bluuebunny/crossref_metadata_2025 huggingface dataset:
Experience with Allogeneic Leukocyte Immunization (Ai) for Implantation Failure in the In‐Vitro Fertilization Program
Authors: Kling Chrisitane , Magez‐Zunker Julia , Jenisch Stefan , Kabelitz Dietrich
Published: March 2003

Immunological mechanisms might contribute to the repeated implantation failure in the in‐vitro fertilization program. AI might be a valuable therapeutic option in this situation. Here we summarize our extensive experience with this immunotherapy. We conducted a retrospective analysis of all couples who were referred to our outpatients department from 1996 to 1998 for AI after two or more embryo transfers (ET). Six hundred and eighty‐six couples (89%) continued IVF/ICSI and were followed up for 2–3 years. On average patients entered AI after 3–4 IVF–ET at an age of 33 years and completed IVF/ICSI treatment after another 2 ET; 2.6 embryos per ET were transferred according to the German embryo protection law. The cumulative birth rate war 38% after IVF transfer and 42% when spontaneous pregnancies were included. Until the age of 36 years the rates of viable pregnancies and birth were improved by a quarter in the first 6 months as compared to the second half year (33.3% versus 26.3%, 26.0% versus 19.6%) and to the German IVF Registry of 1998. Couples who had undergone more than three ET before AI seemed to benefit most. Our data suggest that AI is a valuable adjuvant therapy with a temporary effect when 3–4 IVF/ICSI transfers have been unsuccessful. The underlying immunomodulatory mechanisms require further investigation.
Measurement of peripheral natural killer cells at the time of spontaneous abortions
Authors: Paparistidis N. , Chioti A. , Papadopoulou C. , Tsekoura C. , Genas V. , Graphou O. , Balafoutas C. , Alafaki‐Tzannatou C. , Varla‐Leftherioti M.
Published: March 2003

Increased peripheral NK cells in aborting women are considered as a predictor of immunological miscarriages. Here, we investigated the diagnostic value of the NK count at the time of abortion.

Peripheral NK cells were counted by flow cytometry in 44 women undergoing therapeutic termination of first trimester missed pregnancy (A = 24) or elective abortion (B = 20). Histology of the extracted material was performed and immunological lesions (villitis, intervillositis, vascular thrombosis, increased fibrinoid necrosis) were recorded. Twenty first trimester pregnant women (C1) and 20 nonpregnant women (C2) served as controls. In 20 women of groups A and B (A1 = 10, B2 = 10) count was repeated after 5 days.

At abortion time, the NK percentage and number did not differ between groups A and B (18.18%, 360 versus 15.61%, 374), but there was a statistically significant difference between A and C1 (18.18% versus 13.6%, P < 0.03). When histology negative (11) and positive (8) cases were excluded from groups A and B, respectively, NKs were slightly higher in group A (18.18–19.76%) and significantly lower in B (15.61–13.07%, P < 0.05). At the time of second count, aborters' NKs decreased (A1 = 16.64 versus 13.6 and 10.6 in groups C1 and C2 –P = 0.01, respectively). In group B, NK percentage was equal to that of nonpregnant women (10.94 versus 10.6).

At the time of abortions, NKs increase only in cases of immune‐mediated miscarriages. Their – in time – count may contribute to the diagnosis of immmunological abortions.
Extracellular matrix proteins dependent apoptosis of T Cells in women with a history of recurrent spontaneous abortion
Authors: Jerzak Magorzata , Górski Andrzej , Semczuk Marian , Kasprzycka Monika
Published: March 2003

INTRODUCTION:  The purpose of the study was to determine the role of T cell apoptosis in ECM environment in pregnancy maintenance in women with a history of recurrent spontaneous abortion (RSA).

MATERIALS AND METHODS:  Thirty nonpreg‐ nant women with the history of RSA and 10 normal healthy women with the previous successful pregnancy outcome were studied. In addition, 15 pregnant women with the history of RSA were studied. We studied apoptosis of peripheral blood T cells after culture with mAb OKT‐3 alone and with mAb OKT‐3 and the following ECM: fibronectin and collagen IV. We used Cell Death Detection ELISA. In addition, apoptotic peripheral blood T cells were identified by annexin V‐PE staining protocol using flow cytometry. CD29, CD95 T cells' surface receptors were studied.

RESULTS:  The highest values of enrichment factor: mU of the sample (dying/dead cells) per mU of the corresponding control (viable cells) were observed after peripheral T cells' culture with fibronectin or collagen IV in samples of women with the history of RSA. However, percentage of apoptotic T cells stained by annexin V was significantly lower in RSA women compared to control (P < 0.05). CD95 expression was significantly lower in RSA women compared to control (P < 0.05).

CONCLUSIONS:  Apoptosis of activated cytotoxic T cells might be an interesting possible explanation of successful pregnancy outcome in women with the history of RSA.
Immunogenetic determinants in recurrent miscarriage – evidence from genetic‐epidemiological research
Authors: Christiansen Ole B.
Published: March 2003

Immunological disturbances probably play a major role in the etiology of recurrent miscarriage (RM) – however, a detailed insight in the immunological processes going on is difficult because the most important interactions take place locally in the uterus during pregnancy at the feto–maternal interface. The study of the role of the impact of polymorphic immunogenetic determinants in RM has advantages compared to the study of humoral or cellular immunological factors locally or systemically: genetic polymorphisms can be determined unambiguously using DNA technology, polymorphisms associated with RM are expected to influence immunological processes also locally in the uterus and the finding of an increased prevalence of specific immunogenetic polymorphisms in RM cannot be deduced as a result of RM but may be a cause of the syndrome.

