Three-dimensional reconstructions from non-deparaffinized tissue sections

Three-dimensional (3-D) reconstruction from microscopic images represents a useful tool for the study of biological structures in embryology and developmental biology. However, it is usually necessary to cope with many difficulties connected with the preparation of specimens. In order to minimize mutual displacement of structures in successive sections, the applicability of non-deparaffinized tissue sections for 3-D reconstruction was tested. Chicken embryos were fixed and stained in toto with eosin and then embedded in paraffin. About 30-μm-thick non-deparaffinized serial sections were used for obtaining initial data for 3-D reconstruction of larger stacks of embryonic bodies using either fluorescence or confocal microscope. The same sections served for both collecting optical serial sections of mesonephros as source images for its 3-D reconstruction, and immunohistochemical detection of fibronectin, laminin and vimentin. It was found that sections with retained paraffin preserve the mutual spatial relationships of tissue components as well as provide an excellent differentiation of structure. It makes the process of 3-D reconstruction easier. The localization of the products of immunohistochemical reactions demonstrated the co-localization of fibronectin and laminin in basal laminas and the presence of vimentin in glomeruli and mesenchymal tissue. The use of non-deparaffinized sections represents a less time consuming and more effective alternative to thin histological sections for the purpose of 3-D reconstruction, and enables further application of material.     

Tissue reorganization in response to mechanical load increases functionality

In the rapidly growing field of tissue engineering, the functional properties of tissue substitutes are recognized as being of the utmost importance. The present study was designed to evaluate the effects of static mechanical forces on the functionality of the produced tissue constructs. Living tissue sheets reconstructed by the self-assembly approach from human cells, without the addition of synthetic material or extracellular matrix (ECM), were subjected to mechanical load to induce cell and ECM alignment. In addition, the effects of alignment on the function of substitutes reconstructed from these living tissue sheets were evaluated. Our results show that tissue constructs made from living tissue sheets, in which fibroblasts and ECM were aligned, presented higher mechanical resistance. This was assessed by the modulus of elasticity and ultimate strength as compared with tissue constructs in which components were randomly oriented. Moreover, tissue-engineered vascular media made from a prealigned living tissue sheet, produced with smooth muscle cells, possessed greater contractile capacity compared with those produced from living tissue sheets that were not prealigned. These results show that the mechanical force generated by cells during tissue organization is an asset for tissue component alignment. Therefore, this work demonstrates a means to improve the functionality (mechanical and vasocontractile properties) of tissues reconstructed by tissue engineering by taking advantage of the biomechanical forces generated by cells under static strain.     

Effects of emotional disclosure on psychological and physiological outcomes in patients with rheumatoid arthritis: an exploratory home-based study

The effects of an exploratory, home-based emotional disclosure intervention on psychological and physiological outcomes were assessed in patients with rheumatoid arthritis. Patients were randomly assigned to a disclosure group (n = 19) in which they wrote/talked about traumatic personal experiences, or to a control group (n = 15) in which they wrote/talked about the events of a particular day. Participants undertook these tasks for periods of 20 minutes on 4 consecutive days. The disclosure group demonstrated increases in negative mood and objective markers of disease activity at 1 week post-intervention. However, there were significant trends for the disclosure group to demonstrate minor improvements in mood and stability in disease activity, compared with the control group. These group differences appeared to be due to deteriorations in the control group more than improvements in the disclosure group.     

Surface receptors identify mouse NK1.1+ T cell subsets distinguished by function and T cell receptor type

Natural killer T (NKT) lymphocytes rapidly produce several cytokines, including IL-4 and IFN-gamma, upon activation, and act as regulatory cells at an early interphase of innate and adaptive immune responses. They have been implicated as important elements in diverse immune responses including the regulation of autoimmune disease, the immune response to infections, and the prevention of tumor metastasis. The broad spectrum of their activities suggested that functionally different subsets of NKT cells may exist. We demonstrate two functionally distinct splenic NKT populations identified by the expression of CD49b and CD69, respectively. Each NKT subset was represented by the amplified transgenic NKT cell population in a distinct transgenic mouse line expressing a CD1d-restricted TCR. CD49bhigh CD69- NKT cells, termed NKT1 cells by us, were high producers of IFN-gamma after stimulation, but essentially devoid of IL-4-synthesizing cells. Most NKT1 cells used diverse (non-Valpha14-canonical) TCR. The CD69+ CD49(-/low) NKT cell population, which we term NKT2, produced large quantities of IL-4 and substantial amounts of IFN-gamma upon activation and were dominated by cells using the canonical Valpha14-Jalpha18 T cell receptor. Knowledge of the unique roles of the different NKT cell subsets in specific situations will be essential for our understanding of NKT cell biology.     

