Repolarization of HSC attenuates HSCs failure in Shwachman-Diamond syndrome.

Shwachman-Diamond syndrome (SDS) is a bone marrow failure (BMF) syndrome associated with an increased risk of myelodysplasia and leukemia. The molecular mechanisms of SDS are not fully understood. We report that primitive hematopoietic cells from SDS patients present with a reduced activity of the small RhoGTPase Cdc42 and concomitantly a reduced frequency of HSCs polar for polarity proteins. The level of apolarity of SDS HSCs correlated with the magnitude of HSC depletion in SDS patients. Importantly, exogenously provided Wnt5a or GDF11 that elevates the activity of Cdc42 restored polarity in SDS HSCs and increased the number of HSCs in SDS patient samples in surrogate ex vivo assays. Single cell level RNA-Seq analyses of SDS HSCs and daughter cells demonstrated that SDS HSC treated with GDF11 are transcriptionally more similar to control than to SDS HSCs. Treatment with GDF11 reverted pathways in SDS HSCs associated with rRNA processing and ribosome function, but also viral infection and immune function, p53-dependent DNA damage, spindle checkpoints, and metabolism, further implying a role of these pathways in HSC failure in SDS. Our data suggest that HSC failure in SDS is driven at least in part by low Cdc42 activity in SDS HSCs. Our data thus identify novel rationale approaches to attenuate HSCs failure in SDS.

Serum levels of miR-628-3p and miR-628-5p during the early pregnancy are increased in women who subsequently develop preeclampsia.

OBJECTIVE: Preeclampsia pathogenesis involves imbalances of oxidative stress networks including the heat shock protein (HSP) pathway. Micro-RNAs regulate gene networks associated with preeclampsia. Hsp90 and Runx2 are transcriptional targets of miR-628-3p. Considering that potential participation of hsa-miR-628-3p in PE development is still not elucidated, the aim of this study was to evaluate serum microRNA expression of hsa-miR-628-3p and hsa-miR-628-5p and their association with the preeclampsia development. STUDY DESIGN: A retrospective nested cohort case-control study was conducted. Serum samples from 16 pregnant women who developed preeclampsia (WWD-PE) during the follow-up period were selected and individually matched to that from 18 women in the cohort who had healthy pregnancies without complications (controls). MAIN OUTCOME MEASURES: The levels of hsa-miR-628-3p and hsa-miR-628-5p were measured in serum samples from study groups at 12, 16, and 20 weeks of gestation (WG) using TaqMan probes. Additionally serum levels were measured at the moment of diagnosis, in women with preeclampsia. RESULTS: Serum levels of hsa-miR-628-3p were higher than controls in WWD-PE at 12 WG (RQ = 7.7; P = 0.020), and of hsa-miR-628-5p at 20 WG (RQ = 3.4; P = 0.008). An increase in hsa-miR-628-3p serum levels at 12 WG (RQ = 12.01; P = 0.001) and of hsa-miR-628-5p at 20 WG (RQ = 2.95; P = 0.033) was also observed in women who developed mild preeclampsia, and severe preeclampsia, respectively. CONCLUSIONS: Serum hsa-miR-628-3p and hsa-miR-628-5p were differentially expressed between WWD-PE and controls, suggesting a participation of these miRNAs in the development of preeclampsia. Future studies are needed to validate hsa-miR628-3p and -5p as early predictors of preeclampsia.

Maternal distress and the development of hypertensive disorders of pregnancy.

Despite the implementation of programmes to improve maternal health, maternal and foetal mortality rates still remain high. The presence of maternal distress and its association with the development of pregnancy hypertensive disorders is not well established. The aim of this study was to evaluate the association between maternal distress and the development of hypertensive disorders in pregnancy in a prospective cohort of 321 Mexican women. Symptoms of maternal distressing were evaluated at week 20th of gestation using the General Health Questionnaire. The presence of acute somatic symptoms, social dysfunction, anxiety and insomnia increased the odds of developing a pregnancy hypertensive disorder by 5.1-26.4 times in study population (p values < .05). Our results support the participation of maternal distress in the development of hypertensive disorders of pregnancy. The implementation of effective programmes prioritising risk factors during pregnancy including the presence of maternal distressing factors is recommended. Impact statement What is already known on this subject: Changes in the nervous, endocrine, and immune systems have been observed in pregnant women with distress conditions leading to gestational disorders. What do the results of this study add: The presence of acute somatic symptoms, social dysfunction, anxiety and insomnia increased the developing of hypertensive disorders in Mexican population. What are the implications of these findings for clinical practice and/or further research: These findings may contribute to a better understanding of the role of the maternal stress in the development of hypertensive disorders of pregnancy, and in the implementation of effective programmes for clinical practice prioritising risk factors during pregnancy, including the presence of maternal distressing factors.

