In the era of COVID-19 pandemic, organ transplantation programs were facing serious challenges. T... more In the era of COVID-19 pandemic, organ transplantation programs were facing serious challenges. The lung transplantation donor pool was extremely limited and SARS-CoV-2 viral load assessment has become a crucial part of selecting an optimal organ donor. Since COVID-19 is a respiratory disease, the viral load is thought to be more important in lung transplantations as compared to other solid organ transplantations. We present two challenging cases of potential lung donors with a questionable COVID-19 status. Based on these cases, we suggest that the cycle threshold (Ct) value should always be requested from the laboratory and the decision whether to proceed with transplantation should be made upon complex evaluation of diverse criteria, including the nasopharyngeal swab and bronchoalveolar lavage PCR results, the Ct value, imaging findings and the medical history. However, as the presence of viral RNA does not ensure infectivity, it is still to be clarified which Ct values are associated with the viral viability. Anti-SARS-CoV-2 IgA antibodies may support the diagnosis and moreover, novel methods, such as quantifying SARS-CoV-2 nucleocapsid antigen in serum may provide important answers in organ transplantations and donor selections.
Atherosclerosis pathology is the interplay between high intravascular LDL particle concentration ... more Atherosclerosis pathology is the interplay between high intravascular LDL particle concentration and monocyte/ macrophage presence within the sub-endothelial space of the artery. In this project, phenotypes of macrophages connected with subclinical inflammation in adipose tissue of living kidney donors were studied. Samples of subcutaneous adipose tissue of living kidney donors (n=36) were exposed to collagenase. Stromal vascular fraction (SVF) was eluted from the samples, then labeled with monoclonal antibodies (anti-CD14 and anti-calprotectin), conjugated with fluorochromes and analyzed by flow cytometry. The positive correlation between the number of total macrophages and calprotectin-positive macrophages with BMI in the subcutaneous adipose tissue of postmenopausal women was demonstrated (p<0.05; R=0.43 and p<0.01; R=0.60), whereas no positive correlation in premenopausal women and men was shown. In conclusion, we documented a significant effect of BMI increase on the presence of total macrophages in adipose tissue of postmenopausal women, in contrast to premenopausal women. This difference was much more pronounced when proinflammatory macrophages with membrane-bound calprotectin were analyzed.
Background. Immune response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vacci... more Background. Immune response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination has been recently shown to be impaired in kidney transplant recipients (KTRs), but the underlying factors affecting vaccine effectiveness need to be further elucidated. Methods. In this prospective cohort study, antibodies against S1 and S2 subunits of SARS-CoV-2 were evaluated using an immunochemiluminescent assay (cutoff 9.5 AU/mL, sensitivity 91.2%, and specificity 90.2%) in 736 KTRs, who were previously either naive or infected with SARS-CoV-2 and vaccinated before or after transplantation. Cellular response was analyzed in a subset of patients using an interferon gamma release assay (cutoff 0.15 IU/mL, sensitivity 92%, and specificity 100%). Results. Seroconversion was significantly more impaired in SARS-CoV-2–naive KTRs than in those previously infected (40.1% versus 97.1%; P &lt; 0.001). Mycophenolate use (odds ratio, 0.15; 95% confidence interval, 0.09-0.24; P &lt; 0.001) and depleting therapy in the past year (odds ratio, 0.19; 95% confidence interval, 0.05-0.8; P = 0.023) were found to be among independent factors associated with impaired humoral response. Similarly, the interferon gamma release assay tested in 50 KTRs (cutoff 0.15 IU/mL, sensitivity 92%, specificity 100%) showed that specific T-cell responses against spike protein epitopes are impaired in SARS-CoV-2–naive KTRs, as compared to previously infected KTRs (9.4% versus 90%, P &lt; 0.001). All 35 KTRs vaccinated on the waiting list before transplantation exhibited sustained seroconversion persisting after transplantation. Conclusions. Survivors of coronavirus disease 2019 and those vaccinated while on the waiting list exhibited a marked immune response to mRNA vaccines, contrary to poor response in naive KTRs vaccinated after transplantation (NCT04832841).
The multifunctional cytokine IL-6, which can be locally produced by human bronchial epithelial ce... more The multifunctional cytokine IL-6, which can be locally produced by human bronchial epithelial cells (HBECs), has been found to play a role in IL-4 dependent IgE synthesis. Since the allergic reaction in bronchial asthma is associated with the upregulation of IL-4 and Th2 type of immune response, the purpose of our study was to assess whether IL-4 and related cytokines IL-10 and IL-13 regulate IL-6 release by HBEC s. HBECs were obtained by bronchial brushing, cultured in LHC-9/RPMI 1640. At the third passage the cells were stimulated with cytokines (0.1-20 ng/ml) diluted in unsupplemented media for 24 h. The supernatants were tested for IL-6 content by sandwich ELISA. Unstimulated HBECs produced detectable amounts of IL-6 (368+/-25 pg/ml). Exposure to IL-10 (368+/-22 pg/ml) and IL-13 (395+/-6 pg/ml) resulted in little changes. IL-4 caused a slight but significant increase in IL-6 release (530+/-45 pg/ml), P&lt;0.05, TNFalpha (1657+/-85 pg/ml) and IFNgamma (1953+/-37 pg/ml) showed strong induction of IL-6 release in HBECs (P&lt;0.005 and P&lt;0.001, respectively). Both IL-4 and IL-13 significantly inhibited TNF induced IL-6 release (P&lt;0.01 for both) while augmenting the effect of IFNgamma (P&lt;0.005 and P&lt;0.01, respectively.). IL-10 was without a significant effect. We conclude that Th2-type cytokines IL-4 and IL-13 affect the release of IL-6 by HBECs in response to TNFalpha (inhibition) and IFgamma (augmentation). IL-10 had no effect on the regulation of IL-6 release. Modulation of IL-6 levels by Th2-type cytokines may play a role in allergic reactions through the IL-6 promoting effect on IL-4 mediated IgE production.
