Adverse health outcomes among people who inject drugs who engaged in recent sex work: findings from a national survey.

OBJECTIVES: This study explores trends in sex work among people who inject drugs (PWID) by gender and the relationship between sex work and adverse health outcomes including overdose, injection-site, and blood-borne virus (BBV) infections. STUDY DESIGN: The Unlinked Anonymous Monitoring Survey of PWID is an annual cross-sectional survey that monitors BBV prevalence and behaviours, including transactional sex, among PWID recruited through specialist services in England, Wales, and Northern Ireland. METHODS: Trends in sex work among PWID (2011-2021) were described. Data were analysed to assess differences between PWID who engaged in sex work in the past year (sex workers [SWs]) and those who did not (non-SWs) by gender (Pearson Chi2 tests) (2018-2021). Associations between sex work in the past year and adverse health outcomes were investigated using logistic regression. RESULTS: Between 2011 and 2021, sex work among PWID remained stable, with 31% of women and 6.3% of men who inject, reporting having ever engaged in sex work, and 14% of women and 2.2% of men engaging in sex work in the past year. Between 2018 and 2021, SWs had greater odds of reporting symptoms of an injection-site infection (adjusted odds ratio (aOR): 1.68 [95% confidence interval {CI}: 1.31-2.16], P < 0.001) and reporting overdose (aOR: 2.21 [CI: 1.74-2.80], P < 0.001) than non-SWs had in the past year. Among men, SWs had 243% greater odds of having HIV than non-SWs (aOR: 3.43 [CI: 1.03-11.33], P = 0.043). CONCLUSIONS: Our findings highlight disproportionate vulnerability and intersection of overlapping risk factors experienced by PWID SWs and a need for tailored interventions which are inclusive and low-threshold.

Experiences of professional autonomy among critical care nurses in Kenya: a qualitative study

Exercising autonomy allows nurses to demonstrate their knowledge and skills. However, more information is needed about the critical care nurses' perceptions of their professional autonomy in rural areas. Objective: To explore the perceptions, facilitators and barriers to professional autonomy among critical care nurses in rural Kenya. Methods: The hermeneutic phenomenological study design was used in this study. Data were collected in a critical care unit using a semi-structured interview guide. A sample of 10 participants were recruited. The study was conducted in Nyeri County Referral Hospital. Results: Three themes emerged from the study on the nurses' experiences of professional autonomy, perceptions of autonomy, facilitators of autonomy, and barriers to autonomy. Conclusion: Autonomy undeniably plays a pivotal role in defining the professional status of the nursing profession. Perceptions, facilitators and barriers to professional autonomy form the background of the current professional status. Nurses, therefore, exercise autonomy effectively when it aligns with patient care needs and when a conducive environment supports it.

Talker discontinuity disrupts attention to speech: Evidence from EEG and pupillometry.

Speech is processed less efficiently from discontinuous, mixed talkers than one consistent talker, but little is known about the neural mechanisms for processing talker variability. Here, we measured psychophysiological responses to talker variability using electroencephalography (EEG) and pupillometry while listeners performed a delayed recall of digit span task. Listeners heard and recalled seven-digit sequences with both talker (single- vs. mixed-talker digits) and temporal (0- vs. 500-ms inter-digit intervals) discontinuities. Talker discontinuity reduced serial recall accuracy. Both talker and temporal discontinuities elicited P3a-like neural evoked response, while rapid processing of mixed-talkers' speech led to increased phasic pupil dilation. Furthermore, mixed-talkers' speech produced less alpha oscillatory power during working memory maintenance, but not during speech encoding. Overall, these results are consistent with an auditory attention and streaming framework in which talker discontinuity leads to involuntary, stimulus-driven attentional reorientation to novel speech sources, resulting in the processing interference classically associated with talker variability.

Preliminary indications of the burden of COVID-19 among people who inject drugs in England and Northern Ireland and the impact on access to health and harm reduction services.

