Entourage: the immune microenvironment following follicular lymphoma.

In follicular lymphoma, nonmalignant immune cells are important. Follicular lymphoma depends on CD4+ cells, but CD8+ cells counteract it. We hypothesized that the presence of follicular lymphoma is associated with higher CD4+ than CD8+ cell numbers in the tumor microenvironment but not in the immune system. Using flow cytometry, pre-treatment and follow-up CD4/CD8 ratios were estimated in the bone marrow, blood and lymph nodes of untreated follicular lymphoma patients in two independent data sets (N(1)=121; N(2)=166). The ratios were analyzed for their relation with bone marrow lymphoma involvement. Bone marrows were also investigated with immunohistochemistry. In either data set, the bone marrow CD4/CD8 ratios were higher in bone marrows involved with lymphoma (P=0.043 and 0.0002, respectively). The mean CD4/CD8 ratio was 1.0 in uninvolved and 1.4 in involved bone marrows. Also higher in involved bone marrows were CD4/CD56 and CD3CD25/CD3 ratios. No blood or lymph node ratios differed between bone marrow-negative and -positive patients. Sequential samples showed increased bone marrow CD4/CD8 ratios in all cases of progression to bone marrow involvement. Immunohistochemistry showed CD4+, CD57+, programmed death-1+, forkhead box protein 3+ and CD21+ cells accumulated inside the lymphoma infiltrates, whereas CD8+, CD56+ and CD68+ cells were outside the infiltrates. This study provides evidence in vivo that the microenvironment changes upon follicular lymphoma involvement.

WIN55,212-2 induces cytoplasmic vacuolation in apoptosis-resistant MCL cells.

Cannabinoid receptors 1 (CB1) and/or 2 (CB2) are overexpressed in many types of human malignancies including mantle cell lymphoma (MCL). Agonists to CB1 and CB2 promote ceramide de novo synthesis, p38-mitogen-activated protein kinase-dependent activation of caspase-3 and apoptotic cell death in most MCLs. However, in this report we describe that in some MCLs the response to treatment with cannabinoids decreased cell viability as assessed by metabolic activity but did not involve the caspase-3 cascade or loss of plasma membrane integrity. Both primary cells from one MCL patient and the MCL cell line Granta519 responded to treatment with cannabinoids by formation of cycloheximide-sensitive cytoplasmic vacuoles, but did not enter apoptosis. The persistent expression of mammalian homolog of Atg8 with microtubule-associated protein-1 light chain-3 II (LC3 II) and p62, as well as the lack of protection from chloroquine, indicates that lysosomal degradation is not involved in this cytoplasmic vacuolation process, distinguishing from classical autophagy. Transmission electron microscopy images and immunofluorescence staining of endoplasmic reticulum (ER) chaperone calreticulin showed that the vacuoles were of ER origin and that chromatin remained normal. These features resemble paraptosis-like cell death-a third type of a programmed cell death not previously described in response to cannabinoids.

Group B streptococcus in prosthetic hip and knee joint-associated infections.

The incidence of invasive group B streptococcus (GBS) infections in non-pregnant adults is increasing. Little is known about GBS in periprosthetic joint infections (PJIs). We aimed to analyse the clinical presentation of GBS PJI and its treatment in association with the outcome. The characteristics of 36 GBS PJIs collected from 10 centres were investigated. In 34 episodes, follow-up examination of ≥ 2 years was available, allowing treatment and outcome analysis. Most infections (75%) occurred ≥ 3 months after implantation. Most patients (91%) had at least one comorbidity; 69% presented with acute symptoms and 83% with damaged periprosthetic soft tissue. In 20 of 34 episodes debridement and retention of implant was attempted, but in five of these the prosthesis was ultimately removed. Hence, in 19 (56%) episodes, the implant was removed, including 14 immediate removals. In four episodes the removal was permanent. Penicillin derivatives and clindamycin were the most common antimicrobials administered (68%). In 94% the infection was cured, and in 82% functional mobility preserved. Debridement with implant retention was successful if the duration of symptoms was short, the prosthesis stable, and the tissue damage minor (10/10 vs 3/10 episodes, P = 0.003). Surgery that complied with a published algorithm was associated with a favourable outcome (P = 0.049).

Detection of candidaemia in patients with and without underlying haematological disease.

