[Economic Analysis of Treatment Courses for Patients Over the Age of 80 Years at a Surgical Maximum Care Centre]. / Ökonomische Analyse des Behandlungsverlaufs bei über 80-jährigen Patienten an einer Klinik der chirurgischen Maximalversorgung.

BACKGROUND: The demographic change in Germany with an aging population and the resulting necessity of adequate surgical care for older patients was lately discussed with concern. One major aspect is the estimated higher treatment costs in the care of the elderly. MATERIALS AND METHODS: InEK data from all cases of patients over the age of 80, who were treated and discharged from 2008 to 2012 as inpatients at the Department of General, Visceral, Vascular and Thoracic Surgery at the Charité - Universitätsmedizin Berlin, Campus Mitte, were analysed. Of a total of 13,612 patients 626 patients were over the age of 80. Their lengths of stay, mode of discharge and discharge management as well as costs and reimbursements according to the relevant diagnosis-related groups were analysed. RESULTS: Cases of elderly patients amounted to a stable 5 % of all cases from 2008 until 2012. Their mean length of stay was 14 (median, 9), range, 1-129 days. 80 % of patients could be regularly discharged, 9 % died, 8 % were transferred to another hospital, 2 % discharged into a nursing home and 1 % into a rehabilitation centre. The elderly patients had a patient clinical complexity level of mean 2.84. Costs per day amounted to a mean 778 (median: 627) €, range: 306-7740 €, total costs to 10,686 (median: 5140) €, range: 368-186,059 €. The mean deficit was 491 (median: 176) € per patient, range: - 30,470-75,144 €. The discharge management was significantly different in comparison to patients under the age of 80 with respect to avoidance of discharge at the weekend. CONCLUSION: Patients over the age of 80 are a relevant group in surgery. They have an increased perioperative risk, but patients should not be denied surgery solely because of their age. The perioperative management of the elderly has to be of maximum standardised quality. From an economic perspective it can be stated that elderly patients currently pose no exceptional financial risk to a surgical department, but contribute relevantly to the turnover, whereby special attention has to be paid to an early structured discharge management.

Costs and prolonged length of stay of central venous catheter-associated bloodstream infections (CVC BSI): a matched prospective cohort study.

PURPOSE: Central venous catheter-associated bloodstream infections (CVC BSI) are a common and serious complication among critically ill patients on intensive care units (ICUs), but also result in a financial burden for the health care system. Our aim was to determine the additional costs and length of stay (LOS) of patients with ICU-acquired CVC BSI. METHODS: We used the surveillance method of the German nosocomial infection surveillance system (Krankenhaus Infections Surveillance System, KISS) to find cases of CVC BSI. The associated costs of CVC BSI were estimated as true costs generated within our hospital. We used a matched cohort design, comparing patients with CVC BSI and patients without BSI. The study period was from January to December 2010. Patients were matched by age, sex, and Simplified Acute Physiology Score (SAPS). The LOS in the ICU of control patients needed to be at least as long as that of CVC BSI patients before the onset of CVC BSI. RESULTS: We matched 40 CVC BSI patients to 40 patients without BSI. The median hospital costs for CVC BSI patients were significantly higher than for patients without BSI (60,445 € vs. 35,730 €; p = 0.006) and the CVC BSI patients stayed longer in the hospital than patients without CVC BSI (44 days vs. 30 days; p = 0.110). The median attributable costs per CVC BSI was 29,909 € (p = 0.006) and the median attributable LOS was 7 days (p = 0.006). CONCLUSION: CVC BSI is associated with increased hospital costs and prolonged hospital stay. Hospital management should implement control measurements to keep the incidence of CVC BSI as low as possible.

Bloodstream infection due to extended-spectrum beta-lactamase (ESBL)-positive K. pneumoniae and E. coli: an analysis of the disease burden in a large cohort.

