[History of Immuno-therapy -  from Coley Toxins to Check-points of the Immune Reaction]. / Historie imunoterapie -  od Coley toxinu ke kontrolním bodum imunitní reakce.

Immunotherapy dates back to 1868 when German physicist Busch intentionally infected patients suffering from soft tissue sarcoma with erysipelas. Rapid tumor shrinkage was observed but response was only partial and tumor recurrence subsequently occurred. It was William B. Coley who in 1891 injected a patient with a soft tissue sarcoma with streptococcal cultures. Following a severe attack of erysipelas, the tumor underwent extensive necrosis and the patient remained diseasefree for eight years. The mixture of Streptococcus and other bacteria including Seratia marcescens, Staphylococcus and Escherichia coli was referred to as Coleys toxin and was used for the next 45 years. This first immunotherapy was replaced at the beginning of the 20th century by more exact radiotherapy and later on by first chemotherapy with yperit. However, immunotherapy is a treatment that uses patients own immune system to help fight cancer and as such has several advantages over other treatments. Thus, the next major milestones in immunotherapy came in the middle of the 80s as a) adoptive cell therapy relaying on patients tumor infiltrating lymphocytes, b) injection of recombinant cytokines such as rIL2, c) identification of the first tumorassociated antigens and d) development of tumor specific monoclonal antibodies. It was followed by dendritic cells vaccines. Tremendous progress has been made in the past two decades with regard to understanding the complex interactions between tumors and the immune system and developing innovative ways to manipulate the antitumor immune response. It is recently represented as blockage of immune checkpoint inhibitors.

[Escape Strategies of Tumors from Immune Surveillence]. / Únikové strategie nádoru pozornosti imunitního systému.

Immune system must be able to protect us from foreign dangerous pathogens, but on the other side, it must be able to recognize our own tissues and organs. Activity of the immune system is affected by many positive (stimulatory) and negative (inhibitory) signals. Some of these negative receptors protect us from damage of our tissues at a place of inflammation as it blocks too intensive or longlasting immune reaction. Thereby, they have a physiological protective function against strong inflammatory reaction and possible subsequent autoimmune pathology. However, some of these mechanisms are also utilized by tumors to avoid immune recognition and attention of the immune cells. Other tumor escape mechanisms involve increased production of cytokines and factors which are responsible for immunosuppressive tumor microenvironment where effective immune response is actively blocked. This review summarizes the most frequently used strategies, which are utilized by tumors to avoid immune recognition and/ or killing by the immune cells.

Synthesis of poly[N-(2-hydroxypropyl)methacrylamide] conjugates of inhibitors of the ABC transporter that overcome multidrug resistance in doxorubicin-resistant P388 cells in vitro.

The effects of novel polymeric therapeutics based on water-soluble N-(2-hydroxypropyl)methacrylamide copolymers (P(HPMA)) bearing the anticancer drug doxorubicin (Dox), an inhibitor of ABC transporters, or both, on the viability and the proliferation of the murine monocytic leukemia cell line P388 (parental cell line) and its doxorubicin-resistant subline P388/MDR were studied in vitro. The inhibitor derivatives 5-methyl-4-oxohexanoyl reversin 121 (MeOHe-R121) and 5-methyl-4-oxohexanoyl ritonavir ester (MeOHe-RIT), showing the highest inhibitory activities, were conjugated to the P(HPMA) via the biodegradable pH-sensitive hydrazone bond, and the ability of these conjugates to block the ATP driven P-glycoprotein (P-gp) efflux pump was tested. The P(HPMA) conjugate P-Ahx-NH-N═MeOHe-R121 showed a dose-dependent increase in the ability to sensitize the P388/MDR cells to Dox from 1.5 to 24 µM, and achieved an approximately 50-fold increase in sensitization at 24 µM. The P(HPMA) conjugate P-Ahx-NH-N═MeOHe-RIT showed moderate activity at 6 µM (∼10 times higher sensitization) and increased sensitization by 50-fold at 12 µM. The cytostatic activity of the P(HPMA) conjugate P-Ahx-NH-N═MeOHe-R121(Dox) containing Dox and the P-gp inhibitor MeOHe-R121, both bound via hydrazone bonds to the P(HPMA) carrier, was almost 30 times higher than that of the conjugate P-Ahx-NH-N═Dox toward the P388/MDR cells in vitro. A similar result was observed for P-Ahx-NH-N═MeOHe-RIT(Dox), which exhibited almost 10 times higher cytostatic activity than P-Ahx-NH-N═Dox.

