A human stomach cell type transcriptome atlas.

BACKGROUND: The identification of cell type-specific genes and their modification under different conditions is central to our understanding of human health and disease. The stomach, a hollow organ in the upper gastrointestinal tract, provides an acidic environment that contributes to microbial defence and facilitates the activity of secreted digestive enzymes to process food and nutrients into chyme. In contrast to other sections of the gastrointestinal tract, detailed descriptions of cell type gene enrichment profiles in the stomach are absent from the major single-cell sequencing-based atlases. RESULTS: Here, we use an integrative correlation analysis method to predict human stomach cell type transcriptome signatures using unfractionated stomach RNAseq data from 359 individuals. We profile parietal, chief, gastric mucous, gastric enteroendocrine, mitotic, endothelial, fibroblast, macrophage, neutrophil, T-cell, and plasma cells, identifying over 1600 cell type-enriched genes. CONCLUSIONS: We uncover the cell type expression profile of several non-coding genes strongly associated with the progression of gastric cancer and, using a sex-based subset analysis, uncover a panel of male-only chief cell-enriched genes. This study provides a roadmap to further understand human stomach biology.

Fissure sealants or fluoride varnish? Factors associated with choice of management method for occlusal caries in Public Dental Service in Norway.

PURPOSE: The aim of this practice-based study was to identify factors associated with choice of caries management method in first permanent molars in high-risk children treated in Public Dental Service in Norway. METHODS: The present study was based on practice-based observational data from 366 high-risk children (6-9 years). Caries management of occlusal surface of first permanent molars was used as outcome variable and categorized into "no treatment", "fluoride varnish" or "fissure sealant". Patient-related variables (age, gender, oral hygiene, caries experience at age 5, sugar snacking and dental anxiety), tooth-related variables (upper or lower jaw and diagnosis of occlusal surface) and county were used as independent variables. Data were analysed by descriptive analyses followed by generalized structural equation models (GSEM) and presented as relative risk ratios (RRR) with 95% confidence intervals (CIs). RESULTS: In 319 of the 366 children, both first permanent molars in the same jaw were available for analyses, 276 (87%) had the same diagnosis for both teeth and received the same treatment. Multivariable analysis at patient level showed that age (RRR = 2.42, CI 1.38, 4.23) and caries experience (RRR = 1.39, CI 1.09, 1.77) were associated with higher probability of fissure sealant, while the county variable was significantly associated with lower probability for fluoride varnish use (RRR = 0.03 (0.004, 0.31). CONCLUSION: The majority of high-risk children in PDS received fluoride varnish or fissure sealants on newly erupted occlusal surfaces of first permanent molars. In addition to age and caries experience of the child, county appeared to substantially influence occlusal caries management method.

Psychological impact of the COVID-19 pandemic on dental health personnel in Norway.

BACKGROUND: The SARS-CoV-2 pandemic put a pressure on all healthcare professionals and has affected the delivery of health care services globally. There is a need to understand the impact on different health care professionals in different countries. The aim of the present study was to explore the psychological impact of the pandemic among dental staff in Norway in relation to background characteristics, work situation and preparedness of the service. METHODS: A structured questionnaire sent electronically to dentists, dental hygienists and dental assistants inquired information about the lockdown period in Norway (13 March-17 April 2020). Distributions of background characteristics, perceptions of preparedness and psychological impact were calculated. Exploratory factor analysis was performed, and Structural Equation Models (SEMs) were used to compare psychological impact between dental professionals treating patients versus not during lockdown. RESULTS: Among the 1237 respondents, 58.8% worked clinically with patients. The majority were concerned of becoming infected (71.9%), of infecting others (85.4%) and/or of their family becoming infected (76.9%). Respondents who treated patients felt significantly more insecure about whether having become infected or not. The minority felt discriminated (6.7%), worried about death (11.7%), felt that life was threatening (9.8%) or felt loss of control of their lives (8.9%). More than 80% agreed that their workplace handled the situation well. Four factors were retrieved from the factor analysis. SEMs showed that gender and work experience had a significant effect on the factors Instability, Infection and Concerns. Respondents with work experience ≥10 years were less likely to express fear about Instability and Infection. Personnel reporting that their workplace had adequate equipment were also less concerned, however having adequate equipment did not reduce the factor Loss of control. CONCLUSION: The present study showed a considerable psychological impact of the COVID-19 pandemic on dental personnel in Norway regardless of working clinically with patients or not. However, working with patients increased the insecurity about own infection status and of infecting people close to them. A safe working environment and adequate infection control measures are associated with less fear of infection and feeling of instability.

