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Pro/Ala-rich sequences (PAS) are polypeptides that were developed as a biological alternative to poly-ethylene glycol (PEG) to generate biopharmaceuticals with extended plasma half-life. Like PEG, PAS polypeptides are conformationally disordered and show high solubility in water. Devoid of any charged or prominent hydrophobic side chains, these biosynthetic polymers represent an extreme case of intrinsically disordered proteins. Despite lack of immunogenicity of PAS tags in numerous animal studies we now succeeded in generating monoclonal antibodies (MAbs) against three different PAS versions. To this end, mice were immunized with a PAS#1, P/A#1 or APSA 40mer peptide conjugated to keyhole limpet hemocyanin as highly immunogenic carrier protein. In each case, one MAb with high binding activity and specificity towards a particular PAS motif was obtained. The apparent affinity was strongly dependent on the avidity effect and most pronounced for the bivalent MAb when interacting with a long PAS repeat. X-ray structural analysis of four representative anti-PAS Fab fragments in complex with their cognate PAS epitope peptides revealed interactions dominated by hydrogen bond networks involving the peptide backbone as well as multiple Van der Waals contacts arising from intimate shape complementarity. Surprisingly, Ala, the L-amino acid with the smallest side chain, emerged as a crucial feature for epitope recognition, contributing specific contacts at the center of the paratope in several anti-PAS complexes. Apart from these insights into how antibodies can recognize feature-less peptides without secondary structure, the MAbs characterized in this study offer valuable reagents for the preclinical and clinical development of PASylated biologics., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Objective: To appraise the utility as biomarkers of blood antibodies and immune complexes to neurofilaments and dipeptide repeat proteins, the products of translation of the most common genetic mutation in amyotrophic lateral sclerosis (ALS)., Methods: Antibodies and immune complexes against neurofilament light, medium, heavy chains as well as poly-(GP)-(GR) dipeptide repeats were measured in blood samples from the ALS Biomarkers (n = 107) and the phenotype-genotype biomarker (n = 129) studies and in 140 healthy controls. Target analyte levels were studied longitudinally in 37 ALS cases. Participants were stratified according to the rate of disease progression estimated before and after baseline and C9orf72 genetic status. Survival and longitudinal analyses were undertaken with reference to matched neurofilament protein expression., Results: Compared to healthy controls, total neurofilament proteins and antibodies, neurofilament light immune complexes (p < 0.0001), and neurofilament heavy antibodies (p = 0.0061) were significantly elevated in ALS, patients with faster progressing disease (p < 0.0001) and in ALS cases with a C9orf72 mutation (p < 0.0003). Blood neurofilament light protein discriminated better ALS from healthy controls (AUC: 0.92; p < 0.0001) and faster from slower progressing ALS (AUC: 0.86; p < 0.0001) compared to heavy-chain antibodies and light-chain immune complexes (AUC: 0.79; p < 0.0001 and AUC: 0.74; p < 0.0001). Lower neurofilament heavy antibodies were associated with longer survival (Log-rank Chi-square: 7.39; p = 0.0065). Increasing levels of antibodies and immune complexes between time points were observed in faster progressing ALS., Conclusions: We report a distinctive humoral response characterized by raising antibodies against neurofilaments and dipeptide repeats in faster progressing and C9orf72 genetic mutation carriers ALS patients. We confirm the significance of plasma neurofilament proteins in the clinical stratification of ALS., (© 2021 The Authors. Annals of Clinical and Translational Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association.)

Major Histocompatibility Complex (MHC) class I molecules selectively bind peptides for presentation to cytotoxic T cells. The peptide-free state of these molecules is not well understood. Here, we characterize a disulfide-stabilized version of the human class I molecule HLA-A*02:01 that is stable in the absence of peptide and can readily exchange cognate peptides. We present X-ray crystal structures of the peptide-free state of HLA-A*02:01, together with structures that have dipeptides bound in the A and F pockets. These structural snapshots reveal that the amino acid side chains lining the binding pockets switch in a coordinated fashion between a peptide-free unlocked state and a peptide-bound locked state. Molecular dynamics simulations suggest that the opening and closing of the F pocket affects peptide ligand conformations in adjacent binding pockets. We propose that peptide binding is co-determined by synergy between the binding pockets of the MHC molecule.

Mitogen-activated protein kinase kinase 6 (MKK6) is an essential component of the p38MAPK signaling pathway, which is involved in the modulation of inflammation, cell apoptosis and survival responses in mammals. However, the function of MKK6s in teleosts is still unclear. In this study, a fish MKK6 homolog (CiMKK6) was first identified from the grass carp (Ctenopharyngodon idella), a freshwater fish. CiMKK6 cDNA encodes a putative protein of 357 amino acids that contains conserved structural characteristics of the MKK6 family, including the S&#95;TKc domain, SVAKT motif and DVD site. The deduced CiMKK6 protein exhibits high sequence homology with other reported fish MKK6s and shares the closest relationship with MKK6 from Danio rerio. Quantitative real-time PCR (qRT-PCR) analysis revealed that CiMKK6 mRNA was widely expressed in all tested tissues and stages of embryonic development. Additionally, the transcript levels of CiMKK6 in the intestine were significantly upregulated in response to bacterial muramyl dipeptide (MDP) and L-Ala-γ-D-Glu-meso-diaminopimelic acid (Tri-DAP) stimulation. Moreover, subcellular localization analysis indicated that CiMKK6 was distributed in both the cytoplasm and the nucleus of HEK293T cells. Finally, overexpression of CiMKK6 significantly enhanced the transcriptional activity of the AP-1 reporter gene in HEK293T cells. Overall, these findings may help better clarify the immune function of teleost MKK6s and provide new insight into the immune defense mechanisms of grass carp., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The generation, maturation and remodelling of the extracellular matrix (ECM) are essential for the formation of alveoli during lung development. Alveoli formation is disturbed in preterm infants that develop bronchopulmonary dysplasia (BPD), where collagen fibres are malformed, and perturbations to lung ECM structures may underlie BPD pathogenesis. Malformed ECM structures might result from abnormal protein cross-linking, in part attributable to the increased expression and activity of transglutaminase 2 (TGM2) that have been noted in affected patient lungs, as well as in hyperoxia-based BPD animal models. The objective of the present study was to assess whether TGM2 plays a causal role in normal and aberrant lung alveolarization. Targeted deletion of Tgm2 in C57BL/6J mice increased septal thickness and reduced gas-exchange surface area in otherwise normally developing lungs. During aberrant lung alveolarization that occurred under hyperoxic conditions, collagen structures in Tgm2 -/- mice were partially protected from the impact of hyperoxia, where normal dihydroxylysinonorleucine and hydroxylysylpiridinoline collagen cross-link abundance was restored; however, the lung alveolar architecture remained abnormal. Inhibition of transglutaminases (including TGM2) with cysteamine appreciably reduced transglutaminase activity in vivo, as assessed by N ε -(γ-l-glutamyl)-l-lysine abundance and TGM catalytic activity, and restored normal dihydroxylysinonorleucine and hydroxylysylpiridinoline collagen cross-link abundance under pathological conditions. Furthermore, a moderate improvement in alveoli size and gas-exchange surface density was noted in cysteamine-treated mouse lungs in which BPD was modelled. These data indicate that TGM2 plays a role in normal lung alveolarization, and contributes to the formation of aberrant ECM structures during disordered lung alveolarization., (© 2018 Federation of European Biochemical Societies.)

