Publications by authors named "Petra D Cravens"

32 Publications

CD11cCD88CD317 myeloid cells are critical mediators of persistent CNS autoimmunity.

Proc Natl Acad Sci U S A 2021 Apr;118(14)

Department of Neurology and Neurotherapeutics, University of Texas Southwestern Medical Center, Dallas, TX 75390;

Natalizumab, a humanized monoclonal antibody (mAb) against α4-integrin, reduces the number of dendritic cells (DC) in cerebral perivascular spaces in multiple sclerosis (MS). Selective deletion of α4-integrin in CD11c cells should curtail their migration to the central nervous system (CNS) and ameliorate experimental autoimmune encephalomyelitis (EAE). We generated CD11c.Cre C57BL/6 mice to selectively delete α4-integrin in CD11c cells. Active immunization and adoptive transfer EAE models were employed and compared with WT controls. Multiparameter flow cytometry was utilized to immunophenotype leukocyte subsets. Single-cell RNA sequencing was used to profile individual cells. α4-Integrin expression by CD11c cells was significantly reduced in primary and secondary lymphoid organs in CD11c.Cre mice. In active EAE, a delayed disease onset was observed in CD11c.Cre mice, during which CD11cCD88 cells were sequestered in the blood. Upon clinical EAE onset, CD11cCD88 cells appeared in the CNS and expressed CD317 In adoptive transfer experiments, CD11c.Cre mice had ameliorated clinical disease phenotype associated with significantly diminished numbers of CNS CD11cCD88CD317 cells. In human cerebrospinal fluid from subjects with neuroinflammation, microglia-like cells display coincident expression of (CD11c), (CD88), and (CD317). In mice, we show that only activated, but not naïve microglia expressed CD11c, CD88, and CD317. Finally, anti-CD317 treatment prior to clinical EAE substantially enhanced recovery in mice.
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http://dx.doi.org/10.1073/pnas.2014492118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8040603PMC
April 2021

Limitations of cell-lineage-specific non-dynamic gene recombination in CD11c.CreITGA4 mice.

J Neuroimmunol 2020 07 18;344:577245. Epub 2020 Apr 18.

Department of Neurology and Neurotherapeutics, University of Texas Southwestern Medical Center at Dallas, TX, USA; Neurology Section, VA North Texas Health Care System, Medical Service, Dallas, TX, USA. Electronic address:

Background: The Cre-lox system is a non-dynamic method of gene modification and characterization. Promoters thought to be relatively cell-specific are utilized for generation of cell-lineage-specific gene modifications.

Methods: CD11c.CreITGA4 mice were generated to abolish the expression of ITGA (α4-integrin) in CD11c cells. Ex vivo flow cytometry studies were used to assess the expression of cellular surface markers in different lymphoid compartments and leukocytes subsets after Cre-mediated recombination.

Results: A significant reduction of α4-integrin expression among CD11c cells was achieved in CD11c.CreITGA4 mice in primary and secondary lymphoid tissues. A similar reduction in the expression of α4-integrin was also observed in CD11c cells.

Conclusion: Cre-lox-mediated cell lineage-specific gene deletion is limited by the transient expression of recombination regulating sequences in hematopoietic cell lines. These methodological issues indicate the need to consider when to employ non-dynamic DNA recombination models in animal models of CNS autoimmunity. An experimental algorithm to address the biological complexities of non-dynamic gene recombination is provided.
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http://dx.doi.org/10.1016/j.jneuroim.2020.577245DOI Listing
July 2020

α4-integrin deficiency in B cells does not affect disease in a T-cell-mediated EAE disease model.

Neurol Neuroimmunol Neuroinflamm 2019 07 16;6(4):e563. Epub 2019 Apr 16.

Department of Neurology and Neurotherapeutics (R.Z.H., P.C.C., W.A.M.-L., R.D., V.G., D.T.O., O.S.) and Department of Pathology (E.H.), University of Texas Southwestern Medical Center, Dallas; Department of Pathology and Genomic Medicine (T.N.E.), Houston Methodist Hospital; Neurology Section (O.S.), VA North Texas Health Care System, Medical Service; and Department of Neurology (O.S.), Klinikum Rechts der Isar, Technische Universität München, Germany.

Objective: The 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.

Methods: We generated CD19.Cre α4-integrin mice. EAE was induced by active immunization with myelin oligodendrocyte glycoprotein peptide (MOG). 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.

Results: A 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 MOG-induced disease. The composition of B-cell subsets in the brain, primary lymphoid organs, and secondary lymphoid organs of CD19.Cre α4-integrin mice was unchanged during MOG-induced EAE. Adoptive transfer of purified CD19 B cells from CD19.Cre α4-integrin mice or C57BL/6 wild-type (WT) control mice immunized with recombinant rMOG or ovalbumin into MOG-immunized CD19.Cre α4-integrin mice caused worse clinical EAE than was observed in MOG-immunized C57BL/6 WT control mice that did not receive adoptively transferred CD19 B cells.

Conclusions: Observations made in CD19.Cre α4-integrin mice in active MOG-induced EAE suggest a compartment-specific pathogenic role of CD19 B cells mostly outside of the CNS that is not necessarily antigen specific.
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http://dx.doi.org/10.1212/NXI.0000000000000563DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6481229PMC
July 2019

Defining standard enzymatic dissociation methods for individual brains and spinal cords in EAE.

