Marijke Th. L. Roos

Biphasic kinetics of peripheral blood T cells after triple combination therapy in HIV-1 infection : a composite of redistribution and proliferation

The origin of CD4+ T cells reappearing in the blood following antiretroviral therapy in human immunodeficiency virus type-1 (HIV-1) infection is still controversial. Here we show, using mathematical modeling, that redistribution of T cells to the blood can explain the striking correlation between the initial CD4+ and CD8+ memory T-cell repopulation and the observation that 3 weeks after the start of treatment memory CD4+ T-cell numbers reach a plateau. The increase in CD4+ T cells following therapy most likely is a composite of initial redistribution, accompanied by a continuous slow repopulation with newly produced naive T cells.

Association between CCR5 genotype and the clinical course of HIV-1 infection.

Viral, immune, and host genetic factors may influence the clinical course of HIV-1 infection. High viral load [1, 2], presence of syncytium-inducing HIV-1 [3-5], low T-lymphocyte function [6], and certain HLA types [7, 8] have been associated with rapid disease progression [9]. Several coreceptors for HIV-1 have recently been identified. Syncytium-inducing, T-cell line-adapted HIV-1 variants use the C-X-C chemokine receptor 4, macrophagetropic variants use the C-C chemokine receptor 5 (CCR5), and primary syncytium-inducing viruses can use both [10-16]. Persons who have been exposed to HIV-1 on multiple occasions but remain uninfected seem to be homozygous for a 32-nucleotide deletion (delta32) in the CCR5 gene [17, 18]; this concurs with the idea that macrophage-tropic HIV-1 variants establish new infections [19, 20]. In vitro, HIV-1 replication in cells that were heterozygous for CCR5 delta32 was reduced compared with the level of HIV-1 replication in wild-type cells [18]. Several cohort studies [17, 21-24] have shown a substantial correlation between CCR5 delta32 heterozygosity and delayed disease progression. To further substantiate this finding and to examine the biological principle underlying the protection offered by CCR5 delta32 heterozygosity, we analyzed the role of CCR5 genotype alone and in relation to established progression markers in the clinical course of HIV-1 infection in participants from the Amsterdam Cohort Studies. Methods Study Sample Between October 1984 and March 1986, 961 asymptomatic men who were living in the Amsterdam area and who reported having had at least two homosexual contacts in the preceding 6 months were enrolled in a prospective study on the prevalence and incidence of HIV-1 infection and risk factors for AIDS [25]. In the first serum sample taken, 728 men tested negative for HIV-1 antibodies; 131 of these men underwent seroconversion during the study. The remaining 238 men were positive for HIV antibodies; 5 of these men refused to participate further. Enrollment of seropositive persons was stopped after 6 months (in April 1985). Epidemiologic studies on the incidence of HIV-1 infection [26] showed that infection in seroprevalent homosexual men must have occurred an average of 1.5 years before entry into the Amsterdam Cohort Studies. Therefore, the time of seroconversion for seroprevalent men was set at 1.5 years before study entry. No differences in AIDS-free survival were found between persons who underwent seroconversion during the study and seroprevalent persons by using Kaplan-Meier (P > 0.2) and Cox proportional-hazard analyses in which the development of AIDS was the end point criterion (relative hazard, 1.17 for persons who had seroconversion compared with seroprevalent persons [95% CI, 0.84 to 1.63]). This result suggests a good estimation of the seroconversion date in the latter group. When we restricted our analyses to persons who had seroconversion, relative hazards were similar but less precise than estimates for the group as a whole. Therefore, we used 131 persons who had seroconversion and 233 seroprevalent persons as one study sample. Every 3 months, clinical and epidemiologic data were collected and serum and peripheral blood mononuclear cells were cryopreserved. Most seropositive men (n = 242 [66%]) did not receive early treatment. The remaining 122 men (34%) received zidovudine (70 [19%]), didanosine 10 [3%]), or other antiretroviral therapy (42 [12%]) before AIDS was diagnosed. None of the men received a combination of more than two antiretroviral drugs during our study. The mean age of participants at the time of seroconversion was 34.5 years (range, 19.5 to 57.7 years). By 1 January 1996 (the censor date), 189 men had developed AIDS according to the 1987 definition of AIDS [27] (median follow-up, 5.9 years [range, 0.6 to 12.3 years]), 94 men had not developed AIDS (median follow-up, 10.1 years [range, 0.3 to 13.7 years]), and 81 men were lost to follow-up (median follow-up, 2.0 years [range, 0.6 to 12.5 years]). A nested casecontrol study done using the same group of participants from the Amsterdam Cohort Studies was designed to identify factors that may be correlated with long-term survival. Long-term survivors (n = 23) remained free of clinical diseases for at least 9 years, with a mean CD4+ T-lymphocyte count of more than 400 cells/mm3 in the eighth and ninth year of HIV-1-positive follow-up (median follow-up, 10.8 years [range, 9.1 to 11.1 years]; mean CD4+ T-lymphocyte counts in the ninth year of follow-up, 