Background Promonitor Quick IFX is a lateral flow test (LFT) for the quantification of infliximab (IFX) in human whole blood (finger prick or venous) or serum in 20 minutes. This LFT is based on a sandwich immunoassay to quantify either the reference IFX or biosimilars. Objectives The objective of this study was to perform the required analytical studies to establish the specifications of the product. Methods Clinical and Laboratory Standards Institute (CLSI) guidelines were followed for the evaluation of the analytical specifications of the LFT in whole blood and serum matrices: Linearity (EP-06-A), Detection capability (EP17-A2), Interfering substances (EP07, 3rd Edition) and Intermediate precision (EP05-A3). Results were obtained in combination with the automated portable reader PQreader. A Datamatrix provided with each Promonitor Quick IFX kit contains the calibration information required for the PQreader to measure the Control and Test lines and report the IFX concentration. Results The linear assay range was determined to be 1-58 µg/mL in whole-blood and 0.6-67 µg/mL in serum according to the processes indicated in the Package Insert. The Limit of Blank is 0.8 μg/mL, the Limit of Detection and Lower Limit of Quantification (LLoQ) are 1.1 μg/mL, and the Upper Limit of Quantification (ULoQ) is 15.4 μg/mL. There was no effect on assay performance when each of the following substances were added to samples with 0, 3, and 7 μg/mL of IFX: Haemoglobin (>1000 mg/dL), Bilirubin (>40 mg/dL), Triglycerides (>1500 mg/dL), HAMA (160 AU/mL), Rheumatoid factor (200 IU/mL), EDTA (5.4 mg/mL), Heparin (51 U/mL), Citrate (11.4%), Vedolizumab (60 μg/mL) and Adalimumab (20.25 μg/mL). Repeatability and within-device precision results obtained for the positive samples are shown in Table 1. Table 1. IFX theoretical concentration (μg/mL) Whole blood samples Serum samples Mean observed IFX concentration (μg/mL) Repeatability Within-device precision Mean observed IFX concentration (μg/mL) Repeatability Within-device precision SD CV% SD CV% SD CV% SD CV% 3 3.2 0.6 18 0.8 25 3.6 0.5 15 0.6 17 7 7 1.3 18 1.7 24 8.3 1.4 16 1.8 21 10 9.8 1.8 18 2.7 28 10.9 1.8 16 2.3 21 The negative samples showed a negative result in all the measurements. Conclusion Promonitor Quick IFX is the first LFT available for true Point of Care testing of patients treated with IFX with just a finger prick sample. It provides quick turnaround time to facilitate therapeutic drug monitoring and aid immediate decision making in the doctor office or hospitals with an excellent analytical performance. Disclosure of Interests Amagoia Ametzazurra Employee of: Employee of Progenika Biopharma - Grifols, Javier Pascual Employee of: Employee of Progenika Biopharma - Grifols, Lorena Del Rio Employee of: Employee of Progenika Biopharma - Grifols, Ainara Maguregui Employee of: Employee of Progenika Biopharma - Grifols, Daniel Nagore Employee of: Employee of Progenika Biopharma - Grifols, M. Begoña Ruiz-Argüello Employee of: Employee of Progenika Biopharma - Grifols

BackgroundPromonitor Quick IFX and Promonitor Quick ADL are rapid point of care lateral flow tests (LFT) based on a sandwich immunoassay for the quantification of infliximab (IFX) and adalimumab (ADL), respectively, in human whole blood (finger prick or venous) or serum. These tests are to be used as an aid in Therapeutic Drug Monitoring (TDM) of rheumatic and inflammatory bowel disease patients under anti-TNFα therapy. The international standards (IS) developed by World Health Organization (WHO) for IFX and ADL allow harmonization and comparability among different assays.ObjectivesThe aim of this study, was to show that Promonitor Quick IFX and Promonitor Quick ADL can measure either reference or biosimilar drugs, as well as to evaluate the agreement of Promonitor Quick IFX and Promonitor Quick ADL tests and the WHO IS.MethodsClinical and Laboratory Standards Institute EP10-A3 guidelines were followed to estimate the bias of Promonitor Quick assays when used to quantify IFX or ADL in samples containing the reference drugs, biosimilars or the WHO IS. Briefly, whole blood was spiked with four known concentrations of IFX or ADL, including current clinical decision levels. Ten replicates were measured of each level along two days.Promonitor Quick IFX was evaluated using the reference drug, SB2 and CT-P13 biosimilars, and