lactoferrin and Tuberculosis--Pulmonary

A vaccine that prevents transmission of infection is urgently needed in the fight against tuberculosis (TB). Results of clinical trials have been disappointing. Major problems include lack of biomarkers and understanding of the mechanisms of disease and protection. A more fundamental problem is that the scientific community seldom recognizes that primary and post-primary TB are distinct disease entities. Nearly all vaccine candidates have been designed and tested in models of primary TB, while transmission of infection is mediated by post-primary TB. Post-primary TB is seldom studied because no animal develop complete symptoms of the disease as it exists in humans. Nevertheless, mice, guinea pigs and rabbits all develop infections that at certain points appear to be models of human post-primary TB. Slowly progressive pulmonary TB in immunocompetent mice is an example. It is characterized by an alveolitis with infected foamy macrophages that have multiple characteristics of the human disease. We demonstrated that inclusion of an immune modulating agent, lactoferrin, with a BCG vaccine in this model induced a sustained reduction in lung pathology, but not numbers of organisms in tissue. Since the animals die of expanding pathology, this demonstrates the feasibility of using selected animal models for studies of vaccines against post-primary TB.

Topics: Adjuvants, Immunologic; Animals; BCG Vaccine; Disease Models, Animal; Host-Pathogen Interactions; Humans; Lactoferrin; Lung; Macrophages, Alveolar; Mycobacterium tuberculosis; Tuberculosis, Pulmonary

The airway provides numerous defense mechanisms to prevent microbial colonization by the large numbers of bacteria and viruses present in ambient air. An important component of this defense is the antimicrobial peptides and proteins present in the airway surface fluid (ASF), the mucin-rich fluid covering the respiratory epithelium. These include larger proteins such as lysozyme and lactoferrin, as well as the cationic defensin and cathelicidin peptides. While some of these peptides, such as human beta-defensin (hBD)-1, are present constitutively, others, including hBD2 and -3 are inducible in response to bacterial recognition by Toll-like receptor-mediated pathways. These peptides can act as microbicides in the ASF, but also exhibit other activities, including potent chemotactic activity for cells of the innate and adaptive immune systems, suggesting they play a complex role in the host defense of the airway. Inhibition of antimicrobial peptide activity or gene expression can result in increased susceptibility to infections. This has been observed with cystic fibrosis (CF), where the CF phenotype leads to reduced antimicrobial capacity of peptides in the airway. Pathogenic virulence factors can inhibit defensin gene expression, as can environmental factors such as air pollution. Such an interference can result in infections by airway-specific pathogens including Bordetella bronchiseptica, Mycobacterium tuberculosis, and influenza virus. Research into the modulation of peptide gene expression in animal models, as well as the optimization of peptide-based therapeutics shows promise for the treatment and prevention of airway infectious diseases.

Topics: Animals; Antimicrobial Cationic Peptides; Cathelicidins; Cystic Fibrosis; Defensins; Disease Models, Animal; Humans; Lactoferrin; Muramidase; Proteinase Inhibitory Proteins, Secretory; Proteins; Respiratory System; Toll-Like Receptors; Tuberculosis, Pulmonary; Virus Diseases

Topics: Anaphylaxis; Anti-Glomerular Basement Membrane Disease; Antigen-Antibody Complex; Asthma; Complement System Proteins; Cytotoxicity, Immunologic; Humans; Immunoglobulins; Interferons; Lactoferrin; Leukocytes; Lung; Macrophages; Muramidase; Phagocytosis; Reagins; Respiratory Hypersensitivity; Rhinitis, Allergic, Seasonal; Sarcoidosis; Tuberculosis, Pulmonary

Salivary and serum levels of lactoferrin and ferritin were measured in patients with pulmonary tuberculosis and patients with other nonspecific respiratory diseases. Measurements of lactoferrin in biological media and particularly in the serum of patients with pulmonary tuberculosis proved to be a highly informative test for monitoring the disease course, i.e. for evaluation of inflammatory process activity. Ferritin level can serve as an indicator of tissue destruction during inflammation and of the course of rehabilitation processes.

