Validation of a Muscle-Specific Tissue Image Analysis Tool for Quantitative Assessment of Dystrophin Staining in Frozen Muscle Biopsies.

CONTEXT.­: Duchenne muscular dystrophy is a rare, progressive, and fatal neuromuscular disease caused by dystrophin protein loss. Common investigational treatment approaches aim at increasing dystrophin expression in diseased muscle. Some clinical trials include assessments of novel dystrophin production as a surrogate biomarker of efficacy, which may predict a clinical benefit from treatment. OBJECTIVES.­: To establish an immunofluorescent scanning and digital image analysis workflow that provides an objective approach for staining intensity assessment of the immunofluorescence dystrophin labeling and determination of the percentage of biomarker-positive fibers in muscle cryosections. DESIGN.­: Optimal and repeatable digital image capture was achieved by a rigorously qualified fluorescent scanning process. After scanning qualification, the MuscleMap (Flagship Biosciences, Westminster, Colorado) algorithm was validated by comparing high-power microscopic field total and dystrophin-positive fiber counts obtained by trained pathologists to data derived by MuscleMap. Next, the algorithm was tested on whole-slide images of immunofluorescent-labeled muscle sections from Duchenne muscular dystrophy, Becker muscular dystrophy, and control patients. RESULTS.­: When used under the guidance of a trained pathologist, the digital image analysis tool met predefined validation criteria and demonstrated functional and statistical equivalence with manual assessment. This work is the first, to our knowledge, to qualify and validate immunofluorescent scanning and digital tissue image-analysis workflow, respectively, with the rigor required to support the clinical trial environments. CONCLUSIONS.­: MuscleMap enables analysis of all fibers within an entire muscle biopsy section and provides data on a fiber-by-fiber basis. This will allow future clinical trials to objectively investigate myofibers' dystrophin expression at a greater level of consistency and detail.

Commentary: Roles for Pathologists in a High-throughput Image Analysis Team.

Historically, pathologists perform manual evaluation of H&E- or immunohistochemically-stained slides, which can be subjective, inconsistent, and, at best, semiquantitative. As the complexity of staining and demand for increased precision of manual evaluation increase, the pathologist's assessment will include automated analyses (i.e., "digital pathology") to increase the accuracy, efficiency, and speed of diagnosis and hypothesis testing and as an important biomedical research and diagnostic tool. This commentary introduces the many roles for pathologists in designing and conducting high-throughput digital image analysis. Pathology review is central to the entire course of a digital pathology study, including experimental design, sample quality verification, specimen annotation, analytical algorithm development, and report preparation. The pathologist performs these roles by reviewing work undertaken by technicians and scientists with training and expertise in image analysis instruments and software. These roles require regular, face-to-face interactions between team members and the lead pathologist. Traditional pathology training is suitable preparation for entry-level participation on image analysis teams. The future of pathology is very exciting, with the expanding utilization of digital image analysis set to expand pathology roles in research and drug development with increasing and new career opportunities for pathologists.

Retinal gene expression and visually evoked behavior in diabetic long evans rats.

PURPOSE: Patients with diabetic retinopathy may experience severe vision loss due to macular edema and neovascularization secondary to vascular abnormalities. However, before these abnormalities become apparent, there are functional deficits in contrast sensitivity, color perception, and dark adaptation. The goals of this study are to evaluate early changes (up to 3 months) in retinal gene expression, selected visual cycle proteins, and optokinetic tracking (OKT) in streptozotocin (STZ)-induced diabetic rats. METHODS: Retinal gene expression in diabetic Long Evans rats was measured by whole genome microarray 7 days, 4 weeks, and 3 months after the onset of hyperglycemia. Select gene and protein changes were probed by polymerase chain reaction (PCR) and immunohistochemistry, respectively, and OKT thresholds were measured using a virtual optokinetics system. RESULTS: Microarray analysis showed that the most consistently affected molecular and cellular functions were cell-to-cell signaling and interaction, cell death, cellular growth and proliferation, molecular transport, and cellular movement. Further analysis revealed reduced expression of several genes encoding visual cycle proteins including lecithin/retinol acyltransferase (LRAT), retinal pigment epithelium (RPE)-specific protein 65 kDa (RPE65), and RPE retinal G protein-coupled receptor (RGR). These molecular changes occurred simultaneously with a decrease in OKT thresholds by 4 weeks of diabetes. Immunohistochemistry revealed a decrease in RPE65 in the RPE layer of diabetic rats after 3 months of hyperglycemia. CONCLUSIONS: The data presented here are further evidence that inner retinal cells are affected by hyperglycemia simultaneously with blood retinal barrier breakdown, suggesting that glial and neuronal dysfunction may underlie some of the early visual deficits in persons with diabetes.