534 cells/mm3 [range, 408 to 953 cells/mm3]). Each long-term survivor was matched with two progressors (men who developed AIDS after 2 to 7 years of HIV-1-positive follow-up). Matching was based on mean CD4+ T-lymphocyte count ( 250 cells/mm3) in year 2 of HIV-positive follow-up, HIV-1 serostatus at entry in the cohort study, and age ( 10 years). Use of Polymerase Chain Reaction for CCR5 Genotyping Samples of DNA were available for CCR5 genotyping for 343 of 364 men (94%). Genomic DNA was isolated from cryopreserved peripheral blood mononuclear cells (Qiagen blood kit, Qiagen, Hilden, Germany) and 100 mg of DNA was analyzed by using polymerase chain reaction (PCR) with primers (sense, position 612 to 635 in CCR5, 5-GATAGGTACCTGGCTGTCGTCCAT-3; antisense, position 829 to 850 in CCR5, 5-AGATAGTCATCTTGGGGCTGGT-3) flanking the described 32-nucleotide deletion in the CCR5 gene [17, 18]. Samples were amplified with 1 unit of Taq polymerase (Promega, Madison, Wisconsin) in the provided buffer with a final MgCl2 concentration of 3 mmol/L. Conditions of PCR comprised 5 minutes of denaturation at 95C; 30 cycles of 1 minute at 95C, 1 minute at 56C, and 2 minutes at 72C; and 5 minutes of elongation at 72C in a Perkin Elmer Cetus DNA thermal cycler 480 (Perkin Elmer, Foster City, California). Products of PCR were analyzed by using 2% agarose gel electrophoresis and ethidium bromide staining. Five randomly chosen samples with a reduced product size revealed the described 32-base pair deletion on automatic DNA sequencing (data not shown) [17, 18]. Virologic Assays Cocultivation of HIV-1-positive peripheral blood mononuclear cells with MT2 cells was performed every 3 months to detect syncytium-inducing HIV-1 variants [28, 29]. Serum viral load was measured by using a quantitative HIV-1 RNA nucleic acid-based sequence amplification (Organon Teknika, Boxtel, the Netherlands) with electrochemiluminescent labeled probes [30]. Serum samples obtained approximately 2 years after seroconversion (1 year after seroconversion; mean time point, 2.3 years [range, 1.5 to 3.0 years]) were available for measurement of HIV-1 RNA viral load for 335 of 364 participants (92%). Serum levels of HIV-1 RNA were analyzed after log10 transformation. Numbers of RNA copies that were below the test threshold of quantification were arbitrarily set at 10 (3).0 copies/mL. Immunologic Assays Antibodies to HIV-1 were detected in serum by using a commercial recombinant HIV-1/-2 enzyme immunoassay (Abbott, Chicago, Illinois) and were confirmed with an HIV-1 Western blot IgG assay (version 1.2, Diagnostic Biotechnology Ltd., Singapore, Thailand). Enumeration of CD4+ and CD8+ T lymphocytes was done by using flow cytofluorometry. For seroprevalent persons for whom we estimated the time of seroconversion to have been 18 months before entry into the cohort study, CD4+ T-lymphocyte count was first measured 18 months after the estimated time of seroconversion. Beginning in January 1988, reactivity of T lymphocytes in response to stimulation with CD3 monoclonal antibodies in vitro was routinely determined in whole-blood cultures [31]. The proliferative response measured after 4 days of culture by incorporation of [3H] thymidine was expressed as a percentage of the median values of the responses measured in two to five healthy controls tested on the same day. Statistical Analysis The Fisher exact test was used to compare HIV-1-seronegative participants with HIV-1-seropositive participants for CCR5 genotype distributions. In the casecontrol study, conditional logistic regression was performed to estimate the chance that a CCR5 delta32 heterozygote would be a long-term survivor. The Mann-Whitney U test was used to compare CCR5 delta32 heterozygotes and CCR5 wild-type homozygotes. For each participant, the slope of the decrease in CD4+ T lymphocytes was determined separately by fitting a simple regression line to his CD4+ T-lymphocyte count. At least three CD4+ T-lymphocyte counts had to be available for analysis; this was the case for 66 (97%) of the 68 CCR5 delta32 heterozygotes and 250 (91%) of the 275 CCR5 wild-type homozygotes. A Kaplan-Meier analysis was used to estimate the cumulative incidence of conversion to syncytium-inducing HIV-1 variants in relation to CCR5 genotype. We also estimated the duration of AIDS-free survival in relation to CCR5 genotype for the period during which only non-syncytium-inducing variants were present (conversion to syncytium-inducing HIV-1 was used as a censor criterion) or for the period after conversion to syncytium-inducing HIV-1 variants. A Kaplan-Meier analysis and a Cox proportional-hazards analysis were used to study the predictive value of CCR5 genotype alone or in combination with serum viral RNA load, CD4+ T-lymphocyte count, T-lymphocyte function, and syncytium-inducing phenotype. We evaluated the predictive value of the markers by fitting separate Cox models at 2, 4, 6, and 8 years after seroconversion. Participants were at risk from each specific time point; this method excluded participants who had previously developed AIDS. Because data on HIV-1 RNA load were available approximately 2 years after seroconversion only, data on viral load were not included in the models at 4, 6, and 8 years after seroconversion. All markers were also analyzed as time-dependent covariates. Participants who did not have AIDS were censored at 1 January 1996. Significance in

