It is highly desirable to develop new strategies based on secretomics to more accurately selection of embryos with the highest developmental potential for transfer. Granulocyte-macrophage colony-stimulating factor (GM-CSF) has been reported to promote embryo development and pregnancy establishment. However, the predictive value of GM-CSF in single blastocyst selection remains unclear. This study is to determine the concentration of GM-CSF in human single-blastocyst conditioned medium (SBCM) and to evaluate its association with embryo quality and pregnancy outcome.The patients with ≤38 years of age receiving the first cycle of assisted reproductive therapy were included in this study. The patients who had <4 top-quality embryos formed by the fertilized two pronuclear zygotes on day 3 were excluded. A total of 126 SBCM samples (SBCMs) were included, of which blastocysts from 77 SBCMs were later transferred in subsequent frozen-thawed embryo transfer. The concentrations of GM-CSF were detected by single-molecule array (SIMOA) and analyzed for their possible association with embryo quality and pregnancy outcomes. The top-quality embryo (TQ), positive HCG (HP), clinical pregnancy (CP), and ongoing pregnancy (OP) rates were determined and compared between groups divided based on GM-CSF concentrations.The detection rate of GM-CSF was found to be 50% in all SBCMs. There were significant differences in TQ rate, HP rate, CP rate and OP rate among high concentration group, medium concentration group and low concentration group. Both GM-CSF alone or GM-CSF combined with the morphological score (MS) had a greater AUC of ROC curve than that of MS alone to predict the pregnancy outcome, and GM-CSF combined with MS had the highest AUC.The concentration of GM-CSF in SBCM was detected at fg/ml levels, which was associated with embryo quality and pregnancy outcome. Collectively, GM-CSF may be used as a biomarker for prediction of pregnancy outcome and selection of embryos with high developmental potential for transfer in assisted reproductive technology (ART). The primary goals of assisted reproductive technology (ART) are to perform the single-embryo transfer to achieve high live-birth rates, to minimize the chances of multiple pregnancies, and to attain higher overall successful pregnancy rates. Thus, the selection of the most viable embryos with the best developmental potential for transfer represents a vital integral part of ART . ConventAccumulating studies have suggested that cross-talk between embryo and endometrium, which is mediated by soluble protein and corresponding receptors, is important for embryo growth and implantation . Thus, nSingle-molecule array (SIMOA) is a protein detection method based on digital enzyme-linked immunity , by isolating and detecting single immunocomplexes in arrays of femtoliter-volume wells. SIMOA enables clinically important proteins to be measured at femtogram (fg/ml) concentrations . It has Infertile couples, who underwent IVF or ICSI cycles at the Fertility Center, Shenzhen Zhongshan Urology Hospital between March 2019 and March 2020, were retrospectively enrolled into this study. The inclusion criteria include: 1). females age less than 38 years; 2). undergoing the first cycle of assisted reproductive therapy; 3). the number of top-quality embryos, formed by the fertilized two pronuclear zygotes (2PN), was no less than four on day 3. The exclusion criteria include: 1) abnormal ultrasonography and hysterosalpingogram/hysteroscopy result. The SBCMs of the patients enrolled in this study was collected to do GM-CSF concentration assay. At last, a total of 126 SBCMs from 100 infertile couples were collected, and then all these blastocysts were frozen. Of which, 77 were subjected to embryo transfer operation and the pregnancy outcomes were followed up. For the freeze-thaw single embryo transplantation cycles, endometrium was prepared by either natural protocols or artificial protocols. For natural protocols, embryo transfer carried out on day 5 after ovulation if the endometrial thickness exceeded 7 mm. For artificial protocols, patients received 4, 6 and 8 mg oral oestradiol per day successively. When the thickness of endometrium reached 7–8 mm, progesterone was started and embryo transfer was performed on day 6 after progesterone injection. Luteal phase support was achieved until a pregnancy test was positive and was continued until 3 months of gestation. Maternal serum HCG was measured on 11 days after the embryo transfer and the HCG level higher than 5 IU/L indicated HCG positive (HP). Intrauterine gestational sac detected by transvaginal ultrasound on 30 days after the embryo transfer was considered as a clinical pregnancy (CP). Pregnancy that proceeded beyond 3 months was defined as ongoing pregnancy (OP). This study was approved by the Research Ethics Committee of Shenzhen Zhongshan Urology Hospital .Oocytes were retrieved by an ultrasound-guided method at 36 h after administering human chorionic gonadotropin, and then IVF or ICSI was performed. Fertilization was assessed at 17 ± 1 h after insemination. The fertilized zygotes were cultured in Quinn’s Advantage Cleavage Medium  supplemented with 10% (v/v) serum protein substitute (SAGE BioPharma). The cleaving embryos were evaluated on D3 according to the following criteria: the number of blastomeres, the degree of fragmentation, and the symmetry of the blastomere. In our center, a cleavage stage embryo was defined as a top-quality embryo if it meets one of the following two criteria: 1. the number of blastomeres ≥7 and ≤10 with the degree of fragmentation of <20%; 2. embryos with six symmetrical blastomeres and a degree of fragmentation of <10%. The cleaving embryo was subsequently transferred to the Quinn’s Advantage Blastocyst Medium (SAGE BioPharma) containing 10% (v/v) serum protein substitute and independently cultured until embryos reached the blastocyst stage. The morphological assessment of blastocyst was assessed on D5. The morphological assessment criteria of blastocysts were based on the Gardner system, as described previously . BrieflyAfter blastocysts were frozen, 30 μl SBCM were collected and stored at −80°C until further analysis. Only the SBCMs from the blastocysts which developed on D5 and from top-quality cleavage embryos formed by the fertilized zygote of 2PN were collected. The SBCM without blastocysts under the same condition were collected as negative control (NC) samples (NCs) (n = 6). Furthermore, samples of Quinn’s Advantage Blastocyst Medium without serum protein substitute were also collected (n = 6).The concentration of GM-CSF in SBCMs was measured using a GM-CSF assay kit (Cat No: 102329) following the manufacturer’s protocol  on an HD-1 platform. SBCMs were diluted at 1:4 ratio and were performed in singlicate. The limit of detection (LOD) of GM-CSF is 0.50 fg/ml. SBCMs with GM-CSF concentration lower than LOD were assigned 0.50 fg/ml.p-value of <0.05 was considered statistically significant.Statistical analysis was performed using SPSS version 23 . Data were expressed as mean ± standard deviation (SD) for variables with a normal distribution. Data were presented as median (25th and 75th percentile) for variables with a non-normal distribution. Categorical variables were expressed in ratios and quantities. Statistical differences between groups were determined using the ANOVA test for quantitative data, chi-square test for comparing frequencies. The correlation values between the qualitative datas were calculated by Spearman’s rank correlation coefficient test. To determine the predictive value of GM-CSF for pregnancy outcome, receiver operating characteristic (ROC) curve analyses were performed. The predicted probability of logistic regression model being diagnosed with pregnancy outcome was used as a surrogate marker to construct ROC curve . The areA total of 126 SBCMs were tested, and GM-CSF was detected in 63 SBCMs of them, and the detection rate was 50%. The concentration of GM-CSF in the detected SBCMs was 5.60  fg/ml. GM-CSF was detected in all the NCs and not in the Quinn’s Advantage Blastocyst Medium samples without serum protein substitute. The concentration of GM-CSF in NCs was 3.72  fg/ml Table 1.p = 0.010). The correlation values between GM-CSF concentration group and morphological score (MS) was 0.245 (p = 0.006).To investigate the relationship of GM-CSF and embryo quality, the rate of TQ blastocyst was compared among the groups with different concentration of GM-CSF. Based on the median concentration of GM-CSF in NCs (3.72 fg/ml) and the LOD (0.50 fg/ml), all the 126 blastocysts were divided into three groups: high concentration group , mid concentration group , and low concentration group . As presented in p = 0.029). The CP rate in the HIGH group was significantly higher (80.00%) than that in the MID (50.00%) and LOW (34.48%) group (p = 0.002). The OP rate in the HIGH group was also significantly higher (73.33%) than that in the MID (38.89%) and LOW (20.69%) group (p <0.001). The correlation values between GM-CSF concentration group and HP, CP and OP was 0.302 (p=0.008), 0.402 (p <0.001), and 0.464 (p <0.001), respectively.To further investigate the relationship of GM-CSF and embryo developmental potential, the rates of HP, CP and OP were compared among the groups with different concentration of GM-CSF, respectively. All the 77 blastocysts, which were transferred into maternal uterus, were also divided into three groups based on GM-CSF concentration. As presented in To further analyze the power of GM-CSF in predicting pregnancy outcomes, ROC curve analyses of MS, GM-CSF, and combined indicator with MS and GM-CSF (Combination) for predicting HP, CP, and OP were performed, respectively Figure 1The selection of embryos with the highest implantation potential for transfer is the key to successful pregnancy . To the Although the expression and secretion of GM-CSF in preimplantation embryos have been well-recognized , 10, thein vitro could secrete GM-CSF during development, besides, the concentration of GM-CSF in SBCM was positively correlated with the embryo quality. Therefore, we hypothesized that preimplantation embryos could promote their development through GM-CSF in an autocrine manner. Mechanistically, GM-CSF may function as a survival factor in embryos by promoting glucose uptake in embryos and providing protection from the stress response pathway and apoptosis through up-regulation of the expression of anti-apoptotic factor Bcl-2 . Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.YZ and LD were involved in study design. PC and CH were involved in the organization of the entire project, date analysis and manuscript writing. QS, HZ, FX, ZY, and CW were involved in embryo culture, formulation of clinical sample collection scheme and sample collection. SL and ZL were mainly involved in date analysis. All authors contributed to the article and approved the submitted version.This study was funded by the National Key Research & Developmental Program of China (2018YFC1003900/2018YFC1003904), clinical research special fund of Chinese Medical Association (18010120741), Shenzhen Natural Science Foundation (JCYJ20190813161801676), Basic Research Program of Shenzhen (JCYJ20160427113153295), National Natural Science Foundation of China (21807072) and Sanming Project of Medicine in Shenzhen (SZSM201502035).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The definition of immune tolerance to allogeneic tissue and organ transplants in laboratory animals and humans continues to be the acceptance of the donor graft, rejection of third-party grafts, and specific unresponsiveness of recipient immune cells to the donor alloantigens in the absence of immunosuppressive treatments. Actively acquired tolerance was achieved in mice more than 60 years ago by the establishment of mixed chimerism in neonatal mice. Once established, mixed chimerism was self-perpetuating and allowed for acceptance of tissue transplants in adults. Successful establishment of tolerance in humans has now been reported in several clinical trials based on the development of chimerism after combined transplantation of hematopoietic cells and an organ from the same donor. This review examines the mechanisms of organ graft acceptance after establishment of mixed chimerism  or complete chimerism (self-tolerance), and compares the development of graft versus host disease (GVHD) and graft versus tumor (GVT) activity in complete and mixed chimerism. GVHD, GVT activity, and complete chimerism are also discussed in the context of bone marrow transplantation to treat hematologic malignancies. The roles of transient versus persistent mixed chimerism in the induction and maintenance of tolerance and organ graft acceptance in animal models and clinical studies are compared. Key differences in the stability of mixed chimeras and tolerance induction in MHC matched and mismatched rodents, large laboratory animals, and humans are examined to provide insights into the safety and efficacy of translation of results of animal models to clinical trials. Currently, about 500,000 patients with end stage renal disease are undergoing dialysis in the US of which over 100,000 are on a wait list for a deceased or living donor transplants –4. RecipHowever, even with the use of these potent drug combinations there is continued and progressive loss of the kidney graft due to immune mediated graft rejection: median graft survival of living donor HLA mismatched kidneys is ~15 years, and recipients of deceased donor kidney transplants have graft survival averaging 8-12 years –9.Long-term patient survival after graft loss is poor. In addition to graft rejection, the IS drug regimens themselves cause significant medical comorbidities that include chronic allograft vasculopathy, new onset diabetes after transplant (NODAT), infections, cancer, heart disease, hypertension, renal dysfunction, and osteoporosis and osteopenia –13. It hin utero with the skin graft donor, and in adult mice that had been given injections of genetically disparate bone marrow cells from the skin graft donor strain during the fetal/neonatal period led to the concept of “tolerance” as opposed to the expected “rejection” of the grafts  were present in the recipients –17. SubsAt present the term “mixed chimerism” is used to identify recipients with a mixture of two or more hematopoietic and immune cell lineages in the blood forming and lymphoid tissues –21. NumeStudies with complete chimerism in humans involve recipients who were treated with blood and marrow transplants as curative therapy for a hematologic malignancy. In these patients the donor hematopoietic and immune cells completely replaced the recipient hematopoietic and immune cells –25. The Reports of cancer patients with complete chimerism after bone marrow transplantation and who thereafter developed end stage renal disease (ESRD) accepted a kidney transplant from their bone marrow donor, and without the need for immunosuppressive (IS) drugs , 30. Thein utero, and the inability of the adult chimeras to rapidly reject skin grafts from each other, but not from third parties, formed the conceptual framework for the linkage of chimerism and tolerance . Irradiated adults from one parental strain were given F1 hybrid MHC matched bone marrow transplants followed by skin grafts from the allogeneic parental strain . The useSubsequent studies of acceptance of skin transplants in complete chimeras given lethal TBI, and bone marrow transplants from fully allogeneic strains  also showed acceptance of allogeneic skin grafts in the chimeras . HoweverThe risks of myeloablative radiation and of GVHD after allogeneic bone marrow transplantation were justified for adaptation to humans treated for hematologic malignancies, since these diseases were uniformly lethal , 42. TheAn alternative was to condition recipients of combined organ and bone marrow transplants with non-myeloablative radiation regimens that had been proven safe in humans. Accordingly, the laboratory of Strober and his co-workers at Stanford University studied the non-myeloablative radiation regimen of TLI for application to pre- clinical models of bone marrow transplantation with or without organ transplantation , 44–52. A TLI regimen was developed for use in mice and rats by manufacture of a lead jig that allowed for the irradiation of the spleen, thymus, and lymph nodes and shielded the lungs, skull, liver, portions of the intestines, and marrow in the limbs , 44–46. The TLI regimen developed in mice was adapted for use in rats given combined MHC mismatched bone marrow given intravenously, and heterotopic heart transplants directly anastomosed to the abdominal aorta and vena cava . Lead jiWhen the completely posttransplant regimen was used, mixed chimerism was established in all rat recipients, and the heart transplants were not rejected during an observation period of up to 6 months –60. In fAdditional experiments were performed to determine whether an infusion of purified donor peripheral blood mononuclear cells (PBMC) or purified blood monocytes could substitute for the infusion of donor bone marrow cells in recipients given heart transplants and TLI/ATG conditioning , 59. AltOur further studies used MHC mismatched C57BL/6 (H-2b) and BALB/c (H-2d) mice as either donors or as recipients conditioned with posttransplant TLI/ATG and given combined bone marrow and heart transplants. Four recipient immune cell types were identified that were required for the induction of chimerism and tolerance , 61–63. via production of molecules such as IDO, and arginase-1 as reported previously (The first cells in the chain of interactions in this complex network are Batf-3-/- dependent CD8+DCs that take up apoptotic bodies (efferocytosis) that are produced in great quantities by the TLI procedure Figure 2eviously , 64. In eviously –67. Analeviously .Subsequent to the tolerance studies described above in rodents, several laboratories investigated organ transplantation with bone marrow transplantation using tolerance induction regimens in large animals that were bred and MHC typed in laboratories including dogs, non-human primates, and mini-swine , 69–73. A pretransplant conditioning regimen with non-myeloablative TBI (300 cGy) combined with thymic radiation and ATG allowed for the development of mixed chimerism and acceptance of kidney transplants in MHC mismatched non-human primates given an infusion of donor bone marrow cells at the time of the organ transplant without the need for maintenance IS drugs in the majority of recipients , 72. IntIn conclusion, stable mixed chimerism was not required for acceptance of kidney transplants in both dog and non-human primate studies. However, the stability of mixed chimerism in the MHC matched dogs, and the instability in the MHC mismatched non- human primates suggests that there is likely to be a link between stability and MHC matching. This link has not been studied as yet in either of these large animal models, but has been studied in humans (see below).Although pretransplant TLI conditioning in fully MHC mismatched mice and rats markedly prolonged skin, heart, and kidney transplant survival, tolerance was not achieved without the addition of a hematopoietic cell infusion and the establishment of mixed chimerism , 74–76. In view of the results in the canine study, and the safety of the use of TLI to treat patients with Hodgkin’s disease, pretransplant TLI and posttransplant ATG was used to treat a series of patients given HLA unmatched deceased donor kidney transplants to determine the feasibility of maintaining graft function with low dose (0.1- 0.2mg/kg/day) prednisone monotherapy as maintenance therapy , 79. TLIIn a follow up study, the feasibility of completely withdrawing maintenance prednisone therapy from 3 of the patients given deceased donor kidney transplants and pretransplant TLI was determined . The 3 pA study of 20 patients who received HLA matched kidney transplants, and multiple injections of donor hematopoietic progenitor cells in order to withdraw maintenance IS drugs was performed by investigators at Northwestern University , 83. RecIn order to evaluate the ability of TLI/ATG conditioning to promote the establishment of chimerism in humans Stanford investigators first performed a study of allogeneic hematopoietic cell transplants to treat patients with hematologic malignancies . In thisHLA matched donor cells were collected, purified, and cryopreserved about 6 weeks before kidney transplantation –88. DonoOf the 24 patients withdrawn from IS drugs, 22 had no evidence of rejection with up to 15 years follow up . Two patin vitro. In summary, the 22 of 29 patients developed mixed chimerism and tolerance to their kidney transplants after combined organ and hematopoietic cell transplantation using TLI/ATG conditioning.Ten patients had mixed chimerism that was persistent until the last chimerism test, and 14 patients lost chimerism sometime after the first year posttransplant . DespiteThere were no graft loses due to rejection, and no GVHD, severe neutropenia or thrombocytopenia in the 29 patients enrolled in the protocol –88.Medeor Therapeutics performed a multi-center randomized trial of tolerance induction in 20 experimental HLA matched kidney transplant patients and 10 standard of care patients based on the results of the clinical study report by