Neurol Neuroimmunol Neuroinflamm 2018 Mar 17;5(2):e437. Epub 2018 Jan 17.

Department of Neurology and Neurotherapeutics (R.Z.H., W.A.M.-L., R.D., P.D.C., O.S.), University of Texas Southwestern Medical Center, Dallas; Department of Biostatistics (G.C.), University of Alabama at Birmingham; The Moody Foundation Flow Cytometry Facility (N.L.), Children's Research Institute, University of Texas Southwestern Medical Center, Dallas; Neurology Section (O.S.), VA North Texas Health Care System, Medical Service, Dallas, TX; and Department of Neurology (O.S.), Klinikum rechts der Isar, Technische Universität München, Germany.

Objective: To determine the capacity, effectiveness, efficiency, and reliability of select tissue dissociation methods to isolate mononuclear cells from the CNS of mice with experimental autoimmune encephalomyelitis (EAE).

Methods: As part of an assay qualification, we tested the isolation method Percoll PLUS vs a commercially available enzymatic Neural Tissue Dissociation Kit (Kit), and the enzymes accutase and papain in C57BL/6 mice with active EAE. In a stepwise approach, we applied the following 4 criteria to each dissociation method: (1) mononuclear cell viability post-processing was required to be ≥80% per brain or spinal cord sample, (2) absolute live mononuclear cell numbers was required to be ≥5 × 10 per brain or spinal cord sample of mice with clinical EAE, (3) test-retest reliability had to be verified, and (4) the absolute mononuclear cell numbers in brain and spinal cord had to correlate with the EAE disease course.

Results: Enzymatic dissociations allowed for greatly increased cell yield and specifically allowed for downstream assays from individual brains and spinal cords in C57BL/6 mice with EAE. All enzymatic dissociations provided a more efficient and effective method for isolating mononuclear cells from brains and spinal cord. Only the Kit assay provided a significant correlation between absolute mononuclear cell numbers in the spinal cord and EAE disease severity.

Conclusions: Enzymatic dissociation of CNS tissue of C57BL/6 mice with active EAE with the Kit should be the standard method. The identification of optimized CNS dissociation methods in EAE has the potential to identify cellular events that are pertinent to MS pathogenesis.
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http://dx.doi.org/10.1212/NXI.0000000000000437DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5773844PMC
March 2018

Pathways leading to an immunological disease: systemic lupus erythematosus.

Rheumatology (Oxford) 2017 04;56(suppl_1):i55-i66

The Rheumatic Diseases Division, Department of Internal Medicine, UT Southwestern Medical Center at Dallas, TX, USA.

SLE is a chronic autoimmune disease caused by perturbations of the immune system. The clinical presentation is heterogeneous, largely because of the multiple genetic and environmental factors that contribute to disease initiation and progression. Over the last 60 years, there have been a number of significant leaps in our understanding of the immunological mechanisms driving disease processes. We now know that multiple leucocyte subsets, together with inflammatory cytokines, chemokines and regulatory mediators that are normally involved in host protection from invading pathogens, contribute to the inflammatory events leading to tissue destruction and organ failure. In this broad overview, we discuss the main pathways involved in SLE and highlight new findings. We describe the immunological changes that characterize this form of autoimmunity. The major leucocytes that are essential for disease progression are discussed, together with key mediators that propagate the immune response and drive the inflammatory response in SLE.
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http://dx.doi.org/10.1093/rheumatology/kew427DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5410978PMC
April 2017

IL-12/IL-23p40 Is Highly Expressed in Secondary Lymphoid Organs and the CNS during All Stages of EAE, but Its Deletion Does Not Affect Disease Perpetuation.

PLoS One 2016 25;11(10):e0165248. Epub 2016 Oct 25.

Department of Neurology, University of Texas Southwestern Medical Center at Dallas, Dallas, TX, United States of America.

Background: Interleukin (IL)-12 and IL-23 are heterodimers that share the p40 subunit, and both cytokines are critical in the differentiation of T helper (Th)1 and Th17 cells, respectively. Th1 and Th17 effector cells have been implicated in the pathogenesis of experimental autoimmune encephalitis (EAE), an animal model of the human central nervous system (CNS) autoimmune demyelinating disorder multiple sclerosis (MS). However, ustekinumab, a monoclonal antibody (mAb) against p40 failed to show efficacy over placebo in a phase II clinical trial in patients with MS. The role of p40 in initial T cell priming and maintenance in secondary lymphoid tissues is not yet well understood.

Methods: Active EAE was induced in the B6.129-IL12b strain of p40eYFP reporter mice (yet40 mice), and Th1 and Th17 polarized cells were adoptively transferred into p40-deficient mice. Cellular subsets were phenotyped by multi-parameter flow cytometry, and p40 tissue expression was identified by confocal microscopy.

Results: We show that yet40 mice are susceptible to EAE, and that p40 is highly expressed in secondary lymphoid organs and the CNS during all stages of the disease. Interestingly, p40 expression in the recipient is not required for EAE induction after adoptive transfer of activated and differentiated encephalitogenic Th1 and Th17 cells into p40-deficient mice. Peripheral antagonism of T helper cell trophic factors critical for the differentiation and maintenance of Th1 and Th17 cells ameliorates EAE, indicating that p40 may play a critical role in the induction of CNS autoimmunity but not in its perpetuation.

