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J I Jorquera

Publications and source records attributed to J I Jorquera.

At least 19 recordsLinked to original sources
PubMed2006

Critical factors influencing prion inactivation by sodium hydroxide.

BACKGROUND AND OBJECTIVES: Transmissible spongiform encephalopathies (TSEs) are fatal neurodegenerative diseases caused by aberrantly folded cellular proteins (PrP(Sc); prions) that are generally resistant to conventional pathogen-inactivation techniques. To ensure effective decontamination and inactivation of prions that could be present in source material, we investigated critical factors that influence prion inactivation by NaOH. MATERIALS AND METHODS: A decrease in prion infectivity correlates with the disappearance of the protease-resistant core of PrPSc (PrPRES) observed in biochemical assays. To model prion inactivation, hamster scrapie (strain 263K) brain homogenate (SBH) was incubated for specific periods of time in 0.1 m NaOH at 4 or 18 degrees C, with or without detergent. Neutralized samples were subjected to limited digestion with proteinase K (PK) and then analysed using an endpoint dilution western blot assay and antibody 3F4. Structural changes in prions exposed to NaOH were examined using differential immunoprecipitation. RESULTS: Treatment of SBH with 0.1 m NaOH for 15 min, in the absence of detergent, at 4 and 18 degrees C caused a reduction in the PrP(RES) signal of 3.5 and 4.0 log10 units, respectively, with some residual signal remaining. The presence of the detergent sarkosyl during a 60-min incubation in NaOH further enhanced PrPRES reduction to > or = 4.5 log10 units (i.e. below the limit of detection). NaOH treatment induced conformational changes in PrP that resulted in the exposure of a hidden epitope and enabled prion immunoprecipitation by antibody 3F4. CONCLUSIONS: The use of NaOH can effectively reduce prion levels in an in vitro inactivation assay. After pretreatment of SBH with detergent, NaOH completely eliminates the PrPRES signal. Detergent may liberate lipid membrane-protected PrPSc to improve access to NaOH, which can then inactivate PrPSc by altering its structure. In cases of unidentified exposure to PrPSc during manufacturing, sanitizing procedures combining the use of detergent and NaOH may help to ensure minimal levels of contamination carryover in products.

Animals↗
PubMed2001

Influence of von Willebrand factor on the reactivity of human factor VIII inhibitors with factor VIII.

In order to determine the difference in reactivity of factor (F) VIII inhibitors against the FVIII/von Willebrand factor (vWF) complex and against vWF-deficient FVIII, we investigated a panel of 10 antibodies to FVIII from multitransfused individuals with severe haemophilia A and other pathologies. Immunoblotting of purified FVIII and purified thrombin-cleaved FVIII revealed that in all cases inhibitor epitopes could be localized in the heavy chain (A2 subunit) while in four cases they were also present in the light chain. One of the FVIII inhibitors remained unclassified. The effect on FVIII:C of purified IgG from inhibitor plasmas was tested against a high purity FVIII/vWF concentrate and a monoclonally purified FVIII concentrate with only trace contents of vWF, by two different functional assays. Our results suggest that for those inhibitors showing A2 plus light chain (LC) reactivity, the IgG concentration required to inhibit 50% of FVIII activity in vitro is higher for the FVIII/vWF complex than for the vWF-deficient FVIII. We conclude that there might be a protective role of vWF (at least in vitro) against FVIII inhibitors with A2 and LC subunit specificity.

Antibodies↗
PubMed1999

von Willebrand factor contained in a high purity FVIII concentrate (Fanhdi) binds to platelet glycoproteins and supports platelet adhesion to subendothelium under flow conditions.

