bluuebunny/crossref_metadata_2025 huggingface dataset:
Mammary Glands of Aly Mice: Developmental Changes and Lactation‐Related Expression of Specific Proteins, α‐Casein, GlyCAM‐1 and MAdCAM‐1
Authors: NISHIMURA TOSHIHIDE , KOIKE RYUJI , MIYASAKA MASAYUKI
Published: December 2003

PROBLEM: (1) Aly mouse is an autosomal recessive mutant mouse
that lacks lymph nodes and Peyer's patches. Defects of mammary
glands might explain why these mice fail to nurture their pups. (2)
Expressions of glycosylation‐dependent cell adhesion molecule 1
(GlyCAM‐1) and mucosal addressin cell adhesion molecule‐1 (MAdCAM‐1) in
mammary glands of lactating mice have been suggested, but only scant
information is available. 
METHODS: The thickness and weight of mammary glands were measured
directly. The expression of specific proteins, α‐casein, GlyCAM‐1,
and MAdCAM‐1 in mammary glands was detected by reverse
transcription‐polymerase chain reaction (RT‐PCR). MAdCAM‐1 expression was
also confirmed by immunohistochemistry (IHC). 
RESULTS: Fat pads of mammary glands of homozygous (aly/aly)
mice were small and weighed less than those of heterozygous
(aly/+) mice. An increase in end‐bud development was evident in
aly/aly mice. During pregnancy, growth of the mammary gland
and alveolar formation were noted in both groups. The expression of
α‐casein, GlyCAM‐1 and MAdCAM‐1 was detected in both groups of mice
by RT‐PCR from late‐pregnancy. Immunohistochemically, MAdCAM‐1 was noted
in small vessels around the mammary globules. 
CONCLUSIONS: Our results demonstrated marked changes in the structure of
the mammary glands of aly/aly mice. The expression of
lactation‐related proteins was detected during development, pregnancy,
and lactation in both aly/aly and aly/+ mice.
Incipient Ovarian Failure and Premature Ovarian Failure Show the Same Immunological Profile
Authors: VAN KASTEREN YVONNE M. , VON BLOMBERG MARY , DE KONING CORRY , LAMBALK NILS , VAN MONTFRANS JORIS , SCHOEMAKER JOOP , HOEK ANNEMIEK , KUIK JOOP
Published: December 2003

PROBLEM: Incipient ovarian failure (IOF) is characterized by regular menstrual cycles, infertility and a raised early‐follicular FSH in women under 40. IOF might be a precursor or a mitigated form of premature ovarian failure (POF). Disturbances in the immune system may play a role in ovarian failure.  METHOD OF STUDY: Autoantibodies and lymphocyte subsets were determined in 63 POF patients, 50 IOF patients, and 27 controls.  RESULTS: The prevalence of autoantibodies did not differ between the groups. There was a statistically significant difference in lymphocyte subsets between the control group and the POF group, with the IOF group taking an intermediate position. We found a decrease in percentage of T‐suppressor cells with a rise in T‐helper/T‐suppressor cell ratio, a decrease in natural killer cells, and an increase in B lymphocytes and HLA‐DR positive T cells.  CONCLUSIONS: These data support the concept that IOF is a mitigated form of POF. The question remains whether these changes are the cause or the consequence of the ovarian failure.
Pregnancy as a Model of Controlled Invasion Might Be Attributed to the Ratio of CD3/CD8 to CD56
Authors: ARCK P.C. , HAGEN E. , HILDEBRANDT M. , KLAPP B.F. , HERTWIG K.
Published: December 2003

PROBLEM: Pregnancy can be considered as a model of successfully controlled tissue invasion. Cellular mediated immunity appears to regulate the controlled invasion of fetal trophoblast cells. In endometrium cancer, a dysregulation of invasive malignant cells can be observed. Since immuncompetent cells are known to be involved in recognition and rejection of ‘non‐self’ antigens, we investigated the presence and distribution pattern of CD3, CD8, CD56, and CD68 positive cells in decidua from normal and failing pregancies, compared with malignant and benign endometrium.  METHOD OF STUDY: Decidual tissue from first trimester normal pregancies (NP; n=15) and abortion (AB; n=12), endometrial samples
from premenopausal women (NE; n=8), and endometrioid
adenocarcinoma (EA; n=8) were examined by immunohistochemistry
using monoclonal antibody against large spectrum cytokeratin, and against
the receptors CD3, CD8, CD56 and CD68, respectively. 
RESULTS: In NP, we observed 32.5% CD3, 44.7% CD56, and 22.9%
CD68+ cells. In AB, we found 36.9% CD3, 45.3% CD56, and 17.8%
CD68+ cells. The differences in ratio between normal pregnancy
and abortion were not statistically significant. In NE, we counted 39.5%
CD3, 30.2% CD56 and 30.2% CD68+ cells. In EA, we observed
47.9% CD3, 12.4% CD56 and 39.7% CD68+ cells. The decrease of CD56 positive cells in endometrioid adenocarcinoma was statistically significant. Interestingly, we found 4.1% of cells positive for CD8 in NP, 4.9% in AB, 22.7% in NE, and 48.2% in EA.  CONCLUSIONS: The increase of CD8 cells in NE, and particularely in EA, and decrease of CD56 cells, compared with NP or AB, suggests an interaction between CD8 cells and CD56 cells. Studying different pathological situations in the uterus, such as malignancies or ectopic pregnancies, might help us to understand the mechanisms involved in the maintenance of pregnancy.
Human First‐Trimester Decidua Vascular Density: An Immunohistochemical Study Using VE‐Cadherin and Endoglin as Endothelial Cell Markers
Authors: VAILHÉ BRUNO , ARCK PETRA , KAPP MICHAELA , DIETL JOHANNES
Published: December 2003

