Use of computational biology to compare the theoretical tertiary structures of the most common forms of RhCE and RhD.

BACKGROUND AND OBJECTIVES: Computational biology analyses the theoretical tertiary structure of proteins and identifies the 'topological' differences between RhD and RhCE. Our aim was to identify the theoretical structural differences between the four isoforms of RhCE and RhD using computational biological tools. MATERIALS AND METHODS: Physicochemical profile was determined by hydrophobicity and electrostatic potential analysis. Secondary and tertiary structures were generated using computational biology tools. The structures were evaluated and validated using Ramachandran algorithm, which calculates the single score, p-value and root mean square deviation (RMSD). Structures were overlaid on local refinement of 'RhAG-RhCE-ANK' (PBDID 7uzq) and RhAG to compare their spatial distribution within the membrane. RESULTS: All proteins differed in surface area and electrostatic distance due to variations in hydrophobicity and electrostatic potential. The RMSD between RhD and RhCE was 0.46 ± 0.04 Å, and the comparison within RhCE was 0.57 ± 0.08 Å. The percentage of amino acids in the hydrophobic thickness was 50.24% for RhD while for RhCE it ranged between 73.08% and 76.68%. The RHAG hydrophobic thickness was 34.2 Å, and RhCE's hydrophobic thickness was 33.83 Å. We suggest that the C/c antigens differ exofacially at loops L1 and L2. For the E/e antigens, the difference lies in L6. By contrast, L4 is the same for all proteins except Rhce. CONCLUSION: The physicochemical properties of Rh proteins made them different, although their genes are homologous. Using computational biology, we model structures with sufficient precision, similar to those obtained experimentally. An amino acid variation alters the folding of the tertiary structure and the interactions with other proteins, modifying the electrostatic environment, the spatial conformations and therefore the antigenic recognition.

Primary explants of the postnatal thymus allow the expansion of clonogenic thymic epithelial cells that constitute thymospheres.

BACKGROUND: Thymic epithelial cells (TECs) are responsible for shaping the repertoires of T cells, where their postnatal regeneration depends on a subset of clonogenic TECs. Despite the implications for regenerative medicine, their cultivation and expansion remain challenging. Primary explant cell culture is a technique that allows the seeding and expansion of difficult-to-culture cells. Here, we report a reliable and simple culture system to obtain functional TECs and thymic interstitial cells (TICs). METHODS: To establish primary thymic explants, we harvested 1 mm cleaned fragments of thymus from 5-week-old C57/BL6 mice. Tissue fragments of a complete thymic lobe were placed in the center of a Petri dish with 1 mL of DMEM/F-12 medium supplemented with 20% fetal bovine serum (FBS) and 1% penicillin‒streptomycin. To compare, thymic explants were also cultivated by using serum-free DMEM/F-12 medium supplemented with 10% KnockOut™. RESULTS: We obtained high numbers of functional clonogenic TECs and TICs from primary thymic explants cultivated with DMEM/F-12 with 20% FBS. These cells exhibited a highly proliferative and migration profile and were able to constitute thymospheres. Furthermore, all the subtypes of medullary TECs were identified in this system. They express functional markers to shape T-cell and type 2 innate lymphoid cells repertoires, such as Aire, IL25, CCL21 and CD80. Finally, we also found that ≥ 70% of lineage negative TICs expressed high amounts of Aire and IL25. CONCLUSION: Thymic explants are an efficient method to obtain functional clonogenic TECs, all mTEC subsets and different TICs Aire+IL25+ with high regenerative capacity.

[Psychological study of the dysthymic disorder in the woman]. / Estudio psicológico del trastornodistímico en la mujer.

In this article, we study two dysthymic women who we are treating with psychotherapy in order to reveal the inner components that maintain depressive symptoms. The same findings have been confirmed in other dysthymic patients. The result of the study consisted in discovering a sentimental separation from their love object, while the woman still lives with her partner and while the depressive symptoms are appearing insidiously. This development leads them to the deterioration in the "ideal of love" they sought, that supported their lives and served as an "anchor of their personality. This point of view places classic notion about mourning into doubt.

The 30-kDa band from Salmonella typhimurium: IgM, IgA and IgG antibody response in patients with ankylosing spondylitis.

OBJECTIVE: To determine the association of Salmonella typhimurium antigens with AS by analysing the IgA, IgG and IgM antibody response to the crude lysate and the 30-kDa band from this micro-organism. METHODS: Sera from 28 AS patients, 28 HLA-B27+ healthy relatives, 28 unrelated healthy subjects and 14 RA patients were included. Salmonella typhimurium proteins were electrophoretically separated and blotted onto nitrocellulose sheets for immunodetection with sera from AS patients and unrelated healthy subjects. The electroeluted 30-kDa band (p30) and a crude lysat (StCL) from S. typhimurium were used as antigen to evaluate the IgM, IgA and IgG (total and subclasses) antibody levels by ELISA. An inhibition assay was carried out to confirm the specificity of IgG response to the p30. RESULTS: Twenty out of 28 AS patients (71.4%) and 4 out of 28 unrelated healthy subjects (14.3%) recognized a 30-kDa band from S. typhimurium with IgG antibodies. Six out of 28 AS patients (21.4%) and 4 out of 28 unrelated healthy subjects (14.3%) detected it with IgA antibodies. Recognition of p30 and StCL by both IgA and IgG antibodies was higher in AS patients than in control groups (P = 0.003, <0.001 and 0.003 for IgA and <0.001, 0.003 and 0.006 for IgG). Sera from AS patients have higher percentage of IgG antibodies p30 and IgG3 subclass was higher in AS patients than in control groups. No differences in the IgM response were found. CONCLUSIONS: Data presented suggest the association between the p30 and AS.

Src, Akt, NF-κB, BCL-2 and c-IAP1 may be involved in an anti-apoptotic effect in patients with BCR-ABL positive and BCR-ABL negative acute lymphoblastic leukemia.

BCR-ABL kinase has been observed to be potentially related to leukemic cell development. Adult patients with acute lymphoblastic leukemia (ALL) were evaluated to determine whether presence/absence of BCR-ABL induced differences in activation of Src, PI3K/Akt and NF-κB or in the expression of anti-apoptotic proteins such as BCL-2 and c-IAP1. BCR-ABL positive patients showed a significantly higher activation of Src and Akt compared with BCR-ABL negative patients and healthy donors. BCR-ABL negative patients also showed a significant activation of Src and low levels of Akt activation compared with healthy donors. Both patient groups had increased NF-κB activation and overexpression of BCL-2 and c-IAP1. This is the first study to evaluate concurrently in ALL patients presence/absence of BCR-ABL in relation to activation of Src, Akt and NF-κB and the expression of anti-apoptotic proteins. Results suggest that these proteins may be involved in an anti-apoptotic signaling pathway.