Antibody-mediated status epilepticus: a retrospective multicenter survey.

Background: In recent years, an increasing number of auto-antibodies (AB) have been detected in the CSF and serum of patients with new onset epilepsy. Some of these patients develop convulsive or nonconvulsive status epilepticus (AB-SE), necessitating intensive medical care and administration of multiple antiepileptic and immunomodulatory treatments of uncertain effectiveness. Objectives: In this retrospective multicenter survey we aimed to determine the spectrum of gravity, the duration and the prognosis of the disorder. In addition, we sought to identify the antibodies associated with this condition, as well as determine whether there is a most effective treatment regime. Methods: 12 European Neurology University Clinics, with extensive experience in the treatment of SE patients, were sent a detailed questionnaire regarding symptoms and treatment of AB-SE patients. Seven centers responded positively, providing a total of 13 patients above the age of 16. Results: AB-SE affects mainly women (12/13, 92%) with a variable age at onset (17-69 years, median: 25 years). The duration of the disease is also variable (10 days to 12 years, median: 2 months). Only the 3 oldest patients died (55-69 years). Most patients were diagnosed with anti NMDAR encephalitis (8/13) and had oligoclonal bands in the CSF (9/13). No specific treatment regimen (antiepileptic, immunomodulatory) was found to be clearly superior. Most of the surviving 10 patients (77%) recovered completely or nearly so within 2 years of index poststatus. Conclusion: AB-SE is a severe but potentially reversible condition. Long duration does not seem to imply fatal outcome; however, age older than 50 years at time of onset appears to be a risk factor for death. There was no evidence for an optimal antiepileptic or immunomodulatory treatment. A prospective multicenter study is warranted in order to stratify the optimal treatment algorithm, determine clear risk factors of unfavorable outcome and long-term prognosis.

The antibody response in mice to carrier-free synthetic polymers of Plasmodium falciparum circumsporozoite repetitive epitope is I-Ab-restricted: possible implications for malaria vaccines.

The immunogenicity of a novel synthetic peptide consisting of an average of 40 (Asn-Ala-Asn-Pro) repeats of the circumsporozoite protein of Plasmodium falciparum, (NANP)40, was studied in mice without using any carrier proteins. First, high titers of anti-(NANP)40 antibodies could be obtained after immunization of C57BL/6 mice. These antibodies also reacted with an extract of mosquitoes infected with P. falciparum sporozoites. C57BL/6 nu/nu mice did not produce antibodies against (NANP)40. Secondly, when 14 strains of mice with nine different H-2 haplotypes were immunized with (NANP)40 without carrier, only H-2b mice were found to produce anti-(NANP)40 antibodies, whereas all non-H-2b mice were consistently unresponsive. This response was demonstrated to be I-A-linked by using recombinant and mutant mice. I-Ab [B10.A(5R)] mice produced anti-(NANP)40 antibodies as well as H-2b inbred mice. B6CH-2bm12 I-Ab-mutant mice showed only a very low response. Third, the antibody response against (NANP)40 could be induced in nonresponder mice by immunization with the peptide coupled to a carrier protein. In view of the existence of such an exceptional H-2b restriction in the response to sporozoite synthetic peptides in mice, the triggering of peptide-specific T cell responses in humans receiving sporozoite malaria vaccines might be difficult to achieve.

Antibody-conjugated MHC class I tetramers can target tumor cells for specific lysis by T lymphocytes.

To demonstrate that antibody-guided targeting of antigenic MHC class I-peptide tetramer on tumor cells can render them susceptible to lysis by relevant cytotoxic T lymphocytes (CTL), biotinylated HLA-A*0201/Flu matrix peptide complexes were tetramerized on streptavidin molecules previously coupled to Fab' fragments from monoclonal antibodies (mAb) specific for cell surface markers such as carcinoembryonic antigen (CEA), ErbB-2 or CD20. Flow cytometry analysis showed that coating of the HLA-A2-peptide complexes on the four HLA-A2-negative human cancer lines tested (including a CEA-positive colon carcinoma, an ErbB-2(+) breast carcinoma and two CD20(+) B lymphomas) was entirely dependent upon the specificity of the conjugated antibody fragments. More importantly, HLA-A2-restricted Flu matrix peptide-specific CTL were then found to lyse specifically and efficiently the MHC-coated target cells. These results open the way to the development of new immunotherapy strategies based on antibody targeting of MHC class I-peptide complexes.