Background: Grasses belong to major sources of inhaled allergens. The knowledge of particular mol... more Background: Grasses belong to major sources of inhaled allergens. The knowledge of particular molecules responsible for hypersensitivity is of crucial importance for better understanding of individual differences among single allergic subjects and allergic populations living in various world-areas. Methods: Specific-IgE-antibodies against Phl p 1, Phl p 5, Phl p 7, Phl p 12 were detected in a group of 130 Phleum-allergic-subjects (82 children, 48 adults). Phl p 1 antibodies were detected in most pediatric and adult patients, however, the children were as- sociated with higher RAST classes more often. Anti-Phl p 5-antibodies were found more frequently in adults. An increase was observed in the number of pediatric patients reacting to Phl p 7 and Phl p 12. There were no differences in concentrations of specific-IgE against Phl p 5, Phl p 7 and Phl p 12 depending on age. Almost 10% of allergic children produced antibodies directed exclusively against minor allergens or did not produce specific-IgE-antibodies against tested molecules. Part of the patients reacted to profilin and calcium-binding protein originating from only one source (Phl p 12! Bet v 2 and Phl p 7! Bet v 4). Antibodies against Phl p 1 and Phl p 5 can be used as a marker of allergy to grasses in adult patients. Children reacted exclusively to minor allergens more frequently than adults. Prolonged allergen exposure is evidently necessary to induce sensitization to Phl p 5. A high level of homology between profilins and calcium-binding proteins enables only one allergen to be used for diagnostic purposes but a possibility of a reaction to species-bound epitopes should be taken into account.
Background: Monocytes represent a heterogeneous population of cells subdivided according to the e... more Background: Monocytes represent a heterogeneous population of cells subdivided according to the expression level of membrane antigens. A pro-inflammatory (intermediate/nonclassical) subpopulation of monocytes is defined by expression of CD16. CD163 seems to be characteristically preferentially expressed by immunosuppressive monocytes. The aim of our study was to evaluate the distribution of monocyte subpopulations in 71 patients with kidney allograft transplantation. Results: The phenotype was evaluated by flow cytometry in defined time points. The proportions of peripheral CD14+CD16+ monocytes were downregulated immediately after the kidney transplantation and basiliximab treatment partially attenuated this trend. The transient downregulation of the CD14+CD16+ subpopulation was adjusted to basal values in two months. The proportions of CD14+CD163+ monocytes were transiently upregulated early after the kidney transplantation and remained higher during the first month in most patients. In ATG treated patients, the expansion of CD14+CD163+ monocytes was delayed but their upregulation lasted longer. In vitro data showed the direct effect of ATG and methylprednisolone on expression of CD16 and CD163 molecules while basiliximab did not affect the phenotype of cultured monocytes. Conclusions: We assume from our data that kidney allograft transplantation is associated with modulation of monocyte subpopulations (CD14+CD16+ and CD14+CD163+) partially affected by an immunosuppressive regime used.
Epithelial cells represent an important source of cytokines that may modulate the influx and func... more Epithelial cells represent an important source of cytokines that may modulate the influx and functions of mononuclear phagocytes. The aim of our study was to characterize changes in the gene expression of selected cytokines in human macrophages co-cultured with respiratory epithelial cells. The A549 alveolar type II-like cell line was co-cultured with THP-1 cells (monocyte/macrophage cell line) in filterseparated mode to avoid their cell-cell contact. At different time-points (0, 4, 8, 12 and 24 h), the cells were harvested separately to evaluate their gene and protein expression (IL-1 beta, IL-6, IL-8, IL-10 and GM-CSF). Quantitative RT-PCR analysis showed prominent changes in the THP-1 cytokine gene expression induced by a co-culture with A549 cells. Fourfold upregulation of mRNA expression has been found in 12 genes and 4-fold downregulation in 5 genes as compared to the unstimulated control sample with a p value smaller than 0.05. The induction of inhibin beta A and IL-1 beta mRNA after 12 h and the expression of IL-1 alpha and GM-CSF mRNA after 24 h were the most prominent. When looking at the cytokine levels in culture supernatants, IL-1 beta and IL-8 were induced early (at 8 h) as compared to the release of IL-6 and GM-CSF (at 24 h). We conclude that respiratory epithelial cells constitutively regulate the cytokine gene expression of macrophages located in their environment and might further modulate the release of cytokines by posttranslational pathways.
Introduction. Alemtuzumab [AZ] is a potent lymphodepleting anti CD-52 monoclonal antibody, which ... more Introduction. Alemtuzumab [AZ] is a potent lymphodepleting anti CD-52 monoclonal antibody, which is used as an induction agent in renal transplantation. Early observations suggested that this agent might induce "Prope Tolerance" and reduce graft loss from rejection. The aim of this study was to describe the medium to long-term outcomes of a large cohort of renal transplant recipients following AZ induction. Methods. We retrospectively studied 927 patients [610(65.8%) male, 446(48.1%) living donor, 426(46.0%) caucasian] who received a renal transplant with AZ induction, a tacrolimus based and steroid sparing immunosuppressive regime. Mean follow up was 4.18±2.36 years. Results. Overall patient survival at 1,3 and 5 yrs was 98.3%, 94.9% and 92.5% respectively. Death censored allograft survival was 96.0%, 90.8% and 87.0% at 1,3 and 5yrs and the corresponding rejection free survival was 84.2%, 77.9% and 75.2%. 42/927[4.5%] patients died with a functioning graft.8/927[0.86%] died secondary to cardiac causes, 9[0.97%] from malignancy, 9[0.97%] infection,10 [1.08%] cases were unknown and 6[0.65%] had other defi ned causes. 104/927[11.2%] allografts failed, the causes of which are shown below. Conclusion. Although AZ induction is associated with good patient and allograft survival, rejection remains the leading cause of allograft failure. This is similar to outcomes achieved with other lymphodepleting induction agents such as ATG.
