An association between rs7635818 polymorphism located on chromosome 3p12.3 and the presence of abdominal aortic aneurysm.

The association between gene variant rs7635818 located on chromosome 3p12.3 and abdominal aortic aneurysm (AAA) was not unambiguously determined by the results of genome-wide association studies. The aim of our study was to examine this possible association in the Slovak population, with respect to the presence and severity of AAA.A cross-sectional study was conducted between August 2016 and March 2020. The study included 329 participans, 166 AAA patients and a control group of 163 subjects without confirmed AAA with comparable distribution of genders. The anteroposterior diameter of the abdominal aorta was determined by duplex ultrasonography. AAA was defined as subrenal aortic diameter ≥ 30 mm. DNA samples were genotyped using real-time polymerase chain reaction and subsequent high-resolution melting analysis in presence of unlabelled probe. Genetic models studying the possible association were adjusted to age, sex, smoking, arterial hypertension, diabetes mellitus, creatinine and body mass index (BMI) in multivariate analysis. In the additive model, presence of each C-allele of rs7635818 polymorphism was associated with an almost 50 % increase in probability of developing AAA (OR 1.49; 95 % CI 1.06-2.08; p=0.020). Compared to GG homozygotes, CC homozygotes had more than two times higher risk of developing AAA (OR 2.23; 95 % CI 1.14-4.39; p=0.020). The risk of AAA was also in the recessive model higher for CC homozygotes compared to G-allele carriers (GC/GG) (OR 1.79; 95 % CI 1.01-3.19; p=0.047).The abdominal aortic diameter in CC homozygotes of the rs7635818 polymorphism was 7.66 mm greater compared to GG homozygotes (42.5±22.0 mm vs 34.8±21.3 mm; p=0.022) and 5.88 mm greater compared to G-allele carriers (GC/GG) (42.5±22.0 mm vs 36.6±21.0 mm; p=0.04) in univariate analysis. C-allele variant in rs7635818 G>C polymorphism is associated with a higher probability of developing AAA in the Slovak population.

[Malignant fibrous histiocytoma as a rare cause of acute limb ischemia]. / Malígny fibrózny histiocytóm ako raritná prícina akútnej koncatinovej ischémie.

Malignant fibrous histiocytoma (MFH) represents a rare malignant affection of heart and aorta. Its clinical presentation depends on the localisation, size, degree of invasion and metastasis. Previously, relatively few cases of acute tumour mass embolisation into the visceral and limb arterial system were described in the literature. In the present case study we describe a case of acute ischemia of both lower extremities caused by thromboembolic mass of MFH cells. According to literary sources this tumour type is characterized by poor prognosis as it was in the case of our patient.

Critical care nurses' perceptions of family needs as met.

In this descriptive, exploratory study, nurses' perceptions of family needs as met during the critical care experiences of an adult member were correlated to the families' perception of those same needs as being met. The population consisted of 45 family members in a large county hospital designated as a Level 3 trauma center. Family members of adult patients and registered nurses who were assigned to care for them completed a three-part instrument, which consisted of the Demographics Data Questionnaire, the Critical Care Family Needs Inventory (CCFNI), and the Needs Met Inventory (NMI). Data were analyzed using descriptive and inferential statistics. The top 10 needs perceived by the family members and registered nurses were reported in order of importance during the first 18-24 hours on the CCFNI and NMI. Data were analyzed on all 45 need statements to determine the top 10 needs perceived as important on the CCFNI and perceived as being met on the NMI. A one-way analysis of variance (ANOVA) test was performed on the data and yielded significant differences on three of the items. Linear regression was performed using t test which supported a significant difference on five statements based on critical care nursing years of experiences in critical care. Self-reported or open-ended comments from the family members and nurses were presented.

Differentiation of mouse embryonic stem cells in vitro: III. Morphological evaluation of tissues developed after implantation of differentiated mouse embryoid bodies.

