Disclosing large scale adverse events in the US Veterans Health Administration: lessons from media responses.

OBJECTIVES: We examined print, broadcast and social media reports about health care systems' disclosures of large scale adverse events to develop future effective messaging. STUDY DESIGN: Directed content analysis. METHODS: We systematically searched four communication databases, YouTube and Really Simple Syndication (RSS) feeds relating to six disclosures of lapses in infection control practices in the Department of Veterans Affairs occurring between 2009 and 2012. We assessed these with a coding frame derived from effective crisis and risk communication models. RESULTS: We identified 148 unique media reports. Some components of effective communication (discussion of cause, reassurance, self-efficacy) were more present than others (apology, lessons learned). Media about 'promoting secrecy' and 'slow response' appeared in reports when time from event discovery to patient notification was over 75 days. Elected officials' quotes (n = 115) were often negative (83%). Hospital officials' comments (n = 165) were predominantly neutral (92%), and focused on information sharing. CONCLUSIONS: Health care systems should work to ensure that they develop clear messages focused on what is not well covered by the media, including authentic apologies, remedial actions taken, and shorten the timeframe between event identification and disclosure to patients.

Magnetic resonance imaging-based measures of atlas position: Relationship to canine atlantooccipital overlapping, syringomyelia and clinical signs.

Canine atlantooccipital overlapping (AOO) is characterized by a decreased distance between the dorsal arch of the atlas and the supraoccipital bone. Current diagnostic criteria for this condition are subjective and clinician-dependent; objective criteria are needed to allow a reliable and reproducible diagnosis in clinical and research settings and assess clinical significance. We propose four standardized MRI-based measurements to objectively assess the proximity of the atlas to the foramen magnum. Inclusion criteria for dogs in this study were bodyweight <15 kg, age >5 months, and availability of a complete MRI study performed with the craniocervical junction in extension. Exclusion criteria were space-occupying lesions and poor image quality (i.e. unclear bony margins). Measurements also included blinded determinations of skull type, presence of craniocervical junction anomalies and presence and severity of syringomyelia. Clinical status at the time of imaging was noted. Measurements were obtained in 271 dogs; these were reproducible and reliable. Findings varied by skull type: dolichocephalic dogs had smaller foramen magnum, whereas brachycephalic dogs had more cranially and dorsally positioned atlas bodies in comparison to the other skull types. Measurements also increased with increases in bodyweight. This study demonstrated a close association between AOO, syringomyelia and clinical signs. Toy and small breed dogs (including Cavalier King Charles spaniels) showed higher than previously reported prevalence of AOO; its occurrence was also associated with lower bodyweights within the study population of <15 kg toy/small breed dogs.

Serum insulin-like growth factor-1 (IGF-1) during CF pulmonary exacerbation: trends and biomarker correlations.

INTRODUCTION: Cystic fibrosis (CF) is characterized by low circulating levels of insulin-like growth factor-1 (IGF-1), a hormone produced by the liver that governs anabolism and influences immune cell function. Because treatment of CF pulmonary exacerbation (CFPE) often improves body weight and lung function, we questioned whether serum IGF-1 trends were emblematic of these responses. Initially, we compared serum levels between healthy adults with CF and controls of similar age. We then measured serum IGF-1 throughout the CFPE cycle. We also investigated correlations among IGF-1 and other serum biomarkers during CFPE. METHODS: Anthopometric, spirometric, and demographic data were collected. Serum IGF-1 concentrations were measured by ELISA. RESULTS: CF subjects in their usual state of health had lower serum IGF-1 levels than controls. Serum IGF-1 concentrations fell significantly from baseline at the beginning of CFPE. Treatment with intravenous antibiotics was associated with significant improvement in serum IGF-1 levels, body mass index (BMI), and percent-predicted forced expiratory volume in 1 sec (FEV1 %). At early and late CFPE, serum IGF-1 was directly correlated with FEV1 %, serum iron, hemoglobin concentration, and transferrin saturation (TSAT) and indirectly correlated with alpha-1-antitrypsin. CONCLUSIONS: This study not only supports the paradigm that CF is characterized by IGF-1 deficiency but also that trends in lung function, nutritional status, and serum IGF-1 are related. Improvements in all three parameters after antibiotics for CFPE likely highlight the connection between lung function and nutritional status in CF. Close correlations among IGF-1 and iron-related hematologic parameters suggest that IGF-1 may participate in CF iron homeostasis, another process that is known to be influenced by CFPE.

