Stratum corneum markers of innate and T helper cell-related immunity and their relation to the disease severity in Croatian patients with atopic dermatitis.

BACKGROUND: Atopic dermatitis (AD) presents with the wide spectrum of clinical phenotypes within and between various populations. Recent study showed low frequency of filaggrin loss-of-function (FLG LOF) mutations in Croatian AD patients. At present, there are no data on biomarkers of immune response in Croatian AD patients that might be useful in the selection and monitoring of novel immune therapies. OBJECTIVES: To investigate levels of cytokines of various signature in the stratum corneum (SC) collected from lesional and non-lesional skin of AD patients and healthy controls and to evaluate their relationship with the severity of disease and skin barrier function. METHODS: SC samples were collected from 100 adult patients with moderate-to-severe AD and 50 healthy controls. The levels of 21 cytokines were measured by multiplex immunoassay. We conducted machine learning analysis to assess whether a small number of cytokine measurements can discriminate between healthy controls and AD patients and can predict AD severity (SCORAD). RESULTS: The SC levels of thirteen cytokines representing innate immunity, Th-1, Th-2 and Th-17/22 immune response showed significant differences between healthy and AD skin. Our analysis demonstrated that as few as three cytokines measured in lesional skin can discriminate healthy controls and AD with an accuracy of 99% and that the predictive models for SCORAD did not achieve a high accuracy. Cytokine levels were highly correlated with the levels of filaggrin degradation products and skin barrier function. CONCLUSIONS: Stratum corneum analysis revealed aberrant levels of cytokines representing innate immunity, Th-1-, Th-2- and Th-17/22-mediated immune response in Croatian AD patients. Increased Th-2 cytokines and their strong association with natural moisturizing factor (NMF) can explain low NMF levels despite of low frequency of FLG LOF mutations in Croatian population. Predictive models for SCORAD identified cytokines associated with SCORAD but warrants further investigation.

Filaggrin loss-of-function mutations and levels of filaggrin degradation products in adult patients with atopic dermatitis in Croatia.

BACKGROUND: FLG loss-of-function mutations (FLG LOF) represent the strongest genetic risk factor for atopic dermatitis (AD) and are associated with early-onset and more severe disease. The prevalence of FLG mutations varies greatly across Europe. At present, there are no data on FLG mutation prevalence in Croatian AD patients. OBJECTIVES: To investigate the prevalence of FLG LOF mutations in adult patients with AD and healthy controls. Next to measure the stratum corneum (SC) levels of filaggrin degradation products (NMF), transepidermal water loss (TEWL) and pH in lesional and non-lesional skin. METHODS: We recruited 100 AD patients with moderate to severe disease and 50 healthy controls. They were screened for three FLG mutations (R501X, 2282del4 and R2447X). Samples of the SC for NMF analysis were collected by adhesive tapes. TEWL and skin surface pH levels were determined on the lesional and non-lesional skin. RESULTS: The combined mutation frequency was 4% in the AD group, and all patients with FLG mutations were homozygous carriers. In the control group, no mutations were found. The most common FLG mutation in AD patients was 2282del4 (3%), followed by R501X (1%). As compared to healthy controls, NMF values were strongly reduced in lesional skin; however, no significant difference was found for non-lesional skin. AD patients had elevated TEWL in both lesional and non-lesional skin. The same pattern was observed for pH. CONCLUSIONS: Our study expands understanding of the landscape of FLG mutations in the European population. The low frequency of FLG mutations and similar levels of filaggrin degradation products in healthy controls and in non-lesional skin of AD patients suggest that filaggrin deficiency does not confer a major risk for AD in the Croatian population.

Site-Specific Deuteration of Polyunsaturated Alkenes.

