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For the normal development of pregnancy, a balance between immune tolerance and defense is crucial. However, the mechanisms mediating such a balance are not fully understood. CD83 is a transmembrane protein whose expression has been linked to anti-inflammatory functions of T and B cells. The soluble form of CD83, released by cleavage of the membrane-bound protein, has strong anti-inflammatory properties and was successfully tested in different mouse models. It is assumed that this molecule contributes to the establishment of immune tolerance. Therefore, we postulated that the expression of CD83 is crucial for immune tolerance during pregnancy in mice. Here, we demonstrated that the membrane-bound form of CD83 was upregulated in T and B cells during allogeneic murine pregnancies. An upregulation was also evident in the main splenic B cell subtypes: marginal zone, follicular zone, and transitional B cells. We also showed that there was an augmentation in the number of CD83+ cells toward the end of pregnancy within splenic B and CD4+ T cells, while CD83+ dendritic cells were reduced in spleen and inguinal lymph nodes of pregnant mice. Additionally, B lymphocytes in late-pregnancy presented a markedly higher sensitivity to LPS in terms of CD83 expression and sCD83 release. Progesterone induced a dosis-dependent upregulation of CD83 on T cells. Our data suggest that the regulation of CD83 expression represents a novel pathway of fetal tolerance and protection against inflammatory threats during pregnancy.
Background: Uterine leiomyosarcoma (LMS) is a rare entity amongst malignant gynaecological tumours and is mostly diagnosed after surgery for benign leiomyoma (LM) of the uterus. As minimal invasive surgery is widely used, the morcellation of LM and the uterus is rather common. As there is little known about the impact of the morcellation of LMS on local and distant metastases, as well as overall survival, we carried out a large-scale retrospective study. Methods: A total of 301 LMS cases from the German Clinical Competence Centre for Genital Sarcomas and Mixed Tumours were analysed. We distinguished morcellated and non-morcellated LMS from pT1 and >pT1 tumours. Fine–Gray competing risks regressions and cumulative incidence rates were computed for the time to local recurrence, distant metastases, and patient death. Results: The recurrence free interval in pT1 LMS was significantly lower in the morcellation group with a 2-year cumulative incidence rate of 49% vs. 26% in non-morcellated LMS (p = 0.001). No differences were seen in >pT1 tumours. Distant metastases were more frequently found in non-morcellated pT1 LMS compared to the morcellated cases (5-year cumulative incidence: 54% vs. 29%, p < 0.001). There was no significant difference in time to death between both groups neither in the pT1 stages nor in >pT1 disease. Subdistribution hazard ratios estimated by multivariable competing risks regressions for the morcellation of pT1 LMS were 2.11 for local recurrence (95% CI 1.41–3.16, p < 0.001) and 0.52 for distant metastases (95% CI 0.32–0.84, p = 0.008). Conclusions: Tumour morcellation is not associated with OS for pT1 tumours. The morcellation of pT1 LMS seems to prolong the time to distant metastases whereas local recurrence is more likely to occur after the morcellation of pT1 LMS.
Adaptation mechanisms within the B cell composition for successful human and murine pregnancies.
(2021)
Introduction
A well-balanced immune maternal status is essential for favourable outcome of pregnancy. Due to their complexities, not all immune adaptations that promote tolerance during pregnancy are known. To understand the adaptation of the B cell compartment, we analysed and compared B cell lymphopoiesis in different lymphoid tissues in a number of murine models.
Furthermore, we focused on the humoral immune response during pregnancy. We analysed immunoglobulin profiles in human subjects and mice during pregnancy.
These cellular alterations are subject to the influence of chemokines, among others. Therefore, we assessed serum levels of B cell activation factor to clarify its effects during pregnancy.
