Cytokine, Anti-SARS-CoV-2 Antibody, and Neutralizing Antibody Levels in Conventional Blood Donors Who Have Recovered from COVID-19.

Background: At the beginning of the pandemic, COVID-19 convalescent plasma (CCP) containing anti-SARS-CoV-2 antibodies was suggested as a source of therapy. In the last 3 years, many trials have demonstrated the limited usefulness of CCP therapy. This led us to the hypothesis that CCP could contain other elements, along with the desired neutralizing antibodies, which could potentially prevent it from having a therapeutic effect, among them cytokines, chemokines, growth factors, clotting factors, and autoantibodies. Methods: In total, 39 cytokines were analyzed in the plasma of 190 blood donors, and further research focused on the levels of 23 different cytokines in CCP (sCD40L, eotaxin, FGF-2, FLT-3L, ractalkine, GRO-α, IFNα2, IL-1ß, IL-1RA, IL-5, IL-6, IL-8, IL-12, IL-13, IL-15, IL-17E, IP-10, MCP-1, MIP-1b, PDGF-AA, TGFα, TNFα, and TRAIL). Anti-SARS-CoV-2 antibodies and neutralizing antibodies were detected in CCP. Results: We found no significant differences between CCP taken within a maximum of 180 days from the onset of the first COVID-19 symptoms and the controls. We also made a comparison of the cytokine levels between the low neutralizing antibodies (<160) group and the high neutralizing antibodies (≥160) group and found there were no differences between the groups. Our research also showed no correlation either to levels of anti-SARS-CoV-2 IgG Ab or to the levels of neutralizing antibodies. There were also no significant changes in cytokine levels based on the period after the start of COVID-19 symptoms. Conclusions: No elements which could potentially be responsible for preventing CCP from having a therapeutic effect were found.

Morphological Characteristics of Young and Old Murine Hematopoietic Stem Cell Niches, as Modeled In Vitro.

The hematopoietic stem cell (HSC) niche undergoes detrimental changes with age. The molecular differences between young and old niches are well studied and understood; however, young and old niches have not yet been extensively characterized in terms of morphology. In the present work, a 2D stromal model of young and old HSC niches isolated from bone marrow was investigated using light and scanning electron microscopy (SEM) to characterize cell density after one, two, or three weeks of culturing, cell shape, and cell surface morphological features. Our work is aimed at identifying morphological differences between young and old niche cells that could be used to discriminate between their respective murine HSC niches. The results show several age-specific morphological characteristics. The old niches differ from the young ones in terms of lower cell proliferating capacity, increased cell size with a flattened appearance, increased number of adipocytes, and the presence of tunneling nanotubes. In addition, proliferating cell clusters are present in the young niches but not in the old niches. Together, these characteristics could be used as a relatively simple and reliable tool to discriminate between young and old murine HSC niches and as a complementary approach to imaging with specific cellular markers.

Simple prediction of COVID-19 convalescent plasma units with high levels of neutralization antibodies.

BACKGROUND: Hyperimmune convalescent COVID-19 plasma (CCP) containing anti-SARS-CoV-2 neutralizing antibodies (NAbs) was proposed as a therapeutic option for patients early in the new coronavirus disease pandemic. The efficacy of this therapy depends on the quantity of neutralizing antibodies (NAbs) in the CCP units, with titers ≥ 1:160 being recommended. The standard neutralizing tests (NTs) used for determining appropriate CCP donors are technically demanding and expensive and take several days. We explored whether they could be replaced by high-throughput serology tests and a set of available clinical data. METHODS: Our study included 1302 CCP donors after PCR-confirmed COVID-19 infection. To predict donors with high NAb titers, we built four (4) multiple logistic regression models evaluating the relationships of demographic data, COVID-19 symptoms, results of various serological testing, the period between disease and donation, and COVID-19 vaccination status. RESULTS: The analysis of the four models showed that the chemiluminescent microparticle assay (CMIA) for the quantitative determination of IgG Abs to the RBD of the S1 subunit of the SARS-CoV-2 spike protein was enough to predict the CCP units with a high NAb titer. CCP donors with respective results > 850 BAU/ml SARS-CoV-2 IgG had a high probability of attaining sufficient NAb titers. Including additional variables such as donor demographics, clinical symptoms, or time of donation into a particular predictive model did not significantly increase its sensitivity and specificity. CONCLUSION: A simple quantitative serological determination of anti-SARS-CoV-2 antibodies alone is satisfactory for recruiting CCP donors with high titer NAbs.

