[Show abstract][Hide abstract] ABSTRACT: Sterically stabilized DPPC:CHOL:DSPE-PEG-2000 liposomal formulations of the lipophilic complexes of salinomycin with Na(I), K(I), Mn(II), Co(II), and Ni(II) ions were prepared by film-hydration method at different drug-to-DPPC molar ratios. For the K(I) and Na(I) complexes, optimal loading was established at a drug-to-DPPC molar ratio of 0.5:1, whereas for the Me(II) complexes, it was encountered at 0.1:1. DLS revealed uniform LUV populations (130-160 nm) with monomodal size distribution, further corroborated by AFM. Free and entrapped salinomycinates exhibited cytotoxicity in three human tumor cell lines, whereby the liposomal agents were superior vs. free complexes. DNA-fragmentation and flow cytometric assays showed that the cytotoxicity of free and liposomal salinomycinates is mediated by the induction of apoptosis and G1 arrest. The ability of the carriers to retain the bio-activity of the entrapped cargo gives us reason to conclude that the presented DPPC:CHOL:DSPE-PEG-2000 liposomes are suitable platforms for the salinomycin complexes, needing further evaluation and optimization.
Full-text · Article · Dec 2013 · Journal of Drug Delivery Science and Technology
[Show abstract][Hide abstract] ABSTRACT: House dust mites Dermatophagoides pteronyssinus (Dpt) are among the most frequent causes of allergy symptoms in Europe. Der p 1 is one of the major allergenic compounds of Dpt and the pathological Der p 1-specific B cells play a key role as producers of allergen-binding antibodies. The selective elimination of these cells by artificial protein molecules which inhibit the production of Dpt-recognizing IgE antibodies is a perspective therapeutic goal of allergy. A protein engineered chimeric molecule has been constructed, which binds Der p 1-specific B cells via their BCR and suppresses selectively the production of anti-Der p 1 antibodies via CR1. The synthetic peptide Der p 1 p52-71 and an anti-CD35 monoclonal antibody were used for the construction of Der p 1 chimera. The functional effects of engineered antibodies were analyzed in vitro using PBMCs from allergy patients. Significant inhibition of allergen-specific proliferation and reduction of Der p 1-IgE antibody production were observed after treatment of PBMCs from allergic patients with Der p 1-peptide chimera. Culturing of these PBMCs in the presence of the chimeric molecule increased the percentage of apoptotic (Annexin V-positive) B lymphocytes, but not T lymphocytes.
[Show abstract][Hide abstract] ABSTRACT: A total of 50 patients with chronic lymphocytic leukaemia (CLL), as well as the B-cell leukaemia cell lines MEC-1, JVM-3, and BV-173 were studied in order to assess the incidence of CD13/aminopeptidase N (APN) immunolabelling with a monoclonal antibody 7H5 compared to LeuM7 and to CD13 mRNA levels, and to correlate these data with the cytotoxic and apoptosis-induction activity of the natural phenolic APN inhibitor curcumin. CD13/APN was detected in a significant proportion of B-CLL patients (42/50, 84%), immunolabelled by 7H5 (42/50) ± LeuM7 (10/50). Molecular analysis for CD13 transcripts confirmed these data, resulting in a specific RT-PCR product in CD13 positive cases. Curcumin showed concentration-dependent cytoreductive efficacy and apoptosis-induction activity in all tested cell lines and primary cultures from CLL mononuclear cells. There was a clear tendency for a better response in CD13 positive cases. The incidence of CD13/APN in CLL suggests that the inhibition of APN/CD13 by curcumin may be an effective new molecular target for a more efficient therapy for these patients and warrants further investigations.
No preview · Article · May 2012 · Hematology (Amsterdam, Netherlands)
[Show abstract][Hide abstract] ABSTRACT: Anti-Echinococcus serum immunoglobulin (Ig)E was assessed by the ImmunoCAP system and compared with anti-Echinococcus serum IgG assessed by enzyme-linked immunosorbent assay (ELISA) and Western blot. The ImmunoCAP system revealed very high specificity (one false positive of 110 healthy individuals), low cross-reactivity (one false positive of 58 patients with other diseases) and decreased sensitivity (73.55%). Receiver operating characteristic analysis displayed a beneficial diagnostic value with high accuracy. Comparison of the ImmunoCAP system with ELISA and Western blot showed significantly higher specificity and significantly lower cross-reactivity compared with the ELISA. Examination of sera from 155 patients with cystic echinococcosis (CE) showed varying levels of anti-Echinococcus IgE (range, 0.01-118.33 kUA/L). However, most samples had moderately elevated IgE levels. Analysis of serum-specific IgE revealed significantly higher sensitivity of the ImmunoCAP system and significantly higher antibody levels in hepatic CE compared with pulmonary CE.
