Presence Of Epitopes Research Articles

LA PREDICCIÓN IN SILICO DETERMINA LA PRESENCIA DE EPÍTOPOS INMUNOGÉNICOS ALTAMENTE ESPECÍFICOS EN FRAGMENTOS DE LA PROTEÍNA DE LA MEMBRANA POLIMÓRFICA (PMP17G) DE Chlamydia abortus

<p><strong>Background.</strong> Ovine Enzootic Abortion is a contagious infectious disease caused by a Gram negative and obligate intracellular bacterium, <em>Chlamydia abortus</em>. For field diagnosis, commercial serological tests are used; however, some of these tests show low sensitivity and specificity rates, due to the cross-reactions that the antigens used have against other pathogens. For the most accurate diagnosis, it is necessary to develop tests with more specific antigens such as polymorphic membrane proteins (Pmp's), that allow to determine the presence of specific epitopes using new technologies. <strong>Objective.</strong> To determine <em>in silico</em> the presence of epitopes with specific immunogenic potential against <em>Chlamydia abortus</em> of two fragments of the PMP17G protein. <strong>Methodology.</strong> The cloning and sequencing of the fragments was carried out from a field isolate of <em>Chlamydia abortus</em>, and from the analysis of these sequences, with the help of two bioinformatics software’s. <strong>Results.</strong> Several epitopes from <em>Chlamydia abortus</em> were found, rPOMP90-3 (eight epitopes) and rPOMP90-4 (one epitope). <strong>Implications.</strong> Bioinformatics analysis indicated that both fragments of the protein have the capacity to activate the immune system, which would be useful for the development of diagnostic kits and immunogens. <strong>Conclusions.</strong> The <em>in silico</em> analysis allowed to efficiently predict and identify specific epitopes against <em>Chlamydia abortus</em> in both fragments of the protein. </p>

In silico Analysis of a Novel Peptide Vaccine against Hepatitis B Virus (HBV)

Background : Hepatitis B is a potentially life-threatening liver infection caused by the Hepatitis B virus (HBV). It is a major global health problem and the most serious type of viral hepatitis. It can cause chronic liver disease and puts people at high risk of death from cirrhosis of the liver and liver cancer. HBV is found in highest concentrations in blood and in lower concentrations in other body fluids.
 Methods: Target protein was retrieved from the swissprot database. Epitopes were predicted using the BCEPRED server. After running the BLAST algorithm for the target protein, the template with the best identity was selected. After modeling, target protein is verified by using the swiss model workspace and after this process the obtained target protein is allowed to interact with the MHC which is studied by using patchdock, finally these results were viewed by using the deepview tool.
 Results: The target protein for vaccine development was downloaded from the SwissProt database. Its SwissProt ID was p29178. The protein was isolated from hepatitis B virus genotype G. The virus was isolated from the United States of America. The length of the target protein was found to be 195 amino acids. To confirm that the target protein could be used for vaccine development, the Presence of epitopes in the protein was confirmed using the BCEPRED tool. Results from the SAVS server showed 95.80 of the residues of the protein had an average 3D-1D score greater than 0.2. The protein attained a pass with an ERRAT value of 90.299.
 Conclusion: The present investigation recognized the promising complex formed between the HBV peptide and MHC molecules. All the downloaded MHC molecules were found to interact with the target protein through the formation of hydrogen bonds. Since these interactions are necessary during an immune response to invading pathogens, the target protein would ultimately trigger an immune response if it is administered as a vaccine for Hepatitis B virus genotype G.

Functional genomic characterization of immunogenic gluten proteins from oat cultivars that differ in toxicity for celiac disease