MYC-induced nuclear antigen (MINA) and preeclampsia.

BACKGROUND: Inadequate trophoblast invasion and the subsequent inflammatory response have been implicated in preeclampsia (PE) pathogenesis. Because MYC-induced nuclear antigen (MINA) gene expression is involved in cell proliferation and differentiation, inflammatory response modulation, and the unpaired regulation of which is associated with human diseases, we sought to investigate the connection between MINA and PE. OBJECTIVE: The aim of this study was to evaluate the possible relationship between the MINA rs4857304 variant and susceptibility to PE development as well as to estimate placental MINA gene expression and its association with PE. METHODS: About 242 pregnant women (126 PE cases and 116 controls) were included. MINA genotyping and gene expression were evaluated by quantitative real-time polymerase chain reaction using TaqMan probes. RESULTS: The G/G genotype of the MINA rs4857304 variant was associated with severe PE (p = 0.027, OR = 1.8, 95% CI = 1.8-3.2). Carriers of one G allele of the MINA rs4857304 variant exhibited a 1.7-fold increased risk of severe PE (p = 0.029, 95% CI = 1.1-3.0). MINA was underexpressed in preeclamptic placentas and MINA expression differed between the mild and severe PE groups. Differences in the expression levels of MINA were found among women with the T/T genotype of the rs4857304 polymorphism and carriers of at least one G allele (p = 0.024). CONCLUSION: PE and its severity are associated with the underexpression of placental MINA, and the G/G genotype of the MINA rs4857304 variant may modify the risk of severe PE among the PE cases evaluated.

Regulatory T cells inhibit CD34+ cell differentiation into NK cells by blocking their proliferation.

Graft versus Host Disease (GvHD) remains one of the main complications after hematopoietic stem cell transplantation (HSCT). Due to their ability to suppress effector cells, regulatory T cells (Tregs) have been proposed as a cellular therapy to prevent GvHD, however they also inhibit the functions of natural killer (NK) cells, key effectors of the Graft versus Leukemia effect. In this study, we have explored whether a Tregs therapy will also impact on NK cell differentiation. Using an in vitro model of hematopoietic stem cell (HSC) differentiation into NK cells, we found that activated Tregs led to a 90% reduction in NK cell numbers when added at the time of commitment to the NK cell lineage. This effect was contact dependent and was reversible upon Tregs depletion. The few NK cells that developed in these cultures were mature and exhibited normal functions. Furthermore, adoptive transfer of activated Tregs in rag(-/-) γc(-/-) mice abrogated HSC differentiation into NK cells thus confirming our in vitro findings. Collectively, these results demonstrate for the first time that activated Tregs can inhibit NK cell differentiation from HSC under specific conditions.

Natural Killer Cells Improve Hematopoietic Stem Cell Engraftment by Increasing Stem Cell Clonogenicity In Vitro and in a Humanized Mouse Model.

Cord blood (CB) is increasingly used as a source of hematopoietic stem cells (HSC) for transplantation. Low incidence and severity of graft-versus-host disease (GvHD) and a robust graft-versus-leukemia (GvL) effect are observed following CB transplantation (CBT). However, its main disadvantages are a limited number of HSC per unit, delayed immune reconstitution and a higher incidence of infection. Unmanipulated grafts contain accessory cells that may facilitate HSC engraftment. Therefore, the effects of accessory cells, particularly natural killer (NK) cells, on human CB HSC (CBSC) functions were assessed in vitro and in vivo. CBSC cultured with autologous CB NK cells showed higher levels of CXCR4 expression, a higher migration index and a higher number of colony forming units (CFU) after short-term and long-term cultures. We found that CBSC secreted CXCL9 following interaction with CB NK cells. In addition, recombinant CXCL9 increased CBSC clonogenicity, recapitulating the effect observed of CB NK cells on CBSC. Moreover, the co-infusion of CBSC with CB NK cells led to a higher level of CBSC engraftment in NSG mouse model. The results presented in this work offer the basis for an alternative approach to enhance HSC engraftment that could improve the outcome of CBT.

