Regional Consortia: A Framework for Public Health Laboratory Collaboration and Service Sharing.

Public health laboratories (PHLs) provide specialized testing services for programs focused on the prevention and control of communicable diseases, early detection of congenital disorders, testing for antimicrobial resistance, and identification of environmental contaminants, among other responsibilities. Although national public health programs and partners provide some funding support, training, and technical resources to PHLs, no dedicated funding is provided from federal programs to fully support comprehensive PHL services across the United States or the underlying infrastructure needed for PHLs to provide and ensure their core functions and capabilities. Public health laboratories have begun to rely on a "community of practice" approach to addressing various service needs by creating and formalizing regional consortia, which are organized groups of geographically clustered PHLs that share expertise, capacities, and capabilities to enhance PHL services. The number of states participating in these networks increased from 13 to 48 from 2015 to 2020, including participation by multiple local PHLs and a territorial PHL. These consortia have enabled strengthening of partnerships and collaboration among PHLs to address regional priorities and challenges. We explore the background and evolution of regional consortia, outline some of their practices and activities, review lessons learned from these successful collaborations, and discuss the positive effect they have on the national public health system.

Identification of Missense Extracellular Matrix Gene Variants in a Large Glaucoma Pedigree and Investigation of the N700S Thrombospondin-1 Variant in Normal and Glaucomatous Trabecular Meshwork Cells.

PURPOSE: Primary open-angle glaucoma (POAG) is a complex heterogeneous disease. While several POAG genes have been identified, a high proportion of estimated heritability remains unexplained. Elevated intraocular pressure (IOP) is a leading POAG risk factor and dysfunctional extracellular matrix (ECM) in the trabecular meshwork (TM) contributes to elevated IOP. In this study, we sought to identify missense variants in ECM genes that correlate with ocular hypertensive POAG. METHODS: Whole-genome sequencing was used to identify genetic variants in five members of a large POAG family (n = 68) with elevated IOP. The remaining family members were screened by Sanger sequencing. Unrelated normal (NTM) and glaucomatous (GTM) cells were sequenced for the identified variants. The ECM protein levels were determined by Western immunoblotting and confocal and electron microscopy investigated ECM ultrastructural organization. RESULTS: Three ECM gene variants were significantly associated with POAG or elevated IOP in a large POAG pedigree. These included rs2228262 (N700S; thrombospondin-1 (THBS1, TSP1)), rs112913396 (D563 G; collagen type VI, alpha 3 (COL6A3)) and rs34759087 (E987K; laminin subunit beta 2 (LAMB2)). Screening of unrelated TM cells (n = 27) showed higher prevalence of the THBS1 variant but not the LAMB2 variant, in GTM cells (39%) than NTM cells (11%). The rare COL6A3 variant was not detected. TSP1 protein was upregulated and COL6A3 was down-regulated in TM cells with N700S subject to mechanical stretch, an in vitro method that mimics elevated IOP. Immunofluorescence showed increased TSP1 immunostaining in cell strains with N700S compared to wild-type TM cells. Ultrastructural studies showed ECM disorganization and altered collagen type VI distribution in GTM versus NTM cells. CONCLUSIONS: Our results suggest that missense variants in ECM genes may not cause catastrophic changes to the TM, but over many years, subtle changes in ECM may accumulate and cause structural disorganization of the outflow resistance leading to elevated IOP in POAG patients.

Noncanonical role of transferrin receptor 1 is essential for intestinal homeostasis.

Transferrin receptor 1 (Tfr1) facilitates cellular iron uptake through receptor-mediated endocytosis of iron-loaded transferrin. It is expressed in the intestinal epithelium but not involved in dietary iron absorption. To investigate its role, we inactivated the Tfr1 gene selectively in murine intestinal epithelial cells. The mutant mice had severe disruption of the epithelial barrier and early death. There was impaired proliferation of intestinal epithelial cell progenitors, aberrant lipid handling, increased mRNA expression of stem cell markers, and striking induction of many genes associated with epithelial-to-mesenchymal transition. Administration of parenteral iron did not improve the phenotype. Surprisingly, however, enforced expression of a mutant allele of Tfr1 that is unable to serve as a receptor for iron-loaded transferrin appeared to fully rescue most animals. Our results implicate Tfr1 in homeostatic maintenance of the intestinal epithelium, acting through a role that is independent of its iron-uptake function.