HOXA1 drives melanoma tumor growth and metastasis and elicits an invasion gene expression signature that prognosticates clinical outcome.

Melanoma is a highly lethal malignancy notorious for its aggressive clinical course and eventual resistance to existing therapies. Currently, we possess a limited understanding of the genetic events driving melanoma progression, and much effort is focused on identifying pro-metastatic aberrations or perturbed signaling networks that constitute new therapeutic targets. In this study, we validate and assess the mechanism by which homeobox transcription factor A1 (HOXA1), a pro-invasion oncogene previously identified in a metastasis screen by our group, contributes to melanoma progression. Transcriptome and pathway profiling analyses of cells expressing HOXA1 reveals upregulation of factors involved in diverse cytokine pathways that include the transforming growth factor beta (TGFß) signaling axis, which we further demonstrate to be required for HOXA1-mediated cell invasion in melanoma cells. Transcriptome profiling also shows HOXA1's ability to potently downregulate expression of microphthalmia-associated transcription factor (MITF) and other genes required for melanocyte differentiation, suggesting a mechanism by which HOXA1 expression de-differentiates cells into a pro-invasive cell state concomitant with TGFß activation. Our analysis of publicly available data sets indicate that the HOXA1-induced gene signature successfully categorizes melanoma specimens based on their metastatic potential and, importantly, is capable of stratifying melanoma patient risk for metastasis based on expression in primary tumors. Together, these validation data and mechanistic insights suggest that patients whose primary tumors express HOXA1 are among a high-risk metastasis subgroup that should be considered for anti-TGFß therapy in adjuvant settings. Moreover, further analysis of HOXA1 target genes in melanoma may reveal new pathways or targets amenable to therapeutic intervention.

ß-blockers increase response to chemotherapy via direct antitumour and anti-angiogenic mechanisms in neuroblastoma.

BACKGROUND: The use of ß-blockers for the management of hypertension has been recently associated with significant clinical benefits in cancer patients. Herein, we investigated whether ß-blockers could be used in combination with chemotherapy for the treatment of neuroblastoma. METHODS: Seven ß-blockers were tested for their antiproliferative and anti-angiogenic properties alone, and in combination with chemotherapy in vitro; the most potent drug combinations were evaluated in vivo in the TH-MYCN mouse model of neuroblastoma. RESULTS: Three ß-blockers (i.e., carvedilol, nebivolol and propranolol) exhibited potent anticancer properties in vitro and interacted synergistically with vincristine, independently of P-glycoprotein expression. ß-blockers potentiated the anti-angiogenic, antimitochondrial, antimitotic and ultimately pro-apoptotic effects of vincristine. In vivo, ß-blockers alone transiently slowed tumour growth as compared with vehicle only (P<0.01). More importantly, when used in combination, ß-blockers significantly increased the tumour regression induced by vincristine (P<0.05). This effect was associated with an increase in tumour angiogenesis inhibition (P<0.001) and ultimately resulted in a four-fold increase in median survival, as compared with vincristine alone (P<0.01). CONCLUSION: ß-blockers can increase treatment efficacy against neuroblastoma, and their combination with chemotherapy may prove beneficial for the treatment of this disease and other drug-refractory cancers.

Systematic review: the quality of the scientific evidence and conflicts of interest in international inflammatory bowel disease practice guidelines.