The recognition of pathogen-derived ligands by pattern recognition receptors activates the innate immune response, but the potential interaction of quorum-sensing (QS) signaling molecules with host anti-viral defenses remains largely unknown. Here we show that the Vibrio vulnificus QS molecule cyclo(Phe-Pro) (cFP) inhibits interferon (IFN)-β production by interfering with retinoic-acid-inducible gene-I (RIG-I) activation. Binding of cFP to the RIG-I 2CARD domain induces a conformational change in RIG-I, preventing the TRIM25-mediated ubiquitination to abrogate IFN production. cFP enhances susceptibility to hepatitis C virus (HCV), as well as Sendai and influenza viruses, each known to be sensed by RIG-I but did not affect the melanoma-differentiation-associated gene 5 (MDA5)-recognition of norovirus. Our results reveal an inter-kingdom network between bacteria, viruses and host that dysregulates host innate responses via a microbial quorum-sensing molecule modulating the response to viral infection.

Self-assembled peptide hydrogels are particularly appealing for drug delivery, tissue engineering, and antitumor therapy due to various advantageous features including excellent biocompatibility and biodegradability, defined molecular and higher organized structures, and easy availability. However, the poor mechanical and rheological properties of assembled peptide hydrogels cause difficulties in injection, thus limiting further applications. Herein, injectable peptide-based hydrogels with tunable mechanical and rheological properties were obtained by combination with a positively charged poly peptide (poly-l-lysine, PLL). Electrostatic coupling between PLL and a self-assembling dipeptide (Fmoc-FF) provides a smart switch to enable the fibrous hydrogels to be shear-thinning and self-healing, thus leading to the formation of supramolecular hydrogels with rheological properties suitable for injection. The latter can be flexibly adjusted by merely varying the concentration or the molecular weight of the polypeptide to satisfy a variety of requirements in biological applications. The hydrogels, consisting of helical nanofibers stabilized with disulfide bonds, are prepared and further injected for antitumor therapy. The results demonstrate that the helical fibrous hydrogel, without the addition of antigens, immune regulatory factors, and adjuvants, can activate T cell response and efficiently suppress tumor growth. Therefore, injectable hydrogels self-assembled by a combination of small peptides and biomacromolecules present a great potential for biomedical applications, especially for development of a new type of immuno-responsive materials toward antitumor therapy.

Solanezumab (Eli Lilly) and crenezumab (Genentech) are the leading clinical antibodies targeting Amyloid-β (Aβ) to be tested in multiple Phase III clinical trials for the prevention of Alzheimer's disease in at-risk individuals. Aβ capture by these clinical antibodies is explained here with the first reported mid-region Aβ-anti-Aβ complex crystal structure. Solanezumab accommodates a large Aβ epitope (960 Å(2) buried interface over residues 16 to 26) that forms extensive contacts and hydrogen bonds to the antibody, largely via main-chain Aβ atoms and a deeply buried Phe19-Phe20 dipeptide core. The conformation of Aβ captured is an intermediate between observed sheet and helical forms with intramolecular hydrogen bonds stabilising residues 20-26 in a helical conformation. Remarkably, Aβ-binding residues are almost perfectly conserved in crenezumab. The structure explains the observed shared cross reactivity of solanezumab and crenezumab with proteins abundant in plasma that exhibit this Phe-Phe dipeptide.

Many malignant cells release the NKG2D ligand ULBP2 from their cell surface to evade immunosurveillance by NK cells and CD8 T cells. Although the shedding mechanism remains unclear, various inhibitors of matrix metalloproteinases have been shown to efficiently block the release of soluble ULBP2. The clinical use of these inhibitors, however, is limited because of adverse side effects. Using high-throughput screening technique, we identified a specific inhibitor of phosphatase of regenerating liver 3 (PRL-3) that could reduce the level of soluble ULBP2 in the culture supernatant of various cancer cell lines. Inhibition or gene knockdown of PRL-3 did not reduce ULBP2 shedding, but rather suppressed posttranslational maturation of ULBP2, resulting in intracellular retention of immature ULBP2. We then found that ULBP2 was constitutively associated with heat shock protein HSP60. Complete maturation of ULBP2 required tyrosine phosphorylation of HSP60 which was mediated by PRL-3., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Polyreactive antibodies represent a significant fraction of immune repertoires and play an important role in the immune defense and immune homeostasis. Polyreactive B-cell receptors (BCR), however, are frequently expressed by B-cell lymphomas. It was suggested that polyreactive BCR on lymphoma cells might deliver stimulation signals by binding to various endogenous or exogenous antigens, thus promoting the survival of the malignant cells. In addition to natural polyreactive antibodies, immune repertoires contain antibodies that acquire polyreactivity after exposure to different redox-active substances such as reactive oxygen species, iron ions and heme. Here, we demonstrate that an antibody cloned from a patient's splenic marginal zone B-cell lymphoma acquires physiologically relevant binding affinity to various autoantigens following exposure to heme. We elucidated the mechanisms underlying polyreactive antigen binding. The results obtained in this study imply that antigen-binding receptors expressed on some malignant cells acquire polyreactivity after exposure to redox substances that are released at sites of inflammation or as a result of cellular damage. The acquisition of novel BCR specificities under hemolytic or inflammatory conditions may play an important role in the physiopathology of certain B-cell malignancies., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Background: In the past, numerous methods have been developed for predicting antigenic regions or B-cell epitopes that can induce B-cell response. To the best of authors' knowledge, no method has been developed for predicting B-cell epitopes that can induce a specific class of antibody (e.g., IgA, IgG) except allergenic epitopes (IgE). In this study, an attempt has been made to understand the relation between primary sequence of epitopes and the class of antibodies generated., Results: The dataset used in this study has been derived from Immune Epitope Database and consists of 14725 B-cell epitopes that include 11981 IgG, 2341 IgE, 403 IgA specific epitopes and 22835 non-B-cell epitopes. In order to understand the preference of residues or motifs in these epitopes, we computed and compared amino acid and dipeptide composition of IgG, IgE, IgA inducing epitopes and non-B-cell epitopes. Differences in composition profiles of different classes of epitopes were observed, and few residues were found to be preferred. Based on these observations, we developed models for predicting antibody class-specific B-cell epitopes using various features like amino acid composition, dipeptide composition, and binary profiles. Among these, dipeptide composition-based support vector machine model achieved maximum Matthews correlation coefficient of 0.44, 0.70 and 0.45 for IgG, IgE and IgA specific epitopes respectively. All models were developed on experimentally validated non-redundant dataset and evaluated using five-fold cross validation. In addition, the performance of dipeptide-based model was also evaluated on independent dataset., Conclusion: Present study utilizes the amino acid sequence information for predicting the tendencies of antigens to induce different classes of antibodies. For the first time, in silico models have been developed for predicting B-cell epitopes, which can induce specific class of antibodies. A web service called IgPred has been developed to serve the scientific community. This server will be useful for researchers working in the field of subunit/epitope/peptide-based vaccines and immunotherapy (http://crdd.osdd.net/raghava/igpred/).

