Lytic Assay Research Articles

Hybrid Antimicrobial Enzyme and Silver Nanoparticle Coatings for Medical Instruments

We report a method for the synthesis of antimicrobial coatings on medical instruments that combines the bacteriolytic activity of lysozyme and the biocidal properties of silver nanoparticles. Colloidal suspensions of lysozyme and silver nanoparticles were electrophoretically deposited onto the surface of stainless steel surgical blades and needles. Electrodeposited films firmly adhered to stainless steel surfaces even after extensive washing and retained the hydrolytic properties of lysozyme. The antimicrobial efficacy of coatings was tested by using blades and needles in an in vitro lytic assay designed to mimic the normal application of the instruments. Coated blades and needles were used to make incisions and punctures, respectively, into agarose infused with bacterial cells. Cell lysis was seen at the contact sites, demonstrating that antimicrobial activity is transferred into the media, as well as retained on the surface of the blades and needles. Blade coatings also exhibited antimicrobial activity against a range of bacterial species. In particular, coated blades demonstrated potent bactericidal activity, reducing cell viability by at least 3 log within 1.5 h for Klebsiella pneumoniae, Bacillus anthracis Sterne, and Bacillus subtilis and within 3 h for Staphylococcus aureus and Acinetobacter baylyi. The results confirmed that complex antimicrobial coatings can be created using facile methods for silver nanoparticle synthesis and electrodeposition.

De novo T-lymphocyte responses against baculovirus-derived recombinant influenzavirus hemagglutinin generated by a naive umbilical cord blood model of dendritic cell vaccination

Cancer patients and recipients of hematopoietic stem cell transplantation exhibit a negligible response to influenza vaccine. Toward the goal of addressing this issue, we developed an in vitro model of dendritic cell (DC) immunotherapy utilizing DCs generated from naïve umbilical cord blood (UCB). UCB DCs were loaded with purified rHA protein and used to stimulate autologous T-lymphocytes. Upon recall with HA-loaded autologous DC, a 4–10-fold increase in the number of IFN-γ producing T-lymphocytes was observed in comparison to T-cells stimulated with control DCs. Antigen-specific T-cell functionality was determined by 51Cr lytic assay. Using a peptide library of predicted HA binding epitopes, we mapped an HA-specific, DR15-restricted CD4 T-cell epitope and observed tetramer positive cells. This model demonstrates that HA-specific immune responses might possibly be generated in a de novo fashion and suggests that dendritic cell immunotherapy for the prevention of influenza in populations of immunosuppressed individuals could be feasible.

Role of benzyl alcohol in the prevention of heat-induced aggregation and inactivation of hen egg white lysozyme

The aim of the study was to investigate the stability of a model protein, lysozyme, in the presence of the commonly used preservative benzyl alcohol. Techniques including lytic assay, size exclusion chromatography, circular dichroism, differential scanning calorimetry, native polyacrylamide gel electrophoresis and dynamic light scattering were used to study the overall stability of lysozyme in the presence of benzyl alcohol. The stability of lysozyme against thermal stress was higher in the presence of benzyl alcohol. In the presence of 0.5%, 0.9% and 2% v/v benzyl alcohol, the enzyme showed 33%, 42% and 75% residual activity, respectively, when exposed to 75 degrees C for 2 h, as compared to the 22% activity of control sample. A gradual increase in the size of aggregates was observed for the control sample relative to the samples containing benzyl alcohol, as a result of loss of monomer concentration. The effect was found to be concentration-dependent with 2% benzyl alcohol showing maximum prevention of heat-induced unfolding and aggregation. This effect is remarkable since the thermal transition temperature of the enzyme decreases in the presence of benzyl alcohol. Benzyl alcohol favours the thermal denaturation of lysozyme but stabilizes the lysozyme against the heat-induced aggregation.