Selective deuteration of drugs and biologically relevant molecules is becoming increasingly important in the pharmaceutical industry. Site-selective isotopic reinforcement of polyunsaturated fatty acids (PUFAs) at their bis-allylic sites has been identified as a unique approach in preventing oxidative damage in these molecules, which had been linked to neuronal and retinal diseases, atherosclerosis, and aging. Typical methods for preparation of site-selectively deuterated PUFAs require rather long, laborious, and expensive syntheses. In this report, we disclose a very efficient catalytic protocol for site-specific deuteration of PUFAs and analogous poly-alkenes under exceptional kinetic control. Deuterium oxide (D2O) has been identified not only as a deuterium source but also as a crucial component in the overall reaction mechanism responsible for averting the formation of thermodynamically favored side-products.

Cross-talk between muscle and bone in postmenopausal women with hypovitaminosis D.

OBJECTIVES: Visceral fat is more harmful than subcutaneous fat. Women with high amounts of visceral fat have an increased risk of metabolic syndrome (MetS), insulin resistance and low level of serum vitamin D. METHODS: This was a prospective, randomized and controlled study. Postmenopausal women (n = 101) aged 45-70 years were included. The assessed parameters were: body mass index (BMI), waist circumference (WC), 25-hydroxyvitamin D (25-OHD) status, serum insulin and HOMA-IR, bone mineral density, total fat mass, fat mass index (FMI) measured by DXA, and muscle strength. RESULTS: Significantly lower values of 25-OHD (35.26 ± 1.61 nmol/l) were found in women with MetS compared to those without MetS (47.52 ± 3.47nmol/l) (t = 3.699; p < 0.000). A significant negative correlation was observed between 25-OHD levels and: WC (rhoWC = -0.94; p < 0.004), BMI (rhoBMI = -0.80; p < 0.002), FMI (rhoFMI = -0.31; p < 0.002) and impaired fasting glucose (IFG) (rhoIFG = -0.23; p < 0.001). The percentage of body fat was statistically significantly higher in the group with insufficient 25-OHD (39.6%) and in the group with a deficit of 25-OHD (48.2%), compared with the maximum permitted percentage body fat of 35% for a female (t = -22.63; p < 0.002). The average hand grip strength of these moderately obese participants was 27.4 ± 4.7 kg. It was significantly lower than the normative grip strength for referent range groups (t = -21.65; p < 0.001). CONCLUSION: A low 25-OHD level in postmenopausal women is associated with high body fat, metabolic syndrome, low muscular strength and osteopenia.

Optimisation of breast cancer patients' follow-up - potential way to improve cancer care in transitional countries.

The aim of this analysis was to evaluate adherence of Croatian oncologists to follow-up criteria as suggested by the current national and international guidelines for women with breast cancer receiving adjuvant endocrine therapy. The use of clinical and diagnostic methods was documented in this prospective, non-interventional, multicenter study. A total of 438 post-menopausal patients receiving adjuvant endocrine treatment with non-steroidal aromatase inhibitors were included. Average annual frequency for each clinical and diagnostic method was calculated. Median adjuvant endocrine treatment duration before study recruitment was 10.5 months (interquartile 4.7-26.6). Patients were followed up for an average 23.5 ± 4.9 months. Average number of oncological visits was 5.3. Mammograms were performed at mean annual frequency of 0.7, chest radiographs at 0.5, abdominal ultrasounds at 0.9, breast ultrasounds at 1.2, complete blood counts and chemistry panels at 1.7, carcinoembryonic antigen at 0.8, cancer antigen 15-3 at 1.6, gynaecological examination at 0.3, and densitometry at mean annual frequency of 0.3. In conclusion, among post-menopausal women with breast cancer receiving adjuvant endocrine therapy in this study, more unnecessary and unproven follow-up procedures were done compared to the guidelines' recommendations.

Interferon lambda-2 levels in sputum of patients with pulmonary Mycobacterium tuberculosis infection.

