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PubMed2000

Amphotropic and VSV-G-pseudotyped retroviral vectors transduce human hematopoietic progenitor cells with similar efficiency.

One restriction of retroviral gene transfer into hematopoietic stem cells is the low level of amphotropic virus receptor. In the present study, we examined whether retroviral vectors pseudotyped with the G-protein of vesicular stomatitis virus (VSV) can overcome this restriction. Human progenitor cells purified by magnetic beads and cell sorting were transduced with an amphotropic or VSV-G-pseudotyped retroviral vector containing the truncated human nerve growth factor receptor as a marker gene. Cells were prestimulated with flt-3 ligand, stem cell factor, and interleukin-3 and transduced on fibronectin. Marker gene expression was analyzed by flow cytometry. Transduction efficiencies of amphotropic and VSV-G-pseudotyped virus for CD34+ cells did not differ significantly. Gene transfer into CD34+CD38- cells, which are enriched in more immature progenitors, was not restricted and transfer efficiencies for this subset were also similar for both pseudotypes. The addition of fibronectin improved gene transfer with the amphotropic vector considerably (5- to 19.3-fold, mean 12.6), while the effect on the VSV-G-pseudotype was far less pronounced (1- to 3.9-fold, mean 2.1, P = 0.04). In conclusion, high levels of gene transfer to human hematopoietic progenitors were achieved with an optimized transduction protocol, and transduction efficiencies could not be improved further by the use of VSV-G-pseudotypes.

ADP-ribosyl Cyclase↗
PubMed2006

Subunit architecture of multimeric complexes isolated directly from cells.

Recent developments in purification strategies, together with mass spectrometry (MS)-based proteomics, have identified numerous in vivo protein complexes and suggest the existence of many others. Standard proteomics techniques are, however, unable to describe the overall stoichiometry, subunit interactions and organization of these assemblies, because many are heterogeneous, are present at relatively low cellular abundance and are frequently difficult to isolate. We combine two existing methodologies to tackle these challenges: tandem affinity purification to isolate sufficient quantities of highly pure native complexes, and MS of the intact assemblies and subcomplexes to determine their structural organization. We optimized our protocol with two protein assemblies from Saccharomyces cerevisiae (scavenger decapping and nuclear cap-binding complexes), establishing subunit stoichiometry and identifying substoichiometric binding. We then targeted the yeast exosome, a nuclease with ten different subunits, and found that by generating subcomplexes, a three-dimensional interaction map could be derived, demonstrating the utility of our approach for large, heterogeneous cellular complexes.

Chromatography, Affinity↗
PubMed2004

Non-contact production of oligonucleotide microarrays using the highly integrated TopSpot nanoliter dispenser.

For the first time we report on the production of oligonucleotide microarrays using a highly parallel and highly integrated, pressure driven TopSpot nanoliter dispenser. The system enables non-contact printing of different media like oligonucleotides, DNA or protein solutions. We optimized the printing buffer needed for oligonucleotides microarrays production with respect to two major aspects: microfluidical optimum for droplet dispensing and biochemical coupling efficiency on different commercially available microarray slides. Coefficient of variations (CVs) of generated spot diameters were measured to be smaller than 1% within one single dispensing nozzle and smaller than 1.5% within all 24 parallel nozzles of the printhead for all printing buffers used. No carry-over and no cross-talk was found, in extensive experiments with oligonucleotides. Optimized printing buffer compositions and concentrations for oligonucleotide microarrays were found, as well as optimized coupling protocols. Furthermore, buffers and protocols were adapted to a host of different microarray slides used. With this system, prime critical points of microarray production are solved, leading to high quality high throughput microarray fabrication.

Nanotechnology↗
PubMed2006

The ABC transporter BmrA from Bacillus subtilis is a functional dimer when in a detergent-solubilized state.