Although tumors naturally prime adaptive immune responses, tolerance may limit the capacity to control progression and can compromise effectiveness of immune-based therapies for cancer. Post-proline cleaving enzymes (PPCE) modulate protein function through N-terminal dipeptide cleavage and inhibition of these enzymes has been shown to have anti-tumor activity. We investigated the mechanism by which Val-boroPro, a boronic dipeptide that inhibits post-proline cleaving enzymes, mediates tumor regression and tested whether this agent could serve as a novel immune adjuvant to dendritic cell vaccines in two different murine syngeneic murine tumors. In mice challenged with MB49, which expresses the HY antigen complex, T cell responses primed by the tumor with and without Val-boroPro were measured using interferon gamma ELISPOT. Antibody depletion and gene-deficient mice were used to establish the immune cell subsets required for tumor regression. We demonstrate that Val-boroPro mediates tumor eradication by accelerating the expansion of tumor-specific T cells. Interestingly, T cells primed by tumor during Val-boroPro treatment demonstrate increased capacity to reject tumors following adoptive transfer without further treatment of the recipient. Val-boroPro -mediated tumor regression requires dendritic cells and is associated with enhanced trafficking of dendritic cells to tumor draining lymph nodes. Finally, dendritic cell vaccination combined with Val-boroPro treatment results in complete regression of established tumors. Our findings demonstrate that Val-boroPro has antitumor activity and a novel mechanism of action that involves more robust DC trafficking with earlier priming of T cells. Finally, we show that Val-boroPro has potent adjuvant properties resulting in an effective therapeutic vaccine.

Posttranslational modifications of proteins increase the complexity of the cellular proteome and enable rapid regulation of protein functions in response to environmental changes. Protein ubiquitylation is a central regulatory posttranslational modification that controls numerous biological processes including proteasomal degradation of proteins, DNA damage repair and innate immune responses. Here we combine high-resolution mass spectrometry with single-step immunoenrichment of di-glycine modified peptides for mapping of endogenous putative ubiquitylation sites in murine tissues. We identify more than 20,000 unique ubiquitylation sites on proteins involved in diverse biological processes. Our data reveals that ubiquitylation regulates core signaling pathways common for each of the studied tissues. In addition, we discover that ubiquitylation regulates tissue-specific signaling networks. Many tissue-specific ubiquitylation sites were obtained from brain highlighting the complexity and unique physiology of this organ. We further demonstrate that different di-glycine-lysine-specific monoclonal antibodies exhibit sequence preferences, and that their complementary use increases the depth of ubiquitylation site analysis, thereby providing a more unbiased view of protein ubiquitylation.

Background: Nε-homocysteinyl-lysine (Nε-Hcy-Lys), a product of proteolysis of Nε-homocysteinylated proteins, has been discovered recently. We sought to investigate the presence of Nε-Hcy-Lys in patients on long-term hemodialysis (HD) and its association with markers involved in atherosclerotic vascular disease., Methods: We studied 86 patients on long-term (median, 45 months) HD and 95 apparently healthy controls. Nε-Hcy-Lys and total homocysteine (tHcy) were assayed using high-performance liquid chromatography. Paraoxonase 1 (PON1), asymmetric dimethylarginine (ADMA), folate, 8-isoprostaglandin F2α(8-iso-PGF2α), plasminogen activator inhibitor-1 (PAI-1), C-reactive protein (CRP), together with antibodies against Nε-homocysteinylated albumin and hemoglobin, were also measured., Results: Nε-Hcy-Lys was detected in 15 HD patients (17.4%). Those patients had 3.1-times lower PON1 (p<0.0001), 20% higher ADMA (p<0.0001), 30% higher PAI-1 (p<0.0001), 10% lower total cholesterol (p=0.001) and LDL-cholesterol (p<0.0001), together with 20% lower triglycerides (p<0.0001) compared with subjects without measurable Nε-Hcy-Lys. Nε-Hcy-Lys levels correlated with PON1 (r=-0.62, p<0.0001), ADMA (r=0.58, p<0.0001) and PAI-1 (r=0.59, p<0.0001). Folic acid supplementation, tHcy, folate, autoimmune response to Nε-Hcy-proteins, and oxidative stress were not associated with the presence of Nε-Hcy-Lys. PON1 is the only independent predictor of the presence of Nε-Hcy-Lys in HD patients. None of controls had measurable Nε-Hcy-Lys in serum., Conclusion: The presence of Nε-Hcy-Lys in HD patients is relatively infrequent and associated with lipid profile, endothelial dysfunction and impaired fibrinolysis, regardless of tHcy and folate levels.

Lymphoma is an important disease in dogs and people, with similar biological characteristics. We tested the binding affinity of a peptidomimetic LLP2A, previously shown to bind the alpha4-beta1 integrin on human lymphoma cell lines, to lymphocytes of dogs with spontaneously occurring lymphoma. Fine needle aspirates of lymph nodes from 32 dogs with B-cell lymphoma and 7 dogs with T-cell lymphoma were evaluated using flow cytometry. For B cells, the lowest MFI levels were in unlabeled, non-neoplastic lymphocytes. The highest median fluorescent intensity (MFI) levels occurred in LLP2A-labeled lymphoma cells from dogs that had not received chemotherapy followed by labeled lymphoma cells from dogs that had received chemotherapy. The fluorescence profile of the T-cell samples was similar although many of the differences were not statistically significant, likely due to low sample number. Specifically, LLP2A-labeled T-cell lymphoma cells had a significantly higher MFI compared to unlabeled non-neoplastic lymphocytes. LLP2A affinity was not significantly different in unlabeled and labeled T-cell lymphoma cells, and labeled non-neoplastic lymphocytes. For both B and T cells, labeling with LLP2A tended to increase MFI in both normal and lymphoma cells. Lymphoma cells had higher mean MFI levels than non-neoplastic lymphocytes, and chemotherapy acted to decrease MFI. In summary, these data demonstrate that LLP2A has affinity to canine lymphoma cells and indicates expression of the alpha4-beta1 integrin on these cells. In fact, LLP2A preferentially binds neoplastic B-cells, suggesting that this small molecule may be of use in cross-species clinical trials of targeted therapeutics., (Copyright © 2011 Elsevier B.V. All rights reserved.)

A bacterial strain with putative immunomodulatory properties was isolated from Xi'an hot springs in China. Comparison of 16S rRNA gene revealed a 97% similarity between the tested strain (designated XA-1) and Anoxybacillus kamchatkensis. Two compounds isolated from the secondary metabolites of XA-1 were identified by spectral data (infrared, nuclear magnetic resonance and mass spectrometry) as: (1) cyclo (Gly-L-Pro) and (2) cyclo (L-Ala-4-hydroxyl-L-Pro). Two cyclic dipeptides showed stimulatory properties towards a range of parameters when a dose of 20mg kg(-1) body weight was intraperitoneally injected in naive common carp, Cyprinus carpio. Innate immune parameters (serum SOD, lysozyme and bactericidal activity, and phagocytic activity by peripheral blood leucocytes) along with the expression of two immune-related genes (IL-1β and iNOS) in blood were examined after 7, 14, 21, and 28 days of injection. In the absence of infection, immunomodulators should ideally not affect normal physiology and immunity of the host; possible negative outcomes of activated immune responses in the naive state are discussed. Protection by two bacterial dipeptides was assessed in an intraperitoneal injection challenge trial with live Aeromonas hydrophila. Both compounds reduced mortality, with the highest survival rate observed in the group that received compound 2 (80%) followed by the group that received compound 1 (65%) while control group scored the worse (15%). Elucidation of the involved protective mechanisms in carp requires future studies., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Viruses are known to employ different strategies to manipulate the major histocompatibility (MHC) class I antigen presentation pathway to avoid recognition of the infected host cell by the immune system. However, viral control of antigen presentation via the processes that supply and select antigenic peptide precursors is yet relatively unknown. The Epstein-Barr virus (EBV)-encoded EBNA1 is expressed in all EBV-infected cells, but the immune system fails to detect and destroy EBV-carrying host cells. This immune evasion has been attributed to the capacity of a Gly-Ala repeat (GAr) within EBNA1 to inhibit MHC class I restricted antigen presentation. Here we demonstrate that suppression of mRNA translation initiation by the GAr in cis is sufficient and necessary to prevent presentation of antigenic peptides from mRNAs to which it is fused. Furthermore, we demonstrate a direct correlation between the rate of translation initiation and MHC class I antigen presentation from a certain mRNA. These results support the idea that mRNAs, and not the encoded full length proteins, are used for MHC class I restricted immune surveillance. This offers an additional view on the role of virus-mediated control of mRNA translation initiation and of the mechanisms that control MHC class I restricted antigen presentation in general.

