Effect of an Exclusive Human Milk Diet on the Gut Microbiome in Preterm Infants: A Randomized Clinical Trial.

Importance: The effect of using an exclusive human milk diet compared with one that uses bovine products in preterm infants is uncertain, but some studies demonstrate lower rates of key neonatal morbidities. A potential mediating pathway is the gut microbiome. Objective: To determine the effect of an exclusive human milk diet on gut bacterial richness, diversity, and proportions of specific taxa in preterm infants from enrollment to 34 weeks' postmenstrual age. Design, Setting, and Participants: In this randomized clinical trial conducted at 4 neonatal intensive care units in the United Kingdom from 2017 to 2020, microbiome analyses were blind to group. Infants less than 30 weeks' gestation who had only received own mother's milk were recruited before 72 hours of age. Statistical analysis was performed from July 2019 to September 2021. Interventions: Exclusive human milk diet using pasteurized human milk for any shortfall in mother's own milk supply and human milk-derived fortifiers (intervention) compared with bovine formula and bovine-derived fortifier (control) until 34 weeks' postmenstrual age. Fortifier commenced less than 48 hours of tolerating 150 mL/kg per day. Main Outcomes and Measures: Gut microbiome profile including alpha and beta diversity, and presence of specific bacterial taxa. Results: Of 126 preterm infants enrolled in the study, 63 were randomized to control (median [IQR] gestation: 27.0 weeks [26.0-28.1 weeks]; median [IQR] birthweight: 910 g [704-1054 g]; 32 [51%] male) and 63 were randomized to intervention (median [IQR] gestation: 27.1 weeks [25.7-28.1 weeks]; median [IQR] birthweight: 930 g [733-1095 g]; 38 [60%] male); 472 stool samples from 116 infants were analyzed. There were no differences in bacterial richness or Shannon diversity over time, or at 34 weeks between trial groups. The exclusive human milk diet group had reduced relative abundance of Lactobacillus after adjustment for confounders (coefficient estimate, 0.056; P = .03), but not after false discovery rate adjustment. There were no differences in time to full feeds, necrotizing enterocolitis, or other key neonatal morbidities. Conclusions and Relevance: In this randomized clinical trial in preterm infants using human milk-derived formula and/or fortifier to enable an exclusive human milk diet, there were no effects on overall measures of gut bacterial diversity but there were effects on specific bacterial taxa previously associated with human milk receipt. These findings suggest that the clinical impact of human milk-derived products is not modulated via microbiomic mechanisms. Trial Registration: ISRCTN trial registry identifier: ISRCTN16799022.

Tracheostomy in children is associated with neutrophilic airway inflammation.

BACKGROUND: Tracheostomies in children are associated with significant morbidity, poor quality of life, excess healthcare costs and excess mortality. The underlying mechanisms facilitating adverse respiratory outcomes in tracheostomised children are poorly understood. We aimed to characterise airway host defence in tracheostomised children using serial molecular analyses. METHODS: Tracheal aspirates, tracheal cytology brushings and nasal swabs were prospectively collected from children with a tracheostomy and controls. Transcriptomic, proteomic and metabolomic methods were applied to characterise the impact of tracheostomy on host immune response and the airway microbiome. RESULTS: Children followed up serially from the time of tracheostomy up to 3 months postprocedure (n=9) were studied. A further cohort of children with a long-term tracheostomy were also enrolled (n=24). Controls (n=13) comprised children without a tracheostomy undergoing bronchoscopy. Long-term tracheostomy was associated with airway neutrophilic inflammation, superoxide production and evidence of proteolysis when compared with controls. Reduced airway microbial diversity was established pre-tracheostomy and sustained thereafter. CONCLUSIONS: Long-term childhood tracheostomy is associated with a inflammatory tracheal phenotype characterised by neutrophilic inflammation and the ongoing presence of potential respiratory pathogens. These findings suggest neutrophil recruitment and activation as potential exploratory targets in seeking to prevent recurrent airway complications in this vulnerable group of patients.

Mechanisms affecting the gut of preterm infants in enteral feeding trials: a nested cohort within a randomised controlled trial of lactoferrin.

