Self-assembled dextrin nanogel as protein carrier : controlled release and biological activity of IL-10

Interleukin-10 (IL-10) is an anti-inflammatory cytokine, which active form is a non-covalent homodimer. Given the potential of IL-10 for application in various medical conditions, it is essential to develop systems for its effective delivery. In previous work, it has been shown that a dextrin nanogel effectively incorporated and stabilized rIL10, enabling its release over time. In this work, the delivery system based on dextrin nanogels was further analyzed. The biocompatibility of the nanogel was comprehensively analyzed, through cytotoxicity (lactate dehydrogenase release, MTS, Live and Dead) and genotoxicity (comet) assays. The release profile of rIL-10 and its biological activity were evaluated in vivo, using C57BL/6 mice. Although able to maintain a stable concentration of IL-10 for at least 4 hours in mice serum, the amount of protein released was rather low. Despite this, the amount of rIL-10 released from the complex was biologically active inhibiting TNF-α production, in vivo, by LPSchallenged mice. In spite of the significant stabilization achieved using the nanogel, rIL-10 still denatures rather quickly. An additional effort is thus necessary to develop an effective delivery system for this cytokine, able to release active protein over longer periods of time. Nevertheless, the good biocompatibility, the protein stabilization effect and the ability to perform as a carrier with controlled release suggest that self-assembled dextrin nanogels may be useful protein delivery systems.Contract grant sponsor: Fundacao para a Ciencia e Tecnologia (FCT), PortugalContract grant number: SFRH/BD/27359/2006Contract grant sponsor: FCTContract grant number: PTDC/BIO/67160/2006; SUDOE-FEDERIMMUNONETSOE1/P1/E01

Trypanosome Lytic Factor, an Antimicrobial High-Density Lipoprotein, Ameliorates Leishmania Infection

Innate immunity is the first line of defense against invading microorganisms. Trypanosome Lytic Factor (TLF) is a minor sub-fraction of human high-density lipoprotein that provides innate immunity by completely protecting humans from infection by most species of African trypanosomes, which belong to the Kinetoplastida order. Herein, we demonstrate the broader protective effects of human TLF, which inhibits intracellular infection by Leishmania, a kinetoplastid that replicates in phagolysosomes of macrophages. We show that TLF accumulates within the parasitophorous vacuole of macrophages in vitro and reduces the number of Leishmania metacyclic promastigotes, but not amastigotes. We do not detect any activation of the macrophages by TLF in the presence or absence of Leishmania, and therefore propose that TLF directly damages the parasite in the acidic parasitophorous vacuole. To investigate the physiological relevance of this observation, we have reconstituted lytic activity in vivo by generating mice that express the two main protein components of TLFs: human apolipoprotein L-I and haptoglobin-related protein. Both proteins are expressed in mice at levels equivalent to those found in humans and circulate within high-density lipoproteins. We find that TLF mice can ameliorate an infection with Leishmania by significantly reducing the pathogen burden. In contrast, TLF mice were not protected against infection by the kinetoplastid Trypanosoma cruzi, which infects many cell types and transiently passes through a phagolysosome. We conclude that TLF not only determines species specificity for African trypanosomes, but can also ameliorate an infection with Leishmania, while having no effect on T. cruzi. We propose that TLFs are a component of the innate immune system that can limit infections by their ability to selectively damage pathogens in phagolysosomes within the reticuloendothelial system

Coordinate regulation of tissue macrophage and dendritic cell population dynamics by CSF-1

CSF-1 drives the homeostatic expansion of macrophages within the growing myometrium of pregnant mice by stimulating in situ proliferation and inducing monocyte precursor recruitment from the blood

Increased Monocyte Turnover from Bone Marrow Correlates with Severity of SIV Encephalitis and CD163 Levels in Plasma

Cells of the myeloid lineage are significant targets for human immunodeficiency virus (HIV) in humans and simian immunodeficiency virus (SIV) in monkeys. Monocytes play critical roles in innate and adaptive immunity during inflammation. We hypothesize that specific subsets of monocytes expand with AIDS and drive central nervous system (CNS) disease. Additionally, there may be expansion of cells from the bone marrow through blood with subsequent macrophage accumulation in tissues driving pathogenesis. To identify monocytes that recently emigrated from bone marrow, we used 5-bromo-2′-deoxyuridine (BrdU) labeling in a longitudinal study of SIV-infected CD8+ T lymphocyte depleted macaques. Monocyte expansion and kinetics in blood was assessed and newly migrated monocyte/macrophages were identified within the CNS. Five animals developed rapid AIDS with differing severity of SIVE. The percentages of BrdU+ monocytes in these animals increased dramatically, early after infection, peaking at necropsy where the percentage of BrdU+ monocytes correlated with the severity of SIVE. Early analysis revealed changes in the percentages of BrdU+ monocytes between slow and rapid progressors as early as 8 days and consistently by 27 days post infection. Soluble CD163 (sCD163) in plasma correlated with the percentage of BrdU+ monocytes in blood, demonstrating a relationship between monocyte activation and expansion with disease. BrdU+ monocytes/macrophages were found within perivascular spaces and SIVE lesions. The majority (80–90%) of the BrdU+ cells were Mac387+ that were not productively infected. There was a minor population of CD68+BrdU+ cells (<10%), very few of which were infected (<1% of total BrdU+ cells). Our results suggest that an increased rate of monocyte recruitment from bone marrow into the blood correlates with rapid progression to AIDS, and the magnitude of BrdU+ monocytes correlates with the severity of SIVE

Inhibition of IL-10 Production by Maternal Antibodies against Group B Streptococcus GAPDH Confers Immunity to Offspring by Favoring Neutrophil Recruitment

