abstract: # Introduction
  .
  The finding of antinuclear antibody (ANA) positivity in a healthy individual is usually of unknown significance and in most cases is benign. However, a subset of such individuals is at risk for development of autoimmune disease. We examined demographic and immunological features that are associated with ANA positivity in clinically healthy persons to develop insights into when this marker carries risk of progression to lupus.
  .
  # Methods
  .
  Biological samples from healthy individuals and patients with systemic lupus erythematosus (SLE) were obtained from the Dallas Regional Autoimmune Disease Registry (DRADR). Measurements carried out on serum samples included ANA, extractable nuclear antibodies (ENA) and autoantibody profiling using an array with more than 100 specificities. Whole blood RNA samples from a subset of individuals were used to analyze gene expression on the Illumina platform. Data were analyzed for associations of high ANA levels with demographic features, the presence of other autoantibodies and with gene expression profiles.
  .
  # Results
  .
  Overall, ANA levels are significantly higher in females than in males and this association holds in patients with the autoimmune diseases lupus and rheumatoid arthritis (RA) as well as in healthy controls (HC). Age was not significantly associated with ANA levels and the elevated ANA values could not be explained by higher IgG levels. Another autoantibody, anti- cyclic citrullinated peptide (CCP), did not show gender dimorphism in rheumatoid arthritis (RA) or healthy individuals. The autoantigen array showed significant elevations of other autoantibodies in high ANA HCs. Some of these autoantibodies were directed to antigens in skin and others were related to autoimmune conditions of kidney, thyroid or joints. Gene expression analyses showed a greater prevalence of significantly upregulated genes in HCs with negative ANA values than in those with significant ANA positivity. Genes upregulated in high ANA HCs included a celiac disease autoantigen and some components of the Type I interferon (IFN) gene signature.
  .
  # Conclusions
  .
  Risks for ANA positivity include female gender and organ-specific autoimmunity. Upregulation of skin-specific autoantibodies may indicate that early events in the break of tolerance take place in cutaneous structures. Some of these changes may be mediated by Type I IFN. Blood profiling for expressed autoantibodies and genes has the potential to identify individuals at risk for development of autoimmune diseases including lupus.
author: Quan-Zhen Li; David R Karp; Jiexia Quan; Valerie K Branch; Jinchun Zhou; Yun Lian; Benjamin F Chong; Edward K Wakeland; Nancy J Olsen
date: 2011
institute: 1Department of Immunology, University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd., Dallas TX 75390-9093, USA; 2Department of Medicine, The Division of Rheumatic Diseases, The Simmons Arthritis Center, University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd., Dallas TX 75390-8884, USA; 3Department of Dermatology, University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd., Dallas TX 75390-9069, USA; 4Current address: Division of Rheumatology, Penn State Hershey Medical Center, 500 University Drive, Hershey PA 17033, USA
references:
title: Risk factors for ANA positivity in healthy persons

# Introduction

Antinuclear antibodies (ANAs) are measurable in approximately 25% of the population, and the prevalence of significantly elevated levels may be 2.5% \[1\]. Findings from numerous studies show remarkable consistency across ethnically and racially diverse study populations despite the use of many different methods for ANA measurement. The persistence of this type of autoreactivity in the human population suggests that antinuclear antibodies may be an important component of the normal immune response. Most individuals with a positive ANA do not have an autoimmune disease and most also are unlikely to develop one. This is consistent with the fact that the prevalence of all autoimmune disorders is 5 to 7% \[2\]. Furthermore, the disease that is most closely linked to ANA positivity, systemic lupus erythematosus (SLE), is relatively rare, affecting no more than 1 to 1.5 per 1,000 persons (0.1 to 0.15%) in the United States \[3\]. Nevertheless, since ANA positivity is for all practical purposes a requirement for SLE diagnosis, it must also be assumed that individuals who are in preclinical disease stages are represented in the ANA positive healthy population. Although many consultations for ANA positivity seen in rheumatology practice are not associated with any identifiable pathology, it is also true that if early detection of SLE is to become feasible, focus on the ANA positive population will be necessary.

We have considered the possibility that other blood markers could be used to differentiate benign ANA positivity from that which carries a high risk of autoimmune disease. These markers may include other autoantibodies, since it is well-known that autoantibody positivity increases in quantity and complexity in years preceding a diagnosis of SLE \[4\]. Gene dysregulation in peripheral blood cells has been closely associated with SLE diagnosis and disease status, so changes in gene expression may also signal a condition with enhanced risk.

To address these questions, we studied healthy individuals and patients with autoimmune diseases who had been enrolled in the Dallas Regional Autoimmune Disease Registry (DRADR). A subgroup of healthy controls that were found to have high ANA levels was examined in greater detail using autoantigen and gene expression arrays. The findings suggest the feasibility of identifying risk markers for development of SLE in the setting of ANA positivity, using both demographic features and profiling of autoantibodies and expressed genes in peripheral blood.

# Materials and methods

## Patients and healthy controls

Samples for study were obtained from the Dallas Regional Autoimmune Disease Registry (DRADR). Individuals are classified according to clinical diagnosis or healthy condition at the time of enrollment into the registry. Disease features and the presence of SLE criteria were determined by patient interview and medical record review \[5\]. Blood samples were obtained for banking of serum aliquots and whole blood samples were stored in PaxGene tubes for later isolation of RNA. All samples were maintained at -80°C until use. The overall study group included 1,159 individuals from DRADR: 401 healthy controls (HC) who were negative for current or past autoimmune disease, 116 first-degree relatives (FDR), 294 patients with SLE, 151 patients with less than 4 SLE criteria and considered as having incomplete lupus (ILE), 154 with rheumatoid arthritis (RA) and 43 with other miscellaneous conditions including scleroderma, Sjogren's syndrome, ankylosing spondylitis and vasculitis. More detailed analyses were carried out on a subset of HC individuals with high ANA values (*n* = 18) and these were compared to gender- and age-matched HC with negative ANA values (*n* = 16) and to SLE patients with high ANA levels of \>100 E.U. (*n* = 14). In one experiment, plasma samples from the Dallas Heart Study population, which has been described previously \[1\], were employed. All subjects gave written informed consent for entry into the Dallas Regional Autoimmune Disease Registry. Research carried out under the auspices of this registry has been approved by the UT Southwestern Institutional Review Board.

## Antibody assays

Serum ANA levels were measured by ELISA (Inova, San Diego, CA, USA) using the manufacturer's suggested cut-off of \>20 units to define positive results. Other ELISA kits were used to measure CCP antibodies (Inova), thyroglobulin autoantibodies (Genway Biotech Inc., San Diego, CA, USA) and total IgG (Bethyl Laboratories Inc., Montgomery, TX, USA). The extractable nuclear antibody (ENA) panel for eight additional specificities was a Luminex-based multiplex assay, and positive results were as defined by the manufacturer (Inova). Serum reactivity to a panel of approximately 101 autoantigens and 6 controls was measured on a slide-based array that has been described previously (Additional file 1) \[6\]. Serum samples (1 μL, diluted 1:100) were added to each array in duplicate and autoantibodies were detected with Cy3-labelled anti-human IgG and Cy-5 labeled anti-human IgM simultaneously. Images were generated for analysis and mean fluorescence intensities (MFI) were determined as previously described. Heat maps were generated using Cluster and Treeview software (Michael Eisen, Berkeley CA, USA) \[7\]. On the heat map, intensities higher than the row mean are colored red, those below the mean are green and cells with signals close to the mean are black. Gray was used to denote missing data.

## Gene expression analysis

Total RNA was prepared from 2.5 to 5.0 ml of blood collected in Paxgene tubes that had been stored at -80°C. Purity and concentration of the isolated RNA was determined using Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA) and Nanodrop 1000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA). We used 250 ng of total RNA to generate biotinylated cRNA using a TotalPrep RNA Amplification kit (Ambion, Austin, TX, USA). The samples were applied to Ilumina HumanWG-6 v3.0 Expression Bead Chips (Illlumina Inc., San Diego, CA, USA) following the manufacturer's directions. The resulting array data were analyzed using Ilumina GenomeStudio software (version 3) and statistical analyses were carried out using Partek Genomic Suite (version 6; Partek Inc., Gladstone, MI, USA). Heat maps were generated using the Cluster and Treeview programs \[7\].

## Statistical analyses

Data are presented as mean values and standard errors of the mean. Continuous variables in experiments with three or more groups of samples were analyzed using a one-way ANOVA with Tukey's multiple comparison test for post test analysis of pairs of samples or the Kruskal-Wallis test for data that did not fit a normal distribution. Analyses of experimental data with two comparison groups used an unpaired 2-tailed Student's *t*-test, with Welch's correction for groups with unequal variances or Mann-Whitney U for data that did not fit a normal distribution. Pearson's correlation coefficient was used to compare continuous variables. Discontinuous variables were compared using Fisher's Exact Test. Graph pad PRISM software (version 5.0a; GraphPad Software, La Jolla, CA, USA) was used for data analysis and graphics. *P*-values \<0.05 were considered significant.

# Results

## ANA and autoantibodies

Using the defined cut-off of \>20 ELISA units (EU), 615 individuals out of the 1,159 tested were ANA positive. For the subset of 401 HC, the average ANA was 19.5 EU and 101 individuals were in the positive range. This rate of 25% HC positivity is very close to what we have reported previously \[1\]. Healthy FDRs had a slightly higher overall average ANA (24.4 EU) and a prevalence of ANA positivity of 34%. These values were not significantly different than in the HCs (*P* \>0.07 for both). For the overall group, ANA values were significantly higher in females than in males (Figure 1) and no males had values \>240 EU while 3.2% of females had values in this range (*P* = 0.0030). When the analysis was limited to HC, females again showed significantly higher ANA levels than males (21.4 vs. 15.6 EU; *P* = 0.033) and ANA positivity was also more prevalent in HC females (29%) than in HC males (17%; *P* = 0.014). This corresponds to a relative risk of ANA positivity in healthy females vs. males of 1.21 (95% confidence interval = 1.059 to 1.390). The highest ANA levels were seen almost exclusively in non-Hispanic females (data not shown), although the overall mean values for Hispanic and non-Hispanic females were not significantly different (*P* = 0.7). African-American (AA) HC (*n* = 32) had a higher mean ANA value than non-AAs (27.47 ± 5.8 EU vs. 18.1 ± 5.8 EU), but the difference did not reach statistical significance (*P* = 0.064). Within each gender, AA individuals also had higher values than non-AA individuals (females: 30.38 ± 8.12 EU vs. 20.59 ± 1.80 EU; males 21.09 ± 5.55 EU vs. 15.17 ± 1.31 EU); these differences were also not significant (*P* \>0.1 for each). However, the results are highly suggestive of higher ANA values in AA HC, and the differences might achieve significance in a larger sample size. Patients with SLE did not show gender differences in overall ANA values, although the very highest ANA values were again seen exclusively in females (Figure 1).