The Influence of Heterochronic Non-Myeloablative Bone Marrow Transplantation on the Immune System, Frailty, General Health, and Longevity of Aged Murine Recipients.

The stem cell theory of aging postulates that stem cells become inefficient at maintaining the original functions of the tissues. We, therefore, hypothesized that transplanting young bone marrow (BM) to old recipients would lead to rejuvenating effects on immunity, followed by improved general health, decreased frailty, and possibly life span extension. We developed a murine model of non-myeloablative heterochronic BM transplantation in which old female BALB/c mice at 14, 16, and 18(19) months of age received altogether 125.1 ± 15.6 million nucleated BM cells from young male donors aged 7-13 weeks. At 21 months, donor chimerism was determined, and the immune system's innate and adaptive arms were analyzed. Mice were then observed for general health and frailty until spontaneous death, when their lifespan, post-mortem examinations, and histopathological changes were recorded. The results showed that the old mice developed on average 18.7 ± 9.6% donor chimerism in the BM and showed certain improvements in their innate and adaptive arms of the immune system, such as favorable counts of neutrophils in the spleen and BM, central memory Th cells, effector/effector memory Th and Tc cells in the spleen, and B1a and B1b cells in the peritoneal cavity. Borderline enhanced lymphocyte proliferation capacity was also seen. The frailty parameters, pathomorphological results, and life spans did not differ significantly in the transplanted vs. control group of mice. In conclusion, although several favorable effects are obtained in our heterochronic non-myeloablative transplantation model, additional optimization is needed for better rejuvenation effects.

ABO blood group does not influence the level of anti-SARS-CoV-2 antibodies in convalescent plasma donors.

BACKGROUND: The association of the ABO blood group with COVID-19 disease has been confirmed by several studies, with the blood group A patients being more susceptible and prone to a more severe clinical course of the disease. Additionally, several authors also addressed the association of ABO-types and the levels of anti-SARS-CoV-2 antibodies in convalescents, mostly supporting a theory that the non-O blood group convalescents present with higher levels of anti-SARS-CoV-2 antibodies. STUDY DESIGN AND METHODS: Since previous findings were based on small convalescent cohorts, we quantified the anti-SARS-CoV-2 antibody levels in a total of 3187 convalescent plasma donors with three commercial serological and one standard neutralizing antibody test. The majority of donors had undergone a mild form of the disease and the median time of sampling was 66 days after diagnosis. RESULTS: None of the antibody quantitation results showed any significant association with the ABO blood group types. The same result was evident in the subgroup of vaccinated individuals (n = 370) and the subgroups when stratified according to post-COVID-19 periods (0-60, 60-120, and 120-180 days). CONCLUSION: In conclusion, we found no evidence to confirm that the ABO blood group types influence the level of SARS-CoV-2 antibody response in COVID-19 convalescent plasma donors.

Mixed cultures of allogeneic dendritic cells are phenotypically and functionally stable - a potential for primary cell-based "off the shelf" product generation.

Vaccination against tumors using antigen-pulsed dendritic cell (DC) vaccines has greatly evolved over the last decade, with hundreds of active human clinical trials well on the way. The use of an autologous source for DC-based vaccine therapeutics remains the obvious choice in the majority of clinical studies; however, novel evidence suggests that an allogeneic source of DCs can yield success if administered in the right context. One of the challenges facing successful DC vaccination protocols is the generation of large enough numbers of DCs intended for vaccination and standardization of these procedures. In addition, variations in the quality of DC vaccines due to donor-to-donor variation represent an important therapeutic factor. To this day it has not been shown whether DCs from different donors can readily co-exist within the same co-culture for the extended periods required for vaccine manufacture. We demonstrate that generation of allogeneic DC co-cultures, generated from multiple unrelated donors, allows the preservation of their phenotypical and functional properties in vitro for up to 72 hours. Therefore, in the case of an allogeneic vaccination approach, one could ensure large numbers of DCs generated from a primary cell source intended for multiple vaccinations. By generating large amounts of ex vivo manufactured DCs from multiple donors, this would represent the possibility to ensure sufficient amounts of equipotent "off the shelf" product that could e.g. be used for an entire cohort of patients within a study.

