Secondary Ab Research Articles

Anti-CD20 multivalent HPMA copolymer-Fab′ conjugates for the direct induction of apoptosis

A hybrid biomimetic system comprising high-molecular-weight, linear copolymer of N-(2-hydroxypropyl)methacrylamide (HPMA) grafted with multiple Fab′ fragments of anti-CD20 monoclonal antibody (mAb) was synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization followed by attachment of Fab′ fragments via thioether bonds. Exposure of human non-Hodgkin’s lymphoma (NHL) Raji B cells to the multivalent conjugates resulted in crosslinking of CD20 receptors and commencement of apoptosis. Five conjugates with varying molecular weight and valence (amount of Fab′ per polymer chain) were synthesized. One of the copolymers contained enzyme degradable peptide sequences (GFLG) in the backbone. The multivalency led to higher avidity and apoptosis induction compared to unconjugated whole mAb. Time-dependent studies showed that the cytotoxicity of conjugates exhibited a slower onset at shorter exposure times than mAb hyper-crosslinked with a secondary Ab; however, at longer time intervals the HPMA copolymer conjugates achieved significantly higher biological efficacies. In addition, study of the relationship between the structure of conjugates and Raji B cell apoptosis revealed that both valency and polymer molecular weight influenced biological activities, while insertion of peptide sequences into the backbone was not a factor in vitro.

A faster immunofluorescence assay for tracking infection progress of human cytomegalovirus

Immunofluorescence assay (IFA) is one of the most frequently used methods in the biological sciences and clinic diagnosis, but it is expensive and time-consuming. To overcome these limitations, we developed a faster and more cost-effective IFA (f-IFA) by modifying the standard IFA, and applied this method to track the progression of human cytomegalovirus (HCMV) infection in different cells. The f-IFA that we developed not only saves time, but also dramatically reduces the quantity of antibody (Ab), which will facilitate the application of IFA in clinic diagnosis. f-IFA requires only 15 min for blocking, 10 min incubation for each primary and secondary Abs, followed by 1 min extensive wash after each incubation. Only 25 μl of diluted Ab solution was needed for each coverslip at the primary and secondary Ab incubation steps. In addition, all steps were performed at room temperature. This f-IFA has been applied successfully to follow virion entry (pp65) and expression of viral genes (IE1, UL44, and pp65) in order to track the details of HCMV infection process. We found that ∼0.5% HCMV-infected T98G cells formed multiple-micronuclei (IE1 and nucleus staining) and had virus shedding (pp65 staining) by f-IFA, which could not be detected by the traditional IFA. Our results indicated that f-IFA is a sensitive, convenient, fast, and cost-effective method for investigating the details of virus infection progress, especially HCMV infection. The faster and cost-effective feature with higher sensitivity and specificity implies that f-IFA has potential applications in clinical diagnosis.

Abstract 1574: Quantitation of HLA-A2-MAGE3271-279 complexes on the DC surface improves anticancer vaccine designs