We compared these findings to another autoimmune disease, RA, which is associated with the CCP autoantibody. While RA patients showed significantly higher ANA levels in females than in males (Figure 2), antibodies to CCP did not show gender differences in either the control population represented by the Dallas Heart Study or in RA patients from DRADR (Figure 2). These findings suggest that female gender is a general risk factor for ANA but is not necessarily a correlate with other autoimmune disease-specific antibodies.

Age was not related to ANA positivity in HCs; high values were scattered throughout the age spectrum (R^2^ = 0.01). The high ANA levels in HC also were not explained by overall increases in total IgG as the two measures were not significantly correlated (R^2^ = 0.11; *P* = 0.2).

To further understand high ANA levels in HC, a subgroup analysis was done using HC subjects with ANA values greater than 40 EU as the index group. This value is approximately one standard deviation greater than the overall mean ANA for HC. A total of 18 of the 401 HC, or about 4%, fit this definition of high ANA. Two comparator groups, HC with negative ANA (ANA values \<12 EU) and SLE patients with high ANA (\>100 EU) were generally matched for demographic features including gender, race and ethnicity (Table 1). Autoantibodies on the ENA panel were generally not elevated in the high ANA HC group. Only 3 of the 18 individuals showed positive ENA results, and in all three the lone positive specificity was anti-chromatin.

Demographic features of study groups

| Group | Age (yrs) | ANA (EU) | Female (%) | AA (%) | Hispanic (%) |
|----|----|----|----|----|----|
| HC High ANA (*n* = 18) | 33.8 ± 3.4\* | 78.4 ± 10.0\* | *83* | *22* | 6 |
| HC Low ANA (*n* = 16) | 43.1 ± 3.7 | 7.0 ± 0.6 | *75* | *25* | 0 |
| SLE (*n* = 14) | 38.0 ± 4.0 | 182.4 ± 15.4 | *93* | *43* | 7 |
| *P*\*\* | 0.2 | 0.001^\#^ | 0.4 | 0.4 | 0.6 |

\*Values represent mean and standard error of the mean. \*\**P-*values were calculated by one-way ANOVA or Fisher's exact test. ^\#^ Mean ANA in SLE was significantly higher than in the High ANA HC group (*P* \<0.05). ANA, antinuclear antibodies; SLE, systemic lupus erythematosus.

The three study groups were then compared using the autoantigen array for both IgG and IgM autoantibodies (Figures 3 and 4). Two major IgM clusters showed a tendency for the SLE patients to cluster together, but this was not statistically significant (*P* = 0.08); the high ANA HC individuals had a similar tendency to be in the non-SLE cluster (*P* = 0.06; Figure 3). For IgG, two primary IgG clusters were identified, and all of the SLE patients were in one of these clusters (*P* = 0.009; Figure 4).

Antibodies detected on the arrays were then examined for specificities appearing in HC that were lupus-like. This was defined as overall mean values for a given specificity showing no difference between the high ANA HC and SLE groups (*P* \>0.1) while at the same time having lower values in the low ANA HC group compared to SLE (*P* \<0.1). The antibodies in these groups were examined to choose those with stepwise increases in the groups (low ANA HC \<High ANA HC \<SLE) and to exclude any with distributions that were highly skewed by one or two individuals. This analysis yielded 14 IgGs; 9 of these with the most significant *P*-values (Kruskal-Wallis test) are shown (Figure 5). Four of the nine specificities are directed against autoantigens found in skin tissues (DSG4, MMP1, recombinant human collagen, integrinα6β4) \[8-10\]. Others are associated with autoimmune kidney disease (GBM), thyroid disorders (thyroglobulin), scleroderma (PM/Scl100), and inflammatory arthritis (proteoglycan) \[11\]. A search for autoantibodies that might be associated with a lowered risk of lupus was carried out by choosing specificities that were elevated in high ANA HC but were not high in SLE patients. The ratio of the mean values for the high ANA HC group to the corresponding SLE group mean was calculated, and three specificities had IgG ratios greater than 4.0: Jurkat T cells, gliadin and SK-N-SH (Figure 6). In each of these, a subset of the high ANA HC individuals showed strong reactivity while SLE patients did not have high values. For gliadin, an autoantigen associated with celiac disease, and Jurkat, a measure of anti-T cell and thymocyte antibodies, the distributions were significantly different for the three groups (*P* = 0.042 and 0.033, respectively). Although the antineuronal antibodies measured by SKNSH reactivity did not show significant differences across groups by ANOVA, a dichotomized analysis showed values greater than 400 MFI units were present only in the high ANA HC group (*P* = 0.0242). The four highest values for each of these different autoantibodies represented the same individuals.

An independent ELISA assay was carried out for thyroglobulin (TG) autoantibodies to compare with the findings on the array. Anti-TG levels of the IgG class measured by the two techniques were significantly correlated (*P* = 0.007) and values were significantly higher in the HC high ANA group (1.18 ± 0.23 EU) than the HC low ANA group (0.60 ± 0.07 EU; *P* = 0.030), consistent with the array results.

## Gene expression profiles

Mean gene expression values were compared between the two HC groups defined by ANA status, and this analysis identified 95 dysregulated genes at a significance level of *P* \<0.01. Somewhat surprisingly, the vast majority of these genes (90 out of 95) were upregulated in the ANA-negative group. Of the five genes that were upregulated in the high ANA HC group, the highest (two-fold difference) was *TGM2*, which encodes the celiac disease autoantigen transglutaminase 2.

To detect specificities that might be informative of ANA and diagnosis status, a second analysis was carried out by first comparing the SLE and high ANA HC groups to determine gene specificities that were significantly different at a significance level of *P* \<0.001. This list was then sorted based on the values for the calculated ratio SLE/ANA-High HC. The most highly expressed specificity in this list was *IFI27*, which was 65-fold higher in SLE than in ANA High HC. Overall, 69 genes in this list had greater than two-fold differences between SLE and ANA High HC. These 69 genes were then resorted based on differences in the ratio of ANA High HC/ANA Low HC. The highest ratio in this set was two-fold and out of the top 10 specificities, 7 were in the Type I interferon signature (*IFITM3, MX1, IFI6, IFI44L, ISG15, OAS1, IFIT3*) and one encodes a protein that is activated by dsRNA as might be present in a viral infection (*EIF2AK2*). The specificities that most clearly showed stepwise increases in the three groups going from Low ANA HC to High ANA HC to SLE were *IFITM3* and *MX-1* (Figure 7).

# Discussion

Detecting autoimmune disease in early or preclinical stages is clinically important because the institution of treatment prior to the onset of organ damage has a greater chance to ameliorate or even cure the disease \[12\]. However, early and reliable diagnosis of lupus is a challenge, in large part due to the performance profiles of available diagnostic tests. The optimal test would be sensitive enough to detect all individuals who have a disease while at the same time delivering sufficient specificity to have reasonable predictive probability that the disease is likely. The classic screening test for SLE is the presence in serum of antinuclear antibodies (ANAs) measured by immunofluorescence staining of a cellular substrate \[13\]. ANA positivity is for all practical purposes required to make a diagnosis of lupus since more than 99% of patients with SLE have significant levels of this autoantibody detected at some time during the course of disease. However, since the prevalence of SLE is low, most individuals presenting to a physician with ANA positivity do not in fact have lupus and are not at high risk for developing this disease. But there are few available quantitative and objective measures to establish prognosis for an individual with a positive ANA. This contrasts to the tools that are available for determining the risk of cardiovascular disease, where multifaceted profiles including elements of personal and family history, social habits, body measurements and lab tests can generate a reasonable and very personalized risk prediction for an individual patient \[14\].

In practice, physicians actually do employ some profiling to assess the risk associated with ANA positivity. Young women are more likely to develop lupus than old men, for example, so ANA positivity in the former is of greater concern. The present study confirms our previous observation that female gender is a risk factor for significant ANA positivity \[1\]. This result is also consistent with other findings in healthy control populations including a study of 500 normal individuals in Brazil showing that ANA positivity was almost twice as prevalent in females as in males \[15\]. Similar findings were reported in a rural Canadian population, with the gender difference being greatest at higher ANA levels, as was also noted in the present study \[16\]. The enhanced female risk profile does not appear to extend to the anti-CCP antibody that is associated with RA, another female-predominant autoimmune disorder. Reasons for the association of female gender with strong ANA positivity remain obscure.

Age was not correlated with ANA levels in HCs, which seems to contradict the generally-accepted hypothesis that immunosenescence is associated with increased autoantibody production due to decreased self-regulatory mechanisms. The present findings are consistent, however, with other reports \[15\] and suggest that in a cross-sectional analysis such as this there are many reasons for ANA positivity. The younger individuals may in fact have abnormal immune regulation that predisposes to SLE-like disease while older persons may develop autoreactivity as part of aging immune responses that do not lead to development of pathology.