Methods
For analysis of the human peripheral B cell compartment, peripheral blood samples from age-matched non-pregnant and pregnant women without pregnancy complications, immunological disease or acute/chronic inflammation were collected and sub-classified into four different groups: non-pregnant, and first, second, or third trimester of pregnancy. The experiments, based on a mouse model, were performed with 8-week-old female mice: clinically healthy non-pregnant (CBA/J (H2k)), pregnant mice with normal gestation (BALB/c (H2d) x CBA/J (H2k)), and mice with pregnancy loss (DBA/2J (H2d) x CBA/J (H2k)). Subsequently, peripheral blood mononuclear cells from blood and lymphatic organs were isolated following standard protocols. The B cell analysis was performed by flow cytometry. The immunoglobulin serum levels of the human and murine subgroups were quantitated using Bio-Plex isotyping assay and analysed by a Bio-Plex reader. To quantify B cell activating factor (BAFF) in serum of pregnant and non-pregnant mice a BAFF enzyme-linked immunosorbent assay was used. The concentrations were determined by using a FLUOstar OPTIMA microplate reader. All statistical analyses were performed using the Kruskal–Wallis test with Dunn’s post-test in GraphPad Prism software. P values of < 0.05 were considered statistically significant.
Results
We were able to demonstrate B cell lymphopenia in mice bone marrow downstream of pre-pro B cells, irrespective of pregnancy outcome. The mature bone marrow B cells did not show this adjustment mechanism during normal gestation.
Closer inspection of the splenic tissue revealed expansion and activation of marginal zone B cells in mice with a normal pregnancy. However, this was not observed in mice suffering from pregnancy disturbances. Natural antibodies secreted from marginal zone B cells were also present at higher concentrations in serum of pregnant mice, compared to non-pregnant animals.
We also found significantly higher levels of natural antibodies in serum of pregnant women compared to non-pregnant age-matched controls. Analysis showed significantly lower levels of BAFF in mice with normal pregnancy as compared to non-pregnant mice.
Conclusions
We are able to show mechanisms within the B cell compartment as well as the change within the natural antibodies that might be crucial for successful pregnancy in both humans and mice. Furthermore, BAFF seems to play a central role as a mediator of peripheral B cell compartment and B cell lymphopoiesis in the bone marrow for successful pregnancy.
Introduction:
The amniotic fluid – as the medium surrounding the fetus, it is holding a crucial role in the maintenance and development of a successful pregnancy. While providing mechanical protection to the fetus, it also offers considerable immunological defense. In fact, it is known that the amniotic fluid plays a significant role in the innate immune system, as many of its corresponding substances show substantial antimicrobial function. Also, components of the adaptive immune system, including B cells, have been described within the amniotic fluid. An increase of immune cells in the amniotic fluid in cases of intra-amniotic infection indicates their involvement in inflammation-related pathologies of pregnancy. However, especially B cells in the amniotic fluid have not yet been thoroughly investigated.
The aim of this work is a deeper examination of the B-lymphocytes within the amniotic fluid. Based on the analysis of surface molecules this includes their phenotype, origin and func-tion. In the long term this could substantiate our understanding of intraamniotic inflammation and or infection, which are casually linked with preterm birth, fetal inflammatory response syndrome and fetal morbidity.
This, in turn, could pave the way for potential diagnostic methods and treatments.
Methods:
For all experiments 8-12-weeks-old pregnant mice were sacrificed at day 14 of pregnancy. The amniotic fluid was collected and specific cell subsets were isolated using MACS cell separation. Cells were then co-cultured with a bone marrow stromal cell line and stimulated in vitro.
The analysis of the population distribution and cytokine production was performed by flow cytometry. To analyze IgM-levels in the supernatant of the co culture, ELISA was used. Statistical analysis was performed using GraphPad Prism software.
Results:
The amniotic fluid contains different developmental stages of B cells, which most likely are of fetal origin. This is supported by the expression of paternal surface markers. An extensive proliferation and switch towards a more mature phenotype upon co-culture shows that the immature subsets of amniotic fluid B cells are able to expand and mature in vitro. Amniotic
fluid B cells spontaneously produce IgM and show functional adaption upon in vitro stimula-tion as evidenced by the increase of cell activation markers.
Conclusion:
For the first time a deep investigation of B-cells within the amniotic fluid was performed, covering phenotype and cell functionality. This work shows that there is a B cell compartment within the amniotic fluid, which, to a certain extent, is able to mature and gain functionality when exposed to external stimuli. This supports the hypothesis of the amniotic fluid as crucial immunological line of defense against inflammatory and infectious challenges during pregnancy.
