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Dysregulations in the differentiation of CD4+-regulatory-T-cells (Tregs) and CD4+-responder-T-cells (Tresps) are involved in the development of active systemic lupus erythematosus (SLE). Three differentiation pathways of highly proliferative inducible costimulatory molecule (ICOS)+- and less proliferative ICOS−-CD45RA+CD31+-recent-thymic-emigrant (RTE)-Tregs/Tresps via CD45RA−CD31+-memory-Tregs/Tresps (CD31+-memory-Tregs/Tresps), their direct proliferation via CD45RA+CD31−-mature naïve (MN)-Tregs/Tresps, and the production and differentiation of resting MN-Tregs/Tresp into CD45RA−CD31−-memory-Tregs/Tresps (CD31−-memory-Tregs/Tresps) were examined in 115 healthy controls, 96 SLE remission patients, and 20 active disease patients using six color flow cytometric analysis. In healthy controls an appropriate sequence of these pathways ensured regular age-dependent differentiation. In SLE patients, an age-independently exaggerated differentiation was observed for all Treg/Tresp subsets, where the increased conversion of resting MN-Tregs/Tresps particularly guaranteed the significantly increased ratios of ICOS+-Tregs/ICOS+-Tresps and ICOS−-Tregs/ICOS−-Tresps during remission. Changes in the differentiation of resting ICOS+-MN-Tresps and ICOS−-MN-Tregs from conversion to proliferation caused a significant shift in the ratio of ICOS+-Tregs/ICOS+-Tresps in favor of ICOS+-Tresps and a further increase in the ratio of ICOS−-Tregs/ICOS−-Tresps with active disease. The differentiation of ICOS+-RTE-Tregs/Tresps seems to be crucial for keeping patients in remission, where their limited production of proliferating resting MN-Tregs may be responsible for the occurrence of active disease flares.

Background and Aims An imbalance between CD4+-regulatory T-cells (Tregs) and CD4+-responder T-cells (Tresps) correlates with active disease flares in Systemic Lupus Erythematosus (SLE) patients. Both cell subsets consist of highly proliferative inducible T-cell co-stimulatory molecule expressing (ICOS+-) and less proliferating ICOS--Treg/Tresp cells. Method In this study we aimed to examine the role of age-dependent differentiation for maintaining the ICOS+-Treg/ICOS+-Tresp or ICOS--Treg/ICOS--Tresp balance in SLE remission patients compared to active disease patients and to evaluate the influence of proliferation inhibitor or glucocorticoid therapy concerning the maintenance of these equilibria. Results In 83 healthy volunteers, the ratio of ICOS+-Tregs/ICOS+-Tresps increased significantly with age, while that of ICOS--Tregs/ICOS--Tresps did not change. In 86 SLE remission patients, medication controlled disease activity was associated with an age-independently increased ratio of both ICOS+-Tregs/ICOS+-Tresps and ICOS--Tregs/ICOS--Tresps. In 13 active disease patients this increased ICOS+-Treg/ICOS+-Tresp ratio could not be maintained, due to an age-dependent earlier terminal differentiation of ICOS+-Tregs than of ICOS+-Tresps. The increased ratio of ICOS--Treg/ICOS--Tresp was maintained in active disease patients, but it decreased with age. This was attributable to an age-independent aberrant differentiation of ICOS--Tresps and a resulting loss of age-dependent differentiation of ICOS--Tregs. Especially the administration of proliferation inhibitors (azathioprine or mycophenolic acid) ensured prolonged differentiation and maintenance of functional ICOS+-Tregs with age. There was a similar but weaker effect of this medication on ICOS+-Tresps, causing a significant decrease of these cells with age. The proliferation capacity was neither affected in ICOS+-Tregs/Tresps nor in ICOS--Tregs/Tresps. As a result, the ratio of ICOS+-Tregs/ICOS+-Tresps increased significantly with age and kept the patients in remission. In contrast, glucocorticoids increased the differentiation of ICOS--Tregs especially in younger individuals, but had an adverse effect on their proliferation capacity. Conclusion Our data reveal a crucial role of the ICOS+-Treg/ICOS+-Tresp balance for the maintenance of self-tolerance and avoidance of disease flares in SLE. The used immunosuppressive therapy, especially the administration of proliferation inhibitors, has a decisive effect on the preservation of regular differentiation of all Treg/Tresp subsets with age, and most importantly avoids the terminal differentiation and immune senescence of ICOS+-Tregs.

