Extracellular vesicles in cattle infected with bovine leukaemia virus: isolation and molecular analysis.

Introduction: Exosomes are nanosized lipid bilayer membranous microvesicles, extracellularly released from a variety of mammalian cells. They mediate intercellular signalling by transporting several types of RNA, lipids and proteins and participate in the intercellular exchange of DNA, RNA, micro RNA, proteins and other components. These microvesicles are present in all body fluids in physiological and pathological conditions and reflect the state of the host organism. The aim of the study was the isolation and molecular determination of exosomes in blood and supernatant fluids of bovine dendritic cell cultures infected with bovine leukaemia virus (BLV). Material and Methods: Exosomes were isolated by ultracentrifugation from the blood sera, plasma and supernatant of bovine BLV-infected and uninfected control dendritic cell cultures and their presence was confirmed with scanning electron and transmission electron microscopy. Western blot analysis of the structural BLV glycoprotein 51 (Env) and protein 24 (Gag) and of the tetraspanin exosomal markers CD9, CD63 and flotillin-1 was undertaken in BLV+ and control BLV- cattle. Results: In exosomes of leukaemic cattle both BLV proteins and exosomal markers were detected. In healthy control animals only exosomal markers were determined. Conclusion: Proteins of BLV were released with exosomes and could be transferred into recipient cells as an alternative propagation route not requiring virus infection.

Etiology of Ulcerative Dermal Necrosis (UDN) in Brown Trout (Salmo trutta Morpha trutta)-Preliminary Results.

Every year, ulcerative dermal necrosis (UDN) affects salmonids that spend most of their lives in the sea during their migration to the rivers of northern Poland to spawn. The clinical form of the disease manifests itself in ulcerative skin lesions, which lead to significant weakening of the fish and, in most cases, result in their death. This study was carried out on samples taken from sea trout in the Slupia River in northern Poland. In order to identify the pathogen, experiments on the transmission of the disease were carried out, and additional histopathological, microbiological and electron microscopic examinations were performed. As a result of these studies, it was possible to experimentally transfer the disease from sick to healthy fish. The results indicate a complex etiology of the disease (lack of a clearly defined pathogen), in which the change in the environment from salty to freshwater triggers the related changes in skin physiology, which are the main causes of increased susceptibility to the development of the disease.

The Role of Bcl-2 and Beclin-1 Complex in "Switching" between Apoptosis and Autophagy in Human Glioma Cells upon LY294002 and Sorafenib Treatment.

BACKGROUND: Gliomas are the most malignant tumors of the central nervous system. One of the factors in their high drug resistance is avoiding programmed death (PCD) induction. This is related to the overexpression of intracellular survival pathways: PI3K-Akt/PKB-mTOR and Ras-Raf-MEK-ERK. Apoptosis and autophagy are co-existing processes due to the interactions between Bcl-2 and beclin-1 proteins. Their complex may be a molecular "toggle-switch" between PCD types. The aim of this research was to investigate the role of Bcl-2:beclin-1 complex in glioma cell elimination through the combined action of LY294002 and sorafenib. METHODS: Drug cytotoxicity was estimated with an MTT test. The type of cell death was evaluated using variant microscopy techniques (fluorochrome staining, immunocytochemistry, and transmission electron microscopy), as well as the Bcl-2:beclin-1 complex formation and protein localization. Molecular analysis of PCD indicators was conducted through immunoblotting, immunoprecipitation, and ELISA testing. SiRNA was used to block Bcl-2 and beclin-1 expression. RESULTS: The results showed the inhibitors used in simultaneous application resulted in Bcl-2:beclin-1 complex formation and apoptosis becoming dominant. This was accompanied by changes in the location of the tested proteins. CONCLUSIONS: "Switching" between apoptosis and autophagy using PI3K and Raf inhibitors with Bcl-2:beclin-1 complex formation opens new therapeutic perspectives against gliomas.

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.

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.

Effect of Supplementation with the Combination of Se-Enriched Lentinula edodes Mycelium, Exogenous Enzymes, Acidifiers, Sodium Butyrate and Silicon Dioxide Nanoparticle Feed Additives on Selected Parameters in Calves.