OBJECTIVE: To determine the impact of supplemental bovine lactoferrin on the gut microbiome and metabolome of preterm infants. DESIGN: Cohort study nested within a randomised controlled trial (RCT). Infants across different trial arms were matched on several clinical variables. Bacteria and metabolite compositions of longitudinal stool and urine samples were analysed to investigate the impact of lactoferrin supplementation. SETTING: Thirteen UK hospitals participating in a RCT of lactoferrin. PATIENTS: 479 infants born <32 weeks' gestation between June 2016 and September 2017. RESULTS: 10 990 stool and 22 341 urine samples were collected. Analyses of gut microbiome (1304 stools, 201 infants), metabolites (171 stools, 83 infants; 225 urines, 90 infants) and volatile organic compounds (314 stools, 117 infants) were performed. Gut microbiome Shannon diversity at 34 weeks corrected age was not significantly different between infants in the lactoferrin (mean=1.24) or placebo (mean=1.06) groups (p=0.11). Lactoferrin receipt explained less than 1% variance in microbiome compositions between groups. Metabolomic analysis identified six discriminative features between trial groups. Hospital site (16%) and postnatal age (6%) explained the greatest variation in microbiome composition. CONCLUSIONS: This multiomic study identified minimal impacts of lactoferrin but much larger impacts of hospital site and postnatal age. This may be due to the specific lactoferrin product used, but more likely supports the findings of the RCT in which this study was nested, which showed no impact of lactoferrin on reducing rates of sepsis. Multisite mechanistic studies nested within RCTs are feasible and help inform trial interpretation and future trial design.

Strain-specific impacts of probiotics are a significant driver of gut microbiome development in very preterm infants.

The development of the gut microbiome from birth plays important roles in short- and long-term health, but factors influencing preterm gut microbiome development are poorly understood. In the present study, we use metagenomic sequencing to analyse 1,431 longitudinal stool samples from 123 very preterm infants (<32 weeks' gestation) who did not develop intestinal disease or sepsis over a study period of 10 years. During the study period, one cohort had no probiotic exposure whereas two cohorts were given different probiotic products: Infloran (Bifidobacterium bifidum and Lactobacillus acidophilus) or Labinic (B. bifidum, B. longum subsp. infantis and L. acidophilus). Mothers' own milk, breast milk fortifier, antibiotics and probiotics were significantly associated with the gut microbiome, with probiotics being the most significant factor. Probiotics drove microbiome transition into different preterm gut community types (PGCTs), each enriched in a different Bifidobacterium sp. and significantly associated with increased postnatal age. Functional analyses identified stool metabolites associated with PGCTs and, in preterm-derived organoids, sterile faecal supernatants impacted intestinal, organoid monolayer, gene expression in a PGCT-specific manner. The present study identifies specific influencers of gut microbiome development in very preterm infants, some of which overlap with those impacting term infants. The results highlight the importance of strain-specific differences in probiotic products and their impact on host interactions in the preterm gut.

Systematically quantifying morphological features reveals constraints on organoid phenotypes.

Organoids recapitulate complex 3D organ structures and represent a unique opportunity to probe the principles of self-organization. While we can alter an organoid's morphology by manipulating the culture conditions, the morphology of an organoid often resembles that of its original organ, suggesting that organoid morphologies are governed by a set of tissue-specific constraints. Here, we establish a framework to identify constraints on an organoid's morphological features by quantifying them from microscopy images of organoids exposed to a range of perturbations. We apply this framework to Madin-Darby canine kidney cysts and show that they obey a number of constraints taking the form of scaling relationships or caps on certain parameters. For example, we found that the number, but not size, of cells increases with increasing cyst size. We also find that these constraints vary with cyst age and can be altered by varying the culture conditions. We observed similar sets of constraints in intestinal organoids. This quantitative framework for identifying constraints on organoid morphologies may inform future efforts to engineer organoids.

Necrotising enterocolitis, late-onset sepsis and mortality after routine probiotic introduction in the UK.

