Drainless robot-assisted minimally invasive oesophagectomy-randomized controlled trial (RESPECT).

BACKGROUND: The purpose of this randomized trial is to evaluate the early removal of postoperative drains after robot-assisted minimally invasive oesophagectomy (RAMIE). Evidence is lacking about feasibility, associated pain, recovery, and morbidity. METHODS/DESIGN: This is a randomized controlled multicentric trial involving 72 patients undergoing RAMIE. Patients will be allocated into two groups. The "intervention" group consists of 36 patients. In this group, abdominal and chest drains are removed 3 h after the end of surgery in the absence of contraindications. The control group consists of 36 patients with conventional chest drain management. These drains are removed during the further postoperative course according to a standard algorithm. The primary objective is to investigate whether postoperative pain measured by NRS on the second postoperative day can be significantly reduced in the intervention group. Secondary endpoints are the intensity of pain during the first week, analgesic use, number of postoperative chest X-ray and CT scans, interventions, postoperative mobilization (steps per day as measured with an activity tracker), postoperative morbidity and mortality. DISCUSSION: Until now, there have been no trials investigating different intraoperative chest drain strategies in patients undergoing RAMIE for oesophageal cancer with regard to perioperative complications until discharge. Minimally invasive approaches combined with enhanced recovery after surgery (ERAS) protocols lower morbidity but still include the insertion of chest drains. Reduction and early removal have been proposed after pulmonary surgery but not after RAMIE. The study concept is based on our own experience and the promising current results of the RAMIE procedure. Therefore, the presented randomized controlled trial will provide statistical evidence of the effectiveness and feasibility of the "drainless" RAMIE. TRIAL REGISTRATION: ClinicalTrials.gov NCT05553795. Registered on 23 September 2022.

Bronchodilator Reversibility in the GAN Severe Asthma Cohort.

BACKGROUND AND OBJECTIVE: Positive bronchodilator reversibility (BDR) is a diagnostic criterion for asthma. However, patients with asthma may exhibit a negative BDR response. Aim: To describe the frequency of positive and Negative BDR response in patients with severe asthma and study associations with phenotypic characteristics. METHODS: A positive BDR response was defined as an increase in FEV1 >200 mL and >12% upon testing with a short-acting ß-agonist. RESULTS: BDR data were available for 793 of the 2013 patients included in the German Asthma Net (GAN) severe asthma registry. Of these, 250 (31.5%) had a positive BDR response and 543 (68.5%) a egative BDR response. Comorbidities significantly associated with a negative response were gastroesophageal reflux disease (GERD) (28.0% vs 40.0%, P<.01) and eosinophilic granulomatosis with polyangiitis (0.4% vs 3.0%; P<.05), while smoking history (active: 2.8% vs 2.2%; ex: 40.0% vs 41.7%) and comorbid chronic obstructive pulmonary disease (COPD) (5.2% vs 7.2%) were similar in both groups. Patients with a positive BDR response had worse asthma control (median Asthma Control Questionnaire 5 score, 3.4 vs 3.0, P<.05), more frequently reported dyspnea at rest (26.8% vs 16.4%, P<.001) and chest tightness (36.4% vs 26.2%, P<.001), and had more severe airway obstruction at baseline (FEV1% predicted, 56 vs 64, P<.001) and higher fractional exhaled nitric oxide (FeNO) levels (41 vs 33 ppb, P<0.05). There were no differences in diffusion capacity of the lung for carbon monoxide, single breath (% pred, 70% vs 71%). Multivariate linear regression analysis identified an association between positive BDR response and lower baseline FEV1% (P<.001) and chest tightness (P<.05) and a negative association between BDR and GERD (P<.05). CONCLUSION: In this real-life setting, most patients with severe asthma had a negative BDR response. Interestingly, this was not associated with smoking history or COPD, but with lower FeNO and presence of GERD.

Impact of PI-QUAL on PI-RADS and cancer yield in an MRI-TRUS fusion biopsy population.