Stainless-steel crowns in children: Norwegian and Finnish dentists' knowledge, practice and challenges.

BACKGROUND: Stainless-steel crowns (SSCs) are recommended for restorative treatment of young teeth severely affected by caries, fractures or dental developmental disorders (DDDs). However, despite recommendations and clinical evidence, SSCs are not widely used by general dentists, who favour extraction and more conventional restorations. The present study aimed to investigate the views of and use of SSCs among Norwegian and Finnish dentists. METHODS: The present study was a cross-sectional survey among Norwegian and Finnish dentists. An electronic questionnaire was sent to Norwegian and Finnish dentists asking whether they used SSCs and on which indications. In addition, the questionnaire assessed reasons for non-use and dentists' perceptions regarding advantages and challenges in the use of SSCs, as well as the need for additional training. Distributions of background characteristics, use of and views on SSCs were calculated, and statistical significance of the associations between respondents' background and their answers were evaluated. RESULTS: Of the 574 Norwegian and 765 Finnish respondents, only 12.0% and 12.9% reported to use SSCs, respectively. The most frequently reported barrier reported by those who did not use SSCs was lack of practical training. The most frequent challenge reported by those using SSCs was difficulties in crown adjustment followed by aesthetic issues, and the most frequently reported advantage was that SSCs maintain the function and occlusion. The majority of respondents reported a need for more information and practical training in the use of SSCs, with hands-on course as their most frequently preferred education type. CONCLUSION: Although the value of SSCs for restoring young molars is recognized by Norwegian and Finnish dentists, SSCs are rarely used by general dentists. The majority of the respondents reported lack of training and materials and was interested in receiving more information and education.

Restorative material choices for extensive carious lesions and hypomineralisation defects in children: a questionnaire survey among Finnish dentists.

AIM: This survey assessed Finnish dentists' treatment decisions and choices of restorative materials in selected paediatric dental patient cases, with special emphasis on stainless steel crowns (SSCs). METHODS: A questionnaire with patient descriptions and tooth photographs was e-mailed to members of Finnish Dental Society (n=3,747). The respondents were asked to choose their preferred treatment in cases describing 1) extensive occlusal carious lesion in a primary molar of a cooperative child; 2) an identical lesion, treated under dental general anaesthesia (DGA); and 3) a symptomatic first permanent molar with enamel hypomineralisation (consistent with Molar-Incisor Hypomineralization, MIH) and post-eruptive breakdown. Only responses from dentist treating children were included (final n=765). RESULTS: The majority (47.3%) would have preferred restoration of the extensive primary tooth caries in a normal setting using resin-modified glassionomer cement, and 4.3% by using SSC. The preference of SSC as treatment choice increased to 25.4% upon implementation of DGA. The majority would treat the symptomatic permanent MIH molar with a resin composite restoration (45.0%), while 10.5% suggested SSC. Compared to general dentists, paediatric dentists had a stronger preference for SSCs. CONCLUSIONS: Although the respondents emphasised patient cooperation, but also tooth prognosis and material strength behind their treatment decisions, SSC was an uncommon choice.

Treatment decisions regarding caries and dental developmental defects in children - a questionnaire-based study among Norwegian dentists.

