Correção endovascular de aneurisma de aorta abdominal e artéria ilíaca comum esquerda em paciente com hemofilia C grave

A deficiência do fator XI, também conhecida como hemofilia C, é uma doença hematológica hereditária rara, que se manifesta clinicamente com hemorragia persistente após cirurgias, traumas, menorragias e extrações dentárias. Neste artigo, relatou-se a correção endovascular de um paciente com aneurisma de aorta e de artéria ilíaca comum esquerda em um paciente portador de deficiência major do fator XI (atividade do fator XI inferior a 20%). O procedimento foi realizado com sucesso, com o manuseio do distúrbio da coagulação por meio da infusão de plasma fresco no pré-operatório imediato e no pós-operatório, e controle laboratorial da coagulação do paciente.

[Spontaneous hematomas and isolated prolongation of aPTT]

We report a case of a 64 year old woman with spontaneous hematomas. A prior history of bleeding disorder was not known up to now. The result of investigation of coagulation was an isolated prolonged aPTT that was caused by an inhibitor of factor VIII without underlying disease (idiopathic acquired hemophilia A). Therapy with steroids and cyclophosphamid resulted in partial remission.

Designed ankyrin repeat proteins: a new approach to mimic complex antigens for diagnostic purposes?

Inhibitory antibodies directed against coagulation factor VIII (FVIII) can be found in patients with acquired and congenital hemophilia A. Such FVIII-inhibiting antibodies are routinely detected by the functional Bethesda Assay. However, this assay has a low sensitivity and shows a high inter-laboratory variability. Another method to detect antibodies recognizing FVIII is ELISA, but this test does not allow the distinction between inhibitory and non-inhibitory antibodies. Therefore, we aimed at replacing the intricate antigen FVIII by Designed Ankyrin Repeat Proteins (DARPins) mimicking the epitopes of FVIII inhibitors. As a model we used the well-described inhibitory human monoclonal anti-FVIII antibody, Bo2C11, for the selection on DARPin libraries. Two DARPins were selected binding to the antigen-binding site of Bo2C11, which mimic thus a functional epitope on FVIII. These DARPins inhibited the binding of the antibody to its antigen and restored FVIII activity as determined in the Bethesda assay. Furthermore, the specific DARPins were able to recognize the target antibody in human plasma and could therefore be used to test for the presence of Bo2C11-like antibodies in a large set of hemophilia A patients. These data suggest, that our approach might be used to isolate epitopes from different sets of anti-FVIII antibodies in order to develop an ELISA-based screening assay allowing the distinction of inhibitory and non-inhibitory anti-FVIII antibodies according to their antibody signatures.

Houston History of Medicine Society 2008-2009 Schedule and Abstracts

The National Library of Medicine and the Continuing Legacy of Michael E. DeBakey, M.D. (Stephen B. Greenberg) The Legacy of William Osler: North America’s most famous physician (Robert E. Rakel) A Lady Alone: Elizabeth Blackwell: First American Woman Doctor (Linda Gray Kelley, Charlton) A Mariner with Crippling Arthritis and Bleeding Eyes: The Chronic Arthritis of Christopher Columbus (Frank C. Arnett) Generation C(affeine): A History of Caffeine Consumption and its Medical Implications (Student Essay Contest winners) (Priti Dangayach) Our Artificial Fitness? Relaxed Selection Leads to Medical Dependence (Student Essay Contest winners) Philip Boone Remembering John P. McGovern, M.D. (1921-2007) (Bryant Boutwell) Who Was Albert Schweitzer? (Bryant Boutwell) Disease, Doctors and the Duty to Treat in American History (Thomas R. Cole) Vaccinating Freedom: The African-American Experience of Smallpox Prophylaxis in Old Philadelphia, 1723-1923 (Dayle B. Delancey) The Royal Hemophilia (The Royal Hemophilia)

