Separation and determination of liposomal and non-liposomal daunorubicin from the plasma of patients treated with Daunoxome

Several liposomal formulations of anthracyclines have been developed recently and are currently used in the clinical setting. We describe a technique of separation and quantification of the liposomal and non-liposomal forms of daunorubicin in the plasma of patients treated with DaunoXome, a liposomal formulation of daunorubicin. The method we propose is based upon the property of liposomes to cross reversed-phase C₁₈ silicagel cartridges without being retained, while non-liposomal drug is retained on the stationary phase and is eluted with methanol. Extraction of liposomal and non-liposomal daunorubicin from plasma, therefore, is performed in two steps. This technique is rapid, can be automated in order to handle large series of samples, and the plasma can be frozen after sampling by addition of glycerol. The recovery of liposomal daunorubicin as well as the precision, linearity and accuracy of the technique appear satisfactory for pharmacokinetic purposes.     

TLR7 ligands induce higher IFN-alpha production in females

IFN-alpha exercises multiple immune modulatory and antiviral activities and has been suggested to play a critical role in the pathogenesis of systemic lupus erythematosus (SLE). Plasmacytoid dendritic cells (pDCs) release IFN-alpha upon TLR7 and TLR9 ligation. With respect to the nine times higher incidence of SLE in women and the clinical use of synthetic TLR ligands as novel immune adjuvants, we analyzed IFN-alpha and TNF-alpha production in healthy human individuals. Blood samples were incubated with synthetic TLR7 and TLR9 ligands. In three independent groups (n(1) = 120, n(2) = 101, and n(3) = 123), analysis revealed a capacity of female PBLs to produce significantly higher IFN-alpha levels after TLR7 stimulation (p(1) < 0.0000001, p(2) < 0.0000001, and p(3) < 0.0001) compared with male PBLs. In contrast, no sex differences were evident after TLR9 stimulation. TNF-alpha production after TLR7 stimulation and also total pDC numbers were not different between females and males. X-inactivation escape of the TLR7 gene was investigated in monoclonal B cell lines and, independently, in pDCs after cell sorting and single-cell picking, indicating regular silencing of one TLR7 allele in females. Additionally, exogenous 17beta-estrogen and estrogen receptor antagonism did not indicate a significant role on TLR7-induced IFN-alpha production. Our data reveal for the first time a profound sex-dependent pathway of TLR7-induced IFN-alpha with higher production in females. These findings may explain the higher prevalence of SLE in females and the reported decreased therapeutic efficacy of synthetic TLR7 ligands in male individuals.     

Encapsulation in liposomal nanoparticles enhances the immunostimulatory,adjuvant and anti-tumor activity of subcutaneously administered CpG ODN

Immunostimulatory oligodeoxynucleotides (ODN) containing cytosine-guanine (CpG) motifs are powerful stimulators of innate as well as adaptive immune responses, exerting their activity through triggering of the Toll-like receptor 9. We have previously shown that encapsulation in liposomal nanoparticles (LN) enhances the immunostimulatory activity of CpG ODN (LN-CpG ODN) (Mui et al. in J Pharmacol Exp Ther 298:1185, 2001). In this work we investigate the effect of encapsulation on the immunopotency of subcutaneously (s.c.) administered CpG ODN with regard to activation of innate immune cells as well as its ability to act as a vaccine adjuvant with tumor-associated antigens (TAAs) to induce antigen (Ag)-specific, adaptive responses and anti-tumor activity in murine models. It is shown that encapsulation specifically targets CpG ODN for uptake by immune cells. This may provide the basis, at least in part, for the significantly enhanced immunostimulatory activity of LN-CpG ODN, inducing potent innate (as judged by immune cell activation and plasma cytokine/chemokine levels) and adaptive, Ag-specific (as judged by MHC tetramer positive T lymphocytes, IFN-γ secretion and cytotoxicity) immune responses. Finally, in efficacy studies, it is shown that liposomal encapsulation enhances the ability of CpG ODN to adjuvanate adaptive immune responses against co-administered TAAs after s.c. immunization, inducing effective anti-tumor activity against both model and syngeneic tumor Ags in murine tumor models of thymoma and melanoma.     