OBJECTIVE: To compare necrotising enterocolitis (NEC), late-onset sepsis (LOS), focal intestinal perforation (FIP) and mortality in infants from a single neonatal unit before and after probiotic introduction. DESIGN: Retrospective review of infants <32 weeks admitted January 2009-December 2012 (no probiotic) and January 2013-December 2017 (routine probiotics). Infants included were admitted before day 3, and not transferred out before day 3. NEC, LOS and FIP were defined with standard definitions. PATIENTS: 1061 infants were included, 509 preprobiotic and 552 postprobiotic. Median gestation, birth weight and antenatal steroid use did not differ, and proportions of extremely low birthweight infants were similar (37% and 41%). RESULTS: Overall unadjusted risk of NEC (9.2% (95% CI 7.1 to 12.1) vs 10.6% (95% CI 8.2 to 13.4), p=0.48), LOS (16.3% (95% CI 13.2 to 19.6) vs 14.1% (95% CI 11.5 to 17.4), p=0.37) and mortality (9.2% (95% CI 7.1 to 12.1) vs 9.7% (95% CI 7.6 to 12.6), p=0.76) did not differ, nor proportion of surgical NEC. In multiple logistic regression, accounting for gestation, birth weight, antenatal steroid, maternal milk, chorioamnionitis and sex, probiotic receipt was not significantly associated with NEC (adjusted OR (aOR) 1.08 (95% CI 0.71 to 1.68), p=0.73), LOS or mortality. In subgroup (645 infants) >28 weeks, aOR for NEC in the probiotic cohort was 0.42 (95% CI 0.2 to 0.99, p=0.047). FIP was more common in the probiotic cohort (OR 2.3 (95% CI 1.0 to 5.4), p=0.04), not significant in regression analysis (2.11 (95% CI 0.97 to 4.95), p=0.05). CONCLUSIONS: Probiotic use in this centre did not reduce overall mortality or rates of NEC, LOS or FIP but subgroup analysis identified NEC risk reduction in infants >28 weeks, and LOS reduction <28 weeks.

Pre-procedural Preparation and Sedation for Gastrointestinal Endoscopy in Patients with Advanced Liver Disease.

Gastrointestinal endoscopy in patients with advanced liver disease poses various challenges, a major one being procedural sedation and its associated considerations. While sedation during endoscopy can improve patient comfort, decrease anxiety, and facilitate procedural completion, in patients with advanced liver disease, it is also associated with substantial and unique risks due to alterations in drug metabolism and other factors. As such, the choice of sedative agent(s) and related logistics may require careful inter-disciplinary planning and individualized considerations. Furthermore, a large proportion of agents require dose reductions and particular monitoring of the vital signs, level of consciousness, and other indices. In the present review, we provide a contemporary overview of procedural sedation considerations, commonly used intravenous sedatives, and second-line as well as novel sedatives for gastrointestinal endoscopy in patients with advanced liver disease.

Secretory immunoglobulin A in preterm infants: determination of normal values in breast milk and stool.

BACKGROUND: IgA and its secretory form sIgA impact protection from infection and necrotising enterocolitis but little is known about quantities in preterm mums own milk (MOM) or infant stool, onset of endogenous production in the preterm gut, and what affects these. METHODS: We measured by ELISA in MOM and stool from healthy preterm infants total IgA and sIgA longitudinally and additionally in MOM fresh, refrigerated, frozen, and after traversing feeding systems. RESULTS: In 42 MOM (median gestation 26 weeks), we showed total IgA levels and sIgA were highest in colostrum, fell over 3 weeks, and were not impacted by gestation. Median IgA values matched previous term studies (700 mcg/ml). In MOM recipients stool IgA was detected in the first week, at around 30% of MOM quantities. Formula fed infants did not have detectable stool IgA until the third week. Levels of IgA and sIgA were approximately halved by handling processes. CONCLUSIONS: MOM in the 3 weeks after preterm delivery contains the highest concentrations of IgA and sIgA. Endogenous production after preterm birth occurs from the 3 week meaning preterm infants are dependent on MOM for IgA which should be optimised. Routine NICU practices halve the amount available to the infant. IMPACT: (Secretory) Immunoglobulin A (IgA) is present in colostrum of maternal milk from infants as preterm as 23-24 weeks gestational age, falling over the first 3 weeks to steady levels similar to term. Gestation at birth does not impact (secretory) IgA levels in breast milk. IgA is present in very preterm infant stools from maternal milk fed infants from the first week of life, but not in formula milk fed preterm infants until week three, suggesting endogenous production from this point. Refrigeration, freezing, and feeding via plastic tubing approximately halved the amount of IgA available.

Responsiveness to perturbations is a hallmark of transcription factors that maintain cell identity in vitro.

