The Effect of Long-Term Treatment with Granulocyte Colony ...

Scand. J. Immunol. 52, 298±303, 2000

The Effect of Long-Term Treatment with Granulocyte ColonyStimulating Factor on Hematopoiesis in HIV-Infected Individuals S. D. NIELSEN,* T. U. SéRENSEN,* H. ALADDIN,² A. K. ERSBéLL,³ L. MATHIESEN,* H. ULLUM,² J. GERSTOFT,² J. O. NIELSEN* & B. K. PEDERSEN² *Department of Infectious Diseases, 144, Hvidovre Hospital, 2650 Hvidovre, ²Department of Infectious Diseases, Rigshospitalet, 2200 Copenhagen N, ³Department of Animal Science and Animal Health, Royal Veterinary and Agricultural University, 1870 Frederiksberg C, Denmark (Received 7 February 2000; Accepted in revised form 30 April 2000)

Nielsen SD, Sùrensen TU, Aladdin H, Ersbùll AK, Mathiesen L, Ullum H, Gerstoft J, Nielsen JO, Pedersen BK. The Effect of Long-Term Treatment with Granulocyte Colony-Stimulating Factor on Hematopoiesis in HIV-Infected Individuals. Scand J Immunol 2000;52:298±303 This randomized, placebo-controlled trial examine the long-term effect of granulocyte colony-stimulating factor (G-CSF) on absolute numbers of CD34‡ progenitor cells and progenitor cell function in human immunode®ciency virus (HIV)-infected patients. G-CSF (300 mg ®lgrastim) or placebo was given three times weekly for 12 weeks to 30 HIV-infected patients that had been treated with HAART for at least 24 weeks and not yet achieved CD4 counts above 350 CD4‡ cells/ml. Blood samples were collected at weeks 0, 2, 4, 8, and 12, and again 12 weeks after termination of the G-CSF treatment. Signi®cant increase in absolute numbers of circulating CD34‡ cells was detected in the treatment group (P ˆ 0.006). The function of progenitor cells was examined in vitro using a colony-forming unit (CFU) assay, and increase in the number of CFU/ml was detected (P ˆ 0.005). In order to estimate the effect of G-CSF on in vivo function of progenitors the whiteblood count was determined. Signi®cant increase in white-blood count was found (P < 0.001), while hemoglobin and platelet count decreased (P ˆ 0.001 and P ˆ 0.013, respectively). Signi®cant increase in the CD4 count occurred, but correlation between the numbers of progenitors and the CD4 count was not found. These data suggest that G-CSF mainly increases the number and differentiation of myeloid progenitors. Dr S. D. Nielsen, Department of Infectious Diseases, 144, Hvidovre Hospital, 2650 Hvidovre, Denmark. E-mail: [email protected]

INTRODUCTION The hematopoietic growth factor G-CSF is used in the clinical management of HIV-infected patients with neutropenia to increase the absolute neutrophil count [1±4]. Furthermore, several studies have demonstrated that hematopoietic progenitor cells (CD34‡ cells) can be mobilized by short-term G-CSF treatment in HIV-infected patients [5±11]. In those studies HIV-infected patients were treated with G-CSF for 3 to 6 days and the absolute number of CD34‡ cells increased by two to 37fold [8±11]. Interestingly, the CD4 count in HIV-infected patients treated with G-CSF increase as well [1±3, 9, 11±13], and in one study an increase in the number of naive CD4‡ cells was found [9]. In addition, short-term G-CSF administration may partially restore the in vitro interleukin (IL)-2 production and lymphocyte proliferative response in blood from HIV-infected q 2000 Blackwell Science Ltd

