Noninvasive diagnostic imaging using machine-learning analysis of nanoresolution images of cell surfaces: Detection of bladder cancer.

We report an approach in diagnostic imaging based on nanoscale-resolution scanning of surfaces of cells collected from body fluids using a recent modality of atomic force microscopy (AFM), subresonance tapping, and machine-leaning analysis. The surface parameters, which are typically used in engineering to describe surfaces, are used to classify cells. The method is applied to the detection of bladder cancer, which is one of the most common human malignancies and the most expensive cancer to treat. The frequent visual examinations of bladder (cytoscopy) required for follow-up are not only uncomfortable for the patient but a serious cost for the health care system. Our method addresses an unmet need in noninvasive and accurate detection of bladder cancer, which may eliminate unnecessary and expensive cystoscopies. The method, which evaluates cells collected from urine, shows 94% diagnostic accuracy when examining five cells per patient's urine sample. It is a statistically significant improvement (P < 0.05) in diagnostic accuracy compared with the currently used clinical standard, cystoscopy, as verified on 43 control and 25 bladder cancer patients.

Potential use of serum insulin-like growth factor 1 and E-cadherin as biomarkers of colorectal cancer.

AIM: Despite many efforts, reliable biomarkers for the prediction and diagnosis of colorectal cancer (CRC) are still missing. Insulin-like growth factor 1 (IGF-1) and E-cadherin are recognized as potential biomarkers, but their diagnostic capacity is largely unexplored in CRC. The aim of this work is to investigate IGF-1 and E-cadherin levels with respect to various characteristics of CRC and to estimate their diagnostic potential. METHOD: Seventy CRC patients and 75 healthy individuals were enrolled. IGF-1 and E-cadherin were determined using enzyme-linked immunosorbent assay. The predictive and diagnostic capacities of IGF-1 and E-cadherin were estimated by logistic regression analysis and by determination of the area under the receiver operating characteristic (ROC) curve (AUC). RESULTS: Concentrations of IGF-1 were lower (P = 0.019) while levels of E-cadherin were higher (P < 0.001) in CRC patients than in controls. IGF-1 concentration decreased in parallel with age and progression of CRC (P = 0.023). Also, IGF-1 was higher in men with CRC than in women (P = 0.003). E-cadherin levels were unaffected by variations in either anthropometric characteristics of CRC patients, or localization, grade and stage of the tumour. Both IGF-1 and E-cadherin were independently associated with CRC (P = 0.040; P < 0.001, respectively). The diagnostic accuracy of IGF-1 was estimated as acceptable (AUC = 0.757; P < 0.001), while the diagnostic accuracy of E-cadherin was outstanding (AUC = 0.954; P < 0.001). CONCLUSIONS: Decreased IGF-1 and increased E-cadherin levels were found in CRC patients. IGF-1, but not E-cadherin, concentrations differed according to age, gender and stage of CRC. Both markers were independently associated with the presence of the disease, while E-cadherin demonstrated high diagnostic accuracy.

Raman microscopy for noninvasive imaging of pharmaceutical nanocarriers: intracellular distribution of cationic liposomes of different composition.

Nanotechnology is playing an increasing role in targeted drug delivery into pathological tissues. Drug-loaded pharmaceutical nanocarriers can be delivered into diseased sites by passive targeting (spontaneous accumulation of nanocarriers in the areas with affected vasculature) or by active targeting (via site-specific ligands attached to the surface of drug-loaded nanocarriers). Subsequent level of targeting requires cellular internalization of nanocarriers and their specific association with certain individual cell organelles. The control over intracellular distribution of pharmaceutical nanocarriers requires effective and noninvasive methods of their visualization inside cells. In an attempt to enhance cellular internalization of pharmaceutical nanocarriers and their association with mitochondria specifically, we have prepared three types of cationic liposomes and investigated their intracellular distribution. The analysis was performed using Raman microspectroscopy in order to provide morphological information as well as biochemical signatures of the sample. It was demonstrated that Raman microscopy allows evaluation of the extent of mitochondrial association depending on the liposome composition.

Novel medical therapies of recurrent and metastatic gastroenteropancreatic neuroendocrine tumors.

