Polymers of functionalized diaminopropionic acid are efficient mediators of active exogenous enzyme delivery into cells.

Delivery of active protein especially enzyme is one of the major therapeutic challenge. Replacing or substituted invalid/improper acting protein offer fast and effective treatment of disease. Herein, we describe the synthesis and properties of biotinylated peptidomimetics consisting of oxoacid-modified 2,3, L-diaminopropionic acid residues with guanidine groups on its side chains. Electrophoretic analysis showed that the obtained compounds interact with FITC-labeled streptavidin or a streptavidin-ß-galactosidase hybrid in an efficient manner. Complexes formed by the abovementioned molecules are able to cross the cell membranes of cancer or healthy cells and show promising compatibility with live cells. Analysis of ß-galactosidase activity inside the cells revealed surprisingly high levels of active enzyme in complex-treated cells compared to controls. This observation was confirmed by immunochemical studies in which the presence of ß-galactosidase was detected in the membrane and vesicles of the cells.

Conjugate of Enkephalin and Temporin Peptides as a Novel Therapeutic Agent for Sepsis.

Antimicrobial peptides (AMPs) exhibit a wide spectrum of actions, ranging from a direct bactericidal effect to multifunctional activities as immune effector molecules. The aim of this study was to examine the anti-inflammatory properties of a DAL-PEG-DK5 conjugate composed of a lysine-rich derivative of amphibian temporin-1CEb (DK5) and dalargin (DAL), the synthetic Leu-enkephalin analogue. Detailed study of the endotoxin-neutralizing activity of the peptide revealed that DAL-PEG-DK5 interacts with LPS and the LPS binding protein (LBP). Moreover, DAL-PEG-DK5 prevented dimerization of TLR4 at the macrophage surface upon LPS stimulation. This inhibited activation of the NF-κB signaling pathway and markedly reduced pro-inflammatory cytokine production. Finally, we showed that aggregation of DAL-PEG-DK5 into amyloid-like structures induced by LPS neutralized the endotoxin proinflammatory activity. Consequently, DAL-PEG-DK5 reduced morbidity and mortality in vivo, in a mouse model of endotoxin-induced septic shock. Collectively, the data suggest that DAL-PEG-DK5 is a promising therapeutic compound for sepsis.

The correlation between the concentration of hepcidin in serum and the occurrence of insulin resistance and hyperandrogenemia in women with polycystic ovary syndrome.

OBJECTIVE: Scarce clinical and experimental studies suggest that hepcidin can be a protein participating in the development of metabolic disorders, while its synthesis and concentration in the circulation outside of the iron metabolism parameters can be influenced by hormones. The aim of the present study was to determine the correlation between the concentration of hepcidin in serum and the occurrence of insulin resistance and hyperandrogenemia in women with PCOS. PATIENTS AND METHODS: Five groups of women with PCOS were divided based on: correct body mass (17 without hyperandrogenemia and insulin resistance - G1; 17 with hyperandrogenemia and without insulin resistance - G2; 11 without hyperandrogenemia and with insulin resistance - G3; 10 with hyperandrogenemia and insulin resistance - G4), metabolic and hormonal parameters and selected markers of iron metabolism. RESULTS: Serum glucose levels were significantly higher in the group G3 than G1 and in the group G4 than G1 and G2. Serum insulin levels and HOMA-IR values were significantly higher in the groups G3 and G4 than G1 and G2. Serum androstenedione levels were significantly higher in the group G2 than G1 and G3 than G2. Serum transferrin levels were significantly lower in the group G1 than in the reaming study groups. CONCLUSIONS: It has been demonstrated that insulin resistance and hyperandrogenemia appear to be the factors decreasing the concentration of transferrin circulation, but not the remaining parameters of the iron metabolism in the studied women. No relationship between the concentration of hepcidin circulation and other studied parameters of the iron metabolism and the parameters of the carbohydrate metabolism was discovered. Androstenedione can stimulate hepcidin synthesis in women with PCOS with correct body mass.