[The cost study of first- line treatment of metastatic colorectal carcinoma with bevacizumab- containing regimen in the Czech Republic]. / Analýza nákladu na 1. linii lécby metastatického kolorektálního karcinomu pri podání rezimu s bevacizumabem -  data z reálné klinické praxe v Ceské republice.

BACKGROUND: Bevacizumab, a humanized monoclonal IgG antibody against the vascular endothelial growth factor (VEGF), is reimbursed in combination with chemotherapy for the first and subsequent line treatment of patients with metastatic colorectal cancer (mCRC) in the Czech Republic. However, its high cost is a potentially limiting factor. We assessed the cost of bevacizumab in the treatment of mCRC in a comprehensive cancer center. PATIENTS AND METHODS: A total of 218 patients were included in our analysis. Cost data (examination, medication, hospitalization) were collected since the initiation of bevacizumab treatment to any tumor response (RECIST criteria: complete response -  CR, partial response -  PR, stable disease -  SD, progressive disease -  PD) and/ or to death. Minimal followup for all patients was 28 months. Costs were valued in Czech crowns (CZK) and converted to EUR (1€ = 25.14 CZK). RESULTS: PD was recorded in 194 patients (89% of patients). The mean cost of treatment to PD (median TTP 9.1 months) was 1,002,076.30 CZK (39,859.84 EUR). The majority of costs to PD was made by medication -  917,048.60 CZK (36,477.67 EUR) per patient. The mean cost to response PR, CR or SD was 1,105,823.10 CZK (43,986.60 EUR) after median 9.8 months of treatment (recorded for 21 patients), medication formed 1,023,827.70 CZK (40,725.05 EUR). During the study, 170 patients (78%) died. The mean of the total costs since initiation of treatment to death (median OS 18.8 months) was 1,338,874.20 CZK (53,256.70 EUR) -  out of that, medication was 1,184,251.10 CZK (47,106.25 EUR) per patient. CONCLUSION: Targeted bio-logical therapy is the largest part of the costs of mCRC therapy. Cost of bevacizumab made up to 69% of costs to PD -  687,608.20 CZK ( 27,351.20 EUR ) per patient. The majority of the total cost was formed by targeted drug therapy (bevacizumab in 1st line therapy, cetuximab and panitumumab in 2nd and 3rd line therapy); 58% of total costs since initiation of treatment to death -  778,233.80 CZK (30,956 EUR) per patient.

[Cost of acute heart failure related readmissions]. / Náklady na rehospitalizaci pacientu s akutním srdecním selháním.

AIM OF STUDY: To assess direct in-patient cost and length of stay in the intensive care unit (ICU) and the standard cardiology unit in acute heart failure (AHF) readmissions. RESULTS: Out of 1 759 patients hospitalized with acute heart failure, 223 patients were readmitted to Faculty Hospital Brno-Bohunice (Czech Republic) during study period (61.4% male; mean age 71.2 years) with mean total cost CZK 85 120 (Euro 3 095) per length of stay 9.2 days and interventions. Comparing to the first hospitalization of study cohort (223 pts.) the decrease was recorded in mean room rate, length of stay and need of ICU stay (from 48% to 42% pts.), nevertheless ICU stay increased (from 3.7 days to 4.1 days). The growth of mean cost was recorded in both procedures in angiology (the decrease in number of coronary angiography which is cheaper was more remarkable than PCI decrease in readmitted patients) and arrhythmology (including device: pacemaker, ICD, CRT) which made 57.5% of total readmission costs. CONCLUSION: The difference in mean in-patient cost between the first and second hospitalization was 18%. The antiarrhytmic procedures had the most significant impact on total readmission cost and its variability, butwe assume that these procedures will reduce within next readmissions and their impact will weaken as in angiology procedures.

Synergistic action of doxorubicin bound to the polymeric carrier based on N-(2-hydroxypropyl)methacrylamide copolymers through an amide or hydrazone bond.

