The Use of Combinatorial Hexapeptide Ligand Library (CPLL) in Allergomics.

The recent progress of proteomic protocols led to more efficient protein extraction and concentration procedures to remove nonprotein interfering compounds present in the starting material and to increase the concentration of underrepresented proteins. Combinatorial hexapeptide ligand libraries (CPLL) were recently applied to both plant- and animal-derived tissues for capturing the low- and very low-abundance allergens. Several IgE-binding proteins which were previously absent or poorly represented by using conventional proteomics tools have been detected and characterized through a CPLL-based approach. In the present chapter, a protocol based on improved protein extraction and enrichment by CPLL, allowing the immunochemical characterization of several "hidden allergens" in cypress pollen, is described in detail.

Preclinical Antitumor Efficacy of BAY 1129980-a Novel Auristatin-Based Anti-C4.4A (LYPD3) Antibody-Drug Conjugate for the Treatment of Non-Small Cell Lung Cancer.

C4.4A (LYPD3) has been identified as a cancer- and metastasis-associated internalizing cell surface protein that is expressed in non-small cell lung cancer (NSCLC), with particularly high prevalence in the squamous cell carcinoma (SCC) subtype. With the exception of skin keratinocytes and esophageal endothelial cells, C4.4A expression is scarce in normal tissues, presenting an opportunity to selectively treat cancers with a C4.4A-directed antibody-drug conjugate (ADC). We have generated BAY 1129980 (C4.4A-ADC), an ADC consisting of a fully human C4.4A-targeting mAb conjugated to a novel, highly potent derivative of the microtubule-disrupting cytotoxic drug auristatin via a noncleavable alkyl hydrazide linker. In vitro, C4.4A-ADC demonstrated potent antiproliferative efficacy in cell lines endogenously expressing C4.4A and inhibited proliferation of C4.4A-transfected A549 lung cancer cells showing selectivity compared with a nontargeted control ADC. In vivo, C4.4A-ADC was efficacious in human NSCLC cell line (NCI-H292 and NCI-H322) and patient-derived xenograft (PDX) models (Lu7064, Lu7126, Lu7433, and Lu7466). C4.4A expression level correlated with in vivo efficacy, the most responsive being the models with C4.4A expression in over 50% of the cells. In the NCI-H292 NSCLC model, C4.4A-ADC demonstrated equal or superior efficacy compared to cisplatin, paclitaxel, and vinorelbine. Furthermore, an additive antitumor efficacy in combination with cisplatin was observed. Finally, a repeated dosing with C4.4A-ADC was well tolerated without changing the sensitivity to the treatment. Taken together, C4.4A-ADC is a promising therapeutic candidate for the treatment of NSCLC and other cancers expressing C4.4A. A phase I study (NCT02134197) with the C4.4A-ADC BAY 1129980 is currently ongoing. Mol Cancer Ther; 16(5); 893-904. ©2017 AACR.

Enfortumab Vedotin Antibody-Drug Conjugate Targeting Nectin-4 Is a Highly Potent Therapeutic Agent in Multiple Preclinical Cancer Models.

The identification of optimal target antigens on tumor cells is central to the advancement of new antibody-based cancer therapies. We performed suppression subtractive hybridization and identified nectin-4 (PVRL4), a type I transmembrane protein and member of a family of related immunoglobulin-like adhesion molecules, as a potential target in epithelial cancers. We conducted immunohistochemical analysis of 2,394 patient specimens from bladder, breast, lung, pancreatic, ovarian, head/neck, and esophageal tumors and found that 69% of all specimens stained positive for nectin-4. Moderate to strong staining was especially observed in 60% of bladder and 53% of breast tumor specimens, whereas the expression of nectin-4 in normal tissue was more limited. We generated a novel antibody-drug conjugate (ADC) enfortumab vedotin comprising the human anti-nectin-4 antibody conjugated to the highly potent microtubule-disrupting agent MMAE. Hybridoma (AGS-22M6E) and CHO (ASG-22CE) versions of enfortumab vedotin (also known as ASG-22ME) ADC were able to bind to cell surface-expressed nectin-4 with high affinity and induced cell death in vitro in a dose-dependent manner. Treatment of mouse xenograft models of human breast, bladder, pancreatic, and lung cancers with enfortumab vedotin significantly inhibited the growth of all four tumor types and resulted in tumor regression of breast and bladder xenografts. Overall, these findings validate nectin-4 as an attractive therapeutic target in multiple solid tumors and support further clinical development, investigation, and application of nectin-4-targeting ADCs. Cancer Res; 76(10); 3003-13. ©2016 AACR.