PURPOSE: To test the inter-reader agreement of the Prostate Imaging Quality (PI-QUAL) score for multiparametric prostate MRI and its impact on diagnostic performance in an MRI-ultrasound fusion biopsy population. PATIENTS AND METHODS: Pre-biopsy multiparametric (T2-weighted, DWI, and DCE) prostate MRIs (mpMRI) of 50 patients undergoing transrectal ultrasound-guided MRI-fusion (MRI-TRUS) biopsy were included. Two radiologists independently assigned a PI-QUAL score to each patient and assessed the diagnostic quality of individual sequences. PI-RADS categories were assigned to six regions per prostate (left and right: base/mid-glandular/apex). Inter-reader agreement was calculated using Cohen's kappa and diagnostic performance was compared by the area under the receiver operating characteristics curve (AUC). RESULTS: In 274 diagnostic areas, the malignancy rate was 62.7% (22.5% clinically significant prostate cancer, ISUP ≥ 2). Inter-reader agreement for the diagnostic quality was poor for T2w (kappa 0.19), fair for DWI and DCE (kappa 0.23 and 0.29) and moderate for PI-QUAL (kappa 0.51). For PI-RADS category assignments, inter-reader agreement was very good (kappa 0.86). Overall diagnostic performance did not differ between studies with a PI-QUAL score > 3 compared to a score ≤ 3 (p = 0.552; AUC 0.805 and 0.839). However, the prevalence of prostate cancer was significantly lower when the PI-QUAL score was ≤ 3 (16.7% vs. 30.2%, p = 0.008). CONCLUSION: PI-QUAL has only a limited impact on PI-RADS diagnostic performance in patients scheduled for MRI-TRUS fusion biopsy. However, the lower cancer prevalence in the lower PI-QUAL categories points out a risk of false-positive referrals and unnecessary biopsies if prostate imaging quality is low.

1H, 13C, and 15N backbone chemical shift assignments of the apo and the ADP-ribose bound forms of the macrodomain of SARS-CoV-2 non-structural protein 3b.

The SARS-CoV-2 genome encodes for approximately 30 proteins. Within the international project COVID19-NMR, we distribute the spectroscopic analysis of the viral proteins and RNA. Here, we report NMR chemical shift assignments for the protein Nsp3b, a domain of Nsp3. The 217-kDa large Nsp3 protein contains multiple structurally independent, yet functionally related domains including the viral papain-like protease and Nsp3b, a macrodomain (MD). In general, the MDs of SARS-CoV and MERS-CoV were suggested to play a key role in viral replication by modulating the immune response of the host. The MDs are structurally conserved. They most likely remove ADP-ribose, a common posttranslational modification, from protein side chains. This de-ADP ribosylating function has potentially evolved to protect the virus from the anti-viral ADP-ribosylation catalyzed by poly-ADP-ribose polymerases (PARPs), which in turn are triggered by pathogen-associated sensing of the host immune system. This renders the SARS-CoV-2 Nsp3b a highly relevant drug target in the viral replication process. We here report the near-complete NMR backbone resonance assignment (1H, 13C, 15N) of the putative Nsp3b MD in its apo form and in complex with ADP-ribose. Furthermore, we derive the secondary structure of Nsp3b in solution. In addition, 15N-relaxation data suggest an ordered, rigid core of the MD structure. These data will provide a basis for NMR investigations targeted at obtaining small-molecule inhibitors interfering with the catalytic activity of Nsp3b.

Subcutaneous immunotherapy with purified Der p1 and 2 suppresses type 2 immunity in a murine asthma model.

BACKGROUND: Allergen-specific immunotherapy can induce long-term suppression of allergic symptoms, reduce medication use, and prevent exacerbations of allergic rhinitis and asthma. Current treatment is based on crude allergen extracts, which contain immunostimulatory components such as ß-glucans, chitins, and endotoxin. Use of purified or recombinant allergens might therefore increase efficacy of treatment. AIMS: Here, we test application of purified natural group 1 and 2 allergens from Dermatophagoides pteronyssinus (Der p) for subcutaneous immunotherapy (SCIT) treatment in a house dust mite (HDM)-driven mouse model of allergic asthma. MATERIALS AND METHODS: HDM-sensitized mice received SCIT with crude HDM extract, a mixture of purified Der p1 and 2 (DerP1/2), or placebo. Upon challenges, we measured specific immunoglobulin responses, allergen-induced ear swelling response (ESR), airway hyperresponsiveness (AHR), and inflammation in bronchoalveolar lavage fluid (BAL) and lung tissue. RESULTS: ESR measurement shows suppression of early allergic response in HDM-SCIT- and DerP1/2-SCIT-treated mice. Both HDM-SCIT and DerP1/2-SCIT are able to suppress AHR and eosinophilic inflammation. In contrast, only DerP1/2-SCIT is able to significantly suppress type 2 cytokines in lung tissue and BAL fluid. Moreover, DerP1/2-SCIT treatment is uniquely able suppress CCL20 and showed a trend toward suppression of IL-33, CCL17 and eotaxin levels in lung tissue. DISCUSSION: Taken together, these data show that purified DerP1/2-SCIT is able to not only suppress AHR and inflammation, but also has superior activity toward suppression of Th2 cells and HDM-induced activation of lung structural cells including airway epithelium. CONCLUSIONS: We postulate that treatment with purified natural major allergens derived from HDM will likely increase clinical efficacy of SCIT.

