A Cellular atlas of the human fallopian tube reveals the metamorphosis of secretory epithelial cells during the menstrual cycle and menopause.

The fallopian tube, connecting the uterus with the ovary, is a dynamic organ that undergoes cyclical changes and is the site of several diseases, including serous cancer. Here, we use single-cell technologies to construct a comprehensive cell map of healthy pre-menopausal fallopian tubes, capturing the impact of the menstrual cycle and menopause on different fallopian tube cells at the molecular level. The comparative analysis between pre- and post-menopausal fallopian tubes reveals substantial shifts in cellular abundance and gene expression patterns, highlighting the physiological changes associated with menopause. Further investigations into menstrual cycle phases illuminate distinct molecular states in secretory epithelial cells caused by hormonal fluctuations. The markers we identified characterizing secretory epithelial cells provide a valuable tool for classifying ovarian cancer subtypes. Graphical summary: Graphical summary of results. During the proliferative phase (estrogen high ) of the menstrual cycle, SE2 cells (OVGP1 + ) dominate the fallopian tube (FT) epithelium, while SE1 cells (OVGP1 - ) dominate the epithelium during the secretory phase. Though estrogen levels decrease during menopause, SE post-cells (OVGP1 + , CXCL2 + ) make up most of the FT epithelium.

Active matrix metalloproteinase-8 and periodontal bacteria-interlink between periodontitis and inflammatory bowel disease?

BACKGROUND: The aim of this study was the investigation of concentration and prevalence of selected periodontal pathogenic bacteria and concentration of active matrix metalloproteinase-8 (aMMP-8) within a group of patients with inflammatory bowel diseases (IBD) and to compare the results with a group of healthy control subjects (HC). METHODS: Fifty-nine IBD patients with Crohn`s disease (CD, n = 30) or ulcerative colitis (UC, n = 29) and 59 HC were included in this cross-sectional study. Based on periodontal probing depth (PD) and clinical attachment level (CAL), periodontitis was classified as healthy/mild, moderate, or severe. aMMP-8 was analyzed from gingival crevicular fluid using enzyme linked immunosorbent assay. Eleven selected periodontal pathogenic bacteria were analyzed in subgingival plaque samples using polymerase chain reaction. RESULTS: IBD patients showed higher CAL (P < 0.01), more severe periodontitis (P = 0.04), gingival bleeding (P < 0.01) and aMMP-8 concentration (P < 0.01) than HC. Only in CD, increasing severity of periodontitis was associated with an increase in aMMP-8 concentration (P = 0.02). The prevalences of Eubacterium nodatum and Eikenella corrodens were significantly lower in IBD compared to HC (P = 0.01). Additionally, the prevalence of Eikenella corrodens was significantly higher in CD compared to the UC group (P = 0.04). Further statistically significant differences in selected bacteria between IBD and HC or CD and UC groups could not be found (P > 0.05). CONCLUSIONS: The results reveal changes in host immune response of IBD patients in terms of aMMP-8. Only in CD increasing aMMP-8 was associated with severity of periodontal disease. The role of periodontal pathogenic bacteria in the interrelationship between IBD and periodontitis remains unclear.

Clinical multicenter evaluation of a new FXa-based Antithrombin assay.

