipients in both cohorts demonstrated a reduction in the intensity of the dominant DSA at last follow-up (P =1.0). A reduction in the number of dnDSAs occurred in 58% vs 62% of the IVIG and control cohorts, respectively (P = .81). Post-dnDSA, acute rejection occurred in 8% of the IVIG vs 42% in the control group (P = .06). Forty-two percent of IVIG-treated vs 49% of control recipients had a deterioration in function from first dnDSA until most recent follow-up (P = .81). Actuarial graft survivals were equivalent between groups. Conclusions IVIG treatment of dnDSA in recipients with stable graft function had no impact on DSA clearance or MFI reduction, but this outcome may also be owing to sample size. Larger studies or alternate dosing regimens may be required to determine if there is any role for the use of IVIG as a treatment for dnDSA.

Newly emerged pathogens such as SARS-CoV-2 highlight the urgent need for assays that detect levels of neutralizing antibodies that may be protective. We studied the relationship between anti-spike ectodomain (ECD) and anti-receptor binding domain (RBD) IgG titers, and SARS-CoV-2 virus neutralization (VN) titers generated by two different in vitro assays using convalescent plasma samples obtained from 68 COVID-19 patients, including 13 who donated plasma multiple times. Only 23% (16/68) of donors had been hospitalized. We also studied 16 samples from subjects found to have anti-spike protein IgG during surveillance screening of asymptomatic individuals. We report a strong positive correlation between both plasma anti-RBD and anti-ECD IgG titers, and in vitro VN titer. Anti-RBD plasma IgG correlated slightly better than anti-ECD IgG titer with VN titer. The probability of a VN titer ≥160 was 80% or greater with anti-RBD or anti-ECD titers of ≥1:1350. Thirty-seven percent (25/68) of convalescent plasma donors lacked VN titers ≥160, the FDA-recommended level for convalescent plasma used for COVID-19 treatment. Dyspnea, hospitalization, and disease severity were significantly associated with higher VN titer. Frequent donation of convalescent plasma did not significantly decrease either VN or IgG titers. Analysis of 2,814 asymptomatic adults found 27 individuals with anti-RBD or anti-ECD IgG titers of ≥1:1350, and evidence of VN ≥1:160. Taken together, we conclude that anti-RBD or anti-ECD IgG titers can serve as a surrogate for VN titers to identify suitable plasma donors. Plasma anti-RBD or anti-ECD titer of ≥1:1350 may provide critical information about protection against COVID-19 disease.

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 remains a global threat with few proven efficacious treatments. Transfusion of convalescent plasma collected from donors who have recovered from COVID-19 disease has emerged as a promising therapy and has been granted emergency use authorization by the U.S. Food and Drug Administration (FDA). We recently reported results from interim analysis of a propensity-score matched study suggesting that early treatment of COVID-19 patients with convalescent plasma containing high titer anti-spike protein receptor binding domain (RBD) IgG significantly decreases mortality. We here present results from 60-day follow up of our cohort of 351 transfused hospitalized patients. Prospective determination of ELISA anti-RBD IgG titer facilitated selection and transfusion of the highest titer units available. Retrospective analysis by the Ortho VITROS IgG assay revealed a median signal/cutoff (S/C) ratio of 24.0 for transfused units, a value far exceeding the recently FDA-required cutoff of 12.0 for designation of high titer convalescent plasma. With respect to altering mortality, our analysis identified an optimal window of 44 hours post-hospitalization for transfusing COVID-19 patients with high titer convalescent plasma. In the aggregate, the analysis confirms and extends our previous preliminary finding that transfusion of COVID-19 patients soon after hospitalization with high titer anti-spike protein RBD IgG present in convalescent plasma significantly reduces mortality.

The optimal timeframe for donating convalescent plasma to be used for COVID-19 immunotherapy is unknown. To address this important knowledge deficit, we determinedin vitrolive-virus neutralizing capacity and persistence of IgM and IgG antibody responses against the receptor-binding domain and S1 ectodomain of the SARS-CoV-2 spike glycoprotein in 540 convalescent plasma samples obtained from 175 COVID-19 plasma donors for up to 142 days post-symptom onset. Robust IgM, IgG, and viral neutralization responses to SARS-CoV-2 persist, in the aggregate, for at least 100 days post-symptom onset. However, a notable acceleration in decline in virus neutralization titers ≥160, a value suitable for convalescent plasma therapy, was observed starting 60 days after first symptom onset. Together, these findings better define the optimal window for donating convalescent plasma useful for immunotherapy of COVID-19 patients and reveal important predictors of an ideal plasma donor, including age and COVID-19 disease severity score.One Sentence SummaryEvaluation of SARS-CoV-2 anti-spike protein IgM, IgG, and live-virus neutralizing titer profiles reveals that the optimal window for donating convalescent plasma for use in immunotherapy is within the first 60 days of symptom onset.