Conclusion: Our data may explain why ustekinumab did not ameliorate paraclinical and clinical disease in patients with MS. In patients with already established disease, activated antigen-specific encephalitogenic CD4+ T cells are likely already differentiated, and are not dependent on p40 for maintenance. A clinical trial of longer duration with anti-p40 mAbs or other forms of pharmacological p40 antagonism, or sequential anti-p40 therapy following T cell depletion may show a benefit by affecting de novo generation of autoimmune T cells.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0165248PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5079572PMC
June 2017

Behavioural and neurological symptoms accompanied by cellular neuroinflammation in IL-10-deficient mice infected with Plasmodium chabaudi.

Malar J 2016 08 24;15(1):428. Epub 2016 Aug 24.

Department of Microbiology and Immunology, University of Texas Medical Branch, 301 University Blvd., Galveston, TX, 77555, USA.

Background: Cerebral malaria is one of the most severe complications of Plasmodium falciparum infection and occurs mostly in young African children. This syndrome results from a combination of high levels of parasitaemia and inflammation. Although parasite sequestration in the brain is a feature of the human syndrome, sequestering strains do not uniformly cause severe malaria, suggesting interplay with other factors. Host genetic factors such as mutations in the promoters of the cytokines IL-10 and TNF are also clearly linked to severe disease. Plasmodium chabaudi, a rodent malaria parasite, leads to mild illness in wildtype animals. However, IL-10(-/-) mice respond to parasite with increased levels of pro-inflammatory cytokines IFN-γ and TNF, leading to lethal disease in the absence of sequestration in the brain. These mice also exhibit cerebral symptoms including gross cerebral oedema and haemorrhage, allowing study of these critical features of disease without the influence of sequestration.

Methods: The neurological consequences of P. chabaudi infection were investigated by performing a general behavioural screen (SHIRPA). The immune cell populations found in the brain during infection were also analysed using flow cytometry and confocal microscopy.

Results: IL-10(-/-) mice suffer significant declines in behavioural and physical capacities during infection compared to wildtype. In addition, grip strength and pain sensitivity were affected, suggestive of neurological involvement. Several immune cell populations were identified in the perfused brain on day 7 post-infection, suggesting that they are tightly adherent to the vascular endothelium, or potentially located within the brain parenchyma. There was an increase in both inflammatory monocyte and resident macrophage (CD11b(hi), CD45(+), MHCII(+), Ly6C(+/-)) numbers in IL-10(-/-) compared to wildtype animals. In addition, the activation state of all monocytes and microglia (CD11b(int), CD45(-), MHC-II(+)) were increased. T cells making IFN-γ were also identified in the brain, but were localized within the vasculature, and not the parenchyma.

Conclusions: These studies demonstrate exacerbated neuroinflammation concurrent with development of behavioural symptoms in P. chabaudi infection of IL-10(-/-) animals.
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http://dx.doi.org/10.1186/s12936-016-1477-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4995805PMC
August 2016

The neonatal CNS is not conducive for encephalitogenic Th1 T cells and B cells during experimental autoimmune encephalomyelitis.

J Neuroinflammation 2013 May 24;10:67. Epub 2013 May 24.

Department of Neurology and Neurotherapeutics, University of Texas Southwestern Medical Center at Dallas, Dallas, TX 75390-9036, USA.

Multiple sclerosis (MS) is thought to be a CD4+ T cell mediated autoimmune demyelinating disease of the central nervous system (CNS) that is rarely diagnosed during infancy. Cellular and molecular mechanisms that confer disease resistance in this age group are unknown. We tested the hypothesis that a differential composition of immune cells within the CNS modulates age-associated susceptibility to CNS autoimmune disease. C57BL/6 mice younger than eight weeks were resistant to experimental autoimmune encephalomyelitis (EAE) following active immunization with myelin oligodendrocyte glycoprotein (MOG) peptide (p) 35-55. Neonates also developed milder EAE after transfer of adult encephalitogenic T cells primed by adult or neonate antigen presenting cells (APC). There was a significant increase in CD45+ hematopoietic immune cells and CD45+ high side scatter granulocytes in the CNS of adults, but not in neonates. Within the CD45+ immune cell compartment of adults, the accumulation of CD4+ T cells, Gr-1+ and Gr-1- monocytes and CD11c+ dendritic cells (DC) was identified. A significantly greater percentage of CD19+ B cells in the adult CNS expressed MHC II than neonate CNS B cells. Only in the adult CNS could IFNγ transcripts be detected 10 days post immunization for EAE. IFNγ is highly expressed by adult donor CD4+ T cells that are adoptively transferred but not by transferred neonate donor cells. In contrast, IL-17 transcripts could not be detected in adult or neonate CNS in this EAE model, and neither adult nor neonate donor CD4+ T cells expressed IL-17 at the time of adoptive transfer.
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http://dx.doi.org/10.1186/1742-2094-10-67DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3679999PMC
May 2013

Developmental maturation of innate immune cell function correlates with susceptibility to central nervous system autoimmunity.

Eur J Immunol 2013 Aug 3;43(8):2078-88. Epub 2013 Jun 3.

Department of Neurology, Technische Universität München, Munich, Germany.

