The oncofetal protein, 5T4, is a suitable target for antibody-guided anti-cancer chemotherapy with calicheamicin.

The oncofetal protein, 5T4, is a tumor-associated protein displayed on the cell membrane of various carcinomas. This molecule is a promising target for anti-tumor vaccine development and for targeted therapy with staphylococcus exotoxin. The potential use of 5T4 as a target for antibody-guided chemotherapy has not been demonstrated. We report oncolytic efficacy and selectivity in vitro and in vivo with immuno-conjugates of calicheamicin (CM) and the anti-5T4 antibody, H8. CM is a potent cytotoxic drug that causes double strand breaks in DNA. Conjugates of CM and H8 were constructed with acid-labile as well as acid-stabile linkers. In vitro, when applied to monolayers of 5T4(+) cells, CM-conjugates targeting 5T4 were consistently more toxic than either free drug or a non-binding control CM-conjugate. This difference was less pronounced on 5T4-deficient cells. In vivo, four 5T4-positive subcutaneous tumor models were treated with conjugates. Efficacy was demonstrated by reduction of tumor growth relative to controls treated with drug vehicle. To evidence selectivity, the efficacy of the anti-5T4 conjugates was compared to the efficacy of H8, a mixture of H8 and calicheamicin, calicheamicin alone or calicheamicin conjugated to the anti-CD33 antibody, hP67.6. In addition, the efficacy and selectivity of an acid-labile conjugate of H8 was evaluated in an orthotopic model for 5T4(+) lung cancer. Increased survival following treatment was used as a parameter of efficacy. Calicheamicin conjugates of H8 were effective and selective in all the examined tumor models. Differences in efficacy between the acid-labile and acid-stabile conjugates depended on the investigated tumor model.

Isolation of a high affinity scFv from a monoclonal antibody recognising the oncofoetal antigen 5T4.

The oncofoetal antigen 5T4 is a 72 kDa glycoprotein expressed at the cell surface. It is defined by a monoclonal antibody, mAb5T4, that recognises a conformational extracellular epitope in the molecule. Overexpression of 5T4 antigen by tumours of several types has been linked with disease progression and poor clinical outcome. Its restricted expression in non-malignant tissue makes 5T4 antigen a suitable target for the development of antibody directed therapies. The use of murine monoclonal antibodies for targeted therapy allows the tumour specific delivery of therapeutic agents. However, their use has several drawbacks, including a strong human anti-mouse immune (HAMA) response and limited tumour penetration due to the size of the molecules. The use of antibody fragments leads to improved targeting, pharmacokinetics and a reduced HAMA. A single chain antibody (scFv) comprising the variable regions of the mAb5T4 heavy and light chains has been expressed in Escherichia coli. The addition of a eukaryotic leader sequence allowed production in mammalian cells. The two 5T4 single chain antibodies, scFv5T4WT19 and LscFv5T4, described the same pattern of 5T4 antigen expression as mAb5T4 in normal human placenta and by FACS. Construction of a 5T4 extracellular domain-IgGFc fusion protein and its expression in COS-7 cells allowed the relative affinities of the antibodies to be compared by ELISA and measured in real time using a biosensor based assay. MAb5T4 has a high affinity, K(D)=1.8x10(-11) M, as did both single chain antibodies, scFv5T4WT19 K(D)=2.3x10(-9) M and LscFv5T4 K(D)=7.9x10(-10) M. The small size of this 5T4 specific scFv should allow construction of fusion proteins with a range of biological response modifiers to be prepared whilst retaining the improved pharmacokinetic properties of scFvs.

One-stage treatment of congenital dislocation of the hip in children three to ten years old. Functional and radiographic results.