We have investigated polymorphisms in the genes coding for HLA‐DR, HLA‐DQ, HLA‐G and mannan‐binding lectin (MBL) in patients with RM and in controls and investigated the impact of RM‐associated polymorphisms on the outcome of future pregnancies. Furthermore, we have investigated the impact of the gender of the first child on future pregnancy outcome in patients with secondary RM.

The case–control studies and the prospective studies provide evidence that specific maternal class II HLA alleles or genotypes, specific combinations of HLA‐G alleles in the RM couples, genes coding for low MBL concentrations and male‐specific minor histocompatibility antigens (HY‐antigens) play a role in the pathogenesis of RM.
Psychoemotional stress and enodcrino‐immunological pathways during pregnancy
Authors: Arck P.C. , Joachim R. , Knackstedt M. , Douglas A. , Klapp B.F.
Published: March 2003

A wealth of information has been published on the effect of stress on the nervous, endocrine and immune systems during the past two decades. Further, stress is known to be abortogenic in murine and human pregnancies. It is logical to suspect that stress might cause abortion by altering the endocrine and/or immune system, and thus leading to a hampered or failing pregnancy maintenance. Lowered levels of progesterone did indeed occur as a result of stress and there was little change in cortisol or prolactin. Adequate levels of progesterone exert an antiabortive effect by induction of a Th2 biased immune response an by controlling NK activity. In stressed mice, increased levels of the abortogenic cytokine TNF‐α, IFN‐γ and IL‐12 were found in decidua in early pregnancy and this was associated with decreased levels of pregnancy‐protective TGF‐β2‐related suppressive activity in uterine decidua later in pregnancy. Interestingly, the progesterone derivative dydrogesterone abrogates stress triggered abortion by inducing a Th2 biased local immune response. In summary, the current study represents an important step towards bridging the longstanding assumption of vaguely defined negative effects of stress on pregnancy outcome with the mainstream of current endocrine‐immunological research by showing that stress may indeed influence pregnancy outcome along a putative ‘brain decidua axis’.
Parvovirus B19 – A common infectious agent causing problems during pregnancy
Authors: Modrow Susanne
Published: March 2003

Parvovirus B19 is the aetiological agent of one of the classical diseases of childhood, the rash illness fifth disease (erythema infectiosum). Beside this usually harmless illness parvovirus B19 was, however, found to be associated with a variety of severe diseases, mainly arthritis, anemia, thrombo‐ and granulocytopenia, hepatitis and myocarditis. In these cases parvovirus B19 may persist over long periods of time in the peripheral blood or in individual organs of the patients. During pregnancy B19‐infections may result in spontaneous abortions, hydrops fetalis, and intrauterine fetal death. The detection of viral DNA and antibodies in the maternal serum and precise ultrasound surveillance of the allows an early diagnosis of the development of hydropic symptoms in the fetus. In most cases in time intrauterine blood transfusions result in the birth of sound children. Since about half of the young adults are not immunologically protected parvovirus B19‐infections should be assumed as an important reason for virus‐correlated problems during pregnancy. Based on these epidemiological data that were accumulated during the past years clinicians should pay more attention to B19‐infections and the mode of viral transmission.
IL‐1 Activity is Expressed Differently During Pregnancy in the Rat Uterine Artery than in Aortic or Uterine Tissues
Authors: Huleihel Mahmoud , Leiberman Joseph R. , Yohay David , Holcberg Gershon , Katz Miriam , Mazor Moshe
Published: March 2003

PROBLEM: Uterine artery was shown to be unique in its capacity to change in size and function during pregnancy. As interleukin‐1 (IL‐1) was shown to be involved in reproduction processes, the aim of this study was to determine the levels of IL‐1 activity of the uterine artery tissue in pregnant rat.

METHOD OF STUDY: Nine virgins and nine midpregnant rats were selected. Both uterine arteries were obtained, together with reference tissues from aorta and uterus. The levels of IL‐1 were examined in the above tissues after culturing with media alone (control; CT), and media that contained stimulants like tumor necrosis factor‐alpha (TNF‐α) or lipopolysaccharide (LPS). IL‐1‐like activity was evaluated by its capacity to promote the culture growth of 1A‐5 and cytotoxic T lymphocyte derived (CTLD) cell lines. This activity was expressed as optical density (OD)/mg protein of the examined organ.

RESULTS: Uterine artery tissue, of pregnant rats, cultured in medium alone produced significantly higher levels of IL‐1 than uterine artery of virgin animals under the same conditions (16.2 S.E. 1.3 versus 0.6 S.E. 0.05 OD/mg protein, respectively; P < 0.02). Stimulation of uterine artery in vitro by LPS and TNF increased their capacity to secrete IL‐1. In comparison with uterine artery, aorta produced higher levels of IL‐1 in virgin rats compared with pregnant rats (13.6 S.E. 1.2 versus 1.6 S.E. 0.1; P < 0.02). Stimulation of aorta tissues (from both virgin and pregnant rats) with LPS, in vitro, significantly decreased their capacity to secrete IL‐1 (P < 0.04). Stimulation of aorta tissues from virgin rats with TNF‐α, in vitro, did not change their capacity to secrete IL‐1 activity. However, stimulation of aorta tissues from pregnant rats with TNF‐α decreased the secretion of bioactive IL‐1. The levels of IL‐1 produced by uterine tissues from virgin and pregnant rats were similar, and stimulation with either LPS or TNF‐α significantly decreased their capacity to secrete IL‐1 (P < 0.04).

CONCLUSIONS: The high level of IL‐1 activity detected during pregnancy in the uterine artery may suggest its unique involvement in the changes occurring throughout pregnancy in those blood vessels.
Uterine Chemokines in Reproductive Physiology and Pathology
Authors: KAYISLI UMIT A. , MAHUTTE NEAL G. , ARICI AYDIN
Published: March 2003

PROBLEM: Chemokines are increasingly recognized as important regulators of uterine function.