INTRODUCTION: The determination of functional Antithrombin is a central part of thrombophilia screening. In this multicenter study, a new FXa-based method (INNOVANCE® Antithrombin) was evaluated on four different analyzers. METHODS: The INNOVANCE Antithrombin method was evaluated by precision and reference interval studies and by comparing the new method with established methods through parallel measurement of samples from 249 patients and 151 apparently healthy individuals. RESULTS: The INNOVANCE Antithrombin assay demonstrated on all analyzers repeatability coefficients of variation (CVs) ≤ 3.2% and within-device and between-run CVs ≤ 6.9%. The reference intervals of all analyzers are comparable with 2.5th percentiles between 80% and 85% of normal. The INNOVANCE Antithrombin and the FIIa-based Berichrom® AT III (A) methods demonstrated good concordance with correlation coefficients of r = 0.908 or higher. The INNOVANCE Antithrombin method demonstrated furthermore an excellent comparability with the STA® Antithrombin III assay and an acceptable comparability with the Coamatic® LR Antithrombin assay. The patients with congenital deficiency (n = 31) were identified with all assays except for the patients carrying the P41L heparin-binding site mutation, which was only identified with the INNOVANCE Antithrombin and the STA Antithrombin III methods. CONCLUSION: The INNOVANCE Antithrombin assay has high sensitivity for Antithrombin deficiencies and is reliable, precise and suitable for routine clinical use.

Long- and short-term in vitro D-dimer stability measured with INNOVANCE D-Dimer.

In vitro D-dimer stability in plasma is widely assumed, but has not yet been documented by systematic studies using samples covering a wide range of D-dimer. We investigated the short- and long-term stability of D-dimer in clinical citrated plasma samples with normal and pathological levels. The short-term stability was analysed by measuring D-dimer fresh, after storage of plasma for 4 hours at room temperature (RT) and after an additional 24 h storage at +2 to +8 degrees C (n=40). Long-term stability samples (n=40) were measured fresh and after storage for 19, 25 and 36 months at < or =-60 degrees C. The effect of repeated freezing was analysed by measuring samples (n=50) fresh and after four consecutive freeze-thaw cycles. D-dimer was measured on the BCS System using the INNOVANCE D-Dimer assay (Siemens Healthcare Diagnostics Products GmbH, Marburg, Germany). D-dimer values at baseline ranged from 0.23-22.2 mg/l FEU. The mean percentage change after storage for 4 hours at RT and additional 24 hours at +2 to +8 degrees C was +3.8% and +2.7%, respectively. The mean percentage change after frozen storage for 19, 25 and 36 months at < or =-60 degrees C was -11.7%, -4.8% and -9.3%, respectively. The small decrease of D-dimer values after frozen storage was not time-dependent. Repeated freezing did not significantly alter D-dimer values (mean change < or =5%). The data demonstrate stability of D-dimer in plasma prior to freezing for up to 4 hours at RT and for up to 24 hours at +2 to +8 degrees C as well as in plasma stored for up to three years at < or =-60 degrees C.

Structural basis for autoantibody recognition of phosphatidylserine-beta 2 glycoprotein I and apoptotic cells.

Apoptotic cells contain nuclear autoantigens that may initiate a systemic autoimmune response. To explore the mechanism of antibody binding to apoptotic cells, 3H9, a murine autoantibody with dual specificity for phospholipids and DNA, was used. H chain mutants of 3H9 were constructed, expressed as single-chain Fv (scFv) in Escherichia coli, and assessed for binding to phosphatidylserine, an antigen expressed on apoptotic cells. Both 3H9 and its germline revertant bound to dioleoyl phosphatidylserine in ELISA, and binding was enhanced by beta 2 glycoprotein I (beta 2GPI), a plasma protein that selectively binds to apoptotic cells. Higher relative affinity for DOPS-beta 2GPI was achieved by the introduction of Arg residues into the 3H9 H chain variable region at positions previously shown to mediate DNA binding. Specificity of the two structurally most diverse scFv for apoptotic cells was shown by flow cytometry, and two populations of scFv-bound cells were identified by differences in propidium iodide staining. The results suggest that, in autoimmunity, B cells with Ig receptors for apoptotic cells and DNA are positively selected, and that the antibodies they produce have the potential to affect the clearance and processing of apoptotic cells.

Seasonal influences on in vitro fertilization and embryo transfer.