Dialysis patients have deficiencies regarding the generation of immune responses and show an increased susceptibility for infections. Persisting uremic conditions are made responsible for the increased aging of their immune system. In this study, we analyzed whether age-related differences in the differentiation of both recent-thymic-emigrant-(RTE)-regulatory (Tregs) and RTE-responder T cells (Tresps) into CD31--memory Tregs/Tresps led to differences in the suppressive activity of naive and memory Tregs on autologous Tresps between healthy volunteers and dialysis patients. We found that regardless of age, the differentiation of RTE-Treg/Tresps into CD31--memory-Treg/Tresps was significantly increased in dialysis patients. By analyzing the age-related differences in the differentiation of Tregs/Tresps, we saw that in healthy volunteers RTE-Tregs differentiate via CD31+-memory Tregs into CD31--memory Tregs, which may strengthen the suppressive activity of the total Treg pool. In contrast RTE-Tresps of healthy volunteers differentiate via mature naive (MN)-Tresps into CD31--memory-Tresps, which may weaken the reactivity of the total Tresp pool. Our data revealed that this normal differentiation via MN-Tresps was lost in dialysis patients, suggesting that their Tresps are less sensitive to Treg-mediated immunosuppression. Functional analysis of Tregs on autologous Tresps showed an increasing suppressive activity with age in healthy individuals, who therefore may have a lower risk of developing autoimmune diseases but owing to decreased reactivity of their Tresps are more likely to suffer from infections. In contrast, dialysis patients exhibited a decreasing suppressive activity with age, owing to strengthened Tresp reactivity, which could explain the higher prevalence of chronic inflammatory conditions in these patients.

Background: Modified immune cells (MICs) are mononuclear cells that gain immunosuppressive properties after incubation with the proliferation inhibitor mitomycin C. We recently showed that syngeneic MIC therapy controlled experimental autoimmune encephalitis (EAE) (1). In addition, allogeneic MIC therapy prevented rejection in rat heart and hindlimb as well as pig kidney transplantation (2). Objectives: We now wanted to translate these encouraging findings to the prevention and treatment of lupus nephritis. Methods: Splenocytes of syngeneic NZB/W F1 (BWF1) donor mice were incubated with mitomycin C and injected into recipient’s tail vein after matching for age and disease activity. Group 1 received no MIC therapy, group 2 standard-dose MIC therapy with 1.5x108/kg BW once and group 3 repeated MIC therapy with 1.5x108/kg BW at weeks 1, 2 and 3. Group 4 received MIC infusions before disease onset as preemptive treatment approach. Disease activity was monitored by loss of body weight, protein excretion, serum creatinine and dsDNA antibodies. Combined primary endpoint was day 40 post-treatment, protein excretion ≥3g/l for 2 consecutive weeks and >30% loss of original body weight. Histopathology with PAS and HE staining was performed to assess degree of lupus nephritis. Regulatory cell subsets were measured in peripheral blood. Results: MIC therapy prevented the progression of fatal lupus nephritis in BWF1 mice. Protein excretion, serum creatinine and dsDNA antibodies were lower in standard-dose (group 2) and preemptive (group 4) groups compared to control group (group 1) whereas repeated MIC therapy after disease onset had no effect (Figure 1A-E). The primary endpoint was reached significantly more often in control group (group 1, 67%) compared to treatment groups 2 (14%), 3 (14%) and 4 (0%) (Figure 1F). Renal architecture was preserved in different MIC treatment groups (groups 2-4) with decreased glomerular and tubular damage scores (Figure 1G). Most importantly, frequencies of CD8+CD25+FoxP3+ regulatory T cells and CD19+CD5+CD1dhigh regulatory B cells were significantly higher in MIC-treated (groups 2-4) compared to control animals of group 1, whereas double negative T cells were markedly reduced (Figure 1H). Conclusion: MIC therapy inhibits progression of active lupus nephritis. Interestingly, preemptive MIC therapy was even able to prevent onset of disease with no significant disease activity at completion of the study. In accordance with our previous pre-clinical EAE experiments (1) and a first in-human clinical trial in living-donor kidney transplantation (TOL-1 study), MIC therapy was able to induce an in-vivo induction of regulatory cell subsets. This clinically applicable cell therapeutic approach may control lupus nephritis by specifically silencing deleterious autoimmune responses. References: [1] Terness P, Oelert T, et al. Mitomycin C-treated dendritic cells inactivate autoreactive T cells: toward the development of a tolerogenic vaccine in autoimmune diseases. Proc Natl Acad Sci USA. 2008 Nov 25;105(47):18442–7. [2] Jiga LP, Ehser S, Kleist C, Opelz G, Terness P. Inhibition of heart allograft rejection with mitomycin C-treated donor dendritic cells. Transplantation. 2007 Feb 15;83(3):347–50. Disclosure of Interests: Claudius Speer Grant/research support from: Research Grant from TolerogenixX GmbH, Daniela Kim Grant/research support from: Research Grant from TolerogenixX GmbH, Christian Kleist Shareholder of: TolerogenixX GmbH, Anita Schmitt Shareholder of: TolerogenixX GmbH, Michael Schmitt: None declared, Claudia Sommerer: None declared, Andrea Steinborn: None declared, Florian Kalble: None declared, Christian Nusshag: None declared, Lei Wang Grant/research support from: Research Grant from TolerogenixX GmbH, Employee of: TolerogenixX GmbH, Alexander Kunz Grant/research support from: Research Grant from TolerogenixX GmbH, Employee of: TolerogenixX GmbH, Hanns-Martin Lorenz: None declared, Martin Zeier: None declared, Christian Morath Shareholder of: TolerogenixX GmbH, Matthias Schaier Shareholder of: TolerogenixX GmbH

