Polyfunctionality of the CXCR4/CXCL12 axis in health and disease: Implications for therapeutic interventions in cancer and immune-mediated diseases.

Historically the chemokine receptor CXCR4 and its canonical ligand CXCL12 are associated with the bone marrow niche and hematopoiesis. However, CXCL12 exhibits broad tissue expression including brain, thymus, heart, lung, liver, kidney, spleen, and bone marrow. CXCR4 can be considered as a node which is integrating and transducing inputs from a range of ligand-receptor interactions into a responsive and divergent network of intracellular signaling pathways that impact multiple cellular processes such as proliferation, migration, and stress resistance. Dysregulation of the CXCR4/CXCL12 axis and consequent fundamental cellular processes, are associated with a panoply of disease. This review frames the polyfunctionality of the receptor at a molecular, physiological, and pathophysiological levels. Transitioning our perspective of this axis from a single gene/protein:single function model to a polyfunctional signaling cascade highlights the potential for finer therapeutic intervention and cautions against a reductionist approach.

Novel multiparameter correlates of Coxiella burnetii infection and vaccination identified by longitudinal deep immune profiling.

Q-fever is a flu-like illness caused by Coxiella burnetii (Cb), a highly infectious intracellular bacterium. There is an unmet need for a safe and effective vaccine for Q-fever. Correlates of immune protection to Cb infection are limited. We proposed that analysis by longitudinal high dimensional immune (HDI) profiling using mass cytometry combined with other measures of vaccination and protection could be used to identify novel correlates of effective vaccination and control of Cb infection. Using a vaccine-challenge model in HLA-DR transgenic mice, we demonstrated significant alterations in circulating T-cell and innate immune populations that distinguished vaccinated from naïve mice within 10 days, and persisted until at least 35 days post-vaccination. Following challenge, vaccinated mice exhibited reduced bacterial burden and splenomegaly, along with distinct effector T-cell and monocyte profiles. Correlation of HDI data to serological and pathological measurements was performed. Our data indicate a Th1-biased response to Cb, consistent with previous reports, and identify Ly6C, CD73, and T-bet expression in T-cell, NK-cell, and monocytic populations as distinguishing features between vaccinated and naïve mice. This study refines the understanding of the integrated immune response to Cb vaccine and challenge, which can inform the assessment of candidate vaccines for Cb.

Alginate encapsulant incorporating CXCL12 supports long-term allo- and xenoislet transplantation without systemic immune suppression.

Islet transplantation represents a potentially curative approach for individuals with Type I Diabetes. The requirement for systemic immune suppression to control immune-mediated rejection of transplanted islets and the limited human islet supply represent significant roadblocks to progress for this approach. Islet microencapsulation in alginate offers limited protection in the absence of systemic immunosuppression, but does not support long-term islet survival. The chemokine, CXCL12, can repel effector T cells while recruiting immune-suppressive regulatory T cells (Tregs) to an anatomic site while providing a prosurvival signal for beta-cells. We proposed that coating or encapsulating donor islets with CXCL12 would induce local immune-isolation and protect and support the function of an allo- or xenograft without systemic immune suppression. This study investigated the effect of alginate microcapsules incorporating CXCL12 on islet function. Islet transplantation was performed in murine models of insulin-dependent diabetes. Coating of islets with CXCL12 or microencapsulation of islets with alginate incorporating the chemokine, resulted in long-term allo- and xenoislet survival and function, as well as a selective increase in intragraft Tregs. These data support the use of CXCL12 as a coating or a component of an alginate encapsulant to induce sustained local immune-isolation for allo- or xenoislet transplantation without systemic immunosuppression.

T-lymphocyte development and models of thymopoietic reconstitution.

T-cell development occurs in the thymus and involves a complex stepwise differentiation process from hematopoietic progenitor cell through to the emergence of positively selected, non-self reactive naïve T cells, which emigrate from the organ into the blood stream. This process takes place in the three-dimensional context of a delicate latticework of thymic stromal cells, including epithelial-derived cells and dendritic cells that assist in the development of the mature T cell. The details of the steps in T-cell development are now more comprehensively understood and furthermore in vitro systems are in development to reiterate this process in vitro. The clinical impact of this work is clearly the understanding of immune reconstitution in immune compromised patients (e.g., those with HIV/AIDS or post transplantation) and the development of novel techniques for regenerating the spectrum of T-cell function in these individuals.

Changing patterns of presentations of patients with HIV-related disease at a tertiary referral centre and its implications for physician training.