In humans, there are three members of type III interferon family, designated as IFN-λ1 (IL-29), IFN-λ2 (IL-28A) and IFN-λ3 (IL-28B). Bacterial infections, including Mycobacterium tuberculosis infection, trigger the expression of interferon I and II genes, but little is known about their effect on IFN-λ genes. It has been found that M. tuberculosis infection in A549 lung epithelial cells stimulate upregulation of IFN-λ2 genes in vitro. The aim of this study was to determine the IFN-λ2 levels in sputum of patients with active pulmonary tuberculosis comparing with levels in healthy persons sputum and sputum of persons with latent tuberculosis. IFN-λ2 level in the samples was quantified with sandwich enzyme-linked immunoassay kit for quantization of human IFN-λ2 (IL-28A). The IFN-λ2 level in sputum of patients with pulmonary tuberculosis was significantly higher comparing with IFN-λ2 levels in healthy group (P = 0.001) and latent tuberculosis group (P = 0.005). Logistic regression analysis has shown that IFN-λ2 level in sputum is related to culture result (OR = 4.14; 95% CI = 1.28-17.5; P = 0.019). There was the correlation between Bartlett score and IFN-λ2 levels in group with pulmonary tuberculosis (r = 0.641; P < 0.01). In conclusion, we have found that the inflammatory cells in sputum can be source of IFN-λ after receptor stimulation by M. tuberculosis components.

"V"-technique as a method of breast conserving surgery in multifocal and T2 invasive breast cancers situated in the upper outer quadrant.

OBJECTIVE: To assess the oncologic and cosmetic outcomes in women with multifocal breast cancers or T2 tumours with diameters larger than 3 cm situated in the upper outer quadrant who were treated with "V"-technique. METHODS: From July 1999 till June 2003, 44 conserving surgeries with "V"-technique using a local rotational flap were performed. Localization of tumours was in the upper outer quadrant. All solid tumours were larger than 3 cm in diameter In all patients, axillary lymph node dissection was performed. All the patients received postoperative radiotherapy. Mean follow-up was 58 months. RESULTS: Out of 44 conserving surgeries with "V"-technique, an adequate distance of tumour from the margins was obtained in 84.1% (37/44). Out of 37 patients who underwent conserving surgery with this technique, the cosmetic result was favourable in 83.78% (31/37). None of these patients had a corrective surgery such as reduction mammaplasty or mastopexy. Mean weight of excised tissue was 215 g. The 5-year local recurrence rate was 10.8%. The 5-year metastasis-free survival rate was 81.1%. The 5-year overall survival rate was 86.5%. CONCLUSIONS: Surgical treatment of multifocal and T2 breast cancers larger than 3 cm in diameter situated in the upper outer quadrant and performed with "V"-technique gives a good aesthetic result and enables a wide resection of breast tissue around the tumour

New prognostic index to predict survival in patients with cancer of unknown primary site with unfavourable prognosis.

AIMS: To identify independent prognostic factors in patients with cancer of unknown primary site (CUP) who do not belong to prognostically favourable subsets, and to develop a prognostic index for predicting survival in these patients. MATERIALS AND METHODS: In this prospective study, univariate and multivariate analyses of prognostic factors were conducted in a population of 145 patients with CUP in two clinical institutions. Subsets of patients with favourable prognostic features and those requiring well-defined treatment were excluded. RESULTS: The 1-year overall survival rate for all patients was 42% and the median overall survival was 330 days. Overall survival was significantly related to the following pre-treatment prognostic factors: poor Eastern Cooperative Oncology Group performance status (ECOG PS)>or=2, presence of liver metastasis, elevated serum lactate dehydrogenase (LDH), high white blood cell count, anaemia, age>or=63 years, and prolonged QTc interval in electrocardiography (ECG). In multivariate analysis, four independent adverse prognostic parameters were retained: elevated LDH (hazard ratio 2.21; 95% confidence interval 1.41-3.47; P=0.001), prolonged QTc interval (hazard ratio 2.10; 95% confidence interval 1.28-3.44; P=0.003), liver metastasis (hazard ratio 1.77; 95% confidence interval 1.11-2.81; P=0.016) and ECOG PS>or=2 (hazard ratio 1.69; 95% confidence interval 1.05-2.73; P=0.03). We developed a prognostic index for overall survival based on the following subgroups: good prognosis (no or one adverse factor), intermediate prognosis (two adverse factors) and poor prognosis (three or four adverse factors). The median overall survival for the three subgroups was 420, 152 and 60 days, respectively, P<0.0001. CONCLUSIONS: This study validated previously identified important prognostic factors for survival in patients with CUP. Prolonged QTc was additionally identified as a strong adverse prognostic factor. We developed a simple prognostic index using performance status, LDH, presence of liver metastasis and QTc interval in ECG, which allowed assignment of patients into three subgroups with divergent outcome.