METHODS OF STUDY: The following is a review of uterine chemokines, especially monocyte chemotactic protein (MCP)‐1, interleukin (IL)‐8, and regulated‐upon‐activation normal‐T‐cell‐expressed and ‐secreted (RANTES) protein, in reproductive physiology and pathology.

RESULTS: It is increasingly clear that IL‐8, MCP‐1, RANTES and their receptors are produced by endometrial, myometrial, and trophoblast cell types in a timed and co‐ordinated manner. In addition to the regulation of leukocyte migration and function, uterine chemokines also display specific roles in endometrial angiogenesis, apoptosis, proliferation, and differentiation. IL‐8, MCP‐1 and RANTES are regulated by local growth factors and cytokines such as tumor necrosis factor‐α (TNF‐α), interferon‐γ, and IL‐1. IL‐8 takes part in cervical ripening and parturition. IL‐8, MCP‐1 and RANTES are also found at high levels in the peritoneal fluid of women with endometriosis.

CONCLUSION: Co‐ordination of chemokine–chemokine receptor interactions plays an important role in the menstrual cycle and successful pregnancy. Moreover, unbalanced chemokine expression contributes to pathologic conditions typified by uncontrolled cellular proliferation, migration and invasion.
Preeclampsia: A Multifactorial Disease Resulting from the Interaction of the Feto‐maternal HLA Genotype and HCMV Infection
Authors: CARREIRAS M. , MONTAGNANI S. , LAYRISSE Z.
Published: March 2003

PROBLEM: To clarify the possible influence of human leukocyte antigen (HLA) mother–child genotypes and human cytomegalo virus (HCMV) presence on the development of preeclampsia.

METHODS OF STUDY: One hundred and four DNA samples from mothers with preeclampsia, mothers with a normal history of pregnancies and their neonates were tested by polymerase chain reaction–sequence specific oligonucleotide probes (PCR–SSOP) for HLA‐A, ‐G, ‐DRB1, ‐DQA1, ‐DQB1 alleles. The HCMV sequences were analyzed using a PCR–SSOP method and the four primers described by Chou (Chou S: J Clin Microb 1992; 30:2307–2310).

RESULTS: Compared with their respective controls, a significant increase of DRB1*07 among neonates (Pc=0.05) and of DRB1*07 and/or DRB1*06 among pre‐eclamptic mothers (Pc=0.003, RR=8,5) was found. When HCMV sequences were detected in pre‐eclamptic mothers carrying those phenotypes the RR increased up to 40. Furthermore, the fetal inheritance of a maternal HLA‐G*0104 increased the risk for the appearance of the disease (RR=30; P=0.025).

CONCLUSION: The results suggest that the presence of alleles HLA‐G*0104, DRB1*07/06, HCMV sequences and the fetal inheritance of maternal G*0104, should be considered as conditioning factors for the development of preeclampsia.
Expression of Intracellular Th1 and Th2 Cytokines in Women with Recurrent Spontaneous Abortion, Implantation Failures after IVF/ET or Normal Pregnancy
Authors: NG SIU CHUI , GILMAN‐SACHS ALICE , THAKER POONAM , BEAMAN KENNETH D. , BEER ALAN E. , KWAK‐KIM JOANNE
Published: March 2003

PROBLEM: We aimed to investigate absolute counts of intracellular T helper 1 (Th1) and Th2 cytokine expressing T‐cell subpopulations in women with three or more recurrent spontaneous abortions (RSA), multiple implantation failures after in‐vitro fertilization and embryo transfer (IVF/ET) (three or more) or during normal pregnancy.

METHOD OF STUDY: Absolute cell counts and percentages of CD3+, CD3+/CD4+, and CD3+/CD8+ T‐cell populations expressing intracellular cytokines [interferon‐gamma (IFN‐γ), tumor necrosis factor‐alpha (TNF‐α), interleukin (IL)‐4 and IL‐10] was studied by four‐color flow cytometry in 15 RSA and 13 implantation failure patients. Eighteen fertile non‐pregnant and 47 normal pregnant women were also compared with regard to intracellular cytokine expression.

RESULTS: Interleukin‐10 producing CD3+/CD8+ T‐cell counts were significantly lower in women with RSA (P < 0.05) and implantation failures (P < 0.05), and TNF‐α producing CD3+/CD4+ T‐cell counts were higher in women with RSA (P < 0.05) and implantation failures (P < 0.005) than those of non‐pregnant fertile controls. During normal pregnancies, first trimester IL‐4 expressing CD3+, CD3+/CD4+ T‐cell counts (P < 0.05) and IFN‐γ expressing CD3+ T‐cell counts (P < 0.05) were significantly higher than those of third trimester (P < 0.05). First trimester TNF‐α expressing CD3+/CD8+ T‐cell counts were significantly higher than those of second and third trimester women (P < 0.05). However, there are no differences in cytokine expression between non‐pregnant and first trimester pregnant women.