AbstractIntroduction:Oat human consumption has increased due to its nutritional value and its health benefits. Oat is a rich source of protein that contains high level of minerals, lipids, β-glucan, a mixed-linkage polysaccharide, which forms an important part of oat dietary fiber, and also contains various other phytoconstituents like flavonoids and sterols among others. Different pharmacological activities have been reported on oats like antioxidant, anti-inflammatory, antidiabetic or anticholesterolaemic.The safety of oats in a gluten-free diet has been a topic of debate for several years. Previous studies suggested that oats may induce the immunological response in celiacs and others confirmed the impossibility of consuming oats habitually by its toxicity. Our research group found oat cultivars with different immunotoxic potential against G12 monoclonal antibody that may explain the different clinical responses observed in patients suffering from celiac disease. In this study we have characterized by massive sequencing the transcriptomes of non-toxic and toxic varieties.Materials and Methods:The transcriptomes of both oat varieties were sequenced by Illumina HiSeq™2000. To assemble the contents, criteria of overlap > 40% and similarity > 95% were used. The functional annotations were inferred by similarity to Uniprot reference proteins. The minimum similarity threshold required for annotating a transcript was a BLAST e value minor than 10-10. Uniref90 was used for the selection of annotated proteins.Results:We have found 17 and 11 locus in the non-toxic and toxic varieties, respectively. We selected a set of 239983 reference proteins downloaded from Uniprot belonging to the taxonomic nodes BEP clade. Only proteins representative of Uniref90 clusters were used. The identification of immunotoxic epitopes in the coding sequences were determined by alignment with the T-cell recognized canonicals, encompassing one to three mismatches. We identified a total of 24 epitopes with an average of 2 modifications in the genome of the toxic variety with respect to the non-toxic. The epitope variants DQ2.5-ave-1β and DQ2.5-glia-α3 were the most repeated.Discussion:The presence of epitopes in the toxic oat variety that are not present in non-toxic variety could be related with the immunotoxic potential found in our previous assays and also with the different clinical responses in celiacs consuming oats. Preliminary results suggest that a depth study based on searching epitopes found in toxic oat variety could help to the identification of real oat varieties available for celiac patients, and therefore, their incorporation in improvement programs to obtain commercial lines without toxicity.

Persistent presence of outer membrane epitopes during short- and long-term starvation of five Legionella pneumophila strains

BackgroundLegionella pneumophila, the causative agent of Legionnaire’s disease, may enter a viable but non-culturable (VBNC) state triggered by environmental stress conditions. Specific outer-membrane epitopes of L. pneumophila are used in many diagnostic applications and some of them are linked to important virulence-related factors or endotoxins. However, it is not clear how the presence and status of these epitopes are influenced by environmental stress conditions. In this study, changes of outer membrane epitopes for monoclonal antibodies (mAb) from the Dresden panel and the major outer membrane protein MOMP were analysed for five L. pneumophila strains during short- and long-term starvation in ultrapure water.ResultsWith ELISA and single cell immuno-fluorescence analysis, we could show that for most of the investigated mAb-strain combinations the total number of mAb-stained Legionella cells stayed constant for up to 400 days. Especially the epitopes of mAb 3/1, 8/5, 26/1 and 20/1, which are specific for L. pneumophila serogroup 1 subtypes, and the mAb 9/1, specific for serogroup 6, showed long-term persistence. For most mAb- stained cells, a high percentage of viable cells was observed at least until 118 days of starvation. At the same time, we observed a reduction of the fluorescence intensity of the stained cells during starvation indicating a loss of epitopes from the cell surface. However, most of the epitopes, including the virulence-associated mAb 3/1 epitope were still present with high fluorescence intensity after 400 days of starvation in up to 50% of the starved L. pneumophila population.ConclusionsThe results demonstrate the continuous presence of outer membrane epitopes of L. pneumophila during short-term and long-term starvation. Thus, culture-independent mAb-based diagnostic and detection tools, such as immuno-magnetic separation and microarray techniques are applicable for both L. pneumophila in the culturable and the VBNC state even after long-term starvation but nevertheless require careful testing before application. However, the mere presence of those epitopes is not necessarily an indication of viability or infectivity.

Abstract 319: Design of Novel Chimeras to Provide Insight Into Structure/Function Activity of Apolipoprotein E3 and Apolipoprotein AI

