Immunophenotype of peripheral blood natural killer cells and IL‐10 serum levels in relation to Helicobacter pylori status

Recent findings suggest that NK (Natural Killer) cells may directly modulate the antimicrobial immune responses. In this study, we performed immunophenotypic analysis of peripheral blood NK cells with regard to CD56, CD16, Nkp46, and CD25 markers, as well as IL‐10 levels quantification in the sera samples of asymptomatic, H. pylori (Hp)‐infected or uninfected individuals, and combined these results with our previous findings on lymphocyte cytotoxic activity. Twenty healthy volunteers [10 Hp(?);10 Hp(+)] were included in the study. The percentages of classic lymphocytes (CD3⁺) and NK cells (CD3^?CD56⁺, CD3^?Nkp46⁺, CD3^?CD16⁺) with or without CD25 receptor were evaluated by fluorochrome‐conjugated monoclonal antibody staining and flow cytometry analysis. IL‐10 quantification was performed by enzyme‐linked immunosorbent assay‐ELISA. Our study showed elevated levels of IL‐10 and higher NK cell numbers of both CD3^?CD56⁺CD25⁺ and CD3^?Nkp46⁺CD25⁺ phenotypes, as well as CD3⁺CD25⁺ classic lymphocytes in Hp(+) compared with Hp(?) individuals. No differences between Hp(?) and Hp(+) individuals were found either in total number of classic lymphocytes or NK cell subtypes. Our data suggest that in Hp(+) donors, there is a domination of lymphocytes and NK cells co‐expressing CD25 marker, which might be influenced by the regulatory IL‐10. This phenomenon may be a result of H. pylori adaptation to a changing environment in vivo leading to a chronic infection and lack of severe gastric pathologies.     

Diagnostic difficulties of paroxysmal symptoms in a boy with Parry-Romberg syndrome

Parry-Romberg syndrome is characterized by progressive unilateral facial atrophy affecting subcutaneous tissue, cartilage and bone structures. Headache attacks and epilepsy are commonly associated with this syndrome but the underlying pathophysiology is still unknown. A case of a 12-year-old boy with Parry-Romberg syndrome and syringomyelia suffering from severe headache attacks and epileptic seizures is reported herein.Headache attacks were associated with bilateral autonomic symptoms and hyperventilation and were usually followed by complex partial and sometimes by secondary generalized tonic seizures. Detailed neuroimaging examinations were performed (magnetic resonance imaging [MRI] of the head, orbits, and spinal cord, MR angiography, and MR spectroscopy of the cerebellum). The EEG pattern revealed localized discharges contralaterally to the affected side. Antiepileptic treatment with carbamazepine was instituted with minimal effect. Modification of treatment (replacement with oxcarbazepine) was successful.In the reported patient interesting correlation of headache attacks, autonomic symptoms and epileptic seizures was observed. Additionally we believe it is the first report of coincident syringomyelia and Parry-Romberg syndrome.     

Age-dependent stimulatory effect of desipramine and fluoxetine pretreatment on metastasis formation by B16F10 melanoma in male C57BL/6 mice

Although recent data may provide theoretical support for the preventive use of antidepressants in cancer patients, so far no study has demonstrated the clinical benefits of such strategies in the general population of cancer patients [39, 41]. Moreover, an association between antidepressant use and the risk of tumor promotion could neither be excluded nor established.The aim of this study was to compare the effect of desipramine (a tricyclic antidepressant, TCA) and fluoxetine (a selective serotonin reuptake inhibitor, SSRI) on tumor growth of the mouse B16F10 transplanted melanoma in “young” 6–9 month old and “aged” 18–23 month old male C57BL/6 mice. Drugs were administered daily at a dose of 10 mg/kg, ip, for two weeks and tumor cells were inoculated 2 h after the last antidepressant administration. Control animals were treated with saline. Tumor growth was significantly slower in aged than in young saline-treated control animals. Pretreatment with desipramine dramatically promoted metastasis formation and increased mortality rate but inhibited primary tumor growth in young males. On the other hand, both antidepressants increased primary tumor growth in aged animals, whereas metastasis was only moderately promoted. To determine the effect of antidepressant drug pretreatment and tumor progress on some parameters of cell-mediated immunity (proliferative activity and cytokine production by splenocytes) and angiogenesis, vascular endothelial growth factor (VEGF) and metalloproteinase (MMP)-9 plasma levels were established. The prometastatic effect of desipramine in young animals was connected with an increase of VEGF and MMP-9 plasma levels.     

