Oncogenic KRAS-associated gene signature defines co-targeting of CDK4/6 and MEK as a viable therapeutic strategy in colorectal cancer.

Therapeutic strategies against KRAS mutant colorectal cancers are developed using cell line models, which do not accurately represent the transcriptome driven by oncogenic KRAS in tumors. We sought to identify a KRAS-associated gene signature from colorectal tumors to develop a precise treatment strategy. Integrative analysis of quantitative KRAS mutation detection and matched gene expression profiling in 55 CRC bulk tumors was carried out to define a gene signature enriched in CRC tumors with high KRAS mutation. The KRAS-associated gene signature identified exhibits functional enrichment in cell cycle and mitosis processes, and includes mitotic transcription factor, FOXM1. Combination treatment of CDK4/6 inhibitor Palbociclib and MEK inhibitor PD0325901 was tested in KRAS-mutant, BRAF-mutant CRC, normal colon epithelial lines and xenografts models to determine their efficacy and toxicity and to monitor the changes in the gene signature. Inhibiting CDK4/6, an upstream regulator of FOXM1, and MEK synergistically depleted FOXM1 and KRAS-associated gene signature, suggesting that CDK4/6 and MEK regulate the KRAS gene signature. The combined inhibition of CDK4/6 and MEK elicited a robust therapeutic response in KRAS-dependent and BRAF-mutant CRC, both in vitro and in vivo and this correlated with downregulation of the KRAS-associated gene signature. Our preclinical study demonstrated the efficacy of Palbociclib and PD0325901 combinatorial treatment selectively in KRAS-dependent and BRAF-mutant CRC but not in normal colon epithelial cells. The KRAS-associated gene signature could facilitate the identification of responsive metastatic CRC to this therapeutic strategy in clinical settings.

[Prevention of the development and importation of infections in members of the Army of the Czech Republic participating in foreign missions]. / Prevence vzniku a importu nákaz u vojáku Armády Ceské republiky v souvislosti s plnením ukolu v zahranicí.

Over the last years, Czech soldiers became involved in several foreign missions. In 2001-2003 more than 4000 persons participated in such missions. Since their activities are typically associated with high health risks including that of acquiring infectious diseases, the significance of prevention is underlined. Preventive antiepidemic measures taken in soldiers of the Army of the Czech Republic before their deployment and upon return home are described. Prior to being dispatched on a foreign mission any soldier is screened at the department for occupational diseases at the military hospital. Such a screening enables to identify persons with impaired health who are unfit for the mission. Upon return home any soldier is screened for infectious diseases. The scope of the screening varies with geographical area where the mission took place. In the case of missions to areas at high epidemiological risk such as Afghanistan and Iraq, substantially enlarged laboratory screening is conducted with an emphasis on serodiagnosis of infectious diseases. An important step in the prevention of infectious diseases is vaccination prior to mission deployment. The vaccination schedule has developed over years to currently include vaccination against tetanus, viral hepatitis A and B, typhoid fever, type A and C meningococcal meningitis and poliomyelitis and is being further modified in agreement with the latest recommendations and known risks. Soldiers can also be vaccinated against other diseases such as influenza and rabies. The prevention of infectious diseases as described in this article is based on travel medicine applied to the military environment.

Screening for mutations of 21-hydroxylase gene in Hungarian patients with congenital adrenal hyperplasia.

Congenital adrenal hyperplasia (CAH) is a group of autosomal recessive disorders, causing impaired secretion of cortisol and aldosterone from the adrenal cortex, with subsequent overproduction of adrenal androgens. The most common enzyme defect causing CAH is steroid 21-hydroxylase deficiency. To determine the mutational spectrum in the Hungarian CAH population, the CYP21 active gene was analyzed using PCR. A total of 297 Hungarian patients with 21-hydroxylase deficiency are registered in the 2nd Department of Pediatrics, Budapest, Hungary, and their clinical status was evaluated. Blood samples for CYP21 genotype determination could be obtained from 167 patients (representing 306 unrelated chromosomes and 56.2% of the total group of patients). Eight of the most common mutations were screened [In2 (intron 2 splice mutation), I172N, Del (Del: apparents large gene conversion), Q318X, R356W, 1761Tins, ClusterE6, V281L] using allele-specific amplification. The most frequent mutation in the Hungarian CAH population was found to be In2. Our results have shown a good genotype/phenotype correlation in case of most mutations; the In2 mutation is associated mostly with the severe form of the disease, whereas I172N was expressed in a wide spectrum of phenotypes. 1999)

Regulation of endothelin release from human brain microvessel endothelial cells.

