M. Altamura

Antimicrobial and immunoregulatory functions of lactoferrin and its potential therapeutic application.

Lactoferrin is an iron-binding glycoprotein present in various secretions (e.g. milk, tears, saliva, pancreatic juice, etc.). It is also stored in specific granules of polymorphonuclear granulocytes from which it is released following activation. Lactoferrin exerts a bactericidal activity by damagingthe outer membrane of Gram-negative bacteria, as well as immunoregulatory functions by decreasing the release of interleukin-1 (IL-1), IL-2 and tumor necrosis factor- (TNF-) and enhancing monocyte and natural killer cell cytotoxicity. Lactoferrin binds with high affinity to lipid A, the toxic moiety of the lipopolysaccharide, or endotoxin from Gram-negative bacteria. Lipopolysaccharide interaction with monocytes/macrophages results in the production and release of TNF- , that plays an important role in inducing septic shock. In this respect, it has recently been demonstrated that lactoferrin inhibits the lipopolysaccharide interaction with CD14 on monocytes/macrophages by competition with the lipopolysaccharide binding protein. Therefore, besides its bactericidal activity, lactoferrin may also act by neutralizing the toxic effects of lipopolysaccharide and this protective role against endotoxin lethal shock has been demonstrated in animal models. Moreover, in vitro and in vivo neutralization of endotoxin by a human lactoferrin-derived peptide was also reported and lactoferrin or lactoferrin-derived peptides could represent useful tools for the treatment of endotoxin-induced septic shock. The recent production and characterization of monoclonal antibodies against different epitopes of human lactoferrin, including monoclonal antibodies selectively neutralizinglactoferrin binding to lipid A, may allow a better elucidation of the consequence of lactoferrin-lipopolysaccharideinteraction.

FISH IMMUNOLOGY. I. BINDING AND ENGULFMENT OF CANDIDA ALBICANS BY ERYTHROCYTES OF RAINBOW TROUT (SALMO GAIRDNERI RICHARDSON)

ABSTRACT The role of fish erythrocytes (FE) as phagocytic cells has poorly been investigated, until now. Here, we have focussed our attention on the interplay between rainbow trout (Salmo gairdneri Richardson) erythrocytes and Candida albicans (CA). At the same time, the intervention of autologous head kidney macrophages (MØ) in the CA processing by FE has been studied. Data show that CA particles bind to FE, which, in turn, are able to engulf but not kill them. In the presence of MØ, a decrease of FE with bound CA occurs and, in some microscopic images, FE form rosettes with MØ. Phagocytosis of CA is higher in rosetting MØ than in non-rosetting ones. According to our findings, it appears that FE represent a reservoir of engulfed CA and rosetting is an efficacious phenomenon of presentation of pathogens to MØ, where an effective clearance of them can take place.

Diazepam Inhibits Phagocytosis and Killing Exerted by Polymorphonuclear Cells and Monocytes from Healthy Donors. In Vitro Studies

The effect of a benzodiazepine (BDZ), diazepam on human polymorphonuclear cell (PMN) and monocyte phagocytosis and killing from healthy volunteers has been evaluated. Diazepam is able to inhibit in vitro both functions exerted by PMN and monocytes at 10(-5) and 10(-6) M concentrations/ 4 x 10(6) phagocytes. 10(-7) M concentration was not effective in all the instances. These results are discussed for their possible clinical implications, since previous studies have shown that in patients with phobic disorder there is evidence for reduced phagocytosis and killing capacities.