PURPOSE: The study was aimed to investigate the influence of time factors (day, week, month) on pregnancy rates and in vitro fertilization (IVF) and embryo transfer (ET) parameters. METHODS: 8,184 IVF-ET cycles, taking place in two IVF centers from 1992 to 1999, were analyzed. Multiple logistic and linear regression methods were performed as statistical analyses. RESULTS: Oocyte pickup on Tuesdays achieved a significantly higher mean number of oocytes (median: 7) and the highest pregnancy rate (PR) (33.4%) per ET There was a significant variation over the year, with the lowest PR/ET in July (25.71%) and the highest in December (35.5%). Concerning outcomes, the age factor and the number of embryos transferred had the highest significant (p < 0.0001) influence. CONCLUSIONS: The observed weekly rhythm for oocyte pick-ups is certainly due to preprogrammed ovarian stimulation used in our IVF programs. Age as well as the number of embryos transferred are the main influencing factors on a positive outcome and more predictive than seasonal aspects.

Editors and editing of anti-DNA receptors.

Receptor editing is a means by which immature bone marrow B cells can become self-tolerant. Rearrangements of heavy (H) and/or light (L) chain genes are induced by encounter with autoantigens to change the specificity from self to nonself. We have developed site-directed transgenic mice (sd-tg) whose transgenes code for the H chain of antibodies that bind DNA. B cells that express the transgenic H chain associate mainly with four of the 93 functional Vkappa genes of the mouse. Numerous aspartate residues that might inhibit DNA binding by the V(H) domain distinguish these L chain Vkappa sequences, but engaging these Vkappa editors often requires multiple rearrangements. Among the edited B cells is a subset of multispecific cells that express multiple receptors. One consequence of multispecificity is partial autoreactivity; these multispecific B cells may contribute to autoimmunity.

Number and size of antral follicles as predictive factors in vitro fertilization and embryo transfer.

PURPOSE: The purpose of the study was to evaluate whether number and size of antral follicles can predict the outcome of in vitro fertilization-embryo transfer. METHODS: A total of 113 patients were prospectively included into this study. After 19 days of down-regulation, number and size of follicles were determined by using recent three-dimensional transvaginal ultrasound technology. Before application of gonadotropin, all follicles had been defined as antral follicles. According to size, antral follicles were categorized into four different groups: group I included antral follicles < 5 mm, group II follicles 5-10 mm; group III 11-20 mm; and group IV > 20 mm. Pregnant and non-pregnant patients were compared in terms of their number of antral follicles of group I-IV. These four groups were then compared regarding implantation rate, number of retrieved oocytes, endometrium thickness, and age. RESULTS: Pregnant patients showed an significant higher number of follicles with the size between 5 and 10 mm (P = 0.04). A significant correlation was found between number of retrieved oocytes and antral follicle size of 5-10 mm (P = 0.0001). Antral follicles with a diameter between 5 and 10 mm decreased significantly with age (P = 0.008). In group III and IV, a significant correlation was found between antral follicle size (P = 0.016) and serum estradiol level after gonadotropin-releasing hormone-agonist down-regulation (P = 0.011). CONCLUSIONS: We demonstrated that patients with a higher number of follicles between 5 and 10 mm showed a significantly higher pregnancy rate, whereas patients with a dominant number of antral follicles > 11 mm have a higher cancellation rate due to ovarian low response.

[Painfulness of transvaginal follicle aspiration for oocyte retrieval in in-vitro-fertilization]. / Schmerzhaftigkeit vaginaler Punktionen im Rahmen der In-vitro-Fertilisation.

