Density measurement of Demodex canis by qPCR and analysis of serum cytokine levels in dogs with different clinical forms of demodicosis.

OBJECTIVES: To quantify (by qPCR) the density of Demodex canis mites in the skin of dogs with demodicosis and in healthy dogs, as well as measuring the serum concentrations of interleukin (IL)-1ß, IL-6, IL-8, IL-10, IL-12, and tumour necrosis factor-alfa (TNF-α). METHODS: Fifty-four dogs were divided into three groups: localized demodicosis (LD, n = 16), generalized demodicosis (GD, n = 22), and control group (CG, n = 16). All dogs were subjected to skin scraping, blood collection, and skin biopsy. DNA extraction was performed and the parasite density was established by qPCR. Serum cytokine concentrations were obtained by flow cytometry. RESULTS: The median number of mites in the skin of the GD (6.2 × 104 copies/µL) and LD dogs (1.2 × 104 copies/µL) was statistically higher than that in the CG dogs (8.7 × 102 copies/µL). Whereas there were no significant differences in median IL-1ß, IL-8, IL-10, IL-12, and TNF-α levels among the study groups, there was a statistically higher IL-6 concentration in the LD dogs than in the healthy dogs. CONCLUSIONS: According to our results, qPCR is an effective method for measuring the density of D. canis in the canine integument. In addition, the activation of the acute-phase immune response in localized demodicosis can be induced by IL-6 activity.

Human colostrum action against Giardia lamblia infection influenced by hormones and advanced maternal age.

Children are more susceptible to Giardia lamblia infection. Cells and hormones contained in human colostrum have an immunoprotective action against giardiasis, but the effects of advanced maternal age on these components are poorly understood. This study analyzed the colostrum of older women to determine melatonin and cortisol levels besides the participation of these hormones on the functional activity of phagocytes against G. lamblia. Colostrum samples were collected from younger (18 to 35 years old) and older (over 36 years old) lactating women. Colostrum samples were subjected to melatonin and cortisol determination, immunophenotyping, quantification of superoxide release, and assessment of phagocytic rate and microbicidal activity of phagocytes treated with hormones and in the presence of G. lamblia. Colostrum from mothers of advanced age contained higher melatonin and cortisol levels and a lower rate of cells expressing CD14+ and CD15+. In the colostru of these older mothers, melatonin increased superoxide release by phagocytes. In both groups, superoxide release by phagocytes treated with cortisol was higher in the presence of G. lamblia. In colostrum from mothers of advanced age, mononuclear (MN) phagocytes treated with melatonin showed higher phagocytosis of G. lamblia and higher microbicidal index. In younger mothers, MN and polymorphonuclear (PMN) colostrum phagocytes exhibited higher rates of G. lamblia elimination when treated with both melatonin and cortisol. In older mothers, cortisol and melatonin regulation for the functional activity of colostrum phagocytes against G. lamblia may represent an additional defense mechanism, relevant for the protection and treatment of parasitic infections in breastfed children.

Herbal Mixture Adsorbed to Polyethylene Glycol Microspheres Induces Apoptotic Effects on Breast Cancer Cells.

BACKGROUND: The combination of medicinal plants and polymeric matrices raised the possibility of obtaining new drugs to treat a number of diseases, including cancer. Breast cancer is one of the most frequent diseases among women, but the effective treatments is still a challenge. METHODS: Thus, this study investigated the effect of herbal mixture adsorbed to PEG microspheres on MCF-7 human breast cancer cells and these cells in co-culture of blood MN cells. The MCF-7 cells and the blood mononuclear [MN] cells from volunteer donors. MN cells, MCF-7 cells and co-culture [MN and MCF-7 cells] were pre-incubated for 24 h with or without herbal mixture [HM], polyethylene glycol [PEG] microspheres or herbal mixture adsorbed in PEG microspheres [PEG-HM]. Viability, intracellular calcium and apoptosis were determined by flow cytometry. The herbal mixture adsorbed in PEG microspheres reduces the viability of MCF-7 cells. RESULTS: The lowest viability índex was observed in co-culture incubated with herbal mixture adsorbed to PEG microspheres. MN cells, MCF-7 cells and co-culture showed higher intracellular Ca2+ release when incubated with herbal mixture adsorbed to PEG microspheres. The apoptosis index was higher in MCF-7 cells that were treated with herbal mixture. The highest apoptosis index was observed in coculture of these cells incubated with herbal mixture adsorbed to PEG microspheres. CONCLUSION: These data suggest that herbal mixture adsorbed by PEG microspheres has apoptotic effects on human MCF-7 breast cancer cells and is therefore a possible therapeutic alternative.

