Secretory role for human uterodomes (pinopods): secretion of LIF.

The differentiation of human endometrial epithelium is a dynamic event, which occurs throughout the menstrual cycle in preparation for pregnancy. The appearance of uterodomes (pinopods) in this regard was first introduced in rodents with an established pinocytotic function, whereas little evidence was available in humans in this context. This study was undertaken to identify the potential physiological roles of uterodomes in the implantation process. To address this, endometrial biopsies from early, mid- and late luteal phases of the menstrual cycle of 23 fertile female patients with regular menses were used. Scanning and transmission electron microscopies (SEM and TEM) as well as immunofluorescence and immunogold TEM were performed to study the morphological changes and the expression pattern of leukaemia inhibitory factor (LIF) at uterodomes. Our results illustrated a high level of LIF expression in the human uterodomes, which was colocalized with the well-known biochemical markers of exocytosis, including syntaxin-1, 25-kDa synaptosomal protein (SNAP-25) and vesicle-associated membrane protein-2 (VAMP-2). Our morphological and immunocytochemical findings illustrated a secretory function for human uterodomes for the first time. In conclusion, this novel function for uterodomes provides an important clue in detection of their physiological function(s) during the process of the plasma membrane transformation.

Comparison between partial agonist (ME3412) and antagonist (alosetron) of 5-hydroxytryptamine 3 receptor on gastrointestinal function.

Therapeutic use of 5-hydroxytryptamine 3 (5-HT(3)) receptor antagonists for diarrhoea-predominant irritable bowel syndrome may be accompanied by constipation. We hypothesized that ME3412, 5-chloro-2-(1,4-diazacycloheptan-1-yl)-7-methylbenzoxazole, a novel partial agonist of the 5-HT(3) receptor, would minimize constipation without reducing antidiarrhoeal activity. Receptor binding studies showed that ME3412 is highly selective for the human 5-HT(3) receptor (K(i) = 1.51 nmol L(-1)). A 5-HT(3) receptor agonist, 2-methyl-5-HT, caused contractile response in the isolated guinea-pig ileum and accelerated secretion in the guinea-pig colonic mucosal preparation. ME3412 and 5-HT(3) receptor antagonist, alosetron, antagonized the 2-methyl-5-HT-induced responses with similar potency in insurmountable and surmountable manner, respectively. ME3412 caused weak agonism in isolated ileum strips and also in the colonic mucosa with intrinsic activity of 0.09 and 0.59, respectively. In conscious dogs, alosetron (3 microg kg(-1) i.v.) suppressed the migrating motor complex (MMC), whereas a relatively high dose (300 microg kg(-1)) of ME3412 was required for inhibition of MMC. ME3412 and alosetron suppressed 5-HT induced-diarrhoea in mice. In contrast, ME3412 did not significantly affect colonic propulsion compared with alosetron. These results imply that the partial agonist may relieve diarrhoea with low risk of inducing constipation.

The role of alpha(5)beta(1)-integrin in the IGF-I-induced migration of extravillous trophoblast cells during the process of implantation.

The role of integrins in the processes of adhesion and migration makes them attractive potential participants in the complex events of embryo implantation and placentation. Recently, the role of the alpha(v)beta(3)-integrin pathway was shown in the insulin-like growth factor-I (IGF-I)-stimulated migration of extravillous trophoblast (EVT) cells. This study was designed to investigate the role of alpha(5)beta(1)-integrin in this respect. Using cultured EVT cells, migration assays were carried out for IGF-I-treated or untreated cells in the presence or absence of the GRGDSP and GRGESP hexapeptides, alphaIR3, and a blocking antibody against alpha(5)beta(1)-integrin. Immuno-electron microscopy and immunofluorescent staining were performed to localize the distribution of alpha(5)beta(1)- and alpha(v)beta(3)-integrins, Rab5a, paxillin, phospho-FAK (pFAK), and vinculin. The results showed that IGF-I-induced migration of EVT cells was abolished following treatment with GRGDSP hexapeptide, alphaIR3, and a blocking antibody against alpha(5)beta(1)-integrin. Further, statistical analysis showed that the area-related numerical density of the alpha(5)beta(1)-integrin in the perinuclear regions was significantly higher than in the cell extensions. Immunocytochemical experiments demonstrated an up-regulation in internalization rate of alpha(5)beta(1)-integrin in IGF-I-stimulated EVT cells. Furthermore, alpha(5)beta(1)-integrin exhibited co-localization with Rab5a, but not with alpha(v)beta(3)-integrin, pFAK, paxillin, and vinculin at the focal adhesions of the EVT cells. Taken together, these findings suggest an essential role for alpha(5)beta(1)-integrin in IGF-I-promoted migration of EVT cells. It is possible therefore that IGF-I-induced internalization of alpha(5)beta(1)-integrin may be an important event during the migration of EVT cells in the complex processes of implantation and placentation.

