New therapeutic targeting of Alzheimer's disease with the potential use of proline-rich polypeptide complex to modulate an innate immune response - preliminary study.

BACKGROUND: The lack of effective treatment for Alzheimer's disease (AD) stems mainly from the incomplete understanding of AD causes. Neuroinflammation has emerged as an important component of AD pathology, and a vast number of experimental and clinical data indicated a crucial role for the activation of the innate immune system in disease promotion and symptom progression. METHODS: Clinical examinations of AD patients in a different stage of disease severity in correlation with the measurement of two innate immune reactions, i.e., peripheral blood leukocyte (PBLs) resistance to viral infection (vesicular stomatitis virus, VSV) ex vivo, and cytokines: TNF-α, IFN-γ, IL-1ß, and IL-10, production with enzyme-linked immunosorbent assay (ELISA), have been investigated during this preliminary study before and after 4 weeks of oral treatment with dietary supplement proline-rich polypeptide complex (PRP) (120 µg of PRP/day). The potential effect of PRP on the distribution of PBLs' subpopulations has been specified. RESULTS: We have found a deficiency in innate immune response in AD patients. It was demonstrated for the first time that the degree of PBLs resistance to VSV infection was closely related to the stage of clinical severity of AD. Our study showed significant differences in cytokine production which pointed that in AD patients innate immune mechanisms are impaired. Administration of PRP to our patients increased innate immune response of PBLs and declined pro- and anti-inflammatory cytokine production, thus subduing the excessively developed inflammatory response, especially among patients with high severity of AD. PRP did not exhibit a pro-proliferative activity. It was showed, however, significant influence of PRP on the distribution of PBLs' subpopulations. CONCLUSION: The findings mentioned above might be crucial in the context of potential application of immunomodulatory therapy in AD patients and indicated PRP as a potential target for future treatments in neuroinflammatory diseases like AD.

Immune Consequences of in vitro Infection of Human Peripheral Blood Leukocytes with Vesicular Stomatitis Virus.

BACKGROUND: Oncolytic vesicular stomatitis virus (VSV) can be delivered intravenously to target primary and metastatic lesions, but the interaction between human peripheral blood leukocytes (PBLs) and VSV remains poorly understood. Our study aimed to assess the overall immunological consequences of ex vivo infection of PBLs with VSV. METHODS: Phenotypic analysis of lymphocyte subsets and apoptosis were evaluated with flow cytometry. Caspase 3/7 activity was detected by luminescence assay. Virus release was evaluated in a murine cell line (L929). Gene expression and cytokine/chemokine secretion were assessed by real-time PCR and multiplex assay, respectively. RESULTS: Ex vivo infection of PBLs with VSV elicited upregulated expression of RIG-I, MDA-5, tetherin, IFITM3, and MxA. VSV infection triggered rapid differentiation of blood monocytes into immature dendritic cells as well as their apoptosis, which depended on caspase 3/7 activation. Monocyte differentiation required infectious VSV, but loss of CD14+ cells was also associated with the presence of a cytokine/chemokine milieu produced in response to VSV infection. CONCLUSIONS: Systemic delivery is a major goal in the field of oncolytic viruses. Our results shed further light on immune mechanisms in response to VSV infection and the underlying VSV-PBL interactions bringing hope for improved cancer immunotherapies, particularly those based on intravenous delivery of oncolytic VSV.

Age- and disease-related innate immunity of human leukocytes ex vivo.

Two mechanisms of innate immunity, i.e. resistance to viral infection and the production of cytokines by leukocytes, were compared in blood isolated from four groups of donors: healthy young (19-35 years old), healthy elderly (over 60), elderly Alzheimer's disease (AD) patients, and elderly patients with alimentary tract cancer (CA). Peripheral blood leukocytes (PBLs) were isolated by gradient centrifugation in Gradisol G. The degree of resistance was calculated from the kinetics of vesicular stomatitis virus (VSV) replication in the PBLs. Cytokine (TNFα, IFNα, IFNγ, IL-12, and IL-10) levels were determined by ELISA. The antiviral resistance of the PBLs varied, but a difference was observed only between the young and elderly groups and not between the healthy elderly controls and those with AD or cancer. Differences observed in all the groups concerned the ability and intensity of cytokine production. The most impressive results were obtained for spontaneous TNF and IFNα release. While TNF was released spontaneously by the PBLs of the elderly CA patients and the young healthy group, it was usually undetected in the AD and only sometimes in the healthy elderly group. Leukocytes isolated from the elderly groups responded to VSV infection with more intense IFNα and IFNγ production than the younger group.

Inhibition of vesicular stomatitis virus replication in the course of HIV infection in patients with different stages of immunodeficiency.