Therapeutic targets for olive pollen allergy defined by gene markers modulated by Ole e 1-derived peptides.

Two regions of Ole e 1, the major olive-pollen allergen, have been characterized as T-cell epitopes, one as immunodominant region (aa91-130) and the other, as mainly recognized by non-allergic subjects (aa10-31). This report tries to characterize the specific relevance of these epitopes in the allergic response to olive pollen by analyzing the secreted cytokines and the gene expression profiles induced after specific stimulation of peripheral blood mononuclear cells (PBMCs). PBMCs from olive pollen-allergic and non-allergic control subjects were stimulated with olive-pollen extract and Ole e 1 dodecapeptides containing relevant T-cell epitopes. Levels of cytokines were measured in cellular supernatants and gene expression was determined by microarrays, on the RNAs extracted from PBMCs. One hundred eighty-nine differential genes (fold change >2 or <-2, P<0.05) were validated by qRT-PCR in a large population. It was not possible to define a pattern of response according the overall cytokine results but interesting differences were observed, mainly in the regulatory cytokines. Principal component (PCA) gene-expression analysis defined clusters that correlated with the experimental conditions in the group of allergic subjects. Gene expression and functional analyses revealed differential genes and pathways among the experimental conditions. A set of 51 genes (many essential to T-cell tolerance and homeostasis) correlated with the response to aa10-31 of Ole e 1. In conclusion, two peptides derived from Ole e 1 could regulate the immune response in allergic patients, by gene-expression modification of several regulation-related genes. These results open new research ways to the regulation of allergy by Oleaceae family members.

Effects of Lactobacillus plantarum on production performance, immune characteristics, antioxidant status, and intestinal microflora of bursin-immunized broilers.

Examples of probiotics that can promote host health by improving its intestinal microbial balance and intestinal immunity belong to the genus Lactobacillus. Bursin (BS) is a peptide isolated from the bursa of Fabricius for use as an adjuvant for a variety of immunogens. To investigate the synergistic effects of Lactobacillus plantarum (LP) dietary supplementation and BS immunization on production performance, immune characteristics, antioxidant status, and intestinal microflora in broilers, we randomly allocated 200 1-day-old broilers of mixed sex into 4 treatments in a 2 × 2 factorial arrangement (LP-/BS-, LP-/BS+, LP+/BS-, LP+/BS+) for 42 days. BS immunization enhanced immune response by increasing serum total immunoglobulin G concentration and interleukin-6 concentration, promoted antioxidant capacity by increasing catalase activities in serum and liver and by decreasing serum malondialdehyde (MDA) content at 42 days of age (DOA), and enriched intestinal microflora diversity. LP supplementation enhanced immune response by increasing interleukin-2 concentration at 42 DOA; promoted antioxidant capacity by increasing liver catalase activities, increasing glutathione peroxidase activities in serum and liver at 21 DOA, and decreasing serum MDA content at 42 DOA; promoted intestinal microflora composition by decreasing total aerobes and Escherichia coli counts at 21 DOA, by increasing total anaerobes count at 21 DOA, and by increasing Lactobacillus spp. and Bifidobacterium spp. counts at both 21 and 42 DOA. The interactions between BS and LP had a significant effect on daily body mass gain and feed conversion ratio in the starter period (1-21 DOA); on interleukin-2 concentration and liver MDA content at 21 DOA; and on thymus index, peripheral lymphocyte proliferation, and E. coli counts at 42 DOA. Overall, these data suggest that the combination of LP dietary supplementation and BS immunization promoted the production performance, immune characteristics, antioxidant status, and intestinal microflora of broilers.

Unique and cross-reactive T cell epitope peptides of the major Bahia grass pollen allergen, Pas n 1.