Background: Glutamine (Gln) may become conditionally indispensable during critical illness. The short-term metabolic effects of enteral versus parenteral Gln supplementation are unknown in this clinical setting., Objectives: We studied metabolic effects of intravenous (i.v.) alanyl-Gln dipeptide (AG) supplementation and enteral (e.n.) AG supplementation on plasma Gln concentration, antioxidant status, plasma lymphocyte subset number, gut permeability and nitrogen balance in adult critically ill patients requiring tube feeding compared to a control group not receiving Gln supplementation., Methods: In a double-blind, pilot clinical trial, 44 medical and surgical ICU patients received identical Gln-free tube feedings 24 h/day and were randomized to either isonitrogenous control (n=15), e.n. AG (n=15) or i.v. AG (n=14) groups (AG). Twelve patients were discontinued from the study. The goal AG dose was 0.5 g/kg/day. Biochemical and metabolic endpoints were measured at baseline and on day 9 (plasma Gln, antioxidant indices, lymphocyte subsets; serum IGF-1 and IGF-binding protein-3; intestinal permeability). Nitrogen balance was determined between study days 6 and 8., Results: Illness severity indices, clinical demographics, enteral energy and nitrogen intake and major biochemical indices were similar between groups during study. Plasma Gln was higher in the i.v. AG (565+/-119 microM, mean+/-SEM) vs the e.n. AG (411+/-27 microM) group by day 9 (p=0.039); however, subjects in the i.v. AG group received a higher dose of AG (i.v. AG 0.50 versus e.n. AG 0.32+/-0.02 g/kg/day; p<0.001). E.n. AG subjects showed a significant increase in plasma alpha-tocopherol levels over time and maintained plasma gamma-tocopherol concentrations. There were no differences between groups for plasma concentrations of vitamin C, glutathione, malondialdehyde (MDA), T-lymphocyte subsets, intestinal permeability or nitrogen balance., Conclusions: This study showed that alanyl-Gln administration by enteral or parenteral routes did not appear to affect antioxidant capacity or oxidative stress markers, T-lymphocyte subset (CD-3, CD-4, CD-8) number, gut barrier function or whole-body protein metabolism compared to unsupplemented ICU patients requiring enteral tube feeding. Enteral Gln appeared to maintain plasma tocopherol levels in this pilot metabolic study.

A novel anticancer vaccine candidate built on a nonpeptidic scaffold has been synthesized. Four S-Tn tumor-associated glycomimetic antigens have been clustered onto a calix[4]arene scaffold bearing an immunoadjuvant moiety (P3CS). The immunogenicity of the synthetic construct has been investigated by immunization of mice in vivo. ELISA assay has evidenced that the tetravalent construct stimulates a higher production of anti-Tn antigen IgG antibodies when compared to an analogous monovalent compound. This result is ascribable to an antigen cluster effect and makes the reported vaccine candidate a good mimic of the natural motifs present on the mucine surface.

Purpose: Bispecific antibody (bsMAb) pretargeting procedures use divalent hapten-peptides to stabilize the binding of the hapten-peptide on tumor cells by a process known as the affinity enhancement system. The goal of this study was to determine if a divalent hapten-peptide could induce apoptosis by cross-linking bsMAb bound to CD20., Methods: Three forms of bsMAbs were prepared by coupling the IgG, F(ab')2, or Fab' of a humanized anti-CD20 antibody to a Fab' of a murine antibody directed against the hapten histamine-succinyl-glycine (HSG). A recombinant bsMAb with divalent binding to CD20 and monovalent binding to HSG was also examined. Induction of apoptosis on SU-DHL-6, RL, and Ramos cells was examined by propidium iodide staining, caspase-3 activation, and mitochondrial membrane potential collapse, and compared with induction by cross-linking an anti-CD20 IgG with an antispecies antibody., Results: The various forms of bsMAb had differing baseline levels of apoptosis in the absence of the divalent HSG peptide. The addition of the divalent HSG peptide significantly increased the level of apoptosis seen with the Fab'xFab' bsMAb by 2.2- to 3.9-fold, as well as the F(ab')2xFab', IgGxFab', and the recombinant bsMAbs by approximately 1.5-fold., Conclusions: The addition of a divalent HSG peptide to various forms of bispecific anti-CD20 MAbs could enhance apoptotic signaling in several lymphoma cells. This effect was more consistently measured when the orientation of the anti-hapten-binding arm of the bsMAb was well defined, such as in the Fab'xFab' and recombinant forms of bsMAb.

Noopept, a peptide analog of piracetam, enhanced phagocytic activity of mouse peritoneal macrophages, stimulated humoral and cellular immune response to various antigens, and markedly increased spontaneous proliferative activity of splenocytes. In animals with secondary immune deficiency caused by cyclophosphamide, noopept exhibited immunocorrector properties.

Chronic lung infection by opportunistic pathogens, such as Pseudomonas aeruginosa and members of the Burkholderia cepacia complex, is a major cause of morbidity and mortality in patients with cystic fibrosis. Outer membrane proteins (OMPs) of gram-negative bacteria are promising vaccine antigen candidates. In this study, we evaluated the immunogenicity, protection, and cross-protection conferred by intranasal vaccination of mice with OMPs from B. multivorans plus the mucosal adjuvant adamantylamide dipeptide (AdDP). Robust mucosal and systemic immune responses were stimulated by vaccination of naive animals with OMPs from B. multivorans and B. cenocepacia plus AdDP. Using a mouse model of chronic pulmonary infection, we observed enhanced clearance of B. multivorans from the lungs of vaccinated animals, which correlated with OMP-specific secretory immunoglobulin A responses. Furthermore, OMP-immunized mice showed rapid resolution of the pulmonary infection with virtually no lung pathology after bacterial challenge with B. multivorans. In addition, we demonstrated that administration of B. multivorans OMP vaccine conferred protection against B. cenocepacia challenge in this mouse infection model, suggesting that OMPs provide cross-protection against the B. cepacia complex. Therefore, we concluded that mucosal immunity to B. multivorans elicited by intranasal vaccination with OMPs plus AdDP could prevent early steps of colonization and infection with B. multivorans and also ameliorate lung tissue damage, while eliciting cross-protection against B. cenocepacia. These results support the notion that therapies leading to increased mucosal immunity in the airways may help patients with cystic fibrosis.

Adamantylamide L-alanyl-D-isoglutamine (AdDP) is a synthetic adjuvant which belongs to the family of the desmuramyl peptides. AdDP exerts its adjuvant properties when it is administered either by the parenteral or by the mucosal route, leading to the elicitation of strong humoral responses at both the systemic and the mucosal levels. However, very little is known about the effect of AdDP on cellular immunity. Here we demonstrate that AdDP is able to stimulate cellular responses, which are characterized by the release of gamma interferon by CD8+ T cells when they are restimulated with a major histocompatibility complex class I-restricted peptide and strong in vivo lymphocyte-mediated cytotoxic activity. The capacity of AdDP to stimulate the elicitation of both cellular and humoral adaptive responses makes this adjuvant a promising tool for the development of mucosal vaccine formulations.