The replication of vesicular stomatitis virus (VSV) in isolated human leukocytes has been used to measure the level of nonspecific antiviral immunity. However, during infection with some pathogens, the main effect observed is caused by interaction between the pathogen and VSV. This was also noted in advanced stages of HIV infection, when an inverse association between HIV viral load and VSV replication was found. The mutual effect was markedly stronger than the correlation between the VSV replication level and CD4(+) T-cell count. Since successful antiretroviral therapy is associated with a decrease in HIV viremia to undetectable levels, the effect of such therapy on VSV replication was expected and confirmed in this investigation. In fact, increased VSV titers were observed together with decreased HIV viral load, particularly in the case of efficient therapeutic schemes, for example those including lopinavir/ritonavir. The results showed that VSV replication capacity reflected the progression of HIV infection. Moreover, the presence of interferon in the plasma of AIDS patients was found to be only partially responsible for the inhibition of VSV replication. The results suggest a specific HIV-VSV interaction, whether direct or indirect. Thus the VSV replication assay may be applied in evaluating the stage of HIV infection.

Effect of donepezil on innate antiviral immunity of human leukocytes.

BACKGROUND: The effect of donepezil on two mechanisms of innate immunity: leukocyte resistance to viral infection and cytokine production was studied. METHODS: The degree of natural resistance of human peripheral blood leukocytes (PBLs) was determined by studying the kinetics of vesicular stomatitis virus (VSV) replication. A titer of 0-1 log TCID(50) indicated complete resistance, 2-3 log partial resistance, and >4 lack of resistance. Cytokine levels were determined with use of ELISA test. NFkappaB activation was assayed by immunocytochemical staining. RESULTS: Preliminary study of VSV replication in the PBLs of Alzheimer's disease patients showed a high sensitivity to infection, except of PBL those under donepezil therapy. The PBL resistance stimulated us to study the effect of donepezil on innate immunity. Donepezil inhibited VSV replication in the leukocytes of healthy blood donors but influence on infection in L929 and A549 cells was not shown. The effect was dose dependent and individually differentiated. The production of TNFalpha and IFNs was reduced in infected leukocytes in a dose-dependent manner in the PBLs of the healthy blood donors and of AD patients. NFkappaB activation was also reduced by donepezil. CONCLUSIONS: Donepezil regulate two mechanisms of innate immunity of leukocytes: resistance to viruses and cytokine production.

Diverse production of interferons alpha, beta, and gamma by airway leukocytes of asthmatics with regard to cigarette smoking and corticosteroid treatment.

The production of interferon-alpha(IFN-alpha), IFN-beta, and IFN-gamma by airway leukocytes from induced sputa (IS) of asthmatics was investigated. The groups consisted of 32 corticosteroid-free asthmatics (A), with 13 nonsmokers (nS) and 19 smokers (S), and 30 inhaled corticosteroid-treated asthmatics (cA) with 14 nS and 16 S. The control healthy group (H) comprised 11 nS and 15 S. The levels of IFNs in media from cultures of IS leukocytes were assessed by ELISA. The cells of the smokers produced lower amounts of IFN-alpha than those of nonsmokers in groups H, A, and cA (p = 0.0417, 0.0002, 0.0495, respectively) and significantly higher amounts of IFNbeta than nonsmokers in groups H (p = 0.0044) and cA (p = 0.0007). No differences in the levels of IFN-gamma were observed between S and nS in groups H (p = 0.8148), A (p = 0.8339), and cA (p = 0.0722). In the entire group of smokers, smoking indices correlated negatively with IFN-alpha (R(S) = -0.4374, p = 0.0006), and positively with IFN-beta (R(S) = 0.4239, p = 0.0009). There was no correlation with IFN-gamma (R(S) = 0.0471, p = 0.7004). The results suggest that production of IFNs by the airway leukocytes of cA may be modified by cigarette smoking toward deficient production of IFN-alpha and excess production of IFN-beta, which may have implications in the pathophysiology of asthma.

Cytokine production by human leukocytes with different expressions of natural antiviral immunity and the effect of antibodies against interferons and TNF-alpha.

INTRODUCTION: Two activities of innate antiviral immunity were studied: the resistance of human peripheral blood mononuclear cells (PMBCs) ex vivo to viral infection and the production of cytokines. MATERIALS AND METHODS: Samples of blood were taken from healthy blood donors and from persons with frequent infections of the upper respiratory system. PMBCs were isolated by gradient centrifugation. Vesicular stomatitis virus (VSV) was used as the indicatory virus to infect PMBCs. The cytokines: IFN, TNF, and IL-6 were titrated by biological methods and IL-10 by ELISA. RESULTS: Blood donors were divided for two groups: those with VSV-resistant and those with VSV-sensitive PMBCs and secretion of cytokines by them was compared. The resistant PMBCs produced more cytokines than the sensitive ones. A statistically significant difference, was found only in the case of the IFNs. To examine the contribution of IFNs and TNF in maintaining resistance, leukocytes from both groups were treated with specific anti-cytokine antibodies. The authors' previous study showed that the elimination of spontaneous IFN-alpha, IFN-beta, IFN-gamma, and TNF-alpha from resistant leukocytes resulted in increased VSV replication This indicates the important role of cytokines. In VSV-sensitive PMBCs, anti-IFN-alpha showed the opposite effect (decreased virus replication). In the absence of spontaneous IFN-alpha, disturbances in cytokine production were observed. CONCLUSIONS: Complete resistance of PMBC to VSV infection is accompanied by higher cytokine release, The paradoxical effect of anti-IFN-alpha on virus replication in leukocytes sensitive to viral infection may be attributed to changes in the cytokine profile balance, i.e. high TNF production by VSV-infected leukocytes and a complete reduction of IL-6 production.

