An Increase in HSF1 Expression Directs Human Mammary Epithelial Cells toward a Mesenchymal Phenotype.

HSF1 is a well-known heat shock protein expression regulator in response to stress. It also regulates processes important for growth, development or tumorigenesis. We studied the HSF1 influence on the phenotype of non-tumorigenic human mammary epithelial (MCF10A and MCF12A) and several triple-negative breast cancer cell lines. MCF10A and MCF12A differ in terms of HSF1 levels, morphology, growth in Matrigel, expression of epithelial (CDH1) and mesenchymal (VIM) markers (MCF10A are epithelial cells; MCF12A resemble mesenchymal cells). HSF1 down-regulation led to a reduced proliferation rate and spheroid formation in Matrigel by MCF10A cells. However, it did not affect MCF12A proliferation but led to CDH1 up-regulation and the formation of better organized spheroids. HSF1 overexpression in MCF10A resulted in reduced CDH1 and increased VIM expression and the acquisition of elongated fibroblast-like morphology. The above-mentioned results suggest that elevated levels of HSF1 may direct mammary epithelial cells toward a mesenchymal phenotype, while a lowering of HSF1 could reverse the mesenchymal phenotype to an epithelial one. Therefore, HSF1 may be involved in the remodeling of mammary gland architecture over the female lifetime. Moreover, HSF1 levels positively correlated with the invasive phenotype of triple-negative breast cancer cells, and their growth was inhibited by the HSF1 inhibitor DTHIB.

Component-Resolved Evaluation of the Risk and Success of Immunotherapy in Bee Venom Allergic Patients.

Venom immunotherapy (VIT) is the only efficient therapy for the Hymenoptera insect venom allergy. Immunotherapy with bee venom is encumbered with a higher risk of systemic side effects and/or therapeutic failures. The objective of the study was to assess if specific profiles of molecular IgE (Immunoglobulin E) responses are associated with an increased risk of systemic side effects and/or the treatment's inefficacy. The study group numbered 64 bee venom allergic patients (BVA) who received venom immunotherapy modo ultra-rush (VIT-UR), (f/m: 32/32, mean age 43.4 ± 17.2). In total, 54.84% of them manifested allergic reactions of grades I-III (acc. to Mueller's scale), while 48.66% manifested reactions of grade IV. In all the patients, IgE against bee venom extract, rApi m 1 and tryptase (sBT) were assessed. In 46 patients, assessments of IgE against rApi m 2, 3, 5, 10 were also performed. BVA patients manifesting cardiovascular symptoms (SYS IV0) showed higher levels of both sIgE-rApi m 5 (p = 0.03) and tryptase (p = 0.07) than patients with SYS I−III. Systemic adverse events during VIT with bee venom were more frequent in the induction phase than in the maintenance phase: 15.22% vs. 8.7%. In BVA patients who experienced systemic adverse events during VIT, higher concentrations of sIgE-rApi m 5 (p < 0.05), rApi m 1 (p = 0.009), and sBT (p = 0.019) were demonstrated. We conclude that higher levels of sIgE against rApi m 1, rApi m 5, and tryptase many constitute a potential marker of the severity of allergic reactions and therapeutic complications that can occur during VIT with bee venom.

Transcriptome Analysis of Cells Exposed to Actinomycin D and Nutlin-3a Reveals New Candidate p53-Target Genes and Indicates That CHIR-98014 Is an Important Inhibitor of p53 Activity.

Co-treatment with actinomycin D and nutlin-3a (A + N) strongly activates p53. Previously we reported that CHIR-98014 (GSK-3 kinase inhibitor), acting in cells exposed to A + N, prevents activation of TREM2-an innate immunity and p53-regulated gene associated with Alzheimer's disease. In order to find novel candidate p53-target genes and genes regulated by CHIR-98014, we performed RNA-Seq of control A549 cells and the cells exposed to A + N, A + N with CHIR-98014 or to CHIR-98014. We validated the data for selected genes using RT-PCR and/or Western blotting. Using CRISPR/Cas9 technology we generated p53-deficient cells. These tools enabled us to identify dozens of candidate p53-regulated genes. We confirmed that p53 participates in upregulation of BLNK, APOE and IRF1. BLNK assists in activation of immune cells, APOE codes for apolipoprotein associated with Alzheimer's disease and IRF1 is activated by interferon gamma and regulates expression of antiviral genes. CHIR-98014 prevented or inhibited the upregulation of a fraction of genes stimulated by A + N. Downregulation of GSK-3 did not mimic the activity of CHIR-98014. Our data generate the hypothesis, that an unidentified kinase inhibited by CHIR-98014, participates in modification of p53 and enables it to activate a subset of its target genes, e.g., the ones associated with innate immunity.

