Bernard Ferrua

Reverse ELISPOT assay for clonal analysis of cytokine production. II. Enumeration of interleukin-1-secreting cells by amplified (avidin-biotin anti-peroxidase) assay.

Detection of cytokine-producing cells can be accomplished by reverse modifications of the ELISPOT assay using cytokine-specific unconjugated and enzyme-labelled antibodies as solid phase capture system and detecting reagents, respectively. However, in certain situations where the secreted cytokine is produced in minute amounts such as in the case of interleukin-1 (IL-1), the sensitivity of the indicator immunoenzyme system employed may be insufficient to permit detection of the corresponding secreting cells. We have developed a novel immunoenzyme amplification procedure that involves the use of a biotinylated secondary anti-enzyme antibody reagent to enhance the signal provided by the primary enzyme-labelled antibody conjugate. Following addition of enzyme-conjugated avidin, ELISPOT assay wells are developed with a suitable chromogen substrate yielding spots located at the former position of cells secreting the analyte under study. As a model system, the detection of IL-1 beta-secreting cells by human peripheral blood monocytes is described.

Differential regulation of IL 6, IL 1 A, IL 1β and TNFα production in LPS-stimulated human monocytes: Role of cyclic AMP

Abstract Interleukin 6 (IL 6), IL 1α, IL β and tumor necrosis factor (TNF)α are four cytokines induced in monocytes by lipopolysaccharide (LPS); however, it is unclear whether the mechanisms which control their production are similar. In this study, we report the effects of prostaglandin E2 (PGE2), and two other cAMP-elevating agents, dibutyryl cAMP and 3-isobutyl-1-methylxanthine, on the in vitro LPS-induced production of IL 6, IL 1α, IL 1β and TNFα by human monocytes. The production of these four cytokines was found to be selectively regulated in monocytes, by increases in intracellular cAMP levels. In effect, such agents enhanced, in a dose-dependent manner, both extracellular and cell-associated IL 6 production by LPS-stimulated monocytes. In contrast, it was confirmed, using the same samples, that these cAMP-elevating agents inhibit both extracellular and cell-associated TNFα production in a dose-dependent manner. IL 1α and IL 1β production, measured by means of specific immunoreactive assays, were not significantly modified. Kinetic analysis showed that the potentiating effect of cAMP on IL 6 production, along with its inhibiting effect on TNFα production, could be seen as early as 1 hr after LPS stimulation. These results demonstrate that IL 6, TNFα, IL 1α and IL 1β production can be differently modulated by an agent, PGE2, which is produced simultaneously by LPS-stimulated monocytes. Such differential autocrine modulation may play an important role in the regulation of the production of cytokines participating in immune and inflammatory responses.

Inhibition of phorbol ester-induced cell activation in microgravity

T lymphocytes and monocytes were exposed to microgravity and activated to produce interleukin 2 and interleukin 1, respectively. When Jurkat T cells were triggered with monoclonal antibodies directed against the CD3/T cell receptor complex in the presence of THP-1 monocytes used as accessory cells, cell-to-cell contacts took place in microgravity leading to normal production of interleukin 2 and interleukin 1, as compared to ground controls. In contrast, when cells were individually stimulated by soluble substances including a protein kinase C activating phorbol ester, the production of interleukin 1 and interleukin 2 was dramatically inhibited during microgravity exposure. This result indicates that microgravity may affect the cellular target of phorbol ester.

A century of leishmaniasis in Alpes-Maritimes, France.

Health decision-makers working in Africa often need to act for millions of people over large geographical areas on little and uncertain information. Spatial statistical modelling and Bayesian inference have now been used to quantify the uncertainty in the predictions of a regional, environmental risk map for Loa loa (a map that is currently being used as an essential decision tool by the African Programme for Onchocerciasis Control). The methodology allows the expression of the probability that, given the data, a particular location does or does not exceed a predefined high-risk threshold for which a change in strategy for the delivery of the antihelmintic ivermectin is required.

CCL5 neutralization restricts cancer growth and potentiates the targeting of PDGFRβ in colorectal carcinoma.

Increased CCL5 levels are markers of an unfavourable outcome in patients with melanoma, breast, cervical, prostate, gastric or pancreatic cancer. Here, we have assessed the role played by CCL5/CCR5 interactions in the development of colon cancer. To do so, we have examined a number of human colorectal carcinoma clinical specimens and found CCL5 and its receptors over-expressed within primary as well as liver and pulmonary metastases of patients compared to healthy tissues. In vitro, CCL5 increased the growth and migratory responses of colon cancer cells from both human and mouse origins. In addition, systemic treatment of mice with CCL5-directed antibodies reduced the extent of development of subcutaneous colon tumors, of liver metastases and of peritoneal carcinosis. Consistently, we found increased numbers of CD45-immunoreactive cells within the stroma of the remaining lesions as well as at the interface with the healthy tissue. In contrast, selective targeting of CCR5 through administration of TAK-779, a CCR5 antagonist, only partially compromised colon cancer progression. Furthermore, CCL5 neutralization rendered the tumors more sensitive to a PDGFRβ-directed strategy in mice, this combination regimen offering the greatest protection against liver metastases and suppressing macroscopic peritoneal carcinosis. Collectively, our data demonstrate the involvement of CCL5 in the pathogenesis of colorectal carcinoma and point to its potential value as a therapeutic target.