Moraxella catarrhalis causes acute otitis media in children and lower respiratory tract infections in adults and elderly. In children the presence of antibodies against the highly conserved outer membrane protein CD correlates with protection against infection, suggesting that this protein may be useful as a vaccine antigen. However, native CD is difficult to purify, and it is still unclear if recombinant CD (rCD) is a valid alternative. We performed a side-by-side comparison of the immunogenicities and efficacies of vaccine formulations containing native CD and rCD with adamantylamide dipeptide as the mucosal adjuvant. Intranasal vaccination of mice stimulated the production of high CD-specific antibody titers in sera and of secretory immunoglobulin A in mucosal lavages, which cross-recognized both antigens. While vaccination with native CD increased the number of interleukin-2 (IL-2)- and gamma interferon-producing cells, rCD mainly stimulated IL-4-secreting cells. Nevertheless, efficient bacterial clearance was observed in the lungs of challenged mice receiving native CD and in the lungs of challenged mice receiving rCD (96% and 99%, respectively). Thus, rCD is a promising candidate for incorporation in vaccine formulations for use against M. catarrhalis.

N-Acetylaspartylglutamate (NAAG) is a neuropeptide found in high concentrations in the brain. Using whole-cell recordings of CA1 pyramidal neurons in acute hippocampal slices, we found that either (i) the application of exogenous NAAG or (ii) an increase of endogenous extracellular NAAG, caused by the inhibition of its catabolic enzyme glutamate carboxypeptidase II (GCP II), resulted in a significant reduction in the amplitude of the isolated NMDA receptor (NMDAR) component of the evoked excitatory postsynaptic current (EPSC). Conversely, reduction of endogenous extracellular NAAG caused by either (i) perfusion with a soluble form of pure human GCP II or (ii) affinity purified antibodies against NAAG, enhanced the amplitude of the isolated NMDAR current. Bath application of GCP II inhibitor induced a progressive loss of spontaneous NMDAR miniatures. Furthermore, NAAG blocked the induction of long-term potentiation at Schaffer collateral axons-CA1 pyramidal neuron synapses. All together, these results suggest that NAAG acts as an endogenous modulator of NMDARs in the CA1 area of the hippocampus.

Circulating Vgamma2Vdelta2 T-cell populations in healthy human beings are poised for rapid responses to bacterial or viral pathogens. We asked whether Vgamma2Vdelta2 T cells use the Toll-like receptor (TLR) family to recognize pathogen-associated molecular pattern molecules and to regulate cell functions. Analysis of expanded Vgamma2Vdelta2 T-cell lines showed the abundant presence of TLR2 mRNA, implying that these receptors are important for cell differentiation or function. However, multiple efforts to detect TLR2 protein on the cell surface or in cytoplasmic compartments gave inconsistent results. Functional assays confirmed that human Vgamma2Vdelta2 T cells could respond to the TLR2 agonist (S)-(2,3-bis(palmitoyloxy)-(2RS)-propyl)-N-palmitoyl-(R)-Cys-(S)-Ser(S)-Lys4-OH trihydrochloride (Pam3Cys), but the response required coincident stimulation through the gammadelta T-cell receptor (TCR). Dually stimulated cells produced higher levels of cytoplasmic or cell-free gamma interferon and showed increased expression of the lysosome-associated membrane protein CD107a on the cell surface. A functional TLR2 that requires coincident TCR stimulation may increase the initial potency of Vgamma2Vdelta2 T-cell responses at the site of infection and promote the rapid development of subsequent acquired antipathogen immunity.

Nod1 is a member of family of intracellular proteins that mediate host recognition of bacterial peptidoglycan. To characterize immune responses mediated by Nod1, synthetic ligand compounds possessing enhanced ability to stimulate Nod1 were developed to study the function of Nod1. Stimulation of epithelial cells with Nod1 stimulatory molecules induced chemokines and other proinflammatory molecules that are important for innate immune responses and recruitment of acute inflammatory cells. Administration of Nod1 ligands into mice induced chemokines and recruitment of acute inflammatory cells, an activity that was abolished in Nod1-null mice. Microarray analysis revealed that Nod1 stimulation induces a restricted number of genes in intestinal epithelial cells compared with that induced by tumor necrosis factor (TNF) alpha. Nod1 stimulation did not induce TNFalpha, interleukin 12, and interferon gamma, suggesting that the primary role of Nod1 is to induce the recruitment of immune cells. These results indicate that Nod1 functions as a pathogen recognition molecule to induce expression of molecules involved in the early stages of the innate immune response.

Light-dependent redistribution of transducin between the rod outer segments (OS) and other photoreceptor compartments including the inner segments (IS) and synaptic terminals (ST) is recognized as a critical contributing factor to light and dark adaptation. The mechanisms of light-induced transducin translocation to the IS/ST and its return to the OS during dark adaptation are not well understood. We have probed these mechanisms by examining light-dependent localizations of the transducin-alpha subunit (Gtalpha)in mice lacking the photoreceptor GAP-protein RGS9, or expressing the GTPase-deficient mutant GtalphaQ200L. An illumination threshold for the Gtalpha movement out of the OS is lower in the RGS9 knockout mice, indicating that the fast inactivation of transducin in the wild-type mice limits its translocation to the IS/ST. Transgenic GtalphaQ200L mice have significantly diminished levels of proteins involved in cGMP metabolism in rods, most notably the PDE6 catalytic subunits, and severely reduced sensitivity to light. Similarly to the native Gtalpha, the GtalphaQ200L mutant is localized to the IS/ST compartment in light-adapted transgenic mice. However, the return of GtalphaQ200L to the OS during dark adaptation is markedly slower than normal. Thus, the light-dependent translocations of transducin are controlled by the GTP-hydrolysis on Gtalpha, and apparently, do not require Gtalpha interaction with RGS9 and PDE6.

Transglutaminases (TGs) posttranslationally modify proteins by transamidation of specific polypeptide bond glutamines. This reaction results in deamination, polyamine incorporation or the formation of isopeptide bonds. Transglutaminase activity in the brain is increased in several neurodegenerative diseases. Because insoluble inclusions occur in these neurodegenerative diseases, it has been hypothesized that transglutaminase contributes to the formation of the inclusions by catalyzing the formation of isopeptide bonds resulting in crosslinked, insoluble protein aggregates. To demonstrate a role for transglutaminase in the formation of these inclusions, the primary approach has been to show increased immunoreactivity with antibodies that recognize the isopeptide bonds. However, the specificity of these antibodies for isopeptide crosslinks within or between proteins has not been clearly established. In this report we demonstrate that the two most commonly used isopeptide antibodies do not specifically recognize the isopeptide bonds formed by transglutaminase when they are within or between proteins.