The overproduction of nitric oxide associated with neutrophilic predominance is relevant to airway mycotic infections in asthmatics undergoing prolonged glucocorticoid treatment.

The complex relationship between the local inflammatory response and the spread of airway mycosis during prolonged glucocorticoid therapy in bronchial asthma patients remains unclear. We assessed the ability of airway leukocytes to produce nitric oxide (NO) in relation to differential inflammatory cell counts, levels of asthma severity, and coexisting airway mycotic infections. The study was carried out on leukocytes from the induced sputa (IS) of 14 patients with asthma complicated by mycotic airway infections undergoing prolonged glucocorticoid therapy (group FcA). Three groups of subjects without airway fungal infections were also studied: 18 glucocorticoid-treated asthmatics (group cA), 11 steroid-free asthmatics (group A), and 13 healthy control subjects (group H). In group FcA, both the level of spontaneous production of NO and the percentages of neutrophils in the IS were significantly higher than in all the remaining groups. Additionally, a significant positive correlation was noticed between the NO levels and both the percentages of neutrophils in the IS and the symptom intensity scores. The results suggest a possible predominant role of neutrophils in the overproduction of NO related to asthma severity and coexisting fungal infections in glucocorticoid-treated patients.

Age related natural antiviral non-specific immunity of human leukocytes.

BACKGROUND: The purpose of our study was to examine the dependence of innate antiviral immunity on age and sex in human leukocytes. MATERIAL/METHODS: Innate antiviral immunity was measured by using the direct method of infection of leukocytes with vesicular stomatitis virus (VSV), which was selected as the indicatory virus for detection of immunity. The lack of VSV replication by infected leukocytes (0-1 log TCID50) was taken as an indicator for complete immunity; a low level of VSV replication (2-3 log) for partial immunity; and a high VSV titer (4 or more log) for the no or very low immunity. RESULTS: The kinetics of VSV replication was studied in leukocytes isolated from 127 individuals ranging in a age from 0 to 89 years. Individual differentiation in the kinetics of VSV replication indicated differing degrees of innate immunity even in newborns. Age-related differences in natural immunity were observed: low immunity in newborns, highest in the age group 31-40, and reduced in the age group >60. Sex dependent innate immunity was shown in the group of aged persons, as innate immunity was higher in women than in men. CONCLUSIONS: Innate immunity of leukocytes develops to age 30-40, after which immunity is gradually reduced. Sex dependence was observed only in the group of elderly persons, where women expressed higher immunity; this is probably a reason for their statistically greater longevity.

Comparison of leukocyte populations from bronchoalveolar lavage and induced sputum in the evaluation of cellular composition and nitric oxide production in patients with bronchial asthma.

Bronchoalveolar lavage (BAL) or induced sputum (IS) techniques may provide leukocytes for the evaluation of airway inflammatory response in bronchial asthma. The aim of the present study was to compare features of leukocyte populations obtained by the two different methods regarding the cell types and their activity in patients with bronchial asthma. The nitric oxide (NO) level released from the cells was measured as a marker of their activity. Pulmonary leukocytes were obtained from the BAL and IS of 11 asthmatic patients in stable condition at the time of the study. The BAL and IS leukocyte populations varied in cell count and NO production. Macrophages were the predominant leukocyte population in BAL (median (Me) = 83.0%, range 67.9-88.4%), whereas sputum sediments were found to consist mainly of neutrophils (Me = 55.7%, range 29.0-64.9%). The IS leukocytes released much more NO (p = 0.0022) than the BAL leukocytes. In spite of these quantitative differences, a similar pattern of NO production was observed in BAL and in IS cells. Both BAL and IS leukocyte populations produced almost the same amounts of NO before and after lipopolysaccharide stimulation (p = 0.9063, p = 0.4801, respectively). Furthermore, a slight positive correlation Spearman's rank (RS) = 0.5578, p = 0.0594) was noticed between the neutrophil percentages and NO levels produced by BAL cells, whereas in IS a statistically significant correlation between the percentage of neutrophils and the levels of NO (RS = 0.6643, p = 0.0184) was observed. In conclusion, the BAL and IS leukocyte populations are different in cell type, their size and activity. Depending on the asthma severity and the type of cells needed in a study, either BAL or IS specimens may be chosen as a source of pulmonary leukocytes. The use of IS as a noninvasive technique is supposed to be potential value particularly in the study of the airway inflammatory response mediated mainly by neutrophils, i.e. during and/or after exacerbation of the disease. Based on our results, a possible contribution of neutrophils in the production of NO in the airways of asthmatic patients can be proposed apart from other cells such as macrophages.