[Therapy of moderate cognitive impairment and asthenia in patients with cerebrovascular pathology: results of a prospective observational study]. / Terapiya umerennykh kognitivnykh narushenii i astenii u patsientov s tserebrovaskulyarnoi patologiei: rezul'taty mnogotsentrovoi otkrytoi prospektivnoi nablyudatel'noi programmy.

OBJECTIVE: Obtaining additional data on the efficacy and safety of the drug Prospekta in the treatment of moderate cognitive impairment (MCI) and asthenia in patients with cerebrovascular disease (CVD). MATERIAL AND METHODS: A prospective observational study in more than 40 Russian cities enrolled 232 patients (mean age 61.5±10.0 years) with mild cognitive impairment (MCI), asthenia on ongoing basic nootropic therapy. The presence of MCI was confirmed by the Montreal Cognitive Assessment Scale (MoCA), asthenia - by 10-point Visual Analog Scale (VAS). All patients were prescribed the nootropic medication Prospekta 2 tablets 2 times a day for 8 weeks in addition to the therapy they received. Ultrasound Doppler sonography of the main arteries of the head and magnetic resonance imaging (MRI) of the brain were also assessed. At the end of treatment, the Clinical Global Impression Efficacy Index (CGI-EI) was assessed and the safety of the treatment was evaluated. RESULTS: The baseline severity of cognitive impairment according to the MoCA scale was 21.6 points, severity of asthenia according to the VAS was 6.3 points. According to Doppler flowmetry findings, hemodynamically significant stenosis was revealed in 105 (49.3%) patients, and narrowing of the main vessels without changes in hemodynamic parameters was revealed in 108 (50.7%) patients. According to MRI results, single vascular lesions in the brain matter were detected in 102 (44.0%) patients. The medications with nootropic effect were administered to 144 (62.1%) patients. A positive therapeutic response as improvement of cognitive functions was seen in 93.3% of patients after 8 weeks of taking Prospekta, including 39.4% of patients who had cognitive functions restored to the normal level. No side effects were registered during the observational study. CONCLUSIONS: The nootropic medication Prospekta is effective and safe in treatment of MCI in patients with asthenia with CVD, and improves cognitive function in patients with asthenia with CVD, both in monotherapy and in combination with other nootropic agents.

Not all glucocorticoid-induced obesity is the same: differences in adiposity among various diagnostic groups of Cushing syndrome.

The cAMP signaling pathway is implicated in bilateral adrenocortical hyperplasias (BAHs), which are often associated with ACTH-independent Cushing syndrome (CS). Although CS is invariably associated with obesity and is frequently associated with PKA signaling defects, we recently reported that its different forms appear to also present with variable weight gain and adiposity. The present study was aimed at characterizing further the phenotypic and molecular differences in periadrenal adipose tissue (PAT) among patients with subtypes of CS, by anthropometric/biochemical analyses and quantification of PKA expression and activity in BAHs in comparison to a non-CS group with aldosterone producing adenomas (APAs). Glucocorticoid levels, serum parameters, and BMI were analyzed among a larger patient cohort including those with different forms of CS, APAs, and Cushing disease. Abdominal CT scans were available for a small subset of patients examined for fat distribution. PAT collected during adrenalectomy was assayed for PKA activity, cAMP, and PKA expression. BMI and BMI z-score were lower in adults with PPNAD with PRKAR1A mutations and in pediatric patients with PPNAD with and without PRKAR1A mutations, respectively. Patients with PPNAD had higher cAMP levels in PAT and different fat distribution. Thus, PKA activity in PAT differed between CS diagnostic groups. Increased cAMP and PKA activity may have contributed to phenotypic differences among subtypes of CS. In agreement with the known roles of cAMP signaling in the regulation of adiposity, patients with PPNAD were less obese than other patients with CS.

Comparison of the effects of PRKAR1A and PRKAR2B depletion on signaling pathways, cell growth, and cell cycle control of adrenocortical cells.