Neuroendocrine tumors (NETs) of the gastrointestinal tract and pancreas are slow-growing but commonly advanced malignancies with increasing incidence and prevalence. While locoregional disease can be effectively managed with resection, treatment of recurrent, progressive or metastatic disease has until recently been limited to palliative embolization and cytoreducitve surgery, with cytotoxic chemotherapeutic agents being the last resort. However, novel molecular targeted therapies inhibiting malignant cell proliferation and neoangiogenesis, as well as new cytotoxic chemotherapy drugs and somatostatin analogues, are all being investigated for their potential use in advanced neuroendocrine tumors. Long-acting release forms of octreotide have been shown to not only improve symptoms in carcinoid syndrome but to also delay progression of gastrointestinal NETs. On the other hand, phase III trials have demonstrated everolimus (with octreotide) and sunitinib to increase progression-free survival in pancreatic NETs. Use of bevacizumab has also shown promise in a phase II study, and results of an ongoing phase III trial comparing it to interferon are eagerly expected. Use of radiolabeled somatostatin analogues is still under investigation, though several phase II studies are encouraging. New cytotoxic agents, most notably temozolomide and capecitabine, are already in use, but their relative effectiveness compared to streptozocin in pancreatic NETs is yet to be determined.

Accuracy and reproducibility of two scales in causality assessment of unexpected hepatotoxicity.

WHAT IS KNOWN AND OBJECTIVE: There is almost no published information about reliability of scales for causality assessment in hepatotoxicity at pharmacovigilance centres. The aim of this study was to compare two commonly used scales in cases of unexpected hepatotoxicity, in evaluating their accuracy and reproducibility at pharmacovigilance centres (in signal detection). METHODS: Two scales [Council for International Organizations of Medical Sciences or Rousel Uclaf Causality Assessment Method (CIOMS/RUCAM) and NARANJO] were compared in 19 cases of unexpected hepatotoxicity reported during 2004-2009. Data of the cases (with initial clinical assessments) were collected by a network of medical specialists using a structured reporting form. Later, two independent observers assessed each case using both scales. The accuracy and reproducibility of the scales were analysed by Kappa weighted (Kw) test. RESULTS: Both scales (CIOMS/RUCAM vs. NARANJO) showed moderate agreement with the initial clinical assessments (accuracy) for observer A (Kw: 0·56 vs. 0·60) and substantial agreement for observer B (Kw: 0·72 vs. 0·70), with high agreement between observers (Kw: 0·84 vs. 0·67). Both observers (A vs. B) found low agreement between scales (Kw: 0·21 vs. 0·50), with lower scores for the CIOMS/RUCAM scale in 11 and nine cases, respectively. For an early perception of unexpected serious reactions, the scale is more useful if it is not asked for 'previous knowledge' and if it gives higher causality score. WHAT IS NEW AND CONCLUSION: The CIOMS/RUCAM scale showed similar accuracy, but better reproducibility (agreement between observers) than the NARANJO scale, and therefore is recommended for use at pharmacovigilance centres. Fine-tuning of the CIOMS/RUCAM method could contribute to better detection of unexpected hepatotoxicity.

Lipid profile and lipid oxidative modification parameters in the first trimester of high- risk pregnancies - possibilities for preeclampsia prediction.

OBJECTIVES: The goal of this study was to investigate metabolic changes in lipids and oxidative stress parameters in the first trimester of pregnancy with the more specific aim of estimating the significance and strength of researched parameters in the prediction of preeclampsia. DESIGN AND METHODS: The study included 87 high-risk pregnant (HRG) female subjects, 14 with developed preeclampsia (PEC) and 43 healthy pregnant female subjects matched for gestational age (CG). Thiobarbituric acid-reactive substances (TBARS) concentration, lipid hydroperoxides (LOOH), pro-oxidant antioxidant balance (PAB) and total oxidative status (TOS) were measured as oxidative stress markers, while total antioxidant capacity (TAC) was measured as an antioxidative defense parameter. The Atherogenic Index of Plasma (AIP) was calculated as the base 10 logarithm of the ratio of the plasma concentration of triglycerides (TG) to the plasma concentration of high-density lipoprotein cholesterol (HDL-C), with each concentration expressed in mmol/L. RESULTS: The results have shown that lipid indices, especially AIP, were significantly higher in the first trimester of HRG (p < 0.001) and PEC (p < 0.001). Oxidative stress parameters were significantlly higher, while TAC was significantly lower in HRG vs. CG [0.7 ± 0.15 vs 1.1 ± 0.16; (p < 0.001)] and in PEC [0.6 ± 0.12 vs 1.1 ± 0.16; (p < 0.001)] vs. CG. Also, in the HRG, results have shown an independent association of AIP with the preeclampsia development (p < 0.05), while placental growth factor did not show the expected level of significance (p = 0.648). Analysis of the Receiver Operating Characteristics (ROC) curves indicated that certain parameters included in the research model have very good diagnostic accuracy for preeclampsia (AUC = 0.856). CONCLUSIONS: AIP is associated with high-risk pregnancies. Furthermore, our results firmly underscored AIP as a potential marker for preeclampsia prediction.

