PS Antigen Research Articles

Purification technology design, biochemical and immunological characteristics of the recombinant chimeric antigen for evaluation of T cell immunity against coronavirus infection

Diagnosis of specific T cell immunity to the antigenic determinants of SARS-CoV-2 in patients seems to be an increasingly important task due to accumulation of data about the role of T cell immune response in course of coronavirus infection and clearance of SARS-CoV-2 in case of secondary infection. Previously, we designed the recombinant CorD_PS antigen for evaluation of T cell antiviral immunity, containing conservative and immunogenic sequences of structural proteins of the SARS-CoV-2 coronavirus. E. coli CorD_PS producing strain with stable expression of the recombinant CorD_PS antigen was obtained. Aim of the present work is to design a laboratory technology for the production of recombinant antigen CorD_PS, to control the quality of the obtained chimeric protein and to study its immunological activity. Development of the cultivation conditions for E. coli CorD_PS cells was carried out in conical flasks in LB-M_Km medium at 37 °C, then scaled in a fermenter with a volume of 30 liters. Expression was induced by the addition of IPTG. Expression was controlled in culture lysates in 12% SDS-PAGE. The resulting biomass was lysed using an ultrasonic disintegrator with followed by centrifugation. The compositions of lysing and solubilizing buffers were selected, as well as conditions for refolding of the recombinant protein. Cation exchange (SP-sepharose), hydrophobic (Butylsepharose) and exclusive (Sephacryl S-200 HR) chromatography were used sequentially to purify the dissolved protein. Protein impurities in the preparation were determined by reverse-phase HPLC and electrophoresis in 12% SDS-PAGE, residual lipopolysaccharides were determined using a gel-thrombin variant of the LAL test, residual proteins of the producer strain were determined by enzyme immunoassay, residual DNA of the producer strain was determined by binding with PicoGreen dye. Specificity was controlled by indirect enzyme immunoassay. The evaluation of cytokine production by CD4+T lymphocytes in response to their stimulation by recombinant antigen ex vivo was performed on a flow cytofluorimeter. The biomass yield during cultivation of E. coli CorD_PS in a 30L fermenter was up to 20 g/L for 4 hours of 0.1 mM IPTG induction. Sequential washing of inclusion bodies from bacterial cellular components and their subsequent solubilization in a buffer containing 8 M urea allowed to obtain a solution of denatured antigen with a concentration of 10 mg/mL. The efficiency of refolding by dilution was 75%. After three stages of chromatographic purification, protein samples with a concentration of 1.2-1.4 mg/mL, HPLC purity of 98.43%, corresponding to key quality parameters according to the OFS.1.7.1.0007.15, were obtained. The recombinant antigen showed specific binding to a sample of the First WHO International Standard and sample Company Reference Standard No. 3 of anti- SARS-CoV-2 human immunoglobulins in a specified concentration range. CD4+T lymphocytes effectively responded to recombinant antigen treatment by increasing IFNγ production. The optimal concentration of recombinant coronavirus antigen was 5 μg/mL. The developed technological process makes it possible to obtain 5-7 grams of coronavirus recombinant CorD_PS antigen in one cultivation cycle in 30 liters of fermentation medium with key quality parameters according to the OFS.1.7.1.0007.15. As a result of specific immunological activity studies of the recombinant coronavirus antigen CorD_PS, the concept of its possible use as a diagnostic tool for determining the formation of a T cell immune response was confirmed.

Designing structure and <i>E. coli</i> strain-producer bearing SARS-CoV-2 N, S, M, E protein-related sequence antigen

T-cell immune response is extremely important in protecting human body from diverse viral infections. It is known that it can ensure viral clearance and complete recovery in patients with humoral immunodeficiency. COVID-19 patients were found to have T-cell response primarily directed against SARS-CoV-2 structural S, M, N, E proteins, with nucleocapsid protein being most conserved. To assess patients immunity against coronavirus infection and evaluate an effectiveness of vaccine candidates, it is necessary to develop an optimal diagnostic antigen to evaluate arising T-cell response against SARS-CoV-2 antigenic determinants. A diagnostic test to determine host specific susceptibility to SARS-CoV-2 infection should target conserved regions of global SARS-CoV-2 variants. The study was aimed to develop a structure of an antigen bearing conserved and immunogenic sequences derived from SARS-CoV-2 structural proteins and to obtain an Escherichia coli producer strain containing a recombinant protein to be subsequently used for assessing antiviral T-cell immunity. Developing of the antigen was performed in silico: TepiTool and NetMHCIIpan were used to predict and identify high affinity epitopes spanning SARS-CoV-2 E, M, N, S proteins and MHC II binding. Several variants of recombinant antigen proteins were constructed, from which one was selected based on its physicochemical properties: isoelectric point, hydrophobicity index and aliphatic index, as well as 3D representation built by using the I-TASSER. The sequence was synthesized and cloned into the pET24a(+) vector. The resulting plasmid pCorD_PS was transformed into E. coli DH5 followed by Rosetta (DE3). The strain-producer of the recombinant E. coli protein CorD_PS was assessed for the presence and stability of IPTG-induced antigen protein expression and elimination of recombinant coronavirus antigen-bearing plasmid. Based on the study data, an antigen was developed consisting of conserved regions from SARS-CoV-2 S, M, N, E proteins. A 53 kDa recombinant protein was predicted to be stable in aqueous solutions with isoelectric point of 9.56 potentially allowing to simplify protein purification from E. coli cells. Plasmid DNA pCorD_PS (6695 bp) encoding final recombinant coronavirus antigen cloned into pET24a(+) vector was obtained. A stable, productive E. coli CorD_PS strain was obtained. The obtained strain-producer resulting in recombinant E. coli CorD_PS antigen is stable allowing to move on to design antigen purification technique and further develop SARS-CoV-2-specific diagnostic test system.

