Steve Patterson

Polyvalent dendrimer glucosamine conjugates prevent scar tissue formation

Dendrimers are hyperbranched macromolecules that can be chemically synthesized to have precise structural characteristics. We used anionic, polyamidoamine, generation 3.5 dendrimers to make novel water-soluble conjugates of D(+)-glucosamine and D(+)-glucosamine 6-sulfate with immuno-modulatory and antiangiogenic properties respectively. Dendrimer glucosamine inhibited Toll-like receptor 4–mediated lipopolysaccharide induced synthesis of pro-inflammatory chemokines (MIP-1α, MIP-1β, IL-8) and cytokines (TNF-α, IL-1β, IL-6) from human dendritic cells and macrophages but allowed upregulation of the costimulatory molecules CD25, CD80, CD83 and CD86. Dendrimer glucosamine 6-sulfate blocked fibroblast growth factor-2 mediated endothelial cell proliferation and neoangiogenesis in human Matrigel and placental angiogenesis assays. When dendrimer glucosamine and dendrimer glucosamine 6-sulfate were used together in a validated and clinically relevant rabbit model of scar tissue formation after glaucoma filtration surgery, they increased the long-term success of the surgery from 30% to 80% (P = 0.029). We conclude that synthetically engineered macromolecules such as the dendrimers described here can be tailored to have defined immuno-modulatory and antiangiogenic properties, and they can be used synergistically to prevent scar tissue formation.

Flexibility and cooperation among dendritic cells.

Ever since it was proposed that distinct subsets of dendritic cells induce distinct subsets of T cells, immunologists have been struggling to reconcile those conclusions with other data. New experiments now illuminate a different interpretation.

Comparative loss and maturation of peripheral blood dendritic cell subpopulations in African and non-African HIV-1-infected patients.

ObjectivesTo quantify the percentage of the two major subpopulations of blood dendritic cells (DC) in HIV-1-seropositive Ugandan individuals infected with non-clade B viruses and compare this with that seen in clade B HIV-1 infected non-African individuals. DC maturation/activation status was also investigated via the expression of CD86. MethodsThe percentage of blood DC was quantified by using flow cytometry. DC were identified as the lineage (CD3, CD14, CD16, CD19, CD20, CD56)-negative, HLA-DR-positive population and the two major subpopulations were differentiated by CD11c expression. ResultsThe percentage of blood DC was reduced significantly in HIV-1-seropositive African individuals when compared with controls (0.21 and 0.39% respectively). A similar reduction was also seen in non-African patients residing in the UK (0.19% compared with 0.36% for controls). However, there was no selective loss in either CD11c-positive or CD11c-negative subpopulations. The percentage of blood DC expressing CD86 was significantly greater in HIV-1-seropositive individuals when compared with controls and the increased expression was largely confined to CD11c-negative DC. ConclusionsAfricans infected with non-clade B HIV-1 showed similar reductions in the percentage of blood DC to non-Africans infected with clade B viruses. There was no selective loss of either DC subpopulation, suggesting that the ability of DC to acquire and present antigens or to produce interferon-α may both be impaired in HIV-1 infection.

Chemokine Receptor Expression on Mucosal Dendritic Cells from the Endocervix of Healthy Women

Dendritic cells (DCs) may be an initial target of human immunodeficiency virus (HIV) during heterosexual transmission. An analysis of DCs in the intraepithelial layer of the endocervix of the female genital tract from healthy women showed that ~20% expressed CD1a, and 30% expressed cutaneous leukocyte antigen (CLA). Langerin, a molecule associated with Langerhans DCs, was on CD1a-positive and -negative DCs and on CLA-positive cells. CCR5 and CXCR4 were detected on CD1a-positive and -negative cervical DCs. These findings suggest that DCs in the genital tract are potential targets for macrophage-tropic and lymphotropic strains of HIV.

Evaluation of the cervical cytobrush sampling technique for the preparation of CD45+ mononuclear cells from the human cervix.

A cytobrush technique developed to prepare mononuclear cells from the intraepithelial layer of the endocervix has been evaluated. Specimens yielded approximately 4-6x10(6) cells, of which 10-15% were CD45+. Between 10% and 15% of these CD45+ cells were mononuclear leukocytes. The non-leukocyte cell fraction exhibited high levels of autofluorescence and for flow cytometry analysis, it was necessary to exclude these cells by gating. Macrophages constituted approximately 60% and T lymphocytes, 40% of the mononuclear cells in cytobrush samples. The CD4/CD8 T-cell ratio was similar to that observed in blood. In 9 of 13 specimens, B lymphocytes constituted less than 1% of the mononuclear cell fraction suggesting that the mononuclear cells were derived from the intraepithelial compartment rather then the deeper lamina propria. Lack of B lymphocytes also indicates minimal blood contamination in these samples, a conclusion supported by labelling for the red blood cell (RBC) glycoprotein glycophorin A. However, the need to monitor all samples for possible blood contamination was indicated by 4 of 13 samples in which B lymphocytes accounted for 2-8% of the mononuclear cells.