Correction: FOXO3a represses VEGF expression through FOXM1-dependent and -independent mechanisms in breast cancer.

In the published version of this article, the images for cytoplasmic and nuclear FGF7 in MDA-MB-231 cells were duplicated and mistaken for total FGF7 in SKBR-3 and MDA-MB-231 cells.

Expression of heat shock protein 70 (HSP70) in patients with colorectal adenocarcinoma--immunohistochemistry and Western blot analysis.

The role of heat shock protein 70 (HSP70) expression has been investigated in various types of tumors. There are only little and controversial data about its clinical relevance in colorectal carcinoma, one of the most common carcinomas observed in humans. In this study we investigated expression of HSP70 in human colonic carcinoma and possible correlation with clinicopathology. To assess patterns (cytosolic and membrane) of HSP70 expression, the 48 surgically removed colorectal adenocarcinomas and 12 normal colonic and rectal mucosal samples were examined by immunohistochemistry and Western-blot. According to results of immunohistochemistry, expression of cytoplasmic HSP72 was significantly higher in colorectal carcinoma compared with normal and adjacent mucosa (p<0.01). In addition, there was significant increase in HSP72 expression in lymph node-positive compared to node-negative group (p<0.001). Dukes C2 stage of colonic cancer showed significantly higher immunohistochemical score than Dukes B2 and B1 stage groups (p< 0.05 i.e. p< 0.02). There was no relation between expression of HSP72 and degree of tumor differentiation. Using Western blot analyses, we noticed elevated levels of cytosolic HSP70 in colorectal cancer cells compared to normal. Densitometric analysis of blots of plasma membrane HSP70 expression has shown decrease in colorectal cancer cells compared to normal mucosa. According to our results, overexpression of HSP72 in malignant tissues of patients with colorectal carcinoma is related to tumor progression, suggesting that these proteins could play an important role not only in tumorigenesis but also in the development of drug resistance. Further research is necessary to clarify the mechanisms responsible for differential HSP70 expression as well as its definitive role in colorectal cancer.

Alkene-assisted cis-to-trans isomerization of non-conjugated polyunsaturated alkenes.

Complex [Cp*Ru(NCMe)3][PF6], 1a, has been identified as a cis-to-trans isomerization catalyst of various non-conjugated cis-polyalkenes under exceptional kinetic control as no alkene conjugation was observed. According to the experimental and theoretical data, the cis-trans isomerization occurred via an alkene-assisted mechanism in which one cis-double bond always served as an anchoring site. Using a combination of multinuclear NMR spectroscopic evidence and mathematical methods it was possible to determine the extent of trans isomerization.

Matrix-assisted laser desorption and ionization time-of-flight (MALDI-TOF) mass spectrometry in lipid and phospholipid research.

The interest in the analysis of lipids and phospholipids is continuously increasing due to the importance of these molecules in biochemistry (e.g. in the context of biomembranes and lipid second messengers) as well as in industry. Unfortunately, commonly used methods of lipid analysis are often time-consuming and tedious because they include previous separation and/or derivatization steps. With the development of "soft-ionization techniques" like electrospray ionization (ESI) or matrix-assisted laser desorption and ionization time-of-flight (MALDI-TOF), mass spectrometry became also applicable to lipid analysis. The aim of this review is to summarize so far available experiences in MALDI-TOF mass spectrometric analysis of lipids. It will be shown that MALDI-TOF MS can be applied to all known lipid classes and the characteristics of individual lipids will be discussed. Additionally, some selected applications in medicine and biology, e.g. mixture analysis, cell and tissue analysis and the determination of enzyme activities will be described. Advantages and disadvantages of MALDI-TOF MS in comparison to other established lipid analysis methods will be also discussed.

