Expression of KB cell alkaline phosphatase isoenzymes during growth in immunosuppressed LEW rats.

Alkaline phosphatase isoenzyme expression of human tumor xenografts was studied by the growing of KB cells in immunosuppressed neonatal LEW rats. In culture these cells produced the oncoamniotic (FL) isoenzyme as the major form and the Regan isoenzyme as a minor fraction as well as a "hybrid" that shared properties of both of the other isoenzymes. Despite a reduction in specific activity, this isoenzyme pattern was essentially unchanged during in vivo growth. KB cells "pretreated" in culture with the glucocorticoid prednisolone in hyperosmolal medium exhibited a decrease in the levels of the oncoamniotic (FL) isoenzyme and an increase in the Regan isoenzyme. During growth of pretreated cells in vivo, a time-dependent resumption in the expression of the oncoamniotic (FL) isoenzyme was associated with the disappearance of the Regan isoenzyme. This shows that the expression of the oncoamniotic (FL) isoenzyme is not restricted to human tumor cells monophenotypic with respect to alkaline phosphatase.

Multiple effects of glucocorticoids and hyperosmolality on isoenzyme expression in cultured KB cells.

The KB cell line, though indicted as a HeLa-contamined line, is a useful in vitro model for the study of the regulation of isoenzyme expression. KB cells produced three isoenzymic forms of alkaline phosphatase. When KB cells were grown in the presence of prednisolone and/or in hyperosmolar medium, the total enzyme activity was reduced. This change in activity was coupled with specific alterations in the proportion of each isoenzyme. The slow-moving form (identified biochemically and immunologically as the heat-stable, placental, Regan isoenzyme) was substantially increased by the steroid hormone and/or hyperosmolality. The fast-moving form [identified as the intestine-like, amnion (FL) isoenzyme] was strikingly diminished by either treatment. The intermediate form (tentatively referred to as "hybrid," inasmuch as it shared properties of the other two isoenzymes) was decreased only when KB cells were grown in hyperosmolar medium containing prednisolone. These results corroborated the notion that these stimuli cause the induction of increased levels of the heat-stable Regan alkaline phosphatase only. They also point out the necessity of performing isoenzyme analysis when one is investigating the regulation of this enzyme.

Coproduction of Regan isoenzyme and carcinoembryonic antigen in HCT-8 cells.

The presence of the Regan isoenzyme of alkaline phosphatase was demonstrated in the carcinoembryonic antigen-producing human cell line HCT-8 Biochemical, histochemical, immunologic, and electrophoretic methods were used as criteria for the identification and characterization of this isoenzyme.

Evidence for altered gene regulation in HeLa cells returned to culture after growing in immunosuppressed rats.

HeLa TCRC-1, is a monophenotypic cell line for the production of the carcinoplacental Regan isoenzyme of alkaline phosphatase. It has been previously reported that, when this cell line is grown in immunosuppressed rats, there is an alteration of phenotypic expression in that an isoenzyme that is referred to as oncoamnion (FL) becomes the dominant enzyme form. This report describes the isoenzyme regulation after these cells are returned to culture for at least three months. These cells in culture demonstrate a density-dependent alteration in isoenzyme profiles. The Regan isoenzyme is the dominant isoenzyme form in new, sparsely populated cultures, while the oncoamnion isoenzyme predominates in the later high-density stages of growth. Prednisolone induction of enzyme activity is found to be most effective in the early stages of growth, and the previously reported specificity of hormone action on isoenzymes produced by cancer cells is maintained. Enzyme induction is restricted to the Regan isoenzyme, while hormone-mediated diminution in enzyme activity is confined to the oncoamnion (FL) isoenzyme.

Regulatory controls of oncotrophoblast proteins and developmental alkaline phosphatases in cancer cells.

The two oncotrophoblast proteins, Regan isoenzyme (placental-type alkaline phosphatase) and human chorionic gonadotrophin, are readily studied oncodevelopmantal gene products in human cancer patients and in three experimental model systems. The latter consists of (a) HeLa sublines TCRC-1 and TCRC-2, which produce Regan and non-Regan isoenzymes, (b) HEp-2 and FL amnion cell lines as models for the reciprocal expression of developmental genes, and (c) modulation in vivo of developmental gene expression in HeLa cells. In the case of the third model, for example, HeLa TCRC-1 cells grow in immunosuppressed rats to form a tumor nodule, which expresses a new oncoamnion (FL) isoenzyme, while the Regan isoenzyme disappears. Return of the tumor cells to cell culture medium results in a disappearance of the oncoamnion (FL) species and the reappearance of Regan isoenzyme. This interesting model is expected to bridge the interpretation of experiments done in cell culture with observations made on tumors of cancer patients. Most helpful in interpretation of all these studies has been a chronology of early development. It appears that the counterparts of a number of tumor proteins appear as early as gametogenesis and as late as 10 weeks of gestation.

