Fetal Lymphocytes Research Articles

Результати комплексних пренатальних обстежень за наявності дисбалансу хромосоми 21 у плода

Purpose - to characterize the cases of chromosome 21 imbalance in fetuses of high-risk pregnant women, to describe prenatal cytogenetic diagnosis and associated ultrasound findings - structural anomalies and soft markers. Materials and methods. The results of complex prenatal examination of high-risk pregnant women in the Department of Fetal Medicine of the SI “Institute of Pediatrics, Obstetrics and Gynecology named after academician O.M. Lukyanova of the NAMS of Ukraine” during 5 years period were analyzed: results of ultrasound and cytogenetic exams of 200 cases of chromosome 21 imbalance in the fetus. Results. Among 4,748 prenatal cytogenetic testing of fetuses of high-risk women abnormal karyotypes with an imbalance of chromosome 21 were found in 200 (4.21%) cases. In the majority of cases Down's syndrome (DS) was diagnosed (n=199; 99.5%), in 1 (0.2%) case - deletion of chromosome 21. In DS, regular trisomy of chromosome 21 was found in 191 (95.97%) cases, translocant forms - in 5 (2.51%) cases, in 1 (0.5%) case there was there was an additional sex chromosome, and in 1 (0.5%) - a variant with two cell lines. In 1 (0.5%) case limited placental mosaicism was present, with a false negative result that required verification by the analysis of fetal lymphocytes obtained during cordocentesis. Advanced maternal age (≥35 years) was registered in less than half of fetal DS cases (n=92/199, 46.2%). The main indications for invasive procedures and their types are described. It was demonstrated that ultrasound findings (structural anomalies and/or “soft” markers for chromosomal pathology) were present in 78% of fetal chromosome 21 imbalance; nevertheless in 22% ultrasound findings were absent, and the indication for invasive procedures were advanced maternal age or isolated changes of biochemical markers. Conclusions. Chromosome 21 imbalance in fetuses is mainly represented by trisomy, cases of chromosome 21 deletion are extremely rare. Targeted ultrasound examination is important for screening and diagnosis of fetal chromosomal abnormalities. High-risk pregnant women shouldn’t be reassured in case of “normal” ultrasound exam results. Under certain conditions, it is advisable to perform placental biopsy in the II trimester of pregnancy, but only in specialized tertiary centers or departments. The research was carried out in accordance with the principles of the Helsinki Declaration. The study protocol was approved by the Local Ethics Committee of the participating institution. The informed consent of the patient was obtained for conducting the studies. No conflict of interests was declared by the authors.

Fatty acid composition and metabolic partitioning of α-linolenic acid are contingent on life stage in human CD3+ T lymphocytes.

Immune function changes across the life course; the fetal immune system is characterised by tolerance while that of seniors is less able to respond effectively to antigens and is more pro-inflammatory than in younger adults. Lipids are involved centrally in immune function but there is limited information about how T cell lipid metabolism changes during the life course. We investigated whether life stage alters fatty acid composition, lipid droplet content and α-linolenic acid (18:3ω-3) metabolism in human fetal CD3+ T lymphocytes and in CD3+ T lymphocytes from adults (median 41 years) and seniors (median 70 years). Quiescent fetal T cells had higher saturated (SFA), monounsaturated fatty acid (MUFA), and ω-6 polyunsaturated fatty acid (PUFA) contents than adults or seniors. Activation-induced changes in fatty acid composition differed between life stages. The principal metabolic fates of [13C]18:3ω-3 were constitutive hydroxyoctadecatrienoic acid synthesis and β-oxidation and carbon recycling into SFA and MUFA. These processes declined progressively across the life course. Longer chain ω-3 PUFA synthesis was a relatively minor metabolic fate of 18:3ω-3 at all life stages. Fetal and adult T lymphocytes had similar lipid droplet contents, which were lower than in T cells from seniors. Variation in the lipid droplet content of adult T cells accounted for 62% of the variation in mitogen-induced CD69 expression, but there was no significant relationship in fetal cells or lymphocytes from seniors. Together these findings show that fatty acid metabolism in human T lymphocytes changes across the life course in a manner that may facilitate the adaptation of immune function to different life stages.