菖远胶囊给大鼠长期大剂量用药的安全性评价

目的：研究菖远胶囊对大鼠长期大量用药后的安全性影响。方法：通过对大鼠连续灌胃菖远胶囊13周、26周和停药4周,分别与正常对照组比较血液、生化、病理学检查结果。结果：①菖远胶囊各剂量组给药后5～30 min动物自主活动有所减弱,30 min后自主活动逐渐恢复正常,各剂量组大小便颜色偏深,高剂量组比较明显,停止给药后,上述症状均恢复正常;②大鼠血液细胞学、生化学指标中有个别指标在给药初期（13周）出现明显差异,继续给药至26周,未见明显异常,停止给药后恢复正常;高剂量组动物体重有一定降低,个别脏器质量及指数增大,停药4周后均能恢复正常;病理检查结果示,菖远胶囊各剂量组主要脏器未见由药物引起的病理学改变;整个实验期间各组动物未见死亡;③大鼠长期给予菖远胶囊的安全剂量为4.0 g/kg。结论：菖远胶囊对动物的毒性较低,是值得开发的一个中药新药。     

Single nucleotide polymorphisms (SNPs) in human lactoferrin gene.

The lactoferrin protein possesses antimicrobial and antiviral activities. It is also involved in the modulation of the immune response. In a normal healthy individual, lactoferrin plays a role in the front-line host defense against infection and in immune and inflammatory responses. Whether genomic variations, such as single nucleotide polymorphisms (SNPs), have an effect on the structure and function of lactoferrin protein and whether these variations contribute to the different susceptibility of individuals in response to environmental insults are interesting health-related issues. In this study, the lactoferrin gene was resequenced as part of the Environmental Genome Project of the National Institute of Environmental Health Sciences, which operates within the National Institutes of Health. Ninety-one healthy donors of different ethnicities were used to establish common SNPs in the exons of the lactoferrin gene in the general population. The data will serve as a basis from which study the association of lactoferrin polymorphism and disease.     

Exogenous IFN-alpha administration reduces influenza A virus replication in the lower respiratory tract of rhesus macaques

To determine the role of innate immune responses in controlling influenza A virus replication, rhesus macaques (RM) were administered pegylated IFN-alpha prior to virus challenge. Systemic and mucosal pegylated IFN-alpha administration induced expression of the interferon-stimulated genes (ISG) MxA and OAS in the airways. RM treated with IFN-alpha 24 hours prior to influenza virus challenge had significantly lower peak vRNA levels in the trachea compared to untreated animals. In addition to blunting viral replication, IFN-alpha treatment minimized the weight loss and spike in body temperature after influenza infection of RM. These results confirm the importance of IFN-alpha induced innate immune responses in the rapid control of influenza A virus replication in primates.     

FCgammaRII (CD32)-dependent induction of interferon-alpha by serum from patients with lupus erythematosus

Interferon-alpha (IFN-alpha) is detected in the serum of 70-80% of patients with systemic lupus erythematosus (SLE). Furthermore, soluble factors in SLE serum can induce peripheral blood mononuclear cells (PBMC) to produce IFN-alpha. The purpose of this work was to investigate the mechanism of this IFN-alpha induction. In eleven of fifteen SLE serum samples, an IFN-alpha inducing activity was detected, whereas serum from healthy controls, patients with other autoimmune disease and patients with viral infections were ineffective under the same conditions. After gel filtration of the serum, the inducing activity was found in the same fraction as IgG. The IFN-alpha inducing activity was inhibited by native monoclonal antibodies to the receptors for the Fc portion of IgG: FcgammaRIIA/C and FcgammaRIIB subclasses (CD32) and by their F(ab)'2 fragments. Purified Fc fragments of human IgG were also effective in abolishing the IFN-alpha-inducing activity. Since no anti-CD32 autoantibodies were found in SLE serum, this IFN-alpha-inducing activity may be due to immune complex antibodies. Such results may allow better understand the origin of endogenous IFN-alpha, which has a deleterious effect on the course of this autoimmune disease. The inhibition of this function by the CD32 antibody could lead to new therapeutic approach in SLE.     

Pegylated interferon-alpha 2a treatment of chronic SIV-infected macaques.

BACKGROUND: In vitro and clinical observations in HIV-infected patients receiving interferon alpha therapy have shown a reduction in HIV loads. Limited investigations have explored the innate or adaptive immune responses of IFN-alpha on SIV replication in vivo. METHODS: Seven chronically infected rhesus macaques were given pegylated IFN-alpha 2a (n = four) or saline (n = three) injections once weekly for 14 weeks. Weekly peripheral blood samples were taken for safety parameters, viral load determinations, and measurements of innate and adaptive immune responses. RESULTS: Pharmacokinetic measurements demonstrated therapeutic peg-IFN-alpha levels for the initial period of therapy and IFN-alpha inducible antiviral molecules were increased sporadically in the PBMC mRNA of the treatment group. Despite the demonstrable effect of the IFN-alpha injections, the treatment had no effect on plasma viral RNA levels. CONCLUSIONS: This work demonstrates that while short term IFN-alpha therapy induces innate antiviral immunity, it does not dramatically enhance or suppress viral replication. However, studies in the SIV model to determine therapeutic potential of chronic IFN-alpha therapy for the treatment of HIV will require macaque specific cytokines.     