Introduction: Idiopathic pulmonary fibrosis (IPF) is a serious disease with unknown etiology, whe... more Introduction: Idiopathic pulmonary fibrosis (IPF) is a serious disease with unknown etiology, where an influence of cytokine gene polymorphisms is presumed. We compared HRCT alveolar (AS) and interstitial score (IS) and histoptahologic score with IL-4, and IL-4RA gene polymorphisms in IPF patients. Subjects and methods: IPF was diagnosed in 46 patients according to ATS/ERS consensus statement. 43 patients had evaluable HRCT investigations, 14 patients had surgical lung biopsy. HRCT scans were evaluated using IS and AS scales by Gay et al. The histopathological evaluation of lung biopsies comprised: myofibroblast foci (MF), inflammation, eosinophils, granulomas and Ashcroft criteria for fibrosis grading. The IL-4 (-1098) (-590) (-33) and IL-4 RA +1902 gene polymorphisms were characterized utilizing a PCR- SSP method. Results: AS was higher in IL-4 haplotypes 1 TTC and TTT carrierrs (p=0.0423). Ashcroft score was more advanced in IL-4 haplotype 2 GCC (p= 0,013) and MF counts were higher in TCC carriers (p=0,0736). IL-4 RA +1902 A1 G and IL-4 -590 A1 T correlated with higher AS (p=0,0335; p=0,0123). Ashcroft score was higher in IL-4 -1098 A2 G and IL-4 -33 A1 T carriers (p= 0,0443, p=0,0915). Conclusions: We assume that IL-4 and IL-4RA polymorphisms might influence HRCT and histopathological phenotype of IPF. The correlation of functionally relevant IL-4 genes polymorphisms (especially IL-4 -33 T) with AS could mean, that new alveolar lesions with continuing fibrosis are more pronounced in these polymorphisms carriers. The positive correlation of IL-4 -33 A1 T with Ashcroft score might support a hypothesis of fibrogenetic role of IL-4 in IPF.
Objectives: MRP8/14 is a heterodimer of two myeloid calcium-binding proteins associated with diff... more Objectives: MRP8/14 is a heterodimer of two myeloid calcium-binding proteins associated with different types of acute inflammatory processes. We studied MRP8/14 together with procalcitonin (PCT) serum levels in order to diagnose infectious complications or the rejection process affecting kidney or heart allograft. Methods: A total of 419 serum samples was evaluated. MRP8/14 levels were measured by ELISA (BMA Biomed), PCT by a sensitive immunoluminiscent assay ILMA (Brahms Diagn.) Results: Both parameters showed very low basal levels in healthy subjects (range 303-1,660 ng/ml of MRP8/14; less than 0.08 ng/ml of PCT). A rapid increase in serum levels occurred in response to bacterial infections (MRP8/14 up to 6,230 ng/ml; PCT up to 297 ng/ml). Serum PCT concentration remained low in the presence of kidney allograft rejection, where MRP8/14 levels were increased. An uncomplicated outcome of kidney or heart transplantation did not change basal serum MRP8/14 and PCT levels. Conclusions: We conclude that 1) both MRP8/14 and PCT are very sensitive markers of complications in organ transplant recipients (normal values in uncomplicated outcome) 2) combination of both parameters is useful to discriminate between rejection (increased MRP8/14 with normal PCT) and systemic bacterial infection (both parameters increased).
Background: Steroid avoidance in immunosuppression in kidney transplantation offers several metab... more Background: Steroid avoidance in immunosuppression in kidney transplantation offers several metabolic advantages, however it is associated with higher early acute rejection rate. Cellular and molecular mechanisms of this phenomenon remain poorly understood. Methods: In this single center observational study, low-risk kidney transplant recipients randomized into large multicenter prospective ADVANCE trial with steroid avoidance/early withdrawal and center standard of care treated patients were monitored for 12 months. The expressions of 28 transcripts, associated with alloimmune response and operational tolerance, were evaluated in the peripheral blood using RT-qPCR at 0, 7, 14, 90 and 365 postoperative days (POD) and in the protocol graft biopsy at 3-months while lymphocyte subpopulations were analyzed by flow-cytometry within the follow-up. Results: Both steroid avoidance and withdrawal regimens were associated with significantly higher granzyme B (GZMB) transcript at POD 14 and perforin 1 (PRF1) transcript at POD 7. The higher interleukin 2 (IL-2) expression at POD 7 was detected only in the steroid avoidance group. Initial steroids decreased the expression SH2D1B transcript at POD14 and there were no further differences in other operational tolerance transcripts among groups. The statistically significant decrease in absolute numbers of peripheral NK cells in the first 14 days was observed in the standard of care group only. There were no differences in analyzed intrarenal transcripts in 3months biopsies among groups. Conclusions: The enhanced expression of some of Th1 associated transcripts and limited effects on NK cells of steroid avoidance immunosuppression suggest higher susceptibility for early acute rejection.
American Journal of Physiology-lung Cellular and Molecular Physiology, Sep 1, 1997
The airway epithelial cell may play a role as an effector cell, releasing various cytokines and e... more The airway epithelial cell may play a role as an effector cell, releasing various cytokines and extracellular matrix components in immune responses, inflammation, and wound repair processes, thus contributing to cytokine &quot;networks.&quot; The cytokines transforming growth factor (TGF)-beta and interleukin (IL)-4 are though to have pivotal roles in airway diseases, with IL-4 having proinflammatory actions and TGF-beta generally regarded to mediate repair and to attenuate immune responses. In asthma, where IL-4 and TGF-beta are thought to contribute to the inflammatory process and repair, respectively, interactions between these cytokines are likely to be of importance. Therefore, we studied the potential interaction of both cytokines by measuring IL-8 and fibronectin release by cultured human bronchial epithelial cells (HBECs). IL-4 is capable of inducing IL-8 release from HBECs. This effect of IL-4 can be blocked by the concurrent presence of the cytokine TGF-beta. In contrast, TGF-beta had a modest inconsistent stimulatory effect on IL-8 release by itself and had no effect on the IL-8 release induced by tumor necrosis factor (TNF)-alpha. An antagonistic effect of IL-4 and TGF-beta was also observed on HBEC fibronectin release. TGF-beta stimulated fibronectin release, and IL-4 was able to inhibit this. This effect was not due to a redistribution of fibronectin but appeared to be due to a true reduction in synthesis. Consistent with this, IL-4 and TGF-beta effects on IL-8 and fibronectin release were paralleled by changes in mRNA levels. The ability of TGF-beta to block IL-4-induced IL-8 release is certainly not the only mechanism to inhibit IL-8 release because dexamethasone was capable of inhibiting both TNF-alpha- and IL-4-induced release of IL-8. These results indicate that TGF-beta and IL-4 can have mutually inhibitory effects. The balance determined by this reciprocal inhibition may play an important role in regulating inflammation repair and in diseases such as asthma.
