Inhibitor recurrence after immune tolerance induction: a multicenter retrospective cohort study.

BACKGROUND: Immune tolerance induction (ITI) in patients with congenital hemophilia A is successful in up to 70%. Although there is growing understanding of predictors of response to ITI, the probability and predictors of inhibitor recurrence after successful ITI are not well understood. OBJECTIVES: To determine the association of clinical characteristics, particularly adherence to factor VIII (FVIII) prophylaxis after ITI, with inhibitor recurrence in patients with hemophilia A who were considered tolerant after ITI. METHODS: In this multicenter retrospective cohort study, 64 subjects with FVIII level < 2% who were considered successfully tolerant after ITI were analyzed to estimate the cumulative probability of inhibitor recurrence using the Kaplan-Meier method. The association of clinical characteristics with inhibitor recurrence was assessed using logistic regression. RESULTS: A recurrent inhibitor titer ≥ 0.6 BU mL(-1) occurred at least once in 19 (29.7%) and more than once in 12 (18.8%). The probability of any recurrent inhibitor at 1 and 5 years was 12.8% and 32.5%, respectively. Having a recurrent inhibitor was associated with having received immune modulation during ITI (odds ratio [OR] 3.8, 95% confidence interval [CI] 1.2-22.4) and FVIII recovery of < 85% at the end of ITI (OR 2.6, 95% CI 1.3-5.9) but was not associated with adherence to post-ITI prophylactic FVIII infusion (OR 0.5, 95% CI 0.06-4.3). CONCLUSIONS: The use of immune modulation therapy during ITI and lower FVIII recovery at the end of ITI appear to be associated with an increased risk of inhibitor recurrence after successful ITI. Adherence to post-ITI prophylactic FVIII infusions is not a major determinant of recurrence.

Pediatric transplantation: managing bleeding.

There has been extraordinary progress over the last half-century in the field of medical transplantation in which tissue, organs, or body parts from one human are placed into another. Solid organ transplants have allowed thousands of children with otherwise devastating inherited or acquired disorders to survive. Depending upon the clinical situation, there are many specific peri-transplant issues that must be carefully addressed to optimize outcomes. Although surgical, immunologic, and infectious concerns are usually in the forefront, important aspects regarding hemostasis frequently arise. The number of solid organs that can be successfully transplanted in children has expanded over the last decades and includes kidney, liver, heart, lung, intestine, pancreas, and thymus. Bleeding complications may occur in the setting of organ failure prior to transplantation, during the surgical procedure, or in the post-transplant setting, and can results in significant morbidity. This report will focus on preventing and managing non-surgical-related bleeding complications in children undergoing liver, heart, kidney transplantation, in whom there are often unique aspects of coagulation to be considered.

Organized sports participation and the association with injury in paediatric patients with haemophilia.

INTRODUCTION: With the wide availability of factor and the routine use of prophylaxis boys with haemophilia are now able to participate in regular physical activity, including organized sports. Current guidelines vary regarding specific recommendations for sports participation and concerns remain regarding safety. AIM: To determine if participation in organized sports is associated with an increased risk for injury in paediatric subjects with haemophilia. METHODS: Retrospective single-centre cohort study from January 1, 2008 to December 31, 2010 in male subjects ages 10-18 years with a factor VIII (FVIII) or FIX level <40%. The number of injuries per subject and participation in organized sports was recorded. RESULTS: 48 male subjects with a mean age of 14.3 ± 2.6 years (range: 10-18.8) were included; 64.6% (31/48) FVIII deficiency, 54.2% (26/48) severe haemophilia, 18.8% (9/48) moderate and 27.1% (13/48) mild. The majority [62.5% (30/48)] of subjects participated in at least one season of organized sport. There were 77 injuries in 36/48 (75%) subjects. The mean number of injuries per subject was 1.6 ± 1.5. There was no statistical difference in the mean number of injuries (P = 0.44) or target joint formation (P = 0.52) between the subjects who participated in organized sports compared to those who did not. CONCLUSION: In this study, participation in organized sports by boys with haemophilia, ages 10-18 years, is common and not associated with an increased number of injuries or the development of a target joint. As injuries occurred equally in both groups, concerted efforts should be directed at reducing injuries in all patients.