Mucosal chemokine adjuvant supports heterologous protection against Delta and Omicron VOCs in the context of a SARS-CoV-2 wild-type DNA vaccine

Abstract Emerging variants of concern (VOCs) and waning vaccine-induced immunity challenge SARS-CoV-2 control. Directing vaccine-induced humoral and cell-mediated responses to mucosal surfaces could enhance vaccine efficacy and potentially reduce transmission. We immunized mice with DNA plasmids expressing a wild-type SARS-CoV-2 spike protein (pS) alone, or with the DNA-encoded mucosal chemokine adjuvant cutaneous T cell-attracting chemokine (pCTACK). Mice co-immunized with pS and pCTACK exhibited increased spike specific IFNy T cell responses compared to those receiving the pS alone. While both groups showed similar levels of spike receptor binding domain (RBD)-specific IgG in their serum, in the mucosa, co-immunization with pCTACK enhanced RBD-specific IgA, and cecal extracts from pCTACK co-immunized animals neutralized pseudotyped viruses. When ACE2 transgenic mice were immunized, rested for 3 months, and challenged with a heterologous SARS-CoV-2 Delta variant, co-immunization with pCTACK supported 100% survival while immunization with wild-type pS antigen alone supported 60% survival. Enhanced protection from morbidity was also observed as 100% of pCTACK co-immunized animals did not exhibit infection-induced weight loss. When animals were immunized similarly and challenged with Omicron BA.2, pCTACK co-delivery resulted insignificantly decreased viral loads compared to pS-only immunization. These studies demonstrate that mucosal chemokine adjuvants can direct vaccine-induced responses to specific immunological sites with significant impact on heterologous challenge. These data have significant implications for the design of improved vaccines targeting SARS-CoV-2. This work was supported by NIH NCI award T32 CA09171 (ENG) and NIH NIAID award T32-AI-055400 (EMP) and The Wistar Institute coronavirus discovery fund with additional support from CEPI/Inovio Pharmaceuticals.

Antibody enhanced HPLC for serotype-specific quantitation of polysaccharides in pneumococcal conjugate vaccine

Bacterial infection remains as one of the major healthcare issues, despite significant scientific and medical progress in this field. Infection by Streptococcus Pneumoniae (S. Pneumoniae) can cause pneumonia and other serious infectious diseases, such as bacteremia, sinusitis and meningitis. The pneumococcal capsular polysaccharides (CPS) that constitute the outermost layer of the bacterial cell are the main immunogens and protect the pathogen from host defense mechanisms. Over 90 pneumococcal CPS serotypes have been identified, among which more than 30 can cause invasive pneumococcal diseases that could lead to morbidity and mortality. Multivalent pneumococcal vaccines have been developed to prevent diseases caused by S. Pneumoniae. These vaccines employ either purified pneumococcal CPSs or protein conjugates of these CPSs to generate antigen-specific immune responses for patient protection. Serotype-specific quantitation of these polysaccharides (Ps) antigen species are required for vaccine clinical dosage, product release and quality control. Herein, we have developed an antibody-enhanced high-performance liquid chromatography (HPLC) assay for serotype-specific quantitation of the polysaccharide contents in multivalent pneumococcal conjugate vaccines (PCVs). A fluorescence-labeled multiplex assay format has also been developed. This work laid the foundation for a serotype-specific antigen assay format that could play an important role for future vaccine research and development.