Identifying the particular transcription factors that maintain cell type in vitro is important for manipulating cell type. Identifying such transcription factors by their cell-type-specific expression or their involvement in developmental regulation has had limited success. We hypothesized that because cell type is often resilient to perturbations, the transcriptional response to perturbations would reveal identity-maintaining transcription factors. We developed perturbation panel profiling (P3) as a framework for perturbing cells across many conditions and measuring gene expression responsiveness transcriptome-wide. In human iPSC-derived cardiac myocytes, P3 showed that transcription factors important for cardiac myocyte differentiation and maintenance were among the most frequently upregulated (most responsive). We reasoned that one function of responsive genes may be to maintain cellular identity. We identified responsive transcription factors in fibroblasts using P3 and found that suppressing their expression led to enhanced reprogramming. We propose that responsiveness to perturbations is a property of transcription factors that help maintain cellular identity in vitro. A record of this paper's transparent peer review process is included in the supplemental information.

Use of omic technologies in early life gastrointestinal health and disease: from bench to bedside.

Introduction: At birth, the gastrointestinal (GI) tract is colonized by a complex community of microorganisms, forming the basis of the gut microbiome. The gut microbiome plays a fundamental role in host health, disorders of which can lead to an array of GI diseases, both short and long term. Pediatric GI diseases are responsible for significant morbidity and mortality, but many remain poorly understood. Recent advancements in high-throughput technologies have enabled deeper profiling of GI morbidities. Technologies, such as metagenomics, transcriptomics, proteomics and metabolomics, have already been used to identify associations with specific pathologies, and highlight an exciting area of research. However, since these diseases are often complex and multifactorial by nature, reliance on a single experimental approach may not capture the true biological complexity. Therefore, multi-omics aims to integrate singular omic data to further enhance our understanding of disease.Areas covered: This review will discuss and provide an overview of the main omic technologies that are used to study complex GI pathologies in early life.Expert opinion: Multi-omic technologies can help to unravel the complexities of several diseases during early life, aiding in biomarker discovery and enabling the development of novel therapeutics and augment predictive models.

Genetic screening for single-cell variability modulators driving therapy resistance.

Cellular plasticity describes the ability of cells to transition from one set of phenotypes to another. In melanoma, transient fluctuations in the molecular state of tumor cells mark the formation of rare cells primed to survive BRAF inhibition and reprogram into a stably drug-resistant fate. However, the biological processes governing cellular priming remain unknown. We used CRISPR-Cas9 genetic screens to identify genes that affect cell fate decisions by altering cellular plasticity. We found that many factors can independently affect cellular priming and fate decisions. We discovered a new plasticity-based mode of increasing resistance to BRAF inhibition that pushes cells towards a more differentiated state. Manipulating cellular plasticity through inhibition of DOT1L before the addition of the BRAF inhibitor resulted in more therapy resistance than concurrent administration. Our results indicate that modulating cellular plasticity can alter cell fate decisions and may prove useful for treating drug resistance in other cancers.

Development, Implementation, and Evaluation of a Telemedicine Preoperative Evaluation Initiative at a Major Academic Medical Center.

BACKGROUND: With health care practice consolidation, the increasing geographic scope of health care systems, and the advancement of mobile telecommunications, there is increasing interest in telemedicine-based health care consultations. Anesthesiology has had experience with telemedicine consultation for preoperative evaluation since 2004, but the majority of studies have been conducted in rural settings. There is a paucity of literature of use in metropolitan areas. In this article, we describe the implementation of a telemedicine-based anesthesia preoperative evaluation and report the program's patient satisfaction, clinical case cancellation rate outcomes, and cost savings in a large metropolitan area (Los Angeles, CA). METHODS: This is a descriptive study of a telemedicine-based preoperative anesthesia evaluation process in an academic medical center within a large metropolitan area. In a 2-year period, we evaluated 419 patients scheduled for surgery by telemedicine and 1785 patients who were evaluated in-person. RESULTS: Day-of-surgery case cancellations were 2.95% and 3.23% in the telemedicine and the in-person cohort, respectively. Telemedicine patients avoided a median round trip driving distance of 63 miles (Q1 24; Q3 119) and a median time saved of 137 (Q1 95; Q3 195) and 130 (Q1 91; Q3 237) minutes during morning and afternoon traffic conditions, respectively. Patients experienced time-based savings, particularly from traveling across a metropolitan area, which amounted to $67 of direct and opportunity cost savings. From patient satisfaction surveys, 98% (129 patients out of 131 completed surveys) of patients who were consulted via telemedicine were satisfied with their experience. CONCLUSIONS: This study demonstrates the implementation of a telemedicine-based preoperative anesthesia evaluation from an academic medical center in a metropolitan area with high patient satisfaction, cost savings, and without increase in day-of-procedure case cancellations.