The cyclic AMP/protein kinase A signaling cascade is one of the main pathways involved in the pathogenesis of adrenocortical tumors. The PKA R1A and R2B proteins are the most abundant regulatory subunits in endocrine tissues. Inactivating mutations of PRKAR1A are associated with Carney complex and a subset of sporadic tumors and the abundance of R2B protein is low in a subset of secreting adrenocortical adenomas. We previously showed that PRKAR1A and PRKAR2B inactivation have anti-apoptotic effects on the adrenocortical carcinoma cell line H295R. The aim of this study was to compare the effects of PRKAR1A and PRKAR2B depletion on cell proliferation, apoptosis, cell signaling pathways, and cell cycle regulation. We found that PRKAR2B depletion is compensated by an upregulation of R1A protein, whereas PRKAR1A depletion has no effect on the production of R2B. The depletion of either PRKAR1A or PRKAR2B promotes the expression of Bcl-xL and resistance to apoptosis; and is associated with a high percentage of cells in S and G2 phase, activates PKA and MEK/ERK pathways, and impairs the expression of IkB leading to activate the NF-κB pathway. However, we observed differences in the regulation of cyclins. The depletion of PRKAR1A leads to the accumulation of cyclin D1 and p27kip, whereas the depletion of PRKAR2B promotes the accumulation of cyclin A, B, cdk1, cdc2, and p21Cip. In conclusion, although the depletion of PRKAR1A and PRKAR2B in adrenocortical cells has similar effects on cell proliferation and apoptosis; loss of these PKA subunits differentially affects cyclin expression.

Threat bias in mice with inactivating mutations of Prkar1a.

Anxiety disorders are associated with abnormalities in the neural processing of threat-related stimuli. However, the neurobiological mechanisms underlying threat bias in anxiety are not well understood. We recently reported that a Prkar1a heterozygote (Prkar1a(+/-)) mouse with haploinsufficiency for the main regulatory subunit (R1α) of protein kinase A (PKA) exhibits an anxiety-like phenotype associated with increased cAMP signaling in the amygdala. Prkar1a(+/-) mice provide a novel model to test the direct effect of altered PKA expression and subsequent anxiety-like behavioral phenotype on the response to threat. We hypothesized that Prkar1a(+/-)mice would exhibit a bias in threat detection since increased amygdala activity during emotional stimuli is associated with a maladaptive response. We measured behavior and PKA activity in brain areas after exposure to predator or control odor exposure in male Prkar1a(+/-) and wild-type (WT) littermates. Indeed, there were significant differences in the behavioral response to threat detection; WT mice showed the expected response of decrease in exploratory behavior during predator vs. control odor exposure, while Prkar1a(+/-) mice did not alter their behavior between conditions. Basal and total PKA activity was independently associated with genotype, with an interaction between genotype and threat condition. Prkar1a(+/-) mice had higher PKA activity in amygdala and ventromedial hypothalamus in response to predator odor. In contrast, WT mice had higher PKA activity in amygdala and orbitofrontal cortex after exposure to control odor. Dysregulated PKA activity in the amygdala-prefrontal cortex circuitry in Prkar1a(+/-) mice is associated with behavioral phenotype of anxiety and a bias for threat. This is likely related to a failure to inhibit the amydgala response, which is an effect of the genotype. These results suggest that the alteration in PKA signaling in Prkar1a(+/-) mice is not ubiquitous in the brain; tissue-specific effects of the cAMP/PKA pathway are related to threat detection and fear sensitization.

PRKAR1A gene analysis and protein kinase A activity in endometrial tumors.