Topics: Biomarkers; Ferritins; Humans; Inflammation; Lactoferrin; Pneumonia; Saliva; Tuberculosis, Pulmonary

Lactoferrin is an iron binding glycoprotein possessing multiple immune modulatory activities, including ability to affect macrophage cytokine production, promote maturation of T- and B-lymphocyte and immature dendritic cells, and enhance the ability of macrophages and dendritic cells to stimulate antigen-specific T-cells. These characteristics of lactoferrin suggested that it could function as an effective adjuvant enhance efficacy of the BCG, the current vaccine for tuberculosis disease. Admix of lactoferrin to the BCG vaccine promoted host protective responses that surpasses activity of the BCG vaccine alone as determined by decreasing pulmonary pathology upon challenge with virulent Mycobacterium tuberculosis (MTB). This study builds on previous reports by examining the effectiveness of the lactoferrin adjuvant comparing primary vaccination versus an immunization schedule with a booster administered at 8 weeks. BCG/lactoferrin vaccinating, given once or twice, demonstrated an improvement in pulmonary disease compared to both the BCG vaccinated and non-immunized groups. The splenic recall profiles showed a difference in cytokine production induced by mycobacterial antigen from splenocytes isolated from mice immunized with BCG/lactoferrin once or twice. Production of IL-17 is increased in the BCG/lactoferrin 2× group compared to the primary vaccinated group. Both BCG/lactoferrin vaccinated group exhibited increase production of IFN-γ compared to the non-immunized group and decreased production of IL-10 compared to the group vaccinated with only BCG. This study illustrates that the adjuvant activity of lactoferrin to enhance BCG efficacy occurs whether the vaccination regimen is a single delivery or combined with a booster, leading to enhanced host protection and decreased disease manifestation.

Topics: Adjuvants, Immunologic; Animals; Bacterial Load; BCG Vaccine; Colony Count, Microbial; Cytokines; Disease Models, Animal; Female; Immunization, Secondary; Lactoferrin; Liver; Lung; Mice; Mice, Inbred C57BL; Mycobacterium tuberculosis; Spleen; Tuberculosis, Pulmonary; Vaccination

The ability of lactoferrin to provide protection and decrease immunopathology in infectious diseases was evaluated using an aggressive aerosol model of Mycobacterium tuberculosis (MTB) infection. C57BL/6 mice were challenged with MTB strain Erdman and treated with 0.5% bovine lactoferrin added to the drinking water starting at day 0 or day 7 post-infection. Mice were sacrificed at three weeks post-challenge and evaluated for organ bacterial burden, lung histopathology, and ELISpot analysis of the lung and spleen for immune cell phenotypes. Mice given tap water alone had lung log10 colony forming units (CFUs) of 7.5 ± 0.3 at week 3 post-infection. Lung CFUs were significantly decreased in mice given lactoferrin starting the day of infection (6.4 ± 0.7), as well as in mice started therapeutically on lactoferrin at day 7 after established infection (6.5 ± 0.4). Quantitative immunohistochemistry using multispectral imaging demonstrated that lung inflammation was significantly reduced in both groups of lactoferrin treated mice, with decreased foamy macrophages, increased total lymphocytes, and increased numbers of CD4+ and CD8+ cells. ELISpot analysis showed that lactoferrin treated mice had increased numbers of CD4 + IFN-γ+ and IL-17 producing cells in the lung, cells that have protective functions during MTB infection. Lactoferrin alone did not alter the proliferation of MTB in either broth or macrophage culture, but enhanced IFN-γ mediated MTB killing by macrophages in a nitric oxide dependent manner. These studies indicate that lactoferrin may be a novel therapeutic for the treatment of tuberculosis, and may be useful in infectious diseases to reduced immune-mediated tissue damage.

