Michał Zimecki

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‐α), 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‐α (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‐α 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.

Immunostimulatory activity of lactotransferrin and maturation of CD4− CD8− murine thymocytes

Human milk lactotransferrin at a concentration ranging from 1 to 10 micrograms/ml stimulated up to 5 times the humoral immune response to sheep red blood cells, expressed as the number of plaque-forming cells, when injected into mice 3 h before immunization. Further, lactotransferrin-treated thymocytes given intravenously into mice, enhanced the immune response to sheep red blood cells to the same extent as IL-1. In vitro, studies showed that CD4- CD8- thymocytes incubated with lactotransferrin and added to the splenocyte cultures, increased the immune response to sheep red blood cells. Flow cytometry analysis studies indicated that, after an overnight incubation with human lactotransferrin, CD4- CD8- thymocytes acquired the CD4 antigen characteristic for the helper cell phenotype. Taken together, these results suggest that lactotransferrin stimulates the immune response by a process which involves the promotion of T cell differentiation.

Orally administered lactoferrin restores humoral immune response in immunocompromised mice

Cyclophosphamide (CP) is an anti-tumor drug commonly used in the chemotherapy of human cancer and autoimmune diseases. In our previous studies, we have demonstrated that lactoferrin (LF), given orally to CP-immunosuppressed mice, could reconstitute a T cell mediated immune response by the renewal of the T cell population. The aim of this present study was to evaluate the effects of LF on humoral responses in mice treated with cyclophosphamide. We demonstrate that a single, sublethal dose of cyclophosphamide (400 mg/kg body weight) profoundly inhibited the humoral immune response of CBA mice to sheep red blood cells (SRBC), as measured by the number of antibody forming cells (AFC) in the spleen after 5 weeks following CP treatment. Administration of 0.5% bovine LF in drinking water for 5 weeks partially reconstituted the AFC number (30-40% of the control values, but 7-10x more than in CP-treated controls). Determination of T and B cell levels in the spleens by flow cytometry revealed that the content of CD3+ and CD4+ as well as Ig+ splenocytes was elevated in the immunocompromised mice treated with LF. In addition, the number of peritoneal macrophages was partially restored following LF treatment. Evaluation of the proliferative response to concanavalin A (ConA) and pokeweed mitogen (PWM) demonstrated that the diminished reactivity of splenocytes from CP-treated mice was significantly enhanced by LF. In summary, we conclude that the prolonged, oral treatment of immunocompromised mice with LF led to partial reconstitution of the humoral response, associated with elevation of T and B cell and macrophage content and the proliferative response of splenocytes to mitogens.

Synthesis and pharmacological screening of derivatives of isoxazolo[4,5-d]pyrimidine

A number of derivatives of isoxazolo[4,5-d]pyrimidine were prepared with structures similar to that of purine. Condensation of the hydrazide of 4-amino-5-benzoylisoxazolo-3-carboxylic acid 2 with ethyloxalyl chloride followed by cyclization gave 3-oxdiazolo-[1,3,4]-4-amino-5-benzoylisoxazole 7 which, upon cyclization with acetonitrile followed by reactions with different amines, gave derivatives of isoxazolo[4,5-d]pyrimidine 9 and 10d-g. Compounds 8g and 10f were tested for their effects on the immune response in the mouse model. Both compounds significantly inhibited the humoral immune response in vivo to sheep erythrocytes at a dose of 100 microg, whereas in the delayed type hypersensitivity assay a suppressive activity was shown only by compound 10f. In addition, compound 8g inhibited and compound 10f stimulated the proliferative response of mouse splenocytes to concanavalin A. The results indicated that compound 10f was a universal inhibitor of the immune response, while compound 8g selectively suppressed the humoral immune response.

Lactoferrin immunomodulation of DTH response in mice.

