Failure of rituximab to induce immune tolerance in a boy with severe haemophilia A and an alloimmune factor VIII antibody: a case report and review of the literature.

We report the use of rituximab (MabThera); Roche Grenzach-Wyhlen, Germany) in a 6-year-old boy with severe haemophilia A and a high titre alloimmune factor VIII (FVIII) antibody, which had failed to respond to standard immune tolerance therapy. Rituximab was administered in 4 weekly doses with concurrent high-dose i.v. immunoglobulin (Flebogamma); Grifols, Barcelona, Spain) followed by daily high-dose recombinant FVIII concentrate (Recombinate); Baxter, CA, USA). Despite a fall in CD20 positive cell count to undetectable levels the inhibitor persisted. We discuss the possible reasons for failure of immune tolerance induction and review the literature concerning the use of rituximab for this indication.

Recombinant activated factor VII (rFVIIa/NovoSeven) in intractable haemorrhage: use of a clinical scoring system to predict outcome.

BACKGROUND AND OBJECTIVES: Recombinant activated factor VII (rFVIIa/NovoSeven) has been advocated in the treatment of life-threatening haemorrhage, but appropriate clinical indications remain uncertain. The aim of this study was to detect factors predictive of outcome and to incorporate them into a prognostically significant scoring system. MATERIALS AND METHODS: Thirty-six patients received rFVIIa for uncontrolled surgical, traumatic or obstetric bleeding in the Northern Region of the UK over a 45-month period. Clinical, laboratory and outcome data were examined. Characteristics of survivor and non-survivor groups were compared. A prognostic scoring system was evaluated retrospectively according to the presence of coagulopathy, renal impairment, hypothermia, greater than 10 units of red cell transfusion, advanced age and obstetric indication, with patients allocated to low, intermediate and high-risk groups. RESULTS: Clinical response occurred in 26 patients (72%) with a reduction in prothrombin time and blood product requirements. Death occurred in 19 (53%). Four patients (11%) suffered thrombotic events. Survivors were younger than non-survivors and less likely to have coagulopathy, renal impairment or hypothermia at the time of administration. Survivors were more likely to have had an initial clinical response in terms of an immediate reduction in haemorrhage. Non-survivors were transfused a greater number of red cell units prior to administration. Survival varied according to prognostic score; low-risk patients had a survival rate of 85%, intermediate-risk patients had a survival rate of 50% and high-risk patients had a survival rate of 18%. CONCLUSIONS: FVIIa has a role in the cessation of haemorrhage, but may not improve survival. Use of a clinical scoring system may help to predict outcome.

Warfarin reversal.

Warfarin is the most commonly used oral anticoagulant in the UK. It is associated with few side effects apart from haemorrhage. The most appropriate way to reverse the anticoagulant effect of warfarin depends on the clinical circumstances. In serious bleeding, rapid reversal is required, whereas in minor bleeding or asymptomatic over anticoagulation, a more leisurely approach is usually appropriate. This review discusses the current approaches to warfarin reversal in clinical practice. The development of a uniform approach to warfarin reversal in the Northern Region is described.

Successful high dose therapy for relapsed mediastinal large B cell lymphoma following surgical repair of anterior chest wall defect.

We describe a man with relapsed large B cell mediastinal lymphoma and associated infected large anterior chest wall defect who required high dose salvage therapy for his underlying disease. An initial mediastinotomy wound, associated with recurrent sepsis, had developed into an abscess, then fistula and eventually a large anterior chest wall defect. Safe use of salvage chemotherapy required reconstructive surgery consisting of a pedicled muscle flap. The subsequent high dose chemotherapy was carried out without complications and 15 months later the patient is alive and well.

Hemorrhagic lymphadenopathy as a presenting feature of primary al amyloidosis.

Lymphadenopathy associated with hemorrhage as a presenting feature of primary (AL) amyloidosis has not previously been described. We report two such cases one of whom had an acquired factor X and IX deficiency. The clinical presentations were characterized by sudden spontaneous enlargement of lymph nodes followed by partial regression. In both cases significant delay in diagnosis, and hence treatment, occurred due to the mode of presentation. One patient died with rapidly progressive disease but the other has had an excellent response to therapy with high-dose melphalan (HDM, 200 mg/m2) and peripheral blood stem cell rescue. AL amyloid should be considered in all patients presenting with hemorrhagic lymphadenopathy.