OBJECTIVE: The aim of the study was to describe the impact of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic on people who inject drugs (PWID) in England, Wales and Northern Ireland. STUDY DESIGN: This is a cross-sectional Unlinked Anonymous Monitoring (UAM) Survey of PWID. METHODS: People who had ever injected psychoactive drugs were recruited to the UAM Survey by specialist drug/alcohol services in England, Wales and Northern Ireland. From June 2020, in addition to providing a dried blood spot sample and completing the UAM behavioural questionnaire, participants were asked to complete an enhanced coronavirus disease 2019 (COVID-19) questionnaire. Preliminary data are presented to the end of October and were compared with data from the 2019 UAM Survey, where possible. RESULTS: Between June and October, 288 PWID were recruited from England and Northern Ireland. One in nine (11%; 29/260) PWID reported testing positive for SARS-CoV-2 or experiencing COVID-19 symptoms. Fifteen percent (26/169) reported injecting more frequently in 2020 than in 2019; cocaine injection in the preceding four weeks increased from 17% (242/1456) to 25% (33/130). One in five PWID (19%; 35/188) reported difficulties in accessing HIV and hepatitis testing, and one in four (26%; 47/179) reported difficulties in accessing equipment for safer injecting. CONCLUSIONS: Our preliminary findings suggest that PWID have experienced negative impacts on health, behaviours and access to essential harm reduction, testing and treatment services owing to the COVID-19 pandemic. Continued monitoring through surveillance and research is needed to understand the subsequent impact of COVID-19 on blood-borne virus transmission in this population and on health inequalities.

Are there missed opportunities for vaccinating against hepatitis B among people who inject drugs in the UK?

Sustaining the impact of hepatitis B virus (HBV) vaccination on incidence and prevalence of HBV infection requires increasing and maintaining the uptake of vaccine among those at risk. In recent years, the level of vaccine uptake among people who inject drugs (PWID) in the UK has levelled-off. Data (2015-2016) from the national unlinked-anonymous monitoring survey of PWID, an annual survey that collects data from PWID across England, Wales and Northern Ireland, were used to examine HBV vaccine uptake. Data from participants who had injected drugs during the previous year were used to investigate sources of hepatitis B vaccine doses as well as factors associated with vaccine uptake. Among the 3175 anti-HBc-negative participants, 3138 (99%) reported their vaccination status; 23% (714) reported no vaccine uptake. Among those not vaccinated, 447 (63%) reported being sexually active and 116 (16%) reported sharing needles and syringes. Majority of those not vaccinated reported accessing services in the previous year that could have provided hepatitis B vaccine doses. These missed opportunities for vaccinating of PWID indicate a need for additional targeted interventions.

Low levels of hepatitis C diagnosis and testing uptake among people who inject image and performance enhancing drugs in England and Wales, 2012-15.

INTRODUCTION: People injecting image and performance enhancing drugs (IPEDs) have traditionally not been perceived as being at high risk of hepatitis C virus (HCV) infection. However, recent studies indicate the HCV antibody (anti-HCV) prevalence in this group is 10-times that in the general population. HCV testing uptake and undiagnosed infections are examined using data from a voluntary unlinked-anonymous survey. METHOD: People injecting IPEDs across England and Wales completed a short bio-behavioural survey (2012-15). Anti-HCV status and self-reports of HCV testing were used in the analysis. RESULTS: The participants median age was 31 years, 98% were men, 14% had also injected psychoactive drugs and the anti-HCV prevalence was 4.8% (N=564). Among those who had never injected psychoactive drugs the anti-HCV prevalence was 1.4%; among those who had recently injected psychoactive drugs (preceding 12 months) prevalence was 39% and among those who had done this previously 14% (p<0.001). Overall, 37% had been tested for HCV: among those who had recently injected psychoactive drugs 78% had been tested, as had 56% of those who had injected psychoactive drugs previously; 33% of those never injecting psychoactive drugs were tested (p<0.001). Overall, 44% of those with anti-HCV were aware of this; however, only 14% of those who had never injected psychoactive drugs were aware. CONCLUSIONS: One-in-twenty people who inject IPEDs have anti-HCV. HCV infections among those who had never injected psychoactive drugs were mostly undiagnosed, though this group had a lower prevalence. Targeted HCV testing interventions are also needed for those injecting IPEDs.

