Maciej Kotowski

Circulating Stem Cell Populations in Preterm Infants: Implications for the Development of Retinopathy of Prematurity

OBJECTIVE To investigate the association among different circulating stem cell (SC) populations, the levels of selected growth factors and chemokines regulating SC migration in the peripheral blood, and the incidence of retinopathy of prematurity (ROP). METHODS We evaluated 88 participants in this study: 29 preterm infants with ROP, 29 preterm infants without ROP, and 30 healthy full-term infants. Peripheral blood samples collected 10 weeks after delivery were analyzed using flow cytometry, immunofluorescence, real-time reverse transcriptase-polymerase chain reaction, and enzyme-linked immunosorbent assay. The following cell populations were analyzed: (1) lin⁻CXCR4(+)CD45⁻ (enriched in very small embryonic-like SCs), (2) lin⁻CXCR4(+)CD45(+) (enriched in hematopoietic SCs), and (3) CD34(+)CD133(+)CD144(+) (early endothelial progenitor cells) [lin indicates lineage]. The concentrations of vascular endothelial growth factor, basic fibroblast growth factor, hepatocyte growth factor, and stromal cell-derived factor 1 were measured in the plasma. RESULTS The very small embryonic-like SCs and early endothelial progenitor cells expressing neural and endothelial markers were significantly increased in the preterm infants. The number of early endothelial progenitor cells in the peripheral blood was significantly greater in the preterm infants with ROP than in the preterm infants without ROP. An accompanying increase in the concentrations of vascular endothelial growth factor and hepatocyte growth factor was found in the peripheral blood of the preterm infants with ROP. No significant associations were found between hematopoietic SCs and ROP or prematurity. CONCLUSIONS The increased number of early endothelial progenitor cells along with elevated levels of vascular endothelial growth factor and hepatocyte growth factor in preterm infants with ROP suggest that circulating vasculogenic factors may play a role in the development and progression of ROP. The increased number of very small embryonic-like SCs in preterm infants suggests that the development of immature tissues and organs, including the retina, may require a contribution of circulating SCs.

Circulating endothelial progenitor cells in premature infants: is there an association with premature birth complications?

Abstract Background: The most common morbidities in preterm infants are associated with vascular pathology. Endothelial progenitor cells (EPCs) have been implicated in repair of the vasculature, but their role in the pathogenesis of prematurity complications is not clear. Objectives: We prospectively investigated an association between the number of EPCs circulating in blood during delivery as well as 2 and 6 weeks afterwards, the level of growth factors regulating their migration/homing, and the incidence of premature birth complications. Patients and methods: The study groups consisted of 90 preterm and 52 full-term infants. Early-EPCs (CD133+CD34+CD144+) and late-EPCs (CD133–CD34+CD144+) were analysed in cord blood (CB) and peripheral blood (PB). Results: We found higher early- and late-EPC counts in the CB of premature infants compared with full-term babies. The number of circulating early- and late-EPCs was inversely associated with the Apgar score of preterm infants. A positive association between the early-EPC count and the risk of respiratory distress syndrome, retinopathy of prematurity, bronchopulmonary dysplasia, and infections was found. Nevertheless, multivariate analysis revealed that a higher number of EPCs was not an independent predictor of prematurity complications, which were directly related to lower gestational age. The EPC count in full-term infants maintained a constant, relatively low level over the 6-week follow-up, whereas the EPC population in preterm infants gradually decreased during this period. Furthermore, the number of CB late-EPCs in preterm infants positively correlated with VEGF concentration. Conclusions: EPCs may play a considerable role in vascular development in preterm infants.

Circulating hematopoietic stem cell count is a valuable predictor of prematurity complications in preterm newborns

