Overruling of Procalcitonin-Guided Antibiotics for Lower Respiratory Tract Infections in Primary Care: Ancillary Study of a Randomized Controlled Trial.

BACKGROUND: Lower respiratory tract infections (LRTIs) in primary care are a promising target for antibiotic stewardship. A clinical trial in Switzerland showed a large decrease in antibiotic prescriptions with procalcitonin guidance (cut-off < 0.25 µg/L) compared with usual care. However, one-third of patients with low procalcitonin at baseline received antibiotics by day 28. AIM: To explore the factors associated with the overruling of initial procalcitonin guidance. DESIGN AND SETTING: Secondary analysis of a cluster randomized trial in which patients with an LRTI were included. METHOD: Using the characteristics of patients, their disease, and general practitioners (GPs), we conducted a multivariate logistic regression, adjusted for clustering. RESULTS: Ninety-five out of 301 (32%) patients with low procalcitonin received antibiotics by day 28. Factors associated with an overruling of procalcitonin guidance were: a history of chest pain (adjusted OR [aOR] 1.81, 95% confidence interval 1.03-3.17); a prescription of chest X-ray by the GP (aOR 4.65, 2.32-9.34); a C-reactive protein measured retrospectively above 100 mg/L (aOR 7.48, 2.34-23.93, reference ≤ 20 mg/L); the location of the GP practice in an urban setting (aOR 2.27, 1.18-4.37); and the GP's number of years of experience (aOR per year 1.05, 1.01-1.09). CONCLUSIONS: Overruling of procalcitonin guidance was associated with GPs' socio-demographic characteristics, pointing to the general behavioral problem of overprescription by physicians. Continuous medical education and communication training might support the successful implementation of procalcitonin point-of-care tests aimed at antibiotic stewardship.

Primary HSV-2 Infection Complicated by Radiculomyelitis in a Young Immunocompetent Female Patient with Inherited Chromosomally Integrated HHV-6: A Case Report.

BACKGROUND: HSV-1, HSV-2 and VZV are alpha Herpesviruses, neurotropic viruses that are associated with various neurologic complications upon primary infection or reactivation. Cases of myelitis and radiculomyelitis are rare and appropriate etiologic diagnoses can be tricky. CASE PRESENTATION: Here we describe the case of a young immunocompetent woman who developed painful and extended vesicular genital lesions, with subsequent radiculomyelitis. HSV-1/-2 PCRs in the cerebrospinal fluid were misleadingly negative, whereas HHV-6 PCR was positive. Positive anti-HSV-2 IgM and IgG in serum was consistent with HSV-2 primary infection. On the other hand, the detection of HHV-6 DNA was explained by inherited chromosomally integrated HHV-6. The clinical course was favorable with high-dose IV acyclovir and corticosteroids. CONCLUSION: HSV-2-related radiculomyelitis is a rare clinical entity, which can be difficult to diagnose. In this case report, the causative virus was not detected in the patient's CSF, whereas HHV-6 DNA, non-pathogenic in this situation, was paradoxically positive. The diagnosis was based on the clinical features typical for HSV-2 primary infection, confirmed by the serology results. The delay between the genital lesions and the appearance of the radiculomyelitis, along with the absence of HSV-2 detection in the CSF, suggests a possible immuno-mediated physiopathological process. As for the HHV-6 DNA detection in the patient's CSF, it was explained by inherited chromosomally integrated HHV-6. This case illustrates how both negative and positive clinical virology results need careful interpretation according to the clinical findings.

Rhinovirus Infections among Hematopoietic Stem Cell Transplant Recipients: A Pre-Transplant Dilemma?