68Ga-PSMA 11 ligand PET imaging in patients with biochemical recurrence after radical prostatectomy - diagnostic performance and impact on therapeutic decision-making.

OBJECTIVE: To evaluate the diagnostic performance of [68Ga]Ga-PSMAHBED-CC conjugate 11 positron emission tomography (PSMA-PET) in the early detection of metastases in patients with biochemical recurrence (BCR) after radical prostatectomy (RP) for clinically non-metastatic prostate cancer, to compare it to CT/MRI alone and to assess its impact on further therapeutic decisions. MATERIAL AND METHODS: We retrospectively assessed 117 consecutive hormone-naïve BCR patients who had 68Ga-PSMA 11 PET/CT (n = 46) or PET/MRI (n = 71) between May 2014 and January 2017. BCR was defined as two PSA rises above 0.2 ng/ml. Two dedicated uro-oncological imaging experts (radiology/nuclear medicine) reviewed separately all images. All results were presented in a blinded sequential fashion to a multidisciplinary tumorboard in order to assess the influence of PSMA-PET imaging on decision-making. RESULTS: The median time from RP to BCR was 36 months (IQR 16-72). Overall, 69 (59%) patients received postoperative radiotherapy. Median PSA level at the time of imaging was 1.04 ng/ml (IQR 0.58-1.87). PSMA-positive lesions were detected in 100 (85.5%) patients. Detection rates were 65% for a PSA value of 0.2 to <0.5 ng/ml, 85.7% for 0.5 to <1, 85.7% for 1 to <2 and 100% for ≥2. PSMA-positive lesions could be confirmed by either histology (16%), PSA decrease in metastasis-directed radiotherapy (45%) or additional information in diffusion-weighted imaging when PET/MRI was performed (18%) in 79% of patients. PSMA-PET detected lesions in 67 patients (57.3%) who had no suspicious correlates according to the RECIST 1.1 criteria on MRI or CT. PSMA-PET changed therapeutic decisions in 74.6% of these 67 patients (p < 0.001), with 86% of them being considered for metastases-directed therapies. CONCLUSIONS: We confirm the high performance of PSMA-PET imaging for the detection of disease recurrence sites in patients with BCR after RP, even at relatively low PSA levels. Moreover, it adds significant information to standard CT/MRI, changing treatment strategies in a significant number of patients.

Intravenous immunoglobulin increases plasma viscosity without parallel rise in blood pressure.

WHAT IS KNOWN AND OBJECTIVE: Intravenous immunoglobulin (IVIg) is a commonly used therapy for autoimmune disease, but may cause chronic hypertension and thrombosis. We determined whether: (i) IVIg systematically affects blood pressure in the short term; (ii) acute changes in plasma viscosity because of IVIg correlate with blood pressure effects; (iii) effects of IVIg on acute blood pressure are related to baseline blood pressure or hypertension status and (iv) IVIg influences plasma markers of inflammation, anticardiolipin antibodies and homocysteine as additional putative prothrombotic risk factors. METHODS: Twenty adults with autoimmune neurological disease who received a course of IVIg were evaluated immediately before and after each infusion, on every day of the course. Blood pressure, pulse and the following haematological parameters were determined: plasma viscosity, erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), haematocrit, fibrinogen, interleukin-6 (IL-6), homocysteine and anticardiolipin positivity. RESULTS: Intravenous immunoglobulin caused both acute and cumulative rises in plasma viscosity across a treatment course, but no concordant changes in blood pressure. There was also no correlation between individual blood pressure changes and viscosity, baseline blood pressure or hypertension status. Levels of IL-6 rose across the course of therapy, but the acute-phase reactants CRP and fibrinogen did not. One patient developed anticardiolipin antibodies during therapy. WHAT IS NEW AND CONCLUSION: Individual courses of IVIg do not systematically raise blood pressure. Where IVIg is found to cause hypertension, this does not appear to be due to a direct effect of IVIg on plasma viscosity.