patients [1, 14], although one study has shown a decrease in these functions [12]. New antiretroviral therapies combining inhibitors of HIV-1 protease and reverse transcriptase (highly active antiretroviral therapy, HAART) are very ef®cient at reducing viral replication in HIV-infected patients [15±19]. Furthermore, HIV-infected patients treated with HAART achieve increase in CD4 count and improvement in immune functions, but immune reconstitution is often not complete. Thus, most HIV-infected patients receiving HAART only achieve increase in their CD4 count in the magnitude of 100±200 cells/ml, and the naive CD4‡ cells increase slowly [15, 17, 18, 20, 21]. A decrease in number or function of hematopoietic progenitor cells might contribute to CD4‡ T-cell depletion in HIV infection [22, 23]. We have previously demonstrated that short-term G-CSF treatment increases the number of both CD34‡ and CD4‡ cells in HIV-infected

G-CSF and Hematopoiesis individuals [9]. In addition, we recently found that administration of G-CSF for 12 weeks increased the number of CD4‡ cells [12]. In the present study, we examine the possible mechanisms underlying the long-term effect of G-CSF on T cells. The effects of G-CSF on CD34‡-progenitor cells and progenitor-cell function as well as the possible relationship between progenitor-cell function and the number of T cells are examined. PATIENTS AND METHODS Patients and study design. The study is a randomized, placebo-controlled trial, and G-CSF or placebo was given to HIV-infected patients that had been treated with HAART for at least 24 weeks and not yet achieved CD4 counts above 350 CD4‡ cells/ml. A total of 30 HIV-infected patients (26 men, 4 women) were included in this study [12]. Entry data are given in Table 1. Patients had received HAART for a median of 31 months (range 20±34 months). Each patient received subcutaneous

injections with either G-CSF (300 mg ®lgrastim, Amgen, Thousand Oaks, CA, USA) or placebo three times a week for 12 weeks. Three patients in the G-CSF group were noncompliant owing to the discomfort with injecting themselves or side-effects (bone pain or ¯u-like symptoms) and were withdrawn from the study. The patients were examined and blood samples were collected at enrolment (week 0), and again on weeks 4, 12, and 24. Blood collected in tubes containing EDTA was used to obtain a full blood count and for ¯ow cytometry. Additional blood samples were drawn into tubes containing heparin to obtain peripheral blood mononuclear cells (PBMC) by means of density-gradient centrifugation. The study was approved by the Danish Board of Health and the local ethical committee. Informed consent was obtained from all patients after the nature and consequences of the study had been fully explained. Flow cytometry. Flow cytometric analyses were performed essentially as described [12, 24]. Brie¯y, 100 ml of blood was incubated with 10 ml of ¯uorescence-conjugated monoclonal antibodies (MoAbs) at room temperature for 15 min. Erythrocytes were lysed with 2 ml FACS Lysing solution (Becton Dickinson Immunocytometry Systems (BD), San Jose,

Table 1. Entry data of the HIV-positive patients 27 in this randomized, double-blind and placebocontrolled study of the effect of G-CSF Patient no. G-CSF treatment group G-CSF 1 G-CSF 2 G-CSF 3 G-CSF 4 G-CSF 5 G-CSF 6 G-CSF 7 G-CSF 8 G-CSF 9 G-CSF 10 G-CSF 11 G-CSF 12 mean, G-CSF Placebo group placebo 1 placebo 2 placebo 3 placebo 4 placebo 5 placebo 6 placebo 7 placebo 8 placebo 9 placebo 10 placebo 11 placebo 12 placebo 13 placebo 14 placebo 15 mean, placebo

299

CD4 count cells/ml

HIV RNA log10 copies/ml

311 212 240 233 216 347 273 90 344 234 54 288 237

2.38 1.30 1.30 3.71 4.88 2.94 1.30 1.30 4.10 1.79 1.34 1.30 2.31

ritonavir, AZT, 3TC indinavir, AZT, 3TC indinavir, AZT, 3TC ritonavir, AZT, 3TC indinavir, 3TC, D4T indinavir, 3TC, D4T ritonavir, 3TC, D4T ritonavir, AZT, 3TC indinavir, 3TC, D4T ritonavir, saquinavir, D4T, ddI ritonavir, 3TC, D4T indinavir, AZT, 3TC