PRKAR1A codes for the type 1a regulatory subunit (RIα) of the cAMP-dependent protein kinase A (PKA), an enzyme with an important role in cell cycle regulation and proliferation. PKA dysregulation has been found in various tumors, and PRKAR1A-inactivating mutations have been reported in mostly endocrine neoplasias. In this study, we investigated PKA activity and the PRKAR1A gene in normal and tumor endometrium. Specimens were collected from 31 patients with endometrial cancer. We used as controls 41 samples of endometrium that were collected from surrounding normal tissues or from women undergoing gynecological operations for other reasons. In all samples, we sequenced the PRKAR1A-coding sequence and studied PKA subunit expression; we also determined PKA activity and cAMP binding. PRKAR1A mutations were not found. However, PKA regulatory subunit protein levels, both RIα and those of regulatory subunit type 2b (RIIß), were lower in tumor samples; cAMP binding was also lower in tumors compared with normal endometrium (P<0.01). Free PKA activity was higher in tumor samples compared with that of control tissue (P<0.01). There are significant PKA enzymatic abnormalities in tumors of the endometrium compared with surrounding normal tissue; as these were not due to PRKAR1A mutations, other mechanisms affecting PKA function ought to be explored.

Comparison of aldosterone production among human adrenocortical cell lines.

Several human adrenocortical cell lines have been used as model systems for aldosterone production. However, these cell lines have not been directly compared with each other. Human adrenal cell lines SW13, CAR47, the NCI-H295 and its sub-strains and sub-clones were compared with regard to aldosterone production and aldosterone synthase (CYP11B2) expression. Culture media was collected 48 h after incubation, aldosterone secretion was measured and the data were normalized to the amount of cell protein. RNA was isolated for microarray analysis and quantitative RT-PCR (qPCR). The cell lines with the highest aldosterone production were further tested with regard to angiotensin II (Ang II) stimulation. Neither aldosterone nor CYP11B2 transcript were detected in SW13 or CAR47 cells. The aldosterone production by the NCI-H295, H295A, H295R-S1, H295R-S2, H295R-S3, HAC13, HAC15 and HAC50 were 119, 1, 6, 826, 18, 139, 412, and 1 334 (pmol/mg protein/48 h), respectively. H295A and H295R-S1 expressed less CYP11B2 than the commonly used H295R-S3 cells; while NCI-H295, H295R-S2, HAC13, HAC15 and HAC50 expressed 24-, 14-, 3-, 10-, and 35-fold higher CYP11B2 compared with the H295R-S3 cells. When treated with Ang II, NCI-H295, H295R-S2, HAC13, HAC15 and HAC50 showed significantly higher aldosterone production than the basal level (p<0.05). A comparison of the available human adrenal cell lines indicates that the H295R-S2 and the clonal cell lines, HAC13, HAC15 and HAC50 produced the highest levels of aldosterone and responded well to Ang II.

[Biological age and the pain syndrome at diabetic polyneuropathy].

Patients with diabetic polyneuropathy were examined to study their biological age, rate of aging and pain syndrome. More rapid rate of aging was revealed in patients with diabetic polyneuropathy and pain syndrome. Using Duloxetin and Gabapentin is reliable to decrease the display of pain syndrome in diabetic polyneuropathy patients. Against the background of pain syndrome therapy the rate of aging is noticed to decrease along with the regression of pain syndrome. The medicines of Duloxetin and Gabapentin which are used during depression and epilepsy protect against aging.

The role of germline AIP, MEN1, PRKAR1A, CDKN1B and CDKN2C mutations in causing pituitary adenomas in a large cohort of children, adolescents, and patients with genetic syndromes.

The prevalence of germline mutations in MEN1, AIP, PRKAR1A, CDKN1B and CDKN2CI is unknown among pediatric patients with pituitary adenomas (PA). In this study, we screened children with PA for mutations in these genes; somatic GNAS mutations were also studied in a limited number of growth hormone (GH) or prolactin (PRL)-secreting PA. We studied 74 and 6 patients with either isolated Cushing disease (CD) or GH- or PRL-secreting PA, respectively. We also screened four pediatric patients with CD, and four with GH/PRL-secreting tumors who had some syndromic features. There was one AIP mutation (p.Lys103Arg) among 74 CD patients. Two MEN1 mutations that occurred in patients with recurrent or difficult-to-treat disease were found among patients with CD. There was one MEN1 and three AIP mutations (p.Gln307ProfsX104, p.Pro114fsX, p.Lys241X) among pediatric patients with isolated GH- or PRL-secreting PA and one additional MEN1 mutation in a patient with positive family history. There were no mutations in the PRKAR1A, CDKN1B, CDKN2C or GNAS genes. Thus, germline AIP or MEN1 gene mutations are frequent among pediatric patients with GH- or PRL-secreting PA but are significantly rarer in pediatric CD; PRKAR1A mutations are not present in PA outside of Carney complex.

