Biomedical subjects

S D Nielsen

Publications and source records attributed to S D Nielsen.

At least 19 recordsLinked to original sources

PubMed2003

Thymic size in preterm neonates: a sonographic study.

AIM: To assess the variation in size of the thymus in vivo in preterm neonates and to identify relations between thymic size and gestational age (GA), birthweight, occurrence of postnatal infections and maternal alcohol and tobacco intake during pregnancy. METHODS: Eighty preterm neonates with a GA between 24 and 36 wk, and a birthweight between 490 and 4110 g were examined between days 0 and 19 after birth. The thymic size was assessed by sonography as a volume estimate, the so-called thymic index (Ti). The median Ti was 5.2 (1.2-17.9). Ti was positively correlated with birthweight and GA and negatively correlated with occurrence of postnatal infection (p < 0.01, p = 0.03, p = 0.05, R2 = 0.68). A correlation between thymic size and maternal alcohol and tobacco intake was not demonstrated. CONCLUSION: It is possible to assess the size of the thymus by sonography in very low-birthweight and preterm neonates. A normal range for Ti in preterm neonates has been established. The sonographic method is a safe and effective technique for measuring the size of the thymus in preterm infants.

Alcohol Drinking↗

PubMed2001

Impaired progenitor cell function in HIV-negative infants of HIV-positive mothers results in decreased thymic output and low CD4 counts.

Hematologic and immunologic functions were examined in 19 HIV-negative infants of HIV-positive mothers and 19 control infants of HIV-negative mothers. Control infants were selected to match for gestational age, weight, and mode of delivery. Cord blood was obtained from all infants and used for flow cytometric determination of lymphocyte subsets, including the naive CD4 count. Furthermore, to determine thymic output, cord blood mononuclear cells were used for determination of T-cell receptor excision circles (TRECs). Evaluation of progenitor cell function was done by means of colony-forming cell assay and fetal thymic organ cultures (FTOCs). Lower naive CD4 counts (459.3 +/- 68.9 vs 1128.9 +/- 146.8 cells/microL, P <.001) and reduced thymic output in infants of HIV-positive mothers were found (frequency of CD4(+) cells with TRECs was 3.6% +/- 0.7% compared with 14.3% +/- 2.2% in controls, P <.001). In combination with lower red blood cell counts in infants of HIV-positive mothers, this finding suggested impairment of progenitor cell function. Indeed, progenitors from infants of HIV-positive mothers had decreased cloning efficiency (15.7% +/- 2.6% vs 55.8% +/- 15.9%, P =.009) and seemed to generate fewer T cells in FTOCs. In conclusion, lower numbers of naive CD4(+) cells and reduced thymic output in HIV-negative infants of HIV-positive mothers may be due to impaired progenitor cell function.

Anti-HIV Agents↗

PubMed2000

[Treatment of HIV infections with antiretroviral drugs and recombinant interleukin-2].

The clinical effect of combination antiretroviral therapy against HIV-infection is indisputable, but the current treatment does not produce complete immuno-restitution. Many HIV-positive patients change treatment, because of side effects and virological failure. Owing to the limited number of treatment combinations, supplementary treatment is greatly needed. Intermittent subcutaneous rIL-2 treatment plus antiretroviral combination therapy results in a selective and long-lasting induction of CD4+ cells in 70-80% of HIV-patients and lowers the amount of replication competent virus in blood and lymph nodes. The expanded cell population consists of both naive cells and memory cells with the ability to respond to antigenic stimulation. It is not known whether the rise in the number of CD4+ cells reflects a better clinical outcome. This question is currently under investigation in two global phase III trials, namely the SILCAAT and the ESPRIT studies.

Anti-HIV Agents↗

PubMed2000

The effect of long-term treatment with granulocyte colony-stimulating factor on hematopoiesis in HIV-infected individuals.

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 immunodeficiency virus (HIV)-infected patients. G-CSF (300 microg filgrastim) 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/microl. Blood samples were collected at weeks 0, 2, 4, 8, and 12, and again 12 weeks after termination of the G-CSF treatment. Significant increase in absolute numbers of circulating CD34+ cells was detected in the treatment group (P = 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 = 0.005). In order to estimate the effect of G-CSF on in vivo function of progenitors the white-blood count was determined. Significant increase in white-blood count was found (P < 0.001), while hemoglobin and platelet count decreased (P = 0.001 and P = 0.013, respectively). Significant 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.