PURPOSE: The burden of extended-spectrum beta-lactamase (ESBL)-positive Enterobacteriaceae (ESBL-E) is growing worldwide. We aimed to determine the financial disease burden attributable to ESBL-positive species in cases of bloodstream infection (BSI) due to K. pneumoniae and E. coli. METHODS: We conducted a cohort study on patients with BSI due to K. pneumoniae or E. coli between 2008 and 2011 in our institution. Data were collected on true hospital costs, length of stay (LOS), basic demographic parameters, underlying diseases as Charlson comorbidity index (CCI) and ESBL positivity of the pathogens. Multivariable regression analysis on hospital costs and length of stay was performed. RESULTS: Overall we found 1,851 consecutive cases of ESBL-E BSI, 352 (19.0%) cases of K. pneumoniae BSI and 1,499 (81.0%) cases of E. coli BSI. Sixty-six of E. coli BSI (18.8%) and 178 of K. pneumoniae BSI (11.9%) cases were due to ESBL-positive isolates, respectively (p = 0.001). 830 (44.8%) cases were hospital-onset, 215 (61.1%) of the K. pneumoniae and 615 (41.0%) of the E. coli cases (p < 0.001). In-hospital mortality was overall 19.8, 25.0% in K. pneumoniae cases and 18.5% in E. coli cases (p = 0.006). Increased hospital costs and length of stay were significantly associated to BSI with ESBL-positive K. pneumoniae. CONCLUSION: In contrast to BSI due to ESBL-positive E. coli, cases of ESBL-positive K. pneumoniae BSI were associated with significantly increased costs and length of stay. Infection prevention measures should differentiate between both pathogens.

Molecular signatures of cortical expansion in the human fetal brain.

The third trimester of human gestation is characterised by rapid increases in brain volume and cortical surface area. A growing catalogue of cells in the prenatal brain has revealed remarkable molecular diversity across cortical areas.1,2 Despite this, little is known about how this translates into the patterns of differential cortical expansion observed in humans during the latter stages of gestation. Here we present a new resource, µBrain, to facilitate knowledge translation between molecular and anatomical descriptions of the prenatal developing brain. Built using generative artificial intelligence, µBrain is a three-dimensional cellular-resolution digital atlas combining publicly-available serial sections of the postmortem human brain at 21 weeks gestation3 with bulk tissue microarray data, sampled across 29 cortical regions and 5 transient tissue zones.4 Using µBrain, we evaluate the molecular signatures of preferentially-expanded cortical regions during human gestation, quantified in utero using magnetic resonance imaging (MRI). We find that differences in the rates of expansion across cortical areas during gestation respect anatomical and evolutionary boundaries between cortical types5 and are founded upon extended periods of upper-layer cortical neuron migration that continue beyond mid-gestation. We identify a set of genes that are upregulated from mid-gestation and highly expressed in rapidly expanding neocortex, which are implicated in genetic disorders with cognitive sequelae. Our findings demonstrate a spatial coupling between areal differences in the timing of neurogenesis and rates of expansion across the neocortical sheet during the prenatal epoch. The µBrain atlas is available from: https://garedaba.github.io/micro-brain/ and provides a new tool to comprehensively map early brain development across domains, model systems and resolution scales.

Brain Charts for the Rhesus Macaque Lifespan.

Recent efforts to chart human brain growth across the lifespan using large-scale MRI data have provided reference standards for human brain development. However, similar models for nonhuman primate (NHP) growth are lacking. The rhesus macaque, a widely used NHP in translational neuroscience due to its similarities in brain anatomy, phylogenetics, cognitive, and social behaviors to humans, serves as an ideal NHP model. This study aimed to create normative growth charts for brain structure across the macaque lifespan, enhancing our understanding of neurodevelopment and aging, and facilitating cross-species translational research. Leveraging data from the PRIMatE Data Exchange (PRIME-DE) and other sources, we aggregated 1,522 MRI scans from 1,024 rhesus macaques. We mapped non-linear developmental trajectories for global and regional brain structural changes in volume, cortical thickness, and surface area over the lifespan. Our findings provided normative charts with centile scores for macaque brain structures and revealed key developmental milestones from prenatal stages to aging, highlighting both species-specific and comparable brain maturation patterns between macaques and humans. The charts offer a valuable resource for future NHP studies, particularly those with small sample sizes. Furthermore, the interactive open resource (https://interspeciesmap.childmind.org) supports cross-species comparisons to advance translational neuroscience research.

Novel use of the wild species Cephalaria joppensis for silage preparation and its nutritive value for feeding lactating dairy cows.

