Susanne Dam Nielsen

Association between Larger Thymic Size and Higher Thymic Output in Human Immunodeficiency Virus-Infected Patients Receiving Highly Active Antiretroviral Therapy

To examine the impact of thymic size on immune recovery in patients with human immunodeficiency virus (HIV) infection, the thymus was visualized, using computed tomographic scans, in 25 HIV-infected patients who had received highly active antiretroviral therapy (HAART) for 6-18 months and had levels of viremia <500 copies/mL. For comparison, 10 control subjects were included in the study. Total and naive CD4+ cell counts were determined by flow cytometry. To determine thymic output, the number of CD4+ cells containing T cell receptor excision circles (TRECs) was measured. Qualitative immune recovery was evaluated by determination of CD4+ T cell receptor repertoire in 19 of the HIV-infected patients. Larger thymic size was associated with higher CD4+ cell counts (r=0.498; P=.011) and higher CD4+ TREC frequency (r=0.652; P<.001). Furthermore, patients with abundant thymic tissue seemed to have broader immunologic repertoires, compared with patients with minimal thymic tissue (P=.054). These findings suggest that thymopoiesis is ongoing in the adult thymus and contributes to immune reconstitution in HIV-infected patients receiving HAART.

Dose–response study of probiotic bacteria Bifidobacterium animalis subsp lactis BB-12 and Lactobacillus paracasei subsp paracasei CRL-341 in healthy young adults

Objective:This study was performed to investigate the dose–response effects of supplementation with Bifidobacterium animalis subsp lactis (BB-12) and Lactobacillus paracasei subsp paracasei (CRL-431) on blood lipids, recovery from feces and bowel habits. Changes of the fecal microflora was analyzed in the 1010 CFU/day probiotic and placebo group.Design:The study was designed as a randomized, placebo-controlled, double-blinded, parallel dose–response study.Subjects:Healthy young adults (18–40 years) were recruited by advertising in local newspapers. Of the 75 persons enrolled, 71 (46 women, 25 men, mean age 25.6 years (range 18–40 years)) completed the study.Intervention:The volunteers were randomly assigned into five groups receiving either placebo or a mixture of the two probiotics in the concentration of 108, 109, 1010 or 1011 CFU/day in 2 weeks run-in period, 3 weeks intervention and 2 weeks wash-out. Diary reporting bowel habits and well being (abdominal bloating, flatulence and headache) was kept for all 7 weeks and blood lipids, fecal recovery of BB-12 and CRL-431, as well as fecal microflora was tested before, immediately and 2 weeks after intervention.Results:The fecal recovery of BB-12 increased significantly (P<0.001) with increasing dose. In the group receiving 1011 CFU/day BB-12 was recovered from 13 out of 15 volunteers. CRL-431 was not recovered in any of the fecal samples. Supplementation with probiotics did not change the fecal bacterial composition. A significant linear increase in fecal consistency (looser stool) with increasing probiotic dose (P=0.018) was observed. No overall dose–response effect was found on the blood lipids. High doses of probiotics were well tolerated.Conclusion:A dose-related recovery of BB-12 from feces was observed.Sponsorship:The study was sponsored by Chr. Hansen A/S, Hoersholm, Denmark.

Impact of diet on the intestinal microbiota in 10-month-old infants.

Objective: To investigate whether diet influences the composition of the intestinal microbiota in 10-month-old infants. Patients and Methods: Fecal samples were collected from sixty-five 10-month-old infants participating in a randomized 2 × 2 intervention study comparing cows milk (CM) with infant formula (IF) with or without fish oil (FO) supplement. Infants randomized to CM received a daily iron supplement. Bacterial DNA was extracted from the feces. Polymerase chain reaction was performed with primers targeting the V3 and V6–8 region of the 16S rRNA gene and analyzed by denaturing gradient gel electrophoresis (DGGE). Cluster analysis of the DGGE gels was performed by use of the Pearson correlation coefficient. Results: Samples from infants receiving CM clustered differently than did those from the IF group in the V3-based DGGE gels (P < 0.001) and showed a different distribution with or without FO in the CM group (P = 0.001) but not in the IF group (P = 0.39). Repeat analysis with the V6–8-based DGGE gels showed the same pattern, although the V3 gels had 2.5 times as many bands as the V6–8 gels. Conclusions: Consumption of CM or IF has a decisive influence on the composition of the intestinal microbiota. Supplementation with FO showed an effect on the microbiota only in the CM group. We speculate that these differences could be influenced by the intake of iron and n-3 polyunsaturated fatty acids, respectively.

Increased levels of regulatory T cells (Tregs) in human immunodeficiency virus-infected patients after 5 years of highly active anti-retroviral therapy may be due to increased thymic production of naive Tregs.