CONCLUSION: Absolute counts of IFN‐γ, IL‐4, and TNF‐α expressing T cells decrease with the progress of gestation (third trimester) during normal pregnancies. In women with implantation failures, absolute cell counts of TNF‐α expressing CD3+/4+ cells reflects the presence of dominant Th1 immune response. A significantly increased Th1 cytokine expression may be the underlying immune etiology for reproductive failures.
Heat Shock Fusion Protein gp96‐Ig Mediates Strong CD8 CTL Expansion in vivo
Authors: ŠTRBO NATAŠA , YAMAZAKI KOICHI , LEE KELVIN , RUKAVINA DANIEL , PODACK ECKHARD R.
Published: March 2003

Štrbo N, Yamazaki K, Lee K, Rukavina D, Podack ER. Heat shock fusion protein gp96‐Ig mediates strong CD8 CTL expansion
in vivo.
AJRI 2002; 48:220–225 © Blackwell Munksgaard, 2002

PROBLEM: As shown previously, gp96‐Ig peptide complexes secreted by an ovalbumin transfected tumor (EG7) mediate strong, specific tumor immunity through a CD4 T cell independent CD8+ CTL response. In this study, we set out to develop a system to quantitatively determine the CD8 CTL response to gp96‐Ig and to evaluate the influence of an established wild type tumor.

METHODS: Secreted heat shock protein gp96‐Ig was constructed by replacement of the endoplasmic reticulum retention signal with the Fc portion of IgG1, transfected into EG7 (EG7‐gp96‐Ig) and used to induce CD8+ CTL expansion in vivo. Adoptively transferred, ovalbumin specific T‐cell receptor (TCR) transgenic CD8+ cells (OT‐1) responded with clonal expansion to the immunization with EG7‐gp96‐Ig. OT‐1 expansion was quantitated with Kb‐peptide‐tetramers by flow cytometry.

RESULTS: In response to primary immunization with EG7‐gp96‐Ig, OT‐1 expand from an initial frequency of 0.5 to 25% of all CD8 cells, and to 50% of all CD8 cells after a booster immunization. Endogenous ovalbumin specific CD8 cells also expand strongly. Antigen specific effector function was measured by enzyme‐linked immunosorbent spot‐forming cell assay (ELISPOT) for interferon‐γ (IFN‐γ). While effector function was strongly induced by secreted gp96‐Ig, not all expanded OT‐1 produce IFN‐γ. EG7 does not cause OT‐1 expansion, but rather induces anergy. If OT‐1 are transferred into wild type EG7 tumor bearing mice to induce anergy of OT‐1, immunization with EG7‐gp96‐Ig can partly overcome unresponsiveness.

CONCLUSIONS: We conclude that secreted gp96‐Ig is a powerful mediator of specific CD8+ CTL responses in vivo. Secretory gp96 mimics release of gp96 by damaged or necrotic cells that is able to activate dendritic cells without CD4 help. Gp96‐Ig associated peptides have not been selected by binding to major histocompatibility complex (MHC). Specific immunization by secreted gp96‐Ig therefore is expected to occur also in allogeneic settings.
Distinct Pathways for Constitutive Endocytosis of Fully Conformed and Non‐conformed Ld Molecules
Authors: MAHMUTEFENDIĆ HANA , KUČIĆ NATALIA , LUČIN PERO
Published: March 2003

PROBLEM: To characterize the constitutive internalization of major histocompatibility complex (MHC) class I molecules, we have studied the expression of completely conformed (full) and unconformed (empty) Ld molecules on non‐polarized murine P815 cells.

METHODS OF STUDY: Spontaneous endocytosis of Ld molecules was induced by cycloheximide, an inhibitor of protein synthesis, and their disappearance from the cell surface was determined by flow cytometry. In order to investigate the mechanism of internalization, a palette of inhibitors of endocytosis and vesicular transport was used.

RESULTS: Inhibitors of clathrine endocytosis did not influence the internalization of Ld molecules. Inhibitors of caveolar endocytosis and inhibitors of endolysosomal degradation prevented down‐regulation of empty, but not of full Ld molecules.

CONCLUSIONS: Empty Ld molecules are internalized mostly by caveolar endocytosis and full Ld molecules use a different pathway, neither clathrine‐mediated nor caveolar. After internalization, full Ld molecules are probably degraded and empty Ld molecules recycle between endosomal compartment and the cell surface before they enter into the degradation compartment.
Questioning the Th1/Th2 Paradigm in Reproduction: Peripheral Levels of IL‐12 are Down‐Regulated in Miscarriage Patients
Authors: ZENCLUSSEN ANA C. , FEST STEFAN , BUSSE PETRA , JOACHIM RICARDA , KLAPP BURGHARD F. , ARCK PETRA C.
Published: March 2003

Zenclussen AC, Fest S, Busse P, Joachim R, Klapp BF, Arck PC. Questioning the Th1/Th2 paradigm in reproduction: peripheral levels of IL‐12 are down‐regulated in miscarriage patients. AJRI 2002; 48:245–251 © Blackwell Munksgaard, 2002

PROBLEM: It has been postulated that a T helper (Th)1 response is associated with pregnancy failure, whereas a Th2 response contributes to pregnancy maintenance. However, this Th1/Th2 dichotomy has recently been hypothesized to be an oversimplification. To prove this novel hypothesis, we investigated the levels of the Th1‐inducer cytokine interleukin (IL)‐12 in immunocompetent cells of patients with normal pregnancies (NP) and spontaneous abortion (SA).

METHODS: Presence of intracellular IL‐12 was evaluated in CD8+ and CD56+ blood and decidual lymphocytes as well as in monocytes and granulocytes by flow cytometry from NP and SA individuals. IL‐12 serum levels were measured by enzyme‐linked immunosorbent assay (ELISA). We further investigated the effect of recombinant human (rh) IL‐12 on the production of interferon (IFN)‐γ and tumor necrosis factor (TNF)‐α in peripheral leukocytes ex vivo.

RESULTS: In patients suffering from SA we observed lower percentages of IL‐12 in lymphocytes, monocytes and granulocytes derived from peripheral blood and decidua, compared with women with normally progressing pregnancies. No differences could be observed when evaluating the levels of IL‐12 in the granulocyte population. The IL‐12 serum levels were below the ELISA sensitivity limit. Ex vivo stimulation of the peripheral blood cells with increasing doses of IL‐12 resulted in a significant decrease of IFN‐γ+, whereas levels of TNF‐α+ in lymphocytes were unaffected.

