Tumor regression during radiotherapy for non-small cell lung cancer patients using cone-beam computed tomography images.

PURPOSE: Previous literature has reported contradicting results regarding the relationship between tumor volume changes during radiotherapy treatment for non-small cell lung cancer (NSCLC) patients and locoregional recurrence-free rate or overall survival. The aim of this study is to validate the results from a previous study by using a different volume extraction procedure and evaluating an external validation dataset. METHODS: For two datasets of 94 and 141 NSCLC patients, gross tumor volumes were determined manually to investigate the relationship between tumor volume regression and locoregional control using Kaplan-Meier curves. For both datasets, different subgroups of patients based on histology and chemotherapy regimens were also investigated. For the first dataset (n = 94), automatically determined tumor volumes were available from a previously published study to further compare their correlation with updated clinical data. RESULTS: A total of 70 out of 94 patients were classified into the same group as in the previous publication, splitting the dataset based on median tumor regression calculated by the two volume extraction methods. Non-adenocarcinoma patients receiving concurrent chemotherapy with large tumor regression show reduced locoregional recurrence-free rates in both datasets (p < 0.05 in dataset 2). For dataset 2, the opposite behavior is observed for patients not receiving chemotherapy, which was significant for overall survival (p = 0.01) but non-significant for locoregional recurrence-free rate (p = 0.13). CONCLUSION: The tumor regression pattern observed during radiotherapy is not only influenced by irradiation but depends largely on the delivered chemotherapy schedule, so it follows that the relationship between patient outcome and the degree of tumor regression is also largely determined by the chemotherapy schedule. This analysis shows that the relationship between tumor regression and outcome is complex, and indicates factors that could explain previously reported contradicting findings. This, in turn, will help guide future studies to fully understand the relationship between tumor regression and outcome.

Osteoradionecrosis of the mandible after radiotherapy for head and neck cancer: risk factors and dose-volume correlations.

Background: The project aimed at determining the incidence of mandibular osteoradionecrosis (ORN) after radiotherapy, possible risk factors, and mandibular dose-volume effects in a large cohort of head and neck cancer patients (HNC). Methods: The cohort consisted of 1224 HNC patients treated with 66-68 Gy in 2007-2015 predominantly with IMRT. ORN cases were defined from clinical observations at follow-up and through hospital code diagnostics after oral-maxillofacial surgery and cross-checked with the national Danish Head and Neck Cancer database. In a nested case-control study, patients with ORN cases were matched with two controls (1:2) and pre-RT dental procedures including surgery to the mandible were documented. Multivariable Cox regression analysis was applied using demographic and treatment variables including dental procedures, smoking and tumor characteristics, and combined with dosimetric data. Mean mandibular dose (Dmean) was pre-selected for the multivariable model. Results: ORN was recorded in 56 cases (4.6%) with a median time to event of 10.9 months (range 1.8-89.7) after RT, 90% occurred within 37.4 months. Median follow-up time was 22 months (0.3-95). Average Dmean was significantly higher in the ORN event cohort and significant dose-volume differences were observed for population mean DVH doses between 30 Gy and 60 Gy. In univariable analysis, smoking (HR = 1.69; CI 1.14-2.5), pre-RT surgery/tooth extraction (HR = 2.76; 1.48-5.14), and several dosimetric parameters including Dmean (HR = 1.05, 1.02-1.08) were all significantly associated with ORN. Dmean and surgery/tooth extraction remained significant predictors of ORN in multivariable analysis, HR = 1.04 (CI 1.01-1.07) and HR = 2.09 (CI 1.1-3.98), respectively, while smoking only retained its significance in an interaction analysis with pre-RT dental procedures. Conclusion: The onset of ORN of the mandible was early (median 10.8 months) and the incidence low (4.6%) after IMRT in HNC cancer patients. Surgery to the mandible and pre-RT tooth extraction, tobacco smoking, and treatment dose were associated with the development of ORN.

Relationship between patient and physician-rated xerostomia and dose distribution to the oral cavity and salivary glands for head and neck cancer patients after radiotherapy.

