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Biomedical subjects

M H Kroll

Publications and source records attributed to M H Kroll.

At least 19 recordsLinked to original sources
PubMed2004

von Willebrand factor binding to platelet glycoprotein Ib-IX-V stimulates the assembly of an alpha-actinin-based signaling complex.

BACKGROUND: Pathological shear stress induces platelet aggregation that is dependent on von Willebrand factor (VWF) binding to glycoprotein (Gp)Ib-IX-V and phosphatidylinositol 3-kinase activation. We tested the hypothesis that pathological shear stress stimulates phosphatidylinositol 3,4,5-trisphosphate (PIP3) synthesis by directing the assembly of a molecular signaling complex that includes class IA phosphatidylinositol 3-kinase (PI 3-KIA). METHODS: Platelets were subjected to 120 dynes cm-2 shear stress in a cone-plate viscometer. Resting and sheared platelets were lyzed, immunoprecipitations of PI 3-KIA performed, or lipids extracted for PIP3 measurements. alpha-Actinin was incubated with phosphatidylinositol 4,5-bisphosphate (PIP2), immunoprecipitated, and used as a substrate for in vitro PI 3-KIA activity. RESULTS: Pathological shear stress induces biphasic PIP3 production. In resting platelets, PI 3-KIA associates with alpha-actinin and PIP2. After exposure to shear stress, alpha-actinin and PIP2 rapidly disassociate from PI 3-KIA. PI 3-KIA then gradually re-associates with PIP2 and alpha-actinin, and this complex becomes linked to GpIb alpha through the cytoskeleton. PIP3 production and the observed changes in the association between alpha-actinin, PIP2, and PI 3-KIA are inhibited when VWF binding to GpIb alpha is blocked. In a cell-free system, alpha-actinin binds PIP2 and when the alpha-actinin-PIP2 complex is added to platelet PI 3-KIA, PIP3 production is stimulated. CONCLUSIONS: These results suggest that pathological shear-induced VWF binding to GpIb-IX-V stimulates PIP3 production through the assembly of an alpha-actinin-based complex that colocalizes PI 3-KIA with substrate PIP2.

Actinin↗
PubMed2001

Glycoprotein Ib/IX/V binding to the membrane skeleton maintains shear-induced platelet aggregation.

The extracellular domain of glycoprotein (Gp) Ibalpha serves as the von Willebrand factor (vWf) receptor that triggers shear stress-dependent platelet aggregation. Its intracellular domain associates with actin-binding protein-280 (filamin 1a) that binds directly to filamentous actin, thereby linking the membrane skeleton to GpIbalpha. We examined the functional significance of GpIbalpha interactions with actin during platelet aggregation in response to 120 dyn/cm(2) shear stress. Lysates of resting and sheared platelets were centrifuged at approximately 13,000xg for 15 min, and GpIbalpha was immunoprecipitated from the lysate supernatant. GpIbalpha and coimmunoprecipitated proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotted with antibodies specific for GpIbalpha and actin. We observed a significant increase in the amounts of actin coimmunoprecipitating with GpIbalpha as platelets aggregated in response to shear stress. Actin/GpIbalpha interactions reached a maximum after 90 s of shear stress. Monoclonal antibody (mAb) blockade of vWf binding to GpIbalpha inhibited shear stress-induced platelet aggregation and actin associating with GpIbalpha. Pretreatment of platelets with cytochalasin D resulted in the inhibition of actin binding to GpIbalpha in sheared platelets and in an increase in the rate and magnitude of platelet disaggregation. These data indicate that shear stress causes changes in the association between GpIbalpha and the actin-based membrane skeleton. The increased interaction between GpIbalpha and the actin-based membrane skeleton results from shear-induced vWf binding to GpIbalpha and is mechanoprotective in that it maintains shear-induced aggregation of activated platelets.

Actins↗
PubMed2000

Cytoplasmic domains of GpIbalpha and GpIbbeta regulate 14-3-3zeta binding to GpIb/IX/V.