PROBLEM: Angiogenesis and vasculogenesis appear to be of critical importance for the success of pregnancy. Recent data have emphasized that pregnancy complications, such as abortion or pre‐eclampsia, are linked with vascular pathologies. The aim of this study was to quantify human first‐trimester decidua microvascular density, using two novel, highly specific endothelial cell markers, VE‐cadherin and endoglin.  METHOD OF STUDY: We collected decidua from women undergoing termination of normal pregnancies. VE‐cadherin and endoglin were localized by immunohistochemistry. The blood vessel densities detected by VE‐cadherin or endoglin‐stainings were microscopically quantified per mm2.  RESULTS: Endothelial cells in first‐trimester human decidua both express VE‐cadherin and endoglin. The microvascular density detected by VE‐cadherin‐staining varied from 32.2±1.7 in decidua basalis, to 30±0.6 in decidua parietalis. For the endoglin‐staining, the values varied from 37.5±3 in decidua basalis, and 26.7±1.2 in decidua parietalis.  CONCLUSIONS: Our data shows that both VE‐cadherin and endoglin are good candidates to highlight the decidual endothelial cells, and to quantify the blood vessels density of endometrium.
Stimulation of Uterine Cell Cytokine Production By Ovarian Hormones
Authors: DELOIA J.A. , STEWART‐AKERS A.M. , BREKOSKY J. , KUBIK C. , STEWART‐AKERS A.M.
Published: December 2003

PROBLEM: Although leukocytes do not possess significant numbers of ovarian steroid hormone receptors, their numbers in the endometrium vary consistently, relative to the menstrual cycle. The possibility that cell types within the endometrium express leukocyte‐attracting genes in response to ovarian hormones was investigated.  METHOD OF STUDY: Endometrial biopsies were collected 10 days post‐leutinizing hormone surge; the cell types were separated and cultured individually for 5 days in the presence of increasing amounts of estrogen or progesterone. Following culture, RNA was collected from cells and reverse‐transcription‐polymerase chain reaction was used to determine relative levels of gene expression of monocyte chemotactic proteins (MCP)‐1, ‐2, and ‐3, and interleukin (IL)‐12p35 and p40.  RESULTS: Although both endometrial stroma and glands were able to make MCP mRNA, steady‐state levels of gene expression did not vary significantly relative to hormone treatment. The same was found for the p35 molecule of the IL‐12 gene; however, differences were observed for the p40 subunit.  CONCLUSIONS: Within the human endometrium, chemokines other than MCP and IL‐12 are most likely responsible for cycle‐related leukocyte recruitment.
Interleukin‐6 and Soluble Interleukin‐6 Receptor Serum Levels in Recurrent Spontaneous Abortion Women Immunized with Paternal White Cells
Authors: ZENCLUSSEN ANA CLAUDIA , MARGNI RICARDO , ILEANA  , MALAN BOREL  , KORTEBANI GRACIELA , MAZZOLLI ALICIA
Published: December 2003

PROBLEM: Recurrent spontaneous abortion (RSA) could be interpreted as the cause for the incapacity of the mother to recognize paternal antigens to produce the desired protective response. The practise of alloimmunization was introduced in an attempt to induce in the mother the production of an alloimmune response; some authors proposed an association between cytokines and RSA. The production of IL6 and its soluble receptor (sIL6R) before and after lymphocyte immunotherapy was evaluated in sera of 33 patients suffering from two or more RSA, and in sera of 47 women with normal pregnancy.  METHOD OF STUDY: The immunization of RSA patients was achieved by injection of four doses of 105 mononuclear cells (MNC) from the husband, at weekly intervals, before pregnancy. The IL6 and sIL6R levels were measured using sandwich ELISAs and the results evaluated by Tukey–Kramer multiple comparison‐tests.  RESULTS: Our data show no significant differences between IL6 and sIL6R serum levels of normal pregnant women and RSA pregnant women with white‐cell immunization before pregnancy. In contrast, the sera of pregnant RSA patients without allogeneic therapy show higher values. We also found significant differences between IL6 levels in non‐pregnant RSA women with and without immunotherapy.  CONCLUSION: These results show that the alloimmunization with paternal white cells leads the serum IL6 and sIL6R‐levels to the values observed in the course of normal pregnancy, suggesting a role for IL6 and sIL6R in the modulation of the immune response's quality.
Endocrine–Immune Interaction: Alterations in Immune Function Resulting From Neonatal Treatment with a GnRH Antagonist and Seasonality in Male Primates
Authors: MANN DAVID R. , AKINBAMI MUKAILA A. , ANSARI AHMED A. , GOULD KENNETH G. , LUNN STEPHEN F. , FRASER HAMISH M.
Published: December 2003

PROBLEM: The effect of neonatal gonadotropin releasing hormone (GnRH) antagonist (Ant) treatment and seasonality on immune system development and function was investigated in male primates.  METHOD OF STUDY: Neonatal male rhesus monkeys and marmosets were treated with Ant, and its effect on immune system morphology, circulating lymphocyte subsets, and cell‐ and humorally‐mediated immune responses was assessed during development. In adult rhesus monkeys, we correlated seasonal changes in immune function with circannual fluctuations in immunoactive hormones.  RESULTS: In neonatal marmosets, Ant reduced the number of B cells and T cells in the thymic medulla and T cells in the periarterial lymphatic sheaths (PALS) of the spleen. Ant also altered the development of, but did not permanently impair, the proliferative index (PI) of blood lymphocytes to mitogens. In vitro treatment of control lymphocytes with GnRH analogues altered their response to these proliferative agents. In neonatal rhesus monkeys, Ant treatment increased the frequency of clinical problems, lowered circulating levels of lymphocytes, total T cells, CD8+ T cells and B cells, and altered the PI of lymphocytes to mitogens. As adults, the cell‐ and humorally‐mediated immune responses remained impaired. We also documented seasonal fluctuations in the prevalence of diseases, circulating immune cells and immune function in rhesus monkeys. The number of cases of campylobacteriosis and shigellosis was lowest in the winter and highest in the spring. Circulating numbers of white blood cells (WBC) and neutrophils and the PI of lymphocytes to mitogens were higher in the winter than in the summer. Natural killer cell activity also varied with season. Cortisol and leptin secretion exhibited circannual rhythms, rising in concert with decreasing photoperiod and increasing testicular activity in the fall. Conversely, prolactin levels declined with decreasing photoperiod and then rose in the spring.  CONCLUSION: Neonatal exposure of male primates to Ant appears to alter early postnatal programming of immune function. In the rhesus monkey, immune function shows seasonal fluctuations that may be driven by circannual changes in the secretion of immunoactive hormones.
Fertilization‐Related Sperm Antigens and their Immunocontraceptive Potentials
Authors: NAZ RAJESH K.
Published: December 2003