Estimation of male-to-female ratios of mutation rate in primates and rodents

Haldane (1935) developed a method for estimating the male-to-female ratio of mutation rate ($\alpha$) by using sex-linked recessive genetic disease, but in six different studies using hemophilia A data the estimates of $\alpha$ varied from 1.2 to 29.3. Direct genomic sequencing is a better approach, but it is laborious and not readily applicable to non-human organisms. To study the sex ratios of mutation rate in various mammals, I used an indirect method proposed by Miyata et al. (1987). This method takes advantage of the fact that different chromosomes segregate differently between males and females, and uses the ratios of mutation rate in sequences on different chromosomes to estimate the male-to-female ratio of mutation rate. I sequenced the last intron of ZFX and ZFY genes in 6 species of primates and 2 species of rodents; I also sequenced the partial genomic sequence of the Ube1x and Ube1y genes of mice and rats. The purposes of my study in addition to estimation of $\alpha$'s in different mammalian species, are to test the hypothesis that most mutations are replication dependent and to examine the generation-time effect on $\alpha$. The $\alpha$ value estimated from the ZFX and ZFY introns of the six primate specise is ${\sim}$6. This estimate is the same as an earlier estimate using only 4 species of primates, but the 95% confidence interval has been reduced from (2, 84) to (2, 33). The estimate of $\alpha$ in the rodents obtained from Zfx and Zfy introns is ${\sim}$1.9, and that deriving from Ube1x and Ube1y introns is ${\sim}$2. Both estimates have a 95% confidence interval from 1 to 3. These two estimates are very close to each other, but are only one-third of that of the primates, suggesting a generation-time effect on $\alpha$. An $\alpha$ of 6 in primates and 2 in rodents are close to the estimates of the male-to-female ratio of the number of germ-cell divisions per generation in humans and mice, which are 6 and 2, respectively, assuming the generation time in humans is 20 years and that in mice is 5 months. These findings suggest that errors during germ-cell DNA replication are the primary source of mutation and that $\alpha$ decreases with decreasing length of generation time. ^

HIV and human herpesvirus 8 co-infection across the globe: Systematic review and meta-analysis.

HIV-infection is an important risk factor for developing Kaposi sarcoma (KS), but it is unclear whether HIV-positive persons are also at increased risk of co-infection with human herpesvirus 8 (HHV-8), the infectious cause of KS. We systematically searched literature up to December 2012 and included studies reporting HHV-8 seroprevalence for HIV-positive and HIV-negative persons. We used random-effects meta-analysis to combine odds ratios (ORs) of the association between HIV and HHV-8 seropositivity and conducted random-effects meta-regression to identify sources of heterogeneity. We included 93 studies with 58,357 participants from 32 countries in sub-Saharan Africa, North and South America, Europe, Asia, and Australia. Overall, HIV-positive persons were more likely to be HHV-8 seropositive than HIV-negative persons (OR 1.99, 95% confidence interval [CI] 1.70-2.34) with considerable heterogeneity among studies (I(2) 84%). The association was strongest in men who have sex with men (MSM, OR 3.95, 95% CI 2.92-5.35), patients with hemophilia (OR 3.11, 95% CI 1.19-8.11), and children (OR 2.45, 95% CI 1.58-3.81), but weaker in heterosexuals who engage in low-risk (OR 1.42, 95% CI 1.16-1.74) or high-risk sexual behavior (OR 1.66, 95% CI 1.27-2.17), persons who inject drugs (OR 1.66, 95% CI 1.28-2.14), and pregnant women (OR 1.68, 95% CI 1.15-2.47), p value for interaction <0.001. In conclusion, HIV-infection was associated with an increased HHV-8 seroprevalence in all population groups examined. A better understanding of HHV-8 transmission in different age and behavioral groups is needed to develop strategies to prevent HHV-8 transmission.

