Relapse Prevention in Major Depressive Disorder After Successful Acute Electroconvulsive Treatment: a 6-month Double-blind Comparison of Three Fixed Dosages of Escitalopram and a Fixed Dose of Nortriptyline - Lessons from a Failed Randomised Trial of the Danish University Antidepressant Group (DUAG-7).

INTRODUCTION: Electroconvulsive treatment (ECT) is an effective treatment for severe depression but carries a risk of relapse in the following months. METHODS: Major depressive disorder patients in a current episode attaining remission from ECT (17-item Hamilton Depression Rating Scale (HAM-D17) score≤9) received randomly escitalopram 10 mg, 20 mg, 30 mg or nortriptyline 100 mg as monotherapies and were followed for 6 months in a multicentre double-blind set-up. Primary endpoint was relapse (HAM-D17≥16). RESULTS: As inclusion rate was low the study was prematurely stopped with only 47 patients randomised (20% of the planned sample size). No statistically significant between-group differences could be detected. When all patients receiving escitalopram were compared with those receiving nortriptyline, a marginal superiority of nortriptyline was found (p=0.08). One third of patients relapsed during the study period, and one third completed. DISCUSSION: Due to small sample size, no valid efficacy inferences could be made. The outcome was poor, probably due to tapering off of non-study psychotropic drugs after randomisation; this has implications for future study designs. ClinicalTrials.gov Identifier: NCT00660062.

Deep muscle-proteomic analysis of freeze-dried human muscle biopsies reveals fiber type-specific adaptations to exercise training.

Skeletal muscle conveys several of the health-promoting effects of exercise; yet the underlying mechanisms are not fully elucidated. Studying skeletal muscle is challenging due to its different fiber types and the presence of non-muscle cells. This can be circumvented by isolation of single muscle fibers. Here, we develop a workflow enabling proteomics analysis of pools of isolated muscle fibers from freeze-dried human muscle biopsies. We identify more than 4000 proteins in slow- and fast-twitch muscle fibers. Exercise training alters expression of 237 and 172 proteins in slow- and fast-twitch muscle fibers, respectively. Interestingly, expression levels of secreted proteins and proteins involved in transcription, mitochondrial metabolism, Ca2+ signaling, and fat and glucose metabolism adapts to training in a fiber type-specific manner. Our data provide a resource to elucidate molecular mechanisms underlying muscle function and health, and our workflow allows fiber type-specific proteomic analyses of snap-frozen non-embedded human muscle biopsies.

Do we agree about when patients are psychotic?

OBJECTIVE: To investigate into the use of the term 'psychotic' as defined by ICD-10 or by the concept of impaired reality testing, among psychiatric staff members. METHOD: Questionnaire investigation using 11 short case vignettes. RESULTS: Responses were received from 266 psychiatric staff members: psychiatrists, nursing staff and psychologists. When using ICD-10, patients were identified as psychotic with a sensitivity ranging from 90% to 55%. Specificity ranged from 60% to 75%. According to the concept of impaired reality testing, all three groups showed a sensitivity of about 60%, whereas specificity ranged from 65% to 50%. The combined use of the terms correlated significantly with responses regarding indication for legal detention for psychiatrists and nursing staff. CONCLUSION: In identifying a patient as 'psychotic' a broad concept of impaired reality testing was widely used particularly in cases with legal issues. Psychotic symptoms, however, were identified with high sensitivity and specificity.

DNA-binding proteins in Yoshida ascites tumor fluid.