During the initial months of calves' lives, the young animals are exposed to bacterial and viral infections, and during this period, crucial physiological changes take place in their organisms. Offering calves feed additives that will have a beneficial influence on their organisms and improve their growth while reducing the morbidity rate is the optimal task of feeding. This is the first study to investigate the effect of experimental supplementation for calves with the combination of two feed additives­one containing Lentinula edodes enriched with selenium (Se), and the second containing pancreatic-like enzymes, fat-coated organic acids, sodium butyrate, and silicon dioxide nanoparticles­on the serum Se concentration, selected immune parameters, and the average daily gains in the calves. During the study, the serum Se concentration was examined by means of inductively coupled plasma mass spectrometry, and the immunoglobulin and cytokine concentrations with ELISA assays. The white blood cell (WBC) count with leukocyte differentiation was examined with the use of a hematological analyzer, and the percentages of subpopulations of T lymphocytes and monocytes, phagocytic activity, and oxidative burst of monocytes and granulocytes with the use of a flow cytometer. The average daily gains of the calves were also evaluated. In summary, the supplementation of the experimental calves with the combination of two feed additives resulted in significantly higher serum Se concentrations, and the immune systems of the calves were not suppressed while the examined feed additives were being delivered. Although not statistically significant, some positive effects on the calves were seen: a tendency towards the improvement of some of the immune parameters evaluated, and a tendency for higher average daily gains in the calves.

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.

Alaria alata in Terms of Risks to Consumers' Health.

Alaria alata flukes are cosmopolitan parasites. In Europe, the definitive hosts are red foxes (Vulpes vulpes), wolves (Canis lupus), and raccoon dogs (Nyctereutes procyonoides), as well as animals that belong to the Felidae family. Intermediate hosts, such as snails and frogs, are the sources of infection for definitive hosts. The developmental stages of A. alata mesocercariae may occur in paratenic hosts, including many species of mammals, birds, and reptiles, as well as in wild boars (Sus scrofa), which are important from the zoonotic point of view. Because there are no regulations concerning the detection of A. alata in meat, this fluke is usually detected during official obligatory Trichinella spp. inspections. However, a method dedicated to A. alata detection was developed. The growing popularity of game and organic meat has led to an increased risk of food-associated parasitic infections, including alariosis, which is caused by the mesocercarial stage of A. alata. The aim of this article is to highlight the problem of A. alata as an emerging parasite, especially in the terms of the increasing market for game and organic meats that have been processed with traditional methods, often without proper heat treatment.

Involvement of PI3K Pathway in Glioma Cell Resistance to Temozolomide Treatment.

The aim of the study was to investigate the anticancer potential of LY294002 (PI3K inhibitor) and temozolomide using glioblastoma multiforme (T98G) and anaplastic astrocytoma (MOGGCCM) cells. Apoptosis, autophagy, necrosis, and granules in the cytoplasm were identified microscopically (fluorescence and electron microscopes). The mitochondrial membrane potential was studied by flow cytometry. The activity of caspases 3, 8, and 9 and Akt was evaluated fluorometrically, while the expression of Beclin 1, PI3K, Akt, mTOR, caspase 12, and Hsp27 was determined by immunoblotting. SiRNA was used to block Hsp27 and PI3K expression. Cell migration and localization of Hsp27 were tested with the wound healing assay and immunocytochemistry, respectively. LY294002 effectively diminished the migratory potential and increased programmed death of T98G and MOGGCCM. Autophagy was dominant in MOGGCCM, while apoptosis was dominant in T98G. LY294002 with temozolomide did not potentiate cell death but redirected autophagy toward apoptosis, which was correlated with ER stress. A similar effect was observed after blocking PI3K expression with siRNA. Transfection with Hsp27 siRNA significantly increased apoptosis related to ER stress. Our results indicate that inhibition of the PI3K/Akt/mTOR pathway sensitizes glioma cells to apoptosis upon temozolomide treatment, which was correlated with ER stress. Hsp27 increases the resistance of glioma cells to cell death upon temozolomide treatment.

Blood Counts, Biochemical Parameters, Inflammatory, and Immune Responses in Pigs Infected Experimentally with the African Swine Fever Virus Isolate Pol18_28298_O111.

This study aimed to indicate the influence of infection caused by genotype II African swine fever virus (ASFV)-isolate Pol18_28298_O111, currently circulating in Poland, on blood counts, biochemical parameters, as well as inflammatory and immune responses. Blood and sera collected from 21 domestic pigs infected intranasally with different doses of virulent ASFV were analysed. The infection led to variable changes in blood counts depending on the stage of the disease with a tendency towards leukopenia and thrombocytopenia. The elevated C-reactive protein (CRP) concentrations and microscopic lesions in organs confirmed the development of the inflammation process, which also resulted in an increased level of biochemical markers such as: Aspartate transaminase (AST), creatine kinase (CK), creatinine, and urea. Antibodies could be detected from 9 to 18 days post infection (dpi). Two survivors presented the highest titer of antibodies (>5 log10/mL) with a simultaneous increase in the lymphocyte T (CD3+) percentage-revealed by flow cytometry. Results confirmed a progressive inflammatory process occurring during the ASFV infection, which may lead to multiple organs failure and death of the majority of affected animals.

The percentage of iNKT cells among other immune cells at various clinical stages of laryngeal cancer.

