Defective neutrophil motility and recurrent infection. In vitro and in vivo effects of levamisole.

Eighteen patients with primary abnormalities of neutrophil chemotaxis are described. The most common clinical presentation was one of recurrent upper respiratory tract infection (nine patients) or recurrent pyoderma (seven patients), and two children had a history of oropharyngeal candidiasis and recurrent skin sepsis. Of these eighteen patients, sixteen had intrinsic polymorphonuclear leucocyte (PMN) defects as shown by diminished random migration and movement towards endotoxin-activated serum. PMN chemotaxis towards casein was, however, normal. In nine out of the latter patients, there was an associated inability of the serum to generate chemotactic factors. PMN from two adult patients, both suffering from recurrent boils, moved normally both in random and directed systems, but sera from these patients contained heat-stable inhibitors of neutrophil chemotaxis. In vitro levamisole treatment (10(-3) M) markedly improved the PMN function. When patients were treated with levamisole, however, no clinical response was noted, although PMN movement improved in a number of cases.     

The in vitro effects of propranolol and atenolol on neutrophil motility and post-phagocytic metabolic activity.

Propranolol at concentrations of 1 x 10(-6) to 1 x 10(-4) M consistently increased neutrophil motility as measured in Boyden chambers. The effects were not due solely to stimulation of random migration and chemokinesis but also of directional motility. Propranolol, over a similar concentration range, caused inhibition of post-phagocytic cell metabolic activity (hexose monophosphate shunt, nitro-blue tetrazolium reduction and protein iodination) without any detectable effect on the ingestion rate of Candida albicans. Atenolol had no effect on any of these neutrophil functions. Both drugs were without effect on glycolysis and intracellular cyclic AMP levels. Propranolol however, at concentrations which stimulated cell motility, caused increased intracellular cyclic GMP levels. It is suggested that propranolol may stimulate neutrophil motility by promoting increased intracellular cyclic GMP levels or by decreasing neutrophil superoxide production.     

Prevention of peroxidase mediated inhibition of neutrophil motility and lymphocyte transformation by levamisole, OMPI, sodium aurothiomalate, indomethacin and tolmetin in vitro

The effects of sodium aurothiomalate, levamisole, its active metabolite OMPI and the anti-inflammatory agents indomethacin and tolmetin on neutrophil motility and post-phagocytic hexose monophosphate shunt activity, superoxide and H2O2 generation and myeloperoxidase (MPO) mediated iodination of Candida albicans were investigated in vitro. All five agents caused stimulation of neutrophil random motility and migration towards the leucoattractants f-met-met-phe and EAS. Only levamisole caused inhibition of H2O2 and superoxide production, which was associated with inhibition of HMS activity and not related to superoxide scavenging activity. All five agents caused inhibition of MPO mediated iodination of C. albicans. The relationship between inhibition of peroxidase mediated iodination and enhanced motility was further investigated using the horseradish peroxidase (HRP) H2O2/iodide system. Incubation of neutrophils with this system caused inhibition of neutrophil motility. However in the presence of the various drugs neutrophils were protected from inhibition of motility by the HRP/H2O2/iodide system. Further experiments showed that lymphocyte transformation to mitogens was also inhibited by the HRP/H2O2/iodide system. Incubation of lymphocytes with the various drugs prior to exposure to HRP/H2O2/iodide protected the lymphocyte mitogenic responsiveness.     

Increased leucoattractant binding and reversible inhibition of neutrophil motility mediated by the peroxidase/H2O2/halide system: effects of ascorbate, cysteine, dithiothreitol, levamisole and thiamine.

