IFN-gamma-enhanced allergen penetration across respiratory epithelium augments allergic inflammation

BACKGROUND: Respiratory allergen contact is the critical event in the elicitation and boosting of allergen-specific immune responses, as well as in the induction of immediate and late inflammatory reactions. OBJECTIVE: We sought to investigate the influence of various factors of allergic inflammation on the integrity and barrier function of respiratory epithelium for allergens. METHODS: We cultured the human bronchial epithelial cell line 16HBE14o- in a transwell culture system as a surrogate of intact respiratory epithelium and used purified iodine 125-labeled recombinant major birch pollen allergen (rBet v 1) to study the extent, kinetics, and factors influencing transepithelial allergen penetration. RESULTS: Culture supernatants from activated allergen-specific T H 1 clones decreased transepithelial resistance. A screening of various factors (histamine, IFN-gamma, IL-1beta, IL-2, IL-3, IL-4, IL-5, IL-8, IL-12, and TNF-alpha) identified IFN-gamma as a potent factor capable of reducing epithelial barrier properties and enhancing transepithelial allergen penetration. Increased submucosal allergen concentrations caused by IFN-gamma-mediated reduction of epithelial barrier function provoked a more than 7-fold augmentation of histamine release from sensitized basophils. CONCLUSION: These results demonstrate that the T H 1 cell-derived cytokine IFN-gamma facilitates allergen penetration through the respiratory epithelium and thereby can aggravate allergic inflammation.     

Enhanced expression of Fas-associated proteins in decidual and trophoblastic tissues in pregnancy-induced hypertension

PROBLEM: To determine if feto-placental tissues from gestations complicated by pregnancy-induced hypertension (PIH) have altered expression of Fas-associated proteins. METHOD OF STUDY: The expression of several Fas-related proteins was determined in fetal membranes, decidua, and placentas obtained from PIH-affected (n = 12, age range 32-36 weeks) and normal (n = 6, age range 37-41 weeks) gestations. Paraffin-embedded tissue sections were stained with specific monoclonal antibodies to Fas, Fas ligand (FasL), caspase-3, and bax. RESULTS: We observed greater expression of Fas and FasL in amnion and decidua from PIH-affected gestations than in normal controls. Intense staining was observed only in the perivascular endothelium (caspase-3) and in decidual cells (bax) from PIH gestations. CONCLUSION: Differential expression of Fas-related proteins in fetal membranes, decidua, and placentas from PIH-affected gestations is consistent with increased apoptosis, and suggests activation of the Fas/FasL pathway in a tissue-specific manner.     

C1-esterase inhibitor blocks T lymphocyte proliferation and cytotoxic T lymphocyte generation in vitro

We have previously shown that activated C1s complement and activated T cells cleave beta2-microglobulin (beta2m) in vitro leading to the formation of desLys58 beta2m. This process can specifically be inhibited by C1-esterase inhibitor (C1-inh). Furthermore we showed that exogenously added desLys58 beta2m in nanomolar amounts to a one-way allogenic mixed lymphocyte culture (MLC) increased the endogenous production of IL-2 and the generation of allo-specific cytotoxic T lymphocytes. C1-inh was purified from fresh human plasma and added to human or murine MLC and mitogen-stimulated lymphocyte cultures grown in the presence of complement-inactivated serum. Read-outs were cell proliferation, lymphokine production and development of T cell-mediated cytotoxicity. We found that addition of C1-inh to MLC and mitogen- exposed murine and human lymphocyte cultures inhibited proliferation, the development of allospecific cytotoxic activity, and changed the endogenous production of IL-2, IL-4, IL-10, IL-12 and IFN-gamma. These data clearly demonstrate a regulatory function of C1-inh on T cell- mediated immune functions.      

Human mini-chromosomes in mouse embryonal stem cells

We have introduced human mini-chromosomes of 4 Mb and approximately 15 Mb in size into mouse embryonal stem cells. Although these human mini- chromosomes are stable in hamster and chicken cells, they re-arrange or segregate aberrantly in the embryonal stem cells and are rapidly lost in the absence of selection. However, one of the mini-chromosomes re- arranged, acquired mouse centromeric sequences and was then stably maintained for at least 60 population doublings in culture. This mini- chromosome, which is 4 Mb in size, is a candidate for a mouse germ line chromosome vector.      

