Recombinant forms of Gerbich blood group antigens: expression and purification.

Recombinant forms of normal glycophorin C (GPC), carrying the high frequency Gerbich blood group antigens, and its natural deletion mutants of Yus and Ge type (all combined with oligohistidyl tag) were expressed in CHO and COS 7 cells. The stable expression of all recombinant forms of GPC in CHO cells was obtained, but the level of expression was low and detectable only by flow cytometry. The high level of transient expression of GPC recombinant forms in COS 7 cells allowed their purification on Ni-NTA-agarose. The purified recombinant GPC and mutants of Yus and Ge type behaved in SDS-PAGE similarly to normal GPC forms from RBC membranes. The recombinant GPC.Yus and GPC.Ge mutants appeared as diffuse bands, suggesting the similar heterogeneity of glycosylation that was observed in natural GPC.Yus and GPC.Ge glycoproteins. The flow cytometry analysis of the transfected CHO and COS 7 cells showed that binding of anti-GPC monoclonal antibodies to GPC variants was accordant with the known fine specificity of these antibodies. The obtained recombinant forms of GPC carrying common Gerbich antigens may be useful in serology, and also as model molecules for structure-function studies.     

An immunoblotting procedure for screening glycophorins and band 3 protein in the same blots: Identification of glycophorin and band 3 variant forms

An immunoblotting procedure is described which makes it possible to screen multiple blood samples for the presence of glycophorin and band 3 variant forms with altered electrophoretic mobility. The procedure can be simplified by using whole red blood cell hemolysates instead of membranes for SDS-polyacrylamide gel electrophoresis. The use of hemolysates also has the advantage that antigens sensitive to proteolysis are not degraded in vitro. The same nitrocellulose blots were used for immunoenzymatic detection of glycophorins with a set of anti-glycophorin monoclonal antibodies, and for autoradiographic detection of band 3-derived bands with ¹²⁵I-labeled anti-band 3 monoclonal antibody. The screening of 157 Caucasian blood samples revealed the presence of a slower-migrating form of band 3 in seven cases and variant glycophorin in one case. The variant glycophorin exhibited the features of hybrid glycophorin of B-A type.     

The Influence of Recombinant Human Erythropoietin on Apoptosis and Cytokine Production of CD4+ lymphocytes from Hemodialyzed Patients

Recombinant human erythropoietin (rhEPO) treatment of hemodialyzed (HD) patients normalizes the altered phenotype of CD4⁺ lymphocytes and restores the balance of Th1/Th2 cytokines. We decided to test how the presence of rhEPO in cell culture modulates cytokine production of CD4⁺ lymphocytes in HD patients with stable hemoglobin level and expression of activation antigens of stimulated CD4⁺ lymphocytes similar to those observed in healthy individuals. We also tested whether the presence of rhEPO in cell culture protects stimulated CD4⁺ lymphocytes of HD patients from apoptosis. Peripheral blood mononuclear cells (PBMC) of HD patients were stimulated with an immobilized anti-CD3 antibody with or without addition of rhEPO. The percentage of apoptotic CD4⁺ lymphocytes and the level of Th1/Th2 cytokines in culture supernatants were measured with flow cytometry. HD patients showed a decrease in the percentage of apoptotic CD4⁺ cells after stimulation with the anti-CD3 antibody combined with rhEPO. The level of IFN-γ and IL-10 was increased while the level of TNF-α was decreased in the presence of rhEPO in cell culture from HD patients. These results confirm the role of rhEPO signaling in T lymphocytes of HD patients.     

Differences in Monocyte Subsets and Monocyte-Platelet Aggregates in Acute Myocardial Infarction—PreliminaryResults

Background

Monocyte-platelet interaction may favor the development of a proatherogenic monocyte phenotype. It is still uncertain which of the 3 monocyte subpopulations interact with platelets to form monocyte-platelet aggregates (MPAs) in acute myocardial infarctions. The aim of our study was to evaluate the monocyte subsets, the percentage of MPAs and the involvement of monocyte subsets in MPA formation among patients with ST-elevation myocardial infarction (STEMI) and non-ST-elevation myocardial infarction (NSTEMI), and compared to patients with stable angina (SA).