Resolving Interference of Direct Oral Anticoagulants (DOACs) on Routine Coagulation, Thrombophilia, and Lupus Anticoagulant Testing Using Activated Carbon

Background: Direct (anti-IIa and anti-Xa) oral anticoagulants (DOACs) have a significant impact on various coagulation test results. As the correct interpretation of these tests is mandatory to prevent misclassification and subsequent clinical consequences, withholding treatment could be necessary, with the associated risk of thrombosis.Aims: To evaluate the performance of the activated carbon DOAC-Remove (5-Diagnostics, Basel, Switzerland) in extracting DOACs from plasma samples and its effect on various routine and esoteric coagulation test results.Patients, Materials and Methods: Left-over plasmas from patients treated or not with DOAC obtained in the routine laboratory workload were evaluated. Briefly, 0.8 mL of plasma sample was incubated with 1 tablet of the activated carbon for 10 min at room temperature using a rotating shaker. After a 2 min-centrifugation at 2000x g and room temperature, the supernatant was pipetted before being analyzed. Tests were performed before and after such incubation. DOACs were measured using either a specific direct thrombin inhibitor or an anti-Xa assay with specific calibrations. Routine coagulation tests (PT, aPTT, and factor(F)V, FVIII:C, FIX, fibrinogen, and D-dimer) were performed, as well as thrombophilia panel [antithrombin, protein C (PC, chromogenic and clotting assays), PS (free PS antigen and clotting assays)], and lupus anticoagulant (LA) panel [silica clotting time (SCT), and dilute Russell venom clotting time (dRVVT)]. All assays were performed using reagents from Werfen (Bedford, MA, USA) on the ACL TOP 700 analyzer. As the distribution of data was not normal, test results were compared using non-parametric tests.Results: We evaluated a total of 756 plasma samples, obtained from patients treated with dabigatran (n=139, median concentration 129 ng/mL [range:18-905]), rivaroxaban (n=157, median concentration 159 ng/mL [range:19-815]), or apixaban (n=155, median concentration=154 ng/mL (range:11-510), and from 305 patients with various disease states and not on DOAC including 35 patients on coumadin and 32 patients on heparin (UFH: n=18 ; LMWH: n=14).In untreated patients, the DOAC-Remove had no significant impact on test results (n>30 for each parameter), except for D-dimer, free PS and FVIII:C. However, the mean biases, evaluated according to Bland-Altman, were below the accepted limits, and changes would not have had any clinical relevance.In the plasma from patients treated on DOAC, the DOAC-Remove eliminated all the three DOACs, with levels far below the detection limit of the techniques after a 10 min-incubation, leading to a dramatic correction of the DOAC-induced prolongations of PT and APTT, as well as of their lowering effect on FV, FVIII and FIX activities. The same applied to the DOAC-induced elevation of PC and PS anticoagulant activities (clotting assays), and antithrombin activity in the plasma from patients on rivaroxaban and apixaban.DRVVT screen/confirm ratios, which were above the normal ranges in 45% of the patients on dabigatran (n=31), and 78% of the patients on rivaroxaban (n=32), were normalized in most samples after a 10 min-incubation with the carbon and remained positive in only 10% of the patients on dabigatran and 12% of the patients on rivaroxaban. The impact of the DOACs on the SCT screen/confirm ratio was less noticeable with baseline positive test results in only one of the samples from patients on dabigatran (n=31), and in 2 patients on rivaroxaban (n=32). Treatment with the carbon leaded to a correction in the positive sample from patient on dabigatran and in one of the two positive samples from patients on rivaroxaban. Both dRVVT and SCT test results were within the normal range in the plasma from 32 patients on apixaban before and after treatment with the activated carbon.As expected, fibrinogen, PC activity evaluated using a chromogenic assay, free PS antigen concentration, VWF:RCo and VWF:Ag were not affected by any of the 3 DOACs, and test results were not significantly different before and after incubation with the activated carbon.Conclusion: The DOAC-Remove had no effect on test results obtained in the plasma from untreated patients and from patients on traditional anticoagulants. It effectively removed all 3 tested DOACs from the plasma of treated patients, allowing an accurate measurement of routine and esoteric coagulation tests in DOAC treated patients without withholding the treatment. DisclosuresNo relevant conflicts of interest to declare.

Evaluation of a Commercial Field Test to Detect African Swine Fever.

African swine fever (ASF) is one of the most important and complex infectious diseases affecting pigs ( Sus scrofa ). The disease has been present in Sardinia, Italy, since 1978. Factors influencing the presence of the disease on the island are the presence of illegally bred pigs, uncontrolled movements of animals, and local traditions. Implementation of public health programs is essential for controlling ASF. The use of new diagnostic techniques on both wild boar (WB) and illegally bred pigs would provide tools for faster and more inexpensive control of the disease. We evaluated a commercial serological test kit (Pen-side [PS]) for use in the field. We sampled 113 hunter-harvested WB during the 2014-15 season, collecting blood and lung samples to conduct serological analyses and to screen for the ASF virus. Although the sensitivity (81.8%) and specificity (95.9%) of tests performed in the field were reduced compared to the same test in laboratory, they nevertheless allowed for rapid diagnosis and reduced unnecessary carcass destruction. The test, conducted in the field, was less expensive than in the laboratory and required less manpower. Therefore, we conclude that the combined use of antibody PS test and antigen PS test may be a valuable emergency management method during an outbreak as well as a useful tool for conducting regular monitoring activities as a preventive policy.

