Long-term clinical and surrogate marker effects of subcutaneous intermittent interleukin-2 without antiretroviral therapy in HIV-infected patients.

OBJECTIVES: Subcutaneous administration of interleukin-2 (IL-2) has been shown to increase CD4 counts in HIV-infected patients. It remains unclear whether this effect is associated with a clinical benefit. PATIENTS AND METHODS: We conducted a long-term follow-up in the cohort of the UK-Vanguard study in which three groups of 12 antiretroviral-naive subjects with CD4 cell counts >350 cells/mm(3) received no treatment or IL-2 at either 4.5 or 7.5 MIU twice daily in 5 day cycles, respectively. RESULTS: Mean follow-up was 376 weeks. IL-2 therapy was associated with a higher area under the curve of CD4 cell count change from baseline at week 48 but not thereafter. HIV-RNA levels were unaffected. Highly active antiretroviral therapy (HAART) was initiated after a mean of 172, 175 and 152 weeks in the control group, low-dose and high-dose IL-2 treatment group, respectively, a statistically non-significant difference. There was a tendency to start HAART soon after discontinuation of IL-2 therapy which may have been triggered by the steep decay of CD4 counts. There were two serious adverse events in the control group, seven in the low-dose IL-2 group and eight in the high-dose IL-2 group. No pattern of disease was detected, making an association with IL-2 therapy unlikely. CONCLUSIONS: We could detect neither a benefit of IL-2 therapy after week 48 nor delayed initiation of HAART. This is currently the longest follow-up data comparing IL-2 therapy with no therapy in antiretroviral-naive HIV-infected patients and does not show a persistent benefit of the intervention.

Affordable CD4 T-cell enumeration for resource-limited regions: a status report for 2008.

BACKGROUND: The global struggle with human immunodeficiency virus (HIV) and the battle to develop affordable CD4 T-cell counting technology are both unfulfilled goals in 2008. The need for such instrumentation is more critical now as implementation of antiretroviral therapy (ART) is in progress in many resource limited regions. Major scaling-up efforts in rural situations are difficult to implement without laboratory infrastructure. CD4 T-cell counting is especially critical when trying to reach individuals with HIV to have them enrolled in ART as soon as they qualify for treatment based on CD4 count. METHOD: This review covers both the chronological evolution and the scientific milestones of technological development of affordable immunophenotyping. It is more focused on flow cytometry but does consider the potential contribution by digital image cytometry. RESULTS: Thus far flow cytometry offered only modest progress toward affordable immunophenotyping. A list with desirable features is offered for side by side comparison. Digital image cytometry has yet to show its enormous affordable market potential. CONCLUSIONS: It is possible to develop truly affordable, portable flow cytometry but it is not here yet. There are some hopeful signs as there are innovative and practical technical components appearing at regular intervals. However, so far the technical breakthroughs have been fragmented efforts without any attempts to consider intercorporate collaboration to optimize critical mass and synergy. The smaller players in the industry have made some progress toward meeting the monumental needs in Africa and Asia. Digital image cytometry may well be the ultimate winner in the affordable technology race.

Clinical application of a rapid lung-orientated immunoassay in individuals with possible tuberculosis.

BACKGROUND: Immunological ex vivo assays to diagnose tuberculosis (TB) have great potential but have largely been blood-based and poorly evaluated in active TB. Lung sampling enables combined microbiological and immunological testing and uses higher frequency antigen-specific responses than in blood. METHODS: A prospective evaluation was undertaken of a flow cytometric assay measuring the percentage of interferon-gamma synthetic CD4+ lymphocytes following stimulation with purified protein derivative of Mycobacterium tuberculosis (PPD) in bronchoalveolar lavage fluid from 250 sputum smear-negative individuals with possible TB. A positive assay was defined as >1.5%. RESULTS: Of those who underwent lavage and were diagnosed with active TB, 95% (106/111) had a positive immunoassay (95% CI 89% to 98%). In 139 individuals deemed not to have active TB, 105 (76%) were immunoassay negative (95% CI 68% to 82%). Of the remaining 24% (34 cases) with a positive immunoassay, a substantial proportion had evidence of untreated TB; in two of these active TB was subsequently diagnosed. Assay performance was unaffected by HIV status, disease site or BCG vaccination. In culture-positive pulmonary cases, response to PPD was more sensitive than nucleic acid amplification testing (94% vs 73%). The use of early secretory antigen target-6 (ESAT-6) responses in 71 subjects was no better than PPD, and 19% of those with culture-confirmed TB and a positive PPD immunoassay had no detectable response to ESAT-6. CONCLUSIONS: These findings suggest that lung-orientated immunological investigation is a potentially powerful tool in diagnosing individuals with sputum smear-negative active TB, regardless of HIV serostatus.

