Immune Cells and Immunosenescence.

Aging is associated with progressive loss of physiological integrity, leading to impaired physical and mental functions as well as increased morbidity and mortality. With advancing age, the immune system is no longer able to adequately control autoimmunity, infections, or cancer. The abilities of the elderly to slow down undesirable effects of aging may depend on the genetic background, lifestyle, geographic region, and other presently unknown factors. Although most aspects of the immunity are constantly declining in relation to age, some features are retained, while e.g. the ability to produce high levels of cytokines, response to pathogens by increased inflammation, and imbalanced proteolytic activity are found in the elderly, and might eventually cause harm. In this context, it is important to differentiate between the effect of immunosenescence that is contributing to this decline and adaptations of the immune system that can be quickly reversed if necessary.

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.

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.

Lactoferrin accelerates reconstitution of the humoral and cellular immune response during chemotherapy-induced immunosuppression and bone marrow transplant in mice.

Experimental evidence from previous studies supports the conclusion that orally administered lactoferrin (LF) restores the immune response in mice treated with a sublethal dose of cyclophosphamide (CP). The aim of this study was to elucidate potential benefit of LF in mice undergoing chemotherapy with busulfan (BU) and CP, followed by intravenous (i.v.) injection of bone marrow cells. CBA mice were treated orally with busulfan (4 mg/kg) for 4 consecutive days, followed by two daily doses of CP delivered intraperitoneally (i.p.) at a dose of 100 mg/kg and reconstituted next day with i.v. injection of 10(7) syngeneic bone marrow cells. One group of these mice was given LF in drinking water (0.5% solution). After treatment, mice were immunized with ovalbumin (OVA) to subsequently measure delayed type hypersensitivity responsiveness and with sheep red blood cells to determine humoral immunity by evaluation of splenic antibody-forming cells. As expected, both humoral and cellular immune responses of mice that were treated with these chemotherapeutic agents was markedly impaired. Here we report that this impairment was remarkably attenuated by oral administration of LF. Humoral immunity fell to levels that were 66-88% lower than that of untreated animals. Humoral immunity of LF-treated animals was equivalent to that of untreated mice within 1 month. Cellular immune responses were inhibited by chemotherapy treatment to a lesser degree, reaching levels that were approximately 50% lower than those of untreated animals. Again, LF mitigated this decrease, resulting in responses that were only slightly lower than those observed in untreated animals. Furthermore, when mice were given a lethal dose of BU (4 x 25 mg daily doses, i.p.) followed by a bone marrow transplant, LF caused enhanced lympho-, erythro-, and myelopoiesis in the bone marrow and appearance of transforming splenic lymphoblasts, similar to effects caused by administration of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF). In summary, our study suggests that LF may be a useful agent to accelerate restoration of immune responsiveness induced by chemotherapy in bone marrow transplant recipients.

Protective effects of lactoferrin in Escherichia coli-induced bacteremia in mice: relationship to reduced serum TNF alpha level and increased turnover of neutrophils.

OBJECTIVE AND DESIGN: Previous studies demonstrated that lactoferrin (LF), given intravenously (i.v.), 24 h before lethal Escherichia coli ( E. coli) infection, protects mice against mortality. The aim of this investigation was to determine whether downregulation of serum TNF alpha activity and increase of neutrophil number in the circulation and bone marrow by LF could contribute to the protective action of LF against E. coli-induced sepsis. MATERIAL AND SUBJECTS: CBA female mice, 10-12 week old, weight 20-22 g, were used. TREATMENT: Mice were given 10 mg LF i.v. either 2 h or 24 h before i.v. administration of lethal dose of E. coli (5 x 10(8)). METHODS: Serum activities of TNF alpha and IL-1 were determined by bioassays 2 h following E. coli or LF injection. The blood and bone marrow smears were stained with Giemsa and May-Grünwald reagents and reviewed histologically. RESULTS: LF given 24 h before E. coli caused a 60% reduction of TNF alpha released into circulation. However, pretreatment of mice with LF 2 h before bacterial challenge resulted in strong (15 fold) increase of TNF alpha serum level. Analysis of bone marrow cell composition revealed a significant increase in neutrophil lineage cell content (myelocytes, bands and mature neutrophils) following 24 h pretreatment with LF (51.8% of the total cell count), versus PBS control (32.7%) and 2 h LF pretreatment (35.8%). The percentage of neutrophils (bands and mature forms) in the peripheral blood rose to 47.4% versus 32% and 32%, respectively. Intravenous administration of LF increased also interleukin 1 (IL-1) concentration in the circulation of noninfected mice. CONCLUSIONS: This investigation has added more information regarding the mechanism of the protective action of LF in E. coli-induced bacteremia by revealing the phenomenon of accelerated neutrophil recruitment and down-regulation of E. coli-induced TNF alpha serum level.