Anti-HIV Agents↗

PubMed2000

Decrease in immune activation in HIV-infected patients treated with highly active antiretroviral therapy correlates with the function of hematopoietic progenitor cells and the number of naive CD4+ cells.

This study was conducted to determine the impact of immune activation, cytokine production and apoptosis on the naive CD4+ cell count and the function of hematopoietic progenitor cells during the initial phase of highly active antiretroviral therapy (HAART). Blood samples from 11 HIV-infected patients were collected prior to HAART and after 4 and 12 weeks of therapy. Flow cytometry was used to determine the naive CD4+ count and activated T cells. The cloning efficiency of progenitor cells was determined using a colony-forming cells assay. Finally, apoptosis and cytokine production were determined. During the study period, the naive CD4+ count and the cloning efficiency increased significantly. Immune activation was found in HIV-infected patients and decreased during HAART. The level of immune activation correlated negatively with both the naive CD4+ count and the function of progenitor cells. A negative correlation was found between apoptosis and the naive CD4+ count. Alterations in cytokine production during HAART or correlation between cytokine production and the naive CD4+ count or the cloning efficiency of progenitor cells were not detected. In conclusion, immune activation in HIV-infected patients treated with HAART is inversely correlated with the function of progenitor cells and the naive CD4+ count.

Anti-HIV Agents↗

PubMed2000

FLT3 ligand preserves the uncommitted CD34+CD38- progenitor cells during cytokine prestimulation for retroviral transduction.

Before stem cell gene therapy can be considered for clinical applications, problems regarding cytokine prestimulation remain to be solved. In this study, a retroviral vector carrying the genes for the enhanced version of green fluorescent protein (EGFP) and neomycin resistance (neo(r)) was used for transduction of CD34+ cells. The effect of cytokine prestimulation on transduction efficiency and the population of uncommitted CD34+CD38- cells was determined. CD34+ cells harvested from umbilical cord blood were kept in suspension cultures and stimulated with combinations of the cytokines stem cell factor (SCF), FLT3 ligand, interleukin-3 (IL-3), IL-6, and IL-7 prior to transduction. Expression of the two genes was assessed by flow cytometry and determination of neomycin-resistant colonies in a selective colony-forming unit (CFU) assay, respectively. The neomycin resistance gene was expressed in a higher percentage of cells than the EGFP gene, but there seemed to be a positive correlation between expression of the two genes. The effect of cytokine prestimulation was therefore monitored using EGFP as marker for transduction. When SCF was compared to SCF in combination with more potent cytokines, highest transduction efficiency was found with SCF and IL-3 and IL-6 (5.05% +/- 0.80 versus 2.66% +/- 0.53 with SCF alone, p = 0.04). However, prestimulation with SCF in combination with IL-3 and IL-6 also reduced the percentage of CD34+ cells (p = 0.02). Then, prestimulation with SCF and FLT3 ligand was compared. Significant difference in transduction efficiency was not found. Interestingly, FLT3 ligand seemed to preserve the population of CD34+CD38- cells compared to SCF (16.56% +/- 2.02 versus 9.39% +/- 2.35, p = 0.03). In conclusion, prestimulation with potent cytokine combinations increased the transduction efficiency, but reduced the fraction of CD34+ cells. Importantly, the use of FLT3 ligand seemed to preserve the population of uncommitted cells.

ADP-ribosyl Cyclase↗

PubMed1999

Safe sorting of GFP-transduced live cells for subsequent culture using a modified FACS vantage.

BACKGROUND: A stream-in-air cell sorter enables rapid sorting to a high purity, but it is not well suited for sorting of infectious material due to the risk of airborne spread to the surroundings. METHODS: A FACS Vantage cell sorter was modified for safe use with potentially HIV infected cells. Safety tests with bacteriophages were performed to evaluate the potential spread of biologically active material during cell sorting. Cells transduced with a retroviral vector carrying the gene for GFP were sorted on the basis of their GFP fluorescence, and GFP expression was followed during subsequent culture. RESULTS: The bacteriophage sorting showed that the biologically active material was confined to the sorting chamber. A failure mode simulating a nozzle blockage resulted in detectable droplets inside the sorting chamber, but no droplets could be detected when an additional air suction from the sorting chamber had been put on. The GFP transduced cells were sorted to 99% purity. Cells not expressing GFP at the time of sorting did not turn on the gene during subsequent culture. Un-sorted cells and cells sorted to be positive for GFP showed a decrease in the fraction of GFP positive cells during culture. CONCLUSIONS: Sorting of live infected cells can be performed safely and with no deleterious effects on vector expression using the modified FACS Vantage instrument.