Apolipoprotein (apo) E3 and apoAI are exchangeable apolipoproteins that play a dominant role in regulating plasma cholesterol levels and are considered anti-atherogenic. ApoE3 (299 residues, ~34 kDa) serves as a ligand for the low density lipoprotein receptor (LDLr) family of proteins that mediate cellular uptake and clearance of plasma lipoproteins. The ability of apoE3 to function as a ligand for LDLr resides in the N-terminal domain (NT) of apoE3. ApoAI (243 residues, ~28 kDa) lowers cholesterol levels through its ability to promote cholesterol efflux from peripheral tissues such as macrophages, forming HDL, which transports cholesterol back to the liver. Residues located in the C-terminal domain (CT) of apoAI mediate this function of promoting cholesterol efflux. The objective of this study is to generate chimeric apolipoproteins composed of apoE3 and apoAI to better understand the role of the C-terminal tail of the parent proteins. Two chimeras were generated by “domain swapping”: apoE3-NT/apoAI-CT and apoAI-NT/apoE-CT. They were overexpressed in E. coli, isolated and purified. Western blot analysis revealed the presence of epitopes from both parent proteins. Circular dichroism spectroscopy revealed that the α-helical content of apoE3-NT/apoAI-CT and apoAI-NT/E-CT was 43 and 54 %, respectively, which is comparable to that of the parent proteins. Chemical denaturation was used to probe for changes in protein stability. Addition of CT of apoE3 to NT of apoAI conferred more stability to apoAI, while addition of CT of apoAI to NT of apoE3 had no significant effect on apoE3. The rate of apolipoprotein-induced conversion of DMPC vesicles to discoidal structures was followed to assess their lipid binding capability. ApoE3-NT/AI-CT displayed a rate constant that was ~10x higher compared to apoE3, while apoAI-NT/apoE-CT showed a rate constant similar to that of apoAI. Whereas apoE3-NT/apoAI-CT elicits the ability to bind to the LDLr, apoAI-NT/apoE-CT did not. Lastly, both chimeras promote ABCA1-mediated cholesterol efflux from J774 macrophages, with the efflux capability being 2-fold greater for apoAI-NT/apoE-CT. These results show that CT of apoAI can promote lipid binding of apoE3, while CT of apoE3 can improve cholesterol efflux ability of apoAI.

Safety and toxicological evaluation of a novel, water-soluble undenatured type II collagen

This study was conducted to determine the broad-spectrum safety of a novel, water-soluble undenatured type II collagen (NEXT-II) derived from chicken sternum cartilage. The presence of epitope in NEXT-II was confirmed by using a commercial kit. The acute oral LD50 of NEXT-II was found to be greater than 5000 mg/kg bw in rats, while the single-dose acute dermal LD50 was greater than 2000 mg/kg bw. The primary dermal irritation index (PDII) of NEXT-II was found to be 1.8 and classified as slightly irritating to the skin. In primary eye irritation studies, the maximum mean total score (MMTS) of NEXT-II was observed to be 7.3 and classified as minimally irritating to the eye. Long-term safety studies were conducted in dogs over a period of 150 d, and no significant changes were observed in body weight, heart rate, respiration rate and blood chemistry. NEXT-II does not induce mutagenicity in the bacterial reverse mutation test in five Salmonella typhimurium strains either with or without metabolic activation. Furthermore, two experiments were conducted to assess the potential of NEXT-II to induce mutations with and without metabolic activation at the mouse lymphoma thymidine kinase locus using the cell line L5178Y. No biologically relevant increase of mutants was observed. Also, no dose-dependent toxicity was observed. Furthermore, colony sizing showed no clastogenic effects induced by NEXT-II under the experimental conditions. These studies demonstrated the broad spectrum of safety of NEXT-II.

Engineering, conjugation, and immunogenicity assessment of Escherichia coli O121 O antigen for its potential use as a typhoid vaccine component

State-of-the-art production technologies for conjugate vaccines are complex, multi-step processes. An alternative approach to produce glycoconjugates is based on the bacterial N-linked protein glycosylation system first described in Campylobacter jejuni. The C. jejuni N-glycosylation system has been successfully transferred into Escherichia coli, enabling in vivo production of customized recombinant glycoproteins. However, some antigenic bacterial cell surface polysaccharides, like the Vi antigen of Salmonella enterica serovar Typhi, have not been reported to be accessible to the bacterial oligosaccharyltransferase PglB, hence hamper development of novel conjugate vaccines against typhoid fever. In this report, Vi-like polysaccharide structures that can be transferred by PglB were evaluated as typhoid vaccine components. A polysaccharide fulfilling these requirements was found in Escherichia coli serovar O121. Inactivation of the E. coli O121 O antigen cluster encoded gene wbqG resulted in expression of O polysaccharides reactive with antibodies raised against the Vi antigen. The structure of the recombinantly expressed mutant O polysaccharide was elucidated using a novel HPLC and mass spectrometry based method for purified undecaprenyl pyrophosphate (Und-PP) linked glycans, and the presence of epitopes also found in the Vi antigen was confirmed. The mutant O antigen structure was transferred to acceptor proteins using the bacterial N-glycosylation system, and immunogenicity of the resulting conjugates was evaluated in mice. The conjugate-induced antibodies reacted in an enzyme-linked immunosorbent assay with E. coli O121 LPS. One animal developed a significant rise in serum immunoglobulin anti-Vi titer upon immunization.