Effects of lactoferrin on IL-6 production by peritoneal and alveolar cells in cyclophosphamide-treated mice.

Previous studies have shown that oral treatment with lactoferrin (LF) restores the immune response in cyclophosphamide (CP) immunocompromised mice. The aim of the present investigation was to determine the regulatory ability of LF on the production of interleukin 6 (IL-6) in peritoneal and alveolar cells, derived from CP-treated mice. CBA mice were injected with a single, intraperitoneal (i.p.) dose of CP (350 mg/kg body weight) followed by LF administered in drinking water (0.5% solution) for 21 days. The control counterparts were given water. Peritoneal and alveolar cells were isolated from mice and the production of IL-6, both spontaneous and lipopolysaccharide (LPS) induced, was determined in 24h cell cultures using a bioassay. The results showed increased production of IL-6 in both CP-treated mice and in mice given, in addition, LF. The administration of LF alone led also to an increase in IL-6 production by the cell cultures. Intravenous (i.v.) administration of LPS resulted in a significant increase in IL-6 serum levels in CP and CP/LF but not in LF-treated mice. Analysis of cell type composition in the peritoneal cavity revealed a strong increase in mastocyte and neutrophil content in CP and CP/LF-treated groups. Our findings suggest that enhanced IL-6 production in CP and CP/LF-treated mice may contribute to reconstitution of immune system function in immunocompromised mice.

Bovine lactoferrin decreases histopathological changes in the liver and regulates cytokine production by splenocytes of obstructive jaundiced rats.

OBJECTIVE AND DESIGN: The aim of this study was to evaluate effects of bovine lactoferrin (BLF) on histopathological changes in the liver of 14 day obstructive jaundiced (OJ) rats and production of tumor necrosis factor (TNF-alpha) and interleukin 6 (IL-6) by splenocytes from 7- and 14-day OJ rats. MATERIAL AND SUBJECTS: In the study 50 male rats of the Buffalo strain (170-270 g, mean 230 g) were used. TREATMENT: Rats were given 10 mg BLF in 0.5 ml saline daily, using a stomach tube. BLF was applied 2 days before operation and for 13 days following operation. METHODS: The specimens of liver were prepared using standard techniques. Sections, 5 mm thick were stained with hematoxylin and eosin and reviewed histologically. Microscopic estimation using semiquantitive 4-grade scale was used for evaluation of liver changes. For cytokine measurement splenocyte cultures were stimulated with 5 microg/ml lipopolysaccharide (LPS). After overnight incubation the activities of TNFalpha and IL-6 were determined using bioassays. For statistical evaluation of data the nonparametric Mann-Whitney test was applied. RESULTS: In rats with 14-day OJ, treated with BLF, the pathological changes in the liver were markedly reduced, in particular foci of necrosis with disseminated lymphocytes, cellular necrobiosis, bile duct proliferation and dilation. Neither proliferation of fibrous and reticular connective tissue nor activation of Kupffer cells was revealed. In the 7-day OJ rats treatment with BLF caused significant inhibition of both spontaneous (mean 253, median 275, vs mean 160, median 148 pg/ml, p=0.002) and LPS-induced TNF-alpha production (mean 4967, median 4102, vs mean 2291, median 2234 pg/ ml, p = 0.004) in the splenocyte cultures. The spontaneous (mean 120, median 81, vs mean 43, median 26 pg/ml, p=0.005) as well as LPS-induced IL-6 production (mean 422, median 378, vs mean 293, median 230 pg/ml, p = 0.025) were also lowered. On the other hand, in 14-day OJ, BLF upregulated cytokine production, in particular spontaneous (mean 148, median 158, vs mean 338, median 196, p = 0.001) and LPS-induced TNF-alpha (m ean 1331, median 1507, vs mean 2239, median 1707 pg/ml, p = 0.027). CONCLUSION: We conclude that oral administration of BLF is beneficial in alleviating deleterious effects of OJ.