Antibody chemical reactivity: Beneficial and pathogenic roles

Antibodies (Abs) to autoantigens and foreign antigens (Ags) mediate, respectively, various pathogenic and beneficial effects. Abs express enzyme-like nucleophiles that react covalently with electrophiles. A subpopulation of nucleophilic Abs expresses proteolytic activity, which can inactivate the Ag permanently. This thesis shows how the nucleophilicity can be exploited to inhibit harmful Abs or potentially protect against a virus. ^ Inactivation of pathogenic Abs from Hemophilia A (HA) patients by means of nucleophile-electrophile pairing was studied. Deficient factor VIII (FVIII) in HA subjects impairs blood coagulation. FVIII replacement therapy fails in 20-30% of HA patients due to production of anti-FVIII Abs. FVIII analogs containing electrophilic phosphonate group (E-FVIII and E-C2) were hypothesized to inactivate the Abs by reacting specifically and covalently with nucleophilic sites. Anti-FVIII IgGs from HA patients formed immune complexes with E-FVIII and E-C2 that remained irreversibly associated under conditions that disrupt noncovalent Ab-Ag complexes. The reaction induced irreversible loss of Ab anti-coagulant activity. E-FVIII alone displayed limited interference with coagulation. E-FVIII is a prototype reagent suitable for further development as a selective inactivator of pathogenic anti-FVIII Abs. ^ The beneficial function of Abs to human immunodeficiency virus type 1 (HIV-1) was analyzed. HIV-1 eludes the immune system by rapidly changing its coat protein structure. IgAs from noninfected subjects hydrolyzed gp120 and neutralized HIV-1 with modest potency by recognizing the gp120 421-433 epitope, a conserved B cell superantigenic region that is also essential for HIV-1 attachment to host cell CD4 receptors. An adaptive immune response to superantigens is generally prohibited due to their ability to downregulate B cells. IgAs from subjects with prolonged HIV-1 infection displayed improved catalytic hydrolysis of gp120 and exceptionally potent and broad neutralization of diverse CCR5-dependent primary HIV isolates attributable to recognition of the 421-433 epitope. This indicates that slow immunological bypass of the superantigenic character of gp120 is possible, opening the path to effective HIV vaccination. ^ My research reveals a novel route to inactivate pathogenic nucleophilic Abs using electrophilic antigens. Conversely, naturally occurring nucleophilic Abs may help impede HIV infection, and the Abs could be developed for passive immunotherapy of HIV infected subjects. ^

Prevalence and determinants of hepatitis B co-infection among a United States Veterans Administration cohort with hepatitis C

Background and aim. Hepatitis B virus (HBV) and hepatitis C virus (HCV) co-infection is associated with increased risk of cirrhosis, decompensation, hepatocellular carcinoma, and death. Yet, there is sparse epidemiologic data on co-infection in the United States. Therefore, the aim of this study was to determine the prevalence and determinants of HBV co-infection in a large United States population of HCV patients. ^ Methods. The National Veterans Affairs HCV Clinical Case Registry was used to identify patients tested for HCV during 1997–2005. HCV exposure was defined as two positive HCV tests (antibody, RNA or genotype) or one positive test combined with an ICD-9 code for HCV. HCV infection was defined as only a positive HCV RNA or genotype. HBV exposure was defined as a positive test for hepatitis B core antibodies, hepatitis B surface antigen, HBV DNA, hepatitis Be antigen, or hepatitis Be antibody. HBV infection was defined as only a positive test for hepatitis B surface antigen, HBV DNA, or hepatitis Be antigen within one year before or after the HCV index date. The prevalence of exposure to HBV in patients with HCV exposure and the prevalence of HBV infection in patients with HCV infection were determined. Multivariable logistic regression was used to identify demographic and clinical determinants of co-infection. ^ Results. Among 168,239 patients with HCV exposure, 58,415 patients had HBV exposure for a prevalence of 34.7% (95% CI 34.5–35.0). Among 102,971 patients with HCV infection, 1,431 patients had HBV co-infection for a prevalence of 1.4% (95% CI 1.3–1.5). The independent determinants for an increased risk of HBV co-infection were male sex, positive HIV status, a history of hemophilia, sickle cell anemia or thalassemia, history of blood transfusion, cocaine and other drug use. Age >50 years and Hispanic ethnicity were associated with a decreased risk of HBV co-infection. ^ Conclusions. This is the largest cohort study in the United States on the prevalence of HBV co-infection. Among veterans with HCV, exposure to HBV is common (∼35%), but HBV co-infection is relatively low (1.4%). There is an increased risk of co-infection with younger age, male sex, HIV, and drug use, with decreased risk in Hispanics.^

Pathway Semantics: An Algebraic Data Driven Algorithm to Generate Hypotheses about Molecular Patterns Underlying Disease Progression

