Characterization of specific IgE response in vitro against protein and drug allergens using atopic and normal donors.

BACKGROUND: As the incidence of allergy to different compounds increases in society, the need to understand and characterize specific IgE responses becomes obvious. Different cell culture systems have been evaluated for their ability to support such IgE secretion. METHODS: One system employed human peripheral lymphocytes (PBL) from normal donors stimulated with anti-CD3 activated T cells with or without the presence of allergens like benzylpenicillin (BP) and Phlenum pratense (PP). Secretion of IgE was analyzed in ELISA and compared to the IgG response to the nonallergenic antigen tetanus toxoid (TT). Another system employed stimulation of T and B cells with a heterotope, consisting of a T helper cell epitope derived from TT, and a B cell allergen epitope derived from BP. The specific IgE secretion was compared, using lymphocytes from normal as well as BP-allergic donors. RESULTS: Anti-CD3 stimulated T cells supported BP-specific IgE secretion in six of 11 normal donors. This response was inhibited in four donors and enhanced in two donors by the addition of the BP-allergen to the culture. In contrast, addition of the protein allergen (PP) or antigen (TT) to the same culture system inhibited both IgE and IgG synthesis in all experiments. Cells from the majority (10/16) of the BP-allergic donors failed to produce BP-specific IgE in vitro, when cultured in the presence of allergen. CONCLUSIONS: An allergen specific immune response is readily generated in vitro. The differential response against benzylpenicillin between different donor categories most probably reflects the level of pre-exposure to this allergen in vivo.

High frequency of LOH, MSI and abnormal expression of FHIT in gastric cancer.

The FHIT gene is a putative tumour suppressor gene. In this study, we analysed a set of 50 gastric tumours for alterations of FHIT, and found 38 of 45 tumours (84%) exhibiting loss of heterozygosity (LOH) within the FHIT gene. We used both nested Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) and single step RT-PCR to analyse the FHIT transcripts and found 34 of 39 (87%) tumours and seven of the 11 (64%) corresponding non-cancerous tissues showed low or aberrant expression of FHIT mRNA and the appearance of the aberrant FHIT transcripts depended on the conditions of the RT-PCR. In these aberrant transcripts, frequent deletions and/or insertions were detected by direct sequencing. All breakpoints for deletions and insertions were at splicing sites. All insertions came from the adjacent introns, whose appearance was completely in accordance with the 'GU-AG' rule for pre-mRNA splicing. It may be suggested that an alternative splicing mechanism functions in the formation of these aberrant transcripts. The fragile nature of FRA3B within the FHIT gene could be responsible for the formation of the aberrant mRNA. Negative or reduced Fhit expression was detected in 39 of 50 tumours (78%). Moreover, an association was found between abnormal Fhit expression and positive node status (P=0.012). Thirteen of 48 tumours (27%) displayed microsatellite instability (MSI), among which 10 tumours also showed MSI within the FHIT gene. Furthermore, we detected an association between MSI and negative node status (P=0.02). We conclude that the abnormalities of FHIT, presumably associated with the unstable nature of FRA3B within the FHIT gene, are involved in the carcinogenesis of gastric cancer, and lack of mismatch repair (MMR) could possibly promote its alteration in a subset of gastric tumours.

Alterations of E-cadherin and beta-catenin in gastric cancer.

BACKGROUND: The E-cadherin-catenin complex plays a crucial role in epithelial cell-cell adhesion and in the maintenance of tissue architecture. Perturbation in the expression or function of this complex results in loss of intercellular adhesion, with possible consequent cell transformation and tumour progression. METHODS: We studied the alterations of E-cadherin and beta-catenin in a set of 50 primary gastric tumours by using loss of heterozygosity (LOH) analysis, gene mutation screening, detection of aberrant transcripts and immunohistochemistry (IHC). RESULTS: A high frequency (75%) of LOH was detected at 16q22.1 containing E-cadherin locus. Three cases (6%) showed the identical missense mutation, A592T. This mutation is not likely to contribute strongly to the carcinogenesis of gastric cancer, because a low frequency (1.6%) of this mutation was also found in 187 normal individuals. We also detected a low frequency (0.36%, 0%) of this mutation in 280 breast tumours and 444 other tumours, including colon and rectum, lung, endometrium, ovary, testis, kidney, thyroid carcinomas and sarcomas, respectively. We also analyzed the aberrant E-cadherin mRNAs in the gastric tumours and found that 7 tumours (18%) had aberrant mRNAs in addition to the normal mRNA. These aberrant mRNAs may produce abnormal E-cadherin molecules, resulting in weak cell-cell adhesion and invasive behaviour of carcinoma cells. Reduced expression of E-cadherin and beta-catenin was identified at the frequency of 42% and 28%, respectively. Specially, 11 tumours (22%) exhibited positive cytoplasmic staining for beta-catenin IHC. An association was found between reduced expression of E-cadherin and beta-catenin. Moreover, an association was detected between reduced expression of E-cadherin and diffuse histotype. CONCLUSION: Our results support the hypothesis that alterations of E-cadherin and beta-catenin play a role in the initiation and progression of gastric cancer.