BACKGROUND: Current knowledge on treatment strategies and choice of restorative materials when treating deep caries or severe dental developmental defects (DDDs) in young individuals is scarce. Therefore, the aim was to investigate Norwegian dentists´ treatment decisions and reasons for treatment choice when treating deep caries in primary teeth and severe DDDs in permanent teeth in children. METHODS: A pre-coded questionnaire was sent electronically to all dentists employed in the Public Dental Service (PDS) in Norway (n = 1294). The clinicians were asked about their background characteristics and how often they registered DDDs. Three clinical cases were presented to the dentists and asked to prioritize treatment options and reasons for their choice. RESULTS: After three reminders, 45.8% of the dentists answered. Most clinicians were general practitioners (96.3%), females (77.9%), under 41 year-olds (59.4%), graduated in 2001 or later (61.1%), and representing all regions of Norway. The respondents registered molar incisor hypomineralisation (MIH), other DDDs and dental fluorosis (DF) frequently, 523 (91.1%), 257 (44.8%) and 158 (27.5%), respectively. In case 1a with severe dental caries in a primary molar, the preferred treatment was resin-modified glass ionomer cement (RMGIC) (58.3%), followed by glass ionomer cement (GIC) (17.9%) and zinc oxide-eugenol (ZOE) (13.2%). Extraction, compomer or stainless steel crowns (SSC) were preferred by 0.9, 0.7 and 0.4%, respectively. In case 1b, which was identical to case 1a, but treated under general anaesthesia, the preferred treatment alternatives were RMGIC (37.1%), resin composite (RC) (17.6%) and GIC (17.2%). Extraction and SSC were chosen by 15.1 and 7.2%, respectively. In case 2, showing a severely hypomineralised and symptomatic first permanent molar, the dentists preferred RC (38.4%), followed by RMGIC (26.6%) and GIC (19.0%). Extraction and SSC were chosen by 8.7 and 5.4%, respectively. The treatment choices were not significantly affected by the dentists' background characteristics. The reasons for dentists' treatment decisions varied for each patient case; patient cooperation, prognosis of the tooth and own experience were the dominant reasons. CONCLUSIONS: A notable disparity in treatment choices was shown indicating that Norwegian dentists evaluate each case individually and base their decisions on what they consider best for the individual patient.

Fissure sealants or fluoride varnish? Routines and attitudes among dental health personnel in Norway.

PURPOSE: To investigate routines and attitudes among dentists and dental hygienists concerning use of fissure sealants and fluoride varnish for non-operative management of occlusal caries. METHODS: All dentists and dental hygienists working in child dental care in three counties in Norway were invited to answer a questionnaire on routines for use of fissure sealants and fluoride varnish. Nine statements regarding attitudes towards use of sealants were scored using a five-point Likert scale. Multivariable logistic regression analyses were performed to assess indicators associated with reported routines for use of sealants and varnish. The study was approved by the Norwegian Centre for Research Data. RESULTS: In total 142 of 189 (75%) dentists and dental hygienists answered the questionnaire. The majority of the respondents, n = 83 (59%), reported to prefer fissure sealants while fluoride varnish was preferred by 57 (41%) of the respondents. Frequent use of fissure sealants was reported by 58 (41%) and frequent use of varnish by 104 (74%) of the respondents. Most (n = 104, 74%), used sealants on specific indications, and 89 (64%) opened fissures only when suspecting dentine caries. Preferred method and routines for occlusal caries management differed between counties (p < 0.05). Almost all clinicians agreed with the statement that sealants are protective against caries, while statements regarding costs, technique sensitivity and children's cooperation revealed some concerns regarding fissure sealing. CONCLUSIONS: Fissure sealants were the preferred method for occlusal caries management despite reported concerns related to technical aspects and patient cooperation. County-level variation in frequency of sealant use was observed.

Untargeted screening for novel autoantibodies with prognostic value in first-episode psychosis.

Immunological and inflammatory reactions have been suggested to have a role in the development of schizophrenia, a hypothesis that has recently been supported by genetic data. The aim of our study was to perform an unbiased search for autoantibodies in patients with a first psychotic episode, and to explore the association between any seroreactivity and the development of a Diagnostic and Statistical Manual of Mental Disorders, fourth edition (DSM-IV) disorder characterized by chronic or relapsing psychotic symptoms. We collected plasma samples from 53 patients when they were treated for their first-episode psychosis, and 41 non-psychotic controls, after which the patients were followed for a mean duration of 7 years. Thirty patients were diagnosed with schizophrenia, delusional disorder, schizoaffective disorder, bipolar disorder or a long-term unspecified nonorganic psychosis during follow-up, whereas 23 patients achieved complete remission. At the end of follow-up, plasma samples were analyzed for IgG reactivity to 2304 fragments of human proteins using a multiplexed affinity proteomic technique. Eight patient samples showed autoreactivity to the N-terminal fragment of the PAGE (P antigen) protein family (PAGE2B/PAGE2/PAGE5), whereas no such autoreactivity was seen among the controls. PAGE autoreactivity was associated with a significantly increased risk of being diagnosed with schizophrenia during follow-up (odds ratio 6.7, relative risk 4.6). An immunohistochemistry analysis using antisera raised against the N-terminal fragment stained an unknown extracellular target in human cortical brain tissue. Our findings suggest that autoreactivity to the N-terminal portion of the PAGE protein family is associated with schizophrenia in a subset of patients with first-episode psychosis.