BackgroundCOVID-19 disease, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has spread globally, and no proven treatments are available. Convalescent plasma therapy has been used with varying degrees of success to treat severe microbial infections for more than 100 years.MethodsPatients (n=25) with severe and/or life-threatening COVID-19 disease were enrolled at the Houston Methodist hospitals from March 28 – April 14, 2020. Patients were transfused with convalescent plasma obtained from donors with confirmed SARS-CoV-2 infection and had been symptom free for 14 days. The primary study outcome was safety, and the secondary outcome was clinical status at day 14 post-transfusion. Clinical improvement was assessed based on a modified World Health Organization 6-point ordinal scale and laboratory parameters. Viral genome sequencing was performed on donor and recipient strains.ResultsAt baseline, all patients were receiving supportive care, including anti-inflammatory and anti-viral treatments, and all patients were on oxygen support. At day 7 post-transfusion with convalescent plasma, nine patients had at least a 1-point improvement in clinical scale, and seven of those were discharged. By day 14 post-transfusion, 19 (76%) patients had at least a 1-point improvement in clinical status and 11 were discharged. No adverse events as a result of plasma transfusion were observed. The whole genome sequencing data did not identify a strain genotype-disease severity correlation.ConclusionsThe data indicate that administration of convalescent plasma is a safe treatment option for those with severe COVID-19 disease. Randomized, controlled trials are needed to determine its efficacy.

Newly emerged pathogens such as SARS-CoV-2 highlight the urgent need for assays that detect levels of neutralizing antibodies that may be protective. We studied the relationship between anti-spike ectodomain (ECD) and anti-receptor binding domain (RBD) IgG titers, and SARS-CoV-2 virus neutralization (VN) titers generated by two differentin vitroassays using convalescent plasma samples obtained from 68 COVID-19 patients, including 13 who donated plasma multiple times. Only 23% (16/68) of donors had been hospitalized. We also studied 16 samples from subjects found to have anti-spike protein IgG during surveillance screening of asymptomatic individuals. We report a strong positive correlation between both plasma anti-RBD and anti-ECD IgG titers, andin vitroVN titer. Anti-RBD plasma IgG correlated slightly better than anti-ECD IgG titer with VN titer. The probability of a VN titer ≥160 was 80% or greater with anti-RBD or anti-ECD titers of ≥1:1350. Thirty-seven percent (25/68) of convalescent plasma donors lacked VN titers ≥160, the FDA-recommended level for convalescent plasma used for COVID-19 treatment. Dyspnea, hospitalization, and disease severity were significantly associated with higher VN titer. Frequent donation of convalescent plasma did not significantly decrease either VN or IgG titers. Analysis of 2,814 asymptomatic adults found 27 individuals with anti-RBD or anti-ECD IgG titers of ≥1:1350, and evidence of VN ≥1:160. Taken together, we conclude that anti-RBD or anti-ECD IgG titers can serve as a surrogate for VN titers to identify suitable plasma donors. Plasma anti-RBD or anti-ECD titer of ≥1:1350 may provide critical information about protection against COVID-19 disease.

ObjectiveThe goal of this study was to investigate the role of CD 19+B cells within the brain and spinal cord during CNS autoimmunity in a peptide-induced, primarily T-cell–mediated experimental autoimmune encephalomyelitis (EAE) model of MS. We hypothesized that CD19+B cells outside the CNS drive inflammation in EAE.MethodsWe generated CD19.Cre+/−α4-integrinfl/flmice. EAE was induced by active immunization with myelin oligodendrocyte glycoprotein peptide (MOGp35-55). Multiparameter flow cytometry was used to phenotype leukocyte subsets in primary and secondary lymphoid organs and the CNS. Serum cytokine levels and Ig levels were assessed by bead array. B-cell adoptive transfer was used to determine the compartment-specific pathogenic role of antigen-specific and non–antigen-specific B cells.ResultsA genetic ablation of α4-integrin in CD19+/−B cells significantly reduced the number of CD19+B cells in the CNS but does not affect EAE disease activity in active MOGp35-55-induced disease. The composition of B-cell subsets in the brain, primary lymphoid organs, and secondary lymphoid organs of CD19.Cre+/−α4-integrinfl/flmice was unchanged during MOGp35-55-induced EAE. Adoptive transfer of purified CD19+B cells from CD19.Cre+/−α4-integrinfl/flmice or C57BL/6 wild-type (WT) control mice immunized with recombinant rMOG1-125or ovalbumin323-339into MOGp35-55-immunized CD19.Cre+/−α4-integrinfl/flmice caused worse clinical EAE than was observed in MOGp35-55-immunized C57BL/6 WT control mice that did not receive adoptively transferred CD19+B cells.ConclusionsObservations made in CD19.Cre+/−α4-integrinfl/flmice in active MOGp35-55-induced EAE suggest a compartment-specific pathogenic role of CD19+B cells mostly outside of the CNS that is not necessarily antigen specific.

