Mitotic figure counts are significantly overestimated in resection specimens of invasive breast carcinomas.

Several authors have demonstrated an increased number of mitotic figures in breast cancer resection specimen when compared with biopsy material. This has been ascribed to a sampling artifact where biopsies are (i) either too small to allow formal mitotic figure counting or (ii) not necessarily taken form the proliferating tumor periphery. Herein, we propose a different explanation for this phenomenon. Biopsy and resection material of 52 invasive ductal carcinomas was studied. We counted mitotic figures in 10 representative high power fields and quantified MIB-1 immunohistochemistry by visual estimation, counting and image analysis. We found that mitotic figures were elevated by more than three-fold on average in resection specimen over biopsy material from the same tumors (20±6 vs 6±2 mitoses per 10 high power fields, P=0.008), and that this resulted in a relative diminution of post-metaphase figures (anaphase/telophase), which made up 7% of all mitotic figures in biopsies but only 3% in resection specimen (P<0.005). At the same time, the percentages of MIB-1 immunostained tumor cells among total tumor cells were comparable in biopsy and resection material, irrespective of the mode of MIB-1 quantification. Finally, we found no association between the size of the biopsy material and the relative increase of mitotic figures in resection specimen. We propose that the increase in mitotic figures in resection specimen and the significant shift towards metaphase figures is not due to a sampling artifact, but reflects ongoing cell cycle activity in the resected tumor tissue due to fixation delay. The dwindling energy supply will eventually arrest tumor cells in metaphase, where they are readily identified by the diagnostic pathologist. Taken together, we suggest that the rapidly fixed biopsy material better represents true tumor biology and should be privileged as predictive marker of putative response to cytotoxic chemotherapy.

Traitement du décollement de rétine du pseudophaque: étude rétrospective comparant la vitrectomie sans indentation au traitement ab-externo [Vitrectomy without scleral buckle versus ab-externo approach for pseudophakic retinal detachment: comparative retrospective study].

PURPOSE: Retinal detachment (RD) is a major complication of cataract surgery, which can be treated by either primary vitrectomy without indentation or the scleral buckling procedure. The aim of this study is to compare the results of these two techniques for the treatment of pseudophakic RD. PATIENTS AND METHODS: The charts of 40 patients (40 eyes) treated with scleral buckling for a primary pseudophakic RD were retrospectively studied and compared to the charts of 32 patients (32 eyes) treated with primary vitrectomy without scleral buckle during the same period by the same surgeons. To obtain comparable samples, patients with giant retinal tears, vitreous hemorrhage, and severe preoperative proliferative vitreoretinopathy (PVR) were not included. Minimal follow-up was 6 months. RESULTS: The primary success rate was 84% in the vitrectomy group and 82.5% in the ab-externo group. Final anatomical success was observed in 100% of cases in the vitrectomy group and in 95% of cases in the ab-externo group. Final visual acuity was 0.5 or better in 44% of cases in the vitrectomy group and 37.5% in the ab-externo group. The duration of the surgery was significantly lower in the ab-externo group, whereas the hospital stay tended to be lower in the vitrectomy group. In the vitrectomy group, postoperative PVR developed in 3 eyes and new or undetected breaks were responsible for failure of the initial procedure in 2 eyes. CONCLUSION: Primary vitrectomy appears to be as effective as scleral buckling procedures for the treatment of pseudophakic RD.

Body temperature regulation and outcome after cardiac arrest and therapeutic hypothermia.

