Judah I. Rosenblatt

High-speed, three-dimensional kinematic analysis of the normal wrist.

Carpal kinematics during a wrist flexion/extension motion using high-speed videodata acquisition was investigated. A cadaver forearm was stabilized, allowing unconstrained excursion of the wrist for passive range of motion (ROM). The extensor and flexor pairs of the wrist were looped together and a 1-lb weight was attached to each pair, simulating synergistic muscle tension. Capitate/radius and third metacarpal/radius angles were calculated to determine which measurement would be best for determining global wrist angle. The average difference in capitate/radius and third metacarpal/radius angles at each respective flexion/extension wrist angle for all wrists was 1.1 degrees +/- 1.6 degrees (the maximum difference was 4 degrees). Hence, the capitate-third metacarpal joint can be considered rigid. Capitate/lunate motion as described by capitate-radius Euler angles ranged from -16.9 to 23.5 with total capitate/lunate motion of 40.5 (35%) in the 114 degrees total global wrist ROM measured. Radius/lunate motion as described by lunate-radius angle ranged from -8.2 to 48.4 with total radius/lunate motion of 56.5 (49%) in the 114 degrees total global wrist ROM measured. During global wrist motion, the radiolunate joint contributes more motion in flexion than the capitolunate joint and the capitolunate joint contributes more motion in extension than the radiolunate joint. The instantaneous screw axes (ISAs) were calculated for each third metacarpal position with respect to the radius. The average distance difference between ISAs for the 4 wrists tested was -1.23 +/- 14.97 pixels. The maximum distance was 56.51 pixels and the minimum was -24.09 pixels. This new combination of motion analysis and 3-dimensional reconstructions of computed tomography images affords a high-speed, dynamic analysis of kinematics. It shows that during wrist flexion/extension, normal carpal kinematics does not have an ISA fixed in or limited to the capitate. In addition, the ISA data provide evidence that translational motion is a real and measurable component of normal carpal motion. These findings alter the understanding of carpal kinematics obtained from the results of previous studies which suggested that the center of rotation was fixed in the capitate.

Prediction of mortality from catastrophic burns in children

BACKGROUND We previously developed a model to predict survival in massive paediatric burns (>80% total body surface area [TBSA]). This model included not only demographic variables, but also variables obtained throughout the hospital course. We aimed to prospectively validate our model for accuracy of outcome prediction. METHODS We admitted 33 paediatric burn patients with burns greater than 80% TBSA. We recorded age, burn size, inhalation injury, resuscitation, packed-cell volume at admission, base deficit, serum osmolarity, sepsis, inotropic support, platelet count, creatinine, and ventilator dependency. We entered these data into our previous models. RESULTS 20 male and 13 female children with mean age 7.6 (SD 1) years with TBSA burns of 88% (SD 1; full thickness 86% [SD 1]) were admitted. Mortality was 39.4% (13 of 30). When all variables were integrated into our final model, we predicted outcome with 97% accuracy. When we used a model based only on demographic characteristics of age, burn size, and presence of inhalation injury, outcome was correctly predicted in only 51% of patients. CONCLUSION We show prospectively that mortality in severely burned children can be reliably estimated at a burn centre, and that outcome cannot be reliably predicted on the basis of demographic and injury characteristics alone. These data suggest that all severely burned children should be given a course of treatment before consideration of treatment futility.

Age-associated changes in SAPK/JNK and p38 MAPK signaling in response to the generation of ROS by 3-nitropropionic acid.