Background De novo donor-specific antibodies (dnDSA) after renal transplant are associated with acute rejection (AR) and graft loss, yet most recipients with dnDSA have stable function and no AR. We assessed whether the persistence of dnDSA increased the risk of a detrimental outcome. Methods A single-center review of renal transplant recipients monitored for dnDSA at multiple time points post-transplant. An Isolated dnDSA was defined as one positive dnDSA and no additional positive tests, whereas ≥2 positive dnDSA was defined as persistent dnDSA. Results Of 708 recipients, 22% developed dnDSA, of whom 64% had persistent dnDSA. At median follow-up of 35 (range 12-74) months, there were fewer episodes of AR in the isolated dnDSA vs the persistent dnDSA group (2% vs 22%; P

Background: Human leukocyte antigen (HLA) genes on chromosome 6p21 encode the major histocompatibility complex (MHC). MHC molecules are expressed on all nucleated cells and are essential to the immune system's ability to distinguish healthy native cells from foreign or abnormal ones. Specialized antigen presenting cells display MHC molecules loaded with foreign peptides to initiate T cell responses. HLA sequencing variability is critical to function; studies have shown even single allelic changes can alter the ability of MHC molecules to recognize cognate T cell receptors. Given the role of MHC molecules in identifying cells as "self", associations between HLA sequence variation and autoimmunity is a research focus. Like other autoimmune disorders, association between ITP and HLA genes has been investigated. Prior studies are limited by outdated HLA typing methods, genetically homogenous study populations, and lack of validation studies. Preexisting exome data has historically not been used to provide accurate HLA sequencing information given the polymorphic, highly repetitive sequence in this region. This study applied a novel bioinformatics pipeline, xHLA, to an existing set of whole exome sequencing in a cohort of chronic pediatric ITP patients. xHLA, reported by Xie et al. in 2017, yields high-resolution, two field HLA typing for HLA-A, -B, -C, -DQB1, -DPB1, and -DRB1. A prior genetic analysis of chronic ITP patient whole exome data identified a missense variant in BTNL2, a gene which plays a role in peripheral regulatory T cell induction. This variant (rs143211074) is upregulated in chronic ITP versus healthy controls (minor allele frequency= 4.17% in ITP v. 0.54% in controls, p=1.43x10-4). Linkage of rs143211074 and HLA alleles was examined in this study. Methods: Whole exome sequencing data for a cohort of 272 chronic ITP patients was processed using the xHLA method. In order to eliminate sequencing variability related to ethnicity alone, initial analyses were focused on Caucasians only (n=170), using Eigenvectors for principle components analysis. The frequency of individual HLA alleles and HLA haplotypes for Caucasians from the National Marrow Donor Program (NMDP) Be The Match Registry® were compared to the ITP cohort. HLA haplotypes were inferred using the NMDP HaploStats program. A chi-squared test was utilized to compare nonparametric categorical data using GraphPad Prism version 8.0.1 for Windows, GraphPad Software, San Diego, California, USA, www.graphpad.com. A Bonferroni correction was applied and a p-value of Results: 12 HLA alleles had increased frequency in the chronic ITP cohort versus controls. (Table 1) The BTNL2 variant, rs143211074, was found in linkage disequilibrium with the HLA-DRB1*04:02, HLA-DQB1*03:02 haplotype. Of the 10 patients heterozygous for the variant allele, 80% had the HLA-DRB1*04:02, HLA-DQB1*03:02 haplotype. Both patients homozygous for rs143211074 were also homozygous for the HLA-DRB1*04:02, HLA-DQB1*03:02 haplotype. In the entire cohort, this haplotype was identified in 3.63% of ITP patients, and only in 1.27% of controls. Our group recently published that a positive direct antiglobulin test is associated with chronic ITP and need for second line treatments. Of the patients with the HLA-DRB1*04:02, HLA-DQB1*03:02 haplotype with DAT testing performed (n=9), 66% had a positive DAT test and 33% had a negative DAT result. Of those without the HLA-DRB1*04:02, HLA-DQB1*03:02 haplotype who had DAT testing, 17.2% had a positive DAT and 82.8% had a negative test (p=0.0026). Conclusions: HLA alleles are seen at increased frequency in a cohort of Caucasian chronic ITP patients. The HLA-DRB1*04:02, HLA-DQB1*03:02 haplotype is linked to a variant previously identified in BTNL2. Coupled with prior data suggesting that a positive DAT result is associated with chronic disease and use of second line agents, this presence of the HLA-DRB1*04:02, HLA-DQB1*03:02 haplotype may also be linked to refractory disease. Confirmation of the accuracy of the xHLA method in a cohort of patients with both exome data and traditional HLA typing is underway. In the future, a separate cohort of chronic and acute ITP patients will undergo next generation sequencing and have similar HLA typing performed as an independent validation set. Table 1 Disclosures Despotovic: Novartis: Research Funding; Amgen: Research Funding; Dova: Honoraria.

