Partial Balint's syndrome and left homonymous hemianopsia presenting after resection of a right occipito-parietal glioblastoma.

A 66-year-old left-handed male was admitted to our acute inpatient rehabilitation (AIR) unit following a resection of the right occipito-parietal glioblastoma. He presented with symptoms of horizontal oculomotor apraxia, contralateral optic ataxia and left homonymous hemianopsia. We diagnosed this patient with partial Bálint's syndrome (BS)- oculomotor apraxia, optic ataxia but not simultanagnosia. BS is typically caused by bilateral posterior parietal lesions, but we here describe a unique case due toresection of a right intracranial tumor. A short AIR stay allowed our patient to learn how to compensate for visuomotor and visuospatial deficits, and improved his quality of life significantly.

Social media interruption affects the acquisition of visually, not aurally, acquired information during a pathophysiology lecture.

Poor academic performance from extensive social media usage appears to be due to students' inability to multitask between distractions and academic work. However, the degree to which visually distracted students can acquire lecture information presented aurally is unknown. This study examined the ability of students visually distracted by social media to acquire information presented during a voice-over PowerPoint lecture, and to compare performance on examination questions derived from information presented aurally vs. that presented visually. Students ( n = 20) listened to a 42-min cardiovascular pathophysiology lecture containing embedded cartoons while taking notes. The experimental group ( n = 10) was visually, but not aurally, distracted by social media during times when cartoon information was presented, ~40% of total lecture time. Overall performance among distracted students on a follow-up, open-note quiz was 30% poorer than that for controls ( P < 0.001). When the modality of presentation (visual vs. aural) was compared, performance decreased on examination questions from information presented visually. However, performance on questions from information presented aurally was similar to that of controls. Our findings suggest the ability to acquire information during lecture may vary, depending on the degree of competition between the modalities of the distraction and the lecture presentation. Within the context of current literature, our findings also suggest that timing of the distraction relative to delivery of material examined affects performance more than total distraction time. Therefore, when delivering lectures, instructors should incorporate organizational cues and active learning strategies that assist students in maintaining focus and acquiring relevant information.

Unraveling the cell-type dependent radiosensitizing effects of gold through the development of a multifunctional gold nanoparticle.

The radiosensitizing efficacy of gold is well established, however, there remain several significant barriers to the successful clinical translation of nano-sized gold particles (AuNPs). These barriers include: retaining stability in relevant biological sera, demonstrating effectiveness at clinically relevant AuNP concentrations and identifying the biological context where significant benefit is most likely to be achieved. Herein we have developed a AuNP preparation, stress-tested to provide effective protection from salt and serum mediated agglomeration. Furthermore, the core AuNP is co-functionalized with two biologically derived peptides designed to enhance endocytosis and promote endosomal escape, thus maximizing intracellular AuNP surface area. In summary, these investigations demonstrate restored AuNP internalization using the co-functionalized preparation that generated significant radiosensitization, in both in vitro and in vivo models, at clinically viable treatment concentrations. Furthermore, we have identified an underpinning biological mechanism in the inherent radical scavenging capacity that could be used to predict radiosensitizing efficacy.

Igß ubiquitination activates PI3K signals required for endosomal sorting.

A wealth of in vitro data has demonstrated a central role for receptor ubiquitination in endocytic sorting. However, how receptor ubiquitination functions in vivo is poorly understood. Herein, we report that ablation of B cell antigen receptor ubiquitination in vivo uncouples the receptor from CD19 phosphorylation and phosphatidylinositol 3-kinase (PI3K) signals. These signals are necessary and sufficient for accumulating phosphatidylinositol (3,4,5)-trisphosphate (PIP3) on B cell receptor-containing early endosomes and proper sorting into the MHC class II antigen-presenting compartment (MIIC). Surprisingly, MIIC targeting is dispensable for T cell-dependent immunity. Rather, it is critical for activating endosomal toll-like receptors and antiviral humoral immunity. These findings demonstrate a novel mechanism of receptor endosomal signaling required for specific peripheral immune responses.

