Radiolabelled polymeric IgA: from biodistribution to a new molecular imaging tool in colorectal cancer lung metastases.

By radiolabelling monomeric (m) and polymeric (p) IgA with technetium 99m (99mTc), this study assessed IgA biodistribution and tumour-targeting potency. IgA directed against carcinoembryonic antigen (CEA), a colorectal cancer marker, was selected to involve IgA mucosal tropism. Ig was radiolabelled with 99mTc-tricarbonyl after derivatisation by 2-iminothiolane. 99mTc-IgA was evaluated by in vitro analysis. The biodistributions of radiolabelled anti-CEA mIgA, pIgA and IgG were compared in normal mice. Anti-CEA pIgA tumour uptake was studied in mice bearing the WiDr caecal orthotopic graft. IgA radiolabelling was obtained with a high yield, was stable in PBS and murine plasma, and did not alter IgA binding functionality (Kd ≈ 25 nM). Biodistribution studies in normal mice confirmed that radiolabelled pIgA - and to a lesser extent, mIgA - showed strong and fast mucosal tropism and a shorter serum half-life than IgG. In caecal tumour model mice, evaluation of the anti-CEA-pIgA biodistribution showed a high uptake in lung metastases, confirmed by histological analysis. However, no radioactivity uptake increase in the tumoural caecum was discerned from normal intestinal tissue, probably due to high IgA caecal natural tropism. In microSPECT/CT imaging, 99mTc-IgA confirmed its diagnostic potency of tumour in mucosal tissue, even if detection threshold by in vivo imaging was higher than post mortem studies. Contribution of the FcαRI receptor, studied with transgenic mouse model (Tsg SCID-CD89), did not appear to be determinant in 99mTc-IgA uptake. Pre-clinical experiments highlighted significant differences between 99mTc-IgA and 99mTc-IgG biodistributions. Furthermore, tumoural model studies suggested potential targeting potency of pIgA in mucosal tissues.

Ternary polysaccharide complexes: Colloidal drug delivery systems stabilized in physiological media.

Chitosan-hyaluronan (HYA) polyelectrolyte complexes (PECs) were designed to maintain their colloidal stabilities in physiological ionic strength and pH, via a new concept of ternary complexes. This strategy relied on the formation of a binary PEC between chitosan and a strong polyacid, dextran sulphate (DS) or heparin (HEP), and further functionalization with HYA. The major parameter leading to stabilized colloids was a high ratio of the degrees of polymerization of chitosan versus the strong polyacid. The process afforded either positive or negative particles when HYA was used in default or in excess (vs. chitosan) for the functionalization of the binary complexes. The most stable formulations were loaded with an antiretroviral drug tenofovir (TF), and could be surface functionalized with targeting IgAs. In vitro, the cationic TF loaded ternary complexes exhibited an inhibition of infection of PBMCs by the HIV-1 virus, superior to the free drug.

Phenotypic diversity and selection maintain Leishmania amazonensis infectivity in BALB/c mouse model.

Leishmania are protozoan parasites that show remarkable diversity, as revealed by the various clinical forms of leishmaniasis, which can range from mild skin lesions to severe metastatic cutaneous/mucosal lesions. The exact nature and extent of Leishmania phenotypic diversity in establishing infection is not fully understood. In order to try to understand some aspects of this diversity, we subcutaneously infected BALB/c mice with first and second generation subclones of a L. amazonensis strain isolated from a patient (BA125) and examined in vivo lesion growth rate and antimony susceptibility. In vivo fast-, medium- and slow-growing subclones were obtained; moreover, fast-growing subclones could generate slow-growing subclones and inversely, revealing the continuous generation of diversity after passage into mice. No antimony-resistant subclone appeared, probably a rare occurrence. By tagging subclone cells with a L. amazonensis genomic cosmid library, we found that only a very small number of founding cells could produce lesions. Leishmania clones transfected with in vivo selected individual cosmids were also diverse in terms of lesion growth rate, revealing the cosmid-independent intrinsic characteristics of each clone. Our results suggest that only a few of the infecting parasites are able to grow and produce lesions; later, within the cell mixture of each lesion, there coexist several parasite populations with different potentialities to grow lesions during the next infection round. This may reflect a sort of programmed heterogeneity of individual parasites, favoring the survival of some individuals in various environmental conditions.