Immune reconstitution pneumonitis following Pneumocystis carinii pneumonia in HIV-infected subjects.

An HIV-infected man presented with a pneumonic illness following an episode of treated Pneumocystis carinii pneumonia (PCP). He had a rise in his CD4 count from 4 to 125 cells/microL on antiretroviral therapy prior to the onset of the second respiratory event. Bronchoalveolar lavage (BAL) revealed no pathogen, although a CD4 lymphocytosis in addition to a highly unusual population of rapidly proliferating CD8 cells was demonstrated. Following 2 weeks of steroid and anti-pneumocystis therapy, a repeat bronchoscopy demonstrated that the expression of these markers had returned to low values. This second respiratory illness, which may have arisen as a consequence of the regenerating immune response reacting to residual P. carinii antigen in the lung, is apparently not rare. When we reviewed our case notes, five further individuals were identified that had started antiretroviral therapy following an episode of PCP and subsequently developed a self-limiting pneumonitis for which no pathogen was identified on bronchoscopy.

Affordable CD4+ T cell counts by flow cytometry. II. The use of fixed whole blood in resource-poor settings.

We tested the feasibility and precision of affordable CD4+ T cell counting in resource-poor settings using a recently standardised fixative, TransFix in whole blood (WB) by flow cytometry (FCM). The precision of the assays was established under optimal conditions for single-platform FCM such as the volumetric CytoronAbsolute and the bead-based FACSCan. Fresh WB samples from HIV-seropositive and seronegative patients were tested in Tanzania and South Africa, fixed and sent to the UK for reanalysis 7 days later. Correlation, bias and limits of agreements were analysed by linear regression and the Bland-Altman test. Absolute CD4+ T cell counts remained stable for at least 10 days when TransFix was added to WB in 1:10 dilution at 20-25 degrees C, and for 7 days when added in 1:10 or 1:5 dilution to samples stored to mimic 'tropical' conditions at 37 degrees C. Higher temperatures such as 42 degrees C were tolerated for only short periods since the recovery had decreased to 63% by day 3. The reproducibility of lymphocyte subset analysis remained unchanged by TransFix with coefficient of variations <6% for all T cell subsets. Absolute CD4+ T cell counts and CD4+ T cell % values on fixed samples in the UK showed a high correlation with the results using fresh samples in Tanzania (r=0.993 and 0.969, respectively) and with the samples handled in Johannesburg (r=0.991 and 0.981) with minimal bias. Primary CD4 gating using only a single CD4 antibody also remained accurate in TransFixed samples (r=0.999). Thus, TransFix permits optimal fixation and transport of WB samples in the developing world for FCM to local regional laboratories and for quality assurance in international centres. When used together with inexpensive primary CD4 gating, TransFix will allow reliable and affordable CD4+ T cell counting by FCM in resource-poor settings.

Study protocol for the evaluation of the potential for durable viral suppression after quadruple HAART with or without HIV vaccination: the QUEST study.

PURPOSE: By protecting and stimulating HIV-specific CD4 cell responses, treatment of primary HIV infection (PHI) with potent quadruple HAART could lead to prolonged suppression of HIV replication after cessation of antiretroviral therapy. The QUEST trial investigates this hypothesis and aims to determine whether addition of a therapeutic vaccine to HAART increases the likelihood of prolonged viral suppression compared to HAART alone. METHOD: 148 patients with PHI were recruited. Participants were treated with open-label HAART for at least 76 weeks. Participants with sustained viremia <50 copies/mL were randomized to one of three 5-month, double-blinded study treatment groups: HAART alone, HAART + ALVAC-HIV (vCP1452), or HAART + ALVAC-HIV (vCP1452) + Remune. After a further month of HAART alone, all treatment was stopped where plasma HIV-1 RNA remained at <50 copies/mL. Intensive virologic and immunologic monitoring during a 24-week observation period followed treatment interruption. Patients who met treatment reintroduction criteria were offered HAART rescue.

Cytopathology or immunopathology? The puzzle of cytomegalovirus pneumonitis revisited.