investigators at Stanford University . ExperimA feasibility study was performed at Stanford University to assess the safety and efficacy of infusing purified donor CD34+ hematopoietic progenitor cells into 4 patients given kidney transplants from HLA mismatched living donors in order to establish mixed chimerism using posttransplant TLI/ATG conditioning . Two patPurified CD34+ cells (3-5x10^6 cells/kg) obtained from G-CSF mobilized donor peripheral blood were enriched on Miltenyi columns and infused into recipients shortly after the completion of TLI . Donor cThree of 4 patients developed transient mixed chimerism that was lost during the second and third months posttransplant, and none developed GVHD .Immunosuppressive drug withdrawal was attempted in one patient who developed chimerism for about 1 month, and then donor specific unresponsiveness in the MLR after 7 months. IS drug withdrawal was completed at the end of one year without rejection episodes during withdrawal . HoweverA study of 10 patients given combined kidney and bone marrow transplants from HLA haplotype matched living donors was performed by investigators at Harvard University based on preclinical data generated in non-human primates and mini-swine –93. The The follow up clinical study used cyclophosphamide instead of TBI for conditioning along with thymic radiation and anti-CD2 monoclonal antibodies. Maintenance IS drugs posttransplant were used for several months before complete discontinuation in 7 patients –93. NineThe Harvard group examined mechanisms of immune suppression drug withdrawal and maintenance of graft function in their patients receiving HLA-mismatched combined kidney and bone marrow transplantation (CKBMT) that led to transient (< 3 weeks) donor cell chimerism , 97. TheBased on the results of the HLA matched tolerance induction trial using TLI/ATG conditioning, Stanford investigators performed a follow up trial to determine whether immune tolerance and complete IS drug withdrawal could be achieved in 22 recipients of HLA haplotype matched living donor kidney transplants , 88.The Recipients were given 10 doses of 120cGy each of TLI and 5 doses of ATG as in the fully matched study, and an infusion of donor CD34+ cells and a defined dose of T cells that was escalated from 3x106 cells/Kg to 150x106 cells/Kg in order to facilitate mixed chimerism . During In order to achieve the third goal, complete IS drug withdrawal was attempted during the second year in the 10 haplotype matched patients with persistent chimerism at the end of one year on tacrolimus monotherapy , 88. TacTapering to subtherapeutic levels or to discontinuation of tacrolimus was associated with loss of chimerism in the first 6 patients . Three oThe instability of mixed chimerism and dependence of chimerism on continuation of IS drugs after one year in these patients contrasts with the observations of mixed chimerism in MHC mismatched rodents that were conditioned with TLI/ATG and given combined organ and bone marrow transplants , 44, 75.The observation of IS drug dependent mixed chimerism in the patients with HLA mismatched kidney transplants, and the IS drug independent mixed chimerism in MHC mismatched rodents using the same TLI/ATG conditioning regimen suggests that the rodent and human immune systems differ in their responses to the combined transplants. The instability of mixed chimerism in studies of patients given hematopoietic cell transplants to treat hematologic malignancies and anemias based on genetic mutations is consistent with the observations observed in the kidney transplant patients.It is possible that the low levels of T cells chimerism (<20%) observed in the haplotype matched kidney transplant patients at the end of the first year contributed to the instability of chimerism during IS drug tapering during the second year, and that increased levels of T cell chimerism will allow for complete withdrawal of IS drugs without subsequent rejection. Investigators at Stanford are currently intensifying the conditioning regimen to increase the levels of T cell chimerism to determine the impact on the stability of chimerism during taper and discontinuation of IS drugs during the second year posttransplant.Investigators at Northwestern University used a conditioning regimen for their studies of kidney and hematopoietic progenitor transplantation that was developed at Johns Hopkins University for treatment of hematologic malignancies with HLA mismatched bone marrow transplantation . The conThe use of PTCy was translated to a clinical protocol in cancer patients receiving HLA haploidentical bone marrow in a two-cohort study intended to determine if cyclophosphamide could improve engraftment  by mitigfoxp3 + regulatory T cells (T regs), possibly due to the high expression of aldehyde dehydrogenase, an enzyme that metabolizes cyclophosphamide . Two major CD8+ FC subpopulations were described, one is CD56dim/− and the other CD56bright. The majority of CD56dim/− FC were also positive for CD3ϵ and HLA-DR and negative for the dendritic cell markers CD11c and CD123. The CD56dim/− FC comprised approximately half of the FC total. The majority of the CD56bright FC subpopulation were CD19+, CD11c+, CD11b+ and CD3ϵ− and comprised just under half of the FC total .The intensity of host conditioning combined with the composition of cells in the donor inoculum resulted in kidney transplant patients with early and complete conversion to donor chimerism in most recipients –106. ComIt is difficult to assess the contribution of the unique FC population in promoting conversion to complete chimerism and mitigating GVH alloreactivity. In single and multi- center studies of cancer patients that received haploidentical bone marrow transplants using unmanipulated donor cell infusions with pre- and post-transplantation cyclophosphamide, over 90% of recipients attained complete donor chimerism , 108. ThThe Northwestern group reported at least one year of follow-up in 37 patients  treated using their protocol –106. RecAmong the 37 patients, 26 achieved full donor chimerism and 23 (62%) were removed from immune suppression medication. Eight patients with transient chimerism required immune suppression. Two patients failed to establish donor cell chimerism at any level. Two patients had kidney allograft loss during the first year after transplant, and two other patients with full donor chimerism developed immune suppression dependent GVHD one of who died from GVHD –106.The Harvard, Northwestern, and Stanford University protocols vastly differ from one another albeit all use the concept of combining donor hematopoietic cell infusions, host transplant conditioning, and planned immune suppression drug withdrawal from kidney transplant patients. Comparisons of pros and cons are difficult especially given that relatively few patients have been transplanted therefore the accuracy concerning toxicity, complications and outcomes remains somewhat speculative Table 1.The Northwestern approach was akin to traditional allogeneic HCT for hematologic malignancies with predictable and significant toxicities, some life threatening, and attainment of complete donor chimerism. Immune suppression drug withdrawal in the setting of 100% donor chimerism does not reflect HvG tolerance, and a concern with this approach is GVH alloreactivity and development of GVHD in some patients. GVHD was not observed in the Harvard and Stanford protocols.https://clinicaltrials.gov/ct2/show/NCT04571203) as host conditioning is entirely post kidney transplant.The Stanford protocol was well tolerated and associated with few if any toxicities above and beyond a standard of care transplant. The conceptual framework is predicated on the protection afforded by persistent mixed chimerism that prevents bi- directional HvG and GvH reactions. Immune suppression independent persistent mixed chimerism in HLA mismatched transplants was not reliably been achieved , 88. RatThe Harvard protocol was associated with the side effect of “engraftment syndrome” in 9 of 10 patients in the first month posttransplant . The synThe Stanford study showed evidence of donor specific unresponsiveness in HLA matched and mismatched chimeric recipients, but did not assay for clonal deletion –88. The That host conditioning pre-hematopoietic cell infusion is essential for the engraftment of donor cells is beyond reproach. There are dozens of published host conditioning regimens that enable donor cell engraftment, and they vary significantly in the chemotherapy and radiation components included, and in their intensity profile.Defining the intensity of the conditioning regimen is based on the type and dose of chemotherapy and/or radiation administered, the expected duration and severity of pancytopenia , and the requirement for stem cell support  , 110.It is well established in allogeneic HCT cancer recipients that as the intensity of host chemo-radiation conditioning increases, donor hematopoietic cell engraftment is more easily attained and conversion to complete donor cell chimerism is the result , 111. CoNumerous trials in cancer patients compared clinical outcomes that helped to define conditioning dose intensity. During the initial development of reduced intensity conditioning regimens for cancer patients 2 Gy of TBI was compared to 2Gy TBI combined with 90 mg/m2 fludarabine (Flu) . PretranAs the intensity of host conditioning increases, the likelihood of developing clinically significant Grades 3, 4 and 5 adverse event (AE) toxicities increase , 110. ThA transplant conditioning intensity (TCI) scoring system was developed to help investigators standardize nomenclature and allow a comparison of the many differing regimens . For exaIntermediate score regimens were highly predictive of increased early (day +100 and +180) regimen related toxicity and mortality compared to low intensity regimens.The MGH tolerance regimen in their patients without malignancy consisted of 60 mg/kg of cyclophosphamide on days −5 and −4 with respect to transplantation; a humanized anti-CD2 monoclonal antibody  on days −1, 0, and +1, thymic irradiation (700 cGy) on day −1, +/- rituximab 375 mg/m2 days −7 and −2 . In an eThe Northwestern kidney tolerance regimen consists of cyclophosphamide 50mg/kg on days -3 and +3, 2 Gy TBI on day -1, and Fludarabine total dose of 90mg/kg divided equally on days −5, −4, −3 , 106 is This degree of intensity is associated with severe neutropenia, the need for G-CSF administration in all recipients, and the requirement for transfusion support. A “rescue” donor cell graft is not essential yet is highly desirable (required) in order to avoid prolonged blood count recovery and the associated significant health