Safety profile of a single pegylated asparaginase (PEG-ASP) dose in remission induction for acute lymphoblastic leukemia (ALL).

The incorporation of pegylated asparaginase (PEG-ASP) in pediatric and adult acute lymphoblastic leukemia (ALL) protocols remains a worldwide therapeutic approach. However the safety profile remains a challenge, and herein we report the toxicity of an intravenous single dose of 1000 IU/m2 PEG-ASP administered in remission induction for adult ALL patients. Thirty-two patients at median diagnostic age of 32 years (median of 19-65) were included in this analysis. Most patients had B-cell lymphoblastic leukemia (n=26; 78%) and 81% of cases were <55 years at study entry. 75% of patients had <30x109/l leukocyte count at diagnosis and median follow-up was 14 months (range 0.8-69). All grade 3/4 adverse events (AEs) after PEG-ASP administration were observed in 24 patients (75%). The most common grade 3/4 AEs were: decreased fibrinogen (58%), increased bilirubin (31%) and increased GGTP (27%). Clinical manifestations related to PEG-ASP were seen in 9 patients and included: abdominal pain (n=6), thrombosis (n=2), diarrhea (n=1) and pancreatitis (n=1). The median time from PEG-ASP administration to first toxic symptoms was 7 days (range 1-19), and there were also 4 (13%) early induction deaths. All deaths were observed in ≥50-year-old patients after a median of 5 days following PEG-ASP (range 1-9). Three of these four patients had massive obesity. While all expired patients had grade 4 neutropenia and thrombocytopenia at the time of death, sepsis was not present. Administration of PEG-ASP in induction remission for ALL patients resulted in a significant, but mostly reversible hepatotoxicity. This PEG-ASP treatment should be administered with caution for older, obese patients.

Methotrexate in the treatment of mycosis fungoides - a multicenter observational study in 79 patients.

OBJECTIVE: The first report concerning methotrexate (MTX) in the treatment of Mycosis fungoides (MF) was published in 1964 by Wright. The mechanism of MTX action in the treatment of primary cutaneous T-cell lymphoma (CTCL) has been not explained in detail yet (the anti-inflammatory, immunomodulating, immunosuppressive, and cytostatic actions have been under discussion). PATIENTS AND METHODS: This is a retrospective analysis of 79 MF patients in 4 dermatology clinical centers in Poland. Data are presented in terms of the duration, use of MTX, the effectiveness of treatment with MTX in terms of time required to achieve remission, the disease stage, route of administration, age at diagnosis and the dosage. Moreover, the occurrence of side effects depending on the route of administration and duration of therapy with MTX was analyzed. RESULTS: The analysis has revealed that 56 patients (70,9%) had achieved remission on the MTX. The remission began in the 1st month of therapy in 20% of patients, lasted 4 to 6 months in 50% of cases. At least 12 months' remission was confirmed in 25% of patients (2-year-long only in 10% and 3-year-long in 5% of patients). The time to remission was related to the stage of disease at diagnosis as well as to minimal and maximal dose of MTX. The total therapeutic dose of MTX was found important for the course of the disease: higher total dose had prolonged the remission. CONCLUSIONS: Despite the common use of MTX in MF patients, relatively few clinical studies have been published. The response of MF subjects to MTX seems to depend on the stage and, more importantly, the dose of MTX treatment. Methotrexate appears to be an effective treatment at every stage of MF; however, it is not devoided of side effects such as infections and elevated level of aminotransferases, which are most common.

RADON CHAMBER IN THE CENTRAL MINING INSTITUTE-THE CALIBRATION FACILITY FOR RADON AND RADON PROGENY MONITORS.