Diagnosing candidaemia remains difficult despite the development of new diagnostics. We report a direct comparison of three different blood-culture systems and four indirect tests. One hundred and fourteen episodes either with haematological disease and fever despite antibacterials, or with documented invasive candidiasis, were enrolled prospectively. Clinical, para-clinical information and surveillance cultures were obtained. Blood culture was performed using conventional blood-culture bottles, mycosis bottles, and the Isolator 10 lysis centrifugation system. Serum D-arabinitol/L-arabinitol (DA/LA) ratios were determined by gas chromatography mass spectrometry. Antigen, mannan-antigen (Ag) and anti-mannan antibody (Ab) were detected by CandTec, Platelia Candida Ag ELISA and Candida AB/AC/AK kits, respectively. Episodes were classified as proven (n = 24), probable (n = 14), possible (n = 52) or unlikely (n = 24) invasive candidiasis. Candidaemia involved C. albicans (17), C. albicans + C. glabrata (3), C. tropicalis (1) and yeast (1). Mycosis bottles yielded two additional positives and the conventional blood culture yielded one positive not identified by other blood-culture methods. Considering proven and unlikely episodes, respectively, sensitivity and specificity were as follows: mannan-Ag and/or anti-mannan Ab: 83.3%, 78.3%; DA/LA ratio: 41.7%, 86.4%; and CandTec Candida Ag: 66.6%, 70.8%. Lowering the cut-off values to mannan-Ag 0.10 ng/mL and anti-mannan Ab 4 AU/mL, the values were: 100%, 73.9%. Applying the DA/LA ratio to only patients with haematological neutropenia the values were: 75%, 90.5%. Fungal blood culture allowed slightly improved detection of candidaemia. The best indirect test performance was obtained from combined mannan-Ag and anti-mannan Ab detection, especially with lower cut-offs. DA/LA ratio appears to be useful in the context of haematological neutropenia.

Biofilm formation by Propionibacterium acnes is a characteristic of invasive isolates.

Propionibacterium acnes is a common and probably underestimated cause of delayed joint prosthesis infection. Bacterial biofilm formation is central in the pathogenesis of infections related to foreign material, and P. acnes has been shown to form biofilm both in vitro and in vivo. Here, biofilm formation by 93 P. acnes isolates, either from invasive infections (n = 45) or from the skin of healthy people (n = 48), was analysed. The majority of isolates from deep infections produced biofilm in a microtitre model of biofilm formation, whereas the skin isolates were poor biofilm producers (p <0.001 for a difference). This indicates a role for biofilm formation in P. acnes virulence. The type distribution, as determined by sequencing of recA, was similar among isolates isolated from skin and from deep infections, demonstrating that P. acnes isolates with different genetic backgrounds have pathogenic potential. The biofilm formed on plastic and on bone cement was analysed by scanning electron microscopy (EM) and by transmission EM. The biofilm was seen as a 10-mum-thick layer covering the bacteria and was composed of filamentous as well as more amorphous structures. Interestingly, the presence of human plasma in solution or at the plastic surface inhibits biofilm formation, which could explain why P. acnes primarily infect plasma-poor environments of, for example, joint prostheses and cerebrospinal shunts. This work underlines the importance of biofilm formation in P. acnes pathogenesis, and shows that biofilm formation should be considered in the diagnosis and treatment of invasive P. acnes infections.

Evaluation of ex vivo expanded human NK cells on antileukemia activity in SCID-beige mice.

The possibility of using natural killer (NK) cells in treatment of human hematological malignancies has increased in recent years. One factor contributing to this is the introduction of new methods for ex vivo generation of enriched populations of clinical grade NK cells. The objective of the present study was to evaluate the safety and efficacy of human ex vivo expanded clinical grade NK cells against K562 leukemia cells in severe combined immunodeficiency disease (SCID)-beige mice. Irradiated SCID-beige mice were injected intravenously (i.v.) with K562 leukemia cells. Following leukemia cell injection, mice were injected with ex vivo expanded human NK cells. NK cells were followed in vivo and mice monitored for survival from leukemia. Administration of these ex vivo expanded clinical grade NK cells was safe and prevented leukemia development. In conclusion, these results imply possibilities for the use of this NK cell preparation in treatment trials of human hematological malignancies and possibly other forms of cancer.

Genetically modified autoactivated cells expressing intracellular forms of GM-CSF as a model for regulated administration of cytokines.