risks.The Stanford conditioning regimen that uses TLI-ATG falls below the TCI scoring system because the combination of TLI and ATG does not induce clinically significant cytopenia or regimen related organ  toxicity. There is no need for transfusion support. The unexpected 100-day re-hospitalization rate in over 600 cancer patients transplanted from HLA matched and mismatched related and unrelated donors was 25% compared to over 80% for patients that were transplanted using 2 Gy of TBI or 2 Gy TBI-Flu . The 1-yThe assessment of the risk of developing clinically significant AEs and mortality resulting from toxicity of the intensity of host conditioning is based on scores and indices specifically developed to measure the conditioning regimen intensity. Whereas the MGH and Stanford protocols are non-myeloablative, low intensity regimens the Northwestern protocol follows a traditional BMT reduced intensity conditioning (RIC) regimen of intermediate intensity and that is associated with increased regimen related morbidity and mortality .The composition of the donor cell inoculum is also an important determinant in whether engraftment and chimerism is transient, persistent and mixed, or results in conversion to complete door type. The cellular composition contributes to the risks of developing acute and chronic GVHD, and post-transplant infections, and the risk of transplant related mortality –119. DesAs a general consideration, donor cell engraftment is facilitated by increasing the number of donor TNC , and CD34+ and CD3+ T cells infused. The cellular composition should also be considered in combination with the intensity of host conditioning. For example, as the intensity of host conditioning is reduced higher numbers of TNC, CD34+ and CD3+ T cells were required to support donor cell chimerism , 124. UsIn assessing the MGH protocol for patients without malignancy the graft source and cell doses were not specified. Based on the body of their work, however, it is reasonable to presume unmanipulated donor bone marrow harvest products were obtained at the time of kidney harvest. Decades of clinical bone marrow transplantation has confirmed that the interquartile  range for TNC and CD34+ cells in a bone marrow graft is about 1.5-4.0 x108/kg and 2-4.5 x106/kg, respectively, and CD3+ T cells range between 20-40 x106/kg for . When boThe Northwestern protocol uses a unique donor cell composition that is a challenge for the reader to understand because details are considered proprietary to a commercial entity and was based on preclinical murine models. The publications highlight that using the CliniMACS (Miltenyi Biotec) system mature GVHD-producing and antigen- presenting cells are removed while HSC, FC, and progenitor cells are retained. The dose of CD34+ cells range from about 1-16 x106/kg, the dose of α,β T cells appear set at 3.8 x106/kg and FC range from 2-12 x106/kg –106. It in vitro that was restored following depletion of CD4+ Treg cells. The aggregate of data supports the contention that suppression of bi-directional alloimmune responses in mixed chimerism may be enhanced through peripheral cell-based regulation and raises the potential therapeutic options of Treg cells  using the Stanford host TLI-ATG conditioning regimen, a donor graft enriched for CD34+ cells combined with a defined CD3+ T cell dose and a recipient Treg cell infusion in HLA mismatched living donor kidney transplant recipients.There is a panoply of cells in the donor inoculum that appear important in promoting engraftment, and that are not associated with inducing GVHD. In this regard, there is abundant preclinical literature to support that Tregs of donor and recipient origin promote donor hematopoietic engraftment or enable persistent mixed chimerism without inducing GVHD , 130. Theg cells . This coThe addition of donor derived hematopoietic cells imparts new immunologic phenomena not previously considered in clinical organ transplantation with a crossing of a double barrier, there is the known HVG reactions yet this is combined with GVH reactions. Traditionally, high intensity myeloablative and immune suppressive host conditioning regimens administered before the donor cell infusion were to eliminate HVG reactions and facilitate donor cell engraftment, while the need for post-infusion immune suppression mitigated the complications of GVH reactions. With the development of lower intensity regimens in which residual host hematopoietic and immune cell compartments persist, the importance of post grafting immune suppression focused attention to the need to mitigate HVG reactions. The Seattle group used DLA- identical littermate dogs given donor bone marrow cell infusions at clinically relevant doses of 1.9 to 4.4 × 108 TNC/kg within 4 hours of completing low intensity 2 Gy TBI . RecipieThe Northwestern and Stanford protocols albeit very different from one another, use as a backbone post grafting Tacrolimus combined with MMF, in part because the above article so heavily influenced, in the right direction, the field , 104–106in vitro, and in clinical studies to induce immune modulatory effects that include upregulation of Treg cells , siplizumab, in their ten patient published series –93. CD2 eg cells . The speThe Northwestern group used high-dose, post-transplantation cyclophosphamide (PTxCy). Post-transplantation cyclophosphamide represents the culmination of over 50 years of preclinical research dedicated to inducing immunologic tolerance . The usein vitro T cell depletion sufficiently removed enough alloreactive T cells that GVHD was markedly reduced  cells , and virThe removal of Tnaive cells from the donor cell inoculum by CD45RA column depletion is a platform to promote donor cell engraftment with a low risk of clinically significant GVHD even after conversion to complete donor chimerism , 140.Enrichment for CD8+ T memory cells following CD45RA depletion also promoted conversion to complete chimerism with perhaps even lower GVHD risk then a CD45RA depleted graft as CD4+ memory T cells may promote GVHD , 142. AnThe rodent models summarized in this review uniformly demonstrated that mixed chimerism and tolerance were stable after MHC matched and mismatched combined hematopoietic progenitor and organ transplantation , 44–52. In the case of complete chimerism, concordant observations were made in humans and laboratory animals. Complete chimerism was achieved in HLA mismatched patients given combined kidney and hematopoietic progenitor transplants at Northwestern University, and was stable –106. AccThe tolerance induction clinical trials performed at Stanford and Harvard Universities represented a bench-to-bedside translation of preclinical models of mixed chimerism and organ transplantation tolerance to clinical medicine to establish proof of principle that scientific approaches can be used to treat diseases in humans. Each used a center-specific approach to establish mixed chimerism: In one case there is transient, short duration mixed chimerism, in another persistent and often permanent mixed chimerism. There are pros and cons with each approach and when considered as an aggregate they provide insight into some of the fundamental concepts and mechanisms that underlie immune suppression drug withdrawal chimerism-based tolerance protocols.Mixed chimerism in these studies was uniformly above 1% of donor type cells in the recipient blood and lymphoid tissues, and in almost all instances above 10%. This level of chimerism is referred to as “macrochimerism”. In contrast, some studies of organ with or without hematopoietic progenitor transplantation have reported the development of stable “micro-chimerism”. In 1993 Starzl and colleagues reported a small cohort of liver transplant recipients that spontaneously developed very low levels of donor hematopoietic cell chimerism detectable only by high resolution PCR testing that was subsequently termed micro-chimerism (<1% donor type: the liver itself can act as a hematopoietic cell reservoir and bring forth donor hematopoietic progenitor cells after transplantation). Subsequent withdrawal of immune suppression medication with maintenance of normal graft function was seldom accomplished , 145. ThTo provide context to chimerism-based kidney tolerance protocols the lessons learned from more than five decades of clinical allogeneic HCT in more than 1 million cancer patients that received donor grafts should be considered. Patients receiving allogeneic hematopoietic cells are prepared with pre-hematopoietic cell infusion ‘host conditioning’ that consists of chemotherapy alone or in combination with radiation therapy and/or antibodies directed to immune cells. The pretransplant host conditioning is essential to establish donor hematopoietic cell chimerism and serves two critical purposes: to deplete and suppress the recipient immune cells that would otherwise reject the infused donor cells, and to deplete host bone marrow progenitor cells from their marrow niches to create ‘marrow space’ and enable donor stem cell replacement and engraftment.In context of protocols that use donor hematopoietic cells, tolerance is perhaps easiest to conceptualize in the setting of persistent mixed chimerism. With persistent mixed chimerism, donor APCs in the thymus present donor Ag to developing host T cells and induce deletion of alloreactive HVG T cells. Similarly, with mixed chimerism host APCs in the thymus present host self Ag to donor T cells and induce deletion of GVH reactive T cells. This bi-directional clonal deletion establishes central tolerance Figure 4These findings suggest that in the HLA matched setting, 6-months of persistent mixed chimerism may be a sufficient condition to induce long-lasting central tolerance that can control HVG alloreactivity and prevent graft rejection. The contribution and need for peripheral regulation in this setting is less defined. In a few instances however, patients who had had persistent mixed chimerism and subsequently lost the donor cells after immune suppression drug withdrawal, years later had an acute rejection episode. This suggests that central tolerance may not be a permanent state in a few patients or that peripheral regulation was lost.https://clinicaltrials.gov/ct2/show/NCT03734601).In the HLA mismatched Stanford tolerance protocol low dose single drug immune suppression was required for persistent mixed chimerism as complete drug withdrawal resulted in loss of chimerism that was associated with acute rejection episodes , 87. ThiThe key observations of the link between chimerism and transplant tolerance were initially developed in rodents, and then studied in large laboratory animals and humans. Whereas stable mixed chimerism and organ graft acceptance without IS drugs was achieved in most MHC mismatched models of tolerance induction in rodents, it has been achieved only in MHC matched large animals and humans at present. Stable mixed chimeras showed specific immune unresponsiveness in both the HvG and GvH directions. Stable complete chimerism was achieved in MHC mismatched laboratory animals and humans given HCT as treatment for hematologic malignancies or for withdrawal of IS drugs with organ transplant acceptance. In the latter complete chimeric recipients, kidney graft acceptance was based on self-tolerance rather than HvG tolerance. Safety issues associated with a protocol that resulted in complete chimerism remain a clinical concern Table 1 Both authors contributed to the article and approved the submitted version.This work was supported by the National Institute of Health, grant PO1HL075462 and R01 1085024, and the California Institute for Regenerative Medicine, grant Clin2-09439.RL and SS are scientific founders of Medeor Therapeutics. SS is a consult to Medeor Therapeutics.