[Show abstract][Hide abstract] ABSTRACT: A mathematical model for leukopoiesis has been proposed in literature, consisting of a system of ordinary differential equations with two delays. The aim of this study was to verify the assumption that the model is applicable also for T, B and NK cell recovery after stem cell transplantation and to tune the model parameters on the base of clinical data. The numerical tests illustrate the influence of the model parameters to the behavior of the monitored white blood cell populations. They also show that for certain parameter values the numerically obtained behaviour of B cells is in agreement with the clinical data.
[Show abstract][Hide abstract] ABSTRACT: Ambrosia artemisiifolia L. is a leading cause of allergies in many countries. In the last decade it has appeared and spread in Bulgaria as well.
The aims of this study was to collect pollen from ragweed plants in Bulgaria, prepare the first Bulgarian ragweed allergen,
and characterize it. The new allergen was studied in parallel with two other ragweed pollen extracts from the USA and Russia.
The protein profile was studied through isoelectric focusing, and the total allergen activity was studied by a skin prick
test and a basophil activation test. The proteins of the studied extracts were concentrated in the pI region 3.5–5.5 with
an identical band number and location. The allergens showed similar total allergen activity in vivo:14/81 tested patients (17.3 %) were SPT positive either to a single ragweed extract or to all three. The allergens had identical
histogram profiles and caused basophil degranulation above the test cut-off. The analysis of the flow cytometry results by
parallel line bioassay shows linear dose-response relation between the extracts. The observed immunological properties of
the Bulgarian allergen from Ambrosia artemisiifolia provide a possibility to use this product for a reliable diagnosis and effective specific immunotherapy of ragweed allergy
in our country.
KeywordsAmbrosia artemisiifolia L-Allergen preparation-Isoelectric focusing-Total allergen activity
Full-text · Article · Oct 2010 · Central European Journal of Medicine
[Show abstract][Hide abstract] ABSTRACT: To examine the clinical spectrum of complications in pulmonary system and changes of some parameters of humoral and cell mediated
immunity related to heroin overdose. The study includes 16 patients who are long-term heroin abusers with acute heroin and
mixed with other psychoactive drugs intoxications with an average age of 21,5 ± 5.04 years (12 men and 4 woman). All patients
were hospitalized in the Clinic of Toxicology, MHATEM “N.I.Pirogov”, Sofia. We have used clinical, clinico-laboratory, immunological,
chimicotoxicological, instrumental methods. In severe intoxications with heroin and other psychoactive drugs, we observed
pulmonary system complications, i.e. pneumonia, aspiration of gastric contents, noncardiogenic pulmonary edema (NCPE) and
acute respiratory distress syndrome (ARDS.). Of the 16 patients in our study, 3 patients died due to complications. Some changes
in the immune reactivity observed in the study were (1) statistically significant lower mean levels of IgG and (2) tendency
to lower mean levels of IgA, IgM and complement components — C3 in the studied patients in comparison with the values in healthy
people. The changes were more demonstrative in the group with pulmonary complications compared to the group without pulmonary
complications. We observed that the CD4 lymphocytes were significantly less in the studied patients; in addition, a lower
level of CD56-bearing lymphocytes (natural killer /NK/ cells) was observed in comparison to healthy controls. The results
show that the mixture of acute heroin with other psychoactive drugs leads to complications in the pulmonary system and changes
of some parameters of cell-mediated and humoral immunity.
KeywordsIntoxication-Heroin-Psychoactive drugs-Immune reactivity-Noncardiogenic pulmonary edema-Pneumonia-ARDS
No preview · Article · Aug 2010 · Central European Journal of Medicine
[Show abstract][Hide abstract] ABSTRACT: Fetal stem cells possess some intriguing characteristics, which delineate them as promising cellular therapeutics. They are less immunogenic, at lower stage of differentiation and have higher potential for repopulation and migration. Furthermore, the fetal stem cells secrete a set of cytokines and growth factors, which stimulate the regeneration of the recipient tissue. The present study indicated that the adhesive fraction of human fetal liver cells possessed the morphological characteristics of mesenchymal stem cells, as well as potential to differentiate into adipocyte and osteoblast lineages. The immunophenotypic analysis showed that the cells expressed CD13, CD73, CD90 and CD105 (typical for mesenchymal stem cells) and lacked the haematopoietic lineage markers CD34 and CD45. Addressing the issue of the low-temperature storage of the human fetal liver cells, four different methods for cryopreservation were assessed: conventional slow freezing, program freezing and two vitrification protocols. The obtained results demonstrated that the cells were cryotolerant and maintained their properties and differentiation potential after thawing. Program freezing showed to be the most efficient method for cryopreservation of the investigated cells.