The release of high-molecular-weight alkaline phosphatase and leucine aminopeptidase into the media of cultured human cells.

Using exclusion from Sepharose 4B as our criterion, we have found a high-molecular-weight form of alkaline phosphatase and of leucine aminopeptidase which are released into the culture media by the FL amnion cell line. A low-molecular-weight form of leucine aminopeptidase is also found to contribute to the total levels of this enzyme in the media. The levels of these enzymes increased during the growth cycle of the culture, paralleling the increase in cell density, suggesting that the two events may be related. This phenomen in culture suggests a possible explanation for the appearance of similar enzyme forms in patient serum and fluids originating from diseased tissue.

Induction of alkaline phosphatase activity in cultured human intracranial tumor cells.

Alkaline phosphatase activity in several cultured primary human intracranial tumor cells varied over a relatively wide range, and there was no correlation between specific activity and the type of tumor from which the cultures were derived. The enzyme was thermolabile, and its activity was strongly inhibited by l-bromotetramisole, levamisole, and L-homoarginine but not by L-phenylalanine and L-phenylalanyglycylglycine. These are the characteristics of the liver-bone-kidney form of alkaline phosphatase. Prednisolone induced increased levels of enzyme activity in most cultures, and sodium butyrate acted as an inducer in cultures of pituitary adenoma and hemangioblastoma cells. The increase was most pronounced when response cells were exposed to both stimuli simultaneously. The induced alkaline phosphatase had the same properties as the enzyme of cells grown in the absence of inducers. Increased alkaline phosphatase activity was not induced by osmolality changes of the culture medium; this feature appears to be characteristic of cells producing the liver-bone-kidney enzyme form.

Detection of Epstein-Barr virus in CNS lymphomas by in-situ hybridization.

We used an optimized in-situ hybridization technique employing a biotinylated Epstein-Barr (EB) virus sequence, BamH1V (3.1 kb), to detect this sequence in 2 EB virus-infected cell lines (B95-8 and Namalwa) and 8 CNS lymphomas. We obtained a good hybridization signal from cytospins of B95-8 (EB virus productively infected) and Namalwa (EB virus latently infected, 1 copy per cell) cell lines. We were able to detect signal from both cell lines after overnight fixation in 10% formalin and paraffin embedding, but development time in the detection chromogen required longer incubation and the signal intensity was lower than in cytospin cells. We then used the technique to examine formalin-fixed, paraffin-embedded primary CNS lymphoma tissue from 4 patients who were immunocompromised (1 renal transplant, 3 acquired immune deficiency syndrome) and 4 patients who were not. All 4 CNS lymphomas from immunocompromised patients hybridized well with BamH1V, exhibiting a pattern of staining similar to Namalwa cells and nonlytically infected B95-8 cells. There was no relationship between the intensity and degree of reaction and the patients' survival. None of the 4 CNS lymphomas in immunocompetent patients or uninvolved brain showed any reactivity with BamH1V. We suggest that low-abundance targets are detectable in paraffin-embedded tissue by in-situ hybridization using biotinylated probes. Detection of EB viral sequences in CNS lymphomas in immunocompromised patients suggests a role for the virus in the pathogenesis of this tumor.

Plasma membrane localization of alkaline phosphatase in HeLa cells.

The localization of alkaline phosphatase in HeLa cells was examined by electron microscopic histochemistry and subcellular fractionation techniques. Two monophenotypic sublines of HeLa cells which respectively produced Regan and non-Regan isoenzymes of alkaline phosphatase were used for this study. The electron microscopic histochemical results showed that in both sublines the major location of alkaline phosphatase is in the plasma membrane. The enzyme reaction was occasionally observed in some of the dense body lysosomes. This result was supported by data obtained from a subcellular fractionation study which showed that the microsomal fraction rich in plasma membrane fragments had the highest activity of alkaline phosphatase. The distribution of this enzyme among the subcellular fractions closely paralleled that of the 5'-nucleotidase, a plasma membrane marker enzyme. Characterization of the alkaline phosphatase present in each subcellular fraction showed identical enzyme properties, which suggests that a single isoenzyme exists among fractions obtained from each cell line. The results, therefore, confirm the reports suggesting that plasma membrane is the major site of alkaline phosphatase localization in HeLa cells. The absence of any enzyme reaction in the perimitochondrial space in these cultured tumor cells also indicates that the mitochondrial localization of the Regan isoenzyme reported in ovarian cancer may not be a common phenomenon in Regan-producing cancer cells.

Impact of joint impairment on disability-specific domains at four years.