DNA-binding proteins were isolated from Yoshida ascites tumor fluid by chromatography on DNA-cellulose. This fraction represents 1-2% of the total ascites protein. Most of the DNA-binding proteins will bind to phosphocellulose as well. The proteins migrate by agarose gel electrophoresis at pH 8.6 as alpha and beta globulins. Quantitative immunoelectrophoresis revealed the presence of 12-18 proteins. SDS-polyacrylamide electrophoresis indicated molecular weights ranging from 3-10(4) to 10(6). Seven of the proteins were identified by specific immunoprecipitation as beta1-Eglobulin, beta2-glycoprotein I, fibrinogen split product E (fibrinogen E), coagulation factor XIII (factor XIII), alpha2-macroglobulin, IgG and IgM. Alpha1-antichymotrypsin might also be represented. In nuclear extracts of the tumor cells only factor XIII was present. With the exception of fibrinogen E and P5 all recognized DNA-binding proteins are present in normal rat plasma. With increasing tumor age the concentration of fibrinogen E, factor XIII, P5 and IgM increased both in ascites fluid and in plasma, while the concentration of other DNA-binding-proteins decreased or remained constant. Evidence is presented that the DNA- and phosphocellulose binding ascites protein fraction inhibit tumor cell growth. No inhibition was induced by corresponding protein fractions isolated from normal rat plasma.

Altered modulation of prefrontal and subcortical brain activity in newly diagnosed schizophrenia and schizophreniform disorder. A regional cerebral blood flow study.

To measure prefrontal and subcortical activity during a cognitive task, we examined 19 newly diagnosed schizophrenics and patients with schizophreniform psychosis. Seven healthy volunteers served as controls. The patients were drug naive or had received neuroleptics for a few days only. Cerebral blood flow distribution was depicted by single photon emission computed tomography at rest and during activation with the Wisconsin Card Sorting Test. A significant relative activation deficit in the left inferior-prefrontal region was revealed during the Wisconsin Card Sorting Test in the patient group. Furthermore, the patients had impaired striatal suppression on the left side during the cognitive task. The test performance was significantly impaired in the patients. The inability to reduce striatal activity may be due to a lack of corticostriatal feedback during prefrontal activation.

Flow cytometric DNA analysis of breast cancers with predominance of carcinoma in situ: a comparison of the premalignant and malignant components.

Flow cytometric DNA analysis was performed on unfixed frozen tissue samples from 48 cases of invasive breast cancer (IC) with a predominance of ductal carcinoma in situ (DCIS). In 15 cases the samples contained only the DCIS component, in 17 cases only the IC component, whereas in 16 cases separate samples from the DCIS as well as the IC part within the individual lesion were available. In the latter 16 cases, complete or partial accordance in DNA ploidy between DCIS and IC was found in 12 cases, whereas no correspondence could be demonstrated in the remaining 4 cases, possibly due to intratumoral DNA heterogeneity. Comparison of the DNA index distribution in samples of DCIS and IC from the 48 cases showed concordant results except for the DNA hyperdiploid subclass, in which 6 clones were found in the DCIS portion compared to 18 clones in the IC portion. S-phase fractions were also comparable in the two groups. A comparison of the DCIS component from the present series of breast cancers to our previous series of pure DCIS also showed similar results with respect to the DNA index distribution, DNA heterogeneity, and S-phase fraction. No differences could be demonstrated between DCIS with and without invasion. The results indicate that the DNA ploidy pattern of breast cancer, as detected by flow cytometric DNA analysis, is established at the preinvasive stage of carcinogenesis.

T-lymphocyte clones initiated from lesional psoriatic skin release growth factors that induce keratinocyte proliferation.

To investigate whether growth factors derived from T cells in psoriatic lesions are able to stimulate keratinocyte growth, T-cell lines were initiated from lesional psoriasis skin and cloned by limiting dilution. Eight clones with good proliferative capacity out of 40 clones from one patient were stimulated. After 24 h, the conditioned medium was harvested and the growth modulatory effect of the conditioned medium on keratinocytes was assessed. Seven of the eight T-cell clones stimulated keratinocyte growth to an extent ranging from 22% +/- 19 to 64% +/- 9 (mean +/- SD of three experiments) of maximal inducible keratinocyte growth, and one T-cell clone had no effect (-5% +/- 2) on keratinocyte growth. Keratinocyte growth was also induced by T-cell clones obtained from two other patients. Several cytokines were tested in this system to determine which T-cell growth factor may induce the keratinocyte growth. None of the cytokines interferon-g, transforming growth factor-beta, interleukin (IL)-2, IL-3, IL-4, IL-6, IL-8, or granulocyte-macrophage colony stimulating factor alone was found to possibly be responsible for the T-cell-induced keratinocyte growth. Thus the nature of the T-cell keratinocyte growth-promoting stimulus remains to be elucidated.

