Contrast-enhanced imaging in the biological and functional assessment of breast cancer

Contrast-enhanced MRI and ultrasound have emerged as additional imaging modalities in the management of breast cancer. This thesis examines the role these modalities currently play in the surgical management of breast cancer. Ways in which MRI may contribute to staging, diagnosis, treatment and prognosis are investigated. It was demonstrated that small additional enhancing foci on MRI, away from the primary tumour, represent in-situ or invasive cancer foci. Although their resection may result in extended wide local excisions or even unnecessary mastectomies, it was demonstrated that MRI findings do not currently influence the amount of tissue removed during breast conservation surgery. Volumetric analysis of breast MRI was proposed as an accurate objective assessment of the extent of surgery required for a particular tumour. Breast MRI was shown to be useful in the assessment of extent of residual disease during primary medical therapy but not in the detection of axillary lymph node metastases. In the second section of this thesis, the clinical application of pre-operative MRI in providing prognostic as well as diagnostic information was evaluated. Contrast- enhancement with both MRI and ultrasound is believed to depend on tumour angiogenesis but only a weak correlation was demonstrated between contrast- enhancement intensity and tumour angiogenesis. The detection of angiogenesis was applied to Doppler ultrasound using a novel microbubble ultrasound contrast agent (Levovist). Within a multicentre prospective study, Doppler ultrasound was shown to be a powerful discriminator of malignancy in suspected local recurrence. A strong correlation was found between MRI and histological assessment of tumour size but there was no correlation between enhancement intensity and other pathological prognostic variables. This thesis has shown that breast MRI is useful in pre-operative planning of surgery and provides diagnostic as well as limited prognostic information. Future proposed studies to determine the effect of MRI on patient management and patient outcome in breast cancer are considered

Novel techniques for intraoperative assessment of margin involvement

Breast conserving surgery (BCS) is now the standard of care for the majority of women with early stage breast cancer. There is a finite rate of ipsilateral breast tumour recurrence (IBTR) for breast conserving therapy (BCT) with annual rates of less than 1% for specialist breast practices. There has been recent consensus on the definition of an adequate resection margin for both invasive and noninvasive breast cancer treated with BCS, although some variation in margin policy persists with definitions of ‘no tumour at ink’, 1 and 2 mm margin mandates. Despite the development of methods for intraoperative assessment of margins, up to 20% of patients require further surgery (cavity re-excision or completion mastectomy) to achieve clear surgical margins. In the past decade, several novel technologies for intraoperative margin assessment have been explored with the aim of reducing rates of re-operation and its attendant patient anxiety, inconvenience and additional cost. Ongoing studies are addressing the safety, feasibility and cost-effectiveness of these novel technologies relative to methods in routine clinical usage

A Cure for HIV Infection: "Not in My Lifetime" or "Just Around the Corner"?

With the advent and stunning success of combination antiretroviral therapy (ART) to prolong and improve quality of life for persons with HIV infection, HIV research has been afforded the opportunity to pivot towards studies aimed at finding "a cure." The mere idea that cure of HIV might be possible has energized researchers and the community towards achieving this goal. Funding agencies, both governmental and private, have targeted HIV cure as a high priority; many in the field have responded to these initiatives and the cure research agenda is robust. In this "salon" two editors of Pathogens and Immunity, Michael Lederman and Daniel Douek ask whether curing HIV is a realistic, scalable objective. We start with an overview perspective and have asked a number of prominent HIV researchers to add to the discussion

Autocrine Production of β-Chemokines Protects CMV-Specific CD4+ T Cells from HIV Infection

Induction of a functional subset of HIV-specific CD4+ T cells that is resistant to HIV infection could enhance immune protection and decrease the rate of HIV disease progression. CMV-specific CD4+ T cells, which are less frequently infected than HIV-specific CD4+ T cells, are a model for such an effect. To determine the mechanism of this protection, we compared the functional response of HIV gag-specific and CMV pp65-specific CD4+ T cells in individuals co-infected with CMV and HIV. We found that CMV-specific CD4+ T cells rapidly up-regulated production of MIP-1α and MIP-1β mRNA, resulting in a rapid increase in production of MIP-1α and MIP-1β after cognate antigen stimulation. Production of β-chemokines was associated with maturational phenotype and was rarely seen in HIV-specific CD4+ T cells. To test whether production of β-chemokines by CD4+ T cells lowers their susceptibility to HIV infection, we measured cell-associated Gag DNA to assess the in vivo infection history of CMV-specific CD4+ T cells. We found that CMV-specific CD4+ T cells which produced MIP-1β contained 10 times less Gag DNA than did those which failed to produce MIP-1β. These data suggest that CD4+ T cells which produce MIP-1α and MIP-1β bind these chemokines in an autocrine fashion which decreases the risk of in vivo HIV infection