Differential effects of prophylactic, concurrent and therapeutic lactoferrin treatment on LPS-induced inflammatory responses in mice.

Mice injected with endotoxin develop endotoxaemia and endotoxin-induced death, accompanied by the oxidative burst and overproduction of inflammatory mediators. Lactoferrin, an iron binding protein, provides a natural feedback mechanism to control the development of such metabolic imbalance and protects against deleterious effects of endotoxin. We investigated the effects of intraperitoneal administration of human lactoferrin on lipopolysaccharide (LPS)-induced release of tumour necrosis factor alpha (TNF-alpha), interleukin 6 (IL-6), interleukin 10 (IL-10) and nitric oxide (NO) in vivo. Lactoferrin was administered as a prophylactic, concurrent or therapeutic event relative to endotoxic shock by intravenous injection of LPS. Inflammatory mediators were measured in serum at 2, 6 and 18 h post-shock induction. Administration of lactoferrin 1 h before LPS resulted in a rather uniform inhibition of all mediators; TNF by 82%, IL-6 by 43%, IL-10 by 47% at 2 h following LPS injection,and reduction in NO (80%) at 6 h post-shock. Prophylactic administration of lactoferrin at 18 h prior to LPS injection resulted in similar decreases in TNF-alpha (95%) and in NO (62%), but no statistical reduction in IL-6 or IL-10. Similarly, when lactoferrin was administered as a therapeutic post-induction of endotoxic shock, significant reductions were apparent in TNF-alpha and NO in serum, but no significant effect was seen on IL-6 and IL-10. These results suggest that the mechanism of action for lactoferrin contains a component for differential regulation of cellular immune responses during in vivo models of sepsis.

Lactoferrin-induced up-regulation of zeta (zeta) chain expression in peripheral blood T lymphocytes from cervical cancer patients.

Alteration of T-cell-associated signal transduction molecules has recently been implicated in immune suppression in tumour-bearing hosts. Here we report the immunoregulatory effects of human lactoferrin (LF) on zeta-chain expression in peripheral blood T lymphocytes from cervical cancer patients and healthy donors. By quantitative flow cytometry analysis, we demonstrated that the mean zeta-chain expression was significantly higher in freshly-isolated T lymphocytes from healthy donors (69%), compared with the patients (38%). Following 3-day culture under standard conditions, zeta-chain expression in T lymphocytes from the patients increased significantly, whereas it dropped in the cells from healthy donors. Anti-CD3 MoAb as well as LF, significantly increased expression of zeta-chain in T cells both from patients and control subjects. The addition of LF to the anti-CD3 MoAb cell cultures resulted in an even higher stimulation of the zeta-chain expression. The results suggest that, in patients with cervical cancer, zeta-chain defects could be corrected by the therapeutic application of LF.

Lactoferrin regulates the immune responses in post-surgical patients.

The effect of oral administration of lactoferrin (LF) was studied to determine if it could modify post-surgical immune response. The action of LF was evaluated in 18 LF-treated patients vs 28 placebo counterparts. Patients (women and men, mean age 50 years) were given daily oral doses (20 mg each) of LF for 5 consecutive days prior to thyroid surgery. The following immune response parameters were determined in blood samples taken from the patients day before, day after, and 5-7 days following surgery: cell morphology, the proliferative response of peripheral blood mononuclear cells to phytohemagglutinin, and the spontaneous and lipopolysaccharide (LPS)-induced production of tumor necrosis factor alpha (TNF-alpha) and interleukin 6 (IL-6). As a consequence of the thyroid surgery, the total leukocyte count increased on the post-operative day by about 50% in all patients and the percentage of lymphocytes fell by 26 and 35% in the control vs LF-treated group. The content of neutrophils, on the other hand, elevated on day 1 post-operation by 51 and 68%, respectively. The percent of neutrophil precursors was markedly higher in LF-treated patients, particularly on the day before and the day after surgery (4.1 and 4.8 vs 2.5 and 3.7%, respectively). The post-surgical values were, however, comparable in both groups for neutrophils. The proliferative response of lymphocytes showed a slight decrease in the control group and an increase in the LF-treated patients on day 5 post-operation (20% over control group). LPS-induced TNF-alpha production was higher in LF-treated patients both one day before and one day following surgery (28 and 24%, respectively). LPS-induced IL-6 production was comparable in both placebo and LF-treated patients before surgery, however, on day 1 and 5 following surgery, the production of IL-6 was higher in LF-treated patients by 65 and 27%, respectively. Taken together, the data presented in this study revealed an increased immune responsiveness in all patients treated with LF and subjected to thyroid surgery. This suggests that treatment with LF could constitute an effective protective measure against post-surgical complications.