Respiratory viral infections (RVIs) in allogeneic hematopoietic stem cell transplant (allo-HSCT) recipients can be of concern due to the patients' depressed immune status, but few data are available about the significance of a pre-transplant positive testing. In this retrospective observational study, we analyzed a cohort of patients that were transplanted between 1 January 2010 and 31 October 2019 in the Geneva University Hospitals with at least one RVI before or after transplantation. At least one RVI was detected in 319/533 (63.5%) transplanted patients. Rhinoviruses were most frequently identified (37%), followed by human coronaviruses (17.1%), parainfluenza viruses (13.9%), and influenza viruses (9.9%). First infection in the post-transplant period occurred at a mean time of 334 days (SD 338). Specific analysis of a subgroup of 65 patients with pre-transplant RVIs was performed. Among them, 39 (59%) patients had symptoms and 14 (21.2%) had a lower respiratory tract infection. Four patients (6.1%) (three rhinovirus and one influenza) needed an intensive care unit admission, of which, three (4.5%) (two rhinovirus and one influenza) were intubated. The patient with influenza infection diagnosed the day of the transplantation died within the first 30 days of the infection. Two patients with rhinovirus infection died within 3 months of unrelated causes. Our data show that rhinovirus infections are predominant in allo-HSCT patients, including among pre-transplant infections; however, mortality due to pre-transplant RVI is low and was only clearly identified in one patient with influenza infection. RVI within the month preceding allo-HSCT is not associated with direct morbidity or mortality in this cohort.

Clinical sign and biomarker-based algorithm to identify bacterial pneumonia among outpatients with lower respiratory tract infection in Tanzania.

BACKGROUND: Inappropriate antibiotics use in lower respiratory tract infections (LRTI) is a major contributor to resistance. We aimed to design an algorithm based on clinical signs and host biomarkers to identify bacterial community-acquired pneumonia (CAP) among patients with LRTI. METHODS: Participants with LRTI were selected in a prospective cohort of febrile (≥ 38 °C) adults presenting to outpatient clinics in Dar es Salaam. Participants underwent chest X-ray, multiplex PCR for respiratory pathogens, and measurements of 13 biomarkers. We evaluated the predictive accuracy of clinical signs and biomarkers using logistic regression and classification and regression tree analysis. RESULTS: Of 110 patients with LRTI, 17 had bacterial CAP. Procalcitonin (PCT), interleukin-6 (IL-6) and soluble triggering receptor expressed by myeloid cells-1 (sTREM-1) showed an excellent predictive accuracy to identify bacterial CAP (AUROC 0.88, 95%CI 0.78-0.98; 0.84, 0.72-0.99; 0.83, 0.74-0.92, respectively). Combining respiratory rate with PCT or IL-6 significantly improved the model compared to respiratory rate alone (p = 0.006, p = 0.033, respectively). An algorithm with respiratory rate (≥ 32/min) and PCT (≥ 0.25 µg/L) had 94% sensitivity and 82% specificity. CONCLUSIONS: PCT, IL-6 and sTREM-1 had an excellent predictive accuracy in differentiating bacterial CAP from other LRTIs. An algorithm combining respiratory rate and PCT displayed even better performance in this sub-Sahara African setting.

Unexpected associations between respiratory viruses and bacteria with Pulmonary Function Testing in children suffering from Cystic Fibrosis (MUCOVIB study).

BACKGROUND: Various bacterial and viral assemblages composing Cystic Fibrosis (CF) lung microbiota contribute to long-term lung function decline over time. Yet, the impact of individual microorganisms on pulmonary functions remains uncertain in children with CF. METHODS: As part of the 'Mucoviscidosis, respiratory VIruses, intracellular Bacteria and fastidious organisms'' project, children with CF were longitudinally followed in a Swiss multicentric study. Respiratory samples included mainly throat swabs and sputa samples for bacterial culture and 16S rRNA metagenomics and nasopharyngeal swabs for respiratory virus detection by molecular assays. Percentage of predicted Forced Expiratory Volume in one second (FEV1%) and Lung Clearance Index (LCI) were recorded. RESULTS: Sixty-one children, of whom 20 (32.8%) presented with at least one pulmonary exacerbation, were included. Almost half of the 363 nasopharyngeal swabs tested by RT-PCR were positive for a respiratory virus, mainly rhinovirus (26.5%). From linear mixed-effects regression models, P. aeruginosa (-11.35, 95%CI [-17.90; -4.80], p = 0.001) was significantly associated with a decreased FEV1%, whereas rhinovirus was associated with a significantly higher FEV1% (+4.24 95%CI [1.67; 6.81], p = 0.001). Compared to conventional culture, 16S rRNA metagenomics showed a sensitivity and specificity of 80.0% and 85.4%, respectively for detection of typical CF pathogens. However, metagenomics detected a bacteria almost twice more often than culture. CONCLUSIONS: As expected, P. aeruginosa impacted negatively on FEV1% while rhinovirus was surprisingly associated with better FEV1%. Culture-free assays identifie significantly more pathogens than standard culture, with disputable clinical correlation.