Safety, efficacy and cost-effectiveness of home therapy with recombinant activated factor VII in a patient with severe haemophilia A and an anti-factor VIII inhibitor.

We report the use of recombinant activated factor VII (rFVIIa; NovoSeven, Novo Nordisk, Bagsvaerd, Denmark) in a patient with severe haemophilia A and a high-titre anti-factor VIII inhibitor. A home therapy protocol was devised and its cost and efficacy were assessed after 2 years. rFVIIa (90 microg/kg) was self-administered at the onset of symptoms of bleeding. Doses were subsequently repeated at 2-h intervals until adequate haemostasis was achieved. The patient experienced a total of 25 bleeding episodes over the 2-year period, of which 23 were treated with up to three doses of rFVIIa. On two occasions, there was a recurrence of bleeding in the same area within 24 h, necessitating retreatment. There was a reduction in the number of inpatient hospital days compared with the 2 years before the use of rFVIIa and, overall, the cost of rFVIIa treatment was less than that of the previously used activated prothrombin complex concentrate (FEIBA; Immuno Ltd, Vienna, Austria). Our experience suggests that in selected haemophiliacs with inhibitors, home treatment with rFVIIa may be safe and cost-effective.

The biology of interferon-alpha and the clinical significance of anti-interferon antibodies.

The therapeutic indications for Interferons (IFNs) have dramatically increased in number in recent years to include many different diseases of viral, malignant, angiogenic, allergic, inflammatory and fibrotic origin. In particular, the current pandemic of hepatitis C virus infection has further stimulated the requirement for a comprehensive understanding of both the mechanism of action of IFN and the reasons for therapeutic failure. The role of anti-IFN antibodies as a cause of treatment failure has been a particularly controversial area. In this review we will outline the biology and proposed mechanisms of action of IFN-alpha (IFN-alpha) and discuss the incidence, methods of detection and clinical significance of anti-IFN antibodies.

Patterns of hepatitis G viraemia and liver disease in haemophiliacs previously exposed to non-virus inactivated coagulation factor concentrates.

Hepatitis G virus (HGV), a novel flavivirus, has been implicated as a cause of posttransfusion hepatitis. We have performed a longitudinal study in a cohort of haemophiliacs (n = 68) who previously received non-virus inactivated coagulation factor concentrates to assess both patterns of HGV viraemia and any associated liver disease. Hepatitis C virus (HCV) RNA was present in 58/68 and co-infection with human immunodeficiency virus (HIV) was present in 15/68. HGV RNA was detected in 17/68 (25%) samples from the mid-1980s. There was no association between either HIV infection (p = 0.74) or co-infection with a particular HCV genotype (p = 0.62). However, there was a relationship between HGV viraemia and the severity of haemophilia (p = 0.0004) with HGV RNA detected in 5/19, 9/16 and 3/32 patients with mild, moderate and severe haemophilia respectively. A longitudinal study was performed in 15/17 haemophiliacs with HGV viraemia using stored serum samples from the 1980s and 1990s. HGV viraemia persisted in 8/15 and cleared in 7/15 over a variable period of time. A Weibull model was constructed to estimate the duration of HGV viraemia in the study group. The 75th and 90th percentiles for the duration of HGV were estimated to be 8.7 years (95%, confidence interval 4.8-15.7) and 23.6 years (95% confidence interval 11.8-47.1) respectively. Laparoscopic liver inspection/biopsy was performed in 25/68. There was no association between severity of liver disease and HGV viraemia (p = 0.43). This study demonstrates considerable variation in patterns of HGV viraemia in haemophiliacs. We found little evidence to implicate HGV as a major cause of chronic liver disease in haemophiliacs.

Infection with hepatitis G virus among recipients of plasma products.