Suitability analysis for rice growing sites using a multicriteria evaluation and GIS approach in great Mwea region, Kenya.

Land suitability analysis is a prerequisite to achieving optimum utilization of the available land resources. Lack of knowledge on best combination of factors that suit production of rice has contributed to the low production. The aim of this study was to develop a suitability map for rice crop based on physical and climatic factors of production using a Multi-Criteria Evaluation (MCE) & GIS approach. The study was carried out in Kirinyaga, Embu and Mberee counties in Kenya. Biophysical variables of soil, climate and topography were considered for suitability analysis. All data were stored in ArcGIS 9.3 environment and the factor maps were generated. For MCE, Pairwise Comparison Matrix was applied and the suitable areas for rice crop were generated and graduated. The current land cover map of the area was developed from a scanned survey map of the rice growing areas. According to the present land cover map, the rice cultivated area was 13,369 ha. Finally, we overlaid the land cover map with the suitability map to identify variances between the present and potential land use. The crop-land evaluation results of the present study showed that, 75% of total area currently being used was under highly suitable areas and 25% was under moderately suitable areas. The results showed that the potential area for rice growing is 86,364 ha and out of this only 12% is under rice cultivation. This research provided information at local level that could be used by farmers to select cropping patterns and suitability.

Investigating the impact of rice blast disease on the livelihood of the local farmers in greater Mwea region of Kenya.

Rice is the most important cereal crop in Kenya coming third after maize and wheat. It forms a very important diet for a majority of families in Kenya. The demand for rice in Kenya has seen a dramatic increase over the last few years while production has remained low. This is because rice production has been faced by serious constraints notably plant diseases of which the most devastating is rice blast. Rice blast is known to cause approximately 60% -100% yield losses. It is caused by an Ascomycete fungus called Magnaporthe Oryzae. The aim of this study was to investigate the impact of rice blast disease on the livelihood of the local farmers in Greater Mwea region and develop a rice blast disease distribution map using GIS approach. The study methodology employed a questionnaire survey which were subjected to sample population of households in the 7 sections with 70 blocks within Mwea region. The collected data was analysed using SAS Version 9.1. Descriptive statistics were used to summarize the household characteristics, the farm characteristics and the farmers' perceptions of rice blast disease. In the questionnaire, farmers' response on whether they had been affected by rice blast disease and the total production per acreage was used to develop an attribute table with GPS points. The GPS points were interpolated to create a geographical distribution map of rice blast disease. From the research findings almost all the farmers' had awareness and knowledge of rice blast disease, 98% of the farmers interviewed were aware of rice blast disease. Out of the 98% with knowledge and awareness 76% have been affected by the disease, while 24% have never been affected. Farmers attributed rice blast disease to a range of different causes, including excessive use of nitrogen fertilizer, water shortage, lack of proper drainage canal and due to climate change. Majority of the farmers interviewed (72%) did not engage themselves in any other socio-economic activity even after being affected by the rice blast disease. 15% opted to growing horticultural crops, 7% engaged in trading activities while 2% started livestock raring, wage earning and Boda boda business.

Evaluation of surveillance for surgical site infections in Thika Hospital, Kenya.

