Study of X-ray topography using the super-Borrmann effect.

X-ray topography exerting the super-Borrmann effect has been performed using synchrotron radiation to display dislocation images with a high-speed and high-resolution CMOS camera. Forward-transmitted X-rays are positively employed instead of reflected X-rays to reveal dislocations in relatively thick crystals by simultaneously exciting a pair of adjacent {111} planes owing to the super-Borrmann effect. Before the experiment, minimum values of the attenuation coefficients AminP for σ and π polarizations of the incident X-rays in the three-beam case are calculated. Results demonstrate that AminP for both polarizations are almost 20 times larger than those in the two-beam (usual Borrmann effect) case. The transmitted X-rays can be used to confirm the efficacy of taking topographs under the super-Borrmann conditions, as well as under multiple-diffraction conditions. Furthermore, super-Borrmann topographs can be considered for relatively thick crystals, where a conventional Lang X-ray topography technique is difficult to apply.

Expression of Heparin-Binding EGF-Like Growth Factor (HB-EGF) in Bovine Endometrium: Effects of HB-EGF and Interferon-τ on Prostaglandin Production.

Heparin-binding EGF-like growth factor (HB-EGF) regulates several cell functions by binding to its membrane receptor (ErbB1 and ErbB4). Experimental evidences suggest that HB-EGF, prostaglandins (PGs) and interferon-τ (IFN-τ) regulate uterine function for pregnancy establishment in ruminants. In this study, the mRNA expressions of HB-EGF, ErbB1 and ErbB4 in bovine endometrium and the effects of HB-EGF and IFN-τ on PGE2 and PGF2-α production by endometrial cells were investigated. RT-PCR analysis revealed that HB-EGF mRNA was greater at the mid-luteal stage than at the early and regressed luteal stages (p < 0.05). ErbB1 mRNA expression was greater at the mid- and late luteal stages than at the other luteal stages (p < 0.05). IFN-τ increased the expression of HB-EGF, ErbB1 and ErbB4 mRNA in epithelial cells (p < 0.05). HB-EGF did not affect PGF2-α or PGE2 production by bovine endometrial epithelial cells, but increased PGF2-α and PGE2 production by bovine endometrial stromal cells (p < 0.05). IFN-τ significantly decreased HB-EGF-stimulated PGF2-α (p < 0.05), but not PGE2 (p > 0.05) production by stromal cells. These results indicate that HB-EGF and its receptors expression changed in bovine endometrium throughout the oestrous cycle. IFN-τ increased their expression in cultured endometrial cells. HB-EGF and IFN-τ have the ability to regulate PGs production by stromal cells and therefore may play a role in the local regulation of uterine function at the time of implantation in cattle.

Adipose tissue hypoxia induces inflammatory M1 polarity of macrophages in an HIF-1α-dependent and HIF-1α-independent manner in obese mice.

AIMS/HYPOTHESIS: As obesity progresses, adipose tissue exhibits a hypoxic and inflammatory phenotype characterised by the infiltration of adipose tissue macrophages (ATMs). In this study, we examined how adipose tissue hypoxia is involved in the induction of the inflammatory M1 and anti-inflammatory M2 polarities of ATMs. METHODS: The hypoxic characteristics of ATMs were evaluated using flow cytometry after the injection of pimonidazole, a hypoxia probe, in normal-chow-fed or high-fat-fed mice. The expression of hypoxia-related and inflammation-related genes was then examined in M1/M2 ATMs and cultured macrophages. RESULTS: Pimonidazole uptake was greater in M1 ATMs than in M2 ATMs. This uptake was paralleled by the levels of inflammatory cytokines, such as TNF-α, IL-6 and IL-1ß. The expression level of hypoxia-related genes, as well as inflammation-related genes, was also higher in M1 ATMs than in M2 ATMs. The expression of Il6, Il1ß and Nos2 in cultured macrophages was increased by exposure to hypoxia in vitro but was markedly decreased by the gene deletion of Hif1a. In contrast, the expression of Tnf, another inflammatory cytokine gene, was neither increased by exposure to hypoxia nor affected by Hif1a deficiency. These results suggest that hypoxia induces the inflammatory phenotypes of macrophages via Hif1a-dependent and -independent mechanisms. On the other hand, the expression of inflammatory genes in cultured M2 macrophages treated with IL-4 responded poorly to hypoxia. CONCLUSIONS/INTERPRETATION: Adipose tissue hypoxia induces an inflammatory phenotype via Hif1a-dependent and Hif1a-independent mechanisms in M1 ATMs but not in M2 ATMs.