Aerosolized interferon-alpha treatment in patients with multi-drug-resistant pulmonary tuberculosis

Multi-drug-resistant tuberculosis (MDR-TB) has emerged as an obstacle to the control of tuberculosis. Recent data however, suggest that interferon-(IFN)-gamma and IFN-alpha may improve disease evolution in subjects affected with pulmonary tuberculosis caused by multi-resistant (IFN-gamma) and sensitive (IFN-alpha) strains. The mechanisms involved are not known, even though it has been reported that IFN-gamma-secreting CD4+ Th cells may possess antitubercular effects. In addition, IFN-alpha can induce IFN-gamma secretion by CD4+ Th cells, and both types of IFN may stimulate macrophage activities. The aim of this study was to explore the possibility that aerosolized IFN-alpha, administered concomitantly with conventional antitubercular chemotherapy, may improve the course of pulmonary tuberculosis. After six months of directly observed therapy (DOT), seven patients who were non-responders to a second line antitubercular therapy were given an IFN-alpha aerosol (3 MU, three times a week) for two months as adjunctive therapy. All strains were resistant to at least two first-line drugs. After IFN-alpha administration, the patients were followed up for a further six months with the same DOT. Sputum samples were collected monthly during the study period, with the exception of the IFN-alpha administration period, when the observations were performed weekly. High resolution computed tomography (HRCT) chest scans were performed before and after IFN-alpha inhalations. The analysis of the results showed that the mean number of Mycobacterium tuberculosis (Mt) had remained statistically unchanged (p = 0.80) during the first 6 months of DOT. During the following 2 months of IFN-alpha administration, 5 patients became negative (p = 0.02). After the end of treatment a progressive increase in Mt number was observed (p = 0. 02). Sputum cultures remained positive for all patients throughout the study period, although a significant decrease (p = 0.02) in the colony number per culture was observed after adjunctive treatment with IFN-alpha. After stopping administration of IFN-alpha, a significant increase (p = 0.03) in the colony number per culture was noted as well as in Mt numbers. HRCT scans were slightly improved in all patients. These preliminary data suggest that aerosolized IFN-alpha may be a promising adjunctive therapy for patients with MDR-TB. Optimal doses and schedules however, require further studies.     

In vivo and in vitro induction of (2'-5') oligoadenylate synthetase by human interferons in leukocytes from healthy donors and patients with renal cancer and hairy cell leukemia

The effect of in vivo administered interferon-alpha (IFN-alpha) on 2-5-oligoadenylate (A) synthetase activity of peripheral blood mononuclear cells (PBMC) was compared in patients with hairy cell leukemia and renal cell cancer. Basic levels of this enzyme varied from donor to donor, but mean levels were not significantly different in patients with renal cell cancer or hairy cell leukemia compared to healthy donors. After a single injection of 3 x 10(6) IU IFN, these basic levels rose 2- to 8-fold within 12-24 h post-injection and reverted to pretreatment levels after 48 h. The extent of this in vivo stimulation by IFN-alpha was similar in patients with hairy cell leukemia and renal cell carcinoma, and was correlated with down-regulation of IFN-alpha receptors. The in vitro effects of IFN-alpha, -beta and -gamma were compared after 18 h treatment with 10, 10(2) and 10(3) IU/ml of each IFN. Unlike IFN-alpha and -beta, IFN-gamma did not induce 2-5 A synthetase activity in either normal PBMC or hairy cells; these results were related to the effects of the three IFN on proliferative response of normal PBMC to phytohemagglutinin. Our data support the idea that 2-5 A synthetase activity is a marker of biological response to interferon treatment in human cancers.     

Use of the liposomal forms of a terrilytin preparation for the lysis of experimental thrombi

Thrombolytic activity of liposomal terrilytin forms has been experimentally studied. Liposomal terrilytin form (50-280 PU/kg) was administered to rabbits 24 hours after the experimental induction of femoral thrombi. In 43.75% of cases complete lysis of the thrombi was noted, in 37.5% of cases different stages of the lysis of wall thrombi were observed and in 18.75% of cases the thrombi remained. In the control experiments the thrombi remained in 75% of cases, while in 25% of cases spontaneous lysis of the thrombi occurred. Statistically significant hemostasis changes gave evidence of the decrease in the blood clotting activity after oral administration of a liposomal terrilytin form. It has become possible to achieve thrombolytic effect using lower doses of orally administered liposomal terrilytin form, which is both therapeutically and economically important.     