150 281 218 261 251 293 217 331 261 213 180 90 234 170 132 219

1.30 1.30 3.50 1.36 1.30 1.72 1.43 2.20 1.30 1.30 2.05 1.63 1.30 1.30 1.30 1.62

nel®navir, AZT, 3TC ritonavir, AZT, 3TC ritonavir, saquinavir, 3TC, D4T ritonavir, AZT, 3TC indinavir, AZT, 3TC ritonavir, AZT, 3TC indinavir, 3TC, D4T indinavir, AZT, 3TC indinavir, AZT, 3TC indinavir, AZT, 3TC indinavir, 3TC, D4T indinavir, 3TC, D4T indinavir, 3TC, D4T indinavir, 3TC, D4T nevirapine, AZT, 3TC

Antiretroviral treatment

Abbreviations: AZT, zidovudine; D4T, stavudine; ddI, didanosine; 3TC, lamivudine. q 2000 Blackwell Science Ltd, Scandinavian Journal of Immunology, 52, 298±303

300 S. D. Nielsen et al. CA, USA) at room temperature for 10 min, and the samples were washed and resuspended in phosphate-buffered saline (PBS). Samples were analyzed using either a FACScan (BD) or an Epics XL-MCL (Coulter, FL, USA). To determine the percentage of CD34‡ cells in peripheral blood, the ¯uorescence of 75 000 cells was measured (CD34 and isotype controls) using a CD45 gate. The fraction of cells expressing CD34 was multiplied by the white-blood count to determine the absolute number of CD34‡ cells in peripheral blood. To determine the percentage of CD34‡ cells coexpressing CD7, 200±2000 cells were analyzed using a CD34 gate (SSC versus CD34). MoAbs used were isotype control g1-¯uorescein isothiocyanate (FITC)/g1-phycoerythrin (PE), CD34-PE (anti-HPCA-2), CD7-FITC (Leu-9), and CD45-FITC (anti-HLe) from BD. To determine the number of T cells, including the number of naive T cells and the number of T cells expressing activation markers, the ¯uorescence of 5000 cells was measured. To obtain the absolute number of a lymphocyte population, the fraction of cells in a lymphocyte gate expressing lymphocyte markers was multiplied by the lymphocyte count. MoAbs used to determine lymphocytes were isotype controls g1-FITC/g1-PE/g1carcocyanine-5 (Cy5, clone 679.1Mc7; Immunotech, Marseille, France), CD3-Cy5 (clone UCHT1; Dako, Glostrup, Denmark), CD4-PE (clone SFCI2T4D11, Immunotech), CD8-Cy5 (clone DK25, Dako), CD45RAPE (clone 4KB5, Dako), CD45RO-PE (clone UCHL1, Dako), CD62LFITC (clone DREG56, Immunotech), CD25-FITC (clone 2A3, BD), and CD69-PE (Leu-23, BD). Colony assays for progenitor cells. Colony forming cells were grown in methylcellulose medium (Stem Cell CFU Kit; Baxter Healthcare Corporation, Deer®eld, IL, USA) according to the manufacturer's instructions. Brie¯y, 2 ´ 106 PBMC were resuspended in 1 ml dilution medium and mixed with 3 ml CFU-culture medium to allow plating at a concentration of 5 ´ 105 PBMC/ml. Stem-cell factor 100 ng/ml was added (Genzyme, Cambridge, MA, USA), and the cell/methylcellulose suspension was aliqouted in triplicates of 1 ml in 35 mm culture plates (Nunc, Roskilde, Denmark). The plates were incubated for 14 days in a humidi®ed incubator at 37 8C and 5% CO2. Then colony forming unit (CFU)-GM colonies were counted using an inverted microscope (Olympus, Hamburg, Germany). Only colonies of > 50 cells were counted. Thus, the median number of colonies counted in a triplicate was the number of CFU per 5 ´ 105 PBMC. The number of CFU/ml peripheral blood was calculated using the equation: CFU/ml ˆ 2 ´ (CFU/ 5 ´ 105 PBMC) ´ (number of mononuclear cells (monocytes and lymphocytes) ´ 106/ml peripheral blood). Statistical methods. All data points are means (6 standard error of the mean (SEM)). When varying variance across the weeks occurred, logarithmic transformation of the measurements was done prior to further statistical analyses. Pairwise comparisons of differences between the two groups (G-CSF treatment versus placebo) were evaluated using a t-test. At baseline there were no differences between the two groups in any parameter measured. Differences across weeks within the treatment group were analyzed using a repeated measurement analysis. When signi®cant differences between weeks had been identi®ed a pairwise comparison of weeks was performed using a t-test. A 5% signi®cance level was used. Correlation between measurements was calculated using Pearson's correlation coef®cient.

