Diversity and structure of human T cell receptor delta chain genes in peripheral blood gamma/delta-bearing T lymphocytes.

We have investigated the diversity and repertoire of human TCR delta chain variable gene segments in the human peripheral blood CD4- CD8- (double-negative) population, using rearrangement and expression studies and sequence analyses. 20 TCR delta DNA clones were derived from the RNA of bulk-cultured double-negative T cells and their nucleotide sequences determined. These clones can be classified into six different V delta subfamilies. The distribution, however, was uneven in these cells, with 16 of 20 being derived from the V delta 1 (9) and V delta 2 (7) subfamilies. The remaining subfamilies, V delta 3, V delta 4, V delta 5, and V delta 6, were only represented by one clone each. The majority of these subfamilies seem to consist of a single member, in contrast with the closely linked V alpha subfamilies, which, in most cases, consist of multiple members. Our findings suggest that only a limited number of V delta genes are used in human peripheral blood double-negative T cells and that two major V delta subfamilies (V delta 1 and V delta 2) are used more frequently. Sequence comparison of our cDNA clones to V alpha clones indicates that there is no overlap in usage of V alpha and V delta gene segments, except for the V delta 4 (V alpha 6) subfamily. Comparison of the different V delta sequences suggests that the majority of the sequence diversity is concentrated in the junctions between V, D, and J segments and results from extensive N region diversity.

T cell receptor variable gene usage in a specific cytotoxic T cell response. Primary structure of the antigen-MHC receptor of four hapten-specific cytotoxic T cell clones.

The primary structure of the alpha and beta chains of the T cell antigen receptor in four cytotoxic T cell clones specific for N-iodoacetyl-sulfonic-naphthyl-ethylene-diamine (AED)-haptenated target cells displaying a particular class I MHC molecule has been determined. Two of the T cell clones, 8/10-2 and 5/10-20K, recognize AED-modified targets in association with H-2Kb, while the other two clones 5/10-20D and C9 react with AED-modified cells in the context of H-2Db. Comparison of the nucleotide sequences of both the alpha and beta chain cDNAs and their deduced protein sequences indicates that a specific variable gene segment was not used to recognize the hapten and/or class I gene products. Furthermore, there does not appear to be any conserved amino acid residues used in the AED-specific response other than the framework amino acids. However, when the two clones 8/10-2 and 5/10-20D were compared, a striking similarity was seen in the J segments. These two clones that recognize AED in the context of different MHC epitopes used identical J alpha (J alpha 810) and J beta (J beta 2.6) gene segments. C9, specific for AED-Db, shared identical V beta (V beta 6) and J beta gene segments (J beta 1.1) as those of a cytotoxic T cell that recognizes allogeneic targets expressing Db. These data indicate that a simple rule governing the usage of the variable regions of either the alpha or beta T cell receptor (TcR) genes in the recognition of antigen and MHC gene products cannot be formulated. However, subtle similarities can be detected in some situations between the primary structures of the TcR and the targets they recognize.

Investigation of the biomechanical integrity of decellularized rat abdominal aorta.

OBJECTIVES: The loss or damage of an organ or tissue is one of the most common and devastating problems in healthcare today. Tissue engineering applies the principles of engineering and biology toward the development of functional biological replacements that are able to maintain, improve, or restore the function of pathological tissues. The aim of the overall project is to study an already existing method for the decellularization of homograft vascular grafts for use in vascular surgery. MATERIALS AND METHODS: The biomechanical integrity of native and decellularized rat aortas was assessed under uniaxial tension tests. For this purpose, 36 male rats (12 Wistar and 24 Dark Agouti [DA]) were used to excise their abdominal aortas. Twelve of the aortas were tested fresh (Wistar and DA rats), within 24 hours from euthanasia, and the rest were decellularized using a modified protocol (DA rats only). Fresh and decellularized samples (n = 12) were subjected to uniaxial tensile loading to failure, and the recorded stress-strain behaviour of each specimen was assessed in terms of 6 biomechanical parameters. RESULTS: No statistically significant differences were found in any of the biomechanical parameters studied between the decellularized DA rat aorta group and both the native DA and Wistar rat aorta groups (P > .05). Also, no significant difference was shown between the native DA and native Wistar rat aorta groups. CONCLUSIONS: The results from this study have shown that the decellularization protocol did not affect the mechanical properties of the native rat aorta. In addition to this, both native Wistar and native/decellularized DA rat aorta groups shared similar mechanical properties.

A feasibility study of partially accelerated radiotherapy for invasive bladder cancer.

Thirty-nine patients with histologically confirmed invasive bladder carcinoma (T2-3, N0, M0) were treated with a partially accelerated radiotherapy scheme. After 40 Gy/4 weeks of conventional fractionation we have accelerated the treatment in the last week giving two daily fractions of 2 Gy each, 4-6 h apart in the bladder only. Although the follow-up of some of the patients is not very long our results indicate that this relatively short radiotherapeutic scheme is feasible, convenient and probably safe for patients living in remote areas.