Gallium-67 scintigraphy in the diagnosis and management of chronic sarcoid myopathy.

The authors describe a patient with chronic sarcoid myopathy. Except for the presence of left posterior synechia, no other organ involvement was observed. Gallium-67 ((67)Ga) scintigraphy showed many intense nodular uptake areas in both the upper and lower extremities. Treatment with oral prednisolone 30 mg/day resulted in a marked improvement on (67)Ga scintigraphy. This case suggests that (67)Ga scintigraphy is useful for the differential diagnosis of systemic myopathies and also for monitoring the effect of glucocorticoid treatment.

Expression of erythropoietin in human female reproductive organs.

Erythropoietin (Epo) is known to be a lineage specific cytokine which regulates the number of circulating erythrocytes. Most of it is produced in the kidney. Recently, Epo has been reported to be synthesized in the normal brain, placenta, and capillary endothelium. We also have found that uterine endometrium expresses Epo signals in an estrogen-dependent manner, and that Epo contributes to angiogenesis in the endometrium in mice. To clarify the functional activity of Epo in human reproductive organs, we examined Epo signaling in these organs by Southern analysis of RT-PCR products and studied the distribution of substances relevant to Epo signal transduction by immunohistochemistry and Western blotting. Epo mRNA is expressed in the normal human cervix, endometrium and ovary, but it is not always detected in the specimens. Immunohistochemical analysis revealed Epo-receptor (EpoR) protein in: a) the endothelium of vessels, in glandular and surface epithelial cells, in decidual cells of the endometrium, and b) in follicles at various stages including oocytes, granulosa, theca interna cells and lutein cells of the ovary. Moreover, co-expression of JAK2 and phosphotyrosine, which reflects tyrosine phosphorylation via JAK2, and co-expression of EpoR and STAT5, which is a transcriptional factor relevant to mitogenic activity, were seen at these Epo-responsive sites. Western blotting analysis of these organs confirmed the immunohistochemical results. These findings imply that female reproductive organs can produce Epo, and that signal transduction of Epo contributes to the cyclic changes in the female reproductive organs.

Clonal analysis of B cells in the osteoarthritis synovium.

OBJECTIVES: Cellular and humoral immunity to collagen and cartilage proteoglycan were shown in patients with osteoarthritis (OA). Inflammatory infiltration containing T and B lymphocytes and macrophages, which are HLA-DR positive, is often seen in the synovial membrane of patients with OA. An analysis of the DNA restriction enzyme patterns of T lymphocytes from the OA synovium showed an oligoclonal pattern of T cell receptor beta chain gene rearrangements. No similar studies of B cell clonality have previously been performed. This study aimed at determining the clonal characteristics of the B cells in the OA synovium. METHODS: A reverse transcriptase-polymerase chain reaction of the immunoglobulin transcripts of B cells in the synovial membranes from six patients with OA was performed and the products were analysed by a single strand conformation polymorphism analysis. RESULTS: Several dominant bands were seen in all samples and some of the dominant bands were common among the two or three separate regions of each synovial sample. CONCLUSION: Infiltrated B cells are oligoclonal, and an antigen driven immune response may play a part in the progression of the disease process in OA.