CD40 employs p38 MAP kinase in IgE isotype switching.

IgE switching requires the prior induction of C epsilon germline transcripts which is mediated by the concerted binding of STAT-6 and NF kappa B to the C epsilon promoter. These transcription factors are regulated by IL-4 and CD40, respectively. However the latter can effect other signaling pathways and the present study explores the role of p38 MAPK in induction of C epsilon germline transcripts. CD40 and IL-4, both alone and in synergy, were initially shown to activate the C epsilon promoter in a B cell lymphoma cell line. Under the same conditions CD40 caused activation of p38 MAPK, whereas IL-4 was ineffective. The p38 MAPK inhibitor, SB203580, and a dominant negative form of p38 MAPK decreased the CD40 activation of the C epsilon promoter by reducing the ability of CD40 to increase the transactivation potential of NF kappa B. This study suggests that p38 MAPK is crucially important in mediating CD40 activation of NF kappa B which acts to induce C epsilon germline transcripts, ultimately facilitating IgE switching.

FHIT alterations in breast cancer.

The FHIT gene encodes a diadenosine hydrolase and may be involved in growth control pathways of the cell. Studies on protein-protein interactions, cell lines, including tumourigenicity tests, and knockout mice suggest that the Fhit protein is involved in cell proliferation and apoptosis, and might act as a tumour suppressor. In several different cancers, including breast cancer, alterations in the FHIT gene have been detected in high frequency. The most common alterations are: deletions, DNA hypermethylation, abnormal transcripts and reduced expression at RNA and protein level. The FHIT gene is located at the FRA 3B fragile site at chromosome 3p 14.2, and alterations in the FHIT gene and Fhit protein have been found associated with genome instability, particularly in BRCA 2 mutated breast tumours. This paper will focus on some of the functional aspects of the Fhit protein with respect to tumour pathogenesis and on aberrations detected in breast cancer.

Alterations of the FHIT gene in breast cancer: association with tumor progression and patient survival.

Our previous results on breast tumors show that LOH (loss of heterozygosity) at the FHIT locus is associated with reduced Fhit protein expression. We have also shown that LOH at this locus is significantly higher in tumors from patients carrying the BRCA2 999de15 mutation than in tumors without this mutation, presumably because of lack of DNA repair. Here, our aim was to determine the relationship of FHIT LOH with breast tumor progression. Five microsatellite markers located within the FHIT gene were typed in 239 breast tumors and corresponding normal tissue, and the LOH results were compared with clinicopathologic factors and LOH at other chromosome regions. LOH at FHIT is associated with estrogen- and progesterone-negative breast tumors, high S-phase fraction, reduced patient survival, and LOH at chromosome regions 6q, 7q, 8p, 9p, 11p, 11q, 13q, 16q, 17p, 17q, 18q, and 20q. A multivariate analysis shows that LOH at FHIT results in a 60% increased relative risk of dying. We conclude that the loss of FHIT results in growth advantage of breast tumor cells, is associated with unstable genome, and may be of prognostic value.

Similar regions of human chromosome 3 are eliminated from or retained in human/human and human/mouse microcell hybrids during tumor growth in severe combined immunodeficient (SCID) mice.