The Immunocompromised Host: Immune Alterations in Splenectomized Patients and Clinical Implications

The spleen plays a paramount role in the host protection against invading microorganisms. In support of the above concept, in splenectomized patients there is increasing evidence of overwhelming postsplenectomy infections (OPSI). OPSI are caused by Streptococcus pneumoniae in about 80% of cases, but also Gram-negative bacteria are implicated in a certain number of cases. Therapeutically, penicillin and pneumococcal vaccines represent valid therapeutic approaches in Gram-positive OPSI. However, the effectiveness of polyvalent polysaccharide pneumococcal vaccines is still debated and, thus, other therapeutic strategies should be validated for combating OPSI. According to our personal data, a deficit of phagocytic activities and of T helper (h)-1 cells is very frequent in splenectomized patients. In sera, we found reduced levels of both Interferon-gamma and Interleukin (IL)-4. These data are in accordance with the recent observation on the protective role of T cells against S. pneumoniae. In fact, patients deficient in IL-12 develop severe pneumococcal infections and undergo apoptosis of Th(1) cells.

Maturation of fish erythrocytes coincides with changes in their morphology, enhanced ability to interact with Candida albicans and release of cytokine-like factors active upon autologous macrophages.

Erythrocytes from the rainbow trout Salmo gairdneri Richardson (Salmo g.R.) were classified into immature and mature populations, respectively, by measuring longitudinal diameters. More elongated fish erythrocytes (FE), classified as mature cells, were those interacting with Candida albicans (CA) in a higher frequency in terms of either binding to the fungus or its intracellular engulfment. At the same time, in the rosetting phenomenon more elongated mature FE surrounded macrophages (MØ) phagocytosing CA. Finally, FE activated by CA released in the supernatants cytokine‐like factors able to modulate MØ functions. In particular, these active supernatants were analyzed for their capacity to inhibit MØ migration Macrophage Inhibition Factor (MIF) activity and enhance MØ phagocytosis. Both activities were detected in supernatants from CA stimulated FE but not in control supernatants. MIF activity could play a role in the accumulation of MØ in the context of functional rosettes, while the factor enhancing MØ phagocytosis could promote clearance of CA in a more efficacious way.

Immune responses to fungal infections and therapeutic implications.

Host responses to fungi result from a coordinate interplay between innate and adaptative immune system. Neutrophils and monocytes are involved in the non specific clearance of yeasts (e.g. Candida albicans and Cryptococcus neoformans), while T helper 1 type responses are protective via release of interferon gamma. By contrast, T helper 2 responses (IL-4 and IL 10 release) correlate with disease exacerbation and pathology. IL-12 production which enhances T helper 1 type responses seem to exert a beneficial role in the course of Candida infection. In particular, its production from neutrophilis may support memory T helper 1 cell responses of the fungus. With respect to anti-Candida vaccines several approaches are in progress, such as use of heat-killed Candida albicans in combination with adjuvants, purified peptides and proteins and immunogenic peptide-lipid conjugates. Furthermore, exogenous IL-12 may play an important role in inducing a T helper 1 anticandidal response, also replacing neutrophils in neutropenic patients. At the same time, granulocyte-colony stimulating factor has exhibited therapeutic efficacy in experimental and human models of fungal infection.

Effects of Acetyl-L-Carnitine Oral Administration on Lymphocyte Antibacterial Activity and TNF-α Levels in Patients with Active Pulmonary Tuberculosis. A Randomized Double Blind Versus Placebo Study

Acetyl-L-carnitine (ALC), a drug for the treatment of ageing-related neuroendocrine dysfunctions, was orally administered--2 gm/day for 30 days--to 10 patients with active pulmonary tuberculosis (TBC). Lymphocyte-mediated antibacterial activity and serum levels of tumor necrosis factor (TNF)-alpha were evaluated before and after treatment, comparing the values with those of 10 TBC patients receiving placebo. Results show that by day 30, antibacterial activity remained unmodified or increased in ALC-treated subjects, while decreased in the placebo group. No influence of ALC on TNF-alpha levels was detectable. These data suggest that the hosts immune responses to M. tuberculosis infection can be selectively modulated by drugs acting on the neuroendocrine axis.

A comparative study between conventional and laparoscopic cholecystectomy: evaluation of phagocytic and T-cell-mediated antibacterial activities.