OBJECTIVE: Therapeutic temperature modulation is recommended after cardiac arrest (CA). However, body temperature (BT) regulation has not been extensively studied in this setting. We investigated BT variation in CA patients treated with therapeutic hypothermia (TH) and analyzed its impact on outcome. METHODS: A prospective cohort of comatose CA patients treated with TH (32-34°C, 24h) at the medical/surgical intensive care unit of the Lausanne University Hospital was studied. Spontaneous BT was recorded on hospital admission. The following variables were measured during and after TH: time to target temperature (TTT=time from hospital admission to induced BT target <34°C), cooling rate (spontaneous BT-induced BT target/TTT) and time of passive rewarming to normothermia. Associations of spontaneous and induced BT with in-hospital mortality were examined. RESULTS: A total of 177 patients (median age 61 years; median time to ROSC 25 min) were studied. Non-survivors (N=90, 51%) had lower spontaneous admission BT than survivors (median 34.5 [interquartile range 33.7-35.9]°C vs. 35.1 [34.4-35.8]°C, p=0.04). Accordingly, time to target temperature was shorter among non-survivors (200 [25-363]min vs. 270 [158-375]min, p=0.03); however, when adjusting for admission BT, cooling rates were comparable between the two outcome groups (0.4 [0.2-0.5]°C/h vs. 0.3 [0.2-0.4]°C/h, p=0.65). Longer duration of passive rewarming (600 [464-744]min vs. 479 [360-600]min, p<0.001) was associated with mortality. CONCLUSIONS: Lower spontaneous admission BT and longer time of passive rewarming were associated with in-hospital mortality after CA and TH. Impaired thermoregulation may be an important physiologic determinant of post-resuscitation disease and CA prognosis. When assessing the benefit of early cooling on outcome, future trials should adjust for patient admission temperature and use the cooling rate rather than the time to target temperature.

REPETIDO_A new extract of the plant calendula officinalis produces a dual in vitro effect: cytotoxic anti-tumor activity and lymphocyte activation

BACKGROUND Phytopharmacological studies of different Calendula extracts have shown anti-inflammatory, anti-viral and anti-genotoxic properties of therapeutic interest. In this study, we evaluated the in vitro cytotoxic anti-tumor and immunomodulatory activities and in vivo anti-tumor effect of Laser Activated Calendula Extract (LACE), a novel extract of the plant Calendula Officinalis (Asteraceae). METHODS An aqueous extract of Calendula Officinalis was obtained by a novel extraction method in order to measure its anti-tumor and immunomodulatory activities in vitro. Tumor cell lines derived from leukemias, melanomas, fibrosarcomas and cancers of breast, prostate, cervix, lung, pancreas and colorectal were used and tumor cell proliferation in vitro was measured by BrdU incorporation and viable cell count. Effect of LACE on human peripheral blood lymphocyte (PBL) proliferation in vitro was also analyzed. Studies of cell cycle and apoptosis were performed in LACE-treated cells. In vivo anti-tumor activity was evaluated in nude mice bearing subcutaneously human Ando-2 melanoma cells. RESULTS The LACE extract showed a potent in vitro inhibition of tumor cell proliferation when tested on a wide variety of human and murine tumor cell lines. The inhibition ranged from 70 to 100%. Mechanisms of inhibition were identified as cell cycle arrest in G0/G1 phase and Caspase-3-induced apoptosis. Interestingly, the same extract showed an opposite effect when tested on PBLs and NKL cell line, in which in vitro induction of proliferation and activation of these cells was observed. The intraperitoneal injection or oral administration of LACE extract in nude mice inhibits in vivo tumor growth of Ando-2 melanoma cells and prolongs the survival day of the mice. CONCLUSION These results indicate that LACE aqueous extract has two complementary activities in vitro with potential anti-tumor therapeutic effect: cytotoxic tumor cell activity and lymphocyte activation. The LACE extract presented in vivo anti-tumoral activity in nude mice against tumor growth of Ando-2 melanoma cells.

Methylation alterations are not a major cause of PTTG1 misregulation.

BACKGROUND On its physiological cellular context, PTTG1 controls sister chromatid segregation during mitosis. Within its crosstalk to the cellular arrest machinery, relies a checkpoint of integrity for which gained the over name of securin. PTTG1 was found to promote malignant transformation in 3T3 fibroblasts, and further found to be overexpressed in different tumor types. More recently, PTTG1 has been also related to different processes such as DNA repair and found to trans-activate different cellular pathways involving c-myc, bax or p53, among others. PTTG1 over-expression has been correlated to a worse prognosis in thyroid, lung, colorectal cancer patients, and it can not be excluded that this effect may also occur in other tumor types. Despite the clinical relevance and the increasing molecular characterization of PTTG1, the reason for its up-regulation remains unclear. METHOD We analysed PTTG1 differential expression in PC-3, DU-145 and LNCaP tumor cell lines, cultured in the presence of the methyl-transferase inhibitor 5-Aza-2'-deoxycytidine. We also tested whether the CpG island mapping PTTG1 proximal promoter evidenced a differential methylation pattern in differentiated thyroid cancer biopsies concordant to their PTTG1 immunohistochemistry status. Finally, we performed whole-genome LOH studies using Affymetix 50 K microarray technology and FRET analysis to search for allelic imbalances comprising the PTTG1 locus. CONCLUSION Our data suggest that neither methylation alterations nor LOH are involved in PTTG1 over-expression. These data, together with those previously reported, point towards a post-transcriptional level of misregulation associated to PTTG1 over-expression.