Immuno-activation with anti-CD3 and recombinant human IL-2 in HIV-1-infected patients on potent antiretroviral therapy

BACKGROUND A stable reservoir of latently infected, resting CD4 T cells has been demonstrated in HIV-1-infected patients despite prolonged antiretroviral treatment. This is a major barrier for the eradication of HIV by antiretroviral agents alone. Activation of these cells in the presence of antiretroviral therapy might be a strategy to increase the turnover rate of this reservoir. METHODS Three HIV-1-positive patients on potent antiretroviral therapy, in whom plasma viremia had been suppressed to below 5 copies/ml for at least 26 weeks, were treated with a combination of OKT3 (days 1-5) and recombinant human IL-2 (days 2 6). RESULTS The side-effects were fever, headache, nausea, diarrhea, and in one of the patients transient renal failure and seizures. The regimen resulted in profound T cell activation. In one patient plasma HIV-1 RNA transiently increased with a peak at 1500 copies/ml. In the other two patients plasma HIV-1 RNA levels remained below the detection limit, but HIV-1 RNA levels in the lymph nodes increased two- to threefold. All patients developed antibodies against OKT3. CONCLUSION OKT3/IL-2 resulted in T cell activation and proliferation, and could stimulate HIV replication in patients having achieved prolonged suppression of plasma viremia. OKT3/IL-2 therapy was toxic and rapidly induced antibodies against OKT3.

Hypertension in Sub-Saharan Africa: Cross-Sectional Surveys in Four Rural and Urban Communities

Background Cardiovascular disease (CVD) is the leading cause of adult mortality in low-income countries but data on the prevalence of cardiovascular risk factors such as hypertension are scarce, especially in sub-Saharan Africa (SSA). This study aims to assess the prevalence of hypertension and determinants of blood pressure in four SSA populations in rural Nigeria and Kenya, and urban Namibia and Tanzania. Methods and Findings We performed four cross-sectional household surveys in Kwara State, Nigeria; Nandi district, Kenya; Dar es Salaam, Tanzania and Greater Windhoek, Namibia, between 2009–2011. Representative population-based samples were drawn in Nigeria and Namibia. The Kenya and Tanzania study populations consisted of specific target groups. Within a final sample size of 5,500 households, 9,857 non-pregnant adults were eligible for analysis on hypertension. Of those, 7,568 respondents ≥18 years were included. The primary outcome measure was the prevalence of hypertension in each of the populations under study. The age-standardized prevalence of hypertension was 19.3% (95%CI:17.3–21.3) in rural Nigeria, 21.4% (19.8–23.0) in rural Kenya, 23.7% (21.3–26.2) in urban Tanzania, and 38.0% (35.9–40.1) in urban Namibia. In individuals with hypertension, the proportion of grade 2 (≥160/100 mmHg) or grade 3 hypertension (≥180/110 mmHg) ranged from 29.2% (Namibia) to 43.3% (Nigeria). Control of hypertension ranged from 2.6% in Kenya to 17.8% in Namibia. Obesity prevalence (BMI ≥30) ranged from 6.1% (Nigeria) to 17.4% (Tanzania) and together with age and gender, BMI independently predicted blood pressure level in all study populations. Diabetes prevalence ranged from 2.1% (Namibia) to 3.7% (Tanzania). Conclusion Hypertension was the most frequently observed risk factor for CVD in both urban and rural communities in SSA and will contribute to the growing burden of CVD in SSA. Low levels of control of hypertension are alarming. Strengthening of health care systems in SSA to contain the emerging epidemic of CVD is urgently needed.