Programmed death ligand 1 (PD-L1) plays a vital part in DC tolerogenicity induced by IFN-γ.

Interferon-γ (IFN-γ) is the sole representative of type II IFNs, with well recognized role in numerous inflammatory processes. Lately, its significant pleiotropic nature has been recognized in many scenarios, where IFN-γ contributes to maintenance or induction of tolerogenic responses in context of various immune cell types. In this manuscript we demonstrate, that IFN-γ-mediated induction of programmed death ligand 1 (PD-L1) on human monocyte-derived dendritic cells (DCs) represents an important tolerogenic aspect in immunological network of type II IFNs. When fully differentiated, immature DCs were treated with increasing concentrations of IFN-γ there was no sign of maturation, as revealed by CD80, CD83 and CD86 expression. In terms of co-stimulatory receptor response, we did observe a dose-dependent increase in CD40 expression. Phenotypic analysis of inhibitory molecules revealed that PD-L1 expression is particularly sensitive to IFN-γ, as its expression can be induced almost 10-fold in comparison to non-treated DCs. Functional analysis of such PD-L1high DCs revealed significant immunosuppressive properties in a mixed lymphocyte reaction with whole or memory CD4+ T cells. When IFN-γ treated DCs were co-cultured with naive CD4+CD45RA+ T cells, they induced an increased percentage of CD4+CD25+CD127-FoxP3+ Tregs. Inhibition of PD-1/PD-L1 axis using neutralizing anti-PD-L1 mAbs, reversed the immunosuppressive effect of IFN-γ-treated DCs to suppress CD4+ T cell proliferation and to induce Tregs. In summary, our findings demonstrate the importance of IFN-γ-mediated tolerogenic effects, exerted on DCs by inducing increased expression of PD-L1, which enhances their regulatory function.

Application of the 3R principles: Vertebrae as an additional source of murine bone-marrow cells.

Experimental murine models are an essential tool in the field of bone marrow (BM) transplantation research. Therefore, numerous mice are required to obtain a sufficient number of BM cells, which is in contrast with the Reduction principle of the 3R principles. The selection of the cell source and the isolation protocol are therefore critical in obtaining a sufficient yield of cells for experiments. Nowadays, the vertebrae are already used as an extra source of BM cells to enrich the number of isolated cells from the long bones and ilia (LBI), when needed. Yet, little is known if BM cells from LBI and vertebrae share the same characteristics and can be pooled together for further analysis. Therefore, in this study, we aimed to compare the quantity and characteristics of haematopoietic and stromal cell lines in the BM from the LBI and vertebrae. To count haematopoietic and mesenchymal stem/stromal progenitors, colony-forming unit assays were performed. To determine the expansion capacity of mesenchymal stem/stromal cells (MSCs), cultivation of MSCs and measurement of the expression of surface markers by flow cytometry was performed. The characterisation and enumeration of immune cell populations was also performed by flow cytometry. Here, we show that the vertebrae are a comparable source of BM cells to the LBI regarding the analysed parameters.

Heparin-induced thrombocytopenia: ELISA optical density value and 4T score in correlation with panel donor platelets activation in functional flow cytometric assay.

