scholarly journals A Laser Scalpel for Operations on Hemophiliacs

1977 ◽  
Author(s):  
H. Horoszowski ◽  
U. Seligsohn ◽  
M. Heim ◽  
I. Farin
Keyword(s):  
Factor Viii ◽  
Replacement Therapy ◽  
Hemophilia A ◽  
Operation Time ◽  
Early Ambulation ◽  
Fourth Patient ◽  
Laser Scalpel ◽  
The Mean ◽  
Low Levels ◽  
Hip Contracture

The CO2 laser scalpel has an excellent hemostatic effect producing a minimal parainci-sional necrotic zone. Four severe hemophilia A patients were operated with the laser scalpel. Three underwent knee synovectomy 2 of whom without placing a tourniquet. The fourth patient underwent an operation for hip contracture. Factor VIII levels at the beginning of the operations were gradually reduced from the first operation to the fourth as follows: 100%, 80%, 14.5% and 1.4% respectively. No significant bleeding was observed during any operation. The 3 patients who underwent synovectomy were ambulated as early as 3 days after operation. They were discharged after 20 - 26 days, which is considerably shorter than the mean stay of 39 days in the hospital in 8 patients who had been synovectomized previously by the conventional method. An attempt to stop replacement therapy in the fourth patient on the 6th postoperative day resulted in bleeding. Thus, the advantages of the laser scalpel are: Excellent local hemostasis, tourniquet unecessary, low levels of factor VIII sufficient at operation, early ambulation and discharge. It is not as yet clear whether the amount of postoperative replacement therapy can be reduced. The disadvantages are: Longer operation time (by about a third); smoke produced at theatre; the price of the instrument.

Inactivation-Resistant Recombinant Factor VIII Exhibits Superior Thrombin Generation Capacity in Comparison to Wild-Type and B Domain-Deleted Factor VIII.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1604-1604
Author(s):  
Joshua Russell ◽  
Yesim Dargaud ◽  
Randal J. Kaufman ◽  
Claude Negrier ◽  
Steven W. Pipe
Keyword(s):  
Factor Viii ◽  
Replacement Therapy ◽  
Thrombin Generation ◽  
Hemophilia A ◽  
Specific Activity ◽  
Wild Type ◽  
Whitney Test ◽  
Mann Whitney Test ◽  
Transfer Strategies ◽  
The Mean

Abstract Activated factor VIII (FVIIIa) functions as a cofactor in the intrinsic hemostatic pathway leading to thrombin generation. Recombinant FVIII (rFVIII) has proven effective in replacement therapy for patients with hemophilia A (FVIII deficiency). The activity of FVIIIa in plasma is limited by both spontaneous dissociation of the A2 subunit and by cleavage by activated protein C (APC). Inactivation resistant FVIII (IR8) has been bioengineered to be resistant to both mechanisms of inactivation. The specific activity of purified IR8, as determined by one-stage clotting (aPTT) and two-stage chromogenic assays, was significantly higher (~7 to 20-fold) than that of wild-type (WT)-FVIII and B domain deleted (BDD)-FVIII. The specific activity was calculated based on ELISA antigen results and complemented by Western blots using commercial anti-FVIII antibodies. Since bioengineered IR8 may have altered immunoreactivity with anti-FVIII antibodies, an alternative functional assay was investigated to better characterize its potency. We evaluated WT-FVIII, BDD-FVIII and IR8 via the Calibrated Automated Thrombogram (CAT), a global assay of hemostasis, in platelet-free plasma (PPP) from 6 severe hemophilia A patients (<1 IU/dl FVIII) without inhibitors. The CAT test was chosen because of its ability to offer more valuable insight into the potential clinical value of IR8 than traditional clotting and chromogenic assays. Blood samples were taken into Corn trypsin inhibitor (CTI) to block contact activation and ensure that thrombin generation was triggered exclusively by tissue factor (TF) via the extrinsic hemostatic pathway. In an effort to demonstrate the dose dependency of each concentrate on its thrombin generating capacity, all 3 proteins were added to PPP along with a low TF concentration (1 pM) at varying FVIII activities (0, 25, 50 & 100 IU/dl). At each protein concentration, IR8 showed a significantly higher endogenous thrombin potential (ETP, the area under the thrombin generation curve) and peak height of the thrombin burst compared to either WT-FVIII or BDD-FVIII. The mean ETP values (nM*min) at 100 IU/dl, were WT-FVIII 650 and BDD-FVIII 725 (Mann Whitney test, p=0.69) and IR8 1107 (Mann Whitney test, p=0.04) with a mean ETP for FVIII <1% of 315 used as a control. Similar results were obtained in the presence of 1 nM thrombomodulin, which was added to sensitize the system to the action of APC. Consistent with the increased specific activity of IR8, the mean ETP of IR8 at 25 IU/dl was comparable to that of WT-FVIII at a concentration 4 times greater (100 IU/dl). Furthermore, no significant difference was found between the lag times of IR8 versus WT-FVIII and BDD-FVIII indicating that the advantage of IR8 does not lie in its ability to activate the initiation phase of thrombin generation, but rather in its persistent cofactor activity during the propagation phase of coagulation. These results are encouraging because the development of a rFVIII with markedly increased potency would potentially allow for reduced protein requirements in replacement therapy, thereby reducing costs and possibly decreasing inhibitor antibody development and would improve the efficacy of hemophilia A gene therapy without necessitating large improvements in genetic transfer strategies.