The application of cytokines for immunotherapy is frequently hampered by undesirable side effects. To avoid systemic effects, cytokines can be directly expressed in the target cells by using gene transfer. However, the uncontrolled cellular secretion of cytokines could still exert some undesirable bystander effects. Therefore, it is important to develop additional methods for a more restricted administration of cytokines. Recently, using the murine granulocyte-macrophage colony-stimulating factor (mGM-CSF), we have demonstrated that cytokines can be targeted to different subcellular compartments as stable and biologically active proteins. This model could be used as a method of highly restricted administration of cytokines. Here, as model for the proof of principle, we have used a cell line (DA-3) strictly dependent on mGM-CSF for growth and demonstrated that these cells acquired autonomous growth after gene modification with plasmids encoding either extracellular or intracellular forms of mGM-CSF. Cell lines expressing secreted forms of mGM-CSF displayed the highest rates of autonomous growth and released substantial amounts of mGM-CSF. However, cell lines expressing intracellular forms of mGM-CSF also acquired autonomous growth induced by a mechanism of restricted autocrine stimulation and did not release detectable mGM-CSF to the extracellular medium. Cocultivation experiments of DA-3 cell lines expressing intracellular mGM-CSF with unmodified cells showed that there was no activation of the bystander cells. Taken together, these results support the concept that genes encoding intracellular cytokines may be used to provide the desired effect of cytokines on the target cells while avoiding the side effects of their uncontrolled secretion.

Effect of clonal and serotype-specific properties on the invasive capacity of Streptococcus pneumoniae.

The present study compares the molecular epidemiology of Streptococcus pneumoniae causing invasive disease and carriage, respectively, in one geographic area (Stockholm, Sweden) during a specific point in time (the year 1997). A total of 273 invasive isolates (257 from adults and 16 from children) obtained from the 2 major hospitals in Stockholm, as well as 246 nasopharyngeal isolates recovered from children attending 16 day-care centers in the Stockholm area, were analyzed by serotyping, molecular typing (by pulsed-field gel electrophoresis and multilocus sequence typing), and antibiotic susceptibility testing. Of the 34 different serotypes plus nontypeable strains identified in the present study, 12 were never found among the 246 colonizing isolates, whereas only 3 were never found among the 273 invasive isolates. The isolates formed 2 major classes: 1 class that was found mainly among invasive isolates (type 1, 4, 7F, and 9V isolates) and was clonally highly related and 1 class that caused invasive disease but was also common in carriage (including type 6A, 6B, 14, and 19F isolates) and was genetically more diverse. Clones were found that belonged to the same serotype but had different abilities to cause invasive disease. Also, isolates belonging to the same clone were found, although they had different capsules because of serotype switch, and were found to have the same disease potential. Hence, properties associated with a particular clonal type, in addition to capsular serotype, are likely to be important for the potential of pneumococci to cause invasive disease.

Expansion of natural killer (NK) and natural killer-like T (NKT)-cell populations derived from patients with B-chronic lymphocytic leukemia (B-CLL): a potential source for cellular immunotherapy.

B-cell chronic lymphocytic leukemia (B-CLL) is the most common leukemia in the Western world. It is currently an incurable disease, making new treatment options such as immunotherapy desirable. Monoclonal antibodies (Mabs) to surface antigens of the tumor cell is one option. Administration of cytotoxic cells such as natural killer (NK) and natural killer-like T (NKT) cells expanded in vitro might be a useful treatment modality alone or in combination with MAbs. A limiting step in the development of successful cellular immunotherapy has been the availability of appropriate cytotoxic cells. Here, we report the feasibility of expanding populations of the human killer cells, CD3-CD56+ NK and CD3+CD56+ NKT cells, from peripheral blood mononuclear cells (PBMCs) of B-CLL patients. The influence of tumor B cells on the in vitro expansion of killer cells was assessed by depleting B cells from PBMCs by microbead separation before culture. The 21-day cultures from both B-cell- and non-B-cell-depleted PBMC showed a marked expansion of NK cells, and also of T cells, among which almost half had the NKT phenotype. Depletion of B cells before culture did not change the expansion rates of NK and NKT cells significantly. In patients with progressive B-CLL, NK cell expansion capacity was improved after fludarabine treatment when compared to samples obtained before treatment. Repeated samples of PBMCs from individual untreated patients with both indolent and progressive disease cultured under identical conditions gave similar NK cell expansion rates. Expanded killer cell populations had cytotoxic function against the NK-sensitive target K562 cell line and expressed high levels of Granzyme B. From our studies, we conclude that NK cells as well as NKT cells from the peripheral blood of B-CLL patients can be expanded, and that these cells have cytotoxic capacity.

High level of cannabinoid receptor 1, absence of regulator of G protein signalling 13 and differential expression of Cyclin D1 in mantle cell lymphoma.