Introduction: Xerostomia is a frequent complication after curative intended radiotherapy (RT) for head and neck squamous cell carcinoma (HNSCC). Assessment of xerostomia is commonly done by the physician. The aim of this study is to investigate the relation between patient and physician-rated xerostomia and to predict the degree of xerostomia from patients with self-reported xerostomia based on delivered doses to the oral cavity, parotid, and submandibular glands. Material and methods: During a 2-year period, consecutive HNSCC patients attending the follow-up clinic were included. All included patients had self-reported xerostomia, and completed the disease-specific EORTC QLQ-H&N35 questionnaire. The physician assessed the degree of xerostomia with the DAHANCA toxicity scale and was blinded for the EORTC score. Oral cavity, parotid, and submandibular glands (OAR) were delineated on the planning CT according to international guidelines. DVH were extracted from treatment plans. Logistic regression tested the relation between mean doses, patient characteristics, and xerostomia scores. Differences between DVH values and scoring of xerostomia were analyzed with a Kruskal-Wallis test. The relation between xerostomia and dose distributions was further investigated using principal component analysis (PCA). Results: In total, 109 patients were included in the study. A weak correlation was seen between patient and physician-rated toxicity (p = .001), however, in general patients reported more toxicity than physicians. For EORTC score ≥2, the multi-variable analysis was significant for doses to the oral cavity, tobacco status and use of xerogenic medication. Neither the DVH analysis nor the PCA found any clear distinction between xerostomia scores for EORTC or DAHANCA and investigated OARs. Conclusion: Patients tended to report higher scores of xerostomia than the physician. PCA indicated a complex relation between doses to the OAR and xerostomia scores, showing e.g., that reducing doses in one organ was on the expense of increased dose to another organ.

Fluid shift from intravascular compartment during fetal red blood cell transfusion.

OBJECTIVES: Intrauterine transfusion imposes a considerable burden on the fetal circulation by increasing volume and pressure, and a fluid shift from the fetal circulation occurs even during the procedure. The aim of this study was to quantify the intraprocedural fluid shift and to investigate the effect of procedural and fetal characteristics on this fluid shift. METHODS: In 95 alloimmunized pregnancies, we calculated fluid shift at the first intrauterine transfusion by determining initial and final blood volumes. We evaluated the association of the fluid shift with the speed and volume of the transfusion, the severity of anemia and the presence of hydrops. RESULTS: Of the included fetuses, 11 were mildly hydropic and four were severely hydropic. A mean fluid shift of 36% of the transfused volume was found. Fluid shift related positively to transfused volume (P < 0.001). The percentage fluid shift of transfused volume was inversely related to the speed of transfusion (mL/kg/min) (P < 0.041) and was not related to the severity of anemia (P = 0.55) or to hydrops (P = 0.66). It was found that younger fetuses had been unintentionally subject to high volumes and speeds of transfusion relative to their size. CONCLUSIONS: Around one-third of the transfused volume is lost from the intravascular compartment during the procedure of intrauterine transfusion. There is a large variation between fetuses, partly explained by the volume and speed of the transfusion. Neither severity of anemia nor hydrops plays a clear-cut role, and thus other factors may explain the variation in fluid shift. The probability that hematocrit will still increase after transfusion, as a result of a continuing fluid shift, should be considered in transfusion policy. Advice is given on gestational age-adjusted speed of transfusion.

Prediction of radiation-induced mucositis of H&N cancer patients based on a large patient cohort.

PURPOSE/OBJECTIVE: Radiation-induced mucositis is a severe acute side effect, which can jeopardize treatment compliance and cause weight loss during treatment. The study aimed to develop robust models to predict the risk of severe mucositis. MATERIALS/METHODS: Mucosal toxicity scores were prospectively recorded for 802 consecutive Head and Neck (H&N) cancer patients and dichotomised into non-severe event (grade 0-2) and severe event (grade 3+) groups. Two different model approaches were utilised to evaluate the robustness of the models. These used LASSO and Best Subset selection combined with 10-fold cross-validation performed on two-thirds of the patient cohort using principal component analysis of DVHs. The remaining one-third of the patients were used for validation. Model performance was tested through calibration plot and model performance metrics. RESULTS: The main predicted risk factors were treatment acceleration and the first two principal dose components, which reflect the mean dose and the balance between high and low doses to the oral cavity. For the LASSO model, gender and current smoker status were also included in the model. The AUC values of the two models on the validation cohort were 0.797 (95%CI: 0.741-0.857) and 0.808 (95%CI: 0.749-0.859), respectively. The two models predicted very similar risk values with an internal Pearson coefficient of 0.954, indicating their robustness. CONCLUSIONS: Robust prediction models of the risk of severe mucositis have been developed based on information from the entire dose distribution for a large cohort of patients consisting of all patients treated H&N for within our institution over a five year period.

