Epidemiological profile of pain and non-steroid anti-inflammatory drug use in collegiate athletes in the United States.

BACKGROUND: Although athletic endeavours are associated with a high amount of physical stress and injury, the prevalence of pain is underreported in the sports medicine literature with only a few studies reporting pain on collegiate athletes or exploring sex difference of pain. Impact of pain on athlete availability, training and performance can be mitigated when key epidemiological information is used to inform adequate pain management strategies. This study aims to 1) provide an epidemiological profile of self-reported pain experienced by the National Collegiate Athletic Association (NCAA) athletes by sex during the first half of the 2019 season, 2) describe their self-reported non-steroidal anti-inflammatory drug (NSAID) use. METHODS: Online survey was completed by athletes at three NCAA institutions from 1 August to 30 September 2019. Descriptive statistics were used to describe player demographic data, self-reported pain and self-reported NSAID use. Pain incidence proportion were calculated. RESULTS: Two hundred thirty female athletes and 83 male athletes completed the survey. Self-reported pain incidence proportion for female athletes was 45.0 (95% CI 41.5-48.5) vs 34.9 (95% CI 29.4-40.4) for male athletes. Majority of the athletes did not report pain (55% female vs 62% male) during the first half of the 2019 season. Female athletes reported pain in their back (35%), knee (26%), and ankle/foot (23%) whilst male athletes reported pain in their knee (35%), back (28%), and shoulder (24%). Of all athletes, 28% female vs 20% male athletes reported currently taking NSAIDs. Of athletes that reported pain, 46% female vs 38% male athletes currently took NSAIDs. 70% female vs 61% male athletes self-purchased NSAIDs, and 40% female vs 55% male athletes consumed alcohol. CONCLUSIONS: Half of female athletes and one in three male athletes reported pain. Most commonly back, knee and foot/ankle pain and knee, back and shoulder pain was reported in female and male athletes respectively. One in four female athletes and one in five male athletes use NSAIDs for pain or prophylactic purpose. Majority self-purchase these medications indicating need for health literacy interventions to mitigate potential adverse effects.

A homozygous ADAMTS2 nonsense mutation in a Doberman Pinscher dog with Ehlers Danlos syndrome and extreme skin fragility.

An eight-week old Doberman Pinscher was diagnosed with Ehlers Danlos syndrome based on the dog's hyper-mobile carpal, tarsal and stifle joints and abnormal skin. The skin was loose and hyper-elastic with several wounds and large atrophic scars. The dog was euthanized after a severe degloving injury from minimal trauma. A whole-genome sequence, generated with DNA from the dog's blood, contained a rare, homozygous C-to-T transition at position 2408978 on chromosome 11. This transition is predicted to alter the ADAMTS2 transcript (ADAMTS2:c.769C>T) and encode a nonsense mutation (p.Arg257Ter). Biallelic ADAMTS2 mutations have caused a type of Ehlers Danlos syndrome known as dermatosparaxis in other species.

Post-transplant lymphoproliferative disorders are not associated with IgG4 sclerosing disease.

BACKGROUND: Although the majority of post-transplant lymphoproliferative disorder (PTLD) cases are associated with Epstein-Barr virus (EBV), 20-42% of cases are EBV negative (EBV-N). The antigenic stimulus that drives EBV-N PTLD is unknown, but is likely heterogeneous. A common feature of PTLD, regardless of EBV status, is an abnormal polytypic lymphoplasmacytic infiltrate. Immunglobulin-G4 (IgG4) syndrome is also characterized by a polytypic lymphoplasmacytic infiltrate with a predominance of IgG4-positive (IgG4-P) plasma cells. METHODS: We investigated the possibility of an association between EBV-N PTLD and IgG4 syndrome. Of 33 evaluated PTLD cases, 9 (27%) were EBV-N. EBV-N PTLD cases showed longer transplantation-to-diagnosis times than EBV-positive cases. RESULTS: A single patient had a preceding benign duodenal biopsy with focally prominent IgG4-P plasma cells; however, no clinical data supported IgG4 syndrome, precluding an association between IgG4 syndrome and subsequent EBV-N PTLD in this patient. CONCLUSION: As none of 29 evaluable cases of PTLD (including all 9 EBV-N cases) were associated with an increase in IgG4-P plasma cells, IgG4 syndrome does not appear to play a role in the etiology of EBV-N PTLD. The significance of these findings and the current understanding of the etiology of EBV-N PTLD are discussed.