Leukemic T cells from patients with cutaneous T-cell lymphoma demonstrate enhanced activation through CDw60, CD2, and CD28 relative to activation through the T-cell antigen receptor complex.

Antigen-dependent activation of T cells occurs through the T-cell antigen-receptor complex (TCR/CD3). Antigen-independent T-cell activation may occur through the surface molecules CDw60, CD2, and CD28. We wished to determine whether these antigen-independent T-cell-activation pathways could be involved in proliferation of leukemic T cells from patients with cutaneous T-cell lymphoma (CTCL). Whereas CDw60 was only expressed on 28% +/- 7% (mean +/- SEM) of blood T cells obtained from healthy control subjects (n = 4), CDw60 was expressed on 94% +/- 3% of blood T cells obtained from patients with CTCL (n = 4). Dual color immunofluorescence microscopy of the T-cell infiltrate in involved skin of these patients demonstrated that almost 100% of the T cells expressed CDw60. Not only did T cells in the patients with CTCL express CDw60, but triggering of the T cells with anti-CDw60 resulted in enhanced proliferation relative to anti-TCR/CD3 and mitogenic lectins. Other antigen-independent pathways also appeared highly active in the T cells from patients with CTCL because enhanced proliferation relative to anti-TCR/CD3 or mitogenic lectins was found when anti-CD2 or anti-CD28 plus phorbol ester was used as stimulant. Despite the brisk proliferation induced by anti-CDw60, anti-CD2, or anti-CD28, T cells from the patients did not produce detectable amounts of gamma-interferon. The inability to produce gamma-interferon correlates with our finding of absent (n = 3) or weak (n = 1) intercellular adhesion molecule-1 expression in the lesional keratinocytes in these patients. In conclusion, T cells of patients with CTCL demonstrate elevated expression of a T-cell-independent signaling molecule CDw60 and respond to antigen-independent activating signals.

Flow cytometric deoxyribonucleic acid analysis of granulosa cells aspirated from human ovarian follicles. A new method to distinguish healthy and atretic ovarian follicles.

Granulosa cell aspirates from human ovarian follicles were analyzed by flow cytometry to determine the fraction of cells in the DNA S-phase of the mitotic cell cycle. The aim of the study was to evaluate if the percentage of granulosa cells in S-phase (the S-fraction) could be used to indicate whether a follicle was healthy or atretic. A highly significant relationship was found between the S-fraction and the concentration of estradiol in the follicular fluid (r = 0.6, P less than 0.001). More than 85% of the follicles having an S-fraction of 16% or greater contained intrafollicular levels of estradiol equal to or greater than 200 ng/ml and had a low androstenedione:estradiol ratio. Conversely, 95% or more of the follicles that had an S-fraction of less than 16% contained low estradiol (less than 200 ng/ml) and had a high androstenedione to estradiol ratio. We conclude that flow cytometric DNA measurements on follicular aspirates provide a reliable and rapid method by which to distinguish healthy and atretic ovarian follicles. Since only a small fraction (less than 5%) of an entire granulosa cell population is required for S-phase analysis, the technique allows the majority of cells to be immediately available or other biochemical studies. Moreover, since excision of ovarian tissue is avoided, the technique may be acceptable for studies on women with normal ovarian function but who are undergoing laparotomy or laparoscopy for some reason.