BackgroundThe frequency of preterm labour has risen over the last few years. Hence, there is growing interest in the identification of markers that may facilitate prediction and prevention of premature birth complications. Here, we studied the association of the number of circulating stem cell populations with the incidence of complications typical of prematurity.MethodsThe study groups consisted of 90 preterm (23–36 weeks of gestational age) and 52 full-term (37–41 weeks) infants. Non-hematopoietic stem cells (non-HSCs; CD45-lin-CD184+), enriched in very small embryonic-like stem cells (VSELs), expressing pluripotent (Oct-4, Nanog), early neural (β-III-tubulin), and oligodendrocyte lineage (Olig-1) genes as well as hematopoietic stem cells (HSCs; CD45+lin-CD184+), and circulating stem/progenitor cells (CSPCs; CD133+CD34+; CD133-CD34+) in association with characteristics of prematurity and preterm morbidity were analyzed in cord blood (CB) and peripheral blood (PB) until the sixth week after delivery. Phenotype analysis was performed using flow cytometry methods. Clonogenic assays suitable for detection of human hematopoietic progenitor cells were also applied. The quantitative parameters were compared between groups by the Mann–Whitney test and between time points by the Friedman test. Fisher’s exact test was used for qualitative variables.ResultsWe found that the number of CB non-HSCs/VSELs is inversely associated with the birth weight of preterm infants. More notably, a high number of CB HSCs is strongly associated with a lower risk of prematurity complications including intraventricular hemorrhage, respiratory distress syndrome, infections, and anemia. The number of HSCs remains stable for the first six weeks of postnatal life. Besides, the number of CSPCs in CB is significantly higher in preterm infants than in full-term neonates (p < 0.0001) and extensively decreases in preterm babies during next six weeks after birth. Finally, the growth of burst-forming unit of erythrocytes (BFU-E) and colony-forming units of granulocyte-macrophage (CFU-GM) obtained from CB of premature neonates is higher than those obtained from CB of full-term infants and strongly correlates with the number of CB-derived CSPCs.ConclusionWe conclude that CB HSCs are markedly associated with the development of premature birth complications. Thus, HSCs ought to be considered as the potential target for further research as they may be relevant for predicting and controlling the morbidity of premature infants. Moreover, the observed levels of non-HSCs/VSELs circulating in CB are inversely associated with the birth weight of preterm infants, suggesting non-HSCs/VSELs might be involved in the maturation of fetal organism.

Exercise-induced mobilisation of endothelial progenitor cells in patients with premature coronary heart disease

BACKGROUND Endothelial progenitor cells (EPC) derive from bone marrow and participate in both endothelial regeneration and development of new blood vessels. EPC also play a role in the atherosclerotic process, and their number correlates negatively with the presence of classical risk factors. AIM To evaluate circulating EPC count and their exercise-induced mobilisation in patients with premature coronary artery disease (CAD). METHODS The study group included 60 patients with stable CAD diagnosed before 45 years of age. The control group consisted of 33 healthy age- and gender-matched volunteers. Venous blood was sampled 3 times in order to assess circulating EPC count immediately before an exercise test (EPC 0) and at 15 min (EPC 15) and 60 min (EPC 60) after the exercise test. RESULTS Circulating EPC count in the study group at rest and at 15 min after exercise was comparable (2.1 vs. 2.1 cell/μL, p = 0.35) and increased significantly at 60 min after exercise in comparison to resting values (2.1 vs. 3.2 cell/μL, p < 0.00001). In the control group, circulating EPC count increased significantly at 15 min after exercise (2.0 vs. 3.5 cell/μL, p < 0.0001) but later decreased at 60 min after exercise, although it remained greater than at rest (2.7 vs. 2.0 cell/μL, p < 0.0002). Circulating EPC count at rest and at 60 min after exercise was comparable in the two groups (2.1 vs. 2.0 cell/μL, p = 0.96; and 3.2 vs. 2.7 cell/μL, p = 0.13, respectively) but it was significantly lower in the study group compared to the control group at 15 min after exercise (2.1 vs. 3.5 cell/μL, p < 0.00001). Circulating EPC count at rest and at 15 min after exercise did not correlate with the number of stenosed coronary arteries but at 60 min after exercise it was greater in patients with one-vessel disease compared to those with two- or three-vessel disease (4.2 vs. 3.4 cell/μL, p = 0.01; and 4.2 vs. 2.3 cell/μL, p = 0.00003). However, no difference in circulating EPC count was seen at 60 min after exercise between patients with two- or three-vessel disease (3.4 vs. 2.3 cell/μL, p = 0.3). CONCLUSIONS 1. Circulating EPC count at rest is comparable between subjects with premature atherosclerosis and healthy volunteers. 2. A single bout of physical exercise causes a significant increase in circulating EPC count in both groups, but the dynamics of exercise-induced EPC mobilisation is different, with delayed exercise-induced EPC mobilisation in subjects with premature CAD. 3. The extent of atherosclerotic coronary lesions does not influence circulating EPC count at rest.