Human blood neutrophils manifested markedly decreased motility following exposure to the horseradish peroxidase (HRP)/H2O2/halide system in vitro. These cells were protected from this inhibitory effect (of the HRP/H2O2/halide system) by inclusion of concentrations in the reaction system of ascorbate, cysteinee, levamisole and thiamine which stimulate neutrophil migration and inhibit activity of the HRP/H2O2/halide system. The reversible nature of the oxidative inhibition of migration was demonstrated by exposing neutrophils to the HRP/H2O2/halide system for 15 min followed by washing to remove the components of the peroxidative system, and subsequent addition of ascorbate, cystein, levamisole, thiamine and the reducing agent, dithiothreitol. Neutrophils so treated completely recovered normal or increased motility induced by the leucoattractants endotoxin-activated serum or synthetic chemotactic tripeptide f-met-leu-phe. This reversible loss of migratory responsiveness following exposure of neutrophils to the HRP/H2O2/halide system was not associated with decreased cell viability or adherence. However, membrane oxidation was accompanied by increased uptake of radiolabelled f-met-leu-phe and degranulation. The increased leucoattractant uptake was decreased by ascorbate, levamisole and thiamine. These agents also prevented oxidation of the neutrophil membrane by the HRP/H2O2/halide system as measured indirectly by inhibition of iodination.     

Differential regulation of in vitro humoral and cellular immune responsiveness in Brugia pahangi-infected jirds.

Patent filarial infection is associated with the downregulation of parasite-specific immune reactivity. In the present study, the relationship between in vitro parasite antigen-specific humoral and cellular immune responsiveness was investigated in Brugia pahangi-infected jirds and in jirds immunized with soluble antigens. Spleen cells from B. pahangi-immunized jirds or from jirds with prepatent infections mounted significant in vitro proliferative and antibody responses to B. pahangi. The antigen concentration which elicited optimal antibody production was 10- to 10,000-fold lower than that required to stimulate optimal blastogenesis. Lymph node cells from both immunized and infected jirds consistently produced lower levels of parasite-specific antibody than spleen cells, yet generated higher proliferative responses to filarial antigen. A dissociation between in vitro antibody production and proliferation was also observed in experiments with spleen cells from microfilaremic jirds; spleen cells from patent animals did not proliferate when stimulated with B. pahangi antigen, but did produce significant levels of parasite-specific antibody. Depletion of adherent or histamine receptor-bearing cells restored the proliferative reactivity of spleen cells from microfilaremic jirds, but had limited effects on antibody production. In admixture experiments, spleen cells from microfilaremic animals suppressed the proliferative responsiveness of cells from keyhole limpet hemocyanin (KLH)-immunized jirds to KLH by 38%, but had no effect on KLH-specific antibody production. The present results support the hypothesis that parasite-specific cellular and humoral reactivity are differentially regulated in experimental filariasis.     

Assessment of oral ascorbate in three children with chronic granulomatous disease and defective neutrophil motility over a 2-year period

Two brothers and their sister with chronic granulomatous disease, elevated levels of serum IgE and defective neutrophil motility were treated with a single oral daily dose of 1 g sodium ascorbate as a supplement to prophylactic trimethoprim--sulphamethoxazole therapy for 2 years. Laboratory tests of neutrophil functions were performed prior to ascorbate therapy and repeated at 1-monthly intervals for 6 months and at 6-monthly intervals thereafter. Introduction of ascorbate to the therapeutic regimen was accompanied by slight increases in neutrophil hexose monophosphate shunt activity and staphylocidal activity and good improvement of neutrophil motility in all three children. The improved staphylocidal activity was not due to ascorbate-mediated inhibition of neutrophil or serum catalase activities or to detectable increases in superoxide and H2O2 production or activity of the MPO/H2O2/halide system. Both male children have remained free from obvious infection since ascorbate was added to their therapeutic regimen; their sister has experienced one urinary tract infection during a period when treatment with prophylactic co-trimoxazole and ascorbate was inadvertently stopped. All three children have gained weight.     

In vivo effect of levamisole on cellular and humoral immunity in normal chickens.

