Engineered human Tmpk fused with truncated cell-surface markers: versatile cell-fate control safety cassettes.

Cell-fate control gene therapy (CFCGT)-based strategies can augment existing gene therapy and cell transplantation approaches by providing a safety element in the event of deleterious outcomes. Previously, we described a novel enzyme/prodrug combination for CFCGT. Here, we present results employing novel lentiviral constructs harboring sequences for truncated surface molecules (CD19 or low-affinity nerve growth factor receptor) directly fused to that CFCGT cDNA (TmpkF105Y). This confers an enforced one-to-one correlation between cell marking and eradication functions. In-vitro analysis demonstrated the full functionality of the fusion product. Next, low-dose 3'-azido-3'-deoxythymidine (AZT) administration to non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice injected with transduced clonal K562 cells suppressed tumor growth; furthermore, one integrated vector on average was sufficient to mediate cytotoxicity. Further, in a murine xenogeneic leukemia-lymphoma model we also demonstrated in-vivo control over transduced Raji cells. Finally, in a proof-of-principle study to examine the utility of this cassette in combination with a therapeutic cDNA, we integrated this novel CFCGT fusion construct into a lentivector designed for treatment of Fabry disease. Transduction with this vector restored enzyme activity in Fabry cells and retained AZT sensitivity. In addition, human Fabry patient CD34(+) cells showed high transduction efficiencies and retained normal colony-generating capacity when compared with the non-transduced controls. These collective results demonstrated that this novel and broadly applicable fusion system may enhance general safety in gene- and cell-based therapies.

Evaluation of human papillomavirus-consensus primers for HPV detection by the polymerase chain reaction.

Cervical cancer is one of the most frequently found cancers in women and appears to have a viral aetiology. Substantial evidence points to the human papillomaviruses (HPV) as the infectious agents and there is considerable interest in identifying and accurately typing the viruses. Since HPVs now comprise more than 100 different HPV types, the polymerase chain reaction (PCR) has been the preferred methodology for virus identification and typing on isolated DNA. In that context, five commonly employed PCR consensus primers have been evaluated for the detection and typing of HPV. The five consensus primer pairs were derived from the consensus sequences of either the L1 and E1 open reading frames. All primers exhibited approximately equal sensitivity, as defined by the ability to detect HPV DNA, on a series of standard HPV DNA-containing preparations. However, the five primer pairs performed differently on 24 HPV-positive and 34 HPV-negative samples obtained from cervical scrapes which had been typed by type-specific PCR for HPV 6/11, 16, 18 and 33. The values for agreement between identification of samples by a HPV type-specific PCR and the consensus primer PCR were 78, 84, 91, 93 and 98%. Three samples, which were positive with only one of the five consensus primer pairs and were negative with the PCR for HPV types 6/11, 16, 18 and 33, contained other HPV sequences or HPV-related sequences as determined by DNA sequence analysis. To our knowledge, this report represents the first extensive comparison of five different consensus primers in a polymerase chain reaction for the detection of HPV. Our results suggest that PCR typing for human papillomaviruses requires more than one consensus primer pair to identify all HPV-infected samples.

The measurement of ambulatory impairment in multiple sclerosis.

The objective of this study was to examine the relationships between continuous measures of ambulatory impairment in MS patients and their ordinal counterparts. Much of the disability caused by MS is due to ambulatory impairment. The Expanded Disability Severity Scale (EDSS) and the Ambulation Index (AI) are ordinal measures of MS severity based largely on the maximal distance subjects can walk (Dmax) and the time to walk 8 m (T8), respectively. At EDSS levels 6.0 to 7.0 and AI levels 3 to 6, scores are defined more by the use of ambulatory aids, rather than by Dmax or T8. We determined Dmax (up to 500 m), T8, the EDSS score, and the AI in 237 ambulatory MS patients. The maximal distance subjects could walk and T8 were strongly related to their ordinal counterparts (Spearman r = 0.65 and 0.91, respectively), but the continuous measures showed considerable variability within EDSS and AI levels that the ordinal scales did not reflect. Most of the variability occurred at EDSS levels 6.0 to 7.0 and AI levels 3 to 6. Because the use of an aid did not clearly predict Dmax or T8, many patients in these ranges had better ambulatory function based on the continuous measures than those with less disability according to the ordinal scales. We found that Dmax and T8 provide more precise information about ambulatory impairment in MS than do the EDSS and AI, allowing better discrimination of differences between patients and potentially greater sensitivity to detect therapeutic effects in clinical trials.

Agreement study between two laboratories of immunofluorescence as a confirmatory test for human immunodeficiency virus type 1 antibody screening.

A total of 114 serum specimens from 76 blood donors, 21 patients with acquired immune deficiency syndrome or acquired immune deficiency syndrome-related complex, 7 multiply transfused patients, 3 hemophiliacs, and 7 others were tested for anti-human immunodeficiency virus type 1 (HIV-1) antibody by enzyme immunoassay (EIA) and Western blot (WB) and then blindly tested by immunofluorescence (IF), independently, in two separate laboratories. The IF technique used acetone-fixed HIV-1-infected E cells and uninfected HUT-78 cells mixed at a 1:3 ratio in one spot on a glass slide and uninfected HUT-78 cells (to assess nonspecific fluorescence) alone in a second spot. Of 114 serum specimens, 85 were repeat EIA positive, and 21 of these were WB positive. A total of 129 of 134 of the IF results (included were 20 duplicates) were identical between laboratories, for a Kappa agreement statistic of 0.93. All five IF results discordant between laboratories were EIA repeat positive and WB negative. Included in the study were eight WB-indeterminate sera, of which five blood donor serum specimens and one hemophiliac serum specimen were IF negative and two acquired immune deficiency syndrome serum specimens were IF positive. As a confirmatory test for HIV-1 antibodies, IF provided a faster alternative or supplementary test for confirming EIA results.