Machine learning and statistical prediction of fastball velocity with biomechanical predictors.

In recent years, one of the most important factors for success among baseball pitchers is fastball velocity. The purpose of this study was to (1) to develop statistical and machine learning models of fastball velocity, (2) to identify the strongest predictors of fastball velocity, and (3) to compare the models' prediction performances. Three dimensional biomechanical analyses were performed on high school (n = 165) and college (n = 62) baseball pitchers. A total of 16 kinetic and kinematic predictors from the entire pitching sequence were included in regression and machine learning models. All models were internally validated through ten-fold cross-validation. Model performance was evaluated through root mean square error (RMSE) and calibration with 95% confidence intervals. Gradient boosting machines demonstrated the best prediction performance [RMSE: 0.34; Calibration: 1.00 (95% CI: 0.999, 1.001)], while regression demonstrated the greatest prediction error [RMSE: 2.49; Calibration: 1.00 (95% CI: 0.85, 1.15)]. Maximum elbow extension velocity (relative influence: 19.3%), maximum humeral rotation velocity (9.6%), maximum lead leg ground reaction force resultant (9.1%), trunk forward flexion at release (7.9%), time difference of maximum pelvis rotation velocity and maximum trunk rotation velocity (7.8%) demonstrated the greatest influence on pitch velocity. Gradient boosting machines demonstrated better calibration and reduced RMSE compared to regression. The influence of lead leg ground reaction force resultant and trunk and arm kinematics on pitch velocity demonstrates the interdependent relationship of the entire kinetic chain during the pitching motion. Coaches, players, and performance professionals should focus on the identified metrics when designing pitch velocity improvement programs.

Verification of High-Rate Vertical Loading Laboratory Skeletal Fractures by Comparison with Theater Injury Patterns.

For the current study, an existing theater injury data set was compared to component and whole body experiments meant to replicate the theater high rate vertical loading environment. The theater injury data set was derived from real world events that were within the design range of the Warrior Injury Assessment Manikin. A qualitative and quantitative assessment of the whole body fracture patterns was developed to determine whether the laboratory loading was correctly representing the resulting injuries seen in theater Underbody Blast (UBB) events. Results indicated that most of the experimental test fracture patterns were similar to the theater injuries for Abbreviated Injury Scale body regions of interest (lower extremities, pelvis, and spine); however, some of the body regions had higher similarity scores compared to others. Whole body fracture distribution was less similar than the component tests because of differences in injury distributions. The lower extremity whole body similarity was lower than spine and pelvis similarity. This analysis was able to identify some experimental tests that might not represent theater loading. In conclusion, this analysis confirmed that some laboratory testing produced skeletal injury patterns that are seen in comparable theater UBB events.

Clinical, metabolic, and molecular genetic characterization of hereditary methemoglobinemia caused by cytochrome b5 reductase deficiency in 30 dogs.

Genotype-phenotype correlations of humans and dogs with hereditary methemoglobinemia are not yet well characterized. We determined total hemoglobin and methemoglobin (MetHb) concentrations, cytochrome b5 reductase (CYB5R) enzyme activities, genotypes, and clinical signs in 30 dogs with persistent cyanosis without cardiopulmonary disease. Erythrocytic CYB5R enzyme activities were low in all dogs assayed. Owner-reported quality of life ranged from subclinical to occasional exertional syncope. Two previously reported and two novel CYB5R3 missense variants were identified among the methemoglobinemic cohort and were predicted to impair enzyme function. Two variants were recurrent: a homozygous Ile194Leu substitution was found in Pomeranians and other small dogs, and a homozygous Arg219Pro change occurred predominately in pit bull terriers. The other two variants were Thr202Ala and Gly76Ser substitutions in single dogs. Of the two common CYB5R3 genotypes, Arg219Pro was associated with a more severe metabolic phenotype. We conclude that CYB5R3 deficiency is the predominate cause of canine hereditary methemoglobinemia. Although this finding is unlikely to alter the clinical approach to hereditary methemoglobinemia in dogs, it demonstrates the possibility of how genotype-phenotype cohort analysis might facilitate precision medicine in the future in veterinary medicine.