BACKGROUND: Serological assays for the diagnosis of heparin-induced thrombocytopenia (HIT) detect both platelet-activating and platelet non-activating anti-heparin/platelet factor 4 (PF4) antibodies and have therefore a limited positive predictive value. Functional assays confirm the presence of platelet-activating antibodies but require platelets from healthy donors, whose response to patient serum can differ. Our aim was to investigate the correlation between the level of anti-heparin/PF4 antibodies, 4T score, and the extent of panel donor platelet activation in the functional assay. MATERIALS AND METHODS: In total, 38 sera from enzyme immunoassays (ELISA) positive patients were tested against panel platelets obtained from 10 healthy, randomly selected donors, using our routine flow cytometry functional test for CD62P expression. Levels of anti-heparin/PF4 antibodies from medical and surgical patients and 4T pretest probability scores (where available) were correlated with the number of activated panel platelets. RESULTS: Sera with low ELISA optical density (OD) values (0.4-1) activated on average 5.6, sera with intermediate ELISA OD values (>1-2.5) activated on average 7.3, and sera with high ELISA OD values (>2.5) activated on average 8.6 out of 10 panel platelets. One serum with low 4T score did not activate donor platelets, 12 sera with intermediate 4T score activated on average 6.3 donors, 8 sera with high 4T score activated on average 8.5 panel platelets. DISCUSSION: Sera with higher ELISA OD values activated platelets from a higher number of platelet donors, independently of patient type (medical or surgical). The average number of activated panel platelets increased with rising 4T score. Results indicate that both donor platelet reactivity and quantity of anti-heparin/PF4 antibodies affect the result of the functional assay, meaning special attention is needed in platelet donor selection when testing sera with low levels of antibodies.

Effects of heterochronic, non-myeloablative bone marrow transplantation on age-related behavioural changes in mice.

Experiments using heterochronic parabionts, i.e. young and old animals connected surgically and hence developing a shared circulation, have shown that blood-borne factors, transferred from young to old mice and vice versa, play a role in influencing a range of health outcomes associated with advanced age. Previous work has explored the contributory role of plasma-derived factors in mediating such parabiotic effects, including those on aging-associated neural and behavioural impairments. Here, we wanted to identify possible influences that blood-borne cellular factors may have on age-related behavioural phenotypes. Towards this end, we subjected old BALB/c H-2d mice to repetitive non-myeloablative bone marrow transplants (BMT) from young donor animals and assessed effects on behaviour and cognition. We detected expected age-related alterations in our behavioural assays but did not discern any obvious differences between old BMT mice and old control animals. Our study represents the first to look at possible behavioural and cognitive effects of heterochronic, non-myeloablative BMT. Future work should extend this study by including additional behavioural tests in the analysis, addressing whether beneficial effects of BMT may be detectable on other genetic backgrounds and reconciling our findings with those achieved by myeloablative BMT.

Recent discoveries in dendritic cell tolerance-inducing pharmacological molecules.

Dendritic cells (DCs) represent one of the most important biological tools for cellular immunotherapy purposes. There are an increasing number of phase I and II studies, where regulatory or tolerogenic DCs (TolDCs) are utilized as negative vaccines, with the aim of inducing tolerogenic outcomes in patients with various autoimmune or chronic-inflammatory diseases, as well as in transplant settings. The induction of tolerogenic properties in DCs can be achieved by altering their activation state toward expression of immunosuppressive elements and/or by achieving resistance to maturation, which leads to insufficient co-stimulatory signal delivery and inability to efficiently present antigens. In the past, one of the most efficient ways to induce DC tolerance has been the application of selected pharmacological agents which actively induce a tolerogenic transcription program or inhibit major pro-inflammatory transcription factors such as Nf-κB. Important examples include immunosuppressants such as different corticosteroids, vitamin D3, rapamycin and others. The quality of TolDCs induced by different approaches is becoming a vital issue and recent evidence suggests substantial heterogeneity between variously-generated TolDCs as evidenced by their transcriptomic profile and function. The possibility of various "flavors" of TolDCs encourages future research in discovery of Tol-DC inducing agents to enrich various ways of DC manipulation. This would enable a broader range of tools to manipulate DC toward specific characteristics desirable in different disease settings. In recent years, several novel small molecules have been identified with the capacity to promote DC tolerogenic characteristics. In this review, we will present and discuss these novel findings and also highlight novel understandings of tolerogenic mechanisms by which DC tolerogenicity is induced by already established agents.

How Could We Slow or Reverse the Human Aging Process and Extend the Healthy Life Span with Heterochronous Autologous Hematopoietic Stem Cell Transplantation.