Shear stress causes the platelet glycoprotein (Gp) Ib/IX/V to bind to von Willebrand factor, resulting in platelet adhesion. GpIb/IX/V also functions to stimulate transmembranous signaling, leading to platelet activation and the expression of a ligand-receptive GpIIb-IIIa complex. The highly conserved cytoplasmic domain of GpIbalpha binds directly to a dimeric 14-3-3 adapter protein zeta isoform. To explore structural determinants of GpIb/IX/V binding to 14-3-3zeta, the authors examined 14-3-3zeta interactions with GpIbalpha and GpIbbeta in heterologous cells and platelets. Truncations of GpIbalpha at amino acid 542 or 594, or deletions of residues 542 through 590, inhibited binding of 14-3-3zeta. Deletion of GpIbalpha from Trp(570) to Ser(590) eliminated 14-3-3zeta binding, and deletion of the sequence from Arg(542)-Trp(570) enhanced binding of 14-3-3zeta to GpIbalpha. All GpIbalpha mutations that eliminated GpIbalpha binding to the GST-14-3-3zeta fusion protein also eliminated GpIbbeta binding to the fusion protein. Forskolin treatment of Chinese hamster ovary cells expressing wild-type GpIbalpha/beta/IX resulted in the phosphorylation of GpIbbeta associated with enhanced binding of GpIbbeta to GST-14-3-3zeta fusion protein and increased 14-3-3zeta coimmunoprecipitated with GpIbalpha. When intact human platelets aggregated in response to 90 dynes/cm(2) shear stress, 14-3-3zeta disassociated from GpIbalpha. Prostacyclin treatment of platelets inhibited shear stress-induced aggregation and the release of 14-3-3zeta from GpIbalpha. These data demonstrate that amino acid residues in the cytoskeletal interaction domains of GpIbalpha regulate 14-3-3zeta binding to GpIbalpha/beta/IX, and suggest that protein kinase A-dependent phosphorylation of GpIbbeta enhances 14-3-3zeta binding to the GpIb/IX/V complex in human platelets. (Blood. 2000;95:551-557)

14-3-3 Proteins↗
PubMed2000

Parathyroid hormone temporal effects on bone formation and resorption.

Parathyroid hormone (PTH) paradoxically causes net bone loss (resorption) when administered in a continuous fashion, and net bone formation (deposition) when administered intermittently. Currently no pharmacological formulations are available to promote bone formation, as needed for the treatment of osteoporosis. The paradoxical behavior of PTH confuses endocrinologists, thus, a model bone resorption or deposition dependent on the timing of PTH administration would de-mystify this behavior and provide the basis for logical drug formulation. We developed a mathematical model that accounts for net bone loss with continuous PTH administration and net bone formation with intermittent PTH administration, based on the differential effects of PTH on the osteoblastic and osteoclastic populations of cells. Bone, being a major reservoir of body calcium, is under the hormonal control of PTH. The overall effect of PTH is to raise plasma levels of calcium, partly through bone resorption. Osteoclasts resorb bone and liberate calcium, but they lack receptors for PTH. The preosteoblastic precursors and preosteoblasts possess receptors for PTH, upon which the hormone induces differentiation from the precursor to preosteoblast and from the preosteoblast to the osteoblast. The osteoblasts generate IL-6; IL-6 stimulates preosteoclasts to differentiate into osteoclasts. We developed a mathematical model for the differentiation of osteoblastic and osteoclastic populations in bone, using a delay time of 1 hour for differentiation of preosteoblastic precursors into preosteoblasts and 2 hours for the differentiation of preosteoblasts into osteoblasts. The ratio of the number of osteoblasts to osteoclasts indicates the net effect of PTH on bone resorption and deposition; the timing of events producing the maximum ratio would induce net bone deposition. When PTH is pulsed with a frequency of every hour, the preosteoblastic population rises and decreases in nearly a symmetric pattern, with 3.9 peaks every 24 hours, and 4.0 peaks every 24 hours when PTH is administered every 6 hours. Thus, the preosteoblast and osteoblast frequency depends more on the nearly constant value of the PTH, rather than on the frequency of the PTH pulsations. Increasing the time delay gradually increases the mean value for the number of osteoblasts. The osteoblastic population oscillates for all intermittent administrations of PTH and even when the PTH infusion is constant. The maximum ratio of osteoblasts to osteoclasts occurs when PTH is administered in pulses of every 6 hours. The delay features in the model bear most of the responsibility for the occurrence of these oscillations, because without the delay and in the presence of constant PTH infusions, no oscillations occur. However, with a delay, under constant PTH infusions, the model generates oscillations. The osteoblast oscillations express limit cycle behavior. Phase plane analysis show simple and complex attractors. Subsequent to a disturbance in the number of osteoblasts, the osteoblasts quickly regain their oscillatory behavior and cycle back to the original attractor, typical of limit cycle behavior. Further, because the model was constructed with dissipative and nonlinear features, one would expect ensuing oscillations to show limit cycle behavior. The results from our model, increased bone deposition with intermittent PTH administration and increased bone resorption with constant PTH administration, conforms with experimental observations and with an accepted explanation for osteoporosis.