The Cytoplasmic Terminus of Kaposi's Sarcoma-Associated Herpesvirus Glycoprotein B Is Not Essential for Virion Egress and Infectivity

Kaposi's sarcoma-associated herpesvirus (KSHV)-encoded glycoprotein B (gB) is an important determinant of viral infectivity and virion egress. A small interfering RNA (siRNA)-based strategy was devised to inhibit KSHV gB gene expression. Transient cotransfection of plasmids constitutively expressing gB and anti-gB siRNAs in 293 cells substantially inhibited gB mRNA levels and protein production. Similarly, transient expression of siRNAs into the primary effusion lymphoma cell line BCBL-1 caused a substantial reduction of gB transcripts and protein synthesis. TaqMan real-time PCR assays against the lytic KSHV gene ORF59 and infectivity assays on 293 cells were employed to assess the effect of inhibiting gB synthesis on virion egress from BCBL-1 cells and infectivity on 293 cells, respectively. These experiments showed that gB was essential for virion egress and infectivity. Transfection of a codon-optimized gB gene with the first 540 nucleotides altered, and therefore not recognized by anti-gB siRNAs that target the native but not the codon-optimized sequence, efficiently rescued virion egress and infectivity in BCBL-1 cells in the presence of siRNAs inhibiting wild-type gB expression. To assess the role of the cytoplasmic domain of gB in virion egress, mutant gB genes were generated specifying carboxyl terminal truncations of 25 and 58 amino acids disrupting two prominent predicted alpha-helical domains associated with virus-induced cell fusion. A third truncation removed the entire predicted cytoplasmic terminus of 84 amino acids, while a fourth truncation removed 110 amino acids, including the terminal most hydrophobic, intramembrane anchoring sequence. Virion egress experiments revealed that all truncated gBs facilitated virion egress from BCBL-1 cells, with the exception of the largest 110-amino-acid truncation, which removed the gB anchoring sequence. Importantly, the gB truncation that removed the entire predicted cytoplasmic domain increased virion egress, suggesting the presence of a egress regulation domain located proximal to the intramembrane sequence within the cytoplasmic domain of gB. All supernatant virions were infectious on 293 cells, indicating that the carboxyl terminus of gB is not essential for either virion egress or virus infectivity.

The decay accelerating factor mutation I197V found in hemolytic uraemic syndrome does not impair complement regulation

Hemolytic uremic syndrome is the clinical triad of thrombocytopenia, microangiopathic hemolytic anaemia and acute renal failure. Cases not associated with a preceding Shiga-like toxin producing Escherichia coli are described as atypical HUS (aHUS). Approximately 50% of patients with aHUS have mutations in one of three complement regulatory proteins, Factor H ( CFH), membrane cofactor protein ( MCP;CD46) or factor I ( IF). A common feature of these three proteins is that they regulate complement by cofactor activity. Decay accelerating factor (DAF; CD55) regulates the complement system by disassociating the alternative and classical pathway convertases. Like CFH and MCP, the gene for DAF lies within the regulators of complement activation (RCA) gene cluster at 1q32. In 1998, we described linkage to this region in families with aHUS which led to the discovery of mutations in CFH and MCP. We therefore genotyped DAF in a panel of 46 aHUS patients including families with linkage to the RCA cluster. A mutation, I197V, was identified in one patient with familial HUS which was not found in 100 healthy controls. Molecular modelling of this mutation shows that the I197V mutation does not reside in an area which would be predicted to be important in decay accelerating activity. The expression of I197V on EBV-transformed B lymphocytes was equivalent to that of wild type controls. There was no significant decrease in decay acceleration activity of the recombinantly produced I197V mutant compared with wild type, as measured by a complement-mediated lytic assay. In conclusion, this study, identifies only one mutation in DAF in 46 patients with aHUS. This mutation, I197V, does not impair complement regulation and cannot be implicated in the pathogenesis of aHUS in this patient. This suggests that the complement regulatory abnormality in aHUS is principally one of deficient cofactor activity rather than of decay acceleration activity.

Evaluation of a rapid microbial detection method via phage lytic amplification assay coupled with Live/Dead fluorochromic stains

To develop a method for rapid detection of bacteria via bacteriophage amplification coupled with exogenous fluorochromic stains. A method for the rapid detection of bacteria was developed which consisted of exposing the sample suspected to contain target cells to host-specific phage. After at least one infection cycle, bacteria known to be infected by the phage (helper cells) were added and the number of nascent phage particles was estimated using the Live/Dead BacLight Bacterial Viability kit. Using Pseudomonas aeruginosa, it was shown that the dead helper cell population following phage infection was proportional to the initial number of target cells present in the original sample. Approximately 1 x 10(1) CFU per ml of P. aeruginosa could be detected within 4 h without the need for enrichment. The phage lytic amplification assay coupled with exogenous fluorochromic stains was able to detect approx. 1 x 10(1) CFU per ml of the target bacterium within 4 h. A method to detect low number of bacterial cells in a sample within 4 h without the need for enrichment was developed.