Cell cycle perturbation by sodium butyrate in tumorigenic and non-tumorigenic human urothelial cell lines assessed by flow cytometric bromodeoxyuridine/DNA analysis.

The effect of sodium butyrate on cell proliferation was studied in eight human urothelial cell lines differing in transformation grade (TGr): Hu 1752 (mortal, TGr I); HCV29 (immortal and tumorigenic, TGr II); HCV29T, T24, T24A, T24B, Hu 961A and Hu 1703He (tumorigenic, TGr III). In all cell lines, except Hu 1752, addition of 4 mM sodium butyrate at 18 h after replating resulted in a significantly decreased population of adherent cells after a further 24-48 h. This might partially be explained by detachment of cells, probably mainly S phase cells, from the substrate in the lines HCV29, HCV 29T, Hu 961A and Hu 1703He. Flow cytometric DNA analysis of the adherent cell population showed that all TGr II and III urothelial cell lines were DNA aneuploid, and that butyrate perturbed the cell cycle distribution in these cell lines, mainly by a decrease of the S phase fraction. Flow cytometric bromodeoxyuridine (BrdUrd)/DNA analysis of continuously BrdUrd labelled cultures, using a 'washless' BrdUrd/DNA staining technique, showed that butyrate inhibited the G0/1-S phase transition, indicated by a delayed depletion of BrdUrd negative G0/1 cells in the cell lines HCV29, HCV29T, T24B, Hu 961A and Hu 1703He. BrdUrd/DNA analysis further showed that butyrate inhibited the G2M-G0/1 phase transition, indicated by a pronounced accumulation of BrdUrd positive G2M cells in the cell lines HCV 29T, T24B, Hu 961A and Hu 1703He. Microscopy of HCV29T and Hu 961A cells indicated that this block did not occur in mitosis. The parental cell line T24 and the cell line T24A did not show an accumulation of BrdUrd negative G0/1 cells or BrdUrd positive G2M cells like that occurring in the derived cell line T24B.

Circadian variation of DNA replication in hamster cheek pouch epithelium analysed by tritiated thymidine labelling, flow sorting and autoradiography: no resting S phase cells in the normal epithelium.

In the normal hamster cheek pouch epithelium, cell proliferation takes place with a pronounced circadian rhythm. We tested our previous hypothesis that all cells having S phase DNA content are actively synthesizing DNA and thus participating in the daily cohort of proliferating cells. We found no evidence of resting S phase cells in the normal epithelium. Using labelling with tritiated thymidine followed by fluorescence activated cell sorting according to DNA content and by autoradiography of the sorted nuclei, it was demonstrated that during the 24 h period almost all cells with mid S phase DNA content were active in DNA synthesis.

Immediate bromodeoxyuridine labelling of unseparated human bone marrow cells ex vivo is superior to labelling after routine laboratory processing.

It is important to evaluate the proliferation of bone marrow cells in several disease conditions and during treatment of patients with for example cytokines. Labelling with bromodeoxyuridine (BrdUrd), immunocytochemical staining with anti-BrdUrd antibody and analysis by flow cytometry provides a reliable and reproducible technique for estimation of the fraction of cells that incorporated BrdUrd into DNA during S-phase. We have compared immediate BrdUrd labelling of unseparated bone marrow cells with the previously used labelling in the laboratory after routine separation of the mononuclear cells. Bone marrow aspirates from seven lymphoma patients without bone marrow involvement were studied with these two methods. We found higher BrdUrd labelling indices (LI) in the mononuclear cells, when cells were labelled immediately. A large variation in LI was found between patients. Our results suggest that ex vivo BrdUrd labelling of bone marrow cells should be performed immediately after aspiration and before separation, because these data are closer to values reported from in vivo labelling with BrdUrd.

Increased cellular hypoxia and reduced proliferation of both normal and leukaemic cells during progression of acute myeloid leukaemia in rats.