Macrolide antibiotics such as azithromycin or clarithromycin are known to have potent antiinflamm... more Macrolide antibiotics such as azithromycin or clarithromycin are known to have potent antiinflammatory and immunomodulatory effects but these properties cannot be widely used due to a risk of bacterial resistance. We studied another polyketide antibiotic, structurally related manumycin A known as a streptomycete derived farnesyltransferase inhibitor with limited antibacterial effects, with respect to its potential regulation of mRNA expression of several genes associated with proinflammatory responses. Downregulation of mRNA for IL-6, TLR-8, IL-1 beta and IL-10 was found in THP-1 cells after 4 h stimulation with TNF alpha in the presence of manumycin A and downregulated TLR-8 and EGR-1 genes were observed after 8 h. Among the genes upregulated in response to manumycin were HMOX-1, TNFRSF10A, IL-1R1, TICAM2, NLRP12 after 4 h and only IL-1R1 after 8 h. Furthermore, manumycin A was found to inhibit IL-1beta, IL-6, and IL-8 production in TNF alpha stimulated THP-1 cells and peripheral blood monocytes in a dose dependent manner (0.25-1 M of manumycin A) without affecting cell viability. Cell viability of blood monocytes decreased by about 30% at manumycin A doses of 2-5 M. Manumycin A also inhibited IL-18 release from THP-1 cells, while in cultures of blood monocytes, this cytokine was not detectable. That manumycin A mediated downregulation of proinflammatory genes in human monocytes confirmed by a measurement of cytokine levels in culture supernatants, together with a very limited effect on cell viability, might suggest potential anti-inflammatory properties of this polyketide antibiotic.
Radiation exposure is known to impair healing in irradiated areas. Fibroblasts play a major role ... more Radiation exposure is known to impair healing in irradiated areas. Fibroblasts play a major role in the production and modification of extracellular matrix in wound repair. Since one important aspect of wound repair is the contraction of the wound, this study investigated the effects of radiation on the ability of fibroblasts to mediate collagen gel contraction in an in vitro model of wound retraction. After irradiation, the cells were detached and suspended in a solution of rat tail tendon collagen. Radiation exposure decreased retraction, and this effect was dose dependent. In order to define the mechanism of reduced gel retraction, we investigated alpha2beta1 cell surface integrin and fibronectin, which are thought to mediate contraction, and prostaglandin E2 (PGE2), which is known to inhibit this process. PGE2 release increased dose responsively following radiation. The cyclooxygenase inhibitor indomethacin could partially restore the contractile activity of irradiated fibroblasts. Fibronectin production in gel culture showed a significant decrease. In contrast, there was no decrease in alpha2beta1 integrin expression in radiated cells. In conclusion, radiation decreases fibroblast-mediated gel contraction. Increased PGE2 production and decreased fibronectin production by irradiated fibroblasts may contribute to this effect and may be in part responsible for poor healing of radiated tissue.
Procalcitonin (PCT) represents a new marker of systemic inflammatory reactions of the body to inf... more Procalcitonin (PCT) represents a new marker of systemic inflammatory reactions of the body to infections. PCT is selectively induced by severe bacterial infections leading to sepsis or multiorgan dysfunction syndrome. The aim of our study was to test PCT as a postoperative infection marker in heart and kidney transplant patients compared with healthy subjects and patients with localized lung-inflammatory processes without a manifest systemic response. PCT concentrations were measured by an immunoluminometric assay (ILMA) in a total of 419 serum samples. Normal serum levels were in the range of 0.08-0.6 ng/ml. Operative trauma associated with heart (not kidney) transplantation induced a transient increase in PCT levels to 7-10 ng/ml with a decline to normal levels within 2-3 days in most patients. Severe bacterial infections dramatically augmented serum PCT concentrations reaching values of 46-297 ng/ml in the most critical periods. Good response to antibiotic therapy was associated with a decline in serum PCT concentrations. Acute rejection or cytomegalovirus infections did not significantly increase the serum PCT levels. Localized pulmonary infections showed either no, or only a limited increase, in the serum PCT levels (max. 7 ng/ml). We conclude from our data that PCT can be used as a sensitive marker to differentiate systemic bacterial infections from other complications in organ transplantation.
Chronic allograft nephropathy (CAN) represents a frequent and irreversible cause of long-term ren... more Chronic allograft nephropathy (CAN) represents a frequent and irreversible cause of long-term renal graft loss. TGF-beta1 is a key profibrogenic cytokine associated with CAN pathogenesis. Because of clinical diagnostic inaccuracy, protocol biopsy has been suggested to be a beneficial method for early CAN detection. Protocol core biopsy was carried out in 67 consecutive cyclosporine-based immunosuppression-treated kidney transplant recipients with stable renal function 12 months after renal transplantation. Biopsy specimens were analyzed morphologically according to Banff-97' criteria and immunohistologically for TGF-beta1 staining. The data obtained were correlated with plasma TGF-beta1 levels and clinical data. CAN (grade I-III) was found in 51 patients (76 %). CAN grade I was found to be the most frequent one (44 %). A normal finding within the graft was made in only 12 patients (18 %). Clinically silent acute rejection Banff IA was present in 4 patients (6 %). In 8 patients (...
Purpose Advanced age is associated with an impaired humoral immune response to SARS-CoV-2 mRNA va... more Purpose Advanced age is associated with an impaired humoral immune response to SARS-CoV-2 mRNA vaccination in kidney transplant recipients (KTR). The mechanisms are, however, poorly understood. Frailty syndrome assessment may determine the most vulnerable population. Methods This study is a secondary analysis of a prospective study (NCT04832841) regarding seroconversion after BNT162b2 vaccination, including 101 SARS-CoV-2 naïve KTR 70 years and older. The Fried frailty components were evaluated, and antibodies against S1 and S2 subunits of SARS-CoV-2 were examined > 14 days after the second dose of BNT162b2 vaccine. Results Seroconversion was observed in 33 KTR. Male gender, eGFR, MMF-free immunosuppression, and a lower frailty score were associated with higher seroconversion rates in univariable regression. Concerning frailty components, physical inactivity had the most negative effect on seroconversion (OR = 0.36, 95% CI 0.14–0.95, p = 0.039). In a multivariable regression adju...