Simian Immunodeficiency Virus (SIV)/Immunoglobulin G Immune Complexes in SIV-Infected Macaques Block Detection of CD16 but Not Cytolytic Activity of Natural Killer Cells

Natural killer cells are components of the innate immune system that play an important role in eliminating viruses and malignant cells. Using simian immunodeficiency virus (SIV) infection of macaques as a model, flow cytometry revealed a gradual loss of CD16+ NK cell numbers that was associated with disease progression. Of note, the apparent loss of NK cells was detected in whole-blood samples but not in isolated peripheral blood mononuclear cells (PBMC), suggesting that an inhibitor(s) of the antibody used to detect CD16, the low-affinity immunoglobulin G (IgG) receptor, was present in blood but was removed during PBMC isolation. (Actual decreases in CD16+ cell numbers in PBMC generally were not detected until animals became lymphopenic.) The putative decrease in CD16+ cell numbers in whole blood correlated with increasing SIV-specific antibody titers and levels of plasma virion RNA. With the addition of increasing amounts of plasma from progressor, but not nonprogressor, macaques to PBMC from an uninfected animal, the apparent percentage of CD16+ cells and the mean fluorescence intensity of antibodies binding to CD16 declined proportionately. A similar decrease was observed with the addition of monomeric IgG (mIgG) and IgG immune complexes (IgG-ICs) purified from the inhibitory plasma samples; some of the ICs contained SIV p27(gag) antigen and/or virions. Of interest, addition of purified IgG/IgG-ICs to NK cell lytic assays did not inhibit killing of K562 cells. These results indicate that during progressive SIV and, by inference, human immunodeficiency virus disease, CD16+ NK cell numbers can be underestimated, or the cells not detected at all, when one is using a whole-blood fluorescence-activated cell sorter assay and a fluorochrome-labeled antibody that can be blocked by mIgG or IgG-ICs. Although this blocking had no apparent effect on NK cell activity in vitro, the in vivo effects are unknown.

Immunosensitization of Tumor Cells to Dendritic Cell-Activated Immune Responses with the Proteasome Inhibitor Bortezomib (PS-341, Velcade)

Proteasome inhibition results in proapoptotic changes in cancer cells, which may make them more sensitive to immune effector cells. We established a murine model to test whether the proteasome inhibitor bortezomib could sensitize established B16 melanoma tumors to dendritic cell (DC)-activated immune effector cells. Day 3-established s.c. B16 tumors had significantly decreased tumor outgrowth when treated with a combination of bortezomib and DC, regardless of whether the DC were loaded or not with a tumor Ag. In vivo Ab-depletion studies demonstrated that the effector cells were NK and CD8+ cells, but not CD4+ cells. NF-kappaB nuclear transcription factor assay and gene-expression profiling of B16 treated with bortezomib was consistent with inhibition of NF-kappaB target genes leading to a proapoptotic phenotype. In vitro lytic assays demonstrated that TNF-alpha, but not perforin, Fas-ligand, or TRAIL, was responsible for bortezomib-sensitized B16 cytotoxicity. In conclusion, the proteasome inhibitor bortezomib can pharmacologically sensitize tumor cells to the lytic effects of DC-activated immune effector cells.