Temperature mapping considerations in the breast with line scan echo planar spectroscopic imaging.

A line-scan echo planar spectroscopic imaging (LSEPSI) sequence was used to serially acquire spectra from 4,096 voxels every 6.4 s throughout the breasts of nine female subjects in vivo. Data from the serial acquisitions were analyzed to determine the potential of the technique to characterize temperature changes using either the water frequency alone or the water-methylene frequency difference. Fluctuations of the apparent temperature change under these conditions of no heating were smallest using the water-methylene frequency difference, most probably due to a substantial reduction of motion effects both within and without the imaged plane. The approach offers considerable advantages over other methods for temperature change monitoring in the breast with magnetic resonance but suffers from some limitations, including the unavailability of lipid and water resonances in some voxels as well as a surprisingly large distribution of water-methylene frequency differences, which may preclude absolute temperature measurement.     

Blood inhibitory capacity toward exogenous plasmin.

Stabilized, active plasmin is a novel thrombolytic for direct delivery to clots. Although it is known that protease inhibitors in plasma inhibit plasmin, the amount of plasmin that can be added to plasma/blood before free plasmin is observed is not clear. Determination of free plasmin activity in plasma using chromogenic substrates represents a challenge due to false-positive signals from plasmin entrapped by alpha2-macroglobulin. Size-exclusion chromatography was used to separate the plasmin-alpha2-macroglobulin complex from uninhibited, free plasmin. In this in-vitro study, exogenous plasmin is effectively inhibited up to 2.4 micromol/l after 5-min incubation with plasma at 37 degrees C. Initially, plasmin was consumed predominantly by alpha2-antiplasmin up to 1.2 micromol/l plasmin. Following exhaustion of alpha2-antiplasmin, plasmin was further consumed by alpha2-macroglobulin up to 2.4 micromol/l plasmin added to human plasma. Whole human blood was found to have an increased inhibitory capacity over that of plasma; free plasmin activity could be measured only above 3.8 micromol/l added plasmin. In conclusion, several mechanisms exist that control plasmin activity in human blood; in addition to alpha2-antiplasmin and alpha2-macroglobulin, blood cells contribute to the inhibition of exogenously administered plasmin. These in-vitro results indicate that doses of plasmin up to approximately 12 mg/kg in humans can be completely inactivated by blood.     

RECURRENT DELETION OF THE REGION ENCODING TWO (Gly-X-Y) REPEATS IN PATIENTS WITH ANHIDROTIC ECTODERMAL DYSPLASIA INDICATES IMPORTANT ROLE FOR COLLAGEN-LIKE DOMAIN OF THE EDA GENE PRODUCT - ECTODYSPLASIN-A

We have investigated the structure of the EDA gene in a patient with the clinical symptoms of anhidrotic ectodermal dysplasia (EDA). Electrophoretic analysis of the amplified fragment of exon 5 of the EDA gene in the affected boy revealed a PCR product that was shorter by ~20 bp than the same fragment obtained from a healthy individual. A similar shortened fragment, in addition to a normal one, also was found in his mother, suggesting that she was the carrier of this mutation. Sequence analysis of the mutated fragment displayed an 18bp deletion, extending either from nucleotide 891 to 908 or from nucleotide 901 to 918. The deleted fragment encodes two (Gly-X-Y) repeats in the collagen-like domain of ectodysplasin-A , thus reducing the number of repeats from 19 to 17. These two mutations increase to 9 the number of deletions in exon 5 described so far. The recurrent character of these mutations indicates that the collagen-like domain of ectodysplasin-A plays a critical role in the structure of this receptor and that the deletion may be responsible for the clinical symptoms of EDA.     