Low mortality from intracranial haemorrhage in paediatric patients with haemophilia.

INTRODUCTION: Intracranial haemorrhage (ICH) in patients with haemophilia has an estimated mortality rate of 20%. Advances in haemophilia care have significantly reduced many bleeding complications but it is unclear if these advances have impacted mortality from ICH. AIM: To determine the in-hospital mortality from intracranial ICH in paediatric patients with haemophilia. METHODS: This retrospective multicentre cohort study utilized the Pediatric Health Information System administrative database with data from 43 paediatric tertiary care hospitals in the United States from January 1, 2002-December 31, 2011. Subjects included were male < 21 years of age with an ICD-9-CM code for haemophilia A or B. ICH events were identified using ICD-9-CM codes. RESULTS: There were 8325 admissions for 3133 male subjects with haemophilia. About 271 (3.3%) admissions had an ICH event in 236 (7.5%) individual subjects. The proportion of ICH events was stable over time (P = 0.13). The median age of ICH was 2 years (interquartile range 0.6-7.3). In 28.4% (77/271) of the ICH events the subject had an inhibitor. Twenty-one deaths occurred in the entire cohort (0.7%). Six (28.6%) of these deaths were in patients with an ICH for an ICH mortality rate of 2.5% (6/236). CONCLUSIONS: Mortality from ICH in paediatric patients has significantly improved from prior estimates of 20% to the current estimate of 2.5%. Unfortunately the rate of ICH events remains constant and further efforts are needed to identify alternative strategies of prevention.

Utility of computed tomography of the head following head trauma in boys with haemophilia.

The most serious site of bleeding for patients with haemophilia is the central nervous system. Intracranial haemorrhage (ICH) in patients with haemophilia can occur spontaneously or following mild head trauma however no guidelines exist for the approach to these patients. The goal of this review was to determine the utility of screening computed tomography (CT) of the head for patients with haemophilia who experience head trauma and to determine if the use of clinical criteria could allow a selective approach to radiographic imaging. In a retrospective study we reviewed the management of head trauma in a cohort of paediatric patients with haemophilia in a single institution. The cohort included males, ages birth to 18 years with haemophilia A or B who were followed at the haemophilia treatment center at The Children's Hospital of Philadelphia from 1994 to 2005. Between the years of 1994 and 2005, 97 patients were evaluated for head trauma for a total of 374 emergency department visits. There were 295 head CT scans performed to identify 9 (3%) episodes of intracranial bleeding. Fifty-six per cent of the patients with intracranial bleeding had no clinical signs or symptoms. The clinical outcome was excellent in all cases with no deaths or reported morbidity. In this cohort, a lack of symptoms and a normal neurological exam did not exclude ICH, especially in patients with severe haemophilia who were evaluated soon after a mild head trauma event suggesting the utility of early head CT imaging.

High mobility group proteins 1 and 2 recognize chromium-damaged DNA.

Chromium (Cr) is a human carcinogen and a potent DNA damaging agent. Incubation of DNA with CrCl3 resulted in dose-dependent binding of Cr to DNA and, at concentrations >20 microM, altered the electrophoretic mobility of a 100 bp oligonucleotide. We also demonstrate that high mobility group (HMG) proteins 1 and 2 bind Cr-damaged DNA (Cr-DNA). Protein binding was lesion density-dependent, with maximal binding to DNA treated with 100 microM CrCl3. HMG2 binds to Cr-DNA with a calculated Kd of approximately 10(-9) M. These proteins also bound DNA obtained from chromate-treated cells. These results suggest that the covalent attachment of Cr to DNA induces alterations in DNA structure which are recognized by HMG1 and HMG2. Therefore, these proteins may function as Cr-damaged DNA recognition proteins in vivo and as a consequence of binding, may play a role in directing the cellular response to Cr-DNA adduct formation.

DNA binding activity of the mammalian translation elongation complex: recognition of chromium- and transplatin-damaged DNA.