Topics: Administration, Oral; Animals; Antitubercular Agents; Cells, Cultured; Colony Count, Microbial; Cytokines; Disease Models, Animal; Drug Evaluation, Preclinical; Female; Inflammation Mediators; Interferon-gamma; Interleukin-17; Lactoferrin; Lung; Macrophages; Mice; Mice, Inbred C57BL; Mycobacterium tuberculosis; Spleen; Th1 Cells; Tuberculosis, Pulmonary

Lactoferrin, an iron binding glycoprotein, possesses multiple immune modulatory activities, including the ability to promote antigen specific cell-mediated immunity. Previous studies showed that adding bovine lactoferrin to the BCG vaccine (an attenuated strain of Mycobacterium bovis Bacillus Calmette Guerin) resulted in increased host protective responses upon subsequent challenge with virulent Erdman Mycobacterium tuberculosis (MTB) in mice. The studies outlined here investigate utility of a novel recombinant human lactoferrin to enhance the BCG vaccine and protect against alveolar injury during experimental MTB infection in mice. Sialylated and non-sialylated forms of the recombinant human lactoferrin (rhLF), glycoengineered in yeast (Pichia pastoris) and expressing humanized N-glycosylation patterns, were examined for their ability to enhance efficacy of the BCG vaccine in a murine TB model system. Results indicated that the sialylated form of the recombinant human lactoferrin generated increased antigen specific recall responses to BCG antigens. Furthermore, augmented protection was demonstrated using the sialylated lactoferrin adjuvant with BCG, resulting in significant reduction in associated pathology following challenge with virulent organisms.

Topics: Adjuvants, Immunologic; Animals; BCG Vaccine; Carrier Proteins; Humans; Immunity, Cellular; Lactoferrin; Mice; Mycobacterium tuberculosis; Pulmonary Alveoli; Recombinant Proteins; Tuberculosis, Pulmonary

The current vaccine for tuberculosis (TB), an attenuated strain of Mycobacterium bovis Bacillus Calmette Guerin (BCG), is effective to prevent childhood onset of the disease, but its efficacy is reduced in adults. One strategy to improve the existing vaccine is to develop more effective adjuvants. Lactoferrin, an iron-binding glycoprotein possessing immune modulatory activities, is a promising adjuvant candidate. The studies presented here examine the effect of lactoferrin to enhance efficacy of the BCG vaccine using a vaccination/challenge protocol (8 weeks boost and challenge at 12 weeks post-boost) that focuses on reduction in development of pathological changes to lung tissue. C57BL/6 and BALB/c mice vaccinated with BCG/lactoferrin exhibited protection upon Mycobacterium tuberculosis (MTB) challenge, showing reduced pulmonary disease pathology and decreased organ bacterial load. In addition, BCG/lactoferrin-treated macrophages isolated from BALB/c mice, which express a relative reduced T(H)1 phenotypic response to MTB antigens compared to the C57BL/6 mouse, were able to activate a higher percentage of IFN-gamma-producing CD4+ splenocytes. Overall, lactoferrin stands as an adjuvant capable of enhancing efficacy of the BCG vaccine through induction of T(H)1 immune responses, even in hosts typically demonstrative of reduced T(H)1 responsiveness to BCG antigens.

Topics: Adjuvants, Immunologic; Animals; Antigens, Bacterial; BCG Vaccine; Enzyme-Linked Immunosorbent Assay; Immunization, Secondary; Lactoferrin; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Tuberculosis, Pulmonary

The defence factors against infection in sputum and bronchoalveolar lavage fluid (BALF) of patients with pulmonary tuberculosis were measured. As the defence factors, lactoferrin, lysozyme and secretory IgA (sIgA) in sputum or BALF of patients with bacilli (+) or (-) tuberculosis were measured and compared. Lactoferrin in sputum was significantly higher in patients with sputum smear positive tuberculosis compared with patients with smear and culture negative tuberculosis. SIgA in sputum was significantly higher in smear negative and culture positive cases compared with culture negative cases. As to the lysozyme in sputum, significant difference was not proved between each group. The level of these factors in BALF did not show significant difference between bacilli (+) and (-) cases. Neither significant correlation was observed among the level of three defence factors in sputum or BALF, nor between the number of leucocyte and tubercle bacilli in sputum. In tubercle bacilli positive group, however, significant positive correlation between the number of leucocyte and lactoferrin in sputum was found. In vitro experiments, high concentration of lactoferrin or lysozyme inhibited the growth of standard strain of tubercle bacilli (H37Rv) and BCG. The results suggest that the measurement of lactoferrin, lysozyme and sIgA in sputum or BALF is useful to determine the clinical activity of tuberculosis.