Vaccine(s) for contraception will provide an attractive alternative to contraception. There has been a renewed interest in defining fertilization‐related sperm antigens that can be used for the development of a contraceptive vaccine. The sperm‐zona pellucida (ZP) recognition and binding constitutes the most important event in the fertilization process, and the molecules involved at this site are attractive candidates for immunocontraception. Extensive research indicates that there are four sperm surface proteins that bind to oocyte ZP3 in humans and they belong to the four molecular regions of 95, 63, 51, and 14–18 kDa, respectively. Our laboratory is actively engaged in cloning and sequencing of cDNAs encoding for these sperm proteins and investigating the applications of recombinant (r) proteins in immunocontraception and infertility. Presently, the cDNAs encoding for the 14–18‐kDa proteins, designated the NZ‐1 and NZ‐2, respectively, and the 51‐kDa protein, designated fertilization antigen, have been cloned and sequenced. Active immunization with the rFA‐1 antigen caused a reversible block/inhibition in fertility of female mice by raising a sperm/testis‐specific immune response. FA‐1 antigen is also involved in human immunoinfertility, and a recent clinical trial indicates that it will have a clinical application in the treatment of immunoinfertile men. The long‐term goal is to employ various recombinant fertilization antigens and/or their bioeffective peptide epitopes in a single formulation, to generate an anti‐sperm vaccine that will be strongly immunogenic and highly efficacious for regulation of fertility.
Ly‐49D Transfected NK Cells Show Reduced Interleukin‐10 Production in Response to H‐2d and Increased Lytic Activity: Implication by Interaction Using Class I MHC Alloantigen+ Target Cells in Pregnancy
Authors: NIESS JAN H. , MARKERT UDO R. , MANUEL JUSTIN , CLARK DAVID A.
Published: December 2003

PROBLEM: Mating of CBA/J (H‐2k) with DBA/2
(H‐2d) males leads to a high rate of spontaneous resorption (about 40%), which is not seen in other mating combinations, such as CBA/J X BALB/c. The activation of natural killer cells (NK cells) seems to be a key mechanism for the maternal–fetal intolerance in allogeneic pregnancy, and recurrent spontaneous abortion. The effect of expression of the NK cell activating receptor Ly49D recognizing BALB/c or DBA/2 class I MHC was investigated.  METHOD OF STUDY: Intracellular interleukin (IL)‐10 production was detected and target cell survival rates were calculated after 22 hr coincubation of rat NK cells transfected, or not, with a murine Ly49D receptor, with either male BALB/c or male DBA/2 splenocytes, by using flow cytometry.  RESULTS: Ly49D negative rat NK cells produced 13.7% more IL‐10 than Ly49D positive rat NK cells, and more splenocytes were killed by Ly49D transfected rat NK cells (survival rate 2.45%) than by Ly49D negative rat NK cells (survival rate 4.36%).  CONCLUSION: After physiological stimulation with BALB/c or DBA/2 splenocytes, rat NK cells are able to synthesize IL‐10. Recognition of mouse splenocyte major histocompatibility complex (MHC) by Ly49D mice receptor decreased IL‐10 production. The observed increase in killing activity might be a result of this phenomenon. NK cell activation via the Ly49D receptor might play an important role in pregnancy failure, but cannot explain why CBA/J X DBA/2 matings are abortion prone, and CBA/J X BALB/c matings are abortion resistant.
Painting Qa‐2 onto Ped slow Preimplantation Embryos Increases the Rate of Cleavage
Authors: McELHINNY ABIGAIL S. , EXLEY GINGER E. , WARNER CAROL M.
Published: December 2003

PROBLEM: Qa‐2 protein, the Ped gene product, is linked to the cell surface by a glycosylphosphatidylinositol (GPI) anchor. Some GPI‐linked proteins can be spontaneously incorporated into the membranes of cells via a technique called “protein painting.” We investigated whether Qa‐2 could be painted onto T cells and embryos and whether the painted protein would be functional.  METHOD OF STUDY: Incorporation of Qa‐2 into the membranes of T cells and embryos was measured by FACScan and Immuno‐PCR, respectively. Function of Qa‐2 was measured by cell proliferation.  RESULTS: Qa‐2 was incorporated by T cells and embryos and was functional.  CONCLUSION: GPI‐linked Qa‐2 protein “painted” onto both T cells and preimplantation embryos is functional, as shown by increased proliferation of T cells after cross‐linking with anti‐Qa‐2 antibody, and increased rate of cleavage division of the embryos.
Blocking Effect of Antisera to Recombinant Zona Pellucida Proteins (r‐ZPA) on In Vitro Fertilization
Authors: HASEGAWA AKIKO , KOYAMA KOJI , TSUBAMOTO HIROSHI , HAMADA YUKARI , KOYAMA KOJI
Published: December 2003

PROBLEM: The zona pellucida is a good target antigen for contraceptive vaccines due to its strong immunogenicity and high tissue specificity. However, this contraceptive effect is inevitably associated with ovarian failure. Therefore, it is necessary to define an epitope of the zona antigen to which the antibody produced inhibits fertilization without any undesirable side effects.  METHOD OF STUDY: The DNA fragment coding for the NH2‐terminal
region of porcine zona pellucida proteins (ZPA) (1–198 amino acids)
and human ZPA (1–206 amino acids) was prepared to produce
recombinant porcine ZPA (r‐ZPA), r‐pZPA1–198 and
r‐hZPA1–206. Using Freund's complete adjuvant,
antisera against these proteins were raised in rabbits. 
RESULTS: The resultant antisera to r‐pZPA1–198 and
r‐hZPA1–206 were cross‐reacted with each other on
enzyme‐linked immunosorbent assay and immunofluorescent staining. The
antiserum to r‐pZPA1–198 inhibited in vitro
fertilization in pigs, but not human sperm binding to the zona pellucida,
while the antiserum to r‐hZPA1–206 inhibited the human
sperm‐binding assay. 
CONCLUSIONS: Antiserum to r‐ZPA inhibited fertilization in the animal
species possessing a homologous amino‐acid sequence as an immunogen. The
recombinant protein, r‐hZPA1–206 seems to be a feasible candidate for the development of contraceptive vaccines for humans.
Effects of LPS and IL‐6 on Oxytocin Receptor in Non‐Pregnant and Pregnant Rat Uterus
Authors: FANG XIN , WONG SUSAN , MITCHELL BRYAN F.
Published: December 2003