Mosaic duplication of 8q24.1q24.3 detected by chromosomal microarray but not karyotyping in two unrelated fetuses with cardiac defects

BackgroundDiscordance between traditional cytogenetic and molecular cytogenetic tests is rare but not uncommon. The explanation of discordance between two genetic methods is difficult but especially important for genetic counseling, particularly for prenatal genetic diagnosis.Case presentationTwo unrelated fetuses were diagnosed with cardiac defects by prenatal ultrasound examination, and invasive cordocentesis was performed to obtain cord blood samples for prenatal genetic diagnosis. For both fetuses, chromosomal microarray analysis (CMA) detected a novel approximately 27-Mb mosaic duplication with a high copy number of approximately six to seven copies on chromosome 8q24.1q24.3 that was not identified by karyotyping. To exclude artificial errors and validate laboratory detection results, multiple procedures including copy number variation sequencing, fluorescence in situ hybridization, and short tandem repeat and single-nucleotide polymorphism genotype comparison were performed, confirming the discordant results between CMA and karyotyping. The potential causes of discordance between CMA and karyotyping using fetal blood lymphocytes are discussed; we suggest that extrachromosomal DNA or cell-free DNA fragmentation originating from certain tumor tissues with 8q24.1q24.3 duplication might deserve further investigation.ConclusionsThis study may be helpful for prenatal evaluation and genetic counseling for subsequent patients with similar mosaic 8q24.1q24.3 duplications. Additionally, more cases and further research are needed to understand whether mosaic 8q24.1q24.3 duplication is associated with certain genetic disorders and to investigate the causes of discordance between molecular and morphological methods.

Fetal Macrophages Exposed to Salmonella Antigens Elicit Protective Immunity Against Overwhelming Salmonella Challenge in A Murine Model.

Despite the evidence for fetal immunization following maternal infection, it remained a mystery how the fetal immune system was primed by vertically-transmitted pathogens or microbial antigens, especially before its full maturation. We previously demonstrated the capacity of fetal macrophages for endocytosing oncoprotein and allergens to bridge towards adaptive immunity in postnatal life. To investigate the immunological consequences of fetal contact with microbial antigens and the role of fetal macrophages in the defense against infection before T-cell development, we exposed gestational day 14 murine fetuses and their macrophages to flagellin and heat-killed Salmonella Typhimurium. Recipients with in utero exposure to Salmonella antigens or adoptive transfer of microbial antigen-loaded fetal macrophages were examined for immune responses to Salmonella antigens and resistance to virulent Salmonella challenge. Fetal exposure to microbial antigens or adoptive transfer of microbial antigen-loaded fetal macrophages could confer antigen-specific adaptive immunity. However, protective immunity against lethal Salmonella challenge was only granted to those receiving heat-killed Salmonella antigens, presenting as heightened recall responses of serum anti-lipopolysaccharide immunoglobulins and interferon-gamma. In immunized recipients surviving Salmonella challenge, their serum transfer to succeeding recipients provided immediate protection from lethal Salmonella challenge in preference to lymphocyte transfer, indicating a more active role of humoral immunity in the prevention of Salmonella invasiveness. Our study sheds insight on the role of fetal macrophages in immunogenicity to transplacental pathogens regardless of fetal lymphocyte maturity, paving the way for fetal macrophage therapies to enhance vaccine responsiveness or increase resistance to pathogenic microorganisms in perinatal life.

Immunoadsorption, Intravenous Immunoglobulins and Rituximab (IIR): Successful New Treatment Approach for Severe Anti K- Alloimmunisation during Pregnancy