Aim Histocompatibility testing is used to predict the risk of hyperacute and accelerated-acute transplant rejection. The presence of HLA-specific antibodies, as detected by single antigen bead testing (SAB), are used in organ allocation through the calculated panel reactive antibody (cPRA) and by assessing the presence of donor HLA-specific antibodies (DSA). The presence of DSA and cPRA are considered in the algorithm for assigning patients to immunosuppression protocols. However, the interpretation of low level antibodies in SAB and DSA testing is challenging. Our aim was to look at antibodies below our current threshold of 2000 MFI but above our analytical threshold of 1000 MFI to determine if there is residual risk related to these antibodies. Methods Patient antibody screens were evaluated with 2000 MFI and 1000 MFI cutoff values. 181 patients were assessed for pre-transplant patient risk category and donor compatibility by determining: the change in cPRA, number of identified HLA specific antibodies between the thresholds, the presence of DSA identified between thresholds and the predicted change in immunosuppression protocols. Living donor kidney transplants with an average of 2 years of follow-up were reviewed. 81 recipients were scored for the presence of DSA and examined for the development of de novo DSA (dnDSA) post-transplant. Biopsy data was also reviewed on 40 living donor kidney transplant recipients to determine the incidence of biopsy-proven rejection. Results The reduction in antibody threshold was associated with an average increase in cPRA of 15% for HLA-Class I and 18% for HLA-Class II which corresponded to detection of 10 additional HLA specificities. We determined that using the 1000 MFI threshold, 15% of patients would have been considered higher risk based on transplant center protocols and would have necessitated increased immunosuppression. Changes in risk category were weakly associated with higher rates of dnDSA post-transplant and biopsy-proven rejection. Conclusions In this study, the development of biopsy-proven rejection was not associated with pre-transplant DSA or post-transplant dnDSA that were between 1000 and 2000 MFI.

Acridine-iminodibenzyl chimeric compounds were previously introduced as a class of cholesterol-redistributing substances with antiprion effects. Here, we show that administration of the lead compound quinpramine to mice with experimental autoimmune encephalitis, an animal model of multiple sclerosis (MS), significantly ameliorates disease in preventive and therapeutic paradigms. Quinpramine treatment decreased the number of inflammatory CNS lesions, antigen-specific T-cell proliferation, and pro-inflammatory cytokines IFNgamma and IL-17. Quinpramine is thus an immunoregulatory drug that is a candidate pharmaceutical for MS.

During the progression of relapsing experimental autoimmune encephalomyelitis (R-EAE), in SJL mice, disease relapses are mediated by T cells specific for non-cross-reactive myelin epitopes, a process termed ‘epitope spreading’. CTLA-4, a negative regulator of T cell function modulates R-EAE, in that CTLA-4 blockade exacerbates clinical R-EAE. Herein, we show that CTLA-4-mediated signaling negatively regulates the dynamic spread of autoreactive T cell responses during the course of autoimmune disease. Anti-CTLA-4 mAb, administration at various points during the progression of R-EAE exacerbated subsequent clinical disease and enhanced T cell reactivity to both inducing and relapse-associated epitopes. In addition, CTLA-4 blockade during acute disease inhibited clinical remission. Thus, CTLA-4-mediated events are critical for intrinsic regulation of epitope spreading during autoimmune disease.