BACKGROUND AND OBJECTIVE: There is evidence suggesting that von Willebrand factor (VWF) from high purity factor VIII concentrates could be of clinical use in the management of patients suffering from VWD. We analyzed structural and functional characteristics of VWF present in a high purity factor VIII concentrate VWFHPC (Fanhdi). The multimeric structure, the ability to bind to platelet GP Ib/IX or GP IIb/IIIa, and the capacity of VWFHPC to promote platelet adhesion on injured vessels were investigated and compared with that present in standard plasma cryoprecipitates [VWFCRYO]. DESIGN AND METHODS: Binding studies were carried out by incubating radiolabeled VWF and washed platelets, which were activated with either ristocetin (1 mg/mL; for GP Ib/IX), or thrombin (2.5 U/mL; for GP IIb/IIIa). Platelet adhesion was assessed in a perfusion system (shear rate = 800 s-1, 10 min) in which the source of VWF was added (at 0.4 or 0.8 U/mL VWF:Ag) to washed platelets and red cells suspended in a human albumin solution. The deposition of platelets onto the perfused subendothelial surface was morphometrically evaluated and expressed as percentage of surface coverage (%SC). RESULTS: The VWFHPC (152 Units VWF:RCof/mg protein; VWF:RCof/VWF:Ag = 0.97), lacked only a small proportion of high-molecular-weight multimers present in VWFCRYO. Binding affinities (Kd values, nM) of VWFHPC were similar to those of VWFCRYO (5.3 +/- 0.86 vs 5.2 +/- 0.95, for GP Ib/IX; and 11.6 +/- 2.7 vs 15.4 +/- 1.7 for GPIIb-IIIa). A slightly, though not significantly, higher binding capacity for these receptors (Bmax values, molecules/pit) was obtained for VWFHPC. The %SC in perfusions in the presence of albumin was < 10%. Addition of VWFHPC or VWFCRYO significantly increased the %SC, with values of 27.1 +/- 4.9 and 17.5 +/- 2.8%, respectively with 0.4 U/mL (p < 0.004 and p < 0.02 vs albumin); and 30.8 +/- 4.9% and 20.03 +/- 4.1%, respectively, at 0.8 U/mL (p < 0.001 and p < 0.02 vs albumin). INTERPRETATION AND CONCLUSIONS: Our data show that VWF present in the high purity FVIII concentrate Fanhdi retains the functional capacity to bind to GPs Ib/IX and IIb/IIIa and to promote platelet adhesion onto exposed subendothelium.

Albumins↗
PubMed1999

[No ELISA detectable alterations in immunogenicity following dry-hear treatment (72 hours at 80 degrees C) of FANHDI].

OBJECTIVE: Neoantigen formation during heat treatment (HT) of factor VIII:von Willebrand Factor (FVIII:vWF) concentrates may induce an immune response against the modified protein, which may also affect the native protein. We present a comparative in vitro study on the immunogenicity of a dual virally inactivated (solvent-detergent and 80 degrees C 72 hours) high purity FVIII:vWF concentrate (Fanhdi) versus the same product without heat treatment. MATERIAL AND METHODS: For this purpose rabbit antisera were prepared using both Fanhdi and the same product from which the human albumin, used as stabilizer, had been removed (these were both HT products). Also, antisera were prepared against the same products made without the dry-heat treatment step (non-HT products). Antisera were analysed by Elisa. Mixtures of antisera with increasing amounts of product (incubation-absorption in liquid phase) were assayed in plates coated with HT and non-HT products. RESULTS: The binding of antibodies against HT products to ELISA plates coated with HT products, could be blocked (in a saturable manner) with non-HT products, following liquid phase incubation. These results strongly suggest the absence of neonantigens. Furthermore, the binding of antibodies against non-HT products to ELISA plates coated with non-HT products, could be blocked (also in a saturable manner) with HT products. This result indicates that there is no epitope loss. CONCLUSIONS: The data obtained in these studies suggest that the heat treatment of viral inactivation as applied in Fanhdi, does not give rise to any major alteration in immunogenicity of the product. The data from clinical and drug surveillance studies carried out with Fanhdi do not show any indication of an increase in the frequency of inhibitors.

Animals↗
PubMed1998

Functional compensation of the low platelet count by increased individual platelet size in a patient with May-Hegglin anomaly presenting with acute myocardial infarction.

Platelets are known to play a crucial role in normal hemostasis as well as in thrombus formation at sites exposed to blood flow, as in coronary thrombosis. Thus, low platelet count is a strong negative risk factor for the occurrence of arterial thrombosis, such as occurs in acute myocardial infarction. We encountered a patient with May-Hegglin anomaly, presenting with acute myocardial infarction in his sixth decade, even though his platelet counts had always been less than 50 x 10(3)/microl. We investigated the characteristics of his platelets under the effect of shearing and found that shear-induced platelet aggregation and binding of soluble von Willebrand factor (vWF) to platelets could be induced, even when the patient's platelet count was less than 10 x 10(3)/microl, but that virtually no aggregation or vWF binding by normal platelets could be induced by shearing when platelet counts were less than 50 x 10(3)/microl. We conclude that the low platelet counts in a patient with May-Hegglin anomaly can be functionally compensated for by larger individual platelets, in view of the vWF-dependent platelet thrombus formation occurring under the effect of blood flow and that that is why most patients with May-Hegglin anomaly do not have a bleeding tendency, even though their platelet counts are very low.