The effect of Ureaplasma diversum activated mononuclear leukocytes on the development and interferon-tau production by bovine IVF-derived embryos.

Ureaplasma diversum is an opportunistic pathogen of the bovine genital tract causing herd outbreaks of granular vulvitis, abortion and infertility. Early embryonic death probably contributes to reduction of the reproductive performance in cows, however, pathogenesis of the disease remains obscure. The aim of the study was to examine whether activation of mononuclear leukocytes by U. diversum may affect embryo development and IFN-tau production. Bovine peripheral blood mononuclear leukocytes were cultured with U.diversum antigen for 24 h. The levels of IL-1, TNF-alpha, NO and GM-CSF in the cell culture supernatants were measured. IVF-derived embryos were cultured in the presence of supernatants from activated leukocytes. The development of embryos until day 6 postinsemination and the rate of morulae/blastocysts were determined. IFN-tau production in supernatants of cultured embryos was examined by inhibition of a virally-induced cytopathic effect. The results showed that U. diversum stimulated mononuclear leukocyte production of IL-1, TNF-alpha and NO. Supernatants from U. diversum-activated cells did not impair the rates of the embryo development and blastocyst formation. The products of activated leukocytes increased the IFN-tau production by cultured blastocysts. This suggest that U. diversum infection provides leukocyte-mediated signals for developing embryos for generation of additional production of cytokine - an important component of innate immunity.

Differential effects of heparin on NO and tumor necrosis factor-alpha production in bovine blood mononuclear cells stimulated with Salmonella typhimurium lipopolisaccharide.

We investigated the influence of heparin, one of the extracellular matrix (ECM) components, on nitric oxide (NO) and tumor necrosis factor-alpha (TNF-alpha) production by bovine peripheral blood mononuclear cells (PBMC) and monocytes left to adhere for 2 (freshly adherent monocytes) and 48 h (resting monocytes), activated with Salmonella typhimurium lipopolysaccharide (LPS). After 24-h stimulation with LPS, heparin (100 microg/ml) increased (by about 40%) NO production by peripheral blood mononuclear cells and by freshly adherent monocytes. However, it did not change NO synthesis by the resting monocytes. Unlike its influence on NO level, heparin diminished TNF-alpha production by PBMC and monocytes stimulated with LPS. Microscopical examination of PBMC stained with biotin-labeled heparin, showed that both lymphocytes and monocytes were able to bind this glycosaminoglycan. We suggest that heparin, as a component of ECM, modulates the early response of monocytes to exogenous stimuli.

Lactoferrin regulates proliferative response of human peripheral blood mononuclear cells to phytohemagglutinin and mixed lymphocyte reaction.

The aim of this study was to investigate the effects of lactoferrin (LF) on the proliferative response of human peripheral blood mononuclear cells (PBMC) induced by phytohemagglutinin (PHA) and alloantigens in a two-way mixed lymphocyte reaction (MLR). The proliferative responses were measured by MTT colorimetric assay and presented as a proliferation index (PI) or as changes in PI caused by LF relative to PHA or MLR controls. We found that the effects of LF in both experimental models were differential and dependent on an individual PBMC reactivity, mitogen or alloantigen and LF concentration. Generally, lymphocytes from donors responsive to LF exhibited higher proliferation indices to PHA when compared with non-responsive individuals. Lactoferrin at low doses showed regulatory effects, whereas at higher doses the proliferation was significantly reduced. Mixed lymphocyte reaction was generally inhibited by LF. The results suggest that the differential action of LF might be due to its ability to sense the activation status of lymphocytes.

Reactions of 5-amino-3-methylisoxazole-4-carboxylic acid hydrazide with carbonyl compounds: immunological activity and QSAR studies of products.