Phenotypic diversity and selection maintain Leishmania amazonensis infectivity in BALB/c mouse model

Leishmania are protozoan parasites that show remarkable diversity, as revealed by the various clinical forms of leishmaniasis, which can range from mild skin lesions to severe metastatic cutaneous/mucosal lesions. The exact nature and extent of Leishmania phenotypic diversity in establishing infection is not fully understood. In order to try to understand some aspects of this diversity, we subcutaneously infected BALB/c mice with first and second generation subclones of a L. amazonensis strain isolated from a patient (BA125) and examined in vivo lesion growth rate and antimony susceptibility. In vivo fast-, medium- and slow-growing subclones were obtained; moreover, fast-growing subclones could generate slow-growing subclones and inversely, revealing the continuous generation of diversity after passage into mice. No antimony-resistant subclone appeared, probably a rare occurrence. By tagging subclone cells with a L. amazonensis genomic cosmid library, we found that only a very small number of founding cells could produce lesions. Leishmania clones transfected with in vivo selected individual cosmids were also diverse in terms of lesion growth rate, revealing the cosmid-independent intrinsic characteristics of each clone. Our results suggest that only a few of the infecting parasites are able to grow and produce lesions; later, within the cell mixture of each lesion, there coexist several parasite populations with different potentialities to grow lesions during the next infection round. This may reflect a sort of programmed heterogeneity of individual parasites, favoring the survival of some individuals in various environmental conditions.

Zinc-Stabilized Chitosan-Chondroitin Sulfate Nanocomplexes for HIV-1 Infection Inhibition Application.

Polyelectrolyte complexes (PECs) constituted of chitosan and chondroitin sulfate (ChonS) were formed by the one-shot addition of default amounts of polyanion to an excess of polycation. Key variables of the formulation process (e.g., degree of depolymerization, charge mixing ratio, the concentration, and pH of polyelectrolyte solutions) were optimized based on the PECs sizes and polydispersities. The PECs maintained their colloidal stability at physiological salt concentration and pH thanks to the complexation of polyelectrolytes with zinc(II) ion during the nanoPECs formation process. The PECs were capable of encapsulating an antiretroviral drug tenofovir (TF) with a minimal alteration on the colloidal stability of the dispersion. Moreover, the particle interfaces could efficiently be functionalized with anti-OVA or anti-α4ß7 antibodies with conservation of the antibody biorecognition properties over 1 week of storage in PBS at 4 °C. In vitro cytotoxicity studies showed that zinc(II) stabilized chitosan-ChonS nanoPECs were noncytotoxic to human peripheral blood mononuclear cells (PBMCs), and in vitro antiviral activity test demonstrated that nanoparticles formulations led to a dose-dependent reduction of HIV-1 infection. Using nanoparticles as a drug carrier system decreases the IC50 (50% inhibitory concentration) from an aqueous TF of 4.35 µmol·L(-1) to 1.95 µmol·L(-1). Significantly, zinc ions in this system also exhibited a synergistic effect in the antiviral potency. These data suggest that chitosan-ChonS nanoPECs can be promising drug delivery system to improve the antiviral potency of drugs to the viral reservoirs for the treatment of HIV infection.

IgA Structure Variations Associate with Immune Stimulations and IgA Mesangial Deposition.