Pfizer-BioNTech COVID-19 Vaccine Tolerance in Allergic versus Non-Allergic Individuals.

Individuals with a history of allergy are potentially at risk of suffering from adverse effects after COVID-19 vaccination. We sought to assess the tolerance towards the Pfizer-BioNTech vaccine in allergic patients. To address this issue, we used a questionnaire conducted on-line in a group of medical professionals who were vaccinated with the Pfizer-BioNTech vaccine. A total of 1808 respondents, out of whom 1707 received two doses of the vaccine, returned the questionnaire. Local reactions after injection were more frequent in allergic individuals after both doses (swelling p = 0.0003). Systemic adverse events (AE-SYS) occurred more often after the second than the first dose in both groups (allergic persons: 77.29% vs. 41.06%); vomiting and arthralgia occurred more often in allergic subjects (p = 0.0009). AE-SYS in allergic individuals lasted longer than in non-allergic ones after the first (p = 0.01) and the second dose (p = 0.0009). Allergic reactions after vaccination were reported more frequently in allergic subjects: after the first dose (p = 0.00001) and after the second dose (p = 0.001). Rhinitis was the most frequent symptom observed more often in allergic patients. No severe allergic reactions occurred during the full cycle of vaccination. Although the Pfizer-BioNTech vaccine is tolerated worse by allergic than non-allergic individuals, the occurring adverse symptoms are mild and do not preclude a successful completion of the vaccination cycle. The presence of symptoms suggestive of allergy does not constitute a condition of increased risk of developing clinically significant adverse events following Pfizer COVID-19 vaccination.

HSPA2 Chaperone Contributes to the Maintenance of Epithelial Phenotype of Human Bronchial Epithelial Cells but Has Non-Essential Role in Supporting Malignant Features of Non-Small Cell Lung Carcinoma, MCF7, and HeLa Cancer Cells.

Heat Shock Protein A2 (HSPA2) is a member of the HSPA (HSP70) chaperone family and has a critical role for male fertility. HSPA2 is present in a number of somatic organs. Limited evidence suggests that HSPA2 may be involved in regulating epithelial cell differentiation. HSPA2 also emerged as a cancer-related chaperone; however, no consensus on its functional significance has been reached so far. In this study, we compared the phenotypic effects of HSPA2 deficit in non-transformed human bronchial epithelial cells (HBEC), and in lung, breast, and cervical cancer cells. We used various techniques to inhibit the HSPA2 gene expression in order to examine the impact of HSPA2 deficiency on cell growth, migration, adhesion, and invasion. Our results show that HBEC but not cancer cells are sensitive to HSPA2 deficit. HSPA2 knockdown in HBEC cells impaired their clone-forming ability and adhesiveness. Thus, our results indicate that epithelial cells can rely on a specific activity of HSPA2, but such dependence can be lost in epithelial cells that have undergone malignant transformation.

Synergistic activation of p53 by actinomycin D and nutlin-3a is associated with the upregulation of crucial regulators and effectors of innate immunity.

