Vi Antibody Research Articles

Evaluation of community-based serologic screening for identification of chronic Salmonella Typhi carriers in Vietnam

To determine the utility of screening anti-Vi antibodies to detect chronic Salmonella Typhi carriers in an endemic community. We conducted a community-based serologic survey for anti-Vi antibodies to identify chronic Salmonella Typhi carriers in a typhoid endemic region in Vietnam. We tested sera from 3209 (67.2%) of 4772 eligible adults. The median age was 37 years (range 20-92), 57.3% were female, 4.6% reported a history of typhoid fever and 0.3% reported typhoid vaccination. Anti-Vi antibody titers tested in Vietnam were < 1:40 in 2759 (86.0%), 1:40 in 194 (6.0%), 1:80 in 168 (5.2%), 1:160 in 57 (1.8%), and > or = 1:320 in 31 (1.0%). On re-testing in the USA, an additional 19 sera with titers > or = 1:160 were identified. We collected 589 rectal swabs from 103 (96.3%) of 107 persons with Vi antibody titers > or = 1:160 and 183 swabs from 33 persons with antibody titers < 1:80. No Salmonella Typhi was isolated. Community-based serologic screening is a feasible, but impractical method for identifying chronic Salmonella Typhi carriers. Background levels of anti-Vi antibody titers in this endemic area may be high despite a low prevalence of chronic carriers.

Characterization of Amoeba proteus myosin VI immunoanalog

Amoeba proteus, the highly motile free-living unicellular organism, has been widely used as a model to study cell motility. However, molecular mechanisms underlying its unique locomotion and intracellular actin-based-only trafficking remain poorly understood. A search for myosin motors responsible for vesicular transport in these giant cells resulted in detection of 130-kDa protein interacting with several polyclonal antibodies against different tail regions of human and chicken myosin VI. This protein was binding to actin in the ATP-dependent manner, and immunoprecipitated with anti-myosin VI antibodies. In order to characterize its possible functions in vivo, its cellular distribution and colocalization with actin filaments and dynamin II during migration and pinocytosis were examined. In migrating amoebae, myosin VI immunoanalog localized to vesicular structures, particularly within the perinuclear and sub-plasma membrane areas, and colocalized with dynamin II immunoanalog and actin filaments. The colocalization was even more evident in pinocytotic cells as proteins concentrated within pinocytotic pseudopodia. Moreover, dynamin II and myosin VI immunoanalogs cosedimented with actin filaments, and were found on the same isolated vesicles. Blocking endogenous myosin VI immunoanalog with anti-myosin VI antibodies inhibited the rate of pseudopodia protrusion (about 19% decrease) and uroidal retraction (about 28% decrease) but did not affect cell morphology and the manner of cell migration. Treatment with anti-human dynamin II antibodies led to changes in directionality of amebae migration and affected the rate of only uroidal translocation (about 30% inhibition). These results indicate that myosin VI immunoanalog is expressed in protist Amoeba proteus and may be involved in vesicle translocation and cell locomotion.

Anti–Glycoprotein VI Treatment Severely Compromises Hemostasis in Mice With Reduced α 2 β 1 Levels or Concomitant Aspirin Therapy

Platelet inhibition is a major strategy to prevent arterial thrombosis, but it is frequently associated with increased bleeding because of impaired primary hemostasis. The activating platelet collagen receptor, glycoprotein VI (GP VI), may serve as a powerful antithrombotic target because its inhibition or absence results in profound protection against arterial thrombosis but no major bleeding in mice. Mice lacking (-/-) or expressing half-levels (+/-) of the other major platelet collagen receptor, integrin alpha2beta1, were injected with the anti-GP VI antibody JAQ1 and analyzed on day 5. Anti-GP VI treatment resulted in a marked hemostatic defect in alpha2-/- or alpha2+/- mice, as shown by dramatically prolonged tail bleeding times. Platelet adhesion to collagen was studied in an ex vivo whole-blood perfusion system under high shear conditions. Weak integrin activation by thromboxane A2 (TxA2) receptor stimulation restored defective adhesion of anti-GP VI-treated wild-type but not alpha2-/- or alpha2+/- platelets to collagen. This process required the simultaneous activation of the G(q) and G13 signaling pathways, as demonstrated by use of the respective knockout strains. Conversely, inhibition of TxA2 production by aspirin severely compromised hemostasis in anti-GP VI-treated or GP VI/Fc receptor gamma-chain-deficient but not control mice. Anti-GP VI therapy may result in defective hemostasis in patients with reduced alpha2beta1 levels or concomitant aspirin therapy. These observations may have important implications for a potential use of anti-GP VI-based therapeutics in the prevention of cardiovascular disease.