An Increase in Factor VIII Levels with Increasing Age in a Subgroup of Individuals with Mild Hemophilia A

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2278-2278 ◽  
Author(s):  
Lauren J Lee ◽  
John K. Wu ◽  
Man-Chiu Poon ◽  
Shannon Jackson
Keyword(s):  
Hepatitis C ◽  
Factor Viii ◽  
Medical Records ◽  
Hemophilia A ◽  
Conflicts Of Interest ◽  
Bleeding Disorder ◽  
Physiological Age ◽  
Management Options ◽  
Southern Alberta ◽  
The Mean

Abstract Abstract 2278 Background: Hemophilia A is an X-linked hereditary disorder resulting in absent or reduced levels of functional factor VIII (FVIII:C). The baseline FVIII:C levels are important in the determination of treatment and management options in individuals with mild hemophilia who by definition exhibit FVIII:C levels between 6–40 U/dL. In individuals without hemophilia, FVIII:C increases with physiological age and this trend has not been well characterized in mild hemophilia. This is important to consider in mild hemophilia because treatment with factor concentrate or DDAVP may paradoxically increase the risk of venous or arterial thrombosis in older patients. Objectives: To describe the changes in baseline FVIII:C levels with time in a cohort of pediatric and adult subjects with mild hemophilia registered in the British Columbia Provincial and Southern Alberta Bleeding Disorder programs. Methods: All medical records for subjects with FVIII:C levels 6–40 U/dL registered with the BC Provincial and Southern Alberta Bleeding Disorder programs were reviewed for eligibility. Male subjects with a minimum of 2 FVIII:C level measurements at least 5 years apart were included in the analysis. Retrospective data was extracted from database/medical records, including age, gender, blood group, FVIII mutation, historical FVIII:C levels, historical DDAVP response (>3 fold and/or >50 U/dL), and co-infection (Hepatitis C, B and HIV) status. Linear mixed effects regression models were used to examine time trend of FVIII:C levels and subjects were stratified into 3 groups based on baseline FVIII:C (FVIII:C<15 U/dL, 15–24 U/dL, and ≥25 U/dL). Results: 198 records were reviewed and 116 subjects excluded from the analysis (chart unavailable, n=28; inadequate FVIII:C data, n=79; female, n=9) leaving 82 subjects who met eligibility criteria. The mean age at first FVIII:C measurement was 24.0 years (SD 19.6; Range 0.0–77.8). The mean follow-up time was 16.5 years (SD 19.6; Range 4.8–44.8). There was no observable trend of FVIII:C with time in the primary analysis of the whole cohort (p=0.667). However, for subjects with baseline FVIII:C<15 U/dL, who were ≥25 years of age (n=31), an increase in baseline FVIII:C levels of 0.11 percent per year (p<0.05) was observed. Subjects with baseline FVIII:C<15 U/dL (n=40) also showed less FVIII:C variability between measurements than subjects with FVIII:C≥15 U/dL (n=42). Subjects with baseline FVIII:C≥25 U/dL (n=18) demonstrated a decreasing trend of −0.15 percent per year (p<0.05), however also demonstrated the greatest variability between FVIII:C measurements. Co-infections were present in 24% (n=20) of subjects and included Hepatitis C (n=18), Hepatitis B (n=1), HIV (n=1). 73 subjects had known DDAVP response; >3-fold but ≤50 U/dL (n=11), ≤3-fold but >50 U/dL (n=15), >3-fold & >50 U/dL (n=23), ≤3-fold & ≤50 U/dL (n=24). Conclusion: This study suggests that there is an increase in FVIII:C with time in the subgroup of individuals with mild hemophilia ≥25 years of age and baseline FVIII:C levels <15 U/dL. Further long-term data particularly in the older cohort of subjects is needed to confirm this preliminary finding and further characterize this trend in subgroups with different baseline FVIII:C. Disclosures: No relevant conflicts of interest to declare.