Anti-inflammatory activity of beta2-agonists in primary lung epithelial cells is independent of glucocorticoid receptor.

In patients with asthma and chronic obstructive pulmonary disease, the addition of long-acting beta(2)-agonists (LABA) to glucocorticosteroids (GCS) results in better control than increasing the dose of GCS alone. In smooth muscle cells and fibroblasts, one apparent underlying mechanism involves the ability of LABAs to activate the glucocorticoid receptor (GR). The present study investigates the effects of formoterol (FORM), salmeterol (SALM) and budesonide (BUD) on GR activation in bronchial epithelial cells via tumour necrosis factor-alpha-stimulated granulocyte-macrophage colony-stimulating factor (GM-CSF) release, GR nuclear translocation and GR-regulated reporter gene activity. Both BUD and FORM inhibited GM-CSF release by < or = 50%. The combination of these two drugs, in clinically relevant concentrations, inhibited GM-CSF release by 85% down to unstimulated levels. A similar inhibition was obtained when combining BUD and SALM. The ability of FORM to inhibit GM-CSF synthesis was not altered by small interfering RNA-mediated depletion of GR and FORM nor SALM-induced GR translocation into the cell nucleus. In addition, FORM did not activate GR-regulated reporter gene activity (SALM was not tested), in contrast to the clear effect of BUD. It was concluded that in bronchial epithelial cells, inhibition of granulocyte-macrophage colony-stimulating factor synthesis by formoterol and salmeterol does not act via previously demonstrated glucocorticoid receptor-related mechanisms, suggesting an alternative pathway in these cells.

Primary cytomegalovirus infection in an outpatient setting--laboratory markers and clinical aspects.

BACKGROUND: Occasionally, primary cytomegalovirus (CMV) infection may give rise to more or less severe clinical illness in immunocompetent adults. We retrospectively analyzed cases of acute CMV infection in medical outpatients. PATIENTS AND METHODS: Over a 6-year period, we identified 22 patients with a febrile illness and hepatitis suffering from primary CMV infection. This was diagnosed on the basis of a strongly positive CMV IgM antibody test result and/or CMV IgG seroconversion. Clinical features as well as relevant laboratory results were analyzed. We also tested available samples for CMV glycoprotein B-specific antibodies and CMV IgG avidity and analyzed results of Epstein-Barr virus (EBV)-specific antibody assays. In addition, current age-specific CMV IgG seroprevalence rates were determined using 9,870 routine patient samples. RESULTS: At presentation, all patients complained of malaise and fever higher than 38 degrees C, and many also complained of cephalgia. Most patients who underwent abdominal ultrasonography had an enlargement of the spleen. Most patients had a relative lymphocytosis but only three had a mild leukocytosis. C-reactive protein was only slightly elevated in 13 patients; all 22 patients had elevated levels of alanine aminotransferase (ALT) and lactate dehydrogenase (LDH). Half the patients reported travel to areas outside western Europe, mostly to tropical and subtropical areas, within 3 weeks before onset of illness. Primary CMV infection was confirmed by negative anti-gB antibody test results and the absence of high-avidity CMV antibodies. In contrast, despite past EBV infection demonstrated by positive anti-EBNA-1 results, 15 out of 21 patients tested for EBV markers had positive or nonspecific IgM test results. The overall CMV IgG seroprevalence rate in the routine samples was 64.4%, with marked age-dependent increases. CONCLUSION: CMV is a relevant differential diagnosis in feverish illnesses accompanied by hepatitis in otherwise healthy adults, about 40% of whom are CMV-naïve. Half our patients seem to have acquired their CMV infection abroad, so that a diagnosis of CMV infection needs to be taken into account in travelers, in addition to infectious illnesses more commonly considered in this context, such as dengue or hepatitis A. For diagnosis, both CMV and EBV antibody studies should be performed and the inclusion of assays able to demonstrate past infection is helpful for achieving a definite diagnosis.

Aortopexy in esophageal atresia: long-term experience of a parent support group.

