Identification of transcription factors responsible for dysregulated networks in human osteoarthritis cartilage by global gene expression analysis.

OBJECTIVE: Osteoarthritis (OA) is the most prevalent joint disease. As disease-modifying therapies are not available, novel therapeutic targets need to be discovered and prioritized for their importance in mediating the abnormal phenotype of cells in OA-affected joints. Here, we generated a genome-wide molecular profile of OA to elucidate regulatory mechanisms of OA pathogenesis and to identify possible therapeutic targets using integrative analysis of mRNA-sequencing data obtained from human knee cartilage. DESIGN: RNA-sequencing (RNA-seq) was performed on 18 normal and 20 OA human knee cartilage tissues. RNA-seq datasets were analysed to identify genes, pathways and regulatory networks that were dysregulated in OA. RESULTS: RNA-seq data analysis revealed 1332 differentially expressed (DE) genes between OA and non-OA samples, including known and novel transcription factors (TFs). Pathway analysis identified 15 significantly perturbed pathways in OA with ECM-related, PI3K-Akt, HIF-1, FoxO and circadian rhythm pathways being the most significantly dysregulated. We selected DE TFs that are enriched for regulating DE genes in OA and prioritized these TFs by creating a cartilage-specific interaction subnetwork. This analysis revealed eight TFs, including JUN, Early growth response (EGR)1, JUND, FOSL2, MYC, KLF4, RELA, and FOS that both target large numbers of dysregulated genes in OA and are themselves suppressed in OA. CONCLUSIONS: We identified a novel subnetwork of dysregulated TFs that represent new mediators of abnormal gene expression and promising therapeutic targets in OA.

Statistical shape modeling of proximal femoral shape deformities in Legg-Calvé-Perthes disease and slipped capital femoral epiphysis.

INTRODUCTION: The current understanding of morphological deformities of the hip such as femoroacetabular impingement (FAI), Legg-Calvé-Perthes disease (LCPD), and slipped capital femoral epiphysis (SCFE) is based on two-dimensional metrics, primarily involving the femoral head, that only partially describe the complex skeletal morphology. OBJECTIVE: This study aimed to improve the three-dimensional (3-D) understanding of shape variations during normal growth, and in LCPD and SCFE, through statistical shape modeling. DESIGN: Thirty-two patients with asymptomatic, LCPD, and SCFE hips, determined from physical and radiographic examinations, were scanned using 3-D computed tomography (CT) at a voxel size of (0.5-0.9 mm)(2) in-plane and 0.63 mm slice thickness. Statistical shape modeling was performed on segmented proximal femoral surfaces to determine modes of variation and shape variables quantifying 3-D shape. In addition, conventional variables were determined for all femora. RESULTS: Proximal femur shape was described by eight modes of variation and corresponding shape variables. Statistical shape variables were distinct with age and revealed coordinated, growth-associated differences in neck length-to-width ratio, femoral head medialization, and trochanter protrusion. After size and age-based shape adjustment, diseased proximal femora were characterized by shape variables distinct from those of asymptomatic hips. The shape variables defined morphology in health and disease, and were correlated with certain conventional variables of shape, including neck-shaft angle, head diameter, and neck diameter. CONCLUSION: 3-D quantitative analyses of proximal femoral bone shape during growth and in disease are useful for furthering the understanding of normal and abnormal shape deviations which affect cartilage biomechanics and risk of developing osteoarthritis.

New tricks with old dogs: personalised medicine and clinical trials.

We provide a didactic example of how clinical trials can accommodate individualised patient information relative to design and analysis.

Sonographic monitoring of mass effect in stroke patients treated with hypothermia. Correlation with intracranial pressure and matrix metalloproteinase 2 and 9 expression.