The cytostatic effects of polymeric conjugates based on N-(2-hydroxypropyl)methacrylamide copolymers (PHPMA) and containing doxorubicin bound through amide and hydrazone bonds (mixed conjugates) were compared with the cytostatic effects of monoconjugates containing drug bound through an amide or hydrazone bond. One group of mixed conjugates was formed from two comonomers containing doxorubicin bound to the methacryloyl group through a spacer and an amide (DOX(AM)) or hydrazone (DOX(HYD)) bond via copolymerization with HPMA. A second group of mixed conjugates was formed from two different interconnected HPMA copolymers, one containing DOX(AM) and the other DOX(HYD), forming a high-molecular-weight branched structure. The third mixed polymeric system was a simple mixture of monoconjugates DOX(AM)-PHPMA and DOX(HYD)-PHPMA. Simultaneous treatment with all mixed forms of the polymeric derivatives of doxorubicin significantly increased antitumor efficacy after application of monoconjugates, suggesting a synergizing effect that could be used in designing new doxorubicin-containing therapeutic systems.

Surface modification of polyimide sheets for regenerative medicine applications.

In the present work, two strategies were elaborated to surface-functionalize implantable polyimide sheets. In the first methodology, cross-linkable vinyl groups were introduced on the polyimide surface using aminopropylmethacrylamide. In the second approach, a reactive succinimidyl ester was introduced on the surface of PI. Using the former approach, the aim is to apply a vinyl functionalized biopolymer coating. In the latter approach, any amine containing biopolymer can be immobilized. The foils developed were characterized in depth using a variety of characterization techniques including atomic force microscopy, static contact angle measurements, and X-ray photoelectron spectroscopy. The results indicated that both modification strategies were successful. The subcutaneous implantation in mice indicated that both modification strategies resulted in biocompatible materials, inducing only limited cellular infiltration to the surrounding tissue.

Polymer nitric oxide donors potentiate the treatment of experimental solid tumours by increasing drug accumulation in the tumour tissue.

The delivery of nitric oxide (NO) specifically to solid tumours was explored in this study as a strategy to augment the passive accumulation of nanomedicines in tumours induced by the Enhanced Permeability and Retention (EPR) effect. An increase in accumulation was achieved by the binding of the chemical precursor of NO, based on an organic nitrate, to a water-soluble synthetic polymer drug carrier. Four structurally different N-(2-hydroxypropyl)methacrylamide (HPMA)-based polymer NO donors were synthesized. Depending on their chemical structure, two of these donors were hydrolytically stable, while two rapidly released the parent nitrate under acidic conditions, mimicking the intracellular environment. The polymer NO donors were shown to overcome the drawbacks related to low-molecular-weight NO releasing compounds, namely systemic toxicity, lack of site specificity, and fast blood clearance. The NO donors showed intracellular NO release upon incubation with tumour cells. In vivo, they potentiated the EPR effect, resulting in an increased accumulation of polymer-bound cytotoxic drug doxorubicin (Dox) in EL4 T-cell lymphoma inoculated in mice. This led to a better therapeutic outcome in the treatment of lymphoma with the high-molecular-weight polymer conjugates carrying Dox but not in the treatment with the free Dox. The localized augmentation of the EPR effect via the tumour-specific NO delivery system can be viewed as a promising strategy to potentiate polymer-based tumour therapy without increasing systemic toxicity.

The comparison of in vivo properties of water-soluble HPMA-based polymer conjugates with doxorubicin prepared by controlled RAFT or free radical polymerization.

