Lactoferrin Prevents Susceptibility of WEHI 231 Cells to Anti-Ig-Induced Cell Death Promoting Cell Differentiation.

Immature B cells are susceptible to apoptosis due to ligation of surface immunoglobulin receptors. The WEHI 231 cell line represents a useful model to study the mode of action of factors preventing apoptosis. In this work we investigated the protective effects of multi-species lactoferrins in anti-mouse Ig-induced WEHI 231 cell death. Bovine milk-derived lactoferrin (bLF), recombinant human lactoferrin expressed in Chinese hamster ovary cells - rhLF(CHO) or in human endothelial kidney cells - rhLF(HEK), and recombinant mouse lactoferrin expressed in Chinese hamster ovary cells - rmLF(CHO), were used. Goat-anti-mouse Ig antibodies were used to induce cell apoptosis. Survival of WEHI 231 cells in culture was measured using the colorimetric MTT method. Expression of signalling molecules and subunits of interleukin 2 receptor was determined by the RT PCR method. The results showed that anti-mouse Ig antibodies inhibited cell growth in a dose-dependent manner. The lactoferrins alone had no effect on the cell survival. The cells exposed to LFs, prior to anti-Ig treatment, were rescued to a significant degree from cell death. Determination of the signalling molecule expression revealed almost complete suppression of caspase-3 and NF-κB1 by bLF in untreated cells, as well as deep suppression of caspase-3, block of Fas, and 4-fold increase of NF-κB1 in cells incubated with bLF prior to anti-Ig treatment. In addition, differential changes in the expression of interleukin 2 subunits upon bLF treatment were found, indicating a process of cell differentiation. In conclusion, we showed that LF-induced cell differentiation in immature B-cell line WEHI 231 was correlated with partial protection of the cells from anti-Ig-induced cell death.

A new specific and useful tool in differential diagnosis of periodontitis.

These studies were performed to confirm the diagnostic value of objective factors routinely assessed during clinical examination in an aim to distinguish between patients with chronic and aggressive periodontitis. Moreover, these factors were verified with subjective description of symptoms reported by patients. To obtain the appointed targets multinominal logistic regression analysis was used. Sixty three patients, aged from 21 to 56 years, treated in the Department of Periodontology of Wroclaw Medical University, were examined as to their oral hygiene and periodontal status. We determined Approximal Plaque Index (API), Papilla Bleeding Index (PBI), probing pocket depths of less than 4 millimetres, furcation involvement, patient's age and the time of appearance of the first symptoms of the disease. The measured data of each parameter was divided into two groups with the use of standard deviation and the multinominal logistic regression analysis. The obtained statistical results proved high clinical value of the studied parameters and gave evidence that the established protocol consisting of five objective factors and one subjective factor used in our clinical practice allowed for good diversification of patients into the two mentioned types of disease.

Alternative therapies in antibiotic-resistant infection.

CASE REPORT: A 24-year-old woman suffering from post-influenza otitis media infection was initially treated with several series of a steroid (Elocon) and a combination of steroids and antibiotics (Atecortin, Dicortineff) without significant medical benefit. The isolated bacterial strains were identified as Staphylococcus homis and Staphylococcus epidermidis. Specific phage therapy applied sequentially over a period of three weeks resulted only in a partial reduction in inflammation and limited improvement in overall health condition. Oral application of lactoferrin (LF; 50-mg daily oral doses for seven days with two-week intervals) led to a complete clearance of both bacterial strains and full recovery of the patient. The recovery was associated with increased myelopoiesis and a sustained elevation of serum endogenous LF. In conclusion, specific bacteriophage therapy combined with the administration of lactoferrin proved to be effective in the treatment of antibiotic-resistant external ear infection.

Alterations of the expression of T-cell-related costimulatory CD28 and downregulatory CD152 (CTLA-4) molecules in patients with B-cell chronic lymphocytic leukaemia.