The article presents the advantages of the radon chamber with volume of 17 m3, that belongs to Silesian Centre for Environmental Radioactivity and its applicability for calibration of equipment designed to measure the radon concentration and its short-lived decay products. The chamber can be operated under controlled conditions in the range from -20 to 60°C and relative humidity from 20 to 90%. There is also discussed the influence of aerosol concentration and their size distribution on the calibration results. When calibrating the measuring devices in an atmosphere with a large contribution of ultrafine particles that are defined as particles with diameter <0.1 µm, their sensitivity may decrease by tens of percent.

MEASUREMENTS OF AIRBORNE CONCENTRATIONS OF RADON AND THORON DECAY PRODUCTS.

Liquid scintillation counting (LSC) is a measuring technique, broadly applied in environmental monitoring of radionuclides. One of the possible applications of LSC is the measurement of radon and thoron decay products. But this method is suitable only for grab sampling. For long-term measurements a different technique can be applied-monitors of potential alpha energy concentration (PAEC) with thermoluminescent detectors (TLD). In these devices, called Alfa-2000 sampling probe, TL detectors (CaSO4:Dy) are applied for alpha particles counting. Three independent heads are placed over the membrane filter in a dust sampler's microcyclone. Such solution enables simultaneous measurements of PAEC and dust content. Moreover, the information which is stored in TLD chips is the energy of alpha particles, not the number of counted particles. Therefore, the readout of TL detector shows directly potential alpha energy, with no dependence on equilibrium factor, etc. This technique, which had been used only for radon decay products measurements, was modified by author to allow simultaneous measurements of radon and thoron PAEC. The LSC method can be used for calibration of portable radon decay products monitors. The LSC method has the advantage to be an absolute one, the TLD method to measure directly the (dose relevant) deposited energy.

Lower melanin content in the skin of type 1 diabetic patients and the risk of microangiopathy.

BACKGROUND: Various skin diseases are commonly observed in diabetic patients. Typical biophysical properties of diabetic skin such as lower skin elasticity, decreased water content in stratum corneum, increased itching and sweating disturbances are reported. The aim of the study was to examine the distribution and intensity of skin pigmentation in diabetic patients in correlation with the metabolic control and with presence of microangiopathy. MATERIAL AND METHODS: The study was conducted on 105 patients (42 men and 63 women, median age 31), with type 1 diabetes (DM1). The control group of 53 healthy individuals (22 men and 31 women) was age- and sex-matched. Skin pigmentation was measured at 3 different locations of the body (cheek, dorsal surface of a forearm and dorsal surface of a foot) using Mexameter® MX 18. We calculated melanin index (MI) by the meter from the intensities of absorbed and reflected light at 880 nm. RESULTS: Patients with DM1 had lower MI on the foot (173.2 ± 38.8 vs. 193.4 ± 52.7, p=0.016) as compared to controls. In the univariate analysis cheek MI was negatively related to HbA1c level (ß=-4.53, p=0.01). Forearm MI was negatively associated with daily insulin dose (ß=-0.58, p=0.01), BMI (ß=-3.02, p<0.001), waist circumference (ß=-0.75, p=0.009), serum TG concentration (ß=-18.47, p<0.001) and positively with HDL cholesterol level (ß=15.76, p=0.02). Diabetic patients with hypertension had lower foot MI values (ß=-18.28, p=0.03). Lower MI was associated with the presence of diabetic neuropathy (ß=-18.67, p=0.04) and retinopathy (ß=-17.47, p=0.03). CONCLUSIONS: In conclusion, there seems to be loss of melanocytes in type 1 diabetes. The melanin content is related to glycemic control of diabetes and obesity. The lower melanin content the higher possibility of microangiopathy. This is a first report in the literature devoted to distribution of melanin in the skin of type 1 diabetic patients.

Substrate specificity of human matriptase-2.