Two conjugates of anticancer drug doxorubicin (Dox) covalently bound by the hydrolytically degradable hydrazone bond to the polymer carrier based on water-soluble N-(2-hydroxypropyl) methacrylamide (HPMA) copolymers were synthesized and their properties were compared, namely their behavior in vivo. The polymer carriers differed in dispersity due to different methods of synthesis; the carrier with relatively high dispersity (HD) was prepared by free radical polymerization (Mw=29,900 g/mol, D=1.75) and the carrier with low dispersity (LD) by controlled radical polymerization (Mw=30,000 g/mol, D=1.13). Both polymer-Dox conjugates showed prolonged blood circulation and tumor accumulation of the drug in comparison with the free drug; e.g. the tumor-to-blood ratio for the polymer-bound Dox was 3-5 times higher. The LD polymer-Dox conjugate exhibited moderately higher tumor accumulation than the HD one at a dose of 1x15 mg Dox (eq.)/kg. Also, their anti-tumor activity did not differ when injected at this dose. However, the increase of the dose to 1x25 mg Dox (eq.)/kg resulted in the enhanced therapeutic activity of the conjugates, especially of the LD one with 100% of long-term survivals. The dispersity of polymer drug carriers influenced the tumor accumulation rate, which affected the overall anti-cancer activity of polymer-drug conjugates.

Immunocompatibility and biocompatibility of cell delivery systems.

Immunoisolation therapy overcomes important disadvantages of implanting free cells. By mechanically blocking immune attacks, synthetic membranes around grafted cells should obviate the need for immunosuppression. The membrane used for encapsulation must be biocompatible and immunocompatible to the recipient and also to the encapsulated graft. The ability of the host to accept the implanted graft depends not only on the material used for encapsulation, but also on the defense reaction of the recipient, which is very individual. Such a reaction usually starts as absorption of cell-adhesive proteins, immunoglobulins, complement components, growth factors and some other proteins on the surface of the device. The absorption of proteins is difficult to avoid, but the amount and specificity of absorbed proteins can be controlled to some extent by selection and modification of the device material. If the adsorption of proteins to the surface of the implanted material is reduced, the overgrowth of the device with fibroblast-like and macrophage-like cells is also reduced. Cell adhesion at the surface of the implanted device is, in addition to the selected polymeric material, greatly influenced by the device content. Xenografts trigger a more vigorous inflammatory reaction than allografts, most probably due to the release of antigenic products from encapsulated deteriorated and dying cells which diffuse through the membrane and activate adhering immune cells. There is an evident effect of autoimmune status on the fate of the encapsulated graft. While encapsulated xenogeneic islets readily reverse streptozotocin-induced diabetes in mice, the same xenografts are short-functioning in NOD autoimmune diabetes-prone mice. Autoantibodies, to which most devices are impermeable, are not involved. Among the cytotoxic factors which are responsible for the limited survival of the encapsulated graft the most important are cytokines and perhaps some other low-molecular-weight factors released by activated macrophages at the surface of the encapsulating membrane.

Effect of age on antibody responses in low responder C57BL/10ScSn and high responder A/J strains of mice.

The IgM antibody response to sheep red blood cells (SRBC) appeared significantly earlier in A/J strain mice (already in 10-day-old animals), but after 21 days responses were higher in B10 mice. These differences disappeared after reaching adulthood and IgM responses after either primary or secondary immunization were thereafter comparable in these strains. High-responder A/J mice made significantly more IgG antibodies than low-responder B10 mice from 21 days of age and strong differences lasted until the age of 19 months, when IgG antibody production was again similar. Potentiation of IgM formation by simultaneous application of 10 micrograms of LPS was higher in B10 mice until 19 months of age. On the other hand, potentiation of the IgG response was markedly high in B10 mice only in adult animals (3 months). Thereafter the potentiation was higher in A/J mice. The onset of Ig secretion in A/J mice was at 15 days and markedly increased at the age of 30 days. Levels of immunoglobulin synthesis remained extremely low in B10 mice. Age-related changes in IgG antibody production generally correlated with the decline of MHC class II antigen expression on peritoneal macrophages in these strains.

A possibility to overcome P-glycoprotein (PGP)-mediated multidrug resistance by antibody-targeted drugs conjugated to N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer carrier.

N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers containing doxorubicin (DOX) and different targeting moieties were developed with the aim of specific chemotherapy. Two of them, HPMA-conjugated DOX and galactosamine-targeted DOX, are in phase II clinical trials in the U.K. We studied the effect of conjugates with different targeting moieties (anti-CD71, antithymocyte globulin, anti-CD4, transferrin) on human or mouse multidrug resistance (MDR) cell lines (CEM/VLB, P388-MDR). It was shown that targeting decreases the level of MDR for DOX and the level of MDR depends on the targeting moiety used. The combination of these conjugates with chemosensitisers (cyclosporin A, D, G) restored almost completely the sensitivity of MDR cell lines to that of parental sublines. These results suggest that different intracellular trafficking of these conjugates (in membrane-limited organelles) in contrast to free diffusion for low molecular weight compounds might partially overcome P-glycoprotein (Pgp)-mediated MDR. We also report here the development of biodegradable HPMA hydrogels suitable for prolonged release of the cytostatic drug and chemosensitiser as a potential approach to overcome MDR mediated by Pgp.