Abstract Background: Dendritic cells (DC) loaded with tumor antigen (TA)-derived peptides are used as vehicles for anti-cancer vaccines. HLA class-I restricted short synthetic peptides are commonly used to load DC for therapy. However, randomized clinical trials have failed to confirm the efficacy of DC-based therapies. We hypothesized that new methods for the quantification of peptide loading of DC are needed to improve the overall response rate to anti-tumor vaccines. Methods: An unlabeled monoclonal antibody (Ab), 12B6 Ab, recognizing HLA-A2-MAGE3271-279 complexes was generated by S. Ferrone. Monocyte-derived DC were generated from HLA-A2+ buffy coats of healthy donors in media containing IL-4 and GM-CSF. DC harvested on day 5 of culture were matured by further 48-h exposure to LPS or Interferon-α (IFN-γ). The HLA-A2+ cell line (T2) deficient for LMP2, TAP1 and TAP2 was maintained in a complete RPMI medium. Peptides were synthesized at the University of Pittsburgh Peptide Synthesis facility. An HLA-A2+ CTL clone specific for MAGE3271-279 was used in ELISPOT assays. Cells were stained with 12B6 Ab followed by a PE-labeled secondary Ab. Flow cytometry was used to measure mean fluorescence intensity (MFI) of the HLA class-I-peptide complexes and the loading kinetics. HLA class-I-peptide complexes were dissociated by treatment of cells with citric acid, pH 3.0. Results: The 12B6 Ab reacted with HLA-A2+DC or T2 cells loaded with MAGE3271-279 but not with HLA-A2− DC loaded with MAGE3271-279 or cells loaded with irrelevant peptides. The MFI obtained for the complexes positively correlated with the concentration of MAGE3271-279 used for loading. Maximum levels of HLA-A2-MAGE3271-279 were detectable on DC after 30min loading, while optimal loading of T2 cells required 4h incubation. The levels of detectable complexes correlated with the total expression levels of HLA-A2 molecules on the DC surface. However, donors with comparable levels HLA-A2 expression showed different levels HLA-A2-MAGE3271-279 expression after loading with the same peptide concentration. The MFI for HLA-A2-MAGE3271-279 on DC, but not on T2 cells, was increased when peptide loading was performed after low pH treatment. This treatment did not affect expression of co-stimulatory molecules on DC. Importantly, the peptide-specific stimulation of CTL generated with mDC loaded after low pH treatment resulted in a significantly increased percentage of IFN-γ secreting T cells. Conclusion: The 12B6 Ab is specific for HLA-A2-MAGE3271-279 complexes and allows for their quantification on the surface of DC. Loading of DC was increased by the removal of HLA class-I-self antigen complexes prior to the addition of the TA-derived exogenous peptide. The treated DC showed a higher density of HLA-A2-MAGE3271-279 by flow cytometry and were more effective in priming MAGE3271-279 specific CTL. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1574. doi:1538-7445.AM2012-1574

PI3 Kinase δ Is a Key Regulator of Synoviocyte Function in Rheumatoid Arthritis

Class I phosphoinositide 3 kinase (PI3K) δ is a promising therapeutic target for rheumatoid arthritis (RA) because of its contribution to leukocyte biology. However, its contribution in fibroblasts has not been studied as a mechanism that contributes to efficacy. We investigated the expression and function of PI3Kδ in synovium and cultured fibroblast-like synoviocytes (FLS). Immunohistochemistry demonstrated that PI3Kδ is highly expressed in RA synovium, especially in the synovial lining. Using quantitative PCR and Western blot analysis, we found that PI3Kδ mRNA and protein expression is higher in RA than in osteoarthritis (OA) synovium. PI3Kδ was also expressed in cultured FLS, along with PI3Kα and PI3Kβ, whereas PI3Kγ was not detectable. PI3Kδ mRNA expression was selectively induced by inflammatory cytokines tumor necrosis factor (TNF) and interleukin-1 (IL-1) but not by growth factors platelet-derived growth factor (PDGF) and transforming growth factor β (TGFβ). The use of inhibitors that block individual PI3K isoforms, including the novel selective PI3Kδ inhibitor INK007, showed that PI3Kδ is required for PDGF- and TNF-induced Akt activation. PI3Kδ inhibition also diminished PDGF-mediated synoviocyte growth and sensitized cells to H(2)O(2)-induced apoptosis. These data are the first documentation of increased PI3Kδ expression in both RA synovium and cultured synoviocytes. Furthermore, these are the first data demonstrating that PI3Kδ is a major regulator of PDGF-mediated fibroblast growth and survival via Akt. Thus, targeting PI3Kδ in RA could modulate synoviocyte function via anti-inflammatory and disease-altering mechanisms.