PROBLEM: Little is known regarding the regulation of the timing of parturition. Recent evidence suggests an interaction between the immune system and uterine contractility in late gestation.  METHOD: Pregnant rats were treated with LPS in vivo in attempts to
establish a model of premature parturition induced by the
pro‐inflammatory response. Uterine explants were incubated in vitro to determine the effects of IL‐6 on uterine synthesis of oxytocin (OT) and its receptor (OTR).  RESULTS: LPS injection was quite toxic to pregnant rats and gave extremely variable results. In animals that delivered, there was a marked increase in the uterine concentrations of OTR and OTR mRNA. There was no consistent effect regarding the timing of parturition. IL‐6 caused a significant increase in the concentration of OTR mRNA in uterine explants from pregnant rats but not in tissues from non‐pregnant animals.  CONCLUSION: Rat uterine concentrations of OTR are regulated by IL‐6. Pro‐inflammatory cytokines may stimulate uterine contractility in late gestation rat uterine tissues through a mechanism involving stimulation of OTR.
Infection of Polarized Primary Epithelial Cells from Rat Uterus with Chlamydia trachomatis: Cell–Cell Interaction and Cytokine Secretion
Authors: KAUSHIC CHARU , GRANT KATIE , CRANE MARDI , WIRA CHARLES R.
Published: December 2003

PROBLEM: The objective of this study was to examine the susceptibility of rat uterine epithelial cells (UEC) to infection with Chlamydia
trachomatis and to study the epithelial–stromal interactions
following infection. 
METHOD OF STUDY: UEC were isolated from adult rats and grown in culture.
Polarized, confluent monolayers of UEC were infected with 106
IFU/well C. trachomatis (MoPn). In order to confirm infection,
MoPn was labeled with a fluorescent tracking dye, PKH‐26, and then used
in epithelial cell infections. Transepithelial resistances were measured
prior to and following infection to test the effect of Chlamydia
on the integrity of the epithelial monolayers. In other experiments,
polarized epithelial cultures were infected in the presence and absence
of stromal cells. Media was collected from the apical and basolateral
compartments of the cultures before and after infection and analyzed for
cytokines IL‐1α and TNF‐α. 
RESULTS: Epithelial cell cultures infected with PKH‐26 labeled MoPn were
examined 4–5 days later. Bacterial inclusions were detected inside
epithelial cells indicating infection had occurred. Co‐localization of
PKH‐26 labeled bacteria with FITC‐labelled anti‐Chlamydia antibody
on the epithelial cells confirmed infection. No changes were found in
resistance across the monolayers of epithelial cells in the presence or
absence of infection. ELISA results indicate that UEC secrete IL‐1α
constitutively in vitro. Stromal cells secrete very little
IL‐1α. When stromal cells were co‐incubated with epithelial cells
there was a decrease in the amount of IL‐1α secreted by epithelial
cells 48 hr post‐infection. On the other hand, maximum TNF‐α was
found in stromal cells, both with and without infection. Epithelial
cells, in these studies made very little TNF‐α. 
CONCLUSIONS: These results show that primary rat epithelial cells can be
infected with Chlamydia in vitro. Epithelial and stromal cells
from uteri of adult rats make IL‐1α and TNF‐αin vitro
both prior to and following infection with Chlamydia. This system can be used to analyze the role played by epithelial–stromal interactions in providing protection on this mucosal surface.
Alteration of Cytokine Production in Follicular Cystic Ovaries Induced in Mice by Neonatal Estradiol Injection
Authors: DESHPANDE ROHINI R. , CHAPMAN JOHN C. , MICHAEL SANDRA D. , DESHPANDE ROHINI R. , CHANG MING‐DER Y.
Published: December 2003

PROBLEM: Neonatal estradiol injections in mice lead to follicular cystic ovaries that are similar to ovaries in patients with polycystic ovarian syndrome (PCOS). The present study examined ovarian cytokine production following neonatal estradiol injection.  METHOD OF STUDY: Female (C3H/HeJ×129/HeJ)F1 mice were injected daily with 20 μg 17β‐estradiol from 0–3 days postpartum. At intervals, animals were sacrificed to determine ovarian architecture, circulating levels of estradiol, ovarian and peritoneal machrophage cytokine production, and ovarian P450 aromatase enzyme mRNA levels.  RESULTS: Similar to PCOS, our results show that neonatally estradiol‐injected mice have lower levels of circulating estrogen that are correlated with decreased mRNA levels of P450 aromatase enzyme. Our data also show that follicular cystic ovaries have increased tumor necrosis factor (TNF)‐α and interleukin (IL)‐6 production. This increase in TNF‐α and IL‐6 production is also observed in peritoneal macrophages of estradiol‐injected mice.  CONCLUSION: The present study showed that neonatal estrogen injection in mice has an overall systemic effect on cytokine production. We speculate that increased cytokine production may alter certain important steps in follicular maturation, ultimately contributing to ovarian dysfunction.
Autoimmunity in Endometriosis: Relevance to Infertility
Authors: MATHUR SUBBI P.
Published: December 2003

PROBLEM: Endometriosis is a complicated multi‐factorial disease. Genetic and immunologic factors play a key role in its pathophysiology. We were the first to describe endometrial and ovarian autoimmunity in women with endometriosis in 1981.