Bacteriophages↗

PubMed1999

Improvement of lymphocyte proliferation in human immunodeficiency virus infection after recombinant interleukin-2 treatment.

In this study, the effect of recombinant interleukin-2 (rIL-2) on the function of peripheral blood mononuclear cells (PBMC) from human immunodeficiency virus (HIV)-infected patients was examined. Using polymerase chain reaction (PCR), an impaired ability of PBMC from 8 patients to respond upon mitogen stimulation with expression of IL-2 and IL-2 receptor (IL-2R) messenger ribonucleic acid (mRNA) was found compared with healthy donors (p = 0.02 and p = 0.05, respectively). Flow cytometry was used to determine the expression of p55 interleukin-2 alpha-receptor (CD25) after phytohaemagglutinin (PHA)-stimulation. Induced CD25 expression in response to stimulation was lower in patient cells than in donor cells (in CD4+ (p = 0.01) and in CD8+ (p = 0.03)). After rlL-2 treatment, the functionality of ex vivo expanded PBMC from patients was restored to the level found in donors. Finally, the induced gene expressions for IL-2 and IL-2R were positively correlated (p < 0.0001), suggesting that the activation of the IL-2 and IL-2R genes in humans may share a common activation pathway, as has been found in monkeys infected by simian immunodeficiency virus (SIV). These results indicate the existence of a reversible IL-2 and IL-2R defect at the pretranscriptional or transcriptional level in PBMC from patients. This may help explain the T-cell anergy found during HIV-infection.

Adult↗

PubMed1999

Treatment with granulocyte colony-stimulating factor decreases the capacity of hematopoietic progenitor cells for generation of lymphocytes in human immunodeficiency virus-infected persons.

An obstacle to stem cell gene therapy for AIDS is the limited numbers of hematopoietic progenitors available. Granulocyte colony-stimulating factor (G-CSF) is used for mobilization of progenitors, but little is known about the functional characteristics of mobilized progenitors, and immature and T cell progenitors may not be mobilized. This study examined the effect of G-CSF on the function of progenitors. Ten human immunodeficiency virus-infected patients received G-CSF (filgrastim, 300 microgram/day) for 5 days. Absolute numbers of immature and T cell progenitors did not increase. The ability of CD34+ progenitor cells to generate lymphocytes was examined by use of thymic organ cultures. The mean number of lymphocytes generated per CD34+ cell on day 0 was 0.72 and on day 4 was 0.09 (P<.003). The number of CD4+ cells generated per CD34+ cell was significantly reduced after G-CSF treatment. Thus, G-CSF increased the number of mature progenitor cells but did not increase T cell progenitors.

Animals↗

PubMed1998

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.

Adult↗

PubMed1998

Effect of granulocyte colony-stimulating factor (G-CSF) in human immunodeficiency 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.

The potential of granulocyte colony-stimulating factor (G-CSF) to mobilize CD4 cells and/or CD34 cells for use in gene therapy or to support antiretroviral therapy was examined. Ten human immunodeficiency virus-infected patients were treated with G-CSF (300 microg/day) for 5 days. Numbers of CD4 and CD34 cells were measured. To examine the numbers of naive and memory type CD4 cells, CD4 cell coexpression of CD45RA and CD45RO was measured. Functionality of mobilized CD4 cells was examined by use of the proliferation assay and interleukin-2 ELISA. The number of CD34 cells increased from 1.50 to 20.01/microL (P < .002). The CD4 cell count increased from 236 to 452/microL (P < .002). The CD45RA/CD45RO ratio increased from 0.50 to 0.57 (P < .03). Mobilized CD4 cells were functionally intact. In conclusion, G-CSF induced increases in numbers of CD34 cells and CD4 cells in HIV-infected patients. Furthermore, the fraction of naive CD4 cells increased. These findings have implications for the design of immunotherapy or gene therapy protocols.

Adult↗

PubMed1998

Spontaneous silencing of humanized green fluorescent protein (hGFP) gene expression from a retroviral vector by DNA methylation.