Aberrant BCR-ABL transcript with intronic insertion in a patient with philadelphia chromosome-positive chronic myeloid leukemia: implications for disease progression.

The BCR-ABL fusion gene is important for the leukemogenesis of chronic myeloid leukemia (CML). A relationship between types of BCR-ABL transcripts in CML and clinical features has been proposed. We present here a patient with CML who carried an aberrant BCR-ABL transcript with an intronic sequence insert. A 26-year-old woman was diagnosed as having Philadelphia chromosome (Ph) positive CML. Reverse transcription polymerase chain reaction detected an atypically large BCR-ABL mRNA transcript. Sequencing revealed a 589bp insertion consisting of a 5' portion of BCR intron b2 and a 3' portion of ABL intron 1b between BCR exon b2 and ABL exon a2. Although the typical b2a2 transcript was undetectable initially, it appeared after intensive chemotherapy. The aberrant transcript presumably arose as a result of a lack of splicing, and chemotherapy might modify the disease course by selecting the subpopulation of the CML clone expressing typical BCR-ABL mRNA dominantly.

Primary Sjögren's syndrome complicated by sarcoidosis and psoriasis vulgaris.

Abstract Primary Sjögren's syndrome (SS), sarcoidosis (SA), and psoriasis vulgaris (PV) are all chronic diseases of unknown etiology. Recent studies suggest that activated T cells play a central role in their pathogenesis. We describe a case of a Japanese woman with primary SS complicated by SA and PV. To our knowledge, this is the first case in which these three diseases coexist. Although these three disorders may have a common immunopathogenic mechanism, the extreme rarity of their coexistence suggests that distinct etiological mechanisms are also involved and appear to play an important role in triggering and developing each disease.

Sequence and expression analyses of mu and delta transcripts in patients with Waldenström's macroglobulinemia.

Waldenström's macroglobulinemia (WM) is a malignant lymphoplasmo-proliferative disorder with monoclonal pentameric immunoglobulin (Ig)M production. The most consistent feature of clonal B cells in the bone marrow (BM) and/or lymph nodes of patients with WM is the presence of pleomorphic B-lineage cells at different stages of maturation, such as small lymphocytes, lymphoplasmacytoid cells, and plasma cells. Monoclonal lymphocytes express mu chains with or without delta chains. A recent DNA analysis of WM tumor clones showed WM to be derived from B cells that have been selected by antigen at a relatively late stage of differentiation. To further clarify the origin of WM tumor cells, we analyzed the variable (V) domain sequences of tumor derived mu and delta transcripts. The expression of delta transcripts was also examined in peripheral blood (PB) and BM using the reverse transcriptase polymerase chain reaction (RT-PCR) combined with a single-strand conformation polymorphism (SSCP) analysis. The sequences were identical among the mu and delta transcripts in each patient and the level of somatic mutation in the VH regions expressed by tumor cells was in the same range as that of IgM-only B cells and IgM(+)IgD(+) memory B cells. In our previous RT-PCR-SSCP analysis, a single dominant band of the mu isotype was observed in BM and PB in all patients. However, common dominant bands in BM and PB were detected in only one patient in a delta transcript analysis. In the rest of the patients, monoclonal delta transcripts were only detected in BM. Our results suggest that a normal counterpart of WM cells is somatically mutated IgM(+)IgD(+) and/or IgM-only B cells and the expression patterns of monoclonal mu and delta transcripts differ between BM and PB in some cases of WM.

CP6679, a new injectable cephalosporin. Part 1: synthesis and structure-activity relationships.

A series of cephalosporins bearing a 5,5-fused ring system, an (imidazo[5,1-b]thiazolium-6-yl)methyl group, at the C-3 position were synthesized and evaluated for in vitro antibacterial activities. CP6679 (1s) and its analogues showed potent antibacterial activities against gram-positive and gram-negative bacteria, including Pseudomonas aeruginosa. They were also highly active against methicillin-resistant Staphylococcus aureus (MRSA). CP6679 (1s) showed more potent antibacterial activity than ceftazidime (CAZ) or cefpirome (CPR) against Pseudomonas aeruginosa and MRSA.

