Modulation of thrombin-induced platelet aggregation by inhibition of calpain by a synthetic peptide derived from the thiol-protease inhibitory sequence of kininogens and S-(3-nitro-2-pyridinesulfenyl)-cysteine

Thrombin-induced platelet aggregation has been suggested to play an important role in reocclusion following thrombolytic therapy of angioplasty for treatment of myocardial infarction. We previously demonstrated that aggregation of washed platelets by thrombin is accompanied by cleavage of aggregin, a putative ADP receptor, and that these events are indirectly mediated by calpain, expressed on the surface of the external membrane. High-molecular-mass kininogen (HK) contains, in its heavy chain, domain 2, which is responsible for its action as a potent inhibitor of platelet calpain. Domain 3 of the heavy chain of HK directly inhibits binding of thrombin to platelets, confounding mechanistic studies using the entire molecule. Moreover, HK, a protease of 120 kDa, is unsuitable as a potential pharmacological agent. The highly conserved sequence Gln-Val-Val-Ala-Gly, present in HK and its evolutionary precursors, the cystatins, is thought to be involved in the binding of cysteine proteases but is, itself, not inhibitory. An affinity analog, Phe-Gln-Val-Val-Cys(Npys)-Gly-NH2(Npys, 3-nitro-2-sulfenylpyridine), P1, corresponding to the thiol-protease-binding sequence in HK and containing a ligand, Npys, that can react with the free sulfhydryl group in the active site of calpain, was synthesized. P1 was an irreversible inhibitor of platelet calpain. P1 selectively inhibited thrombin-induced aggregation of washed platelets and platelets in plasma, but did not inhibit the aggregatory effects of other platelet agonists. P1 did not inhibit the amidolytic activity and coagulant activity of thrombin. Unlike HK, P1 did not inhibit binding of thrombin to washed platelets. P1 did not inhibit thrombin-induced platelet-shape change. P1 neither raised intracellular levels of cAMP nor did it interfere with the ability of thrombin to antagonize the rise in intracellular levels of cAMP induced by iloprost, an analog of prostaglandin I2. The design and synthesis of P1 could leave to the development of a new class of inhibitors that selectively block thrombin-induced platelet aggregation while sparing other functions of this pathophysiological protease and without inhibiting the action of other platelet agonists.     

Inhibition of tumor cell invasion through matrigel by a peptide derived from the domain II region in urinary trypsin inhibition

Urinary trypsin inhibitor (UTI) has a multipotent inhibitory effect on proteases such as trypsin, chymotrypsin, plasmin, human leukocyte elastase, or hyaluronidase. UTI can bind easily to its receptors on various types of tumor cells (human ovarian cancer HOC-I cells, human choriocarcinoma SMT-cc1 cells, and murine Lewis lung carcinoma 3LL cells). Our results show that the UTI receptors of some tumor cells have a possible role in modulating plasmin activity on the cell surface and prevention of tumor cell invasion and metastasis (H. Kobayashi et al., J. Biol. Chem., 269; 20642-20647, 1994). UTI interacts with tumor cells as a negative modulator of the invasive cells. We investigated whether this effect may be mediated by UTI binding to the cell surface receptors. In addition, the role of peptide sequences from each UTI domain and their interaction with tumor cells were investigated. UTI derivatized with biotin or FITC was taken up by tumor cells in a dose-dependent manner. This cell association was inhibited with a monoclonal antibody D1, which specifically recognizes NH2 terminus (domain I) of UTI. The binding was inhibited by fluid phase UTI, but not HI-8, COOH terminus (domain II) of UTI, suggesting that UTI binds to cells through a site in the UTI domain I. Furthermore, we found that UTI, HI-8 and a number of peptides containing Arg-Gly-Pro-Cys-Arg-Ala-Phe-Ile promoted the inhibition of tumor cell invasion. This site corresponds to the plasmin-inhibiting domain within HI-8. The possibility that UTI binding to tumor cells might be involved in the prevention of tumor cell invasion in vitro was excluded since HI-8, lacking domain I, promotes the inhibition of tumor cell invasion with essentially the same affinity as UTI. All these data allow us to conclude that inhibition of tumor cell invasion is mediated by domain II, which possesses anti-plasmin activity.     