We reviewed the results of operative treatment of congenital dislocation of the hip in eighteen children (twenty-five hips) whose average age at the time of the index operation was six years and four months (range, three years to nine years and eleven months). None of the patients had had previous treatment of the dislocation. Preliminary traction was used for five patients (six hips), and open reduction and femoral shortening was performed in all hips. The functional result was assessed, according to the Iowa hip-rating system, after an average duration of follow-up of ten years and six months (range, six years and two months to sixteen years and ten months). Sixteen hips had an excellent result; seven, a good result; and two, a fair result. The average limb-length discrepancy was 0.8 centimeter (range, zero to four centimeters), and the average foot-progression angle was 11 degrees (range, 0 to 30 degrees) of external rotation. According to Severin's classification of the radiographic appearance, seven hips had an excellent result; eleven, a good result; four, a fair result; and three, a poor result. Four of eleven hips that had evidence of osteonecrosis of the proximal part of the femur had a severe deformity, and one patient had radiographic evidence of moderate degenerative osteoarthrosis when she was sixteen years old. On the basis of this review, we suggest that a one-stage operative procedure consisting of open reduction, femoral shortening, and pelvic osteotomy (if necessary) for previously untreated congenital dislocation of the hip in children who are three to ten years old can result in remodeling of the acetabulum and a functional hip.

Transpiration and whole-tree conductance in ponderosa pine trees of different heights.

Changes in leaf physiology with tree age and size could alter forest growth, water yield, and carbon fluxes. We measured tree water flux (Q) for 14 ponderosa pine trees in two size classes (12 m tall and ∼40 years old, and 36 m tall and ∼ 290 years old) to determine if transpiration (E) and whole-tree conductance (g t) differed between the two sizes of trees. For both size classes, E was approximately equal to Q measured 2 m above the ground: Q was most highly correlated with current, not lagged, water vapor pressure deficit, and night Q was <12% of total daily flux. E for days 165-195 and 240-260 averaged 0.97 mmol m-2 (leaf area, projected) s-1 for the 12-m trees and 0.57 mmol m-2 (leaf area) s-1 for the 36-m trees. When photosynthetically active radiation (I P) exceeded the light saturation for photosynthesis in ponderosa pine (900 µmol m-2 (ground) s-1), differences in E were more pronounced: 2.4 mmol m-2 (leaf area) s-1 for the 12-m trees and 1.2 mmol m-2 s-1 for the 36-m trees, yielding g t of 140 mmol m-2 (leaf area) s-1 for the 12-m trees and 72 mmol m-2 s-1 for the 36-m trees. Extrapolated to forests with leaf area index =1, the 36-m trees would transpire 117 mm between 1 June and 31 August compared to 170 mm for the 12-m trees, a difference of 15% of average annual precipitation. Lower g t in the taller trees also likely lowers photosynthesis during the growing season.

Reliance on stored water increases with tree size in three species in the Pacific Northwest.

In tall old forests, limitations to water transport may limit maximum tree height and reduce photosynthesis and carbon sequestration. We evaluated the degree to which tall trees could potentially compensate for hydraulic limitations to water transport by increased use of water stored in xylem. Using sap flux measurements in three tree species of the Pacific Northwest, we showed that reliance on stored water increases with tree size and estimated that use of stored water increases photosynthesis. For Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco), water stored in xylem accounted for 20 to 25% of total daily water use in 60-m trees, whereas stored water comprised 7% of daily water use in 15-m trees. For Oregon white oak (Quercus garryana Dougl. ex Hook.), water stored in xylem accounted for 10 to 23% of total daily water use in 25-m trees, whereas stored water comprised 9 to 13% of daily water use in 10-m trees. For ponderosa pine (Pinus ponderosa Dougl. ex Laws.), water stored in xylem accounted for 4 to 20% of total daily water use in 36-m trees, whereas stored water comprised 2 to 4% of daily water use in 12-m trees. In 60-m Douglas-fir trees, we estimated that use of stored water supported 18% more photosynthesis on a daily basis than would occur if no stored water were used, whereas 15-m Douglas-fir trees gained 10% greater daily photosynthesis from use of stored water. We conclude that water storage plays a significant role in the water and carbon economy of tall trees and old forests.

Use of a custom retrograde intramedullary rod for the management of distal femoral nonunion: a report of two cases.

Nonunion of the distal femur is a rare injury that is difficult to manage. A variety of surgical implants and techniques are available. We present an alternative mode of treatment for supracondylar femoral nonunion using a custom retrograde titanium femoral nail (Biomet; Warsaw, IN) to successfully achieve union in two cases. We believe the added length, custom modifications, and enhanced stability allowed better fixation in an osteopenic distal femur.

Developing a Respiratory Protection Program. Understanding the written elements.