We have constructed a functional murine leukemia virus (MLV)-derived retroviral vector transducing two genes encoding the autofluorescent humanized green fluorescent protein (hGFP) and neomycin phosphotransferase (Neo). This was done to determine whether hGFP could function as a marker gene in a retroviral vector and to investigate the expression of genes in a retroviral vector. Surprisingly, clonal vector packaging cell lines showed variable levels of hGFP expression, and expression was detected in as few as 49% of the cells in a clonally derived culture. This indicated that hGFP expression was silenced in individual cells. This silencing could be diminished by selective culturing of the vector packaging cells with the neomycin analog G418 and was reduced by a 3-day treatment with the demethylating agent 5-azacytidine. The 5-azacytidine effect was transient, and hGFP expression in the vector packaging cells returned to untreated control levels within 2 weeks. Using flow cytometric analysis, hGFP expression was detected in up to 15% of transduced MT4 cells (a CD4+ lymphocytic cell line) after coculturing with packaging cells for 4 days. A 3-day postcoculture treatment with 5-azacytidine was shown to increase the hGFP-expressing MT4 cells from either 10.4% to 11.6% or 3.7% to 4.8%, corresponding to an increase in observed transduction efficiencies of 12% and 30%, respectively. These results indicate that silencing of gene expression from a retroviral vector may result from DNA methylation and occurs rapidly after transduction.

Animals↗

PubMed1997

In vitro separation and expansion of CD4 lymphocytes from HIV-infected individuals without activation of HIV infection.

In order to offer a gene therapy-based treatment against AIDS, it is likely to be necessary to harvest and culture CD4 cells from HIV-positive patients without activating the HIV infection. We have used a magnetic cell sorting (MACS) system to enrich CD4 cells. Using positive selection, CD4 cells from a total of 14 patients were enriched from a mean percentage of CD4 cells in PBMC of 18% to 91% CD4 cells in the enriched cell fraction. Furthermore, we found that this separation did not lead to an increase in viral load. The MACS performed equally well on cells from HIV-positive patients and HIV-negative donors. CD4 cells from HIV-positive patients were readily expanded with PHA; 19-fold by day 10, 50-fold by day 20, and 156-fold by day 25. However, CD4 cells from HIV-positive patients grew at a slower rate than CD4 cells from HIV-negative donors. The expanded CD4 cells showed a high degree of CD4 expression and no loss of polyclonality. Only in two of six cultures were we able to detect HIV-antigen production, and using an LTR-PCR and an RT assay, we did not find activation of the HIV infection during the culture period. Thus, the method described separates and expands CD4 cells from HIV-positive patients without activation of the HIV infection.

Adult↗

PubMed1997

Gene therapy of T helper cells in HIV infection: mathematical model of the criteria for clinical effect.

This paper presents a mathematical analysis of the criteria for gene therapy of T helper cells to have a clinical effect on HIV infection. The analysis indicates that for such a therapy to be successful, it must protect the transduced cells against HIV-induced death. The transduced cells will not survive as a population if the gene therapy only blocks the spread of virus from transduced cells that become infected. The analysis also suggests that the degree of protection against disease-related cell death provided by the gene therapy is more important than the fraction cells that is initially transduced. If only a small fraction of the cells can be transduced, transduction of T helper cells and transduction of haematopoietic progenitor cells will result in the same steady-state level of transduced T helper cells. For gene therapy to be efficient against HIV infection, our analysis suggests that a 100% protection against viral escape must be obtained. The study also suggests that a gene therapy against HIV infection should be designed to give the transduced cells a partial but not necessarily total protection against HIV-induced cell death, and to avoid the production of viral mutants insensitive to the gene therapy.

Cell Death↗

PubMed1997

Recombinant human granulocyte colony-stimulating factor increases circulating CD34-postive cells in patients with AIDS.

In a gene therapy-based treatment of AIDS, it would be desirable to have as many transduced target cells as possible. A limiting factor is the number of target cells. In this study, we investigated whether it was possible to increase the absolute number of one possible target cell, i.e., the circulating hematopoietic progenitor cells (CD34 cells) in patients with AIDS, using the recombinant human granulocyte colony-stimulating factor (G-CSF). Eight patients with AIDS were treated with G-CSF for neutropenia (< 1.0 x 10(9)/l). Treatment consisted of daily subcutaneous injections with 300 micrograms G-CSF for 3-5 consecutive days. Within 5 days of initiation of G-CSF therapy, an increase in the absolute neutrophil count (ANC) was seen in all patients. There was a median increase in ANC from 0.4 to 3.4 x 10(9)/l. In addition, G-CSF treatment significantly increased the absolute number of CD34 cells. The median increase in CD34 cells was from 0.8 to 2.2 x 10(6)/l. Finally, using a highly sensitive HIV-1 RNA PCR, we found that treatment of AIDS patients with G-CSF did not lead to enhanced HIV replication. These observations indicate that G-CSF may be used to mobilize CD34 cells in patients with AIDS, e.g., for a gene therapy protocol.