Background: Fetal red blood cells (RBC), erythroid and megakaryopoietic progenitor cells start to express the Kell (K) antigen already during the 10th gestational week (GW). Maternal anti-Kell alloimmunization (allo-anti-K-Ab) causes early (< 20th GW) and severe hemolytic disease of the fetus/newborn (HDFN) accompanied by erythroaplasia and potential thrombocytopenia. Therefore, prevention/delay of HDFN is highly relevant. Current treatment options include intra-uterine transfusions (IUT) of the fetus in case of suspected anemia, i.v. high-dose immunoglobulins (IVIG), plasma exchange and immunoadsorption (IA). Their efficacy often remains unsatisfactory and procedure related complications might occur. We present our new triple-treatment approach, combining IA + IVIG + Rituximab (Rtx) (IIR), which we successfully applied in 2 high-risk patients with allo-anti-K-Ab. Methods: The first patient was a 24 y, para 1, gravida 2, with an allo-anti-K-AB titer of 1:2000 in the 15th GW, at first presentation. Duplex-sonography showed no signs of fetal anemia. The second patient was a 40 y para 1, gravida 2. She presented with an allo-anti-K-Ab titer of 1:8000 in the 20th GW. Duplex-sonography revealed signs of severe anemia of fetus, requiring immediate IUT. Fetal K- genotyping (RT-PCR) using cell-free fetal DNA from maternal plasma confirmed a K- positive status in both fetuses. Duplex sonography monitoring was executed weekly, including the assessment of arteria cerebri media peak systolic velocity (ACM PSV). After having received informed consent of patients, we started the treatment at 14th and 21st GW in our first and second patient, respectively. IIR treatment cycles (1 cycle = 21 days) comprised 6x IA (each processing 2x the patient's plasma volume, weeks 1 + 2, every other day), followed by IVIG (1g/kg body weight) and i.v. Rtx (375 mg/m2) after the last IA of each cycle. This resulted in a total of 6 and 5 cycles for the first and second patient, respectively. We omitted Rtx for the last cycles in both patients. Results: IIR treatment resulted in a significant reduction of maternal anti K-titer to minimum of 1:32 in both patients, with a fluctuating course. In case 1 we performed a cordocentesis in the 30th GW because of an increased ACM PSV. The blood count of the fetus showed no anemia (Hb: 132 g/L) and only minimal Thrombocytopenia (145 G/L). The fetus in case 2 had already duplex- sonographic signs for a severe anemia at first presentation. Diagnostic cordocentesis confirmed a severe anemia (Hb 47 g/L) and also severe thrombocytopenia (21 G/L). Fetal alloimmune thrombocytopenia was ruled out. The first IUT of RBC has been performed right before starting IIR. Two weeks later a second IUT of RBC was necessary (fetal Hb 54 g/L), but thrombocytopenia was completely reversed (Tc: 352 G/l). The third and last IUT of RBC was performed 5 weeks later (Hb 99 g/L). Thereafter, no other signs of fetal anemia were detected. Patient one showed nausea/headache after her first IVIG-application and hypotonia/sinustachykardia during IA of the 5th cycle. IIR-treatment and IUT revealed no further adverse events. We continued treatment in both cases until the 32nd GW. Both neonates were born healthy in the 38th and 37th GW, respectively. The neonate in case 1 presented a mild anemia (Hb: 131 g/l), but normalized his Hb from 2nd day onwards. Both newborns showed a positive direct antiglobulin test (DAT), elution verified an anti-K-Ab, and their RBC were serologically positive for K. Both cases had normal Bilirubin and LDH levels, but a haptoglobin <0.1 g/l at birth. Both developed an icterus neonatorum during the first days after birth and needed phototherapy, Hb remained normal at all times. Flow cytometry of fetal lymphocytes showed a B-cell decrease (5.2%) in the first case and completely missing B-cells in the second case. Rtx level was low (2.13 µg/ml) in maternal plasma and neg. in the newborn of the first case. In the second case Rtx levels of the newborn and mother were 15,87 µg/ml and 4,5 µg/ml, respectively. In both cases, breastmilk showed no Rtx but positivity for anti-K-Ab (IgG). We observed no hemolysis of the newborns, while both were on breastfeeding. Conclusion: High-titer anti-Kell alloimmunisation causes early onset and severe HDFN and may be associated with severe thrombocytopenia. Treatment of affected pregnant women by using the Bern IIR-protocol was effective and safe in both severely affected patients. Disclosures Rovo: Novartis:Honoraria, Research Funding;CSL Behring:Research Funding;AG Alexion:Research Funding;Celgene:Honoraria, Other: financial support for congresses and conferences travel;BMS:Other: financial support for congresses and conferences travel;AstraZeneca:Other: financial support for congresses and conferences travel;Sanofi:Other: financial support for congresses and conferences travel;Amgen:Other: financial support for congresses and conferences travel;Roche:Other: financial support for congresses and conferences travel. OffLabel Disclosure: Rituximab is used for B-cell depletion in different malignant and non-malignant diseases. The off-label use concerns ist use in pregnant women with alloimmunisation.

