Peter Ebbesen

Cell-Dependent Differential Cellular Uptake of PNA, Peptides, and PNA-Peptide Conjugates

Peptide nucleic acid (PNA) oligomers were conjugated to cell-penetrating peptides: pAnt, a 17-residue fragment of the Drosophila protein Antennapedia, and pTat, a 14-amino acid fragment of HIV protein Tat. A 14-mer PNA was attached to the peptide by disulfide linkage or by maleimide coupling. The uptake of (directly or indirectly, via biotin) fluorescein-labeled peptides, PNAs, or PNA-peptide conjugates was studied by fluorescence microscopy, confocal laser scanning microscopy, and fluorometry in five cell types. In SK-BR-3, HeLa, and IMR-90 cells, the PNA-peptide conjugates and a T1, backbone-modified PNA were readily taken up (2 microM). The PNA was almost exclusively confined to vesicular compartments in the cytosol. However, the IMR-90 cells also showed a weak diffuse staining of the cytoplasm. In the U937 cells, we observed a very weak and exclusively vesicular staining with the PNA-peptide conjugates and the T(lys)-modified PNA. No evident uptake of the unmodified PNA was seen. In H9 cells, both peptides and the PNA-peptide conjugates quickly associated with the membrane, followed by a weak intracellular staining. A cytotoxic effect resulting in artificial staining of the cells was observed with fluoresceinated peptides and PNA-peptide conjugates at concentrations above 5-10 microM, depending on cell type and incubation time. We conclude that uptake of PNAs in many cell types can be achieved either by conjugating to certain peptides or simply by charging the PNA backbone using lysine PNA units. The uptake is time, temperature, and concentration dependent and mainly endocytotic. Our results also show that proper controls for cytotoxicity should always be carried out to avoid misinterpretation of visual data.

Induction of Adipocyte‐Like Phenotype in Human Mesenchymal Stem Cells by Hypoxia

Human mesenchymal stem cells (hMSCs) have the capacity to differentiate along several pathways to form bone, cartilage, tendon, muscle, and adipose tissues. The adult hMSCs reside in vivo in the bone marrow in niches where oxygen concentration is far below the ambient air, which is the most commonly encountered laboratory condition. The study reported here was designed to determine whether oxygen has a role in the differentiation of hMSCs into adipocytes. Indeed, when exposed to atmosphere containing only 1% of oxygen, the formation of adipocyte‐like phenotype with cytoplasmic lipid inclusions was observed. The effect of hypoxia on the expression of adipocyte‐specific genes was determined by real‐time reverse transcription polymerase chain reaction. Interestingly, neither of the two central regulators of adipogenesis—the transcription factors peroxisome proliferator‐activated receptor γ2 (PPAR‐γ2) and ADD1/SREBP1c—was induced. Furthermore, hypoxia did not have any effect on the transcription of early (lipoprotein lipase) or late (aP2) marker genes. By the same token, neither of the mature adipocyte‐specific genes—leptin and adipophilin—was found responsive to the treatment. High level of induction, however, was observed with the PPAR‐γ–induced angiopoietin‐related gene, PGAR. The lack of an adipocyte‐specific transcription pattern thus indicates that despite accumulation of the lipid, true adipogenic differentiation did not take place. In conclusion, hypoxia appears to exert a potent lipogenic effect independent of PPAR‐γ2 maturation pathway.

The Human Umbilical Cord Blood: A Potential Source for Osteoblast Progenitor Cells