Murine B cell proliferation and protection from apoptosis with an antibody against a 105-kD molecule: unresponsiveness of X-linked immunodeficient B cells.

We established a novel monoclonal antibody, RP/14, that can protect B cells from apoptosis induced by irradiation or dexamethasone. A molecule recognized by RP/14 (the RP antigen) was expressed on B cells with B220bright, IgMdull, and IgDbright. Immunoprecipitation experiments revealed that RP/14 recognized a monomeric protein with an approximate molecular mass of 105 kD. Stimulation of B cells with RP/14 for 48 h induced B cell proliferation and blastogenesis. In contrast to B cells of wild-type mice, X-linked immunodeficient (XID) B cells did not proliferate upon stimulation with RP/14, although the RP antigen was expressed to the same extent as that of wild-type B cells. These results suggest that the RP antigen-mediated signaling pathway is important for rescuing B cells from apoptosis and is deficient in XID B cells.

Multi-colony stimulating activity of interleukin 5 (IL-5) on hematopoietic progenitors from transgenic mice that express IL-5 receptor alpha subunit constitutively.

The interleukin 3 (IL-3), IL-5, and granulocyte/macrophage colony-stimulating factor receptors consist of a cytokine-specific alpha subunit and the common beta subunit. Whereas IL-3 stimulates various lineages of hematopoietic cells, including multipotential progenitors, IL-5 acts mainly as an eosinophil lineage-specific factor. To investigate whether the lineage specificity of IL-5 is due to restricted expression of the IL-5 receptor alpha subunit (IL-5R alpha), we generated transgenic mice that express the mouse IL-5R alpha constitutively by phosphoglycerate kinase promoter. The transgenic mouse expressed IL-5R alpha ubiquitously, and the bone marrow cells formed various types of colonies, including multi-lineage colonies, in response to IL-5. IL-5 also supported formation of both multi-lineage and blast cell colonies from dormant progenitors of the 5-fluorouracil-treated transgenic mice. The cells composing the blast cell colony gave rise to many colonies including multi-lineage colonies when they were replated in secondary culture containing either Il-5 or IL-3. There was no significant difference in replating efficiency or in types of secondary colonies between IL-5- and IL-3-stimulated cultures. Conversely, the cells from the IL-3-induced blast cell colonies of the transgenic mice proliferated in response to either IL-3 or IL-5. Thus, the development of the progenitors can be equally supported by either IL-5 or IL-3, suggesting that intracellular signals from the IL-3R can be replaced by those from IL-5. These results strongly suggest that the lineage specificity of IL-5 is mainly due to the restricted expression of IL-5R alpha.

Reconstitution of the functional receptors for murine and human interleukin 5.

The murine interleukin 5 receptor (mIL-5R) is composed of two distinct subunits, alpha and beta. The alpha subunit (mIL-5R alpha) specifically binds IL-5 with low affinity. The beta subunit (mIL-5R beta) does not bind IL-5 by itself, but forms the high-affinity receptor with mIL-5R alpha. mIL-5R beta has been revealed to be the mIL-3R-like protein, AIC2B which is shared with receptors for IL-3 and granulocyte/macrophage colony-stimulating factor. We demonstrated here the reconstitution of the functional receptors for murine and human IL-5 on the mouse IL-2-dependent cell line, CTLL-2. CTLL-2 was transfected with the cDNAs for mIL-5R alpha and/or AIC2B. Only CTLL-2 transfectant expressing both mIL-5R alpha and AIC2B expressed the high-affinity receptor and proliferated in response to murine IL-5. Then CTLL-2 was transfected with the cDNAs for hIL-5R alpha and/or KH97 (beta c), the human homologue of AIC2B. Though beta c did not contribute much to binding affinity of hIL-5R, only CTLL-2 transfectant expressing both hIL-5R alpha and beta c proliferated in response to human IL-5. These results showed that the beta subunit is indispensable in IL-5 signal transduction. We further investigated the function of IL-5-specific alpha subunit in transmitting IL-5 signals. Mutant mIL-5R alpha, which lacks its whole cytoplasmic domain, was transfected into mouse IL-3-dependent cell line, FDC-P1 expressing AIC2B intrinsically. The resulting transfectant did not respond to IL-5, though the transfectant expressed the high-affinity IL-5R, indicating that the cytoplasmic portion of the alpha subunit also has some important role in IL-5-mediated signal transduction.