In the era of COVID-19 pandemic, organ transplantation programs were facing serious challenges. T... more In the era of COVID-19 pandemic, organ transplantation programs were facing serious challenges. The lung transplantation donor pool was extremely limited and SARS-CoV-2 viral load assessment has become a crucial part of selecting an optimal organ donor. Since COVID-19 is a respiratory disease, the viral load is thought to be more important in lung transplantations as compared to other solid organ transplantations. We present two challenging cases of potential lung donors with a questionable COVID-19 status. Based on these cases, we suggest that the cycle threshold (Ct) value should always be requested from the laboratory and the decision whether to proceed with transplantation should be made upon complex evaluation of diverse criteria, including the nasopharyngeal swab and bronchoalveolar lavage PCR results, the Ct value, imaging findings and the medical history. However, as the presence of viral RNA does not ensure infectivity, it is still to be clarified which Ct values are associated with the viral viability. Anti-SARS-CoV-2 IgA antibodies may support the diagnosis and moreover, novel methods, such as quantifying SARS-CoV-2 nucleocapsid antigen in serum may provide important answers in organ transplantations and donor selections.
Atherosclerosis pathology is the interplay between high intravascular LDL particle concentration ... more Atherosclerosis pathology is the interplay between high intravascular LDL particle concentration and monocyte/ macrophage presence within the sub-endothelial space of the artery. In this project, phenotypes of macrophages connected with subclinical inflammation in adipose tissue of living kidney donors were studied. Samples of subcutaneous adipose tissue of living kidney donors (n=36) were exposed to collagenase. Stromal vascular fraction (SVF) was eluted from the samples, then labeled with monoclonal antibodies (anti-CD14 and anti-calprotectin), conjugated with fluorochromes and analyzed by flow cytometry. The positive correlation between the number of total macrophages and calprotectin-positive macrophages with BMI in the subcutaneous adipose tissue of postmenopausal women was demonstrated (p<0.05; R=0.43 and p<0.01; R=0.60), whereas no positive correlation in premenopausal women and men was shown. In conclusion, we documented a significant effect of BMI increase on the presence of total macrophages in adipose tissue of postmenopausal women, in contrast to premenopausal women. This difference was much more pronounced when proinflammatory macrophages with membrane-bound calprotectin were analyzed.
Background. Immune response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vacci... more Background. Immune response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination has been recently shown to be impaired in kidney transplant recipients (KTRs), but the underlying factors affecting vaccine effectiveness need to be further elucidated. Methods. In this prospective cohort study, antibodies against S1 and S2 subunits of SARS-CoV-2 were evaluated using an immunochemiluminescent assay (cutoff 9.5 AU/mL, sensitivity 91.2%, and specificity 90.2%) in 736 KTRs, who were previously either naive or infected with SARS-CoV-2 and vaccinated before or after transplantation. Cellular response was analyzed in a subset of patients using an interferon gamma release assay (cutoff 0.15 IU/mL, sensitivity 92%, and specificity 100%). Results. Seroconversion was significantly more impaired in SARS-CoV-2–naive KTRs than in those previously infected (40.1% versus 97.1%; P &lt; 0.001). Mycophenolate use (odds ratio, 0.15; 95% confidence interval, 0.09-0.24; P &lt; 0.001) and depleting therapy in the past year (odds ratio, 0.19; 95% confidence interval, 0.05-0.8; P = 0.023) were found to be among independent factors associated with impaired humoral response. Similarly, the interferon gamma release assay tested in 50 KTRs (cutoff 0.15 IU/mL, sensitivity 92%, specificity 100%) showed that specific T-cell responses against spike protein epitopes are impaired in SARS-CoV-2–naive KTRs, as compared to previously infected KTRs (9.4% versus 90%, P &lt; 0.001). All 35 KTRs vaccinated on the waiting list before transplantation exhibited sustained seroconversion persisting after transplantation. Conclusions. Survivors of coronavirus disease 2019 and those vaccinated while on the waiting list exhibited a marked immune response to mRNA vaccines, contrary to poor response in naive KTRs vaccinated after transplantation (NCT04832841).
The multifunctional cytokine IL-6, which can be locally produced by human bronchial epithelial ce... more The multifunctional cytokine IL-6, which can be locally produced by human bronchial epithelial cells (HBECs), has been found to play a role in IL-4 dependent IgE synthesis. Since the allergic reaction in bronchial asthma is associated with the upregulation of IL-4 and Th2 type of immune response, the purpose of our study was to assess whether IL-4 and related cytokines IL-10 and IL-13 regulate IL-6 release by HBEC s. HBECs were obtained by bronchial brushing, cultured in LHC-9/RPMI 1640. At the third passage the cells were stimulated with cytokines (0.1-20 ng/ml) diluted in unsupplemented media for 24 h. The supernatants were tested for IL-6 content by sandwich ELISA. Unstimulated HBECs produced detectable amounts of IL-6 (368+/-25 pg/ml). Exposure to IL-10 (368+/-22 pg/ml) and IL-13 (395+/-6 pg/ml) resulted in little changes. IL-4 caused a slight but significant increase in IL-6 release (530+/-45 pg/ml), P&lt;0.05, TNFalpha (1657+/-85 pg/ml) and IFNgamma (1953+/-37 pg/ml) showed strong induction of IL-6 release in HBECs (P&lt;0.005 and P&lt;0.001, respectively). Both IL-4 and IL-13 significantly inhibited TNF induced IL-6 release (P&lt;0.01 for both) while augmenting the effect of IFNgamma (P&lt;0.005 and P&lt;0.01, respectively.). IL-10 was without a significant effect. We conclude that Th2-type cytokines IL-4 and IL-13 affect the release of IL-6 by HBECs in response to TNFalpha (inhibition) and IFgamma (augmentation). IL-10 had no effect on the regulation of IL-6 release. Modulation of IL-6 levels by Th2-type cytokines may play a role in allergic reactions through the IL-6 promoting effect on IL-4 mediated IgE production.