IgA1 mesangial deposition is the hallmark of IgA nephropathy and Henoch-Schönlein purpura, the onset of which often follows infections. Deposited IgA has been reported as polymeric, J chain associated, and often, hypogalactosylated but with no information concerning the influence of the IgA repertoire or the link between immune stimuli and IgA structure. We explored these issues in the α1KI mouse model, which produces polyclonal human IgA1 prone to mesangial deposition. Compared with mice challenged by a conventional environment, mice in a specific pathogen-free environment had less IgA deposition. However, serum IgA of specific pathogen-free mice showed more galactosylation and much lower polymerization. Notably, wild-type, α1KI, and even J chain-deficient mice showed increased polymeric serum IgA on exposure to pathogens. Strict germfree conditions delayed but did not completely prevent deposition; mice housed in these conditions had very low serum IgA levels and produced essentially monomeric IgA. Finally, comparing monoclonal IgA1 that had different variable regions and mesangial deposition patterns indicated that, independently of glycosylation and polymerization, deposition might also depend on IgA carrying specific variable domains. Together with IgA quantities and constant region post-translational modifications, repertoire changes during immune responses might, thus, modulate IgA propensity to deposition. These IgA features are not associated with circulating immune complexes and C3 deposition and are more pertinent to an initial IgA deposition step preceding overt clinical symptoms in patients.

Zinc-stabilized colloidal polyelectrolyte complexes of chitosan/hyaluronan: a tool for the inhibition of HIV-1 infection.

Zinc(ii) stabilized polyelectrolyte nano-complexes (PECs) of chitosan and hyaluronan (HYA) were designed as safe and efficient drug delivery systems. HIV-1 reverse transcriptase inhibitor tenofovir (TF) was quantitatively encapsulated and the particle interface could be functionalized in PBS with targeting proteins such as anti-α4ß7 immunoglobulin A. Chitosan-HYA nanoPECs were non-cytotoxic on human peripheral blood mononuclear cells (PBMCs), within the investigated nanoparticle concentrations. A dose-dependent reduction of the HIV-1 infection of PBMCs co-cultured with the nanocarriers was observed. Even more interestingly, a synergistic effect was evidenced with the nanocarriers by comparing the IC50 (50% inhibitory concentration) value of the aqueous TF solution (4.35 µmol L-1) with that of TF loaded nanoPECs (1.71 µmol L-1) and anti-α4ß7 IgA functionalized TF/nanoPECs (1.01 µmol L-1). This effect could be attributed to the presence of zinc(ii) in the formulation of the colloids. All these data establish that the zinc(ii) stabilized chitosan-HYA nanoPECs can be potentially efficient and safe colloidal delivery system candidates for enhancing antiviral activities in the treatment of HIV infection and AIDS.

Anti-CD20 Immunoglobulin G Radiolabeling with a 99mTc-Tricarbonyl Core: In Vitro and In Vivo Evaluations.

In recent years, the diagnostic and therapeutic uses of radioisotopes have shown significant progress. Immunoglobulin (Ig) appears to be a promising tracer, particularly due to its ability to target selected antigens. The main objective of this study is to optimize and assess an Ig radiolabeling method with Technetium 99m (99mTc), an attractive radioelement used widely for diagnostic imaging. Monoclonal anti-CD20 IgG was retained to study in vitro and in vivo radiolabeling impact. After IgG derivatization with 2-iminothiolane, IgG-SH was radiolabeled by an indirect method, using a 99mTc-tricarbonyl core. Radiolabeling stability was evaluated over 24h by thin-layer chromatography. IgG integrity was checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis coupled with Western blot and autoradiography. The radiolabeled Ig's immunoaffinity was assessed in vitro by a radioimmunoassay method and binding experiments with cells (EL4-hCD20 and EL4-WT). Biodistribution studies were performed in normal BALB/c mice. Tumor uptake was assessed in mice bearing EL4-hCD20 and EL4-WT subcutaneous xenografts. With optimized method, high radiolabeling yields were obtained (95.9 ± 3.5%). 99mTc-IgG-SH was stable in phosphate-buffered saline (4°C and 25°C) and in serum (37°C), even if important sensitivity to transchelation was observed. IgG was not degraded by derivatization and radiolabeling, as shown by Western blot and autoradiography results. 99mTc-anti-CD20 IgG-SH immunoaffinity was estimated with Kd = 35 nM by both methods. In vivo biodistribution studies for 48h showed significant accumulation of radioactivity in plasma, liver, spleen, lungs and kidneys. Planar scintigraphy of mice bearing tumors showed a significant uptake of 99mTc-anti-CD20 IgG-SH in CD20+ tumor versus CD20- tumor. Radiolabeling of derivatized IgG with 99mTc-tricarbonyl was effective, stable and required few antibody amounts. This attractive radiolabeling method is "antibody safe" and preserves Ig affinity for antigen, as shown by both in vitro and in vivo experiments. This method could easily be used with noncommercial IgG or other antibody isotypes.