Other clues are available to suggest approaches to stratifying risk in the ANA-positive population. One is the well-recognized presence of other autoantibodies that accumulate prior to SLE diagnosis \[4\]. The prevalence of any one of these is low, however. For example, ribosomal P autoantibodies are highly specific for SLE but are present in less than one-third of patients \[17\]. This finding predicts that if autoantibodies are included in risk profiling, multiplexed assays will be required. One such approach that is available clinically, the ENA panel, was not useful in the high ANA HC individuals in the present study because very few positives were found. This result is consistent with our previous experience indicating that only the high ANA positives are likely to have ENA specificities \[1\] and even then, the prevalence of other autoantibodies in HCs is very low. The alternative approach which has been applied in the present study is to greatly expand the repertoire of autoantibodies that can be probed by use of an autoantigen array, which in other studies has been shown to provide novel insights into expressed autoantibody repertoires \[18\]. The array data in the present study revealed increased autoreactivity in a group of high ANA HCs. One of the elevated autoantibodies was thyroglobulin, and since autoimmune thyroid disease is probably about 10-fold more common than SLE \[19\], this result suggests that a significant proportion of the ANA positivity seen by rheumatologists is related to thyroid autoimmunity. Longitudinal studies suggest that thyroid autoreactivity, especially in women, may be predictive of thyroid dysfunction \[20\]. Autoantibodies to cartilage proteoglycan can be measured in several systemic and joint-specific rheumatic diseases including Sjogren's Syndrome, rheumatoid arthritis, lupus and ankylosing spondylitis \[21\], suggesting that undetected or preclinical joint inflammation may contribute to ANA positivity. The relative increase in skin autoreactivity in the high ANA HC group might be related to a relative enrichment for skin antigens on the arrays, so this finding should be interpreted with caution. However, it does raise the interesting question of whether some early autoimmune events might take place in cutaneous structures. Transient ANA positivity has been observed in patients with polymorphous light eruption \[22\] and exposure to sun in susceptible individuals can trigger major organ-damaging lupus \[23\]. The interface dermatitis that characterizes the pathology in SLE as well as in other skin disorders may precede a diagnosis of lupus \[24\] and is associated with activation of the Type I interferon gene signature \[25\]. The triggering of this set of genes in skin, however, is not limited to inflammatory disease but can also occur as part of the immune response to viruses, raising the question of whether cutaneous reactions to environmental agents in susceptible hosts might generalize to a systemic response.

The autoantibody arrays show that although the autoreactivity spectrum in SLE is broad, not all specificities are elevated. Upregulated autoantibodies to gliadin, and to T lymphocytes and neuroblastoma cells were present only in the ANA high HC group, and not in the SLE patients. This result confirms in part our previous report that gliadin autoreactivity is associated with incomplete forms of lupus that are associated with myopathies \[26\]. Whether these autoantibodies are actively protective and lower the risk of lupus or alternatively are predictive of other autoimmune diseases developing in these individuals will require further longitudinal investigation.

Upregulated genes observed in high ANA HC individuals include some in the Type I IFN signature that are associated with SLE \[27,28\]. While some of these genes, notably *IFI27*, were only elevated in SLE, others such as *MX-1* showed an intermediate level in the High ANA HC group. In addition to being the most highly-upregulated of the IFN genes in our sample, other data suggest that *IFI27* is relatively more specific for lupus than at least some of the other IFN-inducible genes. A recent study demonstrated that *IFI27* is more likely to be upregulated in lupus than in another autoimmune condition, idiopathic thrombocytopenic purpura \[29\]. The specific functions of many of the proteins associated with IFN-related genes are obscure, but *MX-1* is closely associated with the response to the influenza virus, so upregulation of this gene in normal individuals might reflect the ubiquitous exposure to this pathogen.

The present data suggest possible components of a lupus risk profile. As in the cardiovascular risk profiling equations, gender will be a factor but in lupus the risk will be associated with females rather than males. The age category will be inverted from that of cardiovascular disease, with greater weight given to younger ages. The lupus risk is correlated with ANA levels, not just positivity, with values in the upper quartile having a two- to three-fold elevation of risk \[1\], so this will be an important component. Other autoantibodies that may add to risk include those that are clinically well-known like anti-dsDNA and anti-Sm, as well as other novel specificities including the skin determinants identified in the present study. On the other hand, antineuronal, anti-thyroid or gliadin autoantibodies might steer attention away from SLE towards other autoimmune disorders. Elevated expression of genes related to the Type I IFN signature is likely to add points to the risk equation.

This study has several limitations. One is the lack of information regarding use of medications, especially hormones, by the HC. Whether administration of estrogen in the form of oral contraceptives or postmenopausal replacement therapy might induce high ANA levels in a healthy individual cannot be ascertained from our data. Another limitation is the cross-sectional design which does not permit insights into changes that evolve over time. And it would be of interest to determine reactivity to foreign antigens such as infectious agents to further interpret the significance of the autoreactive responses. Ultimately, validation of risk profiles will require longitudinal studies.

Finally, since SLE is a relatively rare disorder, the probability of finding a new onset patient is low even after ANA positivity has been identified. One approach to increasing the likelihood of useful results would be to follow individuals who already have some of the identified risk profile components. For example, studies could be carried out in individuals who have been sent for ANA testing for any reason. The very fact that the individual sought medical attention and that an ANA was ordered is likely to increase the pre-test probability of disease. Such an approach has shown that in the population of individuals sent for rheumatoid factor testing, the pre-test probability for RA is 17% \[30\].

# Conclusions

This study shows that it may be possible to identify the small percentage of ANA positive individuals who are at risk for development of SLE. Characterization of protein and gene expression profiles accompanying ANA positivity has potential to enable more precise risk quantification and ultimately to identify pre-clinical stages of this disease that would allow early and definitive treatment.

# Abbreviations

ANA: antinuclear antibody; CCP: cyclic citrullinated peptide; DRADR: Dallas Regional Autoimmune Disease Registry; ENA: extractable nuclear antibody; EU: ELISA units; FDR: first degree relative; HC: healthy controls; IFN: interferon; ILE: incomplete lupus; MFI: mean fluorescence intensity; RA: rheumatoid arthritis; SLE: systemic lupus erythematosus; TG: thyroglobulin

# Competing interests

NJO has equity interest in ArthroChip LLC and has received research grants from Medimmune, Novo Nordisk, Genentech/Roche and Human Genome Sciences. DRK has received a research grant from Human Genome Sciences. All other authors declare that they have no competing interests.

# Authors' contributions

QZL supervised the protein and gene expression array assays and analyses. DRK organized the patient registry. JQ organized and performed laboratory assays. JZ performed the protein array assays, while YL performed data analyses for protein and gene expression array assays. VKB coordinated patient recruitment for the registry. BFC contributed to design and validation of the autoantibody array. EKW conceived of the study design and NJO assembled the patient cohorts and supervised clinical phenotyping. QZL, DK, EKW and NJO wrote the manuscript with input and consensus from all authors. All authors read and approved the final manuscript.

# Supplementary Material

###### Additional file 1

**Components of the Autoantigen Array**. This table lists the components of the array along with sources of the autoantigens.

Click here for file

## Acknowledgements

This work was supported by NIH/NIAMS P50AR0455503. We appreciate the assistance of Azza Mutwally, Alice Cox, Sarmistha Sen, Justin Ribault and Pratik Doshi for enrolling and evaluating the individuals enrolled in DRADR. Michelle Christadoss and Ferdicia Carr-Johnson processed the samples and carried out the laboratory assessments. Jessica Mayeux assisted with data analysis. Heidi Jacobe MD shared with us a set of skin-specific autoantigens for inclusion in the protein arrays, and we appreciate her input. Thanks also to Chandra Mohan MBBS, PhD, for helpful discussions and support.

The data presented in this publication have been deposited in the NCBI Gene Expression Omnibus with accession number GSE 24706 and may be accessed online \[31\].


date: 2018-09
title: 3 in 5 babies not breastfed in the first hour of life Breastfeeding within an hour after birth is critical for saving newborn lives

**31 July 2018 News Release Geneva** - An estimated 78 million babies – or three in five – are not breastfed within the first hour of life, putting them at higher risk of death and disease and making them less likely to continue breastfeeding, say UNICEF and WHO in a new report. Most of these babies are born in low- and middle-income countries.

The report notes that newborns who breastfeed in the first hour of life are significantly more likely to survive. Even a delay of a few hours after birth could pose life-threatening consequences. Skin-to-skin contact along with suckling at the breast stimulate the mother's production of breastmilk, including colostrum, also called the baby's 'first vaccine', which is extremely rich in nutrients and antibodies.

"When it comes to the start of breastfeeding, timing is everything. In many countries, it can even be a matter of life or death," says Henrietta H. Fore, UNICEF Executive Director. "Yet each year, millions of newborns miss out on the benefits of early breastfeeding and the reasons – all too often – are things we can change. Mothers simply don't receive enough support to breastfeed within those crucial minutes after birth, even from medical personnel at health facilities."

Breastfeeding rates within the first hour after birth are highest in Eastern and Southern Africa (65%) and lowest in East Asia and the Pacific (32%), the report says. Nearly 9 in 10 babies born in Burundi, Sri Lanka and Vanuatu are breastfed within the first hour. By contrast, only two in 10 babies born in Azerbaijan, Chad and Montenegro do so.\*

"Breastfeeding gives children the best possible start in life," says Dr Tedros Adhanom Ghebreyesus, WHO Director-General. "We must urgently scale up support to mothers – be it from family members, health care workers, employers and governments, so they can give their children the start they deserve."

Capture the Moment, which analyzes data from 76 countries, finds that despite the importance of early initiation of breastfeeding, too many newborns are left waiting too long for different reasons, including:

-   Feeding newborns food or drinks, including formula: Common practices, such as discarding colostrum, an elder feeding the baby honey or health professionals giving the newborn a specific liquid, such as sugar water or infant formula, delay a newborn's first critical contact with his or her mother.

-   The rise in elective C-sections: In Egypt, caesarean section rates more than doubled between 2005 and 2014, increasing from 20% to 52%. During the same period, rates of early initiation of breastfeeding decreased from 40% to 27%. A study across 51 countries notes that early initiation rates are significantly lower among newborns delivered by caesarean section. In Egypt, only 19% of babies born by C-section were breastfed in the first hour after birth, compared to 39% of babies born by natural delivery.

-   Gaps in the quality of care provided to mothers and newborns: The presence of a skilled birth attendant does not seem to affect rates of early breastfeeding, according to the report. Across 58 countries between 2005 and 2017, deliveries at health institutions grew by 18 percentage points, while early initiation rates increased by 6 percentage points. In many cases, babies are separated from their mothers immediately after birth and guidance from health workers is limited. In Serbia, the rates increased by 43 percentage points from 2010 to 2014 due to efforts to improve the care mothers received at birth.

Earlier studies, cited in the report, show that newborns who began breastfeeding between two and 23 hours after birth had a 33% greater risk of dying compared with those who began breastfeeding within one hour of birth. Among newborns who started breastfeeding a day or more after birth, the risk was more than twice as high.