Full-text · Article · May 2010 · Cell Biology International
[Show abstract][Hide abstract] ABSTRACT: Development of Echinococcus granulosus in the intermediate host is ac-companied by a release of different allergens leading to the synthesis of specific IgE antibodies (sIgE) in the serum. The aim of the present study was to analyse the relation of sIgE to age, severity of developed allergic reactions and cyst localization in patients with cystic echinococcosis. Sera from 125 individuals (66 males and 59 females, median age of 46 years, range between 10-80 years) with histologically proven diagnosis were analysed. According to cyst localization 87 patients were with hepatic echinococcosis, 17 were with pulmonary echinococcosis and 21 were with multiorgan affection or rare localizations. The serological examination was performed quantitatively by ELISA with automatic system ImmunoCap 100. Positive results were detected in 94 (75.2%) of the patients. Increased sIgE were detected in 16 out of 18 (88.9%) children and in 78 (72.9%) out of 107 adults. Out of 125 patients 16 (12.8%) had allergic reactions - one negative and 15 positive for sIgE. Comparison between hepatic and pulmonary echinococcosis showed positive results in 68 (78.2 %) out of 87 patients with hepatic echinococcosis, and in 11 (64.7 %) out of 17 patients with pulmonary echinococcosis. The present study showed lack of correlation between the age and the amount of sIgE and between the amount of sIgE and the presence of allergic reactions in patients with cystic echinococcosis. The synthesis of sIgE was lower in pulmonary than in hepatic echinococcosis.
No preview · Article · Jan 2010 · Comptes rendus de l'Académie bulgare des sciences: sciences mathématiques et naturelles
[Show abstract][Hide abstract] ABSTRACT: For the present the immunotherapy with bee venom is the only established form of treatment which modifies disease progress by altering underlying immune responses.The aim of this study is to follow the immunological parameters - the size of skin prick test (SPT), the percentage of allergen-specific basophil degranulation and the quantities of allergen-specific IgE and IgG4 in patients with insect allergy during immunotherapy with Bulgarian bee venom allergen and to compare these results with those from beekeepers, who tolerate multiple bee stings.Three groups - allergic patients under immunotherapy with bee venom allergen, non-allergic beekeepers, and clinically health controls were examined. The skin tests and flow cytometry basophil test FastImmune, were performed with bee venom allergen. The quantity of serum allergen-specific IgE and IgG4 were measured using ImmunoCAP system.Immunotherapy decreases the skin reactivity assessed by SPT with bee venom allergen. This treatment does not change considerably the rate of basophil degranulation and the levels of allergen-specific IgE. The therapy leads to significant increase in bee venom specific IgG4 (from 0.71 to 3.19 μgA/L) - which resembles the immunological status of non-allergic beekeepers. The average quantity of allergen-specific IgG4 for them is 6.27 μgA/L.Immunotherapy with bee venom may be considered immunologically relevant when it is found significant increase (at least 3 to 5 fold according to study) in allergen-specific IgG4 antibodies.
