Evaluation of monocyte-derived dendritic cells, T regulatory and Th17 cells in chronic myeloid leukemia patients treated with tyrosine kinase inhibitors

Immunotherapy with dendritic cells (DC) may constitute a new and advantageous option for patients with chronic myeloid leukemia (CML) who respond to therapy with tyrosine kinase inhibitors (TKI), but do not reach complete cytogenetic or molecular remission. In this study, we evaluated the immunophenotype of DC generated from monocytes (Mo-DC) of patients with CML and the influence of TKI therapy on the results of CML-DC generation. We also measured the percentages of T regulatory cells (Tregs) as well as Th17 cells in 19 untreated patients suffering from CML, and in 28 CML patients treated with TKI. We found that DC can be reliably generated from the peripheral blood CD14+ cells of untreated CML patients. But we observed a persistent expression of CD14 monocyte marker on DC from CML patients, together with lower percentages of Mo-DC with expression of CD1a (p = 0.002), CD80 (p = 0.0005), CD83 (p = 0.0004), and CD209 (p = 0.02) compared to healthy donors. There was an adverse correlation between WBC count and the percentage of Mo-DC with co-expression of CD80 and CD86 (R = –0.63; p = 0.03). In patients treated with TKI, we observed higher efficacy of DC generation in seven-day cultures, compared to untreated patients. Expression of CD209 on DC was higher in patients treated with TKI (0.02). The duration of TKI therapy correlated adversely with MFI for CD1a (R = –0.49; p = 0.006) and positively with MFI for CD83 (R = 0.63; p = 0.01). Percentages of CD4+CD25highFoxP3+ cells (p = 0.0002) and Th17 cells (p = 0.02) were significantly higher in untreated CML patients compared to healthy controls. There was a significant correlation between the percentage of Treg cells and the percentage of peripheral blood basophiles (R = 0.821; p = 0.02). There were no changes in Tregs or Th17 cell percentages in CML patients after six months of TKI therapy. However, the expression of intracellular IL-17 in Th17 cells correlated negatively with the time of TKI therapy in the whole group of treated patients (R = –0.516; p = 0.04). We noted a correlation between IL-6 serum level and peripheral blood WBC count (R = 0.492; p = 0.04). There was also an inverse correlation between the serum level of IL-6 and the duration of TKI therapy (R = –0.66; p = 0.03). Taken together, our data shows that mature DC can be generated from CML patients treated with TKI, and that the yield of Mo-DC is higher in patients treated with TKI than in patients with active disease. This should encourage further trials with DC immunotherapy in patients with cytogenetic response after TKI therapy. We also found increased frequencies of T regulatory and Th17 cells in CML patients, which might suggest their potential role in immunity against this disease. Further studies are needed to determine if manipulation of these cell populations might improve the results of DC immunotherapy. (Folia Histochemica et Cytobiologica 2011; Vol. 49, No. 1, pp. 153–160

Generation of dendritic cells from peripheral blood monocytes from patients with chronic lymphocytic leukemia using GM-CSF, IL-4 and TNF does not induce their tolerogenic properties