Mouse embryonic stem cells (ES) were allowed to differentiate in a liquid culture system. After 2-3 weeks, complex cystic embryoid bodies developed. These bodies were composed of several structures identified as cardiac muscle and yolk sac blood islands as well as cup-shape compartments containing a mixed population of hematopoietic stem cells. When these cystic embryoid bodies were implanted into adult mice, either subcutaneously or under the kidney capsule, they developed into various tissues. These included bone, blood vessels, cardiac muscle, nerves, and skin with hair follicles. In addition, highly differentiated, complicated tissues resembling intestinal epithelium with mucus glands or salivary glandular tissue were derived. The ES tissues from these in vitro developed embryoid bodies developed quickly within 2 to 3 weeks of implantation. This is in contrast to a minimal of 6 weeks for teratocarcinomas derived from embryonic carcinoma cells and/or the direct implantation of undifferentiated embryonic stem cells. Moreover, we found that there are different types of tissue developed upon different sites of implantation. The data suggest a local environment and/or growth factors are influential for ES tissue development. This system provides a possible means to purify and identify stem cells that give rise to specific tissues, and to study the factors regulating the commitment of these stem cells.

Immunoglobulin D-deficient mice can mount normal immune responses to thymus-independent and -dependent antigens.

To examine the in vivo function of IgD we generated mice deficient for IgD by gene targeting. The IgD-mice show a reduced B-cell compartment with 30-50% less B cells in the spleen and lymph nodes but show a normal pre-B-cell compartment. The surface-IgD- B cells express two to three times more surface IgM than B cells of control animals. Serum concentrations of the immunoglobulin isotypes of IgD- mice are almost normal, indicating that surface-IgD expression is not necessary for class switching of B cells. Immunization experiments showed that IgD- mice could respond well to thymus-dependent and -independent antigens. After immunization normal germinal centers developed in the IgD- mice. These data suggest that IgD is not necessary for the induction of immune responses but may be important in homeostasis of cells in the B-cell compartment.

Follicular dendritic cells in non-Hodgkin-lymphoma express adhesion molecules complementary to ligands on neoplastic B-cells.

In non-Hodgkin-lymphoma (NHL) with nodular growth patterns, follicular dendritic cells (FDC) form a spherical network which contains neoplastic B-cells. In order to dissect the basis of this close FDC/B cell association, the antigenic profile of adhesion molecules expressed by individual FDC and NHL-B-lymphocytes was evaluated. FDC isolated from NHL were found to express C3bi receptors (CD11b), the very late antigen (VLA) alpha-5- and alpha-6-chain (CD49e, CD49f), and the intercellular adhesion molecule-1 (ICAM-1; CD54). Only a percentage of the FDC population was positive for the VLA beta-1- and alpha-3-chain (CD29, CD49c), the vitronectin receptor (CD51) and the vascular cell adhesion molecule-1 (VCAM-1). B-cells obtained from the lymph nodes of patients with centroblastic-centrocytic lymphoma expressed several ligands complementary to the adhesion molecules detected on FDC. These include LFA-1 alpha- and beta-chains (CD11a, CD18), and ICAM-1 (CD54). Surprisingly, monoclonal lymphocytes in the peripheral blood of patients with a leukaemic course of this lymphoma entity were devoid of these antigens. It seems likely then that neoplastic B-cells without CD11a, CD18 and CD54 surface molecules are unable to associate with FDC and now invade other compartments. Thus, the adhesive interactions which do occur between FDC and NHL-B-cells may directly influence the peculiar growth pattern and spread of centroblastic-centrocytic lymphoma.

Development of autoimmune disease in SCID mice populated with long-term "in vitro" proliferating (NZB x NZW)F1 pre-B cells.