The microenvironmental changes in the bone marrow, spleen and liver during progression of the transplantable promyelocytic leukaemia in the Brown Norwegian rat (BNML) have been studied. We used flow cytometry to estimate cellular hypoxia and proliferation based on in vivo pulse-labelling with a mixture of 2-nitroimidazole linked to theophylline (NITP) and bromodeoxyuridine (BrdUrd). The leukaemic cells were identified with the RM124 antibody. In rats inoculated with leukaemic cells the fraction of RM124+ cells was significantly increased from day 20 onwards in the spleen and from day 27 in the bone marrow and liver, reaching a level of 65-87% in these organs at day 32. At day 32, the NITP+ fraction of RM124+ cells had increased significantly in the bone marrow and spleen to 88% and 90%, respectively. The corresponding fractions of NITP+ normal cells reached 63% and 65%, respectively. From day 13 to day 32, the DNA-synthesizing (BrdUrd+) fraction of RM124+ cells in the bone marrow decreased significantly from 52% to 25%, and of normal cells from about 20% to 6%. In the bone marrow and spleen at day 27 and 32, the S-phase and G2/M-phase fractions according to DNA content were higher for the NITP+ than for the NITP- cells. This could partly be explained by an impaired cell cycle progression due to hypoxia. Nevertheless, we found indications of leukaemic cells that were simultaneously labelled with NITP and BrdUrd, in the bone marrow and spleen. These latter findings suggest that in contrast to normal cells some of the leukaemic cells can proliferate even during hypoxia, and this subpopulation may consequently renew and expand the leukaemic cell load.

Cloning and sequencing of a cDNA encoding the canine HSP27 protein.

The nucleotide (nt) sequence encoding a 27-kDa heat-shock protein (HSP27) was determined from cDNAs cloned from a canine smooth muscle library. The primary structure deduced from the nt sequence reveals a 209-amino-acid protein having 86-89% identity with human, mouse, rat and hamster small HSP. Similar to human HSP27, the canine protein contains three Ser residues that are potential MAPKAP kinase II substrates.

Flowcytometric evaluation of the DNA distribution in isolated pancreatic islets from normal and diabetic mice.

Pancreatic islets from C57BL/KsJ-db/db, and +/+ mice were isolated by collagenase. After isolation the islets were transferred to a hypotonic ethidiumbromide solution with 0.3% of the detergent Nonidet P40. After vortexing, the samples were analyzed in a BioPhysics Cytofluorograf 4802A. The DNA histograms were divided into 2c, 2-4c, 4c and 8c fractions under the assumption of a constant rate of DNA synthesis during the 2-4c phase. In comparison with normal mice, we found that diabetic mice had a lower fraction of 2c nuclei and a higher fraction of 2-4c, 4c, and 8c nuclei. These results obtained by flow cytometry are in agreement with results obtained by 3HTdR incorporation and by cytophotometry on histological sections.

In B-cell chronic lymphocytic leukaemia chromosome 17 abnormalities and not trisomy 12 are the single most important cytogenetic abnormalities for the prognosis: a cytogenetic and immunophenotypic study of 480 unselected newly diagnosed patients.