Feature identification and location experiment.

The feature identification and location experiment (FILE) senses radiation from the earth in spectral bands centered at 0.65 and 0.85 micrometers and compares ratios of the reflected solar radiation in the two wavelengths to make real-time classification decisions about four primary features: water, vegetation, bare land, and a cloud-snow-ice class. The radiance ratio classification algorithm successfully made automatic data-selection decisions. The classification image obtained on the mission is providing information needed to evaluate the FILE algorithm and system performance.

Azithromycin Fails to Prevent Accelerated Airway Obliteration in T-bet-/- Mouse Lung Allograft Recipients.

BACKGROUND: Cellular and molecular mechanisms of acute and chronic lung allograft rejection have yet to be clearly defined, and obliterative bronchiolitis (OB) remains the primary limitation to survival in lung transplant recipients (LTRs). We have previously shown that T-bet-deficient recipients of full major histocompatibility complex (MHC)-mismatched, orthotopic left lung transplants develop accelerated obliterative airway disease (OAD) in the setting of acute cellular rejection characterized by robust alloimmune CD8+ interleukin (IL)-17 and interferon (IFN)-γ responses that are attenuated with neutralization of IL-17. Azithromycin has been shown to be beneficial in some LTRs with bronchiolitis obliterans syndrome/OB. Here, we evaluated the effects of azithromycin on rejection pathology and T-cell effector responses in T-bet-/- recipients of lung transplants. METHODS: Orthotopic left lung transplantation was performed in BALB/c → B6 wild type or BALB/c → B6 T-bet-/- strain combinations as previously described. Mice treated with azithromycin received 10 mg/kg or 50 mg/kg subcutaneously daily. Lung allograft histopathology was analyzed at day 10 or day 21 post-transplantation, and neutrophil staining for quantification was performed using anti-myeloperoxidase. Allograft mononuclear cells were isolated at day 10 for T-cell effector cytokine response assessment using flow cytometry. RESULTS: We show that while azithromycin significantly decreases lung allograft neutrophilia and CXCL1 levels and attenuates allospecific CD8+ IL-17 responses early post-transplantation, OAD persists in T-bet-deficient mice. CONCLUSIONS: Our results indicate that lung allograft neutrophilia is not essential for the development of OAD in this model and suggest allospecific T-cell responses that remain despite marked attenuation of CD8+ IL-17 are sufficient for obliterative airway inflammation and fibrosis.

Effect of combined treatment with interleukin-3 and interleukin-6 on 4-hydroperoxycyclophosphamide-mediated reduction of glutathione levels and cytotoxicity in normal and leukemic bone marrow progenitor cells.

4-Hydroperoxycyclophosphamide (4-HC) is widely used as an ex vivo bone marrow purging agent for acute myeloid leukemia (AML) blasts. We have determined the effect of a combined treatment with interleukin 3 (IL-3) plus IL-6 on 4-HC cytotoxicity against normal (CFU-GEMM) versus AML (L-CFU) bone marrow progenitor cells. Following an 18 h exposure to IL-3 plus IL-6, treatment with 4-HC in conjunction with IL-3 and IL-6 for one hour resulted in a significantly greater inhibition of L-CFU versus CFU-GEMM colony growth. In addition, treatment with IL-3 plus IL-6 reduced the inhibitory effects of higher concentrations of 4-HC on CFU-GEMM but not L-CFU growth. IL-3 and IL-6 did not protect the self-renewing, clonogenic, AML blast progenitor cells from the cytotoxic effects of 4-HC. While the total intracellular glutathione (GSH) levels were not significantly different between untreated normal bone marrow mononuclear cells (NBMMC) and AML blasts, greater intracellular GSH-S transferase activity was observed in the NBMMC. 4-HC produced a marked reduction in GSH levels in NBMMC as well as AML blasts. But treatment with IL-3 plus IL-6 in conjunction with 4-HC resulted in significantly higher GSH levels in NBMMC. These differences in intracellular GSH levels and GST activity may offer an explanation for the differential protective effects of IL-3 plus IL-6 treatment against the cytotoxic effects of 4-HC on CFU-GEMM colony growth.