HIV-1 Nef Binds the DOCK2–ELMO1 Complex to Activate Rac and Inhibit Lymphocyte Chemotaxis

The infectious cycle of primate lentiviruses is intimately linked to interactions between cells of the immune system. Nef, a potent virulence factor, alters cellular environments to increase lentiviral replication in the host, yet the mechanisms underlying these effects have remained elusive. Since Nef likely functions as an adaptor protein, we exploited a proteomic approach to directly identify molecules that Nef targets to subvert the signaling machinery in T cells. We purified to near homogeneity a major Nef-associated protein complex from T cells and identified by mass spectroscopy its subunits as DOCK2–ELMO1, a key activator of Rac in antigen- and chemokine-initiated signaling pathways, and Rac. We show that Nef activates Rac in T cell lines and in primary T cells following infection with HIV-1 in the absence of antigenic stimuli. Nef activates Rac by binding the DOCK2–ELMO1 complex, and this interaction is linked to the abilities of Nef to inhibit chemotaxis and promote T cell activation. Our data indicate that Nef targets a critical switch that regulates Rac GTPases downstream of chemokine- and antigen-initiated signaling pathways. This interaction enables Nef to influence multiple aspects of T cell function and thus provides an important mechanism by which Nef impacts pathogenesis by primate lentiviruses

Acquisition of direct antiviral effector functions by CMV-specific CD4+ T lymphocytes with cellular maturation

The role of CD4+ T cells in the control of persistent viral infections beyond the provision of cognate help remains unclear. We used polychromatic flow cytometry to evaluate the production of the cytokines interferon (IFN)-γ, tumor necrosis factor (TNF)-α, and interleukin (IL)-2, the chemokine macrophage inflammatory protein (MIP)-1β, and surface mobilization of the degranulation marker CD107a by CD4+ T cells in response to stimulation with cytomegalovirus (CMV)-specific major histocompatibility complex class II peptide epitopes. Surface expression of CD45RO, CD27, and CD57 on responding cells was used to classify CD4+ T cell maturation. The functional profile of virus-specific CD4+ T cells in chronic CMV infection was unique compared with that observed in other viral infections. Salient features of this profile were: (a) the simultaneous production of MIP-1β, TNF-α, and IFN-γ in the absence of IL-2; and (b) direct cytolytic activity associated with surface mobilization of CD107a and intracellular expression of perforin and granzymes. This polyfunctional profile was associated with a terminally differentiated phenotype that was not characterized by a distinct clonotypic composition. Thus, mature CMV-specific CD4+ T cells exhibit distinct functional properties reminiscent of antiviral CD8+ T lymphocytes

Immunization with vaccinia virus induces polyfunctional and phenotypically distinctive CD8+ T cell responses

Vaccinia virus immunization provides lifelong protection against smallpox, but the mechanisms of this exquisite protection are unknown. We used polychromatic flow cytometry to characterize the functional and phenotypic profile of CD8+ T cells induced by vaccinia virus immunization in a comparative vaccine trial of modified vaccinia virus Ankara (MVA) versus Dryvax immunization in which protection was assessed against subsequent Dryvax challenge. Vaccinia virus–specific CD8+ T cells induced by both MVA and Dryvax were highly polyfunctional; they degranulated and produced interferon γ, interleukin 2, macrophage inflammatory protein 1β, and tumor necrosis factor α after antigenic stimulation. Responding CD8+ T cells exhibited an unusual phenotype (CD45RO−CD27intermediate). The unique phenotype and high degree of polyfunctionality induced by vaccinia virus also extended to inserted HIV gene products of recombinant NYVAC. This quality of the CD8+ T cell response may be at least partially responsible for the profound efficacy of these vaccines in protection against smallpox and serves as a benchmark against which other vaccines can be evaluated

Isolation of viable antigen-specific CD8+ T cells based on membrane-bound tumor necrosis factor (TNF)-α expression

Current technology to isolate viable cytokine-producing antigen-specific primary human T cells is limited to bi-specific antibody capture systems, which suffer from limited sensitivity and high background. Here, we describe a novel procedure for isolating antigen-specific human T cells based on their ability to produce tumor necrosis factor (TNF)-α. Unlike many cytokines, TNF-α is initially produced in a biologically active membrane-bound form that is subsequently cleaved by TNF-α converting enzyme (TACE) to release the soluble form of TNF-α. By preventing this cleavage event, we show that TNF-α can be ‘trapped’ on the surface of the T cells from which it originates and directly labeled for viable isolation of these antigen-specific T cells. Together with other existing sorting procedures to isolate activated T cells, this new technique should permit the direct isolation of multi-functional T lymphocytes for further protein and gene expression analyses, as well as a detailed functional assessment of the potential role that TNF-α producing T cells play in the adaptive immune system