RESUMO
The antimicrobial protein lactoferrin (Lf) is present in plasma and in mucosal secretions. Using ELISA we analysed plasma and saliva of HIV-infected patients, patients with AIDS, and healthy controls for the presence of secreted Lf. The plasma Lf levels of AIDS patients (classification C3) were significantly lower (p < 0.001) as compared to asymptomatic and symptomatic HIV infected patients, or controls. In addition, plasma Lf levels closely correlated with neutrophilic granulocyte counts in the HIV-infected patients. Thus, basal plasma Lf levels are likely the result of Lf release by neutrophilic granulocytes. The Candida titres present in the oral cavity were determined in a part of the HIV-infected patient group. As it appeared, the presence of this opportunistic pathogen always coincided with low levels of salivary Lf levels. We conclude that Lf, as part of the nonspecific immune system, might play an important role in the first line of defense against opportunistic microbial infections in AIDS patients.
Assuntos
Síndrome da Imunodeficiência Adquirida/sangue , Lactoferrina/sangue , Infecções Oportunistas Relacionadas com a AIDS/microbiologia , Síndrome da Imunodeficiência Adquirida/imunologia , Síndrome da Imunodeficiência Adquirida/fisiopatologia , Candidíase/microbiologia , Humanos , Lactoferrina/imunologia , Contagem de Leucócitos , Neutrófilos/citologia , SalivaRESUMO
Lactoferrin (LF) is an iron binding glycoprotein that is present in several mucosal secretions. Many biological functions have been ascribed to LF. One of the functions of LF is the transport of metals, but LF is also an important component of the non-specific immune system, since LF has antimicrobial properties against bacteria, fungi and several viruses. This review gives an overview of the present knowledge about the antiviral activities and, when possible, the antiviral modes of action of this protein. Lactoferrin displays antiviral activity against both DNA- and RNA-viruses, including rotavirus, respiratory syncytial virus, herpes viruses and HIV. The antiviral effect of LF lies in the early phase of infection. Lactoferrin prevents entry of virus in the host cell, either by blocking cellular receptors, or by direct binding to the virus particles.
Assuntos
Antivirais/metabolismo , Lactoferrina/metabolismo , Viroses/metabolismo , Animais , Antivirais/química , Antivirais/farmacocinética , Antivirais/farmacologia , Humanos , Lactoferrina/química , Lactoferrina/farmacocinética , Lactoferrina/farmacologia , Leite/química , Leite/virologia , Viroses/dietoterapia , Viroses/virologia , Replicação Viral/efeitos dos fármacosRESUMO
A series of native and chemically derivatized lactoferrins (Lfs) purified from milk and colostrum were assayed in vitro for their anti-HIV and anti-HCMV-cytopathic effects in MT4 cells and fibroblasts respectively. All Lfs from bovine and human milk or colostrum were able to completely block HCMV replication as well as inhibited HIV-1 induced cytopathic effects. Through acylation of the amino function of the lysine residues in Lf, using anhydrides of succinic acid or cis-aconitic acid, negatively charged Lf derivatives were obtained that all showed a strong antiviral activity against the HIV-1 in vitro. Acylated-Lf exhibited a 4-fold stronger antiviral effect on HIV-1 than the parent compound but the activity on HCMV was abolished. Peptide scanning studies indicated that the native Lf as well as acylated Lf strongly bind to the V3 domain of the HIV envelope protein gp120, with Kd values in the same concentration range as the in vitro IC50. Therefore, shielding of this domain, resulting in inhibition of the virus-cell fusion and entry of the virus in MT4 cells is the likely mechanism underlying the anti-HIV activity. In contrast, addition of positive charges to Lf through amination of the proteins resulted in an increased anti-HCMV activity and a loss of anti-HIV activity, with anti-HCMV IC50 values in the low micromolar concentration range. The N-terminal portion of LF appeared essential to this anti-HCMV effect. The specific distribution of positively and negatively charged domains in the molecule appears to be important in both the anti-HIV and anti-HCMV effects.