PURPOSE: The aim of this study was to obtain the parents' view of aortopexy after esophageal atresia. METHODS: A questionnaire was completed by the parents of 24 former patients, now aged from 1 to 15 years (average, 8.9; median, 9.0 years). The respondents were all members of a support group. RESULTS: The median age of the patients receiving aortopexy was 4 months. The 24 procedures were performed in 16 different hospitals. The subjects had experienced a median of 3 apneic attacks. Technical complications occurred in 4 of the 24 children. In 71%, aortopexy was an immediate success. CONCLUSION: Despite its low success rate compared with centers with large cumulative experience, 90% of parents were satisfied with the result.

Cytokine-induced activation of nuclear factor-kappa B is inhibited by hydrogen peroxide through oxidative inactivation of IkappaB kinase.

Rapid activation of the IkappaB kinase (IKK) complex is considered an obligatory step in the activation of nuclear factor-kappaB (NF-kappaB) in response to diverse stimuli. Since oxidants have been implicated in the regulation of NF-kappaB, the focus of the present study was the activation of IKK by tumor necrosis factor alpha (TNFalpha) in the presence or absence of hydrogen peroxide (H(2)O(2)). Exposure of mouse alveolar epithelial cells to H(2)O(2) was not sufficient to activate IKK, degrade IkappaBalpha, or activate NF-kappaB. In contrast, TNFalpha induced IKK activity rapidly and transiently resulting in IkappaBalpha degradation and NF-kappaB activation. Importantly, in the presence of H(2)O(2), the ability of TNFalpha to induce IKK activity was markedly decreased and resulted in prevention of IkappaBalpha degradation and NF-kappaB activation. Neither tyrosine kinases nor phosphatidylinositol 3-kinases, known regulators of NF-kappaB by oxidants, were involved in IKK inhibition by H(2)O(2). Direct addition of H(2)O(2) to the immunoprecipitated IKK complex inhibited enzyme activity. Inhibition of IKK activity by H(2)O(2) was associated with direct oxidation of cysteine residues present in the IKK complex and occurred only in enzymatically active IKK. In contrast to previously published observations, our findings demonstrate that the oxidant H(2)O(2) reduces NF-kappaB activation by inhibiting activated IKK activity.

Effects of formoterol and budesonide on GM-CSF and IL-8 secretion by triggered human bronchial epithelial cells.

The effect of formoterol, alone and in combination with budesonide, upon tumour necrosis factor-alpha stimulated (10 ng x mL(-1)) human bronchial epithelial cells was investigated. Addition of formoterol (> or = 10(-10) M) reduced granulocyte macrophage-colony stimulating factor (GM-CSF) levels, as assessed by enzyme-linked immunosorbent assay, by 40-50% and increased interleukin (IL)-8 levels by approximately 50%. The effects of formoterol were long lasting (23 h). Budesonide (10(-8) M) reduced the amounts of both cytokines (GM-CSF and IL-8) by 40%. Simultaneous addition of formoterol and budesonide reduced GM-CSF levels approximately 75%, while IL-8 levels were decreased approximately 40%, similar to the reduction obtained with budesonide alone. The glucocorticoid receptor (GR) antagonist RU486 did not influence the effect of formoterol, suggesting no involvement of the GR. Formoterol rapidly induced an elevation in intracellular cyclic adenosine monophosphate, which was reduced in the presence of propranolol. In addition, the alterations in cytokine secretion induced by formoterol could be fully blocked by propranolol, demonstrating that these effects are beta2-receptor mediated. In conclusion, the combination of budesonide and formoterol reduces the secretion of granulocyte macrophage-colony stimulating factor to basal levels and counteracts the capacity of formoterol alone to induce interleukin-8 production, modulations which may facilitate improved asthma control.

Experiences of a parent support group with the long-term consequences of esophageal atresia.