Background Modified immune cells (MICs) are mononuclear cells that gain immunosuppressive properties after incubation with the proliferation inhibitor mitomycin C. We showed that syngeneic MIC cell therapy prevented experimental autoimmune encephalitis (EAE) as well as alloreactive rejection in rat heart and hindlimb and pig kidney transplantation. We now aimed to translate these encouraging investigations to prevent murine lupus nephritis and control autoimmunity by MIC therapy. Methods Splenocytes of syngeneic NZB/W F1 (BWF1) mice were harvested from a donor, incubated with mitomycin C and injected into recipients tail vein after matching for age and disease activity. Group 1 received no MIC therapy, group 2 standard-dose MIC therapy with 1.5 × 108/kg BW once and group 3 repeated MIC therapy with 1.5 × 108/kg BW at week 1, 2 and 3, respectively. Group 4 was dosed with repeated MIC therapy before disease onset as preemptive treatment approach. Disease activity was monitored by loss of body weight, protein excretion and serum creatinine. Combined primary endpoint was day 40 post-treatment, protein excretion 3 g/L for 2 consecutive weeks and >30% loss of original body weight. Histopathology with PAS and HE staining was performed to assess degree of lupus nephritis. In the peripheral blood we determined regulatory cell subsets. Results MIC therapy prevented the progression of fatal lupus nephritis in BMF1 mice. Protein excretion and serum creatinine were lower in standard-dose group and preemptive group compared to control group whereas repeated MIC therapy in active disease failed to inhibit both significantly. Accomplishment of combined primary endpoint was significantly increased in group 1 (67%) compared to group 2 (14%), group 3 (14%) and group 4 (0%), respectively. Renal architecture was preserved throughout different MIC treatment groups with decreased glomerular and tubular damage scores. After MIC therapy, frequencies of CD8 +CD25+FoxP3+regulatory T cells and CD19 +CD5+CD1 dhigh regulatory B cells increased significantly, whereas double negative T cells were markedly reduced throughout all treatment groups. Conclusions MIC therapy inhibits progression of active lupus nephritis. Interestingly, preemptive MIC therapy was even able to prevent onset of disease since no significant disease activity was observed at completion of the study. In accordance with our previous pre-clinical EAE experiments and a first in-human clinical trial in living-donor kidney transplantation (TOL-1 study), MIC therapy enables an in-vivo induction of regulatory cell subsets. This clinically applicable cell therapeutic approach may control lupus nephritis by specifically silencing deleterious autoimmune response. Funding Source(s): Research grant from TolerogenixX GmbH Disease activity was assessed after MIC cell therapy by loss of original body weight (A), protein excretion via semiquantitative sticks (B), albuminuria via ELISA (C) and serum creatinine via ELISA (D). Primary endpoint was defined as day 40 post-treatment, protein excretion 3 g/L for 2 consecutive weeks and >30% loss of original body weight (E). Histopathology with PAS and HE staining was performed. Representative PAS staining for different treatment groups and the control group are shown (F). Different regulatory cell subsets as well as DN T cells were determined in peripheral blood. Representative dot plots of regulatory CD19 +CD5+CD1 dhigh B cells, CD8 +CD25+FoxP3+T cells and CD3 +CD4 CD8- double-negative T cells are shown (G).

Problem Recent studies revealed appropriate differentiation of recent thymic emigrant (RTE)-regulatory T cells (Tregs) to be crucial for maintaining healthy pregnancy. Currently, the role of responder T cells (Tresps) is not known. Method of study Six-color flow cytometric analysis was used to detect differences in the differentiation of highly proliferative inducible co-stimulatory (ICOS)+-RTE-Tregs/Tresps and apoptosis-sensitive ICOS−-RTE-Tregs/Tresps into mature naive (MN)-, CD31+-memory and CD31−-memory Tregs/Tresps in women with spontaneous preterm labor (sPL) compared to healthy pregnancy. Results Healthy pregnancy was characterized by an increased differentiation of ICOS+- and ICOS−-RTE-Tregs, as well as ICOS+-RTE-Tresps via CD31+-memory Tregs/Tresps into CD31−-memory Tregs/Tresps. Women with sPL showed an early interruption of RTE-Treg/Tresp differentiation. Instead, ICOS+-MN-Tresps and partly ICOS−-MN-Tregs differentiated into CD31−-memory Tregs/Tresps, causing a significant reduction of both ICOS+-Tregs and ICOS+-Tresps, but an increase of ICOS−-Tresps within total CD4+-T-helper cells. Conclusion Aberrant differentiation of ICOS+-T cells is associated with sPL.