In vitro immunomodulatory effects of microemulsions with levamisole delivery systems on blood phagocytes interacting with Giardia lamblia.

BACKGROUND: Giardiasis is one of the main parasites that infect the gastrointestinal tract of humans, affecting hundreds of millions of people worldwide, particularly in developing countries. Antiparasitics administered to treat giardiasis are inefficient in 20% of the cases, usually because of parasite resistance and side effects. In this scenario, microemulsions are a promising pharmaceutical alternative as carriers of molecules with therapeutic action that stimulate the immune system. METHODS: The study evaluated the effects of a microemulsion delivery system with levamisole hydrochloride on the functional activity of MN phagocytes incubated with G. lamblia. RESULTS: The microemulsion formulated was incorporated with levamisole hydrochloride using distilled water, caprylic/capric triglyceride-Polymol 812®, Sorbitan Oleate-Span 80®, Polysorbate 80 - Tween 80® and 1-butanol. The activity of the microemulsion was analyzed by phagocytosis rate, microbicidal activity, apoptosis rate and intracellular calcium concentration. Phagocytosis rate, microbicidal activity and apoptosis index increased in the microemulsion treatment. The results suggest that the microemulsion improves the therapeutic efficacy of levamisole, increasing the functional activity of phagocytes. CONCLUSIONS: The microemulsion with a levamisole delivery system is therefore an efficient alternative for treating giardiasis, acting as an immunomodulator that probably causes fewer side effects than conventional drugs.

Characterization of Natural Killer Cells and Cytokines in Maternal Placenta and Fetus of Diabetic Mothers.

The present study characterized natural killer cells and cytokines in diabetic mothers, their placenta, and fetus. In the maternal blood from the hyperglycemic groups, the CD16(+)CD56(-) NK cells increased, whereas that of CD16(+)CD56(+) decreased in gestational diabetes mellitus [GDM] group. Cord blood from type 2 diabetes [DM-2] showed a higher proportion of CD16(+)CD56(-) and CD16(-)CD56(+). The placental extravillous layer of GDM and DM-2 showed an increase of CD16(+)CD56(-) cells and, irrespective of region, the proportion of CD16(-)CD56(+) cells was higher in mild gestational hyperglycemia [MGH] and GDM and lower in DM-2. IL-2 was lower in maternal blood and IFN-γ higher in maternal and cord blood from the GDM group. IL-17 was higher in maternal and cord blood from the DM-2 group. The placental extravillous layer of the MGH showed high levels of IL-4, IL-6, IL-10, IL-17, and IFN-γ and low levels of IL-1ß and IL-8, whereas the placental villous layer contained high levels of IL-17 and IFN-γ. The GDM group, irrespective of region, showed higher levels of IL-8. The DM-2 group, irrespective of region, placenta showed high levels of TNF-α, IL-17, and IFN-γ. The hyperglycemia produces an inflammatory environment with a high content of inflammatory cytokines and cells expressing CD16(+).

Intracellular calcium is a target of modulation of apoptosis in MCF-7 cells in the presence of IgA adsorbed to polyethylene glycol.

PURPOSE: Clinical and epidemiological studies have indicated that breastfeeding has a protective effect on breast cancer risk. Protein-based drugs, including antibodies, are being developed to attain better forms of cancer therapy. Secretory IgA (SIgA) is the antibody class in human breast milk, and its activity can be linked to the protective effect of breastfeeding. The aim of this study was to investigate the effect of polyethylene glycol (PEG) microspheres with adsorbed SIgA on MCF-7 human breast cancer cells. METHODS: The PEG microspheres were characterized by flow cytometry and fluorescence microscopy. The MCF-7 cells were obtained from American Type Culture Collection. MCF-7 cells were pre-incubated for 24 hours with or without SIgA (100 ng/mL), PEG microspheres or SIgA adsorbed in PEG microspheres (100 ng/mL). Viability, intracellular calcium release, and apoptosis in MCF-7 cells were determined by flow cytometry. RESULTS: Fluorescence microscopy and flow cytometry analyses revealed that SIgA was able to adsorb to the PEG microspheres. The MCF-7 cells that were incubated with PEG microspheres with adsorbed SIgA showed decreased viability. MCF-7 cells that were incubated with SIgA or PEG microspheres with adsorbed SIgA had increased intracellular Ca(2+) levels. In the presence of SIgA, an increase in the percentage of apoptotic cells was observed. The highest apoptosis index was observed when the cells were treated with PEG microspheres with adsorbed SIgA. CONCLUSION: These data suggest that colostral SIgA adsorbed to PEG microspheres has antitumor effects on human MCF-7 breast cancer cells and that the presence of large amounts of this protein in secreted breast milk may provide protection against breast tumors in women who breastfed.