Pre-B cell lines proliferating for several months on stromal cells in the presence of interleukin 7 (IL-7) were established from fetal liver of (NZB x NZW)F1 mice. They express the B lineage-specific markers PB76, B220, and VpreB, but do not express surface immunoglobulin (sIg). Upon removal of IL-7 from the culture, they differentiate to sIg+ B cells that can then be stimulated by lipopolysaccharide to become IgM-secreting cells. Transfer of these pre-B cell lines into SCID mice leads to hypergammaglobulinemia of IgM (600-900 micrograms/ml), IgG2a (1-3 mg/ml), and IgG3 (300-500 micrograms/ml) for the next 3-5 mo. The spleen appears populated with (NZB x NZW)F1-derived pre-B cells, few B cells, and many IgM and/or IgG-producing plasma cells. In contrast, SCID mice populated with pre-B cell lines of normal (C57BL/6 x DBA/2)F1 mouse fetal liver develop normal levels of serum IgM (approximately 100-300 micrograms/ml), almost no detectable levels of IgG, and no plasma cell hyperplasia. The (NZB x NZW)F1 pre-B cell-populated SCID mice contain elevated serum titers of IgG antinuclear autoantibodies, but no retroviral gp70-specific nor erythrocyte-specific autoantibodies. Up to 20% of the SCID mice develop proteinuria as a consequence of IgG deposits in the kidney glomeruli during a 7-mo period of observation. All signs of autoimmune disease seen in these mice are independent of the sex of the SCID host. This experimental system provides a distinction between the disease-determining (NZB x NZW)F1 genes, which are expressed in the B lymphocyte lineage and cause the development of the disease, from those expressed in other cell lineages which only modulate its progression.

Establishment and characterization of lymphoid and myeloid mixed-cell populations from mouse late embryoid bodies, "embryonic-stem-cell fetuses".

Mouse embryonic stem (ES) cells have the potential to differentiate into embryoid bodies in vitro and mimic normal embryonic development. The "ES fetus" is a specific development at a late stage seen under our culture conditions. We have established several mixed populations from ES fetuses by using combinations of retroviruses carrying different oncogenes (v-abl, v-raf, c-myc), interleukins 2 and 3, and Con A. Six groups of mixed populations were characterized by immunophenotyping. For some groups, transfer of cells into sublethally irradiated mice resulted in the development of macrophages, mature T and B lymphocytes, and plasma cells of donor origin. Thus, these mixed populations may contain immortalized precursors of hematopoietic lineages. These mixed populations should be valuable for defining hematopoietic stem cells and their committed progenitors.

Follicular dendritic cell-dependent adhesion and proliferation of B cells in vitro.

In response to an antigenic challenge, B cells proliferate in germinal centers within secondary lymphoid tissue. Specialized accessory cells, follicular dendritic cells (FDC), and T cells are necessary to drive this reaction. Indirect evidence suggests that FDC provide signals which not only induce B cell proliferation but can rescue B cells programmed to die by apoptosis. An in vitro system was developed to: 1) define the role of FDC and 2) identify molecules involved in this response. Activated, low density B cells and T cells were coisolated with FDC from immune mouse lymph nodes. Upon culturing, large cellular aggregates formed, composed of 1 to 3 FDC interdigitating between 30 to 90 B cells and 1 to 5 T cells. Many of these B cells were undergoing DNA synthesis. Depleting FDC or T cells from the cultures immediately stopped cluster formation and proliferation. Separating clustered vs nonclustered cells revealed that the FDC-associated population remained viable, whereas cells in suspension became apoptotic. The adhesion/activation molecules ICAM-1, LFA-1, and CD44 supported both cluster formation and proliferation. In addition, anti-class II and anti-kappa L chain mAb interfered dramatically with DNA synthesis. This model mimics many of the features of a germinal center and can be used to further study B cell activation, proliferation, and differentiation in vitro.

Signals involved in germinal center reactions.