Background CD4+ T cells are of great importance in the pathogenesis of systemic lupus erythematosus (SLE), as an imbalance between CD4+ regulatory T cells (Tregs) and CD4+ responder T cells (Tresps) causes flares of active disease in SLE patients. In this study, we aimed to find the role of aberrant Treg/Tresp cell differentiation for maintaining Treg/Tresp cell balance and Treg functionality. Methods To determine differences in the differentiation of Tregs/Tresps we calculated the percentages of CD45RA+CD31+ recent thymic emigrant (RTE) Tregs/Tresps and CD45RA+CD31− mature naive (MN) Tregs/Tresps, as well as CD45RA−CD31+ and CD45RA−CD31− memory Tregs/Tresps (CD31+ and CD31− memory Tregs/Tresps) within the total Treg/Tresp pool of 78 SLE remission patients compared with 94 healthy controls of different ages. The proliferation capacity of each Treg/Tresp subset was determined by staining the cells with anti-Ki67 monoclonal antibodies. Differences in the autologous or allogeneic Treg function between SLE remission patients and healthy controls were determined using suppression assays. Results With age, we found an increased differentiation of RTE Tregs via CD31+ memory Tregs and of RTE Tresps via MN Tresps into CD31− memory Tregs/Tresp in healthy volunteers. This opposite differentiation of RTE Tregs and Tresps was associated with an age-dependent increase in the suppressive activity of both naive and memory Tregs. SLE patients showed similar age-dependent Treg cell differentiation. However, in these patients RTE Tresps differentiated increasingly via CD31+ memory Tresps, whereby CD31− memory Tresps arose that were much more difficult to inhibit for Tregs than those that emerged through differentiation via MN Tresps. Consequently, the increase in the suppressive activity of Tregs with age could not be maintained in SLE patients. Testing the Tregs of healthy volunteers and SLE patients with autologous and nonautologous Tresps revealed that the significantly decreased Treg function in SLE patients was not exclusively attributed to an age-dependent diminished sensitivity of the Tresps for Treg suppression. The immunosuppressive therapy reduced the accelerated age-dependent Tresp cell proliferation to normal levels, but simultaneously inhibited Treg cell proliferation below normal levels. Conclusions Our data reveal that the currently used immunosuppressive therapy has a favorable effect on the differentiation and proliferation of Tresps but has a rather unfavorable effect on the proliferation of Tregs. Newer substances with more specific effects on the immune system would be desirable. Electronic supplementary material The online version of this article (10.1186/s13075-018-1778-6) contains supplementary material, which is available to authorized users.

Regulatory T cells (Tregs) were shown to be involved into the pathogenesis of acute rejection after transplantation. The suppressive activity of the total regulatory T cell pool depends on its percentage of highly suppressive HLA-DR(+) -Treg cells. Therefore, both the suppressive activity of the total Treg pool and the extent of HLA-DR expression of HLA-DR(+) -Tregs (MFI HLA-DR) were estimated in non transplanted volunteers, patients with end-stage renal failure (ESRF), healthy renal transplant patients with suspicion on rejection, due to sole histological Bord-R or sole acute renal failure (ARF), and patients with clinically relevant borderline rejection (Bord-R and ARF). Compared to patients with only Bord-R or only ARF, the suppressive activity of the total Treg cell pool was exclusively reduced in patients with clinically relevant Bord-R. In parallel, the HLA-DR MFI of the DR(+) -Treg subset was significantly decreased in these patients, due to a significantly lower proportion of DR(high+) -Tregs, which were shown to have the highest suppressive capacity within the total Treg pool. Our findings clearly demonstrate that the determination of the HLA-DR MFI of the HLA-DR(+) -Treg subset allows a highly sensitive, specific and non-invasive discrimination between patients with clinically relevant Bord-R (Bord and ARF) and patients with subclinical rejection or other causes of transplant failure.

Methylprednisolone is widely used to improve immune suppression in transplanted patients threatened by acute rejection. Recently, we showed that the suppressive activity of a Treg cell population depends decisively on their percentage of highly suppressive HLA-DR(high+)-Treg cells, which are strongly reduced in rejecting transplant patients. In order to examine whether the composition of the total CD4(+)CD127(low+/-)FoxP3(+)-Treg cell pool with different Treg-subsets (DR(high+)CD45RA(-)-Tregs, DR(low+)CD45RA(-)-Tregs, DR(-)CD45RA(-)-Tregs, DR(-)CD45RA(+)-Tregs) is affected by methylprednisolone bolus therapy we compared the percentages of these four different Treg cell subsets in transplant patients with biopsy proven rejection before and after steroid bolus therapy (n=23). In patients treated with steroid bolus therapy, the percentage of the naïve DR(-)CD45RA(+)-Tregs was significantly decreased, whereas the percentage of the DR(+)CD45RA(-)-Tregs was significantly increased. By that, the strongest increase was detected for the most suppressive DR(high+)CD45RA(-)-Tregs. However, these effects were only temporarily and closely associated to the duration of the bolus therapy. Our results suggest that besides various anti-inflammatory effects on cells of the adaptive and innate immune system, methylprednisolone also has the capacity to enhance the suppressive activity of the total Treg cell pool by increasing its percentage of highly differentiated and highly suppressive DR(high+)CD45RA(-)-Tregs.