Biological Activity of Anti-CD20 Multivalent HPMA Copolymer-Fab’ Conjugates

High-molecular-weight, branched N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers were synthesized and conjugated with Fab' fragments of the anti-CD20 antibody, 1F5. This produced multivalent conjugates with varying valency (amount of Fab' per macromolecule) targeted to the B-cell antigen CD20. The apoptotic activity of the conjugates was screened against several B-cell lymphomas with varied expression levels of CD20 (Raji, Daudi, Ramos, Namalwa, and DG-75). The multivalent conjugates had the strongest activity against cells that had the highest expression of CD20 and failed to demonstrate any measurable activity against lymphomas that did not express the antigen. Furthermore, there was an apparent dose-dependent response to treatment with multivalent conjugates. At optimal valence and concentration, the apoptotic activity of HPMA copolymer-Fab' conjugates superseded that of free anti-CD20 Ab that was hyper-cross-linked with a polyclonal, secondary Ab.

Peracetylated N-Acetylmannosamine, a Synthetic Sugar Molecule, Efficiently Rescues Muscle Phenotype and Biochemical Defects in Mouse Model of Sialic Acid-deficient Myopathy

Distal myopathy with rimmed vacuoles/hereditary inclusion body myopathy (DMRV/hIBM), characterized by progressive muscle atrophy, weakness, and degeneration, is due to mutations in GNE, a gene encoding a bifunctional enzyme critical in sialic acid biosynthesis. In the DMRV/hIBM mouse model, which exhibits hyposialylation in various tissues in addition to muscle atrophy, weakness, and degeneration, we recently have demonstrated that the myopathic phenotype was prevented by oral administration of N-acetylneuraminic acid, N-acetylmannosamine, and sialyllactose, underscoring the crucial role of hyposialylation in the disease pathomechanism. The choice for the preferred molecule, however, was limited probably by the complex pharmacokinetics of sialic acids and the lack of biomarkers that could clearly show dose response. To address these issues, we screened several synthetic sugar compounds that could increase sialylation more remarkably and allow demonstration of measurable effects in the DMRV/hIBM mice. In this study, we found that tetra-O-acetylated N-acetylmannosamine increased cell sialylation most efficiently, and in vivo evaluation in DMRV/hIBM mice revealed a more dramatic, measurable effect and improvement in muscle phenotype, enabling us to establish analysis of protein biomarkers that can be used for assessing response to treatment. Our results provide a proof of concept in sialic acid-related molecular therapy with synthetic monosaccharides.

Heligmosomoides polygyrus Elicits a Dominant Nonprotective Antibody Response Directed against Restricted Glycan and Peptide Epitopes

Heligmosomoides polygyrus is a widely used gastrointestinal helminth model of long-term chronic infection in mice, which has not been well-characterized at the antigenic level. We now identify the major targets of the murine primary Ab response as a subset of the secreted products in H. polygyrus excretory-secretory (HES) Ag. An immunodominant epitope is an O-linked glycan (named glycan A) carried on three highly expressed HES glycoproteins (venom allergen Ancylostoma-secreted protein-like [VAL]-1, -2, and -5), which stimulates only IgM Abs, is exposed on the adult worm surface, and is poorly represented in somatic parasite extracts. A second carbohydrate epitope (glycan B), present on both a non-protein high molecular mass component and a 65-kDa molecule, is widely distributed in adult somatic tissues. Whereas the high molecular mass component and 65-kDa molecules bear phosphorylcholine, the glycan B epitope itself is not phosphorylcholine. Class-switched IgG1 Abs are found to glycan B, but the dominant primary IgG1 response is to the polypeptides of VAL proteins, including also VAL-3 and VAL-4. Secondary Ab responses include the same specificities while also recognizing VAL-7. Although vaccination with HES conferred complete protection against challenge H. polygyrus infection, mAbs raised against each of the glycan epitopes and against VAL-1, VAL-2, and VAL-4 proteins were unable to do so, even though these specificities (with the exception of VAL-2) are also secreted by tissue-phase L4 larvae. The primary immune response in susceptible mice is, therefore, dominated by nonprotective Abs against a small subset of antigenic epitopes, raising the possibility that these act as decoy specificities that generate ineffective humoral immunity.