Highly active antiretroviral therapy (HAART) has been shown to be highly effective in controlling HIV-related disease progression. Our objective was to determine whether HAART had altered the spectrum of HIV-related disease presentations at a tertiary medical referral centre and if a change in the clinical presentations of HIV-infected individuals to the hospital had impacted on physicians' training. A retrospective study which examined all admissions of HIV-infected patients identified between 1 October 1996 to 30 September 1998 using a hospital-designed computer database was undertaken at the Beth Israel Deaconess Medical Center (BIDMC) tertiary medical referral centre. All medical residents were surveyed in order to assess their knowledge of HIV-associated admissions and their confidence treating HIV-infected patients. There were significant changes in the admitting diagnosis for HIV-related illness between 1996 and 1998. Admissions for opportunistic infections (OIs) declined whereas admissions with bacterial infections increased significantly. Use of HAART remained stable between the 2 years of the study. Physicians' overestimated the use of HAART and only 8% of residents felt very comfortable taking care of an HIV-infected patient. In conclusion, the spectrum of presentations with HIV-related disease to a tertiary referral centre continues to change in the HAART era and impacts on physicians' experience of the management of HIV disease.

Tissue source dictates lineage outcome of human fetal CD34(+)CD38(-) cells.

OBJECTIVE: The translocation from fetal liver hematopoiesis to secondary organs occurs during the second trimester of human gestation. It has been hypothesized that stem cells migrate and acquire lineage potential based on cues specific to the adopted microenvironment. We evaluated primitive hematopoietic cell populations in the fetal human to determine if lineage restriction precedes or follows translocation to sites of hematopoietic activity including thymus, spleen, bone marrow, and liver. METHODS: Sets of hematopoietic tissues from individual second-trimester human abortuses were used to compare and quantitate the lineage outcome of immunophenotypically primitive cells from each of the hematopoietic organs using ex vivo myeloid and lymphoid differentiation systems. RESULTS: Despite uniformity in immunophenotype, functional capabilities were highly restricted by the tissue of origin and alteration in the ex vivo differentiation context did not lead to a change in differentiation outcome. CONCLUSION: Translocation of primitive cells from fetal liver to tissues of mature hematopoietic activity is associated with tissue-specific, quantitative changes in differentiation potential that are unresponsive to alternative differentiation environments. These data suggest that multipotentiality is lost prior to or upon stem-cell migration in the developing human. It is not persistent with residence in a secondary hematopoietic organ.

Efficient generation of human T cells from a tissue-engineered thymic organoid.

Biocompatible inorganic matrices have been used to enhance bone repair by integrating with endogenous bone architecture. Hypothesizing that a three-dimensional framework might support reconstruction of other tissues as well, we assessed the capacity of a tantalum-coated carbon matrix to support reconstitution of functioning thymic tissue. We engineered a thymic organoid by seeding matrices with murine thymic stroma. Co-culture of human bone marrow-derived hematopoietic progenitor cells within this xenogeneic environment generated mature functional T cells within 14 days. The proportionate T-cell yield from this system was highly reproducible, generating over 70% CD3+ T cells from either AC133+ or CD34+ progenitor cells. Cultured T cells expressed a high level of T-cell receptor excision circles (TREC), demonstrating de novo T lymphopoiesis, and function of fully mature T cells. This system not only facilitates analysis of the T-lymphopoietic potential of progenitor cell populations; it also permits ex vivo genesis of T cells for possible applications in treatment of immunodeficiency.

Extracellular calcium elicits a chemokinetic response from monocytes in vitro and in vivo.

Recruitment of macrophages to sites of cell death is critical for induction of an immunologic response. Calcium concentrations in extracellular fluids vary markedly, and are particularly high at sites of injury or infection. We hypothesized that extracellular calcium participates in modulating the immune response, perhaps acting via the seven-transmembrane calcium-sensing receptor (CaR) on mature monocytes/macrophages. We observed a dose-dependent increase in monocyte chemotaxis in response to extracellular calcium or the selective allosteric CaR activator NPS R-467. In contrast, monocytes derived from mice deficient in CaR lacked the normal chemotactic response to a calcium gradient. Notably, CaR activation of monocytes bearing the receptor synergistically augmented the transmigration response of monocytes to the chemokine MCP-1 in association with increased cell-surface expression of its cognate receptor, CCR2. Conversely, stimulation of monocytes with MCP-1 or SDF-1alpha reciprocally increased CaR expression, suggesting a dual-enhancing interaction of Ca(2+) with chemokines in recruiting inflammatory cells. Subcutaneous administration in mice of Ca(2+), MCP-1, or (more potently) the combination of Ca(2+) and MCP-1, elicited an inflammatory infiltrate consisting of monocytes/macrophages. Thus extracellular calcium functions as an ionic chemokinetic agent capable of modulating the innate immune response in vivo and in vitro by direct and indirect actions on monocytic cells. Calcium deposition may be both consequence and cause of chronic inflammatory changes at sites of injury, infection, and atherosclerosis.