METHODS OF STUDY: ELISA and Western blot analysis. It is accepted that women with endometriosis have autoimmunity to endometrial, ovarian and nuclear antigens. We have now established that:

The endometrial autoantigens to which the endometriosis patients have autoimmune reactions are endometrial transferrin and alpha 2‐HS glycoprotein.

Levels of antibodies to both these proteins are specifically elevated in women with endometriosis, thus making them important candidates for developing an endometrial antibody assay for a non‐invasive diagnosis of endometriosis.

Levels of transferrin and alpha 2‐HS glycoprotein are significantly elevated in the peritoneal fluid of women with endometriosis.

Antibodies to transferrin and alpha 2‐HS glycoprotein also inhibit in vitro sperm motility.

CONCLUSION: These data strongly suggest the possibility of endometrial autoantibodies playing an important role in the infertility often associated with endometriosis.
A Method for the Dispersal and Characterization of Leukocytes from the Human Female Reproductive Tract
Authors: WHITE HILLARY D. , PRABHALA RAO H. , HUMPHREY SHIRLEY L. , CRASSI KAREN M. , RICHARDSON JAN M. , WIRA CHARLES R.
Published: December 2003

PROBLEM: The isolation of human female reproductive tract (RT) cells that maintain viability and are representative of the entire population is essential for a thorough evaluation of mucosal immunity in the reproductive tract mucosa. Here, we describe the isolation of RT cells in high yields and with high viability from the Fallopian tube, uterine endometrium, endocervix, ectocervix and vagina.  METHOD OF STUDY: This cell dispersion method uses an enzyme cocktail composed of pancreatin, hyaluronidase, and collagenase (PHC), and employs a 250‐μm mesh screen to facilitate cell dispersion.  RESULTS: The yields of cells isolated per gram of tissue in the presence of this PHC cocktail were compared and found to be strikingly higher relative to the yields obtained with other enzyme cocktails or in the absence of enzymes. Flow cytometry was used to characterize leukocyte subsets isolated from uterine endometrium in the presence of the various enzyme cocktails. The common leukocyte antigen marker CD45, pan T‐cell marker CD3, monocyte/macrophage marker CD14 and B‐cell marker CD19 were retained after exposure to the PHC cocktail of enzymes. The expression of CD8 and CD4 was lost after exposure to added enzymes but regained after culture overnight.  CONCLUSION: These studies demonstrate the feasibility of using enzymatic digestion for the isolation of whole populations of Fallopian tube, endometrial, cervical and vaginal cells, including leukocyte subsets in high yields, and provide a foundation for investigating mucosal immune cell function in the human female RT.
Tumor Stroma as the Main Source of Inhibin Production in Ovarian Epithelial Tumors
Authors: ZHENG WENXIN , LU JEAN J. , LUO FENG , HSIEH JOHN , ZHANG CHUNYING , CHANG LILLY , CHO MICHAEL M. , STANCZYK FRANK Z. , ZHENG WENXIN , WANG CHUN‐YEH
Published: December 2003

PROBLEM: Elevated serum inhibin levels have been found in ovarian cancer patients; however, the source of the elevated inhibin is uncertain. Previous studies of activin in human ovarian cancer suggest that activin may promote the growth of the tumor. The aims of this study were to examine the source of elevated inhibin from ovarian epithelial tumors (OETs) and to preliminarily investigate the role of the gonadotropin–inhibin/activin relationship in the development of OET.  METHOD OF STUDY: The protein and mRNA expression of α and βA subunits of inhibin/activin were examined by immunohistochemistry (IHC) and reverse transcription polymerase chain reaction (RT‐PCR) in 120 OETs, including 30 benign cystadenomas, 30 borderline tumors, and 60 carcinomas. Stromal and epithelial cells were microdissected from 23 OETs to further examine the expression of α and βA subunits by RT‐PCR. Dimeric inhibin A and activin A production were measured by using the two‐site ELISA from three OET cell lines in culture under treatment of follicle‐stimulating hormone (FSH) and luteinizing hormone (LH).  RESULTS: βA subunit was expressed in the epithelial component of 100% of the cystadenomas, in 80% of borderline tumors, and in 75% of the carcinomas, but not in tumor stroma. Inhibin α expression was not found in the epithelium of all OETs studied, but focal inhibin α immunoreactivity was seen in the tumor stroma (mainly luteinized stromal cells) in the majority of cases. Dimeric activin A was produced by all of the three OET cell lines with a 1.5–1.9‐fold increment after FSH stimulation. However, activin A production was not augmented by LH treatment. No inhibin A was produced by the three OET cell lines with or without gonadotropin stimulation.  CONCLUSIONS: The stroma of OET is the major source in the production of inhibin α (monomer). Dimeric inhibin A production may be the result of combined efforts of the tumor stroma (α subunit) and epithelium (βA subunit). Cellular, compartmental expression of inhibin and activin subunits may play a role in the development of OET, although the mechanism remains undefined. The unopposed activin A production stimulated by FSH in OET cell lines suggests that activin production may represent one of the cellular mechanisms of growth promotion by FSH.
Up‐Regulation of Epidermal Growth Factor‐Receptors (EGF‐R) by Nicotine in Cervical Cancer Cell Lines: This Effect May Be Mediated by EGF
Authors: MATHUR RAJESH S. , MATHUR SUBBI P. , YOUNG ROGER C.
Published: December 2003