Co-expression of several molecular mechanisms of multidrug resistance and their significance for paclitaxel cytotoxicity in human AML HL-60 cells.

Overexpression of P-glycoprotein (PGP), MRP or LRP has been characterized as the 'proximal', while overexpression of the anti-apoptosis Bcl-2 or Bcl-xL relative to the pro-apoptosis Bax protein has been recognized as the 'distal' mechanism of multidrug resistance in human AML cells. In the present studies, we examined whether these mechanisms can co-exist in human AML HL-60 cells. We also determined how these mechanisms would affect the accumulation and cytotoxicity of a PGP substrate, such as Taxol (paclitaxel). For this, immunoblot analyses were performed to determine the expression of PGP, MRP, Myc, Bcl-2, Bcl-xL and Bax on either the multidrug-resistant HL-60 sublines created under the selection pressure of doxorubicin (HL-60/AR), paclitaxel (HL-60/TAX1000) or vincristine (HL-60/VCR), or sublines created by transfection and overexpression of the bcl-2 (HL-60/Bcl-2) or bcl-xL gene (HL-60/Bcl-xL). As compared to the control HL-60, HL-60/AR cells possess high MRP while HL-60/TAX1000 and HL-60/VCR cells express high levels of the mdr-1 encoded PGP. In addition, these multidrug-resistant cells possess 1.5- to 2.5-fold higher Bcl-2, while their Bax and Myc levels are similar to those in the control HL-60 cells. HL-60/TAX1000 and HL-60/VCR cells also express three- and 2.5-fold higher Bcl-xL levels. PGP, but not MRP, overexpression significantly impaired paclitaxel accumulation and paclitaxel-induced apoptosis, as well as reduced its cytotoxic effects as determined by the MTT assay. In contrast, enforced and much higher expression of Bcl-2 in HL-60/Bcl-2 (five-fold) or Bcl-xL in HL-60/Bcl-xL cells (10-fold) significantly reduced paclitaxel-induced apoptosis and the loss of cell viability, without affecting its intracellular accumulation. These results confirm the possibility of co-expression of multiple mechanisms of multidrug resistance in human leukemic cells which had been selected by exposure to a single drug. The results also indicate that MRP overexpression does not confer resistance against paclitaxel. In addition, these findings suggest that, for Bcl-2 and Bcl-xL, enforced overexpression to high levels is necessary to induce paclitaxel resistance in HL-60 cells.

Intracellular metabolism of Ara-C and resulting DNA fragmentation and apoptosis of human AML HL-60 cells possessing disparate levels of Bcl-2 protein.

We examined the effects of high intracellular levels of Bcl-2 on the metabolism and DNA incorporation of high-dose Ara-C (HIDAC) as well as on Ara-C-induced DNA strand breaks and apoptosis of human AML HL-60 cells. HL-60/Bcl-2 and HL-60/neo cells were created by retrovirally transfecting the human AML HL-60 cells with the pZip-bcl-2 and pZip-neo plasmids, respectively. As compared to HL-60/neo, HL-60/Bcl-2 cells contained significantly higher (approximately 10-fold) p26Bcl-2, but equivalent levels of Bax and undetectable levels of Bcl-xL. HIDAC (10 or 100 microM for 4 h) produced the kilobase size and internucleosomal DNA fragmentation associated with apoptosis in HL-60/neo but not in HL-60/Bcl-2 cells. Significantly greater loss of survival (by MTT assay) and flowcytometric and morphologically recognizable apoptosis were observed in HL-60/neo cells. HIDAC did not affect Bcl-2 levels in either cell type. The intracellular accumulation of Ara-CTP relative to dCTP, Ara-C DNA incorporation and Ara-C-induced early DNA damage in the form of strand breaks (detected by alkaline elution assay) were not significantly different between HL-60/Bcl-2 and HL-60/neo cells. In addition, HIDAC treatment caused similar DNA synthesis inhibition in the two cell types. These results indicate that high intracellular levels of Bcl-2 operate distally to inhibit the final apototic cell death pathway by preventing the conversion of HIDAC-induced early DNA damage into lethal DNA fragmentation associated with apoptosis.