C-reactive protein as an adverse prognostic marker for men with castration-resistant prostate cancer (CRPC): Confirmatory results

We previously reported that higher serum concentrations of C-reactive protein (CRP) are associated with shorter survival in men with castration-resistant prostate cancer (CRPC). To confirm this finding in an independent data set, we used 119 CRPC patients enrolled in 6 phase II clinical trials and examined the relationship of CRP, alkaline phosphatase, hemoglobin, age, ECOG PS, and prostate specific antigen (PSA) with survival. Median follow-up was 19.7 months (0.9–98.5 months), and 89% have died. After analyzing the form of the risk function using the generalized additive model method, univariate and multivariate Cox proportional hazard models were used to assess associations between baseline individual categorical and continuous variables. Quartiles of CRP were: 0–1.0, 1.1–4.9, 5.0–17.0, and 17.1–311 mg/L. In a Cox multivariate model, log 2 (CRP) (HR 1.106, P = 0.013) as well as hemoglobin and alkaline phosphatase were independently associated with survival, confirming that higher CRP is associated with shorter survival in CRPC. Since CRP is a marker of inflammation, this finding suggests that inflammation may play an important role in the natural history of advanced prostate cancer. CRP is a readily measurable biomarker that has the potential to improve prognostic models and should be validated in a prospective clinical trial.

Stability of coagulation assays performed in plasma from citrated whole blood transported at ambient temperature

Many preanalytical variables affect the results of coagulation assays. A possible way to control some of them would be to accept blood specimens shipped in the original collection tube. The aim of our study was to investigate the stability of coagulation assays in citrated whole blood transported at ambient temperature for up to two days after specimen collection. Blood samples from 59 patients who attended our haematology outpatient ward for thrombophilia screening were transported at ambient temperature (outdoor during the day, indoor overnight) for following periods of time: <1 hour, 4-6, 8-12, 24-28 and 48-52 hours prior to centrifugation and plasma-freezing. The following coagulation tests were performed: PT, aPTT, fibrinogen, FII:C, FV:C, FVII:C, FVIII:C, FIX:C, FX:C, FXI:C, VWF:RCo, VWF:Ag, AT, PC activity, total and free PS antigen, modified APC-sensitivity-ratio, thrombin-antithrombin-complex and D-dimer. Clinically significant changes, defined as a percentage change of more than 10% from the initial value, were observed for FV:C, FVIII:C and total PS antigen starting at 24-28 hours, and for PT, aPTT and FVII:C at 48-52 hours. No statistically significant differences were seen for fibrinogen, antithrombin, or thrombin-antithrombin complexes (Friedman repeated measures analysis of variance). The present data suggest that the use of whole blood samples transported at ambient temperature may be an acceptable means of delivering specimens for coagulation analysis. With the exception of factor V and VIII coagulant activity, and total PS antigen all investigated parameters can be measured 24-28 hours after specimen collection without observing clinically relevant changes.

Subclass distribution of natural salivary IgA antibodies against pneumococcal capsular polysaccharide of type 14 and pneumococcal surface adhesin A (PsaA) in children

A number of studies have shown that the ratio of IgA1 and IgA2 subclasses in secretions can depend upon the nature of the antigen inducing their production. In order to evaluate the effect of the nature of the antigen on the subclass distribution of the naturally occurring salivary IgA antibodies against Streptococcus pneumoniae, we used enzyme immunoassay to measure the levels of natural IgA, IgA1 and IgA2 antibodies to pneumococcal capsular polysaccharide type 14 (PS14) and pneumococcal surface adhesin A (PsaA) in saliva of children during their first 2 years of life. The sum of anti-PS14 and anti-PsaA IgA1 and IgA2 correlated significantly with the antigen-specific total IgA, which showed that IgA1 and IgA2 add up to IgA. IgA1 was the predominant subclass for both antigens. The median of anti-PS14 and anti-PsaA IgA1 was higher than that of IgA2, and the antigen-specific IgA1 was found in a larger proportion of samples than IgA2. The ratio of IgA1 to IgA2 (IgA1/IgA2 ratio) was lower for anti-PS14 than for anti-PsaA, suggesting that the PS antigen induced more IgA2 than the protein antigen. The possible impact of the IgA subclass distribution on protection of mucosal surfaces by natural or vaccine-induced antibodies needs to be determined.