Mantle cell lymphoma (MCL) is a moderately aggressive B-cell lymphoma that responds poorly to currently used therapeutic protocols. In order to identify tumour characteristics that improve the understanding of biology of MCL, analysis of oligonucleotide microarrays were used to define specific gene expression profiles. Biopsy samples of MCL cases were compared to reactive lymphoid tissue. Among genes differentially expressed in MCL were genes that are involved in the regulation of proliferation, cell signalling, adhesion and homing. Furthermore, some genes with previously unknown function, such as C11orf32, C2orf10, TBC1D9 and ABCA6 were found to be differentially expressed in MCL compared to reactive lymphoid tissue. Of special interest was the high expression of the cannabinoid receptor 1 (CB1) gene in all MCL cases analysed. These results were further confirmed at the cellular and protein level by immunocytochemical staining and immunoblotting of MCL cells. Furthermore, there was a reduced expression of a regulator of G protein signalling, RGS13 in all MCLs, with a complete absence in the majority of cases while present in control lymphoid tissue. These results were further confirmed by PCR. Sequencing of the RGS13 gene revealed changes suggesting polymorphisms, indicating that downregulation of the expression of RGS13 is not related to mutations, but may serve as a new specific marker for MCL. Moreover, comparison between individual cases of MCL, revealed that the CCND1 gene appears to be differently expressed in MCL cases with high vs low proliferative activity.

Clonal analysis of Streptococcus pneumoniae nonsusceptible to penicillin at day-care centers with index cases, in a region with low incidence of resistance: emergence of an invasive type 35B clone among carriers.

The nasopharyngeal carriage rate of potential respiratory pathogens was studied in 36 index children with a pneumococci nonsusceptible to penicillin (PNSP), in 595 healthy children, and in 123 personnel at 16 day-care centers (DCCs) with index cases in the Stockholm area, an urban area with a low incidence of antibiotic resistant pneumococci, during the winter of 1997-1998. The spread and clonality of PNSP, Haemophilus influenzae and Moraxella catarrhalis, were studied by analyzing antibiotic susceptibility and serotype, and for PSNP also by using pulsed-field electrophoresis (PFGE) and multilocus sequence typing (MLST). In contrast to the low carriage rate found among the adult contacts (2%), 40% of the children harbored pneumococci, of which 20% were PNSP. Nasopharyngeal colonization decreased with age. The 49 PNSP isolates consisted of 20 clones, of which 10 could be identified in more than one child attending the same or different DCCs. In five DCCs, we observed a spread of PNSP from the index case. A novel PNSP clone of type 35B, found to cause invasive disease in several states in the United States, was found to emerge among several carriers at two DCCs . A high proportion of PNSP isolates were multiresistant to antibiotics (34%), which has implications for treatment regimens, even in a country like Sweden where the proportion of PNSP currently is low (3-4%). One PNSP clone of type 9V found among the carriers, has been shown to cause invasive disease in Sweden as well as in other countries, suggesting that one reason for the occurrence of invasive PNSP clones may be their ability to colonize and spread among healthy carriers. Other internationally spread antibiotic resistant pneumococcal clones found were of types 9V, 19F, and 23F.

Cryptosporidium parvum and Giardia intestinalis in calf diarrhoea in Sweden.

The objective of this study conducted in 75 herds was to investigate the presence and significance of Criptosporidium parvum and Giardia intestinalis in Swedish dairy calves in comparison with rotavirus, coronavirus and Escherichia coli K99+. The farmers were asked to collect faecal samples from each heifer calf that had diarrhoea between birth and 90 days of age, and also from a healthy calf of the same age. In total, 270 samples were collected and analysed. C. parvum, either alone or together with G. intestinalis and/or rotavirus, was detected in 16 (11%) and 6 (5%) of the samples from diarrhoeic and healthy calves, respectively. Even though a higher proportion of diarrhoeic calves shed C. parvum, the difference between the groups was not statistically significant (p = 0.067), possibly due to the low number of positive samples. G. intestinalis was found in 42 (29%) of the diarrhoea samples and in 29 (23%) of the samples from healthy calves. Rotavirus and coronavirus were demonstrated in 24% and 3% of the diarrhoea samples, respectively, whereas E. coli K99+ was only found in samples from 2 healthy calves. C. parvum and G. intestinalis were found in samples from calves 7 to 84 days of age and during all seasons. The results confirm that C. parvum is present in Swedish dairy herds and might have clinical significance. G. intestinalis was the most common agent found but the importance of this parasite remains unclear. Both parasites have suggested zoonotic potential and thus warrant further attention. In addition, rotavirus is a major pathogen in neonatal enteritis in Sweden, whereas coronavirus and E. coli K99+ seem to be of less importance.