Target Joint Bleeding in Pediatric Patients with Hemophilia Α Receiving Twice Weekly Prophylaxis with a Pegylated Full-Length Recombinant Factor VIII with Extended Half-Life

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3778-3778
Author(s):  
Amy L. Dunn ◽  
Alexis A. Thompson ◽  
Werner Engl ◽  
Marlies Sharkhawy ◽  
Brigitt E. Abbuehl
Keyword(s):  
Factor Viii ◽  
Board Of Directors ◽  
Half Life ◽  
Research Funding ◽  
Pediatric Patients ◽  
Hemophilia A ◽  
Point Estimate ◽  
Severe Hemophilia ◽  
Advisory Committees ◽  
The Mean

Abstract Introduction: Patients with hemophilia A are at risk for acute bleeding which may affect muscles and other soft tissues but characteristically involves joints. Prophylaxis with factor VIII (FVIII) is the optimal treatment to prevent bleeding into joints and, when begun at a young age, may prevent arthropathy. BAX 8551, a polyethylene glycol (peg)ylated, full-length, recombinant FVIII built on ADVATE2, demonstrated extended half-life, efficacy, and safety for prophylaxis and treatment of bleeding in patients with severe hemophilia A. Methods: A phase 3, prospective, uncontrolled, multicenter study was performed in pediatric patients with severe hemophilia A without history of inhibitors. To be eligible, patients aged <6 years had to have ≥50, those aged 6 to <12 years ≥150 previous exposure days (EDs) to FVIII. Patients received twice weekly infusions of 50 ±10 IU/kg of BAX 855 over a period of 6 months or ≥50 EDs. The prevalence of target joints, defined as a single joint with ≥3 spontaneous bleeding episodes in any consecutive 6-month period, was assessed at baseline. Annualized rates of target joint bleeds and the course of target joints were evaluated by age (<6 and 6 to <12 years). The study was performed in accordance with the principles of the Declaration of Helsinki of the World Medical Association. Results:Sixty-six patients were treated with a mean (SD) BAX 855 dose of 51.1 (5.5) IU/kg at a mean (SD) frequency of 1.8 (0.2) infusions/week. Fourteen of 66 patients (21.2%), 3/32 (9.4%) in the younger and 11/34 (32.4%) in the older cohort, had a total of 23 target joints at screening. The number of target joint bleeds decreased during a mean (SD) of 48.5 (7.7; median: 49.0) prophylactic EDs/patient. Five of 66 (7.6%) patients had at least 1 target joint bleed, 1/32 (3.1%) in the younger and 4/34 (11.8%) in the older cohort. The point estimate for the mean (95% CI) annualized rate of target joint bleeds was 0 (0 - infinity; median: 0) compared to an annualized rate of all joint bleeds of 1.1 (0.6 - 1.9; median: 0) and an annualized rate of all bleeds of 3.0 (2.2 - 4.2; median: 2.0) (Table 1). The point estimate for the mean (95% CI) annualized bleeding rate (ABR) in 52 patients without target joints was 2.9 (2.0 - 4.2; median: 2.0) and was similar in 14 patients with target joints at screening at 3.5 (1.9 - 6.6; median: 2.1). In the younger cohort, the ABR was lower in patients with than those without target joints. However, the number of patients <6 years with target joints (N = 3) was too small to draw any conclusions (Table 1). During BAX 855 prophylaxis, no new target joints developed in any patient. Ten of 14 patients had at least 1 target joint revert to a non-target joint. In 8 of these 10 patients, 4 with 1 and 4 with 2 target joints, all target joints resolved. Conclusions:These results suggest that twice weekly infusion of BAX 855 is effective in the prevention of bleeding into target joints and may revert target to non-target joints in pediatric patients with severe hemophilia A. 1BAX 855 (Baxalta US Inc., now part of Shire) is licensed in the US and Japan under the trade name ADYNOVATE. 2ADVATE is a trade mark of Baxalta US Inc., now part of Shire. Disclosures Dunn: NovoNordisk: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Kedrion: Research Funding; Pfizer: Research Funding; CSL Behring: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Biogen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Baxalta (now part of Shire): Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Octapharma: Research Funding. Thompson:Eli Lily: Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Research Funding; bluebird bio: Consultancy, Research Funding; ApoPharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; Mast: Research Funding; Amgen: Research Funding; Baxalta (now part of Shire): Research Funding. Engl:Shire: Employment, Equity Ownership. Sharkhawy:Baxalta (now part of Shire): Employment. Abbuehl:Baxalta (now part of Shire): Employment.