The elongation factor complex, EF-1H, serves an essential function in protein biosynthesis in eukaryotic cells, although the role of EF-1H in other physiological processes is unknown. In this report, we demonstrate that three components of EF-1H (EF-1 beta, EF-1 delta, EF-1 gamma) bind to DNA modified with chromium (Cr), a potent DNA-damaging agent and an established human carcinogen. The EF-1H complex also binds to transplatin modified DNA but not to cisplatin-modified DNA. These results demonstrate that the EF-1H complex has functional DNA binding activity and is capable of recognizing the distortions in DNA structure resulting from the covalent binding of Cr and transplatin to DNA.

Effects of benzene metabolite treatment on granulocytic differentiation and DNA adduct formation in HL-60 cells.

Reactive metabolites of benzene (BZ) play important roles in BZ-induced hematotoxicity. Although reactive metabolites of BZ covalently bind to DNA, the significance of DNA adduct formation in the mechanism of BZ toxicity is not clear. These studies investigated the covalent binding of the BZ metabolites hydroquinone(HQ) and 1,2,4-benzenetriol(BT) using the DNA [32P]postlabeling method and explored the potential relationship between DNA adduct formation and cell differentiation in human promyelocytic leukemia (HL-60) cells, a model system for studying hematopoiesis. Maturation of HL-60 cells to granulocytes, as assessed by light and electron microscopy, was significantly inhibited in cells that were pretreated with HQ or BT prior to inducing differentiation with retinoic acid (RA). The capacity of RA-induced cells to phagocytose sheep red blood cells (RBC) and to reduce nitroblue tetrazolium (NBT), two functional parameters characteristic of mature, differentiated neutrophils, was also inhibited in cells pretreated with HQ or BT. These BZ metabolite treatments induced DNA adduct formation in HQ- but not in BT-treated cells. These results indicate that whereas HQ and BT each block granulocytic differentiation in HL-60 cells, DNA adducts were observed only following HQ treatment. Thus DNA adduct formation may be important in HQ but not in BT toxicity.

In vivo effects of chromium.

The production of reactive oxygen species on addition of hexavalent chromium (potassium dichromate, K2Cr2O7) to lung cells in culture was studied using flow cytometer analysis. A Coulter Epics Profile II flow cytometer was used to detect the formation of reactive oxygen species after K2Cr2O7 was added to A549 cells grown to confluence. The cells were loaded with the dye, 2',7'-dichlorofluorescein diacetate, after which cellular esterases removed the acetate groups and the dye was trapped intracellularly. Reactive oxygen species oxidized the dye, with resultant fluorescence. Increased doses of Cr(VI) caused increasing fluorescence (10-fold higher than background at 200 microM). Addition of Cr(III) compounds, as the picolinate or chloride, caused no increased fluorescence. Electron paramagnetic resonance (EPR) spectroscopic studies indicated that three (as yet unidentified) spectral "signals" of the free radical type were formed on addition of 20, 50, 100, and 200 microM Cr(VI) to the A549 cells in suspension. Two other EPR "signals" with the characteristics of Cr(V) entities were seen at field values lower than the standard free radical value. Liver microsomes from male Sprague-Dawley rats treated intraperitoneally with K2Cr2O7 (130 mumole/kg every 48 hr for six treatments) had decreased activity of cytochromes P4503A1 and/or 3A2, and 2C11. Hepatic microsomes from treated female Sprague-Dawley rats, in contrast, had increased activities of these isozymes. Lung microsomes from male Sprague-Dawley rats had increased activity of P4502C11.

Nephrotoxicity of the 1:1 acrolein-glutathione adduct in the rat.