The report urges governments, donors and other decision-makers to adopt strong legal measures to restrict the marketing of infant formula and other breastmilk substitutes.

The WHO and UNICEF-led Global Breastfeeding Collective also released the 2018 Global Breastfeeding Scorecard, which tracks progress for breastfeeding policies and programmes. In it, they encourage countries to advance policies and programmes that help all mothers to start breastfeeding in the first hour of their child's life and to continue as long as they want.

Available from: <http://www.who.int/news-room/detail/31-07-2018-3-in-5-babies-not-breastfed-in-the-first-hour-of-life>


date: 2017-05
title: "DEPRESSION: LET'S TALK" SAYS WHO, AS DEPRESSION TOPS LIST OF CAUSES OF ILL HEALTH

**30 MARCH 2017 \| GENEVA -** Depression is the leading cause of ill health and disability worldwide. According to the latest estimates from WHO, more than 300 million people are now living with depression, an increase of more than 18% between 2005 and 2015. Lack of support for people with mental disorders, coupled with a fear of stigma, prevent many from accessing the treatment they need to live healthy, productive lives.

The new estimates have been released in the lead-up to World Health Day on 7 April, the high point in WHO's year-long campaign "Depression: let's talk". The overall goal of the campaign is that more people with depression, everywhere in the world, both seek and get help.

Said WHO Director-General, Dr Margaret Chan: "These new figures are a wake-up call for all countries to re-think their approaches to mental health and to treat it with the urgency that it deserves."

One of the first steps is to address issues around prejudice and discrimination. "The continuing stigma associated with mental illness was the reason why we decided to name our campaign Depression: let's talk," said Dr Shekhar Saxena, Director of the Department of Mental Health and Substance Abuse at WHO. "For someone living with depression, talking to a person they trust is often the first step towards treatment and recovery."

**Urgent need for increased investment**

Increased investment is also needed. In many countries, there is no, or very little, support available for people with mental health disorders. Even in high-income countries, nearly 50% of people with depression do not get treatment. On average, just 3% of government health budgets is invested in mental health, varying from less than 1% in low-income countries to 5% in high-income countries.

Investment in mental health makes economic sense. Every US\$ 1 invested in scaling up treatment for depression and anxiety leads to a return of US\$ 4 in better health and ability to work. Treatment usually involves either a talking therapy or antidepressant medication or a combination of the two. Both approaches can be provided by non-specialist health-workers, following a short course of training, and using WHO's mhGAP Intervention Guide. More than 90 countries, of all income levels, have introduced or scaled-up programmes that provide treatment for depression and other mental disorders using this Intervention Guide.

Failure to act is costly. According to a WHO-led study, which calculated treatment costs and health outcomes in 36 low-, middle- and high-income countries for the 15 years from 2016-2030, low levels of recognition and access to care for depression and another common mental disorder, anxiety, result in a global economic loss of a trillion US dollars every year. The losses are incurred by households, employers and governments. Households lose out financially when people cannot work. Employers suffer when employees become less productive and are unable to work. Governments have to pay higher health and welfare expenditures.

**Associated health risks**

WHO has identified strong links between depression and other noncommunicable disorders and diseases. Depression increases the risk of substance use disorders and diseases such as diabetes and heart disease; the opposite is also true, meaning that people with these other conditions have a higher risk of depression.

Depression is also an important risk factor for suicide, which claims hundreds of thousands of lives each year. Said Dr Saxena: "A better understanding of depression and how it can be treated, while essential, is just the beginning. What needs to follow is sustained scale-up of mental health services accessible to everyone, even the most remote populations in the world."

Depression is a common mental illness characterized by persistent sadness and a loss of interest in activities that people normally enjoy, accompanied by an inability to carry out daily activities, for 14 days or longer.

In addition, people with depression normally have several of the following: a loss of energy; a change in appetite; sleeping more or less; anxiety; reduced concentration; indecisiveness; restlessness; feelings of worthlessness, guilt, or hopelessness; and thoughts of self-harm or suicide.

Available from: <http://www.who.int/mediacentre/news/releases/2017/world-health-day/en/>


abstract: Retinal neurodegeneration is an early event in the pathogenesis of diabetic retinopathy (DR). Somatostatin (SST) is an endogenous neuroprotective peptide that is downregulated in the diabetic eye. The aim of the study was to test the usefulness of topical administration of SST in preventing retinal neurodegeneration. For this purpose, rats with streptozotocin-induced diabetes mellitus (STZ-DM) were treated with either SST eye drops or vehicle for 15 days. Nondiabetic rats treated with vehicle served as a control group. Functional abnormalities were assessed by electroretinography (ERG), and neurodegeneration was assessed by measuring glial activation and the apoptotic rate. In addition, proapoptotic (FasL, Bid, and activation of caspase-8 and caspase-3) and survival signaling pathways (BclxL) were examined. Intraretinal concentrations of glutamate and its main transporter glutamate/aspartate transporter (GLAST) were also determined. Treatment with SST eye drops prevented ERG abnormalities, glial activation, apoptosis, and the misbalance between proapoptotic and survival signaling detected in STZ-DM rats. In addition, SST eye drops inhibited glutamate accumulation in the retina and GLAST downregulation induced by diabetes mellitus. We conclude that topical administration of SST has a potent effect in preventing retinal neurodegeneration induced by diabetes mellitus. In addition, our findings open up a new preventive pharmacological strategy targeted to early stages of DR.
author: Cristina Hernández; Marta García-Ramírez; Lidia Corraliza; Jimena Fernández-Carneado; Josep Farrera-Sinfreu; Berta Ponsati; Águeda González-Rodríguez; Ángela M. Valverde; Rafael SimóCorresponding author: Cristina Hernández, <firstname.lastname@example.com>.
date: 2013-07
references:
title: Topical Administration of Somatostatin Prevents Retinal Neurodegeneration in Experimental Diabetes

Diabetic retinopathy (DR) has been classically considered to be a microcirculatory disease of the retina. However, there is growing evidence to suggest that retinal neurodegeneration is an early event in the pathogenesis of DR that participates in the microcirculatory abnormalities that occur in DR (1–8). We have contributed to this field by demonstrating that the main hallmarks of retinal neurodegeneration (apoptosis and glial activation) are already present in the retinas of diabetic donors without any microcirculatory abnormalities in ophthalmoscopic examinations performed during the year before death (9–11).

Glutamate is the major retinal excitatory neurotransmitter for the photoreceptor-bipolar-ganglion cell circuit. However, elevated glutamate levels in the retina (which result in excessive stimulation) are implicated in the so-called "excitotoxicity," which leads to neurodegeneration (12). The excitotoxicity of glutamate is the result of overactivation of ionotropic glutamate receptors, which have been found to be overexpressed in rats with streptozotocin-induced diabetes mellitus (STZ-DM) (13,14). Apart from glutamate, oxidative stress (15), advanced glycation end product receptor upregulation (16,17), and renin-angiotensin system activation (18–20) play an essential role in retinal neurodegeneration induced by diabetes mellitus. Finally, recent evidence indicates that diabetes mellitus–induced downregulation of neuroprotective factors synthesized by the retina is also involved in the neurodegenerative process of the diabetic eye (21). On these bases, it is reasonable to hypothesize that therapeutic strategies based on neuroprotection will be effective in preventing or arresting DR development.

Somatostatin (SST) is one of the most important neuroprotective factors synthesized by the retina, retinal pigment epithelium (RPE) being its main source in the human eye. The human retina produces significant amounts of SST, as deduced by the strikingly high levels reported within the vitreous fluid (22,23). Besides SST, its receptors (SSTRs) are also expressed in the retina, with SSTR1 and SSTR2 being the most widely expressed (24). The production of both SST and its receptors simultaneously suggests a relevant autocrine action in the human retina. SST acts as a neuromodulator in the retina through multiple pathways, including intracellular Ca^2+^ signaling, nitric oxide function, and glutamate release from the photoreceptors. Apart from neuroprotection, SST has potent antiangiogenic properties and regulates various ion/water transport systems (24). Therefore, SST seems to be essential in preventing both proliferative DR (PDR) and diabetic macular edema (DME).

In the early stages of DR, there is a downregulation of SST that is associated with retinal neurodegeneration (9). In fact, it has recently been reported that intravitreal administration of SST and SST analogs protects the retina from AMPA-induced neurotoxicity (25). In addition, the lower expression of SST in RPE and neuroretina is associated with a dramatic decrease of intavitreal SST levels in both DME (26) and PDR (22,23). As a result, the physiological role of SST in preventing both fluid accumulation within the retina and neovascularization could be reduced, and consequently, the development of DME and PDR would be favored. For all these reasons, SST replacement treatment can be considered a new target not only for preventing the neurodegenerative process but also for more advanced stages of DR such as DME and PDR.

When the early stages of DR are the therapeutic target, it would be inconceivable to recommend in the clinical practice an aggressive treatment such as intravitreous injections. On the other hand, the use of eye drops has not been considered an appropriate route for the administration of drugs aimed at preventing or arresting DR because of the general assumption that they do not reach the posterior chamber of the eye. However, there is emerging evidence showing the capacity of a lot of drugs to reach the retina in pharmacologically effective concentrations, at least in animal models (27).

On this basis, the aim of the current study was to determine whether topical administration of SST prevents the retinal neurodegenerative process that occurs in STZ-induced diabetic rats. In addition, the effects of SST eye drops on apoptotic/survival signaling pathways and retinal glutamate levels have been examined. Finally, the possibility that SST acts through preventing glutamate accumulation by upregulating glutamate/aspartate transporter (GLAST) has also been explored.

# RESEARCH DESIGN AND METHODS

A total of 24 male Sprague-Dawley rats were obtained from Charles River Laboratories (Senneville, Quebec, Canada). A minimum acclimatization period of 14 days preceded treatment. STZ (60 mg/kg) was administered to 8-week-old (*n* = 16) rats by intravenous injection via tail vein on day −2 prior to starting SST treatment. The remaining eight rats were injected with vehicle and served as a nondiabetic control group. Animals with blood glucose levels \>250 mg/dL the day after STZ administration were considered diabetic. On day −1, all animals were weighed and randomly assigned to treatment groups (vehicle or SST) using a computer-based randomization procedure. Randomization was performed by stratification using body weight as the parameter.