Many of the features observed in the in vitro cultures discussed in this review coincide with characteristics described for an in vivo germinal center response. FDC and T cells are required to maintain B-cell proliferation which is confined to a finite amount of time (i.e. less than 2 wk). Large cellular aggregated form which contain many blasting cells undergoing DNA synthesis. In addition to proliferation, apoptosis is also occurring in the cultures but appears to be limited to the population which is not in contact with the FDC. The system can be driven by specific antigen, suggesting that clonal expansion is occurring. As in other immunological systems, there is an important role for adhesion molecules both for cluster formation and DNA synthesis. Antigen processing and presentation is a major event since blocking this through several mechanisms ends the stimulation. The role of T cells is essential both in vivo and in vitro; however, their exact contribution is still not well understood. It is interesting that blocking IL4 usage either by neutralizing the molecule or its receptor by monoclonal antibodies has no effect on the system. Which interleukins are important for germinal centers remains on open question. Evidence continues to accumulate on the important role of FDC and the molecules they express. Not only are the immune complexes an essential part, but it seems that molecules yet to be defined have an effect. For many practical reasons these have remained a mystery, but using our various systems we are attempting to reveal them. Two intriguing questions which remain include: 1. the molecular nature of the signalling between the FDC and B cell; and 2. how does the FDC retain the antigen in a native form for such long periods of time? An understanding of both mechanisms will provide us with a better appreciation for the events leading to a germinal center response and the immunological phenomenon referred to as memory.

Proliferation of non-Hodgkin-lymphoma lymphocytes in vitro is dependent upon follicular dendritic cell interactions.

Follicular dendritic cells (FDC) are located within the B-cell follicles of non-malignant lymphatic tissues and within non-Hodgkin-lymphomas (NHL) derived from the germinal centre or the mantlezone. The interactions between FDC and non-neoplastic B-cells have been extensively investigated but so far no data on functional studies with FDC isolated from lymphoma tissue are available. Using an enzyme cocktail to digest lymph nodes from patients with NHL followed by density centrifugation, single cell suspensions enriched in FDC and B-lymphocytes were obtained. In these preparations FDC formed small cellular clusters with an average of five neoplastic lymphocytes for every FDC. Immunocytochemistry with Ki67 revealed that after 24 h of culture 23.7% of the cells within the clusters were in late G1 to M phase. In contrast, only 10.2% of the lymphoma cells scattered outside the clusters were in these activated stages. As visualized by autoradiography, after 72 h of incubation the rate of proliferation was 16.8 times higher for the lymphoma cells involved in cluster formation as compared to those lymphocytes not associated with FDC. The data indicate that in vitro FDC from NHL lymph nodes form a microenvironment favourable for the activation and proliferation of lymphoma cells. The search for cytokines secreted by FDC in lymphoma tissue is under way.

Chemotaxis of germinal center B cells in response to C5a.

An infiltrate of B cells and plasma cells is characteristic of certain chronic inflammatory lesions. However, mechanisms involved in the local accumulation of these cells have not been established. Efforts to demonstrate that B cells from normal animals can migrate in response to inflammation-induced chemoattractants have been inconclusive. The objective of this study was to determine if murine germinal center (GC) B cells could respond chemotactically to a C5a gradient. On successive days after secondary immunization, draining lymph nodes were harvested and the activated GC B cells isolated. These GC B cells were placed in modified Boyden chambers, incubated for 3 h and the distance the leading front of cells migrated through the filters was determined. The results show that GC B cells migrated to factors in zymosan- and lipopolysaccharide-activated serum. The migratory response demonstrated distinct kinetics. Cells isolated between 2 to 4 days after secondary immunization migrated, whereas cells isolated at day 0 and beyond day 6 did not. Checkerboard analysis revealed that the migratory response was attributable to both chemokinesis and chemotaxis. Anti-C5 inhibited the migration of day-3 GC B cells implicating C5 in the migration mechanism. Studies using recombinant C5a established that this C5 fragment was chemotactically active. In conclusion, GC B cells generally were not chemotactically active. However, at a particular stage of maturation B cells in the GC become responsive to C5a as a chemotactic agent. Thus, B cells from normal animals may respond chemotactically, and C5a may play a role in recruitment of recently activated B cells into inflammatory sites.