Objective: Glutamine has stimulatory effects on lymphocytes and mucosa cells in vitro and, when given with parenteral nutrition, has been shown to improve the clinical course of patients after bone marrow transplantation and in the critically ill. This study investigated the clinical and immunologic effects of parenteral glycyl-glutamine supplementation in patients with acute leukemia receiving intensive conventional chemotherapy without bone marrow transplantation., Methods: A randomized, double-blind, controlled study compared a standard glutamine-free parenteral nutrition with a glycyl-glutamine-supplemented parenteral nutrition (Glamin, Baxter, Erlangen, Germany) containing 20 g of glutamine in adult patients with acute myeloid leukemia undergoing myelosuppressive chemotherapy. Clinical end points included the duration of neutropenia and the incidence and duration of neutropenic fever. To analyze the effects of glutamine on immunocompetent cells, CD4+ and CD8+ T cells and HLA-DR expression on monocytes were assessed by flow cytometry throughout the treatment course., Results: Fifty-four adult patients entered the study and were randomized. In 45 of 127 chemotherapy cycles, parenteral nutrition was given, and 40 cycles (20 with and 20 without glutamine) were evaluated for comparison. The median durations of neutropenia were 18 d (range, 9-29 d) in the glutamine group and 22.5 d (range, 13-48 d) in the control group (P = 0.052), whereas the median durations of neutropenic fever were 5.5 d (range, 0-13 d) and 5 d (range, 0-31 d), respectively (P = 0.74). Using Kaplan-Meier analysis and controlling for the type of chemotherapy, we found a significantly faster neutrophil recovery in patients receiving glutamine than in the control group (P = 0.040) in patients receiving a high-dose cytarabine regimen. There was no significant difference in the recovery of CD4+ or CD8+ lymphocytes or monocyte activation between groups., Conclusion: In patients with acute myeloid leukemia requiring parenteral nutrition, glycyl-glutamine supplementation could hasten neutrophil recovery after intensive myelosuppressive chemotherapy. However, no impact of glutamine on neutropenic fever or other criteria of immunologic recovery was detected.

Recent immunological approaches have greatly helped understanding the significance of advanced glycation end products (AGEs) in age-related diseases. However, immunohistochemical localization of AGEs in tissues or their quantitative determination in biological fluids sometimes yields inconsistent results among different investigators. Since these differences might be caused by the heterogeneity of the AGE antibodies, a systematic mapping of their epitope recognition pattern would help in the evaluation of these different results. For this purpose, an N-terminally acetylated combinatorial dipeptide library with 400 positionally defined dipeptides was modified by glucose to yield AGEs, which are thought to be present on the protein side chains. Using this library, we have characterized six different AGE antibodies in respect to their immunoreactivity towards AGE-modified dipeptides. All antibodies predominantly recognized only one AGE-modified amino acid side chain (and not a particular dipeptide). In most cases, arginine- and lysine- derived AGEs, but also some epitopes on asparagine and on heterocyclic amino acids were recognized. Very interestingly, the immunization of different animals with the same antigen produced AGE antibodies with a totally different epitope recognition pattern. Defining the antigen recognition pattern with this method might help in the quality control of polyclonal AGE antibodies for immunodetection. In addition, defined AGE antibodies (as neutralizing antibodies) could also help to define "signal-active" AGEs in terms of specific AGE-mediated cellular responses.

Polyclonal antibodies were produced to detect the coccidiostat nicarbazin. Due to structural constraints of the active component of nicarbazin, dinitrocarbanilide (DNC), three different compounds that shared a common substructure with DNC were used as antigen mimics. The compounds (N-succinyl-L-alanyl-L-alanyl-L-alanine 4-nitroanilide (SAN), L-glutamic acid gamma-(p-nitroanilide) (GAN) and p-nitrosuccinanilic acid (NSA)) were conjugated to a carrier protein and used in the immunisation of rabbits. Five different polyclonal sera were produced and consequently characterised. The antibodies exhibited an IC(50) range of 2.3-7.6 ng/ml using a competitive ELISA procedure. Serum from one rabbit, R555, exhibited an IC(50) of 2.9 ng/ml for DNC and cross-reactivity studies showed that this serum was specific for DNC and did not cross-react with other coccidiostats such as halofuginone, toltrazuril or ronidazole.

Vancomycin resistance is currently a major healthcare problem. The development of a catalytic monoclonal antibody (mAb) that hydrolyzes the D-Ala-D-Lac depsipeptide provides a potentially novel antibiotic strategy. A phosphonate hapten design was used to program antibody catalysis. The characteristics of the hapten were shown to be important for obtaining a viable immune response and several catalytic mAbs that cleave a peptidoglycan model substrate. The best mAb afforded a >500-fold rate enhancement over background.

In the search for more potent and less toxic immunomodulators, adamantylamide dipeptide (AdDP) was synthesized by the covalent union of amantadine with the L-alanyl-D-isoglutamine residue of muramyldipeptide (MDP). The present experiments demonstrate the ability of AdDP, co-administered with a protein immunogen, to raise or enhance a humoral response in immunized animals. BALB/c mice were immunized either by the intraperitoneal (ip) or oral route with ovalbumin (Ova) alone or combined with either AdDP or CpG oligonucleotide (ODN-CpG), a proved adjuvant. A clear adjuvant dose-response relationship was observed on the increment of Ova-specific serum antibody titers when AdDP was used as adjuvant, irrespectively of the administration route. The IgG isotype analysis showed that AdDP promotes a consistent increment in IgG1 antibodies associated with a dominant Th2 response pattern. When administered by the oral route, AdDP was at least as efficient as ODN-CpG as adjuvant. Similar results were obtained in rabbits immunized by the oral route, suggesting that the adjuvanticity of AdDP is not restricted to the murine system. In conclusion, AdDP was shown to be a powerful and non-toxic adjuvant at both systemic and mucosal levels, which makes it a promising tool for vaccine development.

The anti-glutathione antibody scFv 20C9, which we previously isolated from a human synthetic phage antibody scFv library [Hirose, M., Hayano, T., Shirai, H., Nakamura, H., and Kikuchi, M. (1998) Protein Eng. 11, 243-248], was expressed in the E. coli pET system and purified by sequential chromatography on Ni and glutathione-conjugated affinity resins. The purified scFv 20C9 antibody was characterized for its binding affinity for several glutathione derivatives by the BIACORE system. Although GSH, GSSG, and gamma-Glu-Cys could bind to the immobilized antibody, this was not the case for Cys-Gly, l-Glu, l-Cys, l-Gly, or several other glutathione derivatives such as gamma-Glu-Ser-Gly. The results suggest that a gamma-glutamic acid and sulfur atom are important for scFv 20C9 antibody recognition of glutathione. This is the first report to indicate that an scFv antibody can recognize a region as small as a dipeptide., (Copyright 2001 Academic Press.)

The human spliceosomal Sm B/B' proteins are essential for the biogenesis of the snRNP particles. B/B' proteins contain several clusters of the PPPPGM/IR sequence, which occurs within the C-terminus of Sm B/B'. This sequence is very similar to the PPPPPGHR sequence of the cytoplasmic tail of the CD2 receptor and closely resembles the class II of SH3 ligands, suggesting a similarly important role. We report that a monoclonal antibody (3E10) against the PPPPPGHR sequence recognizes spliceosomal Sm B/B' proteins. Proteins that are specifically immunoprecipitated by 3E10 include Sm B, B', D1, D2, D3, E, F, and G. However, unlike Y12 and other anti-Sm immunoprecipitates, 3E10 immunoprecipitates appear to lack the U1 snRNP-specific proteins A and C and U snRNAs. These findings indicate that 3E10 recognizes a subset of Sm protein core and suggest the presence of snRNA-free Sm protein complex(es) in vivo. We propose that the epitope binding for 3E10 may become unaccessible upon interactions of Sm proteins and their subsequent incorporation into the core particles. The Sm proline-rich sequences may have an important role in mediating protein-protein interactions necessary for the proper snRNP core assembly or function, or both. To our knowledge, 3E10 is the first well characterized mAb specific for a subclass of polyproline-arg motif recognizing Sm B/B' and CD2 proteins. 3E10 antibody can be used to further characterize the nature of protein components in the snRNA-free Sm subcore protein complex(es) that are formed during the snRNP core assembly steps.

