Diagnostic Tools for Inborn Errors of Human Immunity (Primary Immunodeficiencies and Immune Dysregulatory Diseases).

PURPOSE OF REVIEW: The purpose of this review is to provide an overview of diagnostic testing in primary immunodeficiency and immune dysregulatory disorders (PIDDs), particularly focusing on flow cytometry and genetic techniques, utilizing specific examples of PIDDs. RECENT FINDINGS: Flow cytometry remains a vital tool in the diagnosis and monitoring of immunological diseases. Its utility ranges from cellular analysis and specific protein quantitation to functional assays and signaling pathway analysis. Mass cytometry combines flow cytometry and mass spectrometry to dramatically increase the throughput of multivariate single-cell analysis. Next-generation sequencing in combination with other molecular techniques and processing algorithms has become more widely available and identified the diverse and heterogeneous genetic underpinnings of these disorders. As the spectrum of disease is further clarified by increasing immunological, genetic, and epigenetic knowledge, the careful application of these diagnostic tools and bioinformatics will assist not only in our understanding of these complex disorders, but also enable the implementation of personalized therapeutic approaches for disease management.

Hydroxysteroid 17-Beta Dehydrogenase Type 10 Disease in Siblings.

Hydroxysteroid 17-beta dehydrogenase type 10 (HSD10) deficiency (HSD10 disease) is a rare X-linked neurodegenerative condition caused by abnormalities in the HSD17B10 gene. A total of 10 mutations have been reported in the literature since 2000. Described phenotypes include a severe neonatal or progressive infantile form with hypotonia, choreoathetosis, seizures, cardiomyopathy, neurodegeneration, and death, as well as an attenuated form with variable regression. Here we present the second report of a c.194T>C (p.V65A) mutation in two half-brothers with a clinical phenotype characterized by neurodevelopmental delay, choreoathetosis, visual loss, cardiac findings, and behavioral abnormalities, with regressions now noted in the older sibling. Neither has experienced a metabolic crisis. Both of the siblings had normal tandem mass spectroscopy analysis of their newborn screening samples. The older brother's phenotype may be complicated by the presence of a 3q29 microduplication. Diagnosis requires a high index of suspicion, as the characteristic urine organic acid pattern may escape detection. The exact pathogenic mechanism of disease remains to be elucidated, but may involve the non-dehydrogenase functionalities of the HSD10 protein. Our report highlights clinical features of two patients with the less fulminant phenotype associated with a V65A mutation, compares the reported phenotypes to date, and reviews recent findings regarding the potential pathophysiology of this condition.Summary Sentence Hydroxysteroid 17-beta dehydrogenase type 10 (HSD10) disease (HSD10 disease) is a rare X-linked neurodegenerative condition with a variable clinical phenotype; diagnosis requires a high index of suspicion.

Comparison of Body Composition Assessed by Dual-Energy X-Ray Absorptiometry and BMI in Current and Former U.S. Navy Service Members.

BACKGROUND: Little is known of the diagnostic accuracy of BMI in classifying obesity in active duty military personnel and those that previously served. Thus, the primary objectives were to determine the relationship between lean and fat mass, and body fat percentage (BF%) with BMI, and assess the agreement between BMI and BF% in defining obesity. METHODS: Body composition was measured by dual-energy X-ray absorptiometry in 462 males (20-91 years old) who currently or previously served in the U.S. Navy. A BMI of ≥ 30 kg/m2 and a BF% ≥ 25% were used for obesity classification. RESULTS: The mean BMI (± SD) and BF% were 28.8 ± 4.1 and 28.9 ± 6.6%, respectively, with BF% increasing with age. Lean mass, fat mass, and BF% were significantly correlated with BMI for all age groups. The exact agreement of obesity defined by BMI and BF% was fair (61%), however, 38% were misclassified by a BMI cut-off of 30 when obesity was defined by BF%. CONCLUSIONS: From this data we determined that there is a good correlation between body composition and BMI, and fair agreement between BMI and BF% in classifying obesity in a group of current and former U.S. Navy service members. However, as observed in the general population, a significant proportion of individuals with excess fat are misclassified by BMI cutoffs.