Aged↗
PubMed2000

Multicenter study of whole-blood creatinine, total carbon dioxide content, and chemistry profiling for laboratory and point-of-care testing in critical care in the United States.

OBJECTIVES: To introduce a creatinine biosensor and a total carbon dioxide content (TCO2) method for whole-blood measurements, to evaluate the clinical performance of a new transportable analyzer that simultaneously performs these two and six other tests (Na+, K+, Cl-, glucose, urea nitrogen, and hematocrit), and to assess the potential of the new analyzer for point-of-care testing in critical care by comparing results obtained by nonlaboratory personnel and by medical technologists. DESIGN: Multicenter sites compared whole-blood measurements with the transportable analyzer to plasma measurements from the same specimens with local reference instruments. One site compared whole-blood results produced by nonlaboratory personnel vs. medical technologists and evaluated day-to-day and within-day precision at the point of care. SETTINGS AND PATIENTS: Four medical centers in the United States. Venous and arterial specimens from 710 critically ill patients with a variety of diagnoses. Point-of-care testing in the emergency room and operating room. RESULTS: The linear regression analyses at the four medical centers showed the following: creatinine (a) slope, 0.91 to 1.22, (b) y intercept, -0.07 to 0.15 mg/dL, and (c) r2, 0.77 to 1.00; and TCO2: (a) slope, 0.64 to 1.00, (b) y intercept, 1.36 to 9.6 mmol/L, and (c) r2, 0.52 to 0.72 (yi, whole-blood analyses; xi, plasma reference measurements). Bland-Altman plots also were used to assess multicenter creatinine and TCO2 results. Of the other analytes, K+, glucose, and urea nitrogen had the highest r2-values. For the eight chemistry profile tests performed at the point of care (yi, nonlaboratory personnel results; xi, medical technologist results), the average value of r2 was 0.96 (SD 0.08) in the operating room and 0.96 (SD 0.06) in the emergency room, and mean paired differences (yi - xi) were not statistically or clinically significant. Precision was acceptable. CONCLUSIONS: The performance of the creatinine biosensor and the TCO2 method was acceptable for whole-blood samples. Comparisons of whole-blood results from the transportable analyzer and plasma results from the local reference instruments revealed analyte biases that may be attributed to differences between direct whole-blood analyses and indirect-diluted plasma measurements and other factors. Performance of nonlaboratory personnel and medical technologists was equivalent for point-of-care testing in critical care settings. The whole-blood analyzer should be useful when patient care demands immediate results.

Biosensing Techniques↗
PubMed2000

Standardization of lipoprotein reporting.

We wanted to ascertain whether the current format of lipid laboratory reports seemed adequate to promote identification and treatment of patients with dyslipidemia. In a random survey of lipid laboratory reports from 25 laboratories, we found great inconsistencies among reporting formats and contents. Fewer than half the laboratories correctly reported the ranges for cholesterol, only 4 correctly reported ranges for high-density lipoprotein cholesterol, only 2 correctly reported ranges for triglycerides, and none presented low-density lipoprotein cholesterol ranges in terms of risk factors for coronary heart disease. Reports typically were disjointed and difficult to read. The current practice of reporting results for lipid panels is confusing and does not follow the National Cholesterol Education Program (NCEP) guidelines. We recommend that reporting of results be standardized, and a "model" standardized report is presented herein, based on consensus from a team of experts. The standardized report uses current recommendations for ranges, follows the flowcharts of the NCEP guidelines, and takes the patient's clinical condition (the number of risk factors and the presence of coronary heart disease) into consideration. Standardizing lipid reports should decrease confusion and perhaps increase application of the guidelines and patient compliance with treatment.