MS is an inflammatory CNS disorder, which typically occurs in early adulthood and rarely in children. Here we tested whether functional maturation of innate immune cells may determine susceptibility to CNS autoimmune disease in EAE. Two-week-old mice were resistant to active EAE, which causes fulminant paralysis in adult mice; this resistance was associated with an impaired development of Th1 and Th17 cells. Resistant, young mice had higher frequencies of myeloid-derived suppressor cells and plasma-cytoid DCs. Furthermore, myeloid APCs and B cells from young mice expressed lower levels of MHC class II and CD40, produced decreased amounts of proinflammatory cytokines, and released enhanced levels of anti-inflammatory IL-10. When used as APCs, splenocytes from 2-week-old mice failed to differentiate naive T cells into Th1 and Th17 cells irrespective of the T-cell donor's age, and promoted development of Treg cells and Th2 cells instead. Adoptive transfer of adult APCs restored the ability of 2-week-old mice to generate encephalitogenic T cells and develop EAE. Collectively, these findings indicate that the innate immune compartment functionally matures during development, which may be a prerequisite for development of T-cell-mediated CNS autoimmune disease.
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http://dx.doi.org/10.1002/eji.201343338DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4136766PMC
August 2013

CCL2 upregulation triggers hypoxic preconditioning-induced protection from stroke.

J Neuroinflammation 2012 Feb 16;9:33. Epub 2012 Feb 16.

Department of Neurological Surgery, Washington University School of Medicine, 660 S, Euclid Ave,, Box 8057, St, Louis, MO 63110, USA.

Background: A brief exposure to systemic hypoxia (i.e., hypoxic preconditioning; HPC) prior to transient middle cerebral artery occlusion (tMCAo) reduces infarct volume, blood-brain barrier disruption, and leukocyte migration. CCL2 (MCP-1), typically regarded as a leukocyte-derived pro-inflammatory chemokine, can also be directly upregulated by hypoxia-induced transcription. We hypothesized that such a hypoxia-induced upregulation of CCL2 is required for HPC-induced ischemic tolerance.

Methods: Adult male SW/ND4, CCL2-null, and wild-type mice were used in these studies. Cortical CCL2/CCR2 message, protein, and cell-type specific immunoreactivity were determined following HPC (4 h, 8% O2) or room air control (21% O2) from 6 h through 2 weeks following HPC. Circulating leukocyte subsets were determined by multi-parameter flow cytometry in naïve mice and 12 h after HPC. CCL2-null and wild-type mice were exposed to HPC 2 days prior to tMCAo, with immunoneutralization of CCL2 during HPC achieved by a monoclonal CCL2 antibody.

Results: Cortical CCL2 mRNA and protein expression peaked at 12 h after HPC (both p < 0.01), predominantly in cortical neurons, and returned to baseline by 2 days. A delayed cerebral endothelial CCL2 message expression (p < 0.05) occurred 2 days after HPC. The levels of circulating monocytes (p < 0.0001), T lymphocytes (p < 0.0001), and granulocytes were decreased 12 h after HPC, and those of B lymphocytes were increased (p < 0.0001), but the magnitude of these respective changes did not differ between wild-type and CCL2-null mice. HPC did decrease the number of circulating CCR2+ monocytes (p < 0.0001) in a CCL2-dependent manner, but immunohistochemical analyses at this 12 h timepoint indicated that this leukocyte subpopulation did not move into the CNS. While HPC reduced infarct volumes by 27% (p < 0.01) in wild-type mice, CCL2-null mice subjected to tMCAo were not protected by HPC. Moreover, administration of a CCL2 immunoneutralizing antibody prior to HPC completely blocked (p < 0.0001 vs. HPC-treated mice) the development of ischemic tolerance.

Conclusions: The early expression of CCL2 in neurons, the delayed expression of CCL2 in cerebral endothelial cells, and CCL2-mediated actions on circulating CCR2+ monocytes, appear to be required to establish ischemic tolerance to focal stroke in response to HPC, and thus represent a novel role for this chemokine in endogenous neurovascular protection.
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http://dx.doi.org/10.1186/1742-2094-9-33DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3298779PMC
February 2012

Lymph node-derived donor encephalitogenic CD4+ T cells in C57BL/6 mice adoptive transfer experimental autoimmune encephalomyelitis highly express GM-CSF and T-bet.

J Neuroinflammation 2011 Jun 24;8:73. Epub 2011 Jun 24.

Department of Neurology, University of Texas Southwestern Medical Center at Dallas, TX, USA.