This study determines levels of regulatory T cells (Tregs), naive Tregs, immune activation and cytokine patterns in 15 adult human immunodeficiency virus (HIV)‐infected patients receiving prolonged highly active anti‐retroviral therapy (HAART) who have known thymic output, and explores if naive Tregs may represent recent thymic emigrant Tregs. HIV‐infected patients treated with HAART with a median of 1 and 5 years were compared with healthy controls. Percentages of Tregs (CD3+CD4+CD25+CD127low), naive Tregs (CD3+CD4+CD25+CD45RA+) and activation markers (CD38+human leucocyte antigen D‐related) were determined by flow cytometry. Forkhead box P3 mRNA expression and T cell receptor excision circles (TREC) content in CD4+ cells were determined by polymerase chain reaction and cytokines analysed with Luminex technology. Levels of Tregs were significantly higher in HIV‐infected patients compared with controls, both after 1 and 5 years of HAART (P < 0·001), despite fully suppressed HIV‐RNA and normalization of both CD4 counts, immune activation and cytokine patterns. Furthermore, levels of naive Tregs were elevated significantly in HIV‐infected patients (P < 0·001) and were associated with thymic output measured as the TREC frequency in CD4+ cells (P = 0·038). In summary, Treg levels in HIV‐infected patients are elevated even after 5 years of HAART. Increased thymic production of naive Tregs may contribute to higher Treg levels in HIV‐infection.

Regulatory T cells in human immunodeficiency virus-infected patients are elevated and independent of immunological and virological status, as well as initiation of highly active anti-retroviral therapy

Infection with human immunodeficiency virus (HIV) causes a dysregulation of the immune system. This is caused by HIV‐specific as well as non‐specific mechanisms and has not been explained fully. In particular, knowledge is lacking about the potential role of host‐mediated immunosuppressive mechanisms. During recent years it has become evident that a subpopulation of T cells [T regulatory (Tregs)] play a major role in sustaining tolerance to self‐antigens. To investigate the influence of initiation of highly active anti‐retroviral therapy (HAART) on the Treg level in HIV‐infected patients we have conducted a prospective study enrolling treatment‐naive HIV‐infected patients just prior to starting treatment with HAART, measuring levels of Tregs by flow cytometry and mRNA expression of forkhead box P3 (FoxP3) at weeks 0, 4, 12 and 24 of treatment. In this prospective study neither the percentage of CD4+CD25high+ nor the expression of FoxP3 changed significantly during 24 weeks of HAART. Furthermore, HIV patients have higher Tregs measured as percentages of CD4+CD25high+ cells paralleled by higher levels of FoxP3 compared with healthy controls. The elevated level of Tregs was found to be independent of both immunological and virological status, indicating that initiation of HAART has minor effects on the Treg level in HIV‐infected patients.

Highly active antiretroviral therapy normalizes the function of progenitor cells in human immunodeficiency virus-infected patients.

CD34 cells from human immunodeficiency virus (HIV)-infected persons have been described to be impaired in function. The effect of highly active antiretroviral treatment (HAART) on the function of CD34 cells in HIV-infected patients was examined. Numbers and function of CD34 cells from 11 HIV-infected patients were determined prior to HAART and after 2, 4, 8, and 12 weeks of therapy. The mean number of colony-forming units (cells) per milliliter (cfu/mL) was 15.0 prior to HAART vs. 109.8 in healthy controls (P<.001). During HAART, the number of cfu/mL increased to 100.3 (P<.001). This increase in cfu/mL eliminated the differences between HIV-infected patients and controls. Significant increases in numbers of CD34 cells were not detected. Of importance, the cloning efficiency of CD34 cells increased from 1.7% prior to therapy to a peak at 18.7% (P=.003). In conclusion, HAART normalized CD34 cell function in HIV-infected patients and thus might allow de novo production of T lymphocytes from progenitor cells.

Granulocyte Colony-Stimulating Factor Increases CD4+ T Cell Counts of Human Immunodeficiency Virus-Infected Patients Receiving Stable, Highly Active Antiretroviral Therapy: Results from a Randomized, Placebo-Controlled Trial

Thirty human immunodeficiency virus (HIV)-infected patients with CD4+ T cell counts <350 cells/mm3 who had received stable, highly active antiretroviral therapy (HAART) for at least 24 weeks were randomized to receive either placebo or granulocyte colony-stimulating factor (G-CSF; 0.3 mg/mL 3 times a week) for 12 weeks. Blood samples were collected at specified time points. G-CSF treatment enhanced the total lymphocyte count (P=.002) and increased CD3+ (P=.005), CD4+ (P=.03), and CD8+ (P=.004) T cell counts as well as numbers of CD3-CD16+CD56+ NK cells (P=.001). The increases in CD4+ and CD8+ cell counts resulted from increases in CD45RO+ memory T cells and cells expressing the CD38 activation marker. Lymphocyte proliferative responses to phytohemagglutinin and Candida antigen decreased, whereas NK cell activity and plasma HIV RNA did not change during G-CSF treatment. After 24 weeks, all immune parameters had returned to baseline values. This study suggests that G-CSF treatment of HIV-infected patients receiving stable HAART increases the concentration of CD4+, CD8+, and NK cells without inducing changes in the virus load.