Mitochondrial dysfunction has been identified as a major source of oxidative stress in aged tissues. In this study we asked whether activities of components of the SAPK/JNK and p38 MAPK stress response signaling pathways are indicative of oxidative stress in aged mouse livers and whether these pathways are responsive to oxidative stress generated by 3-nitropropionic acid (3-NPA), an inhibitor of complex II (succinic dehydrogenase). We asked whether (a) aging affects the basal activity of the SAPK/JNK stress signaling pathway; (b) specific isoforms of JNK, i.e. 46 or 54 kDa JNKs are activated by 3-NPA; (c) aging affects the response of this signaling pathway to 3-NPA; (d) there is a cross pathway activation of JNK or p38 MAPK by upstream activators. Our studies have shown that although their protein pool levels are not altered, the basal JNK activities using c-Jun as substrate is elevated. Furthermore, in aged livers, JNK activity is induced to a greater extent and takes longer to recover from 3-NPA treatment. The activities of the upstream activators of JNKs, MAP kinase kinase (MKK) 4 and 7, are also elevated in livers of aged C57BL/6 male mice. These activator kinases, which are induced (phosphorylated) by 3-NPA in young livers, are not inducible by this inhibitor in aged livers. In fact, these proteins are highly phosphorylated in the control aged livers and are dephosphorylated in response to 3-NPA. Finally, we demonstrate for the first time that MKK7 serves as an upstream activator of p38 MAPK and that MKK3 and MKK6 activates 54 kDa JNK2 in aged liver. Our studies suggest that failure to respond to 3-NPA may be indicative of the susceptibility of aged tissue to oxidative stress, supporting our hypothesis that aged tissues (especially liver) develop a state of chronic stress even in the absence of a challenge.

Assessment of butadiene exposure in synthetic rubber manufacturing workers in Texas using frequencies of hprt mutant lymphocytes as a biomarker.

1,3-Butadiene (BD), which is used to manufacture synthetic rubber, is a mutagen and carcinogen. Because past occupational exposures have been associated with an increased risk of leukemia, there has been a dramatic reduction in workplace exposure standards. The health benefits of these reduced levels of occupational exposure to BD will be difficult to evaluate using relatively insensitive traditional epidemiological studies; however, biomarkers can be used to determine whether there are genotoxic effects associated with recent exposures to BD. In past studies of BD-exposed workers in Southeast Texas, we observed an increase in the frequency of lymphocytes with mutations in a reporter gene, hprt. Frequencies of hprt mutant cells correlated with air levels of BD and with the concentration of a BD metabolite in urine. Average exposures to 1-3 parts per million (p.p.m.) of BD were associated with a threefold increase in hprt variant (mutant) frequencies (Vfs). We now report results from a follow-up study of workers in a synthetic rubber plant in Southeast Texas. Thirty-seven workers were evaluated on three occasions over a 2-week period for exposure to BD by the use of personal organic vapor monitors and by determining the concentration of a BD metabolite in urine. The frequency of hprt mutants was determined, by autoradiography, with lymphocyte samples collected 2 weeks after the final exposure measurement. Based on their work locations, the study participants were assigned to high-exposure (N=22) or low-exposure (N=15) groups. The BD exposure, +/-standard error, of the workers in the high-exposure group (1.65+/-0.52 p.p.m.) was significantly greater than the low-exposure group (0.07+/-0.03 p.p.m.; P<0.01). The frequency of hprt mutant lymphocytes was also significantly different in the two groups (high, 10.67+/-1.5 x 10(-6); low, 3.54+/-0.6 x 10(-6); P<0.001). The concentration of the urine metabolite was greater in the high-exposure group, but the difference was not significant. The correlation coefficient between hprt Vf and BD exposure levels was r=0.44 (CI(95), 0.11-0.69; P=0.011). This study reproduced the findings from a previous study at this plant. Although studies of butadiene-exposed workers in other countries have not detected an effect of exposure on frequencies of hprt mutant lymphocytes, we have repeatedly observed this result in our studies in Texas.