Regulatory T cells (Tregs) exert a key role in tolerance induction to the semi-allogeneic fetus. Currently, it is not known whether immunological rejection processes are involved in the induction of normal term or irresistible preterm labor. In this study, we examined whether there were differences in the percentage of the total CD4 þ CD127 low þ/ � CD25 þ FoxP3 þ -Treg-cell pool, its suppressive activity and its composition with distinct Treg subsets (HLA-DR low þ -, HLA-DR high þ -, HLA-DR � - and naive CD45RA þ -Tregs) between preterm and term laboring women. We found that its percentage was decreased neither in term nor in preterm laboring women. Its suppressive activity was strongly diminished in preterm laboring women and to a lesser extent in spontaneously term laboring women. During the normal course of pregnancy, its composition changed in such a way that the percentage of naive CD45RA þ -Tregs increased while the percentage of the highly suppressive HLA-DR low þ - and HLA-DR high þ Tregs decreased significantly until term. With the onset of spontaneous term labor this phenomenon was reversed and reached significant values postpartum. In addition, we confirmed that both the decreased percentage of HLA-DR þ -Tregs within the total Treg-cell pool and their decreased level of HLA-DR expression (depending on the percentage of HLA-DR low þ - and HLA-DR high þ Tregs) had a reducing effect on the suppressive activity of the total Treg cell pool in preterm laboring women. However, spontaneous term delivery was associated with increasing percentages of HLA-DR þ -Tregs and increasing HLA-DR expression of this Treg subset. Therefore, it becomes apparent that the mechanisms inducing term or preterm labor may be completely different.

Dysregulations concerning the composition and function of regulatory T cells (T(regs)) are assumed to be involved in the pathophysiology of complicated pregnancies. We used six-colour flow cytometric analysis to demonstrate that the total CD4(+) CD127(low+/-) CD25(+) forkhead box protein 3 (FoxP3)(+) T(reg) cell pool contains four distinct T(reg) subsets: DR(high+) CD45RA(-), DR(low+) CD45RA(-), DR(-) CD45RA(-) T(regs) and naive DR(-) CD45RA(+) T(regs). During the normal course of pregnancy, the most prominent changes in the composition of the total T(reg) cell pool were observed between the 10th and 20th weeks of gestation, with a clear decrease in the percentage of DR(high+) CD45RA(-) and DR(low+) CD45RA(-) T(regs) and a clear increase in the percentage of naive DR(-) CD45RA(+) T(regs). After that time, the composition of the total T(reg) cell pool did not change significantly. Its suppressive activity remained stable during normally progressing pregnancy, but decreased significantly at term. Compared to healthy pregnancies the composition of the total T(reg) cell pool changed in the way that its percentage of naive DR(-) CD45RA(+) T(regs) was reduced significantly in the presence of pre-eclampsia and in the presence of preterm labour necessitating preterm delivery (PL). Interestingly, its percentage of DR(high+) CD45RA(-) and DR(low+) CD45RA(-) T(regs) was increased significantly in pregnancies affected by pre-eclampsia, while PL was accompanied by a significantly increased percentage of DR(-) CD45RA(-) and DR(low+) CD45RA(-) T(regs). The suppressive activity of the total T(reg) cell pool was diminished in both patient collectives. Hence, our findings propose that pre-eclampsia and PL are characterized by homeostatic changes in the composition of the total T(reg) pool with distinct T(reg) subsets that were accompanied by a significant decrease of its suppressive activity.

Little information exists concerning the role of fetal regulatory T cells (Tregs) during intrauterine development. We examined whether complications such as reduced birth weight or the occurrence of preterm labor were associated with deficiencies in the number or in the immunosuppressive activity of Tregs in the fetal circulation. Their total number did not change during normal or complicated pregnancy. In contrast, their level of FoxP3 expression decreased continuously with gestational age and was significantly reduced in the presence of spontaneous term, but not preterm labor. In small for gestational age (SGA) neonates, FoxP3 expression was constantly decreased when compared to age matched healthy neonates. In accordance with the low FoxP3 expression, the suppressive activity of the Tregs from spontaneously term delivered and from SGA babies was significantly reduced. We propose that the level of FoxP3 expression in the fetal Tregs may be a potential regulator of their suppressive activity.