Effect of lactoferrin on Helicobacter felis induced gastritis.

Lactoferrin possesses antibiotic, antiinflammatory, and immune-modulating properties that may be active against the gastritis-, ulcer- and cancer-inducing bacterium Helicobacter pylori. In vitro testing of bovine and human lactoferrin by several laboratories has shown significant bacteriostatic and bactericidal activity. Subsequent in vivo testing of bovine lactoferrin in animal models of H. pylori infection has shown beneficial effects of this agent. Our laboratory has utilized a mouse model that is infected with the feline strain of this bacterium, H. felis. The resulting gastritis that develops in this model and the effects of bovine lactoferrin and recombinant human lactoferrin (from Aspergillus niger var. awamori, Agennix Inc., Houston, Tex.) treatment were assessed by various measures. Infected animals treated with orally administered lactoferrin showed reversals in all parameters. In addition, when recombinant human lactoferrin was used in combination with low doses of amoxicillin or tetracycline, there was an enhancement in gastritis-reducing activity. Possible mechanisms for these effects of lactoferrin are discussed. Lactoferrin has significant, orally active in vivo actions and should be further investigated for clinical situations involving Helicobacter infections where it may have utility when administered alone and also when given in combination with established antibiotic agents.     

Interferon alpha augments activation-induced T cell death by upregulation of Fas (CD95/APO-1) and Fas ligand expression.

Interferon alpha (IFN-alpha) plays a prominent role in the therapy of a variety of diseases. The Fas/FasL system is crucial for the cytotoxic function and the peripheral elimination of activated T lymphocytes (ATC) by a mechanism referred to as activation-induced cell death (AICD). Recent studies suggest a link between IFN-alpha, the 2', 5'- oligoadenylate system and apoptosis. We therefore asked whether IFN-alpha is able to regulate the Fas/FasL pathway and thereby affects AICD. Peripheral blood mononuclear cells (PBMC), purified T cells and ATC of healthy volunteers were stimulated with various agents and the influence of IFN-alpha on Fas/FasL was assessed by mRNA and protein studies. The proportion of ATC undergoing AICD or anti-Fas-induced apoptosis was determined by FITC-annexin V staining and propidium iodide uptake. IFN-alpha upregulated mRNA expression of Fas and FasL in activated PBMC. Furthermore the concentration of the soluble form of FasL (sFasL) was increased in PBMC and T cells co-stimulated with IFN-alpha and various agents, whereas Fas surface expression was enhanced by IFN-alpha alone. IFN-alpha enhanced apoptosis induced by anti-Fas antibody and augmented AICD via the Fas/FasL pathway. IFN-alpha-regulated AICD may contribute to lymphopenia observed during IFN-alpha therapy. Our data further support that IFN-alpha is a multifunctional cytokine with profound effects on the immune cascades.     

IFN-alpha priming results in a gain of proinflammatory function by IL-10: implications for systemic lupus erythematosus pathogenesis

Interleukin-10 is a predominantly anti-inflammatory cytokine that inhibits macrophage and dendritic cell function, but can acquire proinflammatory activity during immune responses. We investigated whether type I IFNs, which are elevated during infections and in autoimmune diseases, modulate the activity of IL-10. Priming of primary human macrophages with low concentrations of IFN-alpha diminished the ability of IL-10 to suppress TNF-alpha production. IFN-alpha conferred a proinflammatory gain of function on IL-10, leading to IL-10 activation of expression of IFN-gamma-inducible, STAT1-dependent genes such as IFN regulatory factor 1, IFN-gamma-inducible protein-10 (CXCL10), and monokine induced by IFN-gamma (CXCL9). IFN-alpha priming resulted in greatly enhanced STAT1 activation in response to IL-10, and STAT1 was required for IL-10 activation of IFN-gamma-inducible protein-10 and monokine induced by IFN-gamma expression in IFN-alpha-primed cells. In control, unprimed cells, IL-10 activation of STAT1 was suppressed by constitutive activity of protein kinase C and Src homology 2 domain-containing phosphatase 1. These results demonstrate that type I IFNs regulate the balance between IL-10 anti- and proinflammatory activity, and provide insight into molecular mechanisms that regulate IL-10 function. Gain of IL-10 proinflammatory functions may contribute to its pathogenic role in autoimmune diseases characterized by elevated type I IFN levels, such as systemic lupus erythematosus.     

