Discrimination of normal and abnormal prothrombin and protein C in plasma using a calcium ion-inhibited monoclonal antibody to a common epitope on several vitamin K-dependent proteins

Vitamin K deficiency or administration of vitamin K antagonists results in the biosynthesis of abnormal des-gamma-carboxy forms of the vitamin K-dependent proteins. Monoclonal antibody H-11 binds several vitamin K- dependent proteins at a determinant that includes the first two residues of gamma-carboxyglutamic acid. Antibody H-11 binds fully carboxylated prothrombin and protein C in the presence of EDTA but binding is inhibited by the divalent metal ions, calcium, magnesium, and manganese. By contrast, des-gamma-carboxy prothrombin and protein C bind antibody H-11 the same in the presence of EDTA or calcium ion. Antibody H-11 thus appears to bind a conserved antigenic site containing gamma-carboxyglutamic acid that in the presence of divalent metal ion undergoes a conformational transition. This ability of antibody H-11 to bind des-gamma-carboxy prothrombin and protein C in the presence of calcium ion allowed the development of an immunoassay for these proteins in plasma. Prothrombin and protein C from stably anticoagulated individuals receiving warfarin were characterized by their ability to bind antibody H-11 in the presence of calcium ion. Binding of prothrombin and protein C to antibody H-11 in the presence of calcium correlated temporally with warfarin administration. The inability of calcium ion to inhibit binding of antibody H-11 to abnormal prothrombin and protein C in plasma suggests that the circulating forms of both proteins following warfarin administration cannot undergo the metal ion-dependent conformational transition that includes sequence residues 1 through 12.     

Airway pressure and transpulmonary pressure during high-frequency oscillation for acute respiratory distress syndrome

High-frequency oscillation is a novel form of ventilation increasingly being used to treat refractory hypoxic respiratory failure resulting from acute lung injury or acute respiratory distress syndrome. Although there is no known relationship between airway pressure and transpulmonary pressure during conventional mechanical ventilation, no study has attempted to determine transpulmonary pressure during high-frequency oscillation.

BACKGROUND:

High-frequency oscillation (HFO) is used for the treatment of refractory hypoxic respiratory failure.

OBJECTIVE:

To demonstrate that the mean transpulmonary pressure (P_L) cannot be inferred from mean airway pressure (mPaw).

METHODS:

In seven patients already undergoing HFO for refractory acute respiratory distress syndrome, esophageal pressure (Pes) was measured using an esophageal balloon catheter. Pleural pressure (Ppl) and P_L were calculated from Pes.

MAIN RESULTS:

In the seven patients (mean [± SD] age 59±9 years) treated with HFO at 5±1 Hz and amplitude 75±10 cmH₂O, the mPaw was 27±6 cmH₂O, Ppl was 9±6 cmH₂O and P_L was 18±11 cmH₂O. Successful catheter placement and measurement of Pes occurred in 100% of subjects. There was no correlation between P_L and mPaw. The majority of subjects required hemodynamic support during the use of HFO; the frequency and degree of support during the study period was no different than that before the study.

CONCLUSION:

The present report is the first to describe measuring Pes and calculating Ppl during HFO for acute respiratory distress syndrome. While both current guidelines and recent trials have titrated treatment based on mPaw and oxygenation, there is wide variability in P_L during HFO and P_L cannot be predicted from mPaw.     

Monitoring of the neonate undergoing MR imaging: technical considerations. Work in progress

Instrument monitoring of vital signs in neonates undergoing magnetic resonance (MR) imaging can be difficult because of the unique environmental restrictions imposed by the imager. The authors present their experience with monitoring more than 50 newborn infants and discuss the interaction of monitoring devices with the MR imager. Several MR-compatible monitors allow continuous evaluation of body temperature, heart rate, blood pressure, and auscultation of heart sounds and respiration in mechanically ventilated infants. Signal-to-noise (S/N) ratio measurements taken during imaging of the head of an infant with these monitors in place did not differ appreciably from the ratio obtained during imaging without monitors. Tip angles should be optimized to account for widely varying head size among neonates, since adverse monitoring effects are significantly compounded by improper tip angle adjustment.     

A sensitive ELISPOT assay to detect low-frequency human T lymphocytes

We extended the sensitivity of the ELISPOT assay by including an antigen-driven proliferation step prior to a final restimulation with antigen and irradiated antigen presenting cells (APCs). This improved sensitivity made the modified ELISPOT assay better suited to the detection of rare or low frequency T lymphocytes than the standard ELISPOT assay or alternatives such as limiting dilution analysis or in situ hybridization. Use of ELISA-grade plastic or polyvinylidene difluoride (PVDF) plates for the detection of different cytokines improved the signal-to-noise ratio for counting cytokine spots, and use of video computer imaging software improved objective quantitation. Analysis of antigen-reactive peripheral blood mononuclear cells (PBMC) from multiple sclerosis (MS) patients using both the traditional and our modified ELISPOT assay demonstrate a >10-fold increase in numbers of myelin basic protein (MBP)-responsive T cells detected (an average of less than 1 spot forming cell (SFC) per 2×10⁵ PBMC with the standard assay compared to 19 SFC per 2×10⁵ PBMC with the modified assay). In addition, the modified ELISPOT assay could be performed with frozen PBMC, which permitted greater flexibility in sample processing, multiple use of a single sample as an internal standard, and simultaneous analysis of samples collected at different time points. This modified ELISPOT assay has many applications, including analysis of cytokine profiles in rare T cell populations, identification of antigen-responsive individuals as PBMC donors for T lymphocyte cloning or for therapeutic intervention, and assessment of vaccine or therapeutic efficacy as a surrogate clinical marker.     