Accumulation of common clonal T cells in multiple lesions of sarcoidosis.

BACKGROUND: T cells recognizing as yet unknown antigens (Ags) are considered to play an important role in the development and perpetuation of the disease process of sarcoidosis. Several studies have shown that T cells that bear a limited T-cell receptor (TCR) repertoire may play an important role in this disorder. However, regarding variable (V) gene repertoire usage, the results differ among various reports. One reason for such inconsistency may be due to the materials used in these studies. Most studies analyzed the T-cell repertoire in the sarcoid lung. However, clonal expansion of pulmonary T cells, probably due to the activation by inhaled exogenous Ags, was observed and such expansion may seriously influence the repertoire analysis. MATERIALS AND METHODS: Reverse transcriptase-polymerase chain reaction and subsequent single-strand conformation polymorphism analysis were used for the analysis of TCR repertoire. To exclude unrelated T-cell clones, we used intramuscular sarcoid nodules and/or lymph node (LN) sarcoid lesions as our materials. We also analyzed sarcoid lesions from different organs and then compared the results. RESULTS: T cells of the same clonality were found to exist in widely separated sites in intramuscular and LN sarcoid lesions in almost all Vbeta subfamilies. Identical T-cell clones were present in the sarcoid lesions from different organs in several Vbeta subfamilies. CONCLUSIONS: Some of the common T-cell clones in separated sites in intramuscular and LN sarcoid lesions and in sarcoid samples from different organs may recognize Ags that are related to the pathogenesis of sarcoidosis.

Diffuse intervertebral disk calcification in a patient with rheumatoid arthritis.

A patient with seronegative rheumatoid arthritis (RA) who presented with intervertebral disk calcification (IDC) of several thoracic and lumbar intervertebral disks in herein described. There was no evidence of any other coexisting diseases such as ochronosis and hemochromatosis, but a remarkable degree of polyclonal hypergammaglobulinemia was observed as a notable finding. Although the appearance of IDC on T1-weighted images on magnetic resonance is controversial, no increased signal intensity was observed in our patient. To the best of our knowledge, this is the first report of IDC in RA.

Primary macroglobulinemia with t(11;18)(q21;q21).

These are the first cases of primary macroglobulinemia (PMG) with t(11;18)(q21;q21) reported in the literature. The first case was a 77-year-old man with macroglobulinemia (serum IgM: 8.36 g/dL). Abnormal lymphoid cells were detected in the blood and bone marrow. Immunologic and karyotypic analyses revealed that abnormal cells were positive for surface IgM-k, CD19, and CD20, negative for CD5 and CD10, and all had a t(11;18)(q21;q21). The second case was a 57-year-old woman with macroglobulinemia (serum IgM: 12.0 g/dL). Abnormal lymphoid cells were detected in blood and marrow, and cells were positive for surface IgM-lambda, CD19, and CD20, and negative for CD5 and CD10. Plasma cells bearing cytoplasmic IgM-lambda were increased in pleural fluid. Karyotyping demonstrated t(2;11;18)(q21-23;q21;q21). Rearrangements within BCL2 and YES genes located at 18q21 were not detected. Sixteen other cases with t(11;18)(q21;q21) have been reported in marginal zone B-cell lymphoma. Therefore, our report is in agreement with the finding that part of primary macroglobulinemia is a variant of marginal zone B-cell lymphoma.

Expansion of identical B-cell clones in the bilateral parotid glands and their circulation in the peripheral blood in a patient with Sjögren's syndrome.

Abstract We encountered a patient with Sjögren's syndrome (SS) associated with bilateral parotid salivary gland swelling. Histological analysis of biopsy specimens from both parotid glands showed myoepithelial islands and infiltration of small- and intermediate-sized lymphocytes but no cytological atypia. Using reverse transcriptase-polymerase chain reaction and subsequent single-strand conformational polymorphism analysis, monoclonal B-cell expansion was detected in samples from both right and left parotid glands, bone marrow, and peripheral blood (PB). Our case suggests that the circulating clonal lymphocytes represent clones that can repopulate tissue sites and may contribute to B-cell lymphomagenesis. Detection of monoclonal B cells in PB is therefore considered to be important in monitoring the disease course of SS.