Prognosis of Laboratory-Confirmed Influenza and Respiratory Syncytial Virus in Acute Heart Failure.

Concomitant respiratory viral infections may influence clinical outcomes of acute decompensated heart failure (ADHF) but this association is based on indirect observation. The aim of this study was to evaluate the prevalence and impact of laboratory-confirmed influenza or respiratory syncytial virus (RSV) infection on outcomes in patients hospitalised for ADHF. Prospective cohort of patients hospitalised for ADHF with systematic influenza and RSV screening using real-time PCR on nasopharyngeal swabs. The primary outcome was all-cause mortality or readmission at 90 days. Among 803 patients with ADHF, 196 (24.5%) patients had concomitant flu-like symptoms of influenza. PCR was positive in 45 patients (27 for influenza, 19 for RSV). At 90 days, PCR positive patients had lower rates of all-cause mortality or readmission as compared to patients without flu-like symptoms (HR 0.40, 95% CI 0.18-0.91, p = 0.03), and non-significantly less all-cause mortality (HR 0.30, 95% CI 0.04-2.20, p = 0.24), or HF-related death or readmission (HR 0.36, 95% CI 0.13-0.99, p = 0.05). The prevalence of influenza or RSV infection in patients admitted for ADHF was low and associated with less all-cause mortality and readmission. Concomitant viral infection with ADHF may not in itself be a predictor of poor outcomes. (ClinicalTrials.gov NCT02444416).

Viral co-infections among SARS-CoV-2-infected children and infected adult household contacts.

We evaluated the rates of viral respiratory co-infections among SARS-CoV-2-infected children. Twelve percent of SARS-CoV-2-infected children had viral co-infection with one or more common respiratory viruses. This was significantly more frequent than among their SARS-CoV-2-infected adult household contacts (0%; p=0.028). Compared to the same period the previous year, common respiratory viruses were less frequently detected (12% vs 73%, p<0.001).Conclusion: Despite partial lockdown with school and daycare closure, and consequently similar exposure to common viruses between children and adults, SARS-CoV-2-infected children had more frequent viral respiratory co-infections than their SARS-CoV-2-infected adult household contacts. Circulation of common respiratory viruses was less frequent during the SARS-CoV-2 outbreak when compared to the same period last year, showing the impact of partial lockdown on the circulation of common viruses. What is Known: • Viral respiratory co-infections are frequent in children. • SARS-CoV-2 can be identified alongside other respiratory viruses, but data comparing children and adults are lacking. What is New: • Children infected with SARS-CoV-2 are more likely to have viral respiratory co-infections than their SARS-CoV-2-infected adult household contacts, which is surprising in the context of partial lockdown with schools and daycare closed. • When compared to data collected during the same period last year, our study also showed that partial lockdown reduced circulation of common respiratory viruses.

A community outbreak of Legionnaires' disease in Geneva, Switzerland, June to September 2017.