This study presents a novel method for use of the wild plant species Cephalaria joppensis (CJ) as agricultural forage for ruminants. Domesticated CJ tends to have higher crop mass yield per hectare than a commercial wheat variety (W) but is similar in in vitro dry matter (DM) digestibility. This study was composed of 3 experiments. Experiment 1 aimed to measure effects of ensiling CJ versus W in packed polyethylene-wrapped bales. Three types of ensiled bales were produced for each plant: 1) direct-cut CJ versus W packed solely; 2) direct-cut CJ versus W mixed as sole roughage source together with dietary ingredient and packed in bales to create CJ total mixed ration (CJ-TMR) or W-TMR; 3) CJ silage versus W silage mixed as one-third of dietary roughage source together with two-thirds sorghum (S) silage and additional dietary ingredients and packed in bales to create CJ-S-TMR or W-S-TMR. Data showed that packing and wrapping created anaerobic conditions within the 4 types of TMR bales while reducing pH (4.12 to 4.37). Dry matter loss during ensilage was higher for the 2 types of TMR containing W compared with CJ. Ensilage decreased soluble nitrate content as well as yeast and mold contamination, and the 4 types of TMR bales were characterized by a long outdoor shelf life (3 mo) and high stability under aerobic exposure. Experiment 2 aimed to measure the intake and digestibility by sheep of the 4 types of packed TMR after 90 d of ensiling. Data demonstrated higher voluntary intake of the CJ-TMR compared with the other TMR types. The CJ-TMR was characterized by higher digestibility of DM, crude protein, and neutral detergent fiber components compared with the CJ-S-TMR. Experiment 3 examined intake, digestibility, and milk production by 21 pairs of lactating cows individually fed CJ-S-TMR versus W-S-TMR. Similar intake (21.6 to 22.0 kg/d) and digestibility of DM and crude protein were observed in cows fed the 2 TMR types (68 to 69% and 66 to 68%, respectively). However, neutral detergent fiber and cellulose digestibility were slightly higher in the cows fed W-S-TMR and this was reflected in a small increase in their milk and energy-corrected milk yield (36.5 and 31.4 kg/cow per day, respectively) compared with cows fed CJ-S-TMR (35.5 and 30.4 kg/cow per day, respectively). Results demonstrate that direct-cut CJ used as is, or CJ silage can be included and ensiled in TMR bales for feeding productive ruminants as a substitute for wheat silage.

Volume therapy and cardiocircular function during hyperthermic intraperitoneal chemotherapy.

BACKGROUND: Surgical cytoreduction and simultaneous hyperthermic intraoperative intraperitoneal chemotherapy (HIPEC) for peritoneal carcinomatosis is afflicted with a high incidence of postoperative complications. The knowledge of intraoperative volume therapy during surgery and chemotherapy is limited. On the other hand, the choice of a 'liberal' or 'restrictive' regimen of fluid administration has a deep impact on the postoperative morbidity. The aim of this observational trial was to report detailed data on volume replacement and cardiocircular function during the HIPEC procedure. METHODS: Eighteen consecutive patients undergoing cytoreductive surgery and HIPEC for peritoneal carcinomatosis were enrolled. The intraoperative volume administration was observed as well as the postoperative morbidity and mortality. Cardiofunctional data were assessed by the invasive transthoracic thermodilution technique. RESULTS: The study showed that large amounts of volume (1,240 ml h(-1); range: 810-1,570 ml h(-1)) are given during the HIPEC procedure to replace fluid loss and maintain a stable circulatory function. Signs of a hyperdynamic status during intraoperative intraperitoneal chemotherapy were not found. CONCLUSIONS: During surgical cytoreduction and simultaneous HIPEC, large amounts of volume were administered. HIPEC in itself did not lead to an increased fluid requirement. Further prospective studies with larger populations are needed to investigate whether goal-oriented therapies and a restricted volume regimen can contribute to decrease the postoperative morbidity.

New insights into the pathology of Parkinson's disease: does the peripheral autonomic system become central?