Anti-CD20 IgA can protect mice against lymphoma development: evaluation of the direct impact of IgA and cytotoxic effector recruitment on CD20 target cells.

BACKGROUND: While most antibody-based therapies use IgG because of their well-known biological properties, some functional limitations of these antibodies call for the development of derivatives with other therapeutic functions. Although less abundant than IgG in serum, IgA is the most abundantly produced Ig class in humans. Besides the specific targeting of its dimeric form to mucosal areas, IgA was shown to recruit polymorphonuclear neutrophils against certain targets more efficiently than does IgG1. DESIGN AND METHODS: In this study, we investigated the various pathways by which anti-tumor effects can be mediated by anti-CD20 IgA against lymphoma cells. RESULTS: We found that polymeric human IgA was significantly more effective than human IgG1 in mediating direct killing or growth inhibition of target cells in the absence of complement. We also demonstrated that this direct killing was able to indirectly induce the classical pathway of the complement cascade although to a lesser extent than direct recruitment of complement by IgG. Recruitment of the alternative complement pathway by specific IgA was also observed. In addition to activating complement for lysis of lymphoma cell lines or primary cells from patients with lymphoma, we showed that monomeric anti-CD20 IgA can effectively protect mice against tumor development in a passive immunization strategy and we demonstrated that this protective effect may be enhanced in mice expressing the human FcαRI receptor on their neutrophils. CONCLUSIONS: We show that anti-CD20 IgA antibodies have original therapeutic properties against lymphoma cells, with strong direct effects, ability to recruit neutrophils for cell cytotoxicity and even recruitment of complement, although largely through an indirect way.

Similarity of fine specificity of IgA anti-gliadin antibodies between patients with celiac disease and humanized α1KI mice.

Gliadins, and primarily α-gliadins containing several sequences such as aa 31-49, aa 56-88 (33-mer), aa 57-68, and aa 69-82, are critical in the induction of immune response or toxic reaction leading to the development of celiac disease (CLD). The role of IgA anti-gliadin antibodies (IgA AGA) is unknown. To this end, we prepared several humanized monoclonal IgA AGA using transgenic α1KI mice. Employing Pepscan with overlapping decapeptides of α-gliadin we observed a robust similarity between the specificity of humanized mouse monoclonal IgA AGA and IgA AGA from patients with florid CLD. The common immunodominant region included several sequential epitopes localized in the N-terminal part of α-gliadin (QFQGQQQPFPPQQPYPQPQPFP, aa 29-50, and QPFPSQQPYLQL, aa 47-58). Notably, IgA AGA produced by clones 8D12, 15B9, 9D12, and 18E2 had significant reactivity against sequences localized in the 33-mer, LQLQPFPQPQ (aa 56-65) and PQLPYPQPQPFL (aa 69-80). Humanized mouse monoclonal IgA AGA that have a known specificity are suitable as standard in ELISAs to detect serum IgA AGA of CLD patients and for studying the AGA pathogenic role in CLD, especially for analyzing the translocation of complex of specific IgA antibodies and individual gliadin peptides through enterocyte barrier.