BACKGROUND: Guidelines published by the international gastroenterology societies establish standards of care and seek to improve patient outcomes. AIM: We examined inflammatory bowel disease guidelines (IBD) for quality of evidence, methods of grading evidence and conflicts of interest (COI). METHODS: All 182 guidelines published by the American College of Gastroenterology, American Gastroenterological Association, British Society of Gastroenterology, Canadian Association of Gastroenterology, Crohn's and Colitis Foundation of America and European Crohn's and Colitis Organisation as of 27 September 2012 were reviewed. Nineteen IBD guidelines were found. RESULTS: Eighty-nine per cent (n = 17/19) of the guidelines graded the levels of evidence using seven different systems. Of the 1070 recommendations reviewed, 23% (n = 249) cited level A evidence; 28% (n = 302) level B; 36% (n = 383) level C and 13% (n = 136) level D. The mean age of the guidelines was 4.2 years. In addition, 61% (n = 11/19) of the guidelines failed to comment on COI. All eight articles commenting on COI had conflicts with 81% (n = 92/113) of authors reported an average 11.7 COI. Lastly, there were variations in the recommendations between societies. CONCLUSIONS: Nearly half the IBD guideline recommendations are based on expert opinion or no evidence. Majority of the guidelines fail to disclose any COI, and when commenting, all have numerous COI. Furthermore, the guidelines are not updated frequently and there is a lack of consensus between societal guidelines. This study highlights the critical need to centralize and redesign the guidelines development process.

Chronic inflammation and lung fibrosis: pleotropic syndromes but limited distinct phenotypes.

Experimental models of lung fibrosis have been disappointing in predicting therapeutic responses to a wide variety of interventions in clinical fibrosing lung diseases. There are multiple potential reasons, but this fundamentally calls into question the validity of the models and their fidelity to clinical syndromes. We propose that the clinical diseases associated with pulmonary fibrosis, although manifesting a broad array of widely different clinical presentations and features, result in essentially two distinct phenotypes of fibrosis that we will describe. The most common and problematic of these are not effectively modeled experimentally. In this review, we present several clinical entities as examples of the phenotypic distinctions. The first two represent the extremes: postinflammatory fibrosis observed in hypersensitivity pneumonitis (HP) and dysregulated matrix deposition as observed in idiopathic pulmonary fibrosis (IPF). We also present a third clinical entity, that of lung disease associated with rheumatoid arthritis (rheumatoid lung), representing a condition that can manifest as either phenotype, and offering a potential opportunity to explore the mechanisms underlying the pathogenesis of the two distinct fibrotic phenotypes.

Prevalence of streptococci and increased polymicrobial diversity associated with cystic fibrosis patient stability.

Diverse microbial communities chronically colonize the lungs of cystic fibrosis patients. Pyrosequencing of amplicons for hypervariable regions in the 16S rRNA gene generated taxonomic profiles of bacterial communities for sputum genomic DNA samples from 22 patients during a state of clinical stability (outpatients) and 13 patients during acute exacerbation (inpatients). We employed quantitative PCR (qPCR) to confirm the detection of Pseudomonas aeruginosa and Streptococcus by the pyrosequencing data and human oral microbe identification microarray (HOMIM) analysis to determine the species of the streptococci identified by pyrosequencing. We show that outpatient sputum samples have significantly higher bacterial diversity than inpatients, but maintenance treatment with tobramycin did not impact overall diversity. Contrary to the current dogma in the field that Pseudomonas aeruginosa is the dominant organism in the majority of cystic fibrosis patients, Pseudomonas constituted the predominant genera in only half the patient samples analyzed and reported here. The increased fractional representation of Streptococcus in the outpatient cohort relative to the inpatient cohort was the strongest predictor of clinically stable lung disease. The most prevalent streptococci included species typically associated with the oral cavity (Streptococcus salivarius and Streptococcus parasanguis) and the Streptococcus milleri group species. These species of Streptococcus may play an important role in increasing the diversity of the cystic fibrosis lung environment and promoting patient stability.