Symmetry effects in reversible random sequential adsorption on a triangular lattice.

Reversible random sequential adsorption of objects of various shapes on a two-dimensional triangular lattice is studied numerically by means of Monte Carlo simulations. The growth of the coverage rho(t) above the jamming limit to its steady-state value rho(infinity) is described by a pattern rho(t) = rho(infinity - deltarhoE(beta)[-(t/tau)beta], where E(beta) denotes the Mittag-Leffler function of order beta element of (0, 1). The parameter tau is found to decay with the desorption probability P_ according to a power law tau = AP_(-gamma). The exponent gamma is the same for all shapes, gamma = 1.29 +/- 0.01, but the parameter A depends only on the order of symmetry axis of the shape. Finally, we present the possible relevance of the model to the compaction of granular objects of various shapes.

Oxidative activity of human polymorphonuclear leukocytes stimulated by the long-chain phosphatidic acids.

It has already been suggested that phosphatidic acids (PAs) play an important role in the regulation of signaling pathways involved in the production of reactive oxygen species (ROS) by human polymorphonuclear leukocytes (PMNs). The present study was performed to elucidate the effects of extracellularly added PA-- 1,2-distearoyl- (DSPA) and 1-stearoyl-2-arachidonoyl-sn-glycero-phosphate (SAPA)--on the ROS production and on the elastase release by human PMNs. ROS production was monitored by luminol-amplified chemiluminescence and the elastase activity was measured in the supernatant of the PA-stimulated human PMNs by colorimetric assay. Obtained effects were compared with those of cells stimulated by either a chemotactic tripeptide, phorbol ester or calcium ionophore. Our results show that long-chain PAs at concentrations higher than 3 x 10(-5) mol/l stimulate the ROS production by human PMNs, whereas they were ineffective in promoting the elastase release. The chemiluminescence pattern of the SAPA-stimulated cells exhibited a biphasic curve, whereas cell stimulation with DSPA resulted in a monophasic chemiluminescence curve. Stimulation of the ROS production by PAs in dependence of the fatty acid composition required the activity of protein kinases.

Do electrolyte-containing beverages improve water imbalance in hyponatremic schizophrenics?

BACKGROUND: To determine if ready access to a sodium-containing beverage ameliorates life-threatening water imbalance, we compared Gatorade to water in an ABA design. METHOD: Four male chronic schizophrenic inpatients with symptomatic hyponatremia drank water for 1 week, Gatorade plus water for 3 weeks, and then water again for 1 week. Afternoon plasma osmolality and morning serum sodium levels were used to estimate hydration status, and morning and afternoon urine creatinine concentrations were used to estimate fluid intake. RESULTS: During the baseline phase, subjects were mildly polydipsic and mildly overhydrated in the morning and severely overhydrated in the afternoon. All subjects stated they preferred Gatorade and appeared to consume large quantities of it. Indices of fluid intake and hydration status did not differ between the two control phases, nor between the treatment phase and the other two phases. Frequency of severe hypo-osmolemia was also unaffected. CONCLUSION: Substitution of electrolyte-containing beverages is not likely to prevent water intoxication.

The photoprotein pholasin as a luminescence substrate for detection of superoxide anion radicals and myeloperoxidase activity in stimulated neutrophils.