The overarching goal of the Pathway Semantics Algorithm (PSA) is to improve the in silico identification of clinically useful hypotheses about molecular patterns in disease progression. By framing biomedical questions within a variety of matrix representations, PSA has the flexibility to analyze combined quantitative and qualitative data over a wide range of stratifications. The resulting hypothetical answers can then move to in vitro and in vivo verification, research assay optimization, clinical validation, and commercialization. Herein PSA is shown to generate novel hypotheses about the significant biological pathways in two disease domains: shock / trauma and hemophilia A, and validated experimentally in the latter. The PSA matrix algebra approach identified differential molecular patterns in biological networks over time and outcome that would not be easily found through direct assays, literature or database searches. In this dissertation, Chapter 1 provides a broad overview of the background and motivation for the study, followed by Chapter 2 with a literature review of relevant computational methods. Chapters 3 and 4 describe PSA for node and edge analysis respectively, and apply the method to disease progression in shock / trauma. Chapter 5 demonstrates the application of PSA to hemophilia A and the validation with experimental results. The work is summarized in Chapter 6, followed by extensive references and an Appendix with additional material.

AN ASSESSMENT OF KNOWLEDGE AND ATTITUDES OF GENETIC COUNSELING SERVICES IN U.S. HTCs

Hemophilia is a hereditary bleeding disorder which requires lifelong specialized care. A network of Hemophilia Treatment Centers (HTCs) exists to meet the medical needs of patients affected by hemophilia. Genetic counseling services are an integral part of the HTC model of care; however, many HTCs do not have genetic counselors on staff. As a result, the duty to provide these services must fall to other healthcare providers within the HTC. To assess the knowledge and attitudes of these providers we developed a 49 question survey that was distributed electronically to hematologists and nurses at U.S. HTCs. The survey consisted of a three sections: demographic information, knowledge of hemophilia genetics, and attitudes towards genetic services. A total of 111 complete responses were received and analyzed. The average knowledge score among all participants was 74.8% with a total of 81 participants receiving a passing score of 70% or above. Thirty participants scored below 70% in the knowledge section. In general, attitude scores were high indicating that the majority of hematologists and nurses in HTCs feel confident in their ability to provide genetic counseling services. Over 90% of participants reported that they have some form of access to genetic counseling services at their center. Hematologists and nurses practicing in U.S. HTCs demonstrate sufficient knowledge of the genetics of hemophilia, and they generally feel confident in their ability to provide genetic counseling services to their patients. While their knowledge is sufficient, the average knowledge score was lower than 75%. Certain questions covering new genetic technologies and testing practices were more commonly missed than questions asking about more basic aspects of hemophilia genetics, such as inheritance and carrier testing. Finally, many clinics report having access to a counselor, but it is oftentimes a hematologist or nurse who is providing genetic counseling services to patients. Given the inconsistency in knowledge among providers coupled with the high confidence in one’s ability to counsel patients, it leaves room to question whether information about the genetics of hemophilia is being communicated to patients in the most appropriate and accurate manner.

Coexpression of factor VIII heavy and light chain adeno-associated viral vectors produces biologically active protein

We are interested in using recombinant adeno-associated viral vectors in the treatment of hemophilia A. Because of the size constraints of recombinant adeno-associated viral vectors, we delivered the heavy and light chains of the human factor 8 (hFVIII) cDNA independently by using two separate vectors. Recombinant AAV vectors were constructed that utilized the human elongation factor 1α promoter, a human growth factor polyadenylation signal, and the cDNA sequences encoding either the heavy or light chain of hFVIII. Portal vein injections of each vector alone, a combination of both vectors, or a hFIX control vector were performed in C57BL/6 mice. An ELISA specific for the light chain of hFVIII demonstrated very high levels (2–10 μg/ml) of protein expression in animals injected with the light chain vector alone or with both vectors. We utilized a chromogenic assay in combination with an antibody specific to hFVIII to determine the amount of biologically active hFVIII in mouse plasma. In animals injected with both the heavy and light chain vectors, greater than physiological levels (200–400 ng/ml) of biologically active hFVIII were produced. This suggests that coexpression of the heavy and light chains of hFVIII may be a feasible approach for treatment of hemophilia A.