the WHO IS (NIBSC 16/170).Promonitor Quick ADL was evaluated using the reference drug, ABP501 and SB5 biosimilars, and the WHO IS (NIBSC 17/236).Results were obtained in combination with the automated portable reader PQreader.ResultsBias was estimated by comparing the observed concentration of drug spiked whole blood samples. Each biosimilar was compared to the reference at the different drug levels tested. Results showed that Promonitor Quick IFX and Promonitor Quick ADL are able to measure equivalently any molecule (see Table 1).Table 1.Promonitor Quick IFX bias results in whole blood samples. Each molecule was compared to the reference drug.IFX concentration (μg/mL)Bias (%)CT-P13SB2GP1111WHO IS314%3%4%8%79%0%5%15%101%5%6%8%Promonitor Quick ADL bias results in whole blood samples.Each molecule was compared to the reference drug.ADL concentration (μg/mL)Bias (%)ABP501SB5WHO IS317%2%3%513%3%12%82%2%7%10517%2%Similar results were obtained when serum matrix was used.The accuracy or closeness of the agreement between the result provided by Promonitor Quick IFX and Promonitor Quick ADL and the true value of the measurand was assessed by measuring the IS developed by the WHO (see Table 1).ConclusionPromonitor Quick IFX and Promonitor Quick ADL allow monitoring of patients treated with IFX and ADL, respectively, with just a finger prick sample. Both tests can quantify reference and biosimilar drugs with equivalent results. Moreover, comparable results were obtained with the WHO IS, and thus, demonstrate that Promonitor Quick tests are suited to accurately determine drug levels at the clinical decision points in both whole blood and serum, proving to be an effective and valuable tool in TDM and immediate decision making in the doctor office or hospitals.Disclosure of InterestsAmagoia Ametzazurra Employee of: Employee of Progenika Biopharma - Grifols, Javier Pascual Employee of: Employee of Progenika Biopharma - Grifols, Lorena Del Rio Employee of: Employee of Progenika Biopharma - Grifols, Ane Urigoitia Employee of: Employee of Progenika Biopharma - Grifols, Daniel Nagore Employee of: Employee of Progenika Biopharma - Grifols, M. Begoña Ruiz-Argüello Employee of: Employee of Progenika Biopharma - Grifols

The goal of this study was to validate the use of capillary blood in a real point-of-care (POC) setting for patients under infliximab treatment by using Promonitor Quick lateral flow (LF) tests. Results were compared to the Promonitor ELISA reference technique in serum samples used by centralised laboratories. A prospective, observational study was designed to evaluate the performance of a rapid LF test (Promonitor Quick IFX, Progenika, Spain). 160 infliximab treated rheumatology consecutive patients (400 samples) were recruited in two hospitals in Galicia, Spain. Prior to the infusion, a finger prick sample was obtained and analysed. Anti-infliximab antibodies were also determined with Promonitor Quick ANTI-IFX1-4. Results were read with the automated portable PQreader instrument. Additionally, a serum sample was collected for subsequent comparative analysis with either LF or ELISA tests. Qualitative (positive (PPA) and negative (NPA) agreements) and quantitative (Pearson correlation and bias) performance of the LF test was compared to ELISA, as well as between different specimens following CLSI EP09-A3. Overall agreement between Promonitor Quick IFX finger prick and ELISA test was 91% (88% PPA; 100% NPA). The quantitative comparison showed a good correlation (Pearson correlation coefficient: 0.85 and observed bias: 25%) (Table 1). Similar results were also observed when serum was used with either the LF or the ELISA tests (98% overall agreement, 0.91 correlation coefficient; 6% bias) (Table 1). Overall agreements for visual and automated (PQreader) interpretations with Promonitor Quick ANTI-IFX were 99% and 100% for finger prick and serum specimens, respectively (Table 2). Promonitor Quick can be used to reliably quantify infliximab in capillary blood samples and results are comparable to those obtained with the reference ELISA technique. The use of the rapid POC test with finger prick will allow clinicians to monitor their patients in a fully decentralized mode to aid in the decision making process. PQreader is a sensitive portable equipment to report drug as well as antibody levels in the patient samples.