PURPOSE: The aim of this study was to study long-term effects of esophageal atresia based on data from the world's largest parent support group. METHODS: A questionnaire was completed by 128 former patients, now aged 10 to 34 years (median, 14 years), who were all members of the support group. RESULTS: Postoperative bougienage was performed in 70% of patients. The most frequently associated anomalies were skeletal (44%), with 18% of patients having a winged scapula. In 30% of patients, food became trapped; this entrapment continues, although with decreasing frequency. In 38% of patients, recurrent episodes of food lodging in the esophagus lasted only a few years, whereas 32% of patients never experienced any swallowing problems. The foods most often trapped were apples, meat, and bread. Most trapped food is removed by the induction of vomiting, without medical help. Unusual findings of this study were the avoidance of chocolate and sweets by 9% of patients and the habit of eating unorthodox food combinations in 13% of patients. Forty-six percent of patients have gastroesophageal reflux, and 7% have Barrett's esophagus. The most successful antireflux prevention was abstinence from eating late in the evening. Fundoplication was performed in 16% of patients; however, only 25% of these fundoplication operations were successful immediately. Eighty percent of patients had more than 4 lung infections or bronchitis episodes per year. Forty-four percent make noises, such as wheezing or whistling, when breathing or straining. Eighty-eight percent are of normal height, and 70% are of normal weight. Eighty-nine percent of patients attended regular schools, and 52% were above-average students. CONCLUSIONS: Very few pediatric surgeons have had the opportunity to follow into adulthood such a large group of patients with esophageal atresia. Patients themselves feel lost when they undergo the transition from pediatric to adult medical treatment. A support group is able to provide an information database, which helps overcome the isolation because of the rareness of the disease and the separation of patients into different medical specialties. Surgeons should be aware of the difficulties that patients and parents face in later life and should seek cooperation with a support group. J Pediatr Surg 36:605-610.

Modulation of N-type Ca2+ channels by intracellular pH in chick sensory neurons.

Both physiological and pathological neuronal events, many of which elevate intracellular [Ca2+], can produce changes in intracellular pH of between 0.15 and 0.5 U, between pH 7.4 and 6.8. N-type Ca2+ channels, which are intimately involved in exocytosis and other excitable cell processes, are sensitive to intracellular pH changes. However, the pH range over which N-type Ca2+ channels are sensitive, and the sensitivity of N-type Ca2+ channels to small changes in intracellular pH, are unknown. We studied the influence of intracellular pH changes on N-type calcium channel currents in dorsal root ganglion neurons, acutely isolated from 14-day-old chick embryos. Intracellular pH was monitored in patch-clamp recordings with the fluorescent dye, BCECF, and manipulated in both the acidic and basic direction by extracellular application of NH4+ in the presence and absence of intracellular NH4+. Changes in intracellular pH between 6.6 and 7.5 produced a graded change in Ca2+ current magnitude with no apparent shift in activation potential. Intracellular acidification from pH 7.3 to 7.0 reversibly inhibited Ca2+ currents by 40%. Acidification from pH 7.3 to pH 6.6 reversibly inhibited Ca2+ currents by 65%. Alkalinization from pH 7.3 to 7.5 potentiated Ca2+ currents by approximately 40%. Channels were sensitive to pHi changes with high intracellular concentrations of the Ca2+ chelator, bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid, which indicates that the effects of pHi did not involve a Ca2+-dependent mechanism. These data indicate that N-type Ca2+ channel currents are extremely sensitive to small changes in pHi in the range produced by both physiological and pathological events. Furthermore, these data suggest that modulation of N-type Ca2+ channels by pHi may play an important role in physiological processes that produce small changes in pHi and a protective role in pathological mechanisms that produce larger changes in pHi.

Glucocorticoid receptor mRNA levels in bronchial epithelial cells of patients with COPD: influence of glucocorticoids.

Glucocorticoids (gcs) are known to be effective in the treatment of asthma. In chronic obstructive pulmonary disease (COPD), however, no beneficial effects are demonstrated in most patients. Hypothetically, this may be explained by an overexpressed beta-glucocorticoid receptor (GR) compared to the alpha-GR. The aim of this study was to investigate alpha- and beta-GR mRNA levels and ratios in patients with COPD with or without glucocorticoid treatment. GR and, as a control, metallothionein (MT) 2 mRNA levels were compared between patients with COPD receiving glucocorticoids (COPD + gcs), glucocorticoid naive COPD-patients (COPD - gcs) and non-COPD control patients not using gcs. Bronchoscopy was performed and bronchial epithelial cells were sampled with brushing. Smoking did not influence alpha- and beta-GR levels and ratios, nor the MT2 mRNA expression level. The alpha-GR mRNA expression was lower in the COPD - gcs group than in controls. Both GR forms were higher in the COPD + gcs patients than in the COPD - gcs patients, but not different from the levels measured in the controls. alpha 1/beta-GR mRNA ratios did not differ between the groups and averaged 1.7, suggesting no inhibitory effect of the beta-GR on the alpha 1 form. MT2 levels were upregulated in the COPD + gcs patients as compared to the COPD - gcs group, indicating a pharmacological glucocorticoid effect. In the present study it is demonstrated that basal GR mRNA levels are lower in patients with COPD. Although this needs to be investigated further, this might explain, in part, the non-responsiveness of patients with COPD to gcs.