scholarly journals How we choose factor VIII to treat hemophilia

Blood ◽  
2012 ◽  
Vol 119 (18) ◽  
pp. 4108-4114 ◽  
Author(s):  
Pier Mannuccio Mannucci ◽  
Maria Elisa Mancuso ◽  
Elena Santagostino
Keyword(s):  
Factor Viii ◽  
Replacement Therapy ◽  
Hemophilia A ◽  
Recombinant Dna ◽  
Tolerance Induction ◽  
Coagulation Factors ◽  
Pathogen Transmission ◽  
Recombinant Dna Technology ◽  
Mammalian Cell Cultures ◽  
Main Determinants

AbstractIn high-income countries, the large availability of coagulation factors for replacement therapy of patients with hemophilia A has raised the life expectancy of these lifelong bleeders to that of males from the general population. The practicing clinician is offered a multitude of choices among several commercial brands of factor VIII extracted from human plasma or engineered from mammalian cell cultures by means of recombinant DNA technology. This article has the goal to offer our opinions on how to choose among the different products, that we consider interchangeable relevant to their clinical efficacy in the control of bleeding and safety from pathogen transmission. Hence, the main determinants of our choices are price and the risk of occurrence of factor VIII inhibitory alloantibodies. With this as background, we present the rationale underlying the choices for different categories of patients with severe hemophilia A: previously untreated patients, multiply treated patients, and patients undergoing immune tolerance induction with large doses of factor VIII to eradicate inhibitors. Mention is also made to the possible strategies that should be implemented to make available coagulation factors for replacement therapy in developing countries.

scholarly journals Tratamento de Hemofílicos A Grave Atendidos no Hospital de Hematologia da Fundação HEMOPE, com Inibidor de Fator VIII Através da Imunotolerância

2021 ◽  
Vol 25 (2) ◽  
pp. 252-258
Author(s):  
Amanda Oliveira Marinho ◽  
Abdênego Rodrigues da Silva ◽  
Robson Raion de Vasconcelos Alves ◽  
Magaly do Bom Parto Lopes Vieira Lima ◽  
Antônio Sérgio Alves de Almeida Junior
Keyword(s):  
Factor Viii ◽  
Medical Records ◽  
Neutralizing Antibodies ◽  
Hemophilia A ◽  
Treatment Success ◽  
Neutralizing Antibody ◽  
Severe Hemophilia ◽  
Total Treatment ◽  
The Mean ◽  
Hemofilia A