Diffusion tensor imaging of the optic nerve in multiple sclerosis: association with retinal damage and visual disability.

BACKGROUND AND PURPOSE: There is a well-known relationship between MS and damage to the optic nerve, but advanced, quantitative MR imaging methods have not been applied to large cohorts. Our objective was to determine whether a short imaging protocol (< 10 minutes), implemented with standard hardware, could detect abnormal water diffusion in the optic nerves of patients with MS. MATERIALS AND METHODS: We examined water diffusion in human optic nerves via DTI in the largest MS cohort reported to date (104 individuals, including 38 optic nerves previously affected by optic neuritis). We also assessed whether such abnormalities are associated with loss of visual acuity (both high and low contrast) and damage to the retinal nerve fiber layer (assessed via optical coherence tomography). RESULTS: The most abnormal diffusion was found in the optic nerves of patients with SPMS, especially in optic nerves previously affected by optic neuritis (19% drop in FA). DTI abnormalities correlated with both retinal nerve fiber layer thinning (correlation coefficient, 0.41) and loss of visual acuity, particularly at high contrast and in nerves previously affected by optic neuritis (correlation coefficient, 0.54). However, diffusion abnormalities were overall less pronounced than retinal nerve fiber layer thinning. CONCLUSIONS: DTI is sensitive to optic nerve damage in patients with MS, but a short imaging sequence added to standard clinical protocols may not be the most reliable indicator of optic nerve damage.

Evaluation of patient reporting of adverse drug reactions to the UK 'Yellow Card Scheme': literature review, descriptive and qualitative analyses, and questionnaire surveys.

BACKGROUND: The monitoring of adverse drug reactions (ADRs) through pharmacovigilance is vital to patient safety. Spontaneous reporting of ADRs is one method of pharmacovigilance, and in the UK this is undertaken through the Yellow Card Scheme (YCS). Yellow Card reports are submitted to the Medicines and Healthcare products Regulatory Agency (MHRA) by post, telephone or via the internet. The MHRA electronically records and reviews information submitted so that important safety issues can be detected. While previous studies have shown differences between patient and health-care professional (HCP) reports for the types of drugs and reactions reported, relatively little is known about the pharmacovigilance impact of patient reports. There have also been few studies on the views and experiences of patients/consumers on the reporting of suspected ADRs. OBJECTIVES: To evaluate the pharmacovigilance impact of patient reporting of ADRs by analysing reports of suspected ADRs from the UK YCS and comparing reports from patients and HCPs. To elicit the views and experiences of patients and the public about patient reporting of ADRs. DESIGN: (1) Literature review and survey of international experiences of consumer reporting of ADRs; (2) descriptive analysis of Yellow Card reports; (3) signal generation analysis of Yellow Card reports; (4) qualitative analysis of Yellow Card reports; (5) questionnaire survey of patients reporting on Yellow Cards; (6) qualitative analysis of telephone interviews with patient reporters to the scheme; (7) qualitative analysis of focus groups and usability testing of the patient YCS; and (8) national omnibus telephone survey of public awareness of the YCS. PARTICIPANTS: Patients (n = 5180) and HCPs (n = 20,949) submitting Yellow Card reports from October 2005 to September 2007. Respondents to questionnaire survey (n = 1362). Participants at focus groups and usability testing sessions (n = 40). National omnibus telephone survey (n = 2028). SETTING: The literature review included studies in English from across the world. All other components included populations from the UK; the omnibus survey was restricted to Great Britain. INTERVENTIONS: None. MAIN OUTCOME MEASURES: Characteristics of patient reports: types of drug and suspected ADR reported; seriousness of reports; and content of reports. The relative contributions of patient reports and of HCP reports to signal generation. Views and experiences of patient reporters. Views of members of the public about the YCS, including user-friendliness and usability of different ways of patient reporting. Public awareness of the YCS. Suggestions for improving patient reporting to the YCS. RESULTS: Compared with HCPs, patient reports to the YCS contained a higher median number of suspected ADRs per report, and described reactions in more detail. The proportions of reports categorised as 'serious' were similar; the patterns of drugs and reactions reported differed. Patient reports were richer in their descriptions of reactions than those from HCPs, and more often noted the effects of ADRs on patients' lives. Combining patient and HCP reports generated more potential signals than HCP reports alone; some potential signals in the 'HCP-only' data set were lost when combined with patient reports, but fewer than those gained; the addition of patient reports to HCP reports identified 47 new 'serious' reactions not previously included in 'Summaries of Product Characteristics'. Most patient reporters found it fairly easy to make reports, although improvements to the scheme were suggested, including greater publicity and the redesign of web- and paper-based reporting systems. Among members of the public, 8.5% were aware of the YCS in 2009. CONCLUSIONS: Patient reporting of suspected ADRs has the potential to add value to pharmacovigilance by reporting types of drugs and reactions different from those reported by HCPs; generating new potential signals; and describing suspected ADRs in enough detail to provide useful information on likely causality and impact on patients' lives. These findings suggest that further promotion of patient reporting to the YCS is justified, along with improvements to existing reporting systems. In order of priority, future work should include further investigation of (1) the pharmacovigilance impact of patient reporting in a longer-term study; (2) the optimum approach to signal generation analysis of patient and HCP reports; (3) the burden of ADRs in terms of impact on patients' lives; (4) the knowledge and attitudes of HCPs towards patient reporting of ADRs; (5) the value of using patient reports of ADRs to help other patients and HCPs who are seeking information on patient experiences of ADRs; and (6) the impact of increasing publicity and/or enhancements to reporting systems on the numbers and types of Yellow Card reports from patients. FUNDING: The National Institute for Health Research Health Technology Assessment programme.

