Clinical-scale in vitro expansion preserves biological characteristics of cardiac atrial appendage stem cells.

OBJECTIVES: Cardiac atrial appendage stem cells (CASCs) have recently emerged as an attractive candidate for cardiac regeneration after myocardial infarction. As with other cardiac stem cells, CASCs have to be expanded ex vivo to obtain clinically relevant cell numbers. However, foetal calf serum (FCS), which is routinely used for cell culturing, is unsuitable for clinical purposes, and influence of long-term in vitro culture on CASC behaviour is unknown. MATERIALS AND METHODS: We examined effects on CASC biology of prolonged expansion, and evaluated a culture protocol suitable for human use. RESULTS: In FCS-supplemented medium, CASCs could be kept in culture for 55.75 ± 3.63 days, before reaching senescence. Despite a small reduction in numbers of proliferating CASCs (1.37 ± 0.52% per passage) and signs of progressive telomere shortening (0.04 ± 0.02 kb per passage), their immunophenotype and myocardial differentiation potential remained unaffected during the entire culture period. The cells were successfully expanded in human platelet plasma supernatant, while maintaining their biological properties. CONCLUSIONS: We successfully developed a protocol for long-term culture, to obtain clinically relevant CASC numbers, while retaining their cardiogenic potential. These insights in CASC biology and optimization of a humanized platelet-based culture method are an important step towards clinical application of CASCs for cardiac regenerative medicine.

Suppressed increase in blood endothelial progenitor cell content as result of single exhaustive exercise bout in male revascularised coronary artery disease patients.

Endothelial progenitor cells (EPCs) significantly affect endothelial repair capacity and, hence, cardiovascular disease incidence. In healthy subjects, blood EPC content increases significantly as result of a single maximal exercise test, hereby stimulating endothelial repair capacity. It remains to be shown whether a single exercise positively affects blood EPCs in revascularised coronary artery disease (CAD) patients. From male revascularised CAD patients (n = 60) and healthy volunteers (n = 25) blood samples were collected before and immediately after a maximal cardiopulmonary exercise test. Blood samples were analyzed by optimised flow cytometry methodology for EPC content (CD34+, CD34+ CD133+, CD34+VEGFR2+, CD34+CD133+VEGFR2+, and CD34+CD133-VEGFR2+ cells) and compared between groups. CFU-Hill colonies were additionally assessed. As a result of a maximal exercise test, blood CD34+, CD34+VEGFR2+ (all EPCs), CD34+CD133+, and CD34+ CD133-VEGFR2+ (mature EPCs) cells increased significantly in CAD patients (p < 0.05), but less than in healthy subjects (p < 0.05, and p = 0.06 for CD34+VEGFR2+). CD34+CD133+VEGFR2+ cells (immature EPCs) did not change as result of exercise (p > 0.05). No changes in CFU-Hill colonies as result of exercise were observed. This study shows that blood mature EPCs (CD34+CD133-VEGFR2+) increase significantly as result of a single exercise bout in revascularised CAD patients, but with smaller magnitude compared to healthy subjects. Blood immature EPCs (CD34+CD133+VEGFR2+) did not change significantly as result of exercise.

Haplotype 4 of the multiple sclerosis-associated interleukin-7 receptor alpha gene influences the frequency of recent thymic emigrants.

The receptor for the homeostatic T cell cytokine interleukin-7 (IL-7Ralpha) has recently shown genetic association to multiple sclerosis (MS). To investigate the functional contribution of IL-7Ralpha polymorphisms to the pathogenesis of MS, we correlated the IL-7Ralpha haplotypes with different T cell parameters in a group of MS patients and healthy controls. We show that carriers of one of the four IL-7Ralpha haplotypes (Hap4) show a higher expression of IL-7Ralpha (CD127) on their CD4(+) T cells, compared with noncarriers (P=0.04). Moreover, Hap4 carriers possess higher frequencies of recent thymic emigrants (RTEs, CD31(+)) in both the regulatory T cell (Treg; P=0.007) and conventional T cell (Tconv) population (P=0.0001). This effect is most pronounced within the MS population (Treg, P=0.0077; Tconv, P=0.0007), whereas in healthy controls significance was only reached for Tconv (P=0.043; Treg, P=0.11). Because previous studies showed a decreased RTE-Treg frequency in MS patients compared to healthy subjects, we here conclude that this decrease is localized within the MS population of non-Hap4 carriers. In conclusion, our findings suggest that IL-7Ralpha polymorphisms can influence T cell development and homeostasis, and thereby contribute to the altered immune regulation associated with disease development in patients with MS.