After approval by the Local Ethical Committee, brain microvessel endothelial cells from human cadavers were isolated by enzymatic digestion and gradient centrifugation. Basal levels of endothelin-1 (ET) in the supernatant increased over time (3 h, 18.3 +/- 4.3 pg/ml; 6 h, 31.3 +/- 1.1 pg/ml; 24 h, 88.0 +/- 5.7 pg/ml; 48 h, 86.3 +/- 11.2 pg/ml, mean +/- SD). Tumor necrosis factor-alpha (TNF-alpha) (270 U/ml) increased ET concentration dose-dependently: 3 h, 190 +/- 70%; 24 h, 217 +/- 39%; 48 h, 207 +/- 5%; TNF-alpha at 210 U/ml: 3 h, 137%; 24 h, 170%; 48 h, 212% (values are relative changes from control, run in parallel to the stimulated wells). Interleukin-1 alpha (IL-1 alpha) (38.8 U/ml) also increased ET dose-dependently: (3 h, 129%; 24 h, 161%; 48 h, 212%; IL-1 alpha 1.4 U/ml: 3 h, 116%; 24 h, 122%; 48 h, 180%). Lipoprotein (a) (Lp(a)) had a dual effect on ET, increasing ET in the first 3 h but reducing it by the end of the 48-h observation period. This effect was not dose-dependent in the concentration range tested: Lp(a) 450 micrograms/ml; 3 h, 188%; 24 h, 91%; 48 h, 85%; Lp(a) 360 micrograms/ml: 3 h, 180%; 24 h, 94%; 48 h, 52%). Lp(a) reduced the stimulatory effect of cytokines on ET release. Maximal values at 48 h were TNF-alpha 207%, TNF-alpha + Lp(a) 91%, IL-1 alpha 212%, IL-1 alpha + Lp(a) 64%. In HPLC analysis, the total ET-like immunoreactivity co-eluted with the synthetic human ET standard. A cell culture of human brain microvessel endothelial cells was established. TNF-alpha and IL-1 alpha increased ET secretion, whereas Lp(a) had a dual effect. When given together, Lp(a) reduced the effect of cytokines on ETs.

Brain Na+/K+-ATPase activity in two anoxia tolerant vertebrates: crucian carp and freshwater turtle.

The crucian carp (Carassius carassius) and freshwater turtles (Trachemys scripta) are among the very few vertebrates that can survive extended periods of anoxia. The major problem for an anoxic brain is energy deficiency. In the brain, the Na+/K+-ATPase is the single most ATP consuming enzyme, being responsible for maintaining ion gradients. We here show that the Na+/K+-ATPase activity in the turtle brain is reduced by 31% in telencephalon and by 34% in cerebellum after 24 h of anoxia. Both changes were reversed upon reoxygenation. By contrast, the Na+/K+-ATPase activities were maintained in the anoxic crucian carp brain. These results support the notion that crucian carp and turtles use divergent strategies for anoxic survival. The fall in Na+/K+-ATPase activities displayed by the turtle is likely to be related to the strong depression of brain electric and metabolic activity utilized as an anoxic survival strategy by this species.

Role for adenosine in channel arrest in the anoxic turtle brain.

The remarkable ability of the turtle brain to survive anoxia is based on its ability to match energy demand flexibly to energy production. Earlier studies indicate that reduced ion leakage is an important mechanism for energy conservation during anoxia. We tested the hypothesis that extracellular adenosine plays a role in the reduction of K+ flux (channel arrest) that occurs in the anoxic turtle brain. Changes in extracellular K+ concentration ([K+]o in the in situ brain of the turtle Trachemys scripta were monitored following inhibition of Na+/K(+)-ATPase with ouabain. The time to reach full depolarization ([K+]o plateau) was three times longer in the anoxic brain than in normoxic controls and the initial rate of K+ leakage was reduced by approximately 70%. Superfusing the brain before the during anoxia with the general adenosine receptor blocker theophylline, or the specific adenosine A1 receptor blocker 8-cyclopentyltheophylline, significantly shortened the time to full depolarization in the ouabain-challenged anoxic brain and increased the rate of K+ efflux. The results suggest that adenosine A1 receptors are involved in the expression of anoxia-induced ion channel arrest in the turtle brain.

Contraction of human brain endothelial cells induced by thrombogenic and fibrinolytic factors. An in vitro cell culture model.

BACKGROUND AND PURPOSE: Vasogenic brain edema is a frequent complication of ischemic stroke. The mechanism of the blood-brain barrier opening that underlies the edema formation is poorly understood. In the present study we examined the response of endothelial cells cultured from adult human brain to thrombogenic and fibrinolytic factors that possibly accumulate in the occluded vascular segments in ischemic stroke. METHODS: The changes in the morphology of cultured human brain microvascular endothelial cells were observed by phase-contrast light microscopy and quantified with computerized morphometry. RESULTS: Active proteases (eg, thrombin, plasmin, urokinase) as well as heparin and protamine, but not fibrinogen and antithrombin III, produced significant changes in endothelial cell morphology. Two shape patterns of contraction were observed: protamine treatment resulted in rounded cells with a decrease in both cell perimeter and area, whereas all other agents induced spiderlike cell morphology with increased perimeter and reduced area. The rate of contraction was dose dependent, and at comparable enzyme concentrations plasmin produced faster contraction than thrombin. The observed changes were reversed 3 hours after abrogating the treatment. CONCLUSIONS: In an in vitro model we have demonstrated that factors involved in thrombus formation and dissolution induce endothelial cell contraction, which could affect focally the permeability of the blood-brain barrier by opening paracellular avenues between endothelial cells in vivo. Thus, the genesis of brain edema in thromboembolic stroke or occasionally during fibrinolytic therapy can be attributed in part to the contact of these factors with the microvascular endothelium.

Vibration disappearance threshold may be raised by measuring it.

The influence of duration and intensity of the initial level of vibration on vibration perception threshold was studied in 15 healthy young subjects (aged 27.9 +/- 7.6 years) with a Vibrameter (Somedic AB Sweden). The threshold for increasing vibration corresponded well to the normal values published by the manufacturer. The thresholds for decreasing vibration were measured with three different starting conditions: starting from a low level of vibration, a high level, and from a low level sustained for one minute. Higher intensity and longer duration of the initial vibration raised the disappearance threshold significantly. Vibration disappearance thresholds can be influenced by measuring them. As a result, the investigation of the vibration sense calls for strict control over intensity and duration of the stimuli.