New PA/1220/98-like variant of infectious bronchitis virus in Poland

ABSTRACT

The aim of the present study was to report the first detection of a new infectious bronchitis virus (IBV) variant in Polish commercial flocks which is completely different to any previously known in this region. In 2018, samples from Ross 308 breeding hens aged 35 weeks were delivered for IBV diagnosis. IBV presence was detected, but all attempts to amplify the S gene fragment were negative. The field material was analysed using the Illumina MiSeq platform and a 1073-nt fragment of the S1 coding region was obtained. The gCoV/ck/Poland/516/2018 strain shared only 52.7–58.1% nucleotide identity to any known genotype of IBV and shared the highest identity of 81.4% to the unique North American PA/1220/98 variant. Based on the obtained sequence, a specific molecular test was constructed and used for screening of chicken samples from 35 field cases delivered to our laboratory between 2018 and 2019 for IBV diagnosis. Application of this test enabled detection of another three chicken flocks as positive for this new strain. All positives were identified in commercial layers with egg production problems. To date, the virus has not been detected in broiler chickens. Taking into account the proposed criteria for the definition of a new IBV genotype or lineage, it seems that the detected viruses in Poland, together with the unique North American PA/1220/98 variant, may be classified as separate lineages/genotype in the new IBV classification.

RESEARCH HIGHLIGHTS

• The new IBV variant is distantly related to other known GI–GVII IBV genotypes/lineages.

• It affects long-lived birds causing egg production problems.

• The detected IBV and the unique North American PA/1220/98 variant are candidates for separate lineages in the new GVIII genotype.

     

Down-regulation of V1a vasopressin receptors in the cerebellum after myocardial infarction

Vasopressin V1a receptors (V1aR) were found in the cerebellum but their functional role has not been determined. As V1aR are engaged in the central regulation of the cardiovascular system and anxiogenic behavior and their role increases in the heart failure and stress, we decided to find out whether expression of V1aR is altered after myocardial infarction and chronic stressing. RT-PCR and Western blot analysis were performed to determine V1aR mRNA and protein expression in the cerebellum of four groups of rats (control sham-operated, infarcted, chronically stressed and infarcted chronically stressed). The myocardial infarct was produced by left coronary artery ligation, and chronic stressing by exposing the rat for four weeks to different types of mild stressors. The rats were sacrificed four weeks after the myocardial surgery or sham operation. Expressions of V1aR mRNA and protein were significantly lower in the infarcted and infarcted chronically stressed rats than in the sham-operated controls and chronically stressed not infarcted rats. No significant differences were found between the sham-operated controls and chronically stressed rats and between the infarcted rats and infarcted rats exposed to chronic stressing. It is concluded that V1aR mRNA and protein expressions are significantly down-regulated in the rats with the post-infarct heart failure but they are not affected by mild chronic stressing.     

Early wheezing phenotypes and cognitive development of 3‐yr‐olds. Community‐recruited birth cohort study

Jedrychowski W, Perera FP, Jankowski J, Maugeri U, Mrozek‐Budzyn D, Mroz E, Flak E, Skarupa A, Edwards S, Lisowska‐Miszczyk I. Early wheezing phenotypes and cognitive development of 3‐yr‐olds. Community‐recruited birth cohort study.
Pediatr Allergy Immunol 2010: 21: 550–556.
© 2009 John Wiley & Sons A/S The main purpose of the study was to answer the question whether young children without clinical diagnosis of asthma but experiencing early wheezing disorders and therefore being at high risk of developing asthma may have cognitive deficits. In the ongoing birth cohort study wheezing symptoms were recorded postpartum over two first years of age and subsequently cognitive status of children at the age of 3 yr was assessed with the Bayley Mental Development Index (MDI). In the statistical analysis a wide range of modifying and confounding factors (maternal education, gender of children, prenatal exposure to lead and environmental tobacco smoke (ETS) were considered to assess the independent effect of early wheezing phenotypes on cognitive development of children. The MDI score correlated inversely with the number of wheezing days recorded over 24 months (r = ?0.13, p = 0.007), lead cord blood concentration (r = ?0.12, p = ?0.02), number of siblings (r = ?0.17, p = 0.0006) and the number of cigarettes smoked daily by other household members at home over the pregnancy period (r = ?0.18, p = 0.0002). While the children who experienced wheezing over the first year of age showed deficit of 2 MDI scores (beta coeff. = ?2.31, 95%CI: ?4.63 to 0.02), those with persistent wheezing had the score deficit of 4 points (beta coeff. = ?4.41, 95%CI: ?8.27 to ?0.55). To our knowledge, it is the first report in the iterature showing that early wheezing is associated the cognitive deficit in a community‐recruited very young children. Observed cognitive deficit in early wheezers may be caused by RSV infections or can be related to lower lung function attributed to persistent wheezing, which reducing oxygen supply would affect rapidly developing brain.     