Actinomycin D and nutlin-3a (A + N) activate p53, partly through induction of phosphorylation on Ser392. The death of A549 cells induced by A + N morphologically resembles inflammation-inducing pyroptosis - cell destruction triggered by activated caspase-1. The treatment with A + N (or camptothecin) strongly upregulated caspase-1 and its two activators: IFI16 and NLRP1, however, caspase-1 activation was not detected. A549 cells may have been primed for pyroptosis, with the absence of a crucial trigger. The investigation of additional innate immunity elements revealed that A + N (or camptothecin) stimulated the expression of NLRX1, STING (stimulator of interferon genes) and two antiviral proteins, IFIT1 and IFIT3. IFI16 and caspase-1 are coded by p53-regulated genes which led us to investigate regulation of NLRP1, NLRX1, STING, IFIT1 and IFIT3 in p53-dependent mode. The upregulation of NLRP1, NLRX1 and STING was attenuated in p53 knockdown cells. The upsurge of the examined genes, and activation of p53, was inhibited by C16, an inhibitor of PKR kinase. PKR was tested due to its ability to phosphorylate p53 on Ser392. Surprisingly, C16 was active even in PKR knockdown cells. The ability of C16 to prevent activation of p53 and expression of innate immunity genes may be the source of its strong anti-inflammatory action. Moreover, cells exposed to A + N can influence neighboring cells in paracrine fashion, for instance, they shed ectodomain of COL17A1 protein and induce, in p53-dependent mode, the expression of gene for interleukin-7. Further, the activation of p53 also spurred the expression of SOCS1, an inhibitor of interferon triggered STAT1-dependent signaling. We conclude that, stimulation of p53 primes cells for the production of interferons (through upregulation of STING), and may activate negative-feedback within this signaling system by enhancing the production of SOCS1.

Season of birth is not associated with age at menarche in young adults from Central India.

OBJECTIVES: We aimed to test whether birth season is associated with age at menarche in the sub-tropical climate of Central India where temperature and photoperiod differences between seasons are very small. METHODS: Date of birth and age at menarche were collected for 330 female students of Central University, Sagar. The impact of birth month and birth season on age at menarche was analyzed using ANOVA and time-to-event analysis with the use of the Kaplan-Meier curve. RESULTS: ANOVA, Kaplan-Meier estimation and Cox Proportional Hazard did not show statistically significant differences in age at menarche according to birth month and birth season. CONCLUSIONS: Constant sun exposure during the whole year, related to a stable vitamin D amount and to equal access to fruits and vegetables, may translate into a lack of association between seasons and age at menarche.

Large local reactions to Hymenoptera stings: Outcome of re-stings in real life.

BACKGROUND: Large local reaction to Hymenoptera stings is usually defined as a swelling >10 cm which lasts longer than 24 hours, sometimes associated with erythema, pruritus and blisters. Currently, the risk of subsequent systemic reactions after re-stings is considered low (2%-15%). Therefore, a diagnostic workup in case of large local reaction is often judged unnecessary, as well as adrenaline auto-injector and venom immunotherapy prescription. The aim of this study was to prospectively evaluate the outcome of re-stings in a real-world setting, in patients with a history of one previous large local reaction. METHODS: We consecutively enrolled patients who experienced their first large local reaction (as per EAACI definition), treated with antihistamine and steroids. They were followed for field re-stings and assessed for risk of subsequent systemic reactions. RESULTS: We enrolled 662 patients. Out of the 225 re-stung subjects, 24% did not experience reactions, 52% reported a second large local reaction and 24% had systemic reactions. The risk of subsequent systemic reactions was higher in case of skin test reactivity to Apis mellifera or Vespula species (OR 2.1 and 3.8, respectively), in particular if positive at 0.001 µg/mL concentration (OR 13.4 and 16.5, respectively). CONCLUSIONS: Systemic reactions, after a previous large local reaction, occur more frequently than that reported by literature. After analysing the predictive role of large local reactions for systemic reactions, we demonstrated that an accurate diagnostic workup may be considered, particularly skin tests. Further studies in different countries are needed to confirm these results and large local reaction management.

Increased Efficacy of Stem Cell Chemomobilization with Intermediate-Dose Cytarabine Plus Granulocyte Colony-Stimulating Factor (G-CSF) Compared with G-CSF Alone in Patients with Multiple Myeloma: Results of a Randomized Trial.