Recent studies in aged, neurologically unimpaired subjects have pointed to a specific induction site of the pathological process of Parkinson's disease (PD) in the region of the dorsal glossopharyngeus-vagus complex as well as in the anterior olfactory nucleus. From the lower brainstem, the disease process would then pursue an ascending course and involve more rostral brainstem areas, limbic structures, and eventually the cerebral cortex. One barrier to the acceptance of the caudal medullary structures as the induction site of PD pathology is that not all parts of the nervous system have been investigated for the presence of PD-associated lesions in cases of early asymptomatic PD. Using alpha-synuclein immunostaining, we investigated the brain, the sacral, and thoracic autonomic nuclei of the spinal cord as well as several components of the peripheral autonomic nervous system in a autopsy cohort of 98 neurologically unimpaired subjects aged 64 or more. Our data indicate that the autonomic nuclei of the spinal cord and the peripheral autonomic nervous system belong to the most constantly and earliest affected regions next to medullary structures and the olfactory nerves in neurologically unimpaired older individuals, thus providing a pathological basis for early premotor autonomic dysfunctions at a prodromal stage of PD.

["Fast-track" rehabilitation in thoracic surgery. First experiences with a multimodal, interdisciplinary, and proven perioperative treatment course]. / "Fast-track-Rehabilitation" in der Thoraxchirurgie. Erste Erfahrungen mit einem interdisziplinären, multimodalen, beweisbegründeten und perioperativen Behandlungspfad.

OBJECTIVES: "Fast-track" rehabilitation is a multimodal perioperative treatment concept for accelerating postoperative recovery which has been already used successfully in visceral surgery. Of its use in thoracic surgery however, almost no data exist and the relevance of this concept for pulmonary operations is unknown. PATIENTS AND METHODS: In this prospective study we examined a new perioperative fast-track treatment concept for thoracic surgery and evaluated the results. This program employs detailed information of patients, intensive perioperative respiratory therapy, thoracic peridural analgesia, forced mobilization, and an early start of postoperative normal food intake. RESULTS: Fifty consecutive patients with benign or malignant diseases of the lung aged an average of 64 years (range 22-78) were operated on thoracoscopically (n=15) or with thoracotomy (n=35) and treated perioperatively using the fast-track program. All patients were mobilized beginning 4 h postoperatively and had normal food. The incidence of general postoperative complications was 0% in this study. Postoperative stay lasted 4.5 days (range 1.5-28.5). There was no increase in surgical complications, and 6% of the patients were readmitted. The patients' acceptance of this concept was high. CONCLUSION: Fast-track rehabilitation resulted in a decreased rate of general complications and accelerated rehabilitation in thoracic surgery.

Risk factors for KPC-producing Enterobacteriaceae acquisition and infection in a healthcare setting with possible local transmission: a case-control study.

BACKGROUND: Reports of Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae (KPC-Kp) in Australia were previously uncommon, with cases imported sporadically by travellers from higher prevalence countries. AIM: The study institution reported the first outbreak of KPC-Kp in Australia. The aim of this study was to identify risk factors for KPC-Kp colonization and infection using a matched case-control study. METHODS: The study included all hospitalized patients with KPC-Kp colonization or infection from January 2012 to September 2015. FINDINGS: Thirty-four cases of KPC-producing Enterobacteriaceae (including 31 KPC-Kp cases) were matched with 136 controls. Variables associated with KPC-Kp acquisition included: length of hospital stay >28 days in the past 12 months, prior vancomycin-resistant enterococci (VRE) colonization, central venous catheter (CVC), gastrointestinal disease and invasive procedures. Exposure to broad-spectrum antibiotics was also found to be a significant risk factor. In the multi-variate analysis, three factors independently associated with KPC-Kp acquisition were length of hospital stay >28 days in the past 12 months [odds ratio (OR) 23.6, 95% confidence interval (CI) 4.9-113.3], presence of a CVC (OR 15.4, 95% CI 2.7-86.9), and prior VRE colonization (OR 6.0, 95% CI 1.6-23.2). Very few patients had a history of overseas travel. CONCLUSION: This study demonstrates that patients with prolonged hospital exposure are more likely to acquire KPC-Kp in the setting of a local outbreak, and suggests that risk factors for KPC-Kp acquisition may be shared with those for VRE colonization. Local screening strategies targeting overseas travellers would likely miss many cases. The results of this study will help to inform screening policies for carbapenemase-producing Enterobacteriaceae.

Endocrine alterations associated with extended time interval between estrus and ovulation in high-yield dairy cows.