Active movement of T cells away from a chemokine.

Movement towards or away from a given stimulus guides the directional migration of prokaryotes, simple eukaryotes and neurons. As bi-directional cues may influence entry and exit of immune effector cells from tissue sites, we evaluated the migratory responses of T-cell subsets to varying concentrations of the chemokine stromal cell derived factor-1 (SDF-1). There was selective repulsion of subpopulations of T cells at high concentrations of recombinant SDF-1 or naturally occurring bone marrow-derived SDF-1, which could be inhibited by pertussis toxin and antibody against the chemokine receptor CXCR4. Distinct sensitivity profiles to genistein, herbimycin and 8-Br-cAMP biochemically distinguished movement of cells towards or away from an SDF-1 gradient. In vivo, antigen-induced T-cell recruitment into the peritoneal cavity was reversed by high but not low concentrations of SDF-1. The phenomenon of movement away from a chemokine represents a previously unknown mechanism regulating the localization of mature T cells. It adds to the functional repertoire of chemokines that may participate in immune physiology and may be applied therapeutically to alter the immune response.

Inhibition of human immunodeficiency virus replication and growth advantage of CD4+ T cells and monocytes derived from CD34+ cells transduced with an intracellular antibody directed against human immunodeficiency virus type 1 Tat.

Current clinical gene therapy protocols for the treatment of human immunodeficiency virus type 1 (HIV-1) infection involve the ex vivo transduction and expansion of CD4+ T cells derived from HIV-positive patients at a late stage in their disease (CD4+ cell count <400 cells/mm3). We examined the efficiency of transduction and transgene expression in adult bone marrow (BM)- and umbilical cord blood (UCB)-derived CD34+ cells induced to differentiate into T cells and monocytes in vitro with an MuLV-based vector encoding the neomycin resistance gene and an intracellular antibody directed against the Tat protein of HIV-1 (sFvtat1-Ckappa). The expression of the marker gene and the effects of antiviral construct on subsequent challenge with monocytotropic and T cell-tropic HIV-1 isolates were monitored in vitro in purified T cells and monocytes generated in culture from the transduced CD34+ cells. Transduction efficiencies of CD34+ cells ranged between 22 and 27%. Differentiation of CD34+ cells into T cells or monocytes was not significantly altered by the transduction process. HIV-1 replication in monocytes and CD4+ T cells derived from CD34+ cells transduced with the intracellular antibody gene was significantly reduced in comparison with the degree of HIV replication seen in monocytes and CD4+ T cells derived from CD34+ cells transduced with the neomycin resistance gene alone. Further, T cells and monocytes derived from CD34+ cells transduced with the intracellular antibody gene were demonstrated to express the sFvtat1-Ckappa transgene by RT-PCR and had a selective growth advantage in cultures that had been challenged with HIV-1. These data demonstrate that sFvtat1-Ckappa inhibits HIV-1 replication in T cells and monocytes developing from CD34+ cells and supports the continuing development of a stem cell gene therapy for the treatment of HIV-1 infection.

Isolation and transduction of CD34+ cells from small quantities of peripheral blood from HIV-1-infected patients not treated with hemopoietic growth factors.

A proposed hemopoietic stem cell gene therapy for treatment for HIV infection would involve transduction of CD34+ hemopoietic stem cells with vectors encoding anti-HIV constructs. Peripheral blood has proved to be a useful source of these hemopoietic stem cells and this study exploits this finding. Small quantities of peripheral blood were obtained from HIV-negative patients and HIV-positive patients who were and were not receiving hemopoietic growth factors (HGFs). CD34+ cells were obtained from these samples using a simple technique and scored for frequency of colony type. This demonstrated that HIV-negative patients had the highest frequency of colony-forming units (CFUs). HIV-positive patients not treated with HGFs had a lower frequency of CFUs, but the same colony type distribution as HIV-negative patients. HIV-positive patients treated with HGFs had the lowest frequency of CFUs, but their colony type distribution demonstrated that they had responded to treatment. CD34+ cells selected in this way were also transduced with the murine retroviral MFG vector using a technique that demonstrated transduction efficiencies ranging from 2% to 16% (median, 11.5%). This study simplifies the experimental requirements for development of a hemopoietic stem cell gene therapy for HIV infection and offers the possibility that longitudinal studies could be performed on peripheral blood CD34+ cells from HIV-positive or HIV-negative patients without the need for granulocyte colony-stimulating factor mobilization.