Mobilization of hematopoietic stem cells for patients with multiple myeloma (MM) may be done using either steady-state granulocyte colony-stimulating factor (G-CSF) or a combination of chemotherapy with G-CSF. The goal of this randomized, open-label, phase 3 trial was to compare the efficacy of chemomobilization using intermediate-dose cytarabine (ID-AraC) plus G-CSF with G-CSF alone in patients with MM referred for tandem autologous stem cell transplantation (autoSCT). The percentage of patients with stem cell yield of at least 5 × 106 CD34+ cells/kg was the primary endpoint. Ninety patients were enrolled, including 44 assigned to the ID-AraC arm and 46 in the G-CSF arm. The threshold number of CD34+ cells was reached in 43 patients (98%) in the ID-AraC arm and in 32 patients (70%) in the G-CSF arm (P = .0003). The median number of collected CD34+ cells was 20.2 × 106 cells/kg in the ID-AraC arm versus 5.9 × 106 cells/kg in the G-CSF arm (P < .000001). A single apheresis was sufficient to achieve the required number of harvested CD34+ cells in 37 patients (86%) in the ID-AraC arm and in 13 patients (41%) in the G-CSF arm (P = .00008). The times to both neutrophil and platelet recovery after autoSCT were significantly shorter in the patients mobilized with ID-AraC. This study provides the first evidence of the advantage of chemomobilization over G-CSF monotherapy in terms of efficacy. ID-AraC with G-CSF should be the preferred chemomobilization protocol for patients with MM scheduled to undergo tandem autoSCT.

Genome-wide analysis of gene expression after one year of venom immunotherapy.

Background Insect venom immunotherapy (VIT) is used to protect patients against Hymenoptera insects' venom allergy (HVA), which can result in severe systemic or even life-threatening conditions. Molecular mechanisms triggered by VIT remain largely unknown. Objective To compare genome-wide gene expression of patients with severe HVA prior to VIT and 12 months after. Methods Whole blood RNA samples were analyzed on an expression array. Results from differential expression obtained on a microarray platform were confirmed by quantitative real -time PCR (qRT-PCR). Subsequently we applied unsupervised clustering. Relative blood cell proportions and gene expression profiles were used as an input to csSAM to compute cell specific differential gene expression. Finally, transcription factor enrichment analysis was performed in MotifLab. Results & conclusions Comparison of genome-wide expression patterns for whole blood and qRT-PCR experiments revealed no significantly up and/or down regulated genes. This has been corroborated by unsupervised clustering. We found a significant upregulation of 26 genes in macrophages, of 15 genes in monocytes and 2 genes in T regulatory cells (Tregs). Analysis of the promoter sequences of these upregulated genes revealed a significant over-representation of binding motifs specific for kruppel-like factor 4, retinoic acid receptor gamma, and vitamin D receptor. Our results indicate that changes of gene expression invoked by VIT in peripheral blood may have a too small effect to be detected by conventional biostatistical approaches. When blood cell composition was taken into account we uncovered numerous changes of cell-specific gene expression. Given the regulatory implications we hypothesize that above-mentioned alterations may contribute to activation of anti-inflammatory signals in the innate branch of the immune system.

Percentile charts of twin birthweight.

BACKGROUND: The birthweight of multiples is naturally lower than that of singletons. Given that the incidence of twin pregnancy has risen in recent years, it seems reasonable to create standards of birthweight separately for twins. This could help in the objective assessment of small and large for gestational age twin newborns. The main goal of this study was therefore to construct and present up-to-date birthweight references. METHODS: The present percentile charts for twins are based on a cohort retrospective study of 757 pairs of twins (767 boys and 709 girls) born between weeks 25 and 39 of gestation. Mean and standard deviation were calculated for the subsequent weeks of gestation. Percentiles were read for the subsequent gestational age. The obtained curves were smoothed with a fifth-degree polynomial function. The significance of differences between the 50th percentile values for twins and singletons was estimated using median test. RESULTS: In both sexes, a continuous observable trend occurs of a significantly lower average birthweight for twins. Differences increase with increasing gestational age and are greater in girls. The estimated 50th percentile for twins was greater than the estimated 10th percentile for singletons. This supports the notion of discordant growth as a physiological adaptation that promotes maturity. CONCLUSIONS: Percentile charts for singletons are not applicable for twins. This indicates the importance of applying separate percentile charts for twins, enabling objective evaluation of their health status and identifying deviations from normality.

Establishment and Characterization of the Novel High-Grade Serous Ovarian Cancer Cell Line OVPA8.