A series of 4-imino derivatives of the 5-amino-3-methylisoxazole-4-carboxylic acid hydrazide and 5-amino-3-methylisoxazole[5,4-d]-6,7-dihydropyrimidine has been prepared by condensation of 5-amino-3-methylisoxazole-4-carboxylic acid hydrazide with carbonyl compounds. The resulting products were evaluated for their immunological activities in the models of the humoral and cellular immune responses of mice in vivo and concanavalin A (Con A) and lipopolysaccharide (LPS)-induced splenocyte proliferation. In addition, effects on polyclonal antibody production by human peripheral blood cells in culture were investigated. For all studied compounds we carried out quantum chemical calculations at ab initio B3LYP 6-31G(d, p) level. The stimulatory or inhibitory effects depended strongly on the origin and location of substitunets, which is described in the conclusions and was supported by QSAR studies.

Synthesis and immunotropic properties of 5-substituted 1,5-benzodiazepin-2-ones derivatives in cultures of human peripheral blood cells, Part III.

The reaction of 4-methyl-(A) and 3-methyl-1H-2,3,4,5-tetrahydro-1,5-benzodiazepin-2-one (B) with selected alpha,beta-unsaturated acid chlorides: crotonoyl, cinnamoyl, and 4-nitrocinnamoyl is described. We have also characterized immunotropic activities of these compounds in the proliferative response of human lymphocytes to phytohemagglutinin A (PHA) or to allogeneic cells in one-way mixed lymphocyte reaction (MLR), as well as their action on tumor necrosis factor alpha (TNF-alpha) and interleukin 6 (IL-6) production in peripheral blood mononuclear cells (PBMC) and mixed lymphocyte cultures (MLC). Some of the compounds exhibited regulatory activities in the proliferative response of cells to PHA depending on the reactivity of cells to PHA. The MLR induced proliferation of lymphocytes was moderately inhibited by two selected compounds. The compounds showed also inhibitor properties with regard to lipopolysaccharide (LPS)- and MLR-induced TNF-alpha production. Structure-activity relationship was discussed.

The effect of endotoxin administration on cytokine production in obstructive jaundiced rats.

The aim of this study was to evaluate the effect of lipopolysaccharide (LPS) administration, which mimics a surgical intervention, on the immune status of obstructive jaundiced (OJ) and sham-operated control rats. Rats were given 20 microgram LPS intraperitoneally on day 13 following bile duct ligation or sham surgery. We determined serum levels of tumor necrosis factor alpha (TNF-alpha) and interleukin 6 (IL-6) on day 14 after surgery, and spontaneous as well as LPS-induced production of these cytokines in splenocyte and peritoneal exudate cell (PEC) cultures. We found that IL-6, but not TNF-alpha, serum concentrations were significantly elevated (4-fold) in OJ rats treated with LPS compared with LPS-untreated OJ rats. In sham-operated rats the differences between the respective groups were not significant. The production of TNF-alpha by splenocyte and PEC cultures was depressed in OJ rats treated with LPS; in particular, a very deep decline was observed in the case of spontaneous TNF-alpha production in PEC cultures. In contrast, TNF-alpha production in LPS-untreated and LPS-treated sham-operated rats did not differ. In the case of IL-6 production by splenocytes and PEC cultures, we observed a significant suppression of this cellular function in both OJ and sham-operated rats treated with LPS when compared with the respective controls. In conclusion, the results indicate that the already depressed cytokine production in OJ rats leads to even deeper hyporeactivity following LPS challenge. Lack of TNF-alpha suppression upon LPS treatment in sham-operated rats suggests that surgery-elicited hyporeactivity is mediated by a different mechanism than that leading to immune hyporesponsiveness in OJ. Our findings may explain the relatively high mortality rates observed of OJ patients subjected to surgery.

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.

[IL-6 and TNF-alpha levels in oviductal epithelial cells culture after escherichia coli LPS stimulation]. / IL-6 i TNF-alpha w hodowlach blony sluzowej jajowodów po stymulacji lipopolisacharydem (LPS) Escherichia coli.