Recent clinical trials have established B cell depletion by the anti-CD20 chimeric antibody Rituximab as a beneficial therapy for patients with relapsing-remitting multiple sclerosis (MS). The impact of Rituximab on T cell responses remains largely unexplored. In the experimental autoimmune encephalomyelitis (EAE) model of MS in mice that express human CD20, Rituximab administration rapidly depleted peripheral B cells and strongly reduced EAE severity. B cell depletion was also associated with diminished Delayed Type Hypersensitivity (DTH) and a reduction in T cell proliferation and IL-17 production during recall immune response experiments. While Rituximab is not considered a broad immunosuppressant, our results indicate a role for B cells as a therapeutic cellular target in regulating encephalitogenic T cell responses in specific tissues.

Over the past 2 decades, enormous progress has been made with regard to pharmacotherapies for patients with multiple sclerosis. There is perhaps no other subspecialty in neurology in which more agents have been approved that substantially alter the clinical course of a disabling disorder. Many of the pharmaceuticals that are currently approved, in clinical trials, or in preclinical development were initially evaluated in an animal model of multiple sclerosis, experimental autoimmune encephalomyelitis. Two Food and Drug Administration–approved agents (glatiramer acetate and natalizumab) were developed using the experimental autoimmune encephalomyelitis model. This model has served clinician-scientists for many decades to enable understanding the inflammatory cascade that underlies clinical disease activity and disease surrogate markers detected in patients.

Rituximab is a recombinant chimeric monoclonal antibody against CD20, a molecule expressed on cells of the B-cell lineage. A phase 2 clinical trial recently provided strong evidence of the beneficial effects of rituximab in patients with relapsing-remitting multiple sclerosis. We and other investigators previously demonstrated that rituximab therapy depletes B lymphocytes from peripheral blood and cerebrospinal fluid of patients with relapsing-remitting multiple sclerosis.To determine the effect of rituximab on the presence of B cells in cerebral perivascular spaces. Design, Setting, and Patients Case report from a tertiary academic medical center. Cerebral white matter from autopsy material of a patient with gastrointestinal mantle-cell lymphoma who developed progressive multifocal leukoencephalopathy following rituximab therapy was evaluated by immunohistochemistry. Location-matched brain sections of patients with multiple sclerosis not treated with rituximab, patients without central nervous system disease, and patients with progressive multifocal leukoencephalopathy not associated with rituximab were used as controls.Assessment of the number of B lymphocytes in cerebral perivascular spaces in a patient with gastrointestinal mantle-cell lymphoma treated with rituximab, patients with multiple sclerosis, patients with progressive multifocal leukoencephalopathy not associated with rituximab, and healthy control subjects.We were unable to detect B cells in cerebral perivascular spaces of the patient who developed progressive multifocal leukoencephalopathy following rituximab therapy 8 months after her last dose. In contrast, B cells were detectable in all control brain tissues.To our knowledge, this is the first report to show B-lymphocyte depletion from brain tissue following rituximab therapy. A reduction in B-cell numbers may be an important contributing factor in the pathogenesis of central nervous system infections.

Background Minocycline is an oral tetracycline derivative with good bioavailability in the central nervous system (CNS). Minocycline, a potent inhibitor of matrix metalloproteinase (MMP)-9, attenuates disease activity in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). Potential adverse effects associated with long-term daily minocycline therapy in human patients are concerning. Here, we investigated whether less frequent treatment with long-circulating polyethylene glycol (PEG) minocycline liposomes are effective in treating EAE. Findings Performing in vitro time kinetic studies of PEG minocycline-liposomes in human peripheral blood mononuclear cells (PBMCs), we determined that PEG minocycline-liposome preparations stabilized with CaCl2 are effective in diminishing MMP-9 activity. Intravenous injections of PEG minocycline-liposomes every five days were as effective in ameliorating clinical EAE as daily intraperitoneal injections of minocycline. Treatment of animals with PEG minocycline-liposomes significantly reduced the number of CNS-infiltrating leukocytes, and the overall expression of MMP-9 in the CNS. There was also a significant suppression of MMP-9 expression and proteolytic activity in splenocytes of treated animals, but not in CNS-infiltrating leukocytes. Thus, leukocytes gaining access to the brain and spinal cord require the same absolute amount of MMP-9 in all treatment groups, but minocycline decreases the absolute cell number. Conclusions Our data indicate that less frequent injections of PEG minocycline-liposomes are an effective alternative pharmacotherapy to daily minocycline injections for the treatment of CNS autoimmune diseases. Also, inhibition of MMP-9 remains a promising treatment target in EAE and patients with MS.