BACKGROUND: Bahia grass pollen (BaGP) is a major cause of allergic rhinitis. Subcutaneous allergen-specific immunotherapy is effective for grass pollen allergy, but is unsuitable for patients with moderate to severe asthma due to the risk of anaphylaxis. T cell-reactive but IgE nonreactive peptides provide a safer treatment option. This study aimed to identify and characterize dominant CD4(+) T cell epitope peptides of the major BaGP allergen, Pas n 1. METHODS: Pas n 1-specific T cell lines generated from the peripheral blood of BaGP-allergic subjects were tested for proliferative and cytokine response to overlapping 20-mer Pas n 1 peptides. Cross-reactivity to homologous peptides from Lol p 1 and Cyn d 1 of Ryegrass and Bermuda grass pollen, respectively, was assessed using Pas n 1 peptide-specific T cell clones. MHC class II restriction of Pas n 1 peptide T cell recognition was determined by HLA blocking assays and peptide IgE reactivity tested by dot blotting. RESULTS: Three Pas n 1 peptides showed dominant T cell reactivity; 15 of 18 (83%) patients responded to one or more of these peptides. T cell clones specific for dominant Pas n 1 peptides showed evidence of species-specific T cell reactivity as well as cross-reactivity with other group 1 grass pollen allergens. The dominant Pas n 1 T cell epitope peptides showed HLA binding diversity and were non-IgE reactive. CONCLUSIONS: The immunodominant T cell-reactive Pas n 1 peptides are candidates for safe immunotherapy for individuals, including those with asthma, who are allergic to Bahia and possibly other grass pollens.

Establishment and clinical application of a highly sensitive enzyme immunoassay for determination of N-acetyl-seryl-aspartyl-lysyl-proline.

N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP) is a natural inhibitor of pluripotent hematopoietic stem cell proliferation and is normally found in human plasma. Because AcSDKP is hydrolyzed by the N-terminal active site of angiotensin converting enzyme and partially eliminated in urine, its plasma level is a result of a complex balance between its production, hydrolysis by ACE, and renal elimination. In this study, we attempted to establish an enzyme immunoassay (EIA) for quantifying AcSDKP-like immunoreactive substance (IS), which is applicable for monitoring plasma AcSDKP levels in healthy subjects and patients with chronic renal failure. Using ß-D-galactosidase-labeled Gly-γAbu-SDKP as a marker antigen, an anti-rabbit IgG-coated immunoplate as a bound/free separator and 4-methylumbelliferyl-ß-D-galactopyranoside as a fluorogenic substrate, a highly sensitive and specific EIA was developed for the quantification of AcSDKP-IS in human plasma. The lower limit of quantification was 0.32 fmol/well, and the sharp inhibition competitive EIA calibration curve obtained was linear between 8.0 and 513 fmol/ml. This EIA was so sensitive that only 10 µl plasma sample was required for a single assay. The coefficients of variation (reproducibility) for human plasma concentrations of 0.2 and 2.1 pmol/ml were 7.2 and 7.7%, respectively, for inter-assay and 13.3 and 7.8% for intra-assay comparisons. Plasma AcSDKP-IS level was significantly higher in patients with chronic renal failure (0.92 ± 0.39 pmol/ml) compared with healthy subjects (0.29 ± 0.07 pmol/ml). These results suggest that our EIA may be useful to evaluate plasma AcSDKP level as a biomarker in various patients.

A basic phenylalanine-rich oligo-peptide causes antibody cross-reactivity.

Glycolate oxidase (GO) and ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) are the two enzymes that serve key functions in the photorespiration and photosynthesis of plants. A 2 kDa highly basic phenylalanine-rich oligo-peptide (BOP) binds to the surface of acidic GO via ionic and hydrophobic interactions, forming the GO-BOP complex (GC). Previously, RubisCO was thought to exist as a single species composed of a large (rbc L, 54 kDa) and a small subunit (rbc S, 14 kDa). Here we show for the first time, using 2-DE, SDS-PAGE, immunoassays and amino acid determination, that BOP also interacts with RubisCO and that many RubisCO-BOP complexes (RCs), differing in pI, hydrophobicity and activity, coexist in green leaves. GCs, RCs and crude extract from green leaves analyzed by SDS-PAGE Western blotting showed that BOP exists either in subunit-BOP complexes (GO subunit-BOP, rbc L-BOP and rbc S-BOP etc.), with a wide variation in the number and the position of BOPs bound to each subunit molecular, or alone without a binding partner. When rbc L-BOP and rbc S-BOP were assayed by SDS-PAGE, BOP was dissociated from the subunit and it self-assembled to form 37 different BOP polymers (basic phenylalanine-rich protein) whose molecular weights (M(r)s) ranged from 34.0 to 91.6 kDa, indicating that the M(r) of BOP is about 2 kDa. Thus, the addition of BOP changes the M(r) of the subunit-BOP complexes so minimally that the rbc L and rbc S run at their predicted M(r)s on SDS-PAGE. In summary, the results described here demonstrate that the presence of BOP in complexes (both subunit-BOP complex and protein-BOP complex) can cause cross-reactivity of antibodies against different proteins.