Agglutinating Secretory IgA Preserves Intestinal Epithelial Cell Integrity during Apical Infection by Shigella flexneri.

Shigella flexneri, by invading intestinal epithelial cells (IECs) and inducing inflammatory responses of the colonic mucosa, causes bacillary dysentery. Although M cells overlying Peyer's patches are commonly considered the primary site of entry of S. flexneri, indirect evidence suggests that bacteria can also use IECs as a portal of entry to the lamina propria. Passive delivery of secretory IgA (SIgA), the major immunoglobulin secreted at mucosal surfaces, has been shown to protect rabbits from experimental shigellosis, but no information exists as to its molecular role in maintaining luminal epithelial integrity. We have established that the interaction of virulent S. flexneri with the apical pole of a model intestinal epithelium consisting of polarized Caco-2 cell monolayers resulted in the progressive disruption of the tight junction network and actin depolymerization, eventually resulting in cell death. The lipopolysaccharide (LPS)-specific agglutinating SIgAC5 monoclonal antibody (MAb), but not monomeric IgAC5 or IgGC20 MAbs of the same specificity, achieved protective functions through combined mechanisms, including limitation of the interaction between S. flexneri and epithelial cells, maintenance of the tight junction seal, preservation of the cell morphology, reduction of NF-κB nuclear translocation, and inhibition of proinflammatory mediator secretion. Our results add to the understanding of the function of SIgA-mediated immune exclusion by identifying a mode of action whereby the formation of immune complexes translates into maintenance of the integrity of epithelial cells lining the mucosa. This novel mechanism of protection mediated by SIgA is important to extend the arsenal of effective strategies to fight against S. flexneri mucosal invasion.

Site-specific conjugation of a radioiodinated phenethylamine derivative to a monoclonal antibody results in increased radioactivity localization in tumor.

The preparation of a novel radioiodination reagent, the (aminooxy)acetyl derivative of (p-[125]-iodophenyl)ethylamine, is described. Conventional radioiodination of proteins involves the formation of iodotyrosine residues, but for in vivo applications such as thyroid or stomach immunoscintigraphy, the susceptibility of these residues to tissue dehalogenases constitutes a serious disadvantage. Using our new compound, which has a particularly nonreactive aromatic ring, we confirm and extend studies published by other workers indicating the much greater in vivo stability of iodophenyl compounds compared to the more conventional iodophenolic ones. In addition, the aminooxy group of our reagent gives a stable and specific linkage to aldehyde groups formed by periodate oxidation on the sugar moiety of antibody molecules. In vitro, favorable binding activity and high stability was obtained with a (([125I]iodoaryl)amino)oxy labeled monoclonal antibody directed against carcinoembryonic antigen. In vivo, using paired labeling experiments in nude mice bearing colon carcinoma xenografts, the (([125I]iodoaryl)amino)oxy-MAb (MAb = monoclonal antibody) was compared with the same MAb 131I-labeled by conventional chloramine-T method. Tumor 125I concentration of (arylamino)oxy MAb (measured as percent injected dose per gram) was significantly higher as compared to values obtained with a conventionally labeled 131I antibody. Additionally, thyroid uptake, an indicator of iodine release from the antibody, was up to 25 times lower after injection of 125I-MAb obtained by the new method as compared to the conventionally iodinated 131I-MAb.

Microtubule-depolymerizing agents used in antibody-drug conjugates induce antitumor immunity by stimulation of dendritic cells.