Human matriptase-2 is an enzyme that belongs to the family of type II transmembrane serine proteases. So far there is a limited knowledge regarding its specificity and protein substrate(s). One of the identified natural substrates is hemojuvelin, a protein involved in the control of iron homeostasis. In this work, we describe the synthesis and evaluation of internal quenched substrates using a combinatorial approach. The iterative deconvolution of two libraries to define the specificity of matriptase-2 yielded to the identification of the substrate ABZ-Ile-Arg-Ala-Arg-Ser-Ala-Gly-Tyr(3-NO2)-NH2 with a k(cat)/K(m) value of 4.5 × 10(5) M(-1) × s(-1), i.e. the highest specificity constant reported so far for matriptase-2.

A new proteinase 3 substrate with improved selectivity over human neutrophil elastase.

We report the synthesis and enzymatic studies on a new proteinase 3 intermolecular quenched substrate with enhanced selectivity over neutrophil elastase. Using combinatorial chemistry methods, we were able to synthesize the hexapeptide library with the general formula ABZ-Tyr-Tyr-Abu-X1'-X2'-X3'-Tyr(3-NO2)-NH2 using the mix and split method. The iterative deconvolution of such a library allowed us to obtain the sequence ABZ-Tyr-Tyr-Abu-Asn-Glu-Pro-Tyr(3-NO2)-NH2 with a high specificity constant (kcat/KM=1534×10(3)M(-1)s(-1)) and superior selectivity over neutrophil elastase and other neutrophil-derived serine proteases. Moreover, using the obtained substrate, we were able to detect a picomolar concentration of proteinase 3 (PR3). Incubation of the above-mentioned substrate with neutrophil lysate resulted in a strong fluorescent signal that was significantly reduced in the presence of a PR3 selective inhibitor.

Hybrid analogues of SFTI-1 modified in P1 position by ß- and γ-amino acids and N-substituted ß-alanines.

A series of compounds containing either non-proteinogenic ß-/γ-amino acids or N-substituted ß-alanine residues (ß-peptoid units) in P1 specificity position were synthesized based on the structure of sunflower trypsin inhibitor 1 (SFTI-1). The compounds were synthesized on a solid support; the N-substituted ß-alanines (ßNhlys and ßNhphe) were introduced into a peptidomimetic chain via a two-step approach using acryloyl chloride and an appropriate primary amine. The inhibitory activities were characterized in vitro against bovine α-chymotrypsin or bovine ß-trypsin. Three analogues displayed activity comparable to fully proteinogenic counterparts-monocyclic SFTI-1 and [Phe(5)]SFTI-1. Moreover, all active peptidomimetics were resistant toward proteolytic degradation, even after 24-h incubation at room temperature.

Correction factors for determination of annual average radon concentration in dwellings of Poland resulting from seasonal variability of indoor radon.

The method for the calculation of correction factors is presented, which can be used for the assessment of the mean annual radon concentration on the basis of 1-month or 3-month indoor measurements. Annual radon concentration is an essential value for the determination of the annual dose due to radon inhalation. The measurements have been carried out in 132 houses in Poland over a period of one year. The passive method of track detectors with CR-39 foil was applied. Four thermal-precipitation regions in Poland were established and correction factors were calculated for each region, separately for houses with and without basements.

Substrate specificity and inhibitory study of human airway trypsin-like protease.

Human airway trypsin-like protease (HAT), also referred to as TMPRSS11D, is an important physiological enzyme with the main activity pronounced in an airway. In this work we have described the substrate specificity and selectivity study of the protease, performed by the combinatorial approach. Fluorogenic/chromogenic tetrapeptide library was used for this purpose. The most efficiently hydrolyzed substrates' sequences that we selected were ABZ-Arg-Gln-Asp-Arg(Lys)-ANB-NH(2). The most active inhibitor with C-terminal Arg residue underwent detectable proteolysis action in the presence of 35pM of HAT. Based on the selected sequences the two peptide aldehydes were synthesized and (Abz-Arg-Gln-Asp-Arg(Lys)-H) were found to be an effective HAT inhibitor, working in nanomolar range with inhibition constant 54nM and 112nM, respectively.

Human cancer gene therapy with cytokine gene-modified cells.