Proteína S libre en embarazadas normales: comparación entre dos métodos

Pregnancy is a physiological hypercoagulable state with an increased incidence of thromboembolic phenomena. There is an increase in the concentrations of most clotting factors, a decrease in concentration of some of the natural anticoagulants and reduced fibrinolytic activity. Changes in PS levels have also been reported. To establish referral range values of functional PS and free PS antigen, during the second (2nd T) and third trimester (3rd T) of normal gestation. Forty one normal pregnant women were included in our study, 20 during the 2nd T (22-24 weeks) and 21 during the 3rd T (29-38 weeks). Functional PS was measured by a clot based test and free PS antigen by ELISA. Free PS Antigen was 65.8+/-18.3% during the 2nd T and 62.3+/-16.5% during the 3rd T. The figures for normal controls were 106+/-6.5%. Functional PS was 43.8+/-13.3 and 25.9+/-14.6% during the 2nd T and 3rd T, respectively. The figures for normal controls were 97+/-24% (p <0.001 compared with pregnant women). Free PS antigen did not change from the 2nd to the 3rd T (p=NS), however functional PS fell significantly from the 2nd to the 3rd T (p <0.001) and was significantly lower than free PS antigen in both trimesters (p <0.001). Pregnancy is associated to a decrease in PS. This abnormality is more pronounced for functional PS than free PS antigen and functional PS falls progressively during pregnancy. These assays should not be used to screen for PS deficiency during pregnancy because they could lead to a misdiagnosis.

Biodegradable microspheres containing group B Streptococcus vaccine: Immune response in mice

Objective: This study seeks to show the feasibility of producing a group B Streptococcus (GBS) vaccine, which is capable of producing both a local IgA immune response at the mucosal surface where GBS is colonized and a humoral IgG response, which is capable of transplacental passive immunization. Study Design: Inactivated GBS antigen was microencapsulated in poly (D, L-lactic-co-glycolic acid) (PLG) with a water-in-oil-in-water double emulsion technique. Immunostimulatory synthetic oligodeoxynucleotides containing cytidine-phosphate-guanosine (CpG) motifs were coencapsulated as a potent adjuvant. The ICR strain of mouse was used in these studies. Female mice with normal immune systems were immunized with the PLG microparticles containing GBS type III polysaccharide (GBS PS) vaccine and CpG adjuvant (PLG/GBS/CpG) via the oral, vaginal, or nasal routes or by the intramuscular or intraperitoneal routes. Booster doses were administered 4 weeks after the initial immunization. Vaginal washings and blood samples were obtained 3 weeks after the booster dose and examined for both IgG and secretory IgA (sIgA) GBS antibodies with the use of an enzyme-linked immunoabsorbent assay method. Results: PLG/GBS/CpG microparticles elicited a significantly higher GBS antibody response when compared with nonencapsulated GBS antigen or PLG-encapsulated GBS PS vaccine without the addition of the CpG adjuvant. IgG and secretory IgA (sIgA) antibodies to GBS antigen were documented in both the vaginal washings and blood samples. Conclusion: Preliminary findings indicate that this novel PLG/GBS/CpG vaccine elicited both IgA and IgG antibody responses to the GBS PS antigen studied. This antibody response may provide both protection against maternal GBS colonization and passive transplacental immunization for the fetus and neonate. (Am J Obstet Gynecol 2001;185:1174-9.)

Expression of Protein S in the Murine Heart and Cultured Mouse Cardiomyocytes Is Down-regulated by Cytokines

Protein S (PS), a co-factor of activated protein C, is a vitamin K-dependent anticoagulant protein and is known to be produced extrahepatically. In the present study, the concentration of PS mRNA was determined tissue by tissue in the mouse, and it was high in lung, adrenal and heart as well as in liver. We further investigated the effects of lipopolysaccharide (LPS), tumor necrosis factor-alpha (TNF-alpha), and interleukin-1 (IL-1) on the PS mRNA expression in murine tissues in vivo. Although LPS and TNF-alpha significantly decreased the expression level of PS mRNA in all tissues examined (e. g., lung, liver, heart, and kidney) and the PS antigen level in plasma, the suppressive effect of IL-1 on PS gene expression was limited to heart. More specifically, considerable amounts of PS mRNA and antigen were expressed in a cultured mouse cardiomyocyte cell line, and again, treatment with IL-1 decreased the PS expression in these cells. These observations raise a possibility that the expression of cardiac PS may contribute to the regional anticoagulant potential in heart, and suggest that the decreased PS expression by cytokines may result in an increase in the systemic and/or regional prothrombotic potential under inflammatory conditions.