1. Respirators can be the last defense for the estimated 5 million employees who use them for protection from dusts and fibers, fumes, mists, gases, vapors, and biological hazards. Because of these potentially lethal respiratory hazards, occupational and environmental health nurses need to be able to determine the need for, understand, develop, update, and implement an actionable respiratory protection program (RPP). 2. Regulated per 29 CFR 1910.134, a written RPP becomes the map or guideline process specific to the workplace that needs to be followed to ensure employee protection. 3. The nine required written elements of a RPP include respirator selection; fit testing; respirator use in routine and emergency situations; respirator maintenance and change schedules; ensuring adequate breathing air supply, quantity, and flow for atmosphere supplying respirators; regular evaluation of program effectiveness; medical evaluation; training employees in the respiratory hazards in routine and emergent situations; and training employees in proper use of the respirator. 4. Occupational and environmental health nurses are in a unique position to be a RPP program administrator, its designated licensed health care professional, or an active member of a team implementing the RPP process.

Evidence that decomposition rates of organic carbon in mineral soil do not vary with temperature.

It has been suggested that increases in temperature can accelerate the decomposition of organic carbon contained in forest mineral soil (Cs), and, therefore, that global warming should increase the release of soil organic carbon to the atmosphere. These predictions assume, however, that decay constants can be accurately derived from short-term laboratory incubations of soil or that in situ incubations of fresh litter accurately represent the temperature sensitivity of Cs decomposition. But our limited understanding of the biophysical factors that control Cs decomposition rates, and observations of only minor increases in Cs decomposition rate with temperature in longer-term forest soil heating experiments and in latitudinal comparisons of Cs decomposition rates bring these predictions into question. Here we have compiled Cs decomposition data from 82 sites on five continents. We found that Cs decomposition rates were remarkably constant across a global-scale gradient in mean annual temperature. These data suggest that Cs decomposition rates for forest soils are not controlled by temperature limitations to microbial activity, and that increased temperature alone will not stimulate the decomposition of forest-derived carbon in mineral soil.

Growth and maintenance respiration rates of aspen, black spruce and jack pine stems at northern and southern BOREAS sites.

We measured stem respiration rates during and after the 1994 growing season of three common boreal tree species at sites near the northern and southern boundaries of the closed-canopy boreal forest in central Canada. The growth respiration coefficient (r(g); carbon efflux per micro mole of carbon incorporated in structural matter) varied between 0.25 and 0.76, and was greatest for black spruce (Picea mariana (Mill.) B.S.P.), least for jack pine (Pinus banksiana Lamb.) and intermediate for trembling aspen (Populus tremuloides Michx.). There was a consistent trend for higher r(g) at northern sites than at southern sites. Maintenance respiration rates at 15 degrees C (r(m)) varied from 0.5 to 2.7 nmol C mol(-1) C(sapwood) s(-1). Values of r(m) were high at sapling-stage jack pine sites and mature black spruce sites, and low at mature trembling aspen and mature jack pine sites. We found significant relationships between annual maintenance respiration and sapwood relative growth rate and sapwood volume per unit of stem surface area that explained much of the within-stand and between-stand variability. Because of the large differences in parameter values among sites, we conclude that the use of stand-specific respiratory parameters may improve model predictions of ecosystem process models over the use of generic parameter values.

The molybdenum centers of xanthine oxidase and xanthine dehydrogenase. Determination of the spectral change associated with reduction from the Mo(VI) to the Mo(IV) state.

The UV-visible absorbance change associated with reduction of the molybdenum centers of xanthine oxidase and xanthine dehydrogenase has been determined using a double-difference technique. At pH 8.5, the Mo(VI) minus Mo(IV) difference spectrum seen with xanthine oxidase exhibits a positive feature at 420 nm, having an extinction change of approximately 3,000 M-1 cm-1 as well as evidence for a negative feature below 340 nm. In xanthine oxidase this change is found to exhibit a marked pH dependence, implicating protonation/deprotonation events associated with changes in the molybdenum oxidation state. Application of the double-difference protocol to the respective circular dichroism spectra of xanthine oxidase and xanthine dehydrogenase reveals appreciable CD changes at 420 and 580 nm associated with the reduction of the molybdenum center. The present results demonstrate a direct spectroscopic handle on the molybdenum centers of both xanthine oxidase and xanthine dehydrogenase.