The effect of levamisole in vivo was studied on the PHA and Con A responses of chicken peripheral blood lymphocytes and on the in vivo antibody response to a thymus dependent antigen (BSA) and to a thymus independent antigen (Brucella abortus). Levamisole (0.25 mg/kg) increased significantly both the PHA and Con A responses of chicken blood lymphocytes. The antigens were given at the time of enhanced mitogenic responses and a significant increase was observed in both IgM and IgG antibodies to BSA. In contrast, no effect was obtained on antibody responses to Brucella abortus organisms. The results show that levamisole is able to enhance both humoral and cellular immune responses in normal chickens. The effect is probably mediated by the activation of the T cell function and effects only antibody responses to thymus dependent antigen. These findings confirm and extend the observations regarding the ability of levamisole to modulate immune responses.     

Impairment of cellular but not humoral immune responses in chronic pulmonary and disseminated paracoccidioidomycosis in mice.

Humoral and cellular immune responses were measured during the progression of chronic pulmonary and disseminated paracoccidioidomycosis in mice. The chronic disease was established by pulmonary infection of mice with different doses of the yeast form of Paracoccidioides brasiliensis isolate GAP. Levels of antibodies to P. brasiliensis, detected in serum by immunodiffusion and enzyme-linked immunosorbent assay, directly correlated with the size of the infectious challenge. Significant delayed-type hypersensitivity (DTH) responses to antigen were largely restricted to week 1 after pulmonary infection with intranasally administered high doses (5.0 x 10(6) or 1.1 x 10(7) CFU per inoculum). In vitro lymphoproliferative responses of peripheral blood lymphocytes (PBL) to P. brasiliensis antigens were significant only at 2 weeks after infection with intranasally administered 1.1 x 10(7) CFU. Responses of PBL to concanavalin A were depressed (50% of control response) as early as 8 weeks and reached a nadir at 10 to 18 weeks after infection. Infected mice made antibodies to sheep erythrocytes (SRBC) (10(9) intravenously [i.v.]) normally at all times tested after infection. In contrast, infected mice sensitized to SRBC (10(6) i.v.) had significantly depressed DTH responses to SRBC at 9 and 20 weeks postinfection compared with noninfected mice. These results indicated that in this model, normal humoral responses developed to homologous and heterologous antigens. In contrast, the T cellular immune responses were depressed with progression and chronicity of the disease. Thus, this model closely mimics the immunological findings in human paracoccidioidomycosis.     

Rambam-Hasharon syndrome of psychomotor retardation, short stature, defective neutrophil motility, and Bombay phenotype.

We describe 2 Arab patients, both offspring of unrelated consanguineous matings, with unusual facial appearance, severe mental retardation, microcephaly, cortical atrophy, seizures, hypotonia, dwarfism, and recurrent infections with neutrophilia. Neutrophil motility was markedly decreased but the opsonophagocytic activity was normal. Both patients lack the red blood cell (RBC) H antigen and manifest the Bombay (hh) phenotype. Familial endocardial fibroelastosis and familial tetralogy of Fallot segregated independently in one family. The occurrence of the same syndrome in 2 unrelated families suggests that the various aspects of the disorder are the pleiotropic effects of a single mutation. Homozygosity-by-descent for a deletion involving contiguous genes may explain the findings in this syndrome. Alternatively, a mutation which involves an ubiquitous GDP fucose donor rather than the enzyme (alpha 2-L-fucosyltransferase) or its substrate (glcNAc) may account for the pleiotropic manifestations in this syndrome.     

Anti-pancreatic immunity. In vitro studies of cellular and humoral immune reactions directed toward pancreatic islets