The leishmania ARL-1 and Golgi traffic.

We present here the characterisation of the Leishmania small G protein ADP-Ribosylation Factor-Like protein 1 (ARL-1). The ARL-1 gene is present in one copy per haploid genome and conserved among trypanosomatids. It encodes a protein of 20 kDa, which is equally expressed in the insect promastigote and mammalian amastigote forms of the parasite. ARL-1 localises to the Trans-Golgi Network (TGN); N-terminal myristoylation is essential for TGN localisation. In vivo expression of the LdARL-1/Q74L and LdARL-1/T51N mutants (GTP- and GDP-bound blocked forms respectively) shows that GDP/GTP cycling occurs entirely within the TGN. This is contrary to previous reports in yeast and mammals, where the mutant empty form devoid of nucleotide has been considered as the GDP-blocked form. The dominant-negative empty form mutant LdARL-1/T34N inhibits endocytosis and intracellular trafficking from the TGN to the Lysosome/Multivesicular Tubule and to the acidocalcisomes; these defects are probably related to a mislocalisation of the GRIP domain-containing vesicle tethering factors which cannot be recruited to the TGN by the cytoplasmic LdARL-1/T34N. Thus, besides the functional characterization of a new mutant and a better understanding of ARL-1 GDP/GTP cycling, this work shows that Leishmania ARL-1 is a key component of an essential pathway worth future study.

Internalization and molecular interactions of human CD21 receptor.

The human CD21 is a receptor for cleavage fragments of the third complement component and for Epstein-Barr virus. Previous mutational studies showed that the cytoplasmic domain of CD21 is absolutely required for internalization of either ligand. With the exception of CD19, CD81, Leu-13 and CD35 that can form a complex with CD21 at the cell surface, no other partner that interacts with the hCD21 transmembrane or the cytoplasmic domain was identified. We investigated the internalization capacity of hCD21 tail mutants in the absence of B cell receptor cross-linking by using stable murine B cell transfectants. We provide evidence that at least two internalization motifs are activated when hCD21 binds a monoclonal antibody. In order to identify the cellular proteins that interact with the hCD21 transmembrane and cytoplasmic domains, we combined a mutational mapping with a two-hybrid system approach both in yeast and in mammalian cells. We identified four novel partners that are involved in intracellular trafficking, sorting or cytoskeleton remodeling and we mapped the hCD21 transmembrane and tail subdomains they interact with. We discuss the potential physiological significance of these findings in the context of hCD21 internalization and intracellular trafficking.

Asparagine endopeptidase is not essential for class II MHC antigen presentation but is required for processing of cathepsin L in mice.

Class II MHC molecules survey the endocytic compartments of APCs and present antigenic peptides to CD4 T cells. In this context, lysosomal proteases are essential not only for the generation of antigenic peptides but also for proteolysis of the invariant chain to allow the maturation of class II MHC molecules. Recent studies with protease inhibitors have implicated the asparagine endopeptidase (AEP) in class II MHC-restricted Ag presentation. We now report that AEP-deficient mice show no differences in processing of the invariant chain or maturation of class II MHC products compared with wild-type mice. In the absence of AEP, presentation to primary T cells of OVA and myelin oligodendrocyte glycoprotein, two Ags that contain asparagine residues within or in proximity to the relevant epitopes was unimpaired. Cathepsin (Cat) L, a lysosomal cysteine protease essential for the development to CD4 and NK T cells, fails to be processed into its mature two-chain form in AEP-deficient cells. Despite this, the numbers of CD4 and NK T cells are normal, showing that the single-chain form of Cat L is sufficient for its function in vivo. We conclude that AEP is essential for processing of Cat L but not for class II MHC-restricted Ag presentation.

Membrane specializations and endosome maturation in dendritic cells and B cells.