The senescence of the immune system contributes considerably to the age-related diseases that are the main causes of death after the age of 65. In this study, we present an appealing option for the prevention of immune senescence and slowing or reversing the aging process, which can be achieved by heterochronous autologous hematopoietic stem cell transplantation (haHSCT), where healthy autologous bone marrow stem cells are collected from donors while young, cryopreserved and stored for a long period, and reinfused at a later time when indicated. After reinfusion and homing, these young HSCs could participate in normal hemato- and immunopoiesis and improve several immune functions by expanding the immune- as well as hematopoietic cell repertoire. Several animal studies have already confirmed the feasibility of this procedure, which extended the longevity of the treated animals. If translated to human medicine, haHSCT could prevent or mitigate age-related immune defects and extend the healthy life span. In this review, we describe the concept of haHSCT, recent studies that confirm its feasibility, and discuss the further research needed to translate this heterochronous methodology.

Dendritic Cells Generated in the Presence of Platelet Lysate Have a Reduced Type 1 Polarization Capacity.

Previously, we have shown platelet lysate (PL) can be used as a non-xenogeneic serum supplement for generation of monocyte-derived dendritic cells (DCs). Since DC-based activation protocols are extremely sensitive to microenvironmental changes such as replacement of culture medium, we wanted to examine the behavior of DCs cultured in the presence of PL under various type-1 activation conditions and assess their type 1 polarization capacity. We compared the quality of DCs cultured in 10% PL-supplemented RPMI medium (plDCs) with clinical-grade DCs obtained using commercially available serum-free medium (sfDCs), frequently used in established DC vaccine protocols. The DC maturation protocols consisted of either monophosphoryl lipid A/IFN-γ, poly I:C/TNF-α/IFN-α or poly I:C/R848. In general, plDCs were inferior to sfDCs in most aspects of their functional type 1 polarization characteristics. After maturation, the expression of co-stimulatory, HLA class II and lymph node-homing molecules was strongly up-regulated, with some noticeable differences. The expression of CD80 and CD86 was more extensive on plDCs, which was particularly evident in case of CCR7. However, after observing their functional capacity, plDCs had significantly lower allo-stimulatory capacity both in terms of CD4+ and CD8+ T cell stimulation. The high expression of CCR7 corresponded to higher CCL-19 directed DC migration of plDCs compared to sfDCs. Finally, their capacity to induce granzyme B and IFN-γ production in CD8+ T cells was significantly reduced in comparison to sfDCs. Based on these findings, the use of PL as an alternative serum supplement for generation of monocyte-derived DC anti-tumor vaccines is questionable.Abbreviations: Ag: antigen; CCL: chemokine ligand; CCR: chemokine receptor; DC: dendritic cells; DC-SIGN: dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin; FBS: fetal bovine serum; GMP: good manufacturing practice; IFN: interferon; IL: interleukin; MPLA: monophosphoryl lipid A; PGE: prostaglandin E; pI:C: polyinosinic:polycytidylic acid; pl: platelet lysate; sf: serum free; TLR: toll-like receptor; TNF: tumor necrosis factor.

Synergistic Effects of Interferon-γ and Vitamin D3 Signaling in Induction of ILT-3highPDL-1high Tolerogenic Dendritic Cells.