Strain-dependent increases in retinal inflammatory proteins and photoreceptor FGF-2 expression in streptozotocin-induced diabetic rats.

PURPOSE: Inflammation is thought to play a role in disease progression and vision loss in diabetic retinopathy (DR). However, the level of inflammation and the role of cytokines and growth factors in the early stages of this disease are poorly understood. Streptozotocin (STZ)-induced hyperglycemia in rats is widely used as a model of diabetic retinopathy, and therefore this model was used to better define the inflammatory response and the impact of the genetic background. METHODS: The expression of a panel of 57 inflammatory proteins and growth factors in the retina of three rat strains was compared by using a highly sensitive flow cytometry-based assay. Hyperglycemia was induced in Brown Norway (BN), Long-Evans (LE), and Sprague-Dawley (SD) rats, and protein expression in the retina was measured 4 weeks and 3 months later. RESULTS: The data revealed a subtle, but reproducible, inflammatory response in the retina of SD, but not in those of BN or LE, rats. Upregulation of fibroblast growth factor (FGF)-2 in the photoreceptor nuclear layer coincided with the inflammatory response in SD rats and may constitute a neuroprotective mechanism. Reduced expression of genes involved in the phototransduction pathway indicates altered photoreceptor function. CONCLUSIONS: Taken together, these data show that inflammatory changes in the diabetic rat retina are highly strain dependent, and SD rats exhibit low-level inflammation similar to that observed in diabetic patients. Therefore, SD rats may be a good model for the study of early inflammatory changes in human diabetic retinopathy.

Opposing activities of two novel members of the IL-1 ligand family regulate skin inflammation.

The interleukin (IL)-1 family members IL-1alpha, -1beta, and -18 are potent inflammatory cytokines whose activities are dependent on heterodimeric receptors of the IL-1R superfamily, and which are regulated by soluble antagonists. Recently, several new IL-1 family members have been identified. To determine the role of one of these family members in the skin, transgenic mice expressing IL1F6 in basal keratinocytes were generated. IL1F6 transgenic mice exhibit skin abnormalities that are dependent on IL-1Rrp2 and IL-1RAcP, which are two members of the IL-1R family. The skin phenotype is characterized by acanthosis, hyperkeratosis, the presence of a mixed inflammatory cell infiltrate, and increased cytokine and chemokine expression. Strikingly, the combination of the IL-1F6 transgene with an IL1F5 deficiency results in exacerbation of the skin phenotype, demonstrating that IL-1F5 has antagonistic activity in vivo. Skin from IL1F6 transgenic, IL1F5(-/-) pups contains intracorneal and intraepithelial pustules, nucleated corneocytes, and dilated superficial dermal blood vessels. Additionally, expression of IL1RL2, -1F5, and -1F6 is increased in human psoriatic skin. In summary, dysregulated expression of novel agonistic and antagonistic IL-1 family member ligands can promote cutaneous inflammation, revealing potential novel targets for the treatment of inflammatory skin disorders.

Characterization of the inflammatory response to a highly selective PDE4 inhibitor in the rat and the identification of biomarkers that correlate with toxicity.