Fever of unknown origin: A retrospective study of 52 cases with evaluation of the diagnostic utility of FDG-PET/CT

Abstract Objectives: Fever of unknown origin (FUO) is dynamic in its origin and will be an ongoing challenge to the clinician because of shifting disease epidemiology. Here we present a series of patients with classical FUO admitted to an infectious diseases department during a 5-y period, with an emphasis on the diagnostic utility of 18F-fluorodeoxyglucose positron emission tomography (PET)/computed tomography (CT) in present-day cases of FUO. Methods: Patient records were reviewed retrospectively. Results: A final diagnosis was achieved for 31 of the 52 cases (60%). The final diagnoses of these 31 cases and their distribution in the respective diagnostic categories were: infections 32% (10/31), non-infectious inflammatory disease 55% (17/31), and malignancy 13% (4/31). In our study PET/CT successfully identified an infectious, inflammatory, or neoplastic cause of fever in 10 of the 22 patients (45%) who underwent this scan. Conclusions: During the past decade the proportion of non-infectious inflammatory diseases in FUO series has increased. Based on our findings we recommend: (1) a PET/CT scan be performed early in the diagnostic work-up of patients with FUO, and (2) restraint in performing invasive procedures in patients with FUO in whom no cause of fever has been determined during diagnostic work-up.

The prevalence and clinical significance of intestinal parasites in HIV-infected patients in Denmark

Abstract To investigate the prevalence and clinical significance of intestinal parasites in human immunodeficiency virus (HIV)-infected patients, faecal specimens from 96 HIV-infected patients were submitted to microbiological analyses, including microscopy and polymerase chain reaction for protozoa and enteropathogenic bacteria. Results of microbiological analyses were compared with self-reported gastrointestinal complaints collected using a validated questionnaire. Thirty-two (33%) patients were positive for parasites. However, opportunistic parasites (Isospora and Cryptosporidium) were detected in only 2 instances. Entamoeba dispar was detected in 10 cases, 9 of which represented men who have sex with men (MSM). Despite generally low HIV RNA loads and high CD4+ T-cell counts, 42% of the 76 patients reporting symptoms complained of diarrhoea, 31% of whom were parasite-positive. The presence of diarrhoea was not associated with the presence or absence of parasites; neither was it associated with receiving highly active anti-retroviral therapy (HAART) in general, or protease inhibitors (PI) in particular. A CD4+ T-cell count <200 cells/mm3 was not associated with parasitic infection or with diarrhoea. The data show that diarrhoea is a common symptom among HIV-infected patients in Denmark, but do not indicate that the diarrhoea is due to intestinal parasites.

Expression of the activation antigen CD69 predicts functionality of in vitro expanded peripheral blood mononuclear cells (PBMC) from healthy donors and HIV-infected patients

Gene therapy for AIDS necessitates harvest and expansion of PBMC from HIV‐infected patients. We expanded PBMC from healthy blood donors and HIV‐infected patients for up to 14 days using four expansion protocols: 3 days of phytohaemagglutinin (PHA) stimulation, continuous PHA stimulation, 3 days of stimulation with anti‐CD3 and anti‐CD28, and continuous stimulation with anti‐CD3 and anti‐CD28. Functionality of PBMC was evaluated prior to and after expansion using standard proliferation assay. Phenotype and lymphocyte subset activation defined by expression of CD69 and CD25 were determined using flow cytometry. PBMC from healthy donors and HIV‐infected patients were readily expanded. The best expansion was obtained using stimulation for 3 days. After expansion, functionality of PBMC measured as proliferative response was partly conserved. PBMC expanded with stimulation for 3 days exhibited more preserved functionality than PBMC stimulated continuously (P < 0.03). The mean proliferative response in each of the four different expansion protocols correlated with the mean values of CD69 expression. The proliferative responses from patients and healthy donors expanded with PHA stimulation for 3 days correlated with CD69 expression on CD4 cells (r = 0.68, P < 0.01) and on CD8 cells (r = 0.59, P < 0.03). Furthermore, expression of CD69 reliably predicted which patients and donors had highly conserved functionality after in vitro expansion. Finally, PBMC expanded with PHA stimulation for 3 days were examined for apoptosis. Only a minor fraction was primed for apoptosis, and this fraction could be significantly reduced by addition of IL‐2 to the culture medium (P < 0.05). In conclusion, the feasibility of expanding PBMC from HIV patients was demonstrated. Expanded PBMC had conserved functionality. Finally, after in vitro expansion, expression of the activation antigen CD69 reliably predicted functionality of PBMC.