Systemic Delivery of Allogenic Muscle Stem Cells Induces Long-Term Muscle Repair and Clinical Efficacy in Duchenne Muscular Dystrophy Dogs

Duchenne muscular dystrophy (DMD) is a genetic progressive muscle disease resulting from the lack of dystrophin and without effective treatment. Adult stem cell populations have given new impetus to cell-based therapy of neuromuscular diseases. One of them, muscle-derived stem cells, isolated based on delayed adhesion properties, contributes to injured muscle repair. However, these data were collected in dystrophic mice that exhibit a relatively mild tissue phenotype and clinical features of DMD patients. Here, we characterized canine delayed adherent stem cells and investigated the efficacy of their systemic delivery in the clinically relevant DMD animal model to assess potential therapeutic application in humans. Delayed adherent stem cells, named MuStem cells (muscle stem cells), were isolated from healthy dog muscle using a preplating technique. In vitro, MuStem cells displayed a large expansion capacity, an ability to proliferate in suspension, and a multilineage differentiation potential. Phenotypically, they corresponded to early myogenic progenitors and uncommitted cells. When injected in immunosuppressed dystrophic dogs, they contributed to myofiber regeneration, satellite cell replenishment, and dystrophin expression. Importantly, their systemic delivery resulted in long-term dystrophin expression, muscle damage course limitation with an increased regeneration activity and an interstitial expansion restriction, and persisting stabilization of the dog's clinical status. These results demonstrate that MuStem cells could provide an attractive therapeutic avenue for DMD patients.

Critical Role for CD38-mediated Ca2+ Signaling in Thrombin-induced Procoagulant Activity of Mouse Platelets and Hemostasis

CD38, a multifunctional enzyme that catalyzes the synthesis of intracellular Ca(2+) messengers, cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP), is known to be expressed on platelets. However, the role of CD38 in platelets remains unclear. Our present results show that treatment of platelets with thrombin results in a rapid and sustained Ca(2+) signal, resulting from a coordinated interplay of Ca(2+)-mobilizing messengers, inositol 1,4,5-trisphosphate, cADPR, and NAADP. By dissecting the signaling pathway using various agents, we delineated that cADPR and NAADP are sequentially produced through CD38 internalization by protein kinase C via myosin heavy chain IIA following phospholipase C activation in thrombin-induced platelets. An inositol 1,4,5-trisphosphate receptor antagonist blocked the thrombin-induced formation of cADPR and NAADP as well as Ca(2+) signals. An indispensable response of platelets relying on cytosolic calcium is the surface exposure of phosphatidylserine (PS), which implicates platelet procoagulant activity. Scrutinizing this parameter reveals that CD38(+/+) platelets fully express PS on the surface when stimulated with thrombin, whereas this response was decreased on CD38(-/-) platelets. Similarly, PS exposure and Ca(2+) signals were attenuated when platelets were incubated with 8-bromo-cADPR, bafilomycin A1, and a PKC inhibitor. Furthermore, in vivo, CD38-deficient mice exhibited longer bleeding times and unstable formation of thrombus than wild type mice. These results demonstrate that CD38 plays an essential role in thrombin-induced procoagulant activity of platelets and hemostasis via Ca(2+) signaling mediated by its products, cADPR and NAADP.

Application of flow cytometry to detect ABO blood group antibody levels in rhesus monkeys and cynomolgus monkeys

Similar to humans, most of non-human primates also contain the ABO blood group system. In this study, we sought to evaluate the ABO antibody (Ab) levels in monkeys using a modified flow cytometry method (FCM). The standard commercial human A or B-red blood cells (RBCs) were used as target cells. The binding of target cells and anti-A or B blood group Ab in sera of rhesus or cynomolgus monkeys was detected by flow cytometry after adding secondary specific fluorescence-labeled anti-human IgG or IgM Ab. Human healthy blood donor sera were used as controls. The results revealed that, using clear monkey sera, which were pre-absorbed on normal human type O RBCs to remove non-specific anti-human Abs, the modified FCM gave an accurate detection of ABO Ab levels in monkeys. When compared with the results of human sera, the distribution of ABO Ab levels in monkey sera were significantly lower (P<0.05). We concluded that the modified FCM could be used for the detection of monkey ABO Ab levels. The technique and data will be very valuable for the future establishment of ABO-incompatible organ transplant models in non-human primates, which could improve clinical applications.