SST or vehicle eye drops were administered directly onto the superior corneal surface of each eye using a syringe. One drop (20 µL) of SST (1%; 10 mg/mL) or vehicle (20 µL of 0.9% sodium chloride) was administered once daily for 14 days. On day 15, the animals' eyes were instilled with a drop of SST or vehicle \~1 h prior to necropsy. Therefore, the elapsed time between the onset of diabetes mellitus until necropsy was 16 days.

A dose-response experiment assessing the hallmarks of retinal neurodegeneration (apoptosis and glial activation) was performed using SST eye drops at 10, 2, and 0.5 mg/mL (Table 1).

Experimental design of the dose-efficacy study

![](2569tbl1)

The animals used in this study were treated in accordance with the Association for Research in Vision and Ophthalmology Statement.

## Pharmacokinetic analyses.

A pharmacokinetic analysis after a single topical dose of radiolabeled ^125^I-somatostatin was performed. For this purpose, seven rats were anesthetized and placed in a supine position. After they were placed in the experimental area, 10 µL (2.5 µCi) ^125^I-somatostatin was applied in each eye with a micropipette. The animals were killed at 15, 30, and 60 min and 4, 6, 12 and 24 h after the application of the labeled SST solution. After the animals were killed, the eyes were obtained and gently washed in a volume of 20 mL physiologic saline. A small incision was performed in the rear portion of the eye ball, and the eye content was extracted. The vitreous humor, retina, and choroid-RPE were collected. The sclera was placed in different tubes for independent measurements. All the samples were weighted and counted in the γ counter in order to obtain the percentage of injected dose for each gram of tissue. Samples of blood were also obtained.

In addition, absorption of carboxyfluorecein-labeled SST (CF-SST) eye drops through the ocular structures was also evaluated in 8-week-old Sprague-Dawley rats. For this purpose, three rats were treated with vehicle eye drops in both eyes. Three rats were treated with the fluorescent label (CF) eye drops. The eye drops of CF-SST (10 mg/mL; one drop) were administered to the right eye, and the vehicle was administered to the left eye of three rats. One animal of each group was killed at 10, 30, and 60 min after the application of the corresponding eye drop. The eyes were enucleated, fixed in 4% paraformaldehyde, submerged in optimum cutting temperature Tissue-teck, frozen in liquid nitrogen, and stored at −80°C until microtome sectioned. The whole eyes were cryosectioned to 50-μm sections, mounted with antifade-mounting medium with DAPI, and visualized in fluorescent and confocal microscopes (Olympus). All images were taken with the same settings.

## Ophthalmological examinations.

Slit lamp biomicroscopy, indirect ophthalmoscopy, and intraocular pressure measurement were performed in all animals at baseline and at days 7 and 13 posttreatment.

## Electroretinography.

Electroretinogram evaluations were performed on day –1 before treatment and on day 14 as follows: the animals were dark adapted for at least 1 h prior to electroretinography (ERG) recording and then anesthetized with isoflurane to maintain the anesthesia prior to and during the procedure. Tropicamide (1%) was applied to each eye prior to the test. A contact lens electrode was placed on the surface of each eye, and a needle electrode was placed cutaneously on the head between the two eyes. A cutaneous ground electrode was placed near the base of the tail. Carboxymethylcellulose (1%) drops were applied to the interior surface of the contact lens electrodes prior to their placement on the eyes. A topical anesthetic (proparacaine, 0.5%) was applied to the eyes. The amplitude and implicit time of the b-wave after scotopic flash stimuli were measured. b-wave amplitude and implicit time were measured as defined by the International Society for Clinical Electrophysiology of Vision (28).

## Histopathology.

Eyes were prepared for histopathological examination by being embedded in paraffin wax, sectioned, and stained with hematoxylin-eosin. For each eye, a total of three sagittal sections (one section through the optic nerve, one section \~500 µm medially, and another section \~500 µm laterally from the optic nerve) were evaluated.

### Assessment of glial activation.

Sections of 7-μm thicknesses were obtained from the paraffin blocks and fixed on highly adherent slides (Visionbiosystems; Newcastle upon Tyne, U.K.). They were desparaffined in xylol; rehydrated in 100% ethanol, 90% ethanol, and 75% ethanol; and incubated for 10 min in PBS. Unspecific unions were blocked by incubating the samples for 1 h in PBS 1% BSA and 0.05% Triton X-100. Thereafter, the primary rabbit anti-human glial fibrillar acidic protein (GFAP) antibody (Sigma, Madrid, Spain) diluted in the blocking buffer (1:100) was incubated for 36 h at 4°C. After three washings for 5 min with PBS, the sections were incubated with a secondary human IgG antibody labeled with Alexa Fluor 568 (Invitrogen, Eugene, OR) for 1 h at room temperature. The labeled sections were washed and mounted with fluorescent medium containing DAPI for identifying cell nuclei (Vector Laboratories, Burlingame, CA). Positive sections for GFAP were captured in a confocal microscope (FV1000; Olympus, Hamburg, Germany). Optical sections were obtained with a 568-nm laser for Alexa Fluor 568 and a 405-nm laser for the DAPI (image resolution 1,024 × 1,024 pixels). The degree of glial activation was evaluated by using a score system based on the extent of GFAP staining (29). This scoring system is as follows: negligible staining (score 0), Müller cell endfeet region/ganglion cell layer (GCL) only (score 1), Müller cell endfeet region/GCL plus a few proximal processes (score 2), Müller cell endfeet plus many processes but not extending to outer nuclear layer (ONL) (score 3), Müller cell endfeet plus processes throughout with some in the ONL (score 4), and Müller cell endfeet plus a lot of dark processes from GCL to the outer margin of ONL (score 5).

### Assessment of apoptosis.

The In Situ Cell Death Detection kit (Roche Diagnostics, Mannheim, Germany) was used to evaluate the presence of apoptotic cells. Sections were deparaffined in xylol; rehydrated in 100% ethanol, 90% ethanol, and 75% ethanol; and incubated for 10 min in PBS. Autofluorescence was eliminated after incubation with 0.2% potassium permanganate for 20 min and 1% oxalic acid for 30 s before the TUNEL procedure was started. Positive and negative controls were processed at the same time. Three confocal images were recorded (×40) corresponding to a surface of 317.13 × 317.13 μm for each section. Staining with propidium iodide was done to examine nucleus morphology and discard false positives. The total number of nuclei and nuclei positive for TUNEL were counted with the ImageJ program.

### Proapoptotic and survival signaling.

Proapoptotic signaling pathways were investigated by analyzing several proapoptotic molecules (FasL, caspase-8, total Bid, truncated Bid, and active caspase-3), as well as antiapoptotic markers (BclxL), by Western blot as previously described (30).

## Glutamate quantification.

Quantification of glutamate was performed by liquid chromatography coupled to mass spectrometry. Chromatographic separation was performed on an Agilent 1200 series (Waldbronn, Germany) using an Ascentis Express HILIC column, 50 × 2.1 mm with 2.7 µm particle size from Supelco (Belfonte, PA), maintained at 25°C throughout the analysis, a mobile phase acetonitrile (ACN), and water (50 mmol/L ammonium acetate) with a flow rate of 0.6 mL/min. The volume injected was 10 µL. The mobile phase involved a gradient starting at 87% of ACN, which was maintained for 3 min. Then, from min 3 to 10 the ACN content was decreased to 20% and increased again to 87% at min 12.5. Glutamate was eluted at 6.5 min. The mass detection system was an Agilent 6410 Triple Quad (Santa Clara, CA) using positive electrospray ionization with a gas temperature of 350°C, gas flow rate of 12 L/min, nebulizer pressure of 45 ψ, capillary voltage of 3,500 V, fragmentor of 135 V, and collision energy of 10 V.

## GLAST expression

### RNA isolation and GLAST mRNA quantification.

Total RNA was extracted from retinas with the Rneasy Mini kit with DNAase digestion (Qiagen; IZASA, Madrid, Spain). One microgram of total RNA was used for reverse transcription with TaqMan and random primer reagents (Applied Biosystems, Madrid, Spain). Real-time PCR was performed with the GLAST specific assay for exon 3–4 boundary Rn00570130_m1 (Applied Biosystems), and results were normalized to the expression level of β-actin (Rn00667969_m1) as a housekeeping gene. Relative quantification values were obtained using an ABI Prism 7000 SDS software (Applied Biosystems).

### Immunohistochemistry for GLAST.

Sections of 4 μm were deparaffined in xylene and hydrated in a graded ethanol series. Then, sections were antigen retrieved (sodium citrate 10 mmol/L, pH 6.0) and washed in PBS. Next, sections were incubated in blocking solution (2% BSA, Tween 0.05% PBS) for 1 h at room temperature followed by incubation with primary antibody anti-GLAST (1:200; Abcam, Cambridge, U.K.). After washing, sections were incubated with a fluorescent anti-rabbit Alexa Fluor 488 as a secondary antibody (Life Technologies, Madrid, Spain) in blocking solution for 1 h, washed, and mounted in Vectashield (Vector Laboratories). DAPI was used for nuclear staining. Quantification of fluorescence intensity of images was performed as previously explained for GFAP.

## Statistical analysis.

Normal distribution of the variables was evaluated using the Kolmogorov-Smirnov test. Data were expressed either as means ± SD or as median (range). Comparisons of continuous variables between groups were performed using the unpaired Student *t* test or Mann-Whitney *U* test. For comparisons within groups (ERG of day 14 vs. ERG of day −1), the paired Student *t* test was used. Levels of statistical significance were set at *P* \< 0.05.

# RESULTS

## SST contained in eye drops reached the retina.

The results of pharmacokinetic study are displayed in Fig. 1. After topical ocular administration of ^125^I-somatostatin, the maximum postdose concentration (5% of applied dose per gram of tissue) was detected at 1 h. However, at 15 min most of the ^125^I-somatostatin had already reached the retina. This rapid absorption argues against the permeation of SST through the cornea. For further exploration of this issue, a pharmacokinetic study using eye drops containing CF-SST was performed. This study clearly showed that SST contained in the eye drops reached the retina not through the cornea but by the trans-scleral route, thus bypassing the anterior chamber ([Supplementary Fig. 1](http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db12-0926/-/DC1)).