OBJECTIVE: The aim of this study was to analyze the painfulness of a transvaginal ultrasound-guided follicle punction. MATERIAL AND METHODS: Patients could choose to have either a neuroleptanalgesia or to be supported by the partner or a psychotherapist during the oocyte pick up. Based on a questionnaire, 277 patients tried to describe their level of pain in a horizontal alphanumeric scale divided in 15 categories, while other painful physical interacts could be quantified, too. Furthermore, patients were asked for the cause of sterility, former IVF-ET treatments and whether the partner or the psychotherapist looked after them during the oocyte retrieval. Statistical analysis was performed using a standardized computer program (Stat View, Abacus Concepts, USA). RESULTS: The neuroleptanalgesia reduced the intensity of pain (average value of pain: 1.21 vs 9.26 without analgesia) in a significant way (p = 0.0001). Some patients compared the pain at oocyte retrieval with a bone fracture (8.08) or surgeries (10.12). More painful was a colic (13.67), infections (13.5), some diagnostic surgeries (12.09) or a delivery (11.91). Younger woman and patients with dysmenorrhea felt significantly more pain than others. Neither the presence of the partner or psychotherapist during punction nor the indication, number of previous IVF-ET treatments or a following pregnancy had any influence on the statistics. CONCLUSION: Age as well as the rate of problems with menstruation should help the patient to decide on whether to have the follicle punction with or without anesthesia.

Somatic mutation and light chain rearrangement generate autoimmunity in anti-single-stranded DNA transgenic MRL/lpr mice.

Antibodies to single-stranded (ss)DNA are expressed in patients with systemic lupus erythematosus and in lupus-prone mouse models such as the MRL/Mp-lpr/lpr (MRL/lpr) strain. In nonautoimmune mice, B cells bearing immunoglobulin site-directed transgenes (sd-tgs) that code for anti-ssDNA are functionally silenced. In MRL/lpr autoimmune mice, the same sd-tgs are expressed in peripheral B cells and these autoantibodies gain the ability to bind other autoantigens such as double-stranded DNA and cell nuclei. These new specificities arise by somatic mutation of the anti-ssDNA sd-tgs and by secondary light chain rearrangement. Thus, B cells that in normal mice are anergic can be activated in MRL/lpr mice, which can lead to the generation of pathologic autoantibodies. In this paper, we provide the first direct evidence for peripheral rearrangement in vivo.

The effect of smoking on oocyte quality and hormonal parameters of patients undergoing in vitro fertilization-embryo transfer.

PURPOSE: The aim of the present study was to investigate the influence of smoking on different parameters such as oocyte count, embryo score, and basal hormone values within the scope of in vitro fertilization-embryo transfer (IVF-ET). METHODS: Eight hundred thirty-four women undergoing IVF-ET treatment were classified as smokers or nonsmokers on the basis of questionnaires. Additionally, we divided them into three groups according to their stimulation protocol--"combined stimulation" [I; clomiphene citrate plus human menopausal gonadotropin (hMG)], "ultrashort" [II; gonadotropin releasing hormone agonist (GnRHa) plus hMG or follicle-stimulating hormone (FSH)], and "long downregulation protocol" (III)--and further classified again as smokers or nonsmokers within the groups. RESULTS: In general, smoking patients were significantly (P = 0.0195) younger than nonsmokers and showed a significantly (P = 0.0379) lower embryo score and a tendency (P = 0.0931) to produce fewer oocytes. There was no significant difference concerning the number of normally or pathologically fertilized and transferred oocytes and embryos suitable for cryopreservation. Women who smoked had significantly (P = 0.0112) higher basal 17-beta-estradiol (E2), luteinizing hormone (LH) (P = 0.0001), and dehydroepian-drosteronesulfate (DHEAS) (P = 0.0039) levels, but their basal human prolactin (HPRL) levels were significantly (P = 0.0033) lower than those of nonsmokers. According to the stimulation protocol used, we found the following results. Smoking patients in group I showed a significantly (P = 0.023) lower embryo score and produced fewer oocytes (P = 0.0113), with fewer of them being fertilized (P = 0.0072) and transferred (P = 0.0067). Women who smoked had significantly (P = 0.0002) higher basal LH levels, but their HPRL levels were significantly (P = 0.031) lower than those of nonsmokers. Furthermore, they had a thinner endometrium on the day of embryo transfer (P = 0.0366). In group II we measured significantly elevated basal E2 levels (P = 0.0089) and higher LH values (P = 0.0092) in smokers. Group III showed a trend (P = 0.0565) toward lower HPRL values in smokers. CONCLUSIONS: Although the fertilization rate of oocytes and the pregnancy rate were not significantly different between smokers and nonsmokers, we found significantly alterated hormonal parameters and negatively influenced oocyte parameters, particularly after clomiphene stimulation. So we might consider using only GnRHa protocols for smoking patients. Additionally, we advise our patients to stop smoking before an IVF-ET treatment because of the complex effects of smoking on the reproductive and hormonal system.

