T-cell receptor usage of interleukin-2-responsive peripheral gamma delta T cells.

The majority of human peripheral gamma delta T cells express the V gamma 9 gene in combination with the V delta 2 gene. The diversity of this subset of gamma delta T cells is limited by a preferential usage of the J gamma P gene segment and a highly distinctive junctional motif of the T-cell receptor (TCR) delta chain. We and others have observed that peripheral blood derived V gamma 9+V delta 2+ gamma delta T cells of healthy individuals are activated after stimulation with interleukin-2 (IL-2) in vitro, but only a small percentage of gamma delta T cells subsequently proliferates. To assess whether the proliferating, IL-2-responsive gamma delta T cells represent a selective group of T cells, we have analysed TCR junctional features of IL-2-responsive gamma delta T cells. Out of 30 individuals studied, nine were identified as IL-2-responders and three as IL-2-hyperresponders. The TCR V(D)J gene usage from IL-2 stimulated peripheral blood lymphocytes of these IL-2-(hyper)responsive individuals was analysed. The results showed that in most individuals gamma delta T cells polyclonally expanded after stimulation with IL-2. In two IL-2-hyperresponder individuals, however, a monoclonal expansion of a particular V gamma 9+V delta 2+ gamma delta T cell was found. In one of these individuals, this V gamma 9+V delta 2+ T-cell clone expressed a very rare gamma delta TCR type because of the presence of an Ala within the junctional region at a conserved position relative to V delta framework residues (delta 97), which is very infrequently used by peripheral blood V gamma 9+V delta 2+ cells. This particular clonotype could also be detected in unstimulated PBL samples taken from that individual, and made up for 30% of the total peripheral gamma delta T-cell pool. These data indicate that in general IL-2-responsive V gamma 9+V delta 2+ gamma delta T cells represent a polyclonal population, reflecting in vivo stimulation with multiple antigens or superantigens. In contrast, monoclonal expansions of gamma delta T cells after stimulation with IL-2 can also occur, which may be related to an in vivo stimulation by one particular antigen, rendering this gamma delta T-cell type dominant in the peripheral blood.     

The common missense mutation D489N in TRIM32 causing limb girdle muscular dystrophy 2H leads to loss of the mutated protein in knock-in mice resulting in a Trim32-null phenotype

Mutations in tripartite motif protein 32 (TRIM32) are responsible for several hereditary disorders that include limb girdle muscular dystrophy type 2H (LGMD2H), sarcotubular myopathy (STM) and Bardet Biedl syndrome. Most LGMD2H mutations in TRIM32 are clustered in the NHL β-propeller domain at the C-terminus and are predicted to interfere with homodimerization. To get insight into TRIM32's role in the pathogenesis of LGMD2H and to create an accurate model of disease, we have generated a knock-in mouse (T32KI) carrying the c.1465G > A (p.D489N) mutation in murine Trim32 corresponding to the human LGMD2H/STM pathogenic mutation c.1459G > A (p.D487N). Our data indicate that T32KI mice have both a myopathic and a neurogenic phenotype, very similar to the one described in the Trim32-null mice that we created previously. Analysis of Trim32 gene expression in T32KI mice revealed normal mRNA levels, but a severe reduction in mutant TRIM32 (D489N) at the protein level. Our results suggest that the D489N pathogenic mutation destabilizes the protein, leading to its degradation, and results in the same mild myopathic and neurogenic phenotype as that found in Trim32-null mice. Thus, one potential mechanism of LGMD2H might be destabilization of mutated TRIM32 protein leading to a null phenotype.     

Analysis of T-cell clonality in bone marrow of patients with acquired aplastic anaemia

Acquired aplastic anaemia (AA) represents a state of bone marrow (BM) failure which is characterized by BM hypocellularity and pancytopenia. It has been hypothesized that in some AA patients, bone marrow failure is secondary to the targeted destruction of haemopoietic stem cells by autoreactive T cells. The response of T cells to antigenic stimulation has been shown, in a number of animal models and in autoimmune diseases, to result in the (oligo)clonal expansion of positively reacting T cells. For this reason, we studied the utilization of 24 T-cell receptor-β variable gene segments (TCRBV) and the clonality in BM aspirates and peripheral blood (PB) of seven AA patients. BM from transplant donors served as controls. Determination of TCRBV gene segment usage revealed no significant differences between patients and controls.
Clonality within each family was analysed by single-strand conformation polymorphism (SSCP) analysis. Clonal and clonally predominant bands were seen in BM of three AA patients in five to eight TCRBV families. Clonal rearrangements were encountered less often in BM of control subjects. In conclusion, our results suggest an antigen-driven T-cell response in the BM of predominantly AA patients resulting in oligoclonal T-cell outgrowth.     

The time-distance recorder as a means of improving the accuracy of fetal blood flow measurements

The influence of pulsatile diameter changes on calculation of volume flow has been studied. In vitro studies and an animal study were carried out with a real-time imaging and pulsed Doppler velocity measurement system. For precise pulsatile diameter information a wall motion tracking device was incorporated. Whereas in vitro a high degree of accuracy was found for the measurements of volume flow, this could not be substantiated in the descending aorta of the fetal lamb, in which Doppler volume flow differed between -7.5 and 17% from magnetic volume flow. In a clinical study the relative influence of various diameter approximations on calculated fetal aortic volume flow was assessed in 16 normal third trimester pregnancies. Depending on the selected diameter approximation method it appeared that differences from 19% underestimation to 9% overestimation in calculated volume flow could be obtained when reference was made to volume flow derived from diameter and velocity information.     