Short fertile half-lives of the male and female gametes in the female tract necessitate accurate timing of artificial insemination. We examined the possible association between extension of the estrus to ovulation (E-O) interval and alterations in concentrations of estradiol, progesterone, and the preovulatory LH surge before estrus and ovulation. High-yielding Holstein cows (n = 74 from a total of 106) were synchronized and were examined around the time of the subsequent estrus. They were observed continuously for estrual behavior. Blood samples were collected before and after estrus, and ultrasound checks for ovulation were made every 4 h. About three-quarters of the cows exhibited short (but normal) E-O intervals of 22 to 25 h (25%) or normal intervals of 25 to 30 h (47%); 17% of them displayed a long (but normal) E-O interval of 31 to 35 h, and about 10% exhibited a very long E-O interval of 35 to 50 h. Extended E-O interval comprised estrus-to-LH surge and LH surge-to-ovulation intervals that were both longer than normal. Pronounced changes in hormonal concentrations were noted before ovulation in the very long E-O interval group of cows: progesterone and estradiol concentrations were reduced, and the preovulatory LH peak surge was markedly less than in the other 3 groups. Postovulation progesterone concentrations during the midluteal phase were lesser in the very long and the long E-O interval groups compared with those in the short and normal interval groups. Season, parity, milk yield, and body condition did not affect the estrus to LH surge, LH surge to ovulation, and E-O intervals. The results indicate an association between preovulatory-reduced estradiol concentrations and a small preovulatory LH surge, on the one hand, and an extended E-O interval, on the other hand. Delayed ovulation could cause nonoptimal timing of AI, a less than normal preovulatory LH surge that may be associated with suboptimal maturation of the oocyte before ovulation, or reduced progesterone concentrations before and after ovulation. All may be factors associated with poor fertility in cows with a very long E-O interval.

Early decline in c-myb oncogene expression in the differentiation of human myeloblastic leukemia (ML-1) cells induced with 12-O-tetradecanoylphorbol-13-acetate.

The relationship of oncogene expression to proliferation and differentiation has been examined in a line of human myeloblastic leukemia (ML-1) cells. Proliferating leukemic cells were found to express a 4.3-kilobase cellular homologue (c-myb) of the transforming sequence of avian myeloblastosis virus. A rapid decline in the expression of this transcript was seen in cells induced to differentiate with 12-O-tetradecanoylphorbol-13-acetate. The level of c-myb RNA was decreased by greater than 50% as early as 3 hr after 12-O-tetradecanoylphorbol-13-acetate exposure, and at 8 to 72 hr the reduction was greater than or equal to 4-fold. Subsequent to the decrease in oncogene expression at 3 hr, DNA synthesis began to decline; by 24 hr, cell proliferation had ceased. At this time, monocyte- and macrophage-like cells were beginning to emerge. These findings demonstrate that c-myb is expressed during ML-1 cell proliferation and declines prior to the loss of DNA synthesis that accompanies the differentiation process.

Mechanism of interaction between antineoplastic agents and natural differentiation factors in the induction of human leukemic cell maturation.

DNA-specific agents have the capacity to induce the maturation of ML-1 human myeloblastic leukemia cells to monocyte/macrophages if adequate concentrations of fetal bovine serum or mitogen-stimulated human leukocyte-conditioned medium (CM) are present in the culture medium. Fetal bovine serum and CM contain specific differentiation-inducing factors that, in conjunction with the drugs, bring about cell maturation. To examine the mechanism by which this interactive effect occurs, ML-1 cells were exposed to actinomycin D or daunomycin in various combinations with CM, using concentrations at which neither the drug nor CM, when applied individually, induced maturation to a significant extent. Pretreatment for 3 days with drug followed by treatment for 3 days with CM caused maturation of 75% of the cells, as determined by the appearance of Fc receptors. Other markers of differentiation, including alpha-napthyl acetate esterase, acid phosphatase, and morphology, also reflected the increase in maturation. The simultaneous application of drug and CM was equally effective in inducing differentiation. Pretreatment of the cells with CM followed by treatment with drug failed to induce maturation, whereas pretreatment with CM followed by a second application of CM caused the expression of Fc receptors in 62% of the cells. In contrast, pretreatment with drug followed by a second application of drug did not induce differentiation significantly. These results indicate that the drug sensitizes the cells to respond to concentrations of CM to which they would otherwise be refractive. The drug-induced sensitization is reversible. At sensitizing drug concentrations, cell viability was preserved but, as measured by radiolabeling for 1 h, total RNA synthesis was decreased by 38% and mRNA synthesis by 87%. At these drug concentrations, the synthesis of mRNA specified by all seven oncogenes examined (myb, myc, abl, fos, N-ras, sis, erb B) was decreased by 15-60%. The administration of CM subsequent to drug caused a further decrease of some mRNA levels (c-myb, c-myc) but increased the level of others (c-fos). The drug-induced lowering of mRNA levels is considered to inhibit the synthesis of proteins specifically required for G1-S transit and maintenance of the proliferation program, amplifying, thereby, the maturation signal emitted by the differentiation factors present in serum and in CM. As a result, expression of the maturation program is initiated.