Lithium and hydrocortisone interactions on cell growth and gene expression in human promyelocytic leukemia (HL60).

Lithium (Li) and hydrocortisone (HC) modify gene expression as well as stimulate human hematopoiesis in vivo and in vitro. The responses of blood-forming tissues, to these natural substances individually, are closely similar. However, clonogenic co-culture experiments, using normal human bone marrow cells, have revealed that the effects of Li and HC are not additive but rather mutually inhibitory. Studies of HL-60 cells in suspension indicate that growth of this human promyelocytic leukemic line is enhanced by HC at 10(-6) M, a "therapeutic" plasma concentration. This phenomenon is abrogated by the co-addition of Li at 3 x 10(-4) M, likewise a "therapeutic" plasma concentration. Neither substrate exerts any influence on the morphological or cytochemical features of differentiation which accompany exposure of HL-60 cells to dimethyl sulphoxide (DMSO), retinoic acid (RA), tetradecanoyl phorbol acetate (TPA) or sodium butyrate. In contrast, the expression of c-fms, a cellular proto-oncogene which codes for the CSF-1 (monocyte-macrophage colony-stimulating factor) surface membrane receptor, is modified by Li in the presence of the differentiation induction agents DMSO and RA which promote the development of mature granulocytes from HL-60 cells. These observations afford further insights into the humoral mechanisms which modulate the expression of genes involved in the regulation of hematopoiesis.

Effect of sample holding, cryopreservation, and storage on the human lymphocyte cytogenetic test.

In monitoring occupational populations with the human lymphocyte cytogenetic test, it is not always possible to collect and process matched samples on the same day, even though this would be desirable to control for technical variables. The effects of holding samples for 24 hours at 4 degrees C and 22 degrees C, freezing lymphocytes in dimethylsulfoxide at -180 degrees C, and keeping fixed cells for 6 days at 4 degrees C before slide-making were examined. Final cell count, mitotic index, percentage of cells in first division, percentage of cells with chromosomal aberrations, and sister chromatid exchange per cell were measured in paired cultures. A significant increase in the frequency of cells with chromatid breaks occurred after prolonged fixation, so all other results were obtained from freshly fixed cells. Although holding at 22 degrees C and cryopreservation had significant effects on the mitotic index and entry of lymphocytes into the cell division cycle, the cytogenetic endpoints were not affected by any of the sample manipulations. Thus, samples can be held at 4 degrees C or 22 degrees C for 24 hours, or frozen lymphocytes can be stored for at least a week, without altering the cytogenetic endpoints.

[The right work for every disabled person: the ERTOMIS ability and requirement profiles for the appropriate determination of a workplace]. / Jedem Behinderten die richtige Arbeit--Die ERTOMIS Fähigkeits- und Anforderungsprofile zur angemessenen Bestimmung des Arbeitsplatzes.

In close accordance with the "International Classification of Impairments, Disabilities and Handicaps" issued by the World Health Organisation in 1980, and in cooperation with medical doctors, psychologists, and vocational teachers a reintegration of disabled adults. Instead of only attesting to the rehabilitated person his disabilities this system allows qualified persons to assess by means of a set of 64 evaluation criteria in an "Ability Profile" the abilities available to the disabled after rehabilitation; likewise, using the same 64 criteria it allows one to determine in a "Requirement Profile" those requirements a particular job poses to the corresponding person. The two profiles can be compared, thus providing an easy match between the person's abilities and the abilities necessary to perform the job. The various criteria reflect "elementary abilities" or correspondingly "elementary requirements". By comparison of the two profiles it is possible--in a first rough approach based on correspondingly developed judging abilities of the evaluator--to determine rather accurately whether a certain kind of work, to which a "Requirement Profile" is available, is adequate for a certain disabled person--for whom an "Ability Profile" has also been developed. The question whether the chosen work is fully adequate can thereby be answered with maximum accuracy only at the corresponding workplace by an occupational physician and the other professionals responsible for the employment of the disabled. The ERTOMIS system thus facilitates the job placement of disabled people; it can also help decide on eventual complementary rehabilitation measures directed towards the better adjustment of the disabled to a certain work, or the improved adjustment of that work to the abilities of the disabled. Each disability is unique to each individual; by means of this system system--completed with the personal dossier from which data on the vocational training and experience of the disabled can be taken--it can also be assessed in a way that reflects this important individuality. The criteria describe only abilities and requirements relevant to work performance. If one so chooses, for data protection reasons the "Ability Profile" can be handed out to the disabled person only. No copies of documents may be kept except for the purpose of medical records.

Abilities and requirements profiles: a tool to facilitate reintegration of people with disabilities into employment.

An attempt has been made to construct tools that can be applied for assessment of a disabled individual's residual abilities, and of the requirements of a job for which that person might be considered. The system is based on 63 attributes, which are graded on a 4-point scale. Initial tests revealed 90-95% correspondence between independent assessments. Although more detailed assessment tests are under way, the approach does offer a means for rough preselection of disabled candidates for jobs appropriate to their residual abilities.