Interaction between glucocorticoids and beta2-agonists: alpha and beta glucocorticoid-receptor mRNA expression in human bronchial epithelial cells.

Recent studies have suggested that regular use of beta2-agonists has adverse effects on asthma control, due to the cross-talk between cAMP responsive element binding proteins (CREB) and glucocorticoid receptors (GR). The aim of this study was to investigate the interaction between GR and CREB on cytoplasmic protein level with a gel mobility shift assay and to determine the effect of this interaction on mRNA levels by Northern blot analysis. After exposing human bronchial epithelial cells for 1 hr to either 1 microM terbutaline or budesonide, more binding of CREB and GR, respectively, was observed to their responsive elements in DNA. Simultaneous exposure to terbutaline and budesonide also increased the binding of CREB and GR to DNA. After 4 hr, both alpha and beta GR mRNAs were down-regulated by 1 microM budesonide. Simultaneous addition of 1 microM terbutaline prevented this down-regulation. Adding 100 times more budesonide compared to terbutaline again down-regulated both GR forms, although significantly less compared to the down-regulation induced by 1 microM budesonide alone. Addition of terbutaline to cells already exposed to budesonide did not reverse the GR mRNA expression within 44 hr. Similar results were obtained with metallothionein-2 (MT2) mRNA levels. In conclusion, beta2-agonists interfere with the GR function in human bronchial epithelial cells when given simultaneously, with this being overcome by sequential exposure of the cells to first glucocorticoids and later beta2-agonists.

The fate of self-reactive B cells depends primarily on the degree of antigen receptor engagement and availability of T cell help.

Self-reactive B cells from tolerant double-transgenic (Dbl-Tg) mice coexpressing hen egg lysozyme (HEL) and rearranged anti-HEL immunoglobulin genes have a relatively short life span when compared to normal B cells, irrespective of whether they are exposed to antigen in multivalent membrane-bound form (mHEL-Dbl-Tg mice) or soluble form (sHEL-Dbl-Tg mice). The factors responsible for determining the fate of these B cells after encounter with self-antigen were investigated using a cell-tracking technique in which anti-HEL Ig-Tg spleen cells were labeled with the intracellular dye 5-carboxyfluorescein diacetate-succinimidyl ester (CFSE) and injected either into non-Tg recipients or a variety of HEL-Tg hosts. In non-Tg recipients, HEL-binding B cells persisted in the circulation and could be detected in the follicles of the spleen for at least 5 d. On transfer into either mHEL-Tg or sHEL-Tg hosts, they underwent activation and then rapidly disappeared from the blood and spleen over the next 3 d, consistent with the short life span reported previously. Immunohistology of spleens from sHEL-Tg recipients indicated that the transferred B cells had migrated to the outer margins of the periarteriolar lymphoid sheath (PALS), where they were detectable for 24 h before being lost. The positioning of B cells in the outer PALS depended on a critical threshold of Ig receptor binding corresponding to a serum HEL concentration between 0.5 and 15 ng/ml, but was not restricted to endogenously expressed HEL in that the same migratory pattern was observed after transfer into non-Tg recipients given exogenous (foreign) HEL. Moreover, bone marrow-derived immature Ig-Tg B cells homed to the outer PALS of sHEL-Tg mice and then disappeared at the same rate as mature B cells, indicating that the stage of maturation did not influence the fate of self-reactive B cells in a tolerant environment. On the other hand, HEL-binding B cells transferred into sHEL-Dbl-Tg recipients persisted over the 3-d period of study, apparently due to insufficient availability of antigen, as indicated by the fact that the degree of Ig receptor downregulation on the transferred B cells was much less than in sHEL-Tg recipients. If T cell help was provided to Ig-Tg B cells at the time of transfer into sHEL-Tg recipients in the form of preactivated CD4+ T cells specific for major histocompatibility complex-peptide complexes on the B cell surface, HEL-binding B cells migrated through the outer PALS of the spleen to the follicle, where they formed germinal centers, or to adjacent red pulp, where they formed proliferative foci and secreted significant amounts of anti-HEL antibody. Taken together, these results indicated that the outcome of the interaction between self-antigen and B cells is largely determined by a combination of the degree of receptor engagement and availability of T cell help.

Endotracheal tube obstruction during stereotactic craniotomy.