High-grade serous ovarian carcinoma (HGSOC) is the most frequent histological type of ovarian cancer and the one with worst prognosis. Unfortunately, the majority of established ovarian cancer cell lines which are used in the research have unclear histological origin and probably do not represent HGSOC. Thus, new and reliable models of HGSOC are needed. Ascitic fluid from a patient with recurrent HGSOC was used to establish a stable cancer cell line. Cells were characterized by cytogenetic karyotyping and short tandem repeat (STR) profiling. New generation sequencing was applied to test for hot-spot mutations in 50 cancer-associated genes and fluorescence in situ hybridization (FISH) analysis was used to check for TP53 status. Cells were analyzed for expression of several marker genes/proteins by reverse-transcription polymerase chain reaction (RT-PCR), fluorescence-activated cell sorting (FACS), and immunocytochemistry (ICC). Functional tests were performed to compare OVPA8 cells with five commercially available and frequently used ovarian cancer cell lines: SKOV3, A2780, OVCAR3, ES2, and OAW42. Our newly-established OVPA8 cell line shows morphologic and genetic features consistent with HGSOC, such as epithelial morphology, multiple chromosomal aberrations, TP53 mutation, BRCA1 mutation, and loss of one copy of BRCA2. The OVPA8 line has a stable STR profile. Cells are positive for EpCAM, CK19, and CD44; they have relatively low plating efficiency/ability to form spheroids, a low migration rate, and intermediate invasiveness in matrigel, as compared to other ovarian cancer lines. OVPA8 is sensitive to paclitaxel and resistant to cisplatin. We also tested two FGFR inhibitors; OVPA8 cells were resistant to AZD4547 (AstraZeneca, London, UK), but sensitive to the new inhibitor CPL304-110-01 (Celon Pharma, Lomianki/Kielpin, Poland). We have established and characterized a novel cell line, OVPA8, which can be a valuable preclinical model for studies on high-grade serous ovarian cancer.

Evaluation of proinflammatory and immunosuppressive cytokines in blood and bone marrow of healthy hematopoietic stem cell donors.

INTRODUCTION: Cytokine composition of bone marrow microenvironment in comparison to blood is poorly explored. The goal of this study was to investigate the levels of cytokines present in peripheral blood and bone marrow of healthy hematopoietic stem cells donors. The data obtained on this subject with addition to cytometric analysis can provide new insight into the hematopoietic stem cells microenvironment. METHODOLOGY: Study consisted of cytokine concentration analysis performed by ELISA tests of peripheral blood of healthy peripheral blood stem cells donors and bone marrow of healthy bone marrow donors. Additionally we have tested the expression of CD47 and CD274 proteins on the surface of hematopoietic stem cells by the flow cytometry analysis. RESULTS: The results has shown different composition of analyzed cytokines (IL-1 ß, IL-2, IL-4, IL-6, IL-10, IL-17A, TGF-ß1, IFN-γ and TNF-α) present in bone marrow and blood of stem cells donors. The hematopoietic stem cells in peripheral blood are subjected to higher levels of proinflammatory cytokines whilst the lower level of those cytokines in bone marrow with a very high level of TGF-ß1 which possibly creates a more immunosuppressive environment. The IL-10 level was significantly higher in peripheral blood of PBSC donors after the administration of mobilizing factor (G-CSF). The percentage of CD47+HSCs was significantly higher in bone marrow compared to peripheral blood of mobilized donors.

Reduction of DMSO concentration in cryopreservation mixture from 10% to 7.5% and 5% has no impact on engraftment after autologous peripheral blood stem cell transplantation: results of a prospective, randomized study.