BACKGROUND: Hepatitis G virus (HGV or GBV-C) is a newly discovered human flavivirus distantly related to hepatitis C virus (HCV). Little information is available on its natural history or routes of transmission, although it can be transmitted parenterally. We investigated the prevalence of persistent infection of HGV and HCV in patients exposed to non-virus-inactivated pooled blood products associated with transmission of HCV. METHODS: RNA was extracted from the plasma of 112 patients with haemophilia and 57 with hypogammaglobulinaemia, as well as from 64 different batches of archived coagulation-factor concentrates and immunoglobulins. RNA was reverse transcribed and amplified with primers from the 5' non-coding region of HCV and HGV by a nested polymerase chain reaction (PCR). Viral RNA was quantified by titration of complementary DNA before amplification. FINDINGS: Among non-renumerated UK blood donors HGV infection (detected by PCR) was more common than HCV infection (four [3.2%] of 125 compared with 137 [0.076%] of 180658 in southeast Scotland). Testing of batches of factor VIII and factor IX concentrates prepared without viral inactivation procedures showed high frequencies of contamination with HGV (16 of 17 factor VIII batches positive; six of six factor IX batches positive), with no difference between renumerated and non-renumerated donors. However, among 95 haemophiliacs who had received non-virus-inactivated concentrates, 13 (14%) were positive for HGV compared with 79 (83%) who were positive for HCV. Two of 37 recipients of long-term immunoglobulin replacement therapy were positive for HGV. Virus inactivation of blood products substantially reduced or eliminated contamination by HGV RNA sequences. INTERPRETATION: Despite the extremely high level of HGV contamination of non-virus-inactivated blood products, their use was not associated with high rates of persistent infection in recipients. The infectivity of HGV in blood products may be lower than that of HCV, or the virus may be less able to establish persistent infection in humans. Whatever the case, the high prevalence of active HGV infection in the general population remains difficult to explain.

Development of anti-interferon antibodies and breakthrough hepatitis during treatment for HCV infection in haemophiliacs.

The development of anti-interferon antibodies may lead to treatment failure during interferon therapy. We have studied the development of such antibodies in a group of 39 haemophiliacs receiving interferon-alpha 2a for chronic hepatitis C virus (HCV) infection. Anti-interferon antibodies developed in five (13%) patients and were associated with "breakthrough hepatitis' in three cases. There was an association between the development of anti-interferon antibodies and infection with HCV genotype 3a (P = 0.01). This study suggests that the development of anti-interferon antibodies may lead to treatment failure in a proportion of haemophiliacs with HCV infection. The association with genotype 3a has not previously been reported. Monitoring for the development of breakthrough hepatitis due to anti-interferon antibodies may provide the opportunity to develop strategies to overcome their effects.

Investigation of chronic hepatitis C infection in individuals with haemophilia: assessment of invasive and non-invasive methods.

Hepatitis C virus (HCV) infection is the major cause of chronic liver disease in individuals with haemophilia. A wide spectrum of disease severity is found in this group, ranging from mild hepatitis to cirrhosis. We have studied a cohort of 87 anti-HCV positive haemophiliacs who have been infected with HCV for 10-25 years and assessed the relative value of invasive and non-invasive methods of evaluating liver disease. The severity of liver disease was assessed using ultrasound scan (n = 77), upper GI endoscopy (n = 50), laparoscopic liver inspection (n = 33) and liver biopsy (n = 22). Invasive investigations were performed without any significant bleeding complications. Evidence of severe liver disease was found in approximately 25% of patients. There was agreement between the severity of liver histology and the information derived from the laparoscopic liver inspection, endoscopy and ultrasound in 86%. Co-infection with HIV was significantly associated with more severe liver disease (P = 0.006). This study provides further evidence that liver disease is emerging as a major complication in haemophiliacs and severe liver disease is more common in those co-infected with HIV. We have shown the potential value of laparoscopic liver inspection, in combination with endoscopy and ultrasound, in staging the extent of liver disease, and suggest that most patients may be managed without resorting to liver biopsy.

Interferon treatment for chronic hepatitis C infection in hemophiliacs--influence of virus load, genotype, and liver pathology on response.