HPMA-hydrogels result in prolonged delivery of anticancer drugs and are a promising tool for the treatment of sensitive and multidrug resistant leukaemia.

Treatment of an established BCL1 leukaemia in mice showed that the use of hydrogels is advantageous in comparison with free doxorubicin (DOX), partially due to the different pharmacokinetic profile of the drug release. Pharmacologically active concentrations ranging from 100 to 800 ng/ml were detectable in the bloodstream for more than 4 days when DOX-loaded hydrogels were implanted into mice. Animals treated with free DOX survived for 35 days, survival of hydrogel-DOX treated animals increased up to 60 days and long-term survivors were achieved, when the second hydrogel was implanted 2 weeks after the first one. Hydrogels containing vinblastine (VLB) were ineffective. N-(2-hydroxypropyl)methacrylamide (HPMA) hydrogels were also used in combined therapy against multidrug resistant leukaemia P388-MDR to achieve a synergistic effect of both the cytostatic drug and chemosensitising agent. It was shown that when 4 times the maximal tolerated dose (MTD) of free DOX was incorporated into HPMA-hydrogels, tumour volume was reduced by approximately 50% after implantation of the hydrogel containing DOX and cyclosporine A (CsA) and survival was slightly prolonged.

Detection of mouse cells producing antibodies against the azophenylarsonic group by haemolytic plaque assay.

For detection of mouse cells producing anti-ARS antibody a modified method using ARS-SRBC is described. For optimal results a suitable source of SRBC must be selected. The number of ARS groups per SRBC required for optimal lysis in PFC assay varies, and ARS-SRBC prepared by the method of Ingraham which are suitable for detection of rabbit-PFC give negative results with the mouse system. ARS-SRBC prepared for the PFC assay are unsuitable for the haemagglutination test and vice versa.

Abnormal differentiation of thymocytes induced by free cyclosporine is avoided when cyclosporine bound to N-(2-hydroxypropyl)methacrylamide copolymer carrier is used.

BACKGROUND: The side effects of cyclosporine (CsA)-including nephrotoxicity and abnormal differentiation of thymocytes developing in the thymus-can be decreased or even avoided using targeted conjugates of CsA, where both targeting moiety and drug are bound to water-soluble polymeric carrier based on N-(2-hydroxypropyl) methacrylamide (HPMA). METHODS: Irradiated, syngeneic bone marrow transplanted-mice (BALB/c and A/Ph) were treated intraperitoneally for 4 weeks with 20 mg/kg of free CsA, HPMA-conjugated CsA, or antibody-targeted HPMA-bound CsA. Immunohistology of the thymus was performed together with two-color flow cytometry to detect the effect of different forms of CsA on individual thymocyte subpopulations. RESULTS: . We have shown that free CsA strongly abrogated T-cell development. The appearance of mature thymocytes expressing CD3(high) is almost completely inhibited (1.8%) after free CsA treatment, whereas these cells are well detectable in controls (22%) and HPMA polymer-bound CsA-treated animals (19%). Immunohistological studies have shown acellular rests of the medulla after free CsA treatment, whereas well-stained medullary thymocytes were detected in controls and after exposure to antibody-targeted HPMA. conjugated CsA. CONCLUSIONS: HPMA-conjugates of CsA are generally more specific in their targeting to T lymphocytes. It was found that nonspecific binding of CsA to erythrocytes and plasma lipoproteins is significantly reduced using anti-CD3 targeted, HPMA polymer-bound CsA In addition, the entry of these macromolecules into the thymus is limited-probably due to the blood-thymus barrier-and HPMA conjugates of CsA, unlike free drug, do not abrogate T-cell development in bone marrow transplanted mice.