Detection and Enrichment of Rare Antigen-specific B Cells for Analysis of Phenotype and Function.

B cells reactive with a specific antigen usually occur at a frequency of <0.05% of lymphocytes. For decades researchers have sought methods to isolate and enrich these rare cells for studies of their phenotype and biology. Approaches are inevitably based on the principle that B cells recognize native antigen by virtue of cell surface receptors that are representative in specificity of antibodies that will eventually be secreted by their differentiated daughters. Perhaps the most obvious approach to the problem involves use of fluorochrome-conjugated antigens in conjunction with fluorescence-activated cell sorting (FACS). However, the utility of these methods is limited by cell frequency and the achievable rate of analysis and isolation by electronic sorting. A novel method to enrich rare antigen-specific B cells using magnetic nanoparticles that results in high yield enrichment of antigen-reactive B cells from large starting cell populations is described. This method enables improved monitoring of the phenotype and biology of antigen reactive cells before and following in vivo antigen encounter, such as after immunization or during development of autoimmunity.

The Use of a Pressure-Indicating Sensor Film to Provide Feedback upon Hydrogel-Forming Microneedle Array Self-Application In Vivo.

PURPOSE: To evaluate the combination of a pressure-indicating sensor film with hydrogel-forming microneedle arrays, as a method of feedback to confirm MN insertion in vivo. METHODS: Pilot in vitro insertion studies were conducted using a Texture Analyser to insert MN arrays, coupled with a pressure-indicating sensor film, at varying forces into excised neonatal porcine skin. In vivo studies involved twenty human volunteers, who self-applied two hydrogel-forming MN arrays, one with a pressure-indicating sensor film incorporated and one without. Optical coherence tomography was employed to measure the resulting penetration depth and colorimetric analysis to investigate the associated colour change of the pressure-indicating sensor film. RESULTS: Microneedle insertion was achieved in vitro at three different forces, demonstrating the colour change of the pressure-indicating sensor film upon application of increasing pressure. When self-applied in vivo, there was no significant difference in the microneedle penetration depth resulting from each type of array, with a mean depth of 237 µm recorded. When the pressure-indicating sensor film was present, a colour change occurred upon each application, providing evidence of insertion. CONCLUSIONS: For the first time, this study shows how the incorporation of a simple, low-cost pressure-indicating sensor film can indicate microneedle insertion in vitro and in vivo, providing visual feedback to assure the user of correct application. Such a strategy may enhance usability of a microneedle device and, hence, assist in the future translation of the technology to widespread clinical use.

Loss of anergic B cells in prediabetic and new-onset type 1 diabetic patients.

Although dogma predicts that under normal circumstances, potentially offensive autoreactive cells are silenced by mechanisms of immune tolerance, islet antigen-reactive B lymphocytes are known to play a crucial role in the development of autoimmunity in type 1 diabetes (T1D). Thus, participation of these cells in T1D may reflect escape from silencing mechanisms. Consistent with this concept, we found that in healthy subjects, high-affinity insulin-binding B cells occur exclusively in the anergic naive IgD(+), IgM(-) B-cell (BND) compartment. Antigen receptors expressed by these cells are polyreactive and have N-region additions, Vh usage, and charged complementarity-determining region 3 consistent with autoreactivity. Consistent with a potential early role in autoimmunity, these high-affinity insulin-binding B cells are absent from the anergic compartment of some first-degree relatives and all prediabetic and new-onset (<1 year) T1D patients tested, but return to normal levels in individuals diabetic for >1 year. Interestingly, these changes were correlated by transient loss of the entire BND compartment. These findings suggest that environmental events such as infection or injury may, by disrupting B-cell anergy, dispose individuals toward autoimmunity, the precise nature of which is specified by genetic risk factors, such as HLA alleles.