Experimental autoimmune encephalomyelitis (EAE) is a relevant animal model for the human demyelinating inflammatory disorder of the central nervous system (CNS), multiple sclerosis (MS). Induction of EAE by adoptive transfer allows studying the role of the donor T lymphocyte in disease pathogenesis. It has been challenging to reliably induce adoptive transfer EAE in C57BL/6 (H-2b) mice. The goal of this study was to develop a reproducible and high yield protocol for adoptive transfer EAE in C57BL/6 mice. A step-wise experimental approach permitted us to develop a protocol that resulted in a consistent relatively high disease incidence of ~70% in recipient mice. Donor mice were immunized with myelin oligodendrocyte glycoprotein (MOG)p35-55 in complete Freund's adjuvant (CFA) followed by pertussis toxin (PT). Only lymph node cells (LNC) isolated at day 12 post immunization, and restimulated in vitro for 72 hours with 10 μg/mL of MOGp35-55 and 0.5 ng/mL of interleukin-12 (IL-12) were able to transfer disease. The ability of LNC to transfer disease was associated with the presence of inflammatory infiltrates in the CNS at day 12. Interferon gamma (IFNγ) was produced at comparable levels in cell cultures prepared from mice at both day 6 and day 12 post immunization. By contrast, there was a trend towards a negative association between IL-17 and disease susceptibility in our EAE model. The amount of GM-CSF secreted was significantly increased in the culture supernatants from cells collected at day 12 post immunization versus those collected at day 6 post-immunization. Activated CD4+ T cells present in the day 12 LNC cultures maintained expression of the transcription factor T-bet, which has been shown to regulate the expression of the IL-23 receptor. Also, there was an increased prevalence of MOGp35-55-specific CD4+ T cells in day 12 LNC after in vitro re-stimulation. In summary, encephalitogenic LNC that adoptively transfer EAE in C57BL/6 mice were not characterized by a single biomarker in our study, but by a composite of inflammatory markers. Our data further suggest that GM-CSF expression by CD4+ T cells regulated by IL-23 contributes to their encephalitogenicity in our EAE model.
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http://dx.doi.org/10.1186/1742-2094-8-73DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3161869PMC
June 2011

Memory B cells from a subset of treatment-naïve relapsing-remitting multiple sclerosis patients elicit CD4(+) T-cell proliferation and IFN-γ production in response to myelin basic protein and myelin oligodendrocyte glycoprotein.

Eur J Immunol 2010 Oct;40(10):2942-56

Department of Neurology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.

Recent evidence suggests that B- and T-cell interactions may be paramount in relapsing-remitting MS (RRMS) disease pathogenesis. We hypothesized that memory B-cell pools from RRMS patients may specifically harbor a subset of potent neuro-APC that support neuro-Ag reactive T-cell proliferation and cytokine secretion. To test this hypothesis, we compared CD80 and HLA-DR expression, IL-10 and lymphotoxin-α secretion, neuro-Ag binding capacity, and neuro-Ag presentation by memory B cells from RRMS patients to naïve B cells from RRMS patients and to memory and naïve B cells from healthy donors (HD). We identified memory B cells from some RRMS patients that elicited CD4(+) T-cell proliferation and IFN-γ secretion in response to myelin basic protein and myelin oligodendrocyte glycoprotein. Notwithstanding the fact that the phenotypic parameters that promote efficient Ag presentation were observed to be similar between RRMS and HD memory B cells, a corresponding capability to elicit CD4(+) T-cell proliferation in response to myelin basic protein and myelin oligodendrocyte glycoprotein was not observed in HD memory B cells. Our results demonstrate for the first time that the memory B-cell pool in RRMS harbors neuro-Ag specific B cells that can activate T cells.
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http://dx.doi.org/10.1002/eji.201040516DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3072802PMC
October 2010

Natalizumab and progressive multifocal leukoencephalopathy: what are the causal factors and can it be avoided?

Arch Neurol 2010 Aug;67(8):923-30

Neurology Section, VA North Texas Health Care System, Medical Service, 4500 S Lancaster Rd, Dallas, TX 75216, USA.

Natalizumab (Tysabri) was the first monoclonal antibody approved for the treatment of relapsing forms of multiple sclerosis (MS). After its initial approval, 3 patients undergoing natalizumab therapy in combination with other immunoregulatory and immunosuppressive agents were diagnosed with progressive multifocal leukoencephalopathy (PML). The agent was later reapproved and its use restricted to monotherapy in patients with relapsing forms of MS. Since reapproval in 2006, additional cases of PML were reported in patients with MS receiving natalizumab monotherapy. Thus, there is currently no convincing evidence that natalizumab-associated PML is restricted to combination therapy with other disease-modifying or immunosuppressive agents. In addition, recent data indicate that risk of PML might increase beyond 24 months of treatment.
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http://dx.doi.org/10.1001/archneurol.2010.161DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4157908PMC
August 2010

Silencing Nogo-A promotes functional recovery in demyelinating disease.

Ann Neurol 2010 Apr;67(4):498-507

Department of Neurology, Ohio State University Medical Center, Columbus, OH 43210, USA.

Objective: To determine if suppressing Nogo-A, an axonal inhibitory protein, will promote functional recovery in a murine model of multiple sclerosis (MS).

Methods: A small interfering RNA was developed to specifically suppress Nogo-A (siRNA-NogoA). The siRNA-NogoA silencing effect was evaluated in vitro and in vivo via immunohistochemistry. The siRNA was administered intravenously in 2 models of experimental autoimmune encephalomyelitis (EAE). Axonal repair was measured by upregulation of GAP43. Enzyme-linked immunosorbent assay, flow cytometry, and (3)H-thymidine incorporation were used to determine immunological changes in myelin-specific T cells in mice with EAE.

Results: The siRNA-NogoA suppressed Nogo-A expression in vitro and in vivo. Systemic administration of siRNA-NogoA ameliorated EAE and promoted axonal repair, as demonstrated by enhanced GAP43+ axons in the lesions. Myelin-specific T-cell proliferation and cytokine production were unchanged in the siRNA-NogoA-treated mice.