Celem przedstawionych badań była ocena właściwości tolerogennych komórek dendrytycznych generowanych in vitro z monocytów krwi obwodowej chorych na przewlekłą białaczkę limfocytową (PBL) przy użyciu cytokin, takich jak: GM-CSF i IL-4 i TNF. Nie wykazano istotnie większego stężenia tolerogennych cytokin IL-10 i TGF-β w nadsączach znad hodowli dojrzałych komórek dendrytycznych chorych na PBL w porównaniu ze zdrowymi dawcami i ze stężeniem w surowicy chorych. Nie stwierdzono indukcji komórek T-regulatorowych w hodowlach zawierających autologiczne limfocyty T i wygenerowane komórki dendrytyczne. Oceniano ponadto wewnątrzkomórkową ekspresję dioksygenazy 2,3-indoleaminy (IDO) na poziomie białka w wygenerowanych komórkach dendrytycznych. IDO wpływa na hamowanie proliferacji i indukuje śmierć limfocytów efektorowych T oraz powstawanie limfocytów T regulatorowych. Nie wykazano obecności tego enzymu lub jedynie jego śladową ilość w wygenerowanych komórkach dendrytycznych oraz brak korelacji między odsetkiem limfocytów T regulatorowych, a odsetkiem komórek o fenotypie CD83+IDO+. Wyniki uzyskane w badaniach własnych wskazują, że DC generowane za pomocą GM-CSF, IL-4 i TNF z monocytów krwi obwodowej chorych na PBL nie mają właściwości tolerogennych.The aim of this study was to evaluate the tolerogenic properties of dendritic cells generated in vitro from peripheral blood monocytes in patients with chronic lymphocytic leukemia (CLL) using cytokines such as GM-CSF, IL-4 and TNF. There were no significant differences in the concentration of tolerogenic cytokines, IL-10 and TGF-β in supernatants from cultures of mature dendritic cells of CLL patients and healthy donors and the concentration of mentioned cytokines in the serum of CLL patients. There was no induction of regulatory T cells in cultures containing autologous T cells and dendritic cells generated from CLL patients. We have also evaluated the intracellular expression of indoleamine 2,3-dioxygenase (IDO) protein in generated dendritic cells. IDO inhibits proliferation and induces death of effector T cells, and induces the formation of regulatory T cells. There was no or only a trace amount of this enzyme in generated dendritic cells and the no correlation between the percentage of T regulatory cells, and the percentage of cells with phenotype CD83+IDO+. The results obtained in our study show that DC generated with GM-CSF, IL-4 and TNF have no tolerogenic properties

Treatment of Graves’ disease with methimazole in children alters the proliferation of Treg cells and CD3+ T lymphocytes

Almost all cases of hyperthyroidism in children result from Graves’ disease (GD). Recent studies have confirmed a significant role of T regulatory cells (Tregs) in the development of autoimmune diseases. However, the interactions between T cell responses and Treg proliferation in GD are still poorly understood. The aim of this study was to assess the proliferation of Treg cells (Tregs) and CD3+ T lymphocytes isolated from 50 children with GD before and after treatment with the thyreostatic drug methimazole (MMI). The proliferation rates, measured by methyl-3H-thymidyne incorporation, of CD3+ cells and Tregs stimulated with mitogen phorbol 12-myristate 13-acetate (PMA) were compared with those of unstimulated cells. The proliferation rates of both PMA-stimulated and unstimulated CD3+ cells prior to treatment with MMI were significantly higher than after treatment. Simultaneously, the proliferation rates of both PMA-stimulated and unstimulated Tregs were significantly lower before MMI treatment. Moreover, we observed higher cell proliferation rates of unstimulated and PMA-stimulated Tregs before the initiation of MMI therapy and after treatment in patients who had no relapse of hyperthyroidism. There was a positive correlation between the CD3+ cells proliferation rate before MMI treatment and fT3, as well as fT4 concentration in peripheral blood. The proliferation rates of CD3+ T cells before and after MMI treatment positively correlated with the TSI index. Thus, children suffering from Graves’ disease presented lower Tregs proliferative potential compared with CD3+ T cells. Cocultures of CD3+ T cells and Tregs showed that Tregs were not capable of efficiently inhibiting the proliferation of CD3+ T cells in GD patients. Conclusions. MMI treatment reduced the proliferative activity of CD3+ T cells in pediatric GD patients and increased the proliferation rate of Tregs. We suggest that Treg cells that are partly dysfunctional in GD disease are probably suppressed by CD3+ T cells and that methimazole exerts some immunomodulatory effects. (Folia Histochemica et Cytobiologica 2014, Vol. 52, No. 1, 69–77

Dendritic cells based immunotherapy of patient with chondrosarcoma--case report.

