Lionel G. Filion

Safe use of the CXCR4 inhibitor ALX40-4C in humans

ALX40-4C is a small peptide inhibitor of the chemokine receptor CXCR4 that can inhibit X4 strains of HIV-1. Prior to the discovery of chemokine receptors as the HIV coreceptors, ALX40-4C was used in phase I/II clinical trials to evaluate its therapeutic potential against HIV-1, making ALX40-4C the first anticoreceptor inhibitor to be tested in humans against HIV-1. Patients in the highest dose groups achieved ALX40-4C levels above the effective concentration of the drug for nearly the entire 1-month treatment period. ALX40-4C was well tolerated by 39 of 40 asymptomatic HIV-infected patients, despite the critical role of CXCR4 in normal development and hematopoiesis. No significant or consistent reductions in viral load were observed, but only 12 of the enrolled patients harbored virus types that used CXCR4. We also found that ALX40-4C interacts with the second extracellular loop of CXCR4 and inhibits infection exclusively by blocking direct virus-CXCR4 interactions.

A quick, easy and inexpensive method for the isolation of human peripheral blood monocytes.

A commercial monocyte isolation technique based on the OptiPrep density-gradient medium was up-scaled with respect to sample and reagent volumes. The results of 7 isolations are reported in which the average purity ranged from 87.9 to 96.4%. In all but the initial isolation, monocytes were defined as CD15+ dim CD4+ dim as assessed by flow cytometric analysis; in the first isolation, monocytes were defined by the traditional CD14+ CD4+ dim combination. The mean yield (the number of isolated monocytes relative to the number present in the buffy coat) of all isolations was 26.1%, with the individual yields ranging from 10.8 to 41.4%. The mean number of isolated monocytes per experiment was 3.6 x 10(6) monocytes for those isolations performed using 14 ml of buffy coat/OptiPrep mixture (n = 4). The isolated cells were viable (> 95%) and were not activated, according to HLA-DR expression. This technique is a convenient, tast (less than 2 h), relatively simple, and inexpensive alternative to traditional monocyte isolation techniques. The up-scaled version of this method also results in significantly higher numbers of monocytes per isolation than some traditional techniques. Furthermore, this is the first literature report of the use of the OptiPrep density-gradient medium for the isolation of monocytes.

Radiation damage and immune suppression in splenic mononuclear cell populations

We have examined alterations in all of the major splenic mononuclear cell (SMNC) populations in C57Bl/6 mice following whole‐body irradiation (0–700 cGy) in order to determine which populations may play a role in active immune suppression and/or haematopoietic recovery. A protocol has been established for characterization and differentiation by flow cytometric analysis (FCA) of the major MNC populations in the mouse spleen: T lymphocytes (CD4+ and CD8+ cells), B lymphocytes, natural killer (NK) cells, and monocytes/macrophages. Ionizing radiation caused decreased spleen cellularity and decreased ability of surviving SMNC to respond to mitogen. FCA revealed alterations in the relative composition of the constituent splenic cell populations following irradiation, reflecting differential radiosensitivity, with selective enrichment of NK cells (seven‐fold) and CD4+ T lymphocytes (three‐fold). Enrichment developed during the 7‐day post‐irradiation period. In addition, some MNC became activated in a dose‐ and time‐dependent fashion following whole‐body irradiation, as indicated by expression of CD71, the transferrin receptor. These cells were CD34+ and Thy1.2+, but were CD4− or CD8− as well as CD45− (B cell). The observed increase in NK cells corresponds with a previously reported increase in natural suppressor (NS) cells following total‐lymphoid irradiation (TLI). The balance of recovery‐inhibiting NK cells and recovery‐enhancing CD4+ T lymphocytes following irradiation may reflect or influence the degree of haematopoietic recovery, and may provide an indication of the extent of damage (biological dosimetry).

Expression of IL-10, IL-4 and interferon-gamma in unstimulated and mitogen-stimulated peripheral blood lymphocytes from HIV-seropositive patients

Infection of immune cells with HIV induces dysregulation of cytokines which may play a vital role in HIV pathogenesis. We analysed the expression of T helper type I (Th1) (interferon‐gamma (IFN‐γ)) and Th2 (IL‐4, IL‐10) type cytokines in peripheral blood lymphocytes (PBL) from HIV patients. The semiquantitative reverse transcriptase polymerase chain reaction (RT‐PCR) analysis revealed that IFN‐γ mRNA in unstimulated PBL was significantly decreased and IL‐10 mRNA was significantly upregulated in patients with < 400 CD4+ T cells/mm3 (n= 30) as compared to patients with > 400 CD4+ T cells/mm3 (n= 6) and normal controls (n= 16). In addition. IL‐10 mRNA levels were inversely associated with IFN‐γ expression. Similar results were obtained by measuring IL‐10 production in the supernatants of PBL cultured in vitro without stimulation by employing an enzyme immunosorbent assay (ELISA). However, the levels of IL‐4 and IFN‐7 produced by unstimulated PBL were undetectable by ELISA. Mitogen stimulation of PBL revealed two groups of HIV individuals based on IL‐10 production. PBL from one set of individuals produced low levels of IL‐10 (low IL‐10 producers) whereas the other group produced IL‐10 comparable lo that of normal controls (IL‐10 producers). Production of IL‐4 was significantly reduced in HIV+ individuals with<400 CD4+ T cells/mm3 as compared to the normal controls. However, ability to produce IFN‐γ by mitogen‐stimulated total PBL and CD4+ purified cells was not impaired in HIV+ individuals. These results suggest that unstimulaied and mitogen‐stimulated PBL of HIV+ individuals exhibit dysregulation of Th2 type cytokines which may play a role in HIV immunopathogenesis.