Trophozoites of Entamoeba histolytica express a CD59-like molecule in human colon

In vitro studies have proved the presence of epitopes of CD59 in the surface of trophozoites of Entamoeba histolytica (E. histolytica). However, it has not been proved if CD59 molecules are expressed in the surface during the trophozoites' tissue invasion. The aim of the present study was to determine whether the complement-regulatory protein CD59 is present on trophozoites of E. histolytica in human colon. Eleven specimens of amoebic colitis were studied by immunohistochemistry and electron microscopy techniques with a monoclonal antibody against human CD59 molecule. Our results show that a CD59-like molecule is expressed in trophozoites of E. histolytica found in colonic amebic lesions. Also, a CD59-like molecule was detected by western blot analysis in whole lysate of E. histolytica as well as on the plasma membrane by immunocytochemistry. These results suggest that E. histolytica can use CD59-like protein against the lytic action of membrane attack complex.

Rhamnogalacturonan I in Solanum tuberosum tubers contains complex arabinogalactan structures

A rhamnogalacturonan I polysaccharide was isolated from potato ( Solanum tuberosum cv. Posmo) tuber cell walls and characterised by enzymatic digestion with an endo-β-1 → 4-galactanase and an endo-α-1 → 5-arabinanase, individually or in combination. The reaction products were separated using size-exclusion chromatography and further analysed for monosaccharide composition and presence of epitopes using the LM5 anti-β-1 → 4-galactan and LM6 anti-α-1 → 5-arabinan monoclonal antibodies. The analyses point to distinct structural features of potato tuber rhamnogalacturonan I, such as the abundance of β-1 → 4-galactan side chains that are poorly substituted with short arabinose-containing side chains, the presence of α-1 → 5-arabinan side chains substituted with β-1 → 4-galactan oligomers (degree of polymerisation >4), and the presence of α-1 → 5-arabinans that resist enzymatic degradation. A synergy between the enzymes was observed towards the degradation of arabinans but not towards the degradation of galactans. The effect of the enzymes on isolated RG I is discussed in relation to documented effects of enzymes heterologously expressed in potato tubers. In addition, a novel and rapid method for the determination of the monosaccharide and uronic acid composition of cell wall polysaccharides using high-performance anion exchange chromatography with pulsed amperometric detection is described.

Protective immunization against group B meningococci using anti-idiotypic mimics of the capsular polysaccharide.

Use of the serogroup B meningococcal capsular polysaccharide (MenB CP) as a vaccine is hampered by the presence of epitopes that cross-react with human polysialic acid. As non-cross-reactive, protective capsular epitopes have also been described, we set out to develop protein mimics of one of such epitopes using as a template a highly protective mAb (mAb Seam 3) raised against a chemically modified form of the MenB CP (N-Pr MenB CP). Using phage display, anti-idiotypic single-chain Ab fragments (scFvs) were obtained from spleen cells of mice immunized with the Seam 3 mAb. Two Seam 3-specific scFvs competed with N-Pr MenB CP for binding to either mAb Seam 3 or rabbit Abs present in typing sera. Moreover, in mice and rabbits the scFvs elicited the production of Abs reacting with both N-Pr MenB CP and whole meningococci, but not with human polysialic acid. These scFv-induced Ab responses were boostable and of the Th1 type, as shown by a predominance of IgG2a. In addition, passive immunization with sera from scFv-immunized animals partially protected neonatal mice from experimental infection with group B meningococci. In conclusion, we have produced anti-idiotypic scFvs that mimic a protective MenB CP epitope and may be useful in the development of an alternative group B meningococcal vaccine.

Schistosoma mansoni and Schistosoma haematobium: identification and characterization of glycoconjugate antigens in the hemolymph of infected vector snails.