The Susceptibility to Dental Erosion Differs among Individuals.

Studies of wine tasters and patients with self-induced vomiting have revealed that 30-50% of individuals at high risk do not develop erosive lesions. The aim was to investigate this apparent individual susceptibility to enamel erosion. Two enamel specimens were made from each of 3 premolars from 8 persons (donors). Six acrylic mouth appliances were worn by 6 volunteers (carriers). One specimen from each donor was mounted on each appliance. The carriers wore the appliances for 9 days. The appliances were immersed in 0.01 M HCl for 3 min twice per day to imitate a vomiting/reflux situation. The enamel specimens were analysed by a white-light interferometer to measure enamel loss (in micrometres). The enamel loss varied significantly both between the donor teeth (p = 0.009) and the carriers (p = 0.004). The lesion in the specimen with the largest amount of enamel loss was 4 times as deep as in the specimen with the lowest. In 1 carrier, all specimens displayed enamel loss above the mean, including the specimen from the donor with the most resistant enamel. The variation in susceptibility to erosion among individuals appears to be influenced both by the sustainability of the enamel and by factors in the oral environment. This could explain the variation in prevalence and severity of dental erosions among patients exposed to similar acidic challenges. The results suggest that for certain individuals, only minimal acidic challenges may be sufficient to cause damage to the teeth, while others may never develop dental erosions despite extensive exposure to acid.

The human testis-specific proteome defined by transcriptomics and antibody-based profiling.

The testis' function is to produce haploid germ cells necessary for reproduction. Here we have combined a genome-wide transcriptomics analysis with immunohistochemistry-based protein profiling to characterize the molecular components of the testis. Deep sequencing (RNA-Seq) of normal human testicular tissue from seven individuals was performed and compared with 26 other normal human tissue types. All 20 050 putative human genes were classified into categories based on expression patterns. The analysis shows that testis is the tissue with the most tissue-specific genes by far. More than 1000 genes show a testis-enriched expression pattern in testis when compared with all other analyzed tissues. Highly testis enriched genes were further characterized with respect to protein localization within the testis, such as spermatogonia, spermatocytes, spermatids, sperm, Sertoli cells and Leydig cells. Here we present an immunohistochemistry-based analysis, showing the localization of corresponding proteins in different cell types and various stages of spermatogenesis, for 62 genes expressed at >50-fold higher levels in testis when compared with other tissues. A large fraction of these genes were unexpectedly expressed in early stages of spermatogenesis. In conclusion, we have applied a genome-wide analysis to identify the human testis-specific proteome using transcriptomics and antibody-based protein profiling, providing lists of genes expressed in a tissue-enriched manner in the testis. The majority of these genes and proteins were previously poorly characterised in terms of localization and function, and our list provides an important starting point to increase our molecular understanding of human reproductive biology and disease.

Membranous expression of podocalyxin-like protein is an independent factor of poor prognosis in urothelial bladder cancer.

BACKGROUND: Membranous expression of the anti-adhesive glycoprotein podocalyxin-like (PODXL) has previously been found to correlate with poor prognosis in several major cancer forms. Here we examined the prognostic impact of PODXL expression in urothelial bladder cancer. METHODS: Immunohistochemical PODXL expression was examined in tissue microarrays with tumours from two independent cohorts of patients with urothelial bladder cancer: n=100 (Cohort I) and n=343 (Cohort II). The impact of PODXL expression on disease-specific survival (DSS; Cohort II), 5-year overall survival (OS; both cohorts) and 2-year progression-free survival (PFS; Cohort II) was assessed. RESULTS: Membranous PODXL expression was significantly associated with more advanced tumour (T) stage and high-grade tumours in both cohorts, and a significantly reduced 5-year OS (unadjusted HR=2.25 in Cohort I and 3.10 in Cohort II, adjusted HR=2.05 in Cohort I and 2.18 in Cohort II) and DSS (unadjusted HR=4.36, adjusted HR=2.70). In patients with Ta and T1 tumours, membranous PODXL expression was an independent predictor of a reduced 2-year PFS (unadjusted HR=6.19, adjusted HR=4.60) and DSS (unadjusted HR=8.34, adjusted HR=7.16). CONCLUSION: Membranous PODXL expression is an independent risk factor for progressive disease and death in patients with urothelial bladder cancer.