We present a case report of patient with intracranial chondrosarcoma and attempt to use vaccination of dendritic cells as the salvage therapy. To our knowledge, this is the first case report of DCs vaccination in the head and neck chondrosarcoma. Immunotherapy with allogeneic DCs stimulated with tumor cell lysates in this case was demonstrated to be feasible, safe and well tolerated. Unfortunately we did not observe any clinical or immune response during vaccination. CD4+ and CD8+ regulatory cells could be responsible for ineffectiveness of immunotherapy

CD3+/CD16+CD56+ cell numbers in peripheral blood are correlated with higher tumor burden in patients with diffuse large B-cell lymphoma

Diffuse large B-cell lymphoma is the commonest histological type of malignant lymphoma, andremains incurable in many cases. Developing more efficient immunotherapy strategies will require betterunderstanding of the disorders of immune responses in cancer patients. NKT (natural killer-like T) cells wereoriginally described as a unique population of T cells with the co-expression of NK cell markers. Apart fromtheir role in protecting against microbial pathogens and controlling autoimmune diseases, NKT cells havebeen recently revealed as one of the key players in the immune responses against tumors. The objective of thisstudy was to evaluate the frequency of CD3+/CD16+CD56+ cells in the peripheral blood of 28 diffuse largeB-cell lymphoma (DLBCL) patients in correlation with clinical and laboratory parameters. Median percentagesof CD3+/CD16+CD56+ were significantly lower in patients with DLBCL compared to healthy donors(7.37% vs. 9.01%, p = 0.01; 4.60% vs. 5.81%, p = 0.03), although there were no differences in absolute counts.The frequency and the absolute numbers of CD3+/CD16+CD56+ cells were lower in advanced clinical stagesthan in earlier ones. The median percentage of CD3+/CD16+CD56+ cells in patients in Ann Arbor stages 1–2 was5.55% vs. 3.15% in stages 3–4 (p = 0.02), with median absolute counts respectively 0.26 G/L vs. 0.41 G/L (p == 0.02). The percentage and absolute numbers of CD3+/CD16+CD56+ cells were significantly higher in DL-BCL patients without B-symptoms compared to the patients with B-symptoms, (5.51% vs. 2.46%, p = 0.04;0.21 G/L vs. 0.44 G/L, p = 0.04). The percentage of CD3+/CD16+CD56+ cells correlated adversely with serumlactate dehydrogenase (R= –445; p < 0.05) which might influence NKT count. These figures suggest a relationshipbetween higher tumor burden and more aggressive disease and decreased NKT numbers. But it remains tobe explained whether low NKT cell counts in the peripheral blood of patients with DLBCL are the result of theirsuppression by the tumor cells, or their migration to affected lymph nodes or organs

Ocena stężenia erytromycyny w surowicy krwi z tętnicy pępowinowej

Objectives: The aim of the study was to investigate the effectiveness of erythromycin in preventing intrauterine infection caused by group B streptococcus (GBS). Material and methods: The study included 20 pregnant women with GBS-positive screening or whose laboratory screening was not available, who delivered between April 17, 2013 and July 22, 2013. The women were given 600 mg of erythromycin intravenously. After delivery, blood was drawn in parallel from maternal antecubital vein and umbilical cord artery. Serum erythromycin concentrations were evaluated using enzyme-linked immunosorbent assay (ELISA) kit. Statistical analysis for measurable and non-measurable characteristics were performed, correlation coefficients for each pair of variables were calculated in order to investigate the sought dependence. Results: Mean placental transfer of erythromycin was 2.04%. There was a high correlation between umbilical artery serum and maternal serum erythromycin concentration. Selected variables of mothers in the control group had no effect on serum erythromycin concentration in the umbilical artery. Conclusions: Transplacental transfer of erythromycin is limited (2.04%). Intravenous application of erythromycin at a dose of 600 mg does not allow to achieve the value of MIC50 and MIC90 for erythromycin against strains S. agalactiae in umbilical artery serum, what suggests a compromised efficacy in the treatment of intrauterine fetal infections. At the same time, the placenta seems to be an effective barrier reducing fetal exposure when this macrolide is used to treat maternal infections.Cel: Celem badania była ocena skuteczności erytromycyny w zapobieganiu infekcji wewnątrzmacicznych wywołanych obecnością paciorkowców z grupy B (GBS). Materiał i metody: Badaniem objęto 20 kobiet ciężarnych, z pozytywnym wynikiem badań na nosicielsto GBS lub dla których wyniki badań nie były znane. Porody odbywały się w okresie od 17 kwietnia 2013 roku do 22 lipca 2013 roku. Pacjentkom podawano dożylnie 600 mg erytromycyny. Po porodzie równolegle pobierano próbki krwi z tętnicy pępowinowej oraz od matki z żyły łokciowej. Poziom stężenia erytromycyny oceniano z użyciem testu immunoenzymatycznego (ELISA). Przeprowadzono analizy statystyczne cech mierzalnych i niemierzalnych, obliczono współczynniki korelacji dla poszczególnych par zmiennych w celu zbadania poszukiwanych zależności. Wyniki: Przezłożyskowy transport erytromycyny w badanej grupie wynosił średnio 2,04%. Zaobserwowano wysoką korelację między poziomem stężenia erytromycyny w surowicy krwi matki i w tętnicy pępowinowej. Wybrane zmienne opisujące matkę nie miały wpływu na poziom stężenia erytromycyny w tętnicy pępowinowej. Wnioski: Erytromycyna w ograniczonym stopniu przechodzi przez łożysko (2,04%). Podanie leku w dawce 600 mg nie pozwala na osiagnięcie wartości MIC50 i MIC90 erytromycyny wobec szczepów S. agalactiae w surowicy krwi z tętnicy pępowinowej. Sugeruje to małą skuteczność badanego antybiotyku w leczeniu infekcji wewnątrzmacicznych płodu. Jednocześnie łożysko stanowi skuteczną ochronę płodu, gdy celem jest leczenie infekcji występującej jedynie u matki