Cholesterol↗
PubMed2000

Evaluation of the extent of nonlinearity in reportable range studies.

OBJECTIVES: To extend the polynomial method for evaluating linearity in 2 ways. First, we developed a screen to ascertain whether the data were precise enough to permit a reliable evaluation of linearity and therefore eliminate findings of linearity due to low statistical power. Second, we assessed whether the degree of nonlinearity detected by the polynomial method was clinically relevant using a statistically rigorous method. METHODS: Because we assessed linearity relative to a clinically determined level of importance instead of the default value of zero, we used sampling theory based on the noncentral chi(2) distribution. Using statistical power calculations, we incorporated a screen for imprecision that guarantees that the probability of correctly identifying nonlinear methods is at least 80%. RESULTS: With the described methods, we achieved a sensitivity of at least 80% and a specificity of at least 95%. When the data were too imprecise to achieve a sensitivity of 80%, no determination of linearity was made. This procedure mimics the practice in manual inspection of flagging data that appear imprecise by visual inspection and halting the evaluation. CONCLUSIONS: Formal statistical tests for precision and amount of nonlinearity are advantageous because they allow us to quantify and limit classification errors. By formalizing these various aspects of linearity assessment, we maintain some of the complex features of manual methods while making the linearity assessment feasible to apply to a high volume of assessments and removing the between-analyst variability.

Humans↗
PubMed2000

Mechanism of interference of a polymerized hemoglobin blood substitute in an alkaline phosphatase method.

BACKGROUND: Hemoglobin-based oxygen carriers can cause profound interferences in many analytical procedures. We determined the mechanism of interference in the assay of alkaline phosphatase activity and identified approaches that might be used to correct for this interference. METHODS: Interference of a polymerized hemoglobin blood substitute with the assay of alkaline phosphatase was examined with a Hitachi 917 analyzer and ultraviolet-visible spectrophotometry. RESULTS: Hemoglobin-based oxygen carrier solutions had substantial absorbance at 415 nm, the wavelength of analysis used to measure the formation of 4-nitrophenol. In addition to offsetting the initial absorbance at the analytical wavelength, polymerized hemoglobin gave rise to a strong negative interference plot because of alkali denaturation of the substitute. The same interference mechanism was also observed for native hemoglobin (hemolysate), indicating that the interference was not derived from the polymerization process. The interference can be corrected by implementing a rate-correction procedure, or the interference can be avoided by measurement at 450 nm. CONCLUSIONS: The interference of polymerized hemoglobin in the alkaline phosphatase assay is a result of an absorbance offset caused by alkali denaturation of hemoglobin. The interference can be corrected or avoided by modifying the calculation or the analytical wavelength. The correction strategy may also be applicable to improving the hemolysis index for this method.

Alkaline Phosphatase↗
PubMed1999

The glycoprotein Ib/IX complex regulates cell proliferation.

The glycoprotein (Gp) Ib/IX complex contains three transmembranous leucine-rich repeat polypeptides (GpIbalpha, GpIbbeta, and GpIX) that form the platelet von Willebrand factor (vWF) receptor. GpIb/IX functions to effect platelet adhesion, activation, and aggregation under conditions of high shear stress. GpIb/IX is expressed late in the ontogeny of megakaryocytes, the precursor cell that releases platelets when it reaches its terminal stage of differentiation. Because signal pathways can be reused at different stages of development by integration with different effector pathways and because cellular adhesion through other receptor families often modulates cell growth, the hypothesis that GpIb/IX regulates cell growth was investigated. The surface expression of recombinant GpIbalpha decreases the proliferation of transduced CHO cells. GpIbalpha causes growth arrest in the G1 phase of the cell cycle associated with the induction of the cyclin-dependent kinase inhibitor p21. G1 arrest induced by recombinant GpIbalpha in heterologous cells requires signaling through the 14-3-3zeta binding domain of GpIbalpha and is partially dependent on its engagement by the extracellular ligand vWF. Growth arrest induced by the expression of recombinant GpIb/IX is followed by apoptosis of the transduced cells. The endogenous expression of GpIbalpha in human hematopoietic cells is associated with decreased proliferation. These results suggest that the expression of the GpIb/IX complex regulates megakaryocyte growth.