The presence of non-hematopoietic stem cells in the human umbilical cord blood (hUCB) is debated. In this study, we report the isolation of a population of fibroblast-like cells with osteogenic and adipogenic potential that resembles the stromal stem cells found in the bone marrow. Low-density mononuclear cells isolated from hUCB formed few adherent colonies with fibroblast-like morphology after a few days in culture. At confluence, the polyclonal cell populations were characterized. Using FACS analysis and immunocytochemistry, the cells were found to express HLA-ABC, CD9, vimentin, the b subunit of prolyl-4-hydroxylase, integrins a1(CD49a), integrin a3 (CD49c), integrin a5(CD49e), and cytokeratin 18. Furthermore, the cells expressed constitutively transcripts of osteoblast-specific markers: Cbfa1/Runx2, alkaline phosphatase (AP), and collagen type I, and formed a mineralized matrix in vitro visualized by Alizarin red staining. In the presence of normal horse serum and dexamethasone (10?7 M), the cells formed foci of adipocytes. When the cells were implanted mixed with hydroxyapatite/tricalcium phosphate powder in the subcutis of immunocompromised mice for 8 weeks, they formed osteogenic tissue and a myelosupportive microenviroment that enclosed hematopoietic cells and adipocytes. Our results demonstrate the presence of circulating stem cells with osteogenic and adipogenic differentiation potential in hUCB and may encourage the use of hUCB as a potential source for stem cells to be utilized in cell therapy protocols for various diseases.

Taking advantage of tumor cell adaptations to hypoxia for developing new tumor markers and treatment strategies.

Cancer cells in hypoxic areas of solid tumors are to a large extent protected against the action of radiation as well as many chemotherapeutic drugs. There are, however, two different aspects of the problem caused by tumor hypoxia when cancer therapy is concerned: One is due to the chemical reactions that molecular oxygen enters into therapeutically targeted cells. This results in a direct chemical protection against therapy by the hypoxic microenvironment, which has little to do with cellular biological regulatory processes. This part of the protective effect of hypoxia has been known for more than half a century and has been studied extensively. However, in recent years there has been more focus on the other aspect of hypoxia, namely the effect of this microenvironmental condition on selecting cells with certain genetic prerequisites that are negative with respect to patient prognosis. There are adaptive mechanisms, where hypoxia induces regulatory cascades in cells resulting in a changed metabolism or changes in extracellular signaling. These processes may lead to changes in cellular intrinsic sensitivity to treatment irrespective of oxygenation and, furthermore, may also have consequences for tissue organization. Thus, the adaptive mechanisms induced by hypoxia itself may have a selective effect on cells, with a fine-tuned protection against damage and stress of many kinds. It therefore could be that the adaptive mechanisms may take advantage of for new tumor labeling/imaging and treatment strategies. One of the Achilles’ heels of hypoxia research has always been the exact measurements of tissue oxygenation as well as the control of oxygenation in biological tumor models. Thus, development of technology that can ease this control is vital in order to study mechanisms and perform drug development under relevant conditions. An integrated EU Framework project 2004–2009, termed EUROXY, demonstrates several pathways involved in transcription and translation control of the hypoxic cell phenotype and evidence of cross-talk with responses to pH and redox changes. The carbonic anhydrase isoenzyme CA IX was selected for further studies due to its expression on the surface of many types of hypoxic tumors. The effort has led to marketable culture flasks with sensors and incubation equipment, and the synthesis of new drug candidates against new molecular targets. New labeling/imaging methods for cancer diagnosing and imaging of hypoxic cancer tissue are now being tested in xenograft models and are also in early clinical testing, while new potential anti-cancer drugs are undergoing tests using xenografted tumor cancers. The present article describes the above results in individual consortium partner presentations.

Long-Term Seropositivity for Human T-Lymphotropic Virus Type III in Homosexual Men Without the Acquired Immunodeficiency Syndrome: Development of Immunologic and Clinical Abnormalities: A Longitudinal Study