The procedure of autologous peripheral blood stem cell transplantation (autoPBSCT) requires cryopreservation of cells in a mixture containing dimethyl sulfoxide (DMSO). DMSO is necessary to secure cell viability, however, its infusion may be toxic to stem cell recipient. The aim of this study was to prospectively evaluate the impact of DMSO concentration on engraftment after autoPBSCT.One-hundred-fifty patients were randomly assigned to one of three study arms; their leukapheresis products were cryopreserved in 10%, 7.5% or 5% DMSO. The study groups did not differ with regard to the diagnosis (mainly lymphomas and multiple myeloma), age, conditioning regimen, and the number of transplanted hematopoietic stem cells. 143 patients were treated with autoPBSCT. The frequency of adverse effects during and shortly after infusion was the lowest in 5% DMSO arm (p = 0.02 compared to 10% DMSO). 4 patients died due to infection before the engraftment. The median time to leukocyte and neutrophil recovery was 10 days in all study groups (p = 0.36 and p = 0.2). As well, the median day of platelet recovery was the same for all DMSO concentrations and equaled 15 days (p = 0.61).In view of these results, 5% DMSO mixture may be considered a new standard in cryopreservation of hematopoietic stem cells.

Novel role for the testis-enriched HSPA2 protein in regulating epidermal keratinocyte differentiation.

HSPA2, a poorly characterized member of the HSPA (HSP70) chaperone family, is a testis-enriched protein involved in male germ cell differentiation. Previously, we revealed that HSPA2 is present in human stratified epithelia, including epidermis, however the contribution of this protein to epithelial biology remained unknown. Here, we show for the first time that HSPA2 is expressed in basal epidermal keratinocytes, albeit not in keratinocytes exhibiting features attributed to primitive undifferentiated progenitors, and participates in the keratinocyte differentiation process. We found that HSPA2 is dispensable for protection of HaCaT keratinocytes against heat shock-induced cytotoxicity. We also shown that lentiviral-mediated shRNA silencing of HSPA2 expression in HaCaT cells caused a set of phenotypic changes characteristic for keratinocytes committed to terminal differentiation such as reduced clonogenic potential, impaired adhesiveness and increased basal and confluency-induced expression of differentiation markers. Moreover, the fraction of undifferentiated cells that rapidly adhered to collagen IV was less numerous in HSPA2-deficient cells than in the control. In a 3D reconstructed human epidermis model, HSPA2 deficiency resulted in accelerated development of a filaggrin-positive layer. Collectively, our results clearly show a link between HSPA2 expression and maintenance of keratinocytes in an undifferentiated state in the basal layer of the epidermis. It seems that HSPA2 could retain keratinocytes from premature entry into the terminal differentiation process. Overall, HSPA2 appears to be necessary for controlling development of properly stratified epidermis and thus for maintenance of skin homeostasis.

Digit ratio (2D:4D) and month of birth: A link to the solstitial-melatonin-testosterone effect.

BACKGROUND: Digit ratio (2D:4D) is a sexually dimorphic trait and its determination in utero is influenced by testosterone. The solstitial-melatonin-testosterone hypothesis posits that melatonin inhibits the production of foetal testosterone and melatonin levels are at their lowest in months when light levels are high. AIMS: We test the relationship between 2D:4D, month-of-birth and light levels. STUDY DESIGN: We recruited participants whose year of birth was spread across the 20th Century. SUBJECTS: 323 Polish men and women. OUTCOME MEASURES: Finger lengths, month-of-birth, mean daylight hours per month in and around Poznan, Poland. RESULTS: Our sample was born between 1907 and 1997. In comparison to late-Spring births, late-Autumn births had low right-left 2D:4D (high prenatal testosterone). Regarding light levels, there were significant relationships between low right 2D:4D and right-left 2D:4D (high prenatal testosterone) and long days at the end of the 1st trimester. These relationships were strongest for participants born in the first half of the 20th Century. CONCLUSIONS: Participants born in the late-Autumn and who experienced long days in the 2nd and 3rd prenatal months had low 2D:4D. The effects were strongest for early 20th Century births where photoperiods would be less disrupted by artificial light.

Immunological properties of bone marrow microenvironment 1 year after allogeneic hematopoietic stem cell transplantation.

Regeneration of the bone marrow microenvironment after transplantation of allogeneic hematopoietic stem cells is poorly explored. The goal of our study was to investigate this process focusing on immunologic factors: concentrations of selected cytokines, expression of immunosuppressive proteins CD47 and CD274 on hematopoietic stem cells, and frequency of T regulatory lymphocytes (Tregs). Bone marrow samples were collected before transplantation, on the day of transplantation, and at the 1-year follow-up. As a control group, we used bone marrow from healthy donors. Prior to the conditioning, the percentage of Tregs and concentration of interleukin-10 were higher in the bone marrow of patients than in healthy donors. The conditioning regimen resulted in increased concentrations of interferon-γ and expression of CD274 on hematopoietic stem cells. Twenty-eight days after transplantation, level of Tregs, expression of CD47, and concentration of interleukin-10 and latency-associated peptide 1 were increased compared with the period before conditioning. Starting from day 100 after transplantation, the microenvironment tended to normalize; the level of Tregs and concentrations of most cytokines were similar to values in the bone marrow of healthy donors.