Specific inhibition of granzyme B by parainfluenza virus type 3

T-cell-mediated cytotoxicity is an important means of defense against viral pathogens; however, several viruses possess mechanisms to disrupt cytotoxicity, thereby allowing them to avoid immune clearance. These viruses have been shown to inhibit cytotoxicity by interfering with the capacity of T lymphocytes to specifically recognize infected cells. An alternative mechanism for virally induced cytotoxic dysfunction is identified in this report. We show that parainfluenza virus type 3, a negative-stranded RNA virus, can inhibit cytotoxicity by causing a defect in the cytotoxic effector apparatus. This defect is identified as a selective inhibition of granzyme B mRNA.     

Basic peptides in bee venom, III. Synthesis of peptide fragments from the sequence of the mast-cell-degranulating peptide (author's transl)

The synthesis by conventional methods of the following three peptides is described: MCD(8-11) Boc-His(Trt)-Val-Ile-Lys(Z) (III) MCD(5-7) Boc-Cys(SiPr)-Lys(Z)-Arg(Tos) (IV) and MCD(1-4) Boc-Ile-Lys(Z)-Cys(Trt)-Asn(Mbh) (V). These peptides are fragments of the mast cell degranulating peptide from bee venom. The purity of the fragments synthesized was examined by thin-layer chromatography, amino acid and elementary analysis. Including the fragments Boc-Lys(Z)-Ile-Cys(SiPr)-Gly-Lys(Z) (I) and Boc-Pro-His(Trt)-Ile-Cys(Trt)-Arg(Tos) (II), which were described earlier, the synthesis of the mast-cell-degranulating peptide on a polyethylene-asparagine support appears possible.     

Specific T cell recognition of peptides derived from prostate-specific antigen in patients with prostate cancer

Active, population-based surveillance for invasive group A streptococcal (GAS) disease was conducted in laboratories in metropolitan Atlanta from 1 January 1994 through 30 June 1995. Clinical and laboratory records were reviewed and isolates characterized. One hundred and eighty-three cases of invasive GAS disease were identified (annual incidence, 5.2 cases/100,000). The incidence was highest among blacks (9.7/100,000 per year; relative risk (RR), 1.92; confidence interval (CI), 1.69-2.19; P < .0001) and the elderly, particularly nursing home residents (RR, 13.66; CI, 7.07-26.40; P < .0001). The mean age of patients was 41.3 years (range, 0-95 years). Skin and soft-tissue infections were most common. Mortality was 14.4%; risk of death was significantly higher for patients with streptococcal toxic shock syndrome (STSS) (RR, 9.73; CI, 3.34-29; P = .0008) and individuals infected with M-type 1 (RR, 7.40; CI, 1.5-16; P = .0084). Fourteen percent of invasive GAS infections were STSS and 3% were necrotizing fasciitis. Invasive GAS disease was associated with varicella infection in children (RR, 12.19; CI, 5.58-26.62; P < .0001). M (or emm) types included M1 (16%), M12 (12%), and M3 (11%). Continued study of GAS disease is essential to further define risk factors and risk of secondary cases and to develop effective prevention strategies.     

Prostate-specific antigen: characterization of epitopes by synthetic peptide mapping and inhibition studies

To improve our understanding of the prostate-specific antigen (PSA) antigenic regions, we studied the association targets of one anti-PSA polyclonal antibody and 10 anti-PSA monoclonal antibodies (mAbs). We also examined the ability of the mAbs to inhibit PSA enzymatic activity and block the association of PSA with alpha 1-antichymotrypsin (ACT). Linear epitope mapping with a polyclonal antibody indicated the presence of six major antigenic regions in PSA. Examination of the panel of mAbs established that three of them bind to linear epitopes. Five of the mAbs inhibited > 90% of PSA enzymatic activity. However, inhibition of PSA enzymatic activity and hindrance of PSA-ACT association by mAbs cannot be used to predict whether the mAbs bind to free PSA, the PSA-ACT complex, or both. Some of the mAbs may block PSA-ACT association through peripheral occlusion of the binding site, or through induction of conformational changes in PSA.     