Production of monoclonal antibodies against antitumor cyclohexapeptide RA-VII from Rubia cordifolia and their characterization.

An immunoassay system was established for the estimation of the quantity of an antitumor cyclic hexapeptide RA-VII (1) from Rubia cordifolia L. and R. akane Nakai (Rubiaceae). First, 1 was converted into its hapten, which was then conjugated with a carrier protein to be used as an effective antigen to obtain its monoclonal antibody (MAb). In the resulting conjugate, the molecular ratio between 1 and the carrier protein as assayed by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) was about 5:1. Then, the splenocytes from the mouse immunized with the conjugate were fused with mouse myeloma cells to produce hybridoma, secreting MAb against 1. Two clones were isolated, one producing MAb IgG(1) and the other IgM, both having a κ light chain. The sensitivity and cross-reactivity of the thus obtained MAb were also assayed.

The synthetic TLR2 agonist BPPcysMPEG leads to efficient cross-priming against co-administered and linked antigens.

The property of DC to generate effective CTL responses is influenced by TLR signaling. TLR ligands contain molecular signatures associated with pathogens, have an impact on the antigen processing and presentation by DC, and are being exploited as potential adjuvants. We hypothesized that the TLR2/6 heterodimer agonist S-[2,3-bispalmitoyiloxy-(2R)-propyl]-R-cysteinyl-amido-monomethoxyl polyethylene glycol (BPP), a synthetic derivative of the Mycoplasma macrophage activating lipopeptide-2, is a potent adjuvant for cross-priming against cellular antigens. Systemic administration of BPP-induced maturation of CD8alpha+ DC and CD8alpha- DC in the spleen and resulted in enhanced cross-presentation of intravenously co-administered antigen in mice. In addition, administration of BPP and cell-associated OVA generated an effective CTL response against OVA in vivo in a CD4+ T helper cell-dependent manner, but independent of IFN-alpha. Delivering antigenic peptides directly linked to BPP led to superior CTL immunity as compared to giving antigens and adjuvants admixed. In contrast to other TLR ligands, such as CpG, systemic activation of DC with BPP did not result in shut-down of antigen presentation by splenic DC subsets, although cross-priming against subsequently encountered antigens was reduced. Together, our data provide evidence that BPP is a potent stimulus to generate CTL via cross-priming.

Single-step conjugation of bioactive peptides to proteins via a self-contained succinimidyl bis-arylhydrazone.

This paper describes a method for a single-step, site-specific conjugation of bioactive peptides to proteins that exploits the monitoring advantages provided by the unique UV signature absorbance of a bis-arylhydrazone. The utility of this method is demonstrated by the conjugation of a decapeptide molecular adjuvant, YSFKDMP(MeL)aR (EP67), to two test proteins, ovalbumin (OVA) and bovine serum albumin (BSA), and to proteins expressed on intact influenza virons and fungal arthroconidia (spores) of Coccidioides. Conjugation is accomplished with a version of EP67 in which its N-terminus is modified with succinimidyl-4-benzoylhydrazino-nicotinamide (S4BHyNic) (peptide 7), thus enabling conjugation to these large entities via formation of amide bonds with surface-exposed amino groups. The presence of the strongly absorbing bis-arylhydrazone S4BHyNic (ε(354 nm) = 29 000 L mol(-1) cm(-1)) allows for determination of EP67-to-protein molar substitution ratios (MSR), which are in good agreement with the MSRs determined by amino acid analysis. Conjugation to OVA does not compromise the ability of EP67 to engage C5a receptor bearing antigen presenting cells (APC) as measured by the EP67-mediated release of interleukin-6 (IL-6) from APCs. Mice immunized with the resulting OVA-EP67 vaccine conjugate produce high serum titers of OVA-specific IgG antibodies relative to OVA alone. Also, the conjugation of EP67 does not affect the surface integrity of influenza virons or the biological viability of Coccidioides spores. This method of conjugating bioactive peptides to proteins and other large biological entities may represent a convenient and effective way of generating various bioconjugates for use in mechanistic studies or novel therapeutic entities such as EP67-containing vaccines.

