Origins, Development, Current Challenges and Future Directions with Activated Prothrombin Complex Concentrate for the Treatment of Patients with Congenital Haemophilia with Inhibitors.

Congenital haemophilia A (HA) is caused by deficiency of coagulation factor VIII (FVIII) activity, leading to spontaneous or traumatic bleeding events. While FVIII replacement therapy can treat and prevent bleeds, approximately 30% of patients with severe HA develop inhibitor antibodies that render FVIII replacement therapy ineffective. The bypassing agents (BPAs), activated prothrombin complex concentrate (aPCC) and recombinant activated FVII, first approved in 1977 and 1996, respectively, act to generate thrombin independent of pathways that involve factors IX and VIII. Both may be used in patients with congenital haemophilia and inhibitors (PwHIs) for the treatment and prevention of acute bleeds and quickly became standard of care. However, individual patients respond differently to different agents. While both agents are approved for on-demand treatment and perioperative management for patients with congenital haemophilia with inhibitors, aPCC is currently the only BPA approved worldwide for prophylaxis in PwHI. Non-factor therapies (NFTs) have a mechanism of action distinct from BPAs and have reported higher efficacy rates as prophylactic regimens. Nonetheless, treatment challenges remain with NFTs, particularly regarding the potential for synergistic action on thrombin generation with concomitant use of other haemostatic agents, such as BPAs, for the treatment of breakthrough bleeds and in perioperative management. Concomitant use of NFTs with other haemostatic agents could increase the risk of adverse events such as thromboembolic events or thrombotic microangiopathy. This review focuses on the origins, development and on-going role of aPCC in the evolving treatment landscape in the management of PwHI.

Fifteen years of env C2V3C3 evolution in six individuals infected clonally with human immunodeficiency virus type 1.

The study of the evolution of human immunodeficiency virus type 1 (HIV-1) requires blood samples collected longitudinally and data on the approximate time point of infection. Although these requirements were fulfilled in several previous studies, the infectious sources were either unknown or heterogeneous genetically. In the present study, HIV-1 env C2V3C3 (nt 7029-7315) evolution was examined retrospectively in a cohort of hemophiliacs. Compared to other cohorts, the area of interest here was the infection of six hemophiliacs by the same virus strain, that is, the infecting viruses shared an identical genome. As expected, divergence from the founder sequence as well as interpatient divergence of the predominant virus strains increased significantly over time. Based on the V3 nucleotide sequences, CCR5 usage was predicted exclusively throughout the whole period of infection in all patients. Interestingly, common patterns of viral evolution were detected in the patients of the cohort. Four amino acid substitutions within the V3 loop emerged and persisted subsequently in five (positions 305 and 308 of the HXB2 gp120 reference sequence) and six patients (positions 325 and 328 in HXB2 gp120), respectively. These common changes within the V3 loop are likely to be enforced by HIV-1 specific immune response.

Lack of F8 mRNA: a novel mechanism leading to hemophilia A.

Hemophilia A (HA) is caused by partial or total deficiency of F8 protein activity. In a small group, about 1.8% of patients with HA, no mutation is found in the F8 gene. Among this group, we report here on one patient with severe HA in whom no mRNA of the F8 gene was detected. Using 2 common polymorphisms in F8 exon 14, we were able to show that the same allele shared by the patient, his mother, and his sister was not detected by reverse transcription-polymerase chain reaction (RT-PCR) from total blood mRNA. Skewed X-chromosome inactivation in both the mother and the sister was excluded by studying the methylation profile of the androgen receptor gene (HUMARA locus). These findings strongly suggest that the cause of HA in this patient is either absence or rapid degradation of the F8 mRNA, which points to a novel mechanism leading to HA.

Hepatitis C-associated autoimmunity in patients coinfected with HIV.