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Unrelated umbilical cord blood (UCB) and haploidentical grafts have been used for allogeneic hematopoietic stem and progenitor cell (HSPC) transplantation in patients without a related or non-related human leukocyte antigen (HLA)-matched donor. The less stringent HLA-matching requirement in both sources raises an important possibility for patients in need of urgent transplantation to treat any hematological disease. Selection of the best alternative donor is a difficult task that will depend on donor criteria, center experience, patient disease conditions, and risk, among others. Most comparisons available in scientific publications between both graft sources are obtained from retrospective analysis in wide time windows and a heterogeneous number of patients, types of disease, disease stages, previous treatments, graft source, conditioning regimen, graft vs. host disease (GVHD) approach, and evaluable endpoints. There is also an evident impact of the economic traits since low-income countries must consider less expensive treatments to satisfy the needs of the patients in the most effective possible path. Therefore, haploidentical transplantation could be an appealing option, even though it has not been completely established if any chronic treatment derived from the procedure could become a higher cost. In Colombia, there is a huge experience in UCB transplantation especially in units of pediatric transplantation where benign indications are more common than in adults. Due to the availability of a public UCB bank and HLA high-resolution typing in Colombia, there is a wider inventory of cord blood donors. Unfortunately, we do not have an unrelated bone marrow donor registry, so UCB is an important source along with haploidentical transplantation to consider in decision-making. This minireview focuses on comparing the main issues associated with the use of both HSCP sources and provides tools for physicians who face the difficult decision between these alternative donor sources. Hematopoietic stem and progenitor cell (HSPC) transplantation is a potentially curative treatment that has been used for different disorders that affect hematopoiesis , 2. DepeIt is known that only 30% of the patients who require an HSPC transplant have a fully HLA-matched sibling donor , 9, 10 aThere is an important experience in Colombia with UCB transplantation for pediatric patients and a higher inventory of cord blood donors with HLA high-resolution typing available in the public UCB bank which started 5 years ago . Since tOver the past 5 years, some authors have been pointing out an evident increase of haploidentical donor transplantation procedures and a significant decrease in UCB for allogeneic transplants in Europe and the United States , 12–14. There are some particularities in Latin America regarding this issue. Although HSPC transplantation has been performed for the last 50 years in Europe and the US , in Latiex vivo manipulation) solves the problem of finding an HLA-matching donor and its associated high costs, it represents an appealing option in developing countries and its use is rapidly increasing, as reported in the US and Europe  donor registries. Registries available in Latin American countries are considerably smaller, the searching activities are less organized and take longer times, which is critical for patients with malignant diseases . Since hd Europe .in vivo and ex vivo T-cell depletion techniques  and that cell dose comprises a more impacting factor than HLA matching on clinical outcomes , 5. HoweIn a different scenario, a recent report shows that not only the traditional matching of HLA class I sequences in the peptide-binding region could drive the risk of GVHD or even impact the results of the transplantation. A dimorphism in HLA-B exon 1, which produces two different leader peptides, could provide information regarding relapse and non-relapse mortality risk after UCB transplantation. This confirms the importance and potential of high-resolution HLA typing in UCB graft selection and transplantation .Current HLA-matching protocols could be unbalanced if a comparison is pretended between UCB and haploidentical transplantation clinical outcomes. A low- and intermediate-resolution HLA typing is enough for the latter case, due to the inherent relation between donor and patient. On the other hand, and according to the previously mentioned evidence, allelic-level HLA typing would be more appropriate in a non-related UCB setting, not only for HLA-DRB1 but for all HLA genes. There is a potential improvement of the UCB procedure in this area, which could lead to the obtention of better clinical outcomes if additional characteristics are included in the donor selection process.Although the reduction trend in UCB use aligns with the haploidentical transplantation increase, there are not many studies comparing both HSPC sources in terms of clinical efficacy. In pediatric patients, only two publications were found , 25 whil9/L) in both studies, with a median time of 20 and 16 days compared to haploidentical transplanted patients, with a median time of 13 days in both reports  delayed neutrophil recovery  , 29, 30.www.clinicaltrials.gov was consulted to verify if any ongoing clinical trial was comparing outcomes of transplantation from haploidentical and non-related UCB donors. Three interventional, open-label studies were found, two for hematological malignancies and one for ß-thalassemia. The latter lacks information since 2015 and its current state is unknown. The studies in hematological malignancies were designed to compare in parallel groups double UCB vs. haploidentical transplantation. One of those was terminated due to slow accrual—with only two enrolled patients—while the third one, a phase III active study with 368 enrolled patients, was recently completed and published  at allele-level. After randomization, 172 patients received double-UCB transplantation, and 153 patients were transplanted from a haploidentical donor in a post-transplant cyclophosphamide setting. All patients received a reduced intensity conditioning regimen  than for haploidentical donors (17 days) and is even statistically significant when comparing the cumulative incidence at day 56, although it is not completely clear if this variable assessment is identical to the previous studies mentioned. Platelet recovery, however, remains slower in double UCB (median 42 days) than in haploidentical donors (median 28 days). As for GVHD, there were no significant differences between treatments either in the acute or chronic frame. There were no statistically significant differences between the two treatments in the probability of 2-year PFS (primary endpoint) and 2-year incidence of relapse/progression; nonetheless, the 2-year incidence of non-relapse mortality was significantly higher in double-UCB-transplanted patients, which leads to a lower 2-year overall survival than the haploidentical graft receptors (secondary endpoints). In this case, the most common cause of death in both groups was a recurrent disease, followed by infection. Although no analysis regarding this data is provided there is a difference of only 2% in deaths proportion by infection in UCB transplantation compared to haploidentical (9.15 vs. 7.1%) .To determine which is the most favorable donor alternative between unrelated UCB and haploidentical donors for pediatric and adult HSPC transplantation candidates with any hematological disease is not an easy task, considering the diversity of the results. To the best of our knowledge, there are no published comparisons between the two sources in the treatment of non-malignant diseases. There is also an unexplored possibility comparing haploidentical transplantation with a higher matching UCB donor, considering four HLA loci in high-resolution typing, a condition that may improve the neutrophil reconstitution time, as described by Eapen et al. . It is pAlthough there is not enough evidence favoring haploidentical over UCB transplantation from the clinical perspective, there is an increase of haploidentical transplantation vs. UCB , 13, 14.Finally, it is important to note that just as haploidentical transplantation has improved over time, UCB transplantation may reach better results upgrading the clinical criteria and practice, especially because it has gone through a shorter path in history. Even though the comparisons so far have not been equitable between both sources in terms of HLA matching, which is one of the main success indicators, the lack of statistically significant differences in clinical outcomes strongly suggests that improving the UCB donor selection could definitively raise a new scenario for this source, adding possibilities for those patients lacking a related donor and joining forces with haploidentical transplantation possibilities. It is possible that the reduction of UCB use as an HSPC source compared to the haploidentical donor is not essentially based on research data, but it could be also driven by other conditions, such as the knowledge and skills in the clinical practice in some countries, economic issues, and difficulties of finding a donor.DV and A-MP-A contributed to the initial conception of comparing the two sources for transplantation reviewing the available bibliography. LN-Q, MC, ME, and BC-R contributed with the clinical perspective for comparison. DV wrote the first draft of the manuscript, A-MP-A wrote sections of the manuscript. BC-R had financial support. All authors contributed to manuscript revision, read, and approved the submitted version.This work was supported by the Sistema General de Regalías. Fondo de CTeI, Colombia and Fondo Financiero Distrital de Salud (FFDS). Secretaría Distrital de Salud, Bogotá DC, Colombia. BPIN: 2016000100035.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