Differentiation-induced ML-1 cells as targets for transformation by a chemical carcinogen.

The capacity for continuous proliferation (immortalization) of ML-1 human myeloblastic leukemia cells derives from their sensitivity to growth factors and their insensitivity to differentiation factors (DF) at the limiting concentrations at which these are present in the culture medium. Upon increasing the concentration of DF, or after treatment with DNA-specific anticancer agents, the cells exit the proliferation program and differentiate to monocyte/macrophage-like cells (Y. Honma, C. Honma, and A. Bloch, Cancer Res., 46: 6311-6315, 1986). The study reported here shows that when ML-1 cells, induced to differentiate with DF contained in mitogen-stimulated human leukocyte-conditioned medium (CM) are treated with the carcinogen N-nitroso-N-methylurea, their differentiation program is interrupted and proliferation is resumed at a stably increased rate of growth (doubling time, 25.1 versus 31.3 h). This "step-up" transformation is brought about by only a narrow concentration range of carcinogen, acting at a restricted time interval following differentiation induction. The step-up transformed cells are more sensitive to growth factor signals and less sensitive to DF signals than are untreated ML-1 cells. When retreated with a higher concentration of DF and the same concentration of N-nitroso-N-methylurea, the transformed cells undergo a further decrease in doubling time to 21 h. Differentiation-uninduced ML-1 cells do not respond to treatment with N-nitroso-N-methylurea, indicating that differentiation-induced cells, at an early stage of the maturation process, may be the targets for the carcinogen-mediated transformation.

Tumor suppressor proteins as regulators of cell differentiation.

The products of the tumor suppressor genes are considered to function as specific inhibitors of tumor cell growth. In this communication, we present evidence to show that these proteins inhibit tumor cell proliferation by participating in the activation of tumor cell differentiation. The ML-1 human myeloblastic leukemia cells used in this study proliferate when treated with insulin-like growth factor I and transferrin but differentiate to monocytes when exposed to tumor necrosis factor alpha or transforming growth factor beta1, or to macrophage-like cells when treated with both these cytokines. Initiation of proliferation but not of differentiation was followed by a 20- to 25-fold increase in the nuclear level of the DNA polymerase-associated processivity factor PCNA and of the proliferation-specific transcription factor E2F1. In contrast, induction of differentiation but not of proliferation was followed by a 25- to 30-fold increase in the nuclear level of the tumor suppressor proteins p53 (wild type), pRb, and p130/Rb2 and of the p53-dependent cyclin kinase inhibitor p21/Cip1. p53 and p21/Cip1, respectively, inhibit the expression and activation of PCNA, whereas p130 and pRb, respectively, inhibit the expression and activation of E2F1. As a result, G1-S-associated DNA and mRNA synthesis is inhibited, growth uncoupled from differentiation, and maturation enabled to proceed. Where this function of the tumor suppressor proteins is impaired, the capacity for differentiation is lost, which leads to the sustained proliferation that is characteristic of the cancer cell.

Macromolecular and cell cycle effects of different classes of agents inducing the maturation of human myeloblastic leukemia (ML-1) cells.