Function of the small guanosine triphosphate-binding protein RhoA in the process of implantation.

The Rho family of small GTPases occupies a key position in the control of cell motility and morphology in response to extracellular stimuli. Rho proteins trigger the formation of contractile stress fibers, resulting in regulation of cell motility. We explored the expression and function of RhoA in human endometrium and decidua. RhoA immunoreactivity had a predominantly glandular epithelial distribution in the proliferative phase and midsecretory phase. In decidua, the expression of RhoA was more pronounced in the stromal cells as well as in the glandular epithelium. RhoA protein levels in proliferative phase and midsecretory phase endometrium as well as decidua were evaluated by immunoblotting; a single band of RhoA protein with a molecular mass of 21 kDa was detected in all cell lysates. Cultured human decidual cells were found to have few actin stress fibers. Decidual cells lost their actin stress fibers by the treatment with C3, an exoenzyme produced by Clostridium botulinum, whereas new actin stress fibers appeared in human decidual cells stimulated with lysophosphatidic acid (LPA). Mouse blastocysts became attached to cultured human decidual cells after embryos hatched from the zona pellucida. The majority of hatched blastocysts attached to human decidual cells within 24 h. Blastocysts attached to decidual cells exhibited extensive outgrowth after 48 h in culture. Treatment of decidual cells with C3 exoenzyme or LPA did not affect the rates of hatching and attachment of blastocysts, but outgrowth of embryos on decidual cells was inhibited by C3 exoenzyme treatment in a dose-dependent manner. Contrariwise, addition of LPA to decidual cells dose dependently increased the outgrowth of embryos on decidual cells. These findings suggest that RhoA in decidual cells is important for embryonic development and differentiation after attachment.

Clonal identities and multiple isotype transcripts in hematological diseases revealed by a single-strand conformation polymorphism analysis of the immunoglobulin heavy chain messenger signals.

A single-strand conformation polymorphism (SSCP) analysis of polymerase chain reaction (PCR)-amplified products of immunoglobulin (Ig) heavy chain rearrangements can be used to analyze B cell clonalities and clonal identities of B cells from different samples. However, the usefulness of the PCR-SSCP analysis is not fully assessed in B cell malignancies. For example, we did not know whether the PCR-SSCP method can be used to detect tumor-related clones in peripheral blood of patients with multiple myeloma and Waldenström's macroglobulinemia. In addition, because genomic DNA is used in the PCR-SSCP method, we could obtain no information about the isotype of the expanded B cell clone. In this study, we combined the reverse transcriptase (RT)-PCR of immunoglobulin heavy chain transcripts with an SSCP analysis and thus analyzed eight healthy individuals, five patients with B chronic lymphocytic leukemia, four patients with multiple myeloma and three patients with Waldenström's macroglobulinemia. Clonal B cell populations were detected as discrete bands in the RT-PCR-SSCP analysis that can be readily detected over the background of polyclonal rearrangements. Circulating tumor-related clones were detected in all but one peripheral blood sample from multiple myeloma and Waldenström's macroglobulinemia patients and B cell clones in peripheral blood and bone marrow from these patients showed a similar mobility on SSCP gel. Because the transcripts of different isotypes were separately analyzed, we could thus determine the isotype of B cell clones as well. When monoclonal Igs of different isotypes were detected in the individual samples, we analyzed the relationship of each monoclonal band by excising the band and then further analyzing it by a PCR-SSCP analysis. RT-PCR amplification in conjunction with the SSCP analysis is thus considered to be a useful method to detect and characterize the B cell clones in hematological diseases.

Functional role of arg-gly-asp (RGD)-binding sites on beta1 integrin in embryo implantation using mouse blastocysts and human decidua.