Iron and CF-related anemia: expanding clinical and biochemical relationships.

INTRODUCTION: This cross-sectional study was conducted to assess the relationship between iron levels in the plasma and sputum of cystic fibrosis (CF) patients. METHODS: Demographic, clinical, and iron-related laboratory data were prospectively obtained from 25 patients with stable clinical features and 14 patients with worsened clinical features since their most recent evaluations. RESULTS: Compared to patients with stable clinical features, those who experienced clinical deterioration demonstrated significantly worse lung function and were more frequently malnourished and diabetic. Members of the latter group were also significantly more hypoferremic and had higher sputum iron content than patients with stable clinical features. No significant correlation was found between plasma and sputum iron levels when the groups were analyzed together and separately. CONCLUSIONS: Sputum iron content does not correlate with iron-related hematologic tests. Hypoferremia is common in CF and correlates with poor lung function and overall health.

Hepatitis C testing practices and prevalence in a high-risk urban ambulatory care setting.

Approximately 3.2 million persons are chronically infected with the hepatitis C virus (HCV) in the U.S.; most are not aware of their infection. Our objectives were to examine HCV testing practices to determine which patient characteristics are associated with HCV testing and positivity, and to estimate the prevalence of HCV infection in a high-risk urban population. The study subjects were all patients included in the baseline phase of the Hepatitis C Assessment and Testing Project (HepCAT), a serial cross-sectional study of HCV screening strategies. We examined all patients with a clinic visit to Montefiore Medical Center from 1/1/08 to 2/29/08. Demographic information, laboratory data and ICD-9 diagnostic codes from 3/1/97-2/29/08 were extracted from the electronic medical record. Risk factors for HCV were defined based on birth date, ICD-9 codes and laboratory data. The prevalence of HCV infection was estimated assuming that untested subjects would test positive at the same rate as tested subjects, based on risk-factors. Of 9579 subjects examined, 3803 (39.7%) had been tested for HCV and 438 (11.5%) were positive. The overall prevalence of HCV infection was estimated to be 7.7%. Risk factors associated with being tested and anti-HCV positivity included: born in the high-prevalence birth-cohort (1945-64), substance abuse, HIV infection, alcohol abuse, diagnosis of cirrhosis, end-stage renal disease, and alanine transaminase elevation. In a high-risk urban population, a significant proportion of patients were tested for HCV and the prevalence of HCV infection was high. Physicians appear to use a risk-based screening strategy to identify HCV infection.