INTRODUCTION: Invariant natural killer T (iNKT) cells constitute a small population of immune cells that share functional and phenotypic characteristics of T lymphocytes and NK cells. Due to their involvement in specific and non-specific immune responses, iNKT cells may represent an important component of antitumor and anti-infectious immunity. MATERIAL AND METHODS: Using flow cytometry, we analyzed the percentages of iNKT cells as well as T and B lymphocytes in peripheral blood of 50 laryngeal cancer patients at various clinical stages in comparison to healthy controls (n=15). Moreover, we determined the expression of CD25, CD69 and CD95 antigens on T lymphocytes. RESULTS: The percentage of CD4+/CD3+ T lymphocytes in the controls was higher than in laryngeal cancer patients, both with early and late stages of the disease. The percentage of CD8+/CD3+ T lymphocytes in healthy controls was lower than in patients with early and late clinical stages of laryngeal cancer. Patients with advanced laryngeal cancer showed a lower percentage of iNKT cells and higher frequencies of T regulatory cells (Tregs) than the controls. Advanced clinical stages of laryngeal cancer are associated with impaired activation of lymphocytes. CONCLUSIONS: Our study confirmed that laryngeal cancer cells exert a strong suppressor effect on the immune system of the host. This is reflected by a decrease in the percentage of iNKT cells that are capable of cancer cell elimination, and a concomitant increase in the percentage of Tregs. However, further studies are needed in order to explain the underlying mechanisms of immunosuppression and understand interactions between immune and cancer cells.

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.

Expression of selected regulatory molecules on the CD83+ monocyte-derived dendritic cells generated from patients with laryngeal cancer and their clinical significance.

B7H1 and B7H4 overexpression is associated with inhibition of the immune system in many solid tumors, and altogether with CD200 molecule plays an important role in tumor invasion by promoting malignant transformation. However, there is no report about impact of these molecules on laryngeal squamous cell carcinoma. The objective of the present study was to assess by means of flow cytometry the expression of B7H1, B7H4, CD200, and CD200R on CD83+ monocyte-derived dendritic cells (Mo-DC), pulsed with autologous tumor cell lysates (aTCL) in patients who suffer from G1, G2, or G3 laryngeal carcinoma (LC, n = 60) in comparison to healthy donors (HD, n = 15). It has been demonstrated that median value of the percentages of CD83+ B7H1+, CD83+ B7H4+, and CD83+ CD200+ cells were higher in LC patients than HD (p = 0.041, p ≤ 0.0001, and p = 0.02, respectively). Mean fluorescence intensity (MFI) of CD200, CD200R, B7H1, and B7H4 on the Mo-DC pulsed with aTCL of the patients was also higher than on the Mo-DC of HD (p ≤ 0.0001, p ≤ 0.0001, p = 0.002, and p ≤ 0.0001, respectively). The highest MFI levels of all molecules were noted in grade 3 LC. The aforementioned results prove that there is a relation between the presence of laryngeal cancer and the expression of B7H1, B7H4, CD200, and CD200R regulatory molecules on the CD83+ Mo-DC pulsed with autologous cancer cell lysates. Strong association of LC grade and the tested antigens expression suggests a critical role for these proteins in LC biology.

[The assessment of selected molecules belonging to B7 family on the mature dendritic cells in laryngeal cancer patients]. / Ocena ekspresji wybranych molekul z rodziny B7 na powierzchni dojrzalych komórek dendrytycznych u chorych na raka krtani.

UNLABELLED: B7-H1, B7-H2 and B7-H4 molecules play various roles in the adaptive immune system and are broadly expressed on many cells, especially those localized in cancer tissue. The aim of the study was to assess the surface expression of B7-H1, B7-H2 and B7-H4 molecules on the mature dendritic cells (DC) generated from peripheral blood monocytes of laryngeal cancer patients and healthy donors. MATERIAL AND METHODS: Forty-four male patients treated surgically for squamous cell carcinoma of the larynx were included in the study. Peripheral blood from twelve healthy male donors was used as a control. Mononuclear cells were separated from all individuals by density gradient centrifugation, incubated with anti-CD14 microbeads, and passed through MACS separation columns. The CD14 positive cell population was used to prepare monocyte derived DC. Laryngeal cancer tissue was obtained from patients during surgical treatment and homogenized to prepare tumor cell lysates for further stimulation. We evaluated with the use of flow cytometry method the percentage of cells with an expression and mean fluorescent intensity (MFI) of surface markers, such as: CD83, B7-H1, B7-H2, B7-H4. RESULTS: Our study revealed that the percentage of mature dendritic cells, stimulated with autologous tumor cell lysates, with the expression of B7-H1 molecule in patients was lower than in healthy donors (61.81 ± 25.58% vs 93.02 ± 4.63%, p=0.007). In laryngeal cancer patients, the percentage of CD83+/B7-H2+ cells was higher than in healthy individuals (18.32 ± 10.74% vs 2.89 ± 0.43%, p=0.019). CONCLUSIONS: There is a relation between the presence of laryngeal cancer and the expression of B7 family molecules.