Exploration of basal immune parameters in healthy infants receiving an infant milk formula supplemented with prebiotics.

This double-blind, randomized, placebo-controlled study, aimed to explore the effect of an infant milk formula (IMF) with 6 g/l short-chain galacto- and long-chain fructo-oligosaccharides (scGOS/lcFOS, ratio 9:1) on basal immune parameters in 215 healthy, term infants during the first 26 wk of life. After birth, the infants received breast milk or were randomized to receive an IMF with or without scGOS/lcFOS. Blood samples were collected at the age of 8 wk and 26 wk for the analysis of serum immunoglobulins, lymphocyte subpopulations, and cytokines. The scGOS/lcFOS group and the control group were compared in the statistical analysis. A breast fed group was included as a reference. In total, 187 Infants completed the study. No significant differences were observed between both formula groups in the different studied immune parameters at weeks 8 and 26. This explorative study indicates that supplementation of infant formula with a mixture of prebiotic oligosaccharides did not change the basal level of the measured parameters of the developing immune system in healthy infants with a balanced immune system during the first 6 months of life in comparison to feeding a standard infant formula and in comparison to exclusive breastfeeding.

External quality assessment in the measurement of haemoglobin by blood gas analysers in Belgium.

OBJECTIVE: The Belgian national External Quality Assessment Scheme (EQAS) for haematology organized a survey to assess the reliability of haemoglobin (Hb) measurements with the blood gas analysers (BGAs) currently available in Belgian hospitals. MATERIAL AND METHODS: All hospital laboratories received two specimens of fresh EDTA anticoagulated whole blood and were asked to determine the Hb concentration using both the conventional haematology analyser (HA) and all BGAs in the hospital. Ninety-seven hospital laboratories participated in the study and a total of 166 results were reported. The BGAs used (grouped according to technology) were Rapidlab 845, 855, 865 (Bayer 1, n = 41), Rapidlab 1245, 1265, Rapidpoint 405 (Bayer 2, n = 19), GEM Premier 3000 (Instrumentation Laboratory, IL, n = 13), ABL 500 and 600 series (Radiometer 1, n = 13), ABL 700 and 800 series (Radiometer 2, n = 35), Omni C, S5 (Roche 1, n = 7), Omni 3, 6, 9, S2, S4, S6 (Roche 2, n = 21). RESULTS: For the BGAs from Bayer, Radiometer and Roche, interlaboratory variation ranged from 0.6 % to 4.1 %, indicating good precision and close agreement between centres. A significant negative bias observed on the GEM Premier 3000 using the EDTA anticoagulated blood samples did not appear to be present in fresh heparinized whole blood samples. There was no significant difference in imprecision and bias between Hb measurements on BGA situated in and outside the central laboratory.

Variant t(2;11)(p11;q13) associated with the IgK-CCND1 rearrangement is a recurrent translocation in leukemic small-cell B-non-Hodgkin lymphoma.

Classical t(11;14)(q13;q32) involving IGH-CCND1 is typically associated with aggressive CD5-positive mantle cell lymphoma (MCL). Recently, we identified the IGK variant of this translocation, t(2;11)(p11;q13), in three patients with a leukemic small-cell B-non-Hodgkin lymphoma. In all cases, rearrangements of the IGK and CCND1 genes were demonstrated by fluorescence in situ hybridization. Moreover, we mapped the 11q13 breakpoint of this variant translocation in the 3' region of CCND1 which contrasts with the 5' breakpoints in a standard t(11;14)(q13;q32). Expression of cyclin D1 was shown in two cases analyzed either at diagnosis or during disease progression. All three patients were asymptomatic at presentation and no initial therapy was required. One patient died of a progressive disease 58 months from diagnosis, and two patients showed stable disease after 12 months of follow-up. In two analyzed cases, mutated IGVH genes were identified. Our findings indicate that variant t(2;11)(p11;q13) does not typify a classical MCL but possibly a more indolent leukemic lymphoma originating from an antigen experienced (mutated) B cell.