No Brasil, mais de 80% dos casos de hemofilia são do tipo A. A infusão do concentrado do Fator VIII (FVIII) pode desenvolver anticorpos neutralizantes. O tratamento de escolha para indivíduos que desenvolveram inibidor é a imunotolerância (IT). Este estudo objetivou avaliar a conduta e eficácia do tratamento da IT em pacientes com hemofilia A grave na Fundação de Hematologia e Hemoterapia de Pernambuco (HEMOPE), no período de 2012 a 2017. O estudo foi do tipo descritivo com uma abordagem quantitativa com análise dos prontuários de pacientes com hemofilia A grave. No total de 17 pacientes hemofílicos A grave, 47% ainda continuam na IT, 17,7% tiveram insucesso, 17,7% foram excluídos do tratamento, 11,7% conseguiram sucesso parcial e 5,9% obtiveram sucesso total. A média de idade de detecção do inibidor foi de 4 anos a partir do início do tratamento de infusões, variando de 1 a 14 anos; 7 indivíduos apresentaram o primeiro título de baixa resposta (< 5 UB/mL) e os demais título de alta resposta (> 5 UB/ mL); cerca de 70,58% conseguiram começar a IT com um período de detecção < 5 anos. A falta de uma conduta padrão diante do tratamento resulta em uma eficácia não definida, apesar de todas as limitações do presente estudo já relatadas, um pequeno percentual de pacientes obteve sucesso parcial e total do tratamento, sendo um indicativo de que a IT realizada, atualmente, nos pacientes hemofílicos A grave da Fundação HEMOPE (iniciada há apenas 6 anos) apresenta resultados promissores. Palavras-chave: Fator VIII. Anticorpo Neutralizante. Eficácia. Abstract In Brazil, more than 80% of hemophilia cases are type A. Infusion of Factor VIII concentrate (FVIII) can develop neutralizing antibodies. The treatment of choice for individuals who have developed an inhibitor is immunotolerance (IT). This study aimed to evaluate the conduct and effectiveness of treating IT in patients with severe hemophilia A at Fundação de Hematologia e Hemoterapia de Pernambuco (HEMOPE), from 2012 to 2017. The study was the descriptive type with a quantitative approach with analysis of the patients’ medical records with severe hemophilia A. In the total of 17 hemophiliac A severe patients, 47% are still on IT, 17.7% were unsuccessful, 17.7% were excluded from treatment, 11.7% were partially successful and 5.9% were totally successful. The mean detection age of the inhibitor was 4 years from the beginning of the infusions treatment, ranging from 1 to 14 years; 7 individuals had the first low response titer (<5 UB/mL) and the remaining high response titer (> 5 UB/mL); about 70.58% managed to start IT with a detection period <5 years. The lack of a standard approach to treatment results in an undefined efficacy, although all the limitations of the present study already reported, a small percentage of patients achieved partial and total treatment success, being an indication that the IT currently performed on severe hemophiliac A patients from Fundação HEMOPE (started only 6 years ago) shows promising results. Keywords: Factor VIII. Neutralizing Antibody. Effectiveness

scholarly journals Endogenous factor VIII synthesis from the intron 22–inverted F8 locus may modulate the immunogenicity of replacement therapy for hemophilia A

2013 ◽  
Vol 19 (10) ◽  
pp. 1318-1324 ◽  
Author(s):  
Gouri Shankar Pandey ◽  
◽  
Chen Yanover ◽  
Lisa M Miller-Jenkins ◽  
Susan Garfield ◽  
...  
Keyword(s):  
Factor Viii ◽  
Replacement Therapy ◽  
Hemophilia A ◽  
Endogenous Factor

Polymorphisms in the Promoter Region of the IL-10 Gene Is Associated with Inhibitor Development in Hemophilia A.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 212-212 ◽  
Author(s):  
Jan Astermark ◽  
Johannes Oldenburg ◽  
Anna Pavlova ◽  
Erik Berntorp ◽  
Ann-Kari Lefvert ◽  
...  
Keyword(s):  
Factor Viii ◽  
Replacement Therapy ◽  
Promoter Region ◽  
Hemophilia A ◽  
Strong Association ◽  
Causative Factor ◽  
Factor Viii Gene ◽  
Ca Repeat ◽  
Inhibitor Development ◽  
History Of