This article is related to a research paper entitled “Exploring the charge-transfer chemistry of fluorine-containing pyrazolin-5-ones: The complexation of 1-methyl-3-trifluoromethyl-2-pyrazoline-5-one with five π-acceptors” [J. Mol. Liq. 331 (2021) 115814]  Exploring the charge-transfer (CT) properties of pyrazole derivatives may be useful toward improving their clinical efficacy and expanding the range of their medicinal applications.•Determining the stoichiometry of the complexation between one of the pyrazole derivatives (namely 1-methyl-3-trifluoromethyl-2-pyrazoline-5-one) with different acceptors is particularly important to understand the mode of the interaction of these derivatives toward improving their uses and chemical, physical, and biological applications. These data are useful for researchers applying the CT interaction in the fields of chemistry, biochemistry, physics, biology, medicine, and pharmacology.•The most important and useful approaches to verify the stoichiometry of the complexation between molecules are the spectrophotometric titration method and the Job's continuous variation method. These approaches are easily performed.1−3 M) were each individually mixed with the FP donor, also solubilized in methanol at the same concentration Methanolic solutions of PA, CLA, TFQ, and TCNQ acceptors , DDQ , TFQ , CLA , PA , and 1-methyl-3-trifluoromethyl-2-pyrazoline-5-one (donor)  were obtained from Merck KGaA  and UFC Biotechnology .Analytical-grade TCNQ  was combined with each solubilized acceptor (1 mL) and methanol (3 mL) in 5-mL glass tubes to generate the donor-PA, donor-CLA, donor-TFQ, donor-DDQ, and donor-TCNQ systems.3.CT) for each system.The UV-visible spectra of the free donor, free acceptors, and synthesized complexes were collected at room temperature from 200 to 800 nm using a Perkin-Elmer Lambda 25 UV/Vis spectrophotometer and used to determine the CT band (λ4.CT) of 10 standard solutions with varied donor to acceptor molar ratios (from 4:1 to 1:4) were plotted against the volume of the acceptor in each standard solution.To verify the stoichiometry of the interaction between the donor and each of the acceptors using the spectrophotometric titration method, the absorbances (λ5.CT) of 10 standard solutions with varied molar fractions of donor and acceptor (Cdonor + Cacceptor) were plotted against the molar fraction of the acceptor in each standard solution.To verify the stoichiometry of the interaction between the donor and each acceptor using Job's continuous variation method, the absorbances (λAbdel Majid A. Adam: Data curation, Writing – original draft, Writing – review & editing; Tariq A. Altalhi: Conceptualization, Methodology, Software, Supervision; Hosam A. Saad: Visualization, Investigation, Software, Validation; Moamen S. Refat: Conceptualization, Methodology, Software, Supervision; Mohamed S. Hegab: Visualization, Investigation, Software, Validation.The authors declare that they have no known competing financial interests or personal relationships that could be perceived to have influenced the work reported in this article.


Filifactor significantly enriched in TT. The phylum Actinobacteria; genus Veillonella; and species Granulicatella adiacens, Streptococcus sanguinis, and Veillonella rogosae were significantly enriched in NPT, while the phylum Bacteroidetes; genera Capnocytophaga, Haemophilus, and Prevotella; and seven species, including Capnocytophaga sp., Haemophilus sp., and Neisseria sp., were significantly enriched in TS. In TTs, the abundance of Prevotella intermedia was profoundly higher in the gingiva, while Capnocytophaga gingivalis and Rothia mucilaginosa were enriched in the lining mucosa and tongue. Increasing in abundance from the early tumor stage to the late stage, Solobacterium moorei in TT and Campylobacter sp. strain HMT 044 in TS were positively correlated with OSCC development, suggesting that bacteria were selected by different microenvironments. The correlation between 11 microbial species and 17 pathway abundances was revealed, indicating the potential function of low-abundance bacteria. Overall, our analysis revealed that multiple oral bacterial taxa are associated with a subsequent risk of OSCC and may be used as biomarkers for risk prediction and intervention in oral cancers.Oral microbiota dysbiosis is associated with the occurrence and progression of oral cancer. To investigate the association between the microbiota and risk of oral squamous cell carcinoma (OSCC), we identified the microbial composition of paired tumor (TT)/normal paracancerous tissues (NPT) and saliva (TS) samples in OSCC patients through 16S rRNA gene sequencing. A total of 22 phyla, 321 genera, and 869 species were identified in the oral samples. Paired comparisons revealed significant differences between TT, NPT, and TS groups, with the genus  Annually, more than 447,000 new diagnoses of and nearly 228,000 deaths due to oral cancer are reported worldwide, over 90% of which can be attributed to squamous cell carcinoma (SCC) . SCC is Helicobacter pylori, which is known to promote gastric cancer  were elevated in the saliva of patients with OSCC. S. anginosus, a pathogen that occurs frequently in OSCC tissue specimens, was detected in the dental plaque but not in the saliva of patients , Gemella sp. (G. haemolysans and G. morbillorum), Johnsonella ignava, and Peptostreptococcus stomatis were prevalent in tumor sites, whereas Granulicatella adiacens was prevalent in non-tumor sites.Oral bacteria are most likely linked to oral squamous cell carcinoma (OSCC) . Four co of OSCC . Bacteriy mucosa . Mager epatients . By compBacillus, Enterococcus, Parvimonas, Peptostreptococcus, and Slackia) exhibited significant differences in saliva between epithelial precursor lesions and OSCC patients, and the genera Cloacibacillus, Gemmiger, Oscillospira, and Roseburia were 20 times more abundant in OSCC patients than in healthy controls. Yang et al. compared oral mouthwash samples from OSCC patients and healthy controls and found that the abundances of Fusobacterium periodonticum and Streptococcus constellatus were positively correlated with the development of OSCC, whereas S. mitis, Haemophilus parainfluenzae, and Porphyromonas pasteri were negatively correlated (F. nucleatum and Prevotella intermedia) and genes involved in bacterial chemotaxis, flagellar assembly, and lipopolysaccharide (LPS) biosynthesis were significantly increased in the tumor sites. Fusobacterium and Dialister, was significantly enriched in tumor sites. By comparing fresh OSCC biopsies and epithelial samples of healthy controls collected by swab, F. nucleatum and Pseudomonas aeruginosa, were related to OSCC. P. gingivalis and F. nucleatum were present at higher levels in cancer tissue than in normal tissues of patients with OSCC and were correlated with subgingival plaques. By comparing OSCC tissue, saliva, and mouthwash samples from the same subjects, Acinetobacter and Campylobacter were enriched in OSCC tissues, while Streptococcus and Prevotella were enriched in saliva and mouthwash.With the advent of 16S rRNA high-throughput sequencing, there is growing interest in the possible relationships between changes in the oral microbiota and the risk of oral cancer . Lee et rrelated . Zhao etAccording to a growing number of studies, over 30 genera and a panel of bacterial species have been associated with OSCC . HoweverTo date, comparisons among paired tumor/paracancerous tissues and saliva samples from the same OSCC patients have not been performed. In the current pilot study, we aimed to investigate the association between microbiota and the risk of developing OSCC, as well as to establish the connection between the changes in the microbiota in TT, normal paracancerous tissue (NPT), and saliva (TS). Twenty-three patients with OSCC from Northern China were recruited, and the overall microbiota composition and abundance of specific bacterial taxa in TT, NPT, and TS were identified through 16S rRNA gene sequencing. Furthermore, phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt2)  was usedThe subjects were recruited from 2018 to 2020 with the approval of the Chinese Clinical Trial Registry (ChiCTR2000032543). All participants were not administered any antibiotics 3 months before the study. Written informed consent was obtained from all participants before sample collection. In total, 23 patients with OSCC who had not undergone any treatment, including chemotherapy and/or radiotherapy were enrolled. TNM staging and data on age, sex, alcohol intake, cigarette smoking, oral hygiene, and periodontal status were assessed. Saliva samples were successfully collected from 19 out of the 23 OSCC patients before breakfast without stimulation. Furthermore, TT samples were dissected from the deep site of the tumor during surgery, and the diameter of each sample was larger than 3.0 mm. Concurrently, paired NPT samples harvested at least 2.0 cm away from the tumor edge were also obtained under aseptic conditions. Finally, saliva and tissue samples were aseptically transferred into a screw-cap vial and stored at −80°C immediately after collection.To characterize the differences in the microbiota composition in different tissue and saliva samples, 65 samples were classified into three groups as follows: TT, NPT, and TS.Total genomic DNA was extracted using the TIANamp Swab DNA Kit  after TS samples were vortexed with glass beads and TT/NPT samples were liquid nitrogen-ground. To eliminate any contamination caused by blood stains on the surface of the sterile tissues, surface decontamination of the samples was achieved by washing with PBS. Amplification of the 16S rRNA gene V3–4 region was performed with primers 338F and 806R  using TrRaw paired FASTQ files were imported into QIIME 2 , and thep-values for multiple tests in R. Significantly different features (FDR < 0.05) were further validated using linear discriminant analysis effect size (LEfSe) . Microbiome functions were predicted using PICRUSt2 . Signifi (LEfSe) , and onlWe collected 65 samples from 23 patients, including 23 TT, 23 NPT, and 19 saliva samples from the patients . A totalPANOSIM < 0.01, In patients with OSCC, the evenness (Simpsoneven index) of the TS group was significantly different from that of the TT and NPT groups . PrincipFusobacterium (15.99%), Neisseria (7.9%), Streptococcus (6.92%), Porphyromonas (5.66%), and Haemophilus (4.78%) were identified in the