Total blood volume is maintained in nonhydropic fetuses with severe hemolytic anemia.

OBJECTIVE: Fetal alloimmune anemia is associated with increased blood flow velocities and cardiomegaly. In severe cases, hydrops can develop. We investigated whether the decrease of red blood cell volume is associated with a reduction or expansion of plasma volume. METHODS: In 86 alloimmunized fetuses that received a first intrauterine transfusion, we calculated fetal total blood volumes (i.e. fetoplacental blood volumes) using a dilutional principle of fetal hemoglobin with adult hemoglobin. The relation between total blood volume and estimated fetal weight, severity of anemia and hydrops was analyzed. RESULTS: Gestational age ranged from 17 to 35 weeks. Mean hemoglobin deficit was 6.8 standard deviations (range 2.1-11.7) below the normal mean. Fetal total blood volume was significantly related to estimated fetal weight (p < 0.001). Mean total blood volume in nonhydropic fetuses was 123 ml/kg (n = 74) and in hydropic fetuses 144 ml/kg (n = 12). There was a significant relation between total blood volume per kg body weight and hydrops (p = 0.035); however, there was no relation with severity of anemia (p = 0.94). CONCLUSION: In the human nonhydropic fetus with severe hemolytic anemia, total blood volume is maintained: the decrease in red blood cell volume is thus compensated by an increase in plasma volume. In hydropic fetuses, however, total blood volume seems to be increased. This is in accordance with the hypothesis that congestive heart failure plays a role in the pathophysiology of hydrops in anemic fetuses.

Infant burns: A single institution retrospective review.

Thermal injuries amongst infants are common and a cause of significant mortality and morbidity in South Africa. This has been attributed to the lack of an enabling environment (poverty-related lack of safe living conditions) and the cognitive and physical developmental immaturity of infants, who depend on their surroundings and adults to keep them safe. This is a retrospective observational study of 548 infant admissions over 48 months. Infant was defined as children below 13 months of age. The 548 infants constituted 23% of all paediatric burn admissions of ages 0-12 years. Three hundred and fourteen were males (57%) and 234 (42.7%) females. The infants were divided in a pre-ambulatory group of 143 (26%) infants of 0-6 months and an ambulatory group of 7 months to 12 months consisting of 457 (83.3%). The total body surface area (TBSA) ranged from 2-65%. Seventy-six percent (417 infants) occurred in the home environment. Scalds accounted for 86% (471 infants) and 6% (33 infants) were as a result of flame burns. Non-accidental injuries accounted for 1.2%. The anatomical distributions varied between the pre-ambulatory and ambulatory groups. Conservative management was done in 397 (72.4%) and 101(18.4%) infants underwent surgery. Infection was suspected in 76 (13.5%) infants with positive blood cultures in 15(20%) of the 76. ICU care was received in 46 (8.3%) infants and 15 (32.6%) of these had inhalation injuries. Of the inhalation injuries 11(23.9%) infants underwent mechanical ventilation of an average of 4.4 days. Ventilator associated pneumonia was diagnosed in 8(17%) of the ventilated children. The mortality rate was 0.36%. The surgically treated patients acquired more complications than the conservatively treated group. Special treatment considerations should be considered in this paediatric sub-group.

A review of the peri-operative management of paediatric burns: Identifying adverse events.