A randomized, open-label study of the immunogenicity and reactogenicity of three lots of a combined typhoid fever/hepatitis A vaccine in healthy adults

Background: Travelers are often advised to receive both the typhoid fever and hepatitis A virus (HAV) vaccines, particularly when going to areas where the 2 diseases are endemic. Thus, combined administration of these vaccines could make immunization more acceptable by reducing the number of injections needed.Objective: This study compared the safety profiles and immunogenicity of 3 batches of a combined typhoid fever/HAV vaccine administered using a dual-chamber bypass syringe.Methods: This randomized, open-label study was conducted at 2 university-based travel clinics in Germany and Austria. Subjects received a single IM injection from 1 of 3 batches of the combined vaccine. Blood samples were drawn immediately before and 28 days after vaccination to evaluate the response to the 2 antigens by assessing geometric mean titers (GMTs) and rates of seroconversion and seroprotection. Subjects recorded all adverse events (AEs) occurring during the study period in a diary.Results: Six hundred ten healthy adults were enrolled in the study. Twenty-eight days after vaccination, 90.6% of the study population had protective typhoid Vi antibody titers (≥1 μg/mL) and 100% had protective HAV antibody titers (≥20 mIU/mL). Seroconversion rates and GMTs were not significantly different between the 3 batches. There were no differences with regard to local or systemic AEs between the 3 batches of vaccine. There were no immediate adverse reactions (within 30 minutes of vaccination) and no serious AEs related to vaccination. Of 609 evaluable subjects (1 was lost to follow-up after the first visit), 555 (91.1%) experienced ≥1 local reaction within the first 7 days after vaccination, mainly pain at the injection site (550 [90.3%]), but only 26 (4.3%) described this pain as severe. Vaccine-related headache and mild to moderate asthenia were each reported by 54 subjects (8.9%). Symptoms resolved spontaneously in all cases.Conclusions: The 3 batches of the combined typhoid fever/HAV vaccine administered by dual-chamber bypass syringe were equally well tolerated and effective in healthy adults, and did not differ significantly in terms of GMTs or seroconversion rates.

Beta-adrenergic and antiadrenergic modulation of cardiac adenylyl cyclase is influenced by phosphorylation.

Adenosine protects the myocardium of the heart by exerting an antiadrenergic action via the adenosine A1 receptor (A1R). Because beta 1-adrenergic receptor (beta 1R) stimulation elicits myocardial protein phosphorylation, the present study investigated whether protein kinase A (PKA) catalyzed rat heart ventricular membrane phosphorylation affects the beta 1R adrenergic and A1R adenosinergic actions on adenylyl cyclase activity. Membranes were either phosphorylated with PKA in the absence/presence of a protein kinase inhibitor (PKI) or dephosphorylated with alkaline phosphatase (AP) and assayed for adenylyl cyclase activity (AC) in the presence of the beta 1R agonist isoproterenol (ISO) and/or the A1R agonist 2-chloro-N6-cyclopentyladenosine (CCPA). 32P incorporation into the protein substrates of 140-120, 43, and 29 kDa with PKA increased both the ISO-elicited activation of AC by 51-54% and the A1R-mediated reduction of the ISO-induced increase in AC by 29-50%, thereby yielding a total antiadrenergic effect of approximately 78%. These effects of PKA were prevented by PKI. AP reduced the ISO-induced increase in AC and eliminated the antiadrenergic effect of CCPA. Immunoprecipitation of the solubilized membrane adenylyl cyclase with the use of a polyclonal adenylyl cyclase VI antibody indicated that the enzyme is phosphorylated by PKA. These results indicate that the cardioprotective effect of adenosine afforded by its antiadrenergic action is facilitated by cardiac membrane phosphorylation.

Remodeling of the extracellular matrix through overexpression of collagen VI contributes to cisplatin resistance in ovarian cancer cells

The mechanisms of drug resistance in cancer are poorly understood. Serial analysis of gene expression (SAGE) profiling of cisplatin-resistant and sensitive cells revealed many differentially expressed genes. Remarkably, many ECM genes were elevated in cisplatin-resistant cells. COL6A3 was one of the most highly upregulated genes, and cultivation of cisplatin-sensitive cells in the presence of collagen VI protein promoted resistance in vitro. Staining of ovarian tumors with collagen VI antibodies confirmed collagen VI expression in vivo and suggested reorganization of the extracellular matrix in the vicinity of the tumor. Furthermore, the presence of collagen VI correlated with tumor grade, an ovarian cancer prognostic factor. These results suggest that tumor cells may directly remodel their microenvironment to increase their survival in the presence of chemotherapeutic drugs.