The proper function of the fetal heart is indispensable for the fetal development and the normal fetal growth. For prenatal medicine, Doppler sonography offers the possibility of a non-invasive method to examine the fetal cardiovascular function under normal and pathological circumstances. The role of the Doppler sonography is to identify those fetuses who have a high risk factor for developing a pre- or intrapartual asphyxia and therefore have to be delivered promptly. This study aimed at evaluating the clinical value of the intracardiac Doppler sonography (IDS) and at scrutinizing its usefulness during the supervision of the pregnancy of intrauterine growth restricted (IUGR) fetuses. In a prospective research at the Medical School of Hanover, fetal IDS was applied to 174 pregnant women between the 21 and 37 weeks of gestation (WG). The e-wave and the a-wave, the E/A ratio, and the TVI (time velocity integral) were measured at the atrioventricular (AV) valves. The PV (peak velocity) as well as the TVI were determined at both the aortic and the pulmonary valve. Normal range curves were compiled for all measured parameters. Alongside a control group with untroubled gravidity, which consisted of 153 patients, IUGR fetuses, who formed a collective of 21 patients, were Doppler sonographically examined. While the gestational age advanced, an increase of both the e-wave and the a-wave above the AV-valves could be ascertained, which lead to an E/A ratio

Two different subsets of naturally occurring regulatory T cells (nTregs), defined by their expression of the inducible co-stimulatory (ICOS) molecule, are produced by the human thymus. To examine the differentiation of ICOS(+) and ICOS(-) CD45RA(+) CD31(+) recent thymic emigrant (RTE) T regs during normal pregnancy and in the presence of pre-eclampsia or haemolysis elevated liver enzymes low platelet (HELLP)-syndrome, we used six-colour flow cytometric analysis to determine the changes in the composition of the ICOS(+) and ICOS(-) T reg pools with CD45RA(+) CD31(+) RTE T regs, CD45RA(+) CD31(-) mature naive (MN) T regs, CD45RA(-) CD31(+) and CD45RA(-) CD31(-) memory Tregs. With the beginning of pregnancy until term, we observed a strong differentiation of both ICOS(+) and ICOS(-) CD45RA(+) CD31(+) RTE, but not CD45RA(+) CD31(-) MN T regs, into CD45RA(-) CD31(-) memory T regs. At the end of pregnancy, the onset of spontaneous term labour was associated with a significant breakdown of ICOS(+) CD45RA(-) CD31(-) memory T regs. However, in the presence of pre-eclampsia, there was a significantly increased differentiation of ICOS(+) and ICOS(-) CD45RA(+) CD31(+) RTE T regs into CD45RA(-) CD31(+) memory T regs, wherein the lacking differentiation into CD45RA(-) CD31(-) memory T regs was partially replaced by the increased differentiation of ICOS(+) and ICOS(-) CD45RA(+) CD31(-) MN Tregs into CD45RA(-) CD31(-) memory T regs. In patients with HELLP syndrome, this alternatively increased differentiation of CD45RA(-) CD31(-) MN T regs seemed to be exaggerated, and presumably restored the suppressive activity of magnetically isolated ICOS(+) and ICOS(-) T regs, which were shown to be significantly less suppressive in pre-eclampsia patients, but not in HELLP syndrome patients. Hence, our findings propose that the regular differentiation of both ICOS(+) and ICOS(-) CD45RA(+) CD31(+) RTE T regs ensures a healthy pregnancy course, while their disturbed differentiation is associated with the occurrence of pre-eclampsia and HELLP syndrome.

Problem Gestational diabetes (GD) may be associated with temporarily reduced immune tolerance toward alloantigens for the time of pregnancy. The aim of this study was to assess anti-HLA-class I and -II antibodies as markers for an aberrant immunostimulation in women with GD. Method of study The percentage of anti-HLA-class I and -II antibodies was estimated in women with GD, normal term delivery and fetal distress, which was confirmed by demonstrating low cord blood pH for this patient group. These antibodies may cross the placental barrier and cause interleukin-6 (IL-6) release from fetal monocytes by cross-linking monocytes with antibody-loaded cells. Therefore we estimated the percentage of IL-6-positive monocytes in the fetal circulation of these three patient groups. Results We found a significantly increased percentage of anti-HLA-class II in the circulation of women with GD. In comparison with women with normal term delivery, a significantly increased percentage of IL-6-positive monocytes was detected for women with GD and for women with fetal distress. Significantly decreased cord blood pH were detected for neonates born in the presence of fetal distress but not for neonates born in the presence of GD. Conclusions Our results suggest that GD is associated with an increased humoral immune response against HLA-class II antigens.