(0.95 CD34‡ cells/ml 6 0.29 week 0 versus 4.94 6 1.3 week 4 and 3.63 6 0.97 week 12, P ˆ 0.006, Fig. 1). The function of progenitor cells was examined in vitro using a CFU assay, and the number of CFU per 5 ´ 105 PBMC increased from 32.5 (6 9.2) to a peak at 78.5 (6 12.5) after 4 weeks of treatment (P ˆ 0.002). Thus, the increase in circulating CD34‡ cells was paralleled by an increase in the number of CFU/ml from 123.6 (6 38.0) at week 0 to a peak of 399.4 (6 102.7) after 4 weeks of treatment (P ˆ 0.005, Fig. 1). Both the number of CD34‡ cells and CFU/ml had returned to baseline 12 weeks after termination of G-CSF treatment. The cloning ef®ciency of CD34‡ cells (i.e. (CFU/ml)/(number of CD34 ‡ cells/ml) ´ (100) was unchanged throughout the study period. Effect of long-term G-CSF treatment on hematopoiesis G-CSF is known to increase the number of mature progenitors primed for myelopoiesis. To estimate the effect of G-CSF on in vivo function of progenitors, the white-blood count was determined. Signi®cant increase in white-blood count occurred during G-CSF treatment (P < 0.001, Fig. 2) and was primarily owing to an increase in granulocytes [12]. The number of monocytes seemed to increase as well although this did not reach statistical signi®cance (P ˆ 0.087, Fig. 2). Generation of CFU in vitro is thought to re¯ect the function of myeloid progenitors. As expected, signi®cant correlation was found between the number of CFU/ml and the number of granulocytes (r ˆ 0.45, P < 0.001) and between the number of CFU/ml and the number of monocytes (r ˆ 0.55, P < 0.001). In the treatment group, signi®cant changes occurred in both hemoglobin and platelet count. The mean red blood cell count was 8.5 mmol/l 6 0.2 week 0 and the nadir was 8.0 mmol/l 6 0.3 after 8 weeks of G-CSF treatment (P < 0.001). The decrease in

RESULTS Effect of long-term G-CSF treatment on numbers of progenitor cells During the study period signi®cant increase in absolute numbers of circulating CD34‡ cells was detected in the treatment group

Fig. 1. The number of circulating progenitors in HIV-infected patients treated with either G-CSF (V) or placebo (B) three times weekly for 12 weeks. (A) shows the number of CD34‡ cells/ml peripheral blood, and (B) shows the calculated numbers of CFU/ml of peripheral blood. All results are means (6 SEM).

q 2000 Blackwell Science Ltd, Scandinavian Journal of Immunology, 52, 298±303

G-CSF and Hematopoiesis

Fig. 2. The white-blood count (A) and the number of monocytes (B) in HIV-infected patients treated with either G-CSF (V) or placebo (B) three times weekly for 12 weeks. All results are means (6 SEM).