Improvement of chronic diarrhoea in patients with advanced HIV-1 infection during potent antiretroviral therapy

Background:A substantial number of patients with advanced HIV infection suffer from intractable diarrhoea. The aim of this study was to evaluate whether potent antiretroviral therapy could alleviate such diarrhoea. Methods:In an open randomized study the effect of the HIV protease inhibitor indinavir in combination with nucleoside analogue reverse transcriptase inhibitors on chronic HIV-related diarrhoea was investigated in 14 late-stage (CD4+ lymphocyte count ≤ 50 × 106 cells/l) HIV-infected patients. Data concerning stool frequency, stool consistency and antidiarrhoeal drug use were collected in daily diaries over a 24-week period. Endpoints of the study were reduction of stool frequency, improvement of stool consistency, weight gain, and in case of diarrhoea due to Enterocytozoon bieneusi or Cryptosporidium sp. disappearance of these parasites from stool. Results:Thirteen patients started the study drug indinavir. One patient died after 1 week and one patient withdrew prematurely after 18 weeks. Median stool frequency declined from 5.8 daily at baseline to 2.3 daily after 24 weeks (P = 0.04). Stool consistency improved considerably over the study period: before treatment 56% of stools were watery and 0% were formed; at week 24 these figures were 0 and 35%, respectively. Body weight increased significantly with a median increment of 6.6 kg at week 24 (P = 0.0006). In two out of six patients with microsporidiosis and both patients with cryptosporidiosis, stools were free of parasites at week 24. Five out of six patients who used non-specific antidiarrhoeal medication on a regular basis prior to the study had ceased to do so at the end. Conclusion:The use of potent antiretroviral therapy in patients with advanced HIV infection can improve chronic HIV-related diarrhoea and in some cases lead to disappearance of E. bieneusi and Cryptosporidium sp. from the stools.

Restored humoral immune response to influenza vaccination in HIV-infected adults treated with highly active antiretroviral therapy.

Background:Highly active antiretroviral therapy (HAART) effectively suppresses replication of HIV and is accompanied by an increase in CD4+ T lymphocytes. Whether the increase in CD4+ T lymphocytes in the blood is a reflection of a reconstitution of the immune functions is unknown. We investigated the recovery of the humoral immune response during HAART after immunization with T-cell-dependent influenza vaccine. Methods:Forty-one men and three women infected with HIV and treated with HAART, and 15 healthy hospital staff members were immunized with trivalent influenza subunit vaccine. Antibody titres were determined by haemagglutination inhibiting assay in sera obtained before and 30 days after immunization. Lymphocyte subsets were determined in blood samples taken at the time of vaccination. Results:In all HIV-infected individuals, treatment with HAART caused a median reduction of 2.3 log10 in HIV-1 load. The median increase of CD4+ T lymphocytes after initiation of HAART was 170 × 106/l. The antibody response to influenza antigens was proportional to the number of memory CD4+ T lymphocytes in the blood at the time of vaccination. When a group of patients and healthy controls with approximately similar CD4+ T-lymphocyte counts were considered, the antibody titres after vaccination for influenza strain H1N1 and influenza B did not differ between patients and controls (P = 0.12). Vaccination of patients with a CD4+ T-lymphocyte count of < 200 × 106/l (mean 85 × 106/l) before the start of HAART and with a mean of 282 × 106/l CD4+ T lymphocytes at the time of vaccination as a result of HAART, demonstrated a substantial antibody response whereas patients with a CD4+ T lymphocyte count of < 200 × 106/l (mean 56 × 106/l) not treated with HAART (historical controls), and vaccinated with a similar influenza vaccine, failed to induce an antibody response. Conclusion:The present findings demonstrate a recovery of the humoral immune response to influenza antigens in HIV-infected individuals treated with HAART. This indicates that functional improvement of antigen specific CD4+ T helper cell reponses occurs.