BACKGROUND: Hepatitis C virus (HCV) infection is associated with multiple extrahepatic manifestations. It is unclear to what extent extrahepatic manifestations occur in HIV/HCV coinfection. METHODS: We prospectively assessed cross-sectional frequencies of autoimmune manifestations in HIV/HCV-coinfected patients (n=98), HIV-mono-infected (n=45) and HCV-mono-infected patients (n=78). Diagnostic vasculitis scores, HCV and HIV loads, CD4 cell counts, thyroid-, cardiolipin-, non-organ-specific tissue antibodies (nuclear, smooth muscle, anti-liver-kidney-microsome, neutrophil-cytoplasmic) and cryoglobulins were determined. RESULTS: Synergistic effects of HCV and HIV infection were observed with respect to the prevalence of antibodies against thyroglobulin (HCV infection 15.4%, HIV infection 8.8%, HIV/HCV coinfection 30.6%; P<0.001) and cardiolipin antibodies (HCV infection 9.0%, HIV infection 31%, HIV/HCV coinfection 46%; P<0.001). Cryoglobulinemia type III, was significantly associated with HCV infection (HCV, 25.6%; HIV/HCV, 20.4%) but not with HIV infection (4.4%, P<0.05). Rheumatoid factor was commonly detected in patients with HCV infection (48%), but occurred considerably less frequently in patients with HIV infection (4.4%) or HIV/HCV coinfection (9.5%, P<0.01). CONCLUSION: HIV coinfection appears to differentially modulate the frequency of HCV-related autoimmunity. However, autoimmunity is rarely accompanied by clinical manifestations.

Comparison of GB virus C, HIV, and HCV infection markers in hemophiliacs exposed to non-inactivated or inactivated factor concentrates.

BACKGROUND: Until the mandatory introduction of viral inactivation techniques of blood plasma products in the early 1980s many recipients of these products were infected with various viral pathogens. OBJECTIVES: To determine the rate of transmission of GB virus C/hepatitis G virus (GBV-C/HGV) HCV, and HIV through non-virus-inactivated clotting factor concentrates in hemophiliacs, as well as the relation between amount of administered clotting factor and risk for GBV-C/HGV infection. STUDY DESIGN: In this cross-sectional study, we determined retrospectively the rates of infection markers for GBV-C/HGV, HCV, and HIV in a German cohort of hemophiliacs treated with documented amounts of non-virus-inactivated clotting factor concentrates (group A) and in a second group of hemophiliacs who were treated exclusively with virus-inactivated clotting factor (group B). The presence of anti-virus antibodies was determined by ELISA. Viral RNA was detected by RT-PCR. Markers for viral infections were compared to amounts of administered non-virus-inactivated clotting factor. RESULTS: Among hemophiliacs treated with documented amounts of non-virus-inactivated clotting factor the prevalence for GBV-C/HGV, HCV, and HIV was 40.3%, 98.6%, and 56.3%, respectively. In contrast to HIV, the rate of GBV-C/HGV infections did not increase with increasing amounts of consumed non-inactivated clotting factor. Even in the subgroup of heavily treated hemophiliacs the rate of GBV-C/HGV infection markers did not exceed 45%. CONCLUSIONS: The amount of non-virus-inactivated clotting factor is not predictive for the risk of GBV-C/HGV infection in hemophiliacs. Despite repeated parenteral exposure more than 55% of hemophiliacs were not infected with GBV-C/HGV. Our findings indicate a high frequency of host factors preventing parenteral transmission of GBV-C/HGV.

Serum antibodies against the hepatitis C virus E2 protein mediate antibody-dependent cellular cytotoxicity (ADCC).

BACKGROUND/AIMS: The role of antibody dependent cellular cytotoxicity (ADCC) in HCV infection is unclear at present. Antibodies mediating ADCC are usually directed against viral envelope proteins. As cell surface expression of the HCV envelope E2 protein has been shown, the HCV E2 protein is an especially promising candidate target for ADCC. METHODS: Sera from patients with acute (n=6), self-limited (n=11) and chronic (n=19) HCV infection were analyzed in this study. Sera reacting with cell-bound HCV antigens were examined in a flowcytometric cytotoxicity assay using antigen-coated JOK-1 cells as targets. RESULTS: We found that sera from all stages of HCV infection reacted with cells loaded with HCV E2. E2-specific ADCC was observed in patients with acute (n=3/6), self-limited (n=5/11) and chronic (n=13/19) hepatitis C and was closely related to fluorescence intensity in the E2-binding assay (r=0.67, P<0.001). CONCLUSIONS: We conclude that E2-antibodies from all stages of HCV infection can mediate ADCC. Thus, the role of this process in the pathogenesis of chronic hepatitis C should be further elucidated.

Distribution and effects of polymorphic RANTES gene alleles in HIV/HCV coinfection -- a prospective cross-sectional study.