Over the past few years, many reports have pointed out that open, but not minimally invasive, cholecystectomy was associated with reduced immune functions. Also, after laparoscopic surgery, a reduced impairment of T cell functions and lower levels of proinflammatory cytokines, epinephrine, and norepinephrine were found in comparison with those detected in patients who underwent conventional cholecystectomy. We investigated polymorphonuclear cell- and monocyte-mediated phagocytosis and killing and T-cell-mediated antibacterial activity in 12 patients who underwent open cholecystectomy versus another group of 12 patients who underwent laparoscopic cholecystectomy. Our data show that polymorphonuclear and monocyte killing activities are preserved or are less affected in patients who undergo laparoscopy when compared with patients who undergo conventional operation. On the other hand, in both groups of patients, T-cell-mediated antibacterial activity was significantly reduced in the preoperative period, and, therefore, we could not draw conclusions on the effects of the surgical techniques used on the above immune parameter. The overall data suggest that laparoscopic cholecystectomy is a valid alternative to open surgery because of the moderate postoperative immune suppression and decreased risk of postsurgical infections.

Toll-like receptor-positive cells and recognition of pathogens: how human myeloid dendritic cells respond to in vitro infection with Leishmania infantum.

Dendritic cells (DCs), instructed by the priming signals from microbial factors, can produce interleukin (IL)-12p70 and promote T helper (Th)1 proliferation and interferon (IFN)-gamma production. This event seems to be critical for the containment of infections caused by intracellular pathogens, even including Leishmania infection. In the present in vitro study we have investigated: 1) phagocytic capacities and IL-12 production by human monocyte-derived DCs and macrophages (MØs), infected with Leishmania infantum promastigotes; 2) IFN-gamma production by human CD4+ T cells co-incubated with DCs or macrophages pulsed with live promastigotes. Monocyte-derived myeloid DCs and MØs from healthy donors were infected with live metacyclic Leishmania infantum (MON-1) promastigotes, previously opsonized with 5% autologous serum, at 1:4 cell/parasite ratio. Percentage and index of phagocytosis were calculated after 2, 24 and 48 h of incubation. IL-12 production was evaluated by an ELISA in supernatants from 48 h Leishmania-infected or lipopolysaccharides (LPS)-stimulated DCs and MØs, also in the presence of phytohemagglutinin-activated or inactivated CD4+ T cells. For IFN-gamma production, CD4+ T cells were repeatedly stimulated with DCs or MØs, pulsed with live Leishmania promastigotes or activated with LPS. The number of IFN-gamma-secreting cells was evaluated by an ELISpot assay. Results showed that MØs have a higher phagocytic capacity towards L. infantum promastigotes than DCs. Moreover, unlike MØs, Leishmania-infected DCs were able to release IL-12p70; this production significantly increased in the presence of activated CD4+ T cells. Finally, DCs pulsed with live parasites and added to autologous CD4+ T cells induced a higher number of IFN-gamma-secreting cells than MØs, thus indicating their ability to polarize Th cells toward the Th1 subset. These data indicate that DCs are able to promote protective Th1 immune responses in our experimental model of Leishmania infantum infection, thus representing the grounds for initiating immunoterapeutic and vaccinal strategies.

Successful Treatment of Herpes Simplex Virus (HSV) Recurrent Genital Infection with Recombinant Human (RH) Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF): A Case Report

In the present work, we describe the treatment with rhGM-CSF of a woman affected by HSV recurrent genital infection and not responsive to specific antiviral therapy. The therapeutic regimen consisted of a subcutaneous administration of 300 mg/day of rhGM-CSF for six days. Before treatment with rhGM-CSF, polymorphonuclear cell and monocyte functional capacities and the antibacterial activity exerted by T cells were profoundly depressed. After treatment, a normalization of immune functions and a progressive disappearance of clinical manifestations were observed.