Human Cardiac Mesenchymal Stromal Cells with CD105+CD34- Phenotype Enhance the Function of Post-Infarction Heart in Mice.

AIMS: The aim of the present study was to isolate mesenchymal stromal cells (MSC) with CD105+CD34- phenotype from human hearts, and to investigate their therapeutic potential in a mouse model of hindlimb ischemia and myocardial infarction (MI). The study aimed also to investigate the feasibility of xenogeneic MSCs implantation. METHODS AND RESULTS: MSC isolated from human hearts were multipotent cells. Separation of MSC with CD105+CD34- phenotype limited the heterogeneity of the originally isolated cell population. MSC secreted a number of anti-inflammatory and proangiogenic cytokines (mainly IL-6, IL-8, and GRO). Human MSC were transplanted into C57Bl/6NCrl mice. Using the mouse model of hindlimb ischemia it was shown that human MSC treated mice demonstrated a higher capillary density 14 days after injury. It was also presented that MSC administrated into the ischemic muscle facilitated fast wound healing (functional recovery by ischemic limb). MSC transplanted into an infarcted myocardium reduced the post-infarction scar, fibrosis, and increased the number of blood vessels both in the border area, and within the post-infarction scar. The improvement of left ventricular ejection fraction was also observed. CONCLUSION: In two murine models (hindlimb ischemia and MI) we did not observe the xenotransplant rejection. Indeed, we have shown that human cardiac mesenchymal stromal cells with CD105+CD34- phenotype exhibit therapeutic potential. It seems that M2 macrophages are essential for healing and repair of the post-infarcted heart.

Thymic Activity and T Cell Repertoire Recovery after Autologous Hematopoietic Stem Cell Transplantation Preceded by Myeloablative Radiotherapy or Chemotherapy.

It was previously postulated that pretransplant myeloablative treatment may impair thymopoiesis, contributing in this way to delayed reconstitution of T cells after hematopoietic stem cell transplantation (HSCT). On the other hand, de novo generation of T cells after HSCT requires a competent thymus. Various myeloablative conditioning regimens (total body irradiation [TBI] or high-dose chemotherapy) routinely used in clinical practice may have potentially different impacts on the thymus. However, no comparative study on thymic output and T cell repertoire in autologous (auto)HSCT model has been presented so far. Here we evaluated thymic output and TCR diversity in 45 lymphoma patients submitted to autoHSCT differing in respect to conditioning regimen: high-dose chemotherapy as monotherapy (BEAM, n = 22) or combination of total body irradiation with cyclophosphamide chemotherapy: Cy/TBI (n = 23). Thymic output was assessed before and on days +100, +180, and +365 after autoHSCT by flow cytometric counts of recent thymic emigrant (RTE) cells (CD31(+) CD62L(+) CD45RA(+) CD4(+)) and quantification of signal joint TCR receptor excision circles (sjTRECs) by quantitative PCR. T cell repertoire diversity was analyzed on day +365 after autoHSCT by spectra-typing of the CDR3 region in the TCRVß chain. The BEAM group, in contrast to the Cy/TBI group, manifested significantly higher proportions of RTE cells and sjTREC copy numbers on days +100 and +180. Analysis of TCRVß spectra-types on day +365 revealed more restricted (monoclonal or oligoclonal) T cell repertoires in the Cy/TBI versus BEAM group (48.8% versus 18.2%, P = .0002). In conclusion, the conditioning scheme based on BEAM chemotherapy may be performed with lower risk of thymic destruction and T cell repertoire distortion than Cy/TBI scheme. This finding may help to potentially improve conditioning schemes to efficiently perform myeloablation and maintain active thymopoiesis.