CONCLUSIONS: The classical Th1/Th2 paradigm appears to be insufficient to exclusively explain the causes of pregnancy loss. Our current results render us to requestion the role of Th1 cytokines during pregnancy and suggest some protective function of the Th1‐inducer cytokine IL‐12.
Chemical Pregnancies: Immunologic and Ultrasonographic Studies
Authors: Coulam Carolyn B. , Roussev Roumen
Published: March 2003

Coulam CB, Roussev R. Chemical pregnancies: immunologic and ultrasonographic studies. AJRI 2002; 48:323–328 © Blackwell Munksgaard, 2002

PROBLEM: Implantation of the embryo determines successful from unsuccessful cycles after in vitro fertilization (IVF) and embryo transfer (ET). The purpose of this study was to compare immunologic risk factors among women experiencing implanation failure characterized by a negative pregnancy test after IVF/ET and those experiencing chemical pregnancies. In addition ultrasonographic measurement of gestational sac size from 24 to 35 days from last menstrual period (LMP) were compared between chemical pregnancies and other pregnancy outcomes.

METHODS OF STUDY: Blood samples from 122 women experiencing IVF implantation failure with a negative pregnancy test after ET and 20 women with chemical pregnancies were evaluated for the presence of antiphospholipid antibodies (APA), antinuclear antibodies (ANA), circulating embryotoxins (ETA) and elevated levels of natural killer (NK) cells. Gestational sac size measured from 24 to 35 days form LMP were compared according to pregnancy outcome: term birth (n=46), ectopic pregnancy (n=49), spontaneous abortion (n=56) and chemical pregnancy (n=20).

RESULTS: Women experiencing chemical pregnancies had a higher frequency of APA than women with implantation failure associated with a negative pregnancy test (80% versus 28%, P < 0.0001). The prevalence of ANA, elevated NK cells and ETA was not different between the two groups. The mean gestational sac size from 24 to 35 days from LMP did not differ when chemical pregnancies were compared with pregnancies progressing longer than 35 days. The maximal gestational sac diameter among chemical pregnancies was 3.8 mm.

CONCLUSION: Mechanisms involved in implantation failure associated with a negative pregnancy test may be different from those involved in chemical pregnancies. Chemical pregnancies may be the result of defective angiogenesis.
A refined method for lymphocyte isolation from the placenta
Authors: Miranda Silvia , Pöhlmann Tobias , Förster Katharina , Neundorf Rita , Kaufmann Andreas , Markert Udo R.
Published: March 2003

OBJECTIVES:  Analysis of placental lymphocytesfrom different compartments is crucial for many questions in reproductive immunology. A separation of lymphocytes after tissue disaggregations leads to a mixture of maternal and fetal lymphocytes, which are difficult to distinguish by simple methods. HLA‐A2 is expressed by 29% (Romanians) – 45% (Spanish) of a Caucasian population and might be a marker for this approach.

METHODS/RESULTS:  Extravillous blood is obtained by rinsing a placenta lobus with a special pump and tube system. Such blood is usually contaminated with fetal cells. Lymphocytes are separated by a common density gradient. Lymphocytes from rinsed tissue are isolated by mechanical and enzymatical disaggregation followed by a density gradient. By using this method, fetal and maternal lymphocytes are generally widely mixed. Anti‐HLA‐A2 monoclonal antibodies are produced by a murine hybridoma cell line. In positive individuals 29–53% of lymphocytes were HLA‐A2 positive. In approximately 25% of pregnancies a discordance of HLA‐A2 expression between the mother and her fetus was found. When lymphocytes are isolated from placentae of such combinations, at least HLA‐A2 positive cells can be easily detected by flow cytometry. All positive cells derive from the same individual, whereas the origin of negative cells cannot be determined. By application of a secondary microbeads labelled antimurin antibody, anti‐HLA‐A2 can be also used for magnet activated cell separation and used for enrichment of cells of one single origin.

CONCLUSION:  In c. 25% of pregnancies the use of anti‐HLA‐A2 antibodies allows a distinction of maternal and fetal cells and their respective enrichment.
Immunoglobulins and Cytokines Level in Follicular Fluid in Relation to Etiology of Infertility and their Relevance to IVF Outcome
Authors: HAMMADEH MOHAMAD Eid , ERTAN A. KUBILY , ZEPPEZAUER MICHAEL , BALTES SABINE , GEORG THOMAS , ROSENBAUM PETER , SCHMIDT WERNER
Published: March 2003

OBJECTIVE: The aims of the present study were to (i) determine the presence and concentration of albumin fractions (α1, α2, β, γ), immunoglobulins (IgA, IgG, IgM) and cytokines [interleukin (IL)‐6, IL‐8, granulocyte‐macrophage colony‐stimulating factor (GM‐CSF)] in periovulatory ovarian follicular fluid (FF) of in vitro fertilization (IVF) patients, (ii) examine the relationship between these parameters and the etiology of infertility as well as the IVF outcome and (iii) find out if these parameters in FF could be used as a predictive factor of IVF outcome.

DESIGN: The levels of albumin fractions, immunoglobulin and cytokines were measured from women who underwent IVF therapy for various indications and the results were compared between the patient groups and IVF outcome.

MATERIALS AND METHODS: Follicular fluid was obtained from 160 IVF patients. A total of 79 patients underwent controlled ovarian hyperstimulations (COH) either with follicle‐stimulating hormone (FSH) or HMG. Whereas, the HMG was used for the second set of patients (n=81) – after down regulation with gondotropin‐releasing hormone agonists (Gn‐RHa) – the protein fractions were determined using electrophoresis separation. Immunoglobulins were measured using a commercial kits and the concentration of cytokines was determined by the highly sensitive enzyme‐linked immunosorbent assay (ELISA) methods.