A palmitoyl-tailed sequential oligopeptide carrier for engineering immunogenic conjugates.

The main guideline in designing effective immunogens as vaccine candidates capable of eliciting potent and specific immune responses is to combine B/T cell epitopes and adjuvants as immunostimulators on the same carrier that links the major histocompatibility complex with T cell receptors. Aiming at contributing to the development of carriers for human usage a helicoid type sequential oligopeptide carrier, SOC(n)-II, formed by the repeating tetrapeptide unit (Aib-Lys-Aib-Gly)(n), n=2-7, elongated from the amino-terminus by the palmitoyl group, known for its adjuvanticity, is now presented. The main B cell epitope, PPGMRPP, of the Sm autoantigen against which the majority of antibodies in patients with systemic lupus erythematosus is directed, was coupled to the Lys-N(epsilon)H(2) groups of the carrier in four copies and the resulting conjugate Palm-SOC(4)-II-Sm(4) was subjected to animal immunizations without utilizing any adjuvant. The induced immune response was comparable with that produced when Ac-SOC(4)-II-Sm(4) was administered in animals following the conventional immunization protocol of complete/incomplete Freund's adjuvant. High titers of anti-Palm-SOC(4)-II-Sm(4) antibodies were generated, which recognize the priming immunogenic conjugate, as well as reconstituted Sm mimics but not the carrier alone. It is concluded that Palm-SOC(n)-II carrier is a valuable tool for engineering immunogens eliciting enhanced and specific humoral immune responses.

Postnatal development of kisspeptin neurons in mouse hypothalamus; sexual dimorphism and projections to gonadotropin-releasing hormone neurons.

The neuropeptide kisspeptin has recently been implicated as having a critical role in the activation of the GnRH neurons to bring about puberty. We examined here the postnatal development of kisspeptin neuronal populations and their projections to GnRH neurons in the mouse. Three populations of kisspeptin neurons located in the 1) anteroventral periventricular nucleus (AVPV) and the preoptic periventricular nucleus (PeN), 2) dorsomedial hypothalamus, and 3) arcuate nucleus were identified using an antisera raised against mouse kisspeptin-10. A marked 10-fold (P<0.01), female-dominant sex difference in the numbers of kisspeptin neurons existed in the AVPV/PeN but not elsewhere. Kisspeptin neurons in the AVPV/PeN of both sexes displayed a similar pattern of postnatal development with no cells detected at postnatal day (P) 10, followed by increases from P25 to reach adult levels by puberty onset (P<0.01; P31 females and P45 males). This pattern was not found in the dorsomedial hypothalamus or arcuate nucleus. Dual immunofluorescence experiments demonstrated close appositions between kisspeptin fibers and GnRH neuron cell bodies that were first apparent at P25 and increased across postnatal development in both sexes. These studies demonstrate kisspeptin peptide expression in the mouse hypothalamus and reveal the postnatal development of a sexually dimorphic continuum of kisspeptin neurons within the AVPV and PeN. This periventricular population of kisspeptin neurons reaches adult-like proportions at the time of puberty onset and is the likely source of the kisspeptin inputs to GnRH neurons.

Phase I/II combined chemoimmunotherapy with carcinoembryonic antigen-derived HLA-A2-restricted CAP-1 peptide and irinotecan, 5-fluorouracil, and leucovorin in patients with primary metastatic colorectal cancer.