A cohort study of the prognostic and treatment predictive value of SATB2 expression in colorectal cancer.

BACKGROUND: Special AT-rich sequence-binding protein 2 (SATB2) is a novel diagnostic marker of colorectal cancer (CRC), and loss of SATB2 has been linked to poor survival from the disease. In this study, we validated the prognostic ability of SATB2 expression in a large, prospective CRC cohort. METHODS: Immunohistochemical SATB2 expression was assessed in 527 incident CRC cases from the Malmö Diet and Cancer Study. Kaplan-Meier analysis and Cox proportional hazards modelling were used to explore the impact of SATB2 expression on cancer-specific survival (CSS) and overall survival (OS). RESULTS: High SATB2 expression was associated with a prolonged CSS in the full cohort (hazard ratio (HR)=0.61; 95% CI 0.41-0.92) and in colon cancer (HR=0.39; 95% CI 0.20-0.75), remaining significant in multivariable analysis of colon cancer (HR=0.49; 95% CI 0.25-0.96), with similar findings for OS. In curatively resected stage III-IV patients, a significant benefit from adjuvant and/or neoadjuvant therapy was observed for SATB2 high tumours (P(interaction)=0.037 for OS) and high SATB2 expression in rectal cancer correlated with an enhanced effect of neoadjuvant therapy (P(interaction)=0.033 for OS). CONCLUSION: High SATB2 expression is an independent marker of good prognosis in colon cancer and may modulate sensitivity to chemotherapy and radiation.

Overexpression of podocalyxin-like protein is an independent factor of poor prognosis in colorectal cancer.

BACKGROUND: Podocalyxin-like 1 (PODXL) is a cell-adhesion glycoprotein and stem cell marker that has been associated with an aggressive tumour phenotype and poor prognosis in several forms of cancer. In this study, we investigated the prognostic impact of PODXL expression in colorectal cancer (CRC). METHODS: Using tissue microarrays and immunohistochemistry, PODXL expression was evaluated in 536 incident CRC cases from a prospective, population-based cohort study. Kaplan-Meier analysis and Cox proportional hazards modelling were used to assess the impact of PODXL expression on cancer-specific survival (CSS) and overall survival (OS). RESULTS: High PODXL expression was significantly associated with unfavourable clinicopathological characteristics, a shorter CSS (hazard ratio (HR)=1.98; 95% confidence interval (CI) 1.38-2.84, P<0.001) and 5-year OS (HR=1.85; 95% CI 1.29-2.64, P=0.001); the latter remaining significant in multivariate analysis (HR=1.52; 95% CI 1.03-2.25, P=0.036). In addition, in curatively resected stage III (T1-4, N1-2, M0) patients (n=122) with tumours with high PODXL expression, a significant benefit from adjuvant chemotherapy was demonstrated (p(interaction) =0.004 for CSS and 0.015 for 5-year OS in multivariate analysis). CONCLUSION: Podocalyxin-like 1 expression is an independent factor of poor prognosis in CRC. Our results also suggest that PODXL may be a useful marker to stratify patients for adjuvant chemotherapy.

Novel antigen design for the generation of antibodies to G-protein-coupled receptors.

Antibodies are important tools for the study of G-protein-coupled receptors, key proteins in cellular signaling. Due to their large hydrophobic membrane spanning regions and often very short loops exposed on the surface of the cells, generation of antibodies able to recognize the receptors in the endogenous environment has been difficult. Here, we describe an antigen-design method where the extracellular loops and N-terminus are combined to a single antigen for generation of antibodies specific to three selected GPCRs: NPY5R, B2ARN and GLP1R. The design strategy enabled straightforward antigen production and antibody generation. Binding of the antibodies to intact receptors was analyzed using flow cytometry and immunofluorescence based confocal microscopy on A-431 cells overexpressing the respective GPCR. The antibody-antigen interactions were characterized using epitope mapping, and the antibodies were applied in immunohistochemical staining of human tissues. Most of the antibodies showed specific binding to their respective overexpressing cell line but not to the non-transfected cells, thus indicating binding to their respective target receptor. The epitope mapping showed that sub-populations within the purified antibody pool recognized different regions of the antigen. Hence, the genetic combination of several different epitopes enables efficient generation of specific antibodies with potential use in several applications for the study of endogenous receptors.