Antibody-drug conjugates (ADC) are emerging as powerful treatment strategies with outstanding target-specificity and high therapeutic activity in patients with cancer. Brentuximab vedotin represents a first-in-class ADC directed against CD30(+) malignancies. We hypothesized that its sustained clinical responses could be related to the stimulation of an anticancer immune response. In this study, we demonstrate that the dolastatin family of microtubule inhibitors, from which the cytotoxic component of brentuximab vedotin is derived, comprises potent inducers of phenotypic and functional dendritic cell (DC) maturation. In addition to the direct cytotoxic effect on tumor cells, dolastatins efficiently promoted antigen uptake and migration of tumor-resident DCs to the tumor-draining lymph nodes. Exposure of murine and human DCs to dolastatins significantly increased their capacity to prime T cells. Underlining the requirement of an intact host immune system for the full therapeutic benefit of dolastatins, the antitumor effect was far less pronounced in immunocompromised mice. We observed substantial therapeutic synergies when combining dolastatins with tumor antigen-specific vaccination or blockade of the PD-1-PD-L1 and CTLA-4 coinhibitory pathways. Ultimately, treatment with ADCs using dolastatins induces DC homing and activates cellular antitumor immune responses in patients. Our data reveal a novel mechanism of action for dolastatins and provide a strong rationale for clinical treatment regimens combining dolastatin-based therapies, such as brentuximab vedotin, with immune-based therapies. Cancer Immunol Res; 2(8); 741-55. ©2014 AACR.

Inhibition of glial cell proinflammatory activities by peroxisome proliferator-activated receptor gamma agonist confers partial protection during antimyelin oligodendrocyte glycoprotein demyelination in vitro.

Peroxisome proliferator-activated receptor gamma (PPAR-gamma) is a member of the nuclear hormone superfamily originally characterized as a regulator of adipocyte differentiation and lipid metabolism. In addition, PPAR-gamma has important immunomodulatory functions. If the effect of PPAR-gamma's activation in T-cell-mediated demyelination has been recently demonstrated, nothing is known about the role of PPAR-gamma in antibody-induced demyelination in the absence of T-cell interactions and monocyte/macrophage activation. Therefore, we investigated PPAR-gamma's involvement by using an in vitro model of inflammatory demyelination in three-dimensional aggregating rat brain cell cultures. We found that PPAR-gamma was not constitutively expressed in these cultures but was strongly up-regulated following demyelination mediated by antibodies directed against myelin oligodendrocyte glycoprotein (MOG) in the presence of complement. Pioglitazone, a selective PPAR-gamma agonist, partially protected aggregates from anti-MOG demyelination. Heat shock responses and the expression of the proinflammatory cytokine tumor necrosis factor-alpha were diminished by pioglitazone treatment. Therefore, pioglitazone protection seems to be linked to an inhibition of glial cell proinflammatory activities following anti-MOG induced demyelination. We show that PPAR-gamma agonists act not only on T cells but also on antibody-mediated demyelination. This may represent a significant benefit in treating multiple sclerosis patients.

A novel derivative of the chelon desferrioxamine for site-specific conjugation to antibodies.

We describe the preparation of the modified chelator aminooxyacetyl-ferrioxamine, and the replacement of its iron atom by 67Ga at high specific activity. The aminooxy function of this compound was allowed to react with the aldehyde groups generated by the periodate oxidation of the oligosaccharide of a mouse IgG1 monoclonal antibody (MAb) directed against carcino-embryonic antigen (CEA). The use of the aminooxy group allowed a stable bond to be formed between the chelon and the antibody with no need for reduction. Iron was removed from the ferrioxamine moiety and replaced by 67Ga either before or after conjugation of the chelon to the antibody. In either case the labelled antibody was injected into nude mice bearing a human colon carcinoma having the appropriate antigenicity. Unoxidized antibody, labelled with 125I by conventional methods, was co-injected as an internal control. Additional control experiments were carried out with a non-immune IgG using the same 67Ga-labelled modified chelon as above. The in vivo distribution of the modified antibodies was evaluated at various times between 24 and 96 hr after injection. The methods used were gamma-camera imaging and, more quantitatively, gamma-counting of the various organs after dissection. Interestingly, with the metal-chelon-labelled antibody, the intensity and specificity of tumor labelling was comparable and in some cases superior to the results obtained with radio-iodinated antibody. In particular, there was almost no increase in liver and spleen uptake of radioactive metal relative to radio-iodine, contrary to what has been observed with most antibodies labelled with 111In after conjugation with DTPA.