## Ocular safety of SST treatment.

No significant differences in the cornea, pupil size, or intraocular pressure were observed among the three groups at baseline or during follow-up.

## SST treatment prevents ERG abnormalities.

ERG was performed to assess retinal function. b-wave amplitude was significantly reduced at day 14 in the diabetic group treated with vehicle (Fig. 2A). By contrast, in the control group and in the diabetic group treated with SST eye drops no reduction of b-wave amplitude was observed. b-wave implicit time increased at day 14 in diabetic rats treated with vehicle (Fig. 2B). However, in the control group and in the diabetic group treated with SST eye drops the implicit time did not increase at day 14. Therefore, SST treatment prevented ERG abnormalities caused by diabetes mellitus.

## Neurodegeneration was prevented in diabetic rats treated with SST eye drops

### Glial activation.

The retinas of nondiabetic control rats revealed GFAP immunolabeling in cells and their processes in the inner layer of the retina, especially in the inner limiting membrane, and GCL (GFAP score 0–1) (Fig. 3). According to their location and morphology, these GFAP-positive cells were interpreted as being retinal astrocytes. However, a slight GFAP immunofluorescence in Müller cells could be detected. In the retina of diabetic rats treated with placebo, GFAP expression was prominent along the inner limiting membrane, in Müller cell endfeet, and in Müller cell radial fibers extending through both the inner and outer retina (GFAP score 1–3). Diabetic rats treated with SST eye drops presented significantly lower GFAP score (0–1) than diabetic rats treated with placebo (Fig. 3).

### Retinal apoptosis.

Representative images of the presence of apoptotic cells in the retinas of the three experimental groups are shown in Fig. 4A. Two weeks after the induction of diabetes mellitus with STZ, the whole percentage of retinal apoptotic cells, as well as the percentage of apoptotic cells in retinal layers, was significantly higher in comparison with that observed in retinas from age-matched nondiabetic controls (Fig. 4B). In all groups, apoptosis was highest in the GCL. Diabetic rats treated with SST eye drops presented a significantly lower ratio of apoptosis in all retinal layers than diabetic rats treated with placebo and similar to nondiabetic rats (Fig. 4B).

### Dose response effect.

The effectiveness of SST eye drops at concentrations of 2 mg/mL was similar to that of eye drops at 10 mg/mL concentrations in preventing glial activation and apoptosis. However, the effect of 0.5 mg/mL was significantly lower than that obtained using either 2 or 10 mg/mL (Fig. 5).

### Proapoptotic and survival molecules.

The results of proapoptotic and survival molecules measured in the retina of the three rat groups studied are shown in Fig. 6. The expression of FasL, a proapoptotic component of the death receptor apoptotic pathway, was significantly increased in neuroretinas from diabetic rats compared with control rats. The interaction of FasL with the death receptor Fas/CD95 assembles the death-inducing signaling complex (31). This includes the recruitment of caspase-8. Activation of caspase-8, monitored by the presence of its 18-kDa active fragment, was significantly increased in neuroretinas of diabetic rats compared with controls. These data were reinforced by the detection of elevated levels of truncated Bid fragment in diabetic samples. As a result, neuroretina from diabetic rats displayed a significant activation of the executer caspase-3, monitored by the presence of its 17-kDa active fragment. Treatment with SST eye drops prevented the upregulation of all of these proapoptotic molecules.

On the other hand, a reduction in BclxL, an antiapoptotic member of the Bcl2 family, was found in diabetic retinas in comparison with control retinas. When treated with SST eye drops, BclxL expression was greatly improved (Fig. 6).

## Topical administration of SST prevents the increase of glutamate induced by diabetes mellitus.

Glutamate levels (arbitrary units, median \[range\]) in the diabetic retinas were higher than in nondiabetic retinas (117 \[57–412\] vs. 47 \[27–300\]; *P* = 0.012). In diabetic rats treated with SST eye drops, glutamate concentration (40 \[21–127\]) was significantly decreased in comparison with diabetic rats treated with vehicle (*P* = 0.021) and similar to control rats (*P* = 0.51).

## Upregulation of GLAST expression after SST treatment.

Gene and protein expression of GLAST were downregulated in retinas of diabetic rats (Fig. 7). In diabetic rats treated with SST, this downregulation was prevented and GLAST protein levels were even increased in comparison with nondiabetic rats (Fig. 7).

# DISCUSSION

In the current study, we provide the first evidence that topical administration of SST prevents retinal neurodegeneration in STZ-DM rats. SST eye drops prevented b-wave abnormalities in the ERG (reduction in amplitude and increase in implicit time), which are considered sensitive indicators of DR (32). In addition, topical administration of SST abrogated the characteristic hallmarks of neurodegeneration (glial activation and apoptosis) caused by short-term diabetes mellitus. In this regard, it should be noted that the degree of glial activation and apoptosis in diabetic rats treated with SST eye drops was very similar to that found in nondiabetic rats.

Glial activation is a prominent event in retinal neurodegeneration. Retinal astrocytes normally express GFAP, while Müller cells do not. However, during the course of diabetes mellitus there is an aberrant expression of GFAP by Müller cells (33). Because Müller cells produce factors capable of modulating blood flow, vascular permeability, and cell survival and because their processes surround all the blood vessels in the retina, it seems that these cells play a key role in the pathogenesis of retinal microangiopathy in the diabetic eye (33,34). In this paper, we have found that SST eye drops were able to significantly prevent glial activation or, in other words, most of the aberrant expression of GFAP in Müller cells.

Apart from glial activation, apoptosis is the other hallmark of retinal neurodegeneration. As previously reported (35), we found a significant rate of apoptosis in retinas from STZ-induced diabetic rats in comparison with nondiabetic control rats. In addition, we found a misbalance between apoptotic and survival signaling pathways. In this regard, we demonstrated that the activation of the death receptor pathway, monitored by the elevation in the expression of FasL and the activation of caspase-8, was significantly higher in neuroretina from STZ-induced diabetic rats than in nondiabetic controls. Importantly, our data clearly show increased Bid cleavage in the neuroretina of diabetic rats. This result suggests that in the diabetic neuroretina, the apoptotic signals emerging from the death receptor pathway are amplified to additional activation of the intrinsic apoptotic signaling pathway, resulting in a stronger activation of the executer caspase-3. On the other hand, a significant downregulation of BclxL (an antiapoptotic molecule belonging to the Bcl2 family) was found in diabetic retinas. Notably, topical administration of SST was able to prevent all these abnormalities in proapoptotic/survival signaling.

Apart from the prevention of apoptosis and glial activation, SST eye drops significantly reduced glutamate retinal levels. Rapid removal or inactivation of glutamate is necessary to maintain the normal function of the retina and for avoiding excitotoxicity. Elevated levels of glutamate in the retina have been found in experimental models of diabetes mellitus (36–38), as well as in the vitreous fluid of diabetic patients (12,39). The reasons why diabetes mellitus facilitates extracellular accumulation of glutamate include *1*) an increase of glutamate production by glial cells due to the loss of the Müller cell–specific enzyme glutamine synthetase, which converts glutamate to glutamine (36,37); *2*) a reduction in the retinal ability to oxidize glutamate to α-ketoglutarate (37); *3*) the death of neurons with subsequent release of intracellular contents; and *4*) the impairment of glutamate removal from extracellular space by the glial cells (12). Regarding this later point, in the current study we provide evidence that GLAST, the main glutamate transporter expressed by Müller cells (40), is significantly decreased in STZ-induced diabetic rats. More importantly, SST eye drops not only completely prevent GLAST downregulation induced by diabetes mellitus but even lead to a significant increase in GLAST expression (mRNA and protein). These findings can contribute to our understanding of the underlying mechanisms involved in the retinal reduction of AMPA-induced neurotoxicity reported for SST and SST analogs administered intravitreally (25).

The study of the mechanisms linking neurodegeneration with early vascular abnormalities is crucial for understanding how neuroprotection could also exert beneficial effects in diabetic microangiopathy. SST may inhibit angiogenesis directly through SST receptors present on endothelial cells (41) and also indirectly through the downregulation of vascular endothelial growth factor (VEGF) (42,43) or by the inhibition of postreceptor signaling events not only of VEGF but also of other peptide growth factors such as IGF-1, epidermal growth factor, and platelet-derived growth factor (44). The potential effects of SST in abrogating microvascular impairment are beyond the scope of the current study, and specific studies on this issue are urgently needed.

Given the essential role of neurodegeneration in the pathogenesis of DR, it is reasonable to hypothesize that therapeutic strategies based on the neuroprotective effects of SST would be effective in preventing or arresting DR development. The observation that circulating growth hormone–IGF-1 production is reduced by SST has been the basis for proposing systemic administration of SST analogs for treating DR. However, this concept has not been supported by clinical intervention trials. The main concerns of systemic administration of SST are as follows: *1*) the paracrine effects of SST synthesized by the retina involve SSTR subtypes other than SSTR2, which mediates growth hormone inhibition and for which octreotide presents high affinity, and *2*) SST analogs do not cross the blood-retinal barrier and would have access only where there is disruption of the blood-retinal barrier, thus limiting the amount of the drug reaching the retinal target tissues. For these reasons and given that a downregulation of retinal production of SST occurs in diabetic retina, a replacement using local administration of the natural peptide seems a reasonable approach. In this regard, the neuroprotective effects of topical administration of brimonidine and nerve growth factor have already been reported in experimental models (45–47). These findings open up the possibility of developing a topical therapy targeting the early stages of DR in which the use of the only currently established therapies such as laser photocoagulation or intravitreal injections of corticosteroids or anti-VEGF agents are inappropriately invasive. In addition, topical administration of drugs limits their action to the eye and minimizes the associated systemic effects, resulting in higher patient compliance (48). Therefore, topical therapies could revolutionize the care of diabetic patients (49). In this regard, a phase II-III, randomized controlled clinical trial (EUROCONDOR-278040) to assess the efficacy of SST and brimonidine administered topically to prevent or arrest DR has been approved by the European Commission in the setting of the FP7-HEALTH-2011.

Finally, the route by which SST contained in the eye drops reaches the retina deserves a brief comment. The pharmacokinetic study revealed a rapid absorption of ^125^I-SST, which was unexpected for a compound that reached the retina diffusing through the cornea. The study performed using CF-SST showed that the absorption of SST eye drops was mainly by the trans-scleral route.