The authors present a case of endotracheal tube (ETT) obstruction during stereotactic computer-assisted volumetric resection of an enlarging left thalamic mass in a prone patient. The head was fixed in a stereotactic COMPASS head frame and the patient positioned on transverse hip and shoulder rolls. The prone position was achieved with attention given to ensure an adequate distance between the sternum and mentum. At the time of dural incision, the patient's peak airway pressure and end-tidal CO2 tension increased markedly whereas the O2 saturation remained stable at 99%. It immediately became clear that the patient's torso had moved cephalad resulting in ETT obstruction, despite the continued integrity of head fixation. We emphasize the need for proper and safe patient positioning, the placement of a bite block between the molar teeth, and particularly in obese patients, the need to tape the patient's torso securely to the operating room table. In addition, it may be necessary to place the operating room table in reverse Trendelenberg position to prevent cephalad movement of the torso. This case represents an unusual mechanism of ETT obstruction, as normally the latter is caused by flexion of the head rather than by cephalad movement of the torso.

K-ras point mutations in routinely processed tissues: non-radioactive screening by single strand conformational polymorphism analysis.

AIMS: To develop a non-radioactive method to screen routinely fixed, paraffin wax embedded specimens for the occurrence of point mutations; to evaluate the single strand conformational polymorphism (SSCP) analysis technique for the detection of K-ras point mutations as a result of electrophoretic mobility shifts. METHODS: DNA was extracted from archival specimens of colon cancer and from established colon cancer cell lines with known point mutations. A K-ras gene fragment containing codons 12 and 13 of exon 1 was amplified with the polymerase chain reaction (PCR). Denatured DNA fragments were run on 10% polyacrylamide gels under non-denaturing conditions. After electrophoresis DNA was blotted and the single stranded DNA was detected using a digoxigenin labelled ras probe. The nature of the detected point mutations was identified and confirmed by sequencing and hybridisation with oligonucleotides using 32P labelling. RESULTS: Wild type and aberrant alleles were detected caused by mobility shifts after electrophoresis of the PCR products. Commonly occurring mutations in the K-ras gene--in the first two positions of codon 12--could easily be detected in DNA from archival paraffin wax embedded colon cancer tissue. In all the colon tumour samples studied wild type gene alleles were also found, presumably derived from normal cells in the specimen. CONCLUSIONS: The SSCP method permits rapid non-radioactive screening of adenomas or carcinomas for the occurrence of point mutations in the K-ras gene. But if a mutation is detected by an electrophoretic mobility shift, its identification requires confirmation by sequencing or oligonucleotide hybridisation.

A [Na+]o-independent, pHo-dependent mechanism for reduction of intracellular [Ca2+] after influx through Ca2+ channels in mouse pituitary cells.

The effect of extracellular pH (pHo) on the duration of calcium-dependent chloride currents (ICl(Ca] was studied in voltage clamped AtT-20 pituitary cells. ICl(Ca) was activated by Ca2+ influx through plasma membrane Ca2+ channels, which were opened by step depolarization to voltages between -20 and +60 mV. Increasing pHo from 7.3 to 8.0 reversibly prolonged ICl(Ca) tail currents in perforated patch recordings from cells bathed in both Na(+)-containing and Na(+)-free solutions. This prolongation was prevented in standard whole cell recordings when the pipette solution contained 0.5 mM EGTA. The effects of raised pHo were not due to alteration of intracellular pH, since tail current prolongation still occurred when intracellular pH was buffered at 7.3 with 80 mM HEPES. The prolongation of ICl(Ca) at pHo 8 could not be accounted for by a direct action on Ca2+ channels, since tail currents were prolonged when pHo was changed rapidly during the tail current, after all Ca2+ channels were closed. The effects of increasing pHo on ICl(Ca) also could not be explained by a direct action on Cl- channels, since changing to pHo 8 did not prolong Cl- tail currents when intracellular Ca2+ concentration [( Ca2+]i) was fixed by EGTA in whole cell recordings. Raising pHo did, however, prolong depolarization-evoked [Ca2+]i transients, measured directly with the Ca2+ indicator dye, fura-2. Taken together, these data demonstrate the presence of a Na(+)-independent, pHo-sensitive mechanism for reduction of [Ca2+]i after influx through Ca2+ channels. This mechanism is associated with the plasma membrane, and is active on a time scale that is relevant to the duration of single action potentials in these cells. We suggest that this mechanism is the plasma membrane Ca2+ ATPase.