Severe stroke leads to subsequent cerebral oedema. Patients with severe stroke develop midline shift (MLS) which can be measured by transcranial duplex sonography (TCD). We measured MLS with TCD in 30 patients with large infarction in the territory of the middle cerebral artery (MCA). All of the examined patients had intracranial pressure (ICP) measure devices and the ICP at the time of the TCD was recorded. MLS was also determined on CT scan on day 4. Ten of the 30 patients were treated with hypothermia. We also determined matrix metalloproteinase 2 (MMP2) and matrix metalloproteinase 9 (MMP9) in serum by zymography. MLS measured by TCD correlated significantly with MLS on CT. In addition there was a strong correlation between the ICP measured at the time of TCD and MLS. In patients treated with hypothermia MLS was less pronounced. MMP9 and MMP2 showed a characteristic time course and had strong associations with MLS. We confirm earlier reports that TCD is a reliable noninvasive method for serially monitoring patients with intracranial lesions. Hypothermia reduces MMP9 activity as well as MLS. TCD may reduce the need for repetitive CT scans in neurological critically ill patients.

Human recombinant erythropoietin promotes differentiation of murine megakaryocytes in vitro.

To determine if erythropoietin affects megakaryocytopoiesis, we measured acetylcholinesterase (AchE) activity, a marker of the murine megakaryocytic lineage, after the addition of human recombinant erythropoietin to serumless murine bone marrow cultures. Erythropoietin increased AchE activity substantially. Moreover, when the hormone was added to serumless cultures of 426 isolated single megakaryocytes derived from megakaryocytic colonies, erythropoietin induced a significant increase in the diameters of these cells. From a Bayesian analysis of the likelihood that some megakaryocytes increased in DNA content during the culture period, we estimate that 61% of the cells increased in ploidy. These data indicate that the action of erythropoietin is not restricted to the erythroid lineage.

Statistical approach to immunosuppression classification using lymphocyte surface markers and functional assays.

We analyzed results of 22 in vitro parameters of immunocompetence in 72 cancer patients and 73 healthy controls. We then applied three statistical methodologies (discriminant analysis, logistic regression analysis, and recursive partitioning) in an effort to select the best predictors of immunosuppression. Using either of two definitions of immunosuppression (deviation by more than 1 standard deviation from the control mean on any assay, or having a diagnosis of advanced cancer), the same variables were selected. The best predictors were percentage of lymphocytes, percentage of suppressor cells, pokeweed mitogen stimulation, percentage of Ia+ cells, and number of helper cells. By all three methods, immunosuppressed and immunocompetent individuals were selected with 95 to 97% accuracy using a decision tree with these five tests as variables. In a cohort of individuals with incomplete data, the three methods still accurately classified the two groups with 70 to 83% accuracy. We conclude that a much smaller battery of tests can be used to identify immunosuppressed individuals for purposes of evaluation of responses to immune modulating agents.

Superiority of an acid-labile daunorubicin-monoclonal antibody immunoconjugate compared to free drug.

We conjugated the chemotherapy agent daunorubicin to the anti-T-cell monoclonal antibody T101 using an active ester intermediate of the acid-labile linker cis-aconitate anhydride. By converting carbohydrate hydroxyl groups on the antibody to amines prior to conjugation, average drug to antibody ratios of 25:1 were achieved with retention of cytotoxicity and only minimal loss of immunoreactivity. The pH sensitivity of the linkage was confirmed. The preparation was cytotoxic for antigen-bearing cells but not antigen-negative cells, even up to 48-h incubation in vitro. Specific cytotoxicity was apparently mediated through the endocytosis of the intact T101 immunoconjugate and the release of the active drug in the lysosomal compartment. Athymic mice bearing human tumor xenografts who received a single injection of the immunoconjugate had less tumor growth and more tumor regressions than animals receiving antibody alone, drug alone, or a mixture of drug plus antibody. This approach appears promising for further investigation.

Preclinical trials with combinations and conjugates of T101 monoclonal antibody and doxorubicin.