Two carbohydrate epitopes were identified by monoclonal antibodies (KCS and E2) and characterized with respect to their immunoreactivity, monosaccharide structure, and location. Immunofluorescence demonstrated the presence of both epitopes on the surfaces of sporocysts, cercariae, and miracidia of Schistosoma mansoni, Schistosoma haematobium, and Schistosoma japonicum. However, spatial distribution and density of expression varied among species and developmental stages, and neither epitope was detectable on adult worm surfaces. Both glycans were found in the hemolymph of infected, but not uninfected, intermediate snail hosts. The presence of epitopes in hemolymph, as well as in schistosome eggs, is species-specific for KCS, recognizing only S. mansoni, and partly specific for E2, which reacted predominantly with S. haematobium. Immunoaffinity purification of target antigens for KCS and E2 from hemolymph of infected Biomphalaria and Bulinus, respectively, followed by carbohydrate composition analysis revealed a high content of fucose in both glycans. Methylation analysis demonstrated exclusively terminal fucose for the target antigen of KCS and terminal as well as internal fucose for the one of E2. Removal of terminal fucose abolished reactivity with both monoclonal antibodies. Both glycans are different from previously characterized schistosome carbohydrates. Their biological function(s) remain to be defined.

Alternate Translation Occurs within the Core Coding Region of the Hepatitis C Viral Genome

The majority of hepatitis C virus (HCV) isolates contain an open reading frame (ORF) overlapping with the core coding sequences in the +1 frame, which was assumed to be untranslated. We present evidence supporting the expression of this ORF (designated core+1 ORF) via novel translation mechanisms. First, fusion of the luciferase gene with the HCV-1 core+1 ORF followed by in vitro translation resulted in the synthesis of a chimeric protein (core+1-luciferase) that exhibited approximately 54% luciferase activity relative to the positive control (core-luciferase). Second, antisera raised against two different synthetic core+1 peptides recognized the previously identified p16 (but not p21) core protein band expressed from HCV-1, indicating the presence of epitopes from the core+1 ORF within the p16 protein. Third, HCV-positive sera specifically recognized lysates of Escherichia coli cells expressing recombinant core+1 protein, suggesting the presence of anti-core+1 antibodies in HCV-infected patients. Finally, luciferase tagging experiments designed to assess for -1 frameshifting combined with site-directed mutagenesis experiments supported the presence of +1/-1 ribosomal frameshift translation mechanisms within the core coding region. In conclusion, our data provide evidence for novel translation mechanisms within the core coding region and demonstrate the expression of the core+1 ORF, at least for some HCV isolates.

Apical Na+/H+ exchange near the base of mouse colonic crypts

Chu, Jingsong, Shaoyou Chu, and Marshall H. Montrose. Apical Na /H exchange near the base of mouse colonic crypts. Am J Physiol Cell Physiol 283: C358–C372, 2002; 10.1152/ajpcell.01380.2000.—Colonic crypts can absorb fluid, but the identity of the absorptive transporters remains speculative. Near the crypt base, the epithelial cells responsible for vectorial transport are relatively undifferentiated and often presumed to mediate only Cl secretion. We have applied confocal microscopy in combination with an extracellular fluid marker [Lucifer yellow (LY)] or a pH-sensitive dye (2 ,7 -bis(2carboxyethyl)-5(6)-carboxyfluorescein) to study mouse colonic crypt epithelial cells directly adjacent to the crypt base within an intact mucosal sheet. Measurements of intracellular pH report activation of colonocyte Na /H exchange in response to luminal or serosal Na . Studies with LY demonstrate the presence of a paracellular fluid flux, but luminal Na does not activate Na /H exchange in the nonepithelial cells of the lamina propria, and studies with LY suggest that the fluid bathing colonocyte basolateral membranes is rapidly refreshed by serosal perfusates. The apical Na /H exchange in crypt colonocytes is inhibited equivalently by luminal 20 M ethylisopropylamiloride and 20 M HOE-694 but is not inhibited by luminal 20 M S-1611. Immunostaining reveals the presence of epitopes from NHE1 and NHE2, but not NHE3, in epithelial cells near the base of colonic crypts. Comparison of apical Na /H exchange activity in the presence of Cl with that in the absence of Cl (substitution by gluconate or nitrate) revealed no evidence of the Cl -dependent Na /H exchange that had been previously reported as the sole apical Na /H exchange activity in the colonic crypt. Results suggest the presence of an apical Na /H exchanger near the base of crypts with functional attributes similar to those of the cloned NHE2 isoform.

Why are some proteins allergens?