Frequencies of Circulating Cytolytic, CD45RA+CD27−, CD8+ T Lymphocytes Depend on Infection with CMV

Viral infections may cause serious disease unless the adaptive immune system is able to clear the viral agents through its effector arms. Recent identification and functional characterization of subpopulations of human CD8+ T cells has set the stage to study the correlation between the appearance of particular subsets and common viral infections during childhood, i.e., EBV, CMV, varicella-zoster virus (VZV), and the attenuated measles-mumps-rubella (MMR) vaccine strains. In a cohort of 220 healthy children we analyzed lymphocytes and subpopulations of CD4+ and CD8+ T cells. The presence of the cytolytic CD45RA+CD27− subset of CD8+ T cells correlated with prior CMV infection as defined by seroconversion (p < 0.0001). The number of this CD8+ T cell subset remained stable during follow-up over 3 years in 40 children. The CD45RA+CD27− subset of CD8+ T cells first appeared during acute CMV infection and subsequently stabilized at an individual set-point defined by age and immunocompetence. The functional importance of these cells in CMV surveillance was reflected by their increased numbers in immunosuppressed pediatric kidney transplant patients. Preferential expansion of CD8+CD45RA+CD27− cytolytic T cells seems unique for CMV.

Changing virus-host interactions in the course of HIV-1 infection

Acquired immunodeficiency syndrome (AIDS) patients may present with various clinical symptoms related to severe immunodeficiency resulting from persistent infection with the human immunodeficiency virus-l (HIV-1). CD4+ T-helper (Th) cells are an important target for HIV (Klatzmann et al. 1984, Dalgleish et al. 1984, McDougal et al. 1985), and loss of these eells in relatively late stages of HIV infection is well documented and known to be predictive for progression (Meibye et al. 1986, Fahey et al. 1984). In addition to depletion of Th eells, leukocytes from AIDS patients display a variety of functional defects finally resulting in a general disturbance of immune reactivity that includes almost all leukocyte functions. At that stage, the patient is extremely susceptible to diseases related to a variety of intracellular pathogens but also has a moderately increased risk for pyogenic infections particularly with encapsulated bacteria. Moreover, in addition to Kaposis sarcomas, opportunistic neoplasia frequently develop (Fauci 1988). One feature of HIV-1 is its great variability with respect to biological properties such as syncytium inducing (SI) capacity, replication rate and cytotropism (ChengMayer et al. 1988, Asjo et al. 1986, Von Briesen et al. 1987, Evans et al. 1987, Tersmette et al. 1988). HIV-1 isolates recovered from peripheral blood mononuclear cells of asymptomatic subjects are able to grow in phytohemagglutinin (PHA)-stimulated primary blood lymphocytes (PBL) but, in contrast to isolates

OKT3 and IL-2 treatment for purging of the latent HIV-1 reservoir in vivo results in selective long-lasting CD4+ T cell depletion.

Activation of resting T cells has been proposed to purge the reservoir of HIV-1-infected resting CD4+ T cells. We therefore treated three HIV-1-infected patients on antiretroviral therapy with OKT3, a CD3 monoclonal antibody, and recombinant human IL-2. Here we report the profound and partially long-lasting host responses induced by the OKT3 and IL-2 treatment. OKT3/IL-2 induced a strong but transient release of plasma cytokines and chemokines. The percentage CD4+ and CD8+ cells in the blood expressing the activation marker CD38 transiently increased to almost 100%, and in lymph nodes we “observed” a 10-fold increase in the number of dividing Ki67+ cells and increased numbers of apoptotic cells. Following OKT3/IL-2 treatment, a long-lasting depletion of CD4+ cells in the peripheral blood and lymph nodes occurred, suggesting the physical deletion of these cells. Increases in CD4+T cell numbers during the two year followup period were due mainly to increased memory cell numbers. CD8+ cells were also depleted in the blood, but less severely in lymph nodes, and returned to baseline levels within several weeks.

Interferon (IFN)-beta treatment enhances CD95 and interleukin 10 expression but reduces interferon-gamma producing T cells in MS patients

Abstract Interferon (IFN)-β has been shown to favorably alter the disease course of relapsing-remitting multiple sclerosis (RRMS) patients. Although its mode of action is still unclear, there is ample evidence from in vitro studies that IFN-β directly modulates the function of immune cells. We analyzed here the effects of IFN-β treatment on immune functions in vivo in a group of 25 RRMS patients who received IFN-β (8 MIU) on alternate days. At baseline and at 1, 3 and 6 months from the start of the treatment, parameters for differentiation and activation states of both monocytes and T lymphocytes were assessed. A transient increase was seen in plasma (p) interleukin (IL)-10 level whereas pIL-12 (p40) was not affected. A similar change was found in the ability of monocytes to secrete these cytokines in vitro. Notably, patients who in vitro readily responded to IFN-β with enhanced IL-10 production had the highest pIL-10 levels. Concerning T-cell differentiation, flowcytometric analysis of cytokine production showed that treatment with IFN-β moderately decreased the mean percentages of CD8pos T cells producing IL-2 and IFN-γ and CD8neg T cells producing IL-4 (p