Background: Grasses belong to major sources of inhaled allergens. The knowledge of particular mol... more Background: Grasses belong to major sources of inhaled allergens. The knowledge of particular molecules responsible for hypersensitivity is of crucial importance for better understanding of individual differences among single allergic subjects and allergic populations living in various world-areas. Methods: Specific-IgE-antibodies against Phl p 1, Phl p 5, Phl p 7, Phl p 12 were detected in a group of 130 Phleum-allergic-subjects (82 children, 48 adults). Phl p 1 antibodies were detected in most pediatric and adult patients, however, the children were as- sociated with higher RAST classes more often. Anti-Phl p 5-antibodies were found more frequently in adults. An increase was observed in the number of pediatric patients reacting to Phl p 7 and Phl p 12. There were no differences in concentrations of specific-IgE against Phl p 5, Phl p 7 and Phl p 12 depending on age. Almost 10% of allergic children produced antibodies directed exclusively against minor allergens or did not produce specific-IgE-antibodies against tested molecules. Part of the patients reacted to profilin and calcium-binding protein originating from only one source (Phl p 12! Bet v 2 and Phl p 7! Bet v 4). Antibodies against Phl p 1 and Phl p 5 can be used as a marker of allergy to grasses in adult patients. Children reacted exclusively to minor allergens more frequently than adults. Prolonged allergen exposure is evidently necessary to induce sensitization to Phl p 5. A high level of homology between profilins and calcium-binding proteins enables only one allergen to be used for diagnostic purposes but a possibility of a reaction to species-bound epitopes should be taken into account.
Background: Monocytes represent a heterogeneous population of cells subdivided according to the e... more Background: Monocytes represent a heterogeneous population of cells subdivided according to the expression level of membrane antigens. A pro-inflammatory (intermediate/nonclassical) subpopulation of monocytes is defined by expression of CD16. CD163 seems to be characteristically preferentially expressed by immunosuppressive monocytes. The aim of our study was to evaluate the distribution of monocyte subpopulations in 71 patients with kidney allograft transplantation. Results: The phenotype was evaluated by flow cytometry in defined time points. The proportions of peripheral CD14+CD16+ monocytes were downregulated immediately after the kidney transplantation and basiliximab treatment partially attenuated this trend. The transient downregulation of the CD14+CD16+ subpopulation was adjusted to basal values in two months. The proportions of CD14+CD163+ monocytes were transiently upregulated early after the kidney transplantation and remained higher during the first month in most patients. In ATG treated patients, the expansion of CD14+CD163+ monocytes was delayed but their upregulation lasted longer. In vitro data showed the direct effect of ATG and methylprednisolone on expression of CD16 and CD163 molecules while basiliximab did not affect the phenotype of cultured monocytes. Conclusions: We assume from our data that kidney allograft transplantation is associated with modulation of monocyte subpopulations (CD14+CD16+ and CD14+CD163+) partially affected by an immunosuppressive regime used.
Epithelial cells represent an important source of cytokines that may modulate the influx and func... more Epithelial cells represent an important source of cytokines that may modulate the influx and functions of mononuclear phagocytes. The aim of our study was to characterize changes in the gene expression of selected cytokines in human macrophages co-cultured with respiratory epithelial cells. The A549 alveolar type II-like cell line was co-cultured with THP-1 cells (monocyte/macrophage cell line) in filterseparated mode to avoid their cell-cell contact. At different time-points (0, 4, 8, 12 and 24 h), the cells were harvested separately to evaluate their gene and protein expression (IL-1 beta, IL-6, IL-8, IL-10 and GM-CSF). Quantitative RT-PCR analysis showed prominent changes in the THP-1 cytokine gene expression induced by a co-culture with A549 cells. Fourfold upregulation of mRNA expression has been found in 12 genes and 4-fold downregulation in 5 genes as compared to the unstimulated control sample with a p value smaller than 0.05. The induction of inhibin beta A and IL-1 beta mRNA after 12 h and the expression of IL-1 alpha and GM-CSF mRNA after 24 h were the most prominent. When looking at the cytokine levels in culture supernatants, IL-1 beta and IL-8 were induced early (at 8 h) as compared to the release of IL-6 and GM-CSF (at 24 h). We conclude that respiratory epithelial cells constitutively regulate the cytokine gene expression of macrophages located in their environment and might further modulate the release of cytokines by posttranslational pathways.
Introduction. Alemtuzumab [AZ] is a potent lymphodepleting anti CD-52 monoclonal antibody, which ... more Introduction. Alemtuzumab [AZ] is a potent lymphodepleting anti CD-52 monoclonal antibody, which is used as an induction agent in renal transplantation. Early observations suggested that this agent might induce "Prope Tolerance" and reduce graft loss from rejection. The aim of this study was to describe the medium to long-term outcomes of a large cohort of renal transplant recipients following AZ induction. Methods. We retrospectively studied 927 patients [610(65.8%) male, 446(48.1%) living donor, 426(46.0%) caucasian] who received a renal transplant with AZ induction, a tacrolimus based and steroid sparing immunosuppressive regime. Mean follow up was 4.18±2.36 years. Results. Overall patient survival at 1,3 and 5 yrs was 98.3%, 94.9% and 92.5% respectively. Death censored allograft survival was 96.0%, 90.8% and 87.0% at 1,3 and 5yrs and the corresponding rejection free survival was 84.2%, 77.9% and 75.2%. 42/927[4.5%] patients died with a functioning graft.8/927[0.86%] died secondary to cardiac causes, 9[0.97%] from malignancy, 9[0.97%] infection,10 [1.08%] cases were unknown and 6[0.65%] had other defi ned causes. 104/927[11.2%] allografts failed, the causes of which are shown below. Conclusion. Although AZ induction is associated with good patient and allograft survival, rejection remains the leading cause of allograft failure. This is similar to outcomes achieved with other lymphodepleting induction agents such as ATG.