A peptide sequence from platelet factor 4 (CT-112) is effective in the treatment of type II collagen induced arthritis in mice

OBJECTIVE: Platelet factor 4 (PF-4) is a critical alpha chemokine in inflammation and injury responses, with multiple effects upon cellular activities. Discrete peptide sequences of the PF-4 molecule have been shown to retain biological activity. Our aim was to examine the influence of the PF-4 derived octapeptide (CT-112; TTSQVRPR) on type II collagen induced arthritis in mice, to determine if this peptide exhibited antiinflammatory properties. METHODS: DBA/1 mice were treated with CT-112 from either the time of immunization with type II collagen or from the initial onset of arthritis. RESULTS: CT-112 both prevented the development of arthritis in mice treated prophylactically and reduced progression of disease in animals treated therapeutically, and was active when delivered by either subcutaneous injection or oral gavage. No marked immunosuppressive effects were observed during CT-112 treatment, with only moderate decrease in antibody levels and mitogen responses. A significant reduction of the circulating levels of IL-1 was a consistent finding in mice treated therapeutically with CT-112. CONCLUSION: These data suggest PF-4 derived octapeptide exerts antiinflammatory effects of experimental arthritis in mice.     

A polymorphism observed in the experimentally successful peptide vaccine sequence derived from Theileria sergenti piroplasm major surface antigen (p33)

A polymorphism in the experimentally successful peptide vaccine sequence (EVVWKEKKEVKDLDA, amino acids 134-148) derived from the 33 kDa piroplasm major surface antigen (p33) of Theileria sergenti was examined. The vaccine sequences obtained by PCR amplification and sequencing of the p33 gene from a total of 15 parasite-infected cattle blood samples collected from 4 prefectures through Hokkaido to Kumamoto revealed the two major sequences (Ikeda and Chitose stock types) either of which was identified in all samples. Since the peptide vaccine develops the parasite species- or stock-specific immunity in the animals, an application of the two major peptide sequences as cocktailed vaccine should be evaluated for a practical use of this strategy to controlling T. sergenti infection in Japan.     

Inhibition of the cAMP-dependent protein kinase by synthetic A-helix peptides

The catalytic subunit of the cAMP-dependent protein kinase from Dictyostelium discoideum, PkaC, displays the same properties as its mammalian counterpart, except for being about twice as large in size. Sequence comparisons indicated the presence of a conserved alpha-helix (A-helix) within the N-terminal region of PkaC which could potentially establish close contacts with the catalytic core [Véron, M., et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 10618-10622]. We show in this report that a synthetic peptide with the A-helix sequence inhibits PKA activity, whereas unrelated peptides display no inhibitory activity. The inhibition seems competitive with respect to the kemptide substrate rather than due to binding to a secondary site. We further show by amino acid replacements that the last lysine of the A-helix sequence is involved in this specific inhibition. A model is proposed for the possible role of the A-helix.     

Cross-clade p17 peptide recognition by antisera to HGP-30, a 30-amino acid synthetic peptide antigen from HIV type 1 p17

HGP-30, a 30-amino acid synthetic peptide analog of HIV-1SF2 p17 (aa 86-115), was used to immunize both mice and humans. Since the amino acid sequence of HGP-30 is relatively conserved among different HIV-1 strains and clades, experiments were carried out to determine if antisera obtained by immunizing animals and humans can recognize HGP-30-related peptide consensus sequences belonging to different clades. Results show that antisera from mice immunized with HGP-30 can recognize clade B and C and to a lesser degree clade A and E consensus sequences of HIV-1, in addition to recognizing HGP-30 sequence. The cross-clade recognition was higher in mouse sera obtained on day 42 than on day 14 or 28. MPL/SE and Novasomes were better adjuvants than alum in inducing antibodies that showed cross-clade recognition and IgG2a and IgG2b antibody isotypes. Similar cross-clade recognition was observed in several sera from humans immunized with an HGP-30/KLH/alum formulation. The human sera from HGP-30-immunized subjects evaluated for cross-clade recognition of HGP-30 peptides were from subjects whose cells showed significant protection from HIV infection on virus challenge in the hu-PBL-SCID mouse model. These studies suggest that HGP-30 may be useful as a candidate vaccine antigen for populations in countries with prevalence of different HIV clades.     