PURPOSE: Eight confirmed cases of Legionnaires’ disease were identified at the Geneva University Hospitals between 28 July 2017 and 02 August 2017, leading to a detailed outbreak investigation. METHODS: Legionnaires’ disease cases were defined according to Swiss and European (ELDSNet) consensus guidelines. An outbreak investigation task force was put in place. Patients were interviewed, when feasible, with a standard questionnaire. A Legionella pneumophila urinary antigen test was performed in all cases. Lower respiratory tract (LRT) specimens were collected for culture, polymerase chain-reaction (PCR) assay, monoclonal antibody subtyping and sequenced-based typing (SBT). Multiple environmental samples were collected. Case geographical mapping was performed and local meteorological data were obtained. RESULTS: Thirty-four confirmed cases of Legionnaires’ disease were identified between 20 June 2017 and 16 September 2017, including 28 patients living in the Canton of Geneva and 6 cases in neighbouring cantons and France. The case fatality rate was 8.8%. The urinary antigen test was positive in 32/34 (94.1%) cases. Among the 17/34 (50%) cases with available LRT specimens, 8 (47.1%) were culture/PCR positive, 5 (29.4%) were PCR positive only, and 4 (23.5%) were culture/PCR negative. Monoclonal antibody subtyping and SBT on 12 samples allowed subtype identification of 8 samples, with a predominance of L. pneumophila serogroup-1 subtype-France/Allentown ST23 among clinical isolates. A specific city area was identified as a possible outbreak epicentre in 25/34 (73.5%) cases, although molecular analysis of clinical and environmental specimens revealed heterogeneous subtypes of L. pneumophila. CONCLUSIONS: In this largest documented outbreak of Legionnaires’ disease in Switzerland, we report prompt outbreak identification, leading to timely initiation of a detailed, well-orchestrated clinical and epidemiological investigation.

Dengue fever in Dar es Salaam, Tanzania: clinical features and outcome in populations of black and non-black racial category.

BACKGROUND: Although the incidence of dengue across Africa is high, severe dengue is reported infrequently. We describe the clinical features and the outcome of dengue according to raceduring an outbreak in Dar es Salaam, Tanzania that occurred in both native and expatriate populations. METHODS: Adults with confirmed dengue (NS1 and/or IgM on rapid diagnostic test and/or PCR positive) were included between December 2013 and July 2014 in outpatient clinics. Seven-day outcome was assessed by a visit or a call. Association between black race and clinical presentation, including warning signs, was assessed by logistic regression adjusted for age, malaria coinfection, secondary dengue and duration of symptoms at inclusion. The independent association between demographic and comorbidities characteristics of the patients and severe dengue was evaluated by multivariate logistic regression that included potential confounders. RESULTS: After exclusion of 3 patients of mixed race, 431 patients with dengue (serotype 2, genotype Cosmopolitan) were included: 241 of black and 190 of non-black race. Black patients were younger (median age 30 versus 41 years; p < 0.001) and attended care after a slightly longer duration of symptoms (median of 2.9 versus 2.7 days; p = 0.01). Malaria coinfection was not significantly different between black (5%) and non-black (1.6%) patients (p = 0.06). The same proportion of patients in both group had secondary dengue (13 and 14%; p = 0.78). Among warning signs, only mucosal bleed was associated with race, black race being protective (adjusted OR 0.44; 95% CI 0.21-0.92). Overall, 20 patients (4.7%) presented with severe dengue. Non-black race (adjusted OR 3.9; 95% CI 1.3-12) and previously known diabetes (adjusted OR 43; 95% CI 5.2-361) were independently associated with severe dengue. CONCLUSIONS: Although all patients were infected with the same dengue virus genotype, black race was independently protective against a severe course of dengue, suggesting the presence of protective genetic or environmental host factors among people of African ancestry. The milder clinical presentation of dengue in black patients might partly explain why dengue outbreaks are under-reported in Africa and often mistaken for malaria. These results highlight the need to introduce point-of-care tests, beside the one for malaria, to detect outbreaks and orientate diagnosis. TRIAL REGISTRATION: Clinicaltrials.gov Identifier: NCT01947075 , retrospectively registered on the 13 of September 2014.

Prognostic value of quickSOFA as a predictor of 28-day mortality among febrile adult patients presenting to emergency departments in Dar es Salaam, Tanzania.

