Severe XLP Phenotype Caused by a Novel Intronic Mutation in the SH2D1A Gene.

We describe here a novel c.137 + 5G > A intronic mutation in the SH2D1A gene of the signaling lymphocyte activation molecule (SLAM)-associated protein (SAP) in association with Epstein-Barr virus (EBV)-induced fatal infectious mononucleosis (FIM) in an 8-year-old male patient and his 3-year-old step brother. The mother and the maternal grandmother of the boys are healthy and heterozygous for this sequence variant. Genetic sequencing of blood-cell-derived cDNA in the younger patient revealed a 22 bp deletion in the SH2D1A cDNA. Immunoblot and flow cytometry analysis performed in this younger patient showed the lack of SAP protein expression in peripheral blood lymphocytes. These data suggest that the novel c.137 + 5G > A mutation results in loss of function of SAP protein and leads to typical X-linked lymphoproliferative disease phenotype. We propose that intron 1 and the c.137 + 5G may be the most frequent intronic hot spot for SH2D1A splicing mutation.

Host response to EBV infection in X-linked lymphoproliferative disease results from mutations in an SH2-domain encoding gene.

X-linked lymphoproliferative syndrome (XLP or Duncan disease) is characterized by extreme sensitivity to Epstein-Barr virus (EBV), resulting in a complex phenotype manifested by severe or fatal infectious mononucleosis, acquired hypogammaglobulinemia and malignant lymphoma. We have identified a gene, SH2D1A, that is mutated in XLP patients and encodes a novel protein composed of a single SH2 domain. SH2D1A is expressed in many tissues involved in the immune system. The identification of SH2D1A will allow the determination of its mechanism of action as a possible regulator of the EBV-induced immune response.

Anti-TNF-alpha antibody (infliximab) therapy supports the recovery of eNOS and VEGFR2 protein expression in endothelial cells.

The aim of this study is to investigate the effect of sera obtained from patients of Crohn's disease treated by anti-TNF-alpha antibody (Infliximab) on the expression of endothelial nitric oxide synthase (eNOS) and vascular endothelial growth factor receptor-2 (VEGFR2) protein in human umbilical vein endothelial cells (HUVEC) cultured in vitro. HUVEC was cultured in the presence of sera derived from patients before and after treatment, or from healthy individuals. Effects of sera on the expression of eNOS and VEGFR2 were monitored by determination of mRNA and protein levels using real time quantitative PCR and Western blot analysis, respectively. The serum of Crohn's patients contained elevated levels of TNF-alpha (34±1.80 pg/mL), which resulted in a decrease in the protein level of eNOS in HUVEC with a simultaneous induction of VEGFR2. Infliximab treatment normalized the expression level of these proteins by decreasing TNF-alpha level, particularly in those cases when clinical healing was also recorded, and it also conferred restitution of the level of angiogenic cytokines. Results suggest that altered angiogenesis possibly contributes to the initiation and perpetuation of inflammatory processes in inflammatory bowel disease (IBD). Endothelial dysfunction, a selective feature of Crohn's disease is beneficially affected by intravascular TNF-alpha neutralization.

'Gain of function' phenotype of tumor-derived mutant p53 requires the oligomerization/nonsequence-specific nucleic acid-binding domain.

Tumor-derived p53 mutants can transcriptionally activate a number of promoters of genes involved in cellular proliferation. For this transactivation, mutant p53 does not use the wild-type p53 DNA-binding site, suggesting a mechanism of transactivation that is independent of direct DNA binding. Here we describe our analysis of the domain requirements for mutant p53 to transactivate promoters of the human epidermal growth factor receptor (EGFR), human multiple drug resistance 1 (MDR-1) and human proliferating cell nuclear antigen (PCNA) genes. We also report the identification of a structural domain required for the 'gain of function' property of mutant p53-281G. 'Gain of function' is measured as the tumorigenicity (in nude mice) of 10(3) murine cells expressing mutant p53 constitutively. We have generated internal deletion mutants of p53-281G deleting conserved domains I, II, III, IV and V, individually. We have also generated one deletion mutant eliminating amino acids 100 through 300 that removes four of the five conserved domains (II - V); another mutant, p53-281G del 393-327, deletes the oligomerization and nonsequence-specific nucleic acid-binding domains of p53. For the EGFR and MDR-1 promoters, all these mutants have significantly lower transactivation ability than intact p53-281G. These deletion mutants, however, significantly activated the pCNA promoter, suggesting that the mechanism of transactivation of the PCNA promoter is different from that of the EGFR and MDR-1 promoters. When expressed constitutively in 10(3) cells, p53-281G del 393-327 was found to be defective in inducing tumor formation in nude mice although intact p53-281G was very efficient. Thus, our results suggest that structural domains near the C-terminus are needed for 'gain of function'.