The effect of various classes of differentiation-inducing agents on macromolecular synthesis was studied in a human myeloblastic leukemia cell line (ML-1). Antineoplastic drugs such as 1-beta-D-arabinofuranosylcytosine, daunorubicin, and actinomycin D caused early inhibition of DNA synthesis, which generally preceded the accrual of differentiation markers. In contrast, retinoic acid and conditioned medium from mitogen-stimulated leukocytes caused a delayed decline in DNA synthesis, which accompanied the appearance of maturing morphology. With 12-O-tetradecanoylphorbol-13-acetate, the decline in DNA synthesis was temporally linked to the onset of maturation, and this agent evidenced some properties of both the antineoplastic agents and the more physiological inducers, retinoic acid and conditioned medium. Antineoplastic agents and conditioned medium, when applied simultaneously, induced differentiation in an additive or synergistic manner, simulating the effects of 12-O-tetradecanoylphorbol-13-acetate. RNA and protein synthesis continued during maturation induced with all these agents, although a partial reduction in RNA synthesis was observed at later time points (greater than or equal to 24 hr). Agents incapable of inducing differentiation, such as cordycepin and cycloheximide, were characterized by a lack of sustained inhibition of DNA synthesis and/or by early (3 hr) inhibition of RNA or protein synthesis. The decline in DNA synthesis caused by the inducing agents was accompanied by decreased cell cycle progression, cells accumulating largely in G1 phase. With daunorubicin and actinomycin D, block of the G1-S transition was evident at 24 hr, whereas with conditioned medium and retinoic acid, accumulation in G1 occurred in a progressive fashion, greater than 77% of cells residing in this phase on Day 6. Maximal inducing doses of 12-O-tetradecanoylphorbol-13-acetate (greater than 80% differentiation) caused an accumulation of cells in G1, as well as an accumulation of cells with a G2-M-phase DNA content (approximately 40%). These observations indicate that early inhibition of DNA synthesis, with sparing of RNA and protein synthesis, is characteristic of the differentiation-inducing antineoplastic drugs examined. These agents may induce differentiation by inhibition of the proliferation path, whereas conditioned medium and retinoic acid may act by the stimulation of differentiation paths. Differentiation can be enhanced by the simultaneous application of agents targeting both of these paths.

Biological and biochemical effects of 2'-azido-2'-deoxyarabinofuranosylcytosine on human tumor cells in vitro.

2-azido-2'-deoxyarabinofuranosylcytosine (Cytarazid), recently synthesized, was found to inhibit the in vitro growth of several human cell lines by 50% at concentrations ranging from 0.06 to 0.2 microM and to prevent the replication of herpes simplex virus types 1 and 2 by 98% at 50 microM. As determined with HeLa cells, the inhibition of cell growth was partially prevented by 2'-deoxycytidine (dCyd) and cytidine but not by uridine or thymidine. Cytarazid proved resistant to deamination by human cytidine/dCyd deaminases purified from acute myelocytic leukemia blast cells and from liver, a property reflected in the inability of tetrahydrouridine to enhance the cytotoxicity of the compound. Cytarazid served as a substrate for cytoplasmic dCyd kinase partially purified from human peripheral chronic lymphocytic leukemia blast cells. At a concentration of 0.4 mM, the nucleoside analog was phosphorylated 2.6 times more effectively by this enzyme than was dCyd, the Km for Cytarazid being 250 microM. In intact HeLa cells, the triphosphate derivative of Cytarazid was the major drug metabolite formed. In these cells, the analog interfered with the incorporation of radiolabeled thymidine into DNA at a concentration and a time interval at which the incorporation of uridine into RNA and amino acids into protein was not inhibited, suggesting that interference with DNA synthesis is a primary drug effect. Further analysis showed that Cytarazid triphosphate interferes with DNA synthesis in intact HeLa cell nuclei and that it inhibits both the alpha- and beta-DNA polymerases purified from HeLa cells in a manner competitive with deoxycytidine triphosphate, with Ki's of 0.6 and 0.7 microM, respectively. Cytarazid triphosphate was not able to replace deoxycytidine triphosphate for the synthesis of DNA in either intact nuclei or in cell-free preparations; but, in the cell-free assay system, the compound was found to interfere with primer-template activity.

Antitumor activity of cytidine dialdehyde and its effects on nucleotide and nucleic acid synthesis.