High levels of p26BCL-2 oncoprotein retard taxol-induced apoptosis in human pre-B leukemia cells.

In human leukemic cells clinically relevant concentrations of taxol have been demonstrated to induce the biochemical and morphologic hallmarks of apoptosis (Leukemia 1993;7:563-568). Since overexpression of the bcl-2 gene has been reported to retard apoptosis due to a variety of anticancer agents, we examined and compared taxol-induced intracellular microtubular bundling and apoptosis in pre-B human leukemia 697 cells and their counterparts which have been transfected with and overexpress cDNA derived from the bcl-2 gene. Treatment with 0.1 or 1.0 mumol/l taxol for 24 h resulted in internucleosomal DNA fragmentation and morphologic features of apoptosis in 697 cells, but not in 697/BCL-2 cells. However, indirect immunofluorescent staining with anti-tubulin antibody revealed that taxol treatment produces stable microtubule bundles resistant to calcium-mediated disassembly in 697, as well as 697/BCL-2 cells. In addition, taxol-induced microtubule bundling was associated with a marked accumulation of the two cell types in the G2/M phase of the cell cycle. Following exposure to taxol, when 697 cells were washed and kept in drug-free medium, they showed rapid onset of apoptosis followed by loss of cell viability and a decline in cell numbers. In contrast, identically treated 697/BCL-2 cells kept in drug-free medium remained in a growth arrested state, but showed little evidence of apoptosis for up to 4 days. They eventually demonstrated features of apoptotic cell death and loss of viability between 5 and 7 days. This was not accompanied by a decrease in p26BCL-2 levels. Anti-phosphotyrosine or anti-MAP kinase immunoblot analyses of proteins isolated from taxol-treated 697 and 697/BCL-2 cells failed to show any difference in tyrosine phosphorylation of cellular proteins. Therefore, our findings indicate that in 697/BCL-2 cells, high levels of p26BCL-2 significantly delay taxol-induced endonucleolytic internucleosomal DNA fragmentation and apoptosis, but do not affect taxol-induced microtubule bundling or cell cycle growth arrest. The delayed onset of taxol-induced DNA fragmentation and apoptosis in 697/BCL-2 cells without down-regulation of p26BCL-2 levels suggests that an alternative mechanism of taxol-mediated apoptosis might be triggered which is unimpeded by high p26BCL-2 levels, or taxol-induced prolongation of mitotic arrest may lead to the inactivation or inhibition of that mechanism by which p26BCL-2 is able to block apoptosis.

Characterization of a human myeloid leukemia cell line highly resistant to taxol.

Taxol-resistant sublines of HL-60 myeloid leukemia cells (HL-60/TAX100 and HL-60/TAX1000) have been isolated in vitro by subculturing in progressively higher concentrations of taxol. HL-60/TAX100 and HL-60/TAX1000 cells are capable of continuous growth in the presence of 0.1 microM and 1.0 microM taxol, respectively, and the IC50 (50% growth inhibitory dose) values for taxol for the two sublines are 0.34 and 2.44 microM as compared to 3.1 nM for the parent HL-60 cells. HL-60/TAX100 and HL-60/TAX1000 cells display a variable degree of cross-resistance to taxotere, vincristine and doxorubicin, but are sensitive to the antimetabolite Ara-C. Both HL-60/TAX100 and HL-60/TAX1000 cells over-express MDR-1 m-RNA and the membrane efflux multidrug transporter P-glycoprotein (PGP), as determined by Western blot and immunofluorescence labeling with anti-PGP antibodies. Consequently, exposure of the taxol-resistant cells to [3H]taxol or daunomycin results in the accumulation of significantly lower levels of the two drugs. Co-treatment with cyclosporine (0.5 microgram/ml) or verapamil (10 microM) partially overcomes taxol resistance in HL-60/TAX1000 cells. Following treatment with clinically relevant concentration of taxol (1.0 microM for 24 h), HL-60 but not HL-60/TAX1000 cells display intracellular microtubular bundling, markedly enhanced accumulation of the cells in G2/M phase of cell-cycle and internucleosomal DNA fragmentation associated with apoptosis which is independent of bcl-2 gene expression. These taxol-resistant myeloid leukemia cells may serve as in vitro experimental models for examinating strategies which may have potential applicability for overcoming taxol resistance.