At-sea trial of 24-h-based submarine watchstanding schedules with high and low correlated color temperature light sources.

United States Navy submariners have historically lived with circadian disruption while at sea due to 18-h-based watchschedules. Previous research demonstrated that circadian entrainment improved with 24-h-based watchschedules. Twenty-nine male crew members participated in the study, which took place on an actual submarine patrol. The crew were exposed, first, to experimental high correlated color temperature (CCT = 13,500 K) fluorescent light sources and then to standard-issue fluorescent light sources (CCT = 4100 K). A variety of outcome measures were employed to determine if higher levels of circadian-effective light during on-watch times would further promote behavioral alignment to 24-h-based watchschedules. The high CCT light source produced significantly higher circadian light exposures than the low CCT light source, which was associated with significantly greater 24-h behavioral alignment with work schedules using phasor analysis, greater levels of sleep efficiency measured with wrist actigraphy, lower levels of subjective sleepiness measured with the Karolinska Sleepiness Scale, and higher nighttime melatonin concentrations measured by morning urinary 6-sulfatoxymelatonin/creatinine ratios. Unlike these diverse outcome measures, performance scores were significantly worse under the high CCT light source than under the low CCT light source, due to practice effects. As hypothesized, with the exception of the performance scores, all of the data converge to suggest that high CCT light sources, combined with 24-h watchschedules, promote better behavioral alignment with work schedules and greater sleep quality on submarines. Since the order and the type of light sources were confounded in this field study, the results should only be considered as consistent with our theoretical understanding of how regular, 24-h light-dark exposures combined with high circadian light exposures can promote greater behavioral alignment with work schedules and with sleep.

Decrease in CD8+ lymphocyte number and altered cytokine profile in human prostate cancer.

The tumor microenvironment is comprised of multiple cell types arranged in a three-dimensional structure. Interactions amongst the various cell components play an important role in neoplasia, including the inflammatory reaction that occurs as part of the host response. In this study, the regional lymphocyte subpopulations and cytokine profiles associated with prostate cancer were examined using a quantitative imaging approach and expression microarray analysis. Lymphocytes were measured in four different epithelial phenotypes in prostate cancer specimens: carcinoma; prostatic intraepithelial neoplasia (PIN); benign prostate hyperplasia (BPH); and normal epithelium. The data indicate that CD8 positive, cytotoxic T lymphocytes are significantly decreased in regions adjacent to hyperplasia and carcinoma as compared to normal epithelium and PIN. In contrast the relative number of CD4 positive and CD20 positive lymphocytes did not change markedly. Parallel mRNA expression array analysis of the normal and tumor microenvironments identified a distinct cytokine profile in cancer, with 24 dysregulated genes in tumor epithelium and nine altered in tumor-associated stroma. Overall, these data indicate that the spatial distribution of CD8 positive, cytotoxic T lymphocytes is dysregulated in human prostate glands that contain cancer, and cytokine profiles are altered at the mRNA level.

Increased matrix metalloproteinase activation in esophageal squamous cell carcinoma.

BACKGROUND: Esophageal squamous cell carcinomas (ESCC) are usually asymptomatic and go undetected until they are incurable. Cytological screening is one strategy to detect ESCC at an early stage and has shown promise in previous studies, although improvement in sensitivity and specificity are needed. Proteases modulate cancer progression by facilitating tumor invasion and metastasis. In the current study, matrix metalloproteinases (MMPs) were studied in a search for new early detection markers for ESCC. METHODS: Protein expression levels of MMPs were measured using zymography in 24 cases of paired normal esophagus and ESCC, and in the tumor-associated stroma and tumor epithelium in one sample after laser capture microdissection (LCM). MMP-3 and MMP-10 transcripts in both the epithelium and stroma in five cases were further analyzed by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). RESULTS: Gelatin zymography showed bands corresponding in size to MMP-2, MMP-3, MMP-9, and MMP-10 enzymes in each of the 24 cancer cases. MMP levels tended to be higher in tumors than paired normal tissue; however, only the 45 kDa band that corresponds to the activated form of MMP-3 and MMP-10 was strongly expressed in all 24 tumors with little or no expression in the paired normal foci. LCM-based analysis showed the 45 kDA band to be present in both the stromal and epithelial components of the tumor microenvironment, and that MMP-3 and MMP-10 mRNA levels were higher in tumors than paired normal tissues for each compartment. CONCLUSIONS: Increased levels of MMPs occur in ESCC suggesting their up-regulation is important in esophageal tumorigenesis. The up-regulated gene products have the potential to serve as early detection markers in the clinic.