PURPOSE: We conducted a phase I/II randomized trial to evaluate the clinical and immunologic effect of chemotherapy combined with vaccination in primary metastatic colorectal cancer patients with a carcinoembryonic antigen-derived peptide in the setting of adjuvants granulocyte macrophage colony-stimulating factor, CpG-containing DNA molecules (dSLIM), and dendritic cells. EXPERIMENTAL DESIGN: HLA-A2-positive patients with confirmed newly diagnosed metastatic colorectal cancer and elevated serum carcinoembryonic antigen (CEA) were randomized to receive three cycles of standard chemotherapy (irinotecan/high-dose 5-fluorouracil/leucovorin) and vaccinations with CEA-derived CAP-1 peptide admixed with different adjuvants [CAP-1/granulocyte macrophage colony-stimulating factor/interleukin-2 (IL-2), CAP-1/dSLIM/IL-2, and CAP-1/IL-2]. After completion of chemotherapy, patients received weekly vaccinations until progression of disease. Immune assessment was done at baseline and after three cycles of combined chemoimmunotherapy. HLA-A2 tetramers complexed with the peptides CAP-1, human T-cell lymphotrophic virus type I TAX, cytomegalovirus (CMV) pp65, and EBV BMLF-1 were used for phenotypic immune assessment. IFN-gamma intracellular cytokine assays were done to evaluate CTL reactivity. RESULTS: Seventeen metastatic patients were recruited, of whom 12 completed three cycles. Therapy resulted in five complete response, one partial response, five stable disease, and six progressive disease. Six grade 1 local skin reactions and one mild systemic reaction to vaccination treatment were observed. Overall survival after a median observation time of 29 months was 17 months with a survival rate of 35% (6 of 17) at that time. Eight patients (47%) showed elevation of CAP-1-specific CTLs. Neither of the adjuvants provided superiority in eliciting CAP-1-specific immune responses. During three cycles of chemotherapy, EBV/CMV recall antigen-specific CD8+ cells decreased by an average 14%. CONCLUSIONS: The presented chemoimmunotherapy is a feasible and safe combination therapy with clinical and immunologic efficacy. Despite concurrent chemotherapy, increases in CAP-1-specific T cells were observed in 47% of patients after vaccination.

Comparative study of antioxidant properties of immunoregulatory peptides.

We studied antioxidant properties of immunofan, bursin, cyclobursin, thymopoietin II fragment, glycine, and Siberian ginseng. Experiments were performed in 2 model systems: Fe(2+)-induced oxidation of multilamellar phospholipid liposomes in a heterogeneous water-lipid system and oxidation of luminol induced by alpha,alpha'-azo-bis(isobutyramidine dihydrochloride) in a homogenous aqueous system. By the ability to entrap lipid peroxyl radicals, antioxidant activity of substances decreased in the following order: Siberian ginseng extract>bursin>cyclobursin>thymopoietin II fragment>immunofan, glycine. Siberian ginseng extract and thymopoietin II fragment interacted with Fe(2+), which contributed to elimination of catalyst of lipid peroxidation from the system. The ability of substances to interact with aqueous peroxyl radicals and luminol radicals decreased in the following order: Siberian ginseng extract>thymopoietin II fragment>immunofan>glycine, cyclobursin, bursin. Substances with high antioxidant activity improved the state of the endogenous antioxidant system and protected cells from oxidative stress. They entrapped reactive oxygen species formed in the cytoplasm, modulated free radical processes, and regulated the synthesis of bioactive molecules.

Epitope-enhanced conserved HIV-1 peptide protects HLA-A2-transgenic mice against virus expressing HIV-1 antigen.

HIV epitopes may have developed to be poor immunogens. As a counterapproach HIV vaccine strategy, we used epitope enhancement of a conserved HIV reverse transcriptase (RT) epitope for induction of antiviral protection in HLA-A2-transgenic mice mediated by human HLA-A2-restricted CTLs. We designed two epitope-enhanced peptides based on affinity for HLA-A2, one substituted in anchor residues (RT-2L9V) and the other also with tyrosine at position 1 (RT-1Y2L9V), and examined the balance between HLA binding and T cell recognition. CTL lines and bulk cultures in two HLA-A2-transgenic mouse strains showed that RT-2L9V was more effective in inducing CTL reactive with wild-type Ag than RT-1Y2L9V, despite the higher affinity of the latter, because the 1Y substitution unexpectedly altered T cell recognition. Accordingly, RT-2L9V afforded the greatest protection in vivo against a surrogate virus expressing HIV-1 RT mediated by HLA-A2-restricted CTL in a mouse in which all CTL are restricted to only the human HLA molecule. Such antiviral protection has not been previously achieved with an HLA epitope-enhanced vaccine. These findings define a critical balance between MHC affinity and receptor cross-reactivity required for effective epitope enhancement and also demonstrate construction and efficacy of such a component of a new generation vaccine.

Antibody recognition of chiral surfaces. Enantiomorphous crystals of leucine-leucine-tyrosine.