oral samples  in NPT, and the genera Campylobacter, Gemella, Filifactor, and Catonella were highly enriched in TT  in NPT, and Aggregatibacter segnis, Campylobacter showae, and G. morbillorum were highly enriched in TT  and NPT  from the same patients showed diversity  were significantly enriched in TT compared with NPT  .PANOSIM < 0.01, Filifactor and Peptostreptococcus were highly enriched in TT , 6 genera, and 5 species were significantly enriched in NPT, while the phylum Bacteroidetes, 5 genera, and 13 species were significantly enriched in TS , includiTwenty-eight and two pathways were separately enriched in NPT and TS  with seven different pathways , lining mucosa (bucca and mouth floor), and specialized mucosa (tongue). Four genera and 13 species were observed to be separately enriched in each of the three types of tumor mucosa . C. gingpathways .R > 0.68) with four different pathways  and 10 genera and 16 species separately enriched in each of the three sites . Most ofpathways .Treponema sp. and Leptotrichia sp., while Prevotella sp. and Capnocytophaga sp. were enriched in TS  of oral cancer. In the early tumor stage (I/II), both TT and NPT had higher abundances of ed in TS . In the ed in TS . The cored in TS ; for exaOral cancer is a highly complex, multifactorial disease associated with the dysbiosis of the oral microbiota. A comprehensive study of the oral microbiota is essential for understanding the pathogenesis of oral cancer. Previous studies have shown higher alpha diversity in saliva  and tissp = 0.004)  , we did = 0.004) , whereasfindings . FurtherT and TS , 3, 5. Al plaque , and the disease . C. showdontitis , Crohn’sdontitis , and coldontitis . G. morbdontitis  and is hdontitis . TherefoPeptostreptococcus and Filifactor were enriched in TT samples. At the species level, we confirmed that F. naviforme, P. stomatis, and N. flavescens were significantly enriched in TT. F. naviforme is known to be associated with the development and progression of OSCC  and Bacteroidetes  was remarkably higher in TS samples . Consist of OSCC . Two repn in NPT . Additioogenesis . These fPrevotella, Acinetobacter, Pseudomonas, and Fusobacterium in the tongue, oropharynx, gingiva, and bucca of patients with oral cancer. Here, our results revealed that in TTs, Lautropia and Rothia were enriched in the tongue, while Peptococcus and Tannerella were separately enriched in the gingiva and lining mucosa  under accession number OEX003132.All the data generated or analyzed during this study are included in this published article and its The studies involving human participants were reviewed and approved by the Chinese Clinical Trial Registry (ChiCTR2000032543). The patients/participants provided their written informed consent to participate in this study.TZ and HZ designed the study. KY, SZ, DZ, LH, and TZ collected all the saliva and tissue samples. YW and HZ performed the measurements and data analysis. KY, TZ, and HZ wrote and edited the manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Not every neonate with congenital Zika virus (ZIKV) infection (CZI) is born with microcephaly. We compared inflammation mediators in CSF  between ZIKV-exposed neonates with/without microcephaly (cases) and controls. In Brazil, in the same laboratory, we identified 14 ZIKV-exposed neonates during the ZIKV epidemic (2015–2016), 7(50%) with and 7(50%) without microcephaly, without any other congenital infection, and 14 neonates (2017–2018) eligible to be controls and to match cases. 29 inflammation mediators were measured using Luminex immunoassay and multidimensional analyses were employed. Neonates with ZIKV-associated microcephaly presented substantially higher degree of inflammatory perturbation, associated with uncoupled inflammatory response and decreased correlations between concentrations of inflammatory biomarkers. The groups of microcephalic and non-microcephalic ZIKV-exposed neonates were distinguished from the control group . Between controls and those non-microcephalic exposed to ZIKV, IL-1β, IL-3, IL-4, IL-7 and EOTAXIN were the top CSF markers. By comparing the microcephalic cases with controls, the top discriminant scores were for IL-1β, IL-3, EOTAXIN and IL-12p70. The degree of inflammatory imbalance may be associated with microcephaly in CZI and it may aid additional investigations in experimental pre-clinical models testing immune modulators in preventing extensive damage of the Central Nervous System. In 2016, a causal relationship between ZIKV infection during pregnancy and microcephaly was strongly suspected2, when a serious epidemic sparked a major concern given the hundreds of microcephalic neonates born in Brazil3. Subsequently, several studies have shown that ZIKV has high Central Nervous System (CNS) tropism and is harmful when the CNS is immature4.A widespread epidemic of Zika virus (ZIKV) infection was reported in South America in 20155. Leptomeningeal and cerebral parenchymal inflammation was described with varying intensity and distribution5. In 2018, an immunohistochemical analysis of neural parenchyma tissues from 8 deceased neonate/stillbirth babies, 4 with and 4 without ZIKV infection, showed significantly higher expression of several cytokines receptors in ZIKV-positive microcephaly cases, suggesting that inflammation activation can aggravate the neuroinflammatory response and consequently increase CNS damage in neonates with fetal neural ZIKV infection and microcephaly6. Although extensive research has been done focusing on understanding the mechanisms of ZIKV-induced congenital microcephaly, there are still gaps. In a case-series of neonates with laboratory-confirmed CZI, 13% were born without microcephaly7. Additionally, neonates exposed to ZIKV during fetal life may be born with increased protein concentrations in cerebrospinal fluid (CSF), particularly in cases with microcephaly or abnormal neuroimaging findings8. A previous investigation enrolling 6 ZIKV-infected pregnant women who delivered during the study course showed that infants with congenital CNS deformities had significantly higher levels of IL-18 and IP-10 but lower levels of hepatocyte growth factor than those without such abnormalities born to ZIKV-infected mothers9. In a recent study that investigated concentrations of mediators of inflammation in umbilical cord blood of neonates, Vinhaes et al. have demonstrated that individuals with ZIKV-associated microcephaly exhibit an increased systemic inflammation profile compared to those that were exposed to ZIKV but did not develop such clinical outcome10. Whether imbalanced immune activation in peripheral blood is associated with changes in inflammation in CSF in the context of ZIKV microcephaly is still unknown.In 2017, microcephaly was revealed on postmortem examination of 7 neonates with congenital ZIKV infection (CZI)In this context, we compared concentrations of inflammatory mediators in the CSF of neonates exposed to ZIKV during pregnancy (cases), with or without microcephaly, and neonates who were not exposed to ZIKV during pregnancy, without other congenital infection and without microcephaly (control group).3 (n = 19), CSF protein > 132 mg/dL (n = 9), CSF RBC count > 1000/mm3 (n = 8), and ventricular tap (n = 1). Among 24 neonates further evaluated after interviewing mothers, 10 (41.7%) were excluded due to untreated maternal syphilis (n = 9) and CNS hemorrhage (n = 1). The reasons for performing LP in controls were: sepsis (n = 6), maternal syphilis (n = 5), seizure (n = 1), fever without source (n = 1), and acute maternal cytomegalovirus infection (n = 1). Congenital syphilis and congenital cytomegalovirus infections were ruled out as all of them had negative syphilis serological tests  and the neonate born to the woman with gestational acute cytomegalovirus infection had urine negative cytomegalovirus polymerase chain reaction during the first week of life. Therefore, the study group comprised 14 cases and 14 controls. Their baseline characteristics are presented in Supplementary Table Out of 16 cases, 2 did not have enough CSF sample. Then, 14 remained in our study, 7 (50%) with and 7 (50%) without microcephaly, 11 likely ZIKV-infected during the 1st trimester and 3 likely ZIKV-infected during the 2nd trimester of pregnancy. Out of these 3 cases, 2 did and 1 did not have microcephaly. Overall, 85 neonates were evaluated to be included in the control group, out of which 61 (71.8%) were excluded due to: age > 4 days (n = 45), CSF WBC count > 8/mmTable To access the inflammatory changes in CSF related to CZI, we examined the expression of 29 inflammation mediators soluble proteins, stratified according to presence/absence of microcephaly in neonates exposed to ZIKV during pregnancy and in controls Fig. . Unsuper14   Fig. . Next, wNotably, CSF inflammatory parameters are modified in neonates exposed to ZIKV infection during pregnancy, according to presence or absence of congenital microcephaly. To the best of our knowledge, this is the first time in which such findings are reported from ZIKV-exposed neonates with and without microcephaly and controls.16. Nevertheless, the immunopathologic pathways driven by CSF infection which leads to microcephaly remain poorly understood. We performed a detailed investigation of the inflammatory and immune activation profile in CSF of neonates with probable CZI.The mechanism by which ZIKV invades CSF crossing the placental and blood–brain barriers has been elucidated18. Herein, neonates with ZIKV-associated microcephaly exhibited general increase in concentrations of pro-inflammatory mediators compared to controls. These neonates presented higher levels of IL-1β, IL-4, IL-10 and VEGF as previously shown in the Flavivirus model17, in addition to IFN-γ. The IFN pathway has been associated with protection of trophoblastic and non-trophoblastic cells from CZI19 and with viral replication in human placental macrophages20, with higher expression of type I IFN. Despite possible antiviral response mediated by IFN to ZIKV, other important IFN-related cytokines, as IP10 (CXCL10) levels had tendency to decrease in our analysis in both neonates with or without microcephaly, whereas concentrations of IFN-α2 displayed a tendency to decrease in CSF from microcephalic neonates and increase in those without ZIKV-associated microcephaly. Neonates with microcephaly possibly express other myeloid activation signals resulting in disturbances in the IFN pathway. Additional mechanistic studies are needed to test this hypothesis.Our exploratory analyses demonstrated the CSF inflammatory profile from neonates with ZIKV-associated microcephaly and delineated a biosignature able to distinguish ZIKV-exposed neonates from