A new extract of the plant Calendula officinalis produces a dual in vitro effect: cytotoxic anti-tumor activity and lymphocyte activation

BACKGROUND Phytopharmacological studies of different Calendula extracts have shown anti-inflammatory, anti-viral and anti-genotoxic properties of therapeutic interest. In this study, we evaluated the in vitro cytotoxic anti-tumor and immunomodulatory activities and in vivo anti-tumor effect of Laser Activated Calendula Extract (LACE), a novel extract of the plant Calendula Officinalis (Asteraceae). METHODS An aqueous extract of Calendula Officinalis was obtained by a novel extraction method in order to measure its anti-tumor and immunomodulatory activities in vitro. Tumor cell lines derived from leukemias, melanomas, fibrosarcomas and cancers of breast, prostate, cervix, lung, pancreas and colorectal were used and tumor cell proliferation in vitro was measured by BrdU incorporation and viable cell count. Effect of LACE on human peripheral blood lymphocyte (PBL) proliferation in vitro was also analyzed. Studies of cell cycle and apoptosis were performed in LACE-treated cells. In vivo anti-tumor activity was evaluated in nude mice bearing subcutaneously human Ando-2 melanoma cells. RESULTS The LACE extract showed a potent in vitro inhibition of tumor cell proliferation when tested on a wide variety of human and murine tumor cell lines. The inhibition ranged from 70 to 100%. Mechanisms of inhibition were identified as cell cycle arrest in G0/G1 phase and Caspase-3-induced apoptosis. Interestingly, the same extract showed an opposite effect when tested on PBLs and NKL cell line, in which in vitro induction of proliferation and activation of these cells was observed. The intraperitoneal injection or oral administration of LACE extract in nude mice inhibits in vivo tumor growth of Ando-2 melanoma cells and prolongs the survival day of the mice. CONCLUSION These results indicate that LACE aqueous extract has two complementary activities in vitro with potential anti-tumor therapeutic effect: cytotoxic tumor cell activity and lymphocyte activation. The LACE extract presented in vivo anti-tumoral activity in nude mice against tumor growth of Ando-2 melanoma cells.