Comparison and evaluation of Osiris and Sirscan 2000 antimicrobial susceptibility systems in the clinical microbiology laboratory.

The Osiris and Sirscan 2000 systems are two semiautomated systems that can be used to read and interpret the results on disk diffusion agar plates. They are both used for determination of susceptibility to antimicrobial agents. The present study compared both systems versus the NCCLS standard method of visual reading with a ruler. Both inpatient and outpatient samples with a total of 315 nonfastidious gram-negative strains were obtained. In total, 3724 organism-antimicrobial agent combinations that fulfilled the NCCLS guidelines for disk diffusion susceptibility testing were evaluated prospectively. The results obtained with both systems in comparison with those obtained by the classical nonautomated means of interpretation were excellent, with correlation coefficients of 0.96 for both systems. The overall agreements for susceptibility interpretation were 96.56 and 96.24% with the Osiris and Sirscan systems, respectively. Very major errors were obtained for 8 (1.07%) and 10 (1.34%) organism-antimicrobial agent combinations with the Osiris and Sirscan systems, respectively. In addition, major errors were obtained for 2 (0.07%) and 6 (0.21%) combinations with the Osiris and Sirscan systems, respectively. Minor errors were obtained for 118 and 124 organism-antimicrobial agent combinations with the Osiris and Sirscan systems, respectively. Overall, both the Osiris system and the Sirscan system are comparable and reliable systems for determination of interpretative categories from the zone diameters of standard disk diffusion test plates.

Rothia dentocariosa, endocarditis and mycotic aneurysms: case report and review of the literature.

Rothia dentocariosa is a rare cause of endocarditis. It occurs most frequently in patients with prior heart conditions. Although the clinical course is typically subacute, it has a high rate of complications. In particular, the reported incidence of mycotic aneurysms is as high as 25%. Penicillin is the treatment of choice, but additional complications may necessitate prompt surgical intervention. As far as we know, this paper reports the first case of repeated subarachnoid hemorrhages due to R. dentocariosa endocarditis.

Streptococcus pneumoniae-infected aneurysm extending from a persistent lobar pneumonia: case report and review of the literature.

Presented here is the case of a 63-year-old patient with a Streptococcus pneumoniae-infected aneurysm extending from a persistent lobar pneumonia of the left lung into the thoracic aorta. The patient was successfully treated with surgery and high-dose penicillin, and he remained well at 6-month follow-up. A review of the English-language literature over the past 25 years revealed 22 cases of mycotic or infected aortic aneurysms due to Streptococcus pneumoniae; however, none of these cases resulted in a positive outcome for the patient. The characteristics of these cases are discussed.

Natural killer (NK) cell leukaemia in a patient with a B cell non-Hodgkin's lymphoma.

We describe a 74-year-old woman with the diagnosis of natural killer (NK)-cell leukaemia and autoimmune pathology. Four years previously, a diffuse large B cell non-Hodgkin's lymphoma had been diagnosed and treated effectively. Although NK-cell leukaemia has been thought to be a distinct highly aggressive clinicopathological entity, our case shows no further evolution at the present time. As far as we know, this association has not been previously described in the literature.

Peripheral blood T and B lymphocyte subpopulations in infants with acute respiratory syncytial virus brochiolitis.