Cardioprotective effects of PCSK9 inhibitor evolocumab against doxorubicin-trastuzumab sequential treatments and ipilimumab-induced cardiotoxicity: the role of MyD88/NF-KB/mTORC1 pathways

Abstract Introduction Inhibition of proprotein convertase subtilisin/kexin type 9 (PCSK9) has emerged as a novel therapy to treat hypercholesterolaemia and related cardiovascular diseases. Evolocumab, a PCSK9 inhibitor, reduced the risk of cardiovascular events in patients with atherosclerotic cardiovascular diseases when added to maximally tolerated statin therapy (± ezetimibe), and recent data from the ODYSSEY OUTCOMES trial indicate that alirocumab added to maximally tolerated statin therapy (± other lipid-lowering drugs) reduces the risk of cardiovascular events in patients with a recent acute coronary syndrome. Purpose Considering the expression of PCSK9 in heart tissue, we aimed to study for the first time the direct biochemical effects of evolocumab in cardiomyocytes during exposure to doxorubicin, trastuzumab, their sequential treatments, and immune checkpoint inhibitor ipilmumab. Methods Human fetal cardiomyocytes (HFC cell line) were exposed to subclinical concentration of doxorubicin, trastuzumab, sequential treatment of both ( all 100 nM), alone or in combination with evolocumab (50 nM) for 48h. In another experiment, in co-coltures of human fetal cardiomyocytes and lymphocytes, we incubated ipilimumab (200 nM) alone or in combination with evolocumab for 48h. After the incubation period, we performed the following tests: determination of cell viability, through analysis of mitochondrial dehydrogenase activity, study of lipid peroxidation (quantifying cellular Malondialdehyde and 4-hydroxynonenal), intracellular Ca2+ homeostasis. Moreover, pro-inflammatory studied were also performed (activation of NLRP3 inflammasome; expression of TLR4/MyD88; mTORC1 Fox01/3a; transcriptional activation of p65/NF-κB and secretion of cytokines involved in cardiotoxicity (Interleukins 1β, 8, 6). Results Evolocumab co-incubated with doxorubicin alone or in sequence with trastuzumab exerts cardioprotective effects, enhancing cell viability of 35–43% compared to untreated cells (p<0,05 for all); Cardiomyocytes co-incubated withevolocumab and ipilimumab (in co-colture of cardiomyocytes and lymphocytes) reduces significantly the cardiotoxicity phenomena through MyD88/NF-KB/cytokines axis and mTORC1 Fox01/3α mediated mechanisms. Conclusion We demonstrated, for the firts time, that PCSK9 inhibitor evolocumab exerts direct effects in cardiomyocytes during doxorubicin, trastuzumab and ipilimumab mediated cardiotoxicity turning on a new light on its possible use in the management of the cardiotoxic effects of antineoplastic drugs in cancer patients Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): This work was funded by the “Ricerca Corrente” grant from the Italian Ministry of Health

The secrets of placental mosaicism: clinical observation of the full form of confined placental trisomy 22 and literature review

Literature reports of placental mosaicism, including trisomy 22, were analyzed. The chance of correlation of placental aneuploidy with fetus aneuploidy, also the probability of complications in pregnancy and fetal growth restriction and postnatal patients growth in the cases of confined placental mosaicism, were demonstrated. The case of prenatal diagnosis of confined placental mosaicism of trisomy 22 with favorable outcome is presented. The necessity of cytogenic assay of amniocytes and fetal lymphocytes in the case of placental heteroploidy diagnosis was emphasized.

Study of acetylated histone h3k9 – an active chromatin mark – in chromosomes from adult and fetal human lymphocytes

Background: Incorrect epigenetic modifications of the human genome may result in epigenetic disorders, thus, highlighting the necessity of studying chromosome epigenetic patterns in human development. Aim of the study: A comparative analysis of acetylated histone H3K9 (AcH3K9) patterns in human metaphase chromosomes from the lymphocytes of adults and fetuses. Materials and methods: The immunocytochemical detection of AcH3K9 in the metaphase chromosomes from PHA-stimulated peripheral lymphocytes of 13 adults and cord blood lymphocytes of 10 fetuses at 20-22 weeks of gestation. Results: Both in the chromosomes of the adults and the fetuses, AcH3K9 accumulated in the R- and T-, but not G-bands and avoided the regions of pericentromeric heterochromatin of the chromosomes 1, 9 and 16. When comparing the adult and the fetal chromosomes, different levels of AcH3K9 were revealed in a few bands: 2q31, 5p13, 5p15 and 16p13 had higher level of Н3К9 acetylation in adults, in contrast to 9q13 which was hyperacetylated in fetuses. Conclusion: The АсН3К9 distribution in metaphase chromosomes is band-specific and is similar between the adults and the fetuses, excluding a few bands with different acetylation levels.