Characterization of a genetically engineered inactivation-resistant coagulation factor VIIIa

Individuals with hemophilia A require frequent infusion of preparations of coagulation factor VIII. The activity of factor VIII (FVIII) as a cofactor for factor IXa in the coagulation cascade is limited by its instability after activation by thrombin. Activation of FVIII occurs through proteolytic cleavage and generates an unstable FVIII heterotrimer that is subject to rapid dissociation of its subunits. In addition, further proteolytic cleavage by thrombin, factor Xa, factor IXa, and activated protein C can lead to inactivation. We have engineered and characterized a FVIII protein, IR8, that has enhanced in vitro stability of FVIII activity due to resistance to subunit dissociation and proteolytic inactivation. FVIII was genetically engineered by deletion of residues 794-1689 so that the A2 domain is covalently attached to the light chain. Missense mutations at thrombin and activated protein C inactivation cleavage sites provided resistance to proteolysis, resulting in a single-chain protein that has maximal activity after a single cleavage after arginine-372. The specific activity of partially purified protein produced in transfected COS-1 monkey cells was 5-fold higher than wild-type (WT) FVIII. Whereas WT FVIII was inactivated by thrombin after 10 min in vitro, IR8 still retained 38% of peak activity after 4 hr. Whereas binding of IR8 to von Willebrand factor (vWF) was reduced 10-fold compared with WT FVIII, in the presence of an anti-light chain antibody, ESH8, binding of IR8 to vWF increased 5-fold. These results demonstrate that residues 1690–2332 of FVIII are sufficient to support high-affinity vWF binding. Whereas ESH8 inhibited WT factor VIII activity, IR8 retained its activity in the presence of ESH8. We propose that resistance to A2 subunit dissociation abrogates inhibition by the ESH8 antibody. The stable FVIIIa described here provides the opportunity to study the activated form of this critical coagulation factor and demonstrates that proteins can be improved by rationale design through genetic engineering technology.

Successful use of recombinant factor VII in massive hemoptysis due to community-acquired pneumonia

Recombinant activated factor VII (rFVIIa) is a powerful hemostatic agent developed for use in hemophilia. It has been used increasingly in life-threatening hemorrhage in a variety of other settings in which conventional medical or surgical therapy is unsuccessful. This report describes the successful use of rFVIIa for pulmonary hemorrhage due to a focal bleeding source in a regional hospital where bronchial artery embolization or surgery were not available. rFVIIa may be a useful temporizing measure in the unstable patient with pulmonary hemorrhage without coagulopathic bleeding when conventional treatment. is not immediately available.

Delivery and Scavenging of Nucleic Acids by Polycationic Polymers

Electrostatic interaction is a strong force that attracts positively and negatively charged molecules to each other. Such an interaction is formed between positively charged polycationic polymers and negatively charged nucleic acids. In this dissertation, the electrostatic attraction between polycationic polymers and nucleic acids is exploited for applications in oral gene delivery and nucleic acid scavenging. An enhanced nanoparticle for oral gene delivery of a human Factor IX (hFIX) plasmid is developed using the polycationic polysaccharide, chitosan (Ch), in combination with protamine sulfate (PS) to treat hemophilia B. For nucleic acid scavenging purposes, the development of an effective nucleic acid scavenging nanofiber platform is described for dampening hyper-inflammation and reducing the formation of biofilms.

Non-viral gene therapy may be an attractive alternative to chronic protein replacement therapy. Orally administered non-viral gene vectors have been investigated for more than one decade with little progress made beyond the initial studies. Oral administration has many benefits over intravenous injection including patient compliance and overall cost; however, effective oral gene delivery systems remain elusive. To date, only chitosan carriers have demonstrated successful oral gene delivery due to chitosan’s stability via the oral route. In this study, we increase the transfection efficiency of the chitosan gene carrier by adding protamine sulfate to the nanoparticle formulation. The addition of protamine sulfate to the chitosan nanoparticles results in up to 42x higher in vitro transfection efficiency than chitosan nanoparticles without protamine sulfate. Therapeutic levels of hFIX protein are detected after oral delivery of Ch/PS/phFIX nanoparticles in 5/12 mice in vivo, ranging from 3 -132 ng/mL, as compared to levels below 4 ng/mL in 1/12 mice given Ch/phFIX nanoparticles. These results indicate the protamine sulfate enhances the transfection efficiency of chitosan and should be considered as an effective ternary component for applications in oral gene delivery.