Standard B presentation vs. contrast-enhanced ultrasound (US-CE). A comparison of usefulness of different ultrasonographic techniques in the evaluation of the echo structure and size of haematomas inpost-renal transplant patients: A preliminary report.

BACKGROUND: During routine ultrasonographic examination in B presentation, performed as a standard diagnostic procedure during the early post-operative period, the most important problem with the interpretation of the images of perirenal haematoma is their ability to change in time. The aim of this work was to assess the echogenicity and the size of perirenal haematomas in patients after kidney transplant during routine examinations in B presentation and during examinations enhanced with a contrast medium (CE-US). MATERIAL/METHODS: Thirty-seven patients after kidney transplant were examined using standard examination in B presentation. Sixteen patients (7 women and 9 men) with isoechogenic and hypoechogenic areas visualized within the renal parenchyma, who were suspected of perirenal haematoma, underwent a CE-US examination after intravenous administration of sculpture hexafluoride (dose: 2.4 ml/examination). Using time-intensity curves (TIC), changes in the values were analysed for two areas of interest (ROI): in the renal parenchyma and in the areas identified during standard US as haematomas. Identical examination protocols and dynamic data loops allowed the acquisition of identical kidney cross-sections and enabled measuring the echogenicity and thickness of the abnormalities at the same location. RESULTS: During the routine B presentation examination, the average difference between haematoma and the renal cortex was 5 dB. When performing US-CE examination, a significantly greater difference in echogenicity was observed and reached 31 dB. In six patients, the size of haematomas was comparable using both techniques, whereas in ten patients lesions visualized in B presentation were smaller than in the US-CE examination. CONCLUSIONS: The US-CE examination demonstrated a greater, statistically significant, difference in the echogenicity of perirenal haematomas compared to the routine examination in B presentation. This method enabled a more detailed assessment of the size of haematomas in the perirenal space that appeared during early post-operative period.

[The evaluation of apoptosis in peripheral blood lymphocytes in laryngeal cancer patients]. / Ocena apoptozy limfocytów krwi obwodowej u chorych leczonych z powodu raka krtani.

UNLABELLED: The laryngeal cancer is the most often cancer among others in head and neck region. It occurs mostly among 55 and 69. Its development depends on immunological state of the body. Vitality of the immunological system cells was considered due to growth, treatment sensitivity and prognosis of some neoplasms. The aim of this work were estimation and comparison the phenomenon of lymphocytes T and B apoptosis in laryngeal cancer patients treated with surgery and radiotherapy. MATERIAL AND METHODS: The material were 30 patients hospitalized in The Department of Otolaryngology Medical University of Lublin. They all were treated with surgery or surgery and radiotherapy. Apoptosis was estimated on different stages of the treatment process. All samples were examined with the flow cytometry method. The control group were 21 patients hospitalized because of the suspicion of the apnea syndrome, which wasn't confirmed with polysomnographic examination. RESULTS: Results of this study show significantly increasing percentage of peripheral blood apoptotic B (CD19+) cells caused by surgical treatment. The results considering radiotherapy showed different influence on the phenomenon of immunological cells apoptosis, still those results weren't significant. CONCLUSIONS: The surgical treatment causes increased amount of apoptotic peripheral blood lymphocytes.

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.

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, and remains incurable in many cases. Developing more efficient immunotherapy strategies will require better understanding of the disorders of immune responses in cancer patients. NKT (natural killer-like T) cells were originally described as a unique population of T cells with the co-expression of NK cell markers. Apart from their role in protecting against microbial pathogens and controlling autoimmune diseases, NKT cells have been recently revealed as one of the key players in the immune responses against tumors. The objective of this study was to evaluate the frequency of CD3(+)/CD16(+)CD56(+) cells in the peripheral blood of 28 diffuse large B-cell lymphoma (DLBCL) patients in correlation with clinical and laboratory parameters. Median percentages of 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 stages than in earlier ones. The median percentage of CD3(+)/CD16(+)CD56(+) cells in patients in Ann Arbor stages 1-2 was 5.55% 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 serum lactate dehydrogenase (R= -445; p 〈 0.05) which might influence NKT count. These figures suggest a relationship between higher tumor burden and more aggressive disease and decreased NKT numbers. But it remains to be explained whether low NKT cell counts in the peripheral blood of patients with DLBCL are the result of their suppression by the tumor cells, or their migration to affected lymph nodes or organs.

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.