A patient with the hepatorenal syndrome treated with a liver transplant — case description with duties of the nursing staff

Transplantacja wątroby (LTx) jest leczeniem z wyboru w przypadku zespołu wątrobowo-nerkowego (HRS). Pacjenci oczekujący na przeszczep w stanie zdekompensowanej marskości wątroby z niewydolnością nerek stanowią grupę potencjalnych biorców wysokiego ryzyka rozwoju poważnych powikłań i zgonu w okresie oczekiwania na przeszczep. W pracy przedstawiono przypadek pacjenta z niewydolną marską wątrobą w przebiegu toksycznego jej uszkodzenia, u którego doszło do rozwoju HRS typu I. Chorego zakwalifikowano do pilnego przeszczepu wątroby. W okresie oczekiwania na zabieg poddany był hemodializom. Po transplantacji nastąpiła normalizacja parametrów wątrobowych i nerkowych. W dalszym okresie potransplantacyjnym stężenie kreatyniny obniżyło się do 0,6–1,0 mg/dl. U chorego w okresie oczekiwania na transplantację dominowały problemy wynikające z marskości wątroby oraz niewydolności nerek: żółtaczka, wodobrzusze, obrzęki kończyn dolnych, wyniszczenie, encefalopatia, zaburzenia krzepnięcia, zaburzenia hemodynamiczne, bezmocz. Zadania podejmowane przez personel pielęgniarski zależały od zmieniającego się stanu zdrowia pacjenta i stosowaną terapią mającą na celu utrzymanie go przy życiu i zapewnienie mu opieki do czasu uzyskania przeszczepu.Liver transplantation (LTx) is the first-line treatment for hepatorenal syndrome (HRS). Patients awaiting LTx with decompensated cirrhosis and renal failure are a group of potential recipients with a high risk of developing serious complications and death in the period of expecting for a transplant. The work presents the case of a patient with cirrhosis and a failing liver due to toxic-induced damage; the patient developed HRS type 1. The patient required urgent LTx. During the waiting period s/he undergone haemodialyses. After LTx, there was a normalization in the hepatic and renal parameters. In the ensuing post-transplantation period, the concentration of creatine went down to 0,6–1,0 mg/dl. During waiting for treatment, the patient suffered chiefly from conditions related to cirrhosis and renal failure: jaundice, abdominal dropsy, edema of lower extremity, emaciation, encephalopathy, coagulation disorder, hemodynamic disorder, and anuria. The activities of medical personnel were subject to the patient’s changing condition and employed therapy that was to keep the patient alive and provide safety until the transplantation

Comparison of Selected Immune Parameters in a Single Infection and Co-Infection with Infectious Pancreatic Necrosis Virus with Other Viruses in Rainbow Trout