Most data concerning immunopathogenetic mechanisms involved in respiratory syncytial virus (RSV) infection are derived from animal studies. In infants with RSV bronchiolitis the target organ i.e. the airway is hard to explore. We looked for specific alterations in peripheral blood lymphocyte subpopulations in infants hospitalized for RSV bronchiolitis. Flow cytometric analysis with a large panel of monoclonals was performed on peripheral blood lymphocytes in thirty-two infants (mean age: 4.9 months) admitted for RSV bronchiolitis. Data collected on admission were compared with age-matched control values and also with results obtained at the end of the first week of hospitalization. Differences between age-groups (older or younger than 4 months) and between clinical subgroups (clinical severity score more or less than 6) were looked for. In the group of infants as a whole, regardless of age and clinical score the number of CD4+ cells on admission was significantly elevated compared to normal values for age (p<.0001) including a high fraction of the naive suppressor-inducer subpopulation (CD4+/CD45RA+) and a low fraction of the reciprocal memory helper-inducer subpopulation (CD4+/CD29+). Within the CD8+ cell population the number of T cells with cytotoxic activity (CD8+/S6F1+) was significantly elevated (p<.0001) as were other types of cytotoxic cells. A significant decrease (p<.0001) in the proportion of the precursor/suppressor-effector subpopulation (CD8+/S6F1-) was seen. Absolute numbers and percentages of CD19+ B cells were significantly elevated (p<.0001) with a significant increase in the CD5+ subfraction (p<.0001) as well as in the CD10+ subfraction (p<.0001). In the older age group immunophenotypic cytotoxicity was more pronounced with increased clinical score. During recovery the CD45RA+:CD29+ ratio tended to normalize within the CD4+ T cells. Within the B lymphocyte subsets significant increase in the CD19+/CD5+ fraction (p<.05) was seen. We conclude that there are significant changes in the number of peripheral blood lymphocyte subsets in infants with RSV bronchiolitis as compared to age-related controls. We hope that present data could be useful in further exploration of RSV immunology in humans. A possible link between RSV bronchiolitis and the subsequent development of atopy is mentioned.

Lymphocyte subpopulations in healthy newborn infants: comparison of cord blood values with values five days after birth.

Significant differences (p < 0.0001) were demonstrated in lymphocyte subpopulations both in cord blood and in venous blood samples obtained at day 5 from the same healthy infants. Numbers of T lymphocytes increased, especially CD4+/CD45RA+ cells, whereas numbers of B lymphocytes and natural killer cells decreased without changes in CD8+ and other cytotoxic cells.

In vitro susceptibility of Capnocytophaga species to 29 antimicrobial agents.

A hemoglobin-supplemented medium composed of Columbia agar base supplemented with 1% hemoglobin and 1% Polyvitex was used to investigate the in vitro activity of 29 antimicrobial agents against Capnocytophaga species. Clindamycin was the most active agent, with all strains being inhibited by 0.06 microgram/ml or less. Amoxicillin-clavulanic acid and imipenem were the most active among the beta-lactam antibiotics (MIC for 90% of strains tested [MIC90], 0.50 microgram/ml); other very active drugs were BMY 28142, cefpirome, cefotaxime, ceftazidime, and ceftriaxone (MIC90, 0.06 to 0.50 micrograms/ml), although at least one strain showed resistance to each of these antibiotics (MIC, greater than or equal to 16 micrograms/ml). Ciprofloxacin was the most active among the quinolones, with all strains being inhibited by 0.50 microgram/ml. The MICs of the other four drugs ranged from 0.12 to 4 micrograms/ml. Ampicillin, penicillin G, ticarcillin, aztreonam, and temocillin were moderately active (MIC90, 1 to 8 micrograms/ml; MIC range, less than or equal to 0.03 to greater than 128 micrograms/ml). All strains were uniformly resistant to the aminoglycosides, polymyxin B, vancomycin, trimethoprim, and amphotericin B. Three strains produced beta-lactamase. No significant difference was found between the susceptibility of strains isolated from various sources or patients.

Elliptocytosis and schistocytosis in myelodysplasia: report of two cases.

Marked elliptocytosis and schistocytosis are described as unusual manifestations of haematopoietic dysplasia in two patients. The first patient, whose history was negative for inherited haemolytic anaemias, presented these prominent features on his first admission; 22 months later he developed an acute myeloblastic leukaemia. In the second patient, followed since 4 years for an autoimmune thrombocytopenic purpura, elliptocytosis and schistocytosis appeared 17 months before a pancytopenia established. The patient is now on follow-up and is treated for a refractory anaemia. In both cases bone marrow examinations revealed the typical criteria for myelodysplasia and this diagnosis was confirmed by cytogenetic analysis.