BACKGROUND: Burn injuries are common in poverty-stricken countries. The majority of patients with large and complex burns are referred to burn centres. Of the children who qualify for admission, according to burn admission criteria, about half require some kind of surgical procedure to obtain skin cover. These range from massive full-thickness fire burns to skin grafts for small, residual unhealed wounds. Burn anaesthetic procedures are of the most difficult to perform and are known for high complication rates. Reasons include peri-operative sepsis, bleeding, issues around thermoregulation, the hypermetabolic state, nutritional and electrolyte issues, inhalation injuries and the amount of movement during procedures to wash patients, change drapes and access different anatomical sites. The appropriate execution of surgery is therefore of the utmost importance for both minor and major procedures. OBJECTIVE: To review the peri-operative management and standard of surgical care of burnt children. METHODS: This was a retrospective review and analysis of standard peri-operative care of burnt children at Red Cross War Memorial Children's Hospital, Cape Town, South Africa. A total of 558 children were operated on and supervised by the first author. Factors that could adversely affect surgical and anaesthetic outcomes were identified. RESULTS: There were 257 males and 301 females in this study, with an average age of 50.1 months and average weight of 19.5 kg. The total body surface area involved was 1 - 80%, with an average of 23.5%. Inhalational injury was present in 11.3%, pneumonia in 13.1%, wound sepsis in 20.8%, and septicaemia in 9.7%, and organ dysfunction in more than one organ was seen in 6.1%. The average theatre temperature during surgery was 30.0°C. Core temperatures recorded at the start, halfway through and at completion of surgery were 36.9°C, 36.8°C and 36.5°C, respectively. The average preoperative and postoperative haemoglobin levels were 11.28 g/dL and 9.64 g/dL, respectively. Blood loss was reduced by the use of clysis from 1.5 mL/kg/% burn to 1.4 mL/kg/% burn. Adverse intraoperative events were seen in 17.6% of children. CONCLUSION: Burn surgery is a high-risk procedure and comorbidities are common. Anaesthesia and surgery must be well planned and executed with special reference to temperature control, rapid blood loss, preceding respiratory illnesses and measures to reduce blood loss.

Cell scattering of SK-N-MC neuroepithelioma cells in response to Ret and FGF receptor tyrosine kinase activation is correlated with sustained ERK2 activation.

The c-ret proto-oncogene encodes a receptor tyrosine kinase which plays an important role in kidney and enteric nervous system development. Germline mutations in c-ret are responsible for the dominantly inherited cancer syndromes, multiple endocrine neoplasia types 2A and 2B and familial medullary thyroid carcinoma as well as the developmental disorder Hirschsprung's disease. Using SK-N-MC neuroepithelioma cells stably transfected with an EGFR/Ret chimeric receptor, we have studied cellular consequences and signalling events following activation of exogenous EGFR/Ret and endogenous FGF and PDGF receptor tyrosine kinases in cells of neuroectodermal origin. Here we report that Ret activation led to cell scattering, growth inhibition and loss of anchorage-independent growth. Basic FGF, but not PDGF, evoked similar responses in those cells. Nevertheless, activation of all three receptor tyrosine kinases led to ERK2 activation. Analysis of the kinetics of ERK2 activation and downstream events revealed that Ret and FGF receptor activation led to sustained ERK2 activation and SRE transactivation, while PDGF treatment led to transient ERK2 activation and failed to induce SRE transactivation. Our results suggest that sustained, but not transient ERK2 activation may be involved in cell scattering.

Pax4 regulatory elements mediate beta cell specific expression in the pancreas.

Pax4 is a member of the Pax (Pax, paired box) family of transcription factors with restricted expression and essential functions in the developing pancreas. Pax4 deficient mice do not develop pancreatic beta and delta cells and thus die after birth due to diabetes. In this study using transgenic mouse technology we report the identification and characterization of a 0.9 kb DNA fragment in the 5'-region of the gene, which by itself is able to direct Pax4 expression in the endocrine pancreas, recapitulating the beta-cell-specific in vivo expression pattern of Pax4. The 0.9 kb DNA fragment contains an area spanning 407 base pairs that is highly conserved between human and mouse showing 88% identity. This promoter region contains sequence motifs that have been shown to be involved in beta-cell-specific expression of insulin, Pdx1 and islet amyloid polypeptide (IAPP). In addition, we determined a previously undescribed 5'intron.

Transcriptional regulation of retinoic acid receptor beta in retinoic acid-sensitive and -resistant P19 embryocarcinoma cells.