Animals↗
PubMed1999

An algorithm for finding the linear region in a nonlinear data set.

Finding the linear reportable range is an important procedure for each method in clinical chemistry. One is often called upon to limit the reportable range in order to find the linear region. Limiting the reportable range by visual techniques is subjective, may introduce bias and is not programmable. Using Kroll and Emancipator's polynomial method for linearity, we compare the residuals of a test to determine whether eliminating a point from one end or the other of the data set worsens or improves the data sets' linearity. In an example of urinary cortisol, the root mean squares of the residuals improve by 2% when the lowest point is removed, 39% when the highest point is removed and 82% when the two highest points are removed. The latter data set is the most linear.

Algorithms↗
PubMed1999

Biological variation of glucose and insulin includes a deterministic chaotic component.

Serial data of glucose and insulin values of individual patients vary over short periods of time; this phenomenon has been called biological variation. The classic homeostatic control model assumes that the physiological mechanisms maintaining the concentrations of glucose and insulin are linear. The only deviations over a short period of time one should observe are in relation to a glucose load or major hormonal disturbance. Otherwise, the values of these analytes should be constant and any variations seen are due to random disturbances. We investigated previously published serial data (three for glucose and one for insulin) with nonlinear analytical methods, such as embedding space, correlation dimension, Lyapunov exponents, singular value decomposition and phase portraits, as well as linear methods, such as power spectra and autocorrelation functions. The power spectra failed to show dominant frequencies, but the autocorrelation functions showed significant correlation, consistent with a deterministic process. The correlation dimension was finite, around 4.0, the first Lyapunov exponent was positive, indicative of a deterministic chaotic process. Furthermore, the phase portraits showed directional flow. Therefore, the short-term biological variation observed for analytes arises from nonlinear, deterministic chaotic behavior instead of random variation.

Blood Glucose↗
PubMed1999

AIDS-related Kaposi's sarcoma involving bone and bone marrow.

AIDS-related Kaposi's sarcoma rarely involves bone or bone marrow. Computed tomography of the abdomen and pelvis of an AIDS patient with lower back pain and bilateral limb edema revealed multiple lesions involving liver, spleen, and axial skeleton. Bone marrow examination of the involved iliac crest revealed Kaposi's sarcoma. Pathologic diagnosis is important so that appropriate treatment can be prescribed.

AIDS-Related Opportunistic Infections↗
PubMed1998

AIDS-associated Kaposi's sarcoma.

In less than two decades, the disease mechanisms have been elucidated and hypotheses for innovative treatment have been developed. Our understanding of the sarcoma's pathogenesis has led directly to general knowledge of the physiology and pathology of angiogenesis. Hence, it can be expected that new treatments for all cancer patients may someday emerge from clinical intervention trials for the AIDS-related disease.

Acquired Immunodeficiency Syndrome↗
PubMed1997

Bone to total alkaline phosphatase ratios improve sensitivity and specificity of bone alkaline phosphatase immunoassays.

OBJECTIVES: Evaluate the ability of two bone alkaline phosphatase (ALPB) immunoassays (Ostase, Hybritech Inc and Alkphase-B, Metra Biosystems) to clinically differentiate between osseous and non-osseous ALP sources. DESIGN AND METHODS: Specimens from patients with either liver or bone disease (Paget's disease or metastatic cancer) were analyzed by both methods. RESULTS: There was a good correlation between these two assays. Values for ALPB, whether determined as a concentration by the Ostase assay or as an activity by the Alkphase-B assay, were similar for subjects with liver disease or bone disease. However, total ALP (ALPT) activity was higher in liver disease compared to bone. When ALPB was expressed in relation to ALPT, ratios were significantly greater in subjects with bone disease than in those with liver disease. ALPB/ALPT ratios improved the specificity of the Ostase assay from 52% to 86% and the Alkphase-B assay from 58% to 74%. CONCLUSIONS: These two ALPB assays have good analytical performance and their clinical utility can be enhanced by expressing ALPB values in relation to ALPT activity.

Adult↗
PubMed1997

Nitric oxide induces heme oxygenase-1 gene expression and carbon monoxide production in vascular smooth muscle cells.