Assuntos
Infecções por Citomegalovirus/tratamento farmacológico , Citomegalovirus/efeitos dos fármacos , Infecções por HIV/tratamento farmacológico , HIV-1/efeitos dos fármacos , Animais , Bovinos , Linhagem Celular , Citomegalovirus/fisiologia , Fibroblastos/virologia , HIV-1/fisiologia , Humanos , Replicação Viral/efeitos dos fármacosRESUMO
Cell therapy strategies that use adult peripheral blood-derived CD34⺠progenitor cells are hampered by low cell numbers and the infrequent cellular incorporation into the neovasculature. Hence, the use of CD34⺠cells to treat ischaemic diseases is under debate. Interaction between CD34⺠cells and CD14⺠cells results in superior endothelial differentiation of CD14⺠cells in vitro, indicating that cell therapy approaches utilizing both CD34⺠and CD14⺠cells may be advantageous in therapeutic neovascularization. Here, human CD34⺠and CD14⺠cells were isolated from adult peripheral blood and implanted subcutaneously into nude mice, using matrigel as the carrier. Combined implantation of human CD34⺠and CD14⺠cells resulted in superior neovascularization, compared to either cell type alone, albeit incorporation of human cells into the murine vasculature was not observed. Human CD34⺠and CD14⺠cells produced and secreted a pentad of pro-angiogenic mediators, such as HGF, MCP-1 and IL-8, bFGF and VEGFa in monoculture. The production and secretion of pro-angiogenic mediators by CD14⺠cells was highly amplified upon incubation with conditioned medium from CD34⺠cells. In vivo, neovascularization of matrigel implants did not rely on the endothelial differentiation and incorporation of CD34⺠or CD14⺠cells, but depended on the paracrine effects of IL-8, MCP-1, HGF, bFGF and VEGFa secreted by implanted cells. Administration of this growth factor/cytokine pentad using matrigel as a carrier results in cell recruitment and microvessel formation equal to progenitor cell-induced neovascularization. These data provide new insights on neovascularization by cell therapy and may contribute to new strategies for the treatment of ischaemic diseases.
Assuntos
Antígenos CD34/metabolismo , Transplante de Células , Receptores de Lipopolissacarídeos/metabolismo , Neovascularização Fisiológica , Comunicação Parácrina , Animais , Colágeno/farmacologia , Combinação de Medicamentos , Humanos , Laminina/farmacologia , Masculino , Camundongos , Camundongos Nus , Neovascularização Fisiológica/efeitos dos fármacos , Comunicação Parácrina/efeitos dos fármacos , Proteoglicanas/farmacologiaRESUMO
CD34+ progenitor cells hold promise for therapeutic neovascularization in various settings. In this study, the role of human peripheral blood CD34+ cells in neovascularization and inflammatory cell recruitment was longitudinally studied in vivo. Human CD34+ cells were incorporated in Matrigel, implanted subcutaneously in nude mice, and explanted after 2, 4, 7, or 14 days. Cell-free Matrigels served as controls. Histochemical analyses demonstrated that neovascularization occurred almost exclusively in CD34+ implants. Cellular and capillary density were increased in cell-loaded Matrigels after 2 days and further increased at 14 days. Human CD34+ cells did not incorporate in neovessels, but formed vWF+/CD31+/VEGF+ cell clusters that were present up to day 14. However, CD34+ cells induced host neovascularization, as demonstrated by increased presence of murine CD31+ and vWF+ vasculature from day 7 to 14. Moreover, recruitment of murine monocytes/macrophages was significantly enhanced in CD34+ implants at all time points. Gene expression of chemotactic cytokines MCP-1 and IL-8 was detected on CD34+ cells in vitro and confirmed immunohistochemically in cell-loaded explants at all time points. Our data indicate that human CD34+ cells, implanted in a hypoxic environment, generate an angiogenic niche by secreting chemotactic and angiogenic factors, enabling rapid neovascularization, possibly via recruitment of monocytes/macrophages.
