The genetic characterization
of sampled populations is a critical step in designing and interpreting
complex disease-mapping studies. One aspect that recently has been emphasized
is the impact of population stratification on association studies, since
even low-level differentiation—for example, between European populations
with FST close to one percent—can
lead to false-positive results and failure to detect genuine association
in large-scale studies. Few studies have investigated the level of genetic
differentiation within typical outbred Western populations, despite the
initiation of national sample collections, such as the United Kingdom's
UK Biobank, for large-scale genetic association studies. The authors,
therefore, explored the level of genetic differentiation within the Scottish
population of 5 million people, which, in most respects, is representative
of the wider population of the United Kingdom. Like the United Kingdom,
Scotland has large urban and small rural subpopulations and includes a
number of geographically isolated island communities in which greater
differentiation might be expected. The proportion of large Western populations
living in rural versus urban communities within Europe and North America
varies from 21 percent (United States) to 33 percent (Canada), with both
England/Wales (28%) and Scotland (32%) lying in the middle of the range.
Rural populations are typically more stable and homogeneous than the dense
urban populations that constitute the population majority. This raises
the question of whether there are genetically detectable rural-urban differences
that might influence association-study design. The authors used short
tandem repeat markers to explore fine-scale genetic structure and to examine
the extent of linkage disequilibrium (LD) within national subpopulations.
They studied 955 unrelated individuals of local ancestry from nine Scottish
rural regions and the urban center of Edinburgh, as well as 96 unrelated
individuals from the general population in the United Kingdom. Despite
little overall differentiation on the basis of allele frequencies, there
were clear differences among subpopulations in the extent of pairwise
LD, measured between a subset of X-linked markers. These reflected presumed
differences in the depths of the underlying geneologies within these subpopulations.
The authors concluded that there are strategic advantages in studying
rural subpopulations, in terms of increased power and reduced cost, that
are lost by sampling across regions or within urban populations. Similar
rural-urban contrasts are likely to exist in many other populations with
stable rural subpopulations, which could influence the design of genetic
association studies and national biobank data collections.
Vitart V, Carothers AD, Hayward C, et al. Increased
level of linkage disequilibrium in rural compared with urban communities:
a factor to consider in association-study design. Am
J Hum Genet. 2005;76:763-772.
Reprints: Dr. Veronique Vitart, Medical Research
Council Human Genetics Unit, Western General Hospital, Crewe Rd., Edinburgh
EH4 2XU, United Kingdom; firstname.lastname@example.org
Microalbuminuria is the excretion of albumin in urine at levels of 20 to 200 μg/minute or 30 to 300 mg/24 hours for timed collections and 30 to 300 mg/g of creatinine for random or spot urine samples. In type 1 and type 2 diabetes mellitus, microalbuminuria is considered the earliest marker of diabetic nephropathy. Type 1 diabetics have been shown to develop nephropathy in more than 30 percent of cases. Approximately 25 percent of patients with type 2 diabetes mellitus have been shown to develop nephropathy five to 10 years after diagnosis. Diabetic nephropathy is the most common cause of end-stage renal disease in the United States and Europe. Microalbuminuria has also been shown to be a marker for increased risk of cardiovascular morbidity and mortality in diabetic and nondiabetic patients. It is important to detect microalbuminuria early so medical professionals can initiate antiangiotensin therapy to stop or slow the progression of diabetic nephropathy. The development of automated immunoassay methods has allowed medical professionals to measure accurately and precisely the low concentrations of albumin in urine associated with microalbuminuria. However, a series of studies have shown that current immunoassay methods might underestimate the amount of intact albumin in urine from diabetic subjects, especially at excretion rates of less than 100 μg/minute. These studies compared various immunoassay methods with a new high-performance liquid chromatography (HPLC) assay that uses size-exclusion chromatography. This method was cleared by the Food and Drug Administration for measuring albumin in urine. The authors conducted a study to evaluate the analytic performance of this new assay and compare it with a conventional microalbumin immunoassay in healthy and diabetic subjects. The need for robust reference intervals for this assay, for children and adults, was a primary focus of the study. The authors determined the limit of detection, linearity, imprecision, and pediatric and adult reference intervals, and compared the HPLC assay with an immunoturbidimetric assay. The limit of detection was 3.4 mg/L. The assay was linear from 4 to 240 mg/L. Total imprecision was less than 10 percent from 16 to 206 mg/L. Nonparametric reference intervals were 22 to 250 mg/g for girls, 20 to 130 mg/g for boys, 14 to 62 mg/g for women, and 10 to 37 mg/g for men. Comparison of the albumin/creatinine ratio by HPLC with an immunoturbidimetric method showed positive proportional bias, which decreased with increasing concentrations of albumin. The authors concluded that this HPLC assay for urinary albumin shows acceptable performance and quantifies albumin species that are not detected by immunoassay. Separate reference intervals for children and adults may be necessary.