Infection With Kaposi’s Sarcoma–Associated Herpesvirus Among Families of Patients With Classic Kaposi’s Sarcoma

Classic Kaposi's sarcoma (CKS) primarily affects elderly Mediterranean or Eastern European men. Incidence rates of CKS in Israel are among the world's highest. In practically all cases, antibodies against Kaposi's sarcoma-associated herpesvirus (KSHV) can be detected. A relatively high seroprevalence rate of KSHV in Israel generally correlates with the incidence of CKS. A sexual mode of virus transmission is recognized among homosexual men, whereas the precise transmission routes in the heterosexual population and those with CKS are still unclear. To better assess the transmission routes of KSHV in Israeli patients with CKS and their first-degree relatives as compared with a control group. Serum was collected from all study participants and tested for KSHV antibodies by means of latent and lytic immunofluorescence assays. An open reading frame 65 (ORF65) Western blot assay was applied as a confirmatory tool. Three dermatological departments in Israel. Sixty-four Jewish patients with CKS, 143 of their first-degree relatives, and 186 hospital-based control subjects. Seropositivity to KSHV was detected in 62 (96.9%) of the patients with CKS, in 56 (39.2%) of their first-degree relatives, and in only 21 (11.3%) of the hospital controls (P<.001). The specific relationship with the index patient (spouse, offspring, or sibling) had no significant effect on the prevalence of serpositivity in the family members. Our serologic evidence of familial clustering of KSHV infection suggests a predominantly nonsexual horizontal transmission route of the virus.

Limited Sequence Evolution within Persistently Targeted CD8 Epitopes in Chronic Human Immunodeficiency Virus Type 1 Infection

Studies in acute human immunodeficiency virus type 1 (HIV-1) infection indicate viral evolution under CD8 T-cell immune selection pressure, but the effects of ongoing immune pressure on epitope evolution during chronic infection are not well described. In this study, we performed a detailed longitudinal analysis of viral sequence variation within persistently targeted cytotoxic T-lymphocyte (CTL) epitopes in two HIV-1-infected persons during 6 years of persistent viremia. Responses were quantitated using freshly isolated peripheral blood lymphocytes in direct lytic assays as well as by gamma interferon (IFN-gamma) Elispot assays on cryopreserved cells. Seven targeted epitopes were identified in each person. In the majority of cases, the dominant epitope sequence did not change over time, even in the presence of responses of sufficient magnitude that they were detectable using fresh peripheral blood mononuclear cells in direct lytic assays. Only 4 of the 14 autologous epitopes tested represented potential CTL escape variants; however, in most cases strong responses to these epitopes persisted for the 6 years of study. Although persistent IFN-gamma responses were detected to all epitopes, direct lytic assays demonstrated declining responses to some epitopes despite the persistence of the targeted sequence in vivo. These data indicate limited viral evolution within persistently targeted CD8 T-cell epitopes during the chronic phase of infection and suggest that these regions of the virus are either refractory to sequence change or that persistently activated CD8 T-cell responses in chronic infection exert little functional selection pressure.

Human Herpesvirus 8 (HHV-8) Infection in HIV/AIDS Patients From Santos, Brazil: Seroprevalence and Associated Factors

The goal of this study was to evaluate the seroprevalence of human herpesvirus 8 (HHV-8) infection among HIV-infected individuals from Brazil and the associated risk factors. A cross-sectional survey was carried out with 497 HIV/AIDS outpatients attending the local AIDS Reference Center in Santos (southeastern Brazil) between February 1997 and January 1998 had serum samples screened for anti-HHV-8 antibodies. Patients were considered seropositive whenever reactivity was observed in at least 1 of 3 tests (immunofluorescence assays for latent nuclear and lytic antigens and orf65 recombinant antigen enzyme-linked immunosorbent assay). Overall HHV-8 seroprevalence was 13.9% (95% confidence interval [CI], 10.9-17.6). HHV-8 coinfection was significantly more frequent in men (18.7%; 95% CI, 14.1-23.4) than in women (7.8%; 95% CI, 4.2-11.3) (P < 0.001). According to the mode of HIV acquisition among males, seroprevalence of HHV-8 infection was significantly higher in men who have sex with men when compared with the other groups (32.4% vs. 10.0%, P < 0.001). Multivariate logistic regression revealed HHV-8 infection among men to be independently associated with sexual orientation (adjusted odds ratio [AOR], 5.5 for homosexuals; AOR, 2.8 for bisexuals). No significant risk factor for HHV-8 infection could be demonstrated for HIV-infected women in this cohort, This study provides further evidence that men who have sex with men are at higher risk of HHV-8 infection and shows that the epidemiologic pattern of this infection among HIV/AIDS patients from Santos, Brazil, is similar to that described in other countries with a low incidence of Kaposi's sarcoma.