Gut-associated lymphoid tissue, gut microbes and susceptibility to experimental autoimmune encephalomyelitis.

Gut microbiota and gut-associated lymphoid tissue have been increasingly appreciated as important players in pathogenesis of various autoimmune diseases, including multiple sclerosis. Experimental autoimmune encephalomyelitis (EAE) is an animal model of multiple sclerosis that can be induced with an injection of spinal cord homogenate emulsified in complete Freund's adjuvant in Dark Agouti (DA) rats, but not in Albino Oxford (AO) rats. In this study, mesenteric lymph nodes (MLN), Peyer's patches (PP) and gut microbiota were analysed in these two rat strains. There was higher proportion of CD4(+) T cells and regulatory T cells in non-immunised DA rats in comparison to AO rats. Also, DA rat MLN and PP cells were higher producers of pro-inflammatory cytokines interferon-γ and interleukin-17. Finally, microbial analyses showed that uncultivated species of Turicibacter and Atopostipes genus were exclusively present in AO rats, in faeces and intestinal tissue, respectively. Thus, it is clear that in comparison of an EAE-susceptible with an EAE-resistant strain of rats, various discrepancies at the level of gut associated lymphoid tissue, as well as at the level of gut microbiota can be observed. Future studies should determine if the differences have functional significance for EAE pathogenesis.

Medroxyprogesterone acetate: a steroid with potent progestational activity but low receptor affinity in the guinea pig uterus.

The guinea pig progestin receptor appears to be unique among mammalian receptors studied to date in that it displays a low binding affinity for some biologically potent 17 alpha-substituted progestinss. [3H]Medroxyprogesterone acetate (MPA) was synthesized and used to investigate this dichotomy between binding affinity and biological activity. The comparison of cytoplasmic and nuclear receptor binding characteristics suggested that progesterone and MPA were bound to the same receptor but with different affinities. Following an intravenous injection of 3H-steroids, the plasma level of progesterone was lower than that of MPA at all time points. Correspondingly, for up to 6 hours following steroid administration, progesterone levels were lower in uterine cytoplasm and higher in nuclei than those of MPA. However, by 24 hours, MPA nuclear levels were higher than those of progesterone, in accordance with plasma levels. We conclude that the potent biological activity of MPA relative to progesterone is due in part to its slower rate of metabolism and longer nuclear retention.

The in vivo metabolism of progestins. I. The metabolic clearance rates of progesterone and medroxyprogesterone acetate in the dog.

[3H]Medroxyprogesterone acetate (MPA) was synthesized by selective catalytic tritiation of the delta1-olefinic bond of 6alpha-methyl-17alpha-hydroxy-pregn-1,4-diene-3,20 dione acetate. [14C]MPA was synthesized by acetylation of 6alpha-methyl-17alpha-hydroxy-pregn-4-ene-3,20-dione with [14C]acetic anhydride. These radioactive materials were used to determine the MPA metabolic clearance rates (MCRMPA)and volumes of distribution (VoMPA) in dogs by the single injection technique. The metabolism of progesterone was also studied in the same animals. The MCRMPA (696 +/- 51 l/day) was only one-half that of progesterone (1332 +/- 59). By contrast, the volumes of distribution for the two steroids were similar. The metabolic clearance rates and the volumes of distribution for MPA and progesterone did not change during treatment with amino-glutethimide, a drug which is known to alter steroid metabolism in man.

Synthesis and properties of hydroxyapatite/poly-L-lactide composite biomaterials.

Calcium hydroxyapatite (HAp) and poly-L-lactide (PLLA) were synthesized chemically. The obtained HAp was of high purity and, after special thermal treatment, of high crystallinity as well. Synthesis of PLLA was performed using L-lactide as a monomer and nontoxic initiator. In this way a polymer of large molar weight (about 400,000) was obtained. The HAp and PLLA obtained were used as constituents of the HAp/PLLA composite biomaterial, a potential material for implants. The composite was obtained by mixing completely dissolved PLLA with granules of HAp. The composite was compacted by cold and hot pressing at pressures of 49.0-490.5 MPa and temperatures of 20-184 degrees C. The material obtained at optimum process parameters had a density of 99.6% and compressive strength of 93.2 MPa.