Monocyte-derived IL12, CD86 (B7-2) and CD40L expression in relapsing and progressive multiple sclerosis

Multiple sclerosis has been postulated to be an autoimmune disease in which Th1 immune responses predominate. This response is associated with an increased production of IFNgamma and IL12 produced by T cells and by cells of the monocyte (MO) lineage, respectively. An increased expression of costimulatory molecules by T cells and antigen-presenting cells is also observed. We hypothesized that in relapsing-remitting MS (RRMS) (with or without of IFNbeta treatment) and in secondary progressive patients (SPMS) IL12 and costimulatory molecules (CD80 [B7-1], CD86 [B7-2], CD28, CD40, CD40L) would be differentially produced or expressed by MO or T cells. We performed cross-sectional and longitudinal flow cytometric studies (at monthly intervals) on peripheral blood mononuclear cells (PBMC) or on MO from SPMS or untreated and IFNbeta-treated patients with RRMS. We determined that CD86 and CD40L expression was highest on MO derived from SPMS patients compared to those from RRMS or from healthy controls (HC). In vitro culture of PBMC with recombinant human IL10, a cytokine that may be increased in response to treatment with IFNbeta and that down-regulates CD86 expression, reduced the expression of CD86 on MO derived from RRMS patients to a much higher degree compared to cells derived from SPMS or HC. In vitro secreted IL12 levels from freshly isolated MO from SPMS patients were more than 10-fold higher than either the treated or the untreated RRMS or HC. RRMS patients treated with IFNbeta demonstrated slightly lower levels of MO IL12 secretion. Our data suggest that a key mechanism in the pathogenesis of MS is the increased expression of CD86 and CD40L and the increased production of IL12 during disease progression. Part of the mechanism of action of IFNbeta may be to reduce MO CD86 and CD40L expression and IL12 secretion; failure to do so might signify either a lack of response or a transition to a more progressive phase of illness.

Comparison of interferon-γ-, interleukin (IL)-17- and IL-22-expressing CD4 T cells, IL-22-expressing granulocytes and proinflammatory cytokines during latent and active tuberculosis infection

In this study, we investigated the role and expression of T helper type 17 (Th17) cells and Th17 cytokines in human tuberculosis. We show that the basal proportion of interferon (IFN)‐γ‐, interleukin (IL)‐17‐ and IL‐22‐expressing CD4+ T cells and IL‐22‐expressing granulocytes in peripheral blood were significantly lower in latently infected healthy individuals and active tuberculosis patients compared to healthy controls. In contrast, CD4+ T cells expressing IL‐17, IL‐22 and IFN‐γ were increased significantly following mycobacterial antigens stimulation in both latent and actively infected patients. Interestingly, proinflammatory IFN‐γ and tumour necrosis factor (TNF)‐α were increased following antigen stimulation in latent infection. Similarly, IL‐1β, IL‐4, IL‐8, IL‐22 and TNF‐α were increased in the serum of latently infected individuals, whereas IL‐6 and TNF‐α were increased significantly in actively infected patients. Overall, we observed differential induction of IL‐17‐, IL‐22‐ and IFN‐γ‐expressing CD4+ T cells, IL‐22‐expressing granulocytes and proinflammatory cytokines in circulation and following antigenic stimulation in latent and active tuberculosis.

DETECTION AND IDENTIFICATION OF Legionella SPECIES FROM GROUNDWATERS

Legionellae are opportunistic bacterial pathogens causing Legionnaires’ disease and Pontiac fever and are ubiquitous in surface waters and in infrastructure to contain or distribute water, including pipes, cooling towers, and whirlpool spas. Infection in community-acquired and nosocomial outbreaks is by exposure to contaminated aerosols. Little is known about the presence of legionellae in groundwater. This study used samples from various locations in the United States and Canada to determine if legionellae could be isolated from water and biofilms derived from groundwaters not known to be under the direct influence of surface water. Of the 114 total samples of water and biofilm tested, 29.1% and 28.2% were positive for Legionella by cultivation and polymerase chain reaction (PCR), respectively. Legionellae were found in both warm and colder groundwaters, with more isolates from samples incubated at 30°C than the 35°C conventional temperature for Legionella isolation. The concentration of Legionella found in the water samples ranged from 102 to 105 CFU/L and up to 1.2 × 102 CFU/cm2 in the biofilm. The species of Legionella identified included both known pathogenic species and species that have not yet been identified as human pathogens. Millions of people in Canada, and around the world, rely on groundwater as their source for drinking. This study shows that legionellae are widespread in groundwater and have the potential to seed derived water supplies and biofilms in public distribution systems. This further widens the known sphere of Legionella colonization and the implications of its presence for public health.