Factors Associated With Human Herpesvirus Type 8 Infection in an Injecting Drug User Cohort

Human herpesvirus type 8 (HHV-8) has been infrequently studied in injecting drug user (IDU) populations. To estimate the seroprevalence of HHV-8 and risk factors for infection in a cohort of 2,946 IDUs. In this nested cross-sectional study of 390 IDUs, lytic HHV-8 indirect immunofluorescence assay (IFA) was utilized to estimate the HHV-8 seroprevalence. Multivariate logistic regression was used to assess risk factors for infection. The HHV-8 seroprevalence among the IDUs examined in 1988 was 11.5% (95% confidence interval, 8.5-15.13). HHV-8 seroprevalence in this population was associated with being female (OR = 2.2; = 0.080), having a larger body mass index (OR = 3.0; = 0.053), and history of genital warts (OR = 4.0; = 0.023). Injection of any drug more than daily exhibited an inverse effect on HHV-8 seropositivity (OR = 0.5; = 0.085). The seroprevalence of HHV-8 in this population is similar to that seen in the general population, with risk factors being more consistent with sexual behaviors than injection drug use.

A novel cyanobacteriolytic bacterium, Bacillus cereus, isolated from a Eutrophic Lake

Isolation and screening of cyanobacteriolytic bacteria were carried out. Fifteen strains of cyano-bacteriolytic bacteria were isolated by the double layer method using the cyanobacterium, Microcystis, as a sole nutrient. The isolate, N-14, showing the highest cyanobacteriolytic activity was identified as Bacillus cereus based on the 16S rRNA sequence. Components among the extracellular products in the culture supernatant of B. cereus were responsible for the cyanobacteriolytic activity. Lytic assay tests of culture supernatants indicated that the major substances for lytic activity could be non-proteinaceous, and hydrophilic, heat stable, and with a molecular weight of less than 2 kDa. The highest lytic activity was obtained under alkaline conditions, indicating an advantage for the practical application of water bloom control in eutrophic lakes where the pH is usually in the alkaline region. The lytic substance of B. cereus N-14 were compared with enterotoxins and an emetic toxin produced by a pathogenic strain of B. cereus, and also with a known algicide produced by Bacillus brevis, gramicidin. From these results, the lytic substance seemed to be a novel algicide.

A Novel Cyanobacteriolytic Bacterium, Bacillus cereus, Isolated from a Eutrophic Lake.

Isolation and screening of cyanobacteriolytic bacteria were carried out. Fifteen strains of cyano-bacteriolytic bacteria were isolated by the double layer method using the cyanobacterium, Microcystis , as a sole nutrient. The isolate, N-14, showing the highest cyanobacteriolytic activity was identified as Bacillus cereus based on the 16S rRNA sequence. Components among the extracellular products in the culture supernatant of B. cereus were responsible for the cyanobacteriolytic activity. Lytic assay tests of culture supernatants indicated that the major substances for lytic activity could be non-proteinaceous, and hydrophilic, heat stable, and with a molecular weight of less than 2 kDa. The highest lytic activity was obtained under alkaline conditions, indicating an advantage for the practical application of water bloom control in eutrophic lakes where the pH is usually in the alkaline region. The lytic substance of B. cereus N-14 were compared with enterotoxins and an emetic toxin produced by a pathogenic strain of B. cereus , and also with a known algicide produced by Bacillus brevis , gramicidin. From these results, the lytic substance seemed to be a novel algicide.

Detection of serum antibodies to three different recombinant antigens of human herpesvirus 8 by immunoblotting: seroprevalence studies in Taiwan