Introduction: Idiopathic pulmonary fibrosis (IPF) is a serious disease with unknown etiology, whe... more Introduction: Idiopathic pulmonary fibrosis (IPF) is a serious disease with unknown etiology, where an influence of cytokine gene polymorphisms is presumed. We compared HRCT alveolar (AS) and interstitial score (IS) and histoptahologic score with IL-4, and IL-4RA gene polymorphisms in IPF patients. Subjects and methods: IPF was diagnosed in 46 patients according to ATS/ERS consensus statement. 43 patients had evaluable HRCT investigations, 14 patients had surgical lung biopsy. HRCT scans were evaluated using IS and AS scales by Gay et al. The histopathological evaluation of lung biopsies comprised: myofibroblast foci (MF), inflammation, eosinophils, granulomas and Ashcroft criteria for fibrosis grading. The IL-4 (-1098) (-590) (-33) and IL-4 RA +1902 gene polymorphisms were characterized utilizing a PCR- SSP method. Results: AS was higher in IL-4 haplotypes 1 TTC and TTT carrierrs (p=0.0423). Ashcroft score was more advanced in IL-4 haplotype 2 GCC (p= 0,013) and MF counts were higher in TCC carriers (p=0,0736). IL-4 RA +1902 A1 G and IL-4 -590 A1 T correlated with higher AS (p=0,0335; p=0,0123). Ashcroft score was higher in IL-4 -1098 A2 G and IL-4 -33 A1 T carriers (p= 0,0443, p=0,0915). Conclusions: We assume that IL-4 and IL-4RA polymorphisms might influence HRCT and histopathological phenotype of IPF. The correlation of functionally relevant IL-4 genes polymorphisms (especially IL-4 -33 T) with AS could mean, that new alveolar lesions with continuing fibrosis are more pronounced in these polymorphisms carriers. The positive correlation of IL-4 -33 A1 T with Ashcroft score might support a hypothesis of fibrogenetic role of IL-4 in IPF.
Objectives: MRP8/14 is a heterodimer of two myeloid calcium-binding proteins associated with diff... more Objectives: MRP8/14 is a heterodimer of two myeloid calcium-binding proteins associated with different types of acute inflammatory processes. We studied MRP8/14 together with procalcitonin (PCT) serum levels in order to diagnose infectious complications or the rejection process affecting kidney or heart allograft. Methods: A total of 419 serum samples was evaluated. MRP8/14 levels were measured by ELISA (BMA Biomed), PCT by a sensitive immunoluminiscent assay ILMA (Brahms Diagn.) Results: Both parameters showed very low basal levels in healthy subjects (range 303-1,660 ng/ml of MRP8/14; less than 0.08 ng/ml of PCT). A rapid increase in serum levels occurred in response to bacterial infections (MRP8/14 up to 6,230 ng/ml; PCT up to 297 ng/ml). Serum PCT concentration remained low in the presence of kidney allograft rejection, where MRP8/14 levels were increased. An uncomplicated outcome of kidney or heart transplantation did not change basal serum MRP8/14 and PCT levels. Conclusions: We conclude that 1) both MRP8/14 and PCT are very sensitive markers of complications in organ transplant recipients (normal values in uncomplicated outcome) 2) combination of both parameters is useful to discriminate between rejection (increased MRP8/14 with normal PCT) and systemic bacterial infection (both parameters increased).
Background: Steroid avoidance in immunosuppression in kidney transplantation offers several metab... more Background: Steroid avoidance in immunosuppression in kidney transplantation offers several metabolic advantages, however it is associated with higher early acute rejection rate. Cellular and molecular mechanisms of this phenomenon remain poorly understood. Methods: In this single center observational study, low-risk kidney transplant recipients randomized into large multicenter prospective ADVANCE trial with steroid avoidance/early withdrawal and center standard of care treated patients were monitored for 12 months. The expressions of 28 transcripts, associated with alloimmune response and operational tolerance, were evaluated in the peripheral blood using RT-qPCR at 0, 7, 14, 90 and 365 postoperative days (POD) and in the protocol graft biopsy at 3-months while lymphocyte subpopulations were analyzed by flow-cytometry within the follow-up. Results: Both steroid avoidance and withdrawal regimens were associated with significantly higher granzyme B (GZMB) transcript at POD 14 and perforin 1 (PRF1) transcript at POD 7. The higher interleukin 2 (IL-2) expression at POD 7 was detected only in the steroid avoidance group. Initial steroids decreased the expression SH2D1B transcript at POD14 and there were no further differences in other operational tolerance transcripts among groups. The statistically significant decrease in absolute numbers of peripheral NK cells in the first 14 days was observed in the standard of care group only. There were no differences in analyzed intrarenal transcripts in 3months biopsies among groups. Conclusions: The enhanced expression of some of Th1 associated transcripts and limited effects on NK cells of steroid avoidance immunosuppression suggest higher susceptibility for early acute rejection.
American Journal of Physiology-lung Cellular and Molecular Physiology, Sep 1, 1997
The airway epithelial cell may play a role as an effector cell, releasing various cytokines and e... more The airway epithelial cell may play a role as an effector cell, releasing various cytokines and extracellular matrix components in immune responses, inflammation, and wound repair processes, thus contributing to cytokine &quot;networks.&quot; The cytokines transforming growth factor (TGF)-beta and interleukin (IL)-4 are though to have pivotal roles in airway diseases, with IL-4 having proinflammatory actions and TGF-beta generally regarded to mediate repair and to attenuate immune responses. In asthma, where IL-4 and TGF-beta are thought to contribute to the inflammatory process and repair, respectively, interactions between these cytokines are likely to be of importance. Therefore, we studied the potential interaction of both cytokines by measuring IL-8 and fibronectin release by cultured human bronchial epithelial cells (HBECs). IL-4 is capable of inducing IL-8 release from HBECs. This effect of IL-4 can be blocked by the concurrent presence of the cytokine TGF-beta. In contrast, TGF-beta had a modest inconsistent stimulatory effect on IL-8 release by itself and had no effect on the IL-8 release induced by tumor necrosis factor (TNF)-alpha. An antagonistic effect of IL-4 and TGF-beta was also observed on HBEC fibronectin release. TGF-beta stimulated fibronectin release, and IL-4 was able to inhibit this. This effect was not due to a redistribution of fibronectin but appeared to be due to a true reduction in synthesis. Consistent with this, IL-4 and TGF-beta effects on IL-8 and fibronectin release were paralleled by changes in mRNA levels. The ability of TGF-beta to block IL-4-induced IL-8 release is certainly not the only mechanism to inhibit IL-8 release because dexamethasone was capable of inhibiting both TNF-alpha- and IL-4-induced release of IL-8. These results indicate that TGF-beta and IL-4 can have mutually inhibitory effects. The balance determined by this reciprocal inhibition may play an important role in regulating inflammation repair and in diseases such as asthma.