Improved nontoxic adjuvants, especially adjuvants capable of inducing cell-mediated immunity (CMI), are needed for research in immunology and for development of human and veterinary vaccines. Bovine Lactoferrin, an effector molecule shown to directly participate in host defense, was assessed at various concentrations as an adjuvant component for induction of DTH responses to sheep red blood cells (SRBC). Subcutaneous immunization with Lactoferrin enhanced delayed type hypersensitivity (DTH) in CBA mice in a dose-dependent fashion; DTH responses were most significantly increased when sensitization was accomplished using Lactoferrin at 50 microg/dose and 250 microg/dose. Furthermore, Lactoferrin admixed with suboptimal dose of SRBC enhanced DTH responses by over 17-fold. Peritoneal cells collected from mice intraperitoneally injected with a 100 microg/dose of Lactoferrin demonstrated modest, but significant, production of TNF-alpha, IL-12 and MIP-1alpha when cultured in vitro, compared to saline-injected controls. J774A.1 murine macrophages stimulated with Lactoferrin resulted in increased TNF-alpha protein production, and upregulated IL-12 and IL-15 mRNA. Levels of message for chemokines MIP-1alpha and MIP-2 were also increased in a dose-dependent way. Taken together, these results indicate that Lactoferrin as an adjuvant may stimulate macrophages to generate a local environment likely to push immune responses towards development and maintenance of CMI.

Anticancer activity of newly synthesized azaphenothiazines from NCI’s anticancer screening bank#

The activity of the newly synthesized azaphenothiazines: tricyclic 10-substituted dipyridothiazines 1-9, pentacyclic 6-substituted diquinothiazines 10-22 and hexacyclic diquinothiazinium salt 23 was tested on 55-60 in vitro cell lines. The cell lines included nine types of cancer: leukemia, non-small cell lung cancer, colon cancer, CNS cancer, melanoma, ovarian cancer, renal cancer, prostate cancer and breast cancer (National Cancer Institute, Bethesda, MD, USA). The features of the chemical substituent at the thiazine nitrogen atom confer the anticancer activity of diquinothiazines 10-23. Unexpectedly, the most active of the dipyridothiazines 1-9 was the unsubstituted compound 1 (the substituent is a hydrogen atom). The most cytotoxic compound was the half-mustard derivative 18. The GI(50) value of this compound was -7.06 (corresponding to 40 ng/ml) when tested on the melanoma cell line SK-MEL-5 and -6.0 - -6.62 using cell lines from various cancers including: leukemia (CCRF-CEM), the MOLT-4 cell line, colon cancer (HCT-116), central nervous system cancer (SNB-75 and SF-295), prostate cancer (PC-3), non-small cell lung cancer (NCI-H460 and HOP-92), ovarian cancer (IGROV1 and OVCAR-4) and breast cancer (MDA-MB-460). The ethylene group in the aminoalkylazaphenothiazines is as a good linker and is similar to the propylene and butylene linkers in aminoalkylphenothiazines. To our knowledge, this is the first demonstration of significant azaphenothiazine anticancer activity.

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

AbstractObjective 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. Materials 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 × 108). 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.

Systemic or local co-administration of lactoferrin with sensitizing dose of antigen enhances delayed type hypersensitivity in mice

Lactoferrin (LF), a major defense protein synthesized and stored in granulocytes has been implicated in maintaining immune homeostasis during an insult-induced metabolic imbalance. In this study, we demonstrated that lactoferrin augments the delayed type hypersensitivity (DTH) response to specific antigens in mice. Lactoferrin (LF) was given to mice orally or intraperitoneally (i.p. ) at the time of immunization, or subcutaneously (s.c.) in a mixture with the immunizing doses of the following antigens, sheep red blood cells (SRBC), Calmette-Guerin bacillus (BCG) or ovalbumin (OVA). A DTH reaction was determined 24 h after administration of an eliciting dose of antigen as a specific increase in foot pad swelling. Lactoferrin enhanced DTH reaction to all studied antigens in a dose-dependent manner. Lactoferrin (LF) given to mice in conjunction with antigen administered in an incomplete Freunds adjuvant induced the DTH response at the level of control mice given antigen in a complete Freunds adjuvant. In addition, LF remarkably increased DTH response to a very small, otherwise non-immunogenic SRBC dose. The increase in DTH response was less pronounced for orally administered LF than for any other routes of administration, however, statistically significant augmentation was demonstrated for each antigen studied. Although the costimulatory action of LF was accompanied by the appearance of bovine lactoferrin-specific cellular responses in mice, it is very unlikely that such responses will be generated in humans, since bovine lactoferrin is a dietary antigen to which a tolerance has been acquired. Considering the involvement of LF in generation of stimulatory signals during the induction phase of an antigen specific immune responses, we suggest that LF may be useful for development of safer and more efficacious vaccination protocols.