Promonitor® Quick ANTI-IFX is the only rapid test available for Point of Care (POC) testing of anti-infliximab (IFX) antibodies. The qualitative test is based on Lateral Flow (LF) technology to detect free antibodies to any IFX in human whole blood (capillary or venous), serum or plasma. Detection of anti-Remicade® and anti-Inflectra® (CT-P13) antibodies was previously shown to be equivalent in the capillary (finger prick whole blood) and systemic circulations (regular serum collected by venipuncture for therapeutic drug monitoring (TDM). However, detection of antibodies to Flixabi® biosimilar (SB2, Biogen) had only been proved by ELISA and data were lacking in a LF format. In this study, we compare the performance of the POC LF test to detect anti-Flixabi antibodies with the standard ELISA technique for TDM in IBD patients treated with Flixabi. Trough (n = 202) sera collected at the Erlangen University Hospital (Germany) were analysed, corresponding to 76 IBD patients (46 Crohn’s disease, 26 ulcerative colitis and 4 indeterminate colitis) treated with Flixabi only. Samples were frozen for subsequent testing with ELISA (Promonitor® ANTI-IFX, Progenika, Spain) and with the POC test (Promonitor Quick® ANTI-IFX, Progenika, Spain). The LF test uses the same format as the bridging ELISA. The POC test (LoD=23 AU/ml) results were read visually at 30 min after adding 15 µl of serum, whereas ELISA (LoD=5 AU/ml) quantitative results were categorised as positive or negative to allow comparisons with the POC test. The rapid test correctly detected anti-Flixabi antibodies and showed an almost perfect agreement with the reference ELISA method. 124 out of 202 samples were tested positive for anti-IFX antibodies with the POC test, whereas 144 samples were positive with the ELISA. Positive and negative per cent agreements between ELISA and the POC test were 86.1% and 100%, respectively. Fourteen (70%) out of the 20 discrepancies found were due to anti-Flixabi antibody concentration below the LoD of the POC test. Positive and negative agreements were 95.4% and 100%, respectively (124 LF-positive and 130 ELISA-positive sera) within the common measurement ranges of both techniques. The remaining 6 discrepancies (positive with ELISA and negative with LF) corresponded to samples of 2 patients who were confirmed as true positives by radioimmunoassay. The strong agreement reported here between the Promonitor® Quick ANTI-IFX POC LF test and the standard ELISA method reinforces that the rapid test is suitable for TDM of any IFX drug.

Background: Therapeutic drug monitoring (TDM) of infliximab (IFX) and adalimumab is increasingly being used for the management of patients with inflammatory bowel disease (IBD) [1]. Enzyme-linked immunoassays (ELISA) is one of the most used techniques; however, often requires batching samples which can delay results, and be inconvenient in urgent situations where a quick action is needed. In this study we compare the performance of a new lateral flow (LF) rapid test to detect anti-IFX antibodies with ELISA in IBD patients treated with either Remicade® (RMC) only or CT-P13 only or switchers from RMC to CT-P13. Methods: Serum samples from IBD patients who participated in BIOSIM01, an observational retrospective study, in which patients were treated with either RMC only, or CT-P13 only, or CT-P13 following a switch from RMC, were used. In this study a total of 564 consecutive trough sera (corresponding to 103 patients) were collected just before the infusion to all patients. All samples were frozen for subsequent testing with ELISA (Promonitor® Anti-IFX, Progenika, Spain) and the LF rapid test (Promonitor® Quick Anti-IFX, Progenika, Spain). The rapid test is an immunochromatographic qualitative kit based on LF technology to detect anti-IFX antibodies in patients treated with any IFX molecule in human whole blood (fingerprick or venous) or serum. Antibody test results were read visually at 30 minutes. ELISA quantitative results were categorized as positive or negative to allow comparisons with the rapid test. Results: The rapid test allowed the detection of anti-IFX antibodies in a few minutes with just 15 μL of serum and showed an almost perfect agreement with the comparative ELISA method. Overall, positive and negative percent agreements between ELISA and the LF test were 97.9% (CI95: 96.3–98.8), 96.0% (CI95: 88.9–98.6) and 98.2% (CI95: 96.5–99.0), respectively. No significant differences in the detection of Anti-IFX antibodies against either drug in any of the three cohorts were observed (Table 1). All antibody positive patients showed no detectable IFX levels as measured with ELISA. Table 1. Detection of anti-IFX antibodies in the different cohorts with the LF rapid test and ELISA PPA, Positive Percent Agreement; NPA, Negative Percent Agreement. Conclusions: The almost perfect agreement reported here between the LF and ELISA tests provides support to clinicians and laboratory personnel involved in biological drug monitoring in the use of the rapid test. The rapid test shows detection of antibodies aganist either the innovator or the biosimilar with comparable analytical sensitivity and offers a good solution in case a qualitative quick analysis of anti-IFX antibodies is required. References: [1] Vande Casteele, (2015), Trough concentrations of infliximab guide dosing for patients with inflammatory bowel disease, Gastroenterology, 1320–9