Opsonophagocytic activity against Streptococcus pneumoniae type 19F in allogeneic BMT recipients before and after vaccination with pneumococcal polysaccharide vaccine

Opsonophagocytic activity (OPA) of pneumococcal polysaccharide (Pnc PS) antibodies in vitro, a measure of antibody functional activity, usually correlates with specific IgG antibody concentrations measured by an EIA method. In order to investigate the functional activity of specific Pnc PS antibodies, we determined IgG antibodies to Pnc PS type 19F by an EIA method and OPA against Pnc type 19F by a killing assay in a randomized study, where 23 adult allogeneic BMT recipients were vaccinated with Pnc PS vaccine at 8 months (early group) and 21 recipients at 20 months (late group) after transplantation. Serum samples drawn before BMT, before vaccination, and at 1 and 16 months after vaccination were available from 27, 35, 34 and 30 patients, respectively. The geometric mean anti-Pnc 19F concentrations were 4.3, 1.3, 1.6 and 1.3 microg/ml in the early group and 3.8, 0.9, 0.6 and 0.6 microg/ml in the late group before transplantation, before vaccination, and at 1 and 16 months after vaccination, respectively. OPA (titre > or = 8) was found in 10/27, 5/35, 5/34 and 6/30 patients before BMT, before vaccination, and at 1 and 16 months after vaccination, respectively. The specific IgG antibody concentration and OPA correlated with each other before BMT, and in the early group patients before and at 1 month after vaccination. The results demonstrate that after Pnc PS vaccination allogeneic BMT recipients have antibodies with low functional activity to a Pnc PS antigen associated with low specific IgG responses. There is a need to study new Pnc conjugate vaccines in multi-dose schedules for their capacity to elicit higher specific antibody concentrations with high OPA in BMT recipients.

Prevalence of anti-protein S antibodies in patients with systemic lupus erythematosus.

To determine the prevalence of circulating anti-protein S (anti-PS) antibodies in association with PS deficiency in patients with systemic lupus erythematosus (SLE). Plasma was obtained from blood samples collected from 27 patients with SLE (5 men, 22 women; mean +/- SD age 32 +/- 10 years). Anti-PS antibodies were detected by immunoblotting according to a previously described method. Levels of free and total PS antigen were measured by enzyme-linked immunosorbent assay. PS activity was assayed by a clotting method. Levels of C4b binding protein (C4bBP) were measured by latex immunoassay. Among 27 patients with SLE, 19 (70.4%) had PS activity, and 12 (44.4%) had PS free antigen below the lower limit established for a normal population (mean -2SD). There was good correlation between PS activity and levels of PS free antigen (r = 0.851, P < 0.001), PS total antigen (r = 0.743, P < 0.001), and C4bBP (r = 0.597, P = 0.001). Circulating anti-PS antibodies and antiphospholipid antibodies (aPL) (IgG type) were detected in 7 (25.9%) and 14 (51.9%) of the 27 patients with SLE, respectively. Only 1 patient (3.7%) had both anti-PS antibodies and aPL. PS activities in patients who were positive for anti-PS antibodies (mean 38.7%) tended to be lower than those in patients who were negative for anti-PS antibodies (mean 54.9%), but the difference was not statistically significant. Anti-PS antibodies, independently of aPL, may play a role in the occurrence of PS deficiency in some patients with SLE, with possible effects on the function of PS that do not change the levels of PS antigens.

Protein S Deficiency: A Database of Mutations – Summary of the First Update

Protein S is a vitamin K dependent protein whose inherited deficiency is a well recognized risk factor for venous thrombosis. Its role is to act as activated protein C cofactor in factor Va and VIIIa proteolysis, thus restricting thrombin generation. Its gene lies on chromosome 3, at position 3p11.1-q11.2, and its structural organization has been described. Elucidation of the gene defects responsible for protein S deficiency is proceeding rapidly. A first record of identified mutations was undertaken in 1996 under the auspices of the International Society on Thrombosis and Haemostasis Scientific and Standardization Committee (ISTH SSC) and was published in 1997 (Thromb Haemost 1997; 77: 1201-14). This first database reported mutations identified in 126 protein S-deficient subjects postulated to be detrimental, and 19 mutations that were considered as neutral polymorphisms. The classification proposed by Bertina at the subcommittee meeting in 1991 was used to classify the mutations according to the phenotype observed in deficient subjects, that is type I when both free and total PS antigen levels were decreased, type III when free PS levels were decreased with normal total PS antigen levels, and type II when cofactor activity of PS was decreased while total and free antigen levels were within the normal ranges.