We investigated the potential for additive therapy for malignancy using an anti-human T-cell monoclonal antibody, T101, and the chemotherapy agent doxorubicin (DOX). We compared the efficacy of T101 alone, DOX alone, T101 and DOX covalently linked to dextran to form an immunoconjugate, T101 plus DOX mixed together and injected, T101 and DOX injected separately, and nonspecific murine IgG2A plus DOX mixed together. Inhibition of [3H]thymidine was examined in vitro, and the clinical efficacy of each treatment was tested on human T-cell tumors growing in athymic mice. In vitro experiments confirmed retention of immunoreactivity and cytotoxicity by the immunoconjugate, but it was not superior to DOX alone. In efficacy experiments, all therapeutic arms were superior to placebo treatment (P less than 0.05). However, the best results in the animal tumor model were obtained with T101 mixed with DOX, perhaps because of formation of weak complexes via hydrophobic bonds. This mixture was superior to all other treatments, both by growth curve analysis (P less than 0.05) and by analysis of complete regression of tumor (P less than 0.01). T101 mixed with DOX was superior to a mixture of nonspecific mouse immunoglobulin and DOX and superior to a combination of T101 injected i.v. and DOX injected i.p. The antitumor effect of T101 mixed with DOX was blocked by premodulating the target antigen with T101. These data suggest that further exploration into monoclonal antibody-anthracycline complexes is warranted.

Stability, characterization, and kinetics of 111In-labeled monoclonal antitumor antibodies in normal animals and nude mouse-human tumor models.

Monoclonal antibodies (MoAbs) against carcinoembryonic antigen were successfully radiolabeled with 111In, and the radiopharmaceutical was characterized in vitro and in normal and tumor-bearing mice. The 111In-MoAb proved to be stable in vitro and in vivo under normal conditions, although instability could be induced in vitro with large quantities of iron-free transferrin. Animal distribution studies with 111In-MoAb demonstrated tumor localization superior to 67Ga and pharmacokinetics that were highly similar to those of endogenously labeled 75Se-MoAb. The 111In-MoAb followed first-order kinetics and fit a two-compartmental model when studied in nude mice bearing human colon tumors known to express carcinoembryonic antigen. Significant quantities of radiolabel appeared in tissues other than tumor, with liver and skin having the highest concentrations. Sufficient tumor/background ratios were formed for scanning purposes. The data indicate that 111In-MoAb may prove to be effective as a radiopharmaceutical for tumor imaging.

Alternating combination chemotherapy for stages III and IV ovarian carcinoma.

Thirty-nine previously untreated patients with stages III and IV ovarian carcinoma were treated with debulking surgery, followed by alternating combination chemotherapy with cisplatin, Adriamycin (Adria Laboratories, Columbus, Ohio), and cyclophosphamide (PAC); and hexamethylmelamine, cyclophosphamide, methotrexate, and 5-fluorouracil (HexaCAF). Of 19 patients with measureable disease at the onset of therapy, ten (53%) had at least a partial response to chemotherapy. Seven (18% of total) patients were found to be pathologically free of disease at secondlook surgery, but four patients relapsed 19 to 31 months after initiating therapy. The median progression-free survival period of all 39 patients entered into the study is 12 months, and the median crude survival is 21 months. The PAC/HexaCAF alternating combination chemotherapy regimen may be administered with moderate toxicity, but the treatment results are not superior to those reported for PAC or HexaCAF alone in advanced ovarian carcinoma.

Angiotensin type 1 receptor blockage improves ischemic injury following transient focal cerebral ischemia.

Following cerebral ischemia, i.v. infusion of angiotensin II increases cerebral edema and mortality. Angiotensin type 1 receptor blockage should therefore improve acute cerebral ischemia. Left middle cerebral artery occlusion (120 min) followed by reperfusion was performed with the thread method under halothane anesthesia in Sprague-Dawley rats. Olmesartan (angiotensin type 1 receptor blocker; 0.01 or 0.1mumol/kg/h) was infused i.p. for 7 days following middle cerebral artery occlusion followed by reperfusion. Stroke index score, infarct volume, specific gravity, and brain angiotensin II and matrix metalloproteinases were quantified in the ischemic and non-ischemic hemispheres. Olmesartan treatment improved stroke index score, infarct volume, and cerebral edema in our cerebral ischemia model. In particular, stroke index score, infarct volume, and cerebral edema were reduced even with a low dose of olmesartan that did not decrease blood pressure. Paralleling these effects on cerebral ischemia, olmesartan treatment also reduced the reactive upregulation in brain angiotensin II, matrix metalloproteinase-2, matrix metalloproteinase-9, and membrane type 1-matrix metalloproteinase in the ischemic area. Angiotensin type 1 receptor stimulation may be one of the important factors that cause cerebral edema following cerebral ischemia, and that its inhibition may be of therapeutic advantage in cerebral ischemia.