In the past, interferon (IFN)-γ and vitamin D3 (vit D3) have both been associated with induction of tolerogenic characteristics in human dendritic cells (DCs). Although there are only a few reports on interdependency of their actions, the interplay between IFN-γ and vit D3 has been clearly demonstrated in certain aspects of immune reactivity. Since both agents have been associated with regulation of immune responses, we set out to examine their functional and mechanistic interactions in context of principal regulators of immunity, the DCs. Combined treatment with vit D3 and IFN-γ caused an extensive expression of immunoglobulin-like transcript (ILT)-3 and programmed death ligand (PDL)-1 on γ/D3DCs, significantly greater than that caused by vit D3 alone. Such γ/D3DCs retained all general DC characteristics. After CD40 ligand-induced activation, they produced increased amounts of IL-10 with almost absent production of IL-12p70. On the other hand, the co-stimulatory potential of γ/D3DCs was weak, with cells possessing the capacity to inhibit CD4+ T cell, CD8+ T cell, as well as memory T cell responses. Naive CD4+ T cells stimulated with γ/D3DCs produced increased amounts of IL-10 with concomitantly low IFN-γ production, upon T cell receptor activation. Additionally, γ/D3DCs completely inhibited granzyme B expression by CD8+ T cells. The percentage of FoxP3-positive cells in co-cultures with naive CD4+ T cells was significantly higher where γ/D3DCs were used as stimulators compared to DCs treated with vit D3 alone and it could be partially reversed by PDL-1 blockade. Interestingly, γ/D3DCs were inefficient at suppressing mDC-induced CD4+ T cell proliferation, but were twice as effective as D3DCs at suppressing mDC-induced CD8+ T cell proliferation. Blockade of indoleamine-2,3-dioxygenase did not reduce the tolerogenic phenotype induced by IFN-γ and vit D3 treatment. Examination of signaling pathways activation revealed a tendency toward increased ERK and Akt phosphorylation in γ/D3DCs. Inhibition of MEK/ERK and PI3K/mTOR pathways significantly reduced the expression of ILT-3 and PDL-1 on γ/D3DCs. In summary, we present the first evidence for existing synergy between IFN-γ and vit D3 in shaping a unique tolerogenic DC activation state.

Induction of Tolerogenic Dendritic Cells by Endogenous Biomolecules: An Update.

The importance of microenvironment on dendritic cell (DC) function and development has been strongly established during the last two decades. Although DCs with general tolerogenic characteristics have been isolated and defined as a particular sub-population, it is predominantly their unequivocal biological plasticity, which allows for unparalleled responsiveness to environmental ques and shaping of their tolerogenic characteristics when interacting with tolerance-inducing biomolecules. Dendritic cells carry receptors for a great number of endogenous factors, which, after ligation, can importantly influence the development of their activation state. For this there is ample evidence merely by observation of DC characteristics isolated from various anatomical niches, e.g., the greater immunosuppressive potential of DCs isolated from intestine compared to conventional blood DCs. Endogenous biomolecules present in these environments most likely play a major role as a determinant of their phenotype and function. In this review, we will concisely summarize in what way various, tolerance-inducing endogenous factors influence DC biology, the development of their particular tolerogenic state and their subsequent actions in context of immune response inhibition and induction of regulatory T cells.

The potential of non-myeloablative heterochronous autologous hematopoietic stem cell transplantation for extending a healthy life span.

Aging is a complex multifactorial process, a prominent component being the senescence of the immune system. Consequently, immune-related diseases develop, including atherosclerosis, cancer, and life-threatening infections, which impact on health and longevity. Rejuvenating the aged immune system could mitigate these diseases, thereby contributing to longevity and health. Currently, an appealing option for rejuvenating the immune system is heterochronous autologous hematopoietic stem cell transplantation (haHSCT), where healthy autologous bone marrow/peripheral blood stem cells are collected during the youth of an individual, cryopreserved, and re-infused when he or she has reached an older age. After infusion, young hematopoietic stem cells can reconstitute the compromised immune system and improve immune function. Several studies using animal models have achieved substantial extension of the life span of animals treated with haHSCT. Therefore, haHSCT could be regarded as a potential procedure for preventing age-related immune defects and extending healthy longevity. In this review, the pros, cons, and future feasibility of this approach are discussed.

The tolerogenic role of IFN-γ.

Due to its extremely pleiotropic nature, the complex effects of IFN-γ exerted both on immune and non-immune cell types still remain only partially understood. The longstanding view of IFN-γ as being a predominantly inflammatory cytokine is constantly challenged by increasing demonstrations of its direct or indirect regulatory roles. Interferon-γ can exert tolerogenic effects on both innate and adaptive immune cell types, promoting tolerance of various antigen-presenting cells, and augmenting function and differentiation of regulatory T cells, respectively. Its capacity to induce IDO-competence is not limited to immune cells but extends to other cell types such as mesenchymal stem cells, epithelial cells, and tumors. The pro-inflammatory role of IFN-γ in tumor immune surveillance can backfire by directly inducing inhibitory molecule expression, such as PDL-1, on tumor cells. With increasing knowledge regarding the role of different helper T cell subsets in certain autoimmune diseases, the once contradictory observations of disease attenuation by IFN-γ can now be explained by its opposing interplay with other effector cytokines, particularly IL-17. The paradoxically immunosuppressive role of IFN-γ is also becoming evident in the transplantation setting, and graft-versus-host-disease. In the present review, we will discuss the latest findings that help to elucidate this dual role of IFN-γ at a cellular level, and in various pathophysiological states.