In the present study, we have examined the kinetics and magnitude of expression of the CD28 and CD152 molecules on unstimulated and anti-CD3+rIL-2-stimulated peripheral blood CD4+ and CD8+ T cells in patients with chronic lymphocytic leukaemia (B-CLL) and controls. The mean percentages of both CD3+/CD4+/CD28+ and CD3+/CD8+/CD28+ cells were significantly lower in B-CLL than in controls before culture, decreased rapidly, reaching their lowest levels between 24 and 48 h, and returned to basal levels after 72 h of culture. In controls, the lowest proportions of CD3+/CD4+/CD28+ and CD3+/CD8+/CD28+ cells were found after 24 h and returned to prestimulation levels after 48 h of stimulation. We observed significantly higher proportions of unstimulated CD3+/CD4+/CD152+ and CD3+/CD8+/CD152+ cells in B-CLL patients than in controls. The highest percentages of CD3+/CD4+/CD152+ and CD3+/CD8+/CD152+ cells were observed in controls after 72 h, and in B-CLL patients after 24 h, and remained statistically higher after 48, 72 and 96 h of stimulation. CD152 molecule expression returned to prestimulation levels after 96 h of culture in controls, and after 120 h in B-CLL patients. The abnormal kinetics and levels of CD28 and CD152 expression on T cells in B-CLL may lead to a state of hyporesponsiveness or anergy and could be one of the mechanisms of immune deficiency in this disease.

Osteogenic properties of various HeLa cell lines and the BMP family genes expression.

Heterotopic ossicles were induced in thigh muscles of immunosuppressed mice by implantation of suspension of several HeLa cell lines. These ossicles are the object of our research on osteoporosis. It was found that various HeLa cell lines differ in their potential to induce osteogenesis. In the previous paper we demonstrated the involvement of bone morphogenetic proteins (BMP-4 and BMP-6) secreted by HeLa cells in this phenomenon. In the present paper we try to find out the reason of the heterogeneity of various cell lines regarding the differences in their osteoinductive potencies. By the use of semiquantitative RT-PCR the differences in mRNA expression for several isoforms of BMP proteins in examined HeLa cell lines were found. The presence or absence of some of the BMP isoforms seems to be correlated with the quantity of heterotopically induced mineralised tissues. This was measured by weighing the deposited mineral after digestion of soft tissues surrounding the induced ossicles. This finding is supporting the thesis on high and uncontrolled heterogeneity of various HeLa cell lines used all over the world.

Lactoferrin stimulates killing and clearance of bacteria but does not prevent mortality of diabetic mice.

We have previously shown that bovine lactoferrin (BLF) given intravenously (i.v.) protected mice against a lethal dose of Escherichia coli and strongly stimulated both the clearing and killing activities in liver, lungs, spleen and kidney. Since some studies indicated a reduction of the manifestation of experimental pancreatitis with lactoferrin (LF), we decided to examine the protective activity of BLF against lethal E. coli infection in animals with alloxan (Alx)-induced diabetes. It appeared that 48 h diabetes substantially lowered the killing activity in all four organs as well as the clearing rate of E. coli from the circulation. BLF given i.v. reduced this undesirable effect of diabetes. However, in 10- and 20-day diabetic animals, the diabetes alone stimulated the killing activity in the organs investigated, and upregulated the clearing rate of E. coli from the circulation. Lactoferrin significantly increased both the killing and the clearing activity in these long-term diabetic animals. In some cases the stimulating effect of BLF was very high, suggesting a concerted action of BLF and diabetes in that category of mice. Despite these beneficial effects of BLF and diabetes on the killing process in the investigated organs, the survival time of animals from all the diabetic groups (48 h, 10 and 20 days) was not prolonged by BLF. The protective properties of BLF did not depend on the blood glucose levels in the diabetic animals. BLF partly delayed the development of experimental Alx-induced diabetes, measured by the glucose level, but only if administered shortly after Alx injection. In conclusion, we demonstrated that the state of diabetes alone could increase killing of bacteria in the investigated organs and LF enhanced this process. However, LF had no protective effect against the mortality of diabetic mice infected with a lethal dose of E. coli.

Influence of some factors on immunoassays of human myoglobin.

Six ELISA variants exploiting two monoclonal antibodies, one rabbit antibody and their peroxidase conjugates were applied in assays of purified human myoglobin, apomyoglobin and the protein in human muscle extracts. The myoglobin was accurately determined with monoclonal antibody no. 82 used for coating of ELISA plates while assays performed with monoclonal antibody no. 49 or rabbit antibody used for coating were weak or none. Determinations of human apomyoglobin with ELISA variants were somewhat more sensitive than those of myoglobin. Obtained in this work results were compared with those done using commercial Seratec kit for immunoassay of human myoglobin. Addition to the muscle extracts not only concentrated salts but also acetone, ethanol, sodium dodecyl sulfate or some other denaturing agents markedly increased assays of myoglobin by ELISA with monoclonal antibody no. 49 and antibody no. 82 conjugated with peroxidase. Removal of acetone or ammonium sulfate from extracts resulted in dramatic decrease of the estimated myoglobin. Filtration of the extract through Bio-Gel A5m column did not affect low assays of myoglobin in fractions without pretreatment with acetone. Myoglobin was isolated from human heart extract by immunoaffinity chromatography on Sepharose-antibody no. 82 column and the isolated protein was identified by gel electrophoresis and Western blot.