Identification of EpCAM as a molecular target of prostate cancer stroma.

To delineate the molecular changes that occur in the tumor microenvironment, we previously performed global transcript analysis of human prostate cancer specimens using tissue microdissection and expression microarrays. Epithelial and stromal compartments were individually studied in both tumor and normal fields. Tumor-associated stroma showed a distinctly different expression pattern compared with normal stroma, having 44 differentially expressed transcripts, the majority of which were up-regulated. In the present study, one of the up-regulated transcripts, epithelial cell adhesion activating molecule, was further evaluated at the protein level in 20 prostate cancer cases using immunohistochemistry and a histomathematical analysis strategy. The epithelial cell adhesion activating molecule showed a 76-fold expression increase in the tumor-associated stroma, as compared with matched normal stroma. Moreover, Gleason 4 or 5 tumor stroma was increased 170-fold relative to matched normal stroma, whereas the Gleason 3 tumor area showed only a 36-fold increase, indicating a positive correlation with Gleason tumor grade. Since the stromal compartment may be particularly accessible to vascular-delivered agents, epithelial cell adhesion activating molecule could become a valuable molecular target for imaging or treatment of prostate cancer.

Global expression analysis of prostate cancer-associated stroma and epithelia.

Characterization of gene expression profiles in tumor cells and the tumor microenvironment is an important step in understanding neoplastic progression. To date, there are limited data available on expression changes that occur in the tumor-associated stroma as either a cause or consequence of cancer. In the present study, we employed a 54,000 target oligonucleotide microarray to compare expression profiles in the 4 major components of the microenvironment: tumor epithelium, tumor-associated stroma, normal epithelium, and normal stroma. Cells from 5 human, whole-mount prostatectomy specimens were microdissected and the extracted and amplified mRNA was hybridized to an Affymetrix Human Genome U133 Plus 2.0 GeneChip. Using the intersection of 2 analysis methods, we identified sets of differentially expressed genes among the 4 components. Forty-four genes were found to be consistently differentially expressed in the tumor-associated stroma; 35 were found in the tumor epithelium. Interestingly, the tumor-associated stroma showed a predominant up-regulation of transcripts compared with normal stroma, in sharp contrast to the overall down-regulation seen in the tumor epithelium relative to normal epithelium. These data provide insight into the molecular changes occurring in tumor-associated stromal cells and suggest new potential targets for future diagnostic, imaging, or therapeutic intervention.

Layered expression scanning: multiplex molecular analysis of diverse life science platforms.

With the advent of the genomic era, there is an increasing use of high-throughput techniques to generate transcriptome- and proteome-based profiles of biological specimens. Each of these methodologies offers a unique window into the inner workings of cell and tissue samples. Often, these studies generate large data sets and provide investigators with a substantial number of candidate dysregulated genes and pathways. Follow-up studies are then undertaken to independently validate the original findings and to extend the study to additional samples or more quantitative measurements. Although there are several methods available for these validation efforts, they are often tedious and laborious to perform; thus, additional tools that enable this task are needed. One such approach is layered expression scanning (LES), a new technique developed via a cooperative research and development agreement (CRADA) between the National Cancer Institute and 20/20 GeneSystems, Inc. The technique is based on the movement of biomolecules from a two-dimensional life science platform (histological tissue section, electrophoresis gel, multi-well plate, etc.) through a set of analysis membranes while maintaining the original distribution pattern of the molecules. Each membrane measures one analyte and the data are then mapped back to the original specimen, permitting each component of the life science platform to be studied in detail. LES can be configured in several different ways depending on the goals of the study. In this review, we summarize the use of the LES technique for a variety of biological applications.