The effect of IFN-γ and TGF-ß in the functional activity of mononuclear cells in the presence of Entamoeba histolytica.

BACKGROUND: Entamoeba histolytica (E. histolytica) causes amoebiasis, which is a disease with significant morbidity and mortality. Phagocytic cells and cytokines appear to be important in amoebiasis, but very little is known about the influence of these cells and cytokines in protozoan infections. The aim of this study was to analyse the supernatant of cultures of mononuclear (MN) cells with E. histolytica to determine: 1) the levels of the cytokines IFN-γ and TGF-ß, and 2) the amoebicidal activity of MN cells after incubation with cytokines. METHODS: Blood samples were collected from 30 volunteer donors. The cytokine concentrations in MN cells culture supernatants, superoxide release, leukophagocytosis, amoebicide activity, intracellular calcium release and apoptosis were analysed. RESULTS: The IFN-γ concentrations were 6.22 ± 0.36 and TGF-ß concentrations were 17.01 ± 2.21 in cells-trophozoite culture supernatants. MN cells, independently of cytokines, in the presence of amoeba increase the superoxide release. In the absence of cytokines, the ingestion of MN cells by amoebae was higher. In the presence of IFN- γ or TGF- ß, a lower ingestion of MN cells was observed by amoebae. MN cells treated with cytokines exhibited higher amoebicide and apoptosis indexes. The incubation of cytokines increased the intracellular calcium release by MN cells. CONCLUSIONS: These results suggest that cytokines play a beneficial role for the host by activating MN cells against E. histolytica. The increased death of amoebae during the leukophagocytosis suggests that both cytokines (IFN-γ and TGF-ß) can modulate the functional activity of MN cells and that these cytokines probably are important in the control of amoebic infections.

The role of cytokines in the functional activity of phagocytes in blood and colostrum of diabetic mothers.

Immune response changes induced by diabetes are a risk factor for infections during pregnancy and may modify the development of the newborn's immune system. The present study analyzed colostrum and maternal and cord blood of diabetic women to determine (1) the levels of the cytokines IFN- γ and TGF- ß and (2) phagocytic activity after incubation with cytokines. Methods. Colostrum and maternal and cord blood samples were classified into normoglycemic (N = 20) and diabetic (N = 19) groups. Cytokine levels, superoxide release, rate of phagocytosis, bactericidal activity, and intracellular Ca(2+) release by phagocytes were analyzed in the samples. Irrespective of glycemic status, IFN- γ and TGF- ß levels were not changed in colostrum and maternal and cord blood. In maternal blood and colostrum, superoxide release by cytokine-stimulated phagocytes was similar between the groups. Compared to spontaneous release, superoxide release was stimulated by IFN- γ and TGF- ß in normoglycemic and diabetic groups. In the diabetic group, cord blood phagocytes incubated with IFN- γ exhibited higher phagocytic activity in response to EPEC, and maternal blood exhibited lower microbicidal activity. These data suggest that diabetes interferes in maternal immunological parameters and that IFN- γ and TGF- ß modulate the functional activity of phagocytes in the colostrum, maternal blood, and cord blood of pregnant diabetic women.

Secretory IgA-Fcα receptor interaction modulating phagocytosis and microbicidal activity by phagocytes in human colostrum of diabetics.

The effects of secretory immunoglobulin A (SIgA) interaction with its specific Fcα receptors on colostral phagocytes needs further investigation, especially with respect to diabetic women. Accordingly, we studied the colostrum of hyperglycemic women to assess SIgA interactions with Fcα receptors of macrophages as well as the functional activity of these cells. The women were divided for colostrum sampling according to their glycemic status: normoglycemia (N = 51), mild hyperglycemia (N = 23), and diabetes (N = 25) groups. We determined the FcαR expression, the IgA on the surface and the surface-bound IgA in colostrum macrophages. We also evaluated the superoxide release and bactericidal killing of these cells. Colostral phagocytes expressed FcαR, contained IgA on the surface and are able to bind to purified SIgA. The bactericidal activity of colostral phagocytes from the hyperglycemic women was similar to that of normoglycemic only when SIgA was used as opsonin. Addition of a MoAb anti-human Fcα receptor resulted in a significant decrease of superoxide release and bacterial killing by macrophages when bacteria were opsonized with purified SIgA, suggesting an interaction between SIgA and FcαR. The stimulatory effects of SIgA on the functional activity of phagocytes therefore protect infants, especially of diabetic women, against intestinal infections.