Plasma CD93 concentration is a potential novel biomarker for coronary artery disease.

OBJECTIVES: A common nonsynonymous single nucleotide polymorphism (SNP) in the CD93 gene (rs3746731, Pro541Ser) has been associated with risk of coronary artery disease (CAD). CD93 is a transmembrane glycoprotein, which is detectable in soluble form in human plasma. We investigated whether the concentration of soluble CD93 in plasma is related to risk of myocardial infarction (MI) and CAD, using a case-control study of premature MI (n = 764) and a nested case-control analysis of a longitudinal cohort study of 60-year-old subjects (analysis comprising 844 of 4232 subjects enrolled at baseline). In addition, SNPs in the CD93 gene were studied in relation to plasma CD93 concentration and CD93 mRNA expression. METHODS AND RESULTS: A sensitive and specific enzyme-linked immunosorbent assay was established for determination of the plasma CD93 concentration. Subjects were divided into three groups according to tertiles of the distribution of CD93 concentration. Lower odds ratios for risk of MI and incidence of CAD were observed in the middle CD93 tertile (142-173 µg L(-1) ): odds ratio (95% confidence interval), 0.69 (0.49-0.97) and 0.61 (0.40-0.94), respectively. These associations were independent of traditional CAD risk factors. The minor allele of a SNP in the 3' untranslated region of CD93 (rs2749812) was associated with increased plasma CD93 concentrations (P = 0.03) and increased CD93 mRNA expression levels (P = 0.02). CONCLUSION: The results of the present study suggest that the concentration of soluble CD93 in plasma is a potential novel biomarker for CAD, including MI.

Regional occurrence, high frequency but low diversity of mitochondrial DNA haplogroup d1 suggests a recent dog-wolf hybridization in Scandinavia.

The domestic dog mitochondrial DNA (mtDNA)-gene pool consists of a homogenous mix of haplogroups shared among all populations worldwide, indicating that the dog originated at a single time and place. However, one small haplogroup, subclade d1, found among North Scandinavian/Finnish spitz breeds at frequencies above 30%, has a clearly separate origin. We studied the genetic and geographical diversity for this phylogenetic group to investigate where and when it originated and whether through independent domestication of wolf or dog-wolf crossbreeding. We analysed 582 bp of the mtDNA control region for 514 dogs of breeds earlier shown to harbour d1 and possibly related northern spitz breeds. Subclade d1 occurred almost exclusively among Swedish/Finnish Sami reindeer-herding spitzes and some Swedish/Norwegian hunting spitzes, at a frequency of mostly 60-100%. Genetic diversity was low, with only four haplotypes: a central, most frequent, one surrounded by two haplotypes differing by an indel and one differing by a substitution. The substitution was found in a single lineage, as a heteroplasmic mix with the central haplotype. The data indicate that subclade d1 originated in northern Scandinavia, at most 480-3000 years ago and through dog-wolf crossbreeding rather than a separate domestication event. The high frequency of d1 suggests that the dog-wolf hybrid phenotype had a selective advantage.

Characterization of PrEST-based antibodies towards human Cytokeratin-17.

Antibody-based proteomics efforts depend on validated antibodies to ensure correct annotation of analyzed proteins. We have previously argued that a low sequence identity to other proteins is a key feature for antigens used in antibody generation. Thus, a major challenge for whole-proteome studies is how to address families of highly sequence related proteins within the context of generating specific antibodies. In this study, two non-overlapping parts of human Cytokeratin-17, a protein belonging to the intermediate filament family of highly sequence-related proteins, were selected as a model system to study the specificity and cross reactivity of antibodies generated towards such a target. These recombinantly produced Protein Epitope Signature Tags (PrESTs) were immunized in five rabbits each and the batch-to-batch variations in the obtained immune responses were studied by mapping of linear epitopes using synthetic overlapping peptides. The obtained results showed a similar but not identical immune response in the respective antibody groups with a limited number of epitopes being identified. Immunohistochemical analysis of the affinity purified monospecific antibodies on tissue micro arrays resulted in a general recognition of human cytokeratins for all analyzed binders whereas antibodies identified as binding to the most unique parts of the PrESTs showed the most Cytokeratin-17 like staining. The data presented here support the strategy to use sequence identity scores as the main criteria for antigen selection but also indicate the possibility to instead produce a single antibody recognizing a defined group of proteins when the intended targets overall sequence identity score is too high. This type of group-specific antibodies would be an important tool for antibody-based projects aiming for a complete coverage of the human proteome.