Some parameters influencing immunoassay of human and horse myoglobins.

It was noted that human and horse sera as well as human heart and skeletal muscle homogenates or extracts distinctly decrease immunoassays of purified myoglobins. The assays of homogenate and extract myoglobins could be many times increased by precipitation certain proteins with concentrated ammonium sulfate or sodium chloride. Also in homogenates and extracts incubated for several days increased assays of myoglobins were noted. The obtained results indicate that both myoglobins occur in complex with other tissue component(s).

Investigations on human lutropin from pituitary gland and urine.

Two newly isolated monoclonal anti-beta hLH antibodies did not recognize both human chorionic gonadotropin (hCG) and three synthetic peptide fragments corresponding to beta hLH sequence. These antibodies were applied for ELISA of human luteinizing hormone (hLH), however, sensitivities of these assays were much lower than those with two other monoclonal antibodies obtained earlier in our laboratory. No significant differences between assays of hLH from pituitary and urine with 6 different pairs of monoclonal antibodies (4 anti-beta and 1 anti-alpha-subunit) were noticed. The highest hLH concentration in urine samples collected during 53 menstrual cycles of 6 women was demonstrated at different times of the day. In 14 out of 52 "preovulatory urine" portions, preserved for several weeks, over 80% increase of assayed hLH was shown. The highest assays of hLH from pituitary and urine were noted at pH 7.5-9.0. Using affinity chromatography the whole pituitary hLH was bound to ConA-Sepharose column and was eluted with alpha-methyl-D-mannoside as a single peak. However, a small part of hLH from urine was eluted from the column as non retarded peak. When HPLC analysis with DEAE column was performed, pituitary hLH was separated in two fractions while lutropin from urine was eluted as a single peak.

[Isoforms of luteinizing hormone]. / Izoformy hormonu luteinizujacego.

Luteinizing hormone (LH) is a heterodimeric glycoprotein containing varied amount of sialic acid. This is a reason of numerous LH isoforms called also isohormones. The hormone isoforms were separated usually by gel electrophoresis, isoelectrofocusing or chromatofocusing. They differ in biological and immunological activity. Human and some animals LH isoforms were reviewed. Also some genetic mutants of LH are described. Problems of the human isoforms for pathology and diagnostics are presented.

Antigenicity of human and horse myoglobins.

Rabbit antisera against human myoglobin and horse myoglobin cross-reacted with both myoglobins but only one of them recognized human hemoglobin. Two mouse monoclonal antibodies anti-human myoglobin were obtained, but only one of them (No. 49) cross-reacted with horse myoglobin. Antibody No. 49 and rabbit antibodies reacted also with apo-, FITC- and treated with hydrochloric acid or TPCK-trypsin horse myoglobin, but their binding to myoglobin pretreated with NaOH was reduced. Thirteen peptides overlapping sequence of human myoglobin were synthesized on polyethylene pins. Rabbit and mouse polyclonal antibodies reacted with some of these peptides but no reaction was noted with mouse monoclonal antibodies. Two monoclonal antibodies were applied for specific immunoassay of human myoglobin.

[Human myoglobin (hMb) and its diagnostic value]. / Ludzka mioglobina (hMb) i jej wartosc diagnostyczna.

Composition, properties and occurrence of human myoglobin (hMb) were described. Methods for immunological assay of hMb were presented. Localization of antigenic determinants in myoglobin molecule was shown. Value of assay of hMb in serum and urine for early diagnosis of myocardial infarct and some other muscle diseases was discussed.

[Immuno-contraceptive vaccines]. / Szczepionki immunoantykoncepcyjne.

Results of studies on birth control vaccines accomplished by many research groups within the WHO Special Programe of Research, Development and Research Training in Human Reproduction has been reviewed. The mostly investigated contraceptive vaccines contain human chorionic gonadotropin, and therefore structure, biological role and immunogenicity of the hormone were presented. Also, vaccines obtained on the basis of subunit beta of the human chorionic gonadotropin, its heterodimer with subunit beta of ovine lutropin or C-terminal peptide of beta subunit were described. Results of two phases of clinical trials with immuno-contraceptive vaccines were presented. The use of contraceptive vaccines for treatment of some trophoblastic tumors and the idea of using contraceptive recombinant vaccines were also mentioned.

Human serum immunoglobulins binding to synthetic fragments of human chorionic gonadotropin.