The immune response to malaria in utero.

Malaria causes tremendous early childhood morbidity and mortality, providing an urgent impetus for the development of a vaccine that is effective in neonates. However, the infant immune response to malaria may be influenced by events that occur well before birth. Placental malaria infection complicates one quarter of all pregnancies in Africa and frequently results in exposure of the fetus to malaria antigens in utero, while the immune system is still developing. Some data suggest that in utero exposure to malaria may induce immunologic tolerance that interferes with the development of protective immunity during childhood. More recently, however, a growing body of evidence suggests that fetal malaria exposure can prime highly functional malaria-specific T- and B-cells, which may contribute to postnatal protection from malaria. In utero exposure to malaria also impacts the activation and maturation of fetal antigen presenting cells and innate lymphocytes, which could have implications for global immunity in the infant. Here, we review recent advances in ourunderstanding of how various components of the fetal immune system are altered by in utero exposure to malaria, discuss factors that may tilt the critical balance between tolerance and adaptive immunity, and consider the implications of these findings for malaria prevention strategies.

Study of acetylated histone h3k9 – an active chromatin mark – in chromosomes from adult and fetal human lymphocytes

Background: Incorrect epigenetic modifications of the human genome may result in epigenetic disorders, thus, highlighting the necessity of studying chromosome epigenetic patterns in human development. Aim of the study: A comparative analysis of acetylated histone H3K9 (AcH3K9) patterns in human metaphase chromosomes from the lymphocytes of adults and fetuses. Materials and methods: The immunocytochemical detection of AcH3K9 in the metaphase chromosomes from PHA-stimulated peripheral lymphocytes of 13 adults and cord blood lymphocytes of 10 fetuses at 20-22 weeks of gestation. Results: Both in the chromosomes of the adults and the fetuses, AcH3K9 accumulated in the R- and T-, but not G-bands and avoided the regions of pericentromeric heterochromatin of the chromosomes 1, 9 and 16. When comparing the adult and the fetal chromosomes, different levels of AcH3K9 were revealed in a few bands: 2q31, 5p13, 5p15 and 16p13 had higher level of Н3К9 acetylation in adults, in contrast to 9q13 which was hyperacetylated in fetuses. Conclusion: The АсН3К9 distribution in metaphase chromosomes is band-specific and is similar between the adults and the fetuses, excluding a few bands with different acetylation levels.

Alteration in methylation level at differential methylated regions of MEST and DLK1 in fetus of preeclampsia

ABSTRACTObjectives: Offspring born to preeclamptic women are at high risk for metabolic diseases in later life, but the mechanisms are not known. The purposes of the current investigation were to clarify the changes in DNA methylation at MEST and DLK1 DMRs in fetus of preeclampsia and to explore the possible mechanisms behind the high risk of adult diseases in the offspring of preeclampsia.Methods: Fetal lymphocytes were isolated from umbilical cord blood of 78 women with preeclampsia and 95 women with normal pregnancy. Genomic DNA was extracted and then DNA methylation levels of MEST and DLK1 DMRs were determined by MassARRAY quantitative methylation analysis.Results: The methylation levels were detected in 20 CpG sites of MEST DMR and 16 sites of DLK1 DMR. Methylation changes were significantly different at CPG1, 3, 4, 7.8, 15, 18.19, and 20 of MEST between preeclampsia and normal pregnancy (P = 0.014, 0.001, <0.001, <0.001, = 0.001, = 0.005, and = 0.003, respectively). Significant differences were also observed at CPG 3 and 9 of DLK1 (P = 0.002 and 0.027, respectively). However, overall methylation at these DMRs were not affected.Conclusion: We conclude methylation changes at some CpG sites of MEST and DLK DMRs in preeclamptic group. This may be among the mechanisms behind the high risk of adult diseases in the later life of offspring born to preeclamptic pregnancies.Abbreviations: DMR: Differentially Methylated Region; MEST: Mesoderm Specific Transcript

Developmental origins of inflammatory and immune diseases.