BACKGROUND: Quick Sequential Organ Failure Assessment (qSOFA) is a three-item clinical instrument for bedside identification of sepsis patients at risk of poor outcome. qSOFA could be a valuable triage tool in emergency departments of low-income countries, yet its performance in resource-limited settings remains unknown. The prognostic accuracy of qSOFA for 28-day all-cause mortality in febrile adults treated at the EDs in a low-income country was evaluated. METHODS: Retrospective analysis of a prospective cohort study of consecutive patients (≥18 years) with fever (tympanic temperature ≥38°C and fever ≤7 days) who presented between July 2013 and May 2014 at four emergency departments in Dar es Salaam, Tanzania. Medical history, clinical examination, laboratory and microbiological data were collected to document the cause of fever. Variables for the previous and new sepsis criteria were collected at inclusion and qSOFA, SOFA and SIRS were measured at inclusion. Patients were followed up by phone at day 28. The performance (sensitivity, specificity and area under the receiver operating curve [AUROC]) of qSOFA (score ≥2), SOFA (increase of ≥2 points) and SIRS (≥2 criteria) as predictors of 28-day all-cause mortality was evaluated. RESULTS: Among the 519 patients (median age: 30 years) included in the analysis, 47% were female and 25% were HIV positive. Overall, 85% had a microbiologically and/or clinically documented infection and 15% a fever of unknown origin. The most common site and causes of infections were the respiratory tract (43%), dengue (26%), malaria (6%) and typhoid fever (5%). Twenty-eight-day all-cause mortality was 6%: 3% for patients with a qSOFA <2 and 24% for those with a score ≥2 (absolute difference, 21%; 95% CI 12%-31%). The prognostic accuracy of qSOFA (AUROC 0.80, 95% CI 0.73-0.87) for 28-day mortality was similar to SOFA (AUROC 0.79, 0.71-0.87; p = 0.1) and better than SIRS (AUROC 0.61, 0.52-0.71; p<0.001). CONCLUSIONS: Among patients with fever at emergency departments in Tanzania, qSOFA had a prognostic accuracy for 28-day mortality comparable to SOFA and superior to SIRS. These results support the use of qSOFA as a triage tool to identify patients with sepsis and at risk of poor outcome in resource-limited countries. TRIAL REGISTRATION: Clinicaltrials.gov Identifier: NCT01947075.

Novel and classical human astroviruses in stool and cerebrospinal fluid: comprehensive screening in a tertiary care hospital, Switzerland.

Classical human astroviruses (HAstV) are the third most common cause of non-bacterial acute gastroenteritis. Due to the lack of routine molecular assays, novel HAstV are underdiagnosed and the magnitude of their contribution to clinical disease remains unknown. To better understand their prevalence and the susceptible patient profile, we conducted a comprehensive screening of novel and classical HAstV in stool and cerebrospinal fluid (CSF) samples collected for clinical care in a tertiary care hospital using a specially designed rRT-PCR panel for the detection of novel (MLB1-3 and VA1-4) and classical HAstV. Of the 654 stool samples, 20 were positive for HAstV, and the novel (n=10; 3 MLB1, 4 MLB2; 3 VA2) and classical (n=10) serotypes were equally prevalent. None of the 105 CSF samples were positive. Investigating the patient profile, we found a higher prevalence (P=0.0002) of both novel and classical HAstV in pediatric stool samples (3.4% and 3%, respectively) compared with adult stool samples (0.5% and 0.7%, respectively). Furthermore, all novel and classical HAstV-positive pediatric subjects were ≤four years old, demonstrating similar susceptible populations. Forty-five percent of positive patients were immunocompromised (novel: 40%, classical: 50%). A comparison of novel and classical HAstV-positive cases showed a lower viral load for novel HAstV (P=0.0007) with significantly more upper respiratory symptoms (70% of subjects; P=0.02); this observation may suggest a unique pathogenic pathway. This study confirms the clinical and epidemiological relevance of novel HAstV and identifies a target population in which routine screening may yield clinically valuable information.

A new real-time RT-qPCR assay for the detection, subtyping and quantification of human respiratory syncytial viruses positive- and negative-sense RNAs.

Human respiratory syncytial virus (RSV) is a major health problem and the main cause of hospitalization due to bronchiolitis. RSV is divided into two antigenic subgroups, RSV-A and -B that co-circulate worldwide. Rapid and sensitive detection is desirable for proper patient handling while assessment of viral load may help to evaluate disease severity and progression. Finally RSV subtyping is needed to determine the prevalence and pathogenicity of each RSV subgroup, as well as their sensitivity to treatment. In this study, we took into account the most recent circulating RSV variants and designed two quantitative TaqMan one-step RT-PCR assays to detect and quantify both RSV subgroups separately. Standard dilutions of transcripts of positive and negative polarities were included in the assay validation to assess potential differences in sensitivity on negative-sense genomes and positive-sense RNAs. In addition, RSV detection in respiratory specimens of different types and sampled in different populations was compared to commercially available RSV diagnostic tools. Altogether, the RSV-A and -B assays revealed sensitive and quantitative over a wide range of viral loads, with a slight improved sensitivity of the RSV-B assay on positive sense transcripts, and allowed accurate RSV subtyping. We thus provide a useful tool for both RSV diagnostics and research.