Structural characteristics influencing the carrier function of synthetic branched polypeptides based on poly[Lys-(DL-Ala)3)]backbone.

Effective carrier function of selected representatives of new branched polypeptides covalently coupled with the synthetic monovalent hapten, oxazolone was studied. The effectiveness of oxazolone-synthetic polypeptide conjugates in inducing oxazolone-as well as carrier-specific antibody responses in inbred mice was compared to that of bovine serum albumin (BSA)- and KLH-oxazolone conjugates. The synthetic polypeptides, poly[Lys-(D-Leui-DL-Alam)] (D-LAK), LAK and FAK, as well as the common poly[Lys-(DL-Alam)](AK) core covalently coupled to oxazolone (Ox) induced a T cell-dependent antibody response when repeatedly administered with or without Freund's adjuvant in mice. This was evidenced by: the increasing titer of oxazolone-specific IgG during the course of the memory response; the appearance of all IgG subclasses; the effective oxazolone-specific priming by the conjugates; and the induction of an intense oxazolone- and carrier-specific DTH reaction. Although the oxazolone-specific antibody response was 10-100 times lower than that induced by KLH- or BSA-oxazolone conjugates, it was accompanied by a lower level or no detectable carrier-specific antibody response despite an effective carrier-specific T cell-mediated response. Significant differences were observed between the effectiveness of synthetic polypeptides used as carrier: highest oxazolone-specific antibody titers were observed using the AK, LAK and FAK conjugates. The intensity and specificity of the DTH reaction and antibody response induced by the carrier-oxazolone conjugates suggested that the distinct effectiveness of L- and D-amino acid-containing conjugates (LAK vs D-LAK and FAK vs D-FAK) was dependent on altered B cell recognition of the haptenic group. Circular dichroism (CD) spectra indicating different local orientation of oxazolone, when coupled to L or D side chain-terminating amino acids, support this suggestion.

A new candidate region for the positional cloning of the XLP gene.

X-linked lymphoproliferative disease (XLP) is an inherited immunodeficiency characterised by selective susceptibility to Epstein-Barr virus and frequent association with malignant lymphomas chiefly located in the ileocecal region, liver, kidney and CNS. Taking advantage of a large bacterial clone contig, we obtained a genomic sequence of 197620 bp encompassing a deletion (XLP-D) of 116 kb in an XLP family, whose breakpoints were identified. The study of potential exons from this region in 40 unrelated XLP patients did not reveal any mutation. To define the critical region for XLP and investigate the role of the XLP-D deletion, detailed haplotypes in a region of approximately 20 cM were reconstructed in a total of 87 individuals from 7 families with recurrence of XLP. Two recombination events in a North American family and a new microdeletion (XLP-G) in an Italian family indicate that the XLP gene maps in the interval between DXS1001 and DXS8057, approximately 800 kb centromeric to the previously reported familial microdeletion XLP-D.

Disruption of functions of wild-type p53 by hetero-oligomerization.

We report that a p53 segment (p53 del 1-293) containing the oligomerization domain interferes with the functions of wild-type p53. Wild-type p53 inhibits transcription mediated by human cytomegalovirus (CMV) immediate-early promoter significantly; however, co-expression of p53 del 1-293 drastically reduces this repression. We show that wild-type p53 forms hetero-oligomers with p53 del 1-293 suggesting that the hetero-oligomers are defective in repressing the CMV promoter. A synthetic promoter with p53-binding sites is transactivated significantly by wild-type p53. However, co-expression of p53 del 1-293 drastically reduces this activation. At a high concentration, a deletion mutant of wild-type p53 (del 393-327) defective in oligomerization transactivates efficiently a promoter with synthetic p53-binding sites. This transactivation remains unaffected by co-expression of p53 del 1-293. p53 del 393-327 also fails to hetero-oligomerize with p53 del 1-293 indicating that hetero-oligomerization is necessary for disruption of wild-type p53-mediated transactivation. Immunostaining experiments show that hetero-oligomerization does not lead to changes in localization of nuclear p53 demonstrating that delocalization of p53 is not the reason for inactivation. We also show that co-expression of p53 del 1-293 significantly reduces the G1/S arrest by wild-type p53 suggesting that a proper oligomeric form is necessary for wild-type p53-mediated cell cycle arrest. Thus, our work shows that hetero-oligomerization disrupts wild-type p53's biological functions and suggests a mechanism by which p53 mutants may disrupt functions of wild-type p53.