Angiotensin-converting enzyme inhibition with enalapril slows progressive intima-media thickening of the common carotid artery in patients with non-insulin-dependent diabetes mellitus.

BACKGROUND AND PURPOSE: Whether angiotensin-converting enzyme (ACE) inhibitors have any clinically significant antiatherogenic effects in humans remains unproven. We undertook a prospective randomized clinical trial of 98 patients with non-insulin-dependent diabetes mellitus (NIDDM) to examine the efficacy of ACE inhibition with enalapril for preventing intima-media (IM) thickening of the carotid wall as measured ultrasonographically. METHODS: Ninety-eight NIDDM patients were randomly assigned either to enalapril at 10 mg/d (n=48) or to a control group (n=50); the planned duration of the trial was 2 years. All patients were seen at baseline (study entry) and 2 subsequent formal annual evaluations, in addition to standard clinical management for NIDDM. IM thickening and vascular lumen diameters were determined for all patients on the basis of baseline and 2 subsequent annual evaluations with carotid ultrasonography. We performed an intent-to-treat analysis to assess changes in IM thickening over the course of the study. RESULTS: Annual IM thickening measurements of the right and left common carotid arteries were 0.01+/-0.02 and 0.01+/-0.02 mm/y in the enalapril-treated group and 0.02+/-0.03 and 0.02+/-0.02 mm/y in the control group, respectively (P<0.05). From regression analysis, annual IM thickening was found to be predicted by enalapril use, sex, and insulin use (F(3,94)=3.86, P=0.012). When we controlled for these other variables, enalapril use reduced annual IM thickening of right and left common carotid arteries by 0.01+/-0.004 mm/y relative to the control group over the course of this study. CONCLUSIONS: Long-term treatment with an ACE inhibitor (enalapril) slows progressive IM thickening of the common carotid artery in NIDDM patients.

Rapid differential endogenous plasminogen activator expression after acute middle cerebral artery occlusion.

BACKGROUND AND PURPOSE: During focal cerebral ischemia, the microvascular matrix (ECM), which participates in microvascular integrity, is degraded and lost when neurons are injured. Loss of microvascular basal lamina antigens coincides with rapid expression of select matrix metalloproteinases (MMPs). Plasminogen activators (PAs) may also play a role in ECM degradation by the generation of plasmin or by MMP activation. METHODS: The endogenous expressions of tissue-type plasminogen activator (tPA), urokinase (uPA), and PA inhibitor-1 (PAI-1) were quantified in 10-microm frozen sections from ischemic and matched nonischemic basal ganglia and in the plasma of 34 male healthy nonhuman primates before and after middle cerebral artery occlusion (MCA:O). RESULTS: Within the ischemic basal ganglia, tissue uPA activity and antigen increased significantly within 1 hour after MCA:O (2P<0.005). tPA activity transiently decreased 2 hours after MCA:O (2P=0.01) in concert with an increase in PAI-1 antigen (2P=0.001) but otherwise did not change. The transient decrease in free tPA antigen was marked by an increase in the tPA-PAI-1 complex (2P<0.001). No significant relations to neuronal injury or intracerebral hemorrhage were discerned. CONCLUSIONS: The rapid increase in endogenous PA activity is mainly due to significant increases in uPA, but not tPA, within the ischemic basal ganglia after MCA:O. This increase and an increase in PAI-1 coincided with latent MMP-2 generation and microvascular ECM degeneration but not neuronal injury.