Abstract The development of inhibitory antibodies is a severe and costly complication to replacement therapy occurring in 10–15% of patients with hemophilia A, and the aim of the Malmo International Brother Study (MIBS) is to evaluate host genetic factors associated with this adverse effect of treatment. In the present study, factor VIII mutations, HLA genotypes and polymorphisms of the interleukin IL-1beta, IL-4 and IL-10 genes known to influence antibody production in autoimmune diseases, were analyzed in 164 patients with hemophilia A (120 severe, 30 moderate and 14 mild) belonging to 78 unrelated families. Seventy-seven (47.0%) of the subjects had a history of inhibitors (57 high-responding, 20 low-responding) in 54 unrelated families (34 discordant, 20 concordant siblings). In 24 families, no inhibitor was reported in any of the siblings. Seventy-five patients (45.7%) in 36 families had an inversion. In this group, 40 patients (53.3%) in 28 families had inhibitors (17 concordant, 11 discordant). Weak associations between inhibitor development and the HLA alleles A26 and B44 were found. No association was found with the IL-1beta Taq 1 RFLP alleles in exon 5, and the −590 C/T SNP in the promoter region of IL-4. There was however, a strong association between an allele with 134 bp in one of the CA repeat microsatellites, IL-10G, located in the promoter region of the IL-10 gene, and development of inhibitor. Allele 134 was found in 32 (41.6%) of the patients with inhibitors compared with 12 (13.8%) of the inhibitor negative patients (p<0.001), corresponding to an odds ratio of 4.4 (95% CI 2.1–9.5, p<0.001). The association was consistent in the subgroup of families with severe hemophilia and an inversion of the factor VIII gene (p=0.002). Only one discordant inhibitor family was identified in which the subject without allele 134 developed an inhibitor, and the allele 134 positive brother did not. IL-10 is the first gene located outside the causative factor VIII gene mutation to be associated with inhibitor development in hemophilia and our data indicate this gene to be an important determinant for this side-effect of replacement therapy.

Blood Induced Cartilage Changes in a Hemophilia A Murine Model.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1152-1152
Author(s):  
Amy L. Dunn ◽  
Bahig M. Shehata
Keyword(s):  
Articular Cartilage ◽  
Factor Viii ◽  
Hemophilia A ◽  
Age Groups ◽  
Clotting Factor ◽  
Blue Staining ◽  
Mankin Score ◽  
The Mean ◽  
Bleeding Score ◽  
And Control

Abstract Background: Hemophilia A results from a congenital deficiency of clotting factor VIII. Patients with hemophila are unable to generate adequate thrombin during blood coagulation and therefore have a bleeding diathesis. Degenerative joint disease is the largest source of morbidity for hemophilic patients. The pathophysiology of this process is poorly understood but clinically, repeated bleeding into a joint leads to changes in the synovium, articular cartilage and underlying bone. It is not well known how quickly cartilage changes occur or if animal age has any impact on susceptibility to blood induced cartilage damage. Materials and Methods: The E16 fVIII knockout mouse is a well-established model for hemophilia research. These mice contain a targeted disruption of the factor VIII gene at exon 16 (E16) in a C57BL/6 background. Despite having no detectable circulating fVIII activity they rarely suffer from spontaneous hemarthroses. Therefore hemarthroses was mimicked by injecting 5 μL of autologous whole blood into the hind knee. Six male mice per group in each of three ages; 12, 24 and 52 weeks were utilized. The animals were sacrificed 48 hours after injection. The injected and control contralateral knee joint from each animal was fixed in 10% formalin and then mounted in paraffin. After decalcification, five micrometer sections through the joint were obtained. Joint architecture was examined after hematoxylin and eosin staining. Proteoglycan content of articular cartilage was evaluated after alcian blue staining as part of the modified Mankin score. The pathologic specimens were also scored utilizing Valentino’s visual bleeding score. The Student’s t-test was utilized for significance testing. Results: All mice showed evidence of blood remaining in the injected joint at 48 hours. The mean visual bleeding score was significantly higher for the injected knees versus the control knees of all age groups p<0.001. The mean visual bleeding score was significantly between the 12 and 24 week animals p=0.0112 and the 12 and 52 week animals p=0.0379, but not between the 24 and 52 week animals p=0.5423. The modified Mankin score was significantly higher between the injected and control knees of all age groups as seen in Figure 1 where the mean and standard deviation is shown for 6 animals in each group. Interestingly, one control knee in a 24 week old animal demonstrated synovial hyperplasia and decreased proteoglycan staining but had no evidence of blood in the joint. This likely represented a previous spontaneous hemorrhage. The modified Mankin score was highest for the 12 week old animals and lowest for the 52 week animals but did not achieve statistical significance p=0.5746. Conclusions: Injection of autologous blood mimicked clinical hemarthrosis. Evidence of decreased proteoglycan staining of articular cartilage was evident within 48 hours of blood exposure. Additionally, there was a trend toward more prominent proteoglycan loss in the youngest animals. Figure Figure

Indoleamine 2,3-Dioxygenase and Peripheral Tolerance to Exogenous Factor VIII: A Multi-Centre Pilot Study