During pregnancy, the maternal immune system faces a double dilemma: tolerate the growing semi-allogeneic fetus and at the same time protect the mother and the progeny against pathogens. This requires a fine and extremely regulated equilibrium between immune activation and tolerance. As professional antigen presenting cells, B cells and in particular B-1a B cells, can activate or tolerize T cells and thus participate in the generation or regulation of the immune response. B-1a B cells were involved in the humoral immune response leading to pre-eclampsia, one of the main medical complications during pregnancy. Here we demonstrated that B-1a B cells are additionally involved in cellular immune mechanisms associated with pregnancy complications. Using a mouse model of pregnancy disturbances, we showed that B-1a B cells from animals suffering pregnancy disturbances but not from those developing normal pregnancies induce the differentiation of naïve T cells into Th17 and Th1 cells. This differential role of B-1a B cells during pregnancy seems to be associated with the co-stimulatory molecule CD86 as normal pregnant mice showed lower percentages of CD86 expressing B-1a B cells as compared to pregnant mice developing pregnancy disturbances or to non-pregnant animals. Our data bring to light a new and not explored role of B-1a B cells in the context of pregnancy.
With improvements in breast imaging, mammography, ultrasound and minimally invasive interventions, the detection of early breast cancer, non-invasive cancers, lesions of uncertain malignant potential, and benign lesions has increased. However, with the improved diagnostic capabilities comes a substantial risk of false-positive benign lesions and vice versa false-negative malignant lesions. A statement is provided on the manifestation, imaging, and diagnostic verification of isolated benign breast tumours that have a frequent manifestation, in addition to general therapy management recommendations. Histological evaluation of benign breast tumours is the most reliable diagnostic method. According to the S3 guideline and information gained from analysis of the literature, preference is to be given to core biopsy for each type of tumour as the preferred diagnostic method. An indication for open biopsy is also to be established should the tumour increase in size in the follow-up interval, after recurring discrepancies in the vacuum biopsy results, or at the request of the patient. As an alternative, minimally invasive procedures such as therapeutic vacuum biopsy, cryoablation or high-intensity focused ultrasound are also becoming possible alternatives in definitive surgical management. The newer minimally invasive methods show an adequate degree of accuracy and hardly any restrictions in terms of post-interventional cosmetics so that current requirements of extensive breast imaging can be thoroughly met.
Pregnancy involves adaptations of the cellular composition in utero to establish a functioning fetal-maternal interface. Different subsets of leukocytes populate the endometrium and contribute to tolerance of the fetal allograft while protecting it from potentially threatening infections or rejection. ¬¬Innate lymphoid cells are recently discovered immune cells that, besides the gut, lung and skin, possess immunoregulatory functions in the female reproductive tract, especially during gestation. Although present at the fetal-maternal interface, the dynamics of ILC migration during pregnancy remains poorly investigated. The involvement of homing receptors in ILC migration to the uterus was the main subject of the present work.
First, the expression of homing receptors on ILCs from miscellaneous organs was assessed across the course of murine pregnancy in vivo by means of flow cytometry. Then, their migratory capacity towards pregnancy-relevant chemokines was investigated in vitro. The impact of pregnancy related hormones on the migration and homing of ILCs was then analysed in vitro via migration assays.
The results confirm altered proportions of ILCs in utero and the altered expression of homing receptors in ILCs in pregnancy. Different murine lymphoid organs showed augmented expression of chemokine receptors and decreased levels of homing integrin α4β7 in the first trimester, suggesting enhanced migration patterns of ILCs during early pregnancy. Subsequently, migration assays were used to demonstrate the role of different chemokine ligands in enhancing ILC migration.
Eventually, the alterations in homing receptor expression were correlated with female pregnancy hormones. Progesterone treatment caused similar effects on homing receptor expression in ILCs as observed during early gestation. These results represent the first study evaluating the effect of sex steroid hormones on ILC chemokine receptor distribution.
Taken together, our results indicate the involvement of pregnancy-relevant chemokines, including CCL4, CCL20 and CCL28, in the recruitment of ILCs to the uterus during pregnancy. The data highlight an endocrinological-immune crosstalk in the regulation of ILC homing to the female reproductive tract. Gestation alters chemokine receptor expression in order to regulate the access of immune cell subsets to the fetal-maternal interface. An adequate regulation is highly needed, as a lack or abundance of different subgroups could result in pregnancy complications, including fetal loss, pre-eclampsia or pre-term birth. Thus, the role of ILC chemotaxis to the pregnant uterus and its regulation are of interest in the understanding, prevention and treatment of the clinically relevant obstetric diseases.