Immunogenicity of a new Salmonella Typhi Vi polysaccharide vaccine—vax-TyVi ®—in Cuban school children and teenagers

A randomized, controlled, double blind study was carried out in Cuban children and teenagers aged 9–13 years to evaluate the immunogenicity of vax-TyVi ®— Salmonella Typhi Vi polysaccharide vaccine—with respect control vaccines. Serum samples were taken before and 21 days after the immunization, and ELISA was used for the determination of antibodies to Vi polysaccharide. Subjects who received vax-TyVi ® and TYPHIM Vi™ (Pasteur-Mérieux) showed seroconversion rates of 85.61 and 78.36%, respectively. The geometric mean titer (GMT) values for Vi antibodies induced after vaccination were 6.27 μg/ml (5.40–7.38 μg/ml) and 5.97 μg/ml (5.01–7.10 μg/ml), respectively. In contrast, subjects receiving the tetanus toxoid vaccine showed 0% seroconversion.

Comparative immunohistochemical studies of endometriosis lesions and endometriotic cysts

Objective: To compare immunohistochemical staining patterns in noncystic and cystic endometriosis lesions. Design: Experimental. Setting: Archived pathology material in an academic research environment. Patient(s): Endometriosis tissues from the pathology archives including slide tissue sections and blocks. Intervention(s): None; this was a retrospective study. Main Outcome Measure(s): Immunohistochemical staining of the tissues was performed using anti-bcl-2, anti-p53, anti-matrix metalloproteinase IX, and anti-collagen VI antibodies. Staining was qualitatively assessed in terms of extent and intensity. Result(s): p53 showed no staining in both groups. Anti–bcl-2 stained 100% (30/30) of endometriosis lesions compared with only 23% (7/30) of endometriotic cysts ( P<.0001), and anti–matrix metalloproteinase IX stained 85% (23/27) of endometriosis lesions and only 39% (14/36) of endometriotic cysts ( P=.0003). Anti–collagen VI, however, stained only 6% (2/35) of endometriosis lesions and 75% (21/28) of endometriotic cysts ( P<.0001). Conclusion(s): Compared with endometriosis lesions, endometriotic cysts display different expression of proteins with relative overexpression of collagen VI and underexpression of bcl-2 and metalloproteinase IX. This report is the first comparative immunohistochemical study showing these differences.

Involvement of Glycoprotein VI in Platelet Thrombus Formation on Both Collagen and von Willebrand Factor Surfaces Under Flow Conditions

We studied the role of glycoprotein (GP) VI in platelet adhesion and thrombus formation on the immobilized collagen and von Willebrand factor (vWF) surface under flow conditions. Whole blood obtained from 2 patients with GP VI-deficient platelets and the effects of the Fab of anti-GP VI antibody (Fab/anti-GP VI) were tested. Blood containing platelets rendered fluorescent by mepacrine was perfused on immobilized type I collagen or vWF under controlled wall shear rate. Platelet adhesion and thrombus formation were detected by epifluorescent videomicroscopy. The percentage of surface coverage by the platelets was calculated. Fc receptor gamma-chain and spleen tyrosine kinase (Syk) were immunoprecipitated from the lysate of platelets stimulated by vWF plus ristocetin and then analyzed by antiphosphotyrosine immunoblotting. No platelet attachment was seen on the surface of collagen even after 9 minutes of perfusion of blood at relatively low (100 s(-1)) or high (1500 s(-1)) wall shear rate, either in the case of blood containing GP VI-deficient platelets or in the presence of Fab/anti-GP VI, whereas significant platelet thrombus formation was noted after control blood perfusion. Such interference with the actions of GP VI also reduced firm platelet adhesion on immobilized vWF. vWF-induced tyrosine phosphorylation of GP VI-associated Fc receptor gamma-chain followed by Syk activation occurred in normal platelets, but little activation of Syk occurred in GP VI-deficient platelets. GP VI plays crucial roles in platelet thrombus formation on the surface of collagen under flow conditions in humans and is also involved in the process of firm platelet adhesion on the surface of vWF.