Interpretation: Silencing Nogo-A in EAE promotes functional recovery. The therapeutic benefit appears to be mediated by axonal growth and repair, and is not attributable to changes in the encephalitogenic capacity of the myelin-specific T cells. Silencing Nogo-A may be a therapeutic option for MS patients to prevent permanent functional deficits caused by immune-mediated axonal damage.
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http://dx.doi.org/10.1002/ana.21935DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2929680PMC
April 2010

Pharmacological prion protein silencing accelerates central nervous system autoimmune disease via T cell receptor signalling.

Brain 2010 Feb 9;133(Pt 2):375-88. Epub 2010 Feb 9.

Department of Neurology, University of Texas Southwestern Medical Center at Dallas, TX, USA.

The primary biological function of the endogenous cellular prion protein has remained unclear. We investigated its biological function in the generation of cellular immune responses using cellular prion protein gene-specific small interfering ribonucleic acid in vivo and in vitro. Our results were confirmed by blocking cellular prion protein with monovalent antibodies and by using cellular prion protein-deficient and -transgenic mice. In vivo prion protein gene-small interfering ribonucleic acid treatment effects were of limited duration, restricted to secondary lymphoid organs and resulted in a 70% reduction of cellular prion protein expression in leukocytes. Disruption of cellular prion protein signalling augmented antigen-specific activation and proliferation, and enhanced T cell receptor signalling, resulting in zeta-chain-associated protein-70 phosphorylation and nuclear factor of activated T cells/activator protein 1 transcriptional activity. In vivo prion protein gene-small interfering ribonucleic acid treatment promoted T cell differentiation towards pro-inflammatory phenotypes and increased survival of antigen-specific T cells. Cellular prion protein silencing with small interfering ribonucleic acid also resulted in the worsening of actively induced and adoptively transferred experimental autoimmune encephalomyelitis. Finally, treatment of myelin basic protein(1-11) T cell receptor transgenic mice with prion protein gene-small interfering ribonucleic acid resulted in spontaneous experimental autoimmune encephalomyelitis. Thus, central nervous system autoimmune disease was modulated at all stages of disease: the generation of the T cell effector response, the elicitation of T effector function and the perpetuation of cellular immune responses. Our findings indicate that cellular prion protein regulates T cell receptor-mediated T cell activation, differentiation and survival. Defects in autoimmunity are restricted to the immune system and not the central nervous system. Our data identify cellular prion protein as a regulator of cellular immunological homoeostasis and suggest cellular prion protein as a novel potential target for therapeutic immunomodulation.
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http://dx.doi.org/10.1093/brain/awp298DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2822628PMC
February 2010

Depletion of B lymphocytes from cerebral perivascular spaces by rituximab.

Arch Neurol 2009 Aug;66(8):1016-20

Department of Neurology, University of Texas Southwestern Medical Center at Dallas, Dallas, TX, USA.

Background: 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.

Objective: 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.

Main Outcome Measures: 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.

Results: 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.

Conclusions: 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.
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http://dx.doi.org/10.1001/archneurol.2009.157DOI Listing
August 2009

T-bet is essential for encephalitogenicity of both Th1 and Th17 cells.

J Exp Med 2009 Jul 22;206(7):1549-64. Epub 2009 Jun 22.

Department of Neurology, Ohio State University Medical Center, Columbus, OH 43210, USA.

The extent to which myelin-specific Th1 and Th17 cells contribute to the pathogenesis of experimental autoimmune encephalomyelitis (EAE) is controversial. Combinations of interleukin (IL)-1beta, IL-6, and IL-23 with transforming growth factor beta were used to differentiate myelin-specific T cell receptor transgenic T cells into Th17 cells, none of which could induce EAE, whereas Th1 cells consistently transferred disease. However, IL-6 was found to promote the differentiation of encephalitogenic Th17 cells. Further analysis of myelin-specific T cells that were encephalitogenic in spontaneous EAE and actively induced EAE demonstrated that T-bet expression was critical for pathogenicity, regardless of cytokine expression by the encephalitogenic T cells. These data suggest that encephalitogenicity of myelin-specific T cells appears to be mediated by a pathway dependent on T-bet and not necessarily pathway-specific end products, such as interferon gamma and IL-17.
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http://dx.doi.org/10.1084/jem.20082584DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2715092PMC
July 2009

PEG minocycline-liposomes ameliorate CNS autoimmune disease.

PLoS One 2009 7;4(1):e4151. Epub 2009 Jan 7.

Department of Neurology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas, United States of America.

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 CaCl(2) 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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0004151PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2613526PMC
February 2009

Quinpramine is a novel compound effective in ameliorating brain autoimmune disease.

Exp Neurol 2009 Feb 21;215(2):397-400. Epub 2008 Oct 21.

Department of Neurology, University of Texas Southwestern, Dallas, USA.

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.
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http://dx.doi.org/10.1016/j.expneurol.2008.10.001DOI Listing
February 2009

Decrease in the numbers of dendritic cells and CD4+ T cells in cerebral perivascular spaces due to natalizumab.

Arch Neurol 2008 Dec 13;65(12):1596-603. Epub 2008 Oct 13.

Department of Neurology, University of Texas Southwestern Medical Center at Dallas, Neurology Section, Veterans Affairs North Texas Health Care System, Medical Service, 4500 S Lancaster Rd, Dallas, TX 75216, USA.