During pregnancy, regulatory T cells (Tregs) have a key role in maternal immune tolerance to the semi-allogeneic fetus. Our previous results showed that the naive CD45RA(+)-Treg pool is functionally improved in pregnant women compared with non-pregnant women. Therefore, we examined the thymic output and differentiation of CD45RA(+)CD31(+) recent thymic emigrant (RTE)-Tregs during normal pregnancy and in the presence of preeclampsia. With the onset of pregnancy, the composition of the total CD4(+)CD127(low+/-)FoxP3(+)-Treg pool changed in the way that its percentage of RTE- and CD45RA(-)CD31(+)-memory Tregs decreased strongly, whereas that of the CD45RA(+)CD31(-)-mature naive (MN)-Tregs did not change and that of the CD45RA(-)CD31(-)-memory Tregs increased complementary. Thereby, the ratio of RTE-/MN-Tregs decreased from 1.0 to 0.7 leading to a significant increase in the suppressive activity of the naive CD45RA(+)-Treg pool. This effect was confirmed by re-assembling separated RTE- and MN-Tregs from non-pregnant women in the ratio of pregnant women. The suppressive activity of both separated naive Treg subsets was equally high in non-pregnant and pregnant women, but considerably reduced in preeclampsia patients, who showed significantly increased percentages of CD45RA(-)CD31(+)-memory Tregs, but decreased percentages of RTE- and MN-Tregs. Our results suggest a reduced thymic output of RTE-Tregs during pregnancy, which causes a decrease in the ratio of RTE-/MN-Tregs and thus an increase in the differentiation of RTE-Tregs towards CD45RA(-)CD31(-)-memory Tregs. Presumably, this differentiation of RTE-Tregs, which was impaired in preeclampsia patients, ensures the improved suppressive activity of the CD45RA(+)-naive Treg pool and thus retains the maintenance of pregnancy.

Objectives: We aimed to find answers to the following questions: What are the technical and biological prerequisites for easily obtainable three-dimensional (3D) images? What are the visualization rates for various fetal organ systems? What is the potential for assessing fetal malformations? What are the psychological effects of 3D imaging on the expectant mothers? Methods: Between January and June 1998, 433 pregnant women were prospectively examined with two-dimensional (2D) and 3D sonography. Results: 3D visualization in healthy fetuses was inferior in quality to 2D visualization, which also accounted for the comparison of 3D imaging versus 2 D imaging among fetuses affected with malformations. In only 1 case did 3D imaging yield a slightly better description of the given malformation. This did not result in a different therapeutical approach. Concerning the psychological effect of 3D imaging, a marked approval of the 3D method was recorded. Conclusions: These results show that the image information acquired by 3D ultrasound technology is nearly always inferior to the image information obtained by conventional 2D imaging. 3D imaging can be useful for specific malformations under the condition that these examinations be performed in specific ultrasound departments. Thus, a clearly defined range of indications can be assigned to 3D imaging.

Objective: The aim of this study was to evaluate the clinical utility of a novel 3D scanner system for real-time 3D fetal echocardiography. Method: In a prospective study, 13 single, healthy 20- to 24-week-old fetuses were examined with conventional 2D and real-time 3D echocardiography. The visualization rates and imaging quality of standard cardiac views were compared between both methods. Results: The visualization rates of standard cardiac planes were found to be slightly increased and more easily obtainable in 3D imaging whereas the image quality showed better results with conventional 2D echocardiography. Conclusion: Our data show that real-time 3D fetal echocardiography can be considered a useful tool in the evaluation of the fetal heart with the necessity for further refinement of the resolution quality

Recent studies show that regulatory T cells (Tregs) play an essential role in tolerance induction after organ transplantation. In order to examine whether there are differences in the composition of the total CD4(+)CD127(low+/-)FoxP3(+)- Treg cell pool between stable transplant patients and patients with biopsy proven rejection (BPR), we compared the percentages and the functional activity of the different Treg cell subsets (DR(high+)CD45RA(-)-Tregs, DR(low+)CD45RA(-)-Tregs, DR(-)CD45RA(-)-Tregs, DR(-)CD45RA(+)-Tregs). All parameters were determined during the three different periods of time after transplantation (0-30 days, 31-1,000 days, >1,000 days). Among 156 transplant patients, 37 patients suffered from BPR. The most prominent differences between rejecting and non-rejecting patients were observed regarding the DR(high+)CD45RA(-)-Treg cell subset. Our data demonstrate that the suppressive activity of the total Treg pool strongly depends on the presence of these Treg cells. Their percentage within the total Treg pool strongly decreased after transplantation and remained relatively low during the first year after transplantation in all patients. Subsequently, the proportion of this Treg subset increased again in patients who accepted the transplant and reached a value of healthy non-transplanted subjects. By contrast, in patients with acute kidney rejection, the DR(high+)CD45RA(-)-Treg subset disappeared excessively, causing a reduction in the suppressive activity of the total Treg pool. Therefore, both the monitoring of its percentage within the total Treg pool and the monitoring of the HLA-DR MFI of the DR(+)CD45RA(-)-Treg subset may be useful tools for the prediction of graft rejection.

Regulatory T cells (Tregs) are known to suppress alloimmune responses during pregnancy and post organ transplantation. We demonstrate that a distinct subset of FoxP3(+)DR(+)-Tregs among the total CD4(+)CD127(low+/-)CD25(+)-Treg cell pool is critically involved in preterm labor induction and kidney transplant rejection as well. Compared to healthy pregnancies and non-rejecting kidney recipients, we found that the percentage of the FoxP3(+)DR(+)-Treg subset was not reduced, but that the level of HLA-DR expression of such Tregs was strongly diminished in preterm laboring women and in patients with acute renal allograft rejection. In addition, both patient collectives showed a significantly reduced suppressive activity of their circulating CD4(+)CD127(low+/-)CD25(+)-Treg cell pool. Our findings propose that the FoxP3(+)DR(+)-Treg subset may be decisively responsible for the suppressive activity of the total CD4(+)CD127(low+/-)CD25(+)-Treg cell pool and that the immunologic mechanisms leading to preterm labor necessitating preterm delivery may be similar to those leading to allograft rejection after transplantation.