The use of synthetic peptides in the design of a consensus sequence vaccine for Pseudomonas aeruginosa

Based on the five-year population study of red voles Clethrionomys rutilus Pallas in southern West Siberia, we analysed the distribution of two predominating species of parasites (tapeworms Hymenolepis horrida and immature instars of ticks Ixodes persulcatus) in different demographic groups of the host, and seasonal changes of their incidence in the population. We assessed primary humoral immune response of the voles (splenic antibody-forming cells) to antigenic challenge (injection of sheep erythrocytes) in respect to occurrence of these parasites. It was revealed that infection with H. horrida significantly reduced the numbers of antibody-forming cells in immature summer-born voles. In contrast, immune responses in immature and mature voles, which where parasitized by I. persulcatus at the moment of capture, were significantly higher as compared to non-infected hosts. The possible mechanisms of influence of parasites on variability of immune reactions of voles in the population under study are discussed.     

Structural studies of synthetic peptides dissected from the voltage-gated sodium channel

Peptides representing transmembrane regions of the alpha-subunit of the voltage-gated sodium channel were synthesised and their structures analysed, using 1H NMR and CD, in trifluoroethanol and in dodecylphosphocholine micelles. Sequence analysis suggests that the channel has six regions, S1 to S6, predicted to span the membrane in four homologous domains, designated, I, II, III and IV. Presented here are studies of representatives examples of possible single spanning segments (IS2, IS4, IVS4) and a double spanning segment, IS34, composed of segments IS3 and IS4. In addition, we investigated ISlink56, the putative linker region between segments IS5 and IS6. All of the peptides were found to have predominantly alpha-helical structures in both solvent systems. There was some evidence for bending of the longer helices but there was no discernible evidence for well-defined tertiary structure.     

In vitro induction of primary, antigen-specific CTL from human peripheral blood mononuclear cells stimulated with synthetic peptides

A protocol for in vitro induction of primary, antigen-specific CTL from human peripheral blood mononuclear cells (PBMCs) was developed. Antigen presenting cells (APCs) consisted of Staphylococcus aureus Cowan-I (SAC-I) activated PBMCs treated with a citrate-phosphate buffer at pH 3 to release endogenous peptides bound to surface MHC. This treatment resulted in transient expression of empty class I molecules which could be subsequently stabilized with peptide and beta 2-microglobulin (beta 2m). SAC-I activated PBMCs from HLA-A2.1 normal donors loaded with HBV core 18-27 peptide following acid treatment were used to stimulate PBMCs depleted of CD4+ T cells, in the presence of recombinant interleukin-7 (rIL-7). After 12 days, cells were restimulated with autologous, peptide-pulsed, adherent cells and tested for CTL activity 7 days later. In 23 independent experiments from 13 different HLA-A2.1 donors, this protocol resulted in induction of primary CTL more than 90% of the time. As indicated by both the frequency and magnitude of the response against peptide-sensitized target cells, SAC-I activated PBMCs treated with acid were the most efficient stimulator APC. Thirteen per cent of the cultures generated were capable of lysing target cells transfected with the HBV core antigen and, in general, these CTL cultures exhibited high avidity for the HBV core peptide. This protocol is generally applicable to different antigens and class I alleles, and thus, may be utilized to screen large numbers of peptides to identify human CTL epitopes.     