The long-term effects of seropositivity for human T-lymphotropic virus type III (HTLV-III) on T-lymphocyte subsets and health status were evaluated in longitudinal studies of 250 initially healthy homosexual men. The relative risk of having an inverted T-lymphocyte helper-to-suppressor ratio rose from 14.3-fold among short-term seropositive subjects (less than 19 months) to 46.9-fold among long-term seropositive subjects (greater than 29 months) in comparison with the risk among seronegative subjects. Overall, 91.7% of long-term seropositive men had inverted ratios, compared with 12.9% of seronegative men. None of the seropositive men who developed an inverted ratio later reestablished a normal ratio. Both decreased T-helper cell number and percentage (p = 0.003) and increased T-suppressor cell number and percentage (p = 0.03) were significantly correlated with duration of seropositivity. Among seropositive persons, lymphadenopathy was a highly significant short-term as well as long-term consequence, whereas diarrhea, oral thrush, and herpes zoster were correlated with long-term seropositivity. Overall, 50% of long-term seropositive men compared with 16% of seronegative men developed at least one of five clinical symptoms (p less than 0.003). We conclude that a high proportion of persons infected with HTLV-III will develop measurable immunologic and clinical abnormalities.

Sexual transmission of hepatitis C virus: cohort study (1981-9) among European homosexual men.

OBJECTIVE--To determine the prevalence, incidence, and persistence of positivity for antibodies to hepatitis C virus (anti-HCV) and the potential for sexual transmission of the virus. DESIGN--A cohort analysis covering 1981-9 comparing estimated cumulative incidences of and seroconversion rates for anti-HCV with those of hepatitis B core antibody (anti-HBc) and antibodies to the human immunodeficiency virus (anti-HIV). SETTING--Copenhagen and Aarhus, Denmark. SUBJECTS--259 Male members of a Danish homosexual organisation. MAIN OUTCOME MEASURES--Correlations of prevalence and incidence with a wide range of sexual lifestyle variables. RESULTS--Only four (1.6%) subjects were positive for anti-HCV in 1981. The estimated cumulative incidence of positivity for anti-HCV was 4.1% in 1984 (seroconversion rate during 1981-4 (2.5%)) and remained at 4.1% in 1989 (seroconversion rate nil during 1984-9). In contrast, positivity for anti-HBC rose from 44.0% in 1981 to 52.7% in 1984 (seroconversion rate 15.5%) and 58.8% in 1989 (seroconversion rate 12.9%), and that for anti-HIV rose from 8.8% to 24.0% (seroconversion rate 16.7%) and 30.1% (seroconversion rate 8.0%) respectively. Three anti-HCV positive patients seroreverted three to five years later. None of the anti-HCV positive subjects had had a transfusion and only one gave a past history of intravenous drug use. Variables in sexual lifestyle correlated with the presence of anti-HBc but not with that of anti-HCV. CONCLUSIONS--In contrast with hepatitis B virus and HIV, sexual transmission of hepatitis C virus seems to be a rare event. Furthermore, antibodies to the virus may become undetectable after several years.

Seroepidemiology of HTLV-III antibody in Danish homosexual men: prevalence, transmission, and disease outcome.

Sera taken from 250 Danish homosexual men in December 1981 as part of a prospective study of the acquired immunodeficiency syndrome (AIDS) were examined for the presence of HTLV-III antibody with an enzyme-linked immunosorbent assay. Antibody was present in 22 (8.8%) of the men. Seropositivity was most strongly associated with sexual exposure to men in the United States (relative risk 3.5; p less than 0.007). Increased frequency of anal receptive intercourse was also independently associated with seropositivity (p less than 0.05), but age, years of homosexual experience, number of homosexual partners, and use of nitrite inhalant were not independent risk factors. The frequency of seroconversion from absence to presence of HTLV-III antibody appeared to be about 1% a month in this community during December 1981 to February 1983. Of the 22 men who were originally seropositive, two (9%) subsequently developed AIDS as defined by the Centre for Disease Control and two (9%) others the AIDS related complex. Blood was taken in addition from two of the men to develop AIDS earliest in Denmark (diagnosed 1981) at the same time as the initial survey in 1981; both were seropositive. The spread of HTLV-III from high to low risk areas and the subsequent appearance of illnesses related to AIDS in the seropositive group support the hypothesis that HTLV-III is causally related to the development of AIDS.