Reconstitution of the cellular immune response by lactoferrin in cyclophosphamide-treated mice is correlated with renewal of T cell compartment.

Cyclophosphamide is an alkylating agent used to treat both malignant and non-malignant immune-mediated inflammatory disorders in humans. It is also known as a potent immunosuppressive drug in humans and experimental animals. The aim of this study was to evaluate the effects of oral administration of lactoferrin (LF) on cellular responses and reconstruction of the lymphocyte pool in mice treated with cyclophosphamide (CP). Twelve week-old CBA mice were given a single intraperitoneal (i.p.) dose of CP (400 mg/kg body weight), then were treated per os with seven doses of LF (1 mg/dose) on alternate days. We demonstrated that the magnitude of delayed type hypersensitivity to ovalbumin, strongly diminished by CP action, was reconstituted by LF. Oral LF treatment also resulted in partial recovery of Concanavalin A-induced splenocyte proliferation. Blood profile analysis revealed elevation of leukocytosis by LF in CP-treated mice (from 64.9 to 84.76% of the control value). LF also caused substantial restoration of the percentage of the lymphocyte population in circulating blood (from 43.4 to 60.2% of the control values). LF alone had no effect on the neutrophil/lymphocyte ratio in normal mice, however, the total number of leukocytes decreased by 23.25%. Furthermore, we showed that LF increased the cellularity of spleens isolated from CP-treated mice (from 53.2 to 78.8%) and the content of peritoneal and alveolar macrophages (elevations from 50.6 to 67.3% and from 65.2 to 83.6%, respectively). Lastly, using panning technique, we demonstrated that LF strongly elevated the pool of CD3+ T cells in normal and CP-immunocompromised mice and CD4+ T cell content. In conclusion, we showed for the first time that lactoferrin, given orally to CP-immunosuppressed mice, could reconstitute a T-cell mediated immune response by renewal of the T cell pool.

Recombinant human lactoferrin expressed in glycoengineered Pichia pastoris: effect of terminal N-acetylneuraminic acid on in vitro secondary humoral immune response

Traditional production of therapeutic glycoproteins relies on mammalian cell culture technology. Glycoproteins produced by mammalian cells invariably display N-glycan heterogeneity resulting in a mixture of glycoforms the composition of which varies from production batch to production batch. However, extent and type of N-glycosylation has a profound impact on the therapeutic properties of many commercially relevant therapeutic proteins making control of N-glycosylation an emerging field of high importance. We have employed a combinatorial library approach to generate glycoengineered Pichia pastoris strains capable of displaying defined human-like N-linked glycans at high uniformity. The availability of these strains allows us to elucidate the relationship between specific N-linked glycans and the function of glycoproteins. The aim of this study was to utilize this novel technology platform and produce two human-like N-linked glycoforms of recombinant human lactoferrin (rhLF), sialylated and non-sialylated, and to evaluate the effects of terminal N-glycan structures on in vitro secondary humoral immune responses. Lactoferrin is considered an important first line defense protein involved in protection against various microbial infections. Here, it is established that glycoengineered P. pastoris strains are bioprocess compatible. Analytical protein and glycan data are presented to demonstrate the capability of glycoengineered P. pastoris to produce fully humanized, active and immunologically compatible rhLF. In addition, the biological activity of the rhLF glycoforms produced was tested in vitro revealing the importance of N-acetylneuraminic (sialic) acid as a terminal sugar in propagation of proper immune responses.