Production of an affinity-purified antibody against an aldehyde-treated neurofilament protein for use in immunocytochemistry.

Fixation enhances cellular morphology and reduces loss of molecules during tissue processing. Antibodies against fixation-resistant epitopes are very useful, because they allow an immunocytochemical detection in tissue of better preserved morphology. However, fixatives can alter antigenicity and adversely affect the result of immunohistochemical procedures. To address this problem, this study examined the feasibility of generating antibodies to a paraformaldehyde-fixed antigen for use in immunohistochemical procedures. The large subunit of neurofilament proteins was selected for this study. Crude neurofilament proteins were isolated and separated by SDS-polyacrylamide gel electrophoresis. The large subunit of neurofilaments (NF-H) was electroeluted from the electrophoresis gel and exposed to paraformaldehyde, and used for immunization of a rabbit. The rabbit antiserum was affinity purified on CNBr-sepharose immobilized neurofilament proteins. On Western blots, the antibody reacted with the NF-H protein in a phosphorylation-dependent manner. In aldehyde-fixed cerebellum, the antibody strongly stained axons. In contrast, in alcohol-fixed cryostat sections the immunocytochemical detection was substantially reduced. The procedure presented in this study, involving a simple pretreatment of the immunogen, allows for the generation of an antibody that may be used in immunohistochemical studies where localization of the immunogen may be reduced or even lost by aldehyde fixation.

The Role of Secretory Immunoglobulin A in the Natural Sensing of Commensal Bacteria by Mouse Peyer's Patch Dendritic Cells.

The mammalian gastrointestinal (GI) tract harbors a diverse population of commensal species collectively known as the microbiota, which interact continuously with the host. From very early in life, secretory IgA (SIgA) is found in association with intestinal bacteria. It is considered that this helps to ensure self-limiting growth of the microbiota and hence participates in symbiosis. However, the importance of this association in contributing to the mechanisms ensuring natural host-microorganism communication is in need of further investigation. In the present work, we examined the possible role of SIgA in the transport of commensal bacteria across the GI epithelium. Using an intestinal loop mouse model and fluorescently labeled bacteria, we found that entry of commensal bacteria in Peyer's patches (PP) via the M cell pathway was mediated by their association with SIgA. Preassociation of bacteria with nonspecific SIgA increased their dynamics of entry and restored the reduced transport observed in germ-free mice known to have a marked reduction in intestinal SIgA production. Selective SIgA-mediated targeting of bacteria is restricted to the tolerogenic CD11c(+)CD11b(+)CD8(-) dendritic cell subset located in the subepithelial dome region of PPs, confirming that the host is not ignorant of its resident commensals. In conclusion, our work supports the concept that SIgA-mediated monitoring of commensal bacteria targeting dendritic cells in the subepithelial dome region of PPs represents a mechanism whereby the host mucosal immune system controls the continuous dialogue between the host and commensal bacteria.