Biochemical characterization of recombinant human telomerase

Résumé Les télomères sont les structures ADN-protéines des extrémités des chromosomes des eucaryotes. L'ADN télomérique est constitué de courtes séquences répétitives. L'intégrité des télomères est essentielle pour protéger les extrémités des chromosomes contre les systèmes de dégradations et pour les distinguer des cassures de l'ADN double brin. Parce que la machinerie de la réplication de l'ADN n'est pas capable de répliquer l'extrémité des chromosomes, les télomères raccourcissent au fur et à mesure des cycles de réplication. Dès que les télomères atteignent une longueur critique, leur structure protectrice est perdue. Cela induit un signal de dommage de l'ADN et l'arrêt du cycle cellulaire. Pour contrebalancer le raccourcissement des télomères, les cellules qui s'auto régénèrent, dont les cellules de la moelle osseuse, les lymphocytes activés et 80-90% des cellules cancéreuses, expriment la télomérase. C'est une ribonucléoprotéine qui a la capacité de synthétiser des séquences télomériques par transcription inverse d'une courte séquence contenue dans sa propre sous-unité ARN avec laquelle elle est associée. La télomérase humaine est une enzyme processive au niveau de l'addition des nucléotides et aussi des répétitions télomériques. La télomérase de levure et la télomérase humaine sont toutes deux dimériques et il a été montré que la télomérase humaine recombinante contient deux ARN qui coopèrent pour fonctionner ainsi que deux sous-unités catalytiques. Cependant, il n'a pas encore été montré quel est le rôle de la dimérisation dans l'activité de la télomérase. Afin d'élucider ce rôle, nous avons exprimé, reconstitué et purifié la télomérase humaine dimérique recombinante. Et pour étudier l'effet d'ARN mutants sur l'activité de la télomérase, nous avons développé une méthode pour reconstituer et enrichir en hétérodimères de télomérase. Les hétérodimères contiennent une sous-unité ARN sauvage et une sous-unité ARN mutée au niveau de la séquence de la matrice. Sur l'ARN muté nous avons introduit une étiquette aptamer ARN-S1 puis nous avons purifié la télomérase via l'etiquette Si. Nous avons montré que la dimérisation est essentielle pour l'activité de la télomérase. Nos données indiquent que chaque télomérase du dimère allonge leur substrat, l'ADN télomérique, indépendamment l'une de l'autre à chaque cycle d'élongation mais que l'addition itérative de répétitions télomériques nécessite une coopération entre les deux télomérases du dimère. Nous proposons donc un modèle dans lequel les deux télomérases du dimères se lient et allongent deux substrats télomères et que pendant l'élongation processive les deux enzymes subissent un changement de conformation de manière coordonnée, ce changement va permettre le repositionnement des substrats pour d'autres cycles d'additions de répétitions télomériques. Dyskeratosis congenita est une maladie mortelle due majoritairement au disfonctionnement de la moelle osseuse. Dans la forme autosomale de la maladie, l'ARN de la télomérase contient des mutations. En utilisant notre système de reconstitution, nous avons montré que ces ARN mutés, qui ont perdu leur activité enzymatique dans le cas d'un homodimère de mutants, sont dominant négatifs quand ils sont présents dans les hétérodimères sauvage/mutant. Cet effet trans-dominant négatif pourrait contribuer à la progression de la maladie. Abstract Telomeres are protein-DNA structures at the ends of linear eukaryotic chromosomes. The telomeric DNA consists of tandemly repeated sequences. Telomeric integrity is essential to protect chromosomal ends from nucleolytic degradation and to prevent their recognition as DNA double strand breaks. Due to the inability of the conventional DNA replication machinery to replicate terminal DNA stretches, telomeres shorten with continuous rounds of DNA replication. As soon as telomeres reach a critical length, their protective structure is lost and the deprotected telomeres will induce a DNA damage response leading to cell cycle arrest. To counteract telomere shortening, self-renewing cells, including bone marrow cells, activated lymphocytes and 80-90% of cancer cells express the cellular reverse transcriptase telomerase, which has the capacity to synthesize telomeric repeats by reverse transcription of a short template sequence encoded by its stably associated RNA subunit. Human telomerase is a processive enzyme for nucleotide as well as repeat addition. Both yeast and human telomerase are dimeric enzymes and recombinant human telomerase has been shown to contain two functionally cooperating RNAs and most probably also two protein subunits. However, it has remained unclear how dimerization may contribute to telomerase activity. To study the role of dimerization, we expressed, reconstituted and purified recombinant human telomerase. We also developed a new method to reconstitute and enrich for telomerase heterodimers containing wild-type (wt) and mutant telomerase RNA subunits. To this end we introduced an S1-RNA-aptamer tag into telomerase RNA and purified telomerase reconstituted with a mixture of untagged and tagged RNA via the S1-tag. Using this experimental system, we introduced template mutations in the tagged RNA subunit and examined the effect of mutant RNAs on wt telomerase activity in wt/mutant heterodimers. We obtained evidence that dimerization is essential for telomerase activity. Our data indicate that the two subunits elongate telomere substrates independently of each other during single rounds of elongation, but that iterative addition of telomeric repeats requires cooperation between the two subunits. We suggest a model, in which dimeric telomerases bind and elongate two telomere substrates and that the two subunits undergo coordinated conformational changes during processive elongation that enable repositioning the substrates for subsequent rounds of repeat addition. Dyskeratosis congenita is a multisystemic disease with bone marrow failure as the major cause of death. The autosomal form of this disease was found to harbor mutations in the telomerase RNA. Using our reconstitution system, we tested whether mutant dyskeratosis telomerase RNAs behaved in a dominant negative manner. We observed that dyskeratosis telomerase RNA mutants, which lacked enzymatic activity were dominant negative, when present in wt/ mutant heterodimers. The transdominant negative effect of these mutants may contribute to disease progression.