Cytolytic T cell reactivity to Epstein-Barr virus is lost during in vitro T cell expansion.

In the setting of allogeneic hematopoietic stem cell transplantation, ex vivo culturing of donor T lymphocytes is a necessary step for processes such as gene modification. Often the aim is to enable control of undesired alloreactivity after in vivo administration of the cultured cells. However, it is not fully understood how T cell reactivity against donor and third-party targets is affected by the ex vivo cell culturing process. We have assessed how the activity of anti-Epstein Barr virus (EBV)-specific T lymphocytes from healthy EBV-seropositive donors is affected by in vitro cell culturing. Peripheral blood mononuclear cells (PBMCs) were expanded in X-VIVO 15 culture medium supplemented with 5% human serum. The cells were stimulated by either OKT3 (10 ng/ml) and interleukin (IL)-2 (500 U/ml) or by using anti-CD3/CD28-coated immunomagnetic beads and IL-2 (100 U/ml). Induction of polyclonal EBV-specific cytotoxic T lymphocyte cultures was attempted by stimulation of the in vitro-expanded cells at different time points during the cell expansion process, with pre-established autologous EBV-transformed lymphoblastoid cell lines (LCLs). While EBV-specific cytotoxic T lymphocytes (CTL) were generated from untreated PBMCs of 5 healthy donors, EBV-specific cytotoxicity was significantly decreased or absent in CTL cultures established from in vitro-expanded PBMCs. Our results indicate that the ex vivo cell expansion process itself significantly reduces the activity and/or the number of EBV-specific T cells. Additional stimulation with CD28 antibodies could not prevent this effect. Because T cell depleted bone marrow or stem cell grafts are known to contribute to the development of post transplant lymphoproliferative disorders, this should be taken into consideration if one considers expanding and administering PBMCs in conjunction with a T cell-depleted stem cell grafts.

Winter survival in Sweden of L(3)-stage larvae of the ostrich wireworm Libyostrongylus douglassii.

Libyostrongylus douglassii, a pathogenic trichostrongylid nematode of the ostrich, was recently identified as a potentially important parasite in ostriches on Swedish farms. This parasite is well adapted to the hot and dry climates of sub-Saharan Africa, the natural habitat of the ostrich. The influence of low temperatures in colder climates, such as in Sweden, on free-living stages of L. douglassii is however insufficiently known. In this study, parasite free ostrich faeces were mixed with infective L(3)-stage larvae of L. douglassii, which had been cultured from eggs obtained from two Swedish farms. Samples of the mixture were placed on a grass surface, and analyses for L(3)-stage larvae were performed at regular intervals. The results of the study showed that L(3)-stage larvae may remain alive during a period of at least 97 days on pasture during the winter season in Sweden, even when the temperature within the samples was repeatedly below -0.1 degrees C, and the air temperature varied between -19.6 and +14.9 degrees C. It was concluded that L(3)-stage larvae of L. douglassii probably have the ability to remain viable on pastures during winter in Sweden.

Evaluation of seven different enzyme-linked immunosorbent assays for serodiagnosis of Staphylococcus aureus bacteremia.

OBJECTIVE: Serologic assays for Staphylococcus aureus antibodies were evaluated regarding their ability to differentiate between uncomplicated and complicated S. aureus bacteremia, between S. aureus and non-S. aureus bacteremia, and between S. aureus and non-S. aureus endocarditis. METHODS: Enzyme-linked immunosorbent assays (ELISAs) were performed to measure Ig G antibodies against seven S. aureus antigens (peptidoglycan, teichoic acid, S. aureus ultrasonicate, whole S. aureus cells, alpha-toxin, lipase and capsular polysaccharide) in 129 patients with S. aureus bacteremia (including 51 with endocarditis), 78 patients with non-S. aureus bacteremia (including 27 with endocarditis) and 100 febrile non-bacteremic controls. RESULTS: Whole-cell ELISA was the most sensitive assay. The specificity of all assays was low. Two different combinations of ELISAs for whole cells, teichoic acid,alpha-toxin, lipase and capsular polysaccharide did distinguish between S. aureus and non-S. aureus endocarditis, but not between uncomplicated and complicated S. aureus bacteremia.

A new method for in vitro expansion of cytotoxic human CD3-CD56+ natural killer cells.

