Induction of systemic antitumour resistance with targeted polymers.

Conjugates based on N-(2-hydroxypropyl)methacrylamide (HPMA) represent a new generation of antibody-targeted polymeric anticancer drugs with both cytotoxic and immunoprotecting/immunomobilizing activity. 20-90% of mice that are cured of EL4 mouse T-cell lymphoma, BCL1 mouse B-cell leukaemia and 38C13 mouse B-cell lymphoma by injection of doxorubicin-HPMA conjugate develop a long-lasting memory and systemic antitumour resistance. It is suggested that the main activity of the polymeric drug, directly after application is - due to the high level of the drug - of cytotoxic and cytostatic nature. Thereafter, long-term conjugates persist at low concentration in the circulation, which are capable of mobilizing the defence mechanisms of the host. Until now, seven patients with generalized carcinoma were treated with doxorubicin-HPMA-human-Ig conjugate. Disease stabilization, lasting from 6 to more than 18 months, was recorded.

Acquired and specific immunological mechanisms co-responsible for efficacy of polymer-bound drugs.

We present data providing new evidence that poly[N-(2-hydroxypropyl)methacrylamide] (PHPMA)-bound drugs, unlike free drugs, have both cytostatic and immunomobilizing activity (CIA). Immediately after injection, due to the high level of the drug, the main activity of the polymeric conjugate is cytotoxic and cytostatic. Later on, long-term circulating PHPMA-bound drug, at concentrations lower than its minimal inhibitory levels, mobilizes the defense mechanisms of the host. Cytotoxic and cytostatic effects of drug-PHPMA were repeatedly confirmed. The following data support the concept of the immunomobilizing activity of the N-(2-hydroxypropyl)methacrylamide (HPMA) conjugates: (a) pre-treatment with free drugs (doxorubicin, cyclosporin A) accelerates the appearance of EL4 mouse T-cell lymphoma while a similar pre-treatment with doxorubicin-PHPMA induces limited but definitive mobilization of the host's defense mechanisms; (b) mice cured of EL4 mouse T-cell lymphoma, BCL1 mouse B-cell leukemia and 38C13 mouse B-cell lymphoma by injection of doxorubicin-PHPMA conjugate targeted with monoclonal antibodies (anti-Thy 1.2 for EL4, anti-B1 for BCL1 and anti-CD71 for 38C13) and re-transplanted with a lethal dose of the same cancer cells survive without any treatment considerably longer than control mice; (c) increased NK activity and anti-cancer antibody was detected only in animals treated with doxorubicin-PHPMA conjugate; and (d) considerably increased NK and LAK activity was seen in a human patient treated for generalized breast carcinoma with doxorubicin-PHPMA-IgG.

[GBV-C/HGV, a new virus as one possible cause of hepatitis of unknown etiology]. / Nový virus GBV-C/HGV jako jeden z mozných puvodcu etiologicky nevyjasnených hepatitid.