An Intronic mutation is associated with prolactinoma in a young boy, decreased penetrance in his large family, and variable effects on MEN1 mRNA and protein.

Prolactinomas are rare tumors in prepubertal children. A prolactinoma in a young child may be due to sequence variants in genes that are known to cause these tumors ( MEN1, PRKAR1A, AIP). An 11-year-old boy with a macroprolactinoma was treated with cabergoline and the tumor receded. We studied the patient and his family for genetic causes of this tumor. No mutations were present in the coding sequence of PRKAR1A and AIP. A novel heterozygous substitution (IVS3-7 c>a) was identified in intron 3 of MEN1. We also found an additional PCR amplicon that incorporated the entire intron 3 of the gene (210 bp) in the patient's cDNA. The same amplicon was present with lower intensity in some of the control individuals who were not mutation carriers. Intron 3 harbors an in-frame stop codon and its incorporation is predicted to result in a prematurely terminated protein. We conclude that a novel MEN1 variation was identified in a young boy with prolactinoma and six of his relatives who did not present with prolactinoma or other MEN1 related symptoms. This novel MEN1 variation may be associated with low penetrance of the disease. The IVS3-7 c>a defect is suggested to be pathogenic because it is associated with lower menin levels in the cells of these patients, but its consequences may be mitigated by a variety of factors including changes in transcription and translation of the MEN1 gene.

Autoantibody biomarker opens a new gateway for cancer diagnosis.

The list of cancer markers of current interest has grown considerably, but none of the markers used in clinical work is a true tumor marker. These cancer biomarkers are based on the determination of tumor antigens. Here, we report a single method of autoantibody enzyme immunoassay (EIA) screens for a spectrum of serum tumor markers. A comparison of the autoantibody-based EIA to conventional antigen EIA kits, using receiver operating characteristic (ROC) plots, showed that the autoantibody EIA can significantly enhance the sensitivity and specificity of tumor markers. The detection of serum autoantibodies for a spectrum of serum tumor markers, as demonstrated here, suggests that most, if not all, serum cancer biomarkers produce autoantibodies. A unique autoantibody biomarker screening method, as presented here, might therefore facilitate achieving the accurate and early diagnosis of cancer.

Killing the messenger: antisense DNA and siRNA.

Among the technologies available for gene knockdown RNase H-dependent antisense oligonucleotides and RNAi are very popular. Both offer specificity and efficient knockdown of the genes; both are useful tools to study gene functions. Antisense and RNAi methods share many practical problems such as site selection, toxicity at high concentration, and the difficulty of transfection in certain cell types. We will focus in this review on the most important issues in the development of both methods and their possible use in gene-silencing therapy.

Clinical studies in patients with solid tumors using a second-generation antisense oligonucleotide (GEM 231) targeted against protein kinase A type I.

