Jeffrey K. Actor

Lactoferrin as a natural immune modulator.

Lactoferrin, an iron-binding glycoprotein, is a cell-secreted mediator that bridges innate and adaptive immune function in mammals. It is a pleiotropic molecule that directly assists in the influence of presenting cells for the development of T-helper cell polarization. The aim of this review is to provide an overview of research regarding the role of lactoferrin in maintaining immune homeostasis, in particular as a mediator of immune responses to infectious assault, trauma and injury. These findings are critically relevant in the development of both prophylactic and therapeutic interventions in humans. Understanding these particular effects of lactoferrin will provide a logical framework for determining its role in health and disease.

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.

Interleukin-15 Activates Human Natural Killer Cells to Clear the Intestinal Protozoan Cryptosporidium

Intracellular protozoans of the genus Cryptosporidium are a major cause of diarrheal illness worldwide, but little is known about the mechanisms that control intestinal infection. We have previously demonstrated interleukin (IL)-15 expression in the intestinal mucosa of seronegative symptomatic volunteers after oral challenge with C. parvum, which suggests a role for IL-15 in the control of acute infection. We hypothesize that IL-15 activates an innate cytolytic cell response that contributes to the clearance of initial C. parvum infection. We report here that IL-15 activates peripheral blood mononuclear cells to lyse Cryptosporidium-infected epithelial cells in a dose-dependent manner. Lysis was due to CD3(-)CD16(+)CD56+ cells (i.e., natural killer [NK] cells). Furthermore, flow cytometry revealed that IL-15 increased expression of the activation receptor NKG2D on NK cells, particularly among the CD16Hi cytolytically active cells. Major histocompatibility complex class I-related molecules A and B (MICA and MICB), ligands for NKG2D, were increased after infection of epithelial cell lines and human ileal tissue. These data suggest that IL-15 has an important role in activating an NK cell-mediated pathway that leads to the elimination of intracellular protozoans from the intestines, which is a previously unrecognized feature of NK cell function.

A Humanized Mouse Model of Tuberculosis

Mycobacterium tuberculosis (M.tb) is the second leading infectious cause of death worldwide and the primary cause of death in people living with HIV/AIDS. There are several excellent animal models employed to study tuberculosis (TB), but many have limitations for reproducing human pathology and none are amenable to the direct study of HIV/M.tb co-infection. The humanized mouse has been increasingly employed to explore HIV infection and other pathogens where animal models are limiting. Our goal was to develop a small animal model of M.tb infection using the bone marrow, liver, thymus (BLT) humanized mouse. NOD-SCID/γc null mice were engrafted with human fetal liver and thymus tissue, and supplemented with CD34+ fetal liver cells. Excellent reconstitution, as measured by expression of the human CD45 pan leukocyte marker by peripheral blood populations, was observed at 12 weeks after engraftment. Human T cells (CD3, CD4, CD8), as well as natural killer cells and monocyte/macrophages were all observed within the human leukocyte (CD45+) population. Importantly, human T cells were functionally competent as determined by proliferative capacity and effector molecule (e.g. IFN-γ, granulysin, perforin) expression in response to positive stimuli. Animals infected intranasally with M.tb had progressive bacterial infection in the lung and dissemination to spleen and liver from 2–8 weeks post infection. Sites of infection in the lung were characterized by the formation of organized granulomatous lesions, caseous necrosis, bronchial obstruction, and crystallization of cholesterol deposits. Human T cells were distributed throughout the lung, liver, and spleen at sites of inflammation and bacterial growth and were organized to the periphery of granulomas. These preliminary results demonstrate the potential to use the humanized mouse as a model of experimental TB.

Relationship of Survival, Organism Containment, and Granuloma Formation in Acute Murine Tuberculosis

The relationship among organism growth, immunopathology, and survival was studied in C57BL/6 and A/J mice acutely infected with Mycobacterium tuberculosis (MTB) (Erdman). Although organisms grew at similar rates in the lungs of both mouse strains, A/J mice died prior to 14 days after infection, whereas C57BL/6 mice survived twice as long. The lungs of A/J mice exhibited necrotizing interstitial inflammation and widely distributed acid-fast bacilli without granuloma formation. In contrast, the lungs of C57BL/6 mice had relatively mild interstitial inflammation, which was replaced by focal granulomas, and acid-fast bacilli were primarily within granulomas. MTB induced similar granulomas for A/J and C57BL/6 mice in spleen and liver. In the lung, the A/J mice produced only transient messages for interferon-y (IFN-y), interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), IL-10, and inducible nitric oxide synthase (iNOS). The C57BL/6 mice, in contrast, produced a delayed but sustained response in the lung correlating with granuloma onset and characterized by high induction of IL-6, IFN-gamma, IL-1beta, IL-10, and TNF-alpha. Responses in the liver and spleen were also evaluated. These results demonstrate that histopathology and cytokine response to MTB infection varies among organs in mice. Increased survival during acute infection may, therefore, depend on the ability to contain organisms within granulomas in the lung.