Proliferation of germinal center B lymphocytes in vitro by direct membrane contact with follicular dendritic cells.

In secondary lymphoid organs, follicular dendritic cells (FDC) are located within B cell follicles and germinal centers. Through their cytoplasmic extensions they come into contact with a large number of neighboring lymphocytes. Using an enzyme cocktail to digest human tonsils followed by ultracentrifugation on bovine serum albumin gradients, single cell suspensions were obtained. Immunocytochemistry revealed that 7% of the cells were FDC, 5% T cells, and 5% macrophages. The remaining population were B cells with greater than 95% being of the germinal center phenotype (i.e. CD19-positive, CD39/sIgD negative). After 24 h of culture up to 44% of the lymphocytes were found in clusters centered around FDC. At the start of the culture as well as 24 and 72 h later, between 31 and 55% of the B cells within FDC associated clusters were in late G1 to M phase of the cell cycle. In contrast, less than 10% of the B cells not in contact with FDC (i.e. outside the clusters) were in an activated state. Autoradiography revealed that after three days of incubation the rate of proliferation was 26.2 times higher for the lymphocytes involved in cluster formation as compared to those cells not associated with FDC. Furthermore, the number of viable B cells after a 72 h mitogen-free culture period was determined. By adding FDC to these preparations, 31.9% of the lymphocytes were rescued from dying. These data show that FDC provide a microenvironment which can maintain the viability, activation and proliferation of germinal center B cells in vitro.

Iccosomes and the secondary antibody response.

Iccosomes derived from follicular dendritic cells (FDC) are believed to play an important role in dispersion of antigen necessary for induction of anamnestic responses. Because FDC are aberrant and iccosome release has not been observed in aged mice, we hypothesized that these animals would be impaired in their ability to mount anamnestic responses. To test this, anamnestic responses were compared in aged and control mice. To ensure the presence of functional lymphocytes, some aged mice were reconstituted with T- and B-memory cells obtained from control mice. Anamnestic responses in aged mice were markedly depressed even when given functional memory cells. To more directly relate the impaired antibody response of aged mice to FDC function, antigen-bearing FDC from either aged or control mice were incubated with T- and B-memory cells from control mice to induce an anamnestic response. Antigen retained by FDC from aged mice was much less immunogenic than antigen retained by FDC from control mice. Since iccosome formation does not appear in aged mice, iccosome-like fragments were generated by sonicating FDC from aged mice and tested for their ability to induce an anamnestic response. This procedure restored the ability of antigen retained on FDC from aged mice to induce a normal anamnestic response. These data support the concept that the inability to form and disperse iccosomes contributes to the impaired ability of aged mice to mount anamnestic antibody responses and provides further support for the role of iccosomes in anamnestic responses.

Antigen presentation to B cells.

B lymphocytes encounter antigen within the body, which causes them to undergo clonal expansion, affinity maturation and differentiation to antibody-forming cells. How they come into contact with the immunogen, and the subsequent response induced is the subject of several recent reports.

Novel pathways of antigen presentation for the maintenance of memory.

Follicular dendritic cells (FDC) store native antigen for long periods in lymphoid follicles and so provide a source of continued stimulation for specific B cells. The expression of MHC class II by FDC suggested they might act as antigen-presenting cells for MHC class II-restricted T cells. We show here, however, that the MHC class II molecules found on their surface are not synthesized by the FDC but are picked up from surrounding B cells in germinal centres. Although FDC by themselves cannot present native antigen to T cells, acquired MHC class II-peptide complexes can be recognized by T cells. The true physiological role of FDC seems to be as long-term antigen depots. We demonstrate that antigen localized onto FDC in vivo can be retrieved by antigen-specific B cells, which in turn process and present it to T cells. These presentation pathways are likely to be crucial in both the maintenance of long-term immune responses and the continued survival of memory cells.