Dying cells release nucleic acids (NA) and NA-complexes that activate the inflammatory pathways of immune cells. Sustained activation of these pathways contributes to chronic inflammation related to autoimmune diseases including systemic lupus erythematosus, rheumatoid arthritis, and inflammatory bowel disease. Studies have shown that certain soluble, cationic polymers can scavenge extracellular nucleic acids and inhibit RNA-and DNA-mediated activation of Toll-like receptors (TLRs) and inflammation. In this study, the cationic polymers are incorporated onto insoluble nanofibers, enabling local scavenging of negatively charged pro-inflammatory species such as damage-associated molecular pattern (DAMP) molecules in the extracellular space, reducing cytotoxicity related to unwanted internalization of soluble cationic polymers. In vitro data show that electrospun nanofibers grafted with cationic polymers, termed nucleic acid scavenging nanofibers (NASFs), can scavenge nucleic acid-based agonists of TLR 3 and TLR 9 directly from serum and prevent the production of NF-ĸB, an immune system activating transcription factor while also demonstrating low cytotoxicity. NASFs formed from poly (styrene-alt-maleic anhydride) conjugated with 1.8 kDa branched polyethylenimine (bPEI) resulted in randomly aligned fibers with diameters of 486±9 nm. NASFs effectively eliminate the immune stimulating response of NA based agonists CpG (TLR 9) and poly (I:C) (TLR 3) while not affecting the activation caused by the non-nucleic acid TLR agonist pam3CSK4. Results in a more biologically relevant context of doxorubicin-induced cell death in RAW cells demonstrates that NASFs block ~25-40% of NF-ĸβ response in Ramos-Blue cells treated with RAW extracellular debris, ie DAMPs, following doxorubicin treatment. Together, these data demonstrate that the formation of cationic NASFs by a simple, replicable, modular technique is effective and that such NASFs are capable of modulating localized inflammatory responses.

An understandable way to clinically apply the NASF is as a wound bandage. Chronic wounds are a serious clinical problem that is attributed to an extended period of inflammation as well as the presence of biofilms. An NASF bandage can potentially have two benefits in the treatment of chronic wounds by reducing the inflammation and preventing biofilm formation. NASF can prevent biofilm formation by reducing the NA present in the wound bed, therefore removing large components of what the bacteria use to develop their biofilm matrix, the extracellular polymeric substance, without which the biofilm cannot develop. The NASF described above is used to show the effect of the nucleic acid scavenging technology on in vitro and in vivo biofilm formation of P. aeruginosa, S. aureus, and S. epidermidis biofilms. The in vitro studies demonstrated that the NASFs were able to significantly reduce the biofilm formation in all three bacterial strains. In vivo studies of the NASF on mouse wounds infected with biofilm show that the NASF retain their functionality and are able to scavenge DNA, RNA, and protein from the wound bed. The NASF remove DNA that are maintaining the inflammatory state of the open wound and contributing to the extracellular polymeric substance (EPS), such as mtDNA, and also removing proteins that are required for bacteria/biofilm formation and maintenance such as chaperonin, ribosomal proteins, succinyl CoA-ligase, and polymerases. However, the NASF are not successful at decreasing the wound healing time because their repeated application and removal disrupts the wound bed and removes proteins required for wound healing such as fibronectin, vibronectin, keratin, and plasminogen. Further optimization of NASF treatment duration and potential combination treatments should be tested to reduce the unwanted side effects of increased wound healing time.

Design of a myofascial therapy protocol for the treatment of hemophilic arthropathy of the knee and ankle

Hemophilic arthropathy limits daily life activities of patients with hemophilia, presenting with clinical manifestations such as chronic pain, limited mobility, or muscular atrophy. Although physical therapy is considered essential for these patients, few clinical studies have demonstrated the efficacy and safety of the various physiotherapy techniques. Physical therapy may be useful for treating hemophilic arthropathy by applying safe and effective techniques. However, it is necessary to create protocols for possible treatments to avoid the risk of bleeding in these patients. This article describes the musculoskeletal pathology of hemophilic arthropathy and characteristics of fascial therapy. This systematic protocol for treatment by fascial therapy of knee and ankle arthropathy in patients with hemophilia provides an analysis of the techniques that, depending on their purpose and methodology, can be used in these patients. Similarly, the protocol's applicability is analyzed and the steps to be followed in future research studies are described. Fascial therapy is a promising physiotherapy technique for treating fascial tissue and joint contractures in patients with hemophilic arthropathy. More research is needed to assess the efficacy and safety of this intervention in patients with hemophilia, particularly with randomized multicenter clinical trials