Field Effectiveness of Vi Polysaccharide Typhoid Vaccine in the People's Republic of China

To the Editor—Field investigations in the People’s Republic of China to determine the protective efficacy of typhoid Vi polysaccharide vaccine (Vi vaccine) were done among students attending a middle school [1]. Vi vaccine was offered to students even during an outbreak of typhoid fever. The efficacy was 73% among those immunized before the outbreak and 71% among vaccine recipients during the outbreak. Many vaccine failures during the vaccine efficacy trials might have been caused by infection with Salmonella serotype Typhi strains that were lacking the Vi antigen. Vi-negative Salmonella Typhi cause a disease indistinguishable from that caused by a Vi-positive strain [2]. On their entry to an intracellular niche, Vi-negative Salmonella Typhi are protected from the Vi antibody [3]. Since Vi vaccines would not elicit any cellular immunity, they would not stop intracellular replication of Salmonella Typhi. Vi-negative Salmonella Typhi selected during a slide agglutination test with Vi typing antiserum have been known for several decades. Salmonella Typhi isolates that lacked the ViaB genetic sequences were responsible for an epidemic of multidrug-resistant typhoid fever in Calcutta, India [4]. The role of Vi-negative Salmonella Typhi infections in the People’s Republic of China should be determined immediately, and bacterial field isolates should be characterized fully in a reference laboratory. A high molarity affects the expression of the Vi antigen, rendering an otherwise Vi-positive isolate into an apparently negative one. Genotyping investigations for ViaB sequences on isolates would indicate the role of Vi-negative Salmonella Typhi in any failures with Vi vaccine, in locally produced batches of vaccine [1] and in the conjugated Vi vaccine bound to nontoxic recombinant Pseudomonas aeruginosa exotoxin A [5]. Establishment of ViaB genotyping facilities in the People’s Republic of China would be cost effective, and characterization of Salmonella Typhi isolates would be an asset to antityphoid strategies. Genotyping would indicate the contribution of Vi-negative Salmonella Typhi in partial failures [1, 5] of Vi vaccines.

Synaptotagmin VI Participates in the Acrosome Reaction of Human Spermatozoa

Acrosomal exocytosis is a calcium-dependent secretion event causing the release of the acrosomal contents and the loss of the membranes surrounding the acrosome. The synaptotagmins are a family of calcium-binding proteins that participate in the exocytosis of synaptic vesicles. The ubiquitous synaptotagmin VI isoform was found in human sperm cells by Western blot analysis. Immunocytochemistry at the optical and electron microscopy levels localized the protein to the outer acrosomal membrane. Calcium-triggered acrosomal exocytosis in permeabilized sperm cells was abrogated by a specific anti-synaptotagmin VI antibody, indicating that the protein is required for the process. Moreover, a recombinant fusion protein between glutathione S-transferase and the two calcium and phospholipid binding domains of synaptotagmin VI completely inhibited calcium-triggered exocytosis. Interestingly, phorbol ester-dependent in vitro phosphorylation of this recombinant protein abolished its inhibitory effect. We previously showed that, in permeabilized spermatozoa, addition of active Rab3A triggers acrosomal exocytosis at very low calcium concentration. Rab3A-promoted exocytosis was inhibited by the cytosolic domain of synaptotagmin VI and by the anti-synaptotagmin VI antibody, indicating that synaptotagmin is also necessary for Rab-mediated acrosomal content release. In conclusion, the results strongly indicate that synaptotagmin VI is a key component of the secretory machinery involved in acrosomal exocytosis.

Constitutive expression of the Vi polysaccharide capsular antigen in attenuated Salmonella enterica serovar typhi oral vaccine strain CVD 909.

Live oral Ty21a and parenteral Vi polysaccharide vaccines provide significant protection against typhoid fever, albeit by distinct immune mechanisms. Vi stimulates serum immunoglobulin G Vi antibodies, whereas Ty21a, which does not express Vi, elicits humoral and cell-mediated immune responses other than Vi antibodies. Protection may be enhanced if serum Vi antibody as well as cell-mediated and humoral responses can be stimulated. Disappointingly, several new attenuated Salmonella enterica serovar Typhi oral vaccines (e.g., CVD 908-htrA and Ty800) that elicit serum O and H antibody and cell-mediated responses following a single dose do not stimulate serum Vi antibody. Vi expression is regulated in response to environmental signals such as osmolarity by controlling the transcription of tviA in the viaB locus. To investigate if Vi antibodies can be stimulated if Vi expression is rendered constitutive, we replaced P(tviA) in serovar Typhi vaccine CVD 908-htrA with the constitutive promoter P(tac), resulting in CVD 909. CVD 909 expresses Vi even under high-osmolarity conditions and is less invasive for Henle 407 cells. In mice immunized with a single intranasal dose, CVD 909 was more immunogenic than CVD 908-htrA in eliciting serum Vi antibodies (geometric mean titer of 160 versus 49, P = 0.0007), whereas O antibody responses were virtually identical (geometric mean titer of 87 versus 80). In mice challenged intraperitoneally with wild-type serovar Typhi 4 weeks after a single intranasal immunization, the mortality of those immunized with CVD 909 (3 of 8) was significantly lower than that of control mice (10 of 10, P = 0.043) or mice given CVD 908-htrA (9 of 10, P = 0.0065).