Methylene Blue Usage in Horseshoe Kidney Graft Separation: Case Report

Definitive diagnostics and strict procedures during kidney donor qualification are required. Nowadays, precise and accurate imaging techniques are at hand for every diagnostician. However, many studies have described intraoperative occurrence of horseshoe kidney. Although the harvesting procedure in the case of horseshoe kidney is not technically difficult, graft separation for successful renal transplantation is a challenge. The complex anatomy of malformed organs causes issues during kidney separation. This procedure may lead to damage of the collecting urinary system as well as vascularization damage. Separate graft transplantation is probable when a thin isthmus in a horseshoe kidney is present. Otherwise, poor graft function may occur. We present a technique for horseshoe kidney separation with the use of methylene blue for vascularization determination. The above-mentioned procedure was performed with the methylene blue solution dose injected into a single renal graft artery. Even with the malformed organs thick isthmus, the exact incision line was identified, exposing vascular perfusion asymmetry and allowing precise renal graft separation.

Role of platelets in the modulation of kidney allograft recipients’ immune systems

BACKGROUND Renal transplantation is the most effective method of treatment in end-stage renal disease. Chronic allograft rejection still remains a challenge for transplant physicians. Despite a growing amount of data regarding the role of platelets (PLT) in immunological processes, few reports have correlated number of platelets with transplanted kidney function. We aimed to evaluate the correlation between number of circulating platelets and number of immune system cells, including lymphocytes CD 4+, CD8+, lymphocytes B, monocytes, NK cells, and lymphocytes T reg in kidney transplant recipients with stable graft function. MATERIAL AND METHODS We enrolled 100 kidney transplant recipients (ages 20-78 years) 10 month to 10 years after transplantation. The numbers of platelets (using standard procedure) and immune blood cells were evaluated using flow cytometry. Statistical analysis was performed with Spearman rank correlation. RESULTS We found a negative correlation between number of platelets and number of lymphocytes T reg, and a positive correlation between platelet count and number of other examined immunocompetent cells. CONCLUSIONS The number of PLT correlates with number of cells responsible for induction and effector mechanisms of acquired cellular response.

Bone marrow of multiorgan donors underutilized: implications for improvement of accessibility of hematopoietic cells for transplantations.

Background. The demand for human hematopoietic stem and progenitor cells (HSPCs) for transplantation is increasing. Thus, effective alternative sources of HSPCs are required. Consequently, we sought to expand the accessibility of hematopoietic cells for clinical purposes by the investigation of hematopoietic reconstitution after transplantation of human HSPCs harvested from the bone marrow (BM) of heparinized deceased organ donors (HDODs). Methods. For multipart research comparison, human BM HDODs-, healthy donor-derived, umbilical cord blood nuclear cells, or CD34+ cells were transplanted into sublethally irradiated NOD/SCID mice. Twenty-eight days after transplantation nuclear cells were isolated from the murine BM, spleen, and peripheral blood and were used to quantitatively detect human CD45 antigen by quantitative real-time reverse transcriptase–polymerase chain reaction and flow cytometry. The clonogenic growth of human colony-forming units was also investigated. Results. We found that umbilical cord blood-derived HSPCs showed the greatest transplantation potential in our in vivo model. Interestingly, the transplantation potential of HSPCs collected from the BM of HDODs was of the same quality as cells obtained from healthy BM donors. Conclusion. Based on these results, we conclude that HDODs are a strongly underappreciated source of HSPCs that are ready to use for clinical purposes.

Safety and Feasibility of Lin- Cells Administration to ALS Patients: A Novel View on Humoral Factors and miRNA Profiles