Adamantylamide dipeptide (AdDP), muramyl dipeptide (MDP), and glucan were shown to increase significantly the numbers of granulocyte-macrophage hemopoietic progenitor cells (GM-CFC) in the bone marrow of mice. However, whereas the sera of mice given MDP or glucan were found to stimulate the growth of colonies from GM-CFC in vitro, i.e. to produce a colony-stimulating activity (CSA), administration of AdDP did not lead to this effect. Nevertheless, when serum of mice given AdDP was added to the cultures concomitantly with a suboptimal concentration of mouse interleukin-3 (mIL-3), a broad spectrum hemopoietic stimulator, counts of colonies from GM-CFC were significantly increased, and accelerated growth of the colonies was found as well. This property of AdDP, i.e. its ability to exhibit co-stimulating activity (CoSA) without being able to exhibit CSA, suggests that AdDP acts in hemopoietic tissues differently as compared with the two other immunomodulators studied. It can be hypothesized that the action of AdDP is more specific when compared with its natural related compound, MDP, as well as with glucan. Our findings prove the possibility to stimulate by AdDP the granulopoietic compartment of hemopoiesis and are in agreement with previous observations concerning the absence of systemic side effects of AdDP. Both these qualities of AdDP may be advantageous when pondering over contingent clinical utilization of AdDP as hemopoietic stimulator.

BALB/c mice were immunized against the 19 C-terminal amino acids of the mouse mammary tumour virus [MMTV(SW)] superantigen, either actively with the recombinant ORF19 protein or passively by subcutaneous implantation of 6E1.8 hybridoma cells. Protection against the MMTV(SW) superantigen-induced activation of specific T cells was obtained in both immunizations. Whereas the monoclonal antibody provided complete protection, similar antibody titres showed less effective protection in actively immunized mice. This was due to the presence of a histidine tag on the recombinant ORF19 protein preventing immune recognition of the C-terminal amino acids during immunization. Preincubation of the 6E1.8 monoclonal antibodies with modified ORF19 peptides at their C terminus established specific recognition of the last two amino acids of MMTV(SW) superantigen by this antibody, and showed that these amino acids are critical in the interaction between the MMTV(SW) superantigen and the Vbeta element of the T-cell receptor.

Amphiphilic lipopeptides, such as Pam3CysAlaGly and Pam3CysSerSer, were synthesized and incorporated into liposomes, and their ability to induce the proliferation of BALB/c mouse splenocyte was tested in vitro. When compared to monophosphoryl lipid A (MPL) the following potency order was found: liposomal lipopeptides > liposomal MPL > free (emulsified) lipopeptides. These results strongly depend on the size of the vesicles used: a mitogenic effect was observed only with lipopeptides incorporated within vesicles of diameter < or = 100 nm while lipopeptides in larger vesicles (diameter approximately 300 nm) gave no response. This may be related to the necessity for the liposome-associated lipopeptides to be endocytosed to reach putative intracellular targets. As immunoadjuvanticity seems to be linked to B-lymphocyte activation, the lipopeptides represent attractive alternatives to MPL for the realization of completely synthetic liposome-based peptide vaccine formulations. This was borne out by showing that Pam3CysAlaGly and Pam3CysSerSer, when incorporated in small unilamellar vesicles carrying a covalently conjugated synthetic peptide of sequence IRGERA, corresponding to an epitope of the C-terminal region of histone H3, were able to induce a potent and long-lasting immune response.

To generate conventional or monoclonal antibodies for the serological detection of drugs, antibiotics, toxins and other low molecular mass substances, a suitable and effective adjuvant is needed. Lipopeptides derived from a major component of the bacterial cell wall constitute potent nontoxic and nonpyrogenic immunoadjuvants when mixed with conventional antigens. Here we demonstrate that the synthetic lipopeptide N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2R,S)-propyl]-(R)-cysteinyl- serine (P3CS) coupled to a Th-cell epitope (P3CS-Th) can efficiently enhance the specific immune response against low molecular weight compounds in different species. In the presence of the synthetic lipopeptide P3CS-Th, the peptides which are per se non-immunogenic stimulated a specific humoral immune response in mice after intraperitoneal application. Mixtures containing adjuvants without the Th sequence showed no significant antibody induction. A marked enhancement of the humoral immune response was obtained with the low molecular mass antigens Iturin AL, Herbicolin A and Microcystin (MLR) coupled to poly-l-lysin (MLR-PLL), in rabbits and in chickens. Lipopeptide-Th cell epitope conjugates also constituted adjuvants for the in vitro immunization of either human mononuclear cells or mouse B-cells with MLR-PLL; after fusion of the immunized cultures with the heteromyeloma cell lines CB-F7 or the mouse myeloma cell line SP 2/0, respectively, we observed a significantly increased yield of antibody secreting hybridomas.

Glutamate is the probable neurotransmitter of both retinal and cortical afferents to the cat superior colliculus (SC). The present study shows that glutamate is also contained in many postsynaptic neurons in SC. The distribution, morphology, and ultrastructure of neurons in SC were examined using glutamate antibody immunocytochemistry. Labeled cells were widely distributed throughout, but a specific laminar pattern was evident. Relatively few cells were found in the zonal and upper superficial gray layers (SGL). A dense band of intensely labeled neurons was found within the deep superficial gray and upper optic layers. Many cells were also labeled in the deeper layers. Labeled cells had varied sizes and morphologies. Soma diameters ranged from 9-67 microns, with a mean of 22 microns. Cells with stellate, vertical fusiform, and multipolar morphologies were labeled. Cells in the deep subdivision all had morphologies and sizes typical of projection neurons. To determine if labeled cells in the dense band were also projection neurons, WGA-HRP was injected into the lateral posterior nucleus and these sections were double-labeled with the glutamate antibody. Over one-half of cells in the dense band that were labeled by HRP were also obviously labeled by antibody. At the electron-microscope level, both medium- and large-sized neurons were also labeled by glutamate antibodies. These cells had different but characteristic morphologies.

Biochemical studies provide evidence that the pathway from visual cortex to the superior colliculus (SC) utilizes glutamate as a neurotransmitter. In the present study, we have used immunocytochemistry, visual cortex lesions, and retrograde tracing to show directly by anatomical methods that glutamate or a closely related analog is contained in corticocollicular neurons and terminals. A monoclonal antibody directed against gamma-L-glutamyl-L-glutamate (gamma glu glu) was used to localize glutamate-like immunoreactivity in both the superior colliculus (SC) and visual cortex (VC). Unilateral lesions of areas 17-18 were made in four cats to determine if gamma glu glu labeling was reduced in SC by this lesion. WGA-HRP was injected into the SC of 10 additional cats in order to determine if corticocollicular neurons were also labeled by the gamma glu glu antibody. A distinctive dense band of gamma glu glu immunoreactivity was found within the deep superficial gray and upper optic layers of SC where many corticotectal axons are known to terminate. Both fibers and cells were labeled within the band. Immunoreactivity was also found in cells and fibers throughout the deep layers of SC. Measures of total immunoreactivity (i.e. optical density) in the dense band were made in sections from the SC both ipsilateral to and contralateral to the lesions of areas 17-18. A consistent reduction in optical density was found in both the neuropil and in cells within the dense band of the SC ipsilateral to the lesion. A large percentage of all corticocollicular neurons that were retrogradely labeled by WGA-HRP also contained gamma glu glu. These results provide further evidence that the corticocollicular pathway in mammals is glutamatergic. The results also suggest that visual cortex ablation alters synthesis or storage of glutamate within postsynaptic SC neurons, presumably as a result of partial deafferentation.