A simple, battery-operated, temperature-controlled cuvette for respiration measurements.

We designed a simple, portable, battery-operated, temperature-controlled cuvette to measure respiration of small samples of plant tissue in the field. The cuvette is built around a peltier cell and is controlled with a data logger. The cuvette maintained sample temperature within 0.5 degrees C over a temperature range of 5 to 45 degrees C and operated for 6-8 h from a 12 V 105 ampere-hour "deep-cycle" battery. Based on measurements with this cuvette, we found that, at 15 degrees C, CO(2) efflux from dark respiration of white pine (Pinus strobus L.) foliage was 40% greater during the day than at night.

One hundred and eighty-seven cases of child abuse and neglect.

The case histories of 187 abused and neglected children, admitted to the Royal Alexandra Hospital for Children over a three-year-period, were reviewed. Multiproblem families from the lower socioeconomic groups predominated, reflecting the hospital's geographic catchment area. Eight children died and 30 were made state wards. A high proportion of mothers described problems during the pregnancy, a difficult delivery and neonatal problems. The majority of parents in this study were married and had small families. First-born male infants of young isolated mothers were found to be at greatest risk of child abuse and neglect.

Carbon dioxide/digital subtraction arteriography-assisted transluminal angioplasty.

During a 62-month period, carbon dioxide was used to supplement or completely replace iodinated contrast agents in performing 27 transluminal angioplasties in 26 patients. The arterial segments addressed included the following: renal in two cases, iliac in five, femoral/popliteal in 15, infrapopliteal in two, and combined in three. Indications for intervention included lower extremity gangrene in 11 cases, ischemic ulceration in 10, rest pain in three, claudication in one, and ischemic nephropathy in two. Contraindications to iodinated contrast agents included renal insufficiency resulting from diabetes (n = 20) or ischemic nephropathy (n = 2) and congestive heart failure (n = 4). Eight procedures used carbon dioxide as the sole contrast agent, whereas 19 required supplementation of carbon dioxide with a mean of 39 ml of nonionic contrast medium. Technical success was achieved in 25 procedures with significant hemodynamic improvement in 20 patients. Complications included transient deterioration in renal function in two patients and myocardial infarctions in two. At 30 days 18 patients had demonstrated significant clinical improvement. Patients at high risk for iodinated contrast-related complications may undergo transluminal angioplasty using carbon dioxide/digital subtraction arteriography to reduce or eliminate the need for iodinated contrast agents.

The reductive half-reaction of xanthine oxidase. The involvement of prototropic equilibria in the course of the catalytic sequence.

The pH dependence and solvent isotope sensitivity of three discrete steps in the reductive half-reaction of xanthine oxidase have been investigated. The pH dependence of both kcat/Km from steady-state experiments and kred/Kdfrom rapid reaction experiments with xanthine as substrate indicate that enzyme reacts preferentially with the neutral form of substrate and that an ionizable group in the active site having a pKa of approximately 6.6 must be unprotonated for reaction to take place. The solvent kinetic isotope effect on kred/Kd is 2.4, once a uniform shift on going to D2O of approximately 1 unit for both pKa values is taken into account. The pH dependence of the formation and decay of Ered-P formed in the course the reaction of xanthine oxidase with lumazine has also been examined. Formation of this complex exhibits bell-shaped pH dependence, with pKa values of 6.5 and 7.8, consistent with the results obtained with xanthine. Decay of the Ered-P complex is base-catalyzed with a pKa > 11 and exhibits a small solvent kinetic isotope effect of 1.7 at pH/D 8.5. By contrast, the catalytic intermediate giving rise to the "very rapid" EPR signal that is transiently observed in the course of the reaction of enzyme with the substrate 2-hydroxy-6-methylpurine is found to undergo acid-catalyzed breakdown with an associated pKa < 6. Formation and decay of this species exhibit solvent kinetic isotope effects of 2.0 and 3.5 at pH 10. The results are discussed in the context of a specific reaction mechanism for the reductive half-reaction of xanthine oxidase, in which discrete ionizations associated with the molybdenum center of the active site play critical roles in determining the magnitude of the rate constants by which the Mo(IV)-P and Mo(V)-P intermediates form and decay.