PROBLEM: Over‐expression of epidermal growth factor‐receptors (EGF‐R) has been described in a variety of cancers, including cervical cancer. Nicotine may increase cellular proliferation rates through a mechanism involving EGF or EGF‐R. In this study, we ascertain the effect of EGF antibodies on nicotine‐enhanced proliferation rates in two cervical cancer cell lines.  METHOD OF STUDY: We studied (a) nicotine‐induced increase in the cellular expression of EGF‐R in human papillomavirus (HPV)‐positive ME‐180 and HPV‐negative HT‐3 cervical cancer cell line cultures, using a semi‐quantitative immunofluorescent antibody assay; (b) alterations in cellular proliferation in association with changes in EGF‐R levels; and (c) the EGF‐R mediation by EGF.  RESULTS: Nicotine exposure at physiologically attainable plasma concentrations caused increased expression of EGF‐R in both cervical cancer cell lines. Up‐regulation of EGF‐R was associated with increased cellular proliferation. Decreased expression of EGF‐R was associated with decreased cellular proliferation. These data were consistent with EGF‐R expression as a mechanism for the control of proliferation of the cervical cancer cells. The action of nicotine was abrogated when antibodies to EGF were added, implying that nicotine up‐regulation of EGF‐R may be mediated by EGF.  CONCLUSIONS: Our data show that nicotine‐induced proliferation of cervical cancer cells is mediated through EGF‐R over‐expression and that this action of nicotine utilizes EGF.
Clinical Experience with IVIg Rx in Patients with Prior Failed IVF Pregnancies: Report of 30 Consecutive Patients
Authors: SCHER J. , SALAZAR C.
Published: December 2003

PROBLEM: This study reviews one practitioner's experience with intravenous immunoglobulin (IVIg) therapy in the in‐vitro fertilization (IVF) cycles of 30 patients with previous IVF failures.  METHOD OF STUDY: Thirty patients had undergone 82 prior assisted reproductive technology (ART) cycles (mean 3.9±2 failed ART cycles, median 3.0, range 1–8) yielding one term birth, one loss at 22.5 weeks, and five chemical pregnancies. These patients underwent comprehensive clinical and laboratory evaluation, including immunologic workup, and were accepted for IVIg therapy in their next IVF cycle.  RESULTS: A total of 40 cycles were treated. Twenty‐four (60%) of the IVIg‐treated IVF cycles showed a positive human chorionic gonadotropin test. Comparing the IVIg cycles to the untreated ART cycles, there were no differences in the number of embryos transferred, fertilized embryos, or eggs. Eighty‐six percent of the cases with confirmed implantation delivered; there was one chemical pregnancy, one 20‐week spontaneous fetal death, and one trisomy. Five (24%) of the 21 pregnant patients delivered at 30–36 weeks. The remaining 13 delivered at term. Only three (11%) had no positive immune test.  CONCLUSION: In what may be a selected population of IVF patients (with high incidence of abnormal immune testing), early IVIg therapy may be associated with the improved success of IVF, and the high rate of live birth.
NIH Funding and Career Development Opportunities for Women in Reproductive Immunology
Authors: VOGEL DONNA L.
Published: December 2003

The National Institutes of Health (NIH) has a number of mechanisms available to support research, research training and research career development. The various institutes, centers and special offices within the NIH have, as part of their missions, a diverse array of basic and clinical research goals. Much of the scientific spectrum of reproductive immunology, however, falls within the mission of the National Institute of Child Health and Human Development (NICHD). There have been a number of recent innovations in the preparation, review and funding of NIH grant applications. Specific opportunities, such as fellowships, career awards, and new investigator status, are available for investigators at different career stages. A wealth of information is available on the NIH website at www.nih.gov. NIH staff are available to answer your questions and to provide administrative guidance in preparing grant applications.
Is the Paternal Mononuclear Cells’ Immunization a Successful Treatment for Recurrent Spontaneous Abortion?
Authors: RAMHORST R. , AGRIELLO E. , ZITTERMANN S. , PANDO M. , LARRIBA J. , FAINBOIM L. , IRIGOYEN M. , ETCHEPAREBORDA J.J. , CONTRERAS ORTIZ C. , CORTELEZZI M. , AUGE L. , LOMBARDI E.
Published: December 2003

PROBLEM: Alloimmunization as a treatment for recurrent spontaneous abortion (RSA) is still controversial due to the lack of enough controls to evaluate its effectiveness. The present study was conducted to compare the live birth rate in the presence or absence of immunotherapy.  METHOD OF STUDY: Ninety‐two women with RSA (79 primary [PA] and 13 secondary aborters[SA]) received immunotherapy. Thirty‐seven RSA couples not receiving paternal alloimmunization, constituted the “control” group.  RESULTS: The pregnancy rate in alloimmunized was 58 vs 46% in the control group. The live birth increased from 71% in the controls to 88% after immunotherapy. The alloimmunization induced mixed lymphocyte reaction blocking factors (MLR BFs) in 79% of women. However, they were also present in 83% of immunized women experiencing a new abortion.  CONCLUSION: These results indicate that alloimmunization may be useful in the treatment of RSA.
Anti‐DNA Antibodies Cross‐reacting with Laminin Inhibit Trophoblast Attachment and Migration: Implications for Recurrent Pregnancy Loss in SLE Patients
Authors: QURESHI FAISAL , YANG YAN , JACQUES SUZANNE M. , SCHUGER LUCIA , JOHNSON MARK P. , NAPARSTEK YAAKOV , ULMANSKY RINA
Published: December 2003

PROBLEM: Systemic lupus erythematosus (SLE), an autoimmune disease, is associated with reduced fetal survival, recurrent abortions, and other pregnancy complications. Some of the autoantibodies found in SLE bind to laminins (LNs), which play an important role in the implantation of the fertilized ovum in humans.  METHOD OF STUDY: To elucidate the role of these specific autoantibodies, chorionic villous explants from 6–7‐week‐old human placentas were established as organ cultures on laminin‐1 (LN‐1), collagen IV (CN‐IV) or uncoated culture dishes. The cultures were then exposed to a mouse monoclonal anti‐DNA/anti‐LN‐1 antibody, to human polyclonal lupus antibodies cross‐reacting with LN‐1, a function‐blocking polyclonal antibody to LN‐1, polyclonal antibodies to CN‐IV, or IgG control.  RESULTS: The explants attached to LN‐1 and CN‐IV, but not to uncoated culture dishes. LN‐1 promoted migration of trophoblast, whereas CN‐IV promoted migration of fibroblast‐like cells. Trophoblast attachment and migration were abolished in a dose‐dependent manner by all three antibodies to LN‐1, but not by antibodies to CN‐IV or IgG control. Furthermore, the effect of anti‐LN antibodies was abolished by preincubating them with LN‐1.  CONCLUSIONS: These studies suggest that anti‐DNA antibodies cross‐reacting with LNs may play a role in early pregnancy failure in SLE patients by interfering with placental implantation.
Changes in Cytokine Production During and After Normal Pregnancy
Authors: SHIMAOKA YUKI , HIDAKA YOH , TADA HISATO , AMINO NOBUYUKI , NAKAMURA TAKAFUMI , MURATA YUJI , MITSUDA NOBUAKI , MORIMOTO YASUHIKO
Published: December 2003