Molecular cloning and expression of the human interleukin 5 receptor.

Human interleukin 5 (IL-5) plays an important role in proliferation and differentiation of human eosinophils. We report the isolation of cDNA clones from cDNA libraries of human eosinophils by using murine IL-5 receptor alpha chain cDNA as a probe. Analysis of the predicted amino acid sequence indicated that the human IL-5 receptor has approximately 70% amino acid sequence homology with the murine IL-5 receptor and retains features common to the cytokine receptor superfamily. One cDNA clone encodes a glycoprotein of 420 amino acids (Mr 47,670) with an NH2-terminal hydrophobic region (20 amino acids), a glycosylated extracellular domain (324 amino acids), a transmembrane domain (21 amino acids), and a cytoplasmic domain (55 amino acids). Another cDNA encodes only the extracellular domain of this receptor molecule. Other cDNA clones encode molecules having diversified cytoplasmic domains. COS7 cells transfected with the cDNA expressed a approximately 60-kD protein and bound IL-5 with a single class of affinity (Kd = 250-590 pM). The Kd values were similar to that observed in normal human eosinophils. In contrast to the murine 60-kD alpha chain, which binds IL-5 with low affinity (Kd = approximately 10 nM), the human alpha chain homologue can bind IL-5 with much higher affinity by itself. RNA blot analysis of human cells demonstrated two transcripts (approximately 5.3 and 1.4 kb). Both of them were expressed in normal human eosinophils and in erythroleukemic cell line TF-1, which responds to IL-5. The human IL-5 receptor characterized in this paper is essential for signal transduction, because expression of this molecule in murine IL-3-dependent cell line FDC-P1 allowed these cells to proliferate in response to IL-5.

Identification of a T cell-derived b cell growth factor distinct from interleukin 2.

We report here a factor (B cell growth factor) found in induced supernatants of the mouse thymoma EL4 that co-stimulates with anti-IgM antibodies in short-term cultures of purified B lymphocytes to induce polyclonal B cell proliferation but not antibody-forming cell production. The factor is not mitogenic for resting B cells and interacts with anti-IgM-activated B cells in a non-H-2-restricted manner. Absorption studies and molecular weight analysis reveal the factor is distinct from interleukin 2. This factor synergises with antigen, interleukin 2, and an interleukin 2-free, B cell growth factor-free T cell supernatant that contains T cell-replacing factor to produce erythrocyte-specific plaque-forming cells in cultures of highly purified B cells.

Highly purified murine interleukin 5 (IL-5) stimulates eosinophil function and prolongs in vitro survival. IL-5 as an eosinophil chemotactic factor.

The recent molecular cloning of the complementary DNA encoding T cell--replacing factor (TRF) has demonstrated that a single molecule is responsible for B cell growth factor II (BCGF-II) activity and eosinophil differentiation activity. It has been proposed that this molecule be called interleukin 5 (IL-5). We previously reported that purified rIL-5 supports the terminal differentiation and proliferation of eosinophilic precursors. In this study, we examined the effects of IL-5 on functional activities of mature eosinophils. IL-5 maintained the viability of mature eosinophils obtained from peritoneal exudate cells of mice infected with parasites. It also induced superoxide anion production in a dose-dependent manner. The Boyden's chamber Millipore assay revealed that IL-5 had a marked chemokinetic effect on eosinophils in a dose-dependent manner. Moreover, IL-5 was found to be an eosinophil chemotactic factor by the checkerboard assay. In conclusion, IL-5 is suggested to play an important role in increasing the functional activities of eosinophils as well as their production in allergic and parasitic diseases.

Receptors for T cell-replacing factor/interleukin 5. Specificity, quantitation, and its implication.