The hepatitis C self-management programme: a randomized controlled trial.

Chronic hepatitis C (HCV) infection afflicts millions of people worldwide. While antiviral treatments are effective for some patients, many either cannot or choose not to receive antiviral treatment. Education about behavioural changes like alcohol avoidance and symptom management, in contrast, is universally recommended, particularly in HCV-infected persons from disadvantaged groups where liver risk factors are most prevalent. Self-management interventions are one option for fostering improved HCV knowledge and health-related quality of life (HRQOL). One hundred and thirty-two patients with VA with HCV (mean age of 54.6, 95% men, 41% ethnic minority, 83% unmarried, 72% unemployed/disabled, 48% homeless in last 5 years) were randomized to either a 6-week self-management workshop or an information-only intervention. The weekly 2-h self-management sessions were based on cognitive-behavioural principles and were adapted from an existing self-management programme that has been efficacious with other chronic diseases. HCV-specific modules were added. Outcomes including HRQOL, HCV knowledge, self-efficacy, depression, energy and health distress were measured at baseline and 6 weeks later. Data were analysed using ANOVA. When compared to the information-only group, participants attending the self-management workshop improved more on HCV knowledge (P < 0.001), HCV self-efficacy (P = 0.011), and SF-36 energy/vitality (P = 0.040). Similar trends were found for SF-36 physical functioning (P = 0.055) and health distress (P = 0.055). Attending the self-management programme improved disease knowledge and HRQOL 6 weeks later in this disadvantaged population. The intervention can improve the health of people with hepatitis C, independent of antiviral therapy. Future research will study longer-term outcomes, effects on antiviral treatment and costs.

Cystic endometrial hyperplasia, pseudo-placentational endometrial hyperplasia, and other cystic conditions of the canine and feline uterus.

Cystic lesions in the uteri of bitches and queens arise from the uterine serosa, myometrium, or endometrium and include: serosal inclusion cysts, adenomyosis, endometrial polyps, cystic remnants of mesonephric ducts, and cysts associated with endometrial hyperplasia (both cystic glands and "pseudo-placentational" hyperplasia). Of these, "cystic endometrial hyperplasia (CEH)" is the most common and is frequently associated with pyometra. A second form of endometrial hyperplasia occurs in the bitch; although it was first described over 100 y ago, it is not widely recognized by clinicians or diagnostic pathologists. In this form, the endometrium proliferates in a highly organized manner, remodeling the uterine lining to closely resemble the histology of the endometrium at placentation sites in normal pregnancy. Although this lesion is very different from CEH, it is quite easy to induce in dogs during the luteal phase of their cycles and has been perhaps inappropriately proposed as modeling CEH. This lesion has been referred variously as "deciduoma", endometrial hyperplasia in pseudocyesis, and "maternal placental-like endometrial hyperplasia". An alternative name is suggested that is descriptive and draws attention to the difference between this lesion and CEH; the term pseudo-placentational endometrial hyperplasia (PEH) is proposed. The histopathology and pathogenesis of CEH and PEH are discussed. The objectives of this paper are to review the pathophysiology of cystic lesions of canine uterus, to demonstrate these using subgross photomicrographs taken from natural cases, and to present key diagnostic features of each.

Risk factors for Clostridium difficile-associated diarrhea on an adult hematology-oncology ward.