Epidemiological and experimental animal studies show that suboptimal environments in fetal and neonatal life exert a profound influence on physiological function and risk of diseases in adult life. The concepts of the 'developmental programming' and Developmental Origins of Health and Diseases (DOHaD) have become well accepted and have been applied across almost all fields of medicine. Adverse intrauterine environments may have programming effects on the crucial functions of the immune system during critical periods of fetal development, which can permanently alter the immune function of offspring. Immune dysfunction may in turn lead offspring to be susceptible to inflammatory and immune diseases in adulthood. These facts suggest that inflammatory and immune disorders might have developmental origins. In recent years, inflammatory and immune disorders have become a growing health problem worldwide. However, there is no systematic report in the literature on the developmental origins of inflammatory and immune diseases and the potential mechanisms involved. Here, we review the impacts of adverse intrauterine environments on the immune function in offspring. This review shows the results from human and different animal species and highlights the underlying mechanisms, including damaged development of cells in the thymus, helper T cell 1/helper T cell 2 balance disturbance, abnormal epigenetic modification, effects of maternal glucocorticoid overexposure on fetal lymphocytes and effects of the fetal hypothalamic-pituitary-adrenal axis on the immune system. Although the phenomena have already been clearly implicated in epidemiologic and experimental studies, new studies investigating the mechanisms of these effects may provide new avenues for exploiting these pathways for disease prevention.

Granzyme B-inhibitor serpina3n induces neuroprotection in vitro and in vivo.

BackgroundMultiple sclerosis (MS) is an autoimmune inflammatory and neurodegenerative disease of the central nervous system (CNS). It is widely accepted that inflammatory cells play major roles in the pathogenesis of MS, possibly through the use of serine protease granzyme B (GrB) secreted from the granules of cytotoxic T cells. We have previously identified GrB as a mediator of axonal injury and neuronal death. In this study, our goal was to evaluate the effect of GrB inhibition in the human system in vitro, and in vivo in EAE using the newly isolated GrB-inhibitor serpina3n.MethodsWe used a well-established in vitro model of neuroinflammation characterized by a co-culture system between human fetal neurons and lymphocytes. In vivo, we induced EAE in 10- to 12-week-old female C57/BL6 mice and treated them intravenously with serpina3n.ResultsIn the in vitro co-culture system, pre-treatment of lymphocytes with serpina3n prevented neuronal killing and cleavage of the cytoskeletal protein alpha-tubulin, a known substrate for GrB. Moreover, in EAE, 50 μg serpina3n substantially reduced the severity of the disease. This dose was administered intravenously twice at days 7 and 20 post EAE induction. serpina3n treatment reduced axonal and neuronal injury compared to the vehicle-treated control group and maintained the integrity of myelin. Interestingly, serpina3n treatment did not seem to reduce the infiltration of immune cells (CD4+ and CD8+ T cells) into the CNS.ConclusionOur data suggest further studies on serpina3n as a potentially novel therapeutic strategy for the treatment of inflammatory-mediated neurodegenerative diseases such as MS.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-015-0376-7) contains supplementary material, which is available to authorized users.

Dopamine D2 Receptor Relies upon PPM/PP2C Protein Phosphatases to Dephosphorylate Huntingtin Protein

Striatal dopamine D2 receptor (D2R) relies upon G protein- and β-arrestin-dependent signaling pathways to convey its action on motor control and behavior. Considering that D2R activation inhibits Akt in the striatum and that huntingtin physiological functions are affected by Akt phosphorylation, we sought to investigate whether D2R-mediated signaling could regulate huntingtin phosphorylation. We demonstrate that D2R activation decreases huntingtin phosphorylation on its Akt site. This dephosphorylation event depends upon the Gαi-dependent engagement of specific members of the protein phosphatase metallo-dependent (PPM/PP2C) family and is independent of β-arrestin 2. These observations identify the PPM/PP2C family as a mediator of G protein-coupled receptor signaling and thereby suggest a novel mechanism of dopaminergic signaling.