AIM: Chemokines and their receptors are crucial for immune responses in HCV and HIV infection. RANTES gene polymorphisms lead to altered gene expression and influence the natural course of HIV infection. Therefore, these mutations may also affect the course of HIV/HCV coinfection. METHODS: We determined allele frequencies of RANTES-403 (G --> A), RANTES-28 (C --> G) and RANTES-IN1.1 (T --> C) polymorphisms using real-time PCR and hybridization probes in patients with HIV (n = 85), HCV (n = 112), HIV/HCV coinfection (n = 121), and 109 healthy controls. Furthermore, HIV and HCV loads as well as CD4(+) and CD8(+) cell counts were compared between different RANTES genotypes. RESULTS: Frequencies of RANTES-403 A, RANTES-28 G and RANTES-IN1.1 C alleles were higher in HIV infected patients than in healthy controls (-403: 28.2% vs 15.1%, P = 0.002; -28: 5.4% vs 2.8%, not significant; IN1.1: 19.0% vs 11.0%, P = 0.038). In HIV/HCV coinfected patients, these RANTES alleles were less frequent than in patients with HIV infection alone (15.4% P = 0.002; 1.7%; P = 0.048; 12.0%; not significant). Frequencies of these alleles were not significantly different between HIV/HCV positive patients, HCV positive patients and healthy controls. CONCLUSION: All three RANTES polymorphisms showed increased frequencies of the variant allele exclusively in patients with HIV monoinfection. The finding that the frequencies of these alleles remained unaltered in HIV/HCV coinfected patients suggests that HCV coinfection interferes with selection processes associated with these alleles in HIV infection.

Effects of the CCR5-Delta32 mutation on antiviral treatment in chronic hepatitis C.

BACKGROUND/AIMS: The CC-chemokine receptor (CCR) 5-Delta32 mutation may predispose to chronic liver disease and high level viremia in hepatitis C. However, it is unclear whether CCR5-Delta32 also affects the response to antiviral treatment. METHODS: We determined CCR5 genotypes in patients with hepatitis C treated with either interferon-alpha (N=78) or interferon and ribavirin (N=78). In each group, rates of end of treatment responses (ETRs) and sustained virological responses (SVRs) were compared between CCR5-Delta32 carriers and homozygous CCR5 wildtype patients. RESULTS: ETR and SVR were achieved in 25 and 12 patients with interferon-alpha and in 52 and 45 patients with interferon/ribavirin treatment, respectively. CCR5-Delta32 carriers had significantly lower ETR rates than homozygous CCR5 wildtype patients (10.5 vs. 39.0%; P=0.02), whereas SVR rates only showed a non-significant trend (5.3 vs. 18.6%). Multivariate analysis confirmed CCR5-Delta32 carriage as an independent negative predictor for ETR in interferon-alpha monotherapy (odds ratio: 0.16; 95% confidence limits: 0.032-0.82; P=0.03). In interferon/ribavirin treated patients CCR-Delta32 carriers and CCR5 wildtype patients had similar ETR rates [19.2% vs. 23.1%] and SVR rates [20.0% vs. 21.2%]. CONCLUSIONS: Response rates to interferon-alpha monotherapy are reduced in hepatitis C virus (HCV)-infected patients carrying the CCR5-Delta32 mutation. However, interferon/ribavirin combination treatment may overcome this negative effect of CCR5-Delta32.

Differential expansion of T-cell receptor variable beta subsets after antigenic stimulation in patients with different outcomes of hepatitis C infection.

Persistent antigenic stimulation during chronic hepatitis C may alter the T-cell receptor variable chain beta (TCR BV) repertoire as well as the cytokine responses of hepatitis C virus (HCV)-specific T lymphocytes. We analysed the distribution of the TCR BV subsets 2.1, 3.1, 5.1, 6.1, 8, 13.1, 13.6, 14.1, 17.1, 21.3 in relation to intracytoplasmic expression of interleukin-2, interferon-gamma, interleukin-4 and interleukin-10. Using flow cytometry, CD45RO+ memory T cells of 27 patients with chronic hepatitis C, eight patients with resolved HCV infection and 16 non-HCV-related controls were studied with and without stimulation by the HCV core, NS3, NS4, NS5a and NS5b proteins. Patients with chronic and resolved hepatitis C differed by larger basal TCR BV2.1+, BV6.1+, BV17.1+ and BV21.3+ subsets in chronic hepatitis C, which were correlated to the numbers of T cells with spontaneous interleukin-2 and interferon-gamma production (r=0.51-0.73, P<0.05). Upon HCV-specific stimulation these subsets did not expand, whereas a marked in vitro expansion of TCR BV8+ T cells in response to all HCV proteins was selectively noted in chronic hepatitis C (P<0.05). This expansion of TCR BV8+ memory T cells was significantly correlated to HCV-induced interleukin-10 expression (r=0.58-0.98, P<0.01). Thus, differential involvement of selected TCR BV subsets may be related to the outcome of HCV infection.