Porcine reproductive and respiratory syndrome virus persistence in blood, spleen, lymph nodes, and tonsils of experimentally infected pigs depends on the level of CD8high T cells

Porcine reproductive and respiratory syndrome virus (PRRSV) induces a persistent viral infection suggesting an inefficient cellular immune response. The aim of the study was to evaluate the relationship between viral persistence and cytotoxic cells in blood, spleen, mediastinal lymph nodes (MLN) and tonsils of PRRSV experimentally infected pigs. Groups of four to six specific pathogen-free (SPF) pigs were infected with the LHVA-93-3 isolate, and blood and lymphoid organs were collected from 3 to 60 days post-infection (p.i.). Infectious particles and viral RNA were more or less rapidly eliminated in serum, spleen, lungs and MLN but persisted the longest in tonsils. Lymphocytes CD2+ CD4+, CD2+ CD8high, CD2+ CD8low and NK cells populations were phenotyped and their reactivity to PHA and ConA were tested. Analysis of T cell subsets in blood and lymphoid organs indicated that the percentages of CD2+ CD8+ T cells slightly increased in spleen at 17 days p.i, whereas no changes were observed in CD2+ CD4+ cells in blood or lymphoid organs. However, discrimination of CD8+ cells in CD8high and CD8low subsets revealed that the percentages of CD2+ CD8high cells increased in spleen and blood from 10 to 45 or 60 days p.i. while they transiently increased in MLN and decreased in tonsils. The CD8low/CD8high ratio increased in the blood of PRRSV-infected animals at three days p.i. due to a transient decrease of CD2+ CD8high cells. This same ratio decreased in the spleen of infected pigs from 10 to 45 days p.i. due to an increase of CD2+ CD8high cells. The CD2+ MIL-4+ cell subset (NK cells) was not significantly modified in blood or lymphoid organs. In addition, the ability of lymphoid T cells from blood and lymphoid organs to respond to ConA or PHA stimulation was transiently impaired in blood and spleen during the PRRSV persistent infection. Taken together, these results suggest that, in persistently infected pigs, an impaired CD2+ CD8high cell response in MLN and tonsils favors viral persistence in these organs, in contrast with the response seen in blood and spleen where viral elimination appears to occur sooner.     

Genotype-phenotype correlation in Smith-Magenis syndrome: evidence that multiple genes in 17p11.2 contribute to the clinical spectrum.

PURPOSE: Smith-Magenis syndrome (SMS) is a complex disorder that includes mental retardation, craniofacial and skeletal anomalies, and behavioral abnormalities. We report the molecular and genotype-phenotype analyses of 31 patients with SMS who carry 17p11.2 deletions or mutations in the RAI1 gene. METHODS: Patients with SMS were evaluated by fluorescence in situ hybridization and/or sequencing of RAI1 to identify 17p11.2 deletions or intragenic mutations, respectively, and were compared for 30 characteristic features of this disorder by the Fisher exact test. RESULTS: In our cohort, 8 of 31 individuals carried a common 3.5 Mb deletion, whereas 10 of 31 individuals carried smaller deletions, two individuals carried larger deletions, and one individual carried an atypical 17p11.2 deletion. Ten patients with nondeletion harbored a heterozygous mutation in RAI1. Phenotypic comparison between patients with deletions and patients with RAI1 mutations show that 21 of 30 SMS features are the result of haploinsufficiency of RAI1, whereas cardiac anomalies, speech and motor delay, hypotonia, short stature, and hearing loss are associated with 17p11.2 deletions rather than RAI1 mutations (P<.05). Further, patients with smaller deletions show features similar to those with RAI1 mutations. CONCLUSION: Although RAI1 is the primary gene responsible for most features of SMS, other genes within 17p11.2 contribute to the variable features and overall severity of the syndrome.