IL-21 Receptor Is Critical for the Development of Memory B Cell Responses

Development of long-term humoral immunity, characterized by the formation of long-lived plasma cells (PCs) in the bone marrow and memory B cells, is a critical component of protective immunity to pathogens, and as such it is the major goal of vaccination. However, the mechanisms involved in the generation of long-term humoral immunity remain poorly understood. In this study, we used IL-21R-deficient (IL-21R.KO) mice to examine the role of the IL-21 pathway in the development of the B cell memory response. Primary IgG serum Ab responses to the T cell-dependent Ag 4-hydroxy-3-nitrophenylacetyl (NP) hapten conjugated to chicken γ globulin were delayed in IL-21R.KO mice, but reached normal titers within 3 to 4 wk of immunization. IL-21R.KO mice formed germinal centers and generated normal numbers of PCs in their bone marrow. Additionally, memory B cell formation was similar in wild-type and IL-21R.KO mice. However, NP-specific memory B cells and PCs failed to expand following secondary immunization of IL-21R.KO mice, and consequently, secondary IgG Ab responses to NP hapten conjugated to chicken γ globulin were significantly impaired. These results identify the IL-21 pathway as a critical component of the memory B cell response.

Tetra-O-methyl nordihydroguaiaretic acid (Terameprocol) inhibits the NF-κB-dependent transcription of TNF-α and MCP-1/CCL2 genes by preventing RelA from binding its cognate sites on DNA

BackgroundTetra-O-methyl nordihydroguaiaretic acid, also known as terameprocol (TMP), is a naturally occurring phenolic compound found in the resin of the creosote bush. We have shown previously that TMP will suppress production of certain inflammatory cytokines, chemokines and lipids from macrophages following stimulation with LPS or infection with H1N1 influenza virus. In this study our goal was to elucidate the mechanism underlying TMP-mediated suppression of cytokine and chemokine production. We focused our investigations on the response to LPS and the NF-κB protein RelA, a transcription factor whose activity is critical to LPS-responsiveness.MethodsReporter assays were performed with HEK293 cells overexpressing either TLR-3, -4, or -8 and a plasmid containing the luciferase gene under control of an NF-κB response element. Cells were then treated with LPS, poly(I:C), or resiquimod, and/or TMP, and lysates measured for luciferase activity.RAW 264.7 cells treated with LPS and/or TMP were used in ChIP and EMSA assays. For ChIP assays, chromatin was prepared and complexes precipitated with anti-NF-κB RelA Ab. Cross-links were reversed, DNA purified, and sequence abundance determined by Q-PCR. For EMSA assays, nuclear extracts were incubated with radiolabeled probes, analyzed by non-denaturing PAGE and visualized by autoradiography.RAW 264.7 cells treated with LPS and/or TMP were also used in fluorescence microscopy and western blot experiments. Translocation experiments were performed using a primary Ab to NF-κB RelA and a fluorescein-conjugated secondary Ab. Western blots were performed using Abs to IκB-α and phospho-IκB-α. Bands were visualized by chemiluminescence.ResultsIn reporter assays with TLR-3, -4, and -8 over-expressing cells, TMP caused strong inhibition of NF-κB-dependent transcription.ChIP assays showed TMP caused virtually complete inhibition of RelA binding in vivo to promoters for the genes for TNF-α, MCP-1/CCL2, and RANTES/CCL5 although the LPS-dependent synthesis of IκB-α was not inhibited. EMSA assays did not reveal an effect of TMP on the binding of RelA to naked DNA templates in vitro.TMP did not inhibit the nuclear translocation of NF-κB RelA nor the phosphorylation of IκB-α.ConclusionTMP acts indirectly as an inhibitor of NF-κB-dependent transcription by preventing RelA from binding the promoters of certain key cytokine and chemokine genes.