Assuntos
Antígenos CD34/metabolismo , Células-Tronco Hematopoéticas/imunologia , Inflamação/imunologia , Neovascularização Fisiológica/imunologia , Animais , Antígenos CD34/administração & dosagem , Antígenos CD34/genética , Células Cultivadas , Quimiocina CCL2/metabolismo , Colágeno , Modelos Animais de Doenças , Combinação de Medicamentos , Transplante de Células-Tronco Hematopoéticas/métodos , Humanos , Imuno-Histoquímica , Injeções Subcutâneas , Interleucina-8/metabolismo , Laminina , Macrófagos/citologia , Masculino , Camundongos , Camundongos Nus , Monócitos/citologia , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Proteoglicanas , Fatores de Tempo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
BACKGROUND: In humans the prevalence of asthma is higher among females than among males after puberty. The reason for this phenomenon is not clear. OBJECTIVE: We tested the hypothesis that female mice are more susceptible to the development of allergic asthma than male mice and studied allergic immune responses in the lung. METHODS: We compared allergic airway inflammation, i.e. methacholine (MCh) responsiveness, serum IgE, and cytokines, and the number of the different leucocytes in lungs of male and female BALB/c mice, twice-sensitized to ovalbumin (OVA) and subsequently challenged with OVA (OVA-mice) or phosphate-buffered saline (PBS-mice) aerosols on days 24-26, 30, and 31. RESULTS: OVA challenge significantly increased MCh responsiveness, numbers of eosinophils, CD4(+) T cells, CD4(+)/CD25(+) T cells, B cells, and levels of Thelper (Th)2 cytokines, total, and OVA-specific IgE. There was, however, also an effect of gender, with female mice responding to OVA challenges with higher numbers of eosinophils, CD4(+) T cells, B cells, and levels of IL-4, IL-13, IFN-gamma, total, and OVA-specific IgE than male mice. In contrast, female PBS-mice had significantly lower percentages of regulatory CD4(+)/CD25(+) T cells than males (females 4.2+/-0.2% vs. males 5.3+/-0.1% of CD4(+) T cells, P<0.05). CONCLUSION: Female mice develop a more pronounced type of allergic airway inflammation than male mice after OVA challenge. The reduced percentage of regulatory T cells in the lungs of female PBS-mice may indicate that the level of these cells in the lung during the sensitization phase is important for the development and/or progression of an allergic immune response after multiple OVA challenges.
Assuntos
Asma/imunologia , Pulmão/imunologia , Animais , Asma/patologia , Linfócitos B/imunologia , Testes de Provocação Brônquica , Líquido da Lavagem Broncoalveolar/imunologia , Antígenos CD4/imunologia , Quimiocina CCL5/análise , Eosinófilos/imunologia , Feminino , Imunoglobulina E/análise , Interleucinas/análise , Pulmão/patologia , Masculino , Cloreto de Metacolina/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Ovalbumina/imunologia , Receptores de Interleucina-2/imunologia , Fatores Sexuais , Linfócitos T/imunologia , Células Th2/imunologiaRESUMO
OBJECTIVE: The cytomegalovirus (CMV) antigenemia consists of the detection of CMV pp65 in the nucleus of polymorphonuclear granulocytes (PMN), but it is unclear where and how PMN pick up virus particles or proteins. In an in vitro model for CMV antigenemia we investigated the mechanism of pp65 uptake by PMN that results in its expression in the nucleus. METHODS: A series of inhibitors of different mechanisms was used to study the uptake of pp65 by PMN during coculture with CMV-infected endothelial cells and we performed a morphological analysis by light and transmission electron microscopy. RESULTS: Nocodazole and cytochalasin B inhibited uptake of pp65 by PMN with 59.4 +/- 14.1 and 73.3 +/- 12.7%, respectively. The presence of anti-CMV hyperimmune globulin or lactoferrin during coculture reduced the number of pp65-positive PMN with 45.8 +/- 7.0 or 40.6 +/- 3.2%. Furthermore, a small number of the pp65-positive PMN obtained after coculture had fused to large cells with multilobed nuclei. PMN were observed that enclosed viral particles as well as free viral particles containing PMN in the cytoplasm. CONCLUSION: Fusion of viral particles with PMN and phagocytosis are both involved in the uptake of pp65.