UNLABELLED: The aim of the study was to determine wether Gram-negative bacterial cell membrane lipopolysaccharides (endotoxine) can change the IL-6 and TNF-alpha cytokine concentration synthetized by fallopian tube endothelial cells. For the study 5 normal fallopian tubes from females at their reproductive age who underwent total hysterectomy due to uterine myoma were used. The fallopian tubes specimens (endothelial tissue) 2 mm2 fixed in 0.5 ml DMEM/HAM F-12 (GibcoBRL) solution with 15% FCS, Gentamycin, Fungizone, ITS (GibcoBRL) were incubated at 37 degrees C with 5% CO2. The explants were stimulated by Escherichia coli lipopolysaccharide (LPS) at ascending concentrations 1 ng/mL, 10 ng/mL and 100 ng LPS/mL incubation media. Tissue specimen incubated in a media without LPS were used for control test. IL-6 activity in the supernatants were determined by Van Sinc method, TNF-alpha activity were determined against WEHI-164.13. cells according to Espevik and Nisser-Mayer. The presence of TNF-alpha and IL-6 were confirmed in all the supernatants of the incubated fallopian tubes explants. The LPS stimulation caused a concentration increase in both cytokines. The maximum cytokine concentration level was observed in the incubation stimulated at 1 and 10 ng LPS/mL incubation media. The use of the highest LPS concentration retarded the cytokine production. CONCLUSION: The TNF-alpha and IL-6 cytokines dose-dependent production is caused by the fallopian tubes LPS stimulation.

Differential patterns of cyclosporine A-induced inhibition of humoral and cellular immune responses to sheep erythrocytes in mice.

In this communication we reveal differential inhibitory effects of cyclosporine A (CsA) on generation of the cellular and humoral immune responses to sheep erythrocytes (SRBC) in mice. For the analysis of the regulatory effects of CsA, we analyzed the results of 45 separate experiments performed in recent years where CsA served as a reference drug for our research on various immunoregulatory compounds. The humoral immune response was determined as the number of plaque-forming cells (PFC), and the delayed type hypersensitivity (DTH) was measured by foot pad swelling. We demonstrated that treatment of mice intraperitoneally (ip) with a dose of 100 microg of CsA/mouse, 2 h after immunization resulted in a differential pattern of inhibition of these two types of the immune response depending on the magnitude of the response in a given experiment. In the case of the antibody response (mean number of PFC 2312 median 2200), high PFC numbers were inhibited stronger than low ones; mean values in respective quarters and inhibitory actions were the following: 1,552 (42.3%), 2,049 (52.4%), 2,441 (61.2%) and 3042 (62.5%). In consequence, high, medium and low responses were down-regulated to approximately the same level. Another inhibitory pattern was observed in the DTH model (mean 10.35 units, median 10.8 units), i.e. low DTH responses were suppressed more strongly than high ones. The mean DTH responses and suppression effects in respective quarters were: 7.4 (58.2%), 10.1 (52.7%),11.8 (51.8%) and 13.1 (38.8%). As the result of such CsA action, the DTH response profile was parallel to that of the control response. In summary, the humoral immune response was down-regulated by CsA proportionally to the immune response observed in control mice, while DTH response was inversely proportional. The possible mechanisms of the observed regulatory CsA actions are discussed.

Immunotropic activity of vratizolin (ITCL, Denotivir).

The aim of this study was to evaluate immunotropic properties of vratizolin, a known antiviral drug, in several in vitro and in vivo assays in mouse and human models. We demonstrated that vratizolin exerted strong immunosuppressive actions both in the humoral and cellular immune response to SRBC in mice. The compound affected not only the inductive phase of delayed type hypersensitivity (DTH) but also the effector phase of that response. Vratizolin was effective when given intraperitoneally and orally. The inhibitory action of vratizolin was comparable to that of cyclosporin A (CsA), the reference drug. Vratizolin exhibited also suppressory properties with regard to PHA-induced proliferation of human peripheral blood lymphocytes and that effect exceeded the inhibitory action of CsA. We also showed that vratizolin inhibited to some degree LPS-induced cytokine production in human peripheral blood cultures. The activities of TNF-alpha, IL-1 and IL-6 were inhibited on average by 37, 26 and 35%, respectively. This was in contrast to the effects of CsA which strongly inhibited only IL-1 production. Lastly, we demonstrated that vratizolin markedly inhibited growth of several tumor cell lines. In particular, the compound significantly inhibited growth of mouse leukemia L-1210 and human acute lymphoblastoid leukemia CCRF-CEM cell lines. The presented data suggest that the immunosuppressory action of vratizolin, although similar to that of CsA, is mediated by a different mechanism. The properties of vratizolin, described in this report, indicate that the drug should be further investigated for possible immunosuppressory and antitumor application.