Monoclonal antibodies were selected after immunization with crystals of the tripeptide l-leucine-l-leucine-l-tyrosine. They interact with the tripeptide crystals, but do not interact with the tripeptide molecule, with other crystalline surfaces, or with adsorbed protein. The interactions of two antibodies with crystals of l-Leu-l-Leu-l-Tyr and of its enantiomer d-Leu-d-Leu-d-Tyr were characterized in depth. Antibody 48E is stereoselective and enantioselective: it recognizes only the [011] faces of the l-Leu-l-Leu-l-Tyr crystals, and not the enantiomorphous [011] faces of d-Leu-d-Leu-d-Tyr crystals, or any other faces of either crystal. In contrast, antibody 602E is poorly stereoselective and is not enantioselective: it recognizes the crystals of both enantiomers, interacting with a number of different faces of each. The different recognition patterns are explained on the basis of the nature of the interactions and the structure of the interacting surfaces. Understanding this antibody specificity advances our general understanding of surface recognition and transfer of chiral information across biological interfaces.

Endomorphin and mu-opioid receptors in mouse brain mediate the analgesic effect induced by 2 Hz but not 100 Hz electroacupuncture stimulation.

This work was designed to examine whether brain endomorphins (EM1 and EM2), the endogenous mu-opioid ligands, are involved in electroacupuncture (EA)-induced analgesia in the mice. C57BL/6J mice were given EA for 30 min and the effect of EA-induced analgesia was assessed by radiant heat tail flick latency (TFL). Intracerebroventricular (i.c.v.) injection of mu-opioid receptor antagonist D-Phe-Cys-Tyr-D-Tyr-Orn-Thr-Pen-Thr-NH(2) (CTOP), or antiserum against EM1 or EM2 was performed to see whether EA analgesia could be blocked. The results showed that: (1) i.c.v. injection of CTOP at 25-100 ng dose-dependently antagonized the analgesia induced by EA of 2 Hz, but not 100 Hz. (2) Intracerebroventricular injection of EM1 antiserum (5 ml, 1:1 or 1:10 dilution) dose-dependently antagonized 2 Hz, but not 100 Hz EA analgesia. (3) EM2 antiserum showed similar effect at 1:1 dilution. The results are interpreted to mean that endogenously released EM1 and EM2 and the cerebral mu-receptors are involved in mediating 2 Hz but not 100 Hz EA analgesia in the mice.

Production of monoclonal antibody to deltorphin-I and its immunocytochemical application to adult rat brain and cultured rat brain neurons.

A monoclonal anti-deltorphin-I antibody specifically recognizing its NH2-terminal region was produced. In the adult rat brain sections, it recognized immunoreactive nerve fibers mainly in the bed nucleus of stria terminalis, central nucleus of amygdala, lateral hypothalamus, hippocampus, substantia nigra, periaqueductal gray and locus ceruleus. Occasionally, positive somata were localized in the bed nucleus of stria terminalis, central nucleus of amygdala, supraoptic and periventricular nuclei. In primarily cultured neurons from various brain regions of new-born rats, the antibody immunostained strongly neuronal somata and processes. The abundant DADTI-immunoreactive substance in the cultured neurons promises to provide an alternative pathway to search for the counterpart of deltorphins in mammals.

D-LysAsnProTyr tetrapeptide: a novel B-cell stimulant and stabilized bursin mimetic.

The tetrapeptide I (D-lysine-L-asparaginyl-L-prolyl-L-tyrosine or D-LysAsnProTyr), and analogue sequences, were synthesized and evaluated for the ability to stimulate immune cell subsets. These sequences were selected based on their perceived ability to readily adopt a beta-turn structure. In vitro immunological assays revealed a robust stimulation of mitogen activated B-cell proliferation and a modest to significant stimulation of cytotoxic T lymphocytes (CTLs). Further, this in vitro stimulation of B-cells was accompanied by an in vivo expansion of B-cells in C57BL/6 mice, as demonstrated by immunophenotyping experiments. Interestingly, a conformational analysis of the low energy conformers of I and the endogenous B-cell stimulant bursin (LysHisGlyNH2) shows that these molecules can be superimposed. However, I displayed significantly enhanced physiological stability. For a number of reasons, I may be a particularly useful vaccine adjuvant.