Multiple sclerosis is widely recognized as the most commonly identified cause of progressive neurologic disability in young adults throughout the developed world. The disorder is clinically suspected when patients experience either acute attacks of neurologic compromise or instead are afflicted by a steadily progressive deterioration in functional capabilities. The pathophysiology of acute exacerbations is thought to be related to the development of inflammation and its consequences, within strategic and often discrete central nervous system tract systems. Although a myriad of hypotheses have been formulated to explain the underpinnings of the mechanisms that contribute to both the predilection and triggering of the multiphasic inflammatory events that personify multiple sclerosis, much remains to be done to understand fully the specific set and sequence of events that produce the disease and its cardinal features.

Multiple sclerosis (MS) is the most common human inflammatory, demyelinating and degenerative disorder of the CNS. Based mostly on work in experimental autoimmune encephalomyelitis, CD4(+) T cells were long thought to play the crucial role in MS pathogenesis. Only more recently has it been recognized that other effector cell types, including CD8(+) T cells, gammadelta-T cells and B lymphocytes may also have an important role in disease initiation and perpetuation. The expression of soluble inflammatory mediators, including cytokines and free radicals, may be one of the late pathways mediating CNS tissue damage. In addition, in virtually all patients with MS, an oligoclonal banding pattern of antibodies can be detected in the cerebrospinal fluid (CSF). However, the cause of MS still remains unknown. Specifically, no single foreign or self antigen has been identified to account for clinical disease activity or the presence of surrogate disease markers. All approved pharmacotherapies have anti-inflammatory or immunoregulatory properties and work in early stages of the disease. In a recent clinical trial, BHT-3009, a DNA vaccine encoding full-length human myelin basic protein, was tested in patient with MS. BHT-3009 was safe and well tolerated. In addition, immunization with BHT-3009 induced anti-inflammatory antigen-specific immune changes consisting of a marked decrease in T-cell proliferation of IFN gamma production and a reduction in titers of myelin-specific autoantibodies in the CSF. This review will discuss these intriguing observations and the overall potential of DNA vaccination in MS.

The single antigen bead assay is a widely-used platform for the detection of human leukocyte antigen (HLA)-specific antibodies in both pre- and post-transplant patients. Our regional organ procurement organization recommends a cutoff value of 2000 mean fluorescence intensity (MFI) for the detection of HLA antibodies using the Luminex single antigen bead assay. This cutoff has proven effective for the prevention of HLA-mediated hyperacute rejection; however, this cutoff may be too lenient for the detection of post-transplant donor-specific antibodies. Indeed, we hypothesize that some patients have donor-specific HLA antibody-mediated organ allograft rejection even with a MFI of To define a clinically-relevant DSA MFI cutoff, we analyzed MFI values of 75 runs of a commercially available negative control serum and MFI values of 75 post-transplant patients with negative DSAs. After normalizing the data by log-transformation, we found that the negative control runs had a mean + 3 standard deviation cutoff of 328, and the post-transplant negative DSA patients had a mean + 3 standard deviation cutoff of 969, suggesting that a cutoff of 1000 MFI would be adequate to capture approximately 99% of all negative DSA patients. We applied the 1000 MFI cutoff to the six cases with findings suggestive of ABMR but with no DSAs identified. We found that the highest MFI for donor-specific antigens in these patients was well below the 1000 MFI cutoff. Using a cutoff of 1000 would not have changed the DSA result to positive in these patients. The overall findings suggest that a cutoff of 2000 MFI has been adequate to capture all cases of HLA-mediated antibody-mediated rejection over the period analyzed at our institution.

Initial migration of encephalitogenic T cells to the central nervous system (CNS) in relapsing experimental autoimmune encephalomyelitis (R-EAE), an animal model of multiple sclerosis (MS), depends on the interaction of the alpha4 integrin (VLA-4) expressed on activated T cells with VCAM-1 expressed on activated cerebrovascular endothelial cells. Alternate homing mechanisms may be employed by infiltrating inflammatory cells after disease onset. We thus compared the ability of anti-VLA-4 to regulate proteolipid protein (PLP) 139-151-induced R-EAE when administered either before or after disease onset. Preclinical administration of anti-VLA-4 either to naive recipients of primed encephalitogenic T cells or to mice 1 week after peptide priming, i.e., before clinical disease onset, inhibited the onset and severity of clinical disease. In contrast, Ab treatment either at the peak of acute disease or during remission exacerbated disease relapses and increased the accumulation of CD4(+) T cells in the CNS. Most significantly, anti-VLA-4 treatment either before or during ongoing R-EAE enhanced Th1 responses to both the priming peptide and endogenous myelin epitopes released secondary to acute tissue damage. Collectively, these results suggest that treatment with anti-VLA-4 Ab has multiple effects on the immune system and may be problematic in treating established autoimmune diseases such as MS.