Interest in the cell biology of antigen presentation is centered on dendritic cells (DCs) as initiators of the immune response. The ability to examine primary antigen-presenting cells, as opposed to cell lines, has opened a new window for study of antigen processing and peptide acquisition by Class II major histocompatibility complex (MHC) products, especially where intracellular trafficking of peptide-Class-II complexes is concerned. Here, we review the dynamics of Class II MHC-positive intracellular structures in dendritic cells as well as B cells. We focus on the generation of multivesicular bodies, where Class II MHC products acquire antigenic peptide, on the endosomal transport of peptide-loaded Class II MHC to the cell surface and on the importance of Class II MHC localization in membrane microdomains.

Abortive infection of Lutzomyia longipalpis insect vectors by aflagellated LdARL-3A-Q70L overexpressing Leishmania amazonensis parasites.

Leishmania donovani ADP-ribosylation factor-like protein 3A (LdARL-3A) is a small G protein isolated from the protozoan parasite L. donovani with no defined physiological function. Previously [Cuvillier, A., Redon, F., Antoine, J.-C., Chardin, P., DeVos, T., and Merlin, G. (2000) J Cell Sci 113: 2065-2074] we have shown that overexpression in L. amazonensis promastigotes of the mutated protein LdARL-3A-Q70L, which remains constitutively associated with GTP, leads to the disappearance of the flagellum but does not impair cell viability or growth. Here we report that parasites overexpressing LdARL-3A-Q70L can invade in vitro cultivated macrophages to the same extent as control cells, demonstrating that the flagellum is not necessary for attachment to or engulfment into macrophages. These infections are productive because amastigotes differentiate and multiply. However, aflagellated LdARL-3A-Q70L-overexpressing Leishmania promastigotes could not survive in experimentally infected Lutzomyia longipalpis insect vectors, in contrast to untransfected or native LdARL-3A-overexpressing cells. Overexpression of the native and mutated proteins did not modify in vitro procyclic to metacyclic lipophosphoglycan maturation or differentiation from procyclic to metacyclic promastigotes, nevertheless there is a block in transmission of Leishmania. Better understanding of LdARL-3A pathways, notably those regarding flagellum biogenesis, may lead to the future development of Leishmania-specific drugs, which may stop parasite transmission in nature without affecting other species.

The immunoglobulin heavy-chain locus hs3b and hs4 3' enhancers are dispensable for VDJ assembly and somatic hypermutation.

The more distal enhancers of the immunoglobulin heavy-chain 3' regulatory region, hs3b and hs4, were recently demonstrated as master control elements of germline transcription and class switch recombination to most immunoglobulin constant genes. In addition, they were shown to enhance the accumulation of somatic mutations on linked transgenes. Since somatic hypermutation and class switch recombination are tightly linked processes, their common dependency on the endogenous locus 3' enhancers could be an attractive hypothesis. VDJ structure and somatic hypermutation were analyzed in B cells from mice carrying either a heterozygous or a homozygous deletion of these enhancers. We find that hs3b and hs4 are dispensable both for VDJ assembly and for the occurrence of mutations at a physiologic frequency in the endogenous locus. In addition, we show that cells functionally expressing the immunoglobulin M (IgM) class B-cell receptor encoded by an hs3b/hs4-deficient locus were fully able to enter germinal centers, undergo affinity maturation, and yield specific antibody responses in homozygous mutant mice, where IgG1 antibodies compensated for the defect in other IgG isotypes. By contrast, analysis of Peyer patches from heterozygous animals showed that peanut agglutinin (PNAhigh) B cells functionally expressing the hs3b/hs4-deficient allele were dramatically outclassed by B cells expressing the wild-type locus and normally switching to IgA. This study thus also highlights the role of germinal centers in the competition between B cells for affinity maturation and suggests that membrane IgA may promote recruitment in an activated B-cell compartment, or proliferation of activated B cells, more efficiently than IgM in Peyer patches.