Various hypotheses have been proposed to explain why cytomegalovirus pneumonitis (CMV-P) is frequent and severe in bone marrow transplant patients while remaining rare and mild in HIV infected patients. One hypothesis suggests that CMV-P is an immunopathological condition that is common in bone marrow transplantation (BMT) under the effects of an abnormally regenerating immune system that reacts against CMV infected lung tissue. Such a hypothesis implicates CD4 T lymphocytes as one of the critical cell populations involved in immunopathology and also suggests that this process would be aborted by CD4 T cell deficiency in HIV infection. However, studies correlating the onset of CMV-P with lymphocyte reconstitution following BMT have revealed that CD4 cells are present at very low frequencies in the blood during the early period after transplantation when most cases of CMV-P occur. Furthermore, studies directly investigating bronchoalveolar lavage cell types during episodes of CMV-P in BMT patients have also failed to demonstrate significant CD4 involvement and, instead, have emphasized a predominance of natural killer (NK) cells and CD8 cells. These findings serve as the basis for questioning the validity of a CD4-driven immunopathological model of CMV-P in BMT. On the other hand, a variety of experimental and clinical observations support the protective role of CMV-specific CD3+ CD8 T lymphocytes against CMV in both immunocompetent individuals and BMT patients. In a murine BMT model, adoptive transfer of syngeneic BM cells was associated with massive increases in lung CD8 cells which resulted in the resolution rather than the exacerbation of existing CMV-P. In the light of these findings a more plausible hypothesis for CMV-P in BMT is that during the early period after transplantation adequate protective CD8 responses are absent and an uncontrolled CMV proliferation is allowed to develop. Once a critical viral load is reached a cytokine 'storm' may be triggered in the lung tissue that aggravates direct CMV-associated cytopathic effects. Likely candidates for this process would include the release of tumour necrosis factor-alpha (TNF-alpha) from alveolar macrophages stimulated by interferon-gamma (IFN-gamma) released from NK cells that are reconstituted early after BMT.

Changes in CD4 lymphocyte counts after interruption of therapy in patients with viral failure on protease inhibitor-containing regimens. Royal Free Centre for HIV Medicine.

OBJECTIVE: To describe the short-term changes in CD4 lymphocyte counts after the interruption of antiretroviral HIV therapy in order to increase the understanding of CD4 lymphocyte dynamics, and so that appropriate monitoring strategies can be designed. METHODS: We studied 35 HIV-infected patients with late-stage disease who had therapy interruptions leading to high viral load levels, median greater than 750 000 RNA log10 copies/ml, and in whom two CD4 cell counts (median 28 days apart) were available before beginning a salvage regimen. RESULTS: Overall, there was a substantial decline in CD4 cell counts from a median of 125 to 83 cells/mm3 in the average 28 day period, with median proportionate and absolute losses of 26% and 24 cells/mm3 per month, respectively (P < 0.008). This tended to be greater in individuals studied sooner after interrupting therapy (P = 0.03) and in those with CD4 cell counts above the pre-therapy baseline (P = 0.06). There was a strong negative correlation between the proportionate increase in viral load and the absolute change in CD4 cell count (-0.66, P = 0.0002). CONCLUSION: Patients with relatively advanced HIV infection interrupting antiretroviral therapy after failing a protease inhibitor-containing regimen require frequent monitoring because CD4 cell counts appear to fall quite rapidly, at least in the first few weeks after interruption.

Course of viral load throughout HIV-1 infection.

HIV-1 RNA levels are routinely monitored as part of patient management. However, little is known about the course of HIV-1 RNA levels over the entire period of infection. The aim of this study was to investigate the course of HIV-1 RNA levels in a cohort of men with hemophilia who were observed for up to 17 years after HIV-1 seroconversion, and to assess the risk of HIV disease progression at any HIV-1 RNA level. Viral loads were measured on annual stored serum samples in 107 men with hemophilia A using the Roche Amplicor Monitor assay with non-B primers. On average, HIV-1 RNA levels increased significantly by 0.11 log10 per year over the course of HIV infection. This rate of increase was significantly faster in those who developed AIDS or died over the subsequent 12 to 17 year period, and in those who were older at HIV- 1 seroconversion. The risk of developing AIDS and death remained low when the HIV-1 RNA level was below 4 log10 copies/ml, but increased rapidly thereafter, supporting current guidelines for the initiation of antiretroviral therapy after the viral load has exceeded this level.

Affordable CD4(+) T-cell counts on 'single-platform' flow cytometers I. Primary CD4 gating.