Ebola virus disease diagnosis by real-time RT-PCR: A comparative study of 11 different procedures.

BACKGROUND: The diagnosis of Ebola virus disease relies on the detection of viral RNA in blood by real-time reverse-transcription PCR. While several of these assays were developed during the unprecedented 2013-2015 Ebola virus disease outbreak in West Africa, few were applied in the field. OBJECTIVES: To compare technical performances and practical aspects of 11 Ebola virus real-time reverse-transcription PCR procedures. STUDY DESIGN: We selected the most promising assays using serial dilutions of culture-derived Ebola virus RNA and determined their analytical sensitivity and potential range of quantification using quantified in vitro transcribed RNA; viral load values in the serum of an Ebola virus disease patient obtained with these assays were reported. Finally, ease of use and turnaround times of these kits were evaluated. RESULTS: Commercial assays were at least as sensitive as in-house tests. Five of the former (Altona, Roche, Fast-track Diagnostics, and Life Technologies) were selected for further evaluation. Despite differences in analytical sensitivity and limits of quantification, all of them were suitable for Ebola virus diagnosis and viral load estimation. The Lifetech Lyophilized Ebola Virus (Zaire 2014) assay (Life Technologies) appeared particularly promising, displaying the highest analytical sensitivity and shortest turnaround time, in addition to requiring no reagent freezing. CONCLUSIONS: Commercial kits were at least as sensitive as in-house tests and potentially easier to use in the field than the latter. This qualitative comparison of real-time reverse transcription PCR assays may serve as a basis for the design of future Ebola virus disease diagnostics.

Foxa1 and Foxa2 regulate α-cell differentiation, glucagon biosynthesis, and secretion.

The Forkhead box A transcription factors are major regulators of glucose homeostasis. They show both distinct and redundant roles during pancreas development and in adult mouse ß-cells. In vivo ablation studies have revealed critical implications of Foxa1 on glucagon biosynthesis and requirement of Foxa2 in α-cell terminal differentiation. In order to examine the respective role of these factors in mature α-cells, we used small interfering RNA (siRNA) directed against Foxa1 and Foxa2 in rat primary pancreatic α-cells and rodent α-cell lines leading to marked decreases in Foxa1 and Foxa2 mRNA levels and proteins. Both Foxa1 and Foxa2 control glucagon gene expression specifically through the G2 element. Although we found that Foxa2 controls the expression of the glucagon, MafB, Pou3f4, Pcsk2, Nkx2.2, Kir6.2, and Sur1 genes, Foxa1 only regulates glucagon gene expression. Interestingly, the Isl1 and Gipr genes were not controlled by either Foxa1 or Foxa2 alone but by their combination. Foxa1 and Foxa2 directly activate and bind the promoter region the Nkx2.2, Kir6.2 and Sur1, Gipr, Isl1, and Pou3f4 genes. We also demonstrated that glucagon secretion is affected by the combined effects of Foxa1 and Foxa2 but not by either one alone. Our results indicate that Foxa1 and Foxa2 control glucagon biosynthesis and secretion as well as α-cell differentiation with both common and unique target genes.

Pax6 controls the expression of critical genes involved in pancreatic {alpha} cell differentiation and function.