Multiple, Non-conserved, Internal Viral Ligands Naturally Presented by HLA-B27 in Human Respiratory Syncytial Virus-infected Cells

Cytotoxic T lymphocyte (CTL)-mediated death of virus-infected cells requires prior recognition of short viral peptide antigens that are presented by human leukocyte antigen (HLA) class I molecules on the surface of infected cells. The CTL response is critical for the clearance of human respiratory syncytial virus (HRSV) infection. Using mass spectrometry analysis of complex HLA-bound peptide pools isolated from large amounts of HRSV-infected cells, we identified nine naturally processed HLA-B27 ligands. The isolated peptides are derived from six internal, not envelope, proteins of the infective virus. The sequences of most of these ligands are not conserved between different HRSV strains, suggesting a mechanism to explain recurrent infection with virus of different HRSV antigenic subgroups. In addition, these nine ligands represent a significant fraction of the proteome of this virus, which is monitored by the same HLA class I allele. These data have implications for vaccine development as well as for analysis of the CTL response.

Deregulation of Mitochondrial Membrane Potential by Mitochondrial Insertion of Granzyme B and Direct Hax-1 Cleavage

The cytoplasm and the nucleus have been identified as activity sites for granzyme B (GrB) following its delivery from cytotoxic lymphocyte granules into target cells. Here we report on the ability of exogenous GrB to insert into and function within a proteinase K-resistant mitochondrial compartment. We identified Hax-1 (HS-1-associated protein X-1), a mitochondrial protein involved in the maintenance of mitochondrial membrane potential, as a GrB substrate within the mitochondrion. GrB cleaves Hax-1 into two major fragments: an N-terminal fragment that localizes to mitochondria and a C-terminal fragment that localizes to the cytosol after being released from GrB-treated mitochondria. The N-terminal Hax-1 fragment major cellular impact is on the regulation of mitochondrial polarization. Overexpression of wild-type Hax-1 or its uncleavable mutant form protects the mitochondria against GrB or valinomycin-mediated depolarization. The N-terminal Hax-1 fragment functions as a dominant negative form of Hax-1, mediating mitochondrial depolarization in a cyclophilin D-dependent manner. Thus, induced expression of the N-terminal Hax-1 fragment results in mitochondrial depolarization and subsequent lysosomal degradation of such altered mitochondria. This study is the first to demonstrate GrB activity within the mitochondrion and to identify Hax-1 cleavage as a novel mechanism for GrB-mediated mitochondrial depolarization.

T Cell-Related Costimulatory Molecules in the Conjunctiva of Patients with Severe Allergic Conjunctivitis

We showed previously that costimulatory molecules such as OX40 and 4-1BB participate in the development of murine allergic conjunctivitis (AC). We investigated whether these molecules are expressed in the conjunctiva of patients with severe AC. Giant papillae were collected from 4 patients with severe AC. As a control, bulbar conjunctivas from 4 normal volunteers were used. Frozen sections of the conjunctivas were stained with anti-CD4, anti-CD8, anti-OX40, anti-4-1BB, or control antibodies (Abs) and then incubated with the biotinylated secondary Ab. Alternatively, staining was performed with a biotinylated anti inducible costimulator (ICOS) Ab. After using an avidin-biotin complex kit, the staining levels were graded from 0 to 3. For immunofluorescence analysis, conjunctival sections were stained with primary Abs that had been fluorescently labeled. The conjunctival stroma of all 4 patients with severe AC were infiltrated with CD4+ and CD8+ cells, whereas only 1 control sample contained stromal CD4+ and CD8+ cells. Conjunctivas from patients but none of the control conjunctivas expressed OX40, 4-1BB, and ICOS. The expression levels of these molecules were significantly higher in patients than the control volunteers. Immunofluorescence analysis demonstrated that the OX40 and 4-1BB, but not the ICOS, molecules were expressed by the infiltrating CD4+ and/or CD8+ cells. Together with the data in murine AC, OX40 and 4-1BB are expressed in the conjunctiva of patients with severe AC but not in the control conjunctivas, which suggests that OX40 and 4-1BB participate, at least in part, in the development of severe AC.