Relapsing experimental autoimmune encephalomyelitis (R-EAE) is a CD4+ T cell-mediated demyelinating disease model for multiple sclerosis. Myelin destruction during the initial relapsing phase of R-EAE in SJL mice initiated by immunization with the proteolipid protein (PLP) epitope PLP139–151 is associated with activation of T cells specific for the endogenous, non-cross-reactive PLP178–191 epitope (intramolecular epitope spreading), while relapses in R-EAE induced with the myelin basic protein (MBP) epitope MBP84–104 are associated with PLP139–151-specific responses (intermolecular epitope spreading). Here, we demonstrate that T cells specific for endogenous myelin epitopes play the major pathologic role in mediating clinical relapses. T cells specific for relapse-associated epitopes can serially transfer disease to naive recipients and are demonstrable in the CNS of mice with chronic R-EAE. More importantly, induction of myelin-specific tolerance to relapse-associated epitopes, by i.v. injection of ethylene carbodiimide-fixed peptide-pulsed APCs, either before disease initiation or during remission from acute disease effectively blocks the expression of the initial disease relapse. Further, blockade of B7-1-mediated costimulation with anti-B7-1 F(ab) during disease remission from acute PLP139–151-induced disease prevents clinical relapses by inhibiting activation of PLP178–191-specific T cells. The protective effects of anti-B7-1 F(ab) treatment are long-lasting and highly effective even when administered following the initial relapsing episode wherein spreading to a MBP epitope (MBP84–104) is inhibited. Collectively, these data indicate that epitope spreading is B7-1 dependent, plays a major pathologic role in disease progression, and follows a hierarchical order associated with the relative encephalitogenic dominance of the myelin epitopes (PLP139–151 > PLP178–191 > MBP84–104).

Context DNA β-amyloid 1-42 (Aβ42) trimer immunization was developed to produce specific T helper 2 cell (T H 2)–type antibodies to provide an effective and safe therapy for Alzheimer disease (AD) by reducing elevated levels of Aβ42 peptide that occur in the brain of patients with AD. Objective To compare the immune response in wild-type mice after immunization with DNA Aβ42 trimer and Aβ42 peptide. Design and Intervention Wild-type mice received either 4 μg of DNA Aβ42 trimer immunization administered with gene gun (n = 8) or intraperitoneal injection of 100 μg of human Aβ42 peptide with the adjuvant Quil A (n = 8). Titers, epitope mapping, and isotypes of the Aβ42-specific antibodies were analyzed. Main Outcome Measures Antibody titers, mapping of binding sites (epitopes), isotype profiles of the Aβ42-specific antibodies, and T-cell activation. Results DNA Aβ42 trimer immunization resulted in antibody titers with a mean of 15 μg per milliliter of plasma. The isotype profile of the antibodies differed markedly. A predominant IgG1 antibody response was found in the DNA-immunized mice, indicating a T H 2 type of immune response (IgG1/IgG2a ratio of 10). The peptide-immunized mice showed a mixed T H 1/T H 2 immune response (IgG1/IgG2a ratio of 1) (P Conclusion In this preliminary study in a wild-type mouse model, DNA Aβ42 trimer immunization protocol produced a T H 2 immune response and appeared to have low potential to cause an inflammatory T-cell response.