BACKGROUND: In low-income countries, surgical site infections (SSIs) are a very frequent form of hospital-acquired infection. Surveillance is an important method for controlling SSI but it is unclear how this can best be performed in low-income settings. AIM: To examine the epidemiological characteristics of various components of an SSI surveillance programme in a single Kenyan hospital. METHODS: The study assessed the inter-observer consistency of the surgical wound class (SWC) and American Society of Anesthesiologists (ASA) scores using the kappa statistic. Post-discharge telephone calls were evaluated against an outpatient clinician review 'gold standard'. The predictive value of components of the Centers for Disease Control and Prevention - National Healthcare Safety Network (CDC-NHNS) risk index was examined in patients having major obstetric or gynaecological surgery (O&G) between August 2010 and February 2011. FINDINGS: After appropriate training, surgeons and anaesthetists were found to be consistent in their use of the SWC and ASA scores respectively. Telephone calls were found to have a sensitivity of 70% [95% confidence interval (CI): 47-87] and a specificity of 100% (95% CI: 95-100) for detection of post-discharge SSI in this setting. In 954 patients undergoing major O&G operations, the SWC score was the only parameter in the CDC-NHNS risk index model associated with the risk of SSI (odds ratio: 4.00; 95% CI: 1.21-13.2; P = 0.02). CONCLUSIONS: Surveillance for SSI can be conducted in a low-income hospital setting, although dedicated staff, intensive training and local modifications to surveillance methods are necessary. Surveillance for post-discharge SSI using telephone calls is imperfect but provides a practical alternative to clinic-based diagnosis. The SWC score was the only predictor of SSI risk in O&G surgery in this context.

Prevalence and determinants of human papillomavirus infection and cervical lesions in HIV-positive women in Kenya.

BACKGROUND: We assessed the association of human papillomavirus (HPV) infection and cervical intraepithelial neoplasia (CIN) with various characteristics, CD4 count and use of combination antiretroviral therapy (cART) among HIV-positive women. METHODS: Cross-sectional study of 498 HIV-positive women who underwent HPV PCR-based testing, cytology, and systematic cervical biopsy. RESULTS: In all, 68.7% of women were HPV-positive, 52.6% had high-risk (hr) HPV, and 40.2% multiple type infections. High-risk human papillomavirus-positivity did not vary significantly by age but it was negatively associated with education level. The most frequent types in 113 CIN2/3 were HPV16 (26.5%), HPV35 (19.5%), and HPV58 (12.4%). CD4 count was negatively associated with prevalence of hrHPV (P<0.001) and CIN2/3 among non-users of cART (P=0.013). Combination antiretroviral therapies users (≥2 year) had lower hrHPV prevalence (prevalence ratio (PR) vs non-users=0.77, 95% confidence interval (CI): 0.61-0.96) and multiple infections (PR=0.68, 95% CI: 0.53-0.88), but not fewer CIN2/3. The positive predictive value of hrHPV-positivity for CIN2/3 increased from 28.9% at age <35 years to 53.3% in ≥45 years. CONCLUSION: The burden of hrHPV and CIN2/3 was high and it was related to immunosuppression level. Combination antiretroviral therapies ( ≥2 year) use had a favourable effect on hrHPV prevalence but cART in our population may have been started too late to prevent CIN2/3.

A preliminary comparison of flow cytometry and tube agglutination assays in detecting red blood cell-associated C3d.

This study compared flow cytometric analysis with tube agglutination assays for the detection of red blood cell (RBC)-associated complement and immunoglobulins (Igs). RBCs from 20 patients with reactive tube direct antiglobulin tests (DATs) were evaluated by flow cytometry with anti-C3d, anti-IgG, anti-IgM and anti-IgA. Serial samples were also tested from a patient at risk of passenger lymphocyte haemolysis. Results of flow cytometry and tube assays for anti-IgG were as follows: 12 of 20 samples reactive in both; six of 20 nonreactive in both; two of 20 discordant with a reactive tube and a nonreactive flow cytometry assay. Anti-C3d results showed nine of 20 reactive in both and 11 of 20 discordant with a nonreactive tube and a reactive flow cytometry assay. In the IgM flow cytometry assay, three samples were reactive with anti-IgM. Samples from a group A woman who was transplanted with stem cells from a group B donor showed that on days 3 through 6 post-transplant, the flow cytometry assays for anti-IgG and/or anti-C3d were reactive, whilst the tube assays were nonreactive. In conclusion, flow cytometric analysis is more sensitive than the tube assay for the detection of RBC-associated C3d. Further studies are needed to determine the correlation of C3d levels with clinical sequelae.