T cell-replacing factor (TRF)/IL-5 is a glycosylated polypeptide that acts as a key factor for B cell growth and differentiation. Since IL-5 action is probably mediated by specific cell surface receptor(s), we have characterized the binding of IL-5 to cells using biosynthetically [35S]methionine-labeled IL-5 and 125I-IL-5 that had been prepared using Bolton-Hunter reagent. The radiolabeled IL-5 binds specifically to BCL1-B20 (in vitro line) (a murine chronic B cell leukemic cell line previously shown to differentiate into IgM-secreting cells in response to IL-5) within 10 min at 37 degrees C. There are two classes of binding sites with high affinity (Kd = 66 pM) and low affinity (Kd = 12 nM) for IL-5 and an average number of binding sites for high affinity and for low affinity were 400 and 7,500 per cell, respectively. The specificity of binding of radiolabeled IL-5 has been confirmed by demonstrating that only unlabeled IL-5 and anti-IL-5 mAb but not by IL-1, IL-2, IL-3, IFN-gamma, and GM-CSF inhibit radiolabeled IL-5 binding to BCL1-B20 cells. Treatment of surface-bound radiolabeled IL-5 with bivalent crosslinkers identified a membrane polypeptide of Mr 46,500 to which IL-5 is crosslinked. A variety of cell types have been surveyed for the capacity to bind specifically radiolabeled IL-5 with high affinity. BCL1 cells MOPC104E (murine myeloma cell line) expressed IL-5-R, whereas BAL. 17 and L10 A (B cell lymphoma) did not. T cell line, mastocytoma cell line, or macrophage tumor cell line did not display detectable levels of IL-5-R. were hardly detectable on normal resting B cells but were expressed on LPS-activated B cells, fitting the function of IL-5 that acts on activated B cells for their differentiation into Ig-secreting cells. Intriguingly, early B cell lines (J-87 and T-88) that grow in the presence of IL-5 expressed significant but low numbers of high-affinity binding sites for IL-5. The biological effects of IL-5 were mediated by high-affinity binding sites. The identification and characterization of IL-5-R should provide new insight into the apparent diverse biological activities of IL-5.

Purified interleukin 5 supports the terminal differentiation and proliferation of murine eosinophilic precursors.

Using a clonal culture system, we investigated the hemopoietic effects of purified recombinant IL-5 obtained from conditioned media of transfected Xenopus oocytes. IL-5 alone acted on untreated bone marrow cells and supported the formation of a small number of colonies, all of which were predominantly eosinophilic. However, it did not support colony formation by spleen cells from 5-FU-treated mice, in which only primitive stem cells had survived, while IL-3 and G-CSF did. Eosinophil-containing colonies were formed from these cells in the presence of IL-5 and G-CSF together. In contrast, G-CSF alone did not support any eosinophil colonies. The eosinophilopoietic effect of IL-5 was dose-dependent, and was neutralized specifically by anti-IL-5 antibody. To exclude the possibility of interactions with accessory cells in the same culture dish, we replated a small number (200 cells/dish) of enriched hemopoietic progenitors, obtained from blast cell colonies, which were formed by cultivation of spleen cells from 5-FU-treated mice in the presence of IL-3 or G-CSF. From these replated blast cells, eosinophil colonies were induced in dishes containing IL-5 but not in those containing G-CSF alone. From these findings, it was concluded that IL-5 did not act on primitive hemopoietic cells, but on blast cells induced by IL-3 or G-CSF. IL-5 specifically facilitated the terminal differentiation and proliferation of eosinophils. In this respect, the role of IL-5 in eosinophilopoiesis seems to be analogous to erythropoietin, which promotes the terminal differentiation and amplification of erythroid cells. Moreover, IL-5 maintained the viability of mature eosinophils obtained from peritoneal exudate cells of the mice infected with parasites, indicating mature functional eosinophils carried IL-5 receptors. The synergistic effects of IL-5 and colony-stimulating factors on the expansion of eosinophils is supposed to contribute to the urgent mobilization of eosinophils at the time of helminthic infections and allergic responses.

Transforming growth factor beta induces IgA production and acts additively with interleukin 5 for IgA production.

