Measurement of the ADP:ATP ratio in human leukaemic cell lines can be used as an indicator of cell viability, necrosis and apoptosis.

In this study the relative levels of ADP and ATP have been measured in cells undergoing apoptosis. Using HL60, CEM7, Jurkat and U937 cell lines and cytotoxic agents known to induce apoptosis, there was a significant correlation (P<0.01 for all models) between the ADP:ATP ratio and the degree of apoptosis measured by TUNEL and estimation of the sub G(0) fraction by propidium iodide staining and flow cytometry. The ratio measured in viable proliferating cells was found to be less than 0.11 compared with ratios between 0.11 and 1.0 seen in cells undergoing apoptosis. The higher the percentage of hypodiploidy the greater the ratio. Necrosis induced by heat shock resulted in ADP:ATP ratios in excess of 15.0. When primary cultures of AML blast cells were used, there was again a significant correlation between the ADP:ATP ratio and the degree of hypodiploidy. Recent evidence suggests that apoptosis is accompanied by opening of the mitochondrial permeability pores, leading to disruption of the mitochondrial transmembrane potential (DeltaPsi(m)). This results in caspase activation due to the release of cytochrome c and apoptogenic factors into the cytosol. In five experiments using CEM7 and dexamethasone the mitochondrial transmembrane potential was assessed using the fluorescent cyanine dye JC-1 and flow cytometry. Functioning mitochondria concentrate the JC-1 to produce red fluorescence. Loss of mitochondrial transmembrane potential results in green fluorescence only. The percentage of cells exhibiting red fluorescence correlated positively with the ATP values and negatively with the ADP:ATP ratio.

Glenoid version in children with obstetric brachial plexus palsy.

Glenoid version in a group of 29 children with obstetric brachial plexus paralysis and posterior dislocation of the shoulder was studied by using computed axial tomography (CT). The CT scan in most patients was done before an open release and reduction of the shoulder. A comparison was made between the normal and affected sides in regard to glenoid version and structure. In the study population, there were 16 girls and 13 boys with an average age at the time of initial CT of 2.8 years. Sixteen of the patients had posterior dislocations of the right shoulder, and none was bilateral. In 18 patients, the neurologic lesion was confined to the upper roots of the brachial plexus, with the remaining patients having whole plexus involvement. A significant difference in glenoid version between normal and affected sides was found in these patients. The mean glenoid version for the dislocated side was -29.5 +/- 2.5 degrees and that of the normal side was -6.9 +/- 2.4 degrees. Glenoid structure was different in dislocated shoulders. The glenoid articular surface was observed to be laterally convex in the majority of cases, and in these cases, the posterior rim of the glenoid was often hypoplastic and rounded.

Diagnosis and management of the infected total knee arthroplasty.

While infection in TKA is a relatively infrequent complication, it can be devastating in terms of morbidity and cost. Prevention of infection begins with patient selection. Prior knee sepsis surgery, rheumatoid arthritis, and poor general health may lead to an increased rate of infection. Prophylactic antibiotics, meticulous surgical technique, and control of the intraoperative environment have been shown to be beneficial in prevention of infection after TKA. Diagnosis can be difficult and often is heralded by the onset of pain in a previously pain-free knee. Aspiration is an excellent screening tool and is also beneficial in determining management of potentially infected TKAs. In cases posing a diagnostic dilemma, radiographs and nuclear medicine studies also may prove beneficial as well as intraoperative frozen section. Management is based on chronicity of the infection and fixation of the components. Antibiotic suppression is unlikely to yield a cure but may be indicated in the medically infirm. Debridement with component retention may be used with varying degrees of success, especially in the acute postoperative period. The current treatment of choice for chronic infections in this country is a two-stage revision with interim intravenous antibiotics. This would be expected to yield a cure in approximately 80% of patients. Arthrodesis may be necessary in the patient who is status post-multiple revisions with particular virulent organisms. Resection arthroplasty should be reserved for the older rheumatoid patient with limited functional demands. Finally, amputation should be considered in the patient with life-threatening sepsis or the patient who is status post-multiple revisions with intractable pain and poor bone stock.

Protein synthesis is required for laminin-induced expression of the 67-kDa laminin receptor and its 37-kDa precursor.

The high affinity 67-kDa laminin receptor (67LR) is highly expressed in metastatically active human cancers. A 37-kDa polypeptide has been identified as its precursor (37LRP). Antibodies raised against 37LRP-derived synthetic peptides were used in immunogold electron microscopy and immunoblot studies to assess the effect of laminin on expression of the 67LR and the 37LRP. Laminin (15 micrograms/ml) treatment of suspended A2058 human melanoma cells doubled the expression of both 37LRP and the 67LR. Fibronectin had no effect. There was no effect of laminin on the expression of actin or galectin-3. Cycloheximide treatment of cells prior to laminin abrogated its inducible effect. The results suggest that binding of laminin by cell surface laminin receptors induces synthesis of the 37LRP and mature 67LR, with a consequent delivery to the cell surface of more laminin binding proteins for potentiated attachment of the melanoma cell to the basement membrane during invasion and metastasis.

Cell swelling, blebbing, and death are dependent on ATP depletion and independent of calcium during chemical hypoxia in a glial cell line (ROC-1).

The morphological and biochemical changes that occur during chemical hypoxic injury in a neural cell line were studied in the presence and absence of calcium. Oligodendroglial-glioma hybrid cells (ROC-1) were subjected to inhibitors of glycolytic and oxidative ATP synthesis (chemical hypoxia). Complete respiratory inhibition depleted [ATP] to less than 5% of control by 4 min. Blebs appeared on the cell surfaces and cells began to swell within a few minutes of ATP depletion. A 200% increase in cell volume and bleb coalescence preceded irreversible cell injury (lactate dehydrogenase release) which began at approximately 20 min with 50% cell death by 40 min. In energized cells an equivalent degree of osmotic swelling induced by ouabain inhibition of the Na+, K(+)-ATPase pump did not produce blebbing or cell death. Partial inhibition of respiration decreased [ATP] to approximately 10% of control by 40 min. Blebbing and swelling began at 40 min and bleb coalescence preceded plasma membrane disruption which began at approximately 55 min. ATP depletion, blebbing, swelling, and death followed similar time courses in the presence or absence of extracellular calcium ([Ca2+]e). Intracellular calcium ([Ca2+]i) was measured using fura-2. In calcium-containing medium metabolic inhibition caused a transient increase in resting [Ca2+]i (100 +/- 17 nM) followed by a low steady-state level preceding plasma membrane disruption. Following deenergization in calcium-free medium, [Ca2+]i remained below 60 nM throughout injury and death. These data suggest that decreased ATP initiates a sequence of events including bleb formation and cell swelling that lead to irreversible cell injury in the absence of large increases in [Ca2+]i.