Pholasin, the photoprotein of the common piddock Pholas dactylus, emits an intense luminescence upon oxidation. The contribution of superoxide anion radicals and myeloperoxidase (MPO) to Pholasin luminescence in stimulated neutrophils was investigated. Data on Pholasin luminescence were compared with results of superoxide anion radical generation detected by the cytochrome c test as well as with the release of elastase and MPO. In N-formyl-methionyl-leucyl-phenylalanine (fMLP) stimulated neutrophils, most of the luminescence is caused by superoxide anion radicals, whereas MPO shows only a small effect as shown by coincubation with superoxide dismutase (SOD) as well as potassium cyanide (KCN), an inhibitor of MPO. However, both, O2- and MPO contribute to light emission in fMLP/cytochalasin B and phorbol myristoyl acetate (PMA) stimulated cells. Thus, the kinetics of O2- generation and MPO release can be very well detected by Pholasin luminescence in stimulated neutrophils. Degranulation of azurophilic granules was assessed using an ELISA test kit for released MPO or detection of elastase activity with MeO-Suc-Ala-Ala-Pro-Val-p-nitroanilide in the supernatant of stimulated cells. Both approaches revealed concurrently similar results concerning the amount and kinetics of enzyme release with data of Pholasin luminescence. Both, cytochrome c measurements and Pholasin luminescence indicate that fMLP/cytochalasin B and PMA stimulated neutrophils produce more O2- than fMLP stimulated cells. Thus, Pholasin luminescence can be used to detect, sensitively and specifically, O2- production and MPO release from stimulated neutrophils.

CsCl as an auxiliary reagent for the analysis of phosphatidylcholine mixtures by matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS).

Matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) is mainly used for protein and peptide analysis. However, there is growing evidence that also phospholipids like phosphatidylcholines (PC) can be easily analyzed by MALDI-TOF MS. In MALDI-TOF methodology, the sample is cationized by the addition of inorganic ions. This process is strongly dependent on the corresponding ion concentration. In biological samples various cations are present (mainly H+, Na+ and K+) and, therefore, a mixture of different adducts is formed. Since phospholipids exhibit a wide distribution of different fatty acid residues a considerable peak overlap may occur. This is a major problem since the peak assignment in a mixture will be often unclear. In this paper we demonstrate that this problem can be easily overcome by mixing the analyte with caesium chloride (CsCl). This yields naturally non-occurring Cs+ adducts that are apparent due to the large shift of the molecular mass. The proposed method facilitates the clear assignment of most peaks. Besides that, we will show that CsCl can also be used for the determination of the relative fatty acid composition of a given PC sample. For this purpose naturally occurring mixtures of PCs as well as organic extracts of human lipoproteins-that are mainly composed of PC and sphingomyeline-are used.

Limits for the detection of (poly-)phosphoinositides by matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS).

Matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been recently established as a powerful tool for the analysis of biomolecules. Here, MALDI-TOF MS was used for the detection of (poly-)phosphoinositides (PPI). PPI possess higher molecular weights than other phospholipids and a high phosphorylation-dependent negative charge. Both features affect the MALDI detection limits expressed as the minimum of analyte on the sample plate resulting in a signal-to-noise-ratio of S/N = 5. Using 2,5-dihydroxybenzoic acid (DHB) as matrix the detection limit for phosphatidylinositol (PI) is seven times higher than for phosphatidylcholine (PC) and further increases with increasing phosphorylation or in mixtures with other well-detectable phospholipids. For phosphatidylinositol-tris-phosphate (PIP3) in a mixture with PC, the limit is about 20 times higher than for PI. The consequences for the experimental conditions are discussed. It is advisable to pre-separate PPI from biological lipid mixtures prior to the application of MALDI-TOF MS.

CD30-positive cutaneous anaplastic large cell lymphoma with ichthyosis acquisita.

Anaplastic large cell lymphoma (ALCL) is a high-grade non-Hodgkin's lymphoma recognized by the expression of the CD30 marker and by its morphology. We report an unusual case of ALCL in a 42-year-old woman. For ten years the patient only had pruritic erythematous skin lesions, then a plaque with nodules and ulcers on the right thigh and leg developed, followed by ichthyosis acquisita. The development of ALCL in women and the association of ichthyosis acquisita with this type of lymphoma are uncommon.