Response of embryonic heart to hypoxia and reoxygenation: an in vitro model

OBJECTIVES: To define properly the consequences of oxygen deprivation and readmission for the functioning of the developing heart. METHODS: Spontaneously beating hearts excised from three-day-old chick embryos were loaded with a drop of viscous nontoxic silicone oil and cultured in a special chamber in which variations of PO2 at the tissue level could be strictly controlled. All parts of the hearts were simultaneously submitted to identical changes in PO2. Instantaneous heart rate, myocardial shortening, velocities of contraction and relaxation, and mechanical propagation along the heart tube were determined photometrically. RESULTS: The hearts, submitted to a PO2 ramp (0 to 9.3 kPa) or absolute anoxia, reacted rapidly, reversibly and reproducibly. Under sustained anoxia, ventricular activity stopped after 3.8±0.7 mins (n=4) and then resumed intermittently in the form of tachycardic bursts. Brief anoxia (1 min) provoked tachycardia followed by bradycardia, induced contracture, depressed contractility and retarded atrioventricular propagation. Upon reoxygenation, ventricular contractions ceased suddently for 20±11 s (n=5), whereas a residual atrial activity could persist. The duration of this arrest and the rate of recovery depended on duration of the preceding anoxia. Such a dysfunction constitutes the embryonic analogue of the oxygen paradox observed in adult hearts. Initial impulses, including arrhythmic activity, originated exclusively from the atrium, and no ventricular ectopic beats were detected whatever the conditions of oxygenation. CONCLUSIONS: This in vitro model seems promising for studying the pathophysiological mechanisms associated with hypoxia and reoxygenation in the developing heart.

Progression of auditory discrimination based on neural decoding predicts awakening from coma.

Auditory evoked potentials are informative of intact cortical functions of comatose patients. The integrity of auditory functions evaluated using mismatch negativity paradigms has been associated with their chances of survival. However, because auditory discrimination is assessed at various delays after coma onset, it is still unclear whether this impairment depends on the time of the recording. We hypothesized that impairment in auditory discrimination capabilities is indicative of coma progression, rather than of the comatose state itself and that rudimentary auditory discrimination remains intact during acute stages of coma. We studied 30 post-anoxic comatose patients resuscitated from cardiac arrest and five healthy, age-matched controls. Using a mismatch negativity paradigm, we performed two electroencephalography recordings with a standard 19-channel clinical montage: the first within 24 h after coma onset and under mild therapeutic hypothermia, and the second after 1 day and under normothermic conditions. We analysed electroencephalography responses based on a multivariate decoding algorithm that automatically quantifies neural discrimination at the single patient level. Results showed high average decoding accuracy in discriminating sounds both for control subjects and comatose patients. Importantly, accurate decoding was largely independent of patients' chance of survival. However, the progression of auditory discrimination between the first and second recordings was informative of a patient's chance of survival. A deterioration of auditory discrimination was observed in all non-survivors (equivalent to 100% positive predictive value for survivors). We show, for the first time, evidence of intact auditory processing even in comatose patients who do not survive and that progression of sound discrimination over time is informative of a patient's chance of survival. Tracking auditory discrimination in comatose patients could provide new insight to the chance of awakening in a quantitative and automatic fashion during early stages of coma.

Increased blood glucose variability during therapeutic hypothermia and neurological recovery after cardiac arrest