The human fetal lymphocyte lineage: identification by CD27 and LIN28B expression in B cell progenitors

CD27, a member of the TNFR superfamily, is used to identify human memory B cells. Nonetheless, CD27(+) B cells are present in patients with HIGM1 syndrome who are unable to generate GCs or memory B cells. CD27(+)IgD(+) fetal B cells are present in umbilical cord blood, and CD27 may also be a marker of the human B1-like B cells. To define the origin of naïve CD27(+)IgD(+) human B cells, we studied B cell development in both fetal and adult tissues. In human FL, most CD19(+) cells coexpressed CD10, a marker of human developing B cells. Some CD19(+)CD10(+) B cells expressed CD27, and these fetal CD27(+) cells were present in the pro-B, pre-B, and immature/transitional B cell compartments. Lower frequencies of phenotypically identical cells were also identified in adult BM. CD27(+) pro-B, pre-B, and immature/transitional B cells expressed recombination activating gene-1, terminal deoxynucleotidyl transferase and Vpre-B mRNA comparably to their CD27(-) counterparts. CD27(+) and CD27(-) developing B cells showed similar Ig heavy chain gene usage with low levels of mutations, suggesting that CD27(+) developing B cells are distinct from mutated memory B cells. Despite these similarities, CD27(+) developing B cells differed from CD27(-) developing B cells by their increased expression of LIN28B, a transcription factor associated with the fetal lymphoid lineages of mice. Furthermore, CD27(+) pro-B cells efficiently generated IgM(+)IgD(+) immature/transitional B cells in vitro. Our observations suggest that CD27 expression during B cell development identifies a physiologic state or lineage for human B cell development distinct from the memory B cell compartment.

Prenatal Diagnosis of LAD-I on Cord Blood by Flowcytometry

To optimize a simple flowcytometric technique for Prenatal diagnosis (PND) for Leukocyte adhesions defect (LAD-I) on cordocentesis sample at 18wk gestation. Normal reference ranges for expression of CD18/CD11-integrins in neutrophils and lymphocytes at 18wk of gestation were established by flowcytometry. PND for LAD-I was then performed on the cordocentesis samples in three 'at risk' pregnancies after ruling out maternal contamination. CD18 and CD11a expression on fetal lymphocytes were found to be the most useful parameters for PND of LAD-I. All the three fetuses tested showed normal expression of CD18/CD11-integrins and thus were unaffected. This was confirmed by testing the cord blood (CB) samples after delivery and normal growth and absence of serious infections on follow-up. Flowcytometry offers a rapid and sensitive technique for PND of LAD-I in the absence of facilities for molecular diagnosis. Obstetricians, even in developing countries with modest facilities, can offer considerable relief for the families.

Absence of N addition facilitates B cell development, but impairs immune responses.

The programmed, stepwise acquisition of immunocompetence that marks the development of the fetal immune response proceeds during a period when both T cell receptor and immunoglobulin (Ig) repertoires exhibit reduced junctional diversity due to physiologic terminal deoxynucleotidyl transferase (TdT) insufficiency. To test the effect of N addition on humoral responses, we transplanted bone marrow from TdT-deficient (TdT−/−) and wild-type (TdT+/+) BALB/c mice into recombination activation gene 1-deficient BALB/c hosts. Mice transplanted with TdT−/− cells exhibited diminished humoral responses to the T-independent antigens α-1-dextran and (2,4,6-trinitrophenyl) hapten conjugated to AminoEthylCarboxymethyl-FICOLL, to the T-dependent antigens NP19CGG and hen egg lysozyme, and to Enterobacter cloacae, a commensal bacteria that can become an opportunistic pathogen in immature and immunocompromised hosts. An exception to this pattern of reduction was the T-independent anti-phosphorylcholine response to Streptococcus pneumoniae, which is normally dominated by the N-deficient T15 idiotype. Most of the humoral immune responses in the recipients of TdT−/− bone marrow were impaired, yet population of the blood with B and T cells occurred more rapidly. To further test the effect of N-deficiency on B cell and T cell population growth, transplanted TdT-sufficient and -deficient BALB/c IgMa and congenic TdT-sufficient CB17 IgMb bone marrow were placed in competition. TdT−/− cells demonstrated an advantage in populating the bone marrow, the spleen, and the peritoneal cavity. TdT deficiency, which characterizes fetal lymphocytes, thus appears to facilitate filling both central and peripheral lymphoid compartments, but at the cost of altered responses to a broad set of antigens.Electronic supplementary materialThe online version of this article (doi:10.1007/s00251-011-0543-7) contains supplementary material, which is available to authorized users.