Amino acid residues 140-164 of integrin beta1 comprise an Arg-Gly-Asp (RGD) cross-linking region. The present study was undertaken to study the role of the RGD cross-linking region of integrin beta1 subunit in embryo implantation. Decidual cells attached to fibronectin (FN)-coated dishes. A peptide corresponding to integrin beta1[140-164] (DDL; DYPIDLYYLMDLSYSMKDDLENVKS) inhibited decidual cell attachment to FN-coated dishes in a dose-dependent manner. A variant integrin peptide in which Asp 157 and Asp 158 were replaced by Ala (AAL; DYPIDLYYLMDLSYSMKAALENVKS) did not affect decidual cell attachment to FN. Inhibition by DDL peptide was reversed by prior treatment with an RGD-containing peptide but not by prior treatment with an RGE-containing peptide. Mouse blastocysts became attached to cultured human decidual cells after embryos hatched from the zona pellucida. The majority of hatched blastocysts attached to human decidual cells within 24 h of culture. Blastocysts that attached to decidual cells exhibited extensive outgrowth after 48 h. Treatment of decidual cells with synthetic peptides did not affect the rates of hatching and attachment of blastocysts. The outgrowth of embryos on decidual cells was inhibited by DDL peptide in a dose-dependent manner, but not by AAL peptide. These findings suggest that integrin beta1[140-164] on decidual cells may be important in embryonic development and differentiation following attachment.

IgM heavy chain complementarity-determining region 3 diversity is constrained by genetic and somatic mechanisms until two months after birth.

Due to the greater range of lengths available to the third complementarity determining region of the heavy chain (HCDR3), the Ab repertoire of normal adults includes larger Ag binding site structures than those seen in first and second trimester fetal tissues. Transition to a steady state range of HCDR3 lengths is not complete until the infant reaches 2 mo of age. Fetal constraints on length begin with a genetic predilection for use of short DH (D7-27 or DQ52) gene segments and against use of long DH (e.g., D3 or DXP) and JH (JH6) gene segments in both fetal liver and fetal bone marrow. Further control of length is achieved through DH-specific limitations in N addition, with D7-27 DJ joins including extensive N addition and D3-containing DJ joins showing a paucity of N addition. DH-specific constraints on N addition are no longer apparent in adult bone marrow. Superimposed upon these genetic mechanisms to control length is a process of somatic selection that appears to ensure expression of a restricted range of HCDR3 lengths in both fetus and adult. B cells that express Abs of an "inappropriate" length appear to be eliminated when they first display IgM on their cell surface. Control of N addition appears aberrant in X-linked agammaglobulinemia, which may exacerbate the block in B cell development seen in this disease. Restriction of the fetal repertoire appears to be an active process, forcing limits on the diversity, and hence range of Ab specificities, available to the young.

Establishment of a novel B cell clonality analysis using single-strand conformation polymorphism of immunoglobulin light chain messenger signals.

The remarkable diversity of the complementarity determining region (CDR) 3 of the immunoglobulin (Ig) heavy (H) chain gene rearrangements has been exploited to identify the clonal populations of B cells in B cell malignancies. However, when B cell malignancies of different categories were examined, the overall detection rate was found to be approximately 70%. The development of a simple clonality analysis using Ig light (L) chain CDR3 diversity has been hampered due to the sparseness of knowledge regarding the sequence of Vkappa and Vlambda gene segments and the restriction of L chain CDR3 length. Based on the recently reported Vkappa and Vlambda gene sequences, we designed Vkappa and Vlambda framework 3 consensus primers. We combined the reverse transcriptase polymerase chain reaction (RT-PCR) of IgL chain transcripts with a single-strand conformation polymorphism (SSCP) analysis and then analyzed samples from patients with B cell malignancies. Clonal B cell populations were detected as discrete bands, and identical clones showed a similar mobility in a RT-PCR SSCP analysis. This method was thus found to be a useful supplement to the previously described approach of VH gene amplification for detecting clonal B cell populations. By using SSCP, we were able to determine the clonal identities of B cell expansion in different samples.