Evaluation of decellularization in umbilical cord artery.

Major achievements in creating decellularized whole tissue scaffolds have drawn considerable attention to decellularization as a promising approach for tissue engineering. Developing a tissue-engineered small-diameter (≤2 mm) vascular graft, using decellularized human umbilical arteries (hUAs), for reconstructive surgery is a challenging task. Polymers used in the past, proved unsuitable due to serious adverse effects and autologous vessels are available only in 40% of patients. In this study, histological and proteomic analysis was performed to evaluate the efficiency of two decellularization protocols. In decellularization protocol A, hUAs were incubated in 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS) and sodium dodecyl sulfate (SDS) followed by incubation in alpha minimal essential medium (α-MEM) with foetal bovine serum (FBS) while in decellularization protocol B the hUAs were incubated in Hypotonic Tris and SDS followed by incubation in nuclease solution. Histological analysis of decelullarised hUA with both protocols revealed good preservation of extracellular cell matrix (ECM) proteins and immunofluorescent staining detected collagen I and fibronectin. The DNA content within the hUAs after decellularization with protocol A was 6.2% and with protocol B 17.3%. Proteomic analysis identified cytoplasmic enzymes such as, dehydrogenase X, α-enolase and peptidyl-prolyl cis-trans isomerase A only in native samples, while, cytoskeletal proteins such as a-actin, filamin and ECM proteins like collagens were found both in native and decellularised hUA. In conclusion, both decellularization protocols effectively removed the cellular material while the ECM remained intact. Future studies are warranted to elucidate the specific effects of altered structure-function relationships on the overall fate of decellularized hUAs.

Ocular rotation as a tool for superior oblique tendon exposure.

In several clinical studies involving superior oblique surgery it was noticed that the posterior insertion of the superior oblique tendon can be moved outside from the undersurface of the superior rectus muscle. This was achieved by rotating the eye in a maximum nasal and downwards direction while avoiding intorsion or retraction of the globe. Traction was applied on the superior temporal quadrant of the globe.

Orbital cellulitis due to mucormycosis. A case report.

A case of orbital cellulitis caused by mucormycosis developed in a patient subsequent to cataract extraction and during systemic steroid treatment for postoperative complications. Fatal mucormycosis is a rare disease usually beginning with a subcutaneous inflammatory lesion. As the subsequent development of orbital cellulitis is very rare, little has been published on this subject. In cases of subcutaneous mucormycosis, the diagnosis can easily be made by means of histologic examination of the lesion. However, early diagnosis is difficult in cases with orbital involvement, because the most common cause of orbital cellulitis is bacterial. Thus, orbital cellulitis caused by mucormycosis is often wrongly treated with antibacterial agents only, as histologic examination is neither easy nor part of any routine investigation. Therefore, a combined treatment using antibiotics and antifungal agents in immunusuppressed patients with this disease is advocated.

Clinical trial with diffractive multifocal intraocular lens implantation.

This study presents results obtained after implantation of 25 diffractive multifocal intraocular lenses. The follow-up ranged from six to 12 months. Visual acuity for distance without correction was 0.5 or better in 72% of cases; for near, it was J1 to J3 in 84% of cases. Slight decentration of the lens and pupil size did not affect visual acuity.

Gene order on the short arm of human chromosome 11: regional assignment of the LDH A gene distal to catalase in two translocations.

Leukemic cells with reciprocal translocations involving 11p13 and 14q13 were obtained from two patients with T-cell acute lymphoblastic leukemia and fused with mouse Ltk- cells. DNA from independent hybrid clones was screened by Southern blot and hybridization to molecular probes for the human catalase and Ha-ras-1 genes. Several clones showed segregation of these two genes, indicating the presence of either the der11 or der14 human chromosomes. When DNA from these hybrid clones was examined for the presence of the human genes for calcitonin and gamma-globin, both genes were found to segregate with the Ha-ras-1 gene and the der14 chromosome indicating that they lie distal to catalase. When the hybrid clones were examined for the presence of human lactate dehydrogenase A (LDH A) activity, only those clones containing the der14 chromosome expressed activity indicating that the LDH A gene is also distal to catalase on the short arm of chromosome 11.

Breakpoints in the human T-cell antigen receptor alpha-chain locus in two T-cell leukaemia patients with chromosomal translocations.