Cytokines can impede tumour growth and activate innate and adaptive immune responses, leading to elimination of cancer cells. For many years, it was believed that systemic administration of recombinant cytokines might become a standard treatment of different cancer types. However, due to a high toxicity of therapeutic doses and a low efficacy, even in combination with chemotherapy, this strategy is generally not accepted. On the other hand, cancer gene therapy approaches utilising cells modified with cytokine genes seem to represent a novel promising approach. For the last decade, numerous Phase I and II clinical trials evaluating different therapies based on cytokine gene-modified cells have been carried out. In the early studies, several strategies have been shown to improve clinical outcomes and induce strong antitumour immune responses. Recently, a few prospective, randomised, Phase III clinical trials have been initiated in order to finally determine the efficacy of particular cancer immunogene therapy strategies. This article reviews the present status and perspectives of clinical trials of cancer immunotherapies utilising cytokine gene-modified cells.

The 34th Annual Meeting of the German Society of Immunology with participation of the Polish Society of Experimental and Clinical Immunology.

The 34th Annual Meeting of the German Society of Immunology was held in Berlin on 24 - 27 September 2003. This meeting, organised for the first time in cooperation with the Polish Society for Experimental and Clinical Immunology, gathered 1200 participants, mostly from central Europe. The programme comprised > 30 symposium lectures and > 750 oral and poster presentations. The main concept of this meeting was based on the rule of ABC--Applied, Basic and Clinical immunology. The state-of-the-art lectures devoted to immuno-based therapies provided by experts in the particular fields discussed some well-known therapeutic approaches. However, several workshop presentations demonstrated novel approaches employing biological therapies. These lectures are the focus of these meeting highlights.

Genetically modified dendritic cells--a new, promising cancer treatment strategy?

Dendritic cells (DCs), the most potent antigen-presenting cells (APCs), were discovered almost 30 years ago. Due to the priming of antigen-specific immune responses mediated by CD4+ and CD8+ lymphocytes, DCs are crucial for the induction of adaptive immunity against cancer. Therefore, vaccination of cancer patients with DCs presenting tumour-associated antigens (TAAs) have been believed to be a promising anticancer strategy. Multiple clinical trials have been carried out in order to evaluate the safety and efficacy of cancer vaccines based on antigen-pulsed DCs. However, pulsing of DCs with particular peptides has several disadvantages: i) short-time duration of antigen-major histocompatability complex (MHC) complexes, ii) a requirement for matching defined peptides with MHC complexes and iii) exclusive presentation of single antigen epitopes. Application of gene transfer technologies in the field of DC-based vaccines made possible the development of novel, anticancer immunisation strategies. In several animal models, DCs modified with genes encoding TAA or immunostimulatory proteins have been shown to be effective in the induction of antitumour immune responses. Based on these encouraging results, a first clinical trial of prostate cancer patients vaccinated with gene modified DCs has recently been initiated. In this article, methods used for genetic modification of DCs and anticancer vaccination strategies based on genetically modified DCs are reviewed.

The role for interleukin-12 therapy of cutaneous T cell lymphoma.

Recent phase I and phase II trials using recombinant human interleukin-12 (rhIL-12) for cutaneous T cell lymphoma (CTCL) have been completed. Observations on 32 evaluable patients revealed an overall response rate approaching 50 percent. Biopsy of regressing lesions revealed an increase in numbers of CD8+ and/or TIA-1+ T cells. These results suggest that rhIL-12 may induce lesion regression by augmenting antitumor cytotoxic T cell responses. Future trials will be focused on strategies for further immune enhancement by the concomitant use of additional immune augmenting cytokines with rhIL-12.

Dysregulation of lymphocyte interleukin-12 receptor expression in Sézary syndrome.