Susceptibility to IVM in a field strain of Haemonchus contortus subjected to four treatments in a closed sheep-goat flock in Kenya.

Susceptibility to IVM (IVM) of "strain A" Haemonchus contortus which had been exposed to IVM four times over a 2-year period was compared to IVM susceptibility of "strain C" H. contortus which had no prior field exposure to IVM, by in vivo and in vitro methods. In vivo, the percentage reduction in faecal egg counts (FEC) and the total worm counts (TWC) were compared between control animals (lambs and kids) and animals treated with low dose IVM (20 microg/kg). In vitro susceptibility to IVM was evaluated by larval migration inhibition (LMI) after the two strains of H. contortus were exposed to different concentrations of IVM. The dose response, measured as the proportion of larvae inhibited from migrating, was used to estimate LD(50). Although differences in response to IVM in the in vivo determinations were not significant, "strain A" H. contortus had a significantly higher LD(50) than "strain C" in the LMI assay. Coincident with the conduct of the in vivo experiment, it was observed that "strain A" H. contortus established and survived better than "strain C" in the control lambs.

Comparison of DATs using traditional tube agglutination to gel column and affinity column procedures.

BACKGROUND: Detection of immunoglobulin or complement bound to RBCs by using the DAT is valuable in the diagnosis of immune-mediated hemolytic anemia. Traditionally, the DAT has been performed by tube agglutination using anti-IgG or anti-C3d. The purpose of this study was to compare the tube agglutination DAT to gel microcolumn, affinity microcolumn, and flow cytometric DATs on RBCs coated in vitro and on patient RBC samples. STUDY DESIGN AND METHODS: RBCs from 84 patients were assessed by tube agglutination DAT, one gel microcolumn DAT, and two affinity microcolumn DATs. One affinity microcolumn assay was unmodified and one was modified by the addition of polyspecific antiglobulin or anti-IgG as a secondary antibody. RBCs from 15 of the 84 patients underwent analysis by flow cytometry with fluorescence-labeled anti-IgG. The assays were also compared by using D+ RBCs sensitized with serially adjusted concentrations of anti-D. RESULTS: Both tube agglutination and gel microcolumn DATs were positive in 49 patient samples; both assays were negative in 20 samples, and the results were discordant in 15. Gel microcolumn DATs were more likely than were tube agglutination DATs to detect IgG on RBCs. Affinity microcolumn DATs were less likely than gel microcolumn or tube agglutination DATs to detect IgG on RBCs. Flow cytometry results were the same as gel microcolumn results in 12 of 15 patient samples and the same as tube agglutination results in 13 of 15. Tube agglutination and both affinity microcolumn assays reacted with RBCs coated with anti-D that was diluted 1-in-100. The gel microcolumn and flow cytometry assays reacted with RBCs coated with anti-D diluted 1-in-400. There was no correlation between tube agglutination and gel microcolumn DATs in detecting bound C3d. CONCLUSION: Detection of IgG bound to RBCs was not consistent with the methods described. Gel microcolumn DATs were more sensitive than tube agglutination and affinity microcolumn DATs. Given the varied results of these assays, reference laboratories should not rely on a single method for DATs. More comprehensive testing should be performed when the tube agglutination DAT is negative in a patient with suspected immune-mediated hemolytic anemia. Further comparisons are necessary to determine the proficiency of flow cytometric assays.

Characterization of viable autofluorescent macrophages among cultured peripheral blood mononuclear cells.