Background: Human herpesvirus 8 (HHV-8), a γ-herpesvirus, is associated with Kaposi's sarcoma (KS). A variety of assays for the diagnosis HHV-8 infection have been developed. However, the accuracy of current HHV-8 antibody testing is uncertain, mainly in patients with asymptomatic HHV-8 infection. Methods: We utilized a new immunoblot assay, comprised of three recombinant HHV-8 encoded proteins (ORF16, ORF57, and ORF71) to determine the seroprevalence of HHV-8 in Taiwan. ORF16 encodes a Bcl-2 homologue and is expressed only in the lytic cycle. ORF57 is an immediate–early gene product and can activate gene expression by a post-transcriptional mechanism. ORF71 encodes a viral FLICE inhibitory protein and is a latently expressed protein. We collected 6 KS(+)/HIV(−) patients, 10 KS(−)/HIV(+) individuals, 174 blood donors, and 62 non-KS cancer patients in this study. Results: Among the different populations, ORF57 showed highest reactivities. The frequency of positive results for KS(+)/HIV(−), KS(−)/HIV(+), blood donors, and non-KS cancer patients were 83.3%, 40%, 23%, and 12.9%, respectively. Compared to the widely used lytic immunofluorescence assay, our immunoblotting assay using ORF57 showed higher reactivities among the blood donors. Conclusions: ORF57 is useful for the detection of HHV-8 infection, especially during the asyptomatic infections.

Prime-boost immunization generates a high frequency, high-avidity CD8(+) cytotoxic T lymphocyte population.

Development and expansion of high-avidity T cell populations may be important for the success of immunization strategies against HIV and other pathogens that have presented major problems for vaccine development. We have used tetrameric-MHC complexes ex vivo and lytic assays to show that 'prime-boost' immunization with DNA vaccines and recombinant poxvirus vectors generates high frequencies of cytotoxic T lymphocytes (CTL) that recognize target cells expressing very low levels of specific antigen. These cells persist for at least 6 months at levels representing approximately 10% of the CD8(+) T cell population. Using a novel in vivo assay, we also found that prime-boost immunized animals were capable of eliminating target cells expressing 10- to 100-fold less immunogenic peptide than mice given either vector alone. In addition, viral challenge led to rapid expansion of CTL effectors in prime-boost groups, to levels representing >30% of total CD8(+) T cell numbers. Strategies that generate specific T cells of high avidity, optimizing early detection of infected cells, offer new hope for effective prophylaxis and immunotherapy.

Dendritic cells cross-present latency gene products from Epstein-Barr virus-transformed B cells and expand tumor-reactive CD8(+) killer T cells.

Dendritic cells (DCs) are not targets for infection by the transforming Epstein-Barr virus (EBV). To test if the adjuvant role of DCs could be harnessed against EBV latency genes by cross-presentation, DCs were allowed to process either autologous or human histocompatibility leukocyte antigen (HLA)-mismatched, transformed, B lymphocyte cell lines (LCLs) that had been subject to apoptotic or necrotic cell death. After phagocytosis of small numbers of either type of dead LCL, which lacked direct immune-stimulatory capacity, DCs could expand CD8(+) T cells capable of killing LCLs that were HLA matched to the DCs. Necrotic EBV-transformed, major histocompatibility complex (MHC) class I-negative LCLs, when presented by DCs, also could elicit responses to MHC class II-negative, EBV-transformed targets that were MHC class I matched to the DCs, confirming efficient cross-presentation of LCL antigens via MHC class I on the DC. Part of this EBV-specific CD8(+) T cell response, in both lytic and interferon gamma secretion assays, was specific for the EBV nuclear antigen (EBNA)3A and latent membrane protein (LMP)2 latency antigens that are known to be expressed at low levels in transformed cells. The induced CD8(+) T cells recognized targets at low doses, 1-10 nM, of peptide. Therefore, the capacity of DCs to cross-present antigens from dead cells extends to the expansion of high affinity T cells specific for viral latency antigens involved in cell transformation.

Rapid detection of streptokinase resistance using a bedside lytic assay of dry reagent technology