Contradictory immunohistochemical data have been reported on the localization of N-acetylaspartylglutamate in the rat forebrain, using different carbodiimide fixation protocols and antibody purification methods. In one case, N-acetylaspartylglutamate immunoreactivity was observed in apparent interneurons throughout all allocortical and isocortical regions, suggesting possible colocalization with GABA. In another case, strong immunoreactivity was observed in numerous pyramidal cells in neocortex and hippocampus, suggesting colocalization with glutamate or aspartate. Reconciling these disparate findings is crucial to understanding the role of N-acetylaspartylglutamate in nervous system function. Antibodies to N-acetylaspartylglutamate and a structurally related molecule, N-acetylaspartate, were purified in stages, and their cross-reactivities with protein conjugates of N-acetylaspartylglutamate and N-acetylaspartate were monitored at each stage by solid-phase immunoassay. Reduction of the cross-reactivity of the anti-N-acetylaspartylglutamate antibodies of N-acetylaspartate-protein conjugates to about 1% eliminated significant staining of most pyramidal neurons in the rat forebrain. Utilizing highly purified antibodies, the distributions of N-acetylaspartylglutamate and N-acetylaspartate were examined in several major telencephalic and diencephalic regions of the rat, and were found to be distinct. N-acetylaspartylglutamate-immunoreactivity was observed in specific neuronal populations, including many groups thought to use GABA as a neurotransmitter. Among these were the globus pallidus, ventral pallidum, entopeducular nucleus, thalamic reticular nucleus, and scattered non-pyramidal neurons in all layers of isocortex and allocortex. N-acetylaspartate-immunoreactivity was more broadly distributed than N-acetylaspartylglutamate-immunoreactivity in the rat forebrain, appearing strongest in many pyramidal neurons. Although N-acetylaspartate-immunoreactivity was found in most neurons, it exhibited a great range of intensities between different neuronal types.

The expression of tissue transglutaminase in skeletal tissues is strictly regulated and correlates with chondrocyte differentiation and cartilage calcification in endochondral bone formation and in maturation of tracheal cartilage (Aeschlimann, D., A. Wetterwald, H. Fleisch, and M. Paulsson. 1993. J. Cell Biol. 120:1461-1470). We now demonstrate the transglutaminase reaction product, the gamma-glutamyl-epsilon-lysine cross-link, in the matrix of hypertrophic cartilage using a novel cross-link specific antibody. Incorporation of the synthetic transglutaminase substrate monodansylcadaverine (amine donor) in cultured tracheal explants reveals enzyme activity in the pericellular matrix of hypertrophic chondrocytes in the central, calcifying areas of the horseshoe-shaped cartilages. One predominant glutaminyl substrate (amine acceptor) in the chondrocyte matrix is osteonectin as revealed by incorporation of the dansyl label in culture. Indeed, nonreducible osteonectin-containing complexes of approximately 65, 90, and 175 kD can be extracted from mature tracheal cartilage. In vitro cross-linking of osteonectin by tissue transglutaminase gives similar products of approximately 90 and 175 kD, indicating that the complexes in cartilage represent osteonectin oligomers. The demonstration of extracellular transglutaminase activity in differentiating cartilage, i.e., cross-linking of osteonectin in situ, shows that tissue transglutaminase-catalyzed cross-linking is a physiological mechanism for cartilage matrix stabilization.

Although glutamate is thought to be the neurotransmitter at the invertebrate neuromuscular junction, acetylcholine is accepted as the primary neurotransmitter of the vertebrate motoneurons. N-acetylaspartylglutamate, a dipeptide localized in putative glutamatergic neurons in brain, is also found in high concentrations (> mM) in mammalian motoneurons and the ventral roots of spinal cord. N-acetylaspartylglutamate, which is released from neurons by depolarization in a Ca(2+)-dependent fashion, is implicated in glutamatergic transmission in two ways: it is a partial agonist at NMDA receptors, and it is cleaved to yield extracellular glutamate and N-acetylasparate by the specific peptidase N-acetylated alpha-linked acidic dipeptidase. Given the localization of N-acetylaspartylglutamate in motor neuronal perikarya and axons, we wondered whether N-acetylaspartylglutamate or glutamate cleaved from N-acetylaspartylglutamate by N-acetylated alpha-linked acidic dipeptidase may also play a role in neuromuscular transmission. Here we describe the immunocytochemical detection at the rat neuromuscular junction of N-acetylaspartylglutamate in terminals of motoneurons, of N-acetylated alpha-linked acidic dipeptidase in perisynaptic Schwann cells, and of the NMDAR-1 glutamate receptor subunit on postsynaptic muscle membranes. These results point to a potential role for N-acetylaspartylglutamate at the rat neuromuscular junction. Further, this is the first demonstration of a glutamate receptor protein at vertebrate neuromuscular synapses. Together with other recent findings, our results suggest that glutamate-like molecules are involved in neuromuscular transmission not only in invertebrates but also in veretebrates where they may modulate signaling by acetylcholine.

Two monoclonal antibodies, GLU-1 and A1.6, raised against gamma-L-glutamyl-L-glutamic acid dipeptide (Glu-Glu) and Ca(2+)-dependent ATPase from Paramecium, respectively, recognized the dipeptide Glu-Glu sequence. Whereas the antibodies immunofluorescently stained very few, if any, cytoskeletal fibers in cultured mammalian cells, almost all interphase as well as mitotic spindle microtubules became visible after treatment of cells with carboxypeptidase A. Immunoblot analysis demonstrated intense cross-reaction of the antibodies to the alpha-tubulin subunit. alpha-Tubulin isotypes produced as fusion proteins in bacteria were labeled by both the antibodies only when the proteins did not contain a tyrosine residue at the C terminus, indicating that GLU-1 and A1.6 specifically recognize the detyrosinated form of alpha-tubulin. When microtubule protein purified from brain was probed, not only alpha-but also, to a lesser extent, beta-tubulin were revealed by the dipeptide antibodies. A synthetic tripeptide YED containing one glutamyl group linked to the second residue of the peptide via the gamma position was also recognized by the antibodies. Since this peptide sequence corresponds to the amino acid sequence of polyglutamyated class III beta isotype at amino acid position 437 to 439, it is suggested that GLU-1 and A1.6 are able to recognize the glutamylated form of beta-tubulin. These results indicate that the C-terminal Glu-Glu sequence displays strong antigenicity, and the antibodies recognize the sequence present in the C terminus of the detyrosinated form of alpha-tubulin and the glutamyl side chain of beta-tubulin. Particularly strong immunoreaction was detected with ciliary and flagellar microtubules; thus, stable axonemal microtubules appear to be rich in post-translationally modified tubulin subunits.

The synthetic peptide antigen (Ag) (the primary structure Tyr-Leu-Lys-Asp-Gln-Gln-Leu-Leu-Gly-Ile-Trp-Gly-Cys-Ser-Gly-Lys-Leu-Ile- Cys-Thr derived from the envelope glycoprotein gp41 of the human immunodeficiency virus type 1 (HIV-1) and exerting specificity with all HIV-1-positive sera available in the Czech Republic (and also in a panel of 10,000 sera from WHO)) was conjugated with bovine serum albumin (BSA) and encapsulated into liposomes. Adjuvant activities of liposomes with various lipid compositions were compared with Freund's complete adjuvant (FCA) and with aluminium hydroxide (AL). The immune response to BSA-Ag liposomes with coentrapped adamantylamide dipeptide (AdDP) was comparable with that of FCA in terms of longevity and levels of specific antibodies in mouse sera.