Background Loss of clinical response and infusion reactions to infliximab (IFX) are associated to the development of antibodies to IFX (ATI). ATI detection is a key step of patient management. However, current techniques may require additional patient appointments for sample collection, processing and batching in centralised facilities. Test reporting usually takes several days or weeks impairing effective decision making. Objectives To clinically validate the use of a new rapid test to detect ATI in capillary blood in a real-life point-of-care (POC) setting where patients attend the infusion center for the reference IFX (Remicade®, RMC) or CT-P13 (Inflectra®, IFT, or Remsima®) infusions. Methods PQ-EF1 and PQ-EF2 are prospective, observational studies designed to evaluate and compare the performance of a rapid POC test (CE-marked Promonitor® Quick Anti-IFX, Progenika, Spain) to detect ATI in routine clinical practice in rheumatic and gastroenterology patients treated with the reference IFX or the biosimilar CT-P13 attending the infusion center with the ELISA technique as a reference. The POC test is a qualitative immunochromatographic assay based on lateral flow technology to detect ATI (including biosimilar CT-P13) in either fingerprick or serum or venous whole blood. Consecutive patients (initiating or under maintenance therapy) were recruited and tested in La Paz and Basurto University hospitals with the rapid test in capillary and venous whole blood specimens immediately before the infusion. ATI test results were read visually with the POC test in 30 min, just before the patient started the infusion. Trough sera were also collected for subsequent analysis with the rapid test and benchmarked with Promonitor®-Anti-IFX ELISA (Progenika, Spain). Follow-up time was 6 months. ELISA quantitative results were categorized as positive and negative to allow comparisons with the qualitative rapid test. Results Ninety consecutive patients were recruited (a total of 137 visits in the 6 months follow-up) accounting for a total of 137 sera, 137 fingerpricks and 71 venous whole blood samples. Overall, 8 (8.9%) patients developed ATI (5 ankylosing spondylitis, 1 Crohn9s disease, 1 ulcerative colitis and 1 juvenile idiopathic arthritis). ATI were detected in 5 patients treated with Remicade® and 3 treated with Inflectra®. Overall agreements between fingerprick vs venous whole blood and fingerprick vs serum measured with the rapid POC test were 100% and 99%, respectively. Positive (PPA) and negative (NPA) agreements between the POC test and ELISA were 91% and 99%, respectively. PPA and NPA between the ELISA and the POC test in serum was 100% and 99%, respectively. Conclusions ATI can be reliably detected in either venous or capillary circulation. Results show an almost perfect agreement between specimens and with the reference ELISA technique. ATI measurement with the POC test allows the clinician to detect ATI in a quick and fully decentralised mode facilitating immediate POC decision making. Disclosure of Interest A. Ametzazurra Employee of: Employee of Progenika Grifols, N. Rivera: None declared, A. Balsa: None declared, M. Arreba: None declared, E. Ruiz: None declared, C. Plasencia: None declared, J. Ortiz: None declared, D. Pascual-Salcedo: None declared, M. Muñoz: None declared, C. De Aysa: None declared, M. Allande: None declared, N. Torres Employee of: Employee of Progenika Grifols, A. Hernández Employee of: Employee of Progenika Grifols, X. Recalde Employee of: Employee of Progenika Grifols, A. Martínez Employee of: Employee of Progenika Grifols, D. Nagore Employee of: Employee of Progenika Grifols