The aim of this retrospective observational study is to describe and discuss various complications that can arise after insertion of alloplastic materials in the field of urogynecology that require further surgical interventions in order to manage them or to at least improve the quality of life in those women. We were able to collect data on 77 patients who fulfilled the criteria. Medical history, data of clinical findings, and outcomes were collected and analyzed. The most common complication seen as an indication for resecting slings or meshes was de novo overactive bladder syndrome (40%). Other indications seen were lower urinary tract obstruction or obstructive voiding symptoms (21%), chronic pain (21%), and de novo dyspareunia (13%). 36% of the patients had recurrent symptoms (failure) after insertion of alloplastic materials in the form of urinary incontinence or prolapse, 32% presented with vaginal erosions, 2 women had severe signs of infection with abscess formation, another 3 women had urogenital fistulae. Other rare complications after mesh or sling insertion are perforations of the urinary bladder or urethra. Proper case selection is the key factor. The use of meshes and slings seems justified only in patients with known connective tissue weakness and recurrences after native tissue repair. Otherwise, patients will be exposed to unnecessary risk without any expectable improvement to their quality of life. Most of the complications are mainly caused by wrong and inadequate surgical techniques, wrong indications, or missed diagnosis of the underlying problem. In addition, lack of long-term follow-up is usually the cause behind the negligence towards many complications. Therefore, only experienced physicians should be allowed to perform such procedures, and long-term postoperative follow-up is strongly recommended. As slings and meshes are used for procedures of choice as means to improve quality of life, and not for life threatening situations, there is a need for intensive informed consent. All possible alternatives have to be discussed, as do the pros and cons of selected procedures, even the rare complications. Mesh or sling resection is considered to be an effective solution for the management of such complications. It has shown a high success rate in comparison to conservative treatment, and the majority of patients were satisfied and experienced a big improvement in their quality of life. The most common complication after resection is the recurrence of primary symptoms, either urinary incontinence or prolapse. Major or serious intra- or postoperative complications are very rare. All complications were classified and given a code according to the classification system of the international urogynecological association and the international continence society (IUGA/ICS) on 2011. The applicability and practicability of this code were evaluated, looking for ways to possibly improve it or to identify missing parameters. Many patients had more than one code, a problem that entirely torpedoed the idea of “simple” classification. Some complications are not covered individually in the classification, such as failure and recurrence or overactive bladder syndrome. These complications should be included. Many cases began with the same code, despite having different complications. Further sub-classifications should be considered to enable the reader to easily recognize the complication at hand. Patients who came with complications more than one year after mesh or sling insertion were categorized as (T4), regardless of whether the complication arose after 1 year of after 10. Therefore, sub-classifications in the (T4) category are recommended. The “site” category was not applicable in many cases. Furthermore, it is necessary that the severity of a complication is discernible, and should be mentioned in the code. We did not find any correlation between the code given and patient satisfaction. After re-modification and completion, the IUGA/ICS code could be more practical for clinical use, which would allow for the comparison of complications and make the assessment of adverse effects easier for research purposes.
Gynaecologic immunologic research aims to answer an important question: How does the immune system manage to protect both mother and unborn child while not harming the semi-allogeneic and thus partially unaccustomed fetus? Several distinct adaptions in both the innate and adaptive immune system take place during pregnancy. Alterations in these processes can cause dramatic consequences like pregnancy loss. Here, molecules with immunomodulatory functions can provide possible treatment options. One molecule with the described features emerged as a candidate: The transmembrane molecule mCD83 as well as its soluble form, sCD83. As mCD83 overexpressing cells and cells from pregnant mice showed similar behaviour regarding interleukin-10 secretion and B-cell (BC) development, a contribution of mCD83 in immunologic pregnancy adaptions is possible. Additionally, the soluble form could be a future therapeutic agent in pregnancy disorders, regarding its already shown benefits in therapy of various autoimmune diseases in animal models.
The aim of this work is to evaluate the expression, release and regulation of CD83 in its membrane bound and soluble forms during normal and disturbed pregnancies in mouse models.