GEM 231 is a second-generation antisense oligonucleotide targeted against the RIalpha regulatory subunit of cAMP-dependent protein kinase type I (PKA-I). Excessive expression of PKA-I is associated with cell proliferation and transformation, and increased levels of secreted extracellular PKA (ECPKA) are found in the serum of cancer patients. Preclinical studies have demonstrated single-agent antitumor activity of GEM 231 in a variety of human cancer xenograft models, and additive or synergistic antitumor activity has been observed with taxane and/or camptothecin-based combinations. Based on prior safety (MTD) data demonstrating dose-dependent, reversible, and cumulative transaminitis, and high peak plasma concentration (Cmax)-dependent changes in activated partial thromboplastin time (aPTT) with GEM 231 2-h twice-weekly infusions, an alternative schedule of GEM 231 given as a single agent was evaluated in patients with advanced solid tumors. Fourteen patients (median age approximately 60 yrs) with advanced solid malignancies received a total of 78 weeks of therapy. GEM 231 was infused via a CADD pump at 80 mg/m2/day (d) for 3 d/wk (n = 1), then for 5 d/wk at 80 (n = 3), 120 (n = 8), and 180 mg/m2/d (n = 2). One cycle was defined as 4 weeks of therapy. Apparent dose dependency for the occurrence of transaminitis was readily reversible. At 180 mg/m2/d, 2 of 2 patients had cycle 1 dose-limiting toxicity (DLT) transaminitis. One patient treated at 120 mg/m2/d experienced grade 3 transaminase elevations after 8 weeks of therapy, but when serum transaminase values rapidly improved he resumed treatment at 80 mg/m2/d for 6 weeks until tumor progression was documented. Another patient at 120 mg/m2/d developed grade 3 esophagitis after 3 weeks, limiting further dosing. One patient (lung cancer) demonstrated stable disease for 9 weeks. Overall, plasma aPTT was minimally prolonged and changes were transient, peaked at the end of each infusion, and were not associated with spontaneous bleeding. A constitutive symptom (e.g., low-grade fatigue) was common, cumulative, and reversible following discontinuation of therapy. Serum ECPKA was measured by enzymatic assay and Western blotting from blood drawn at the beginning and end of each infusion. Serum ECPKA levels demonstrated a trend to decline with the treatment. In addition to single agent schedules, combination trials were undertaken to assess safety and possible interaction of GEM 231 with taxanes (paclitaxel, docetaxel), given once every 3 weeks (one cycle). While trials using the 2-h twice-weekly GEM 231 infusions are ongoing, preliminary results from both studies show that it is safe to combine paclitaxel or docetaxel with GEM 231. Overall, it is also feasible to administer GEM 231 in combination with taxane or nontaxane chemotherapy (e.g., camptothecins). Phase I combination studies are currently underway to further explore the clinical, pharmacokinetic, and biologic profile of GEM 231 with chemotherapy.

Protein kinase A: regulation and receptor-mediated delivery of antisense oligonucleotides and cytotoxic drugs.

Protein kinases help regulate eukaryotic cell division. We investigated the regulation of cAMP-dependent protein kinase A (PKA) and casein kinase (CK) type I activity in normal cells and in cancer. To assess this activity in biopsies we suggest a new parameter--the ratio of CK activity and total PKA activity divided by cAMP concentration: CK/PKA/cAMP. In 98 samples of colon mucosa in normal, inflamed, polyp, and adenocarcinoma cells, we found this parameter to be fairly constant in normal conditions and increased 10-fold in colon cancer; the ratio does not depend on the place of biopsy or the patient's age or sex. Experiments with model systems of concanavalin A-stimulated lymphocytes and regenerating rat liver showed that in normal cell proliferation the parameter increases 2-3-fold, as compared to a 30-fold increase in cancer. Unlike normal cells, malignant cells show CK activation and decrease of cAMP; therefore, PKA activity decreases. This suggests a correlation of CK and PKA activity and significant damage to their regulation at pathological changes of tissue proliferation. To further study concerted CK and PKA regulation we used monoclonal antibodies (mAbs) against cAMP-dependent protein kinase regulatory subunit RKII beta. We produced 11 antibodies in three groups: inhibiting, which block cAMP binding with RII beta and inhibit holoenzyme formation (RS6); activating, which enhance cAMP binding and do not affect holoenzyme formation (RS28); and neutral (RS17). To investigate mAb influence on protein kinase regulation in live cells we permeabilized pheochromocytoma PC12 by digitonin. When used at 5-microM concentration for 5 min, digitonin allowed us to deliver mAb into PC12 cells at 30-34-nM concentration, leaving 68-75% viable cells. Protein kinase activity was measured within 0.5 and 4 h after incorporation of mAbs into cells. After 30 min incorporation, mAb RS6 blocked PKA activation in PC12 cells under the influence of cAMP; other mAbs showed no effect. mAb RS6 caused a 4-fold increase of free C subunit activity 4 h after incorporation. mAb RS38 decreased R2C2 activity and did not influence C subunit activity. The change of free C subunit activity caused by mAb incorporation was followed by a synchronized, well-balanced change of CK type I activity, which suggests a correlation between the two phosphorylation systems of cell proteins.