Recruitment of Cbl-b to B cell antigen receptor couples antigen recognition to Toll-like receptor 9 activation in late endosomes.

Casitas B-lineage lymphoma-b (Cbl-b) is a ubiquitin ligase (E3) that modulates signaling by tagging molecules for degradation. It is a complex protein with multiple domains and binding partners that are not involved in ubiquitinating substrates. Herein, we demonstrate that Cbl-b, but not c-Cbl, is recruited to the clustered B cell antigen receptor (BCR) and that Cbl-b is required for entry of endocytosed BCRs into late endosomes. The E3 activity of Cbl-b is not necessary for BCR endocytic trafficking. Rather, the ubiquitin associated (UBA) domain is required. Furthermore, the Cbl-b UBA domain is sufficient to confer the receptor trafficking functions of Cbl-b on c-Cbl. Cbl-b is also required for entry of the Toll-like receptor 9 (TLR9) into late endosomes and for the in vitro activation of TLR9 by BCR-captured ligands. These data indicate that Cbl-b acts as a scaffolding molecule to coordinate the delivery of the BCR and TLR9 into subcellular compartments required for productively delivering BCR-captured ligands to TLR9.

Hydrogel-forming microneedle arrays exhibit antimicrobial properties: potential for enhanced patient safety.

We describe, for the first time, the microbial characterisation of hydrogel-forming polymeric microneedle arrays and the potential for passage of microorganisms into skin following microneedle penetration. Uniquely, we also present insights into the storage stability of these hydroscopic formulations, from physical and microbiological viewpoints, and examine clinical performance and safety in human volunteers. Experiments employing excised porcine skin and radiolabelled microorganisms showed that microorganisms can penetrate skin beyond the stratum corneum following microneedle puncture. Indeed, the numbers of microorganisms crossing the stratum corneum following microneedle puncture were greater than 105 cfu in each case. However, no microorganisms crossed the epidermal skin. When using a 21G hypodermic needle, more than 104 microorganisms penetrated into the viable tissue and 106 cfu of Candida albicans and Staphylococcus epidermidis completely crossed the epidermal skin in 24 h. The hydrogel-forming materials contained no microorganisms following de-moulding and exhibited no microbial growth during storage, while also maintaining their mechanical strength, apart from when stored at relative humidities of 86%. No microbial penetration through the swelling microneedles was detectable, while human volunteer studies confirmed that skin or systemic infection is highly unlikely when polymeric microneedles are used for transdermal drug delivery. Since no pharmacopoeial standards currently exist for microneedle-based products, the exact requirements for a proprietary product based on hydrogel-forming microneedles are at present unclear. However, we are currently working towards a comprehensive specification set for this microneedle system that may inform future developments in this regard.

Monophosphorylation of CD79a and CD79b ITAM motifs initiates a SHIP-1 phosphatase-mediated inhibitory signaling cascade required for B cell anergy.

Anergic B cells are characterized by impaired signaling and activation after aggregation of their antigen receptors (BCR). The molecular basis of this impairment is not understood. In studies reported here, Src homology-2 (SH2)-containing inositol 5-phosphatase SHIP-1 and its adaptor Dok-1 were found to be constitutively phosphorylated in anergic B cells, and activation of this inhibitory circuit was dependent on Src-family kinase activity and consequent to biased BCR immunoreceptor tyrosine-based activation motif (ITAM) monophosphorylation. B cell-targeted deletion of SHIP-1 caused severe lupus-like disease. Moreover, absence of SHIP-1 in B cells led to loss of anergy as indicated by restoration of BCR signaling, loss of anergic surface phenotype, and production of autoantibodies. Thus, chronic BCR signals maintain anergy in part via ITAM monophosphorylation-directed activation of an inhibitory signaling circuit involving SHIP-1 and Dok-1.

Change you can B(cell)eive in: recent progress confirms a critical role for B cells in type 1 diabetes.