As in other embryocarcinoma (EC) cell lines retinoic acid (RA) rapidly induces expression of the nuclear retinoic acid receptor (RAR) beta in murine P19 EC cells, while RAR alpha is expressed constitutively. In the RA-resistant P19 EC-derived RAC65 cells, however, there is no such induction and an aberrant (smaller) RAR alpha transcript is expressed. RAR gamma 1 is expressed at low levels in both cell lines. To study the regulation of the RAR beta gene and the possible involvement of RAR alpha protein in transcriptional activation of the RAR beta gene we transfected these cells with a construct containing a 1.6 kb promoter fragment of the human RAR beta gene fused to the CAT gene. Upon transient assays in P19 EC cells CAT activity is enhanced rapidly by RA, to more than 100-fold in a concentration-dependent fashion. On the contrary no activity can be observed in the RA-resistant RAC65 cells; however, co-transfection of hRAR alpha, hRAR beta or hRAR gamma 1 restores the RA-dependent induction of CAT activity. These results clearly show that RAR alpha and RAR gamma 1 can transactivate the RAR beta gene; that RAR beta can stimulate its own expression and that resistance to RA in RAC65 cells is probably due to the altered RAR alpha transcript present in these cells.

Sensitive detection of squamous cells in bone marrow and blood of head and neck cancer patients by E48 reverse transcriptase-polymerase chain reaction.

In previous studies, we described the selective reactivity of monoclonal antibody E48 with normal squamous and transitional epithelia and their malignant counterparts and the capacity of monoclonal antibody E48 for selective tumor targeting in head and neck cancer patients. Cloning of the E48 encoding cDNA and elucidation of the gene structure enabled the selection of an intron-spanning primer set for the detection of circulating tumor cells in blood and bone marrow of head and neck cancer patients. Extensive optimizations led to a reproducible reverse transcriptase-PCR assay with an internal standard for RNA quality control and an external standard for sensitivity control. In reconstruction experiments, we were able to reach a reproducible sensitivity of one single tumor cell per 7 ml of blood (2 x 10(7) nucleated cells). When applying this method to patient material, we were able to detect positive signal in 35% of the bone marrow samples (0 of 2 stage II, 0 of 4 stage III, 4 of 11 stage IV, and 4 of 6 recurrences) and 10% of the blood samples (2 of 21) of patients with squamous cell carcinoma of the head and neck. The specificity of the method was demonstrated on 29 blood and bone marrow samples of noncancer controls, which were all negative. Our study shows the feasibility of E48 reverse transcriptase-PCR for the detection of squamous cells in nonsquamous tissues.

Evaluation of soluble CD44v6 as a potential serum marker for head and neck squamous cell carcinoma.

In recent years, the measurement of soluble CD44 levels in the circulation of patients with malignant diseases has been introduced as a new and simple diagnostic tool for the detection of human cancer. The high CD44v6 expression in head and neck squamous cell carcinoma (HNSCC) would enable the use of soluble CD44v6 proteins present in the circulation of HNSCC patients as a marker of disease. In the present study, we determined CD44v6 plasma levels using a domain-specific ELISA in healthy volunteers, non-cancer patients, and HNSCC patients before and after surgical removal of the tumor. A difference between the CD44v6 plasma levels of HNSCC patients and controls could not be observed. Moreover, surgical removal of the tumor did not result in a reduction of the CD44v6 plasma level in the HNSCC patients. In addition, the spectrum of soluble v6-containing CD44 proteins present in the plasma of HNSCC patients and controls was determined by immunoprecipitation experiments, but again, tumor-related isoforms could not be distinguished in patient samples. Additional experiments to unravel the biological source of these circulating proteins indicated surprisingly that the v6-containing proteins present in the circulation of healthy individuals are only released in part, if at all, by activated lymphocytes or other nucleated blood cells. Most circulating CD44v6 proteins seem to be derived from the normal epithelial cell compartments, including breast cells, colon cells, and squamous cells. Taken together, these data do not support the use of soluble CD44v6 as a tumor marker in HNSCC or any other tumor type that has developed from tissues producing soluble isoforms.

Embryonic stem cells stably transfected with mRAR beta 2-lacZ exhibit specific expression in chimeric embryos.