Blood Platelets↗
PubMed1997

[Functionality of von Willebrand factor present in Fanhdi: adhesion to the vascular subendothelium in vitro].

PURPOSE: To study the ability of VWF present in a plasma derived high purity factor VIII concentrate (> or = 100 UI FVIII/mg of protein, Fanhdi) to promote deposition and platelet adhesion on the injured vessel wall, as an indicator of the functionality of said VWF. MATERIAL AND METHODS: An in vitro perfusion system was employed. Adequate proportions of platelets and red cells were suspended in a human albumin solution. Aliquots of the studied product were added to obtain levels of 0.4 and 0.8 VWF:AG U/mL in the perfusates. As a comparative group, cryoprecipitate was assayed at similar VWF:Ag doses. Perfusions were performed on rabbit de-endothelialized abdominal aorta segments, placed in annular perfusion chambers, at 37 degrees C and at a flux of 140 mL/min (800 s-1) for 10 min. Results were evaluated as surface covered by platelets and as the percentage ratio between thrombus and surface covered by platelets. FVIII activity was determined by one stage clotting assay. VWF:RCo activity was determined by aggregometry and VWF:Ag by ELISA. RESULTS: The VWF:Ag/FVIII:C ratio of Fanhdi is 1.57, while VWF:Roc/FVIII:C ratio is 1.15. The values of platelet deposition obtained, expressed as times of increase in the surface covered by platelets, considering the basal value (without VWF added) as 1, were: Cryoprecipitate 0.4 = 1.93; Cryoprecipitate 0.8 = 2.21; Fanhdi 0.4 = 2.99; Fanhdi 0.8 = 3.40. The ratios between thrombus and surface covered are 13.23%, 23.84%, 42.23%, 21.93% and 26.25% respectively. CONCLUSIONS: These data show that VWF present in Fanhdi maintains a high degree of functionality, promoting platelet adhesion on subendothelium under flow conditions, after its incorporation into an albumin-platelet-red cell preparation and resulting in a significant increase in platelet adhesion when compared to the VWF-free basal control.

Animals↗
PubMed1996

A modification of the APC resistance test and its application to the study of patients on coumarin therapy.

APC resistance appears to be caused, predominantly, by a mutation in coagulation factor V (nucleotide 1691: G to A). This phenomenon is usually studied by performing APTTs in the absence and presence of added APC. We studied a modification of the assay involving dilution of the test plasma in factor V deficient plasma, to render the assay more factor V specific. This modification was applied to 76 patients with venous thrombosis on coumarin treatment and to 45 controls. Two out of 45 controls (4.4%) showed abnormal results with the modified test. They also showed loss of factor V exon 10 Mnl I restriction site, associated to APC resistance. All remaining controls, with normal functional results by the modified assay, showed normal restriction profile. We detected 9 affected patients (11.8%), one of them homozygous or double heterozygous. In conclusion, the modified assay is very sensitive for factor V dependent APC resistance, and can successfully be applied to patients on coumarin therapy.

Adult↗
PubMed1996

[Development and characterization of a high-purity human factor VIII concentrate submitted to 2 specific virus inactivation treatments (FANDHI)].