Assuntos
Citomegalovirus/metabolismo , Endotélio Vascular/metabolismo , Neutrófilos/metabolismo , Fosfoproteínas/metabolismo , Proteínas da Matriz Viral/metabolismo , Anticorpos Antivirais/farmacologia , Antineoplásicos/farmacologia , Transporte Biológico/efeitos dos fármacos , Fusão Celular , Núcleo Celular/metabolismo , Técnicas de Cocultura , Citocalasina B/farmacologia , Citomegalovirus/isolamento & purificação , Citoplasma/virologia , Endotélio Vascular/virologia , Humanos , Imunoglobulina G/farmacologia , Lactoferrina/farmacologia , Microscopia Eletrônica , Neutrófilos/virologia , Nocodazol/farmacologia , Fagocitose , Fosfoproteínas/análise , Proteínas da Matriz Viral/análiseRESUMO
In vitro, lactoferrin (LF) strongly inhibits human cytomegalovirus (HCMV), which led us to hypothesize that in vivo HCMV might also be inhibited in secretions with high LF concentrations. In breast milk, high viral loads observed as high viral DNA titers tended to coincide with higher LF levels. However, the LF levels did not correlate to virus transmission to preterm infants. The viral load in the transmitting group was highest compared to the nontransmitting group. We conclude that viral load in breast milk is an important factor for transmission of the virus.
Assuntos
Infecções por Citomegalovirus/virologia , Transmissão Vertical de Doenças Infecciosas , Lactoferrina/metabolismo , Leite Humano/virologia , Carga Viral , Aleitamento Materno/efeitos adversos , Citomegalovirus/genética , Infecções por Citomegalovirus/metabolismo , Infecções por Citomegalovirus/transmissão , DNA Viral/análise , Interpretação Estatística de Dados , Feminino , Humanos , Recém-Nascido , Recém-Nascido Prematuro , Leite Humano/metabolismo , Reação em Cadeia da Polimerase , Estudos ProspectivosRESUMO
The role of leukocytes in the in vivo dissemination of cytomegalovirus was studied in this experiment. Rat cytomegalovirus (RCMV) could be transferred to rat granulocytes and monocytes by cocultivation with RCMV-infected fibroblasts in vitro. Intravenous injection of purified infected granulocytes or monocytes resulted in a systemic infection in rats, indicating that our model is a powerful tool to gain further insight into CMV dissemination and the development of new antivirals.
Assuntos
Granulócitos/virologia , Monócitos/virologia , Muromegalovirus/fisiologia , Animais , DNA Viral/sangue , Masculino , Neutrófilos/virologia , RatosRESUMO
Previous studies have shown that acylated plasma and milk proteins with increased negative charge, derived from various animal and human sources, are potent anti-HIV compounds. The antiviral effects seemed to correlate positively with the number of negative charges introduced into the various polypeptides: proteins with a high content of basic amino acids in which all of the available epsilonNH2 groups were anionized yielded the most potent anti-HIV compounds. It remained unclear however whether the total net negative charge of the various derivatized proteins, or rather the charge density on the protein backbone, is essential for the observed anti-HIV activity. Earlier studies have shown that acylated albumins preferentially block the process of HIV/cell fusion through binding to the HIV envelope proteins gp120 and gp41 as well as to the cell surface of the HIV target cells. Some of these polyanionic proteins have been shown to interfere also with the gp120-CD4 mediated virus/cell binding. The relative contribution of these effects to the anti-HIV activity may depend both on the total negative charge introduced as well as the hydrophobicity of the acylating reagent added to the particular proteins. In this study we show that the higher the charge density of the derivatized proteins, the more potent their HIV replication inhibiting effects are. In contrast, the addition of positive charge to the studied plasma and milk proteins through amination resulted in a reduced anti-HIV activity but a clearly increased anti-HCMV activity, with IC50 values in the low micromolar concentration range. Interestingly, native lactoferrin (Lf) was antivirally active against both HIV and HCMV. Acylation or amination of Lf increased the anti-HIV and anti-HCMV activity, respectively. The N-terminal portion of Lf appeared essential for its anti-HCMV effect: N-terminal deletion variants of human Lf were less active against HCMV. Circular dichroism of the modified proteins showed that the secondary structure of the tested proteins was only moderately influenced by acylation and/or covalent attachment of drugs, making these (derivatized) proteins useful candidates as antiviral agents and/or intrinsically active drug carriers. The relatively simple chemical derivatization as well as the abundant sources of blood plasma and milk proteins provides attractive opportunities for the preparation of potent and relatively cheap antiviral agents for systemic or local applications.