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 26-26
Author(s):  
Davide Matino ◽  
Alfonso Iorio ◽  
Marco Gargaro ◽  
Giuseppe Tagariello ◽  
Matteo Luciani ◽  
...  
Keyword(s):  
Factor Viii ◽  
B Cell ◽  
Replacement Therapy ◽  
Hemophilia A ◽  
Immunoblot Analysis ◽  
Fold Change ◽  
Peripheral Tolerance ◽  
Multi Centre Study ◽  
Indoleamine 2,3 Dioxygenase ◽  
And Function

Abstract Abstract 26 Introduction: Clinically relevant antibodies neutralizing the hemostatic effect of fVIII occur in approximately 20–25% of patients with hemophilia A on replacement therapy, and they are associated with significant mortality, morbidity, and a poorer life quality. Many factors predisposing to inhibitor formation have been advocated, but the reasons why some patients develop a neutralizing antibody response to FVIII remains unclear. Evidence suggests that hemophilia-associated mutations (resulting in the absence or severe truncation of the FVIII protein) are associated with the highest risk of inhibitor formation on replacement therapy, as those mutations may have prevented the early development of physiologic tolerance to human fVIII. Recent evidence suggests a role for professional antigen-presenting cells (APC) in the initiation of immunogenic versus tolerogenic responses to fVIII. In particular dendritic cells (DC) actively establish and maintain both central and peripheral tolerance to self. Several mechanisms contribute to DC tolerogenicity and the tryptophan catabolic pathway involving indoleamine 2,3-dioxygenase (IDO) may be at work. IDO is responsible for multiple regulatory effects on immune cells, including inhibition of activated T and B cell proliferation, apoptosis, and lymphocyte differentiation towards T or B cell regulatory phenotypes. The role of IDO and tryptophan catabolytes in immunoreactivity versus tolerance to self prompted us to investigate whether IDO has a role in peripheral tolerance to exogenous fVIII. Objectives: The goal of this multi-centre study was to examine IDO expression and function in peripheral blood mononuclear cells (PBMC) from hemophilic subjects to test the hypothesis that IDO is involved in the modulation of fVIII-specific responses and, particularly, that inhibitor-positive patients might have impaired IDO induction/activity and therefore higher propensity to develop anti-fVIII responses. Methods: Blood was collected from 21 severe (<0.01 IU/mL) hemophilia A patients who provided written informed consent and consisted of 9 inhibitor-positive patients (≥5 Bethesda Units) and 12 inhibitor-free patients (with undetectable inhibitor levels). PBMC were isolated by Ficoll-Paque Plus gradient centrifugation with standard procedures. IDO induction in PBMC was assessed by immunoblot analysis (WB) and Real-Time PCR (mRNA), while IDO activity was analyzed through HPLC evaluation of kynurenine production (the first breakdown product of tryptophan catabolism by IDO). Results: 11 out of 12 patients without inhibitor displayed normal IDO expression and function, while only 1 out of 9 patients with inhibitor had unimpaired IDO competence. Consistent results were obtained by i) immunoblot analysis of protein expression (densitometry units; inhibitor-positive patients, 0.78 ± 0.57; inhibitor-free patients 4.52 ± 2.9; p = 0.002), by ii) RT-PCR for IDO mRNA (mRNA fold change over control; inhibitor-positive patients 1.11 ± 0.54; inhibitor-free patients 3.18 ± 1.85; p = 0.007), and by iii) kynurenine production (kynurenine fold change over control; inhibitor-positive patients 0.86 ± 0.34; inhibitor-free patients 2.3 ± 0.87; p = 0.0002) (fig.1). Conclusions: These preliminary data show that the immunoregulatory enzyme IDO could be involved in peripheral tolerance to factor VIII. Studies with a larger number of patients (combined with studies of IDO gene polymorphism) and the implementation of suitable animal models (currently underway in our laboratory) might yield further insight into any possible relationships between defective IDO function and inhibitor development in hemophilic patients. Ultimately, the level of IDO expression could represent an additional prognostic factor in hemophilic patients on replacement therapy when combined with fVIII gene mutation (high- vs. low- risk) assessment and, possibly, HLA class II molecule expression profiling. Disclosures: No relevant conflicts of interest to declare.

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