Chimerism and gene therapy - Lessons learned from non-conditioned murine bone marrow transplantation models.

OBJECTIVE: Hematopoietic stem and progenitor cells (HSPCs) can be used as a vector for gene therapies. In order to predict the number of HSPCs cells necessary to achieve the target level of chimerism in an autologous setting, syngeneic male bone marrow (BM) cells were transplanted into 35 non-conditioned female BALB/c mice. METHOD: The resulting chimerism was determined at 6-53 weeks using qPCR, cell subpopulation sorting, and colony-forming units (CFU) analysis. RESULTS: After the transplantation of 125.8 ± 2.5 million nucleated BM cells, the BM of recipients contained 20.0 ± 2.8% donor cells, representing a chimerism of 0.16 ± 0.02% per one million transplanted nucleated BM cells. Chimerism levels in the BM, neutrophils, and B cells were comparable, whereas in T cells it was lower, and in CFU was approximately twice greater than in BM. CONCLUSION: By extrapolating our murine data, and data from some previous studies to a human non-conditioned autologous CD34+ HSPC transplantation setting, we conclude that approximately 44 million CD34+ HSPCs would be needed to achieve 20% donor chimerism in a 70-kg human, which could serve as a starting point for the future use of HSCPs in gene and cell therapy.

REGENERATION OF CHRONIC WOUNDS WITH ALLOGENEIC PLATELET GEL VERSUS HYDROGEL TREATMENT: A PROSPECTIVE STUDY.

- The aim of the study was to evaluate the efficacy and safety of the new method of platelet-rich plasma activation in the form of platelet gel, used in the treatment of non-healing chronic lower leg ulcers. The study was prospectively randomized, double blind and placebo controlled. We treated 60 patients (42 males and 18 females, mean age 69.43 years, SD 14.74) with chronic lower leg ulcers of different etiologies. Thirty patients were treated with allogeneic platelet gel and 30 with hydrogel. Both groups were comparable for duration of ulcer and its size. Treatment was repeated once a week for three consecutive weeks and then the last examination was scheduled at 6 months of the first platelet gel application. The t-test was used to analyze independent samples. Healing of chronic wounds with platelet gel was statistically significantly more effective compared to the treatment with hydrogel (p<0.05). At 6 months of platelet gel application, the mean wound area in the experimental group decreased to 35.01% (SD 53.69) of the initial wound size. In the control group, the wound area decreased to 89.95% (SD 71.82) of the initial wound size (p=0.001). The circumference of the wounds diminished to 54.62% (SD 39.85) of the initial value in the experimental group, compared to 91.28% (SD 29.32) in the control group (p<0.001). Allogeneic platelet gel prepared by the new method used in this study was found to be a good treatment option for non-healing chronic wounds when other methods are ineffective.

The level of heparin-induced antibodies in correlation with the result of the flow cytometric functional assay in the patients with suspected HIT.

Heparin can induce the formation of antibodies against a heparin complex with a platelet factor 4 (PF4), leading to platelet activation and the development of heparin-induced thrombocytopaenia (HIT). Because screening ELISA does not discriminate between platelet activating and non-activating anti-heparin/PF4 antibodies, each positive result is confirmed by an additional functional assay. We analysed 1004 sera of patients with suspected HIT. Optical density (OD) values of ELISA-positive results were correlated with the risk for a positive result with our functional flow cytometric assay. Only 10.7% were ELISA positive and 59.8% of those were positive with the functional assay. The positive functional assay was found in 23.4% of patients with OD<1.0, in 57.7% with 1.02.0. Although our results showed that higher ELISA OD values increasethe possibility of the presence of platelet-activating anti-heparin/PF4 antibodies , there is no need for improving ELISA cut-off value for positive result.