[Show abstract][Hide abstract] ABSTRACT: The presently used approaches to silence autoreactive disease-associated B cells act indiscriminately and more specific therapies
are obviously needed. In the present study, we analyze the ability of a chimeric antibody to suppress selectively pathological
autoreactive B-lymphocytes in lupus-prone mice by cross-linking their surface Ig receptors with the inhibitory IgG FcγRIIb
receptors. The chimera was constructed by coupling an immunodominant mouse Histone 1 peptide to a rat monoclonal anti-mouse
CD32 (FcγRIIb) antibody. The administration of these chimeric molecules to MRL/lpr mice with initial and with full-blown disease
resulted in the reduction of the levels of IgG anti-Histone 1 antibodies, of the albuminuria levels, of the size of lymphoid
organs and in prevention of the development of skin lesions. The observed effect was limited to lupus-associated B cells only,
as the treatment did not decrease the IgG antibody response to an administered foreign antigen. This study demonstrates the
possibility to silence selectively autoreactive B cells and to delay the progression of an autoimmune disease using chimeric
Full-text · Article · Mar 2008 · International Immunology
[Show abstract][Hide abstract] ABSTRACT: The introduction of new in vitro methods, recommended by the European pharmacopoeia (J), enrich the characterization and evaluation of the allergens. The standardization of house dust mite allergen (A13 Dermatophagoides pteronyssinus) is based on different methods for determination of its structure and biological activity. Five batches of allergen were standardized by determination of protein nitrogen, by isoelectric focusing and by assessment of major allergen Der p1 by ELISA. The biological activity of the A13 was evaluated by skin prick test (SPT), IgE-binding potential after competitive inhibition and its activation capabilities for in vitro basophil degranulation. These batches, produced from 2002 - 2006, were with identical protein distribution according their isoelectrical points. Der p1 and protein nitrogen quantities varied between 37-87 ng/ml and 39 - 34 mg %. The studied lots showed similar results in the IgE-binding properties in comparison with the reference at 50 % inhibition level and in flow cytometry test for allergen-specific basophil degranulation. All the batches were with similar biological activity and induced skin-allergic reactions, comparable with the in-house reference preparation and histamine hydrochloride standard 1 mg/ml. The data from these new, in vitro methods improve the results from the accepted standardization procedure and will be performed as routine in the evaluation of biological activity of the house dust mite and others allergens.
[Show abstract][Hide abstract] ABSTRACT: Two-flow cytometry methods for quantification of degranulated basophil after allergen-specific activation were discussed. The methods are discerned by used membrane receptors--FcepsilonRI or IL-3Ralpha Our goal was to evaluate the diagnostic potential of the methods and to correlate them to allergen-specific IgE detection and skin prick test (SPT).
Patient's and control's groups were studied with Bulgarian grass pollen allergen B1 simultaneously by flow cytometry kits: Basotest and BD FastImmune test. Allergy diagnosis was based on clinical history and SPT. The determination of specific IgE was performed by ELISA--RIDASCREEN.
There were no significant differences between the patient's results from Basotest and FastImmune (P>0.05). A significant correlation between values, analyzed by Basotest and by FastImmune was found (r=0.88). The sensitivity and specificity of the Basotest, FastImmune, specific IgE and SPT were 85%, 72%, 92% and 92% sensitivity and 100%, 92%, 100% and 85% specificity respectively. The efficiency was between 82% and 97%. There were a significant correlation between the specific IgE and flow cytometry tests: tau=0.92 (Basotest) and tau=0.71 (FastImmune) and a moderate significant correlation between the SPT and the in vitro tests: tau=0.26 (Basotest) and tau=0.31 (FastImmune).
The successful use of the Bulgarian grass pollen allergen B1 and both flow cytometry tests was presented. These methods could be as specific tools for IgE-mediated diagnosis especially FastImmune in the case of low IgE receptor expression.
[Show abstract][Hide abstract] ABSTRACT: We examined the diagnostic potential of two in vitro flow cytometry methods for quantification of the basophile's degranulation after allergen-specific activation, differentiated by the expressed membrane receptors - FcεRI or IL-3Rα. The control group (14 persons) and 56 patients, sensitized to house dust mites, were simultaneously investigated using flow cytometry tests (Basotest and FastImmune) with the Bulgarian mite allergen A13. No significant differences between the patient's results from Basotest and FastImmune with both allergen concentrations - 100 BU and 1BU were revealed (P>0.05). However, a significant positive correlation between the percentages of CD63+ cells after activation with allergen A13 was found: r=0.81 for 100 BU and r=0.78 for 1 BU A13 (Spearman, P<0.05). A threshold of 10% CD63+ for both assays was estimated. The sensitivities of the Basotest, FastImmune, and skin prick test (SPT) were 85%, 73%, and 100%, while the specificities were 91%, 93%, and 93% respectively. The efficiency was between 83 and 97%. There was a moderate significant correlation between the SPT and the two in vitro tests of the higher concentration - 100 BU: tau=0.27 for Basotest and tau=0.38 for FastImmune (Kendall, P<0.05). Our study confirms the use of the Bulgarian allergen A13 with both flow cytometry tests as aspecific tool to diagnose IgE-mediated allergy, especially FastImmune in the case of low IgE receptor expression.