INTRODUCTION. Recent studies suggest that increased blood glucose variability (BGV) is associated with ICU mortality1. Hypothermia is known to induce insulin resistance, thus potentially increasing BGV. No studies however have examined the effect of therapeutic hypothermia (TH) on insulin requirements and BGV. OBJECTIVES. To examine the effect of TH on BGV and its relationship to outcome in patients with coma after cardiac arrest (CA). METHODS. We prospectively studied 132 consecutive comatose CA patients treated with TH (target core temp 33_C for 24 h, using surface cooling). All patients were treated with intravenous insulin (blood glucose target 6-8 mM), according to a written algorithm, with nurse-driven adjustment of insulin dose. For each patient, standard deviation of repeated blood glucose samples was used to calculate BGV. Two time-points, comparable in duration, were studied: TH (stable maintenance phase, i.e. 6-24 h, core temp ± 33_C) vs. Normothermia (NT, i.e. after rewarming, stable normothermic phase, core temp ± 37_C). Mortality and neurological recovery (Glasgow-Pittsburgh Cerebral Performance Categories, CPC, dichotomized as good = CPC 1-2 vs. poor = CPC 3-5) were assessed at hospital discharge. Statistical analysis was performed with ANOVA for repeated measures. RESULTS. Compared to NT, TH was associated with increased intravenous insulin dose (0.8 ± 1.1 vs. 1.6 ± 2 U/h, P\0.0001), higher mean (6.9 ± 1.3 vs. 7.7 ± 1.8 mM, P\0.0001) and maximum (9.1 ± 3.7 vs. 10.9 ± 3.6 mM, P\0.0001) blood glucose, and increased BGV (1.3 ± 1.2 vs. 1.7 ± 1.1 mM, P = 0.004). Increased BGV was strongly associated with mortality (2.5 ± 1.5 mM in non-survivors vs. 1.6 ± 1 mM in survivors, P\0.001) and worse outcome (2.3 ± 1.4 mM in patients with poor vs. 1.5 ± 0.8 mM in those with good neurological recovery, P\0.0001). CONCLUSIONS. Therapeutic hypothermia is associated with increased insulin requirements and higher blood glucose variability,which in turn correlateswithworse prognosis in patientswith post- CA coma. Strategies aimed to maintain stable glycemic profile and avoid blood glucose variability might contribute to optimize the management of TH and may translate into better outcome.

Resuscitated sudden cardiac death caused by left main coronary artery compression by an aneurysm of the sinus of Valsalva.

A 49-year-old woman, without known cardiovascular risk factors. Hoarseness of voice caused by a paralysis of left vocal cord. She was admitted to hospital because of acute coronary syndrome, associated to resuscitated cardiac arrest (asystolia documented) without later neurology sequels. Physical examination was anodyne. Echocardiographic study demonstrated a compatible image with a large left sinus of Valsalva aneurysm (SVA) (Panel A) and mild aortic regurgitation. Cardiac catheterization confirmed the presence of left SVA (Panel B) that produced extrinsic compression of the left main coronary artery (Panels C and D). Repair surgery was made by means of closing the aneurysmal orifice with a patch of dacron. Intra-operatory echocardiographic control study found severe aortic regurgitation, so valvular replacement with 19 mm mechanical prosthesis and extension of the valve annulus with patch of dacron was performed, associated with bypass with safena vein graft to left coronary artery. SVA is a very infrequent cardiac anomaly, generally with silent clinical course until it ruptures. Myocardial ischaemia caused by coronary artery compression is unusual. We described the case of a patient diagnosed of left SVA, whose initial clinical manifestation was the appearance of resuscitated sudden cardiac death in the context of an acute coronary syndrome.

Accidental cross-clamping of the superior vena cava: management and BIS evolution.

The case of a 38-year old man with recurrent adenocarcinoma of the right lung and infiltration of the thoracic wall, who underwent a right pneumonectomy with thoracic wall resection, is described. Dissection of the right pulmonary hilous was extremely difficult due to the previous operations. The superior vena cava (SVC) was accidentally ligated, resulting in profound hypotension and increased venous pressure in the internal jugular vein. Immediate blood aspiration through a 8.5-French introducer sheath lowered the venous congestion. After the SVC was cross-clamped, the Bispectral Index (BIS) acutely decreased to 0 and remained low during the resuscitation. Over the next 5 hours, BIS was directly affected by mean arterial pressure. Four days postoperatively, the patient was neurologically intact.

Delay for admission of unstable hospitalized patients to intensive care unit: a need for a medical emergency team?