Group B Streptococcus (GBS) is the leading cause of neonatal pneumonia, septicemia, and meningitis. We have previously shown that in adult mice GBS glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is an extracellular virulence factor that induces production of the immunosuppressive cytokine interleukin-10 (IL-10) by the host early upon bacterial infection. Here, we investigate whether immunity to neonatal GBS infection could be achieved through maternal vaccination against bacterial GAPDH. Female BALB/c mice were immunized with rGAPDH and the progeny was infected with a lethal inoculum of GBS strains. Neonatal mice born from mothers immunized with rGAPDH were protected against infection with GBS strains, including the ST-17 highly virulent clone. A similar protective effect was observed in newborns passively immunized with anti-rGAPDH IgG antibodies, or F(ab')2 fragments, indicating that protection achieved with rGAPDH vaccination is independent of opsonophagocytic killing of bacteria. Protection against lethal GBS infection through rGAPDH maternal vaccination was due to neutralization of IL-10 production soon after infection. Consequently, IL-10 deficient (IL-10−/−) mice pups were as resistant to GBS infection as pups born from vaccinated mothers. We observed that protection was correlated with increased neutrophil trafficking to infected organs. Thus, anti-rGAPDH or anti-IL-10R treatment of mice pups before GBS infection resulted in increased neutrophil numbers and lower bacterial load in infected organs, as compared to newborn mice treated with the respective control antibodies. We showed that mothers immunized with rGAPDH produce neutralizing antibodies that are sufficient to decrease IL-10 production and induce neutrophil recruitment into infected tissues in newborn mice. These results uncover a novel mechanism for GBS virulence in a neonatal host that could be neutralized by vaccination or immunotherapy. As GBS GAPDH is a structurally conserved enzyme that is metabolically essential for bacterial growth in media containing glucose as the sole carbon source (i.e., the blood), this protein constitutes a powerful candidate for the development of a human vaccine against this pathogen

The clinical significance of inflammatory cytokines in primary cell culture in endometrial carcinoma

Endometrial cancer is the most common malignancy of the female genital tract, and the incidence and mortality rates from this disease are increasing. Although endometrial carcinoma has been regarded as a tissue‐specific disease mediated by female sex steroid pathways, considerable evidence implicates a role for an inflammatory response in the development and propagation of endometrial cancer. We hypothesized that if specific patterns of cytokine expression were found to be predictive of adverse outcome, then selective receptor targeting may be a therapeutic option. This study was therefore undertaken to determine the relationship between cytokine production in primary cell culture and clinical outcome in endometrial adenocarcinoma. Fresh endometrial tissues were fractionated into epithelial and stromal fractions and cultured. After 6–7 days, supernatants were collected and cells enumerated. Batched aliquots were assayed using ELISA kits specific for CSF‐1, GMCSF, G‐CSF, TNF‐α, IL‐6, IL‐8, and VEGF. Data were compared using ANOVA, Fisher's exact, and log rank tests. Increased epithelial VEGF production was observed more often in tumors with Type 2 variants (p = 0.039) and when GPR30 receptor expression was high (p = 0.038). Although increased stromal VEGF production was detected more often in grade 3 endometrioid tumors (p = 0.050), when EGFR expression was high (p = 0.003), and/or when ER/PR expression was low (p = 0.048), VEGF production did not correlated with overall survival (OS). Increased epithelial CSF‐1 and TNF‐α production, respectively, were observed more often in tumors with deep myometrial invasion (p = 0.014) and advanced stage (p = 0.018). Increased CSF‐1 (89.5% vs. 42.9%, p = 0.032), TNF‐α (88.9% vs. 42.9%, p = 0.032, and IL‐6 (92.3% vs. 61.5%, p = 0.052) also correlated with low OS. In Cox multivariate models, CSF‐1 was an independent predictor of low survival when stratified by grade (p = 0.046) and histology (p = 0.050), and TNF‐α, when stratified by histology (p = 0.037). In this study, high CSF‐1, TNF‐α, and IL‐6 production rates identified patients at greatest risk for death, and may signify patients likely to benefit from receptor‐specific therapy

IL-4 directly signals tissue-resident macrophages to proliferate beyond homeostatic levels controlled by CSF-1

Macrophages (M Phi s) colonize tissues during inflammation in two distinct ways: recruitment of monocyte precursors and proliferation of resident cells. We recently revealed a major role for IL-4 in the proliferative expansion of resident M Phi s during a Th2-biased tissue nematode infection. We now show that proliferation of M Phi s during intestinal as well as tissue nematode infection is restricted to sites of IL-4 production and requires M Phi-intrinsic IL-4R signaling. However, both IL-4R alpha-dependent and -independent mechanisms contributed to M Phi proliferation during nematode infections. IL-4R-independent proliferation was controlled by a rise in local CSF-1 levels, but IL-4R alpha expression conferred a competitive advantage with higher and more sustained proliferation and increased accumulation of IL-4R alpha(+) compared with IL-4R alpha(-) cells. Mechanistically, this occurred by conversion of IL-4R alpha(+) M Phi s from a CSF-1-dependent to -independent program of proliferation. Thus, IL-4 increases the relative density of tissue M Phi s by overcoming the constraints mediated by the availability of CSF-1. Finally, although both elevated CSF1R and IL-4R alpha signaling triggered proliferation above homeostatic levels, only CSF-1 led to the recruitment of monocytes and neutrophils. Thus, the IL-4 pathway of proliferation may have developed as an alternative to CSF-1 to increase resident M Phi numbers without coincident monocyte recruitment