Background Antibodies to infliximab (ATI) are associated to loss of response, adverse effects and discontinuation of treatment in rheumatic patients1. However, the clinical relevance of measuring free versus total anti-drug antibodies (free plus drug-complexed antibodies) remains to be established. Objectives To analyse the association between the clinical response and ATI measured as free and total content of immunoglobulins in a cohort of patients with spondyloarthritis. Methods A retrospective pilot study was conducted including 12 patients with spondyloarthropathies (9 ankylosing spondylitis (AS) and 3 spondyloarthritis (SpA)) under maintenance infliximab (IFX) therapy (3–5 mg/kg every 8 weeks). Disease activity was assessed by BASDAI score every 6 months. Trough serum samples were consecutively collected every two months during a follow up period of 1±0.1 years. In total 75 samples were collected (average of 6.3 per patient). Ten patients (83%) received concomitant therapy with methotrexate. IFX levels were measured with Promonitor®-IFX (Progenika-Grifols, Spain). Free and total-ATI were measured with Promonitor® Anti-IFX (Progenika-Grifols, Spain) (PR) and IDKmonitor® Infliximab total ADA (IDK) (Immundiagnostik, Germany) ELISA kits, respectively. Cut-point of PR and IDK ATI assays is 5 and 10 AU/mL, respectively. FDA guidance for comparing two diagnostic tests in the absence of a gold standard was followed2. Results Five (41.7%) and (83.3%) patients were positive for free and total-ATI using PR and IDK assays, respectively. Overall, positive (PPA) and negative (NPA) percent agreements between the two assays were 52%, 16% and 100%, respectively. All samples with positive free-ATI (7) had undetectable IFX levels (0.0±0.0 μg/mL); however, 72% of total-ATI positive samples (43) were also positive for IFX levels (1.7±1.7 μg/mL, min 0.035 μg/mL and max 6.4 μg/mL). Both outcomes are coherent with the intended use of each kit, which also explains the low PPA. However, total-ATI are positive in 40% of the samples even when the patient responds (BASDAI<4), which questions the utility of total-antibodies in a clinical setting. This evidences a low clinical specificity of total-ATI measurement using the cut-off of 10 AU/mL recommended by the manufacturer and therefore that the presence of total-ATI in the presence of detectable drug has little utility. Patients without free-ATI had significantly lower BASDAI than patients with free-ATI (2.7±2.0 without antibodies vs 5.0±0.9 with antibodies, p=0.049), whereas no statistically significant difference was observed between patients when total-ATI were used (3.1±2.2 without antibodies vs 2.9±2.0 with antibodies, p=0.969) (Fig. 1). Conclusions Total-ATI do not correlate with disease activity in spondyloarthritis. The fact that many samples are positive to total-ATI even in patients with low disease activity makes very difficult interpretation of results. The explanation is that the fraction of ATI bound to IFX is not clinically relevant as long as there is free therapeutic drug in circulation. References Ducourau et al. Arthritis Res Ther 2011;13:R105 Statistical guidance on reporting results from studies evaluating diagnostic tests. 2007.FDA Disclosure of Interest A. Ruiz del Agua Employee of: Employee of Progenika Grifols, J. Pascual Employee of: Employee of Progenika Grifols, N. Torres Employee of: Employee of Progenika Grifols, D. Pascual-Salcedo: None declared, A. Martínez: None declared, T. Jurado: None declared, C. Plasencia: None declared, A. Balsa: None declared, B. Ruiz-Argüello Employee of: Employee of Progenika Grifols, A. Maguregui Employee of: Employee of Progenika Grifols, A. Ametzazurra Employee of: Employee of Progenika Grifols, A. Martínez Employee of: Employee of Progenika Grifols, D. Nagore Employee of: Employee of Progenika Grifols

The development of non-invasive diagnostic methods for endometriosis requires sensitive and disease specific biomarkers. Here, we describe the use of aspirated endometrial fluid from women with and without endometriosis as a novel biological sample for biomarker discovery.Differential protein expression profiling of aspirates from women with early endometriosis (n = 14), advanced endometriosis (n = 32) and without evidence of the disease (n = 32) was assessed by two-dimensional gel electrophoresis (2-DE). A biomarker validation study was performed in an independent cohort (early endometriosis n = 6 and advanced endometriosis n = 14, controls n = 15).The analysis resulted in the identification of 31 proteins showing statistically significant differences in expression. The proteins identified are related to cell signalling, cell death and cell movement, processes that may be involved in the onset and/or progression of endometriosis. The differences in expression observed for 14-3-3 (signal transduction) and moesin (cytoskeletal structure) were confirmed in an independent group of endometriosis patients.Endometrial fluid represents a novel sample for proteomic analysis offering reliable, disease specific information on protein expression, facilitating the discovery of biomarkers for endometriosis. The results described here complement previous proteomic studies, providing new endometriosis-related proteins to be validated as diagnostic markers.