hemoglobin lasted throughout the treatment period, but the hemoglobin returned to the baseline at week 24. In contrast, the effect of G-CSF on platelet count was only transient. A signi®cant decrease in the platelet count was found from week 0 to week 2 (227 ´ 109/l 6 13 versus 207 ´ 109/l 6 15, P ˆ 0.013), and after week 2 the platelet count returned to the baseline. Signi®cant correlation was not found between either platelet count or hemoglobin and number of circulating progenitors (r ˆ 0.04 and r ˆ 0.15, respectively). Effect of long-term G-CSF treatment on lymphopoiesis Previous studies have shown increase in the CD4 count in HIVinfected patients treated with G-CSF for short periods of time [1±3, 9, 11±13]. However, G-CSF has also been shown to diminish the capacity of progenitors for generation of T cells [24], and the

301

effect of long-term G-CSF treatment on lymphocyte counts was therefore determined. As reported previously, signi®cant increases in total lymphocytes, CD4‡ cells and CD8‡ cells were detected [12]. Both memory and naive CD4‡ cells seemed to increase, however, signi®cant increase was found for memory CD4‡ cells only (P ˆ 0.040, Fig. 3), and all lymphocyte counts had returned to the baseline at week 24. To determine if the increase in CD4‡ cells was owing to the increase in numbers of progenitor cells, the number of circulating progenitors available for T-cell generation in the thymus, i.e. CD34‡ cells coexpressing of CD7 was determined. There were no signi®cant changes in the fraction or the absolute number of CD34‡ CD7‡ cells during G-CSF treatment. A weak correlation was found between CFU/ml and the CD8 count (r ˆ 0.27, P ˆ 0.014). In contrast, signi®cant correlations between the CD4 count, the naive CD4 count, the naive CD8 count and numbers of circulating CD34‡ cells or CFU/ml were not found. These data indicate that the total number of circulating progenitors does not re¯ect the number of progenitors with capacity to develop into mature lymphocytes in HIV-infected patients treated with G-CSF. Finally, to determine if G-CSF-enhanced proliferation of mature CD4‡ and CD8‡ cells in vivo, the percentage and absolute numbers of CD4‡ and CD8‡ cells coexpressing the activation markers CD25 and CD69 were determined. Signi®cant changes in these parameters were not detected. Furthermore, there were no signi®cant changes in viral load during the study period [12]. DISCUSSION This randomized, placebo-controlled trial comparing G-CSF and placebo demonstrates that long-term treatment with G-CSF increases the numbers of circulating progenitor cells. The in vitro function of progenitor cells remains unchanged. G-CSF treatment results in an increase in white-blood count mainly owing to an increase in granulocytes. Furthermore, G-CSF treatment results in an increase in the CD4 count. However, correlation between the number of circulating progenitors and the CD4 counts was not found.

Fig. 3. The CD4 count in HIV-infected patients treated with either G-CSF (left side of diagram) or placebo (right side of diagram) three times weekly for 12 weeks. The mean number of naive CD4‡ CD45RA‡ CD62L‡ cells is represented by the white boxes and the mean number of memory CD4‡ cells is represented by the hatched boxes. Further information about the changes in lymphocyte counts during G-CSF can be found in [12]. q 2000 Blackwell Science Ltd, Scandinavian Journal of Immunology, 52, 298±303