A calcium channel mutant mouse model of hypokalemic periodic paralysis

Hypokalemic periodic paralysis (HypoPP) is a familial skeletal muscle disorder that presents with recurrent episodes of severe weakness lasting hours to days associated with reduced serum potassium (K⁺). HypoPP is genetically heterogeneous, with missense mutations of a calcium channel (Ca_V1.1) or a sodium channel (Na_V1.4) accounting for 60% and 20% of cases, respectively. The mechanistic link between Ca_V1.1 mutations and the ictal loss of muscle excitability during an attack of weakness in HypoPP is unknown. To address this question, we developed a mouse model for HypoPP with a targeted Ca_V1.1 R528H mutation. The Ca_v1.1 R528H mice had a HypoPP phenotype for which low K⁺ challenge produced a paradoxical depolarization of the resting potential, loss of muscle excitability, and weakness. A vacuolar myopathy with dilated transverse tubules and disruption of the triad junctions impaired Ca²⁺ release and likely contributed to the mild permanent weakness. Fibers from the Ca_V1.1 R528H mouse had a small anomalous inward current at the resting potential, similar to our observations in the Na_V1.4 R669H HypoPP mouse model. This “gating pore current” may be a common mechanism for paradoxical depolarization and susceptibility to HypoPP arising from missense mutations in the S4 voltage sensor of either calcium or sodium channels.     

Evidence for selective in vivo expansion of synovial tissue-infiltrating CD4+ CD45RO+ T lymphocytes on the basis of CDR3 diversity

In this study we have analyzed the TCR V and V_ß regionsat the DNA level in the CD4⁺CD45RO⁺ memory T cell populationof synovial tissue infiltrating T lymphocytes of three rheumatoidarthritis (RA) patients and one patient with chronic arthritis.Cell lines of CD4⁺CD45RO⁺, CD4⁺CD45RO^–, CD8⁺CD45RO⁺ andCD8⁺CD45RO^– T lymphocyte populations were generated followingFACS cell sorting of freshly isolated synovial tissue mononuclearcell infiltrates (STMC) and of freshly isolated peripheral bloodmononuclear cells (PBMC) of these patients. The phenotyplc andmolecular analyses have revealed the following. (I) The TCRrepertoires of tissue infiltrating T lymphocytes in the varioussubsets were extensive on the basis of TCR V gene family usage.(II) Furthermore, each patient displayed individual specificTCR V gene expression patterns in the various STMC and PBMCderived T cell subsets. However, the majority of these arthritispatients manifested increased expression of multiple TCR V genefamilies in the synovial tissue derived CD4⁺CD45RO^– Tcell population when compared with the peripheral blood derivedCD4⁺CD45RO⁺ subset. Of these gene families, we found enhancedexpression of the TCR V7 and V_ß11 gene segments insynovial tissue to be shared by all four patients analyzed.OH) Nucleotlde sequence analysis of the CDR3 regions of a numberof TCR V regions in the CD4⁺CD45RO⁺ T cell subsets has revealedthat the CDR3 regions comprised within synovial tissue derivedTCR V regions differed from those found in peripheral bloodderived TCR V regions. These differences in CDR3 diversity mightbe the consequence of a specific interaction with particularMHC-peptlde complexes expressed at the site of inflammation.(Iv) The CDR3 region analysis also showed individual specificamlno acid motifs within the N-D-N regions of all analyzed TCRV_ß genes derived from PBMC as well as STMC.     

Cutaneous squamous cell carcinoma and p53 codon 72 polymorphism: a need for screening?

The association between human papillomavirus (HPV)-associated cervical cancer and cutaneous squamous cell carcinoma and codon 72 polymorphism in the p53 gene is not unequivocal. Especially, it is not known whether carriers of the arginine form have an increased risk of cancer that necessitates screening. The alternative is that the polymorphism is a tumor marker instead of a risk factor. We set out a case-control study to determine the risk of squamous cell carcinoma of the skin in individuals with the p53 codon 72 arginine genotype in order to establish the possible need for screening. The distribution of the different p53 codon 72 genotypes was examined in 86 subjects with a history of cutaneous squamous cell carcinoma and in 168 controls. Additionally, 121 subjects who had had histologically proven basal cell carcinoma and 108 subjects who had had non-familial malignant melanoma were tested. p53 polymorphism was evaluated by polymerase chain reaction (PCR) using DNA samples from peripheral blood lymphocytes. In a subgroup of patients with squamous cell carcinoma and controls, the presence of epidermodyplasia verruciformis human papillomavirus (EV-HPV) DNA was determined in plucked eyebrow hair. Differences in the distributions of the genotypes among cases and controls were calculated, and univariate and multivariate analyses were performed to assess the risk to develop cutaneous squamous cell carcinoma in the presence of the p53 codon 72 arginine genotype. Frequency distributions of the three different genotypes (homozygous for the arginine allele, heterozygous for the two alleles, and homozygous for the proline allele) were similar among the squamous cell carcinoma group and the control group: 47.1%, 46.0% and 6.9% versus 47.8%, 45.8% and 6.4%, respectively. Statistical analysis showed no significant differences between these groups. In patients with squamous cell carcinoma and controls who harbored EV-HPV DNA in their plucked eyebrow hair, similar results were obtained. The distributions of the p53 codon 72 genotypes in the basal cell carcinoma and malignant melanoma group were also not significantly different from the control group. p53 codon 72 arginine homozygosity does not appear to represent a significant risk factor for cutaneous squamous cell carcinoma and screening seems not to be indicated. Mol. Carcinog. 30:56-61, 2001.