RESULTS: The stimulation regimens used have no effect on albumin (α1, α2, β, γ) and immunoglobulin (IgA, IgG, IgM) concentrations, as no significant difference was observed between the two groups. Besides, no specific relationship was found between the concentration of these investigated parameter in FF and etiology of infertility or fertilization, cleavage and pregnancy rate. Besides, there were no significant differences between the groups for any cytokine investigated. Moreover, there were no correlations between the concentration of IL‐6, IL‐8 and GM‐CSF in FF and steroid hormone concentration in the blood at the day of oocytes retrieval or IVF outcome.

IN CONCLUSION: Total protein, albumin fraction, immunoglobulins and cytokines level in FF of patients undergoing COH for IVF therapy for various etiology of infertility could not be a useful parameter for predicting IVF outcome.
Diversity of Antisperm Antibodies Bound to Sperm Surface in Male Immunological Infertility
Authors: SHIBAHARA HIROAKI , TSUNODA TETSUO , TANEICHI AKIYO , HIRANO YUKI , OHNO AKIKO , TAKAMIZAWA SATORU , YAMAGUCHI CHIEKO , TSUNODA HIROMICHI , SATO IKUO
Published: March 2003

PROBLEM: The presence of antisperm antibodies (ASA) in males can reduce fecundity, however, relationship between the two is disputed. This study was performed to investigate if there is diversity of ASA bound to sperm surface using immunobead test (IBT) combined with complement dependent sperm immobilization test (SIT).

METHODS: The ASA bound to sperm surface were detected using the direct IBT (D‐IBT) in 275 semen samples. In some cases with ASA detected by D‐IBT, sperm immobilizing antibodies bound to sperm surface were also evaluated using direct SIT (D‐SIT).

RESULTS: The incidence of the immunoglobulin G (IgG), IgA, and IgM classes of ASA detected by D‐IBT were 2.5, 1.8, and 0.4%, respectively. Totally, nine (3.3%) infertile men had ASA on the sperm surface. D‐SIT was tested positive in four (66.7%) of six cases with ASA assessed by D‐IBT.

CONCLUSIONS: Some of the sperm‐bound antibodies are associated with complement dependent sperm immobilizing antibodies, indicating that there exists a heterogeneity of sperm‐bound antibodies. This result might be one of the reasons for the controversy about the relationship between ASA and immunological infertility in men.
Different treatment outcomes of two intruded permanent incisors – a case report
Authors: Chan Alex W. K. , Cheung Gary S. P. , Ho May W. M.
Published: March 2003

Abstract – Intrusive luxation involves the displacement of the tooth apically into the alveolar socket. This type of injury represents a very complex wound, involving disruption of the marginal gingival seal, alveolar bone, periodontal ligament fibers, cementum and the neuro‐vascular supply to the pulp, which results in severely compromised healing and possible complications. The case presented is a report of a 60‐year‐old lady who fell and intruded her two maxillary central incisors. The healing outcomes of the two central incisors were markedly different from each other even though they sustained similar injuries.
Consequences of crown fractures with pulpal exposure: histopathological evaluation in dogs
Authors: Harrán‐Ponce Elías , Holland Roberto , Barreiro‐Lois Andrés , López‐Beceiro Ana María , Pereira‐Espinel José Luis
Published: March 2003

Abstract  – When a crown fracture involving pulpal exposure is produced, the therapeutic treatment to be applied depends to a great extent on the general histopathological condition of the exposed pulp. Hence, the objective of this study was to evaluate histopathological and bacteriological changes occurring in dental tissue and periradicular tissue of crown‐fractured teeth with pulpal exposure. Twenty‐four anterior teeth (central and lateral incisors) from the maxillary teeth of four young, adult Mongrel dogs were used. At 48 and 72 h after performing the crown fractures, the animals were sacrificed and the results evaluated. Both observation periods revealed the existence of an area of superficial inflammation with the formation of hyperplastic tissue towards the external surface. Intense neutrophilic infiltrate was observed below it. Mean depth of inflammation was greater at 48 h (4633.33 µm) than at 72 h (3933.33 µm), perhaps coinciding with the bigger pulp chamber opening (×1332.14 µm at 48 h vs. ×479.52 µm at 72 h). Upon approaching the cervical portion, the inflammation became less. Bacterial contamination was constant in all the cases evaluated, worsening the histopathological findings with exposure time. This study demonstrates that when a crown fracture with pulpal exposure is produced, the success in treating it depends partly on how quickly therapeutic treatment is administered.
Long‐term calcium hydroxide as a root canal dressing may increase risk of root fracture
Authors: Andreasen Jens Ove , Farik Ban , Munksgaard Erik Christian
Published: March 2003

Abstract – It has been proposed (Cvek 1992) that immature teeth are weakened by filling of the root canals with calcium hydroxide dressing and gutta‐percha. The aim of the present study was to test the hypothesis that dentin in contact with calcium hydroxide would show a reduction in fracture strength after a certain period of time. Immature mandibular incisors from sheep were extracted and divided into two experimental groups. Group 1: the pulps were extirpated via the apical foramen. The root canals were then filled with calcium hydroxide (Calasept®) and sealed with IRM® cement, and the teeth were then stored in saline at room temperature for 0.5, 1, 2, 3, 6, 9, or 12 months. Group 2: the pulps were extirpated and the root canals were filled with saline and sealed with IRM® cement. The teeth were then stored in saline for 2 months. Intact teeth served as controls and were tested immediately after extraction. All teeth were tested for fracture strength in an Instron testing machine at the indicated observation periods. The results showed a markedly decrease in fracture strength with increasing storage time for group 1 (calcium hydroxide dressing). The results indicate that the fracture strength of calcium hydroxide‐filled immature teeth will be halved in about a year due to the root filling. The finding may explain the frequent reported fractures of immature teeth filled with calcium hydroxide for extended periods.
Strain differences in activity and emotionality do not account for differences in learning and memory performance between C57BL/6 and DBA/2 mice
Authors: Podhorna J. , Brown R. E.
Published: March 2003

