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Biomedical subjects

J E Hansen

Publications and source records attributed to J E Hansen.

At least 55 records · Page 3Linked to original sources
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

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.

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↗
PubMed1998

Inhibition of HIV-1 replication by chimeric phosphorothioate oligodeoxynucleotides applied in free solution.

Oligodeoxynucleotides (ODNs) containing a variable number of 3' and 5' terminal phosphorothioate linkages were applied in free solution to cells infected by HIV-1. ODNs of 28 nt length were applied at up to 5 microM concentration. The ODNs were found to inhibit HIV-1 infection in a dose dependent manner, which correlated with the number of modified linkages (4, 8 and 12, respectively). A target sequence in the HIV-1 rev mRNA, previously reported as sensitive to antisense inhibition by full length phosphorothioate ODNs, only revealed non-sequence dependent inhibition of HIV-1, when tested by these modified chimers.

Base Sequence↗
PubMed1998

Patterns of dissimilarities among instrument models in measuring PO2, PCO2, and pH in blood gas laboratories.

STUDY OBJECTIVES: To ascertain the degree of dissimilarities among blood gas and pH analyzer models of the same and different manufacturers in measurement of PO2, PCO2, and pH using fluorocarbon containing emulsion (FCE) proficiency testing material. DESIGN: Statistically and graphically analyze data from six recent proficiency testing surveys for the 20 more frequently used models of analyzers. SETTING AND PARTICIPANTS: Over a 2-year period, approximately 900 participants from blood gas laboratories in the United States analyzed similar ampules from each of 30 lots. MEASUREMENTS AND RESULTS: Both graphic and statistical comparisons were used to demonstrate differences between manufacturers. For each of the four major manufacturers, comparisons revealed statistically significant differences not only for PO2, but also for PCO2 and pH. Additionally, comparison models within each of the three manufacturers (those with multiple models and > 15 instruments per model represented) disclosed statistically significant dissimilarities among models for each analyte in 115 of 153 model pairings. Previously reported tonometered blood differences among analyzer models for PO2 are qualitatively similar to the differences found in these same models in this FCE study. Model differences are important in research studies and may be clinically important in deciding abnormality, selecting oxygen therapy, or the treatment of patients with respiratory failure or severe respiratory alkalosis. CONCLUSIONS: To minimize the likelihood of misleading clinicians, laboratory directors should consider the degree of dissimilarity among blood gas analyzer models in current use and when changing instrumentation.

Blood Gas Analysis↗
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

O-GLYCBASE version 2.0: a revised database of O-glycosylated proteins.

O-GLYCBASE is an updated database of information on glycoproteins and their O-linked glycosylation sites. Entries are compiled and revised from the literature, and from the SWISS-PROT database. Entries include information about species, sequence, glycosylation sites and glycan type. O-GLYCBASE is now fully cross-referenced to the SWISS-PROT, PIR, PROSITE, PDB, EMBL, HSSP, LISTA and MIM databases. Compared with version 1.0 the number of entries have increased by 34%. Revision of the O-glycan assignment was performed on 20% of the entries. Sequence logos displaying the acceptor specificity patterns for the GalNAc, mannose and GlcNAc transferases are shown. The O-GLYCBASE database is available through WWW or by anonymous FTP.

Algorithms↗
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

Demonstration of peripheral fucose units in N-linked glycans of human immunodeficiency virus type 1 gp 120: effects on glycoprotein conformation.

Fucosylated N-linked glycans are important constituents of membrane glycoproteins, owing to their significance as biologically active ligands for several selectins and their role in modulating protein conformation of viral glycoproteins. The human immunodeficiency virus type 1 (HIV-1) glycoprotein contains more than 30 different glycan structures but so far fucose was found associated solely with the innermost GlcNAc of N-linked glycans. In the present report we determined whether fucose units also were linked to the distal GlcNAc via alpha(1-3) or alpha(1-4) linkages in N-linked glycans of gp 120. [3H]-fucose labelled gp 120 was subjected to endoglycosidase F digestion, releasing diantennary complex type N-linked glycans, but leaving the inner polypeptide-bound carbohydrates, GlcNAc and possibly associated fucose units, intact. Gel filtration of the digested material revealed that [3H]-fucose label was released from gp 120 by this treatment, indicating presence of peripheral fucose units. Furthermore, [3H]-focuse label was also released by treatment of the labelled gp 120 with an alpha-L-fucosidase specifically removing fucose in alpha(1-3) and alpha(1-4) linkages. Altogether the results indicated presence of fucose units linked to peripheral GlcNAc of gp 120 N-linked glycans. We have earlier shown that other peripheral carbohydrate determinants, i.e. beta(1-4)-galactose on N-linked glycans, maintain a correct antigenic conformation of gp 120. Using a coupled ELISA system, where changes in antigenic behaviour of a viral glycoprotein were correlated to stepwise elimination of peripheral monosaccharides from N-linked glycans, we found that treatment of gp 120 with a pan-specific alpha-fucosidase as well as an enzyme specific for alpha(1-3)- or alpha(1-4)-linked fucose disclosed a hidden linear epitope situated in the gp 120 C2 region. The effects of the general fucosidase on epitope exposure was more prominent than those obtained with the enzyme with narrow specificity, suggesting that peripheral and inner fucose units co-operate in the maintenance of gp 120 conformation.