The role of the protein C-thrombomodulin system and fibrinolysis during cardiovascular surgery: Influence of acute preoperative plasmapheresis

Objectives: To assess the benefits of withdrawn autologous plasma, the objective of this study was to investigate whether withdrawal of acutely performed platelet-rich or platelet-poor plasmapheresis allays changes in the protein C-thrombomodulin and fibrinolytic systems after retransfusion secondary to cardiopulmonary bypass (CPB). In addition, the study attempted to determine the influence of acute plasmapheresis (APP) on the protein C-thrombomodulin and fibrinolytic systems as well as on homologous blood consumption and perioperative blood loss in elective aortocoronary bypass patients. Design: The investigation was scheduled as a prospective, randomized, unblinded study. Setting: This single investigation was conducted in the Department of Anaesthesiology and Intensive Care Medicine at a university in Germany. The study protocol was approved by the Ethics Committee of the hospital, and informed consent was obtained. Participants: Sixty male patients scheduled for elective coronary artery bypass grafting with extracorporeal circulation were included in the study. Interventions: APP was performed between induction of anesthesia and incision, collecting either 10 mL/kg of autologous platelet-poor plasma (PPP patients, group 1; n = 20) or the same amount of platelet-rich plasma (PRP patients, group 2; n = 20). Patients of group 3 ( n = 20) had no APP (control group). All patients were maintained on their usual regimen of cardiac drugs until the morning of surgery. To preserve hemodynamic stability and restore the intravascular oncotic pressure, the separated plasma was replaced by infusion of an equal amount of hydroxyethyl starch solution (HES) (6% HES, molecular weight 2 × 10 5, substitution rate 0.5%). In all operations, the same surgical procedure was chosen. For all patients, induction and maintenance of anesthesia were similar, consisting of weight-related doses of fentanyl (35 μg/kg), midazolam (0.65 mg/kg), and pancuronium bromide (0.15 mg/kg). The lungs of all patients were mechanically ventilated during the first 5 hours after the end of the operation. Measurements and Main Results: All patients had serial coagulation studies including antithrombin (AT) III-activity, prekallikrein, factor XII, and immunologic tests such as thrombin-antithrombin III (TAT), fibrinopeptide A (FPA), protein C and S (PC and PS), thrombomodulin (TM), tissue-plasminogen-activator (t-PA), plasminogen-activator-inhibitor (PAI 1), fibrinopeptide Bβ 15–42 (FPBβ 15–42), D-dimers, and hemoglobin and platelet counts determined intraoperatively and postoperatively. Chest tube drainage and transfusion requirements were recorded. APP had no negative effects on the quality of PPP and PRP plasma. The platelet count of the withdrawn plasma was 28 ± 12 × 10 9/L (PPP group) and 245 ± 36 × 10 9/L (PRP group). At the end of the operation (after retransfusion of autologous plasma) and on the morning of the first postoperative day, platelet counts were significantly higher ( p > 0.05) in the PRP than in the PPP and control groups. Plasma concentrations of TAT and FPA increased (ranging from +185% to +340% from baseline values) and AT III-activity, PC, PS, and TM antigen decreased (ranging from −8% to −55% from baseline values) to a different extent for all three groups throughout CPB. t-PA-activity increased with a maximum at the end of CPB (PPP group, 6.9 ± 1.5 IU/mL; PRP group, 3.8 ± 0.8 IU/mL; control group, 10.9 ± 2.8 IU/mL). Fibrin and fibrinogen degradation markers such as D-dimers and FPBβ 15 to 42 occurred in peak concentrations after neutralization of heparin by protamine. Only PRP patients showed baseline concentrations of coagulation parameters the next morning ( p < 0.05). Total postoperative blood loss within the first 24 hours reached 482 ± 273 mL (PRP group), 775 ± 256 mL (PPP group), and 948 ± 342 mL in the control group ( p < 0.05). None of the PRP patients received homologous blood, but one PPP patient received two units and five control patients received nine units of packed red cells ( p < 0.05). Conclusions: The results suggest that in cases of elective cardiac surgery, heparin cannot prevent generation of both thrombin and fibrin throughout CPB and postoperatively. The APP procedure had no negative effect on the quality of autologous plasma. APP-withdrawn PRP did not only increase platelet count effectively but also improved an altered hemostatic system after retransfusion. Because of the beneficial effects of APP-withdrawn PRP, it appears to be an attractive technique, especially in patients in whom withdrawal of autologous whole blood cannot be performed, as well as to reduce allogeneic blood usage and perioperative blood loss.

A major crossreactive idiotype associated with human antibodies to the Haemophilus influenzae b polysaccharide. Expression in relation to age and immunoglobulin G subclass.