This study examined emotionality, activity, learning and memory, as well as the influence of emotionality and activity on learning and memory performance in C57BL/6 and DBA/2 mice using a mouse‐test battery. DBA/2 mice performed more poorly than C57BL/6 mice in complex learning tasks such as the water maze and object recognition tasks. In contrast, C57BL/6 mice showed attenuated habituation to novelty in the open field apparatus and poorer performance in the step‐down passive avoidance task. The C57BL/6 mice were less exploratory and more anxious than the DBA/2 mice. The anxiety score (open arm entries in the elevated plus maze) was significantly correlated with all measures of learning and memory in the object recognition task, and some measures in the passive avoidance and water maze tasks. Analysis of covariance (with open arm entries as a covariate) revealed that some measures on trial 1 of the object recognition task, but not the memory scores on trial 2, were confounded by anxiety. No confounding factors of anxiety were found in the water maze or passive avoidance tasks. Similar results were obtained with the activity scores (line crossing and rearing in the open field). In conclusion, strain differences in activity and anxiety did not account for strain differences in learning and memory performance of C57BL/6 and DBA/2 mice. Nonetheless, the importance of using complete behavioural test batteries should be stressed to ensure that strain differences in learning and memory tasks are not confounded by non‐cognitive factors.
Home‐cage activity in heterogeneous stock (HS) mice as a model of baseline activity
Authors: Mill J. , Galsworthy M. J. , Paya‐Cano J. L. , Sluyter F. , Schalkwyk L. C. , Plomin R. , Asherson P.
Published: March 2003

Behavioral genetic work in humans indicates that clinical hyperactivity is best viewed as the extreme end of activity levels in the population. However, current animal models of hyperactivity are not studied as quantitative traits as they are either knockout models or inbred strains. Furthermore, these animal models generally demonstrate elevated locomotion in novel environments, but not in their home‐cages. This is the opposite of the symptoms seen in the human condition where childhood hyperactivity is generally more pronounced in constant, unstimulating situations. In this study we filmed an outbred population of 44 heterogeneous stock (HS) mice under red light during their active phase, to assess the reliability of individual differences in home‐cage behavior and extract an index of home‐cage activity (HCA) level. We then compared this measure to locomotor behavior in a novel environment — the open‐field. Reliable individual differences in home‐cage behaviors such as running, swinging on bars, and burrowing were found, and principal component factor analysis yielded a general activity factor, which accounted for 32% of the variance and correlated 0.90 with a subjective impression of activity level. The correlation between HCA and locomotor activity in the open‐field was 0.23, which was non‐significant. However, the association with HCA level appeared to increase over the five minutes of the open‐field, presumably as the mice habituated. Furthermore, although mice displaying particularly high and low HCA were indistinguishable early in the open‐field task, they became significantly differentiated over time. We conclude that home‐cage behaviors and the open‐field, after habituation, display good face and construct validity, and may provide a good model of baseline activity for quantitative trait loci (QTL) discovery and functional genomics in the HS mice.
Mice overexpressing CRH show reduced responsiveness in plasma corticosterone after a 5‐HT1A receptor challenge
Authors: Van Gaalen M. M. , Reul J. H. M. , Gesing A. , Stenzel‐Poore M. P. , Holsboer F. , Steckler T.
Published: March 2003

Corticotropin‐releasing hormone (CRH) overproduction and serotonergic dysfunction have both been implicated in a range of psychiatric disorders, such as anxiety and depression, and several studies have shown interactions between these two neurotransmitter systems. In this study, we investigated the effects of CRH challenge on hypothalamo‐pituitary‐adrenal (HPA) axis activity in female transgenic mice overproducing CRH. Furthermore, the effects of mild stress on HPA axis activity and body temperature were investigated in these mice. Pre‐ and post‐synaptic 5‐HT1A receptor function were studied by monitoring body temperature and plasma corticosterone levels after challenge with the 5‐HT1A receptor agonist 8‐hydroxy‐2‐(di‐n‐propyl‐amino)‐tetralin (8‐OH‐DPAT). Hypothermia in response to 8‐OH‐DPAT treatment did not differ between transgenic and wild type mice, indicating unaltered somatodendritic 5‐HT1A autoreceptor function in mice overproducing CRH. In wild type mice 8‐OH‐DPAT increased plasma corticosterone levels, but not in transgenic animals. CRH injection, however, increased corticosterone levels in both groups. These data suggest desensitization of post‐synaptic, but not pre‐synaptic, 5‐HT1A receptors in mice overproducing CRH. These findings resemble those seen in depressed patients following 5‐HT1A challenge, which is in accord with the hypothesized role of CRH in the pathogenesis of depression.
Difference in anxiety and sensitization of the acoustic startle response between the two inbred mouse strains BALB/cAN and DBA/2N
Authors: Plappert C. F. , Pilz P. K. D.
Published: March 2003