BmrA from Bacillus subtilis is a half-size ABC (ATP-binding cassette) transporter involved in multidrug resistance. Although its supramolecular organization has been investigated after reconstitution in a lipid bilayer environment, and shows a dimeric and possibly a tetrameric form, the precise quaternary structure in a detergent-solubilized state has never been addressed. In the present study, BmrA was purified from Escherichia coli membranes using an optimized purification protocol and different detergents. Furthermore, the ATPase activity of BmrA and the quantity of bound lipids and detergent were determined, and the oligomeric state was analysed using SEC (size-exclusion chromatography) and analytical ultracentrifugation. The activity and the quaternary structure of BmrA appeared to be strongly influenced by the type and concentration of the detergent used. SEC data showed that BmrA could be purified in a functional form in 0.05 and 0.01% DDM (n-dodecyl-beta-D-maltoside) and was homogeneous and monodisperse with an R(s) (Stokes radius) of 5.6 nm that is compatible with a dimer structure. Sedimentation-velocity and equilibrium experiments unequivocally supported that BmrA purified in DDM is a dimer and excluded the presence of other oligomeric states. These observations, which are discussed in relation to results obtained in proteoliposomes, also constitute an important first step towards crystallographic studies of BmrA structure.

ATP-Binding Cassette Transporters↗
PubMed2003

A multiparameter flow cytometric analysis of the effect of bexarotene on the epidermis of the psoriatic lesion.

BACKGROUND: A new retinoid, bexarotene (Targretin), was recently investigated in a large multicentre trial for its efficacy and safety in psoriasis. Bexarotene is a novel retinoid X receptor (RXR)-selective ligand. OBJECTIVES: The aim was to study the effect of bexarotene in psoriasis by analysing markers for epidermal differentiation, proliferation and inflammation in epidermal single cell suspensions from lesions of patients with psoriasis treated with various doses of bexarotene. METHODS: Thirty-four patients with moderate to severe plaque psoriasis participated in this study and were assigned in sequence to four different dose regimens: 0.5, 1, 2 and 3 mg kg-1 once daily. Before and after 12 weeks of bexarotene treatment, punch biopsies were taken from lesional skin from which epidermal single cell suspensions were prepared using an optimized thermolysin protocol. A sum of scores was determined for each biopsy site, based on a four-point scale for erythema, induration and desquamation. An improved multiparameter flow cytometric assay was used that enabled simultaneous assessment of epidermal proliferation, various aspects of differentiation and epidermal inflammation. The following variables were measured simultaneously: relative DNA content, relative cell size, keratin (K) 10, K6 and vimentin expression. RESULTS: The psoriasis area and severity index (PASI) and sum of scores for the individual psoriatic lesion each showed a statistically significant decrease of 28% after 12 weeks of bexarotene treatment (P < 0.001). However, no significant dose-response effect was found. The total percentage of K10+ cells showed a significant increase of 43% (P < 0.01). The total population of K6 expressing cells did not show significant changes. Regarding the subpopulations of K6 single, K10 single and K6 and 10 co-expressing cells, a significant increase of 77% was seen in the K10+ K6- cells (P < 0.05), a significant decrease of 33% in K10- K6+ cells (P < 0.01), and no significant changes in the remaining population of K10+ K6+ cells. After 12 weeks of treatment with bexarotene no significant changes in epidermal proliferation and inflammation were shown. CONCLUSIONS: The present study indicates a direct effect of RXR activation by bexarotene on the transition of proliferation-associated keratinization into normal keratinization. Although no direct effect of bexarotene on DNA content in the total K10- cells was shown, further studies on subpopulations within the germinative layer such as stem cells and transit amplifying cells might be worthwhile.

Adult↗
PubMed2002

Topical 5-ALA photodynamic therapy for the treatment of cutaneous T-cell lymphoma.

Therapeutic options for cutaneous T cell lymphoma (CTCL) include topical steroids, topical chemotherapy and phototherapy. Patients with limited disease that is unresponsive to these therapies present a particular challenge. We report successful treatment of a patient with two plaques of CTCL using topical photodynamic therapy (PDT). 5-aminolaevulinic acid (5-ALA) was applied 6-24 h preillumination with 100 J/cm2 red light. Treatment was repeated on four occasions with clinical and histological clearance. ALA-PDT may be a useful addition to the therapeutic options for CTCL. Further studies are required to define optimal treatment protocols.