Differential modulation of cellular and viral promoters by p73 and p53.

p73 has been shown to transcriptionally activate genes positively responsive to wild-type p53. In order to undertake a comparative study of functions of p53 and p73 we have cloned the cDNA of p73 from MCF-7 cells. Adenovirus onco-protein E1A inhibits the transactivation by p73; a deletion mutant of E1A incapable of interacting with p300 and CREB-binding protein (CBP) fails to disrupt the transactivation. Furthermore, CBP increases the transactivation mediated by p73 suggesting that CBP may function as a co-activator and E1A inhibits p73-mediated transactivation by sequestering p300 or CBP. We show that p73 can transcriptionally inhibit a number of cellular and viral promoters. However, wild-type p53, p73 alpha and p73 beta differ in their ability to inhibit transcriptional activity of different promoters. While wild-type p53 inhibits the promoters of the human cytomegalovirus (CMV) immediate-early gene, the long terminal repeat of human immunodeficiency virus type 1 (HIV LTR), human cyclin A (cyc A) gene, and insulin-like growth factor receptor I (IGF-I-R), p73 alpha only inhibits the HIV LTR and cyc A promoters significantly; and p73 beta inhibits the CMV, HIV LTR and cyc A promoters. A mutant of p73 alpha having amino acid substitutions at positions 268 and 300 on the presumptive DNA-binding domain fails to transactivate the p21 promoter but represses the CMV and the HIV LTR promoter quite efficiently showing that the mechanisms of transactivation and repression by p73 are different. Interestingly, p73 alpha transactivates the IGF-I-R promoter, which is inhibited by wild-type p53; p73 beta has no significant effect on this promoter. This is a unique situation where p73 alpha differs from p73 beta as well as p53.

[Architectonics of pulmonary vessels in pulmonary stenosis]. / Architektonika pl'úcnych ciev pri pulmonálnych stenózach.

The authors aimed their work on the study of the architecture of pulmonary blood vessels and blood flow via pulmonary vascular network in patients with Fallot malformation. In 34 children with valvular stenosis of a. pulmonalis and in 12 children with Fallot tetralogy they analysed in detail their angiograms, paying attention to evaluation of the architecture of truncus pulmonalis and its branches, the discharging part of the right ventricle, and the state of the arterial phase of the pulmonary vascular network. On the basis of their results the authors explain the preferential blood distribution in Fallot's tetralogy by the displacement of the outflow part of the right ventricle to the right and by a greater amanation angle between the right branch of a.pulmonalis and its truncus. Better routing of blood into the left lung in patients with pulmonary valvular stenosis is according to the authors due to a greater emanation angle between the left branch of a.pulmonaris and its truncus. The authors analyse also the mechanism of formation of poststenotic dilatation of truncus pulmonaris and the left branch of a.pulmonaris in cases of valvular stenosis in infant age. (Fig. 4, Ref. 12.)

[Disorders of the cardiovascular system in congenital lentiginosis]. / Poruchy kardiovaskulárneho systému pri vrodenej lentiginóze.

The authors investigated during the last 10 years four patients with lentiginosis and affection of the heart. In none of them the symptoms were complete enough to include it under the leopard syndrome. In two patients the authors found changes in the outflow portion of the right ventricle which was in their opinion caused by hypertrophy of the interventricular septum and musculature of the right ventricle. The authors consider the term lentiginocardiomyopathic syndrome suitable for this rare clinical and genetic entity. The authors check patients with lentiginocardiomyopathic syndrome regularly after 6-month intervals. In two children they recorded progression of the heart disease.