The primary toxicity associated with repeated oral administration of the PDE4 inhibitor IC542 to the rat is an inflammatory response leading to tissue damage primarily in the gastrointestinal tract and mesentery. Although necrotizing vasculitis is frequently seen with other PDE4 inhibitors, blood vessel injury was rare following IC542 administration and was always associated with inflammation in the surrounding tissue. The incidence and severity of the histologic changes in these studies correlated with elevated peripheral blood leukocytes, serum IL-6, haptoglobin, and fibrinogen, and with decreased serum albumin. By monitoring haptoglobin, fibrinogen and serum albumin changes in IC542-treated rats, it was possible to identify rats with early histologic changes that were reversible. Since PDE4 inhibition is generally associated with anti-inflammatory activity, extensive inflammation in multiple tissues was unexpected with IC542. Co-administration of dexamethasone completely blocked IC542-induced clinical and histologic changes in the rat, confirming the toxicity resulted from inflammatory response. In addition, IC542 augmented release of the proinflammatory cytokine IL-6 in LPS-activated whole blood from rats but not monkeys or humans. The effect of IC542 on IL-6 release from rat leukocytes in vitro is consistent with the proinflammatory response observed in vivo and demonstrates species differences to PDE4 inhibition.

Characterization of the in vivo function of TNF-alpha-related apoptosis-inducing ligand, TRAIL/Apo2L, using TRAIL/Apo2L gene-deficient mice.

To define the normal physiological role for the TRAIL/Apo2L in vivo, we generated TRAIL/Apo2L gene-targeted mice. These mice develop normally and show no defects in lymphoid or myeloid cell homeostasis or function. Although TRAIL/Apo2L kills transformed cells in vitro, TRAIL/Apo2L(-/-) mice do not spontaneously develop overt tumors at an early age. However, in the A20 B cell lymphoma-transferred tumor model, TRAIL/Apo2L(-/-) mice are clearly more susceptible to death from overwhelming tumor burden, due to increased lymphoma load in the liver. A20 tumors are susceptible to TRAIL/Apo2L killing in vitro, indicating that TRAIL/Apo2L may act directly to control A20 cells in vivo. Despite the fact that TRAIL binds osteoprotegerin and osteoprotegerin-transgenic mice are osteopetrotic, TRAIL/Apo2L(-/-) mice show no evidence of altered gross bone density, and no alterations in frequency or in vitro differentiation of bone marrow precursor osteoclasts. Moreover, leucine zipper TRAIL has no toxicity when repeatedly administered to osteoprotegerin(-/-) mice. Thus, TRAIL/Apo2L is important in controlling tumors in vivo, but is not an essential regulator of osteoprotegerin-mediated biology, under normal physiological conditions.

RIP4 is an ankyrin repeat-containing kinase essential for keratinocyte differentiation.

The epidermis is a stratified, continually renewing epithelium dependent on a balance among cell proliferation, differentiation, and death for homeostasis. In normal epidermis, a mitotically active basal layer gives rise to terminally differentiating keratinocytes that migrate outward and are ultimately sloughed from the skin surface as enucleated squames. Although many proteins are known to function in maintaining epidermal homeostasis, the molecular coordination of these events is poorly understood. RIP4 is a novel RIP (receptor-interacting protein) family kinase with ankyrin repeats cloned from a keratinocyte cDNA library. RIP4 deficiency in mice results in perinatal lethality associated with abnormal epidermal differentiation. The phenotype of RIP4(-/-) mice in part resembles that of mice lacking IKKalpha, a component of a complex that regulates NF-kappaB. Despite the similar keratinocyte defects in RIP4- and IKKalpha-deficient mice, these kinases function in distinct pathways. RIP4 functions cell autonomously within the keratinocyte lineage. Unlike IKKalpha, RIP4-deficient skin fails to fully differentiate when grafted onto a normal host. Instead, abnormal hair follicle development and epidermal dysplasia, indicative of progression into a more pathologic state, are observed. Thus, RIP4 is a critical component of a novel pathway that controls keratinocyte differentiation.