Nosocomial diarrhea caused by Clostridium difficile causes significant morbidity and mortality in an increasing proportion of hospitalized patients annually. This case-control study of patients admitted to the hematology-oncology ward of a tertiary academic medical center over a 2-year period demonstrates that patients with Clostridium difficile-associated diarrhea (CDAD) were 22 times more likely than ward-matched controls with diarrhea to have received any antibiotic either during hospitalization or in the month preceding admission (p < 0.005), and they were nearly three times as likely as controls to have received a cephalosporin during the same period (p < 0.005). Diarrhea among lung cancer patients was approximately three times more likely to be caused by this organism than to be due to other causes (p = 0.04). A trend towards CDAD patients receiving higher numbers of different antibiotics during hospitalization (3.3 vs. 2.6, 95%CI -1.42-0.02, p = 0.06) was noted. Administration of interleukin-2 either during hospitalization or in the 30 days preceding admission was seven times more likely to have occurred in CDAD cases (p = 0.04), raising the question of whether or not this agent increases risk.

Balancing disfigurement and fear of disease progression: Patient perceptions of HIV body fat redistribution.

This study was conducted to identify and describe the perceived morphologic changes of body fat redistribution and related distress among persons taking combination antiretroviral therapy. Six focus group interviews were conducted in four different US cities with men and women (n = 58) who reported antiretroviral-related symptoms of body fat loss and/or gain. Interview data were audiotaped, transcribed verbatim and systematically analysed using inductive techniques. Physical discomfort and impairment and psychological and social distress were reported across sex, sexual orientation and geographic subgroups. While participants acknowledged that antiretroviral drugs were keeping them alive, there was tension between the desire for life-sustaining treatment and optimal quality of life. Some participants engaged in harmful heath behaviours in an attempt to control bodily changes (e.g. non-adherence to antiretroviral regimen). Participants feared that fat loss represented disease progression and worried that visible changes would lead to unintentional disclosure of their HIV status. Although a potential source of support, healthcare providers were commonly perceived as ignoring and, in so doing, discrediting patient distress. Participants recognised the limitations of current lipodystrophy treatment options, yet a cure for the syndrome seemed less important to them in the short term than simply being listened to and the powerful, but oblique sources of distress addressed.

Role of the E45K-reduced folate carrier gene mutation in methotrexate resistance in human leukemia cells.

Resistance to the antifolate methotrexate (MTX) can cause treatment failure in childhood acute lymphoblastic leukemia (ALL). This may result from defective MTX accumulation due to alterations in the human reduced folate carrier (hRFC) gene. We have identified an hRFC gene point mutation in a transport-defective CCRF-CEM human T-ALL cell line resulting in a lysine to glutamic acid substitution at codon 45 (E45K), which has been identified in other antifolate-resistant sublines (JBC 273:30 189, 1998; JBC 275:30 855, 2000). To characterize the role of this mutation in MTX resistance, transfection experiments were performed using hRFC-null CCRF-CEM cells. E45K transfectants demonstrated an initial rate of MTX influx that was approximately 0.5-fold that of CCRF-CEM cells, despite marked protein overexpression. Cytotoxicity studies revealed partial reversal of MTX and raltitrexed resistance in E45K transfectants, while trimetrexate resistance was significantly increased. Kinetic analysis indicated only minor differences in MTX kinetics between wild-type and E45K hRFCs, however, K(i)s for folic acid and 5-formyltetrahydrofolate were markedly reduced for E45K hRFC. This was paralleled by increased folic acid transport and reduced synthesis of MTX polyglutamates. Collectively, the results demonstrate that expression of E45K hRFC leads to increased MTX resistance due to decreased membrane transport and, secondarily, from alterations in binding affinities and transport of folate substrates. However, despite these findings, we could find no evidence of this mutation in 121 childhood ALL samples, suggesting that it does not contribute to clinical MTX resistance in this disease.