BACKGROUND: Inflammatory macrophages that demonstrate intense autofluorescence have been isolated directly from alveolar and peritoneal tissues, but their generation in vitro remains vague. We use flow cytometry to identify a population of autofluorescent macrophages as they arise among nonadherent populations of cultured blood mononuclear cells. METHODS: Cells were obtained from donated blood buffy coats and placed in culture for 14 days. Unstained populations from the cells remaining in suspension were sampled daily using flow cytometry. During the first 5 culture days, a distinct population of autofluorescent cells arose and comprised an average of < or =14% of the total cell population. This population declined to less than 6% by culture day 8. RESULTS: The cells were identified as viable macrophages expressing CD68, lysozyme, and HLA-DR. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) demonstrated a unique cytokine profile with IL-1 alpha expression levels 138-fold higher than those measured in uncultured monocytes. No significant elevation in the levels of other cytokines was identified. Upon replating, the sorted populations became readherent, were able to ingest plastic beads, and remained viable for 6 or more additional weeks in culture without evidence of proliferation or multinucleation. CONCLUSION: Viable autofluorescent macrophage populations arising among cultured peripheral blood may be easily identified and isolated for further study using flow cytometry. Cytometry 44:38-44, 2001. Published 2001 Wiley-Liss, Inc.

Identification of the dombrock blood group glycoprotein as a polymorphic member of the ADP-ribosyltransferase gene family.

Identification of the 25 known human blood group molecules is of fundamental importance for the fields of erythroid cell biology and transfusion medicine. Here we provide the first molecular description of the "Dombrock" blood group system. A candidate gene was identified by in silico analyses of approximately 5000 expressed sequence tags (ESTs) from terminally differentiating human erythroid cells. Transfection experiments demonstrated specific binding of anti-Dombrock and confirmed glycosylphosphatidylinositol membrane attachment. Dombrock expression is developmentally regulated during erythroid differentiation and occurs at highest levels in the fetal liver. Homology studies suggest that the Dombrock molecule is a member of the adenosine 5'-diphosphate (ADP)-ribosyltransferase ectoenzyme gene family. Genotypic comparisons suggest Do(a) versus Do(b) antigenicity results from a single amino acid substitution within an encoded arginine-glycine-aspartic acid (RGD) motif of the molecule.

Protection of cells from complement-mediated attack: CD59 receptor clustering for the entry of macromolecules into hematopoietic cells.

CD59 is a glycosylphosphatidylinositol anchored protein (GPI-protein) that is expressed on surface membranes to protect host cells from complement-mediated attack. CD59 may also serve as a receptor for the endocytosis of macromolecules into nucleated cells. Here we investigate the effects of primary clustering of CD59 with anti-CD59 monoclonal antibody and the secondary clustering of biotinylated anti-CD59 with avidin on red blood cells and erythroleukemic K562 cells. On red blood cells, CD59-targeted antibodies remained evenly distributed on the external membranes. In contrast, clustering, capping and endocytosis of the CD59-targeted complexes was detected on K562 cells. Secondary clustering appeared more efficient and resulted in endosomal localization of the fluorescently labeled complexes within 2 hours. The endocytosis of CD59-bound complexes did not affect K562 cell viability or growth and the surface level of CD59 was constant during the process. These result suggest clustering and subsequent endocytosis of CD59 may enable the entry of macromolecules to the endosomal compartments of hematopoietic cells.

Gene expression in proliferating human erythroid cells.