Aminolevulinic Acid↗
PubMed2005

Spinal muscular atrophy carrier screening by multiplex polymerase chain reaction using dried blood spot on filter paper.

Spinal muscular atrophy (SMA) is a common, often fetal, autosomal recessively inherited disease leading to progressive muscle wasting and paralysis as a result of degeneration of anterior horn cells of the spinal cord. The SMA-determining gene, called the survival of motor neuron gene (SMN), is present on 5q13 in two nearly identical copies, telomeric SMN (SMN1) and centromeric SMN (SMN2). It has been established that SMA is caused by mutations in SMN1 whereas homozygous deletion of SMN2 has apparently no pathological consequences. The aim of this study is to develop an easy and inexpensive method for the isolation of high-quality template DNA from blood samples for SMA carrier screening by multiplex polymerase chain reaction. We have developed a protocol that optimizes detection of the SMN1 copy number in the human genome, producing a specific and sensitive assay using DNA extracted from a dried blood spot on IsoCode paper.

Chromosomes, Human, Pair 5↗
PubMed1993

Differential effect of cryopreservation on natural killer cell and lymphokine-activated killer cell activities.

The use of lymphokine-activated killer (LAK) cell therapy in delayed treatment requires the use of cryopreserved effector cells. The purpose of this study was to determine the optimal cryopreservation protocol for the maintenance of cytotoxic activity in mononuclear cells (MNCs). MNCs were cryopreserved with dimethyl sulfoxide or 1,2-propanediol before and after 3 days of culture with recombinant interleukin 2. The effects of cryopreservation on cell recovery, LAK cell and natural killer (NK) cell cytotoxic activities, and surface antigen markers were studied. Recovery of nonactivated MNCs was higher with 1,2-propanediol than with dimethyl sulfoxide (p < 0.05). Cytotoxic activities, measured with a 51Cr release assay, significantly decreased after thawing, on both activated cells (76.3%; range, 35.8-92.2%) and fresh cells (54.6%; range, 17.5-75.4%). A 6-day kinetic test was used to compare the cytotoxic activity of cryopreserved and fresh cells. The results showed different patterns for NK cells (cryopreserved cells had lower levels of activity than fresh cells) and LAK cells (cryopreserved cells had higher levels of activity than fresh cells). Phenotype changes of effector cells in culture, with and without cryopreservation, were monitored by flow cytometry using monoclonal antibodies. These results were compared with changes in the cytotoxicity of cells with and without cryopreservation. After thawing, there was a decrease in MNCs expressing CD14 and CD56. Recovery of the CD56 marker correlates with increased cytotoxic activity. Despite some loss of NK cell activity, it is concluded that MNCs may be successfully cryopreserved before their use in immunotherapeutic treatment.

CD4-CD8 Ratio↗
PubMed1997

Preapheresis peripheral blood CD34+ mononuclear cell counts as predictors of progenitor cell yield.

BACKGROUND: Peripheral blood progenitor cells, harvested by apheresis after mobilization, provide rapid hematologic recovery after high-dose chemotherapy. However, because harvesting these cells is expensive and time-consuming, there has been much interest in optimizing collection protocols. An investigation was made to determine whether, in this clinical setting, peripheral blood progenitor cell yields may be predicted from preapheresis progenitor cell counts, allowing the length of each procedure to be "fine tuned" to achieve specific target goals. STUDY DESIGN AND METHODS: Preapheresis peripheral blood CD34+ cell and total colony-forming cell counts were assessed before 78 peripheral blood progenitor cell collections from 13 consecutive patients were performed. Preapheresis counts were correlated with actual progenitor cell yields. Factors affecting this correlation were analyzed. RESULTS: With the use of linear regression analysis preapheresis progenitor cell counts were found to correlate significantly but weakly with actual yields per kg of body weight per liter of blood processed (CD34+ cells: r = 0.43; colony-forming cells: r = 0.56). Further analysis revealed two possible causes: 1) circulating progenitor cell concentrations fluctuate widely during harvest, which implies that preapheresis counts are not representative of actual concentrations during apheresis, and 2) the efficiency with which apheresis machines extract mononuclear cells varies greatly between procedures. CONCLUSION: Preapheresis CD34+ and colony-forming cell counts correlated poorly with subsequent yields in this clinical setting, which suggests that it is not practical to use such counts to predict with certainty the length of apheresis needed to achieve a target yield.