Novel Myosin VI Isoform is Abundantly Expressed in Retina

Several forms of sensory deficit have been associated with unconventional myosin defects in humans and other animals. Normal hearing in mammals has been shown to require functional myosin VI (Avraham et al., 1995) and myosin VIIA (Gibson et al., 1995; Liu et al., 1997), and the combined blindness and deafness of Usher syndrome type IB has been shown to be produced by specific defects in myosin VIIA (Weil et al., 1997). Here we report the cloning and characterization of two distinct myosin VI isoforms (FMVIA and FMVIB) initially identified in a degenerate PCR screen of retinal cDNA from the striped bass,Morone saxatilis . Open reading frames for FMVIA and FMVIB encode predicted proteins of 1304 and 1270 amino acids respectively, which are 83% identical at the amino acid level. Both fish isoforms are likewise approximately 83–86% identical to mammalian class VI myosins (Hasson and Mooseker, 1994). Northern blot analysis revealed that FMVIA mRNA is broadly expressed and most abundant in kidney, a pattern similar to that previously reported for mammalian myosin VI. FMVIB expression is dramatically more abundant in retina than in any other tissue examined. Antibodies directed against pig myosin VI (Hasson and Mooseker, 1994) detect a doublet at ∼150 kDa in bass retina and RPE. Since both fish VIA and VIB isoforms share high sequence identity with pig myosin VI within the domain used for antibody production, it seems likely that this antibody crossreacts with both FMVIA and FMVIB. Immunocytochemistry with this same affinity-purified rabbit anti-myosin VI antibody shows that myosin VI isoforms are primarily localized in photoreceptors, horizontal cells and Müller cells in both fish and primate retinas. This report is the first demonstration that two myosin VI genes are expressed in the same organism and the same cell type (RPE). The relatively high abundance of FMVIB expression in retina suggests that it may play an important role in retinal motility events.

Safety and Immunogenicity of Vi Conjugate Vaccines for Typhoid Fever in Adults, Teenagers, and 2- to 4-Year-Old Children in Vietnam

The capsular polysaccharide of Salmonella typhi, Vi, is an essential virulence factor and a protective vaccine for people older than 5 years. The safety and immunogenicity of two investigational Vi conjugate vaccines were evaluated in adults, 5- to 14-year-old children, and 2- to 4-year-old children in Vietnam. The conjugates were prepared with Pseudomonas aeruginosa recombinant exoprotein A (rEPA) as the carrier, using either N-succinimidyl-3-(2-pyridyldithio)-propionate (SPDP; Vi-rEPA(1)) or adipic acid dihydrazide (ADH; Vi-rEPA(2)) as linkers. None of the recipients experienced a temperature of >38.5 degrees C or significant local reactions. One injection of Vi-rEPA(2) into adults elicited a geometric mean (GM) increase in anti-Vi immunoglobulin G (IgG) from 9.62 enzyme-linked immunosorbent assay units/ml (EU) to 465 EU at 6 weeks; this level fell to 119 EU after 26 weeks. In the 5- to 14-year-old children, anti-Vi IgG levels at 6 weeks elicited by Vi-rEPA(2), Vi-rEPA(1), and Vi were 169, 22.8, and 18.9 EU, respectively (P = 0.0001 for Vi-rEPA(1) and Vi with respect to Vi-rEPA(2)). At 26 weeks, the anti-Vi IgG levels for recipients of Vi-rEPA(2), Vi-rEPA(1), and Vi were 30.0, 10.8, and 13.4 EU, respectively (P < 0.001 for Vi-rEPA(1) and Vi with respect to Vi-rEPA(2)); all were higher than the preinjection levels (P = 0. 0001). Vi-rEPA(2) also elicited the highest anti-Vi IgM and IgA levels of the three vaccines. In the 2- to 4-year-old children at 6 weeks following the first injection, Vi-rEPA(2) elicited an anti-Vi IgG level of 69.9 EU compared to 28.9 EU for Vi-rEPA(1) (P = 0.0001). Reinjection increased Vi antibody levels from 69.9 to 95.4 EU for Vi-rEPA(2) and from 28.9 to 83.0 EU for Vi-rEPA(1). At 26 weeks, anti-Vi IgG levels remained higher than those at preinjection (30.6 versus 0.18 for Vi-rEPA(2) and 12.8 versus 0.33 for Vi-rEPA(1); P = 0.0001 for both). Vi vaccine is recommended for individuals of 5 years of age or older. In the present study, the GM level of anti-Vi IgG elicited by two injections of Vi-rEPA(2) in the 2- to 4-year-old children was higher than that elicited by Vi in the 5- to 14-year-old children (30.6 versus 13.4; P = 0.0001). The safety and immunogenicity of the Vi-rEPA(2) conjugate warrant further investigation.