A complete understanding of human erythropoiesis will require a robust description of transcriptional activity in hematopoietic cells that proliferate and differentiate in response to erythropoietin (EPO). For this purpose, we cultured peripheral blood mononuclear cells in the presence or in the absence of EPO and examined the transcriptional profile of those cells arising only in response to EPO. A distinct population of CD71( +) cells that demonstrated an average of six additional doublings in suspension culture and erythroid colony formation in methylcellulose was isolated. Suppression subtractive hybridization of mRNA isolated from those cells permitted the identification of transcribed genes. A summary of 719 expressed sequence tags (ESTs) describing 505 independent transcripts is provided here with a full analysis of each EST available at http://hembase.niddk.nih.gov. Several transcripts that matched genes previously reported in the context of erythroid differentiation including 4 cell surface proteins were expressed at this developmental stage. Active chromatin remodeling was suggested by the identification of 4 histone proteins, 4 high-mobility group proteins, 13 transcription factors, and 6 genes involved in DNA recombination and repair. Numerous genes associated with leukemic translocations were also recognized including topoisomerases I and II, nucleophosmin, Translin, EGR1, dek, pim-1, TFG, and MLL. In addition to known transcripts, 44 novel EST were discovered. This transcriptional profile provides the first genomic-scale description of gene activity in erythroid progenitor cells.

Prospective identification of erythroid elements in cultured peripheral blood.

We have developed a prospective approach to identify the generation of erythroid cells derived from cultured peripheral blood mononuclear cells (PBMC) by monitoring the expression of the cell surface protein CD48. Unpurified populations of PBMC obtained from the buffy coats of normal volunteers were grown in suspension culture in the absence or presence of erythropoietin. A profile of surface CD48 expression permitted a flow cytometric identification of erythropoietin responsive populations at various stages of their maturation. In the absence of erythropoietin (EPO) supplemented media, the CD48- cells represented <5% of the total population of PBMC remaining in culture. In cultures supplemented with 1 U/mL EPO, the mean percentage of CD48- cells increased to 34.7 + 14.9% (p < 0.01) after 14 days in culture. Coordinated CD34 and CD71 (transferrin receptor) expression, morphology, gamma-globin transcription, and colony formation in methylcellulose were observed during the 14-day culture period. Flow cytometric monitoring of bulk cultured PBMC provides a simple and reliable means for the prospective or real-time study of human erythropoiesis.

Stable expression of green fluorescent protein after liposomal transfection of K562 cells without selective growth conditions.

Little is known about the durability of plasmid DNA transgene expression in mammalian cells in the absence of growth selection. For this purpose, we have begun the study of liposomal transfer and expression of plasmid DNA encoding green fluorescent protein (GFP) in human erythroleukemia K562 cells. Detection and selection of GFP expression were accomplished visually and by flow cytometry. GFP expression was noticeable in cells within 4 h of transfection. In nine separate transfections, approximately 20% of the transfected cells expressed GFP with a mean fluorescence 40-50x that of control cells (15 fluorescent units [FU] vs. 0.3 FU) during the first five days after transfection. The percentage of GFP positive cells dropped rapidly to 0.1% by day 14 post-transfection, but fluorescence activated cell sorting on this day resulted in the identification of stable transfectants expressing GFP for an additional 6-12 months in culture. GFP expression is adequate for the identification, isolation and monitoring of stable transfection events after lipid-mediated transfection of eukaryotic cells.

Long-term, stable expression of green fluorescent protein in mammalian cells.

Despite the proven utility of green fluorescent protein (GFP) as a reporter molecule for transient gene expression, the adequacy of this marker for models requiring durable, high-level gene expression has not been fully tested. To address this issue, we performed the transfection of Chinese Hamster Ovary (CHO) cells with plasmid DNA encoding both GFP and neomycin phosphotransferase (neo) cassettes. The expression of GFP was measured after the cells were cultured in the presence or absence of G418-mediated selective pressure. After removal of G418 from the growth medium, the percentage of pooled G418 resistant transfectants which co-expressed the GFP transgene increased or remained unchanged. Flow cytometric and visual isolation of GFP-expressing cells was possible without continued selection in G418. One cloned cell line, C463, maintained high-level green fluorescence for 18 weeks in G418 and an additional 12 weeks in nonselective medium. Our data suggest expression of GFP does not confer a growth disadvantage in mammalian cells.