AIM: To develop a therapeutic human high purity FVIII concentrate, treated with two documented and complementary specific inactivation methods, for the treatment of haemophilia A. MATERIAL AND METHODS: Cryoprecipitate was obtained from human plasma, normal for ALT levels and negative for HBsAg and antibodies to HIV and to HCV. The cryoprecipitate was resuspended and purified with PEG. The PEG precipitate was resuspended and treated with TNBP/Polysorbate 80. The mixture was processed by heparin affinity chromatography. The eluate was concentrated and precipitated with glycine and salts. After resuspension, stabilizers were added and the solution was sterile filtered, dispensed in vials and lyophilized. These final vials were treated at 80 degrees C for 72 hours: FVIII:C, vWF:RCo, vWF:Ag, vWF multimeric structure and the concentration of other plasmatic proteins were analyzed. RESULTS: FVIII: C specific activity was between 1000 and 3000 U/mg (after elimination of vWF, present as a stabilizer and before the addition of human pasteurized albumin). vWF:RCo activity (0.7 U vWF:RCo/U FVIII:C) and the multimeric structure of vWF showed a good degree of conservation. Other plasmatic proteins analyzed were undetectable or at trace amounts. No prekalllikrein, kallikrein or activated coagulation factors activity could be detected. CONCLUSION: The FVIII concentrate described shows a high degree of purity and stability, which makes it very suitable for haemophilia A treatment.

Blood↗
PubMed1996

[Evaluation of viral safety of a high-purity human factor VIII concentrate submitted to 2 specific virus inactivation treatments (FANDHI)].

AIM: To perform a validation study of the production process of a human high purity FVIII concentrate, obtained by affinity chromatography and treated with solvent-detergent and 80 degrees C, 72- hour dry heating in the final vial, in order to demonstrate its viral safety. MATERIAL AND METHODS: The ability to inactivate or eliminate viruses was studied in the steps of PEG precipitation, solvent-detergent treatment (6 h 25 degrees C), affinity chromatography and lyophilization plus heating 80 degrees C for 72 h. HIV and models for hepatitis A, B and C, as well as a model for parvovirus B-19 were employed. The experiments were carried out by spiking the samples at each step with 10% of their volume with the highest titer available virus culture. The samples were processed under validated conditions (mimicking the industrial process) and the residual infectivity was determined (as well as p24 antigen and reverse transcriptase for HIV at the solvent-detergent step). RESULTS: No residual infectivity could be detected for enveloped viruses (HIV and models for hepatitis B and C) after the first minutes of solvent-detergent treatment, which lasts 6 hours. Lyophilization followed by heating 80 degrees C for 72 hours caused complete disappearance of infectivity for the models of hepatitis A and C, before 24 hours of a treatment which lasts 72. Furthermore, lyophilization plus heating reduced infectivity for the models of hepatitis B and parvovirus B-19 by 3.4 and 4.1 logs, respectively. The affinity chromatography reduced infectivity by 7.6 logs for the model of hepatitis B and 2 logs for HIV. PEG precipitation also reduced the infectivity by 3.3 logs for the model of hepatitis A and by 1.2 logs for the model of parvovirus B-19. Taking the process as whole, the study showed cumulative reduction values between 5.3 and > 19 logs of the analyzed viruses. 25 million FVIII units have been transfused so far as FANHDI, with no seroconversion detected. Furthermore, no increase in FVIII inhibitor frequency has been described. CONCLUSION: The FVIII concentrate described shows outstanding viral safety characteristics. These data, together with the preliminary clinical experience after one year usage of the product, indicate that FANHDI is a suitable preparation for haemophilia A treatment.

Blood↗
PubMed1991

Detection of two different anti-factor VIII/von Willebrand factor antibodies of the IgA class in a hemophilic patient with a polyclonal factor VIII inhibitor of the IgG class.

A hemophilic patient treated with Factor VIII (F.VIII) concentrates developed a F.VIII inhibitory activity. The patient's plasma showed 2 anti-F.VIII/von Willebrand Factor (vWF) antibodies (Abs) of the IgG and IgA class respectively. The specific anti-F.VIII/vWF Abs were isolated and resulted to be IgG (20 micrograms/ml) and IgA (35 micrograms/ml). The plasma was subsequently fractionated by Protein A-Sepharose and the IgG and IgA containing fractions were separately analyzed for anti-F.VIII activity. Both fractions exhibited F.VIII inhibitory activity, but that corresponding to the IgA was lower than expected. Regarding the possible existence of a non-inhibitor Ab in the IgA containing sample, plasma was processed through an immunoadsorbent to which a F.VIII devoid vWF preparation had been previously coupled and further fractionated by Protein A-Sepharose, obtaining 2 IgA fractions. One of them exhibited F.VIII inhibitory activity while the other, which reacted with the F.VIII devoid vWF preparation, did not. Therefore, this latter one was considered as a true anti-vWF Ab. The IgG and IgA inhibitors were polyclonal and the IgG one was composed of the 4 IgG subclasses.