The semi-allogeneic pairing of two inbred stems, C57Bl6/J×BALB/c, results in healthy pregnancy and was used to investigate the expression in different stages of pregnancy. Pairing CBA/J females with DBA/2J males results in resorption of fetal units and represents a poor pregnancy outcome mating (PPOM). This model in comparison with CBA/J×BALB/c pairings (presenting a good pregnancy outcome mating (GPOM)) is a model for immunologic pregnancy disturbance. It was used to detect alterations in mCD83 expression and sCD83 release during disturbed pregnancy.
During normal murine pregnancy, mCD83 expression increased with a peak on day 14 of pregnancy on B- and T-cells, while the amount of mCD83 positive cells was elevated at the end of pregnancy. PPOM mice showed higher mCD83 expression and mCD83 positive cell count on various lymphocyte subtypes in comparison to GPOM, while sCD83 levels were lower in PPOM pregnancies. Splenocytes released sCD83 in cell culture, whereby the main part under unstimulated conditions was produced by BC. Progesterone treatment of splenocytes led to a dose dependent mCD83 upregulation on T-cells and reduced mCD83 expression as well as sCD83 release from BC. Culture of splenocytes with tissue inhibitor of metalloproteinases 1 (TIMP1) resulted in elevated sCD83 release and mCD83 expression on BC. Progesterone reduced TIMP1 expression on BC in vitro.
mCD83 expression and sCD83 release showed various alterations during normal murine pregnancy as well as when comparing PPOM with GPOM. Noticeable are in particular a higher mCD83 expression on splenic BC on day 14 of pregnancy. In BC from PPOM, mCD83 expression is higher than on BC from GPOM, while PPOM mice show a lower sCD83 serum level, hinting a problem in the shedding mechanism during PPOM.
Progesterone regulates mCD83 expression on BC via TIMP1 and a yet unknown proteinase, resulting in degradation of mCD83 with lower mCD83 expression and sCD83 release. Here, the resulting expression level may vary depending on the BC surroundings and cell compartmentation.
The results thereby suggest a CD83 involvement in pregnancy and encourage further research on mCD83 expression at the feto-maternal interface as well as sCD83 in human blood and tissue. Especially the sCD83 alterations are of clinical interest, indicating the molecule as potential therapeutical option for pregnancy disturbances.
Introduction: For a successful pregnancy, a set of physiological requirements has to be fulfilled. The mother has to provide enough nutrients and the proper anatomical environment for the developing fetus and protect him and herself against pathogens. The cells of the im-mune system constantly monitor the organism in search for pathogens and mount a response to eradicate the threat. The favourable outcome of an immune response re-lays on the capacity of those cells to recognize structures that shouldn’t be present in the organism and the speed or strength at which the cells react. During pregnancy, however, a fetus is able to establish a firm contact with the endometrium of the mother and then grow for an extended period of time. This “exception to the rule” hides behind a set of fine-tuned regulations of the immune responses which are not completely un-derstood. Though many cell types have been extensively investigated in the past dec-ades, B cells play yet enigmatic roles. The aim of this work is to uncover the events occurring within the B cell development during pregnancy and to study the role of certain subtypes in healthy pregnancy and pregnancy miscarriage. Methods: For all experiments, 8-weeks-old female mice either non-pregnant, having normal preg-nancies or miscarriage were used. Organs were removed and cells isolated using standard protocols. The analysis of the population distribution was performed by Flow Cytometry. For in vitro experiments, specific cell subsets were isolated using MACS Cell Separation. Bio-plex method was used for the assessment of Immunoglobulin isotypes in serum, while CBA Array was the method used to measure cytokine levels in the supernatant of cell cultures. Statistical analysis was done using GraphPad Prism software. Results: Pregnancy had a strong impact on the murine B cell development. The restructuration of the B cell compartment could be appreciated already from the bone marrow progeni-tors, reduced in pregnant mice. Peripheral subsets drastically adapted their develop-mental pathways, with a drift towards the generation of marginal zone B cells. B cells also showed functional adaptations to gravidity, as evidenced by the changes in the immunoglobulin production and immunomodulatory capacity. Conclusions: For the first time a deep investigation of the consequences of pregnancy on the B cell development was performed, covering several aspects of B cell functionality. This work shows that B lymphocyte compartment is remodelled during pregnancy. Aberration of this process may lead to pregnancy complications including miscarriage.