Topics: Bronchoalveolar Lavage Fluid; Humans; Immunoglobulin A, Secretory; Lactoferrin; Muramidase; Mycobacterium tuberculosis; Sputum; Tuberculosis, Pulmonary

The levels of immunoglobulins and other proteins (alpha 2-MG, alpha 1-AT, C3, albumin, transferrin and lactoferrin) were studied in the BAL of 60 patients with different types of pulmonary tuberculosis, 4 patients with sarcoidosis and 7 CNPD patients. The level of most proteins in BAL of the examinees was higher than that reported for healthy subjects. The highest protein levels were noted in CNPD and sarcoidosis patients. The diagnostic importance of the level of alpha 2-MG was established for sarcoidosis. 27 paired BAL-serum specimens from the same patients with pulmonary tuberculosis were investigated for analysis of the mechanisms of protein appearance in BAL. The protein/albumin ratio for most proteins was higher in BAL than in the respective serum. A relatively high level of proteins in the patients' BAL was probably determined by the activation of their local synthesis.

Topics: Albumins; alpha 1-Antitrypsin; alpha-Macroglobulins; Blood Proteins; Bronchoalveolar Lavage Fluid; Humans; Immunoglobulins; Lactoferrin; Lung Diseases; Lung Diseases, Obstructive; Proteins; Sarcoidosis; Serum Albumin; Transferrin; Tuberculosis, Pulmonary

The interrelationships between various components of the non-immune inflammatory response (white cell count, plasma lactoferrin, C-reactive protein, ferritin, iron and iron-binding capacity), were studied serially in a variety of inflammatory conditions including acute lobar pneumonia, active pulmonary tuberculosis, rheumatoid arthritis on gold therapy and sepsis in the face of marrow hypoplasia induced by chemotherapy. Lactoferrin concentrations paralleled the white count in all groups. They were highest in pneumonia and tuberculosis, mildly elevated in rheumatoid arthritis and markedly decreased in neutropenic sepsis. Very high initial lactoferrin concentrations were associated with a poor prognosis in acute pneumonia. C-reactive protein and ferritin concentrations remained elevated through the period of study in acute pneumonia and neutropenic sepsis, while they gradually normalised over weeks in subjects with tuberculosis or rheumatoid arthritis on therapy. In pneumonia and tuberculosis moderate hypoferraemia and a reduced iron-binding capacity were evident. In contrast, a raised percentage saturation was present in neutropenic sepsis, probably related to erythroid marrow suppression. Comparisons between ferritin, lactoferrin and C-reactive protein in the various groups supported the concept that ferritin behaves in part as an acute phase reactant and that hypoferraemia in inflammation is due to deviation of iron into ferritin stores. The suggestion that lactoferrin is responsible for the hypoferraemia and hyperferritinaemia was not supported by the present data. Iron deficiency appeared to limit the hyperferritinaemic response in rheumatoid arthritis, while erythropoietic inhibition by chemotherapy dampened the hypoferraemic response in neutropenic sepsis.

Topics: Arthritis, Rheumatoid; C-Reactive Protein; Ferritins; Humans; Inflammation; Iron; Lactoferrin; Lactoglobulins; Leukocyte Count; Pneumonia; Sepsis; Tuberculosis, Pulmonary

Highly purified lactoferrin was obtained from human breast milk by sequential use of affinity chromatography and isoelectric focusing. IgG antibody to purified lactoferrin was used to develop a sensitive and reproducible enzyme linked immunosorbent assay. Characteristics of the assay included linearity over a wide range of lactoferrin concentration (3.125-200 micrograms/l) and sensitivity (lower range less than 1 microgram/l). The assay can be adapted for use on tissue cytosol as well as plasma. Healthy subjects showed plasma lactoferrin levels ranging from 187.5-450.1 micrograms/l. Pulmonary tuberculosis and acute pneumonia are associated with a 2-3-fold increase in plasma lactoferrin content while neutropenic subjects have markedly depressed lactoferrin concentrations. The assay will be useful for further delineation of lactoferrin and neutrophil function and turnover.

Topics: Antibodies; Arthritis, Rheumatoid; Breast Neoplasms; Cytosol; Edetic Acid; Enzyme-Linked Immunosorbent Assay; Female; Humans; Lactoferrin; Lactoglobulins; Leukopenia; Male; Milk, Human; Pneumonia, Pneumococcal; Tuberculosis, Pulmonary