Safety and effectiveness of high-dose, weight-based factor VIII inhibitor bypassing activity for warfarin-induced life-threatening bleeding.

: Previous studies suggest low, fixed-dose regimens of activated prothrombin complex concentrate [factor VIII inhibitor bypassing activity (FEIBA); 500 U for international normalized ratio (INR) < 5; 1000 U for INR > 5] is effective for reversal of warfarin-induced life-threatening bleeds. Little data are available on the use of high-dose, weight-based FEIBA for this indication. The objective of this study was to evaluate effectiveness and safety of high-dose, weight-based FEIBA (50 U/kg) vs. frozen plasma alone in this population. This was a matched case-control, multicenter retrospective study including patients who received high-dose, weight-based FEIBA or frozen plasma alone for warfarin-induced life-threatening bleeds matched (1 : 1) based on age and bleed location. Forty-eight patients were included in the analysis (24 FEIBA, 24 frozen plasma). The primary endpoint was time to INR less than 1.5 after administration of FEIBA or frozen plasma. Secondary endpoints include rates of thromboembolic events and mortality. Median baseline INR was 3.7 (interquartile range 2.7, 7.30) and 2.9 (2.3, 6.61) in the FEIBA and frozen plasma groups, respectively (P = 0.13). Median FEIBA dose was 4530 (3672, 5028) U. Use of FEIBA resulted in faster time to INR less than 1.5 with a median of 2.5 (1.25, 4.15) vs. 12 (5.6, 28.35) h; (P < 0.0001). Thromboembolic events occurred in nine (16.7%) patients (FEIBA n = 5; plasma n = 4); (P = 1.0). Mortality was similar in both groups (FEIBA 33% vs. frozen plasma 15%; P = 0.2). The use of high-dose, weight-based FEIBA resulted in faster time to reversal of warfarin-induced coagulopathy compared with frozen plasma alone and showed a similar safety profile.

Progression of hair cell ejection and molecular markers of apoptosis in the avian cochlea following gentamicin treatment.

Aminoglycoside treatment induces caspase-dependent apoptotic death in inner ear sensory hair cells. The timing of apoptotic signaling in sensory hair cells following systemic aminoglycoside treatment has not been characterized in vivo. We administered a single subcutaneous injection of the aminoglycoside gentamicin (300 mg/kg) to 12-16-day-old chicks and used immunocytochemical techniques to document the following responses in affected hair cells: T-cell restricted intracellular antigen-related protein (TIAR) translocation from the nucleus to the cytoplasm, cytochrome c release from the mitochondria, caspase-3 activation, nuclear condensation, and an orderly progression of hair cell ejection from the proximal end of the basilar papilla. Hair cells in the proximal tip exhibited TIAR translocation from the nucleus and aggregation into punctate granules in the cytoplasm 12 hours after injection and the response progressed distally. Cytochrome c release from the mitochondria into the cytoplasm and caspase-3 activation were observed in affected hair cells immediately prior to and during ejection. Hair cell ejection occurred between 30 and 54 hours after injection, beginning in the proximal tip and progressing distally. Nuclear condensation accompanied ejection while the loss of: 1) membrane integrity; 2) phalloidin labeling of F-actin; and 3) TO-PRO-1 labeling of nuclear contents occurred within 48 hours following ejection. Our results present a timeline of aminoglycoside-induced inner ear sensory hair cell apoptotic death that includes an 18-hour window between the initial apoptotic response and the later stages of programmed death signaling that accompany ejection and a gradual breakdown of hair cells following ejection.

A molecular mechanism of formalin fixation and antigen retrieval.

Despite the popularity of antigen-retrieval techniques, the precise molecular mechanism underlying the process remains enigmatic. We examined the molecular features underlying the loss of immunoreactivity following formalin fixation, with subsequent recovery by antigen retrieval. To do this, we first created a molecular model using short peptides that mimic the antibody-binding site of common clinical protein targets. The advantage of this model is that we know the amino acid sequence in and around the antibody-binding site. We observed that some, not all, of the peptides exhibited the formalin-fixation and antigen-retrieval phenomenon. Other peptides did not lose their ability to be recognized by antibody, even after prolonged incubation in formalin. A third, intermediate group exhibited the formalin-fixation and antigen-retrieval phenomenon only if another irrelevant protein was mixed with the peptide before fixation. Amino acid sequence analysis indicates that fixation and antigen retrieval are associated with a tyrosine in or near the antibody-binding site and with an arginine elsewhere, implicating the Mannich reaction as important in fixation and antigen retrieval.

Caspase inhibitors promote vestibular hair cell survival and function after aminoglycoside treatment in vivo.

The sensory hair cells of the inner ear undergo apoptosis after acoustic trauma or aminoglycoside antibiotic treatment, causing permanent auditory and vestibular deficits in humans. Previous studies have demonstrated a role for caspase activation in hair cell death and ototoxic injury that can be reduced by concurrent treatment with caspase inhibitors in vitro. In this study, we examined the protective effects of caspase inhibition on hair cell death in vivo after systemic injections of aminoglycosides. In one series of experiments, chickens were implanted with osmotic pumps that administrated the pan-caspase inhibitor z-Val-Ala-Asp(Ome)-fluoromethylketone (zVAD) into inner ear fluids. One day after the surgery, the animals received a 5 d course of treatment with streptomycin, a vestibulotoxic aminoglycoside. Direct infusion of zVAD into the vestibule significantly increased hair cell survival after streptomycin treatment. A second series of experiments determined whether rescued hair cells could function as sensory receptors. Animals treated with streptomycin displayed vestibular system impairment as measured by a greatly reduced vestibulo-ocular response (VOR). In contrast, animals that received concurrent systemic administration of zVAD with streptomycin had both significantly greater hair cell survival and significantly increased VOR responses, as compared with animals treated with streptomycin alone. These findings suggest that inhibiting the activation of caspases promotes the survival of hair cells and protects against vestibular function deficits after aminoglycoside treatment.