302 S. D. Nielsen et al. Neutropenia is common in patients with advanced HIV infection. The cause vary, but neutropenia is often caused by the use of myelotoxic agents directed against HIV, myelosuppressive chemotherapy, or radiation therapy [1, 2, 4, 25]. G-CSF is known to increase the absolute neutrophil count by stimulating proliferation and differentiation of neutrophil precursors including CD34‡ progenitor cells [25±27]. This property of G-CSF has made it popular in the clinical management of HIV-infected patients with neutropenia, and it has been shown that administration of G-CSF prevents neutropenia and reduces infective morbidity in patients with advanced HIV infection [25, 28±30]. G-CSF has been used for several years for mobilization of progenitors in stem-cell transplantations. With the recent efforts to develop gene therapy for the treatment of AIDS, G-CSF has been used to mobilize progenitors in HIV-infected patients [5, 7±11]. A concern with the use of G-CSF is that mobilized progenitors may only contain mature progenitors committed to myelopoiesis. Studies of short-term administration of G-CSF in healthy adults as well as in HIV-infected persons have shown that G-CSF mainly mobilize mature progenitors, but small numbers of immature CD34‡ cells are mobilized as well [24, 31±33]. Furthermore, the capacity for T-cell generation in G-CSF mobilized progenitors is decreased [24]. In the present study, long-term administration of G-CSF allowed an analysis of the in vivo effect of G-CSF on progenitor cell function. As expected, there was an increase in absolute neutrophil count and numbers of monocytes tended to increase as well. The platelet count and red blood cell count decreased. Furthermore, increase in numbers of memory CD4‡ and CD8‡ cells was found with no change in numbers of naive CD4‡ cells [12]. All blood counts had returned to the baseline when monitored 12 weeks after termination of GCSF treatment. These data suggest that long-term administration of G-CSF increases numbers and differentiation of myeloid progenitors while numbers of other lineage speci®c progenitors may decrease or remain unchanged. Previous studies have shown increase in the CD4 count in HIVinfected patients following short-term G-CSF treatment [1±3, 9], and one study showed increase in the naive CD4 count [9]. The mechanism behind the increase in CD4 count is still obscure. The lack of correlation between the CD4 count and the numbers of circulating progenitors suggests that the total number of circulating progenitors does not re¯ect the number of T-cell progenitors which is in agreement with the previous ®nding that G-CSF does not mobilize T-cell progenitors [24]. Furthermore, previous studies do not provide evidence for peripheral proliferation of CD4‡ cells owing to G-CSF treatment [9, 12]. The effect of GCSF treatment on apoptosis was not examined in this study, and it may be possible that G-CSF treatment reduced apoptosis in CD4‡ cells. Thus, it cannot be ruled out that the initial increase in CD4 count is owing to enhanced proliferation and differentiation of T-cell progenitors in the thymus resulting in release of naive CD4‡ cells. However, the long-term effect of G-CSF on the number of memory CD4‡ cells is more likely owing to redistribution of cells from the lymphatics or a combination of all three mechanisms. All lymphocyte counts had returned to the