PURPOSE OF REVIEW: Here we review extant recent findings regarding the multiple roles of B cells in type 1 diabetes (T1D) and discuss how autoreactive B cells may become activated by a breach in B cell tolerance, and thereby initiate disease. Finally, we discuss the use of B cell-targeted therapies for treatment of autoimmunity. RECENT FINDINGS: Anti-CD20-specific depletion of B cells prevents and reverses diabetes in human CD20/non-obese diabetic (NOD) mice. Correspondingly, in nontransgenic NOD mice, B cells are effectively depleted with high dose antimouse CD20 mAbs of varying isotypes, and this also prevents diabetes in more than 60% of the mice when administered early, and significantly delays disease in 15-week-old animals. A separate study revealed that targeting B cells with anti-CD22/cal monoclonal antibody therapy delays diabetes onset in prediabetic NOD mice and restores normoglycemia in new-onset hyperglycemic NOD mice. In humans, a clinical trial of rituximab in new onset type 1 diabetics has yielded promising preliminary findings. SUMMARY: B cells are major players in T1D in humans, and clearly essential for disease development in the NOD mouse model of T1D. In this review, we discuss the silencing of autoreactive B cells and how failure of this process may contribute to autoimmunity. Further, we describe the most recent advances in studies of therapeutic effects of B cell depletion in T1D, and provide recent data indicating the diverse functions by which B cells may mediate disease.

Endocytic sequestration of the B cell antigen receptor and toll-like receptor 9 in anergic cells.

In autoimmune prone murine strains, sequential engagement of the B cell antigen receptor (BCR) on the cell surface and toll-like receptors (TLRs) in late endosomes is necessary and sufficient for secretion of autoantibodies. However, ubiquitous nucleoprotein self-antigens fail to elicit productive TLR activation, and break self-tolerance in anergic DNA-reactive B cells. The mechanisms limiting TLR activation in these cells are largely unknown. Here, we demonstrate that in anergic 3H9/Vkappa8 and Ars/A1 B cells the normal endocytic transit of both the ligated BCR and TLR9 into late endosomes is abrogated. The BCR and TLR9 arrest together just outside late endosomes, indicating that they enter this compartment along a single, regulated endocytic route. Access to late endosomes could be restored by reversing anergy through several methods, including conferring genetic susceptibility to autoimmunity, complementing proximal BCR signaling or by preventing BCR binding to self-antigen. Downstream of the BCR, JNK, which is activated in naive but not anergic B cells, regulated entry into late endosomes. Restoration of BCR and TLR9 endocytic trafficking rescued TLR9 activation by BCR-captured ligands. These results indicate that B cell anergy is reinforced by the exclusion of both TLRs and their BCR captured ligands from subcellular environments necessary for TLR activation.

Expression of CD80/86 on B cells is essential for autoreactive T cell activation and the development of arthritis.

Depletion of B cells in rheumatoid arthritis is therapeutically efficacious. Yet, the mechanism by which B cells participate in the inflammatory process is unclear. We previously demonstrated that Ag-specific B cells have two important functions in the development of arthritis in a murine model of rheumatoid arthritis, proteoglycan (PG)-induced arthritis (PGIA). PG-specific B cells function as autoantibody-producing cells and as APCs that activate PG-specific T cells. Moreover, the costimulatory molecule CD86 is up-regulated on PG-specific B cells in response to stimulation with PG. To address the requirement for CD80/CD86 expression on B cells in the development of PGIA, we generated mixed bone marrow chimeras in which CD80/CD86 is specifically deleted on B cells and not on other APC populations. Chimeras with a specific deficiency in CD80/CD86 expression on B cells are resistant to the induction of PGIA. The concentration of PG-specific autoantibody is similar in mice sufficient or deficient for CD80/86-expressing B cells, which indicates that resistance to PGIA is not due to the suppression of PG-specific autoantibody production. CD80/86-deficient B cells failed to effectively activate PG-specific autoreactive T cells as indicated by the failure of T cells from PG-immunized CD80/86-deficient B cell chimeras to transfer arthritis into SCID mice. In vitro secondary recall responses to PG are also dependent on CD80/86-expressing B cells. These results demonstrate that a CD80/86:CD28 costimulatory interaction between B cells and T cells is required for autoreactive T cell activation and the induction of arthritis but not for B cell autoantibody production.