ON FEBRUARY 12, 2009, WE CELEBRATE THE 200TH anniversary of the birth of Charles Darwin. He has earned the scientific reputation as being the most influential naturalist-biologist of his generation and perhaps of all time. Achievement of his esteemed status resulted initially from studying the geographical distribution of wildlife and fossils collected on his 5-year voyage on the Beagle and concluding that the “transmutation of species” was due to natural selection. In 1858, he was in the process of documenting his research when he received an essay from Alfred Russel Wallace putting forth the same concept of natural selection as the mechanism for generation of new species, resulting in a joint publication that same year. On the Origin of Species was published by Darwin in 1859, making it now the 150th anniversary of its publication. This book proposed evolution as the scientific basis of diversity and species generation produced through the effects of natural selection. Evolution has been proven to be the mechanism for species generation and the engine for change is natural selection, providing the evolved species, beginning from a common organism, adaptation and fitness to maximize reproduction and survival. Dobzhansky put it succinctly by saying that “nothing in biology makes sense except in the light of evolution.” Darwin expressed it eloquently in 1859: There is grandeur in this view of life, with its several powers, having been originally breathed into a few forms or into one; and that, whilst this planet has gone cycling on according to the fixed law of gravity, from so simple a beginning endless forms most beautiful and most wonderful have been, and are being evolved. Two hundred years after Darwin’s birth, molecular genomic analyses of the human genome have been implemented, seeking the genetic basis for natural selection providing biological fitness and also risk of developing disease. Genome-wide association studies (GWASs) seeking gene variations, or single-nucleotide polymorphisms (SNPs), causal of several human diseases have been conducted in recent years. These diseases include autism, schizophrenia, obesity, diabetes, and heart disease. Several GWASs for risk association with neurological diseases,orneuromicstudies,havebeenreported. Increasedrisks foramyotrophic lateral sclerosis,Alzheimerdisease(AD), restless legssyndrome,andmultiplesclerosishavebeenassociated with polymorphisms in specific genes. It was hoped that these observationswouldadvanceanunderstandingof thecausation of inherited, complexpolygenetic,multifactorialneurological diseases. They have been made possible by the publication of thehumangenomeandhaplotypestudies (HapMapanalyses). Awhole-genomeanalysisofsporadicamyotrophiclateralsclerosis was published by Dunckley et al. They identified 10 genetic loci that were associated with sporadic amyotrophic lateral sclerosis in 3 independent series of patients and control subjects.Themostsignificantassociationwithdiseasewasfound foraSNPnearanuncharacterizedgeneknownasFLJ10986on chromosome1.Thus, theydescribedspecificSNPs that areassociated with onset of sporadic amyotrophic lateral sclerosis. Reiman et al conducted a genome-wide survey of 502 627 SNPs to characterize and confirm late-onset AD susceptibility genes. They found that late-onset AD was associated with 6 SNPs from the GRB-associated binding protein 2 (GAB2) gene and a common haplotype encompassing the entire GAB2 gene. The GAB2 gene was overexpressed in pathologically vulnerable neurons, and interference with GAB2 gene expression increased tau phosphorylation. They concluded that GAB2 modifies late-onset AD risk in apolipoprotein E (APOE) e4 allele carriers. Coon et al found a single SNP in linkage disequilibrium to the APOE e4 allele in a neurome-wide association study. Grupe et al described 3 additional SNPs on chromosome 19 in linkage disequilibrium to the APOE e4 locus also in a neurome-wide association study. Liu et al described significant linkage to 3q23 markers associated with AD in a genetically isolated Dutch population. In 2008, Li et al described SNPs associated with risk and ageatonsetofADinaGWASof469 438SNPs.They identified theAPOE linkagedisequilibriumregionasthestrongestgenetic risk factor for AD. They concluded that this event could be a consequence of the coevolution of more than 1 susceptibility allele, includingAPOC1, inthesameregion.Additionalpotential at-riskgenes forADwereof interestandrequire furtherstudies.

Objective To extend our studies on the prolonged and differential effect of natalizumab on T lymphocyte numbers in the cerebrospinal fluid, we investigated the number and phenotypes of leukocytes and the expression of major histocompatibility complex (MHC) classes I and II in cerebral perivascular spaces (CPVS). We hypothesized that natalizumab reduces the number of antigen presenting cells in CPVS. Design A case-control study in which inflammatory cell numbers in the CPVS of cerebral tissue were assessed by immunohistochemical staining. Subjects A patient with multiple sclerosis (MS) who developed progressive multifocal leukoencephalopathy (PML) during natalizumab therapy. Controls included location-matched cerebral autopsy material of patients without disease of the central nervous system, patients with MS not treated with natalizumab, and patients with PML not associated with natalizumab therapy. Results The absolute number of CPVS in the patient with MS treated with natalizumab was significantly lower than in the control groups owing to extensive destruction of the tissue architecture. The expression of MHC class II molecules and the number of CD209 + dendritic cells were significantly decreased in the CPVS of the patient with MS treated with natalizumab. No CD4 + T cells were detectable. Conclusions Our observations may explain the differential and prolonged effects of natalizumab therapy on leukocyte numbers in the cerebrospinal fluid. Published online October 13, 2008 (doi:10.1001/archneur.65.12.noc80051).