The effects of CAMPATH-1H on cell viability do not correlate to the CD52 density on the cell surface.

Graft versus host disease (GvHD) is one of the main complications after hematological stem cell transplantation (HSCT). CAMPATH-1H is used in the pre-transplant conditioning regimen to effectively reduce GvHD by targeting CD52 antigens on T cells resulting in their depletion. Information regarding CD52 expression and the effects of CAMPATH-1H on immune cells is scant and limited to peripheral blood (PB) T and B cells. To date, the effects of CAMPATH-1H on cord blood (CB) cells has not been studied. Here we aimed to analyze CD52 expression and the effects of CAMPATH-1H on fresh or frozen, resting or activated, PB mononuclear cells (PBMC) and CB mononuclear cells (CBMC). In resting state, CD52 expression was higher in CB than PB T cell subsets (653.66 ± 26.68 vs 453.32 ± 19.2) and B cells (622.2±20.65 vs 612.0 ± 9.101) except for natural killer (NK) cells where CD52 levels were higher in PB (421.0 ± 9.857) than CB (334.3 ± 9.559). In contrast, CD52 levels were comparable across all cell types after activation. CAMPATH-1H depleted resting cells more effectively than activated cells with approximately 80-95% of apoptosis observed with low levels of necrosis. There was no direct correlation between cell surface CD52 density and depleting effects of CAMPATH-1H. In addition, no difference in cell viability was noted when different concentrations of CAMPATH-1H were used. CD52 was not expressed on HSC but began to be expressed as the cells differentiate, implying that CAMPATH-1H could potentially affect HSC differentiation and proliferation. Our study provides insightful information, which contributes to the better understanding in the use of CAMPATH-1H as part of the conditioning regime in HSCT.

Differential effects of mycophenolate mofetil and cyclosporine A on peripheral blood and cord blood natural killer cells activated with interleukin-2.

BACKGROUND AIMS: Graft-versus-host disease remains a major cause of death after hematopoietic stem cell transplantation. Cyclosporine (CsA) and mycophenolate mofetil (MMF) have been successfully used alone or in combination as prophylaxis for graft-versus-host disease. Although the effects of these drugs on T cells have been studied, little is known about the effects of both drugs on natural killer (NK) cells. We examined if the sensitivity of umbilical cord blood (CB) NK cells to MMF and/or CsA differs from their adult counterparts. METHODS: An approach that was based on flow cytometry and real-time polymerase chain reaction was used to assess the effects of MMF, CsA and the combination of both drugs on the viability, activation, proliferation and cytotoxicity of peripheral blood (PB) and CB NK cells after culture with interleukin-2. RESULTS: MMF alone or together with CsA induced cell death of CB NK cells but not of PB NK cells. MMF and CsA had differential effects on NK cell activation but significantly reduced proliferation of CB NK cells. MMF reduced perforin expression by PB NK cells, whereas CsA alone or together with MMF drastically decreased degranulation of CB and PB NK cells. However, neither affected cytokine secretion by PB and CB NK cells. CONCLUSIONS: This study showed that CB NK cells were more sensitive to MMF and CsA than were PB NK cells. MMF and CsA had significant effects on NK cells that could jeopardize the beneficial effects of NK cells after hematopoietic stem cell transplantation.

Frozen cord blood hematopoietic stem cells differentiate into higher numbers of functional natural killer cells in vitro than mobilized hematopoietic stem cells or freshly isolated cord blood hematopoietic stem cells.