In summary, we provide first evidence that topical administration of SST has a potent effect in preventing the retinal neurodegenerative process that occurs in the early stages of DR. A preventive effect on the impairment of survival/apoptotic signaling induced by diabetes mellitus and a significant reduction in glutamate-induced excitotoxicity are among the mechanisms by which SST exerts its beneficial actions.

# Supplementary Material

###### Supplementary Data

## ACKNOWLEDGMENTS

This study was supported by grants from the Ministerio de Ciencia e Innovación (SAF2012-35562 and SAF2009-08114), from the European Foundation for the Study of Diabetes, and from the 7th Framework Programme (EUROCONDOR, FP7-278040).

No potential conflicts of interest relevant to this article were reported.

C.H. designed the project, led the analysis, wrote the manuscript, reviewed and edited the manuscript, and approved the final version of the manuscript. M.G.-R. and L.C. led the analysis, reviewed the manuscript, and approved the final version of the manuscript. J.F.-C., J.F.-S., and B.P. contributed to discussion, reviewed the manuscript, and approved the final version of the manuscript. A.G.-R. led the analysis, reviewed the manuscript, and approved the final version of the manuscript. A.M.V. contributed to discussion, reviewed the manuscript, and approved the final version of the manuscript. R.S. designed and coordinated the project, wrote the manuscript, reviewed and edited the manuscript, and approved the final version of the manuscript. C.H. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

The authors thank Dr. O. Yanes (CIBERDEM) for glutamate quantification and Dr. A.R. Carvalho (Ophthalmologic Research Unit, Vall d' Hebron Research Institute) for her technical assistance in obtaining the images of pharmacokinetic analysis.

# REFERENCES


author: J. Glenn MorrisAddress for correspondence: J. Glenn Morris, Jr., Emerging Pathogens Institute, University of Florida, PO Box 100009, Bldg 62, S Newell Dr, Gainesville, FL 32610-0009, USA; email: <firstname.lastname@example.com>
date: 2011-01
references:
title: How Safe Is Our Food?

How safe is our food? Put another way, how much illness in the United States is caused by foodborne pathogens? It sounds like a simple question. Getting a reasonable answer, however, is far from simple. The basic problem lies in the fact that only a small fraction of foodborne disease cases get reported through official (or unofficial) reporting systems. Calculating the "real" rate of foodborne illness requires development of models that use reported cases as a starting point to estimate underlying disease rates. Given the plethora of pathogens that can be transmitted through foodborne routes, this is a complex, and somewhat daunting, process. It is, however, necessary for assessing the safety of foods and developing strategies for disease prevention. The articles by Scallan et al. (*1*,*2*) in this issue represent the latest efforts to develop such estimates of the magnitude of foodborne illness in the United States.

In 1999, Mead et al. (*3*) published initial estimates of foodborne disease in the United States. This landmark undertaking was the first to provide a comprehensive compilation of data from a variety of sources, including the Centers for Disease Control and Prevention (CDC) and the medical literature. It resulted in the often-cited estimates that foodborne pathogens cause 76 million episodes of illness, 325,000 hospitalizations, and 5,000 deaths each year in the United States. (Hereafter, episodes of illness are referred to as illnesses.) During the past decade, these numbers have strongly driven ongoing efforts to implement or reform regulatory systems to protect the public from foodborne illness. However, some aspects of the methods have been criticized, particularly the high degree of uncertainty of particular parameters and thus of the results themselves (*4**–**6*). These concerns have led to requests for CDC to repeat and update the work of Mead et al., using better methods and parameter estimates that more closely reflect current realities.

Now, ≈11 years later, Scallan et al. have produced "Sons of Mead," which include substantial improvements to the methods used by Mead et al. and to the quality and timeliness of data (*1*,*2*). Scallan et al. should be commended, especially for 2 specific improvements: their advanced treatment of statistical uncertainty and variability and their transparent inclusion of voluminous appendixes of data, models, and assumptions. These authors followed the same basic approach as Mead et al. but chose to report their estimates in 2 articles. In the first article, they based their estimates of illnesses caused by 24 major pathogens (e.g., *Salmonella* spp., *Escherichia coli* O157:H7) primarily on data from the Foodborne Diseases Active Surveillance Network (FoodNet) and other pathogen-specific surveillance systems. In the second article, they estimated illnesses caused by unknown (or unspecified) pathogens by subtracting illnesses caused by known pathogens from the annual estimated number of cases of acute gastroenteritis in the US population and adjusting the result by the percentage assumed to be acquired domestically through food. If these 2 estimates are combined, as they were by Mead et al., the new totals are 47.8 million foodborne illnesses, 127,839 hospitalizations, and 3,037 deaths per year in the United States.

When one compares the 1999 and 2010 estimates (76 million vs. 47.8 million illnesses), the immediate response is to ask: Does this mean that food in this country is safer than it was 11 years ago? Unfortunately, the Scallan et al. articles do not enable us to answer this question. The methods, underlying assumptions, and parameter estimates used to generate these new numbers differ sufficiently from those used ≈11 years ago to preclude comparisons. In fact, if one looks simply at rates of overall gastrointestinal illness in the United States, based on FoodNet Population Surveys (*2*), one might infer that overall rates of acute gastrointestinal illness have increased during this period, from 0.49 episodes per person per year in 2000–2001, to 0.54 in 2002–2003, and to 0.73 in 2006–2007 (see \[*7*\] for a discussion of some methodologic issues with regard to the 2006–2007 survey). For the Scallan et al. articles, these 3 numbers were averaged to arrive at a rate of 0.6 episodes of acute gastroenteritis per person per year over the past decade. In contrast, Mead et al. used an estimate of 0.79 episodes of gastroenteritis per person per year, based on FoodNet data but also on older community surveys; they also used a somewhat different definition of acute gastrointestinal illness. This difference in estimated annual rates of acute gastroenteritis, when combined with a lower assumed proportion of gastroenteritis that is foodborne, explains much of the dramatic drop in total annual episodes of foodborne disease. Had Scallan et al. elected to use the 2006–2007 FoodNet estimate of 0.73 cases per person per year rather than use the average of 0.6 cases, their numbers would have been substantially higher and closer to the Mead et al. estimates.

Thus, if we can't use the Scallan estimates for comparison, is there any way to say whether food in the United States is safer now than it was 11 years ago? The best answer to this question comes from the FoodNet system (*7*), an active laboratory-based sentinel surveillance system that was established to monitor the public health impact of the 1995 US Department of Agriculture (USDA) Pathogen Reduction: Hazard Analysis and Critical Control Point (HAACP) System regulations (the first major revision of USDA food safety regulations since 1906). FoodNet provides annual data from designated sentinel surveillance sites on numbers of laboratory-diagnosed cases of 10 predominantly foodborne bacterial and parasitic pathogens; it reports actual case totals, not estimates. Despite year-to-year variability (including significant decreases in incidence of *Shigella* spp. and *E. coli* O157:H7 for 2009) (*8*), the overall trends show an initial drop in incidence of infection with the major bacterial foodborne pathogens after implementation of the 1995 USDA regulations, followed by a leveling off of incidence in subsequent years. One exception is infections caused by *Vibrio* spp., which are increasing, partly because climate change is affecting coastal environments (*9*). Bottom line: with the exception of *Vibrio* spp., things don't seem to be getting worse; however, after the initial decline since the USDA regulatory changes in 1995, one does not see evidence of sustained improvement.

How do numbers from the United States compare with those from Europe and the rest of the world? Again, differences in methods used by Scallan et al. make it difficult, if not impossible, to directly compare these numbers with those being published by other countries, including Canada, Australia, and members of the European Union (*10**–**12*). Although these new estimates cannot be compared directly with previous estimates or with estimates from other countries, these articles nonetheless constitute a necessary starting point for generation of more robust and regularly updated numbers. Looking across time, use of a consistent method, with regular updating of data (ideally annually), would provide a basis for assessing the effect of changes in regulation and other interventions at a national level. Similarly, if the methods are further modified in keeping with current international discussions on standardization of foodborne disease estimates (*13*), direct comparison of US numbers with those from other countries may become possible.

Estimates of the relative burden of disease caused by specific pathogens are crucial for improving our understanding of foodborne illness risks, but they are insufficient on their own. To target interventions (which are almost always food specific), illnesses must be quantified in terms of food–pathogen combinations. Doing so, in turn, requires development of what have been termed food attribution data (*14**,**15*). That is, how much salmonellosis is caused by eating contaminated chicken versus eggs, beef, or pork? How often is beef, compared with produce, the source of infection with *E. coli* O157:H7? Likewise, summary statistics such as number of cases, hospitalizations, and deaths ignore at-risk subpopulations and chronic sequelae such as end-stage renal disease, congenital toxoplasmosis, and irritable bowel syndrome. As such, the World Health Organization and many industrialized countries are increasingly reporting integrated measures of disease, such as disability-adjusted life years, which more fully capture disease symptoms and severities (*13*). Furthermore, to reduce specific foodborne hazards, we need information about the many factors along the complex farm-to-table pathway that can lead to the introduction or amplification of pathogens that contaminate food. This information would also help determine feasibility and efficacy of potential interventions.

As outlined in a recent Institute of Medicine report (*16*), implementation of a modern, risk-based food safety system in the United States will ultimately require much better data and a strong analytic capacity at the federal level that cuts across current agency lines. Although we still have a long way to go to bring our food safety system into the current century, the articles by Scallan et al. are critical steps in the right direction.

Dr Morris is director of the Emerging Pathogens Institute and professor of medicine at the College of Medicine, University of Florida; a member of the Institute of Medicine Food and Nutrition Board; and an associate editor for CDC's Emerging Infectious Diseases journal. He has worked extensively with foodborne pathogens and served on 5 National Academy of Sciences/Institute of Medicine expert committees dealing with food safety.