Interferon-alpha and cancer: mechanisms of action and new perspectives of clinical use

Interferons-alpha (IFN-alpha) are pleiotropic cytokines belonging to type I IFNs, extensively used in the treatment of patients with some types of cancer and viral disease. IFN-alpha can affect tumor cell functions by multiple mechanisms. In addition, these cytokines can promote the differentiation and activity of host immune cells. Early studies in mouse tumor models showed the importance of host immune mechanisms in the generation of a long-lasting antitumor response after treatment of the animals with IFN-alpha/beta. Subsequently, an ensemble of studies based on the use of genetically modified tumor cells expressing specific IFN molecules provided important information on the host-mediated antitumor mechanisms induced by the local production of IFN-alpha. Of note, several studies have then underscored new immunomodulatory effects of IFN-alpha, including activities on T cells and dendritic cells, which may lead to IFN-induced antitumor immunity. In addition, recent reports on new immune correlates in cancer patients responding to IFN-alpha represent additional evidence on the importance of the interactions of IFN-alpha with the immune system for the generation of a durable antitumor response. On the whole, this knowledge suggests the advantage of using these cytokines as adjuvants of cancer vaccines and for the in vitro generation of highly active dendritic cells to be utilized for therapeutic vaccination of cancer patients.     

Induction of CD83+CD14+ nondendritic antigen-presenting cells by exposure of monocytes to IFN-alpha

IFN-alpha is a well-known agent for treatment of viral and malignant diseases. It has several modes of actions, including direct influence on the immune system. We investigated IFN-alpha effects on PBMC in terms of dendritic cell (DC) differentiation, as PBMC are exposed to high IFN-alpha levels during treatment of infections and cancers. We show that in vitro IFN-alpha exposure induced rapid and strong up-regulation of the DC-maturation markers CD80, CD86, and CD83 in bulk PBMC. Consistently, IFN-alpha induced up-regulation of these molecules on purified monocytes within 24 h. Up-regulation of CD80 and CD83 expression was IFN-alpha concentration-dependent. In contrast to GM-CSF + IL-4-generated DCs, most of the IFN-alpha-challenged CD83(+) cells coexpressed the monocyte marker CD14. Despite a typical mature DC immunophenotype, IFN-alpha-treated monocytes conserved phagocytic activity and never acquired a dendritic morphology. In mixed lymphocyte reactions IFN-alpha-treated monocytes were less potent than GM-CSF + IL-4-generated DCs but significantly more potent than untreated monocytes to induce T cell proliferation in bulk PBMC. However, only GM-CSF + IL-4-generated DCs were able to induce a significant proliferation of naive CD4(+) T cells. Notably, autologous memory CD4(+) T cells proliferated when exposed to tetanus toxoid-pulsed IFN-alpha-treated monocytes. At variance with untreated or GM-CSF + IL-4-exposed monocytes, those challenged with IFN-alpha showed long-lasting STAT-1 phosphorylation. Remarkably, CD83(+)CD14(+) cells were present in varicella skin lesions in close contact with IFN-alpha-producing cells. The present findings suggest that IFN-alpha alone promptly generates nondendritic APCs able to stimulate memory immune responses. This may represent an additional mode of action of IFN-alpha in vivo.     

Protection against infections by oral lactoferrin: Evaluation in animal models

It has been reported previously that oral administration of lactoferrin (LF) provides some host-protective effects against infections, cancers, and inflammations. In this review, we focus on the effect of oral LF on various infectious diseases and discuss the mechanism as elucidated in animal models. In the case of infections occurring at sites other than the digestive canal, it is unclear whether oral LF is absorbed from the intestine and exerts its protective effect at the site of infection. In preterm human infants, neonatal pigs, and rats with colitis, it was reported that LF is detectable in various body fluids after oral administration. We could not detect the transport of oral bovine LF into the blood of adult rats without gastrointestinal illness using several techniques, suggesting that there is an extremely low level of transport of LF, if any. Orally administered LF may act at the oro-gastro-intestinal mucosa and aid the defense system against infections through a network of mucosal immunity and systemic immunity. Indeed, it is reported that oral LF increases the number of cells in the leukocyte subset and cytokine (IFN-gamma and IL-18) production in the intestinal mucosa of mice. Regarding systemic immunity, we have observed an increase of leukocyte number, cytokine (IFN-gamma, TNF-alpha, IL-12, and IL-18) production, and effector activity of macrophages in response to LF administration in several animal models. These enhanced immune responses may contribute to eradication of the pathogen, resolution of the symptoms, and maintenance of the homeostasis during infectious diseases.