Using the embryonic stem (ES) cell/chimera approach, we have studied the activity of the mouse retinoic acid receptor beta 2 (mRAR beta 2) promoter during ES cell differentiation and during embryonic development. Stable ES clones were isolated after introduction of a 1.8 kb mRAR beta 2-lacZ expression cassette. LacZ expression in these stable clones was specifically induced by retinoic acid (RA) in a similar fashion as the endogenous RAR beta 2 gene. Following introduction of three different ES clones into blastocysts, an integration-independent mRAR beta 2-lacZ expression pattern was obtained in chimeric embryos similar to that described by in situ hybridization and transgenic studies. Moreover, mRAR beta 2-lacZ expression was also detected at some additional sites not described before, e.g. body wall, ureter, mesonephric duct and optic stalk. Maternal RA administration at 8.5 days of pregnancy extended lacZ expression to more anterior and posterior regions. Transgenic mice were generated from germ-line transmission of the transfected ES cells; expression pattern and changes in expression upon RA induction in these transgenic embryos were identical to those in chimeric embryos. We conclude that by using the ES/chimera approach, the proximal 1.8 kb of the mRAR beta 2 promoter produces a reliable and reproducible expression pattern of the reporter gene, and that the ES cell/chimera approach is invaluable for the study of gene expression and regulation.

Changes in retinoic acid receptor messenger ribonucleic acid levels in the vitamin A-deficient rat testis after administration of retinoids.

Recently, we have reported that retinoic acid (RA), similarly to retinol acetate, is able to reinitiate spermatogenesis in vitamin A-deficient rats. Here, we investigated the expression of RA receptors RAR alpha, RAR beta, RAR gamma, and retinoid X receptor RXR alpha by Northern blot analysis of poly(A)+ RNA of testes of vitamin A-deficient rats before and after reinitiation of spermatogenesis induced by injection of retinol acetate or RA and testes of 21-day-old and 10-week-old normal rats. In the testis of vitamin A-deficient rats 1.9-, 2.8-, and 3.8-kilobase (kb) transcripts of RAR alpha; 2.8- and 3.3-kb transcripts of RAR beta; 1.8-, 2.8-, and 3.4-kb transcripts of RAR gamma; and two transcripts of RXR alpha of 2.5 and 4.8 kb are expressed. When vitamin A-deficient rats receive RA or retinol acetate, a 3-fold increase in the amount of poly(A)+ RNA per testis can be observed after 8 h, while the amounts of glyceraldehyde-3-phosphate dehydrogenase and sulfated glycoprotein-1 mRNA hardly change. Also, the expression of several transcripts of each RAR type is significantly increased from 1.8- up to 3.6-fold. Moreover, additional transcripts of RAR beta and RXR alpha (1.8 and 1.0 kb, respectively) can be detected. In the testes of 21-day-old rats, three transcripts of each RAR type and two RXR alpha transcripts are expressed. In contrast, in the normal adult rat testis the expression of all RARs, if present, is lower than that in the 21-day-old rat testis or the adult vitamin A-deficient rat testis. The expression of all transcripts of each RAR in the testis of 21-day-old rats shows great similarity with the expression in the testis of the vitamin A-deficient rat after replacement of retinol acetate or RA. These changes in expression indicate that RARs and RXR alpha may play a role in the process of proliferation and differentiation of A spermatogonia, which is induced in vitamin A-deficient rats shortly after replacement of RA or retinol acetate.

4-hydroxytamoxifen trans-represses nuclear factor-kappa B activity in human osteoblastic U2-OS cells through estrogen receptor (ER)alpha, and not through ER beta.

Estrogens are important mediators of bone homeostasis, and postmenopausal estrogen replacement therapy is extensively used to prevent osteoporosis. The biological effects of estrogen are mediated by receptors belonging to the superfamily of steroid/thyroid nuclear receptors, estrogen receptor (ER)alpha and ER beta. ER alpha, not only trans-activates target genes in a hormone-specific fashion, but it can also neutralize other transcriptional activators, such as nuclear factor (NF)-kappa B, causing repression of their target genes. A major mechanism by which estrogens prevent osteoporosis seems to be repression of transcription of NF-kappa B target genes, such as the osteoclast-activating cytokines interleukin-6 and interleukin-1. To study the capacity of both ERs in repression of NF-kappa B signaling in bone cells, we first carried out transient transfections with ER alpha or ER beta of the human osteoblastic U2-OS cell line, in which endogenous NF-kappa B was stimulated by tumor necrosis factor alpha. Repression by ER alpha was already observed without 17 beta-estradiol, whereas addition of the ligand increased repression to 90%. ER beta, however, was able to repress NF-kappa B activity only in the presence of ligand. Because it is known that some antiestrogens can also display tissue-specific agonistic properties, 4-hydroxytamoxifen was tested for its capacity in repressing NF-kappa B activity and was found to be active (albeit less efficient than 17 beta-estradiol) and, interestingly, only with ER alpha. The pure antagonist ICI 164,384 was incapable of repressing through any ER subtypes. Deletion analysis and the use of receptor ER alpha/ER beta-chimeras showed that the A/B domain, containing activation function-1, is essential for this suppressive action. Next, we developed stable transfectants of the human osteoblastic U2-OS cell line containing ER alpha or ER beta in combination with an NF-kappa B luciferase reporter construct. In these cell lines, repression of NF-kappa B activity was only mediated through ER alpha and not through ER beta. These findings offer new insights into the specific role of both ER subtypes in bone homeostasis and could eventually help in developing more specific medical intervention strategies for osteoporosis.