Isotopic determination of fibronectin synthesis in humans

Fibronectin is an opsonic protein that, among other functions, activates the reticuloendothelial system. Accurate measurement of its rate of synthesis is necessary to more fully understand its physiological role in normal and pathological conditions. We have determined the rate of fibronectin synthesis in three normal volunteers using a primed-constant infusion of 15N-glycine and 1,2-13C-leucine, and measuring the incorporation of the isotopes into the protein over 5 days of infusion. In nine additional subjects, the fractional synthetic rate (FSR) of fibronectin was calculated during a 24-hour infusion using urinary hippurate and plasma alpha-ketoisocaproic acid enrichment to represent the precursors for incorporation of labeled glycine and leucine, respectively, into fibronectin. The FSR using glycine and leucine was 1.56 +/- 0.14 and 1.29 +/- 0.04 (%/h), respectively, in the 5-day infusion study, and 1.56 +/- 0.10 versus 1.83 +/- 0.09 (%/h), respectively, in the 24-hour study. The results of the 5-day infusion of 15N-glycine justify the use of urinary hippurate to reflect the precursor enrichment for the determination of the FSR of fibronectin during a shorter (less than 24 hour) infusion period.

Some theoretical and practical considerations for multivariate statistical cell classification useful in autologous stem cell transplantation and tumor cell purging

BACKGROUND As flow cytometric data becomes more complex, it becomes increasingly difficult to classify cells using conventional flow cytometry data techniques based on visual classification of the data by user-drawn regions. This paper shows some simple applications of multivariate statistical classification to classify flow cytometric data. METHODS Discriminant Function Analysis (DFA) and Logistic Regression (LR) analysis techniques were evaluated with respect to their potential utility in the problem of detecting human breast cancer cells within normal bone marrow cells. Data sets having defined properties were employed to evaluate the potential utility of these statistical classification techniques whose performance was measured by ROC analysis. RESULTS Two extreme but reasonable situations are presented: (1) data where the separation of cells was obvious by visual inspection and (2) data where major overlaps in the values of the individual FCM parameters made intuitive classification improbable. Both DFA and LR analysis were able to classify the cells of each type with acceptable accuracy and yield. CONCLUSIONS The excellent empirical performance of both DFA and LR techniques, suggests that they offer promising approaches for classifying multiparameter FCM data using objective rules that may represent an improvement over commonly employed ad hoc approaches.

Real-time multivariate statistical classification of cells for flow cytometry and cell sorting: a data mining application for stem cell isolation and tumor purging

Multivariate statistics can be used for visualization of cell subpopulations in multidimensional data space and for classification of cells within that data space. New data mining techniques we have developed, such as subtractive clustering, can be used to find the differences between test and control multiparameter flow cytometric data, e.g. in the problem of human stem cell isolation with tumor purging. They also can provide training data for subsequent multivariate statistical classification techniques such as discriminant function or logistic regression analyses. Using lookup tables, these multivariate statistical calculations can be performed in real-time, and can even include probabilities of misclassification. Thus, the only distinction between off-line classification of cells in data analysis and real-time statistical decision-making for cell sorting is the time limit in which a classification decision must be made. For real-time cell sorting we presently are able to perform these classifications in less than 625 microseconds, corresponding to the time that it takes the cell to travel from the laser intersection point to the sort decision point in a flow cytometer/cell sorter. Statistical decision making and the ability to include the costs of misclassification into that decision process will become important as flow cytometry/cell sorting moves from diagnostics to therapeutics.

Diarrhea in Severely Burned Children

BACKGROUND Diarrhea is a common problem in critically ill patients. Our patients are fed a high-carbohydrate enteral formula. We hypothesized that diarrhea in our patients may be related to the osmotic effects of unabsorbed carbohydrate in the small intestine and colon. METHODS We studied 19 patients, 3 months to 17 years, with burns >40% total body surface area. Each subject was studied weekly for up to 4 weeks postburn. Breath H2 concentration was measured. For the 24-hour period before the breath H2 measurement, the enteral carbohydrate intake, stool volume, and total enteral fluid volume were recorded. At each of several weekly intervals for each subject, the times when stool volume and enteral carbohydrate intake were each maximal were noted. RESULTS Maximal stool volume ranged from 12 to 69 mL/kg/d. At the time point of maximal carbohydrate intake, diarrhea (stool volume >10 mL/kg/d) occurred in 18 of 19 patients, and maximal stool volume occurred in 10 of 19. Breath H2 concentration (ppm/5% CO2; mean +/- SEM) was 5.5 +/- 3.5 at the time of maximal carbohydrate intake, and was 25 +/- 20 at maximal stool volume. There were no correlations among breath H2 concentration, stool volume, enteral fluid intake, and enteral carbohydrate intake. CONCLUSIONS Almost all the subjects had diarrhea over several weeks postburn. The lack of correlation of either carbohydrate intake or breath H2 with stool volume suggests that diarrhea in these patients may be caused by factors other than carbohydrate malabsorption. These data do not support altering nutrition support because of watery diarrhea.