Infectious pancreatic necrosis virus (IPNV) often occurs in an aquatic environment in co-infection with other viruses. In this study, we wanted to investigate the effect of this virus on the course of co-infection with other viruses in rainbow trout. For co-infection we used viral hemorrhagic septicemia virus (VHSV), infectious hematopoietic necrosis virus (IHNV) and salmonid alphavirus (SAV) field strains and infected rainbow trout divided into eight groups; I; IPNV, II; IHNV, III; VHSV, I; SAV, V; IPNV+IHNV, VI; IPNV+VHSV, VII; IPNV+SAV, and the control group. We assessed apoptosis in white blood cells and used a real time RT-PCR to analyze RNA obtained from the internal organs of the fish. During single infection and co-infection the level of expression of immune genes such as interferon and toll-like receptor 3 (TLR-3) was assessed. The highest mortality during the experiment was observed in group III infected by VHSV. The average percentage of apoptotic cells was higher in groups without co-infection, especially in groups II and III. Interferon expression was higher in singly infected groups, the highest being in the heart in group III, while expression of the TLR-3 gene was generally raised in all tested organs in all groups. We found that co-infection with IPNV had a positive impact on the course of infection with the viruses listed because it lowered mortality, reduced apoptosis in co-infected cells, and positively affected fish health

The Effect of Pegbovigrastim Injection on Phagocytic and Oxidative Burst Activities of Peripheral Blood Granulocytes and Monocytes in Calves Challenged with Mycoplasma bovis

Mycoplasma bovis (M. bovis) is an important pathogen affecting cattle, causing various diseases including pneumonia which mainly occurring in calves. Control of M. bovis infections is difficult due to the lack of commercial vaccines in most parts of the world and increasing trends of antimicrobial resistance in field isolates of the pathogen; therefore, it seems reasonable to look for new solutions for the prevention of the infection. Pegbovigrastim is a pegylated form of naturally occurring circulating cytokine in cattle that affects bovine leukocytes and some cell functions. Most studies on pegbovigrastim have focused on reducing the occurrence of mastitis and other diseases occurring during the periparturient period in cows, while this study attempts to use pegbovigrastim in the prevention of respiratory diseases in calves, which are largely caused by M. bovis. Based on previous observations on the immunostimulatory properties of pegbovigrastim in cattle, for the first time, the effect of its injection on the number and phagocytic and oxidative burst activities of peripheral blood granulocytes and monocytes in calves experimentally infected with M. bovis was investigated. Pegbovigrastim administration in the calves significantly stimulated an increase in peripheral blood granulocyte and monocyte counts and phagocytic activity of the cells, especially granulocytes, which was also generally expressed in the course of M. bovis infection. In response to pegbovigrastim administration, a general increase in the oxygen burst activity of the cells was observed. This effect was also shown despite ongoing infection with M. bovis which, taken together, may indicate a beneficial effect of pegbovigrastim injection on the immunity of the affected animals

Protein-Coding Region Derived Small RNA in Exosomes from Influenza A Virus–Infected Cells

Exosomes may function as multifactorial mediators of cell-to-cell communication, playing crucial roles in both physiological and pathological processes. Exosomes released from virus-infected cells may contain RNA and proteins facilitating infection spread. The purpose of our study was to analyze how the small RNA content of exosomes is affected by infection with the influenza A virus (IAV). Exosomes were isolated by ultracentrifugation after hemadsorption of virions and their small RNA content was identified using high-throughput sequencing. As compared to mock-infected controls, 856 RNA transcripts were significantly differentially expressed in exosomes from IAV-infected cells, including fragments of 458 protein-coding (pcRNA), 336 small, 28 long intergenic non-coding RNA transcripts, and 33 pseudogene transcripts. Upregulated pcRNA species corresponded mainly to proteins associated with translation and antiviral response, and the most upregulated among them were RSAD2, CCDC141 and IFIT2. Downregulated pcRNA species corresponded to proteins associated with the cell cycle and DNA packaging. Analysis of differentially expressed pseudogenes showed that in most cases, an increase in the transcription level of pseudogenes was correlated with an increase in their parental genes. Although the role of exosome RNA in IAV infection remains undefined, the biological processes identified based on the corresponding proteins may indicate the roles of some of its parts in IAV replication