Hypopituitarism and hematological abnormalities mimicking myelodysplastic syndrome. Report of four cases

Insufficiency of the pituitary gland and hematological abnormalities may coexist in the context of two syndromes. In the course of some hematopoietic neoplasms, particularly acute leukemias, the pituitary insufficiency may be caused by destruction of the gland either by direct neoplastic infiltration or occlusion of vessels. Alternatively, thy pituitary dysfunction may be associated with but not caused by hematological abnormalities, usually mild peripheral cytopenias. We present four cases of the latter type (1. M/33, pituitary tumor, hypogonadism, hyperprolactinemia, anemia, mild leukopenia with leukocytosis, 2. F/54, pituitary tumor, hyperprolactinemia, thyreotropic and corticotropic insufficiency, anemia, thrombocytopenia, mild neutropenia, 3. F/27, pituitary tumor, diabetes insipidus, hypogonadism, sideropenic anemia, leukopenia, thrombocytopenia, 4. M/24, primary multihormonal insufficiency of the anterior portion of the pituitary gland, neutropenia, microcytosis). Trephine and aspiration bone marrow biopsies revealed topographic and cytological abnormalities partially resembling these found in myelodysplastic syndromes (MDS). Bone marrow cellularity varied markedly between and within the cases, and in three patients abnormal aplastic areas were found. The percentage of hematopoietic stem cells (CD34+) was low in three cases and normal in one case. Pituitary dysfunction may be associated with hematological abnormalities simulating MDS, but showing different, less aggressive clinical course. The proliferative potential of hematopoietic cells is low, the peripheral blood abnormalities are stable, and no patient developed acute leukemia. Detailed bone marrow examination, including the trephine bone marrow biopsy, is useful in differentiation with true MDS, which was also reported in the literature in the patients with pituitary insufficiency.     

A forearm exercise screening test for mitochondrial myopathy

BACKGROUND: The authors hypothesized that impaired oxygen extraction in mitochondrial myopathy (MM) results in a high oxygen saturation in venous effluent blood from working muscle and that this phenomenon can be used as a diagnostic tool for MM. METHODS: Twelve patients with MM, 10 patients with muscular dystrophy, and 12 healthy subjects were studied. All subjects performed intermittent static handgrip exercise (1/2 Hz) at 40% of maximal voluntary contraction (MVC) for 3 minutes. Cubital venous oxygen saturation and brachial artery flow were measured in the exercised arm. RESULTS: Exercise-induced venous oxygen desaturation was smaller in patients with MM (Delta - 7 +/- 5%) than in subjects with muscular dystrophy (Delta - 38 +/- 2%; p = 0.00001) and healthy subjects (Delta - 43 +/- 2%; p = 0.0000002). MVC and exercise blood flow were similar in patients with MM (18 +/- 3 kg; 436 +/- 65 mL/min) and patients with muscular dystrophy (15 +/- 3 kg; 460 +/- 85 mL/min), but were higher in healthy subjects (32 +/- 4 kg; 630 +/- 58 mL/min; p < 0.03). In seven patients with MM and seven patients with McArdle disease, studied with a slightly different protocol, exercise-induced oxygen desaturation was also impaired in MM (Delta - +/- 5%) compared with McArdle disease (Delta - 26 +/- 3%; p = 0.007). CONCLUSION: Oxygen desaturation in venous blood from exercising muscle is markedly lower in patients with mitochondrial myopathy than in subjects with other muscle diseases and healthy subjects, suggesting that a forearm exercise test can be a diagnostic screening tool for mitochondrial myopathy.