The N-α-acetyltransferase NatB, composed in Saccharomyces cerevisiae by the Nat3p and Mdm20p subunits, is an important factor for yeast growth and resistance to several stress agents. However, the expression and functional role of the mammalian counterpart has not yet been analysed. Here, we report the identification of Nat3p human homologue (hNAT5/hNAT3) and the characterization of its biological function. We found that hNAT5/hNAT3 silencing in HeLa cells results in inhibition of cell proliferation and increased sensitivity to the pro-apoptotic agent MG132. Moreover, inhibition of hNAT5/hNAT3 expression induces p53 activation and upregulation of the antiproliferative protein p21(WAF1/CIP1). The changes of the cellular transcriptome after hNAT5/hNAT3 knockdown confirmed the involvement of this protein in cell growth and survival processes. Among the genes differentially expressed, we observed upregulation of several p53-dependent antiproliferative and pro-apoptotic genes. In the c-myc transgenic mice, which is a model of inducible hepatocarcinoma, we found that hNAT5/hNAT3 was upregulated when the tumour was induced. In accordance with this observation, we noticed increased hNAT5/hNAT3 protein level in neoplastic versus non-neoplastic tissue in a high proportion of patients with hepatocellular carcinoma. Consequently, our results suggest that hNAT5/hNAT3 is required for cellular proliferation and can be implicated in tumour growth.

Background: Signal transducers and activators of transcription (STATs) play a critical role in antiviral defence. STAT3 is also important in cell protection against inflammatory damage. STAT proteins are activated by interferons and by hepatoprotective cytokines of the interleukin 6 superfamily, including cardiotrophin 1.Methods: We analysed the status of STATs in hepatitis C virus (HCV) infected livers and the relationship between expression and activation of STATs and HCV replication in Huh7 cells transfected with HCV genomic replicon.Results: STAT3 alpha expression was reduced in HCV infected livers showing an inverse correlation with serum alanine aminotransferase. In patients with HCV infection, nuclear staining for phosphorylated STAT3 was faint in parenchymal cells (although conspicuous in infiltrating leucocytes), in contrast with strong nuclear staining in hepatocytes from control livers. Expression and activation of STAT1 (a factor activated by both interferon (IFN)-alpha and IFN-gamma) were increased in HCV infected livers, particularly in those with high inflammatory activity. Conversely, phosphorylated STAT2 (a factor selectively activated by IFN-alpha) was undetectable in livers with HCV infection, a finding that was associated with marked downregulation of the two functional subunits of the IFN-alpha receptor. HCV replication in Huh7 cells caused STAT3 alpha downregulation and blocked STAT3 phosphorylation by either IFN-alpha or cardiotrophin 1. HCV replication in Huh7 cells also inhibited STAT1 and STAT2 activation by IFN-alpha while there was no impairment of STAT1 phosphorylation by the proinflammatory cytokine IFN-gamma.Conclusions: STAT3 is downregulated in HCV infected livers and in Huh7 cells bearing the full length HCV replicon. HCV replication is associated with impaired Jak-STAT signalling by antiviral and cytoprotective cytokines. These effects may favour viral replication while facilitating the progression of liver disease.

Schizosaccharomyces pombe cdc42(+) regulates cell morphology and polarization of the actin cytoskeleton. Scd1p/Ral1p is the only described guanine nucleotide exchange factor (GEF) for Cdc42p in S. pombe. We have identified a new GEF, named Gef1p, specifically regulating Cdc42p. Gef1p binds to inactive Cdc42p but not to other Rho GTPases in two-hybrid assays. Overexpression of gef1(+) increases specifically the GTP-bound Cdc42p, and Gef1p is capable of stimulating guanine nucleotide exchange of Cdc42p in vitro. Overexpression of gef1(+) causes changes in cell morphology similar to those caused by overexpression of the constitutively active cdc42G12V allele. Gef1p localizes to the septum. gef1(+) deletion is viable but causes a mild cell elongation and defects in bipolar growth and septum formation, suggesting a role for Gef1p in the control of cell polarity and cytokinesis. The double mutant gef1delta scd1delta is not viable, indicating that they share an essential function as Cdc42p activators. However, both deletion and overexpression of either gef1(+) or scd1(+) causes different morphological phenotypes, which suggest different functions. Genetic evidence revealed a link between Gef1p and the signaling pathway of Shk1/Orb2p and Orb6p. In contrast, no genetic interaction between Gef1p and Shk2p-Mkh1p pathway was observed.