The Human Protein Atlas--a tool for pathology.

Tissue-based diagnostics and research is incessantly evolving with the development of new molecular tools. It has long been realized that immunohistochemistry can add an important new level of information on top of morphology and that protein expression patterns in a cancer may yield crucial diagnostic and prognostic information. We have generated an immunohistochemistry-based map of protein expression profiles in normal tissues, cancer and cell lines. For each antibody, altogether 708 spots of tissues and cells are analysed and the resulting images and data are presented as freely available in the Human Protein Atlas (www.proteinatlas.org). The new version 4 of the atlas, including more than 5 million images of immunohistochemically stained tissues and cells, is based on 6122 antibodies, representing 5011 human proteins encoded by approximately 25% of the human genome. The gene-centric database includes a putative classification of proteins in various protein classes, both functional classes, such as kinases or transcription factors and project-related classes, such as candidate genes for cancer or cardiovascular diseases. For each of the internally generated antibodies, the exact antigen sequence is presented, together with a visualization of application-specific validation data, including a protein array assay, western blot analysis, immunohistochemistry and, in most cases, immunofluorescent-based confocal microscopy. The updated version also includes new search algorithms to allow complex queries regarding expression profiles, protein classes and chromosome location. Thus, the presented Human Protein Atlas provides a resource for pathology-based biomedical research, including protein science and biomarker discovery.

Human protein atlas and the use of microarray technologies.

Currently one of the most challenging tasks in biological and medical research is to explore and understand the function of all proteins encoded by the genome of an organism. A systematic approach based on the genome sequences is feasible because the full genome of many organisms presently is available and many more are underway. For the production of expression atlases different strategies are used. Early attempts to acquire information about protein expression levels have focused on the analysis of mRNA levels within different tissues and cell types. Recently, novel strategies to focus directly on protein levels have been developed. To assess global protein expression in a systematic and high-throughput manner, methods based on design of specific affinity ligands to recognize the proteins have been presented. By subsequently using these affinity molecules for detection of the corresponding proteins in a wide range of platforms, important information can be gained. This article focuses on strategies to profile protein levels and in particular the human protein atlas initiative and the use of microarray technologies.

Systematically generated antibodies against human gene products: high throughput screening on sections from the rat nervous system.

Completion of the Human Genome Project and recent developments in proteomics make it possible to systematically generate affinity reagents to a large portion of the proteome. Recently an antibody-based human protein atlas covering many organs including four areas of the brain has been released (www.proteinatlas.org). Due to the heterogeneity, size, and availability of tissue a more thorough analysis of the human brain is associated with considerable difficulties. Here we applied 120 antibodies raised against 112 human gene products to the smaller rat brain, a rodent animal model, where a single section represents a 'superarray' including many brain areas, and consequently allowing analysis of a huge number of cell types and their neurochemicals. Immunoreactive structures were seen in the investigated brain tissue after incubation with 56 antibodies (46.6%), of which 25 (20.8%) showed a clearly discrete staining pattern that was limited to certain areas, or subsets of brain cells. Bioinformatics, pre-adsorption tests and Western blot analysis were applied to identify non-specific antibodies. Eleven antibodies, including such raised against four 'ambiguous' proteins, passed all validation criteria, and the expression pattern and subcellular distribution of these proteins were studied in detail. To further explore the potential of the systematically generated antibodies, all 11 antibodies that passed validation were used to analyze the spinal cord and lumbar dorsal root ganglia after unilateral transection of the sciatic nerve. Discrete staining patterns were observed for four of the proteins, and injury-induced regulation was found for one of them. In conclusion, the study presented here suggests that a significant portion (10%) of the antibodies generated to a human protein can be used to analyze orthologues present in the rodent brain and to produce a protein-based atlas of the rodent brain. It is hoped that this type of antibody-based, high throughput screening of brain tissue from various rodent disease models will provide new information on the brain chemical neuroanatomy and insights in processes underlying neurological pathologies.