The ability of certain proteins to induce an allergic response in susceptible individuals is well established. Symptoms can range from mild erythema or rhinitis, to acute, and possibly fatal, anaphylactic shock. Because such allergic responses require complex interactions between the protein and the immune system, they are notoriously difficult to predict. Nevertheless, it is clear that some proteins are intrinsically more allergenic than others. The challenge for toxicologists is to identify those characteristics that confer on proteins the potential to induce allergic sensitization and allergic disease. Here, we first consider the potential contribution that individual epitopes may make to the allergenicity of a protein. These are the minimal peptide units within proteins that can be recognized by the immune system and are a fundamental requirement for all immune responses, including those resulting in allergic sensitization. It appears that allergens must necessarily contain B-cell epitopes to which immunoglobulin E (IgE) can bind, and T-cell epitopes capable of inducing a type 2 T-lymphocyte response. Nevertheless, it appears doubtful that the presence of appropriate epitopes alone is sufficient to endow a protein with allergenic potential. We therefore consider also the contribution that other features and characteristics of proteins may make to their overall allergenicity. In particular, we consider the effects that resistance to proteolysis, post-translational glycosylation, and enzymatic activity may have. It appears that relative stability in simulated gastric fluid (SGF) sometimes correlates with allergenic activity. However, this is not universally true, and it is known that there are protein allergens, such as some of those associated with oral allergy syndrome, that are unstable. Nevertheless, if stability in SGF is associated with the intrinsic allergenicity of many proteins irrespective of the route of exposure, then this may reflect some more fundamental property of proteins, and possibly their stability in other biologic matrices and/or to intracellular proteases. Post-translational modification appears generally to enhance allergenicity, perhaps by increasing uptake and detection of the protein by the immune system. Some enzymatic activities also enhance allergenicity through what appear to be several different mechanisms, including nonspecific activation of cells participating in the immunologic response. Overall, it appears likely that many factors can contribute to the overall allergenicity of any given protein. Some, such as the presence of epitopes with allergenic potential, may be essential. Others, such as the glycosylation status, resistance to proteolysis, and enzymatic activity, may play a subsidiary but nevertheless critically important role. By better defining the limits within which these factors operate, we can hope to gain a better understanding of the fundamental origins of protein allergenicity, and therefore be in a position to identify and characterize the hazards and risks of allergic disease associated with novel proteins.

Expression of an epitopic region of AspfI, an allergen/antigen/cytotoxin of Aspergillus fumigatus

The gene for an 18 kD allergen/cytotoxin of Aspergillus fumigatus was cloned in pUC-19 vector and expressed in Escherichia coli JM109. Digestion of this gene with AluI resulted in four fragments of 216bp, 120bp, 39bp and 21bp. These fragments were cloned in the Sma-I site of pUC-19. The recombinants thus, generated after transformation in E. coli JM109, were screened using monoclonal antibodies raised against the AspfI. The fusion protein containing 120 bp AluI fragment was recognised by the MoAb indicating presence of epitope(s) in the 120 bp region. The study indicates a viable strategy for identification and expression of an immunologically active domain of a major allergen/antigen of A. fumigatus for the first time.

Genomic, protein homogeneity and antigenic variability of Mycoplasma agalactiae

Eleven strains of Mycoplasma agalactiae differing in pathogenicity, animal species origin and geographic localisation, showed similar chromosome restriction profiles with four endonucleases. However the international reference strain PG2 showed a unique profile. The protein and antigenic variabilities of 31 strains of M. agalactiae were investigated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting performed with naturally infected animal sera and purified antibodies against the 29 kDa protein. Protein profiles were similar but antigenic profiles could be separated into two main groups according to geographic origin: (i) strains isolated in south-west France and (ii) strains from north-east France. Some differences also occurred from strain to strain within each group. The antigenic profile variability found in immunoblotting, originated in two different phenomena: (i) some epitopes were expressed only in strains ofone profile type and (ii) some other epitopes were common to all strains but located on several proteins which differed in number and molecular mass from one strain to another. The presence of epitopes which undergo phase variation in the same lineage of clones from a single cell is discussed.

During AA amyloidogenesis is proteolytic attack on serum amyloid A a pre- or post- fibrillogenic event?