Adult↗
PubMed2005

Mass screening with CT colonography: should the radiation exposure be of concern?

BACKGROUND & AIMS: Computed tomography colonography (CTC), particularly using noncathartic techniques, has the clear potential to increase compliance for colorectal cancer screening. Because the geometry for CTC is highly advantageous, it can be performed with lower radiation doses than almost any other CT examination. If CTC were to become a standard screening tool for the population age 50 years and older, the potential market in the United States would soon be over 100 million people. Therefore, it is pertinent to consider the radiation exposure and any potential radiation risk to the population from such a mass CTC screening program. METHODS: Organ doses from CTC examinations can be estimated with standard techniques. These doses can be applied to organ- and dose-specific radiation cancer risk estimates to estimate the excess cancer risk resulting from the radiation from a paired (supine and prone) CTC examination. RESULTS: The cancer risks associated with the radiation exposure from CTC are unlikely to be zero, but they are small. A best estimate for the absolute lifetime cancer risk associated with the radiation exposure using typical current scanner techniques is about 0.14% for paired CTC scans for a 50-year-old, and about half that for a 70-year-old. These values probably could be reduced by factors of 5 or 10 with optimized CTC protocols. CONCLUSIONS: In terms of the radiation exposure, the benefit-risk ratio potentially is large for CTC.

Colonography, Computed Tomographic↗
PubMed2002

Hepatitis B: progress in the last 15 years.

1. Patients undergoing orthotopic liver transplantation (OLT) for hepatitis B without effective prophylaxis have a high risk for recurrent infection and severe graft damage, leading to death or re-OLT. 2. Long-term prophylaxis with hepatitis B immune globulin (HBIg) significantly reduces the risk for hepatitis B virus (HBV) recurrence and increases survival. Patients with detectable HBV DNA at the time of OLT have a high risk for recurrence despite HBIg prophylaxis. 3. Lamivudine (LAM) therapy for patients with decompensated HBV cirrhosis before OLT results in inhibition of viral replication and clinical improvement. Its efficacy is limited by the frequent emergence of LAM-resistant YMDD mutations. The ideal length of therapy with LAM pre-OLT has not yet been defined. 4. Prophylaxis of HBV recurrence with LAM monotherapy is not recommended because of the reappearance of hepatitis B surface antigen after OLT in approximately 50% of patients. 5. LAM is the best available treatment for patients with established recurrent hepatitis B. Long-term therapy is associated with the emergence of drug-resistant mutants in up to 60% of patients. Severe hepatitis and liver failure have been described among liver transplant recipients with YMDD mutations. 6. Combination therapy with HBIg and LAM prevents HBV recurrence in 90% to 100% of patients who undergo OLT for hepatitis B. The optimal HBIg protocol in the LAM era is yet to be defined. 7. Preliminary studies suggest that adefovir dipivoxil inhibits HBV replication in patients infected with LAM-resistant HBV strains. 8. Fifteen years ago, hepatitis B was regarded as a relative or absolute contraindication for OLT. Today, hepatitis B is a universally accepted indication for OLT.

2-Aminopurine↗
PubMed2002

Botulinum toxin type A (BOTOX) for treatment of migraine.