Adult↗
PubMed1989

[ELISA method for the determination of factor VII antigen].

The low plasma concentration of clotting factor VII makes it difficult to assay its antigenic fraction by the conventional methods of precipitation with specific antigens. Simple and peroxidase-conjugated antisera are currently available from commercial sources, thus allowing one to determine F VII:Ag by enzyme immunoassay. An ELISA method has been developed in this laboratory which provides sensitivity limits about 0.1% of the plasma concentration of F VII and correlates significantly with its functional activity (r = 0.603, n = 44, p less than 0.001). This technique can be highly helpful in characterising molecular variants of F VII, as well as in detecting acquired deficiencies of this factor.

Antigens↗
PubMed1989

[A fast and simple method for obtaining specific anti-von Willebrand factor IgG].

Date expired commercial F.VIII concentrates (1.000 I.U. in 15 ml) were chromatographed through a Sepharose 4B in Tris-HCI (0.15 M, pH = 7.4) column. The void volume of this column was found to constitute an enriched vWF preparation. Half a milliliter of this preparation (protein content: 200 micrograms/ml) was mixed (1: 3) with Freund's complete adyuvant and administered to two rabbits by intramuscular route. A total of 3 doses were administered at days 1, 15 and 21 and the 2 animals were bled at day 30. The serum samples so obtained were pooled and analysed (DGD and IEP) for anti-vWF and anti-NHP activity. Strong anti-vWF specificity could be demosntrated. However, since a weak anti-fibrinogen and anti-IgG contaminant activity was also detected, the pool was filtered through a column containing normal human plasma coupled to a periodate activated Sephacryl S-1.000 immunoadsorbent. The filtrate, which was found to contain only a strong anti-vWF activity, was again processed by affinity chromatography through another Sephacryl immunoadsorbent containing a commercial F.VIII concentrate of the same lot employed for purifying the vWF. The eluate so obtained demonstrated to be constituted only by a pure rabbit IgG with strong anti-vWF specificity and was completely devoid of activity against any other human plasmastic protein. The method was found to be rapid, simple and economic, yielding as much as 5 mg of specific IgG for every 10 ml of antiserum so processed.

Antibody Specificity↗
PubMed1989

[ELISA method using microplates developed with commercial antisera for measuring von Willebrand factor antigen].

The commercial availability of free and peroxidase-conjugated anti-FvW IgG fractions makes it possible to use ELISA methods for FvW: Ag determination in the routine blood coagulation laboratory. A highly sensitive microplate ELISA method was developed in our laboratory (capable of detecting 0.02% FvW: Ag), which proved to be reproducible, quick and not expensive. No significant differences were found between the results attained on different days in 4 control samples (variance analysis), variation coefficient about 12% being observed even for samples with only 11% FvW: Ag. The standard and control sample curves were parallel, which discarded a dilution effect in the assay. The correlation with Laurell's method was r = 0.889 (n = 34, p less than 0.001) and with ristocetin cofactor it was r = 0.677 (n = 19, p less than 0.005).

Antigens↗
PubMed1985

A standardized method for measuring anti-F VIII: C inhibitors in haemophilia A by coagulation inhibition in agarose gel.

Antibodies against factor-VIII coagulant activity can appear in haemophilic patients and, although infrequently, can affect individuals not suffering from Haemophilia A. The Oxford and Bethesda methods are presently the most commonly used techniques for measuring these antibodies. Both methods are time-consuming and not suitable for the screening of large risk groups. An appropriate method for screening these coagulation inhibitors is that described by P. Bird in 1975. It is based on inhibition of the coagulation produced when plasma samples containing inhibitors diffuse in agarose gels mixed with normal platelet rich plasma (PRP). However, this technique is highly dependent on the variability derived from the use of PRP (amount of coagulant factor-VIII, number of platelets, etc.). In an attempt to avoid these disadvantages, Bird's method has been modified by using standardized commercial reagents (lyophilized plasma with 100% factor-VIII coagulant activity, purified fibrinogen, and platelet Factor 3) instead of PRP. The sensitivity reaches 0.8 Bethesda units and the correlation with the Bethesda method is r = 0.964, p less than 0.001. This newly developed method is as simple as Bird's, and appears to be at least, as accurate and reproducible as the Bethesda method.

Blood Coagulation Tests↗