Cystic fibrosis transmembrane conductance regulator in human muscle: Dysfunction causes abnormal metabolic recovery in exercise

Individuals with cystic fibrosis (CF) have exercise intolerance and skeletal muscle weakness not solely attributable to physical inactivity or pulmonary function abnormalities. CF transmembrane conductance regulator (CFTR) has been demonstrated in human bronchial smooth and cardiac muscle. Using (31)P-magnetic resonance spectroscopy of skeletal muscle, we showed CF patients to have lower resting muscle adenosine triphosphate and delayed phosphocreatine recovery times after high-intensity exercise, suggesting abnormal muscle aerobic metabolism; and higher end-exercise pH values, suggesting altered bicarbonate transport. Our objective was to study CFTR expression in human skeletal muscle. We studied CFTR expression in human skeletal muscle by Western blot with anti-CFTR antibody (Ab) L12B4 and demonstrated a single band with expected molecular weight of 168kDa. We isolated the cDNA by reverse transcription polymerase chain reaction and directly sequenced a 975bp segment (c. 3,600-4,575) that was identical to the human CFTR sequence. We showed punctate staining of CFTR in sarcoplasm and sarcolemma by immunofluorescence microscopy with L12B4 Ab and secondary Alexa 488-labeled Ab. We confirmed CFTR expression in the sarcotubular network and sarcolemma by electron microscopy, using immunogold-labeled anti-CFTR Ab. We observed activation of CFTR Cl(-) channels with iodide efflux, on addition of forskolin, 3-isobutyl-1-methyl-xanthine, and 8-chlorphenylthio-cyclic adenosine monophosphate, in wild-type C57BL/6J isolated muscle fibers in contrast to no efflux from mutant F508del-CFTR muscle. We speculate that a defect in sarcoplasmic reticulum CFTR Cl(-) channels could alter the electrochemical gradient, causing dysregulation of Ca(2+) homeostasis, for example, ryanodine receptor or sarco(endo)plasmic reticulum Ca(2+) adenosine triphosphatases essential to excitation-contraction coupling leading to exercise intolerance and muscle weakness in CF.

Modulation of influenza-specific humoral recall responses by CD154 blockade and cyclosporine A (145.28)

Abstract Background: Current methods of immunosuppression are broadly inhibitory and thus associated with an increased risk of opportunistic infections. Here we compare the effect of CD154 blockade (αCD154) and Cyclosporine A (CsA) therapy on secondary responses to influenza, when the first immunization was performed before therapy, as might be done in transplant wait-listed patients. Methods. Sixteen cynomologus monkeys were immunized with influenza vaccine in absence of immunosupression and administered a secondary boost immunization with either αCD154 mAb (IDEC-131), CsA, or no treatment. Ab responses against three influenza antigens (H1N1, H3N2, B) were measured by ELISA. An average fold increase &amp;gt;1.4 was considered positive. Results. 14 of 16 animals responded to the primary immunization (I) performed in absence of therapy. In untreated controls, 4/5 animals mounted a strong secondary response (II). With αCD154, 4/6 animals showed mild to strong IgM and IgG, and 2/6 demonstrated either IgM or IgG responses. In contrast, in CsA-treated animals, only 2/5 produced IgM without IgG, and 3/5 mounted low titers of IgG but no IgM. Conclusions. Relative to CsA, αCD154 only moderately suppressed secondary Ab responses to flu antigens. This suggests that αCD154 treatment spares important facets of adaptive immunity against opportunistic infections, potentially conferring a clinically significant advantage over conventional immunosuppression with respect to common pathogens

Antiviral protection following immunization correlates with humoral but not cell‐mediated immunity