In this study, we assessed the effectiveness of interferon treatment in 31 hemophiliacs with chronic hepatitis C virus (HCV) infection. Interferon alfa-2a (3 MU three times weekly) was administered for 6 months. Response was assessed by both serial alanine transaminase (ALT) and HCV RNA levels measured by a sensitive semiquantitative polymerase chain reaction (PCR) method. HCV genotype was determined by restriction fragment length polymorphism (RFLP), and evidence of changing genotypes during interferon therapy was sought. Severity of liver disease was assessed by both noninvasive and invasive methods, including laparoscopic liver inspection and biopsy. Sustained normalization of ALT levels occurred in eight patients (28%), and seven (24%) became nonviremic as assessed by PCR (<80 HCV/mL). Responders universally cleared HCV RNA within 2 months of starting interferon. Genotype 3a was associated with a favorable response to interferon. No evidence was found for a change in circulating genotype in patients who failed to respond to interferon or who relapsed. This study confirms that response rates to interferon are low in hemophiliacs as compared with other groups with chronic HCV infection. We have also demonstrated that virus load measurement over the first 8 to 12 weeks of treatment is an extremely useful method to identify responders at an early stage.

Interaction of hepatitis B and hepatitis C infection in haemophilia.

The management of haemophilia has been greatly complicated by the clinical sequelae of viral infection acquired through contaminated blood products. Many haemophiliacs have been infected by several viruses and the interaction between these viruses may be complex. In a cohort of 42 anti-HCV positive haemophiliacs, five were also found to be positive for HBsAg. All five were HCV reverse transcriptase/PCR negative compared to the 4/37 (11%) anti-HCV positive haemophiliacs who were HBsAg negative (P = 0.0001). We have identified a striking interaction between hepatitis C (HCV) and hepatitis B (HBV) in haemophiliacs co-infected by these agents, suggestive of the phenomenon of viral interference.

The carboxyl terminus of RAP30 is similar in sequence to region 4 of bacterial sigma factors and is required for function.

Transcription factor beta gamma (RAP30/74) from rat liver was previously shown in biochemical studies to control the binding of RNA polymerase II to promoters by a mechanism analogous to that utilized by bacterial sigma factors, by decreasing the affinity of polymerase for nonpromoter sites on DNA and by increasing the affinity of the enzyme for the preinitiation complex (Conaway, R. C., Garrett, K. P., Hanley, J. P., and Conaway, J. W. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 6205-6209). By constructing and analyzing mutants of beta gamma, we have identified a novel functional domain located in the carboxyl terminus of the gamma (RAP30) subunit. This domain shares sequence similarity with region 4 of bacterial sigma factors; in particular, it exhibits striking similarity to the carboxyl-terminal regions 4.1 and 4.2 of SpoIIIC (Bacillus subtilis sigma k). Evidence from biochemical studies argues that a mutant gamma (RAP30), lacking amino acid sequences similar to sigma homology region 4.2, is able to assemble with the beta (RAP74) subunit to form a mutant beta gamma (RAP30/74) with impaired ability to interact with RNA polymerase II.

Mechanism of promoter selection by RNA polymerase II: mammalian transcription factors alpha and beta gamma promote entry of polymerase into the preinitiation complex.

Productive binding of RNA polymerase II at the core region of TATA box-containing promoters is controlled by the action of the TATA factor and four additional transcription factors, designated alpha, beta gamma, delta, and epsilon, which have each been purified to near homogeneity from rat liver. This process is accomplished in three distinguishable stages. In the first stage (initial complex formation), the core promoter is packaged with the TATA factor into a binary complex that serves as the recognition site for RNA polymerase II. Here we show that, in the second stage (site selection), transcription factors alpha and beta gamma act in combination to promote selective binding of RNA polymerase II to the initial complex. Several lines of evidence argue that alpha and beta gamma function at this stage by a mechanism related to that utilized by bacterial sigma factors. In the third stage, transcription factors delta and epsilon promote assembly of the functional preinitiation complex. Our evidence supports the model that delta and epsilon enter the preinitiation complex and direct formation of stable protein-DNA contacts that anchor the transcription apparatus to the core promoter at sequences near the cap site.