Risk of hepatitis C after immunoadsorption.

An episode of acute hepatitis in a patient with hemophilia during immunoadsorption therapy initially was misinterpreted as a reactivated hepatitis C virus (HCV) infection, but ultimately was shown to be an exogenous reinfection during cohort treatment with another HCV-positive patient. This incident illustrates that policies for the prevention of nosocomial transmission of blood-borne pathogens, especially in cohort treatment units, may need to be reassessed.

Frequency of the HIV-protective CC chemokine receptor 5-Delta32/Delta32 genotype is increased in hepatitis C.

BACKGROUND & AIMS: A homozygous 32-base pair deletion in the CCR5 gene (CCR5-Delta32) protects against human immunodeficiency virus infection (HIV). However, the role of this mutation in other infections, such as hepatitis C virus (HCV) infection, has not been defined. METHODS: We determined the frequency of the CCR5-Delta32 mutation by polymerase chain reaction in anti-HCV(+) (n = 153), anti-HIV(+) (n = 102), and anti-HCV(+)/HIV(+) (n = 130) white patients as well as in 102 healthy blood donors. Then, HIV and HCV loads, aminotransferases, and CD4 and CD8 cell counts were compared between the resulting subsets of CCR5-Delta32/wild-type heterozygotes, CCR5-Delta32, and wild-type homozygotes, respectively. RESULTS: Twelve of 153 (7.8%) anti-HCV-seropositive patients and 1 of 102 (1.0%) healthy blood donors were CCR5-Delta32 homozygous, whereas CCR5-Delta32 homozygosity was absent in anti-HIV(+) and anti-HCV(+)/HIV(+) patients (P < 0.001). The frequency of the CCR5-Delta32 allele was higher in the anti-HCV(+) (16.0%, P < 0.05) and anti-HCV(+)/HIV(+) (12.7%, NS) patients than in healthy blood donors (8.3%) and anti-HIV(+) patients (9.3%), respectively. Anti-HCV(+) CCR5-Delta32 homozygotes occurred 3 times more frequently than expected from the Hardy-Weinberg equation (P < 0.0001) and had significantly higher HCV loads than wild-type patients (P = 0.045). CONCLUSIONS: The increased prevalence of CCR5-Delta32 homozygosity associated with increased viral loads in patients with chronic hepatitis C suggests that the CCR5-Delta32 mutation may be an adverse host factor in hepatitis C.

HCV-specific cytokine induction in monocytes of patients with different outcomes of hepatitis C.

AIM: Cytokine release by macrophages critically determines the type of immune response to an antigen. Therefore, we studied hepatitis C virus (HCV)-specific induction of interleukins-1 beta, -10, -12 (IL-1 beta, IL-10, IL-12), and tumor necrosis factor-alpha (TNF-alpha) in monocytes. METHODS: Intracellular cytokine expression was studied by flow cytometry in 23 patients with chronic hepatitis C, 14 anti-HCV seropositives without viremia and 11 controls after stimulation of peripheral blood mononuclear cells with recombinant core, NS3, NS4, NS5a and NS5b proteins. RESULTS: Patients with HCV viremia revealed greater spontaneous expression of IL-1beta, TNF-alpha, and IL-10. Furthermore, greater than twofold higher IL-10 expression was induced by the HCV antigens in chronic hepatitis C than in the other two groups (P<0.05). In contrast, neither IL-12 nor TNF-alpha was induced preferentially. CONCLUSION: In chronic hepatitis C antigen-specific cytokine induction in monocytes is apparently shifted towards predominant IL-10 induction - not counterbalanced by antiviral type 1 cytokines. This may contribute to persistent viral replication.