Initial phase I and II clinical trials with recombinant human interleukin-12 have demonstrated the therapeutic efficacy of this cytokine in early stage cutaneous T cell lymphoma as compared with more advanced stages such as the leukemic Sézary syndrome. In an effort to optimize the use of recombinant human interleukin-12, using flow cytometry we studied the regulation of the interleukin-12 receptor beta1 (high affinity chain) and beta2 (chain necessary for interleukin-12 signal transduction) on normal volunteer CD4+ and CD8+ T cells and CD4+ and CD8+ cells from eight patients with different degrees of leukemic involvement with Sézary syndrome. The beta1 chain was not readily detectable on resting normal and T cells from Sézary patients, but expression was induced following T cell activation with phytohemagglutinin. Similarly, the beta2 chain was not detectable on resting normal volunteer T cells, but could be induced following phytohemagglutinin stimulation. Moreover, the beta2 chain on normal volunteer T cells was markedly upregulated following short-term culture with interferon-gamma or recombinant human interleukin-12. CD8+ T cells routinely exhibited a greater expression of beta2 than did CD4+ T cells. In marked contrast, both CD4+ and CD8+ T cells from patients with Sézary syndrome and a high tumor cell burden (> 50% circulating atypical Sézary T cells) failed to express the beta2 chain under any culture conditions. Although, culture with anti-interleukin-10 also markedly increased beta2 expression on normal volunteer T cells, this failed to induce expression on either CD4+ or CD8+ T cells from Sézary patients and a high tumor burden. Investigation of patients with Sézary syndrome and a low tumor cell burden (< 15% circulating Sézary T cells) revealed a pattern of beta2 expression that was intermediate between advanced Sézary syndrome and normal volunteers. Both CD4+ and CD8+ peripheral blood T cells from these earlier stage patients were induced to express the beta2 chain, although at a lower frequency of positivity than T cells from normals, following culture with phytohemagglutinin, interferon-gamma, recombinant human interleukin-12, or anti-interleukin-10. These results indicate that short-term culture with interferon-gamma and recombinant human interleukin-12 potently upregulates beta2 chain expression on T cells from normal volunteers, whereas a similar, but less marked effect occurs on T cells from Sézary syndrome patients and a low circulating tumor cell burden. In contrast, the beta2 chain appears to be suppressed on both CD4+ and CD8+ T cells from Sézary patients with a heavy circulating tumor cell burden and it is not induced by interferon-gamma or recombinant human interleukin-12. Therefore, recombinant human interleukin-12 is likely to be most effective for early stage cutaneous T cell lymphoma due to a greater display of beta2 receptors on responding CD8+ anti-tumor cytotoxic T cells.

IL-12 suppression during experimental endotoxin tolerance: dendritic cell loss and macrophage hyporesponsiveness.

Endotoxin tolerance, the transient, secondary down-regulation of a subset of endotoxin-driven responses after exposure to bacterial products, is thought to be an adaptive response providing protection from pathological hyperactivation of the innate immune system during bacterial infection. However, although protecting from the development of sepsis, endotoxin tolerance also can lead to fatal blunting of immunological responses to subsequent infections in survivors of septic shock. Despite considerable experimental effort aimed at characterizing the molecular mechanisms responsible for a variety of endotoxin tolerance-related phenomena, no consensus has been achieved yet. IL-12 is a macrophage- and dendritic cell (DC)-derived cytokine that plays a key role in pathological responses to endotoxin as well as in the induction of protective responses to pathogens. It recently has been shown that IL-12 production is suppressed in endotoxin tolerance, providing a likely partial mechanism for the increased risk of secondary infections in sepsis survivors. We examined the development of IL-12 suppression during endotoxin tolerance in mice. Decreased IL-12 production in vivo is clearly multifactorial, involving both loss of CD11c(high) DCs as well as alterations in the responsiveness of macrophages and remaining splenic DCs. We find no demonstrable mechanistic role for B or T lymphocytes, the soluble mediators IL-10, TNF-alpha, IFN-alphabeta, or nitric oxide, or the NF-kappaB family members p50, p52, or RelB.