Of 560 consecutive, newly diagnosed untreated patients with B CLL submitted for chromosome study, G-banded karyotypes could be obtained in 480 cases (86%). Of these, 345 (72%) had normal karyotypes and 135 (28%) had clonal chromosome abnormalities: trisomy 12 (+12) was found in 40 cases, 20 as +12 alone (+12single), 20 as +12 with additional abnormalities (+12complex). Other frequent findings included abnormalities of 14q, chromosome 17, 13q and 6q. The immunophenotype was typical for CLL in 358 patients (CD5+, Slg(weak), mainly FMC7-) and atypical for CLL in 122 patients (25%) (CD5-, or Slg(strong) or FMC7+). Chromosome abnormalities were found significantly more often in patients with atypical (48%) than in patients with typical CLL phenotype (22%) (P < 0.00005). Also +12complex, 14q+, del6q, and abnormalities of chromosome 17 were significantly more frequent in patients with atypical CLL phenotype, whereas +12single was found equally often in patients with typical and atypical CLL phenotype. The cytomorphology of most of the +12 patients was that of classical CLL irrespective of phenotype. In univariate survival analysis the following cytogenetic findings were significantly correlated to a poor prognosis: chromosome 17 abnormalities, 14q+, an abnormal karyotype, +12complex, more than one cytogenetic event, and the relative number of abnormal mitoses. In multivariate survival analysis chromosome 17 abnormalities were the only cytogenetic findings with independent prognostic value irrespective of immunophenotype. We conclude that in patients with typical CLL immunophenotype, chromosome abnormalities are somewhat less frequent at the time of diagnosis than hitherto believed. +12single is compatible with classical CLL, and has no prognostic influence whereas chromosome 17 abnormalities signify a poor prognosis. In patients with an atypical CLL immunophenotype, chromosome abnormalities including +12complex, 14q+, del 6q and chromosome 17 are found in about 50% of the patients, and in particular chromosome 17 abnormalities suggest a poor prognosis.

Relationship between mood and TSH response to TRH stimulation in bipolar affective disorder.

Moderate to severe depression and mania are associated with a reduced thyroid stimulating hormone (TSH) response to TSH releasing hormone (TRH). Continued reduction of this response after clinical recovery seems indicative of early relapse. The aim of the present study was to test the relationship between mild changes in mood and the TSH response to TRH stimulation in patients with bipolar affective disorder. Nineteen outpatients with bipolar affective disorder were followed prospectively for three years. Every third month, mood symptoms were rated using the 17-item Hamilton Depression Rating Scale (HAMD-17) and the Bech-Rafaelsen Mania Scale (BRMS). A TRH test was performed in connection with each rating session (IV injection of 200 microg TRH), and serum TSH was measured at 0, 20, and 60 min. The maximum TSH response (D-max TSH) and the temporal change in D-max TSH between succeeding rating sessions (DD-max TSH) were determined. Psychometric rating and TRH data were obtained for a total of 198 examinations. The temporal change in mood symptom rating score was negatively correlated with the temporal change in D-max TSH, thus suggesting that increasing severity of mood symptoms was related to a reduced TSH response to TRH stimulation. The temporal change in TSH response to TRH stimulation correlated with the actual score on an overall index of symptom severity. In conclusion, milder fluctuations in mood in bipolar affective disorder seem to correlate with the TSH response to TRH stimulation: Increasing severity of mood symptoms seems to be associated with reduced TSH response.

Human-human hybridoma producing monoclonal antibodies against colorectal cancer-associated antigens.

Lymphocytes from lymph nodes draining the tumor region in patients with colorectal cancer were fused with two different human B-lymphoblastoid cell lines, LICR-LON-HMy-2 (HMy-2) and WI-L2-729-HF2 (729-HF2), to generate hybridomas synthesizing antibodies reacting with tumor-associated antigens. In this way 220 hybridomas were obtained which produce antibody reacting with colon cancer cells. All established clones produced IgM. Four human monoclonal antibodies have been further analyzed. The cell lines producing these antibodies are all hybrids based on DNA analysis. Three of the antibodies (G4146, B9165 and D4213) showed binding to differentiation antigens by immunocytochemical analysis on different cancer cell lines and normal human leucocytes and by immunohistochemical analysis on sections of frozen malignant and normal tissues, while the fourth (F11348) showed a reaction with all cells and tissues tested. Western blots of tumor extracts showed binding of G4146 to two components from colon cancer cells with Mr of 59 K and 61 K, while B9165 bound to a 43 K component and F11348 to several components with Mr from 30 to 200K. D4213 showed no binding in this analysis. The results obtained demonstrate the successful application of hybridoma technology to produce human monoclonals with reactivity to differentiation antigens.