IL-5 Exposure In Utero Increases Lung Nerve Density and Airway Reactivity in Adult Offspring.

Asthma is characterized by airway hyperreactivity and inflammation. In the lungs, parasympathetic and sensory nerves control airway tone and induce bronchoconstriction. Dysregulation of these nerves results in airway hyperreactivity. Humans with eosinophilic asthma have significantly increased sensory nerve density in airway epithelium, suggesting that type 2 cytokines and inflammatory cells promote nerve growth. Similarly, mice with congenital airway eosinophilia also have airway hyperreactivity and increased airway sensory nerve density. Here, we tested whether this occurs during development. We show that transgenic mice that overexpress IL-5, a cytokine required for eosinophil hematopoiesis, give birth to wild-type offspring that have significantly increased airway epithelial nerve density and airway hyperreactivity that persists into adulthood. These effects are caused by in utero exposure to maternal IL-5 and resulting fetal eosinophilia. Allergen exposure of these adult wild-type offspring results in severe airway hyperreactivity, leading to fatal reflex bronchoconstriction. Our results demonstrate that fetal exposure to IL-5 is a developmental origin of airway hyperreactivity, mediated by hyperinnervation of airway epithelium.

Effectiveness of Prehospital Dual Sequential Defibrillation for Refractory Ventricular Fibrillation and Ventricular Tachycardia Cardiac Arrest.

Objective: Dual sequential defibrillation (DSD) - successive defibrillations with two defibrillators - offers a novel approach to refractory ventricular fibrillation (RVF) and tachycardia (VF/VT). While associated with rescue shock success, the effect of DSD upon out-of-hospital cardiac arrest (OHCA) is unknown. We evaluated the association of DSD with survival after refractory VF/VT OHCA. Methods: We used data from a large metropolitan fire-based EMS service. We included all adult OHCA during 2013-2016 with ≥3 standard defibrillations. Physicians authorized subsequent DSD use by two separate defibrillators (PhysioControl LIFEPAK® 12/15) with pads placed anterior-lateral and anterior-posterior. Evaluated outcomes included return of spontaneous circulation (ROSC), survival to hospital admission, survival to 72 hours, and survival to hospital discharge. Using multivariable logistic regression, we evaluated the association between defibrillation type and OHCA outcomes, adjusting for patient demographics and event characteristics. Results: We included 310 patients in the analysis, 71 patients receiving DSD and 239 receiving conventional defibrillation. Patient demographics and event characteristics were similar between both groups. ROSC was lower for DSD than standard defibrillation: 39.4% vs. 60.3%, adjusted OR 0.46 (95% CI: 0.25-0.87). There were no differences in survival to hospital admission (35.2% vs. 49.2%, adjusted OR 0.57 [95% CI: 0.30-1.08]), survival to 72 hours (21.4% vs. 32.3%, adjusted OR 0.52 [95% CI: 0.26-1.10]), or survival to hospital discharge (14.3% vs. 20.9%, adjusted OR 0.63 [95% CI: 0.27-1.45]). Conclusions: Compared with conventional defibrillation, DSD was associated with lower odds of prehospital ROSC. Defibrillation type was not associated with other OHCA endpoints. DSD may not be beneficial in refractory VF/VT OHCA.

Automated Assessment of Existing Patient's Revised Cardiac Risk Index Using Algorithmic Software.