The in vivo effects of combination antiretroviral drug therapy on peripheral blood CD34+ cell colony-forming units from HIV type 1-infected patients.

This study investigated the effects of a combination antiretroviral drug regimen (indinavir and two nucleoside analogs or ritonavir and saquinavir) on the levels of CD34+ colony-forming units (CFU-Cs) in the peripheral blood of HIV-1+ patients. Ten patients who were receiving combination antiretroviral drug therapy were studied and their peripheral blood CD34+ CFU-Cs were measured prior to, 1 month after, and 4 to 6 months after the commencement of therapy. The levels of CD4+ T cells increased significantly in these patients (paired t test, p = 0.0027) and plasma viral load became undetectable in all but one patient studied. Measurements of the CFU-Cs showed that their levels tended to increase on the commencement of therapy, and these levels became significantly higher than baseline by 4-6 months (paired t test, p = 0.0293). Analysis of the different colony phenotype demonstrated that the main contributor to this increase consisted of burst-forming unit erythroid (BFU-E) cells. These data also demonstrated that there was an inverse correlation between the rise in CFU-Cs at 4-6 months compared with CD4+ cell, CD8+ cell, and neutrophil counts, and hemoglobin concentration, at baseline. The demonstrated increase in the levels of CD34+ CFU-Cs suggests that HIV-1 may have an inhibitory effect on these cells in vivo, and that this inhibition may be abrogated by suppression of viral replication.

Inhibition of human immunodeficiency virus replication and growth advantage of CD4+ T cells from HIV-infected individuals that express intracellular antibodies against HIV-1 gp120 or Tat.

Current clinical gene therapy protocols for the treatment of human immunodeficiency virus type 1 (HIV-1) infection often involve the ex vivo transduction and expansion of CD4+ T cells derived from HIV-positive patients at a late stage in their disease (CD4 count <400). These protocols involve the transduction of T cells by murine leukemia virus (MLV)-based vectors encoding antiviral constructs such as the rev m10 dominant negative mutant or a ribozyme directed against the CAP site of HIV-1 RNA. We examined the efficiency and stability of transduction of CD4+ T cells derived from HIV-infected patients at different stages in the progression of their disease, from seroconversion to AIDS. CD4+ T cells from HIV-positive patients and uninfected donors were transduced with MLV-based vectors encoding beta-galactosidase and an intracellular antibody directed against gp120 (sFv 105) or Tat. (sFvtat1-Ckappa). The expression of marker genes and the effects of the antiviral constructs were monitored in vitro in unselected transduced CD4+ T cells. Efficiency and stability of transduction varied during the course of HIV infection; CD4+ T cells derived from asymptomatic patients were transducible at higher efficiencies and stabilities than CD4+ T cells from patients with acquired immunodeficiency syndrome (AIDS). Expression of the anti-tat intracellular antibody was more effective at stably inhibiting HIV-1 replication in transduced cells from HIV-infected individuals than was sFv 105. The results of this study have important implications for the development of a clinically relevant gene therapy for the treatment of HIV-1 infection.

Efficiency of a high-titer retroviral vector for gene transfer into skeletal myoblasts.

BACKGROUND: Genetic transformation of skeletal myoblasts for myocardial repair is dependent on an efficient gene transfer system that integrates the genes of interest into the genome of the target cell and its progeny. The aim of this investigation was to evaluate the use of a new retrovirally based gene transfer system for this purpose. METHODS: MFGnlslacZ retroviral vector, packaged in high-titer, split-genome packaging cell line (FLYA4) was used to transduce the skeletal myoblast cell line L6. L6 cells, cultured in 10% fetal calf serum, were transduced with the MFGnlslacZ vector by means of filtered supernatant from FLYA4 cells. Transduced L6 cells were divided into four groups. Group I cells were fixed as myoblasts 3 days after transduction. Group II cells were allowed to differentiate into myotubes. Group III cells were split every 3 days for 4 months. Group IV cells were split as in group III but then allowed to differentiate into myotubes. All samples were fixed and stained for beta-galactosidase activity. The effects on gene transfer of transforming growth factor-beta, insulin-like growth factor-I, and platelet-derived growth factor were determined by spectrophotometric assay of beta-galactosidase activity in cells transduced in the presence or absence of serum with 0 to 200 ng/ml of each growth factor. RESULTS: Morphometric analysis showed that 66.3% +/- 3% to 69.6% +/- 6% of cells in group I to IV expressed the lacZ reporter gene. In the presence of serum, transforming growth factor-beta significantly inhibited gene transfer, whereas insulin-like growth factor-I and platelet-derived growth factor significantly enhanced gene transfer. In absence of serum, however, only platelet-derived growth factor enhanced retrovirally mediated gene transfer into skeletal myoblasts. CONCLUSION: MFG retroviral vectors packaged in FLYA4 cells are efficient in gene transfer into skeletal myoblasts and result in transgenic expression that is maintained after repeated cell division, differentiation, or both. Platelet-derived growth factor enhances retrovirally mediated gene transfer into skeletal myoblasts.