A new bedside lytic assay using dry reagent technology for rapid (3–5 min) detection of streptokinase resistance (SKR) was recently introduced, which measures lysis onset time (LOT) of whole blood clot in response to high and low streptokinase (SK) concentrations: 100 U/ml (SK100) and 10 U/ml (SK10). SKR was defined by pro longation of LOT, previously correlated with the standard SK Reactivity Test and with clinical outcome of acute myocardial infarction (AMI) SK-treated patients, high SKR when SK100 > 50 seconds and SK10 > 120 seconds; partial SKR when SK10 > 120 seconds. Five prospective clinical groups (325 patients) were screened in cardiac units of four university hospitals. In patients previously treated with SK, the prevalence of SKR was 87% (70% high, 17% partial); in those who had documented streptoco ccal infection, 92% (75% high, 17% partial); and in patients with rheumatic heart disease, 76% (all high). SKR prevalence was 55% (33% high, 22% partial) in those with recent respiratory tract infection. In 225 acute coronary patients, SKR was 28% (21% h igh, 7% partial), and was identical by gender, but was 36% (32% high, 4% partial) in patients ≥ 65 years versus 19% (9% high, 10% partial) in those ≤ 65 years (P< 0.0001).In conclusion, we demonstrated (with a rapid functional assay) the consistence of our results with the expected prevalence of SKR in the groups studied, this points out to the feasibility of pre-therapeutic detection of SKR and choice between t-PA and SK made at bedside without delaying the onset of treatment. As SKR is common among candidates for thrombolysis, pre-therapeutic detection of SKR merits further investigation.

Differences in the recognition by CTL of peptides presented by the HLA-B*4402 and the HLA-B*4403 molecules which differ by a single amino acid.

The HLA-B*4402 and B*4403 molecules differ only at residue 156, which borders the peptide binding site. Strong in vivo allogeneic reactions mediated by cytolytic T lymphocytes (CTLs) were reported in patients who received a bone marrow graft mismatched for these B44 subtypes, indicating that HLA-B*4402 and B*4403 molecules present distinct antigens. This could be due either to the presentation of different sets of antigenic peptides or to the recognition by CTLs of conformational epitopes formed by the MHC molecules alone or in association with antigenic peptides. To address this question, we compared the two B44 subtypes in their presentation to tumor-specific CTLs of three peptides, encoded by genes MAGE-3, MUM-1 and Tyrosinase. The peptides bound with similar affinities to B*4402 or B*4403 molecules, as assessed by lytic competition assays. One HLA-B*4402-restricted and one HLA-B*4403-restricted CTL clone were derived against each peptide. When tested for lysis of B*4402 and B*4403 cells incubated with the antigenic peptides, most CTLs showed a marked preference for one of the two B44 subtypes. Using variant peptides incorporating single alanine substitutions, we compared a given CTLs' recognition of its antigenic peptide presented by both B44 subtypes. Some substitutions, which had no effect on the binding of the peptide, affected its recognition by the same CTL differently on B*4402 and B*4403 molecules. These results imply that the conformations adopted by the same peptide on the two HLA-B44 subtypes are different. We conclude that the B44 subtype specificity of T cells results mostly from distinct conformations adopted by the same peptides in the two B44 molecules. This does not exclude the possibility that in some cases the B44 subtype specificity results from the selective binding of a peptide to one subtype. We found several peptides, different from the three mentioned above, that contain the canonical HLA-B44 binding motif and bind to B*4403 but not to B*4402 molecules.

Expression and characterization of a novel plasminogen activator from Agkistrodon halys venom

A venom gland cDNA library of Agkistrodon halys was constructed and screened with a probe based on the consensus sequence of venomic serine proteases. Next, we determined the sequences of the entire open reading frames of two selected positives which were found to encode novel serine proteases of 234 and 233 amino acids in length and named as Haly-PA and Haly 2, respectively. Upon protein data base search, Haly-PA showed the highest similarity of 82% to the previously characterized plasminogen activator, TSV-PA (Zhang et al. 1995, J. Biol. Chem., 270, 10246–10255). Haly 2 displayed a 78% similarity to β-fibrinogenase (Hung et al. 1994, B. B. R. C., 205, 1707–1715). Haly-PA was successfully expressed using the baculovirus system and secreted into the culture media as a 32 kDa glycoprotein. In the western analysis of snake venom, anti-Haly-PA antibody detected the same size of band indicating that this enzyme is a component of snake venom. Recombinant Haly-PA was purified to homogeneity using the combination of anion exchange and gel filtration column. In the fibrino(geno)lytic assay, recombinant Haly-PA displayed an indirect fibrino(geno)lytic activity depending on the presence of plasminogen and cleaved the plasminogen to generate the active plasmin. These results indicate that Haly-PA is a plasminogen activator and displays fibrino(geno)lytic activity through conversion of plasminogen to plasmin.