Here, we demonstrate the flow cytometric concept of 'primary CD4 gating' utilizing three different CD4 monoclonal antibodies (mAbs) conjugated with five different fluorochromes. CD4(+) lymphocytes were defined by an autogate in a single histogram of CD4 fluorescence intensity (FI) (y-axis) vs. side light scatter (x-axis). A wide range of absolute counts for > 600 individuals, including HIV(+) patients, were compared with those obtained by 'state-of-the-art' single-platform flow cytometers such as the volumetric Ortho CytoronAbsolute and the Becton Dickinson FACSCalibur using TruCount beads. The correlation between CD4 counts obtained with primary CD4 gating and the full test panel on the Ortho Cytoron was excellent (R(2) = 0.999). Bland-Altman statistics showed a mean difference of -2 cells/mm(3) [confidence interval (CI) 95% = -3 to -1; limits of agreement -27 to +23]. In addition to absolute CD4 counts, CD4% values and CD4/CD8 ratios are also frequently requested. To obtain these, lymphocytes need to be counted using scatter gates, and a second tube stained with a CD8 mAb to count CD8(++) lymphocytes can be incorporated. We conclude that primary CD4 gating on single-platform volumetric flow cytometers is one of the most economical and flexible technologies for routine cost-conscious service work, particularly during the follow-up of patients undergoing anti-HIV therapy and/or vaccination in the developing world.

Cytofluorometric methods for assessing absolute numbers of cell subsets in blood. European Working Group on Clinical Cell Analysis.

The enumeration of absolute levels of cells and their subsets in clinical samples is of primary importance in human immunodeficiency virus (HIV)+ individuals (CD4+ T- lymphocyte enumeration), in patients who are candidates for autotransplantation (CD34+ hematopoietic progenitor cells), and in evaluating leukoreduced blood products (residual white blood cells). These measurements share a number of technical options, namely, single- or multiple-color cell staining and logical gating strategies. These can be accomplished using single- or dual-platform counting technologies employing cytometric methods. Dual-platform counting technologies couple the percentage of positive cell subsets obtained by cytometry and the absolute cell count obtained by automated hematology analyzers to derive the absolute value of such subsets. Despite having many conceptual and technical limitations, this approach is traditionally considered as the reference method for absolute cell count enumeration. As a result, the development of single-platform technologies has recently attracted attention with several different technical approaches now being readily available. These single-platform approaches have less sources of variability. A number of reports clearly demonstrate that they provide better coefficients of variation (CVs) in multicenter studies and a lower chance to generate aberrant results. These methods are therefore candidates for the new gold standard for absolute cell assessments. The currently available technical options are discussed in this review together with the results of some cross-comparative studies. Each analytical system has its own specific requirements as far as the dispensing precision steps are concerned. The importance of precision reverse pipetting is emphasized. Issues still under development include the establishment of the critical error ranges, which are different in each test setting, and the applicability of simplified low-cost techniques to be used in countries with limited resources.

The importance of lymphocyte trafficking in regulating blood lymphocyte levels during HIV and SIV infections.

In humans, blood is commonly monitored to provide surrogates of disease progression and assess immune status. However, the varied, rapid and atypical alterations in lymphocyte subsets which may occur in blood in response to pathogens, are not predictive of changes in the bulk of the immune system. A hallmark of human and simian immunodeficiency virus (SIV) infections is the profound loss of blood CD4(+) lymphocytes, a feature widely accepted as being a consequence of direct or indirect viral killing of CD4(+) cells throughout the body. However, in recording declining CD4 counts and CD4/8 ratios in the blood, little attention has been paid to migratory behaviour or the composition and tissue distribution of various lymphocyte subsets. This article compares the lymphocyte subsets in blood and various tissues in normal and virus-infected individuals prior to and following drug treatment and indicates an absence of selective CD4(+) cell decreases or increases, highlighting the importance of lymphocyte trafficking and compartmentalization in regulating blood T cell levels and suggesting a reevaluation of the currently favoured paradigm.

In vivo HIV-1 replicative capacity in early and advanced infection.