uninfected controls. In a Flavivirus infection model, investigations described a cytokine storm with immunological imbalance, with a pro-inflammatory response detected in acute phase22. Reduction in cell growth which culminates in neurological malformations such as microcephaly was observed in an experimental model22. Besides that, the CNS may be directly affected due to an unbalanced local inflammatory response elicited by ZIKV infection which contributes to fetal brain damage23. Results from an immunogenetical study that looked to factors associated with the host phenotype against ZIKV infection suggested that variations at Toll-like receptor 3 and TNF-α genes associated with innate immune responses in pregnant mothers infected with ZIKV and their CZI babies may influence the risk of occurrence of microcephaly24. However, the specific interplay between immunologic factors and microcephaly development remains unclear. Our results revealed that the concentration values of inflammatory markers in CSF alongside the relationships among them are altered in ZIKV-associated microcephaly.The association between microcephaly development and immune activation remains unclear, and the influence of inflammatory changes in such process is still unknown. The presence of ZIKV infection leads to alteration in gene regulation associated with immunological homeostasis, including cell cycle, differentiation and apoptosis in neuronal progenitor cells14. In this setting, increases in network connectivity usually infer augmented inflammation whereas decreases in the number of correlation or presence of negative associations may indicate uncoupling of the immune activation and leading to an imbalanced inflammatory response. In agreement with this idea, Vinhaes et al. recently reported that there is also an inflammatory imbalance in umbilical cord blood of neonates with CZI, and that the magnitude of the immune activation perturbation was inversely correlated with the cephalic perimeter10. Interestingly, in direct contrast with the results presenting here, the network analysis of concentrations of inflammatory mediators reported by Vinhaes et al. showed increased density  in neonates with ZIKV-microcephaly10, whereas in the present study in CSF we found a reduction in connectivity of the inflammatory networks. This discrepancy between results from blood vs. CSF could be consequence of the differential activation of immune responses in distinct compartments, which may involve tissue-specific regulation of immune responses25. Those results, together with the findings of our present study, make us hypothesize that the hyperinflammatory milieu examined by MDP assessment in CSF may represent the immune activation at the CNS driving cytotoxicity related to immunopathology27, further leading to microcephaly. Future studies are needed to direct test our hypothesis in experimental pre-clinical models in which immune modulators could be evaluated as an approach to prevent extensive damage of the CNS and microcephaly. Neonates with probable CZI with microcephaly had strong elevation of the IgM and IgA classes in CSF28. Along with the significant increase in IL-4 levels in CSF of microcephalic neonates described here  appeared in our canonical coefficient model as top markers in the discrimination between both ZIKV-exposed groups and uninfected controls, whereas IL-4 and IFN-α2 appeared to be important only in distinction between those without microcephaly Our study has some limitations. We examined samples obtained from a single time-point, which preclude us from making conclusions about the dynamicity of inflammatory process before birth. The number of individuals investigated was small, but the groups were carefully matched to reduce potential confounding factors. In addition, we have employed multidimensional analyses that have been used for analysis of small datasets as well. We adjusted the results for multiple measurements to minimize the odds of spurious findings. Finally, enterovirus infection was not investigated either in cases or in controls. However, CSF was obtained from all included neonates before maternity unit discharge what makes enterovirus infection improbable.34.In conclusion, our finds extend the current information about CZI and immune activation status of CSF in pediatric patients, with delineation of the inflammatory changes associated with microcephaly. The degree of inflammatory imbalance may be associated with microcephaly driven by cytotoxicity in CZI and it may aid additional investigations in experimental pre-clinical models testing immune modulators in preventing extensive damage of the CNS. Additionally, they can be applied to the prospective evaluation of neonates with other viral congenital infectionsThe study was approved by the Ethics Committee of the Federal University of Bahia under the number CAAE: 59198016.2.0000.5577. All research was performed in accordance with the relevant guidelines and regulations. Mothers were interviewed after informed consent had been signed.This is a case–control study with a retrospective and a prospective component. Neonates exposed (cases), with or without microcephaly, and not exposed (controls) to ZIKV during pregnancy, who underwent lumbar puncture (LP) in the CSF Laboratory, in Salvador, Brazil, were evaluated. All LPs were performed at the discretion of the neonatologist. CSF was obtained from a LP in cases in the beginning of the ZIKV epidemic in Brazil (December/2015-March/2016) due to congenital infection clinical characteristics. The clinical epidemiological criteria to diagnose probable CZI included (1) LP performed during the ZIKV infection epidemic plus (2) ZIKV symptoms during pregnancy reported by mothers resident in the ZIKV epidemic affected area plus (3) negative laboratory tests to investigate other congenital infections . CSF was obtained from a LP in controls between November/2017 and September/2018. CSF was obtained from all included neonates (cases and controls) before maternity unit discharge. All patients were identified in the CSF Laboratory logbook. After reviewing the CSF charts, the medical charts were reviewed, and the respective mothers were interviewed, after informed consent had been signed, to collect prenatal and neonatal data. Information about serological tests (STORCH-HIV-HTLV-HB-HC) from mother and child was searched for, as well as information about neonates’ age, gender, weight, gestational age, 5-min Apgar, head circumference and neuroimaging. After confirmation of patients’ eligibility, the residual CSF kept frozen at − 20 °C was transported to the Gonçalo Moniz Institute (FIOCRUZ), to measure inflammation mediators.36.Sixteen neonates were identified. All mothers reported clinical symptoms from the ZIKV infection during pregnancy , 13 (81.3%) during the first (≤ 13 weeks) and 3 (18.7%) during the second trimester (14–25 weeks). Congenital microcephaly was defined as head circumference lower than 31.9 cm (boys) and 31.5 cm (girls) for full-term (≥ 37 weeks of gestation) neonates and > 2 standard deviations below average for premature ones 3, CSF protein ≤ 132 mg/dl, CSF Red Blood Cell (RBC) count ≤ 1000 cells/mm3, absence of CNS disease, any congenital infection and microcephaly. All controls had negative laboratory tests to investigate congenital infections (STORCH-HIV-HTLV-HB-HC) as well as negative blood, urine, and CSF cultures.Inclusion criteria comprised: age ≤ 4 days, CSF White Blood Cell (WBC) count ≤ 8/mmWe evaluated 29 markers to examine the inflammatory and immune activation profiles. The cytokines interleukin (IL)-1α, IL-1β, IL-1RA, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL10, IL-12p40, IL-12p70, IL-13, IL-15, IL-17A, interferon (IFN)- α2, IFN-γ, tissue necrosis factor (TNF)-α, TNF-β, granulocyte colony-stimulating factor (GCSF), granulocyte macrophage colony-stimulating factor (GMCSF), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), monocyte chemoattractant protein-1 [MCP-1 (CCL2)], interferon gamma induced protein/chemokine (C-X-C motif) ligand 10 [IP-10 (CXCL10)], macrophage inflammatory protein-1 alpha [MIP-1α (CCL3)], MIP-1 beta [MIP-1β (CCL4)] and EOTAXIN (CCL11) were measured using a commercial kit from Luminex  according to the manufacturer’s instructions.U test. The Kruskal–Wallis test with the Dunn’s multiple comparisons ad hoc test were used to compare continuous variables and the Pearson’s chi-square test was used to compare variables displayed as frequency/percentage. The Spearman rank test was used to identify correlations between different cytokines. Correlations with coefficient (rho) > 0.5 and P < 0.05 were included in the network visualization.Data was entered in the Statistical Package for Social Sciences (version 9.0) software and checked twice to ensure correctness. The median values with interquartile ranges were used as measures of central tendency and dispersion. Comparison between two groups was performed by using Mann Whitney A hierarchical cluster analysis (Ward’s method), with 100X bootstrap of log10 transformed and Z-score (row) normalized data was employed to depict the overall expression profile of indicated biomarkers. In such analysis, dendrograms represent the Euclidean distance. All comparisons were two-tailed and pre-specified. Differences with P-values below 0.05 after Holm-Bonferroni’s adjustment for multiple comparisons were considered statistically significant.37. MDP is a novel statistical approach proposed by us to evaluate the degree of inflammatory disturbance in patients with a variety of infectious diseases12. Healthy controls were defined as the “reference” group, and the average level and standard deviation of this reference group were calculated for the CSF concentrations of each inflammatory marker. The MDP score of an individual marker in a given sample “s” was defined by taking the difference in concentration level in sample “s” from the average of the marker in reference group divided by the corresponding standard deviation. The MDP score represents the number of standard deviations from the reference. Individuals who had MDP values > 2 standard deviations from mean value of controls were considered molecularly perturbed. A detailed description of each step used to calculate MDP values is shown in Fig. The molecular degree of perturbation (MDP) in CSF measurements was calculated to infer the level of inflammatory imbalance associated with probable CZI. This method has been detailed previouslyThe statistical analyses were performed using GraphPad Prism 8.0 , STATA 11 , JMP 14.0  and Gephi (version 0.9.2) programs 0.82 with circular layout plug-in.Supplementary Information 1.Supplementary Information 2.