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.
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http://dx.doi.org/10.1001/archneur.65.12.noc80051DOI Listing
December 2008

DNA-based vaccines: the future of multiple sclerosis therapy?

Expert Rev Neurother 2008 Mar;8(3):351-60

VA North Texas Health Care System, Neurology Section, Medical Service, 4500 South Lancaster Road, Dallas, TX 75216, USA.

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.
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http://dx.doi.org/10.1586/14737175.8.3.351DOI Listing
March 2008

Potential risk of progressive multifocal leukoencephalopathy with natalizumab therapy: possible interventions.

Arch Neurol 2007 Feb;64(2):169-76

Neurology Section, Medical Service, Veterans Affairs North Texas Health Care System, Dallas, TX, USA.

Natalizumab (Tysabri) is an effective therapy for multiple sclerosis. Recently, 3 patients who were treated with natalizumab developed progressive multifocal leukoencephalopathy (PML), an opportunistic infection of the brain with the polyomavirus JC. The pathogenesis of natalizumab-associated PML may be different from that of PML not associated with the drug. We reviewed biologically feasible interventions for patients diagnosed as having PML or other infections while receiving natalizumab therapy. Existing interventions include antiviral treatment, immunomodulatory therapies, hematopoietic growth factors, plasma exchange, intravenous immunoglobulins, and leukapheresis and autotransfusion of leukocytes. In addition, we examined the feasibility of experimental therapies, including small interfering RNA, the in vivo use of antiserum, and recombinant natalizumab-blocking molecules. There is only circumstantial evidence that any of the proposed treatments will benefit patients with multiple sclerosis treated with natalizumab who may develop PML. In addition, the expected incidence of PML in this patient population will likely be too low to test any of the proposed interventions in a controlled manner. Because it is currently impossible to identify patients at risk, and thus to prevent PML as a consequence of natalizumab therapy, it is important that neurologists be aware of possible therapeutic interventions.
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http://dx.doi.org/10.1001/archneur.64.2.169DOI Listing
February 2007

T-bet regulates the fate of Th1 and Th17 lymphocytes in autoimmunity.

J Immunol 2007 Feb;178(3):1341-8

Department of Neurology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.

IL-17-producing T cells (Th17) have recently been implicated in the pathogenesis of experimental autoimmune encephalomyelitis (EAE), an animal model for the human disease multiple sclerosis. However, little is known about the transcription factors that regulate these cells. Although it is clear that the transcription factor T-bet plays an essential role in the differentiation of IFN-gamma-producing CD4(+) Th1 lymphocytes, the potential role of T-bet in the differentiation of Th17 cells is not completely understood. In this study, therapeutic administration of a small interfering RNA specific for T-bet significantly improved the clinical course of established EAE. The improved clinical course was associated with suppression of newly differentiated T cells that express IL-17 in the CNS as well as suppression of myelin basic protein-specific Th1 autoreactive T cells. Moreover, T-bet was found to directly regulate transcription of the IL-23R, and, in doing so, influenced the fate of Th17 cells, which depend on optimal IL-23 production for survival. We now show for the first time that suppression of T-bet ameliorates EAE by limiting the differentiation of autoreactive Th1 cells, as well as inhibiting pathogenic Th17 cells via regulation of IL-23R.
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http://dx.doi.org/10.4049/jimmunol.178.3.1341DOI Listing
February 2007

Altered CD4+/CD8+ T-cell ratios in cerebrospinal fluid of natalizumab-treated patients with multiple sclerosis.

Arch Neurol 2006 Oct;63(10):1383-7

Department of Neurology, University of Texas Southwestern Medical Center at Dallas, USA.

Background: Treatment with natalizumab, a monoclonal antibody against the adhesion molecule very late activation antigen 4, an alpha4beta(1) integrin, was recently associated with the development of progressive multifocal leukoencephalopathy, a demyelinating disorder of the central nervous system caused by JC virus infection.

Objective: To test the effect of natalizumab treatment on the CD4(+)/CD8(+) T-cell ratios in cerebrospinal fluid (CSF) and peripheral blood.

Design: Prospective longitudinal study.

Setting: Academic and private multiple sclerosis centers.

Patients: Patients with multiple sclerosis (MS) treated with natalizumab, untreated patients with MS, patients with other neurologic diseases, and human immunodeficiency virus-infected patients.

Main Outcome Measures: CD4(+) and CD8(+) T cells were enumerated in CSF and peripheral blood. The mean fluorescence intensity of unbound alpha4 integrin on peripheral blood CD4(+) and CD8(+) T cells was analyzed before and after natalizumab therapy.

Results: Natalizumab therapy decreased the CSF CD4(+)/CD8(+) ratio of patients with MS to levels similar to those of human immunodeficiency virus-infected patients. CD4(+)/CD8(+) ratios in peripheral blood in patients with MS progressively decreased with the number of natalizumab doses, but they remained within normal limits. Six months after the cessation of natalizumab therapy, CSF CD4(+)/CD8(+) ratios normalized. The expression of unbound alpha4 integrin on peripheral blood T cells decreases with natalizumab therapy and was significantly lower on CD4(+) vs CD8(+) T cells.