A Novel Alternatively Spliced Variant of Synaptotagmin VI Lacking a Transmembrane Domain: IMPLICATIONS FOR DISTINCT FUNCTIONS OF THE TWO ISOFORMS

Synaptotagmins are a family of membrane proteins that are characterized by a single transmembrane region and tandem C2 domains and that are likely to regulate constitutive and/or regulated vesicle traffic. We have shown that a subclass of synaptotagmins (III, V, VI, and X) forms homo- and heterodimers through an evolutionarily conserved cysteine motif at their N termini (Fukuda, M., Kanno, E., and Mikoshiba, K. (1999) J. Biol. Chem. 274, 31421-31427). In this study, we identified a novel alternatively spliced variant of synaptotagmin (Syt) VI that lacks the N-terminal 85 amino acids including the transmembrane region (thus designated as Syt VIDeltaTM). Because it lacks the cysteine motif responsible for self-dimerization, Syt VIDeltaTM could not associate with Syt VI even in the presence of Ca(2+). Despite lacking the transmembrane region, Syt VIDeltaTM can associate with the plasma membrane through the C-terminal 29 amino acids. In adult mouse brain, two closely comigrating bands at M(r) approximately 50,000, which closely corresponded to the molecular weight of recombinant Syt VIDeltaTM, were detected by anti-Syt VI antibody. These immunoreactive bands were found in both soluble and membrane fractions of mouse brain, indicating that they are membrane-associated proteins (Syt VIDeltaTM), but not transmembrane proteins (Syt VI). Expression of Syt VI and Syt VIDeltaTM in PC12 or COS-7 cells indicated that the two molecules have a distinct subcellular distribution: Syt VIDeltaTM is present in the cytosol or is associated with the plasma membrane or internal membrane structures, whereas Syt VI is localized to the endoplasmic reticulum and/or Golgi-like perinuclear compartment. These results suggest that Syt VI and Syt VIDeltaTM may play distinct roles in vesicular trafficking.

Activation of the GP IIb-IIIa Complex Induced by Platelet Adhesion to Collagen Is Mediated by Both α2β1 Integrin and GP VI

alpha2beta1 integrin, CD36, and GP VI have all been implicated in platelet-collagen adhesive interactions. We have investigated the role of these glycoproteins on activation of the GP IIb-IIIa complex induced by platelet adhesion to type I fibrillar and monomeric collagen under static conditions. In the presence of Mg2+, platelet adhesion to fibrillar collagen induced activation of the GP IIb-IIIa complex and complete spreading. Anti-alpha2beta1 integrin and anti-GP VI antibodies inhibited the activation of the GP IIb-IIIa complex by about 40 and 50%, respectively, at 60 min although minimal inhibitory effects on adhesion were seen. Platelet spreading was markedly reduced by anti-alpha2beta1 integrin antibody. The combination of anti-alpha2beta1 integrin with anti-GP VI antibody completely inhibited both platelet adhesion and activation of the GP IIb-IIIa complex. Anti-CD36 antibody had no significant effects on platelet adhesion, spreading, and the activation of the GP IIb-IIIa complex at 60 min. Aspirin and the thromboxane A2 receptor antagonist SQ29548 inhibited activation of the GP IIb-IIIa complex about 30% but had minimal inhibitory effect on adhesion. In the absence of Mg2+, there was significant activation of the GP IIb-IIIa complex but minimal spreading was observed. Anti-GP VI antibody completely inhibited adhesion whereas no effect was observed with anti-alpha2beta1 integrin antibody. Anti-CD36 antibody partially inhibited both adhesion and the activation of the GP IIb-IIIa complex. Platelet adhesion to monomeric collagen, which requires Mg2+ and is exclusively mediated by alpha2beta1 integrin, resulted in partial activation of the GPIIb-IIIa complex and spreading. No significant effects were observed by anti-CD36 and anti-GP VI antibodies. These results suggest that both alpha2beta1 integrin and GP VI are involved in inside-out signaling leading to activation of the GP IIb-IIIa complex after platelet adhesion to collagen and generation of thromboxane A2 may further enhance expression of activated GP IIb-IIIa complexes.