PROBLEM: The systemic T helper 1/T helper 2 (Th1/Th2) cytokine balance during normal human pregnancy is controversial, and observations about the balance in the postpartum period have only been reported for up to 3 months.  METHOD: Whole‐blood, from 83 healthy pregnant women, 80 healthy postpartum women, and 31 healthy non‐pregnant women was stimulated with phorbol 12‐myristate 13‐acetate (PMA) and ionomycin, and the levels of cytokines in the supernatant were measured by enzyme‐linked immunosorbent assay (ELISA).  RESULTS: The production of all measured cytokines decreased during pregnancy, especially in the second trimester. After delivery, interferon‐Γ (IFN‐Γ) and interleukin‐2 (IL‐2) increased from 2 to 11 months postpartum, and IL‐4 increased from 6 to 11 months postpartum.  CONCLUSIONS: These data indicate that 1) decreases in production of both Th1‐ and Th2‐type cytokines during pregnancy may be related to the pregnancy‐induced amelioration of autoimmune diseases; 2) increases in production of both Th1‐ and Th2‐type cytokines in the postpartum period may be related to the postpartum aggravation of autoimmune diseases.
Reconstitution of Humoral Immunity During Pregnancy
Authors: NARITA MITSUO , YAMADA SATOSHI , KIKUTA HIDEAKI , TOGASHI TAKEHIRO
Published: December 2003

BACKGROUND: Recently, increasing attention has been paid to hormonal regulations of the immune system.  MATERIALS AND METHODS: In this study, cord sera and the corresponding maternal sera were obtained at delivery. Sera from pregnant women were obtained at early, middle, and late stages of pregnancy. These sera were tested for titer and avidity of measles or mumps virus‐specific immunoglobulin G (IgG) by means of a single‐dilution, urea‐denaturation enzyme‐linked immunosorbent assay method.  RESULTS: A positive and significant correlation was found between the titer and avidity of the virus‐specific IgG, both in the cord sera and in the maternal sera. This correlation was established already at the early stage of pregnancy. There were no such correlations found in nonpregnant individuals.  CONCLUSIONS: This is the first observation in human subjects that the avidity and concentration of the virus‐specific IgG had a positive and significant correlation. Pregnancy must have some significant effects on the regulation of humoral immunity.
TNFα Expression in Hyperplastic Endometrium
Authors: ZUPI ERRICO , SBRACIA MARCO , MARCONI DANIELA , BRAZOLIN TIZIANO , ARDUINI DOMENICO , ROMANINI CARLO , ATZEI GIOVANNI
Published: December 2003

PROBLEM: Tumor necrosis factor‐alpha (TNFα) is produced by the endometrium, and it has been shown that this cytokine has cyclic variations through the menstrual cycle.  METHOD OF STUDY: In this study we assessed TNFα, estradiol and progesterone levels in the endometrium and serum of 21 patients with recent abnormal uterine bleeding (AUB). Eleven women showed histological diagnosis of endometrial hyperplasia, and ten women showed normal endometrium.  RESULTS: The TNFα value in the serum of patients with hyperplasia and in normal endometrium did not show significant differences (64.8±21.4 vs 77.6±14.8 pg). The amounts of TNFα released by hyperplastic endometrial tissue were significantly higher than in control patients (258.8±78.0 vs 140.6±79.8 pg/g;
P<0.001). Immunohistochemistry of hyperplastic endometria showed a
stronger staining than normal ones. Serum E2 levels in
patients with hyperplastic endometrium were 49.5±31.6 ng/mL, and
in controls were 107.3±66.0 ng/mL. The levels of estradiol
released by hyperplastic endometria were significantly lower than in
normal endometria (152.7±60.0 vs 279.9±178.7 ng/g;
P<0.003).  CONCLUSIONS: Hyperplastic endometria produce higher amounts of TNFα with respect to controls. These findings may explain the frequent bleeding observed in the patients with endometrial hyperplasia, since TNFα promotes apoptosis and endometrial shedding.
Immunoidentification of Gonadotropin Releasing Hormone Receptor in Human Sperm, Pituitary and Cancer Cells
Authors: LEE CHI‐YU GREGORY , HO JOSHUA , CHOW SONG‐NAN , YASOJIMA KOJI , SCHWAB CLAUDIA , MCGEER PATRICK L.
Published: December 2003

PROBLEM: To generate monoclonal antibodies specific to the receptor of gonadotropin releasing hormone (GnRH) for immunoidentification of the GnRH receptor (RGnRH) in human sperm, pituitary and cancer cells.  METHODS: Four oligopeptides corresponding to RGnRH amino acid residues 1–29, 182–193, 195‐206 and 293–306 in the extracellular domains were synthesized, conjugated to hemocyanin from Keyhole Limpet and used as immunogens to generate specific monoclonal antibodies. Enzyme‐linked immunosorbent assay was used for initial screening of hybridomas.  RESULTS: A total of 15 hybrid cell lines secreting RGnRH‐specific monoclonal antibodies were initially established. By using some of these monoclonal antibodies as immunohistochemical probes, RGnRH was localized in the acrosomal region of human sperm, in the anterior pituitary tissue and in cancer cells of human ovarian and cervical origins. RGnRH was shown to have a molecular size of 45,000±5,000 kDa by Western blot assay. Expression of RGnRH mRNA in several human tissues and cancer cells was established by the reverse transcriptase–polymerase chain reaction method.  CONCLUSION: RGnRH‐specific monoclonal antibodies may be a valuable tool of identifying the presence of RGnRH in various normal and malignant human tissues.
Corticosterone Treatment of Pregnant Low Dose Endotoxin‐Treated Rats: Inhibition of the Inflammatory Response
Authors: FAAS M.M. , SLOT K. , KOITER T.R. , SCHUILING G.A.
Published: December 2003