Determination of the parent of origin in nine cases of prenatally detected chromosome aberrations found after intracytoplasmic sperm injection

Prenatal cytogenetic analysis of 71 fetuses conceived by intracytoplasmic sperm injection (ICSI) resulted in the detection of nine (12.7%) chromosome aberrations including two cases of 47,XXY, four cases involving a 45,X cell line and three autosomal trisomies. Molecular analysis of the parental origin of the deleted or supernumerary chromosome was performed by using polymorphic microsatellite markers. Six cases involving a sex chromosome abnormality were found to be of paternal origin while the two trisomic cases that could be analysed were of maternal origin. Two cases involved the same infertile couple who had two consecutive ICSI pregnancies terminated because of a chromosome abnormality. The replaced embryos in both cases originated from a single batch of ICSI fertilized oocytes of which part was used to initiate the first pregnancy and part was cryopreserved and used to initiate the second pregnancy.      

Blood-brain barrier breakdown by cold injury. Polyamine signals mediate acute stimulation of endocytosis, vesicular transport, and microvillus formation in rat cerebral capillaries

Polyamines have been previously implicated in the mediation of blood-brain barrier breakdown induced by cryogenic injury (H Koenig, AD Goldstone, CY Lu, Biochem Biophys Res Commun 116:1039, 1983). We studied acute (less than 5 minute) changes in capillary ultrastructure, microvascular permeability, and the levels of polyamines and their rate regulating synthetic enzyme ornithine decarboxylase (ODC) in rat cerebral cortex after focal cold injury. Microvascular permeability was measured by relative transport of intravenously administered fluorescein. Capillary ultrastructure was studied by quantitative stereology and morphometry after intravenous administration of horseradish peroxidase. Focal cold injury induced a 2.5-, 3.8-, 1.7-, and 1.4-fold increase in the levels of ODC, putrescine, spermidine and spermine, and a 46-fold increase in fluorescein uptake in perilesional cortex. Few capillaries in control cortex contained endocytic pits or horseradish peroxidase-positive vesicles, whereas most capillaries near lesions showed these structures. Cryoinjury induced a 5-fold increase in the relative volume of microvilli and horseradish peroxidase vesicles, a 2.3-fold increase in area of luminal endocytic pits, and a 6.3-fold increase in area of abluminal exocytic pits. The ODC inhibitor alpha-difluoromethylornithine blocked the cryoinjury-induced changes in ODC, polyamines, fluorescein uptake, and capillary ultrastructure. Putrescine negated the effect of alpha-difluoromethylornithine or capillary ultrastructure, and was previously shown to nullify the alpha-difluoromethylornithine effects on polyamines and fluorescein permeability (cited above). These data link rapid changes in ODC and polyamines to blood-brain barrier breakdown, and suggest that the abnormal permeability is associated with an acute, polyamine-mediated stimulation of microvillus formation, endocytosis, and vesicular transport in capillary endothelium.     

Nonhereditary p53 mutations in T-cell acute lymphoblastic leukemia are associated with the relapse phase

We have previously reported that greater than 60% of human leukemic T- cell lines possess mutations in the p53 tumor suppressor gene. To determine whether T-cell acute lymphoblastic leukemia (T-ALL) patient samples possess p53 mutations, we screened peripheral blood-and bone marrow-derived leukemia samples, taken at diagnosis and at relapse, for p53 mutations. Exons 4 through 9 and selected intron regions of the p53 gene were analyzed using polymerase chain reaction-single-strand conformation polymorphism and direct sequencing. p53 mutations were found in 0 of 15 T-ALL diagnosis samples, as compared with 10 of 36 (28%) T-ALL relapse samples. To determine whether p53 mutations play a role in the recurrence (relapse) of T-ALL, two special groups of T-ALL patients were studied: (1) a group of 8 relapse patients whose disease was refractory to chemotherapeutic treatment, and (2) a group of 6 "paired" T-ALL cell samples from patients for whom we possess both diagnosis and relapse samples. Three of 8 relapsed patients (37.5%) whose disease was refractory to the reinduction of remission by chemotherapy possessed missense mutations of the p53 gene. All 3 cases had mutations in exon 5. Among the paired samples, 3 of 6 patients harbored p53 mutations at disease recurrence, but possessed only wild- type p53 alleles at diagnosis. One case had mutation on exon 4, 1 case in exon 5, and 1 case in exon 8 with loss of heterozygosity. These data clearly indicate that recurrence of T-ALL is associated with missense mutations in p53. Our results indicate that (1) mutations of p53 do occur in T-ALL in vivo, and such mutations are associated with the relapse phase of the disease; and (2) p53 mutation is involved in the progression of T-ALL. This conclusion is supported by our observation that the introduction of T-ALL-derived mutant p53 expression constructs into T-ALL cell lines further increases their growth rate in culture, enhances cell cloning in methylcellulose, and increases tumor formation in nude mice.