For etiologically obscure (some 4%) viral hepatitis agents are sought and tested to make elucidation of their cause possible. One of the candidates is since 1995 the newly discovered virus GBV-C/HGV. Despite intense research its relationship to viral hepatitis of obscure origin (VHN) has not been elucidated so far. In the submitted paper the authors attempted to contribute to the elucidation of etiological associations of GBV-C/HGV infection and VHN by comparing the dynamics of markers of the infection in a group of 59 patients with VHN, two control groups exposed to a high risk of parenteral operations and a third comparative group. The first control group comprised 64 patients in a long-term haemodialyzation programme (HD), the second group was formed by 82 patients with haematooncological disease (BD). The third comparative group comprised 22 patients coinfected (CI) with virus of hepatitis C (VHC), or possibly hepatitis B (VHB). The patients with VHN were HBsAg, anti HCV and anti HEV negative. In the majority in the first blood sample transaminases were elevated which was one of the main reasons for examination of GBV-C/HGV RNA. Prevalence of GBV-C/HGV infection, proved by the presence of at least one of the two markers of current or past infection (GBV-C/HGV RNA, antiGBV-C/HGV) was in the compared VHN, HD and BD groups as follows: 88.1%, 59.4% and 43.9%. The frequency of GBV-C/HGV positivity was highest in VHN-76.3%. In control groups HD and BD GBV-C/HGV RNA positivity was substantially lower, 18.8% and 25.6% resp. Long-term continuous viraemia was recorded in patients with VHN in 18.6%. In groups HD and BD it was half that value: 9.3% and 9.18%. In patients with VHN surprisingly after 6.5 months a marked rise of negative findings occurred (5.6x) without the expected increase of antibodies. A similar finding was recorded also in the other groups (HD and BD), incl. CI patients. Disappearance of viraemia was observed most frequently in VHN (55.9%). In groups HD and BD GBV-C/HGV RNA disappeared only in 7.8% and 12.1% resp. In treated patients of the CI group viral RNA was present in 45.5% and it disappeared in 36.4%. On the other hand, seroconversion to antibodies was comparable in VHN, HD and BD (11.9%, 9.4%, 8.5%), only in group CI it was higher (18.2%), obviously in conjunction with treatment of concurrent HCV or HBV infection. Disappearance of viraemia without subsequent seroconversion occurs in GBV-C/HGV infection frequently, the highest rate was observed by the authors in patients with VHN. Disappearance of viraemia does not necessarily imply clearance of GBV-C/HGV but may be due to a change of GBV-C(HGV into a state of persistence without positive laboratory markers of the infection. Persistence of the virus could also be the reason of the assumed conditioned pathogenicity of the virus, and the effect of frequent disappearance of both markers could explain some controversial epidemiological observations when in studies only static data without dynamic associations were used.

Comparison of different immunoassays for CA 19-9.

We compared six routinely employed immunoassay kits: Architect i2000 and AxSYM, Abbott Laboratories; Elecsys 2010, Roche Diagnostics; ELSA, CIS-BioInternational; Immulite 1, Diagnostic Products Corporation; and IRMA-mat, Byk-Sangtec Diagnostica. Using all analytical systems, we measured identical groups of clinical samples completed with selected control samples. The repeatability of measurements (coefficient of variation) ranged from 2.1% (Elecsys 2010) to 6.7% (ELSA). The parameters of Passing-Bablok regression show significant systematic differences among analytical systems. Data from a Bland-Altman diagram suggest that these differences project onto other, still more significant individual differences among individual samples. Though the cut-off values differ between various systems, no similar clinical efficacy appears to be attained. The behavior of individual systems is quite different for identical control materials and does not necessarily duplicate the calibration for biological samples. The results of determining CA 19-9 cannot be extrapolated from one analytical technique to another, even in cases where the same monoclonal antibody is used. Standardization of CA 19-9 measurement systems is necessary to allow use of the results for the purposes of evidence-based medicine.

Immunolocalization of cyclophilin in normal and cyclosporin A-treated human lymphocytes.

A polyclonal rabbit antibody against a protein fraction (10-30 kDa) of human thymuses with a high CsA-binding activity of dominant protein cyclophilin (CPH) was prepared and characterized. In immunoblotting with the cell lysate from JURKAT T cell line, this antibody specifically reacted with 18-kDa protein corresponding to CPH. In indirect immunofluorescence the antibody visualized granular structures in JURKAT cells and human peripheral blood lymphocytes. In JURKAT cells cultivated with 1-2 micrograms of CsA per ml for 7 days a much weaker reaction of the antibody was found, compared with non-treated cells. In some CsA-treated cells the antibody visualized various 'star-like' or filamentous structures. A similar staining pattern has also been obtained in lymphocytes of the patients receiving CsA therapy. Complementary staining with rhodamine-tagged phalloidin revealed changes in F-actin distribution of CsA-treated JURKAT cells. In conclusion, the treatment with CsA induces dramatic changes of CPH cellular distribution, which may take part in the final therapeutic effect of the drug.

A natural specific intracellular ligand as a reagent for cyclosporine blood level determination.

We isolated and characterized a non-immunoglobulin fraction from calf thymuses which was found very active in binding [3H]labelled cyclosporine (CsA). It contains the natural, intracellular receptor cyclophilin, specifically binding to the active site of the CsA molecule, which may also play a part in the immunosuppression of human immunocompetent cells. This material with a high selectivity could be used, instead of specific monoclonal or polyclonal antibodies, to determine CsA levels in patient's blood.