Nadir B cell counts are significantly correlated with the risk of Kaposi's sarcoma

Infection with HIV‐1 is known to impair B cell function. To further elucidate the role of B cells during infection and tumorigenesis, we studied their numbers in cases of AIDS‐related Kaposis sarcoma (KS) during the HAART era. Patients with AIDS‐related KS were identified from a database of 4,480 HIV‐1 positive individuals and the incidence of KS and rate ratio was stratified according to nadir number of B cells, measured as the CD19 count. In an unadjusted model, we observed that lower B cell counts were associated with a statistically significant increased risk of KS development (p < 0.001). We also observed a trend toward increased counts during KS resolution. When adjusted for nadir CD4 count in a multi‐variable model, higher B cell counts were protective against KS development (p = 0.015). These data highlight a potential role for B cells and therefore the humoral immune system in KS aetiopathogenesis.

Expression of the common heat-shock protein receptor CD91 is increased on monocytes of exposed yet HIV-1-seronegative subjects

The significantly higher surface expression of the surface heat‐shock protein receptor CD91 on monocytes of human immunodeficiency virus type‐1 (HIV‐1)‐infected, long‐term nonprogressors suggests that HIV‐1 antigen uptake and cross‐presentation mediated by CD91 may contribute to host anti‐HIV‐1 defenses and play a role in protection against HIV‐1 infection. To investigate this further, we performed phenotypic analysis to compare CD91 surface expression on CD14+ monocytes derived from a cohort of HIV‐1‐exposed seronegative (ESN) subjects, their seropositive (SP) partners, and healthy HIV‐1‐unexposed seronegative (USN) subjects. The median fluorescent intensity (MFI) of CD91 on CD14+ monocytes was significantly higher in ESN compared with SP (P=0.028) or USN (P=0.007), as well as in SP compared with USN subjects (P=0.018). CD91 MFI was not normalized in SP subjects on highly active antiretroviral therapy (HAART) despite sustainable, undetectable plasma viraemia. Data in three SP subjects experiencing viral rebounds following interruption of HAART showed low CD91 MFI comparable with levels in USN subjects. There was a significant positive correlation between CD91 MFI and CD8+ T cell counts in HAART‐naïve SP subjects (r=0.7, P=0.015). Increased surface expression of CD91 on CD14+ monocytes is associated with the apparent HIV‐1 resistance that is observed in ESN subjects.

New insights into the immunology and evolution of HIV

ABSTRACTFewer than one million HIV infected individuals are currently receiving anti-retroviral therapy. The limitations of such treatment have underscored the need to develop more effective strategies to control the spread and pathogenesis of HIV. Typically, naturally occurring protective immune responses provide the paradigm for such development. It is now clear however that HIV can utilise the millieu of an activated immune system to its own replicative advantage. Mobilisation of the immune response, intended to thwart the virus, may instead fuel its dissemination, immune escape and spread. The immense genetic variation of HIV contributes to lack of immune control and the development of progressive disease in the majority of infected, untreated individuals. Further delineation of the intimate interactions between the HIV and the immune system will be critical and recent advances in this direction are discussed.

Ex vivo analysis of HIV-1 co-receptors at the endocervical mucosa of women using oral contraceptives

Combined oral contraceptives may alter the microenvironment of the female genital tract and, thus, influence susceptibility of endocervical cells to HIV‐1 transmission. The mechanism for this effect is unknown but might involve combined oral contraceptive up‐regulation of chemokine receptors on CD4+ endocervical cells. We measured chemokine co‐receptor (CCR5 and CXCR4) expression on cervical intraepithelial CD4+ T lymphocytes, macrophages and dendritic cells using flow cytometry in 32 healthy women, 16 of whom were combined oral contraceptive users and 16 non‐users. All women tested negative for sexually transmitted infections. Combined oral contraceptive users showed a higher proportion of CCR5+ CD4+ T lymphocytes compared with combined oral contraceptive non‐users (P < 0.05). However, expression of both co‐receptors on cervical intraepithelial macrophages and dendritic cells was no different between the two groups. Up‐regulation of CCR5 on cervical intraepithelial CD4+ T lymphocytes offers a potential explanation by which women receiving combined oral contraceptives may be at increased risk of HIV transmission.

Studies on the allostimulatory function of dendritic cells from HCV-HIV-1 co-infected patients.

ABSTRACTThere is increasing recognition of the potential morbidity and mortality associated with HIV-1 and hepatitis C (HCV) co-infection. HIV appears to adversely affect HCV disease while the reciprocal effect of HCV on HIV remains controversial. We therefore studied the effect of co-infection on dendritic cell function versus HIV infection alone, as previous work has shown that HCV impairs dendritic cell (DC) function. HIV-1 positive individuals with HCV were matched for CD4 count, HIV-1 RNA viral load and therapy, to HIV-1 positive patients without HCV. Monocyte-derived DC were generated and mixed leukocyte reactions were performed. We assessed allostimulatory capacity with and without administration of exogenous Th1 cytokines, using thymidine uptake and cell division analyses with the vital dye CFSE. We found that monocyte-derived DC from co-infected individuals showed no significant differences in allostimulatory capacity to ex vivo generated DC from HIV-1 infected individuals without HCV. Unlike the situation with HCV infection alone, this impairment was not reversed by increasing concentrations of either interleukin-2 or -12. Monocyte-derived DC from HIV-1 and HCV co-infected individuals have a similar allostimulatory capacity to DC from matched patients with HIV-1 alone. These findings are compatible with results of prior clinical studies that found no evidence that HCV co-infection altered HIV disease progression and has implications for immunotherapeutic approaches in co-infected individuals.