[Monitoring of etiological agents of acute enteric infections in the Moscow region]. / Monitoring vozbuditelei ostrykh kishechnykh infektsii v regione Moskovskoi oblasti.

The fecal microflora of patients with acute enteric infections (AEI) has been examined on the territory of the Moscow region. The pathogens of high, moderate and low priority levels have been detected. As revealed in this study, shigellae, salmonellae, enteropathogenic and enteroinvasive Escherichia are the etiological agents of bacterial diarrheas on the territory of the Moscow region. The dynamics of the etiological agents of AEI in the region has been established.

[Monitoring of uropathogens and their susceptibility to antibiotics]. / Monitoring uropatogenov i ikh chuvstvitel'nosti k antibiotikam.

Samples of urine collected from patients with complicated urology infection and hospitalized to the Moscow Region Research Clinical Institute in 1986, 1991, 1995 and 1999 were analysed. Of 11,444 samples examined, bacteriuria was estimated in 7143 samples. 9786 strains (29 genus) of bacteria were isolated--56.9 per cent as mono culture and 43.1 per cent as associations. Susceptibility to 21 antibiotic was determined by disk diffusion method for 1607 strains; beta-lactamase production was determined in 198 strains, MIC was determined for 41 antibiotics. Gram-negative rods relative amount among pathogens decreased substantially (84.7 per cent in 1986 against 61.6 per cent in 1999), particularly Enterobacteriaceae (74.7 per cent in 1986 against 41.4 per cent in 1999). Nonfermenting Gram-negative rods (NFGNR) relative amount increased (10.8 per cent against 19.2 per cent), along with Gram-positive cocci (19.8 per cent against 64.2 per cent), particularly coagulasenegative staphylococci (CNS) (10.8 per cent against 35.9 per cent) and enterococci (5 per cent against 16.5 per cent) and candida and fungi (0.5 per cent in 1986 against 15.9 per cent in 1999). At the period 1986-1999 the main pathogens in urology infection were E. coli, Enterobacter spp., NFGNR (including P. aeruginosa), Staphylococcus, CNS, Enterococcus spp. The problem pathogens for urological department were the following: E. coli, Klebsiella spp., Enterobacter spp., Proteus spp., NFGNR including P. aeruginosa, CNS, Enterococcus spp., candida and fungi. At the period 1991-1997 Gram-negative pathogens susceptibility to amikacin, ofloxacin, ciprofloxacin, imipenem, ceftazidime, cefotaxime was not changed in general, Gram-positive cocci (staphylococci and enterococci) retained the same susceptibility to vancomicin, cefamandol and amoxyclave. Staphylococci were also susceptible to amikacin, imipenem, rifampicin, oxacillin, ciprofloxacin, and ofloxacin. Production of beta-lactamase was registered for 38.7 per cent of CNS, 26.5 per cent of E. coli, 38.5 per cent of K. pneumoniae, 25 per cent of P. mirabilis and 55.6 per cent of P. aeruginosa strains.

[Human microflora in the norm and in pathology studied with laser fluorescence]. / Indikatsiia mikroflory cheloveka v norme i patologii s pomoshch'iu lazernoi fluorestsentsii.

On the basis of experimental and clinical data the use of laser fluorescence for indication of microflora in normal and pathological states has been substantiated. Standardized intensity characteristics of fluorescence integrally reflect the presence and activity of the total microflora and its changes in dysbiosis and pyo-inflammatory diseases. The possibility of its use in clinical practice is shown.