Ubiquitinylation of Ig beta dictates the endocytic fate of the B cell antigen receptor.

In both infection and autoimmunity, the development of high-affinity Abs and memory requires B cells to efficiently capture and process Ags for presentation to cognate T cells. Although a great deal is known about how Ags are processed, the molecular mechanisms by which the BCR captures Ag for processing are still obscure. In this study, we demonstrate that the Ig beta component of the BCR is diubiquitinylated and that this is dependent on the E3 ligase Itch. Itch-/- B lymphocytes manifest both a defect in ligand-induced BCR internalization and endocytic trafficking to late endosomal Ag-processing compartments. In contrast, analysis of ubiquitinylation-defective receptors demonstrated that the attachment of ubiquitins to Ig beta is required for endosomal sorting and for the presentation of Ag to T cells, yet, ubiquitinylation is dispensable for receptor internalization. Membrane-bound Ig mu was not detectably ubiquitinylated nor were the conserved lysines in the mu cytosolic tail required for trafficking to late endosomes. These results demonstrate that ubiquitinylation of a singular substrate, Ig beta, is required for a specific receptor trafficking event. However, they also reveal that E3 ligases play a broader role in multiple processes that determine the fate of Ag-engaged BCR complexes.

The role of B cells in animal models of rheumatoid arthritis.

Rheumatoid arthritis is a chronic systemic inflammatory autoimmune disease that affects approximately 1% of the population. Recent studies demonstrate a significant improvement in clinical symptoms in patients treated with Rituximab, an anti-CD20 monoclonal antibody that depletes pro-B cells and mature B cells but not plasma cells. These findings indicate that B cells are an important contributor to the pathogenesis of RA. In this review we will examine the role of B cells in several different murine models of RA. There are a number of antibody-dependent mechanisms by which B cells support inflammatory processes in the joint. However, there are also antibody-independent mechanisms that involve B cell/T cell collaboration where B cells may modulate autoreactive T cell responses. In addition, B cells may be an important source of cytokines that either stimulate or inhibit autoimmune responses. Understanding the role of B cells in RA will provide new and directed therapeutic approaches to the treatment of disease.

Antigen-specific B cells are required as APCs and autoantibody-producing cells for induction of severe autoimmune arthritis.

B cells play an important role in rheumatoid arthritis, but whether they are required as autoantibody-producing cells as well as APCs has not been determined. We assessed B cell autoantibody and APC functions in a murine model of autoimmune arthritis, proteoglycan (PG)-induced arthritis, using both B cell-deficient mice and Ig-deficient mice (mIgM) mice that express an H chain transgene encoding for membrane-bound, but not secreted, IgM. The IgH transgene, when paired with endogenous lambda L chain, recognizes the hapten 4-hydroxy-3-nitro-phenyl acetyl and is expressed on 1-4% of B cells. B cell-deficient and mIgM mice do not develop arthritis after immunization with PG. In adoptive transfer of PG-induced arthritis into SCID mice, T cells from mIgM mice immunized with PG were unable to transfer disease even when B cells from PG-immunized wild-type mice were provided, suggesting that the T cells were not adequately primed and that Ag-specific B cells may be required. In fact, when PG was directly targeted to the B cell Ig receptor through a conjugate of 4-hydroxy-3-nitrophenyl acetyl-PG, T cells in mIgM mice were activated and competent to transfer arthritis. Such T cells caused mild arthritis in the absence of autoantibody, demonstrating a direct pathogenic role for T cells activated by Ag-specific B cells. Transfer of arthritic serum alone induced only mild and transient arthritis. However, both autoreactive T cells and autoantibody are required to cause severe arthritis, indicating that both B cell-mediated effector pathways contribute synergistically to autoimmune disease.