It has been suggested that the immune system may be responsible for the destruction of insulin secreting cells in some types of diabetes. In order to test this hypothesis, we studied the consequences of immune-mediated reactions on the function of pancreatic islet cells in vitro. A model was set up in vitro where mouse pancreatic islet cells are exposed to human lymphocytes or sera + complement then stimulated for the release of insulin or glucagon. A selective inhibition of insulin secretion, but not of glucagon secretion, was observed in the presence of lymphocytes from 37 out of 40 insulin-dependent diabetic (IDD) patients and in the presence of sera (+ complement) from 22 out of 40. Lymphocytes were found inhibitory in almost all patients in both groups, with and without associated autoimmune diseases. In contrast, inhibitory sera were observed almost only in patients with associated autoimmune diseases or recent onset diabetes. The selective inhibition of insulin secretion, but not of glucagon secretion, suggests that lymphocytes or sera may be involved in a destructive process of insulin secreting cells in vivo. This cell-mediated effect depends on direct T lymphocyte cytotoxicity, rather than antibody-dependent cell cytotoxicity, as suggested by the lack of any effect of aggregated immunoglobulins on the reaction. In contrast, when C57BL/6 mice were immunized by mastocytoma cells from a DBA2 strain, their lymphocytes and sera blocked both secretions of insulin and glucagon when incubated in vitro with DBA2 islet cells. This non-selective inhibition may be due to anti-H2 immunity, rather than immunity directed against insulin secreting cells.     

Influence of pathological progression on the balance between cellular and humoral immune responses in bovine tuberculosis

Studies of tuberculosis have suggested a shift in dominance from a T helper type 1 (Th1) towards a Th2 immune response that is associated with suppressed cell-mediated immune (CMI) responses and increased humoral responses as the disease progresses. In this study a natural host disease model was used to investigate the balance of the evolving immune response towards Mycobacterium bovis infection in cattle with respect to pathogenesis. Cytokine analysis of CD4 T-cell clones derived from M. bovis-infected animals gave some indication that there was a possible relationship between enhanced pathogenesis and an increased ratio of Th0 [interleukin-4-positive/interferon-gamma-positive (IL-4(+)/IFN-gamma(+))] clones to Th1 (IFN-gamma(+)) clones. All animals developed strong antimycobacterial CMI responses, but depressed cellular responses were evident as the disease progressed, with the IFN-gamma test failing to give consistently positive results in the latter stages. Furthermore, a stronger Th0 immune bias, depressed in vitro CMI responses, elevated levels of IL-10 expression and enhanced humoral responses were also associated with increased pathology. In minimal disease, however, a strong Th1 immune bias was maintained and an anti-M. bovis humoral response failed to develop. It was also seen that the level of the anti-M. bovis immunoglobulin G1 (IgG1) isotype antibody responses correlated with the pathology scores, whereas CMI responses did not have as strong a relationship with the development of pathology. Therefore, the development and maintenance of a Th1 IFN-gamma response is associated with a greater control of M. bovis infection. Animals progressing from a Th1-biased to a Th0-biased immune response developed more extensive pathology and performed less well in CMI-based diagnostic tests but developed strong IgG1 humoral responses.     

糖尿病肾病患者炎症水平、免疫功能及与肾脏病变的关系

目的:探讨糖尿病肾病(DN)患者炎症水平、免疫功能与肾脏病变的关系。方法:将124 例DN 患者根据24 h尿微量白蛋白排泄率(UAER)随机分为正常白蛋白尿(NA)组35 例,微量白蛋白尿(MA)组45 例和临床白蛋白尿(CP)组44例,另选取35 例健康体检者为对照组,应用放射免疫法测定各组C 反应蛋白(CRP)水平,应用酶联免疫法测定各组白细胞介素(IL)-6、细胞肿瘤坏死因子(TNF) 及体液免疫(IgG、IgA、IgM)水平,采用流式细胞仪测定机体细胞免疫反应(CD4+ Th17、Th17/ Treg 、CD4+ CD25+ Treg)。采用全自动化生化分析仪测定各种血清血肌酐(Scr)、半胱氨酸蛋白酶抑制物C(CYSC)及UAER。结果:NA 组、MA 组、CP 组血清CRP、IL-6、TNF 、Scr、CYSC、UAER 水平显著高于对照组(P<0.05),CP 组血清CRP、IL-6、TNF 、Scr、CYSC、UAER 水平显著高于NA 组、MA 组(P<0.05)。NA 组、MA 组、CP 组血清CD4+ CD25+ Treg、IgG 水平显著低于对照组(P<0.05),而CP 组血清CD4+ CD25+ Treg、IgG 水平显著低于NA 组、MA 组(P<0.05)。NA 组、MA 组、CP 组血清IgA、IgM、Th17/ Treg、CD4+ Th17 水平显著高于对照组(P<0.05)。经Pearson 单因素分析,CRP、IL-6、TNF 与Scr、CYSC、UAER 呈正相关(P<0.05),而与Th17/ Treg 、CD4+ CD25+ Treg、IgG 水平呈负相关(P<0.05)。结论:DN 患者存在微炎状态及免疫功能紊乱的情况,通过控制或消除促炎因素,改善DN 患者免疫功能,对延缓DN 患者病情发生发展有重要的意义。     