Tolerance of guinea-pig airway muscle preparations to relaxant agonists induced by chronic exposure to isoprenaline in vivo.

1 The histamine sensitivities of complete tracheal spiral preparations from guinea-pigs were similar to those of paired half tracheal tissues. 2 Airway muscle preparations from animals chronically treated with isoprenaline showed a significant increase in resting tone and a significant decreased responsiveness to histamine. 3 The paired half tracheal preparations exhibited no significant difference when either their isoprenaline or theophylline sensitivities were compared. 4 Paired half tracheal muscle preparations from chronically treated animals (0.4 mumol, 4.0 mumol, and 40.0 mumol isoprenaline, 3 times daily for 21 days, s.c.) showed a significantly reduced sensitivity to isoprenaline when compared to appropriate controls. 5 Theophylline concentration-effect curves for the paired half tracheal preparations from chronically treated guinea-pigs (4.0 mumol and 40.0 mumol isoprenaline) were significantly reduced compared with appropriate controls. 5 Indomethacin treatment reversed the isoprenaline desensitization induced by chronic treatment (0.4 mumol isoprenaline) but was ineffective in animals that received the higher doses of isoprenaline in vivo.

The actions of Paf-acether (platelet-activating factor) on guinea-pig isolated heart preparations.

Paf-acether (platelet-activating factor) is a phospholipid capable of stimulating platelets to release their granular contents and cause platelet aggregation. When Paf-acether was administered to isolated heart preparations from normal guinea-pigs there was a significant concentration-dependent reduction in coronary flow and contractile force. The high concentration of Paf-acether was equally effective in reducing these cardiac parameters in the presence of atropine. The non-acetylated Paf-acether analogue, 2-lyso Paf-acether, the enantiomer, and a closely related phospholipid 1, alpha-lysophosphatidylcholine palmitoyl, did not affect coronary flow and contractile force, indicating the specificity of Paf-acether. These data demonstrate a potent effect of Paf-acether on cardiac function. Whether or not these effects are direct or mediated through generation of endogenous mediators remains to be established.

The interaction between indomethacin and contractile agents on human isolated airway muscle.

1 Concentration-effect curves to acetylcholine and histamine were produced in fresh human bronchial muscle (2 to 4 h after removal from the patients) and in preparations previously stored at 4 degrees C for 12 h. 2 Sensitivities of fresh human airway muscle preparations to acetylcholine (pD2 value, 5.89 +/- 0.03; n = 4) and histamine (pD2 values, 5.41 +/- 0.03; n = 13) were similar. There was no significant difference in the sensitivities of stored preparations (acetylcholine: pD2 value, 5.70 +/- 0.06; n = 23 and histamine: pD2 value, 5.44 +/- 0.07; n = 16) when compared to the fresh preparations. 3 Indomethacin did not significantly change the basal tone in preparations of either fresh or stored human airway muscle. 4 A low concentration of indomethacin (0.17 muM) significantly reduced responsiveness and sensitivity to histamine in stored bronchi but not in fresh bronchi. The acetylcholine concentration-effect curves were unaltered by exposure to this concentration of indomethacin in either fresh or stored tissues. High concentrations (1.7 muM and 17 muM) depressed the maximal responsiveness of the bronchi to both agonists. 5 These results suggest indirectly that the regulatory role of prostaglandins in human airway muscle may be different from that in other species.