# References


abstract: An approach using 'chemical genetics' has identified small-molecule agonists of the Hedgehog signaling pathway that may lead the way to drugs for chronic degenerative diseases.
author: Jonathan B Weitzman
date: 2002
references:
title: Agonizing Hedgehog

It is a rare treat when a drug discovery program teaches us something about the biology of the process that it attempts to modulate. The paper by Maria Frank-Kamenetsky and colleagues in this issue of the *Journal of Biology*\[1\] presents a compelling example of how the search for therapeutics can provide powerful experimental tools and insights into fundamental biology, blurring the distinction between applied and basic research. By characterizing a small group of chemically similar agonists of the **Hedgehog** signaling pathway, Frank-Kamenetsky *et al*. have been able to propose a new model for how the **Smoothened** component of the Hedgehog-receptor complex works, and to hint at the existence of natural-ligand agonists of the Hedgehog signaling pathway (see 'The bottom line' box for a summary of their work).

# Hedgehog history

Signaling by the Hedgehog (Hh) family of secreted proteins plays a central role in regulating cell differentiation and tissue patterning during development \[2\]. The *hedgehog* gene (*hh*) was first identified by virtue of its role in the specification of positional identity during *Drosophila* embryonic segmentation, and it was subsequently found to control patterning of structures such as the eye and the abdominal cuticle. In mammals there are three *hh* homologs, called ***Sonic Hedgehog***, ***Indian Hedgehog*** and ***Desert Hedgehog*** (*Shh*, *Ihh* and *Dhh*, respectively), which have been implicated in patterning events in a range of developing tissues \[2,3\] (see the 'Background' box).

Recently, signaling by Hh has been shown to be important for patterning of the cerebellum, where it promotes the proliferation of granule neuron precursors. A link between Hh proteins and stem-cell proliferation has raised the enticing possibility that modulating Hh signaling might be relevant for the clinical management of certain degenerative diseases. Indeed, it was recently demonstrated that Shh might be effective in treating peripheral nerve damage or degenerative brain disorders such as **Parkinson's disease**\[4,5\]. Perhaps not surprisingly given its role in development, misregulated Hh signaling has also been implicated in **cancer**\[3\]. Specifically, medulloblastoma and basal cell carcinoma (BCC) are associated with inappropriate activation of Hh signaling \[6,7\]. These observations motivated Frank-Kamenetsky and colleagues to search for small-molecule modulators of the Hh pathway, with the hope that **antagonists** and **agonists** might be used as drugs to treat proliferative or degenerative diseases, and that **small molecules** might prove more amenable to pharmacological delivery than the Hh-family proteins themselves.

# Sending the signal

Hedgehog signaling challenges the way we normally think about signal transduction pathways. Biology is full of examples of extracellular ligands that bind to specific cell-surface receptors, initiating a cascade of biochemical events (often involving protein kinases) that leads to the activation of a transcription factor and the induction of a set of effector genes. But Hedgehog signaling is not so simple. Even the ligand is complicated: the Hh proteins undergo unusual processing and cleavage to generate an extracellular cholesterol-linked peptide that serves as the signaling ligand \[2\]. And the receptor component is far from understood.

The cellular response to Hh is controlled by two transmembrane proteins, **Patched** (Ptc) and Smoothened (Smo). The Ptc protein weaves across the cell membrane twelve times and resembles some transmembrane channels. It acts as a negative regulator of the Hh signal and has been defined as a tumor-suppressor. In contrast, Smo is a proto-oncogene and activates signaling in response to Hh ligand. The Smo protein is a seven-transmembrane receptor that resembles conventional G-protein-coupled receptors (Figure 1a).

It appears that Ptc inhibits Smo, although the precise mechanisms are unclear. Hh stimulation relieves Smo from inhibition by Ptc, leading to the generation of an intracellular signal that culminates in a nuclear transcriptional response (Figure 1b). When Ptc is removed the pathway is constitutively 'on', independent of the Hh ligand, whereas certain mutations in Smo can activate Hh signaling, bypassing Ptc regulation. A heteromeric receptor model has been proposed, in which Hh interacts directly with Ptc and thereby affects the interaction of Ptc with Smo \[8\]. "But things look much more complicated than we had earlier thought," says Philip Beachy (Johns Hopkins University School of Medicine, Maryland, USA). "The previous models are not tenable," and he suggests that alternative models of receptor function must be considered.

# The power of chemical genetics

Frank-Kamenetsky *et al.*\[1\] chose to use **chemical genetics** in an attempt to identify compounds that could interfere with the inhibition of Smo by Ptc, or could activate Smo independent of Ptc, or that might act downstream of Smo. In addition to making effective drug candidates, these molecules might also help to illuminate the complex mechanisms underlying signaling by Hh, Ptc, and Smo (see the 'Behind the scenes' box for more of the background to the work).

The high-throughput screen was elegantly simple. First, Frank-Kamenetsky *et al.* identified a cell line that responds well to Hh stimulation. The introduction of a reporter gene (encoding the firefly luciferase protein) that was turned on by Hh signaling permitted screening for compounds that block or induce signaling by monitoring the expression of the luciferase protein (using a simple luminescence test). They had previously used such a screen to isolate antagonists of Hh signaling, and had demonstrated the effectiveness of some antagonists as potential anti-tumor drugs to treat BCC \[7\]. Their new screen of 140,000 synthetic compounds led to the discovery of a few candidate agonists that could stimulate the reporter gene and mimic Hh activity.

Once the cell-based screen was completed, the chemists took over, synthesizing 300 derivative molecules until they found a few compounds that were related to the previous 'best' agonist but that were a thousand times more effective at eliciting a cellular response, affecting cells when applied in the nanomolar range. "Getting more potent compounds was essential if we were to figure out where the agonists were acting" recalls Jefferey Porter who headed the team at Curis, Inc.

Then the cell biologists began again, studying the effects of the agonists on the proliferation of primary neonatal cerebellar granule neuron precursors. They monitored the incorporation of tritiated thymidine into cultured rat neurons (as a marker of DNA synthesis and hence proliferation) and were pleased to see that the agonists were as effective in this assay as the Hh protein itself. An assay using an explant of embryonic neural plate was used to confirm that the agonists could induce dose-dependent gene expression in neural precursors, just as the Shh protein does.

Having established the effects of the agonists in culture assays, the researchers then turned to an *in vivo* model, feeding the compounds to pregnant mice and following the effects on the phenotypes of embryos lacking *Shh* or *Smo*. The treated embryos displayed activated Hh signaling, demonstrating that the compounds were not toxic and could cross the placental barrier. The developmental defects of *Shh*^-/-^ embryos were rescued by treatment with the agonist but the compound had no effect on Hh signaling in the absence of Smo.

"Once we knew that the agonists were targeting Smo, we wanted to investigate whether they bound to Smo directly and how they activated Hh signaling," says Porter. The cell line that was created for the screen served as a useful tool to test the effects of known antagonists on the function of the agonist. An anti-Hh blocking antibody had no effect, so the agonist must work downstream of the Hh-Ptc interaction. But the agonist was blocked by antagonists that work at the level of Smo or further downstream. "We have similar conclusions," says Beachy, whose group used photo-affinity labeling and cross-linking experiments to show that small-molecule agonists and antagonists bind directly to Smo.

Next, for Frank-Kamenetsky *et al.,* it was time for some careful biochemistry. Analysis of the expression of fusion proteins of Ptc or Smo showed that, unlike the Hh ligand itself, the agonist had no effect on the stability of the Ptc protein. In contrast, both Hh and the agonist could increase the stability of the Smo receptor. Immunoprecipitation experiments with radiolabeled agonist showed that the agonist must bind directly to Smo receptors, and that Hh-signaling inhibitors compete with the agonist for binding. Pharmokinetic analysis provided evidence for a single binding site competition model. Finally, Frank-Kamenetsky *et al*. exploited an oncogenic, constitutively active mutant form of Smo (Smo^act^); the agonists bound equally well to mutant and wild-type forms, whereas the antagonist bound less well to the mutant form.

# Learning lessons from drugs

Frank-Kamenetsky *et al*. have demonstrated convincingly that high-throughput chemical genetics can be used to isolate modulators of a developmental signaling pathway. The agonists that they have generated are efficient and apparently non-toxic mimics of Hh signals and are promising candidates for drugs for regenerative medicine. The authors work at the biotechnology company Curis Inc. (Cambridge, USA), so finding new drugs is obviously their primary objective. But the agonists also provide useful tools for probing the complex Hh-Ptc-Smo signaling pathway.

"A lot of our biological insight is driven by having specific chemical inhibitors, and many drugs are used as tools to dissect signaling systems as a substitute for genetic studies," says Arnon Rosenthal (Rinat Neuroscience Corp., Palo Alto, USA). Beachy agrees: "these compounds, first the plant-derived inhibitor cyclopamine and more recently the agonists, have really helped us to understand Smoothened function." Recent work from Beachy's lab \[9,10\], demonstrating that Ptc suppresses Smo in catalytic manner, has led to speculation that Ptc may function as a transporter protein pumping natural small-molecule Smo modulators across the cell membrane. Rosenthal adds that "good basic research always leads to better medicines, since the more we understand about the mechanisms operating in the body, the better able we are to modulate them rationally."

The data presented by Frank-Kamenetsky *et al*. \[1\] led them to propose a new model for Hh signaling based on the classic 'ternary complex model' that was developed to describe ligand-induced conformational changes of G-protein-coupled receptors \[11\]. According to this model, active and inactive conformations of Smo are selected by the binding of agonists or antagonists at independent sites. Furthermore, the model predicts that Smo binds to a novel effector molecule and it raises the possibility that endogenous ligands, analogous to the newly found agonist, may naturally regulate Smo activity.

Cheryll Tickle (University of Dundee, UK) studies the role of Hh in development and finds the possibility that there are endogenous small-molecule agonists that interact with Smoothened particularly intriguing. "This would mean that we are missing a whole layer of control of Hedgehog signaling in our current models," she notes. Porter adds, "It is tempting to speculate that endogenous molecules act directly on Smo, bypassing Ptc. That is consistent with what we know about how G-protein-coupled receptors work. But at the moment, it's pure speculation."

The study by Frank-Kamenetsky *et al.*\[1\] exploits a dazzling variety of experimental techniques to illustrate the path from high-throughput screening to compound characterization. Biochemists, cell biologists, pharmacologists and chemists have come together to demonstrate effectively that 'drug discovery' can combine all the excitement of 'real' scientific discovery with the satisfaction of isolating compounds that might be used for tomorrow's therapies.

## Figures and Tables