Leucocyte motility in the newborn: determination of spontaneous movement is essential in the in vitro assessment of neutrophil chemotaxis.

Polymorphonuclear leucocytes (PMNs) of the newborn show poor movement in vitro toward a chemotactic stimulus, such as zymosan-activated human serum (ZAS). This may result from a defect either in spontaneous movement or in the response of the cells to the stimulus. To identify the defect we studied chemotaxis, chemokinesis and spontaneous movement of cord blood PMNs by agarose assay and by the leading front modification of the Boyden chamber technique. Under agarose, cord PMNs showed much spontaneous movement. This, associated with poor stimulated movement, indicated a defect in the responsiveness of cord PMNs to ZAS. In the membrane filter, cord PMNs migrated spontaneously, and in the presence of ZAS, significantly less than adult cells. The weak spontaneous movement most probably resulted from poor deformability of cord PMNs and contributed to the weak stimulated movement of these cells in the filter. Our results indicate that the impaired chemotaxis was due to a defect in both responsiveness and deformability of cord PMNs rather than to a defect in their intrinsic ability to move.     

Pentoxifylline enhancement of defective neutrophil function and host defense in neonatal mice.

Decreased neutrophil (PMN) chemotaxis is thought to contribute to the increased morbidity and mortality from infection in newborn infants. Pentoxifylline, a methylxanthine, has previously been shown to augment PMN chemotaxis in vitro. The authors therefore investigated the effects of pentoxifylline on 1) in vitro PMN chemotaxis, 2) in vivo leukocyte accumulation, and 3) protection against Staphylococcus aureus infection in newborn mice. Using a modified Boyden chamber system, they demonstrated that pentoxifylline significantly enhanced neonatal PMN chemotaxis in a dose-dependent manner. Additionally, pentoxifylline was found to increase PMN accumulation in vivo in a proteose peptone-induced peritonitis model. Finally, the survival rate in experimentally induced S aureus infection was 51% in neonatal mice given pentoxifylline, compared with 17% in a control (nonpentoxifylline) group (P less than 0.01). These data demonstrate pentoxifylline modulation of PMN migration and enhancement of host defense against bacterial infection.     

Mtb9.9 protein family: an immunodominant antigen family of Mycobacterium tuberculosis induces humoral and cellular immune responses in mice

Tuberculosis (TB) is a major cause of morbidity and mortality worldwide. This study evaluated the immune response in mice to members of the Mtb9.9 protein family. C57BL/6 mice were immunized with four different Mtb9.9 recombinant proteins to evaluate the humoral and cellular immune responses to each protein. Mouse splenocytes from these mice were stimulated in vitro for 72?h with the Mtb9.9 proteins. Concentrations of the cytokines interferon (IFN)-γ and tumor necrosis α (TNF-α) in the culture supernatants were increased compared with controls. Sera were taken from the same mice at 3 weeks after immunization and contained significantly higher levels of immunoglobulin (Ig)G2c antibody when compared with sera from control mice. Our findings indicate a bias toward T helper (Th)1-type cellular and humoral memory responses against Mtb9.9 recombinant proteins in mice. This study demonstrates that these proteins might be suitable candidates for use as subunit vaccines.     

Experimental murine paracoccidioidomycosis: relationship among the dissemination of the infection, humoral and cellular immune responses.