An open-label study and 2 double-blind, placebo-controlled studies have provided supporting evidence of botulinum toxin type A (BTX-A) as an effective, well-tolerated treatment for migraine. Observed durations of benefit were consistent with known properties of BTX-A. Findings suggest that response may vary by features of preinjection headaches, such as migraine frequency. The precise mechanism by which BTX-A provides pain relief is hypothesized to be related not only to acetylcholine inhibition but also to a blocking action on the parasympathetic nervous system. Additional studies that control factors likely to be related to response may lead to better understanding of the BTX-A effect on migraine and an optimal treatment protocol.

Blepharoptosis↗
PubMed2001

Botulinum toxin type A (BOTOX) for treatment of migraine.

An open-label study and 2 double-blind, placebo-controlled studies have provided supporting evidence of botulinum toxin type A (BTX-A) as an effective, well-tolerated treatment for migraine. Observed durations of benefit were consistent with known properties of BTX-A. Findings suggest that response may vary by features of preinjection headaches, such as migraine frequency. The precise mechanism by which BTX-A provides pain relief is hypothesized to be related not only to acetylcholine inhibition but also to a blocking action on the parasympathetic nervous system. Additional studies that control factors likely to be related to response may lead to better understanding of the BTX-A effect on migraine and an optimal treatment protocol.

Adult↗
PubMed2001

Cryopreservation of Digitalis obscura selected genotypes by encapsulation-dehydration.

Shoot-tips from several genotypes of the cardenolide-producing perennial shrub Digitalis obscura L. were successfully cryopreserved using the encapsulation-dehydration technique. Precultivation on MS medium containing 0.5 M sucrose, followed by 2.5 h dehydration (final weight 30 %) induced shoot regrowth in 42 % of cryopreserved shoot-tips. Cold-hardening of the in vitro cultures before sucrose treatment dramatically increased shoot recovery up to 86 %. The optimized cryopreservation protocol was then employed using different shoot cultures from five D. obscura genotypes. Responses to cryopreservation depended mainly on the genotype, best results being obtained when shoot tips from HU3 and LL11 were used. Prolonged subcultures reduced proliferation rates in both control and cryopreserved HU3 shoot-tips, whereas long-term storage in LN did not affect the shoot recovery rate of the genotype. RAPD markers were employed to evaluate possible somaclonal variation occurring in shoots regenerated through successive subcultures and after cryopreservation. The band patterns revealed differences between the original parent plant and the shoots grown in vitro, especially after a prolonged subculture (84.9 % of matches for HU3 after 16 subcultures vs. 93.4 % for AY3 after 2 subcultures). Nevertheless, match percentages were higher (98.6 % to 99.5 %) when band patterns from subcultured shoots were compared to those obtained from their respective control or frozen progenies indicating that cryopreservation ensure genetic stability of selected Digitalis obscura genotypes.

Acclimatization↗
PubMed2002

Weekly oxaliplatin, high-dose folinic acid and 24h-5-fluorouracil (FUFOX) as salvage therapy in metastatic colorectal cancer patients pretreated with irinotecan and folinic acid/5-fluorouracil regimens.

UNLABELLED: Irinotecan (CPT-11), oxaliplatin (OXA) and different folinic acid(FA) modulated 5-fluorouracil (5-FU) regimens are active as first-and second-line chemotherapy of metastatic colorectal cancer. However, the best palliative sequence of these substances is still unclear. After CPT-11 containing regimens the optimal salvage protocol has not yet been defined. Here, we retrospectively analysed the weekly ambulant combination of OXA with continuous FA/5-FU (FUFOX) after two different CPT-11 containing chemotherapeutic regimens. PATIENTS: During October 1999 and May 2001, 20 patients (median 62; 48-74 years) were included who had disease progression after CPT-11 /bolus FA/5-FU (Saltz; 7 patients, group A) or after FA/5-FU followed by CPT-11 alone (13 patients, group B). OXA(60 mg/m2) was given for 2 hours prior to FA (500 mg/m2) as 2 h-infusion and continuous 5-FU (2.600 mg/m2) for 24 h-infusion on day 1,8,15 and 22 (repeated after week 6). RESULTS: FUFOX was administered 252 times. About 1,203 mg OXA per patient was given. Toxicities NC-CrC grade 3 were observed in 10 patients: diarrhoea (4), mucositis (5), nausea/vomiting (2), anaemia (1), leucopenia (1), thrombopenia (1) and hand-foot-syndrome (1), 3 patients showed minor remissions, II patients stable disease. The median time to progression was 16(0-39) weeks. The median survival from start of FUFOX and from start of any palliative therapy was 33 (5-65) and 99 (44-200) weeks for all patients, respectively. CONCLUSIONS: FUFOX was efficient for additional tumour control in 70% of patients pretreated with CPT-11/5-FU based regimens. Sequential palliative treatment can lead to prolonged survival.