Acquired Immunodeficiency Syndrome↗

PubMed1997

Transduction potential of human retroviruses in highly proliferating small-cell lung cancer cells as well as non-proliferating hematopoietic stem cells.

Direct gene transfer to solid tissues or metastatic cancer cells requires vectors capable of in vivo transduction to specific cells. The predominant retroviral vectors of murine origin are inactivated by human complement, which precludes their use in vivo. Such inactivation does not take place with vectors based on human retroviruses. Murine retroviral vectors are also limited to proliferating cells, which human retroviruses are not. In this study we examined whether or not a vector using components from the human retroviruses HIV-1 and HTLV-1 could infect small-cell lung cancer cells and resting CD34+ hematopoietic stem cells. While HIV-1 itself was unable to infect cells lacking the CD4-membrane molecule, chimeric viral particles (pseudotype virus) with HIV-1 genome and HTLV-1 envelope components were able to infect both CD4-containing lymphocytic cells, CD4-negative tumour cells and hematopoietic stem cells. After infection with the pseudotype vector, the RNA genome was reverse transcribed and integrated. Transduction efficiency and gene expression under the HIV-1 LTR promoter in both tumour and stem cells were found to be of a similar or greater magnitude than in lymphocytic cells. These results suggest that gene transfer targeting proliferating as well as resting cells in vivo may be realized using components from human retroviruses.

Antigens, CD34↗

PubMed1997

Complement-mediated enhancement of HIV-1 infection in peripheral blood mononuclear cells.

We investigated if complement-mediated enhancement of HIV infection occurs in peripheral blood mononuclear cells (PBMC). In 7 experiments, we evaluated the effect of human complement on HIVIIIB infection in vitro. We measured HIV antigen production on day 4 and found that pre-incubation of HIV with complement led to enhanced production of antigen with a median enhancement of 2.5-fold (range 1.1-6.8). This complement-mediated increase in antigen production was statistically significant (p < 0.02). Complement-mediated enhancement of HIV infection was also tested in CD4 cells enriched from PBMC, and CD4 cells persistently gave higher levels of infection enhancement than PBMC. Thus, CD4 cells appear to be sufficient for complement-mediated enhancement of HIV infection to occur. In addition, we tested if it was possible to detect complement-mediated enhancement of primary HIV isolates in PBMC. We tested 3 isolates and found only a minor effect on antigen production (median enhancement 1.2-fold, range 0.6-1.5). Furthermore, addition of HIV-specific antibodies in combination with complement resulted in enhanced antigen production in 2/3 sera tested. However, the combination of complement and antibodies resulted in only a minor increase in enhancement of HIV infection compared to that obtained with complement alone. Finally, we found evidence of complement-mediated enhancement of HIV infection in resting PBMC. In conclusion, we demonstrated that complement-mediated enhancement of HIV infection does occur in vitro in PBMC.

CD4-Positive T-Lymphocytes↗

PubMed1997

The serotonin analogue buspirone increases the function of PBMC from HIV-infected individuals in vitro.

HIV infection is characterized by the loss of CD4+ T cell numbers as well as loss of T cell function leading to severe immunodeficiency. The proliferative capacity of T cells, measured in vitro as response to antigens and mitogens, is severely reduced during HIV infection. An increased level of the intracellular second messenger cAMP has been demonstrated to cause impaired proliferative capacity of PBMC from HIV-infected individuals in vitro. We have identified a serotonin analogue, buspirone, that inhibits the activity of adenylyl cyclase, the enzyme responsible for regulation of intracellular levels of cAMP. Using this inhibitor the proliferative responses of PBMC to a polyclone activator in vitro were increased in 28/30 HIV-seropositive individuals (p < 0.00001). Further, we demonstrated that this was due to proliferation of CD4+ T cells and that buspirone induced expression of IL-2 mRNA.

Adult↗