Therapeutic options for amyotrophic lateral sclerosis (ALS) are still limited. Great hopes, however, are placed in growth factors that show neuroprotective abilities (e.g., nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and vascular endothelial growth factor (VEGF)) and in the immune modulating features, in particular, the anti-inflammatory effects. In our study we aimed to investigate whether a bone marrow-derived lineage-negative (Lin-) cells population, after autologous application into cerebrospinal fluid (CSF), is able to produce noticeable concentrations of trophic factors and inflammatory-related proteins and thus influence the clinical course of ALS. To our knowledge, the evaluation of Lin- cells transplantation for ALS treatment has not been previously reported. Early hematopoietic Lin- cells were isolated from twelve ALS patients’ bone marrow, and later, the suspension of cells was administered into the subarachnoid space by lumbar puncture. Concentrations of selected proteins in the CSF and plasma were quantified by multiplex fluorescent bead-based immunoassays at different timepoints post-transplantation. We also chose microRNAs (miRNAs) related to muscle biology (miRNA-1, miRNA-133a, and miRNA-206) and angiogenesis and inflammation (miRNA-155 and miRNA-378) and tested, for the first time, their expression profiles in the CSF and plasma of ALS patients after Lin- cells transplantation. The injection of bone marrow cells resulted in decreased concentration of selected inflammatory proteins (C3) after Lin- cells injection, particularly in patients who had a better clinical outcome. Moreover, several analyzed miRNAs have changed expression levels in the CSF and plasma of ALS patients subsequent to Lin- cells administration. Interestingly, the expression of miR-206 increased in ALS patients, while miR-378 decreased both in the CSF and plasma one month after the cells’ injection. We propose that autologous lineage-negative early hematopoietic cells injected intrathecally may be a safe and feasible source of material for transplantations to the central nervous system (CNS) environment aimed at anti-inflammatory support provision for ALS adjuvant treatment strategies. Further research is needed to evaluate whether the observed effects could significantly influence the ALS progression.

Activity of Cathepsin B in Serum of Patients after Kidney Transplantation Depends on Glucocorticosteroids Treatment.

BACKGROUND Kidney diseases are characterized by deterioration of the function of this organ, often leading to irreversible failure, where transplantation is the only alternative to permanent dialysis. Proteolytic enzymes, including cathepsins B, cleave the peptide bond by hydrolysis reaction. They are also involved in pathological processes such as carcinogenesis and inflammatory processes. The aim of this study was to determine the activity of cathepsin B in the serum of patients after kidney transplantation and to assess the correlation with glucocorticosteroids treatment. MATERIAL AND METHODS In the study, blood samples of 100 renal transplant recipients were used. The subjects were divided into groups according to the time elapsed since transplantation and the use of steroids in the current and primary treatment. Enzyme activity was measured by spectrofluorometric technique. RESULTS The study showed significant correlations of cathepsin B with the time since renal transplantation (p<0.05) and steroid used in the primary and current treatment. Steroid treatment is associated with a decrease of the activity of cathepsin B in serum. CONCLUSIONS The obtained results show decreasing activity of cathepsin B with longer time elapsed since transplantation. We have shown that steroids decrease activity of cathepsin B after renal transplantation. A significant increase in cathepsin B activity is observed mainly in cancer and atherosclerosis. Decreased activity of cathepsin B is probably due to the stabilizing action of steroids on the lysosomal membrane. The impact of steroid therapy for patients with these diseases appears to be significant.

Evaluation of Renal Graft Function based on Standard Mathematical Formulas

BACKGROUND Creatinine is a standard marker for estimation of the transplanted kidney function. Concentration values are used in mathematical equations for GFR (glomerular filtration rate) calculation, with MDRD (modification diet in renal disease) being most commonly used. Cystatin C is an alternative marker for changes in glomerular filtration, which is also used in eGFR (estimated GFR) formulas. The aim of this study was to reveal eGFR <60 ml/min/1.72 m(2) in a population of patients after renal transplant, with stable graft function, using different formulas. MatERIAL AND METHODS: A group of 100 patients (56 females and 44 males) aged 20-78 years, took part in this study. Renal transplantation was conducted from 10 years to 10 months prior to the study. Estimated GFR was calculated with 4 formulas: MDRD, CKD-EPI (Chronic Kidney Disease Epidemiology Collaboration), CKD-EPI cys (using cystatin C), and CKD-EPI mix (using creatinine and cystatin C). We used electronic calculators available on the National Kidney Foundation and the Nephron Information Center websites. RESULTS The occurrence of eGFR values <60 ml/min/1.73 m(2) was 28% according to MDRD formula, 23% according to CKD-EPI, 25% according to CDK-EPI cys, and 26%according to CDK-EPI mix. CONCLUSIONS Occurrence of GFR <60 ml/min/0.73 m(2) was the highest when calculated by MDRD formula, and the lowest when calculation was done with CDK-EPI. The significant discrepancy with different eGFR formula testing suggests the need for further research to find the best marker and/or formula for graft function estimation.