No preview · Article · Mar 2005 · Clinical Application of Immunology
[Show abstract][Hide abstract] ABSTRACT: For better understanding of engraftment properties after autologous peripheral blood stem cell transplantation (PBSCT), the reconstitution of lymphocyte populations was studied prospectively in 22 transplanted patients, suffering from different malignant proliferations. Percentage and absolute counts of T (CD3+), T helper/inducer (CD3+CD4+). T cytotoxic (CD3+CD8+). B (CD19+) and NK. (CD56+) lymphocytes were followed by flow cytometry in the course of 12 months after PBSCT. A rapid hematopoietic engraftment proceeding to stable long-term reconstitution was observed in all patients. Lymphocyte counts remained low throughout the studied period with a relative prevalence of T cells. The reconstitution of T lymphocytes was associated with an increase of CD3+CD8+ cytotoxic T cells. The share of CD4+ T lymphocytes remained low throughout the studied period, resulting in a low CD4/CD8 ratio. A severe depression of B-lymphopoiesis was detected in the first months after PBSCT that recovered slowly thereafter. In conclusion, the first year following autologous PBSCT was characterized by a rapid hematopoietic recovery and partly impaired immune reconstitution, affecting the regeneration of B lymphocytes and T helper cells.
No preview · Article · Sep 2004 · Clinical Application of Immunology
[Show abstract][Hide abstract] ABSTRACT: Using multicolor flow cytometry, we compared the dynamics of Th1-like (CD7+CD57-) and Th2-like (CD7-CD57+ / CD7-CD57-) CD4 T cell subsets in HIV-1 + patients with good (group A) and transient (group B) response to long-term HAART. At baseline, in comparison to healthy controls an increase of Th2-like CD4 T cells was observed in both groups (p < 0.01) accompanied by a decreased percentage of Th1-like cells (p< 0.001). The Th2/Th1 ratio was significantly higher in group B as compared to group A (p <0.05, M-W). HAART induced the recovery of Th1/Th2 ratio in both groups. However, the recovery of Th1 subset was dominated by naive CD4 T cells in group A (R=0.6, p<0.001), and by central memory CD4 T cells in group B (R=0.5, p<0.001). Besides, the proportion of CD57+ Th2-like cells was significantly higher in patients from group B at baseline and endpoint, possibly reflecting the gradual loss of the memory pool flexibility resulting in the irreversible accumulation of Th2-like cells. Thus, a Th2/Th1 imbalance may further impede immune recovery and compromise the effect of HAART.
No preview · Article · Sep 2004 · Clinical Application of Immunology
[Show abstract][Hide abstract] ABSTRACT: We examined the recovery dynamics of CD8+ T-cell subsets defined by the expression of CD45RA and CD62L surface antigens in HIV-1+ patients with good (group A) and transient (group B) response to HAART. At baseline, the percentage of naive (CD45RA+CD62L+) CD8+ T-cells in both groups were significantly lower than control values (p<0.01) while the percentage of effector memory (CD45RA-CD62L-) CD8+ T-cells were significantly increased in both groups, and it was higher in group A than in group B (p<0.01). In the course of therapy, naive CD8+ T cells recovered with similar dynamics in both groups, and did not differ significantly from healthy controls at the endpoint. The same was valid for central memory (CD62L+ CD45RA-) CD8+ T cells. Naive CD8+ T cells in group B increased at the expense of the terminal effector (CD45RA+CD62L-) CD8+ T subset. In contrast, in group A naive cells replaced mostly the effector memory subset. At the endpoint the good responders group still exhibited a higher percentage of effector memory cells than poor responders and in comparison to controls (p=0.05), probably reflecting a better preserved virus-specific CTL memory pool. Our results show that patients with different response to HAART have a distinct dynamics of the effector memory and terminal effector CD8+ T cell subpopulations. A better preserved cytotoxic T cell memory pool might be a prerequisite for immune restoration following HAART.
No preview · Article · Sep 2004 · Clinical Application of Immunology
[Show abstract][Hide abstract] ABSTRACT: CTL virus-specific responses to HIV, CMV and EBV as well as non-specific and spontaneous activation were studied in HIV+ patients with good, discordant and transient response to long-term HAART. The percentage of functional CTL and their differentiation stage were closely dependant on the extent of viral replication and resulting immune activation. The possible mechanisms that determine the phenotypic structure of virus-specific CTL pool in HIV infection are discussed.