Obsessional disorders during recovery from severe head injury: report of four cases.

Four cases of transient obsessional disorders following severe head injury are described within the context of recovery from acute traumatic brain injury (TBI). Obsessional features following TBI have important treatment implications in brain injury rehabilitation settings, since emergence of this disorder in the acutely brain injured patient poses a significant obstacle to interdisciplinary rehabilitation. Although the numbers of patients described here and in previous reports are too small to draw conclusions about the incidence of obsessional disorders following TBI, these cases illustrate the importance of correctly identifying and treating obsessional symptoms in the brain injured patient.

Foliage, fine-root, woody-tissue and stand respiration in Pinus radiata in relation to nitrogen status.

We measured respiration of 20-year-old Pinus radiata D. Don trees growing in control (C), irrigated (I), and irrigated + fertilized (IL) stands in the Biology of Forest Growth experimental plantation near Canberra, Australia. Respiration was measured on fully expanded foliage, live branches, boles, and fine and coarse roots to determine the relationship between CO(2) efflux, tissue temperature, and biomass or nitrogen (N) content of individual tissues. Efflux of CO(2) from foliage (dark respiration at night) and fine roots was linearly related to biomass and N content, but N was a better predictor of CO(2) efflux than biomass. Respiration (assumed to be maintenance) per unit N at 15 degrees C and a CO(2) concentration of 400 micro mol mol(-1) was 1.71 micro mol s(-1) mol(-1) N for foliage and 11.2 micro mol s(-1) mol(-1) N for fine roots. Efflux of CO(2) from stems, coarse roots and branches was linearly related to sapwood volume (stems) or total volume (branches + coarse roots) and growth, with rates for maintenance respiration at 15 degrees C ranging from 18 to 104 micro mol m(-3) s(-1). Among woody components, branches in the upper canopy and small diameter coarse roots had the highest respiration rates. Stem maintenance respiration per unit sapwood volume did not differ among treatments. Annual C flux was estimated by summing (1) dry matter production and respiration of aboveground components, (2) annual soil CO(2) efflux minus aboveground litterfall, and (3) the annual increment in coarse root biomass. Annual C flux was 24.4, 25.3 and 34.4 Mg ha(-1) year(-1) for the C, I and IL treatments, respectively. Total belowground C allocation, estimated as the sum of (2) and (3) above, was equal to the sum of root respiration and estimated root production in the IL treatment, whereas in the nutrient-limited C and I treatments, total belowground C allocation was greater than the sum of root respiration and estimated root production, suggesting higher fine root turnover or increased allocation to mycorrhizae and root exudation. Carbon use efficiency, the ratio of net primary production to assimilation, was similar among treatments for aboveground tissues (0.43-0.50). Therefore, the proportion of assimilation used for construction and maintenance respiration on an annual basis was also similar among treatments.

Connective tissue structures in clubfoot: a morphologic study.

A morphologic study of connective tissue structures in clubfoot, with special emphasis on the presence of myofibroblasts, was undertaken to investigate the theory of retracting fibrosis as an etiologic factor. Nine idiopathic clubfeet from six patients were studied. Specimens from the medial and lateral capsule, medial and lateral fascia, spring and lacinate ligaments, and flexor digitorum longus, flexor hallucis longus, posterior tibialis, and Achilles tendon sheaths were investigated. Fifty specimens were examined using light microscopy and hematoxylin and eosin staining, and 26 were examined by transmission electron microscopy. Light microscopy failed to reveal any myofibroblast-like cells or any qualitative differences between specimens from capsule, fascia, ligaments, and tendon sheath. Using transmission electron microscopy, the authors identified two cell types: fibroblasts and mast cells. Some fibroblasts contained a network of microfilaments but all lacked microbundles, basal lamina, or plasmalemmal attachment plaques seen in typical myofibroblasts. Mast cells were rarely identified in capsular specimens. The absence of myofibroblast-like cells or typical myofibroblasts in clubfoot connective tissue structures does not support the theory of retracting fibrosis as a likely cause of contracture in idiopathic clubfoot.