BACKGROUND: Previous work in the field of medical informatics has shown that rules-based algorithms can be created to identify patients with various medical conditions; however, these techniques have not been compared to actual clinician notes nor has the ability to predict complications been tested. We hypothesize that a rules-based algorithm can successfully identify patients with the diseases in the Revised Cardiac Risk Index (RCRI). METHODS: Patients undergoing surgery at the University of California, Los Angeles Health System between April 1, 2013 and July 1, 2016 and who had at least 2 previous office visits were included. For each disease in the RCRI except renal failure-congestive heart failure, ischemic heart disease, cerebrovascular disease, and diabetes mellitus-diagnosis algorithms were created based on diagnostic and standard clinical treatment criteria. For each disease state, the prevalence of the disease as determined by the algorithm, International Classification of Disease (ICD) code, and anesthesiologist's preoperative note were determined. Additionally, 400 American Society of Anesthesiologists classes III and IV cases were randomly chosen for manual review by an anesthesiologist. The sensitivity, specificity, accuracy, positive predictive value, negative predictive value, and area under the receiver operating characteristic curve were determined using the manual review as a gold standard. Last, the ability of the RCRI as calculated by each of the methods to predict in-hospital mortality was determined, and the time necessary to run the algorithms was calculated. RESULTS: A total of 64,151 patients met inclusion criteria for the study. In general, the incidence of definite or likely disease determined by the algorithms was higher than that detected by the anesthesiologist. Additionally, in all disease states, the prevalence of disease was always lowest for the ICD codes, followed by the preoperative note, followed by the algorithms. In the subset of patients for whom the records were manually reviewed, the algorithms were generally the most sensitive and the ICD codes the most specific. When computing the modified RCRI using each of the methods, the modified RCRI from the algorithms predicted in-hospital mortality with an area under the receiver operating characteristic curve of 0.70 (0.67-0.73), which compared to 0.70 (0.67-0.72) for ICD codes and 0.64 (0.61-0.67) for the preoperative note. On average, the algorithms took 12.64 ± 1.20 minutes to run on 1.4 million patients. CONCLUSIONS: Rules-based algorithms for disease in the RCRI can be created that perform with a similar discriminative ability as compared to physician notes and ICD codes but with significantly increased economies of scale.

Cdh1 and Pik3ca Mutations Cooperate to Induce Immune-Related Invasive Lobular Carcinoma of the Breast.

CDH1 and PIK3CA are the two most frequently mutated genes in invasive lobular carcinoma (ILC) of the breast. Transcription profiling has identified molecular subtypes for ILC, one of which, immune-related (IR), is associated with gene expression linked to lymphocyte and macrophage infiltration. Here, we report that deletion of Cdh1, together with activation of Pik3ca in mammary epithelium of genetically modified mice, leads to formation of IR-ILC-like tumors with immune cell infiltration, as well as gene expression linked to T-regulatory (Treg) cell signaling and activation of targetable immune checkpoint pathways. Interestingly, these tumors show enhanced Rac1- and Yap-dependent transcription and signaling, as well as sensitivity to PI3K, Rac1, and Yap inhibitors in culture. Finally, high-dimensional immunophenotyping in control mouse mammary gland and IR-ILC tumors by mass cytometry shows dramatic alterations in myeloid and lymphoid populations associated with immune suppression and exhaustion, highlighting the potential for therapeutic intervention via immune checkpoint regulators.

Quanti.us: a tool for rapid, flexible, crowd-based annotation of images.

We describe Quanti.us , a crowd-based image-annotation platform that provides an accurate alternative to computational algorithms for difficult image-analysis problems. We used Quanti.us for a variety of medium-throughput image-analysis tasks and achieved 10-50× savings in analysis time compared with that required for the same task by a single expert annotator. We show equivalent deep learning performance for Quanti.us-derived and expert-derived annotations, which should allow scalable integration with tailored machine learning algorithms.

The GalNAc-T Activation Pathway (GALA) is not a general mechanism for regulating mucin-type O-glycosylation.

Mucin-type O-glycosylation is initiated by the UDP-GalNAc polypeptide:N-acetylgalactosaminyltransferase (GalNAc-T) family of enzymes. Their activity results in the GalNAc α1-O-Thr/Ser structure, termed the Tn antigen, which is further decorated with additional sugars. In neoplastic cells, the Tn antigen is often overexpressed. Because O-glycosylation is controlled by the activity of GalNAc-Ts, their regulation is of great interest. Previous reports suggest that growth factors, EGF or PDGF, induce Golgi complex-to-endoplasmic reticulum (ER) relocation of both GalNAc-Ts and Tn antigen in HeLa cells, offering a mechanism for Tn antigen overexpression termed "GALA". However, we were unable to reproduce these findings. Upon treatment of HeLa cells with either EGF or PDGF we observed no change in the co-localization of endogenous GalNAc-T1, GalNAc-T2 or Tn antigen with the Golgi complex marker TGN46. There was also no enhancement of localization with the ER marker calnexin. We conclude that growth factors do not cause redistribution of GalNAc-Ts from the Golgi complex to the ER in HeLa cells.