Kinetics of Repair of UV-induced DNA Damage in Repair-proficient and -deficient Cells as Determined by Quantitative Polymerase Chain Reaction

Abstract— Advances in methodologies to monitor gene‐specific repair in human cells have facilitated a detailed understanding of the complexity of the nucleotide excision repair system. One of these procedures, quantitative polymerase chain reaction (QPCR), holds significant promise for dissecting the fine structure of the repair of UV‐induced DNA damage. This assay was used to study the repair of UV photoproducts in both actively transcribed and nontranscribed genes from human cells that were capable of (1) repair of both cyclobutane pyrimidine dimers and 6‐4 photoproducts; (2) removal of neither dinners nor 6‐4 photoproducts; (3) strong preferential repair of 6‐4 photoproducts relative to dimers; and (4) severely depressed rates of 6‐4 photoproducts and dimers. Detailed kinetic analyses revealed that repair of both active and inactive genes can be studied with a very fine degree of precision and that the repair status of the cells can easily be detected by use of the procedures described.

The mathematics behind modeling.

The purpose of this chapter has been to furnish insight into the theoretical background on which compartmental modeling software packages are based. To accomplish this goal, only the basic ideas were stressed, avoiding discussion of the intricacies required for efficiency. The object was to remove the mystery from these powerful programs by examining the fundamental ideas which make them tick. The first section was concerned with how compartmental models are built up and how to obtain information concerning the system behavior described by these models. The cornerstone here is to describe a system by determining how it behaves over (typically) very short time periods. This leads to a differential equation description of a compartmental system. Information can be extracted from these equations by returning to their basic meaning, illustrated in their derivation. Computers are ideal for obtaining this information by piecing together the results obtained over short time periods, to find the behavior over long time periods. In an actual situation governed by a compartmental model, its often the case that we may know only the form of the model, but not the values of the rate constants which must be known for its effective use. The second section of the chapter was devoted to the practical problem of determining the rate constants of the model, based on observed data. This is a matter of searching for those values which, in some sense, best fit the data. To attack this problem we need a reasonable criterion to judge how well a proposed model fits the data. We chose to use the total squared deviation, psi, which is the most common such criterion--but not the only reasonable one. The search technique we examined--steepest descent--is based on a simple idea: looking at the total squared deviation criterion geometrically. In graphical terms, the best fit corresponds to finding the low point on a surface, whose height above any point at sea level is computable. If we could imagine the view of someone trying to find the low point from some arbitrarily chosen initial position on this mountain-like surface, we would look around and find the direction where (close by) the mountain drops off most steeply. We would go in that specific direction until we reach a low point, moving only along this initially chosen direction. At this new low point, we could change again to a direction of steepest descent, and keep repeating this process until we make no further effective downward progress. No guarantee in general is made for this process, but it often works. Finally, having found the best values for the rate constants, we must recognize that if the data is affected by factors not explicitly taken into account in the model, the variability induced by these factors precludes a perfect fit. For this reason, it is finally necessary to determine how good the model is, as a description of the data, and how accurate are the fitted rate constants. For the model fit, the sample RMS error (simply related to the total squared deviation, and often the same as the sample standard deviation) may be used. For determining the accuracy of the fitted rate constants, practical methods based on computer software simulations are recommended.