baseline 12 weeks after termination of the G-CSF treatment. These ®ndings question the likelihood that the G-CSF treatment will have clinical bene®ts in non-neutropenic HIV-infected patients. In conclusion, this randomized, placebo-controlled study of the effect of long-term G-CSF treatment in HIV-infected patients has shown an increase in the number of circulating progenitors and increase in the white-blood count. An increase in the CD4‡ count was detected while hemoglobin and the platelet count decreased. All cell counts had returned to the baseline 12 weeks after termination of G-CSF treatment. These data suggest that GCSF mobilize progenitors that are committed for myeloid differentiation while other lineage-speci®c progenitors may decrease or remain unchanged. ACKNOWLEDGMENTS We gratefully acknowledge the patients who participated in the study and nurses Lisbet Skinnes and Astrid Dilling for selecting study patients. This work was ®nancially supported by the 17± 12±1981 Foundation, the John and Birthe Meyer Foundation, the Danish AIDS Foundation, and Amgen. REFERENCES 1 Miles SA, Mitsuyasu RT, Moreno J et al. Combined therapy with recombinant granulocyte colony-stimulating factor and erythropoietin decreases hematologic toxicity from zidovudine. Blood 1991; 77:2109±17. 2 Perrella O, Finelli E, Perrella A, Tartaglia G, Scognamiglio P, Scalera G. Combined therapy with zidovudine, recombinant granulocyte colony stimulating factors and erythropoitin in asymptomatic HIV patients. J Chemother 1996;8:63±6. 3 Stricker RB, Goldberg B. Increase in lymphocyte subsets following treatment of HIV-associated neutropenia with granulocyte colonystimulating factor. Clin Immunol Immunopathol 1996;79:194±6. 4 van der Wouv PA, van Leeuwen R, van Oers RHJ, Lange JMA, Danner SA. Effects of recombinant human granulocyte colonystimulating factor on leucopenia in zidovudine-treated patients with AIDS and AIDS related complex, a phase I/II study. Br J Haematol 1991;78:319±24. 5 Bauer G, Valdez P, Kearns K et al. Inhibition of human immunode®ciency virus-1 (HIV-1) replication after transduction of granulocyte colony-stimulating factor-mobilized CD34‡ cells from HIV1-infected donors using retroviral vectors containing anti-HIV-1 genes. Blood 1997;89:2259±67. 6 Gervaix A, Schwartz L, Law P et al. Gene therapy targeting peripheral blood CD34‡ hematopoietic stem cells of HIV-infected individuals. Hum Gene Ther 1997;8:2229±38. 7 Junker U, Moon JJ, Kalfoglou CS et al. Hematopoietic potential and retroviral transduction of CD34‡Thy-1‡ peripheral blood stem cells from asymptomatic human immunode®ciency virus type-1-infected individuals mobilized with granulocyte colony-stimulating factor. Blood 1997;89:4299±306. 8 Nielsen SD, Dam-Larsen S, Nielsen C, Wantzin P, Mathiesen L, Hansen JES. Recombinant human granulocyte colony-stimulating factor increases circulating CD34-positive cells in patients with AIDS. Ann Hematol 1997;74:215±20.

q 2000 Blackwell Science Ltd, Scandinavian Journal of Immunology, 52, 298±303

G-CSF and Hematopoiesis 9 Nielsen SD, Afzelius P, Dam-Larsen S et al. Effect of granulocyte colony-stimulating factor (G-CSF) in human immunode®ciency virus-infected patients: Increase in numbers of naive CD4 cells and CD34 cells makes G-CSF a candidate for use in gene therapy or to support antiretroviral therapy. J Infect Dis 1998;177:1733±6. 10 Slobod KS, Bennett TA, Freiden P et al. Mobilization of CD34‡ progenitor cells by granulocyte colony-stimulating factor in human immunode®ciency virus type 1-infected adults. Blood 1996; 88:3329±35. 11 Law P, Lane TA, Gervaix A et al. Mobilization of peripheral blood progenitor cells for human immunode®ciency virus-infected individuals. Exp Hematol 1999;27:147±54. 12 Aladdin H, Ullum H, Nielsen SD et al. G-CSF increases CD4‡ Tcell counts in HIV-infected patients on stable HAART. Results from a randomized, placebo-controlled trial. J Infect Dis 2000;181:1148±52. 13 Aladdin H, Ullum H, Katzenstein T, Gerstoft J, Skinhùj P, Pedersen BK. Immunological and virological changes in antiretroviral naive HIV infected patients randomised to G-CSF or placebo simultaneously with initiation of HAART, Scand J Immunol 2000;51:520±525. 14 Hartung T, Pitrak DL, Foote M, Shatzen EM, Verral SC, Wendel A. Filgrastim restores interleukin-2 production in blod from patients with advanced human immunode®ciency virus infection. J Infect Dis 1998;178:686±92. 15 Autran B, Carcelain G, Li TS et al. Positive effect of combined antiretroviral therapy on CD4‡ T cell homeostasis and function in advanced HIV disease. Science 1997;277:112±6. 16 Bisset LR, Cone RW, Huber W et al. Highly active antiretroviral therapy during early HIV infection reverses T-cell activation and maturation abnormalities. AIDS 1998;12:2115±23. 17 Giorgi JV, Majchrowicz MA, Johnson TD, Hultin P, Matud J, Detels R. Immunological effects of combined protease inhibitor and reverse transcriptase inhibitor therapy in previously treated chronic HIV-1 infection. AIDS 1998;12:1833±44. 18 Pakker NG, Notermans DW, De Boer RJ et al. Biphsic kinetics of peripheral blood T cells after triple combination therapy in HIV-1 infection: a compositie of redistribution and proliferation. Nat Med 1998;4:208±14. 19 Wei X, Ghosh SK, Taylor ME et al. Viral dynamics in human immunode®ciency virus type 1 infection. Nature 1995;373:117±22. 20 Connors M, Kovacs JA, Krevat S et al. HIV infection induces changes in CD4‡ T-cell phenotype and depletions within the CD4‡ T-cell repertoire that are not immediately restored by antiviral or immune-based therapies. Nat Med 1997;3:533±40. 21 Notermans DW, Pakker NG, Hamann D et al. Immune reconstitution