Regulation of anti-DNA B cells in recombination-activating gene-deficient mice.

Anti-DNA antibodies are regulated in normal individuals but are found in high concentration in the serum of systemic lupus erythematosus (SLE) patients and the MRL lpr/lpr mouse model of SLE. We previously studied the regulation of anti-double-stranded (ds)DNA and anti-single-stranded (ss)DNA B cells in a nonautoimmune background by generating mice carrying immunoglobulin transgenes coding for anti-DNAs derived from MRL lpr/lpr. Anti-dsDNA B cells undergo receptor editing, but anti-ssDNA B cells seem to be functionally silenced. Here we have investigated how anti-DNA B cells are regulated in recombination- activating gene (RAG)-2-/- mice. In this setting, anti-dsDNA B cells are eliminated by apoptosis in the bone marrow and anti-ssDNA B cells are partially activated.

A solution to the rheumatoid factor paradox: pathologic rheumatoid factors can be tolerized by competition with natural rheumatoid factors.

Rheumatoid factors (RF) associated with arthritic joint erosion are only seen transiently, if at all, in nondiseased individuals. Therefore, a tolerance mechanism must exist that prevents pathologic RF B cells from expressing Abs. Surprisingly, it has been shown that pathologic RF B cells are not tolerized by any previously established tolerance mechanism such as deletion, receptor editing, anergy, or prevention of memory establishment. How are pathologic RF cells tolerized? By simulating the RF response with a cellular automaton model immune system, we demonstrate that pathologic RFs can be tolerized by the novel mechanism of "competitive tolerance" with natural, nonpathologic RFs. We then demonstrate that competitive tolerance can be broken when a sequestered pool of expanding B cells are inappropriately subjected to chronic stimulation (as appears to occur in MRL/lpr mice and in patients with rheumatoid arthritis).

Editing disease-associated autoantibodies.

We have generated site-directed transgenic mice whose transgenes code for anti-DNA antibodies. These antibodies are representative of the lupus-associated anti-DNAs seen in mouse models of autoimmunity and human SLE, and have the usual characteristics of pathogenic autoantibodies. As conventional transgenics in nonautoimmune mice, anti-DNA B cells have been shown to be deleted or inactivated. Autoreactive B cells can also escape negative regulation by a process called receptor editing. Here we describe two combined immunoglobulin H and L chain site-directed transgenic mouse models and characterize their editing phenotypes. One model, 3H9R/Vkappa4R, has a deletion-prone phenotype and undergoes editing, primarily by inactivation of the light chain by leap-frogging events. In the other model, 3H9R/Vkappa8R, B cells are susceptible to anergy and maintain most of their HR and LR chains. These studies clarify the relationship between editing and other mechanisms of tolerance.

A Shannon entropy analysis of immunoglobulin and T cell receptor.