Competency Guidelines for Public Health Laboratory Professionals: CDC and the Association of Public Health Laboratories.

These competency guidelines outline the knowledge, skills, and abilities necessary for public health laboratory (PHL) professionals to deliver the core services of PHLs efficiently and effectively. As part of a 2-year workforce project sponsored in 2012 by CDC and the Association of Public Health Laboratories (APHL), competencies for 15 domain areas were developed by experts representing state and local PHLs, clinical laboratories, academic institutions, laboratory professional organizations, CDC, and APHL. The competencies were developed and reviewed by approximately 170 subject matter experts with diverse backgrounds and experiences in laboratory science and public health. The guidelines comprise general, cross-cutting, and specialized domain areas and are divided into four levels of proficiency: beginner, competent, proficient, and expert. The 15 domain areas are 1) Quality Management System, 2) Ethics, 3) Management and Leadership, 4) Communication, 5) Security, 6) Emergency Management and Response, 7) Workforce Training, 8) General Laboratory Practice, 9) Safety, 10) Surveillance, 11) Informatics, 12) Microbiology, 13) Chemistry, 14) Bioinformatics, and 15) Research. These competency guidelines are targeted to scientists working in PHLs, defined as governmental public health, environmental, and agricultural laboratories that provide analytic biological and/or chemical testing and testing-related services that protect human populations against infectious diseases, foodborne and waterborne diseases, environmental hazards, treatable hereditary disorders, and natural and human-made public health emergencies. The competencies support certain PHL workforce needs such as identifying job responsibilities, assessing individual performance, and providing a guiding framework for producing education and training programs. Although these competencies were developed specifically for the PHL community, this does not preclude their broader application to other professionals in a variety of different work settings.

Interleukin-20 receptor expression in the trabecular meshwork and its implication in glaucoma.

PURPOSE: To determine whether interleukin-20 receptors (IL-20R) are expressed in trabecular meshwork cells and the effect of a T104M mutation in IL-20R2 on downstream cellular functions. METHODS: Evaluation of signal transducer and activator of transcription (STAT)3 phosphorylation and generic matrix metalloproteinase (MMP) activity in primary open angle glaucoma (POAG) dermal fibroblasts (pHDF) with the T104M IL-20R2 mutation were compared with normal human dermal fibroblasts (HDF). Expression of IL-20R1 and IL-20R2 in human trabecular meshwork (HTM) cells was determined by immunohistochemistry and western immunoblotting. RESULTS: A T104M mutation in IL20-R2 was identified in a large POAG family in which the GLC1C locus was originally mapped. pHDFs harboring this mutation had significantly increased phosphorylated STAT3 (pSTAT3) activity compared with normal HDFs. However, stimulation with either IL-19 or IL-20 for 15 min resulted in significantly decreased levels of pSTAT3 in pHDFs compared with controls. Generic MMP activity was significantly decreased in pHDFs compared with controls after stimulation with IL-20 for 24 h. Both IL-20R1 and IL-20R2 receptors were expressed in HTM cells by western immunoblot and immunofluorescence, and they appeared to be up-regulated in response to cytokine treatment. CONCLUSIONS: A T104M mutation in IL-20R2 significantly impacts the function of this receptor as shown by decreased pSTAT3 levels and generic MMP activity. Reduced MMP activity may affect the ability of glaucoma patients to alter outflow resistance in response to elevated intraocular pressure.

Ankyrin repeat and suppressor of cytokine signaling box containing protein-10 is associated with ubiquitin-mediated degradation pathways in trabecular meshwork cells.