Effects of transforming growth factor beta (TGF-beta) on IgA production by LPS-stimulated B cells have been studied. TGF-beta itself could augment polyclonal IgA production in concomitant inhibition of polyclonal IgM and IgG1 production. Furthermore, TGF-beta and IL-5 additively augmented IgA production. TGF-beta exerted its activity early in the culture (by 2 d in a 5-d culture) and IL-5 was required late in the culture. Surface IgA- (sIgA-) B cells responded to TGF-beta for the development of IgA-secreting cells. By contrast, sIgA+ B cells, but not sIgA- B cells, responded to IL-5 for IgA production. These results suggest that TGF-beta has a differential role in the induction of IgA production from IL-5 on murine-activated B cells.

Delayed progression of a murine retrovirus-induced acquired immunodeficiency syndrome in X-linked immunodeficient mice.

The murine acquired immunodeficiency syndrome (MAIDS) caused by defective LP-BM5 murine leukemia virus (MuLV) is a disease that shows severe immunodeficiency with abnormal lymphoproliferation, and hypergammaglobulinemia in susceptible C57BL/6 (B6) mice. To examine the cellular mechanisms of development of MAIDS, we injected LP-BM5 MuLV intraperitoneally into B6 mice bearing the X chromosome-linked immunodeficiency (xid). xid mice lack functionally mature B cells including Ly-1 B cells (also known as B-1 cells). All B6 mice died by 20 wk after LP-BM5 MuLV inoculation. In marked contrast, xid mice have continued to survive without any sign of MAIDS-related symptoms till at least 20 wk after the inoculation. The delayed progression of MAIDS in xid mice appears to depend on xid mutation, according to our experiments using both sexes of (B6.xid x B6)F1 and (B6 x B6.xid)F1 mice. Furthermore, Ly-1 B cells, enriched by a FACS, were shown to integrate the defective genome and appeared to be a major virus-infected B cell population. Our data corroborate that Ly-1 B cells play an important role in the induction and progression of MAIDS.

IL-5 receptor-mediated tyrosine phosphorylation of SH2/SH3-containing proteins and activation of Bruton's tyrosine and Janus 2 kinases.

Interleukin 5 (IL-5) induces proliferation and differentiation of B cells and eosinophils by interacting with its receptor (IL-5R) which consists of two distinct polypeptide chains, alpha and beta (beta c). Although both IL-5R alpha and beta c lack a kinase catalytic domain, IL-5 is capable of inducing tyrosine phosphorylation of cellular proteins. We investigated the role of IL-5R alpha in tyrosine phosphorylation of molecules involved in IL-5 signal transduction, using an IL-5-dependent early B cell line, Y16 and transfectants expressing intact or mutant IL-5R alpha together with intact beta c. The results revealed that the transfectants expressing truncated IL-5R alpha, which entirely lacks a cytoplasmic domain, together with beta c, showed neither protein-tyrosine phosphorylation nor proliferation in response to IL-5. This confirms that IL-5R alpha plays a critical role in protein-tyrosine phosphorylation which triggers cell growth. IL-5 stimulation results in rapid tyrosine phosphorylation of beta c and proteins containing Src homology 2 (SH2) and/or SH3 domains such as phosphatidyl-inositol-3 kinase, Shc, Vav, and HS1, suggesting their involvement in IL-5-mediated signal transduction. IL-5 stimulation significantly enhanced activities of Janus 2 and B cell-specific Bruton's tyrosine kinases (JAK2 and Btk) and increased the tyrosine phosphorylation of JAK2 kinase. These results and recent data on signaling of growth factors taken together, multiple biochemical pathways driven by tyrosine kinases such as JAK2 and Btk are involved in IL-5 signal transduction.

Interleukin-5.

Interleukin-5 is a dimeric cytokine that controls the differentiation of B cells into antibody-secreting cells as well as inducing the growth of Ly-1(CD5)+ B cells and B-cell tumors. It also has a major role of the growth and differentiation of eosinophils. Rapid progress has been made during the last year in the delineation of the structure and activities of interleukin-5, and the molecular nature of its functional receptors. Interleukin-5 exerts pleiotropic activities on various target cells through a high-affinity receptor which is composed of two different polypeptide chains, alpha and beta. The alpha chain binds interleukin-5 with low affinity and the beta chain has been identified as the interleukin-3 receptor-like protein and is also the beta chain of the granulocyte-macrophage colony stimulating factor receptor.