Development of inflammation in proteoglycan-induced arthritis is dependent on Fc gamma R regulation of the cytokine/chemokine environment.

FcgammaRs are specialized cell surface receptors that coordinately regulate immune responses. Although FcgammaR expression is a prerequisite for the development of several immune complex-mediated diseases, the mechanism responsible for FcgammaR-dependent regulation in autoimmunity remains unclear. Therefore, we assessed FcgammaR-dependent regulation of inflammation in proteoglycan-induced arthritis (PGIA) using FcgammaR(-/-) mice. FcgammaRIIb(-/-) mice developed arthritis at an earlier time point and with a greater severity than wild-type (WT) mice. In gamma-chain(-/-) (FcgammaRI(-/-) and FcgammaRIII(-/-)) mice, no clinical or histological evidence of inflammation was observed. Exacerbation of arthritis in FcgammaRIIb(-/-) mice correlated with enhanced PG-specific Ab production, but did not significantly affect PG-specific T cell priming. In gamma-chain(-/-) mice, the absence of arthritis did not correlate with serum Ab responses, as PG-specific Ab production was normal. Although PG-specific T cell proliferation was diminished, spleen cells from gamma-chain(-/-) mice successfully adoptively transferred arthritis into SCID mice. Our studies indicated that the mechanism responsible for FcgammaR regulation of PGIA development was at the level of inflammatory cytokine and beta-chemokine expression within the joint. FcgammaRIIb regulated the development of PGIA by controlling the initiation of cytokine and chemokine expression within the joint before the onset of arthritis, whereas the expression of FcgammaRI and or FcgammaRIII controlled cytokine and chemokine expression late in the development of PGIA during the onset of disease. These results suggest that FcgammaRs are critical for the development of inflammation during PGIA, possibly by maintaining or enhancing inflammatory cytokine and beta-chemokine production.

IL-4 and IL-12 regulate proteoglycan-induced arthritis through Stat-dependent mechanisms.

IL-4, a well-recognized modulator of macrophage activation, is perceived as an anti-inflammatory cytokine; however, under certain circumstances IL-4 may function as a proinflammatory cytokine. We have previously demonstrated that IL-4 treatment of mice with proteoglycan-induced arthritis (PGIA) inhibited the development of disease. To determine whether the capacity of IL-4 to inhibit disease is dependent on IL-4-mediated regulation of IL-12, we assessed the requirement for IL-4 in modulating development of PGIA. Immunization of mice, lacking IL-4 and Stat6, with proteoglycan results in a significant increase in arthritis severity in comparison to wild-type controls, suggesting that arthritis severity is regulated by IL-4 through a Stat6-dependent mechanism. Concomitant with exacerbated disease in IL-4(-/-) mice, there is a significant increase in the systemic production of proinflammatory cytokines IL-12, TNF-alpha, and IFN-gamma and in levels of mRNA transcripts for proinflammatory cytokines and chemokines in joints. Disease is suppressed in Stat4(-/-) mice indicating that elevated levels of IL-12 contribute to exacerbation of arthritis and that suppression is accompanied by reduced levels of IFN-gamma production. In support of this, IFN-gamma(-/-) mice are protected from PGIA and the degree of inflammation is similar to Stat4(-/-) mice. The decrease in disease severity in IFN-gamma(-/-) and Stat4(-/-) mice correlates with diminished TNF-alpha levels but there is no switch to a Th2-type response. Taken together, these results suggest that IL-4 regulates the severity of disease in PGIA by controlling IL-12 production, which in turn regulates the magnitude of IFN-gamma expression through a Stat4-dependent pathway.