Collagen-platelet interaction: Gly-Pro-Hyp is uniquely specific for platelet Gp VI and mediates platelet activation by collagen.

Peptides consisting of a repeat Gly-Pro-Hyp sequence are potent platelet agonists. The aim of this study was: (1) to examine the specificity of this sequence for platelet activation; (2) to confirm its recognition by platelet glycoprotein VI; and (3) to assess with suitable peptides the relative importance of glycoprotein VI and integrin alpha 2 beta 1 in platelet activation by collagen. Peptides were synthesized by standard Fmoc chemistry and tested for their ability to support adhesion of human platelets and HT 1080 cells, induce platelet aggregation, bind integrin alpha 2 subunit A-domain and to cause tyrosine phosphorylation of platelet proteins. (1) Peptides consisting of a repeat Gly-Pro-Pro, Gly-Pro-Ala or Gly-Pro-Arg sequence exhibited little if any platelet-reactivity. (2) The platelet-reactive peptide consisting of a repeating Gly-Pro-Hyp sequence failed to induce tyrosine phosphorylation in glycoprotein VI-deficient platelets. Platelet adhesion to this peptide was inhibited by intact anti-glycoprotein VI antibody and its Fab fragment. The latter inhibited aggregation by the peptide and fibres of both collagens I and III. (3) A peptide containing a 15-mer alpha 2 beta 1-binding sequence in a repeat Gly-Pro-Pro structure supported alpha 2 beta 1-mediated platelet and HT 1080 cell adhesion and bound alpha 2 A-domain, but failed to activate platelets or to induce tyrosine phosphorylation. Conversely, a peptide containing this sequence but with an essential Glu replaced by Ala and inserted in a repeat Gly-Pro-Hyp structure did not recognize alpha 2 beta 1, but was highly platelet activatory. Platelet activation by collagen involves the highly-specific recognition of the Gly-Pro-Hyp sequence by platelet glycoprotein VI. Recognition of alpha 2 beta 1 is insufficient to cause activation. Interaction between collagen and glycoprotein VI is unique since Gly-Pro-Hyp is common in collagens but occurs rarely in other proteins, and glycoprotein VI may be expressed solely by platelets. This sequence could provide a basis for a highly-specific anti-thrombotic reagent to control thrombosis associated with plaque rupture.

Persistence of antibodies to the Salmonella typhi Vi capsular polysaccharide vaccine in South African school children ten years after immunization

Between 10 and 11 years after children were vaccinated with Vi capsular polysaccharide of Salmonella typhi or meningococcal A+C control vaccine in a double blind randomized trial, we traced 83 subjects, aged 16–20 years. A blood sample was taken for determination of Vi antibody titres in both groups by radioimmunoassay. T O and T H titres were also done to assess if the participants had had recent exposure to typhoid fever. Fifty-eight percent of subjects in both groups had protective levels of Vi antibody against Salmonella typhi (a titre greater than 1 μg ml −1). There was no significant difference in the levels of Vi antibodies in the cases versus the controls ( p=0.5). Two of the children who had received meningococcal A+C vaccine had recently had typhoid fever. Our data show that adolescents in typhoid endemic areas have high levels of Vi antibodies regardless of previous vaccination status, suggesting that Vi antibodies are acquired in adolescence by a large percentage of the population in this area. Moreover, Vi vaccination has led to ongoing antibody production in greater than 50% of Vi vaccinated children in an endemic area for a period of 10 years. Ongoing antigenic exposure may have contributed to these antibody levels.