Problem Soluble (s) HLA-G1/G5 molecules may potentially affect immune homeostasis during pregnancy. The aim of this study was to determine changes of sHLA-G1/G5 plasma levels throughout normal pregnancy and to assess its predictive value for the occurrence of characteristic gestation-associated diseases during further course of pregnancy. Method of study sHLA-G1/G5 levels were estimated in plasma samples of 40 non-pregnant women, 291 women throughout normal pregnancy and 236 women affected by different complications. Results In comparison with non-pregnant women sHLA-G1/G5 levels strongly increased during the first trimenon and then decreased continuously toward term. Non-parametric discriminant analysis showed that women with significantly decreased sHLA-G1/G5 levels in the second trimenon had an increased risk of developing preeclampsia and/or intrauterine growth retardation (IUGR) during further course of pregnancy. However, in the third trimenon, sHLA-G1/G5 levels in affected women did not deviate significantly from those of non-affected women. Surprisingly, significantly increased sHLA-G1/G5 levels were detected in third trimenon women with uncontrollable preterm labor, but not in women with prolonged preterm rupture of fetal membranes. Conclusion For the identification of women with an increased risk of IUGR and/or preeclampsia, measurement of sHLA-G1/G5 plasma levels may be a powerful new tool in prenatal diagnostics.

First-trimester screening according to Nicolaides uses maternal age to obtain a common background risk for trisomy 21. The likelihood ratios by nuchal translucency, free beta-human chorionic gonadotropin and pregnancy-associated plasma protein-A are not with respect to maternal age. It was the aim of this study to investigate if likelihood ratios should better take care of it.Pearson's correlation and different models of regression analysis had been performed on the results of 8,116 first-trimester screenings. The total number of pregnancies was subdivided into three subgroups of healthy fetuses (n = 8,038); fetuses with Down's syndrome (n = 46) and fetuses with other genetic abnormalities (n = 32). Statistical testing was applied to each of the three groups.Strong independence from maternal age could be found for each of the first-trimester screening measurement parameter, as well for healthy and as for affected fetuses. Neither Pearson's test nor nonlinear regression models could detect a correlation. Accordingly significance of Pearson's test is not given.First-trimester screening would not be improved by considering the maternal age in the calculation of the likelihood ratios. Therefore the currently used algorithm is adequate. According, to the results, it seems to be proper as well to disregard the maternal age in newer test strategies advanced first-trimester screening (AFS) at all.

Objective Fetal genetic materials in maternal circulation might be potentially used for non-invasive prenatal diagnosis (NIPD). In this study, we quantitatively analysed the intact fetal cells existing in maternal plasma, which would be a special method of NIPD. Study design Eleven samples from preeclamptic patients, two samples from patients with RhD incompatibilities, and 30 samples from normal pregnancies as controls were collected. The intact cells in the plasma fraction were separated by centrifugation at high speed. The intact cell pellets were washed with PBS to remove any cell-free DNA. Results We were able to detect intact fetal cells in 3 out of 11 preeclamptic plasma samples from women carrying male fetuses, and from both plasma samples of RhD incompatibility affected pregnancies. However, intact fetal cells were not detected in all the 16 normal pregnancies with a male fetus, although cell-free fetal DNA was detected in all these cases. Conclusions Intact fetal cells might be present in maternal plasma. However, the rarity of these cells limits their use for reliable, non-invasive prenatal diagnosis. The detection of such cells was successful in some preeclamptic and RhD incompatibility samples, not in the normal controls. Therefore, as opposed to free fetal DNA in maternal blood the use of intact fetal cells does not provide a reliable mode for NIPD.

Objective: We recently showed that both maternal and fetal erythroblast counts are elevated in the peripheral blood of pregnant women with preeclampsia. The purpose of this study was to examine whether this elevation actually occurs before the clinical onset of the disorder. Study Design: Erythroblasts were enriched and enumerated in 97 maternal blood samples obtained in the second trimester, and results were subsequently correlated with pregnancy outcomes. Results: Significantly higher quantities of erythroblasts (mean, 6041.7 vs 928.9; P =.008) were detected in blood samples obtained from women who later acquired preeclampsia (n = 15) than in blood samples from the control cohort (n = 72). Intrauterine growth restriction (n = 10) was not accompanied by a similar rise in erythroblast count. Conclusion: Because a large proportion of the erythroblasts in maternal blood are fetal, our data suggest that fetal-maternal cell traffic is affected early in pregnancies that are later complicated by preeclampsia but not in those affected only by intrauterine growth restriction. (Am J Obstet Gynecol 2001;184:165-8.)