Evidence against a direct role for the induction of c-jun expression in the mediation of drug-induced apoptosis in human acute leukemia cells.

Previous reports have demonstrated that a variety of anticancer drugs, e.g., 1-beta-D-arabinofuranosylcytosine (ara-C), mitoxantrone, etoposide, camptothecin, and cisplatin, induce the expression of c-jun oncogene in leukemic cells prior to producing internucleosomal DNA fragmentation and the morphological features of apoptosis. This has led to the impression that the induction of c-jun expression may be directly involved in the molecular signaling of the final common pathway of programmed cell death or apoptosis. In the present study, we examined the role of c-jun expression in three different settings of anticancer drug-induced apoptosis in human leukemic cells. First, exposure of human myeloid leukemia HL-60 cells to high-dose ara-C for 4 h produced internucleosomal DNA fragmentation preceded by c-jun induction. However, pretreatment of HL-60 cells with staurosporine, a protein kinase C inhibitor, repressed c-jun yet enhanced DNA fragmentation and apoptosis due to ara-C. Second, in human pre-B leukemia 697/BCL-2 cells which are transfected with the cDNA of the bcl-2 oncogene and overexpress p26BCL-2, although ara-C or mitoxantrone treatment caused greater c-jun induction than in the 697/neo cells, significantly reduced endonucleolytic DNA fragmentation and apoptosis was observed in 697/BCL-2 cells. Finally, taxol-induced internucleosomal DNA fragmentation and morphological features of apoptosis in HL-60 cells were not associated with the induction of c-jun expression. These lines of evidence indicate that the induction of c-jun expression may not have a direct role in the molecular signaling of anticancer drug-induced apoptosis, and that the anticancer drug-induced apoptosis can occur by a mechanism that does not involve the induction of c-jun expression.

Effects of modulators of protein kinases on taxol-induced apoptosis of human leukemic cells possessing disparate levels of p26BCL-2 protein.

Taxol-induced polymerization of tubulin into stable microtubules and cell cycle metaphase arrest have been demonstrated to result in internucleosomal DNA fragmentation and morphological features of apoptosis in human leukemia cells. Recent studies have also shown that Taxol-induced apoptosis, but not Taxol-induced microtubular bundling or mitotic arrest, is significantly inhibited in cells that overexpress the bcl-2 gene product p26BCL-2. In the present studies we examined the effects of several modulators of activities of protein kinases on Taxol-induced DNA fragmentation and apoptosis in human pre-B leukemia 697 cells transfected with the cDNA of the bcl-2 gene and expressing high intracellular levels of p26BCL-2 (697/BCL-2 cells). Treatment with 0.1-1.0 microM MTaxol for 24 h produced prolonged mitotic arrest of control 697/neo cells, which had been transfected with the neomycin resistance gene. This resulted in apoptosis-associated large DNA fragments ranging between 5 and 200 kb and internucleosomal DNA fragmentation. Cotreatment with the phorbol ester phorbol dibutyrate (PdBU) significantly reduced Taxol-induced internucleosomal and large DNA fragmentation and inhibited apoptosis of 697/neo cells. In contrast, a combined exposure to Taxol and staurosporine (ST; 5 or 50 ng/ml), a potent inhibitor of protein kinase C and other kinases, significantly increased DNA fragmentation and apoptosis of 697/neo cells. Additionally, in 697/BCL-2 cells, ST partially overcame the suppressive effects of high p26BCL-2 levels on Taxol-induced apoptosis. Cotreatment with the tyrosine kinase inhibitor Genistein (30 microM) markedly inhibited Taxol-induced DNA fragmentation and apoptosis of 697/neo cells. However, it is noteworthy that the modulations of Taxol-induced DNA fragmentation and apoptosis by PdBU, ST, and Genistein occurred without significant effects on Taxol-mediated mitotic arrest of 697/neo cells. These agents also did not affect intracellular p26BCL-2 levels in 697/neo or 697/BCL-2 cells. These findings indicate that Taxol-induced apoptosis can be modulated by agents that affect the activities of protein kinases, and these effects are not mediated by modulations of Taxol-induced mitotic arrest or by alterations of intracellular p26BCL-2 levels.