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.

Apoptosis in Mycobacterium tuberculosis infection in mice exhibiting varied immunopathology

This study examined mechanisms contributing to pulmonary immunopathology following acute Mycobacterium tuberculosis (MTB) infection in vivo in a murine model. A/J and C57BL/6 mice were intravenously infected with MTB (Erdman). Pathological differences were found between strains, unrelated to pulmonary load of bacilli. A/J mice developed progressive interstitial pneumonitis, while C57BL/6 mice maintained granuloma formation. The contribution of FAS and FAS ligand‐mediated apoptosis was assessed via bioluminescent reverse transcription‐polymerase chain reaction (RT‐PCR), immunohistochemical staining, and TUNEL assessment of DNA fragmentation. Cytokine messages for pulmonary tumour necrosis factor‐α (TNF‐α) and interferon‐γ (IFN‐γ), as well as for the lytic molecules perforin and granzyme B, were quantified. Immunohistochemical staining for CD3 receptor was performed to monitor lymphocytic lung infiltration. Soon after infection, A/J mice exhibited increased pulmonary IFN‐γ message, concurrent with the appearance of CD3+ lymphocytes distributed throughout the lung. C57BL/6 mice exhibited perivascular cuffing, with no accompanying increase in IFN‐γ message. A/J mice also had elevated levels of FAS and FAS ligand message and protein early after infection, while the C57BL/6 mice had no increased expression of these molecules. Both strains exhibited qualitatively similar numbers of TUNEL‐positive cells throughout infection, with a marked increase on day 7. Apoptotic cells appeared to co‐localize with acid fast bacilli. It is therefore proposed that apoptosis during initial granuloma formation following MTB infection may occur through a FAS/FAS ligand‐independent pathway. Moreover, a failure of completion of the FAS/FAS ligand‐mediated apoptosis pathway in the A/J mice may contribute to inefficient elimination of lymphocytes, thus further aggravating pulmonary pathology. Copyright

A mutant of Mycobacterium tuberculosis H37Rv that lacks expression of antigen 85A is attenuated in mice but retains vaccinogenic potential.

ABSTRACT The fbpA and fbpB genes encoding the 85A and 85B proteins of Mycobacterium tuberculosis H37Rv, respectively, were disrupted, the mutants were examined for their ability to survive, and the strain lacking 85A (ΔfbpA) was tested for its ability to immunize mice. The ΔfbpA mutant was attenuated in mice after intravenous or aerosol infection, while replication of the ΔfbpB mutant was similar to that of the wild type. Complementation of the fbpA gene in ΔfbpA restored its ability to grow in the lungs of mice. The ΔfbpA mutant induced a stronger expression of pulmonary mRNA messages in mice for tumor necrosis factor alpha, interleukin-1 beta (IL-1β), gamma interferon, IL-6, IL-2, and inducible nitric oxide (NO) synthase, which led to its decline, while H37Rv persisted despite strong immune responses. H37Rv and ΔfbpA both induced NO in macrophages and were equally susceptible to NO donors, although ΔfbpA was more susceptible in vitro to peroxynitrite and its growth was enhanced by NO inhibitors in mice and macrophages. Aerosol-infected mice, which cleared a low-dose ΔfbpA infection, resisted a challenge with virulent M. tuberculosis. Mice subcutaneously immunized with ΔfbpA or Mycobacterium bovis BCG and challenged with M. tuberculosis also showed similar levels of protection, marked by a reduction in the growth of challenged M. tuberculosis. The ΔfbpA mutant was thus attenuated, unlike ΔfbpB, but was also vaccinogenic against tuberculosis. Attenuation was incomplete, however, since ΔfbpA revived in normal mice after 370 days, suggesting that revival was due to immunosenescence but not compensation by the fbpB or fbpC gene. Antigen 85A thus affects susceptibility to peroxynitrite in M. tuberculosis and appears to be necessary for its optimal growth in mice.

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.

Immunopathology of postprimary tuberculosis: increased T-regulatory cells and DEC-205-positive foamy macrophages in cavitary lesions.

Postprimary tuberculosis occurs in immunocompetent people infected with Mycobacterium tuberculosis. It is restricted to the lung and accounts for 80% of cases and nearly 100% of transmission. Little is known about the immunopathology of postprimary tuberculosis due to limited availability of specimens. Tissues from 30 autopsy cases of pulmonary tuberculosis were located. Sections of characteristic lesions of caseating granulomas, lipid pneumonia, and cavitary stages of postprimary disease were selected for immunohistochemical studies of macrophages, lymphocytes, endothelial cells, and mycobacterial antigens. A higher percentage of cells in lipid pneumonia (36.1%) and cavitary lesions (27.8%) were positive for the dendritic cell marker DEC-205, compared to granulomas (9.0%, P < .05). Cavities contained significantly more T-regulatory cells (14.8%) than found in lipid pneumonia (5.2%) or granulomas (4.8%). Distribution of the immune cell types may contribute to the inability of the immune system to eradicate tuberculosis.