The in vivo metabolism of progestins. IV. The metabolic clearance rate and plasma binding of 6alpha-methylpregn-4-ene-3, 20-dione in women.

[3H]6alpha-methylprogesterone (6MP) was synthesized by selective catalytic tritiation of the delta1-olefinic bond of 6alpha-methylpregna-1,4-diene-3,20-dione. The metabolic clearance rate of 6MP (MCR6MP) was determined in 6 women by the single injection technique. The plasma MCR6MP was 4047 +/- 298 L/day (59 +/- 15 L/day/kg) which was higher than the MCR of progesterone and medroxyprogesterone acetate (6alpha-methyl-17alpha-hydroxy-pregna-4-ene-3,20-dione acetate). The high clearance was not due to binding or metabolism of 6MP by red cells. Although 6MP was bound to CBG with a lower affinity than progesterone, this could not entirely explain the high MCR6MP. When considered with the reports of progesterone and medroxyprogesterone acetate clearance, the present studies suggest that the 6alpha-substitution of progesterone leads to an increased rate of steroid metabolism in women.

PIP: In vivo research has shown that the gestagenic potency of MPA (medroxyprogesterone acetate) is greater than that of progesterone in virtually all species tested. It was hypothesized that this was due to a slower rate of metabolism of MPA relative to progesterone. In women, the clearance rate of the 2 substances was found to be similar, suggesting that the functional groups at the 6alpha- and 17alpha-positions might not influence the rate of progestin metabolism in humans as had been suspected. To test this hypothesis, independent effects of the 6alpha-methyl and the 17alpha-acetoxy substitutions on the metabolism of progesterone were examined. For this purpose, 6alpha-methylprogesterone was synthesized for metabolic studies. The clearance rate of this substance, tested by plasma concentrations, was found to be higher than that of either MPA or progesterone. This rate was not attributable to binding or metabolism by red cells. The differences in clearance rates do not seem to be related to steroid-protein interactions in plasma.

In vitro metabolism of progestins. III. The metabolic clearance rate of medroxyprogesterone acetate in monkeys.

3H-medroxyprogesterone acetate (MPA) was synthesized by selective catalytic tritiation of the delta1-olefinic bond of 6alpha-methyl-17alpha-hydroxy-pregna-1,4-diene-3,20-dione acetate. The metabolic clearance rate of MPA (MCRMPA) was determined in 9 female Rhesus monkeys by the single injection technique. The blood MCRMPA was 201 +/- 19 L/day (42.2 +/- 4.0 L/day/kg) which was approximately the same as that reported for progesterone clearance in the monkey. We conclude that a greater biological activity of MPA compared to progesterone cannot be related to its prolonged retention in the blood. Although both MPA and amino-glutethimide alter the rate of steroid metabolism in some species, neither of these agents influences the metabolic clearance rate of 3H-MPA in the monkey.

PIP: 3 H-medroxyprogesterone acetate (MPA) was synthesized by selective catalytic tritiation of the delta 1-olefinic bond of 6alpha methyl-17alpha-hydroxypregna-1,4-diene-3,20-dione acetate. The (MCR-MPA) metabolic clearance rate of MPA was determined in 9 female Rhesus monkeys by the single injection technique. The blood MCR-MPA was 201 + or - 19 L/day (42.2 + or - 4.0 L/day/kg) which was approximately the same as that reported for progesterone clearance in the monkey. We conclude that a greater biological activity of MPA compared to progesterone cannot be related to its prolonged retention in the blood. Although both MPA and aminoglutethimide alter the rate of steroid metabolism in some species, neither of these agents influences the metabolic clearance rate of 3H-MPA in the monkey.

Oxidative degradation of glycosphingolipids revisited: a simple preparation of oligosaccharides from glycosphingolipids.

In order to cleave gangliosides and isolate the oligosaccharide portion, the allylic nature of OH-3 of the sphingenine base was utilized in its selective oxidation to a ketone group by 2,3-dichloro-5,6-dicyanobenzoquinone. Triethylamine treatment of the oxidation products resulted in the beta-elimination of the intact oligosaccharide. The isolation of the pure oligosaccharide from the modified ceramide residue and unreacted ganglioside was obtained by liquid chromatography. Preliminary investigations suggest that the same reaction conditions can be used for an analogous elimination of oligosaccharides linked to the serine or threonine residues of glycoproteins.