PURPOSE: Ankyrin repeat and suppressor of cytokine signaling (SOCS) box containing protein-10 (ASB10) was recently identified as a gene that causes primary open-angle glaucoma. Here, we investigated endogenous ASB10 protein expression in human trabecular meshwork (HTM) cells to provide the first clues to the biologic function of this protein. METHODS: Primary HTM cells were cultured and immunostained with anti-ASB10 and various biomarkers of the ubiquitin-mediated proteasomal and autophagy-lysosomal degradation pathways. Cells were imaged with confocal and high-resolution structured illumination microscopy. Colocalization was quantified using Imaris Bitplane software, which generated a Pearson's correlation coefficient value. Coimmunoprecipitation of ASB10-transfected cells was performed. RESULTS: Immunofluorescence and confocal analysis showed that ASB10 was localized in intracellular structures in HTM cells. Two populations were observed: small, spherical vesicles and larger, less abundant structures. In the ASB10-silenced cells, the number of large structures was significantly decreased. ASB10 partially colocalized with biomarkers of the ubiquitin-mediated proteasomal pathway including ubiquitin and the α4 subunit of the 20S proteasome. However, ASB10 itself was not ubiquitinated. ASB10 also colocalized with numerous biomarkers of specific autophagic structures: aggresomes (histone deacetylase 6 [HDAC6] and heat shock protein 70 [HSP70]), autophagosomes (light chain 3 [LC3] and p62), amphisomes (Rab7), and lysosomes (lysosomal-associated membrane protein 1 [LAMP1]). Pearson coefficients indicated strong colocalization of large ASB10-stained structures with the α4 subunit of the 20S proteasome, K48 and K63-linked ubiquitin antibodies, p62, HSP70, and HDAC6 (Pearson's range, 0.59-0.82). Coimmunoprecipitation assays showed a positive interaction of ASB10 with HSP70 and with the α4 subunit of the 20S proteasome. Super-resolution structured illumination confocal microscopy suggested that the smaller ASB10-stained vesicles aggregated into the larger structures, which resembled aggresome-like induced structures. Treatment of HTM cells with an autophagy activator (MG132) or inhibitors (wortmannin, bafilomycin A1) significantly increased and decreased the number of small ASB10-stained vesicles, respectively. No discernible differences in the colocalization of large ASB10-stained structures with ubiquitin or HDAC6 were observed between dermal fibroblasts derived from a normal individual and a patient with primary open-angle glaucoma carrying a synonymous ASB10 mutation. CONCLUSIONS: Our evidence suggests that ASB10 may play a role in ubiquitin-mediated degradation pathways in TM cells.

The role of the WDR36 gene on chromosome 5q22.1 in a large family with primary open-angle glaucoma mapped to this region.

OBJECTIVE: To determine whether mutations in the WD40-repeat 36 (WDR36) gene are responsible for primary open-angle glaucoma (POAG) that maps to the GLC1G locus in a family with 16 affected family members. METHODS: Ninety-two family members underwent clinical evaluation for POAG on the basis of intraocular pressures, cupping of discs, and visual fields after informed consent was obtained. All 23 exons of WDR36 were sequenced in DNA from 5 affected and 2 unaffected family members. RESULTS: Sixteen family members showed evidence of POAG. A number of sequence variations were identified in family members; most of the variations were previously described single-nucleotide polymorphisms also present in the general population. The 3 new sequence changes were all intronic; 2 were found in only 1 of the family members undergoing screening. CONCLUSIONS: Several polymorphisms, including known single-nucleotide polymorphisms, were identified; however, none of these were consistent with disease-causing mutations. A mutation in a noncoding region of WDR36 may be responsible for POAG in this family, or another gene in this region may be the actual cause of glaucoma in this family. CLINICAL RELEVANCE: The finding that the WDR36 gene is probably not the responsible gene in this family further documents the genetic heterogeneity of POAG.

A large GLC1C Greek family with a myocilin T377M mutation: inheritance and phenotypic variability.

PURPOSE: POAG is a complex disease; therefore, families in which a glaucoma gene has been mapped may carry additional POAG genes. The goal of this study was to determine whether mutations in the myocilin (MYOC) gene on chromosome 1 are present in two POAG families, which have previously been mapped to the GLC1C locus on chromosome 3. METHODS: The three exons of MYOC were screened by denaturing (d)HPLC. Samples with heteroduplex peaks were sequenced. Clinical findings were compared with genotype status in all available family members over the age of 20 years. RESULTS: A T377M coding sequence change in MYOC was identified in family members of the Greek GLC1C family but not in the Oregon GLC1C family. Individuals carrying both the MYOC T377M variant and the GLC1C haplotype were more severely affected at an earlier age than individuals with just one of the POAG genes, suggesting that these two genes interact or that both contribute to the POAG phenotype in a cumulative way.