INTRODUCTION. Patients admitted in Intensive Care Unit (ICU) from general wards are more severe and have a higher mortality than those admitted from emergency department as reported [1]. The majority of them develop signs of instability (e.g. tachypnea, tachycardia, hypotension, decreased oxygen saturation and change in conscious state) several hours before ICU admission. Considering this fact and that in-hospital cardiac arrests and unexpected deaths are usually preceded by warning signs, immediate on site intervention by specialists may be effective. This gave an impulse to medical emergency team (MET) implementation, which has been shown to decrease cardiac arrest, morbidity and mortality in several hospitals. OBJECTIVES AND METHODS. In order to verify if the same was true in our hospital and to determine if there was a need for MET, we prospectively collected all non elective ICU admissions of already hospitalized patients (general wards) and of patients remaining more than 3 h in emergency department (considered hospitalized). Instability criteria leading to MET call correspond to those described in the literature. The delay between the development of one criterion and ICU admission was registered. RESULTS. During an observation period of 12 months, 321 patients with our MET criteria were admitted to ICU. 88 patients came from the emergency department, 115 from the surgical and 113 from the medical ward. 65% were male. The median age was 65 years (range 17-89). The delay fromMETcriteria development to ICU admission was higher than 8 h in 155 patients, with a median delay of 32 h and a range of 8.4 h to 10 days. For the remaining 166 patients, an early MET criterion was present up to 8 h (median delay 3 h) before ICU admission. These results are quite concordant with the data reported in the literature (ref 1-8). 122 patients presented signs of sepsis or septic shock, 70 patients a respiratory failure, 58 patients a cardiac emergency. Cardiac arrest represent 5% of our collective of patients. CONCLUSIONS.Similar to others observations, the majority of hospitalized patients admitted on emergency basis in our ICU have warning signs lasting for several hours. More than half of them were unstable for more than 8 h. This shows there is plenty of time for early acute management by dedicated and specialized team such as MET. However, further studies are required to determine if MET implementation can reduce in-hospital cardiac arrests and influence the morbidity, the length of stay and the mortality.

Mechanisms regulating the proliferative potential of human CD8+ T lymphocytes overexpressing telomerase.

In human somatic cells, including T lymphocytes, telomeres progressively shorten with each cell division, eventually leading to a state of cellular senescence. Ectopic expression of telomerase results in the extension of their replicative life spans without inducing changes associated with transformation. However, it is yet unknown whether somatic cells that overexpress telomerase are physiologically indistinguishable from normal cells. Using CD8+ T lymphocyte clones overexpressing telomerase, we investigated the molecular mechanisms that regulate T cell proliferation. In this study, we show that early passage T cell clones transduced or not with human telomerase reverse transcriptase displayed identical growth rates upon mitogenic stimulation and no marked global changes in gene expression. Surprisingly, reduced proliferative responses were observed in human telomerase reverse transcriptase-transduced cells with extended life spans. These cells, despite maintaining high expression levels of genes involved in the cell cycle progression, also showed increased expression in several genes found in common with normal aging T lymphocytes. Strikingly, late passage T cells overexpressing telomerase accumulated the cyclin-dependent inhibitors p16Ink4a and p21Cip1 that have largely been associated with in vitro growth arrest. We conclude that alternative growth arrest mechanisms such as those mediated by p16Ink4a and p21Cip1 still remained intact and regulated the growth potential of cells independently of their telomere status.

Mitotic functions for SNAP45, a subunit of the small nuclear RNA-activating protein complex SNAPc.

The small nuclear RNA-activating protein complex SNAP(c) is required for transcription of small nuclear RNA genes and binds to a proximal sequence element in their promoters. SNAP(c) contains five types of subunits stably associated with each other. Here we show that one of these polypeptides, SNAP45, also known as PTF delta, localizes to centrosomes during parts of mitosis, as well as to the spindle midzone during anaphase and the mid-body during telophase. Consistent with localization to these mitotic structures, both down- and up-regulation of SNAP45 lead to a G(2)/M arrest with cells displaying abnormal mitotic structures. In contrast, down-regulation of SNAP190, another SNAP(c) subunit, leads to an accumulation of cells with a G(0)/G(1) DNA content. These results are consistent with the proposal that SNAP45 plays two roles in the cell, one as a subunit of the transcription factor SNAP(c) and another as a factor required for proper mitotic progression.