Aged↗
PubMed2003

High-resolution computed tomography techniques in diffuse parenchymal lung disease and their application to clinical practice.

The remarkable ability of high-resolution computed tomography (HRCT) to provide sufficient detail of both normal and abnormal pulmonary anatomy requires high-quality examinations with considerable attention paid to the technique. Despite the use of optimal scanning protocols, there are well-known limitations of sequential scanning that explain the recent interest in the use of multislice spiral CT for diagnosing lung diseases in routine clinical practice. A basic knowledge of the most recent developments in spiral CT technology appears a necessary prerequisite for those clinicians, pulmonologists, internists, or thoracic surgeons who are involved in the management of diffuse infiltrative lung diseases. This chapter reviews the various aspects of multislice spiral CT technology applied to routine clinical evaluation of diffuse infiltrative lung diseases.

Journal Article↗
PubMed2005

Cryopreservation in assisted reproductive technology: new trends.

During the last few years, cryopreservation has become a relevant addition to therapeutic concepts in reproductive medicine. New data and publications have made it difficult to maintain an overview of all of the new developments and their results. The focus of interest more recently, especially with the cryopreservation of human oocytes and human ovarian tissue, has been vitrification as an interesting alternative to slow freezing methods. Even though studies investigating the slow freezing of human mature oocytes have resulted in very different survival rates, it could be an option for donor oocyte programs, in the case of threatened ovarian loss or when there is an objection to embryo freezing. An optimal freezing protocol and later use of thawed human ovarian tissue is still a point of discussion. There are encouraging results regarding different kinds of autotransplantation, and recently the first birth after orthotopic autotransplantation of cryopreserved/thawed human ovarian tissue was described in the literature. Independent of any objections to cryopreservation in general, vitrification is a potential and effective alternative to conventional slow cryopreservation, especially for oocytes and embryos. Vitrification might be also be an option for human ovarian tissue; however this is only in its infancy and requires much additional investigation. Our article discusses new trends and results of actual studies regarding these issues.

Cryopreservation↗
PubMed2006

Obstetrical antiphospholipid syndrome.

Antiphospholipid syndrome (APS) is an autoimmune disease manifested by a thrombotic or obstetrical event in the presence of antiphospholipid antibodies. Obstetrical APS was initially described in the 1950s. Obstetrical features presently include recurrent pre-embryonic and embryonic miscarriage, fetal demise, preeclampsia, intrauterine growth restriction, and possibly placental abruption. Since the first description of obstetrical APS, researchers have unraveled some of the pathophysiology involved in the disease. At present, there are numerous antiphospholipid antibodies that can be measured in human serum, each of which requires evaluation with regard to whether an association with obstetrical events exists. Clinical trials have provided some insight into optimal treatment protocols, but to date, such trials are limited. On the basis of small, randomized trials, live birth rates of approximately 70 to 80% have been reported with the use of low-dose acetylsalicylic acid and heparin. Despite such encouraging results, ongoing pregnancies are fraught with maternal and fetal morbidity. This review will highlight the obstetrical morbidity associated with APS, discuss the limitations of the present criteria for diagnosis, appraise published treatment trials, and summarize directions for future study of obstetrical APS.

Abortion, Habitual↗