Binding and presentation of peptides derived from melanoma antigens MART-1 and glycoprotein-100 by HLA-A2 subtypes. Implications for peptide-based immunotherapy

Cellular immune responses to melanoma-associated Ags are the focus of ongoing studies aimed at developing immunotherapies for treatment of malignant melanoma. Melanoma predominantly affects Caucasians, a population in whom expression of HLA-A2 is prevalent. Among HLA-A2 subtypes, HLA-A*0201 is widely expressed, and HLA-A*0201-restricted, tumor-reactive CTL responses are well studied. We have observed in a group of melanoma patients an unexpectedly high frequency (approximately 20%) of non-HLA-A*0201 subtypes (*0202, *0204, and *0205), and little is known regarding antimelanoma response profiles in patients expressing such subtypes. We analyzed non-HLA-A*0201 peptide response profiles using HLA-A*0201-restricted epitopes from melanoma Ags MART-1/Melan A and glycoprotein 100. Most of these peptides bound to the majority of subtypes tested with 50% inhibitory concentrations less than 500 nM. Recognition of cells pulsed with different peptides (MART-1(27-35), G9(154), and G9(280) Flu M1(58-66)) and expressing different subtype molecules by HLA-A*0201-restricted CTL was limited to only a subset of non-HLA-A*0201 molecules, and the peptide/subtype complexes recognized varied among the effector populations tested. CTL responses elicited from PBL of patients and healthy donors expressing subtypes HLA-A*0202 and HLA-A*0205 suggested significant differences among HLA-A2 subtype function in the context of melanoma Ag presentation. These observations imply the necessity of subtyping patients considered for peptide-based protocols and highlight the need for further study of melanoma-directed cellular responses among patients expressing non-HLA-A*0201 subtypes.     

Inhibition of type I and type II phospholipase A2 by phosphatidyl-ethanolamine linked to polymeric carriers

We have previously shown that cell surface proteoglycans protect the cell membrane from the action of extracellular phospholipase A2 (PLA2) enzymes [Dan, P., Nitzan, D. W., Dagan, A., Ginsburg, I., and Yedgar, S. (1996) FEBS Lett. 383, 75-78]. Cell-impermeable PLA2 inhibitors (ExPLIs) were prepared by linking phosphatidylethanolamine (PE) to polymeric carriers, specifically, carboxymethylcellulose, heparin, or hyaluronic acid. The structure of these inhibitors enables the incorporation of their PE moiety into the membrane while the polymer remains at the membrane surface. In the present study, we show that the ExPLIs are effective inhibitors of the hydrolysis of different phospholipids in biological (Escherichia coli) and model (phospholipid vesicle) membranes, by diverse types of PLA2 enzymes, specifically human recombinant synovial fluid and C. atrox (type II), as well as Naja mocambique and porcine pancreatic (type I) PLA2. It is proposed that the external polymers of the ExPLIs, which are anchored to the membrane by the PE, mimic the naturally occurring cell surface proteoglycans and similarly protect membranes from the action of exogenous PLA2.     

Modeling inhibition of type II units in the dorsal cochlear nucleus

Type II units in the dorsal cochlear nucleus (DCN) are characterized by vigorous but nonmonotonic responses to best frequency tones as a function of sound pressure level, and relatively weak responses to noise. A model of DCN neural circuitry was used to explore two hypothetical mechanisms by which neurons may be endowed with type II unit response properties. Both mechanisms assume that type II units receive excitatory input from auditory nerve (AN) fibers and inhibitory input from an unspecified class of cochlear nucleus interneurons that also receive excitatory AN input. The first mechanism, a lateral inhibition (LI) model, supposes that type II units receive inhibitory input from a number of narrowly tuned interneurons whose best frequencies (BFs) flank the BF of the type II unit. Tonal stimuli near BF result in only weak inhibitory input, but broadband stimuli recruit enough lateral inhibitors to greatly weaken the type II unit response. The second mechanism, a wideband inhibition (WBI) model, supposes that type II units receive inhibitory input from interneurons that are broadly tuned so that they respond more vigorously to broadband stimuli than to tones. Physiological and anatomical evidence points to the possible existence of such a class of neurons in the cochlear nucleus. The model extends an earlier computer model of an iso-frequency DCN patch to multiple frequency slices and adds a population of interneurons to provide the inhibition to model type II units (called 12-cells). The results show that both mechanisms accurately simulate responses of type II units to tones and noise. An experimental paradigm for distinguishing the two mechanisms is proposed.