Abnormalities of the neonatal brain: MR imaging. Part I. Intracranial hemorrhage

The authors prospectively evaluated 82 neonates, ranging in gestational age from 29 to 44 weeks postconception, with magnetic resonance (MR) imaging at 0.6 T. Twenty-two cases of hemorrhage in 15 infants were identified. Ultrasound (US) and computed tomography (CT) were superior to MR in the first few days after parenchymal hemorrhage, since at this time lesions were apparent on only T2-weighted images. After the first 3 days, MR was the single best modality because (a) hemorrhage on CT became imperceptible in the 2d week, whereas the high signal of hemorrhage on MR persisted for 2-11 weeks; (b) MR permitted rough dating of hemorrhage according to changes in signal intensity; and (c) MR was superior in identifying subdural or epidural hemorrhage. Because of the nonspecificity and restricted field of view of US and the inability of CT to depict hemorrhage after 7-10 days, the authors conclude that MR significantly improves the detection of intracranial hemorrhage in neonates.     

Abnormalities of the neonatal brain: MR imaging. Part II. Hypoxic- ischemic brain injury

Eighty-five infants, 82 of whom were 29-44 weeks postconceptional age, were imaged with a 0.6-T magnet. Eight infants had cerebral infarction. In premature neonates with very water, low-intensity white matter on T1-weighted images, ultrasound was better than both computed tomography and magnetic resonance (MR) imaging in depicting parenchymal changes of infarction or edema. However, after 37 weeks gestation, MR imaging was superior. Cerebral atrophy, present in seven infants, was consistent with subarachnoid space widths of 7 mm or more, or subarachnoid space widths of 5-6 mm with ventricular/brain ratios of 0.36 or greater. Delayed myelination was seen in a total of 18 infants with histories of hypoxic-ischemic insult. MR imaging shows promise in the neonatal period. It facilitates recognition of infarcts in full-term infants and may be used to predict abnormal neurologic outcome in infants who have initial delayed myelination.     

Mathematical modeling of platelet survival with implications for optimal transfusion practice in the chronically platelet transfusion- dependent patient

BACKGROUND: It is known that in vivo platelet survival varies as the platelet count changes. Previous attempts at curve fitting fail to predict the decreased platelet survival in thrombocythemia. Therefore, mathematical relations that more closely approximate platelet survival were derived and used in models of platelet transfusion practice. STUDY DESIGN AND METHODS: A differential equation for platelet loss was derived that included a constant (constant homeostatic loss), a first- order term (senescent loss), and a second-order term (one proportional to the square of the platelet concentration and whose contribution is expected to be significant only at higher platelet concentrations). Data derived from this model was compared to platelet survival data in normal, thrombocytopenic, and thrombocythemic patients and to the platelet decay after high-dose chemotherapy. To provide further validation of this model, predicted and actual platelet requirements were calculated or obtained (chart review) in bone marrow patients with uncomplicated thrombocytopenia after ablation and at two platelet- transfusion thresholds (20 and 10 × 10(9)/L). RESULTS: The equations accurately modeled normal, thrombocytopenic, and thrombocythemic platelet survival. Chart review demonstrated a 12.5 percent reduction in platelet transfusion requirements when the transfusion threshold was reduced from 20 to 10 × 10(9) per L. The model predicted a reduction of 14.0 percent. For 100 days of uncomplicated thrombocytopenia and a transfusion threshold of 10 × 10(9) per L, transfusion of 3 units of platelet concentrates compared to a 6-unit pool of platelet concentrates, resulted in a 22-percent savings of platelet units. CONCLUSION: Platelet survival as a function of platelet concentration can be modeled by use of a differential equation. This model challenges current dogma regarding platelet destruction and predicts decreased platelet survival in thrombocythemic patients. The model illustrates that large doses of platelets would result in greater time between transfusions, however, more units of platelets are used. Consideration should be given to the more frequent use of smaller doses of platelets in patients who chronically require platelet transfusion support.     

Gene and other biological therapies for vascular diseases

Summary— Gene transfer and antisense therapy offer novel approaches to the study and treatment of vascular diseases. The localized nature of vascular diseases like restenosis has made the application of genetic material an attractive therapeutic option. Viral and nonviral vectors have been developed to facilitate the entry of foreign DNA or RNA into cells. Vector improvement and production, demonstration of vector safety and demonstration of therapeutic efficacy are among the main present challenges. Various strategies have already been shown to be successful in preventing restenosis in animal models and include: the transfer of the herpes simplex virus thymidine kinase associated with ganciclovir; transfection of the cell cycle regulatory genes encoding for the active form of retinoblastoma gene product (Rb) or the cyclin-dependant kinase inhibitor p21, and antisense therapy. Therapeutic angiogenesis using gene transfer is a new strategy for the treatment of severe limb ischemia. Transfection of DNA encoding for the vascular endothelial growth factor has resulted in increasing collateral flow in animal models of peripheral ischemia. This approach is currently being investigated in a clinical trial in patients with distal ischemia. Other potential targets for genetic treatment in cardiovascular diseases include thrombosis, extracellular matrix synthesis and lipid metabolism.