A critical cytoplasmic domain of the interleukin-5 (IL-5) receptor alpha chain and its function in IL-5-mediated growth signal transduction.

Interleukin-5 (IL-5) regulates the production and function of B cells, eosinophils, and basophils. The IL-5 receptor (IL-5R) consists of two distinct membrane proteins, alpha and beta. The alpha chain (IL-5R alpha) is specific to IL-5. The beta chain is the common beta chain (beta c) of receptors for IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF). The cytoplasmic domains of both alpha and beta chains are essential for signal transduction. In this study, we generated cDNAs of IL-5R alpha having various mutations in their cytoplasmic domains and examined the function of these mutants by expressing them in IL-3-dependent FDC-P1 cells. The membrane-proximal proline-rich sequence of the cytoplasmic domain of IL-5R alpha, which is conserved among the alpha chains of IL-5R, IL-3R, and GM-CSF receptor (GM-CSFR), was found to be essential for the IL-5-induced proliferative response, expression of nuclear proto-oncogenes such as c-jun, c-fos, and c-myc, and tyrosine phosphorylation of cellular proteins including JAK2 protein-tyrosine kinase. In addition, analysis using chimeric receptors which consist of the extracellular domain of IL-5R alpha and the cytoplasmic domain of beta c suggested that dimerization of the cytoplasmic domain of beta c may be an important step in activating the IL-5R complex and transducing intracellular growth signals.

Rap1 is a potent activation signal for leukocyte function-associated antigen 1 distinct from protein kinase C and phosphatidylinositol-3-OH kinase.

To identify the intracellular signals which increase the adhesiveness of leukocyte function-associated antigen 1 (LFA-1), we established an assay system for activation-dependent adhesion through LFA-1/intercellular adhesion molecule 1 ICAM-1 using mouse lymphoid cells reconstituted with human LFA-1 and then introduced constitutively active forms of signaling molecules. We found that the phorbol myristate acetate (PMA)-responsive protein kinase C (PKC) isotypes (alpha, betaI, betaII, and delta) or phosphatidylinositol-3-OH kinase (PI 3-kinase) itself activated LFA-1 to bind ICAM-1. H-Ras and Rac activated LFA-1 in a PI 3-kinase-dependent manner, whereas Rho and R-Ras had little effect. Unexpectedly, Rap1 was demonstrated to function as the most potent activator of LFA-1. Distinct from H-Ras and Rac, Rap1 increased the adhesiveness independently of PI 3-kinase, indicating that Rap1 is a novel activation signal for the integrins. Rap1 induced changes in the conformation and affinity of LFA-1 and, interestingly, caused marked LFA-1/ICAM-1-mediated cell aggregation. Furthermore, a dominant negative form of Rap1 (Rap1N17) inhibited T-cell receptor-mediated LFA-1 activation in Jurkat T cells and LFA-1/ICAM-1-dependent cell aggregation upon differentiation of HL-60 cells into macrophages, suggesting that Rap1 is critically involved in physiological processes. These unique functions of Rap1 in controlling cellular adhesion through LFA-1 suggest a pivotal role as an immunological regulator.

Interleukin 5 and B cell differentiation.

Interleukin-5 (IL-5) stimulates proliferation and differentiation of B cells and eosinophils. IL-5 receptor (IL-5R) comprises alpha and (beta)c chains. IL-5 specifically binds to IL-5Ralpha and induces the recruitment of (beta)c to IL-5Ralpha. JAK2 and JAK1 tyrosine kinases are constitutively associated with hIL-5Ralpha and (beta)c, respectively and activated upon IL-5 stimulation. IL-5 induces tyrosine phosphorylations of cellular proteins including (beta)c and STAT5 and activates Btk. X-linked immunodeficient mice have B-cell-specific defects due to missense mutation of the btk gene. The cytoplasmic proline-rich regions of both IL-5Ralpha and (beta)c are essential for the IL-5 signalling. IL-5 appears to play a critical role in hypereosinophilic syndromes and atopic diseases. The treatment of animals with anti-IL-5 mAb can decrease the enhanced bronchial responsiveness induced by allergen sensitization. Clinical studies provide a strong impetus for investigating the means of modulating IL-5 effects.