Sacroiliitis Causing Salmonella Typhi

Letters to the EditorSacroiliitis Causing Salmonella Typhi Subhash C. AryaMBBS, DCP, DipBact, PhD Subhash C. Arya Centre for Logistical Research and Innovation, M-122 (of Part 2), Greater Kailash-II, New Delhi 110048 India Search for more papers by this author Published Online:1 Jul 1998https://doi.org/10.5144/0256-4947.1998.370SectionsPDF ToolsAdd to favoritesDownload citationTrack citations ShareShare onFacebookTwitterLinked InRedditEmail AboutIntroductionSC Arya, Sacroiliitis Causing Salmonella Typhi. 1998; 18(4): 370-370To the Editor: The clinical and laboratory features of two patients with sacroiliitis caused by Salmonella typhi have been comprehensively described in a recent issue of the Annals.1 While one of the isolates was sensitive to ampicillin, chloramphenicol and co-trimexazole, the other was totally resistant to them. Both strains of the S. typhi responsible for two of the five known cases of sacroiliitis should be characterized for chromosomal electrophoresis pattern, plasmids and Vi phage patterns. Chromosomal DNA from 52 isolates of S. typhi was digested with three restriction endonucleases and subjected to a pulse-field gel electrophoresis (PFGE). The isolates from Papua, New Guinea, were reported to be sensitive to 16 antibiotics, and were devoid of plasmids.2 The PFGE pattern combination of X1S1A1 was manifest in all isolates from patients with fatal typhoid fever, while patients with non-fatal infections exhibited different combinations. PFG electrophoresis performed on two strains from Qatar for their pattern combinations might establish both qualitative and quantitative polymorphism characteristics that might be linked to the potential of S. typhi isolates to cause sacroiliitis.1 Knowledge of such PFGE characteristics might be predictive of aberrant manifestations of S. typhi attributable to their multiplication at rare and unusual sites in the body. It would be desirable to examine these isolates for the presence of plasmids, especially the high-molecular-weight ones commonly associated with antibiotic resistance of S. typhi. They could complicate the interpretation of PFGE patterns through their DNA fragments.3 The two strains differ in their antibiotic sensitivity pattern.1 They might differ in their Vi polysaccharide quantum and phage-typing profile. Many strains of S. typhi have an antigen and cannot be typed by Vi phages. Such strains have been widely known since the 1960s.4 Between 1990 and 1993, of the 1500 strains of S. typhi isolated in South Africa, 125 were Vi-negative and could not be typed by Vi phages.5 In Qatar, where approximately 40 new cases of typhoid are diagnosed annually, a surveillance of Vi-negative S. typhi strains would be desirable to ascertain the possibility of success of Vi-polysaccharide vaccine for S. typhi infection. In all probability, Vi antibodies induced by Vi-polysaccharide vaccine would not be protective against episodes of typhoid fever caused by Vi-polysaccharide S. typhi.ARTICLE REFERENCES:1. Alsoub H. "Sacroiliitis due to Salmonella typhi: a report of two cases" . Ann Saudi Med. 1997; 17: 331–3. Google Scholar2. Thong KL, Passey M, Clegg A, Combs BG, Yassin RM, Pang T. "Molecular analysis of isolates of Salmonella typhi obtained from patients with fatal and nonfatal typhoid fever" . J Clin Microbiol. 1996; 34: 1029–33. Google Scholar3. Pang T. "Molecular analysis of isolates of Salmonella typhoid from patients with fatal or nonfatal or aberrant clinical presentations" . J Clin Microbiol. 1996; 34: 2340. Google Scholar4. International Committee for Enteric Phage Typing. "The geographical distribution of Salmonella typhi and Salmonella paratyphi A and B phages during the period 1 January 1966 to 31 December 1969" . J Hyg Camb. 1973; 71: 59–84. Google Scholar5. "The geographical distribution of Salmonella typhi and Salmonella paratyphi A and B phages during the period 1 January 1990 to 31 December 1994" . 7th International Congress of Applied Bacteriology and Applied Microbiology. Prague, Czechoslovakia, 3-8 July1994. IFEPT Report 1994: 4. Google Scholar Previous article Next article FiguresReferencesRelatedDetails Volume 18, Issue 4July 1998 Metrics History Published online1 July 1998 InformationCopyright © 1998, Annals of Saudi MedicinePDF download

Immunohistochemical investigation on type III and VI collagen organization in human intervertebral discs in the neonatal period.

Nine L5-S1 discs from cadavers following death at ages ranging from 28 weeks of gestation to 1 month after birth were analysed immunohistochemically to investigate type III and VI collagen organization in neonatal discs. The annulus fibrosus stained poorly with the anti-type III antibody. The transition zone and the nucleus pulposus showed a fibrillary framework. No clear pericellular staining was detected. Poor staining with the anti-type VI antibody was detected in the annulus. Single pericellular rings were observed in the transition zone and in the nucleus. The pericellular rings were never multilayered. Type III and VI collagen organization in the newborn is similar to that observed in adults and in the aged. Multiple pericellular rings are never observed. Further research on diseased and healthy intervertebral discs may help clarify which disc changes are age-related and which follow on from degeneration.