Mechanism of γδ T cell-induced human oligodendrocyte cytotoxicity: relevance to multiple sclerosis

Abstract γδ T cells may contribute to the pathogenesis of Multiple Sclerosis (MS) via cytotoxicity directed at the myelin-oligodendrocyte unit. We have previously demonstrated that peripheral blood-derived γδ T cells lyse fresh human oligodendrocytes in vitro. The present work extends these observations to γδ T cells derived from both peripheral blood (PBL) and cerebrospinal fluid (CSF) of MS and non-MS neurological disease controls and addresses the mechanism of cellular cytotoxicity. We found that MS patients contained increased proportions of V δ 1 + γδ T cells in both CSF and PBL samples compared to other neurological disease (OND) controls. Although γδ T cells from all patients were cytotoxic towards Daudi, RPMI 8226, U937, Jurkat, oligodendroglioma and fresh human oligodendrocyte targets, OND-derived, V δ 2 + rich, populations derived from the CSF exhibited greater cytotoxicity towards cell lines (Daudi, RPMI 8226) known to express high levels of heat shock proteins (hsp). To clarify the mechanism(s) of cytotoxicity used by γδ T cells, we first showed that cell-target contact was necessary by the use of physical barriers (transwells), which reduced target cell lysis by at least 75%. The use of Ca 2+ -free media reduced lysis by up to 50%, but fully blocking γδ T cell Perforin release and function by either Ca 2+ chelation (Mg 2 EGTA) or the H + -ATPase inhibitor Concanamycin-A (CMA), completely abrogated the lysis of Fas − /hsp60 high expressing targets (Daudi, U937). However, additional treatment with Brefeldin A was required for the complete inhibition of γδ T cell mediated killing of Fas + expressing Jurkat targets and fresh human brain-derived oligodendrocytes. Inhibition of granzyme activity by an isocoumarin compound reduced cytolysis only slightly. The use of either Brefeldin A or an anti-Fas antibody alone did not significantly affect lysis. These findings suggest that in MS, γδ T cells may utilize either the Fas-mediated or Perforin-based cell cytotoxicity pathways in exerting oligodendrocyte damage, though the Perforin pathway is predominant.

Detection of surface and cytoplasmic CD4 on blood monocytes from normal and HIV-1 infected individuals

CD4 on blood monocytes is generally regarded as being found on a subset of blood monocytes. However, our results show that all monocytes are CD4+ but the number of molecules per cell is lower than T cells. We have performed immunofluorescent (flow cytometry, microscopy) analysis of monocytes from normal donors and HIV-1-infected patients using anti-CD4 (Leu-3a) and the monocyte-specific marker anti-CD14 (Leu-M3) monoclonal antibodies. Specifically: (1) 91% of monocytes from normal individuals show dual positivity for CD14 and CD4 (n = 14) as measured by flow cytometry; (2) 76% of monocytes expressed surface bound CD4 and CD14 when an enhanced two colour immunofluorescence microscopic technique was employed; (3) all CD14+ monocytes stained with an intensity of 3+(-)4+ for cytoplasmic CD4. Few monocytes were CD14- and CD4+. Cell surface CD4 expression was blocked with unconjugated anti-CD4 prior to staining; (4) the staining intensity for cytoplasmic CD4 in T cells was negligible; (5) CD4 expression on monocytes obtained from patients was as observed with normal individuals. The conclusion drawn is that all monocytes are CD4+ and the CD4 expression in monocytes is mainly cytoplasmic. Thus, all monocytes are potentially infectable with HIV-1.

Differential production of IL-10 by T cells and monocytes of HIV-infected individuals: association of IL-10 production with CD28-mediated immune responsiveness

Immune unresponsiveness in HIV‐1 infection can result from impaired signals delivered by the costimulatory CD28‐B7 pathway and the altered production of immunoregulatory cytokines, in particular IL‐10, whose production is altered in HIV‐1 infection. In this study we investigate IL‐10 regulation in T cells and monocytes from HIV+ individuals, and its association with CD28‐mediated T cell proliferation. IL‐10 production as analysed in T cell‐ and monocyte‐depleted peripheral blood mononuclear cells (PBMC), and by intracellular staining at the single‐cell level, reveals a defect in IL‐10 production by CD4+ and CD8+ T cells, whereas monocytes constitute the major IL‐10‐producing cell type. To investigate the impact of IL‐10 on immune responsiveness, CD28‐mediated proliferative responses in HIV+ individuals were correlated with PHA‐induced IL‐10 production. CD4+ T cells expressed CD28, yet exhibited markedly reduced CD28‐mediated cell proliferation. This CD28‐mediated CD4+ T cell proliferation was found to be inversely associated with the levels of PHA‐induced IL‐10 production and could be restored, at least in part, by anti‐IL‐10 antibodies. These results suggest that IL‐10 production is differentially regulated in T cells and monocytes of HIV+ individuals, and that IL‐10 may have a role in inducing immune unresponsiveness by modulating the CD28‐B7 pathway.