The inbred mouse strain BALB has been proposed to be an animal model for pathological anxiety. BALB exhibits a stronger acoustic startle response (ASR) than the ‘less emotional’ inbred strain DBA. Four experiments were conducted to determine whether this strong ASR is due to a higher anxiety level and/or to greater sensitization in BALB than in DBA, with the following results: (1) The ASR to the very first startle stimulus was found to be much stronger in BALB than in DBA, and freezing behavior evoked by startle stimuli was more pronounced in BALB than in DBA. These findings indicate a higher level of anxiety in this strain. (2) ASR amplitudes of BALB initially rose much higher during consecutive startle stimuli and remained at a high level much longer than in DBA. Thereafter, ASR amplitude dropped more slowly and to a lesser degree than in DBA. Startle amplitudes decreased similarly in both strains (strong exponential decrease) only when a low sound pressure level (SPL) was used which elicited approximately the same low ASR in both strains. These results can only be explained by increased sensitization in BALB. (3) The slope of the i/o‐function, which represents the relation between sensory input and motor output, was steeper in BALB than in DBA. As it has been shown recently, sensitization increases the slope of the startle i/o‐function indicating increased sensitization in BALB. It is discussed, however, whether anxiety also contributes to this effect. (4) Footshocks increased the ASR much less in BALB than in DBA, again showing increased sensitization in BALB. Both a higher level of anxiety and greater sensitization therefore determined the greater strength of the ASR in BALB than in DBA.
The structure of individual differences in Heterogeneous Stock mice across problem types and motivational systems
Authors: Locurto C. , Fortin E. , Sullivan R.
Published: March 2003

Sixty Heterogeneous Stock (HS) mice received a battery of six problem‐solving tasks and three control procedures. The problem‐solving tasks included Hebb‐Williams, a place learning task conducted in a plus maze, radial maze, a working memory test following the radial maze, a set of detour problems and a visual non‐matching to sample task. The control procedures consisted of land and water activity measures and a light‐dark test. The correlation matrix derived from these tasks did not exhibit positive manifold, that is, positive correlations across all problem‐solving tasks. Principal components analysis reduced the correlation matrix to four components with eigenvalues exceeding 1.0. Instead of the general factor solution common in the study of human problem‐solving, this component structure appeared more congenial to a modular interpretation, with the four components each explaining approximately the same magnitude of matrix variance.
Lattice organs in y‐cyprids of the Facetotecta and their significance in the phylogeny of the Crustacea Thecostraca
Authors: Høeg J. T , Kolbasov G. A
Published: March 2003

Scanning and transmission electron microscopy (SEM and TEM) were used to study lattice organs in facetotectan y‐cyprids from the White Sea and from Norwegian and Bahamian waters. The larvae represent at least four and possibly five different species of Facetotecta. Y‐cyprids have five pairs of lattice organs in the head shield (carapace) organized into two anterior pairs and three posterior pairs. Both groups of lattice organs are arranged around a large central pore. The facetotectan lattice organs are elongate areas with a longitudinal keel, just as in the Ascothoracida and some Cirripedia Acrothoracica. The terminal pore of the organs is situated posteriorly in all five pairs. TEM confirms that the organs have the same general morphology as in the Cirripedia and Ascothoracida, namely, a cuticular chamber into which project ciliary segments from the chemosensory cells. Unlike Cirripedia the cuticular roof of the chamber lacks any pores. We conclude that five pairs of lattice organs represent an autapomorphy for the Thecostraca, which supports the monophyly of this taxon. In the ground pattern the terminal pore is posterior in all five pairs. The anterior position of the pore in lattice organ pair 2 is apomorphic for the Cirripedia, while within this taxon an anterior position also in pair 1 is apomorphic for a monophylum comprising the Thoracica and the Rhizocephala. Minute pores in the roof of the organs is another apomorphy of the Cirripedia, but its elaboration into pores visible with SEM may have been subject to some homoplasy. Since lattice organs are omnipresent in the settling instar of the Thecostraca they probably serve a critical role for the function of these cypris or cypris‐like larvae.
Outcome evaluation in a surgical intensive care unit in Barbados
Authors: Hariharan S. , Moseley H. S. L. , Kumar A. Y.
Published: March 2003

The performance of the Acute Physiology and Chronic Health Evaluation II scoring system was␣prospectively assessed in the surgical intensive care unit at the Queen Elizabeth Hospital, Barbados. A total of 309 patients admitted consecutively during a 2‐year period (1999–2001) were evaluated. Demographic data, diagnosis, Acute Physiology and Chronic Health Evaluation II score,␣duration of stay and hospital outcome were recorded. The predicted mortality for every patient and the costs incurred were also calculated. The overall observed mortality rate was 15.9% while the mean predicted mortality rate for our case‐mix was 16.4%, which is comparable to results␣from developed countries. The cost incurred per patient was much lower at $13 636 (Barbados), compared to the patients' cost in North America ($60 000 Barbados).
Comparability of the amniotic fluid index and single deepest pocket measurements in clinical practice
Authors: Magann Everett F. , Chauhan Suneet P. , Bofill James A. , Martin James N.
Published: March 2003

Two ultrasound techniques, the amniotic fluid index (AFI) and the single deepest pocket (SDP), are currently used to detect oligohydramnios, predict variable decelerations, risk of Caesarean delivery for fetal distress, Apgar scores, umbilical cord artery pH, perinatal mortality, and cerebral palsy. Both techniques poorly identify oligohydramnios. Both techniques identify pregnancies at risk for variable decelerations, low Apgar scores, and Caesarean delivery for fetal distress. Only the SDP is predictive of a compromised fetus–umbilical artery pH, as a stand‐alone test, has been correlated with perinatal mortality, and as part of the biophysical profile has been linked to cerebral palsy. This brief communication reviews the comparability of these two techniques and which method, if either, is superior in the identification of oligohydramnios, the predictability of these techniques to identify an adverse pregnancy outcome, and the ability to predict cerebral palsy and perinatal mortality.