PROBLEM: Can the endotoxin‐induced inflammatory response, underlying experimental pre‐eclampsia, in pregnant rats be inhibited by corticosterone?  METHOD OF STUDY: On day 10 of pregnancy, rats were implanted with pellets containing 25% corticosterone and 75% cholesterol (n=10) or with
100% cholesterol‐pellets (n=10). On day 14 of pregnancy, rats were infused with either endotoxin (1.0 μg/kg bw) or saline. Three days later, they were sacrificed. Cryostat kidney sections were immunohistologically stained for the presence of neutrophils (PMN) and monocytes (M∅) and the expression of inflammation‐associated adhesion molecules.  RESULTS: In cholesterol‐treated rats, endotoxin significantly increased glomerular numbers of PMN and M∅, glomerular expression of ICAM‐1 and VCAM‐1 and glomerular numbers of LFA‐1 and VLA‐4‐positive cells as compared with saline. Corticosterone treatment significantly inhibited glomerular infiltration of PMN, M∅ and LFA‐1 positive cells after endotoxin infusion. It did not affect glomerular ICAM‐1 or VCAM‐1 expression or numbers of VLA‐4 positive cells.  CONCLUSIONS: It is concluded that pre‐treatment with corticosterone inhibits the low dose endotoxin‐induced glomerular inflammatory reaction in pregnant rats, most likely by inhibiting LFA‐1 expression, thereby decreasing the adhesiveness of inflammatory cells for activated endothelial cells.
Immunocontraceptive Efficacy of Synthetic Peptides Corresponding to Major Antigenic Determinants of Chicken Riboflavin Carrier Protein in the Female Rats
Authors: SUBRAMANIAN SARADA , RADHAKANTHA ADIGA P. , KARANDE ANJALI A.
Published: December 2003

PROBLEM: Earlier studies have demonstrated that antibodies directed towards the N‐terminal (residues 10–17) and C‐terminal (residues 200–207) regions on chicken riboflavin carrier protein (RCP; 219 AA) are effective in pregnancy termination in rodents and sub‐human primates. In the present study, the immunocontraceptive potential of three additional immunodominant sequences comprising of residues 33–49, 64–83 and 130–147 (CYA, CED and CGE peptides, respectively) of chicken RCP was investigated.  METHOD OF STUDY: The three antigenic peptides were synthesized by using Fmoc chemistry. Oligoclonal antibodies were generated in rabbits. Bioneutralizing capacity of these peptides was assessed by passive and active immunoneutralization studies.  RESULTS: All the three peptides‐specific antisera recognized their cognate epitopes on native RCP. When the affinity purified peptide IgG were administered on three consecutive days to pregnant rats (on days 10, 11 and 12), it was observed that the rats injected with CED and CGE‐IgG failed to deliver any pups whereas the animals which received CYA IgG delivered normal pups. Active immunization of fertile female rats with CED or CGE peptide conferred protection from pregnancy.  CONCLUSIONS: These results demonstrate the presence of two additional stretches in chicken RCP which can serve as mini‐vaccines.
Effect of Hepatocyte Growth Factor on Sperm Motility
Authors: KITAMURA MASAYA , MATSUMIYA KIYOMI , NISHIMURA KENZI , YAMANAKA MASAKI , OKUYAMA AKIHIKO , MATSUMOTO KUNIO
Published: December 2003

PROBLEM: Hepatocyte growth factor (HGF) exists abundantly in seminal plasma and its receptor, c‐met, is expressed on spermatozoa. Considering its motogenic activity, we speculated that HGF might affect the movement ability of spermatozoa.  METHODS: Recombinant HGF was added to washed spermatozoa and their movements were analyzed using a computer‐assisted sperm analyzer. The concentration of HGF in the seminal plasma of infertile patients (n =83) was measured by ELISA, and the data were compared with their hormonal profile and semen parameters.  RESULTS: The HGF physiological concentration (1 ng/mL) maintained the motility of sperm after a long incubation, though the difference was not statistically significant. Recombinant HGF did not affect the linearity or frequency of movement, which suggested that it does not evoke the hyperactivation of spermatozoa. The concentration of HGF in seminal plasma did not correlate with any clinical parameter of the patients.  CONCLUSIONS: These findings contradict the theory that HGF controls the movement of sperm. The main role of this axis in the male reproductive system might be maturation in the epididymis.
Immunobiologic Implication of RANTES in Seminal Plasma of Fertile, Infertile and Immunoinfertile Men
Authors: NAZ RAJESH K. , LESLIE MONICA H.
Published: December 2003

PROBLEM: To investigate the presence of Regulated on Activation, Normal T Expressed and Secreted (RANTES) in the seminal plasma of fertile, infertile, and immunoinfertile men in order to determine whether it has any role in fertility/infertility and in the modulation of immunogenicity of the sperm cell.  METHOD OF STUDY: The enzyme‐linked immunosorbent assay was performed by using a commercially available immunoassay kit to measure RANTES levels in seminal plasma of fertile (n=28), male‐factor infertile
(n=35), and immunoinfertile (n=21) patients. The levels
were correlated with various seminal parameters. 
RESULTS: RANTES was detected in the seminal plasma of fertile,
male‐factor infertile, and immunoinfertile men. Its levels differed
significantly (P<0.05) between the fertile and immunoinfertile
groups, with the immunoinfertile group showing an overall decrease of
19.9% in mean values. When RANTES levels were correlated with sperm
concentration, total sperm number, and motility, only the total number of
the immunoinfertile group correlated (r=0.86) significantly
(P=0.03) with the RANTES levels, and none of the other seminal
parameters of any other group correlated
(r=−0.37–0.30) significantly (P>0.05).  CONCLUSIONS: The findings of this study indicate that RANTES may have a role in immunomodulation of antigenicity of sperm cells in the male genital tract before ejaculation, and perhaps also in the female genital tract after intercourse. The exact physiologic, pathopysiologic, and molecular mechanism(s) involved in these processes require further study.