Human sera obtained from healthy individuals, patients with cancer diseases, infertile and pregnant women were investigated for the presence of immunoglobulins (Ig) binding to synthetic fragments corresponding to sequence of human chorionic gonadotropin (hCG). Four synthetic peptides corresponding to some regions of alpha hCG and beta hCG subunits, coupled to polystyrene beads and 16 peptides overlapping sequences of alpha- and beta hCG subunits, synthesized on polyethylene pins, were used. IgG and IgM bound to hCG-synthetic peptides were assayed using goat or mouse monoclonal antibodies anti-human IgG labeled with horseradish peroxidase. Most of the investigated sera contained IgG which bound to examined synthetic peptides, but obtained results were dependent on serum donors. Some relationships between results obtained with peptides synthesized on modified pins according to Geysen et al. method and corresponding peptides synthesized by Merrifield method and coupled to beads were noted.

Stability of human lutropin studied with immunoenzymatic technique.

Characteristics of two monoclonal antibodies against human lutropin and their use in enzyme immunoassays of the hormone are presented. Diluted solution of pituitary lutropin was unstable in buffered saline and could be stabilized with some proteins. Concentration of the lutropin in woman urine, collected during "lutropin-peak", was markedly increased during long time storage at 5 degrees C or at room temperature. Using HPLC it was demonstrated that pituitary lutropin, normal urine lutropin and "increased" urinary lutropin were eluted from ion exchange column almost with the same retention time.

[Human luteinizing hormone (hLH)]. / Ludzki hormon luteinizujacy (hLH).

Heterodimeric composition and amino acid sequence of hLH is reviewed. The hormone isoforms and methods of their assay are summarized. Releasing of the hormone, its control by synthetic antagonists and agonists and their significance for therapy are described. Structure and function of hLH receptor are presented.

Effect of pretreatment of wells in polystyrene plates on adsorption of some human serum proteins.

Coating of Immulon polystyrene plates with 0.2% casein in phosphate buffered saline (PBS) completely abolishes adsorption of human serum proteins to these plates. However, pretreatment of such plates with PBS or 0.15 M NaCl in deionized water (10 microS) before coating makes possible adsorption of human immunoglobulins G (HIgG) and M but not serum albumin (HSA) and transferrin (HTrf). Effect of pretreatment with PBS on adsorption of HIgG is long-term and rather stable. Results of experiments with radioiodinated HIgG, mouse IgG, HSA and human chorionic gonadotropin confirm the peculiar effect of pretreatment with PBS on casein coating. Pretreatment with deionized water, instead of PBS, markedly diminish adsorption but tap or commercial spring waters (> 1000 microS), even without 0.15 M NaCl, affect adsorption similarly to PBS in deionized water. Super pure water (0.05 microS) even with NaCl used for pretreatment does not influence adsorption of HIgG to plates coated with casein. Distinct differences in adsorption of HIgG and HTrf was demonstrated using solutions for ELISA prepared from super pure, deionized and tap waters.

Search for antibodies for immunoenzymatic assay of human lutropin.

Five peptides corresponding to human lutropin (hLH) subunit fragments were synthesized by a solid phase method and their physicochemical characteristics are presented. Antibodies induced in rabbits with 3 peptides of beta hLH fragments coupled to thyrotropin did not bind hLH and human chorionic gonadotropin (hCG). Also 2 synthetic peptides of alpha hLH fragments did not react with rabbit anti-hLH, anti-hCG and anti-alpha hCG antibodies. Using 2 monoclonal anti-alpha hCG and anti-beta hLH antibodies a sensitive sandwich ELISA technique was elaborated. Using this technique stability of hLH was investigated. The ELISA was also applied for assays of urine hLH in normal and ovulation days.

Goat antibodies to amino acid sequences of human chorionic gonadotropin (hCG)

Human chorionic gonadotropin, its two subunits and its conjugate with thyroglobulin were used for immunization. The strongest immunogens were demonstrated to be the intact hormone and beta subunit, the weakest was alpha subunit. Using three peptides corresponding to hormone subunits coupled to Sepharose 4B various antibodies were isolated from goat antiserum by immunoaffinity chromatography. Specificity of the purified antibody preparations was studied. It was demonstrated that enzyme linked immunosorbent assay with two goat antibodies--one for coating of microtiter plates and the other one conjugated with peroxidase--is suitable for sensitive assays of both human chorionic gonadotropin and luteinizing hormone. Specific and sensitive assays were performed using combination of goat antibody anti-122-145-beta hCG and monoclonal antibody anti-alpha hCG.