In vivo involvement of polymorphonuclear neutrophils in Leishmania infantum infection

BackgroundThe role of lymphocytes in the specific defence against L. infantum has been well established, but the part played by polynuclear neutrophil (PN) cells in controlling visceral leishmaniasis was much less studied. In this report we examine in vivo the participation of PN in early and late phases of infection by L. infantum.ResultsPromastigote phagocytosis and killing occurs very early after infection, as demonstrated by electron microscopy analyses which show in BALB/c mouse spleen, but not in liver, numerous PN harbouring ultrastructurally degraded parasites. It is shown, using mAb RB6-8C5 directed against mature mouse granulocytes, that in chronically infected mice, long-term PN depletion did not enhance parasite counts neither in liver nor in spleen, indicating that these cells are not involved in the late phase of L. infantum infection. In acute stage of infection, in mouse liver, where L. infantum load is initially larger than that in spleen but resolves spontaneously, there was no significant effect of neutrophils depletion. By contrast, early in infection the neutrophil cells crucially contributed to parasite killing in spleen, since PN depletion, performed before and up to 7 days after the parasite inoculation, resulted in a ten-fold increase of parasite burden.ConclusionsTaken together these data show that neutrophil cells contribute to the early control of the parasite growth in spleen but not in liver and that these cells have no significant effect late in infection in either of these target organs.

Ciprofloxacin treatment in vivo increases the ex vivo capacity of lipopolysaccharide-stimulated human monocytes to produce IL-1, IL-6 and tumour necrosis factor-alpha.

Because in vitro treatment with quinolones, at pharmacological concentrations, modifies lipopolysac‐charide (LPS) induced production of cytokines by monocytes, we studied the effect of orally administered ciprofloxacin (25 mg/kg) on the capacity of peripheral blood monocyles of healthy volunteers to produce tumour necrosis factor‐alpha (TNF‐α), IL‐1 activity, IL‐1 a, IL‐1β and IL‐6 ex vivoin response to endotoxin stimulation. After 7 days of ciprofloxacin, the extracellular and cellular production of TNF‐α, the cellular production of IL‐1 activity, the extracellular and cellular production of IL‐1α, and the cellular production of IL‐6 increased significantly. Seven days after the end of the treatment, values returned to basal levels or even lower. To our knowledge, this is the first demonstration that ciprofloxacin can modulate in vivo the capacity of human monocytes to react to an inflammatory stimulus such as endotoxin.

Detection of human IL-1α and IL-1β at the subpicomolar level by colorimetric sandwich enzyme immunoassay

Abstract Two separate convenient sandwich enzyme immunoassay methods were developed for measuring the production of the monokines interleukin-1α (IL-1α) and interleukin-1β (IL-1β) from peripheral blood mononuclear cells. Polyclonal antisera raised against the recombinant proteins and selected on the basis of their ability to neutralize IL-1-induced IL-2 secretion were used for coating microtiter plates or preparing peroxidase-Fa′ conjugates. Both techniques were able to accurately and specifically detect monokines from various sources in the sub-picomolar range and were not influenced by compounds currently used for cell activation. A high molecular weight form of IL-1β was demonstrated under certain conditions and the two enzyme immunoassays were successfully applied to the detection of IL-1α and IL-1β present in cell supernatants following stimulation with mitogenic or chemical agents.

Immunisation with DNA encoding Leishmania infantum protein papLe22 decreases the frequency of parasitemic episodes in infected hamsters.

We tested in outbred golden hamsters the protective potential of highly immunogenic Leishmania infantum protein papLe22 which we recently identified. Immunisation was performed using papLe22 cDNA, administered as a single intramuscular injection. The level of antibodies directed against total leishmanial antigens was significantly decreased in the vaccinated hamsters as compared with the controls, indicating that the administration of papLe22 cDNA downregulated the Th2 type response and suggesting that the immune response was reoriented toward the cell-mediated type. The presence of the parasite kDNA in the peripheral blood was systematically detected as early as 3 weeks post infection in all mock-vaccinated hamsters. By contrast, in the vaccinated animals the occurrence of the episodes of Leishmania circulation was reduced by 50%. The immunisation presenting efficacy in this highly susceptible species which develop VL similar in gravity to human and canine disease should prove also efficient in naturally infected hosts. The marked decrease of the frequency of parasite circulation induced by papLe22 cDNA immunisation appears therefore important and potentially able to reduce transmission and thus to control the spread of the disease.

A sandwich method of enzyme-immunoassay. II. Quantification of rheumatoid factor

A sandwich enzyme-immunoassay (EIA) has been applied to the determination of the rheumatoid factor (RF). This non-competeitive assay comprises 3 steps: 1) the RF to be assayed is extracted for the biological medium by an immunosorbent of aggregated IgG linked to cellulose; 2) the solid phase is then incubated with the enzyme-labeled aggregated IgG; 3) the enzymatic activity of the immunosorbent is then measured with a suitable chromogenic reagent. This activity is a direct function of the amount of RF to be assayed. This assay gave reproducible results in the range 0.5-50.0 IU/ml. A good agreement was obtained between the EIA and the Waaler-Rose test but no correlation was obtained with the latex slide-test. This assay permits a quantitation of RF with a good reproducibility (coefficient of variation in the range of 10% for moderately elevated values) and thus allows a closer follow-up of patients. The results do not depend on the interpretation of the technician performing the test, which can be easily automated. Finally, it may detect some RF devoid of agglutinating activity.