Using idiotypic analysis, we examined the variable (V) region diversity of human antibodies specific for the capsular polysaccharide of Haemophilus influenzae b (Hib PS). A goat anti-idiotypic serum (anti-Id) was prepared against anti-Hib PS antibodies isolated from the serum of an adult immunized with Hib PS. The anti-Id bound donor anti-Hib PS antibodies and inhibited Hib PS binding of donor anti-Hib PS. In contrast, the anti-Id did not bind donor or pooled Ig depleted of Hib PS antibodies, nor did it inhibit antigen binding of human antibodies to pneumococcal PS's, meningococcal A PS or diphtheria toxoid. Crossreactive idiotype (CRI), as measured by anti-Id inhibition of Hib PS binding, was found in 74 of 98 subjects (76%) vaccinated with Hib PS at 1.7-57 yr of age. 60 of these 74 subjects had greater than 50% of their serum Hib PS-binding activity inhibited by anti-Id. No correlation was found between age and CRI expression. In subjects showing both IgG1 and IgG2 antibody responses, CRI was most frequently detected in both subclasses (71% of subjects). CRI was limited to either IgG1 or IgG2 in 19% of subjects, a finding suggestive of independent B cell lineages. 13 of 15 infants less than 17 mo of age, who responded to Hib PS-outer membrane protein conjugate vaccine, had greater than 50% of their serum anti-Hib PS antibody activity inhibited by anti-Id. The ability of native Hib PS and Hib PS oligomer to partially inhibit (60 and 35%, respectively) the binding between anti-Id and heterologous anti-Hib PS, indicated that some CRI determinants are in or near the combining site. In summary, our findings demonstrate a highly penetrant and frequently predominant CRI, which is expressed in both infants and adults. The results underscore the limited V region diversity of anti-Hib PS antibodies and indicate that CRI predominance is manifest early in ontogeny and is induced by both TI and TD forms of the Hib PS antigen.

Ability of cell-mediated immune production in systemic immunization of guinea pigs with mucous polysaccharides of human colonic goblet cells (PS antigen)

Ability of cell-mediated immune production in systemic immunization of guinea pigs with mucous polysaccharides of human colonic goblet cells (PS antigen)

High-molecular-weight polysaccharide antigen from Pseudomonas aeruginosa immunotype 2.

Previously, we isolated a high-molecular-weight immunogenic polysaccharide (designated PS) from Pseudomonas aeruginosa immunotype 1 (IT-1). The method which we used was modified to permit the isolation of a similar PS from P. aeruginosa IT-2. This antigen was composed primarily of carbohydrate, had a complex monosaccharide composition, including sugars not found in the lipopolysaccharide, and was nonpyrogenic in rabbits and nontoxic in mice at high doses. This material protected mice from challenges with live homologous cells. P. aeruginosa IT-2 PS gave a line of identity with the O side chain of the lipopolysaccharide, but different from this polysaccharide in molecular weight, chemical composition, and ability to immunize mice actively. Lipopolysaccharide from P. aeruginosa IT-2 contained an immunological determinant not found on P. aeruginosa IT-2 PS, which was detected due to its stability during treatment with dilute alkali. Thus, we recovered a high-molecular-weight PS antigen from P. aeruginosa IT-2, which was serologically identical to the lipopolysaccharide O side chain but was chemically and physically distinct. Also, like P. aeruginosa IT-1 strains, P. aeruginosa IT-2 contains an alkali-stable immunodeterminant on the lipopolysaccharide that may represent a core-like antigen.

Physical and Serologic Properties of an Antigen Prepared from Erythrocytes of Horses with Acute Babesiosis*

SYNOPSIS. By means of precipitation with protamine sulfate, a soluble antigen (PS) was obtained from erythrocytes of horses with acute babesiosis due to Babesia caballi and B. equi. This antigen reacted in gel diffusion tests with sera from horses recovered from acute babesiosis. The PS antigen was found to be muco‐protein, susceptible to destruction by trypsin and taka‐diastase. Analysis of the antigen by paper electrophoresis revealed 2 components which were not present in similar preparations made from erythrocytes of Babesia‐free horses.When the PS antigen was heated in boiling water for 30 minutes, a serologically inactive precipitate was formed; however, the supernate remained serologically active and was termed boiled PS (BPS) antigen. This antigen was polysaccharide in nature; its serologic activity was destroyed by taka‐diastase.In gel diffusion tests with sera of recovered horses, the PS antigen formed 2 lines of precipitation which coalesced in a single line formed between these sera and the BPS antigen. Both PS and BPS antigens reacted with sera of horses recovered from acute babesiosis in the gel‐diffusion test, but not with sera of dogs and rats recovered from acute infection with Babesia canis and Babesia rodhaini, respectively. The serologic specificity of these antigens suggests that they might have application in the serodiagnosis of inapparent Babesia infections of equine animals.