Since recent studies demonstrate that vascular smooth muscle cells synthesize two distinct guanylate cyclase-stimulatory gases, NO and CO, we examined possible regulatory interactions between these two signaling molecules. Treatment of rat aortic smooth muscle cells with the NO donors, sodium nitroprusside, S-nitroso-N-acetyl-penicillamine, or 3-morpholinosydnonimine, increased heme oxygenase-I (HO-1) mRNA and protein levels in a concentration and time-dependent manner. Both actinomycin D and cycloheximide blocked NO-stimulated HO-1 mRNA and protein expression. Nuclear run-on experiments demonstrated that NO donors increased HO-1 gene transcription between 3- and 6-fold. In contrast, NO donors had no effect on the stability of HO-1 mRNA. Incubation of vascular smooth muscle cells with the membrane-permeable cGMP analogues, dibutyryl cGMP and 8-bromo-cGMP, failed to induce HO-1 gene expression. Treatment of vascular smooth muscle cells with NO donors also stimulated the production and release of CO, as demonstrated by the CO-dependent increase in intracellular cGMP levels in coincubated platelets. Finally, incubating vascular smooth muscle cells with interleukin-1 beta and tumor necrosis factor-alpha induced NO synthesis and also significantly increased the level of HO-1 protein. The cytokine-stimulated production of both NO and HO-1 protein in smooth muscle cells was blocked by the NO synthase inhibitor methyl-L-arginine. These results demonstrate that exogenously administered or endogenously released NO stimulates HO-1 gene expression and CO production in vascular smooth muscle cells. The ability of NO to induce HO-catalyzed CO release from vascular smooth muscle cells provides a novel mechanism by which NO might modulate soluble guanylate cyclase and, thereby, vascular smooth muscle cell and platelet function.

Animals↗
PubMed1997

Serum pooling lowers cholesterol recovery.

Matrix effects hinder the transferability of accuracy for cholesterol. A general assumption is that pooled and individual samples yield similar results. We tested the hypothesis that serum-pooling affects the recovery for cholesterol. We pooled 100 serum samples, determining cholesterol of pool and of the individual samples with Hitachi 717 and 914. Over twenty days, we daily determined cholesterol of individual and pooled samples, using a Hitachi 736 and 747 analyzers. For the hundred-sample pool, the pool was 1.1 to 1.5% lower than the individual samples. With the daily pool study, the ratio of 747 to 736 was 1.7% lower for the pooled compared with the individual samples. Therefore, pooling of serum samples causes a decreased recovery, averaging from 1.1-1.7%, and representing 37-57% of the allowable bias for cholesterol (< 3%), and it is thereby significant.

Bias↗
PubMed1997

Linear regression estimation of minimal detectable concentration. Thyrotropin as an example.

BACKGROUND: Minimal detectable concentration is an important analytic feature of certain clinical immunoassays. We believe that accuracy is an important component of the minimal detectable concentration; for a given observed concentration to be meaningful, it should reflect a consistent linear relationship with the amount of analyte actually present. METHODS: To evaluate the minimal detectable concentration, we developed a linearity regression protocol based on accuracy and also accounting for between-run variability. Using serial twofold dilutions of serum samples, we regressed the log of concentration (x) and of dilution (y) with linear, second-, and third-order polynomials. Initially, we evaluated two elements to find the linear region of the dataset, establishing the statistical significance of the beta coefficients with a t test and the reduction of the sum of square of the residuals between the linear regression and the higher-order regressions by means of an F test. As needed, we successively eliminated the lowest point until the linear regression was the best fit. Once we found the best fit, we added the most recently removed point back and calculated the difference between the value predicted by the first-order regression and the observed value. If the difference was not analytically significant, then we considered the point to be part of the linear set; otherwise, it was not included. In either case, the lowest included point was considered to be the minimal detectable concentration. RESULTS: We applied the technique in evaluating two automated systems for serum thyrotropin. One system appeared linear and accurate down to 0.02 mU/L, or better, approximately 77% of the time, and to 0.01 mU/L 68% of the time. The second system was linear infrequently and appeared to be useful down to 0.02 mU/L, or better, only about 20% of the time. CONCLUSIONS: This accuracy-based approach to determining the minimal detectable concentration is an attractive alternative to current empiric approaches, which are based only on interassay variability.

Humans↗