Smallpox was a deadly disease when it was rife yet despite its eradication more than 30 years ago, the possibility of accidental or intentional release has driven research in search of better definitions of correlates of protective immunity. Mousepox, a disease caused by ectromelia virus (ECTV), is arguably one of the best surrogate small animal models for smallpox. Correlates of protection in mousepox are well defined during primary infection, whereas those in a secondary infection, which have definite relevance to vaccination strategies, are less well understood. We previously established that neutralizing antibody (Ab), which is generated far more rapidly during a secondary infection compared with a primary infection, has a key role during a secondary virus challenge. In this study, we show that the route of immunization or the use of homologous or heterologous virus vaccines for immunization does not influence the ability of mice to control high-dose virulent ECTV challenge or to mount a substantial secondary neutralizing Ab response. In contrast, the recall cytotoxic T lymphocyte (CTL) responses generated under these regimes of immunization were varied and did not correlate with virus control. Furthermore, unlike the recall Ab response that was generated rapidly, the kinetics of the secondary antiviral CTL response was no different to a primary infection and peaked only at day 8 post-challenge. This finding further underscores the importance of Ab in conferring protection during secondary poxvirus infection. This information could potentially prove useful in the design of safer and more efficacious vaccines against poxviruses or other diseases using poxvirus vectors.

Mechanism for Amyloid Precursor-like Protein 2 Enhancement of Major Histocompatibility Complex Class I Molecule Degradation

Earlier studies have demonstrated interaction of the murine major histocompatibility complex (MHC) class I molecule K(d) with amyloid precursor-like protein 2 (APLP2), a ubiquitously expressed member of the amyloid precursor protein family. Our current findings indicate that APLP2 is internalized in a clathrin-dependent manner, as shown by utilization of inhibitors of the clathrin pathway. Furthermore, we demonstrated that APLP2 and K(d) bind at the cell surface and are internalized together. The APLP2 cytoplasmic tail contains two overlapping consensus motifs for binding to the adaptor protein-2 complex, and mutation of a tyrosine shared by both motifs severely impaired APLP2 internalization and ability to promote K(d) endocytosis. Upon increased expression of wild type APLP2, K(d) molecules were predominantly directed to the lysosomes rather than recycled to the plasma membrane. These findings suggest a model in which APLP2 binds K(d) at the plasma membrane, facilitates uptake of K(d) in a clathrin-dependent manner, and routes the endocytosed K(d) to the lysosomal degradation pathway. Thus, APLP2 has a multistep trafficking function that influences the expression of major histocompatibility complex class I molecules at the plasma membrane.

Effects of dust and airborne dust components on antibody responses, body weight gain, and heart morphology of broilers

Pathogen-associated molecular patterns (PAMP) such as lipopolysaccharide (LPS), lipoteichoic acid, β-glucans (BGL), and possibly many others are important parts of (fine) dust in animal houses. When intratracheally (i.t.) administered, PAMP affected specific primary and secondary humoral immune responses to concurrently i.t. or systemically administered antigens and BW gain (BWG) of layer chickens. In the present study, we evaluated the effects of i.t. challenge with various PAMP known to be present in dust: LPS, lipoteichoic acid, zymosan-A (containing 1,3 BGL), next to heat-inactivated dust particles as a representative of mechanical stress, a combination of the former components, and NH3 as a chemical component of dust on primary and secondary (total) systemic antibody (Ab) responses and (isotype) IgM and IgG responses to concurrently i.t.-administered human serum albumin (HuSA) in broilers. Birds were challenged via the trachea for 2 consecutive days at 3 and 7 wk of age, respectively. All treatments affected immune responses at several moments, BWG, and heart morphology. β-Glucans and LPS affected the birds most pronounced and for a prolonged period. Intratracheally administered LPS and BGL significantly enhanced primary and secondary total Ab, IgM Ab, and IgG Ab responses to HuSA. All birds that were challenged with dust, PAMP, or NH3 concurrently with HuSA showed a decreased BWG especially after primary, but also after secondary challenge. Weight, width, and length of hearts were enhanced in dust and PAMP-treated birds as well when these birds were challenged with HuSA. The present results indicated that components of dust such as PAMP when i.t. administered affect humoral immune responsiveness of broilers, which may lead to an enhanced status of immune reactivity. Furthermore, our results suggest that the hygienic status of the environment influences BWG and may affect heart morphology, and as a consequence physiology in broilers. The consequences of our findings with respect to dust, (airborne) PAMP, hygienic conditions in the barn, and immune responsiveness of broilers are discussed.