Adoptive transfer of immunocompetent cells may induce anti-tumor effects in vivo. However, a significant obstacle to the development of successful cellular immunotherapy has been the availability of appropriate cytotoxic cells. Among the immunologic effector cells that are considered mediators of anti-tumor effects, those with the highest per-cell cytotoxic capacity express a natural killer (NK) cell phenotype, i.e., CD56(+)CD3(-). However, such cells are normally present only in low numbers in peripheral blood mononuclear cells (PBMCs), lymphokine activated killer (LAK), and cytokine induced killer (CIK) cell preparations. To optimize the expansion of human NK cells, PBMCs were cultured in different serum free medium supplemented with monoclonal anti-CD3 antibodies and interleukin (IL)-2 at varying concentrations. By using Cellgro stem cell growth medium supplemented with 5% human serum and IL-2 (500 U/ml) cells expanded 193-fold (median, range 21-277) after 21 days, and contained 55% (median, range 7-92) CD3(-)CD56(+) cells. The remaining cells were CD3(+) T cells, 22% (median, range 2-68) of which co-expressed CD56. The expanded cell population lysed 26 to 45% of K562 targets in a 1:1 effector to target ratio, signifying substantial cytotoxic efficacy. The described method is a simple and efficient way of expanding and enriching human NK cells. We have termed these high-yield CD3(-)CD56(+) cells cytokine-induced natural killer (CINK) cells.

Otodectic otoacariasis in free-ranging Eurasian lynx in Sweden.

An infestation with Otodectes cynotis, the ear mite of cats and dogs, was observed in three free-ranging Eurasian lynx (Lynx lynx) killed in Sweden. The ear canals were obstructed by waxy secretions and exfoliated epithelium. Histologically, there were hyperkeratosis and acanthosis, and the epithelial surface was overlained by hyperkeratotic and parakeratotic crusts with mites, mite detritus and cerumen. In the subcutis there was a slight to moderate infiltration of lymphocytes and macrophages. The ceruminous glands were bypertrophic and hyperplastic, and there was also an hyperplasia of the sebaceous glands. The lesions seemed to correlate with the degree of infestation. To our knowledge, this is the first report of otoacariasis in free-ranging lynx.

Effects of arsenic trioxide (As2O3) on leukemic cells from patients with non-M3 acute myelogenous leukemia: studies of cytotoxicity, apoptosis and the pattern of resistance.

Arsenic oxide (As2O3) has recently been reported to induce remission in a high percentage of patients with acute promyelocytic leukemia (APL). The aim of this study was to investigate the effects of As2O3 at therapeutic concentrations on cell viability and apoptosis on leukemic cells from patients with non-M3 acute myelogenous leukemia (AML) and to study the resistance profile compared to conventional AML drugs. Cells from 20 patients were exposed to therapeutic concentrations of As2O3 continuously for 96 h. As2O3 reduced the viability in blast cells from all the 20 tested patients compared to unexposed controls (p-value: 0.02 at 0.05 microM; <0.005 at 1.0 microM and higher concentrations). An increase in the apoptotic rate was also seen after incubation with As2O3. Parallel to the incubation with arsenic the in vitro sensitivity to a number of chemotherapeutic agents commonly used in AML was studied. Correlation coefficients for the in vitro sensitivity were highly significant between the conventional AML drugs except for Ara-C. For As2O3, all the correlation coefficients were negative and ranged between -0.05 and -0.51. Furthermore, increased P-gp expression in a multidrug resistant HL-60 cell line did not decrease the sensitivity to As2O3 as compared to the parental cell line. Neither did a P-gp-transfected variant of the K562 cell line show decreased sensitivity to As2O3. We conclude that As2O3 at therapeutic concentrations induces apoptosis and cytotoxic effects in blast cells from patients with non-M3 AML, and that As2O3 differs from conventional AML drugs with respect to the mechanisms that confer resistance to the drugs.

In vivo nuclear delivery of oligonucleotides via hybridizing bifunctional peptides.

Linking proteins directly to nucleic acids has been a complex task. By hybridizing a bifunctional peptide nucleic acid (PNA) consisting of a nucleic acid binding moiety and a nuclear localization signal (NLS) we have previously demonstrated that it is possible to link protein functions directly to nucleic acids containing a PNA target site. By hybridizing fluorescently labeled oligonucleotides to PNA-NLS molecules and subsequently transfecting different organs in vivo we demonstrate an active nuclear translocation of the PNA-NLS/oligonucleotide complex in different mouse organs.