Multiple facets of secretory immunoglobulin A at mucosal surfaces

Résumé : Le condylome acuminé anal (CAA), transmis par contact sexuel, résulte d'une infection par Human Papilloma Virus (HPV). Son traitement chirurgical est grevé d'un taux de récidive de 4-29%. Le but de cette étude était d'identifier une éventuelle corrélation entre type d'HPV présent dans les CAA excisés chirurgicalement et taux de récidive de la maladie, ? Cette étude rétrospective porte sur 140 patients opérés au Centre Hospitalier Universitaire Vaudois de CAA, entre 1990 et 2005. Le diagnostic lésionnel a été confirmé par un examen histomorphologique. Le(s) type(s) d'HPV présent(s) dans ces lésions a été déterminé par Polymerase Chain Reaction (PCR). Les patients ont donné leur accord à cette analyse et complété un questionnaire. Une éventuelle corrélation entre récidive de CAA, type d'HPV et status HIV a été recherchée. HPV 6 et 11 sont les virus les plus fréquemment découverts (51% et 28%, respectivement) chez les 140 patients (123H/17F). Trente-cinq (25%) d'entre eux ont présenté une récidive. HPV 11 était present chez 19 (41%) sujets. Ceci est statistiquement significatif (P<0.05), en comparaison aux autres HPVs. Il n'y a par contre pas de différence significative entre la fréquence de récidive des 33 (24%) patients HIV-positifs et le reste du collectif. HPV 11 est donc associé à un taux de récidive de CAA significativement élevé. Un suivi strict des patients atteints est nécessaire pour identifier une récidive et la traiter sans délai, notamment lorsque HPV 11 est present. Ces résultats innovateurs soulèvent la question de la nécessité de pratiquer une typisation virale systématique sur les lésions excisées. La justification d'une telle attitude demande toutefois encore d'être confirmée.

Relevance of biallelic versus monoallelic TNFRSF13B mutations in distinguishing disease-causing from risk-increasing TNFRSF13B variants in antibody deficiency syndromes.

TNFRSF13B encodes transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI), a B cell- specific tumor necrosis factor (TNF) receptor superfamily member. Both biallelic and monoallelic TNFRSF13B mutations were identified in patients with common variable immunodeficiency disorders. The genetic complexity and variable clinical presentation of TACI deficiency prompted us to evaluate the genetic, immunologic, and clinical condition in 50 individuals with TNFRSF13B alterations, following screening of 564 unrelated patients with hypogammaglobulinemia. We identified 13 new sequence variants. The most frequent TNFRSF13B variants (C104R and A181E; n=39; 6.9%) were also present in a heterozygous state in 2% of 675 controls. All patients with biallelic mutations had hypogammaglobulinemia and nearly all showed impaired binding to a proliferation-inducing ligand (APRIL). However, the majority (n=41; 82%) of the pa-tients carried monoallelic changes in TNFRSF13B. Presence of a heterozygous mutation was associated with antibody deficiency (P&lt; .001, relative risk 3.6). Heterozygosity for the most common mutation, C104R, was associated with disease (P&lt; .001, relative risk 4.2). Furthermore, heterozygosity for C104R was associated with low numbers of IgD(-)CD27(+) B cells (P= .019), benign lymphoproliferation (P&lt; .001), and autoimmune complications (P= .001). These associations indicate that C104R heterozygosity increases the risk for common variable immunodeficiency disorders and influences clinical presentation.

Intestinal bacteria condition dendritic cells to promote IgA production.

Immunoglobulin (Ig) A represents the predominant antibody isotype produced at the intestinal mucosa, where it plays an important role in limiting the penetration of commensal intestinal bacteria and opportunistic pathogens. We show in mice that Peyer's Patch-derived dendritic cells (PP-DC) exhibit a specialized phenotype allowing the promotion of IgA production by B2 cells. This phenotype included increased expression of the retinaldehyde dehydrogenase 1 (RALDH1), inducible nitric oxide synthase (iNOS), B cell activating factor of the tumor necrosis family (BAFF), a proliferation-inducing ligand (APRIL), and receptors for the neuropeptide vasoactive intestinal peptide (VIP). The ability of PP-DC to promote anti-CD40 dependent IgA was partially dependent on retinoic acid (RA) and transforming growth factor (TGF)-beta, whilst BAFF and APRIL signaling were not required. Signals delivered by BAFF and APRIL were crucial for CD40 independent IgA production, although the contribution of B2 cells to this pathway was minimal. The unique ability of PP-DC to instruct naïve B cells to differentiate into IgA producing plasma cells was mainly imparted by the presence of intestinal commensal bacteria, and could be mimicked by the addition of LPS to the culture. These data indicate that exposure to pathogen-associated molecular patterns present on intestinal commensal bacteria condition DC to express a unique molecular footprint that in turn allows them to promote IgA production.