Patients unaware of their HIV status present to inner city accident and emergency department with respiratory complications.

OBJECTIVE: To examine the clinical presentations and management of patients presenting to an accident and emergency (A&E) department with an AIDS defining illness (ADI). METHODS: Presentations of patients in the A&E department with ADI were reviewed retrospectively. The age, sex, ethnic origin, risk factor for HIV infection, route of referral to hospital, presenting complaint, triage category, referral from A&E, admission under medical specialists, diagnosis, and survival from ADI were noted for each patient. RESULTS: 133 patients were registered at St Mary's Hospital in London with ADI during 1994. A significant minority of these patients (25/133) presented to the hospital without prior knowledge of their HIV positive status. Thirty two patients presented to the A&E department with their ADI. Of these, 13/32 (41%) were unaware of the HIV serostatus. All 13 patients had an acute respiratory disease (Pneumocystis carinii pneumonia or pulmonary tuberculosis). In contrast, patients aware of their HIV positive status (19/32) presented to the A&E department with a wide range of non-pulmonary ADI. CONCLUSIONS: The study emphasises the importance of respiratory complications in patients who present with a ADI to emergency departments but are unaware of their HIV positivity. These patients presented solely with Pneumocystis carinii pneumonia or pulmonary tuberculosis, conditions in which early diagnosis and treatment significantly reduce morbidity and mortality.

The rising prevalence of HIV-1 infection in patients attending an inner city accident and emergency department.

The recently published findings of the unlinked anonymous HIV prevalence study in England and Wales showed unchanging HIV prevalence in groups such as homo/bisexual men, and declining rates in non-injecting heterosexual men attending genitourinary medicine clinics. However, this multicentre study did detect a significant rise in seroprevalence rates in pregnant women in England and Wales and sentinel groups within hospitals in London, warning that changing patterns of HIV infection might account for these variable results. In 1992-1993 a seroprevalence study of adult patients attending the accident and emergency department at St. Mary's Hospital in West Central London showed a rate of HIV-1 infection of 1 in 77. We have repeated the seroprevalence study over the same calendar months in 1994-1995 to gain further information about HIV positive patients attending the department and to see whether a change in the patterns of HIV infection in the population served by St Mary's Hospital had occurred.

HIV positive patients first presenting with an AIDS defining illness: characteristics and survival.

OBJECTIVES: To study the presentation and survival of patients who present with their first diagnosis of being HIV positive at the same time as their AIDS defining illness. DESIGN: Retrospective study of patients presenting with AIDS between 1991 and 1993. SETTING: Department of genitourinary medicine, St Mary's Hospital, London. MAIN OUTCOME MEASURES: AIDS defining illness at presentation and survival after diagnosis of AIDS. RESULTS: Between January 1991 and December 1993, 97 out of 436 patients (22%) presented with their first AIDS defining illness coincident with their first positive result of an HIV test (group B). The remaining 339 patients (78%) had tested positive for HIV-1 infection within the previous eight years and had consequently been followed up in clinics before developing their first AIDS defining illness (group A). The two groups of patients did not differ in age and sex distribution, risk factors for HIV-1 infection, nationality, country of origin, or haematological variables determined at the time of the AIDS defining illness. However, the defining illnesses differed between the two groups. Illnesses associated with severe immunodeficiency (the wasting syndrome, cryptosporidiosis, and cytomegalovirus infection) were seen almost exclusively in group A whereas extrapulmonary tuberculosis and Pneumocystis carinii pneumonia were more common in group B. The survival of patients in group B after the onset of AIDS was significantly longer than that of patients in group A as determined by Kaplan-Meier log rank analysis (P = 0.0026). CONCLUSIONS: Subjects who are HIV positive and present late are a challenge to the control of the spread of HIV infection because they progress from asymptomatic HIV infection to AIDS without receiving health care. The finding that presentation with an AIDS defining illness coincident with a positive result in an HIV test did not have a detrimental effect on survival gives insights into the effects of medical intervention on disease progression after a diagnosis of AIDS.