Activated microvessels express vascular endothelial growth factor and integrin alpha(v)beta3 during focal cerebral ischemia.

Both vascular endothelial growth factor (VEGF) and integrin alpha(v)beta3 play roles in angiogenesis. In noncerebral vascular systems, VEGF can induce endothelial integrin alpha(v)beta3 expression. However, it is unknown whether VEGF, like integrin alpha(v)beta3, appears in the initial response of microvessels to focal brain ischemia. Their coordinate expression in microvessels of the basal ganglia after middle cerebral artery occlusion (MCAO) in the nonhuman primate model was examined quantitatively. Cells incorporating deoxyuridine triphosphate (dUTP+) by the polymerase I reaction at 1 hour (n = 3), 2 hours (n = 3), and 7 days (n = 4) after MCAO defined the ischemic core (Ic) and peripheral regions. Both VEGF and integrin alpha(v)beta3 were expressed by activated noncapillary (7.5- to 30.0-microm diameter) microvessels in the Ic region at 1 and 2 hours after MCAO. At 7 days after MCAO, the number of VEGF+, integrin alpha(v)beta3+, or proliferating cell nuclear antigen-positive microvessels had decreased within the Ic region. The expressions of VEGF, integrin alpha(v)beta3, and proliferating cell nuclear antigen were highly correlated on the same microvessels using hierarchical log-linear statistical models. Also, VEGF and subunit alpha(v) messenger ribonucleic acids were coexpressed on selected microvessels. Here, noncapillary microvessels are activated specifically early during a focal cerebral ischemic insult and rapidly express VEGF and integrin alpha(v)beta3 together.

Matrix metalloproteinases increase very early during experimental focal cerebral ischemia.

Microvascular integrity is lost during focal cerebral ischemia. The degradation of the basal lamina and extracellular matrix are, in part, responsible for the loss of vascular integrity. Matrix metalloproteinases (MMPs) may play a primary role in basal lamina degradation. By using a sensitive modification of gelatin zymography, the authors investigated the activity of MMP-2 and MMP-9 in frozen 10-microm sections of ischemic and nonischemic basal ganglia and plasma samples of 27 non-human primates after middle cerebral artery occlusion/reperfusion (MCAO/R) for various periods. The gelatinolytic activities were compared with parallel cell dUTP incorporation in the ischemic zones of adjacent sections. In the brain, the integrated density of MMP-2 increased significantly by 1 hour after MCAO and was persistently elevated thereafter. Matrix metalloproteinase-2 expression was highly correlated with the extent of neuron injury and the number of injured neurons (r = 0.9763, SE = 0.004, 2P < 0.0008). Matrix metalloproteinase-9 expression only was significantly increased in subjects with hemorrhagic transformation. In plasma, only MMP-9 increased transiently at 2 hours of MCAO. These findings highlight the early potential role of MMP-2 in the degradation of basal lamina leading to neuronal injury, and an association of MMP-9 with hemorrhagic transformation after focal cerebral ischemia.

Kinetics of sodium thiosulfate, a cisplatin neutralizer.

Sodium thiosulfate kinetics were studied in eight subjects, six of whom were given the drug as a cisplatin neutralizer. Thiosulfate at a dose of 12 gm/m2 was injected by continuous intravenous infusion over 6 hr. Under these conditions, steady-state plasma concentrations were not achieved and apparent volume of distribution could not be calculated. The drug was eliminated from plasma by first-order kinetics, and the data best fit a one-compartment kinetic model with a t1/2 (mean +/- SD) of 80 +/- 38 min. Total body thiosulfate clearance was 190 +/- 76 ml/min/m2 and renal clearance was 50 +/- 11 ml/min/m2. The plasma elimination t1/2 and renal thiosulfate clearance correlated poorly with clearance of endogenous creatinine. Only 28.5% +/- 9.4% of the thiosulfate was recovered unchanged in the urine. Urinary excretion was rapid, with approximately 95% of recoverable drug eliminated within 4 hr after termination of the infusion. No toxic effects of thiosulfate were observed. These data provide the basis for the rational development of dose schedules when sodium thiosulfate is used as a cisplatin neutralizer.