Macrophage function in high- and low-responder strains of mice.

Unstimulated peritoneal macrophages of the high-responder A/J mice compared to the low-responder B10 strain have a lower number of cells with the different Fc receptor (FcR) subtypes and correspondingly the FcR mediated phagocytosis is also lower. The intraperitoneal immunization with SRBC leads to a prompt increase of FcR expression and of phagocytic and pinocytic activity in the A/J mice, while in the B10 strain the activity of macrophages remains unchanged.

Immunostimulatory effect of cis-diamine dichloroplatinum (II).

Cis-diamine dichloroplatinum (II) (DDP) injected i.p. or i.v. at the dosage of 0.83 mg/kg to BALB/c mice on days 0, 7 and 14 elicited antibodies against proteins (RSA, BGG) and haptens (TNP, FITC) detectable by ELISA or by passive haemagglutination. Significant increase in antibody production was observed with either route of DDP application. A similar set of antibodies against antigens which do not cross-react with the immunizing proteins was observed when mice were immunized with complexes formed by an in vitro incubation of RSA and BGG with DDP. Thus, proteins modified in vivo or in vitro by DDP may stimulate the production of antibodies of unexpected specificities.

IL-2 and IL-3 production in high and low IgG-responding strains of mice.

The antibody response of mouse strain C57Bl/10ScSn (B10) is characterized by a low IgG responsiveness to a number of different antigens. Aberrant function of antigen-presenting cells and/or low activity of the Th cell population have been suggested as the cause of the defect. We studied the production of IL-2 and IL-3 in vitro by unstimulated and ConA-stimulated spleen cells. Unstimulated spleen cells of low-responding B10 mice produce significantly less IL-2 compared with the high-responding A/J mice in both intervals tested, i.e. after 24 and 48 h of in vitro incubation. IL-3 production is low but almost comparable in unstimulated cells of both strains. Stimulation of spleen cells by 5 micrograms/ml of ConA leads to considerably higher production of IL-2 in A/J spleen cells. IL-3 production by ConA-stimulated spleen cells showed the same pattern of activity. This low IL-3 production by B10 cells is most likely due to the low production of IL-2 during Th cell activation and to the limited proliferation of these cells. The low IgG production of B10 spleen cells during the secondary response to SRBC in vitro could be restored by IL-2 added to the medium. 50 U/ml of IL-2 increased the number of anti-SRBC IgG-producing cells 40 times in B10 cells, but only 4 times in A/J cells, so that the IgG production in B10 cells reached the same level as that in the A/J cells without exogenous IL-2. We suppose that the limited IL-2 production in the low-responding strain B10 is the cause of the low IgG responsiveness of these mice.

Immunological problems of polymer-bound drugs.

For the application of polymer-bound drugs (especially in combination with the targeting antibodies), it is essential to know more about the immunogenicity and possible hazards from immune reactions induced by their repeated application. The immune reaction depends on many factors (including structure of antigen, dose of antigen, schedule and way of the application, genetic background of the immunized, i.e., receiving organism) and could be weakened or eliminated by a choice of suitable factors. Problems connected with the immune reaction of the humoral and cellular type (B and T cell involvement) against different polymers and polymer-bound drugs and possible consequences for their clinical use will be discussed.

Changes in some properties and functions of plasma membranes of macrophages from young, adult and aged mice.

We studied age-dependent changes in distribution of receptor-ligand complexes in the lateral plain of plasma membrane after binding of six different lectins and anti H-2a and anti Ias antisera to the surface of mouse peritoneal macrophages. Additionally we examined formation of spontaneous rosettes with sheep red blood cells and the phagocytic and pinocytic activities of these macrophages. Macrophages of young and aged mice exhibited a lower mobility of the studied antigens and lectin receptors (determined as a function of patch and cap formation) and a higher rate of formation of spontaneous rosettes. The percentage of Ia antigens bearing macrophages was very low during early ontogenesis; it increased during adulthood and decreased during ageing. The highest percentage of phagocytizing macrophages was found in adult mice, even thought the phagocytic index (i.e., the average quantity of phagocytized particles in a phagocytizing cell) kept increasing during postnatal life. However, the level of pinocytosis was the same in all three groups.