Conclusions: Natalizumab treatment alters the CSF CD4(+)/CD8(+) ratio. Lower expression of unbound alpha4 integrin on CD4(+) T cells is one possible mechanism. These results may have implications for the observation that some natalizumab-treated patients with MS developed progressive multifocal leukoencephalopathy.
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http://dx.doi.org/10.1001/archneur.63.10.1383DOI Listing
October 2006

Immune surveillance in multiple sclerosis patients treated with natalizumab.

Ann Neurol 2006 May;59(5):743-7

Neurology Section, VA North Texas Health Care System, Medical Service, Dallas, USA.

Objective: Our objective was to test whether natalizumab, an antibody against very late activating antigen (VLA)-4, interferes with central nervous system immune surveillance as assessed by leukocyte cell numbers and cellular phenotypes in cerebrospinal fluid (CSF) and peripheral blood.

Methods: Cell numbers and cellular phenotypes in CSF and peripheral blood were analyzed in multiple sclerosis (MS) patients treated with natalizumab, untreated MS patients, and patients with other neurological disease (OND). JC virus DNA in the CSF and peripheral blood was quantified by kinetic polymerase chain reaction.

Results: CSF leukocyte counts, CD4(+) and CD8(+) T cells, CD19(+) B cells, and CD138(+) plasma cells were significantly lower in natalizumab-treated MS patients compared with OND patients and untreated MS patients. JC virus DNA was not detected in CSF or peripheral blood from natalizumab-treated patients. Six months after cessation of natalizumab therapy, low lymphocyte counts in the CSF persisted. The patient with the highest total leukocyte and CD4(+) and CD8(+)T-cell counts in the CSF experienced a clinical relapse.

Interpretation: These data suggest that natalizumab treatment results in a prolonged decrease of lymphocytes in the CSF and are consistent with the hypothesis that natalizumab impairs immune surveillance of the central nervous system.
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http://dx.doi.org/10.1002/ana.20858DOI Listing
May 2006

Therapeutic potential of small interfering RNA for central nervous system diseases.

Arch Neurol 2005 Dec;62(12):1810-3

Department of Neurology, UT Southwestern Medical Center, Dallas, Tex 75390-9036, USA.

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http://dx.doi.org/10.1001/archneur.62.12.1810DOI Listing
December 2005

Effect of rituximab on the peripheral blood and cerebrospinal fluid B cells in patients with primary progressive multiple sclerosis.

Arch Neurol 2005 Feb;62(2):258-64

Department of Neurology and Center for Immunology, The University of Texas Southwestern Medical Center, Dallas 75390, USA.

Background: Rituximab, an anti-CD20 monoclonal antibody that depletes CD20(+) B cells, has demonstrated efficacy in peripheral neurological diseases. Whether this efficacy can be translated to neurological diseases of the central nervous system with possible autoimmune B-cell involvement remains unknown.

Objective: To determine the effect of rituximab on cerebrospinal fluid B cells in patients with multiple sclerosis.

Design: Four patients with primary progressive multiple sclerosis were treated with rituximab. Cerebrospinal fluid and peripheral blood B-cell subsets were identified by flow cytometry from each patient before and after rituximab treatment.

Results: The B cells in cerebrospinal fluid were not as effectively depleted as their peripheral blood counterparts. Rituximab treatment temporarily suppressed the activation state of B cells in cerebrospinal fluid. The residual B cells underwent expansion after rituximab treatment.

Conclusion: The effect(s) of rituximab on the cerebrospinal fluid B-cell compartment is limited in comparison with the effect(s) on the B cells in the periphery, but this finding will need to be confirmed in a larger group of MS patients.
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http://dx.doi.org/10.1001/archneur.62.2.258DOI Listing
February 2005

Development and activation of human dendritic cells in vivo in a xenograft model of human hematopoiesis.

Stem Cells 2005 Feb;23(2):264-78

Division of Infectious Diseases Y9.206, Department of Internal Medicine, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390-9113, USA.

Dendritic cells (DCs) are derived from CD34+ progenitors and play a central role in the development of immune responses and in tolerance. Their therapeutic potential underscores the need for in vivo models that accurately recapitulate human DC development and function to provide a better understanding of DC biology in health and disease. Using nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice transplanted with human CD34+ cells as a model of human hematopoiesis, we examined DC ontogeny. Progenitors of both myeloid (m) and plasmacytoid (p) DCs were identified in the bone marrow of mice up to 24 weeks after transplant, indicating ongoing and sustained production of DCs after initial engraftment. To determine whether human DCs derived from transplanted stem cells were functional, their response to acute inflammation using lipopolysaccharide (LPS) was examined. Eighteen hours after LPS administration, a dramatic increase in the plasma levels of the human inflammatory cytokines interleukin (IL)-8, IL-10, tumor necrosis factor-alpha, and IL-12p70 was observed. Only mDCs and not pDCs responded in vivo to LPS by upregulating CD86 and CD83. In vivo activation of human mDCs resulted in a substantial increase in the ability of mDCs to induce the proliferation of naive human T cells. Taken together, these data indicate that human CD34+ cells seem to have differentiated appropriately within the NOD/SCID microenvironment into DCs that are developmentally, phenotypically, and functionally similar to the DC subsets found in humans.
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http://dx.doi.org/10.1634/stemcells.2004-0116DOI Listing
February 2005