OBJECTIVE: Previous studies on patients treated with potent antiretroviral therapy have shown that viral clearance rates do not tend to change between early and advanced HIV-1 infection. Our objective was to investigate whether the other major aspect of virus dynamics, viral replicative capacity, does change. In vitro work has indicated that the viral replicative, capacity increases but in vivo evidence has been lacking. METHODS: As an in vivo measure of the viral replicative capacity, we studied the rate of rebound of plasma HIV RNA level during a 1-week therapy interruption in previously untreated patients who had received 2 weeks of antiretroviral therapy. RESULTS: Such therapy in five previously drug-naive patients with high CD4 lymphocyte counts (mean, 611 x 10(6)/l) and five patients with low counts (mean, 49 x 10(6)/l) led to a mean 2.2 log10 copies/ml decrease in plasma HIV-1 levels (from 5-6 log10 copies/ml) in 2 weeks. This was similar in the two groups. Interruption of therapy for the ensuing week resulted in a stable HIV-1 level for approximately 2 days followed by a rebound towards pretherapy level, which was much more marked in the patients with low CD4 cell counts (estimated mean rise 2.22 log10 versus 1.06 log10 copies/ml; P < 0.02). After restarting therapy, HIV RNA levels returned to pre-interruption levels. CONCLUSIONS: These findings need confirmation, but the ability of HIV-1 to replicate in vivo appears to increase during HIV-1 infection. This increased replicative capacity, for which there are several potential explanations, may be the cause of gradual CD4 lymphocyte depletion.

'Naïve' and 'memory' CD4+ T-cells and T-cell receptor (TCR) V beta repertoire dynamics are independent of the levels of viremia following HIV seroconversion.

The progression of 'naive' and 'memory' T-cells and the T-cell receptor Vbeta (TCR Vbeta) repertoire dynamics within the peripheral CD4+ T-cell compartment were studied in individuals following HIV seroconversion. Profound TCR Vbeta repertoire perturbations were observed within the CD4+ T-cell pool in treatment-naive patients regardless of their levels of viremia during the first 6-8 months after seroconversion. The ratio of 'naive' to 'memory' CD4+ T-cells as well as the TCR Vbeta repertoire dynamics did not appear to correlate with absolute numbers of CD4 T-cells.

Flow cytometric analysis of normal B cell differentiation: a frame of reference for the detection of minimal residual disease in precursor-B-ALL.

During the last two decades, major progress has been made in the technology of flow cytometry and in the availability of a large series of monoclonal antibodies against surface membrane and intracellular antigens. Flow cytometric immunophenotyping has become a diagnostic tool for the analysis of normal and malignant leukocytes and it has proven to be a reliable approach for the investigation of minimal residual disease (MRD) in leukemia patients during and after treatment. In order to standardize the flow cytometric detection of MRD in acute leukemia, a BIOMED-1 Concerted Action was initiated with the participation of six laboratories in five different European countries. This European co-operative study included the immunophenotypic characterization and enumeration of different precursor and mature B cell subpopulations in normal bone marrow (BM). The phenotypic profiles in normal B cell differentiation may form a frame of reference for the identification of aberrant phenotypes of precursor-B cell acute lymphoblastic leukemias (precursor-B-ALL) and may therefore be helpful in MRD detection. Thirty-eight normal BM samples were analyzed with five different pre-selected monoclonal antibody combinations: CD10/CD20/CD19, CD34/CD38/CD19, CD34/CD22/CD19, CD19/CD34/CD45 and TdT/CD10/CD19. Two CD19- immature subpopulations which coexpressed B cell-associated antigens were identified: CD34+/CD22+/CD19- and TdT+/CD10+/CD19-, which represented 0.11 +/- 0.09% and 0.04 +/- 0.05% of the total BM nucleated cells, respectively. These immunophenotypes may correspond to the earliest stages of B cell differentiation. In addition to these minor subpopulations, three major CD19+ B cell subpopulations were identified, representing three consecutive maturation stages; CD19dim/CD34+/TdT+/CD10bright/CD22dim/CD45dim /CD38bright/CD20- (subpopulation 1), CD19+/CD34-/TdT-/CD10+/CD22dim/CD45+/CD38bright/ CD20dim (subpopulation 2) and CD19+/CD34-/TdT-/CD10-/CD22bright/CD45bright/ CD38dim/CD20bright (subpopulation 3). The relative sizes of subpopulations 1 and 2 were found to be age related: at the age of 15 years, the phenotypic precursor-B cell profile in BM changed from the childhood 'immature' profile (large subpopulations 1 and 2/small subpopulation 3) to the adult 'mature' profile (small subpopulation 1 and 2/large subpopulation 3). When the immunophenotypically defined precursor-B cell subpopulations from normal BM samples are projected in fluorescence dot-plots, templates for the normal B cell differentiation pathways can be defined and so-called 'empty spaces' where no cell populations are located become evident. This allows discrimination between normal and malignant precursor-B cells and can therefore be used for MRD detection.