Using differential affinity chromatography, antibodies were prepared to the carboxy-terminal and amino-terminal portions of murine SAA. Immunohistochemical examinations of spleen AA amyloid with these antibodies, at both the light and electron microscopic levels, indicated the presence of epitopes from both regions of SAA in these fibrillary deposits. Whole spleen homogenates.from animals at varying periods of time following the induction of amyloidosis, were used in conjunction with electrophoresis and autoradiographic Western blotting to determine the SAA/AA ratio in spleen as a function of AA amyloid induction time. Intact SAA was a consistent constituent of splenic homogenates regardless of the time following induction. Furthermore, over a three to four week period the SAA/AA ratio gradually decreased with time. These morphologic and time course data are more consistent with SAA undergoing proteolytic cleavage after, rather than before, it is incorporated into an AA fibril.

Expression of β4 Integrins in Human Skin: Comparison of Epidermal Distribution with β1-Integrin Epitopes, and Modulation by Calcium and Vitamin D3 in Cultured Keratinocytes

The expression of beta 4 integrins in adult and fetal human skin as well as in cultured keratinocytes was studied by immunodetection with monoclonal antibodies, and compared with that of beta 1 integrins. The distribution of the beta 1 and beta 4 integrin epitopes was entirely different in the adult epidermis. As reported previously by us (J Clin Invest 84:1916, 1989), the beta 1 epitopes were present most prominently at lateral cell-cell contact points, whereas staining with the beta 4 antibody demonstrated a linear staining pattern polarizing to the basal surface juxtaposed to the dermal-epidermal basement membrane. In contrast, in fetal skin, the staining patterns both with beta 1 and beta 4 antibodies were similar and demonstrated the presence of epitopes surrounding the entire cell surface of both basal and suprabasal keratinocytes. Distinct polarization of beta 4 integrin epitopes was noted in cultured keratinocytes maintained in low-calcium (0.15 mM) medium, and the expression of these integrins was also noted in human papilloma virus-transformed keratinocytes. Switch of the cultures to high-calcium (1.2 mM) medium resulted in redistribution of the epitopes to a pattern suggesting their location at under-surface of the cells adjacent to the substratum. This Ca(++)-induced redistribution of beta 4 integrin epitopes could be counteracted by 10(-7) M vitamin D3. Finally, incubation with anti-beta 4 integrin antibody reduced the capacity of keratinocytes to attach to plastic substratum. The results provide further evidence for the role of beta 4 integrins in cell-matrix interactions.

Cellular Proliferative Responses in Squirrel Monkeys Immunized with Recombinant and Synthetic Plasmodium Vivax Circumsporozoite Peptides

The role of circulating peripheral blood momonuclear cells (PBMC) in mediating protective immunity was examined during an immunization trial in Saimiri monkeys. Three engineered constructs representing different but overlapping regions of the circumsporozoite (CS) protein of Plasmodium vivax were used to immunize the Saimiri monkeys. Monkeys were randomly placed into three immunization groups: rPvCS2, rPvCS3, and LCV3 (representing three different but overlapping portions of the P. vivax CS protein) and two control groups: an alum adjuvant control group and an unimmunized control group. Collections of PBMC were made throughout the study at weeks 0, 2, 8, challenge (week 16), and two weeks after challenge. Proliferative responses to all immunogens and pokeweed mitogen were measured in all monkeys. Fourteen of 18 monkeys immunized with either rPvCS2 or rPvCS3 responded on the day of challenge to the appropriate immunogen with a stimulation index less than 2. Immunization with LCV3, which represents the repeat region only, elicited a specific response in only one monkey. However monkeys in both control groups also responded to rPvCS2 and rPvCS3, regardless of immunization, suggesting the presence of epitopes in rPvCS2 and rPvCS3 capable of associating with differing MHC antigens. Furthermore, the frequency of these cells in the periphery was increased by immunization, as demonstrated by a greater number of responding monkeys in the rPvCS2 and rPvCS3 immunized groups.

Collagenous structures present in brain contain epitopes shared by collagen and microtubule-associated protein tau

A novel type of collagenous fibers has been isolated from human brain and characterized by electron microscopy and optical diffraction. It was found that the morphology of the fibers is similar, but not identical, to that of skin collagen. Also, the collagenous fibers show some similarities with the paracrystals that could be assembled in vitro from purified microtubule-associated protein tau. Immunological analyses indicated the presence of epitopes in these collagenous fibers which react with antibodies against collagen and tau.