[Microalbuminuria--a risk indicator for the development of nephropathy in insulin-dependent diabetics]. / Mikroalbuminurie--indikátor rizika vývoje nefropatie u inzulíndependentního diabetu.

The authors submit preliminary results of a prospective study in 65 insulin-dependent diabetics with a varying duration of the disease where they followed up the development of microangiopathic organ changes in relation to the compensation of diabetes and the development of clinically manifest proteinuria or albumin excretion (microalbuminuria). From the results ensues that in recent and postrecent patients the increased albumin excretion is as a rule only a manifestation of metabolically conditioned and treatable changes of renal function and is of minor importance for the prediction of the risk of development of diabetic nephropathy. In patients with prolonged duration of diabetes the position is not unequivocal and if the albumin excretion persists or increases despite intensive insulin treatment it is most probably an indicator of a high risk of development of diabetic nephropathy.

[Methodologic aspects of immunoperoxidase technics]. / Metodické aspekty imunoperoxidázových technik.

In attempt to standardize immunoperoxidase tests many partial methodological experiments were performed and the results discussed. The proved processing included purification of peroxidase conjugates, sequential administering in double indirect immunoperoxidase method (double sandwich). A high level dilution of primary antibodies and lengthening of incubation were recommended. Silicagel and vaseline rim round sections were found advantageous for improving adhesion of paraffin sections.

Microalbuminuria--a marker of the risk of developing nephropathy in insulin-dependent diabetes.

The authors present partial results of a prospective study conducted in 65 insulin-dependent diabetics with varying duration of disease in whom development of micro-angiopathic organ alterations is followed in relation to diabetes compensation and development of clinically manifest proteinuria or to albumin excretion (microalbuminuria). The results suggest that the increase in albumin excretion in recent-onset and non-recent-onset patients is in most cases only an expression of changes in renal function due to metabolism and therapy and apparently of little value in predicting the risk of developing diabetic nephropathy. The situation is not so unambiguous in patients with long duration of diabetes and, in case increased albumin excretion remains unchanged or further increases despite intensive insulin therapy, it may serve most likely as a marker of high risk of developing diabetic nephropathy.

Peroxidase labelled mumps virus antigens and their application in IgM capture immunoassay: first experience.

Three types of mumps virus antigen have been prepared: virion antigen (MV); soluble nucleoprotein (NP) antigen; envelope glycoprotein (eGp) antigen. These antigens were coupled directly with horse-raddish peroxidase and tested by IgM-capture technique against selected human sera. The advantage of the suggested design, in addition to general superiority of the IgM-capture systems, lies in its selectivity achieved by introducing a monoclonal antibody for IgM binding and in time saving due to the use of labelled antigen without the loss of sensitivity and specificity. The assay fulfils the criteria for quick viral diagnostic. Our first experience on application of the labelled antigens in enzyme immunoassay (EIA) is described.

Monoclonal antibodies against horseradish peroxidase.

Four hybridomas making monoclonal antibodies against horseradish peroxidase have been established. Two of them are IgM and two are IgG1 with kappa light chains. Antibody HP-03 has been selected as the most suitable one for preparation of the peroxidase-anti-peroxidase complex. Furthermore, optimal conditions for construction and applications of the PAP complex in immunocytochemical and immunohistochemical methods are shown.

The gamma 1 heavy-chain disease FOR protein is present in two molecular forms.

Heavy chain disease proteins (FOR) were isolated from human plasma. These proteins were also detected immunochemically in the urine of the patient. The proteins were disulphide-linked Fc-like dimers with molar mass 64.2 kg/mol and sedimentation rate S020,W = 0.356 ps (3.56 S). Similar amounts of aspartic acid and pyroglutamic acid were found at the N-terminus. After cyanogen bromide cleavage of the FOR proteins, three peptides were isolated and their amino acid composition and partial amino acid sequence was determined. We suggest that two Fc-like proteins of similar sizes are present in the plasma: (1) the first with N-terminal aspartic acid corresponding to position 221 of gamma 1 EU chain and (2) the second with N-terminal pyroglutamic acid. The first protein and small amounts of related low-molar mass fragments found also in the plasma could be degradation products of the second protein. Evidence is given on structural differences between the FOR proteins and the corresponding portion of the gamma 1 EU chain.