By passaging microcell hybrids (MCHs) containing human chromosome 3 (chr3) on A9 mouse fibrosarcoma background through severe combined immunodeficient (SCID) mice (elimination test), we have previously defined a 1-Mb-long common eliminated region 1 (CER1) at 3p21.3, a second eliminated region (ER2) at 3p21.1-p14 and a common retained region (CRR) at 3q26-qter. In the present work, chr3 was transferred by microcell fusion into the human nonpapillary renal cell carcinoma line KH39 that contained uniparentally disomic chr3. Four MCHs were generated. Compared with KH39, they developed fewer and smaller tumors, which grew after longer latency periods in SCID mice. The tumors were analyzed in comparison with corresponding MCHs by chr3 arm-specific painting, 19 fluorescent in situ hybridization (FISH) probes, and 27 polymorphic markers. Three MCHs that maintained the intact exogenous chr3 in vitro lost one 3p copy in all 11 tumors. Seven of 11 tumors lost the exogenous 3p, whereas four tumors contained mixed cell populations that lacked either the exogenous or one endogenous KH39 derived 3p. In one MCH the exogenous chr3 showed deletions within CER1 and ER2 already in vitro. It remained essentially unchanged in 8/9 derived tumors. The third, exogenous copy of the 3q26-q27 region (part of CRR) was retained in 16/20 tumors. It can be concluded that the human/human MCH-based elimination test identifies similar eliminated and retained regions on chr3 as the human/murine MCH-based test.

Low-frequency magnetic noise in micron-scale magnetic tunnel junctions.

We have observed low-frequency noise due to quasiequilibrium thermal magnetization fluctuations in micron-scale magnetic tunnel junctions (MTJs). This strongly field-dependent magnetic noise occurs within the magnetic hysteresis loops, either as 1/f or Lorentzian (random telegraph) noise. We attribute it to the thermally excited hopping of magnetic domain walls between pinning sites. Our results show that magnetic stability is a crucial factor in reducing the low-frequency noise in small MTJs.

Rapid polarization of Th2 cells during induction of antigen-specific IgE antibodies in vitro.

BACKGROUND: Type 2 T-helper cells (Th2) are involved in the regulation of the humoral immune response against antigens and allergens and directly affect which isotype will be produced. The mechanism that regulates antigen-specific IgE secretion and immune deviation is still not known. OBJECTIVES: To delineate mechanisms behind antigen-specific IgE secretion we have used in vitro immunization and focused on T-cell phenotype and the activation status of the transcription factor NFkappaB. METHODS: Peripheral blood lymphocytes (PBMC) from seronegative donors were immunized in vitro with a peptide consisting of both a T-cell and a B-cell epitope. RESULTS: Antigen-specific IgE antibodies could be detected after a primary immunization, during which T-helper cells secreted type 2 cytokines. Specific IgE was also detected in the secondary immunization, but due to a rapid polarization from Th2 to Th1 phenotype, exogenous IL-4 was required for the specific IgE secretion. Analysis of NFkappaB activation in B and T cells during primary and secondary immunization showed that NFkappaB could be detected in both B and T cells during primary immunization, but was dependent on exogenous IL-4 in the secondary immunization. CONCLUSION: This is the first evidence of antigen-specific IgE induction in vitro using naive B cells, demonstrating the involvement of T-helper cell phenotype and NFkappaB and demonstrates the usefulness of in vitro cultures to study the effect of antigens on human immunocytes.

Analysis of the fragile histidine triad (FHIT) gene in lobular breast cancer.

The fragile histidine triad (FHIT) gene is a candidate tumour suppressor gene in breast and other cancers. We investigated deletions within the FHIT gene in lobular breast cancer and found that 16% of cases showed loss of heterozygosity (LOH) within the gene. We compared LOH within FHIT in lobular and ductal breast tumours and found a significant association between LOH at FHIT and the ductal histological type (P<0.001). To determine whether genomic alteration of the FHIT gene in lobular breast cancer leads to Fhit inactivation we have assessed the level of Fhit expression by immunohistochemical detection and determined that 27% (15 of 55) consecutive sporadic lobular tumours showed negative or reduced Fhit expression. A significant association was found between LOH at the FHIT gene and reduced Fhit expression in lobular and ductal tumours (P=0.025 and P=0.001, respectively). Thus, genetic alterations within the FHIT gene, leading to loss of Fhit protein, may play an important role in the carcinogenesis of a significant number of sporadic lobular breast cancers, even though the apparent frequency of genomic alterations within the gene is lower than in ductal breast cancer.