Specific chromosomal translocations have been observed in several human and animal tumours and are believed to be important in tumorigenesis. In many of these translocations the breakpoints lie near cellular homologues of transforming genes, suggesting that tumour development is partly due to the activation of these genes. The best-characterized example of such a translocation occurs in mouse plasmacytoma and human B-cell lymphoma, where c-myc, the cellular homologue of the viral oncogene myc, is brought into close proximity with either the light- or heavy-chain genes of the immunoglobulin loci, resulting in a change in the regulation of the myc gene. T-cell malignancies also have characteristic chromosomal abnormalities, many of which seem to involve the 14q11-14q13 region. This region has recently been found to contain the alpha-chain genes of the human T-cell antigen receptor. Here we determine more precisely the chromosome breakpoints in two patients whose leukaemic T cells contain reciprocal translocations between 11p13 and 14q13. Segregation analysis of somatic cell hybrids demonstrates that in both patients the breakpoints occur between the variable (V) and constant (C) region genes of the T-cell receptor alpha-chain locus, resulting in the translocation of the C-region gene from chromosome 14 to chromosome 11. As the 11p13 locus has been implicated in the development of Wilms' tumour, it is possible that either the Wilms' tumour gene or a yet unidentified gene in this region is involved in tumorigenesis and is altered as a result of its translocation into the T-cell receptor alpha-chain locus.

Molecular analysis of gene deletion in aniridia--Wilms tumor association.

Hybrid clones were produced from the fusion of Chinese hamster cells and human fibroblasts from a patient with the aniridia-Wilms tumor association (AWTA). The DNA from the parental cells and the hybrid clones was screened by Southern blot and DNA hybridization with probes for the human insulin and Ha-ras-1 genes. Two alleles for the Ha-ras-1 gene were shown to exist in the AWTA cells by restriction fragment length polymorphism. One hybrid clone, containing a single allele for Ha-ras-1 was shown to contain a single chromosome 11 with a cytogenetically visible deletion at 11p13. The DNA from this hybrid contained the human genes for insulin, A gamma-globin, G gamma-globin, Ha-ras-1, and calcitonin, but lacked any human sequences homologous to a human catalase cDNA. This clone was also shown to express human lactate dehydrogenase A (LDH A) activity. These data indicate that the deletion of the affected chromosome in this AWTA patient begins distal to LDH A and includes band 11p13, but does not extend to calcitonin or other genes thought to be located in the distal half of chromosome 11p.

Sequence and organization of the diversity, joining, and constant region genes of the human T-cell delta-chain locus.

In this paper we describe the genomic organization and sequence of the human T-cell receptor delta-chain diversity, joining, and constant genes. There is one delta-chain constant region gene (C delta) located approximately equal to 85 kilobases (kb) upstream of the alpha-chain constant region. The delta-chain constant region consists of four exons, whose organization is very similar to that of the C alpha exons, suggesting that C alpha and C delta may have arisen from a gene duplication event. The first exon encodes most of the extracellular constant domain, the second encodes a hinge-like region, and the third encodes the entire transmembrane segment and intracytoplasmic portion, whereas the last exon contains exclusively 3' untranslated sequences. Three joining segments, J delta 1, J delta 2, and J delta 3, are found approximately equal to 12, approximately equal to 5.7, and approximately equal to 3.4 kb upstream of the first exon of C delta. Two functional diversity gene segments, D delta 1 and D delta 2, which can be productively translated in all three reading frames, are found 1 and 9.6 kb upstream of J delta 1. The presence of two D delta with such potential for diversity may offset the limited repertoire of the J delta and V delta genes. The spacer distribution in the recombinational signals flanking D delta and J delta segments allows recombination with V alpha gene segments; however, examination of delta-chain messages does not indicate that this is the case, suggesting that the delta chain uses unique variable gene segments and raising the question as to the reasons for this phenomenon.

Determination of malondialdehyde by capillary electrophoresis, application to human plasma and relation of its levels with prematurity.

Malondialdehyde (MDA) is considered as the most important marker for monitoring lipid peroxidation, which is strongly associated with the development of serious diseases in adults and premature neonates. In this paper we report a method for determination of free MDA in human plasma using capillary zone electrophoresis. MDA was separated and determined as conjugate with tetrabutylammonium hydrogen sulphate (TBAS). Analysis was performed using 20 mM borare, pH = 9.3, as operating buffer and detection of the MDA-TBAS adduct at 267 nm. The method has a linear range up to 80 microM with a detection limit of 0.2 microM. The method was applied to the analysis of MDA in plasma of healthy adults, normal-gestation infants and of preterm neonates. Plasma proteins were successfully removed following centrifugation through a centricon-3 membrane. Results showed that the method can be easily and accurate applied for the determination of MDA in human plasma and that the level of MDA in pretern neonates is significantly higher (p

Molecular analysis of the antigen receptor of virus-specific cytotoxic T cells and identification of a new V alpha family.

We have determined the primary structure of the alpha/beta-antigen receptor of a lymphocytic choriomeningitis virus (LCMV)-specific cytotoxic T cell clone (P14). The beta chain of the T cell receptor is composed of V beta 8.1, D beta and J beta 2.4 gene segments while the alpha chain uses a member of the V alpha 2 family and J alpha TA31. Southern blot analysis of a panel of six other independent LCMV-specific cytotoxic T cell clones did not reveal any preferential usage of the V alpha 2 or V beta 8 gene families in the cytotoxic LCMV response. Additionally, we have isolated and sequenced cDNA clones derived from the non-functionally rearranged alpha allele of P14. This transcript is composed of a unique V alpha gene segment which belongs to a new V alpha gene family.