Effect of combined treatment with interleukin-3 and interleukin-6 on 4-hydroperoxycyclo-phosphamide-induced programmed cell death or apoptosis in human myeloid leukemia cells.

In autologous bone marrow transplantation in patients with acute myeloid leukemia (AML), 4-hydroperoxycyclophosphamide (4-HC) is a commonly used ex vivo purging agent for leukemic blasts. In the present report, we demonstrate that exposure to high concentrations of 4-HC for 1 hour, as used in ex vivo bone marrow purging, produces internucleosomal DNA fragmentation characteristic of apoptosis, or programmed cell death (PCD), in human myeloid leukemia HL60 cells. Lower concentrations of 4-HC (10, 20, or 50 microM/L) failed to cause this effect, while higher concentrations (> or = 200 microM/L) produced random DNA fragmentation. 4-HC-mediated internucleosomal DNA fragmentation was associated with a marked induction in c-jun and significant reductions in bcl-2 and c-myc oncogene expressions. A combined treatment with interleukin-3 (IL-3) plus IL-6 for 18 hours before an additional, 1-hour concurrent treatment with 4-HC (100 microM/L) significantly increased 4-HC-induced DNA fragmentation as well as colony growth inhibition of HL60 cells. The effects of cotreatment with IL-3 plus IL-6 were also associated with a further, modest decrease in bcl-2 and c-myc and augmentation of c-jun expression. These findings highlight an alternative mechanism of 4-HC-induced leukemic cell death that can be potentially enhanced by cotreatment with IL-3 plus IL-6.

Calcium delivery and time: factors affecting the progression of cellular damage during the calcium paradox in the rat heart.

Using an isolated rat heart preparation we have investigated the influence of calcium delivery and time upon the induction of cellular injury during the calcium paradox. Hearts were subjected to 10 min of calcium depletion. This was followed by calcium repletion for up to 20 min during which time the calcium concentration in the perfusate was varied between 0.025 and 1.00 mmol X litre-1. For the lowest calcium repletion concentration, cumulative leakage of creatine kinase activity was small and linear with time over the 20 min repletion period, and relatively few damaged cells were observed, these being situated around coronary vessels. For calcium concentrations of 0.05 mmol X litre-1 and above the progression of structural injury was dependent on both increasing calcium concentration and time. After 1 min of repletion with 0.10 mmol calcium X litre-1 the percentage damaged cells was 2%, this sharply increased to 95% after 10 min of repletion but without a parallel increase in the profile for creatine kinase leakage. For calcium repletion at 0.05 mmol X litre-1 morphological injury was shown to be highly heterogeneous both within and between hearts. Uniform cellular damage (ie greater than 95%) in the concentration range 0.25 to 1.00 mmol calcium X litre-1 was only seen after 10 min of calcium readmission. Maximal cumulative creatine kinase activity only occurred after 15 to 20 min of repletion with 0.50 and 1.00 mmol calcium X litre-1. Our results show calcium delivery and time can both modulate the progression of cellular injury and allow a dissociation between indices of tissue damage.

Autoradiographic study of the distribution of [3H]- and [14C]-hydrallazine in the rat.