Cytidine dialdehyde inhibited the growth of leukemia L1210 cells in culture at a 50% inhibitory concentration of 3.5 X 10(-5) M and, when administered i.p. at 200 mg/kg daily for 5 days, increased the mean survival of L1210 tumor-bearing mice by up to 171%. Given by the s.c. or i.v. routes, the compound was ineffective. The ethanol adduct of cytidine dialdehyde, although inactive in cell culture, increased the mean survival of L1210 tumor-bearing mice by up to 225% when administered i.p. but was inactive upon s.c. administration. Exposure of L1210 cells in culture for 25 hr to cytidine dialdehyde at the 50% inhibitory concentration increased the ribonucleoside di- and triphosphate pools, slightly increased deoxyadenosine triphosphate, deoxythymidine triphosphate, and deoxyguanosine triphosphate pools, and caused a pronounced increase in the deoxycytidine triphosphate pool. As determined by the rate of pyrimidine precursor incorporation into nucleic acids, this concentration of drug showed no effect on RNA synthesis but caused a reduction in DNA synthesis to 53% of control. Exposure of L1210 cells for 3 to 6 hr to 10(-4) M cytidine dialdehyde, a concentration which inhibits growth completely, effected an increase in the ribonucleoside di- and triphosphate pools and a rapid decrease of the deoxythymidine triphosphate pool. The deoxycytidine triphosphate and deoxyguanosine triphosphate pools decreased more slowly, and the deoxyadenosine triphosphate pool remained slightly elevated. Analysis of the rate of substrate incorporation into nucleic acids showed that this concentration of drug produced an 80% decrease in RNA synthesis and a 75% decrease in DNA synthesis 3 hr after drug exposure. These results suggest that the mechanism of action of cytidine dialdehyde may be due to its initial interference with DNA synthesis followed by a generalized inhibition of DNA, RNA, and protein synthesis at cytotoxic concentrations.

Inhibition of messenger RNA transcriptional activity in ML-1 human myeloblastic leukemia cell nuclei by antiserum to a c-myb-specific peptide.

Antiserum to a synthetic peptide that defines a hydrophilic region within the putative c-myb translation product was prepared in the rabbit. In lysates from exponentially growing ML-1, human myeloblastic leukemia cells, the antiserum ("anti-myb") reacted with five proteins of Mr 58,000, 75,000, 85,000, 90,000 and 105,000. Of these, only p75 and a trace of p85 were detected, by immunoblotting, in extracts derived from ML-1 cell nuclei. The proteins p58, p75 and p90 were present in readily detectable amounts only in the relatively immature myeloid cell lines ML-1 and HL-60, whereas in the more mature myeloid cell line THP-1 and in the lymphoid line BALL-1 only traces of these proteins were found. p85 and p105 were detected in lysates from all cell lines tested, including myeloid and lymphoid leukemia cells and mouse 3T3 cells. In lysates from ML-1 cells induced to differentiate to monocyte/macrophages or to granulocytes, the concentrations of p58 and p75 decreased in parallel with the cell population moving to maturity; in completely mature populations these two proteins were no longer detectable. In ML-1 cells arrested in G1 by serum depletion, the amount of p58 and p75 and to a smaller extent that of p90 was decreased, whereas the concentration of p85 and p105 remained unchanged. In nuclei from exponentially growing ML-1 cells, the antiserum or its derived immunoglobulin fraction ("anti-myb IgG") inhibited mRNA transcriptional activity by 30%. DNA synthesis was not affected. In contrast, in nuclei from differentiated ML-1 cells, the mRNA transcriptional activity was not significantly inhibited by anti-myb IgG. Similarly, in nuclei from ML-1 cells arrested largely in G1 by serum depletion for 2 days, mRNA transcriptional activity was inhibited by only 11%. Upon supplementation with serum, the mRNA transcriptional activity inhibitable by anti-myb IgG increased in parallel with the increasing rate of cell growth. The difference in total mRNA transcriptional activity observed in nuclei from cells of different growth rate was accounted for by the difference in transcriptional activity inhibitable by anti-myb IgG.(ABSTRACT TRUNCATED AT 400 WORDS)

E. coli bacteremia in comparison to K. pneumoniae bacteremia: influence of pathogen species and ESBL production on 7-day mortality.

In a previous study, we demonstrated prolonged length of hospital stay in cases of extended-spectrum beta-lactamase (ESBL)-positive K. pneumoniae bacteremia compared to bacteremia cases due to E. coli (ESBL-positive and -negative) and ESBL-negative K. pneumoniae. The overall mortality was significantly higher in bacteremia cases resulting from ESBL-positive pathogens but also in K. pneumoniae cases disregarding ESBL-production. In order to examine whether pathogen species rather than multidrug resistance might affect mortality risk, we reanalyzed our dataset that includes 1.851 cases of bacteremia.