The objective of this study was to assess the longitudinal impact of joint impairment on overall disability and crossing domain-specific thresholds for physical activity, mobility, dexterity, instrumental activities of daily living (IADL), and activities of daily living (ADL) that are associated with use of long-term care. This 4-year longitudinal study observed 484 persons older than age 60. Logistic regression assessed the contribution of demographics, psychological mediators, lower- and upper-extremity joint impairment, and comorbidities to increased domain-specific self-reported disability above a threshold associated with use of long-term care. Lower-extremity joint impairment and age predicted crossing thresholds by year 4 in physical activity, mobility, IADL, and ADL disability that were associated with use of long-term care. Lower-extremity joint impairment is a strong risk factor for future disability that is associated with use of long-term care.

Skeletal traction for fractures of the femoral shaft in children. A long-term study.

Fifty-four children who had been treated with 90-90 skeletal traction for a fracture of the femoral shaft were examined after an average follow-up of 4.3 years. All of the patients were functioning normally and had a symmetrical range of motion of both hips and knees. A limb-length discrepancy of more than thirteen millimeters was found in three of thirty-nine children who were less than eleven years old and in eight of fifteen who were more than eleven years old. Traction pins that were placed obliquely were associated with a statistically significant (p less than 0.01) and predictable difference in the intercondylar angle (axis of the knee joint) as compared with pins that were placed horizontally. The study showed that pins for skeletal traction should be placed parallel to the axis of the knee joint and that fractures in children who are more than eleven years old should be reduced without overriding.

Transfer of the trapezius for flail shoulder after brachial plexus injury.

Shoulder arthrodesis is often used to treat flail shoulder after a brachial plexus injury, but has a high complication rate and entails loss of passive mobility. We have reviewed 27 patients with brachial plexus injury treated by transfer of the trapezius to the proximal humerus at an average time from injury of 31.3 months. Pre-operatively, all 27 shoulders were subluxated, with an average abduction of 3.5 degrees. Postoperatively, shoulder abduction averaged 45.4 degrees, and subluxation was abolished. All patients were satisfied with their improvement in function. Trapezius transfer is recommended as a simple procedure that requires only a brief period in hospital, allows early rehabilitation, and gives a satisfactory outcome, while retaining passive mobility of the shoulder.

Neurobehavioral changes among shipyard painters exposed to solvents.

Painters in three shipyards, exposed to a wide variety of solvents, were examined. A short battery of performance tests, a detailed occupational history, and a special questionnaire to assess acute (prenarcotic, transitory) and chronic (persistent) neurologic symptoms was administered. The results of the neurobehavioral performance tests demonstrated decrements in central nervous system function in painters when compared with a control group matched for age, sex, race, and education. The prevalence of reported acute neurological symptoms among painters was increased significantly compared to other occupational groups in the same yards; for chronic, persistent symptoms the difference was not statistically significant. Performance test scores were significantly, negatively correlated with chronic symptoms but not with acute symptoms. No significant correlations between performance test scores and duration of solvent exposure or between symptoms and duration of solvent exposure were observed. The reversibility of such symptoms and of decrements in central nervous system function after cessation of exposure is still uncertain.

Snowblower injuries to the hand.

We retrospectively reviewed the records of 62 patients who sustained serious hand injuries caused by snowblowers between 1981 and 1990. Frequency of injuries to digits tended to correlate with length (i.e., middle, index, ring, or small finger or thumb). Damage to tendons did not seem to follow any particular pattern. The majority of victims sustained multiple digital involvement. Complete versus partial amputation followed the same length distribution as did injured digits. Most of the injuries occurred to the dominant hand. When patients were further questioned regarding the circumstances and events leading to their injury, a recurring pattern was found. Most patients described a wet, heavy snow having recently fallen. The majority of the patients who were injured by placing their hands into the exit chute admitted that they were aware the machine was running, but thought that they had a greater clearance to the rotating impeller blade.

Carcinoplacental alkaline phosphatase. Base level and hormone-induced activity associated with events in the cell cycle.

Using HeLa TCRC-1, a cell line which is monophenotypic with respect to the Regan isoenzyme of alkaline phosphatase, we have examined the factors which influence its expression in relation to events of the cell cycle. DNA synthesis is not required for hormone induction of the Regan isoenzyme as in the presence of hydroxyurea, a specific inhibitor of DNA synthesis, we found induction to occur. Additionally, when partially synchronised cells were allowed to leave the S period prior to hormone treatment, and hydroxyurea was added to prevent cells from entering the next S period, hormone induction of the Regan isoenzyme was still observed. This indicates that initiation of expression of hormone-induced carcinoplacental alkaline phosphatase occur prior to the DNA synthetic phase of the cell cycle. We propose a hypothetical two-step mechanism of hormone induction to interpret the present findings in relation to previous results.

Purpura fulminans.

Purpura fulminans is a rare disease that typically begins as a benign infectious process and subsequently progresses to severe sepsis, hypotension, purpura ecchymosis, and disseminated intravascular coagulation. We present an unusual case of an adult who was seen initially with pneumococcal sepsis that subsequently developed into purpura fulminans with major extremity involvement. A multidisciplinary approach is needed in the treatment of this often catastrophic disease.