The paired box homeodomain Pax6 is crucial for endocrine cell development and function and plays an essential role in glucose homeostasis. Indeed, mutations of Pax6 are associated with diabetic phenotype. Importantly, homozygous mutant mice for Pax6 are characterized by markedly decreased ß and δ cells and absent α cells. To better understand the critical role that Pax6 exerts in glucagon-producing cells, we developed a model of primary rat α cells. To study the transcriptional network of Pax6 in adult and differentiated α cells, we generated Pax6-deficient primary rat α cells and glucagon-producing cells, using either specific siRNA or cells expressing constitutively a dominant-negative form of Pax6. In primary rat α cells, we confirm that Pax6 controls the transcription of the Proglucagon and processing enzyme PC2 genes and identify three new target genes coding for MafB, cMaf, and NeuroD1/Beta2, which are all critical for Glucagon gene transcription and α cell differentiation. Furthermore, we demonstrate that Pax6 directly binds and activates the promoter region of the three genes through specific binding sites and that constitutive expression of a dominant-negative form of Pax6 in glucagon-producing cells (InR1G9) inhibits the activities of the promoters. Finally our results suggest that the critical role of Pax6 action on α cell differentiation is independent of those of Arx and Foxa2, two transcription factors that are necessary for α cell development. We conclude that Pax6 is critical for α cell function and differentiation through the transcriptional control of key genes involved in glucagon gene transcription, proglucagon processing, and α cell differentiation.

Pax-6 and c-Maf functionally interact with the alpha-cell-specific DNA element G1 in vivo to promote glucagon gene expression.

Specific expression of the glucagon gene in the rat pancreas requires the presence of the G1 element localized at -100/-49 base pairs on the promoter. Although it is known that multiple transcription factors such as Pax-6, Cdx-2/3, c-Maf, Maf-B, and Brain-4 can activate the glucagon gene promoter through G1, their relative importance in vivo is unknown. We first studied the expression of Maf-B, c-Maf, and Cdx-2/3 in the developing and adult mouse pancreas. Although Maf-B was detectable in a progressively increasing number of alpha-cells throughout development and in adulthood, c-Maf and Cdx-2/3 were expressed at low and very low levels, respectively. However, c-Maf but not Cdx-2/3 was detectable in adult islets by Western blot analyses. We then demonstrated the in vivo interactions of Pax-6, Cdx-2/3, Maf-B, and c-Maf but not Brain-4 with the glucagon gene promoter in glucagon-producing cells. Although Pax-6, Cdx-2/3, Maf-B, and c-Maf were all able to bind G1 by themselves, we showed that Pax-6 could interact with Maf-B, c-Maf, and Cdx-2/3 and activate transcription of the glucagon gene promoter. Overexpression of dominant negative forms of Cdx-2/3 and Mafs in alpha-cell lines indicated that Cdx-2/3 and the Maf proteins interact on an overlapping site within G1 and that this binding site is critical in the activation of the glucagon gene promoter. Finally, we show that specific inhibition of Pax-6 and c-Maf but not Cdx-2/3 or Maf-B led to decreases in endogenous glucagon gene expression and that c-Maf binds the glucagon gene promoter in mouse islets. We conclude that Pax-6 and c-Maf interact with G1 to activate basal expression of the glucagon gene.

The beta-cell specific transcription factor Nkx6.1 inhibits glucagon gene transcription by interfering with Pax6.

The transcription factor Nkx6.1 is required for the establishment of functional insulin-producing beta-cells in the endocrine pancreas. Overexpression of Nkx6.1 has been shown to inhibit glucagon gene expression while favouring insulin gene activation. Down-regulation resulted in the opposite effect, suggesting that absence of Nkx6.1 favours glucagon gene expression. To understand the mechanism by which Nkx6.1 suppresses glucagon gene expression, we studied its effect on the glucagon gene promoter activity in non-islet cells using transient transfections and gel-shift analyses. In glucagonoma cells transfected with an Nkx6.1-encoding vector, the glucagon promoter activity was reduced by 65%. In BHK21 cells, Nkx6.1 inhibited by 93% Pax6-mediated activation of the glucagon promoter, whereas Cdx2/3 and Maf stimulations were unaltered. Although Nkx6.1 could interact with both the G1 and G3 element, only the former displayed specificity for Nkx6.1. Mutagenesis of the three potential AT-rich motifs within the G1 revealed that only the Pax6-binding site preferentially interacted with Nkx6.1. Chromatin immunoprecipitation confirmed interaction of Nkx6.1 with the glucagon promoter and revealed a direct competition for binding between Pax6 and Nkx6.1. A weak physical interaction between Pax6 and Nkx6.1 was detected in vitro and in vivo suggesting that Nkx6.1 predominantly inhibits glucagon gene transcription through G1-binding competition. We suggest that cell-specific expression of the glucagon gene may only proceed when Nkx6.1, in combination with Pdx1 and Pax4, are silenced in early alpha-cell precursors.