Review: Fetal antigens – Identity, origins, and influences on the maternal immune system

Pregnancy induces priming of the maternal cellular and humoral immune systems. The paternally-inherited fetal antigens that influence maternal T and B cells include both major and minor histocompatibility antigens – the same antigens that are problematic in allotransplantation. Animal models have facilitated our understanding of the lymphocyte responses to fetal antigens, and our appreciation of the parallel response in pregnant women is increasing. The physiologic properties of the placenta as well as trafficking of cells between mother and fetus allow ample opportunity for sampling of fetal proteins by the maternal immune system. Here, the current state of knowledge of fetal antigen-specific lymphocyte responses in pregnancy is reviewed.

Funisitis is associated with increased interleukin-10 gene expression in cord blood mononuclear cells in preterm infants ≤32 weeks of gestation

Objectives A systemic inflammatory response after intrauterine funisitis is assumed to be an important priming factor for acute and chronic pulmonary morbidity and neurological impairment in premature infants. Fetal lymphocytes and monocytes modulate the primary immune response. Genetic regulation of the cytokine-mediated process is partially known. The objective of our study was to examine the pro-inflammatory and anti-inflammatory responses in umbilical cord blood mononuclear cells (CBMC) of preterm infants on the transcriptional level. Study design Fifteen preterm infants with a gestational age ≤32 weeks were enrolled in this prospective study. Funisitis was diagnosed in five of the 15 by histological examination. Gene expression of pro-inflammatory cytokines (TNF-α, IL-8, IL-1β and IL-17) and anti-inflammatory cytokines (IL-10 and TGF-β1) was examined in CBMC by real time reverse transcription polymerase chain reaction. Results Gene expression of IL-10 was significantly higher in the funisitis group compared to unexposed controls ( p < 0.008). Expression of TGF-β1, TNF-α, IL-8 and IL-1β did not differ significantly between the funisitis and control group. IL-17 was detectable in only two samples. Conclusions Funisitis is associated with increased IL-10 gene expression in CBMC of preterm infants with a gestational age ≤32 weeks. This might contribute to modulation of postnatal immunoreactions and immunoregulation in these individuals.

Digital Fetal Aneuploidy Diagnosis by Next-Generation Sequencing

Since the identification of fetal lymphocytes in maternal blood in 1969, investigators have endeavored to develop genetics-based noninvasive prenatal diagnostics (NIPD)1 (1). A robust noninvasive approach would augment or potentially supplant amniocentesis and chorionic villus sampling, which, although gold standards, carry a risk of fetal loss. Despite considerable efforts, the use of fetal cells for NIPD has never reached clinical implementation because of their paucity (on the order of a few cells per milliliter of maternal blood) and concerns of fetal cell persistence in the maternal circulation between pregnancies (2). A new avenue was opened in 1997 by the discovery of circulating cell-free fetal DNA in maternal blood (3). Cell-free fetal DNA constitutes between 5% and 10% of the total DNA in maternal plasma and increases during gestation. It rapidly clears from the circulation postpartum, a feature that enhances its attractiveness as a per-pregnancy–specific analyte. Several clinical applications based on the analysis of cell-free fetal DNA have been developed. These assays include determining fetal Rh D status in Rh D-negative women (4), sex in sex-linked disorders (3)(5), and detection of paternally inherited autosomal recessive and dominant mutations (6). In the context of these successes, however, there remains the outstanding challenge of the use of cell-free fetal DNA for the detection of chromosomal aneuploidy, in particular trisomies 21, 18, and 13. A cell-free fetal DNA–based approach for chromosomal aneuploidy must overcome several technical hurdles. By virtue of being cell free, fetal DNA in the maternal plasma cannot be easily analyzed by traditional visualization methods such as fluorescence in situ hybridization. The high “background” of maternal DNA (cell free or from residual cells) further complicates analysis by diluting the fetal genetic information. To address these issues, investigators have worked to identify fetal-specific molecular markers. The origin of circulating cell-free …