Previous metabolic studies in rats have suggested in vivo formation of the acrolein-glutathione (acrolein-GSH) adduct following administration of the highly reactive alpha, beta-unsaturated aldehyde acrolein. Early studies by several investigators demonstrated that similar compounds such as alpha, beta-unsaturated aldehyde-cysteine adducts have toxic (carcinostatic) activity against Ehrlich ascites tumor cells implanted in mice. The current studies investigated the in vivo toxicity associated with the acrolein-GSH adduct in the male Sprague-Dawley rat. The 1:1 acrolein-GSH adduct was synthesized and characterized by physical-chemical methods. Rats given the acrolein-GSH adduct intravenously at 0.5 or 1 mmol/kg developed nephrotoxicity characterized by glucosuria, proteinuria, elevation in serum urea nitrogen, and gross and histologic changes of the kidney. The toxicity was not affected by pretreatment of rats with pyrazole, an alcohol dehydrogenase inhibitor; disulfiram, an inhibitor of aldehyde dehydrogenases; or probenecid, a renal organic anion transport inhibitor. Administration of a similar but nonaldehydic glutathione conjugate, S-n-propylglutathione, did not result in nephrotoxicity in the rat. The nephrotoxicity induced by the acrolein-GSH adduct was inhibited by acivicin, a gamma-glutamyl-transpeptidase inhibitor. These results indicate that the acrolein-GSH adduct requires processing through the first step of the renal mercapturic acid synthesis pathway to be activated to a toxic species.

The effects of alpha, beta-unsaturated aldehydes on hepatic thiols and thiol-containing enzymes.

The effects of series of alpha, beta-unsaturated aldehydes on hepatic glutathione, cytochrome P450, and NADPH-cytochrome c reductase activity were compared with time. Male F-344 rats were dosed with muconaldehyde (36 mumol/kg), acrolein (89 mumol/kg), crotonaldehyde (450 mumol/kg), or the saturated aldehyde propionaldehyde (89 mumol/kg) and terminated 0.5, 4, or 24 hr later. Acrolein or muconaldehyde reduced glutathione to 51 and 75% of controls, respectively, at 4 hr; glutathione returned to control values at 24 hr. Only at 24 hr, acrolein, muconaldehyde, or crotonaldehyde decreased cytochrome P450 to 61, 71, and 67% of control values, respectively; ethylmorphine N-demethylation was decreased to a greater extent, i.e., to 35, 60, and 23% of controls. The reductase activity was unchanged at any time following the treatment with reactive aldehydes which were not hepatotoxic (as shown by glucose 6-phosphatase activity, histological changes, or serum enzymes). Propionaldehyde changed none of these activities. Acrolein (44.5 mumol/kg) given 4 hr prior to phenobarbital (50 mg/kg) for two consecutive days decreased the phenobarbital induction of cytochrome P450 to 45% of phenobarbital alone. This treatment also decreased the 2 alpha, 2 beta, 6 beta, 16 alpha, and 16 beta hydroxylation of testosterone as well as androstenedione formation showing effects on individual cytochrome P450 isozymes. NADPH-cytochrome c reductase induction was not decreased by this treatment, thus indicating that in vivo these changes are due to a mechanism other than generalized inhibition of protein synthesis.

Oral bioavailability of chromium from a specific site.

Analysis of soil from a specific site in New Jersey indicated a low level of sodium and chromium present as a calcium compound. Chromium was then administered orally to young, mature male rats at a level of 240 micrograms/kg for 14 days as chromium-contaminated soil, as CaCrO4, and as an equimolar mixture of the soil and calcium salts for 14 days. The rats were sacrificed 24 hr after the last dosing, and tissues were taken immediately for chromium analysis. Blood, muscle, and liver contained the highest levels of chromium in these animals, although kidney contained the highest concentration per gram of tissue. The total amount of chromium in the tissues was less than 2% of the administered chromium. In a study of the excretion of chromium, the animals were dosed orally for 8 days (with CaCrO4 or contaminated soil, each at the level of 240 mumole Cr/kg), and the chromium in feces and urine was determined on days 1, 2, 7, and 8. After cessation of dosing for 27 days, the same rats were dosed for 2 days at the same level, and chromium in urine and feces was determined for the 2 days. The animals administered the chromium in soil had higher levels of chromium in both urine and feces on all days compared to the group fed the CaCrO4. The total recovery of chromium in any of the 2-day periods was less than 50% of the chromium administered during that period.