22

23

24

25

26

27

28

29 30 31

32

33

q 2000 Blackwell Science Ltd, Scandinavian Journal of Immunology, 52, 298±303

303

after 2 years of successful potent antiretroviral therapy in previously untreated human immunode®ciency virus type 1-infected adults. J Infect Dis 1999;180:1050±6. Clark DR, Repping S, Pakker NG et al. T cell progenitor function during progressive HIV-1 infection and after anti-retroviral therapy, Blood 2000;96:242±249. Nielsen SD, Ersbùll AK, Mathiesen L, Nielsen JO, Hansen JES. Highly active antiretroviral therapy HAART normalizes the function of progenitor cells in human immunode®ciency virus HIV-infected patients. J Infect Dis 1998;178:1299±305. Nielsen SD, Clark DR, Hutchings M et al. Treatment with granulocyte-colony stimulating factor decreases the capacity of hematopoietic progenitor cells for generation of lymphocytes in human immunode®ciency virus-infected persons. J Infect Dis 1999;180: 1819±26. Kuritzkes DR, Parenti D, Ward DJ et al. Filgrastim prevents severe neutropenia and reduces infective morbidity in patients with advanced HIV infection: results of a randomized, multicenter, controlled trial. AIDS 1998;12:65±74. Anderlini P, Przepiorka D, Champlin R, KoÈrbling M. Biologic and clinical effects of granulocyte colony-stimulating factor in normal individuals. Blood 1996;88:2819±25. DuÈhrsen U, Villeval JL, Boyd J, Kannourakis G, Morstyn G, Metcalf D. Effects of recombinant human granulocyte colony-stimulating factor on hematopoietic progenitor cells in cancer patients. Blood 1988;72:2074±81. Ambati BK, Perlman DC, Salomomn N. Outcomes of granulocyte colony-stimulating factor or granulocyte-macrophage colonystimulating factor use in neutropenic patients infected with human immunode®ciency virus. Int J Infect Dis 1998±99;3:70±5. Hartung T. Granulocyte colony-stimulating factor: its potential role in infectious disease. AIDS 1999;13 (Suppl. 2):S3±9. Mitsuyasu R. Prevention of bacterial infections in patients with advanced HIV infection. AIDS 1999;13 (Suppl. 2):S19±23. Fujisaki T, Otsuka T, Harada M, Ohno Y, Niho Y. Granulocyte colony-stimulating factor mobilizes primitive hematopoietic stem cells in normal individuals. Bone Marrow Transplant 1995;16:57±62. Lozano ML, Ortuno F, de Arriba F et al. Effect of rhG-CSF on the mobilization of CD38 and HLA-DR subfractions of CD34‡ peripheral blood progenitor cells. Ann Hematol 1995;71:105±10. Steen R, Tjùnnfjord GE, Grùseth LAG, Heldal D, Egeland T. Ef¯ux of CD34‡ cells from bone marrow to peripheral blood is selective in steady-state hematopoiesis and during G-CSF administration. J Hematother 1997;6:563±73.