In 1970, before any antigen-bound immunoglobulin structure had been solved, Elvin Kabat proposed that regions of high amino acid diversity would be the antigen binding sites of immunoglobulin (Kabat, 1970). Conversely, sites of low variability were proposed to be structural, framework regions. This variability was defined by Wu and Kabat as the number of different amino acids found at a site divided by the relative frequency of the most common amino acid at that site (Wu and Kabat, 1970). Several groups have subsequently devised improvements of Kabat-Wu variability analysis (Litwin and Jores, 1992). While these methods are somewhat better than Kabat-Wu, they still suffer from Kabat-Wu's basic limitation: they account for only the most common one or two amino acids in estimating diversity. This leads to underestimates of low diversities and exaggerations of high diversities. Shannon information analysis eliminates serious bias and is more stable than Kabat-Wu and second generation measures of diversity (Jores et al. 1990; Wu and Kabat, 1970). Statistical reliability can be measured using Shannon analysis, and Shannon measurements can be provided with error estimates. Here we use Shannon's method to analyze the amino acid diversity at each site of T cell receptor Valpha and Vbeta to identify complementarity determining regions and framework sites. Our results reveal that the T cell receptor is significantly more diverse than immunoglobulin-suggesting T cell receptor has more than the previously-discovered four complementarity determining regions. These new complementarity determining regions may represent a larger antigen combining site, additional combining sites, or an evolutionary strategy to avoid inappropriate interaction with other molecules.

A disease-related rheumatoid factor autoantibody is not tolerized in a normal mouse: implications for the origins of autoantibodies in autoimmune disease.

We have analyzed B cell tolerance in a rheumatoid factor (RF) transgenic mouse model. The model is based on AM14, a hybridoma, originally isolated from an autoimmune MRL/lpr mouse that has an affinity and specificity typical of disease-related RFs from this strain. AM14 binds to immunoglobulin (Ig)G2a of the "a" allotype (IgG2aa) and not to IgG2ab. Thus, by crossing the transgenes onto an IgHa (BALB/c) background or to a congenic IgHb (CB.17) background, we could study the RF-expressing B cells when they were self-specific (IgHa) or when they were not self-specific (IgHb). These features make the AM14 model unique in focusing on a true autoantibody specificity while at the same time allowing comparison of autoreactive and nonautoreactive transgenic B cells, as was possible in model autoantibody systems such as anti-hen egg lysozyme. Studies showed that AM14 RF B cells can make primary immune responses and do not downregulate sIgM, indicating that the presence of self-antigen does not induce anergy of these cells. In fact, IgHa AM14 transgenic mice have higher serum levels of transgene-encoded RF than their IgHb counterparts, suggesting that self-antigen-specific activation occurs even in the normal mouse background. Since AM14 B cells made primary responses, we had the opportunity to test for potential blocks to self-reactive cells entering the memory compartment. We did not find evidence of this, as AM14 B cells made secondary immune responses as well. These data demonstrate that a precursor of a disease-specific autoantibody can be present in the preimmune repertoire and functional even to the point of memory cell development of normal mice. Therefore, immunoregulatory mechanisms that normally prevent autoantibody production must exert their effects later in B cell development or through T cell tolerance. Conversely, the data suggest that it is not necessary to break central tolerance, even in an autoimmune mouse, to generate pathologic, disease-associated autoantibodies.

Immunoglobulin heavy chain gene replacement: a mechanism of receptor editing.

We have generated a site-directed transgenic (sd-tg) mouse model in which the JH locus has been replaced with a rearranged VDJ coding for the heavy chain of an anti-DNA antibody. In these mice, B cells expressing the anti-dsDNA specificity are negatively regulated. We observe a novel mechanism for B cell tolerance, receptor editing at the heavy chain locus. In most sd-tg B cells, the inserted anti-DNA VH gene has been replaced by the upstream endogenous VH, or DH, or both genes through recombination with the heptamer embedded at the 3' end of most VH genes. Three types of recombination events have been identified. VH-to-VDJ, DH-to-VDJ, and VH-to-DH-VDJ. Analysis of the junctional sequences revealed features of classical V(D)J rearrangement, namely N sequence addition and nucleotide deletion. A conserved nonamer was found 12 bp upstream of the embedded heptamer. This nonamer may represent a novel recombination signal sequence used for VH editing. The sd-tg model thus provides direct evidence for secondary rearrangement at VH-D-JH. This process may play a role in tolerance by editing autoreactive receptors and may also serve to diversify the VH repertoire.