The distribution of [3H]-hydrallazine (HP) in the rat was investigated using autoradiography from the whole-body, to the electron microscopic level. Intravenous dosing gave rapid and persistent labelling of blood vessels, particularly arteries, whilst radiolabel from orally administered drug was detectable in the vasculature in modest amounts only at 6 h, the longest interval studied. Light microscopic autoradiographs of blood vessels showed silver grains associated with elastic laminae and the marginal region of smooth muscle cells. Analysis of electron microscopic autoradiographs of arteries from rats dosed intravenously (1 h and 6 h) and orally (6 h) revealed the greatest percentage of radiolabel in each case to be associated with the elastic laminae (34.9 to 40.3%). Significant proportions of total radiolabel (between 12.7 and 34.8%) were ascribed to the smooth muscle cells. It is concluded that radiolabel, possibly in the form of intact HP, is accessible to the vascular smooth muscle cells where the vasodilator action of HP is held to be exerted.

Influence of the vascular endothelium on agonist-induced contractions and relaxations in rat aorta.

The influence of the vascular endothelium on agonist-induced contractions and relaxations has been measured using intact segments of rat aorta. Contiguous rubbed segments were used as controls. Angiotensin II, histamine, noradrenaline, U46619 and UK14304 contracted both rubbed and intact tissues. The threshold spasmogenic concentrations of these agonists were lower in rubbed tissues than in intact preparations. The sensitivity and responsiveness of tissues to angiotensin II, histamine, noradrenaline and UK14304 were greater in rubbed than in intact tissues. Acetylcholine and histamine relaxed the established spasms of intact tissues but not those of rubbed preparations, These relaxant effects of acetylcholine were abolished by pre-incubation with haemoglobin. In the presence of prazosin, noradrenaline or UK14304 relaxed established contractions in intact tissues. These effects were antagonized by idazoxan or by pre-incubation with haemoglobin. In intact preparations, idazoxan had no effect on the spasmogenic sensitivity and responsiveness to UK14304. Pre-incubation with haemoglobin augmented the spasmogenic actions of noradrenaline, U46619 or UK14304 in intact tissues, but had no effect on these responses in rubbed preparations. Tissue concentrations of cyclic GMP were greater in intact than in rubbed tissues. A concentration of acetylcholine (10 microM) evoking just maximal mechanical inhibition produced a significant increase in cyclic GMP concentration in intact preparations. However, no detectable changes in cyclic GMP concentration were produced by UK14304 (10 microM) or by acetylcholine (30 nM), concentrations which were equi-effective in inhibiting mechanical activity. In the presence of threshold spasmogenic concentrations of noradrenaline, the contractile effects of angiotensin II were augmented and became comparable to those observed in rubbed preparations. In the presence of greater concentrations of noradrenaline, angiotensin II always produced an additional contraction. It is concluded that the presence of the vascular endothelium limits the spasmogenic action of a variety of agonists. Although spasmogens like noradrenaline and UK14304 can stimulate the release of endothelium-derived relaxing factor (EDRF) via alpha 2-adrenoceptors, the inhibitory effects of EDRF largely result from the spontaneous release of this substance.

Effects of a combination of metoprolol and dazmegrel on myocardial infarct size in rats.

The effects of acute pretreatment with metoprolol, dazmegrel and a combination of these two drugs has been examined on myocardial infarct size in rats. Ischaemic damage was assessed 4 h after coronary artery occlusion in anaesthetized rats and after 48 h of ischaemia in conscious rats. Infarct size was measured histochemically (by using periodic-acid-Schiff diastase reaction for glycogen) and by standard histological examination (haematoxylin and eosin stain). There was some evidence of protection of the myocardium by metoprolol following 4 h of ischaemia (determined histologically) but this was not apparent 48 h after occlusion. When given alone, dazmegrel had no significant effects on infarct size assessed by either method. A clear reduction in the extent of glycogen depletion and histological damage was observed with the combination of metoprolol and dazmegrel 48 h after the onset of ischaemia. This protection was seen to occur in the horizontal plane of the heart, preventing the extension of the infarct towards the posterior wall of the left ventricle and showing some salvage of the epicardial surfaces.