Activin A decreases glucagon and arx gene expression in alpha-cell lines.

Activin A is a potent growth and differentiation factor involved in development, differentiation, and physiological functions of the endocrine pancreas; it increases insulin and pax4 gene expression in beta-cells and can induce transdifferentiation of the exocrine acinar cell line AR42J into insulin-producing cells. We show here that Activin A decreases glucagon gene expression in the alpha-cell lines InR1G9 and alphaTC1 in a dose- and time-dependent manner and that the effect is blocked by Follistatin. This effect is also observed in adult human islets. Glucagon gene expression is inhibited at the transcriptional level by the Smad signaling pathway through the G3 DNA control element. Furthermore, Activin A decreases cell proliferation of InR1G9 and alphaTC1 cells as well as cyclin D2 and arx gene expression, whose protein product Arx has been shown to be critical for alpha-cell differentiation. Overexpression of Arx in Activin A-treated InR1G9 cells does not prevent the decrease in glucagon gene expression but corrects the inhibition of cell proliferation, indicating that Arx mediates the Activin A effects on the cell cycle. We conclude that Activin A has opposite effects on alpha-cells compared with beta-cells, a finding that may have relevance during pancreatic endocrine lineage specification and physiological function of the adult islets.

The zinc finger-containing transcription factor Gata-4 is expressed in the developing endocrine pancreas and activates glucagon gene expression.

Gene inactivation studies have shown that members of the Gata family of transcription factors are critical for endoderm development throughout evolution. We show here that Gata-4 and/or Gata-6 are not only expressed in the adult exocrine pancreas but also in glucagonoma and insulinoma cell lines, whereas Gata-5 is restricted to the exocrine pancreas. During pancreas development, Gata-4 is expressed already at embryonic d 10.5 and colocalizes with early glucagon+ cells at embryonic d 12.5. Gata-4 was able to transactivate the glucagon gene both in heterologous BHK-21 (nonislet Syrian baby hamster kidney) and in glucagon-producing InR1G9 cells. Using gel-mobility shift assays, we identified a complex formed with nuclear extracts from InR1G9 cells on the G5 control element (-140 to -169) of the glucagon gene promoter as Gata-4. Mutation of the GATA binding site on G5 abrogated the transcriptional activation mediated by Gata-4 and reduced basal glucagon gene promoter activity in glucagon-producing cells by 55%. Furthermore, Gata-4 acted more than additively with Forkhead box A (hepatic nuclear factor-3) to trans-activate the glucagon gene promoter. We conclude that, besides its role in endoderm differentiation, Gata-4 might be implicated in the regulation of glucagon gene expression in the fetal pancreas and that Gata activity itself may be modulated by interactions with different cofactors.

Agenesis of human pancreas due to decreased half-life of insulin promoter factor 1.

Neonatal diabetes mellitus can be transient or permanent. The severe form of permanent neonatal diabetes mellitus can be associated with pancreas agenesis. Normal pancreas development is controlled by a cascade of transcription factors, where insulin promoter factor 1 (IPF1) plays a crucial role. Here, we describe two novel mutations in the IPF1 gene leading to pancreas agenesis. Direct sequence analysis of exons 1 and 2 of the IPF1 gene revealed two point mutations within the homeobox in exon 2. Genetic analysis of the parents showed that each mutation was inherited from one parent. Mutations localized in helices 1 and 2, respectively, of the homeodomain, decreased the protein half-life significantly, leading to intracellular IPF1 levels of 36% and 27% of wild-type levels. Both mutant forms of IPF1 were normally translocated to the nucleus, and their DNA binding activity on different known target promoters was similar to that of the wild-type protein. However, transcriptional activity of both mutant IPF1 proteins, alone or in combination with HNF3 beta/Foxa2, Pbx1, or the heterodimer E47-beta 2 was reduced, findings accounted for by decreased IPF1 steady state levels and not by impaired protein-protein interactions. We conclude that the IPF1 level is critical for human pancreas formation.