Adoptive natural killer (NK) cell therapy relies on the acquisition of large numbers of NK cells that are cytotoxic but not exhausted. NK cell differentiation from hematopoietic stem cells (HSC) has become an alluring option for NK cell therapy, with umbilical cord blood (UCB) and mobilized peripheral blood (PBCD34(+)) being the most accessible HSC sources as collection procedures are less invasive. In this study we compared the capacity of frozen or freshly isolated UCB hematopoietic stem cells (CBCD34(+)) and frozen PBCD34(+) to generate NK cells in vitro. By modifying a previously published protocol, we showed that frozen CBCD34(+) cultures generated higher NK cell numbers without loss of function compared to fresh CBCD34(+) cultures. NK cells generated from CBCD34(+) and PBCD34(+) expressed low levels of killer-cell immunoglobulin-like receptors but high levels of activating receptors and of the myeloid marker CD33. However, blocking studies showed that CD33 expression did not impact on the functions of the generated cells. CBCD34(+)-NK cells exhibited increased capacity to secrete IFN-γ and kill K562 in vitro and in vivo as compared to PBCD34(+)-NK cells. Moreover, K562 killing by the generated NK cells could be further enhanced by IL-12 stimulation. Our data indicate that the use of frozen CBCD34(+) for the production of NK cells in vitro results in higher cell numbers than PBCD34(+), without jeopardizing their functionality, rendering them suitable for NK cell immunotherapy. The results presented here provide an optimal strategy to generate NK cells in vitro for immunotherapy that exhibit enhanced effector function when compared to alternate sources of HSC.

Transcription factors involved in the regulation of natural killer cell development and function: an update.

Natural killer (NK) cells belong to the innate immune system and are key effectors in the immune response against cancer and infection. Recent studies have contributed to the knowledge of events controlling NK cell fate. The use of knockout mice has enabled the discovery of key transcription factors (TFs) essential for NK cell development and function. Yet, unwrapping the downstream targets of these TFs and their influence on NK cells remains a challenge. In this review, we discuss the latest TFs described to be involved in the regulation of NK cell development and maturation.

Generation of natural killer cells from hematopoietic stem cells in vitro for immunotherapy.

Natural killer (NK) cells are part of the innate immune system and are an alluring option for immunotherapy due to their ability to kill infected cells or cancer cells without prior sensitization. Throughout the past 20 years, different groups have been able to reproduce NK cell development in vitro, and NK cell ontogeny studies have provided the basis for the establishment of protocols to produce NK cells in vitro for immunotherapy. Here, we briefly discuss NK cell development and NK cell immunotherapy approaches. We review the factors needed for NK cell differentiation in vitro, which stem cell sources have been used, published protocols, challenges and future directions for Good Manufacturing Practice protocols.

The unique profile of cord blood natural killer cells balances incomplete maturation and effective killing function upon activation.

Cord blood (CB) is increasingly used as a source of stem cells for hematopoietic stem cell transplantation, and natural killer (NK) cells may be the effectors of the antileukemic response observed after CB transplantation. Here, we analyzed the phenotype and functions of CB NK cell subsets. We determined that the percentage of NK cells was higher in CB compared with peripheral blood (PB). Furthermore, there was a higher percentage of the CD56(bright) subset in CB. CB NK cells reached a late stage of differentiation, but exhibited higher expression of NKG2A and expressed fewer killer-cell immunoglobulin-like receptors, suggesting an incomplete maturation. CB NK cells highly expressed CXCR4, but did not express L-selectin, highlighting unique homing properties of CB NK cells. CB NK cells proliferated in response to interleukin-2 and degranulated in response to stimulation with tumor cells, but failed to lyse K562 cells in (51)Cr-release assay. CB NK cells exhibited a lower interferon-γ production in comparison with PB NK cells. Culture with IL-2 increased CB NK cell functions. Our study sheds light on CB NK cell properties and highlights the potential of CB as a source of NK cells for immunotherapy.

High-throughput profiling of the humoral immune responses against thirteen human papillomavirus types by proteome microarrays.

We have developed microarrays with all eight proteins encoded by 13 different human papillomavirus types associated with anogenital cancer (HPV-16, -18, -31, -33, -35, -45, and -53), genital warts (HPV-6 and -11), or skin lesions (HPV-1, -2, -4, and -5). We analyzed the seroprevalence of antibodies in 546 patients, which had either cervical carcinomas, or precursor lesions, or which were asymptomatic. All patient groups contained sera ranging from high reactivity against multiple HPV proteins to low or no reactivity. Computational analyses showed the E7 proteins of carcinogenic HPV types as significantly more reactive in cancer patients compared to asymptomatic individuals and discriminating between cancer and HSIL or LSIL patients. Antibodies against E4 and E5 had the highest seroprevalence but did not exhibit differential reactivity relative to pathology. Our study introduces a new approach to future evaluation of the overall antigenicity of HPV proteins and cross-reaction between homologous proteins.