Macrolide antibiotics such as azithromycin or clarithromycin are known to have potent antiinflamm... more Macrolide antibiotics such as azithromycin or clarithromycin are known to have potent antiinflammatory and immunomodulatory effects but these properties cannot be widely used due to a risk of bacterial resistance. We studied another polyketide antibiotic, structurally related manumycin A known as a streptomycete derived farnesyltransferase inhibitor with limited antibacterial effects, with respect to its potential regulation of mRNA expression of several genes associated with proinflammatory responses. Downregulation of mRNA for IL-6, TLR-8, IL-1 beta and IL-10 was found in THP-1 cells after 4 h stimulation with TNF alpha in the presence of manumycin A and downregulated TLR-8 and EGR-1 genes were observed after 8 h. Among the genes upregulated in response to manumycin were HMOX-1, TNFRSF10A, IL-1R1, TICAM2, NLRP12 after 4 h and only IL-1R1 after 8 h. Furthermore, manumycin A was found to inhibit IL-1beta, IL-6, and IL-8 production in TNF alpha stimulated THP-1 cells and peripheral blood monocytes in a dose dependent manner (0.25-1 M of manumycin A) without affecting cell viability. Cell viability of blood monocytes decreased by about 30% at manumycin A doses of 2-5 M. Manumycin A also inhibited IL-18 release from THP-1 cells, while in cultures of blood monocytes, this cytokine was not detectable. That manumycin A mediated downregulation of proinflammatory genes in human monocytes confirmed by a measurement of cytokine levels in culture supernatants, together with a very limited effect on cell viability, might suggest potential anti-inflammatory properties of this polyketide antibiotic.
Radiation exposure is known to impair healing in irradiated areas. Fibroblasts play a major role ... more Radiation exposure is known to impair healing in irradiated areas. Fibroblasts play a major role in the production and modification of extracellular matrix in wound repair. Since one important aspect of wound repair is the contraction of the wound, this study investigated the effects of radiation on the ability of fibroblasts to mediate collagen gel contraction in an in vitro model of wound retraction. After irradiation, the cells were detached and suspended in a solution of rat tail tendon collagen. Radiation exposure decreased retraction, and this effect was dose dependent. In order to define the mechanism of reduced gel retraction, we investigated alpha2beta1 cell surface integrin and fibronectin, which are thought to mediate contraction, and prostaglandin E2 (PGE2), which is known to inhibit this process. PGE2 release increased dose responsively following radiation. The cyclooxygenase inhibitor indomethacin could partially restore the contractile activity of irradiated fibroblasts. Fibronectin production in gel culture showed a significant decrease. In contrast, there was no decrease in alpha2beta1 integrin expression in radiated cells. In conclusion, radiation decreases fibroblast-mediated gel contraction. Increased PGE2 production and decreased fibronectin production by irradiated fibroblasts may contribute to this effect and may be in part responsible for poor healing of radiated tissue.
Procalcitonin (PCT) represents a new marker of systemic inflammatory reactions of the body to inf... more Procalcitonin (PCT) represents a new marker of systemic inflammatory reactions of the body to infections. PCT is selectively induced by severe bacterial infections leading to sepsis or multiorgan dysfunction syndrome. The aim of our study was to test PCT as a postoperative infection marker in heart and kidney transplant patients compared with healthy subjects and patients with localized lung-inflammatory processes without a manifest systemic response. PCT concentrations were measured by an immunoluminometric assay (ILMA) in a total of 419 serum samples. Normal serum levels were in the range of 0.08-0.6 ng/ml. Operative trauma associated with heart (not kidney) transplantation induced a transient increase in PCT levels to 7-10 ng/ml with a decline to normal levels within 2-3 days in most patients. Severe bacterial infections dramatically augmented serum PCT concentrations reaching values of 46-297 ng/ml in the most critical periods. Good response to antibiotic therapy was associated with a decline in serum PCT concentrations. Acute rejection or cytomegalovirus infections did not significantly increase the serum PCT levels. Localized pulmonary infections showed either no, or only a limited increase, in the serum PCT levels (max. 7 ng/ml). We conclude from our data that PCT can be used as a sensitive marker to differentiate systemic bacterial infections from other complications in organ transplantation.
Chronic allograft nephropathy (CAN) represents a frequent and irreversible cause of long-term ren... more Chronic allograft nephropathy (CAN) represents a frequent and irreversible cause of long-term renal graft loss. TGF-beta1 is a key profibrogenic cytokine associated with CAN pathogenesis. Because of clinical diagnostic inaccuracy, protocol biopsy has been suggested to be a beneficial method for early CAN detection. Protocol core biopsy was carried out in 67 consecutive cyclosporine-based immunosuppression-treated kidney transplant recipients with stable renal function 12 months after renal transplantation. Biopsy specimens were analyzed morphologically according to Banff-97' criteria and immunohistologically for TGF-beta1 staining. The data obtained were correlated with plasma TGF-beta1 levels and clinical data. CAN (grade I-III) was found in 51 patients (76 %). CAN grade I was found to be the most frequent one (44 %). A normal finding within the graft was made in only 12 patients (18 %). Clinically silent acute rejection Banff IA was present in 4 patients (6 %). In 8 patients (...
Purpose Advanced age is associated with an impaired humoral immune response to SARS-CoV-2 mRNA va... more Purpose Advanced age is associated with an impaired humoral immune response to SARS-CoV-2 mRNA vaccination in kidney transplant recipients (KTR). The mechanisms are, however, poorly understood. Frailty syndrome assessment may determine the most vulnerable population. Methods This study is a secondary analysis of a prospective study (NCT04832841) regarding seroconversion after BNT162b2 vaccination, including 101 SARS-CoV-2 naïve KTR 70 years and older. The Fried frailty components were evaluated, and antibodies against S1 and S2 subunits of SARS-CoV-2 were examined > 14 days after the second dose of BNT162b2 vaccine. Results Seroconversion was observed in 33 KTR. Male gender, eGFR, MMF-free immunosuppression, and a lower frailty score were associated with higher seroconversion rates in univariable regression. Concerning frailty components, physical inactivity had the most negative effect on seroconversion (OR = 0.36, 95% CI 0.14–0.95, p = 0.039). In a multivariable regression adju...
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