The dissemination of Paracoccidioides brasiliensis cells to the heart, omentum/pancreas, spleen, liver and lungs, assessed by colony forming unit (CFU) counts, the levels of specific antibodies to this fungal agent (by ELISA), and the specific DTH reaction were studied in susceptible (B10.A) and resistant (A/Sn) mice. The animals were infected intraperitoneally with P. brasiliensis yeast cells and were evaluated 2, 4, 12 and 16 weeks later. The most remarkable differences between the two mouse strains were observed 16 weeks after infection, when B10.A mice displayed high numbers of CFU in all examined organs, except the heart, high antibody titres, and depressed DTH response. At this point, A/Sn mice presented low or absent CFU in all organs, low antibody titres and expressive DTH response. The CFU counts were shown to be a reliable parameter to discriminate susceptible from resistant animals. The fungal load in the most affected organs correlated with the antibody titres and was inversely correlated with the intensity of the DTH reaction. The patterns of immune response in this model mimic human paracoccidioidomycosis, in which high specific antibody levels and depressed DTH reactions are found in multifocal and severe forms of the disease.     

Reticuloendothelial Fc receptor function in SLE patients. II. Associations with humoral immune response parameters in vivo and in vitro.

We studied the relationship between reticuloendothelial Fc receptor function and some parameters of the humoral immune response in vivo and in vitro in 18 SLE patients. Fc receptor-mediated immune clearance correlated remarkably well with (a) a decrease of antigen specific IgG after immunization with a primary test antigen (alpha-Helix pomatia haemocyanin) (P less than 0.01) and (b) the spontaneous IgG release in vitro of B cells obtained from peripheral blood (P less than 0.01). These two parameters were significantly interrelated. Reticuloendothelial Fc receptor function was not related to serum IgG levels. The study provides evidence for an association between polyclonal B cell activation and Fc receptor-mediated immune clearance in SLE patients. The possible nature of this association is discussed.     

Suppression of humoral immune response against Herpes simplex virus induced by defective strains,ts- and TK− mutants

Summary Suppression of humoral antibody formation against HSV is not only induced by replicating Herpes simplex virus type 2 (HSV-2) but also by the defective strain ANG and the deletion mutant 1301 of Herpes simplex virus type 1 (HSV-1). Moreover, ts-mutants A, H, K, S, 1201 and 1208 of HSV-1 as well as some ts-mutants of HSV-2 and defective-interfering particles of HSV-1 after high multiplicity of infection-passages induced suppression. Treatment of infected mice with ACG reduced antibody-formation but did not result in suppression. UV-irradiation of the antibody producing strain Len of HSV-1 strongly reduces antibody formation and induces suppression. Experiments using a series of intertypic recombinants showed the suppressing activity to be spread over the whole genome of HSV-2. It is concluded that suppression is induced by more than one region of the genome of HSV-2 and by incomplete replication of HSV-1 and 2.     

The in vitro evaluation of certain neutrophil and lymphocyte functions following the ingestion of 150 mg oral dose of levamisole: assessment of the extent and duration of stimulation of neutrophil chemotaxis, protein iodination and lymphocyte transformation.

Certain functions of human blood neutrophils and lymphocytes were investigated at varying time intervals after the ingestion of a single 150 mg dose of levamisole. The functions tested were neutrophil chemotaxis and post-phagocytic metabolic activity and mitogen-induced DNA and protein synthesis of lymphocytes. It was found that levamisole causes a stimulation of neutrophils motility (cell- and serum-associated) and post-phagocytic hexose monophosphate shunt activity and protein iodination. Increased lymphocyte DNA synthesis, but not protein synthesis, to the mitogen phytohaemagglutinin was observed. The stimulation which was detected almost immediately of these neutrophil and lymphocyte functions was still evident 24 hr later but not at 48 hr, indicating that a single oral dose of levamisole can cause the alteration (stimulation) of leucocyte functions which persists until 24--48 hr after intake of the drug.