Latent class analysis of human herpesvirus 8 assay performance and infection prevalence in sub-Saharan Africa and Malta

Human herpesvirus 8 (HHV‐8) is thought to be highly prevalent in Mediterranean countries and sub‐Saharan Africa, where it causes Kaposis sarcoma in a small proportion of infected immunocompetent persons. However, the lack of serological tests with established accuracy has hindered our understanding of the prevalence, risk factors and natural history of HHV‐8 infection. We tested 837 subjects from Congo, Botswana (mostly young adults) and Malta (elderly adults), using an immunofluorescence assay and 2 enzyme immunoassays (EIAs, to viral proteins K8.1 and orf65). Each assay found HHV‐8 seroprevalence to be high (49–87%) in the African populations and generally lower (9–54%) in Malta. However, there was only modest agreement among tests regarding which subjects were seropositive (3‐way κ, 0.05–0.34). We used latent class analysis to model this lack of agreement, estimating each tests sensitivity and specificity and each populations HHV‐8 prevalence. Using this approach, the K8.1 EIA had consistently high sensitivity (91–100%) and specificity (92–100%) across populations, suggesting that it might be useful for epidemiological studies. Compared with the K8.1 EIA, both the immunofluorescence assay and the orf65 EIA had more variable sensitivity (80–100% and 58–87%, respectively) and more variable specificity (57–100% and 48–85%, respectively). HHV‐8 prevalence was 7% among elderly Maltese adults. Prevalence was much higher (82%) in Congo, consistent with very high Kaposis sarcoma incidence there. Prevalence was also high in Botswana (87% in Sans, an indigenous group, and 76% in Bantus), though Kaposis sarcoma is not common, suggesting that additional co‐factors besides HHV‐8 are needed for development of Kaposis sarcoma. Int. J. Cancer 88:1003–1008, 2000.

Pericellular oxygen depletion during ordinary tissue culturing, measured with oxygen microsensors

Abstract.  Recent research has found important differences in oxygen tension in proximity to certain mammalian cells when grown in culture. Oxygen has a low diffusion rate through cell culture media, thus, as a result of normal respiration, a decrease in oxygen tension develops close to the cells. Therefore, for the purpose of standardization and optimization, it is important to monitor pericellular oxygen tension and cell oxygen consumption. Here, we describe an integrated oxygen microsensor and recording system that allows measurement of oxygen concentration profiles in vertical transects through a 1.6‐mm deep, stagnant, medium layer covering a cell culture. The measurement set‐up reveals that, when confluent, a conventional culture of adherent cells, although exposed to the constant oxygen tension of ambient air, may experience pericellular oxygen tensions below the level required to sustain full oxidative metabolism. Depletions reported are even more prominent and potentially aggravating when the cell culture is incubated at reduced oxygen tensions (down to around 4% oxygen). Our results demonstrate that, if the pericellular oxygen tension is not measured, it is impossible to relate in vitro culture results (for example, gene expression to the oxygen tension experienced by the cell), as this concentration may deviate very substantially from the oxygen concentration recorded in the gas phase.

HTLV-III SEROPOSITIVITY IN EUROPEAN HAEMOPHILIACS EXPOSED TO FACTOR VIII CONCENTRATE IMPORTED FROM THE USA

77 Scottish haemophiliacs and 22 Danish haemophiliacs were serologically tested for antibodies to human T-cell leukaemia virus III (HTLV-III). Since 1979 the Scottish patients had been treated largely with factor VIII concentrate produced in Scotland, whereas all but 2 of the Danish patients had received both locally prepared concentrate and commercial concentrate made from US donor material. 15.6% of Scottish and 59.1% of Danish haemophiliacs were antibody positive (p less than 0.001). None of 11 haemophiliacs not treated in the period 1979-84 was seropositive. 2 (6.7%) of 30 subjects who had been treated with locally produced concentrate only were antibody positive, compared with 23 (39.7%) of 58 subjects who had been treated with commercial concentrate. Among 52 users of both commercially and locally produced factor VIII concentrate, seropositivity was directly correlated with the consumption of commercial concentrate (p less than 0.001) but not locally produced material. These data indicate that European haemophiliacs were exposed to HTLV-III via some factor VIII concentrates obtained from the USA.