In vitro drug testing using hemopoietic cells: goals and limitations.

In vitro drug sensitivity is one of many biologic variables which may predict in vivo drug response. Even if in vitro assays provide relevant data, for some tumors, variable levels of stem-cell origin, differentiation, tumor heterogeneity, or self renewal may be more important than cytotoxicity to proliferating cells. Although ANLL has been used here frequently as a model, it may not be the most appropriate tumor for study. Unlike many cancers, in ANLL, primary drug resistance is unusual, and in relapse, secondary drug resistance is usually incomplete. It has been suggested that in vitro drug sensitivity predicts remissions for patients who do not die of infection or remain aplastic during induction therapy. However, for the majority of patients, this argument acknowledges the overriding importance of biologic variables other than in vitro drug cytotoxicity. For rapidly growing tumors, such as Burkitt's lymphoma, rapid emergence of drug resistance related to disease burden may be the most important response determinant. Perhaps in other tumors, in vitro drug sensitivity will be an independent variable of overriding importance. To determine the role of in vitro drug testing, trials examining in vitro drug sensitivity must meet stringent criteria. The assays should use well-defined and reproducible cultures and drug exposures. The trials must be large enough, contain homogenously treated patients, and use carefully defined response and survival endpoints. Decision rules derived from such trials must be further tested by prospective evaluation. Investigators conducting these trials must be prepared to search for important in vitro results reflecting tumor biology and to analyze in vitro drug sensitivity as only one continuous variable determining in vivo responses. Such trials will be difficult to conduct and expensive. In the final analysis, in vitro assays may find their most important roles as preclinical drug screens and models for in vitro drug resistance. Further insights into molecular genetics of malignant transformation and drug resistance may make such assays obsolete, but for the present, they provide important insights into tumor variability and mechanisms of drug response.

Assessing information in T2-weighted MRI scans from secondary progressive MS patients.

BACKGROUND: Magnetic resonance imaging is used routinely for diagnosing MS and for objective assessment of the extent of disease as a marker of treatment efficacy in MS clinical trials. Nevertheless, in many clinical studies only weak correlations have been reported between MRI findings and clinical outcome measures. PURPOSE: The purpose of this study is to compare clinical outcome measures (neurologic scales) with MRI findings (evaluation of T2-weighted MRI scans using a semiautomated quantitative technique and with an independent assessment by a neurologist) in the context of a randomized clinical trial evaluating the efficacy of cladribine for treatment in secondary progressive MS. METHODS: Baseline, 6-month, and 12-month scans from 41 secondary progressive MS patients were examined and ranked in terms of lesion burden from the quantitative assessment and independently in terms of severity by neurologic evaluation. Comparison is made to monthly Expanded Disability Status Scale (EDSS) and Scripps Neurologic Rating Scale (SNRS) determinations in these patients with a nonparametric statistical procedure. RESULTS: Average rank correlations between any of the MRI assessment procedures and either clinical outcome measure were less than 0.15 in absolute magnitude. The average rank correlation between the two MRI assessment procedures was 0.10. There is only a weak degree of association between the MRI assessment procedures and the clinical parameters, although the study has statistical power in excess of 0.90 to find even a moderate level of association between them. CONCLUSIONS: Disease-related activity in T2-weighted scans of secondary progressive MS patients is a multidimensional construct, and is not summarized adequately solely by quantification of overall lesion burden or by assessment of severity. Neither method of summarizing information from T2-weighted scans is strongly related to measures of the clinical course of disease as assessed by the EDSS or SNRS.

Evaluation of monoclonality of cell lines from sequential dilution assays. Part II.

We extend the analysis in Koziol et al. (J. Immunol. Methods (1987) 105, 139) for the determination of the probability of achieving monoclonality in limiting dilution assays to three or more cloning stages. We also provide a computer program which will carry out this analysis for an arbitrary number of stages.