Frequency of common CYP3A5 gene variants in healthy Polish newborn infants

Cytochrome P450 monooxygenases catalyze the metabolism of approximately 40-60% of widely used drugs with a A6986G CYP3A5 polymorphism determining expresser (A6986, *1) and reduced- expresser (*3) variants with modified drug metabolism activity. In this report, the allele frequency of CYP3A5 *1 and *3 (A6986 or G6986, respectively) was analyzed by the PCR-RFLP technique in a cohort of 200 Polish newborns from the West Pomeranian region. Of the studied group, 1% (n = 2/200) proved homozygous for the CYP3A5*1 allele, 89% (n=178/200) for the *3 allele, and 10% (n = 20/200) were heterozygous for *1/*3.Similar frequencies were found in other Caucasian European populations. This study provides basic genetic data related to the metabolism of drugs, with a narrow therapeutic window in a Polish population.     

Altered Expression of Nuclear Non-Histone Protein (P44/46) in Different Stages of B-Chronic Lymphocytic Leukemia

Our previous data have shown some differences in electrophoretic characteristics of proteins from cellular fractions (nuclear, mitochondrial, microsomal and cytosolic) isolated from peripheral blood mononuclear cells of B-cell chronic lymphocytic leukemia (B-CLL), acute lymphoblastic leukemia (ALL) patients and healthy donors. The main differences were found in electrophoretic patterns of nuclear proteins from normal and leukemia cells, especially in the nuclear mass regions of 36-52, 58-85, and 120-180 kDa. Electrophoretically-specific nuclear non-histone protein in the molecular mass zone 44/46 kDa of cells obtained from the peripheral blood of a B-CLL patient was used to produce rabbit polyclonal antiserum. SDS-polyacrylamide gel electrophoresis as well as immunological techniques (Western blot and immunocytochemistry) indicate that the nuclear protein with a molecular mass of 44/46 kDa is specifically expressed in mononuclear cells from B-CLL patients. The expression of this particular nuclear protein seems to correlate with the progression of the leukemia.     

The cell membrane as the barrier in the defense against nanoxenobiotics: Zinc oxide nanoparticles interactions with native and model membrane of melanoma cells

Currently, we are dealing with ever-increasing pollution of the environment with metal and metal oxide nanoparticles. One type of these, zinc oxide nanoparticles (ZnO-NPs), are increasingly used in areas such as cosmetology, electrical engineering, medicine, and even in the food and textile industries. As a consequence, ZnO-NPs may enter the human body in many ways. Their influence on the body is still not clear. Here, we define the mechanism of the initial toxicity of ZnO-NPs to cells based on interaction with the lipid part of the native and model cell membrane. The selected cell lines react differently to contact with nanoparticles. We found a disruption of the native membranes of B16-F0 cells and to a lesser extent of COLO 679. In turn, the membrane of COLO 679 cells was more peroxidated, and cell viability was much lower. A model of the lipid part of the membrane was created for B16-F0 cells and compared with previously published studies on immune cells. On the basis of physicochemical parameters obtained for individual lipids and a mix representing the native membrane of the tested cells, we concluded that exposure to nanoparticles resulted in a change within the model membranes (specifically with the polar parts of lipids). The greatest interaction has been noticed between ZnO-NPs and zwitterionic phospholipids (PC and PE), cholesterol, and negatively charged phosphatidylglycerol. Assessing the interactions between the membrane and nanoparticles will help to better understand the first steps of its toxicity mechanism.