Animals↗
PubMed1997

Thyroid volume and function after 131I treatment of diffuse non-toxic goitre.

OBJECTIVE: Traditional treatment of diffuse non-toxic goitre are thyroid hormone suppression or surgery. When treating nodular non-toxic goitre with 131I treatment a reduction in thyroid volume to about 50% has been observed. In the present study we evaluated the effect of 131I treatment of diffuse non-toxic goitre. DESIGN: Retrospective study of patients treated for a diffuse non-toxic goitre and followed by evaluation of thyroid volume measured by ultrasound. PATIENTS: Ten selected patients from our out-patient clinic with diffuse non-toxic goitre. MEASUREMENTS: Thyroid volume was measured by ultrasound and thyroid function by serum values of T4, T3, T3 uptake ratio, TSH, TSH receptor antibodies and thyroid peroxidase antibodies (anti-TPO). Measurements were performed before and 1, 3, 6 and 12 months (and 18 months (n = 7), thyroid volume measured in six patients)) after 131I treatment. RESULTS: Thyroid volume declined in all patients from median 41 (range 27-160) ml to 20 (range 9-108) ml over 1 year, a reduction of 47%. One patient developed transient and one persistent hypothyroidism in the follow-up period. Both had elevated anti-TPO levels before treatment (331 and 9185 U/ml) and demonstrated titre increases of 2.5 and 30 times after 3 and 6 months, respectively. Pretreatment values were reached after 1 year. The other eight patients had normal anti-TPO levels and free T4 and T3 indices did not change during follow-up, whereas serum TSH levels demonstrated upward trends within the normal range (P < 0.05). TSH receptor antibodies were normal and remained so in all patients. CONCLUSIONS: 131I treatment of diffuse non-toxic goitre reduces thyroid volume by approximately 50% within 12-18 months. Hypothyroidism, during a limited follow-up period, developed only in patients with positive anti-TPO levels before treatment.

Adult↗
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

Cardiac output estimated noninvasively from oxygen uptake during exercise.

Because gas-exchange measurements during cardiopulmonary exercise testing allow noninvasive measurement of oxygen uptake (Vo2), which is equal to cardiac output (CO) x arteriovenous oxygen content difference [C(a-vDo2),] CO and stroke volume could theoretically be estimated if the C(a-vDo2) increased in a predictable fashion as a function of % maximum Vo2 (Vo2max) during exercise. To investigate the behavior of C(a-vDo2) during progressively increasing ramp pattern cycle ergometry exercise, 5 healthy subjects performed 10 studies to exhaustion while arterial and mixed venous blood were sampled. Samples were analyzed for blood gases (pH, Pco2, Po2) and oxyhemoglobin and hemoglobin concentration with a CO-oximeter. The C(a-vDo2) (ml/100 ml) could be estimated with a linear regression [C(a-vDo2) = 5.72 + 0.105 x % Vo2max; r = 0.94]. The CO estimated from the C(a-vDo2) by using the above linear regression was well correlated with the CO determined by the direct Fick method (r = 0.96). The coefficient of variation of the estimated CO was small (7-9%) between the lactic acidosis threshold and peak Vo2. The behavior of C(a-vDo2), as related to peak Vo2, was similar regardless of cardiac function compared with similar measurements from studies in the literature performed in normal and congestive heart failure patients. In summary, CO and stroke volume can be estimated during progressive work rate exercise testing from measured Vo2 (in normal subjects and patients with congestive heart failure), and the resultant linear regression equation provides a good estimate of C(a-vDo2).

Adult↗
PubMed1997

Thyrotropin receptor antibodies and Graves' disease, a side-effect of 131I treatment in patients with nontoxic goiter.

The use of 131I treatment in patients with benign nontoxic goiter is increasing, and the described side-effects are few. In this paper we describe appearance of TSH receptor antibodies (TRAb) and concomitant development of hyperthyroidism as a side-effect of 131I treatment in patients with nontoxic goiter. In this retrospective study, 191 consecutive patients with 131I-treated nontoxic goiter are described. Nine patients (5%) developed hyperthyroidism 3 months after 131I treatment, and 5 patients (3%) developed radiation thyroiditis within the first month. Frozen sera were analyzed for thyroid peroxidase antibodies (anti-TPO) in 130 patients before 131I treatment. In 21% of these, serum levels of anti-TPO were over 200 U/mL. The complication frequency of Graves'-like hyperthyroidism and hypothyroidism was 51% in patients with elevated anti-TPO (n = 27) and 15% in patients with normal serum anti-TPO levels (P < 0.00005). TRAb, anti-TPO, and thyroglobulin were followed in patients developing hyperthyroidism or radiation thyroiditis and in 10 control patients remaining euthyroid. At the time of 131I treatment, all patients had serum TRAb values within the normal range. Three months after administration of 131I, the patients developing hyperthyroidism had a transient extensive rise in serum TRAb and anti-TPO levels parallel to a rise in the serum free T4 index. In patients developing radiation thyroiditis, serum TRAb values were normal. In control patients, serum TRAb and anti-TPO values were both within the normal range throughout the observation period. In conclusion, hyperthyroidism can be triggered by 131I in patients with nontoxic goiter, not only related to radiation thyroiditis but also as a Graves'-like hyperthyroidism induced by TRAb. Elevated anti-TPO pretreatment is a marker of an increased risk of side-effects to 131I treatment in nontoxic goiter.

Adult↗
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↗