Mental illness and substance use among sheltered homeless persons in lower-density population areas.

OBJECTIVE: The prevalence of mental illness and substance abuse in homeless populations has been studied primarily in large urban areas. This study examines a sheltered homeless population in two counties of lower-density population, Dauphin and Cumberland counties in central Pennsylvania, to assess the prevalence of mental illness and substance abuse. METHODS: A total of 81 homeless adults from nine emergency shelters were interviewed using a structured questionnaire. RESULTS: The estimated lifetime prevalence rate of major depressive disorder was 26.6 percent; 6.4 percent of the sample showed evidence of psychotic thinking. Almost one-third reported previous hospitalization for emotional problems, and about one-third reported a suicide attempt. The estimated lifetime prevalence rate of alcohol or drug abuse or dependence was almost 60 percent. CONCLUSIONS: Although mental illness, especially psychosis, and substance abuse may be somewhat less prevalent among homeless persons in lower-density population areas than in large urban areas, they are nevertheless significant problems.

Spectral detection of micro-metastases and individual metastatic cells in lymph node histology.

The detection of micro-metastases and individual metastatic cells in lymph node tissue by spectral methods is summarized. These methods are based on instrument-based acquisition of thousands of infrared spectra of individual tissue pixels from the tissue section, and analysis of the resulting spectral hypercube by multivariate algorithms. The method of infrared image acquisition, followed by multivariate analysis, is henceforth referred to as Spectral Histopathology (SHP). SHP produces pseudo-color images of tissue sections which reveal details that compare very favorably with images collected from hematoxylin/eosin (H and E) stained tissues in that the same tissue structures are detected. However, the infrared results are based on objective and reproducible measurements and do not depend on subjective interpretation. One of the major topics of this paper is the comparison of spectral patterns observed for the same cancer type from different patients. While this is easy in some tissue types, we found it to be difficult in tissues of very different cellularity, or tissue sections that exhibit high levels of inflammatory response. In both cases, spectral quality will be compromised due to confounding effects resulting from scattering effects. The correction of these effects now permits the direct comparison of different patient samples, and paves the way for diagnostic algorithms for cancer detection to be developed.

[Interactions of the most frequently used drugs in the treatment of angina pectoris]. / Interakcije lekova koji se najcesce koriste u lecenju angine pektoris.

It seems that knowledge and evaluation of drug-interactions with organic nitrates, beta-blockers and calcium-channel blockers are of great importance in relation to patients with chronic diseases, who usually take more than one drug, which is in conjunction with the effect of their ageing. Organic nitrates are safe and the most important drug-group in angina pectoris therapy. Beta-adrenoreceptor blocking agents and calcium-channel blockers are involved in interactions with many different drugs (twenty interactions), but only three interactions are evaluated as interactions of high clinical significance (adrenaline, cimetidine and anti-thyroid agents with beta-blockers, and anti-arrhythmic agents, beta-blockers and cyclosporin with calcium-channel blockers), while the others are of moderate or minimal significance. The most desired interactions of these drugs can be avoided: adjustment of dosage, avoidance of fixed-combination, use of adequate form of drug, or alternative drug and, if necessary, elimination of one drug from therapy. Knowledge of these drug-interactions is important for physician's routine practice either in primary health-care or in hospital conditions.

A novel affinity column for isolation of androgen binding protein from rat epididymis.

An androgen affinity column was synthesized by covalently linking 3-oxo-17beta-hydroxy-5alpha-androstan-17alpha-(6-hexanoic acid) to cyanogen bromide activated Sepharose through a dipropyldiamine side arm. This column was designed to recover androphilic proteins from homogenates rich in nonspecific esterases. An extract of rat epididymis was adsorbed on the affinity column after partial purification by ammonium sulfate precipitation. The column was washed with 1 M KCl and the androgen binding protein eluted with 17beta-hydroxy-5alpha-androstan-3-one resulting in a 1,100-fold increase in specific activity. This protein had the same mobility on polyacrylamide gels and the same estimated molecular weight (135,000 daltons by gel filtration) as androgen binding protein in the original extract. By contrast, electrophoresis on sodium dodecyl sulfate containing gels yielded 2 bands with estimated molecular weights of 42,000 and 47,000 daltons. These observations are consistent with a subunit structure for rat epididymal androgen binding protein.