Screening of the Men1 gene and discovery of germ-line and somatic mutations in apparently sporadic parathyroid tumors.

Hyperparathyroidism is the first manifestation in a majority of multiple endocrine neoplasia (MEN1) patients. To discriminate between sporadic and hereditary parathyroid tumors and characterize MEN1 somatic mutations, we examined MEN1 gene mutations in patients who had undergone surgery for sporadic parathyroid tumors. DNA was extracted from fresh frozen parathyroid tumor specimens from 112 patients as well as from peripheral blood leukocytes from 64 of the 112 patients. Sequence analysis was performed to examine exons 2-10 of the MEN1 gene for mutations. Loss of heterozygosity (LOH) was also examined by an analysis of codon 418 and 541, which lie within a polymorphic region of MEN1. Somatic MEN1 mutations were found in 25 of the 112 patients (22%). Two patients had two point mutations (508del33 and Y341X and 363insT and 1767delT, respectively). A total of 27 mutations were characterized, 20 of which have not been reported previously. There were 7 nonsense mutations, 10 frameshift mutations, 2 splice site deletions, 5 missense mutations, and 3 in-frame mutations. Nineteen mutations (70%) predicted truncation of the menin protein. Germ-line MEN1 mutations were found in 3 of 64 patients (5%) who had no family history of endocrine tumors associated with MEN1, and these patients were identified as MEN1 gene probands. LOH at the MEN1 locus was detected in three parathyroid tumors showing germ-line mutation. LOH was significantly frequent in parathyroid tumors with somatic MEN1 mutations (15 of 22 tumors, 68%) but not in those without germ-line or somatic MEN1 mutations (14 of 51 tumors, 28%; P = 0.0011). Our findings suggest that alterations of both alleles of the MEN1 gene may be associated not only with endocrine tumors of affected MEN1 patients but also with sporadic parathyroid tumors. Germ-line MEN1 gene analysis can distinguish heritable from nonheritable parathyroid tumors, and MEN1 gene evaluation of patients with apparently sporadic parathyroid tumor is recommended before parathyroid surgery.

Role of CD4+ T lymphocytes and interleukin-5 in antigen-induced eosinophil recruitment into the site of cutaneous late-phase reaction in mice.

Previous studies suggested that the eosinophil recruitment into the site of cutaneous late-phase reaction (LPR) was dependent on IgE antibody and mast cells. In this study, we determined the role of CD4+ T cells and CD8+ T cells in causing antigen-induced eosinophil recruitment of LPR in mouse skin. Eosinophil infiltration into the subcutaneous tissue of ovalbumin (OVA)-sensitized BALB/c mice was biphasic, reaching the first peak at 6 h after the subcutaneous challenge with OVA and the second peak at 24 to 48 h. The in vivo depletion of CD4+ T cells by pretreatment with anti-L3T4 monoclonal antibody (mAb) significantly decreased the second peak (at 24 h and 48 h), but not the first peak (at 6 h), of OVA-induced eosinophil infiltration into the skin of OVA-sensitized mice. However, the depletion of CD8+ T cells by pretreatment with anti-Lyt-2 mAb had no significant effect on either the first peak or second peak of OVA-induced cutaneous eosinophilia. Pretreatment with anti-murine interleukin-5 (IL-5) mAb also decreased the second peak, but not the first peak, of OVA-induced cutaneous eosinophilia. In contrast to the inhibitory effects of depletion of CD4+ T cells and of anti-IL-5 mAb on the second peak of antigen-induced cutaneous eosinophilia, disodium cromoglycate and a selective antagonist for platelet activating factor (PAF) CV-6209 decreased the first peak of OVA-induced cutaneous eosinophilia in the mouse. These results indicate that CD4+ T cells, but not CD8+ T cells, cause the second peak of antigen-induced eosinophil recruitment of cutaneous LPR and that IL-5 mediates this eosinophil recruitment. In contrast, the first peak of antigen-induced eosinophil recruitment of cutaneous LPR is mediated by mast cells and PAF.