Owen WE, Roberts WL. Performance characteristics of
an HPLC assay for urinary albumin. Am
J Clin Pathol. 2005;124:219-225.
Reprints: Dr. W. L. Roberts, ARUP Laboratories, 500 Chipeta Way, Salt Lake City, UT 84108
Chronic obstructive pulmonary disease is a common disease worldwide, with significant morbidity, and it uses an abundance of health care resources. In Hong Kong, chronic obstructive pulmonary disease (COPD) was the fourth leading cause of death in 2001, accounting for three percent of all public hospital admissions. The prevalence of chronic bronchitis and emphysema was estimated to be seven percent and two percent, respectively, among those 70 years or older in the general population in Hong Kong. Previous studies have shown that pulmonary function and quality of life were adversely affected by frequent exacerbations, particularly in active smokers. Infectious agents are a major pathogenic factor in exacerbations. Current data on bacteriology related to acute exacerbations of COPD (AECOPD) were mainly derived from Western countries. There is little data from the Asia-Pacific region. Because the spectrum of organisms might be different between countries or regions, it is important to examine the bacteriology responsible for AECOPD, which has important therapeutic implications for the choice of empirical antibiotic treatment for this common condition. Therefore the authors conducted a study to examine the background, clinical manifestations, and infectious etiologies in the sputum specimen in relation to clinical outcome in patients with AECOPD admitted to a tertiary university hospital in Hong Kong. For the retrospective study, all episodes of AECOPD, patient demographics, length of stay, and sputum culture and radiological results for patients admitted in the first half of 2000 were retrieved from hospital records. The authors identified 329 patients with 418 episodes of AECOPD without concomitant pneumonia. The age of the patients was 74.4±8.3 years. The acute hospital length of stay for an episode of AECOPD was 7.3±6.5 days. Haemophilus influenzae was the most common organism found in sputum (23.1%), followed by Pseudomonas aeruginosa (6.3%) and Streptococcus pneumoniae (4.0%). Mycobacterium tuberculosis was found in 1.1 percent of admissions. Presence of organisms in sputum was not associated with hospital length of stay and intensive care unit admissions. In patients whose FEV1 was greater than 50 percent of predicted values, there was a higher chance of positive sputum growth of H. influenzae than those with FEV1 of less than 50 percent (16 of 44 versus 31 of 162 episodes, respectively, P=0.02). The authors concluded that H. influenzae was the most common bacterium isolated in sputum in patients with AECOPD. In areas endemic of tuberculosis, one should exercise caution when using fluoroquinolones for AECOPD.
Ko FWS, Ng TKC, Li TST, et al. Sputum bacteriology
in patients with acute exacerbations of COPD in Hong Kong. Respiratory
Reprints: David S. C. Hui, Dept. of Medicine and Therapeutics,
Prince of Wales Hospital, Chinese University of Hong Kong, 30-32 Ngan
shing St., Shatin, New Territories, Hong Kong; email@example.com
Human platelet factor 4 (PF4), a member of the CXC chemokine superfamily of proinflammatory cytokines, is released as a low-molecular weight (7.8 kDa) protein upon platelet secretion. The soluble levels of these chemokines increase during prolonged storage of platelet concentrates through platelet activation or through cell destruction. Being able to routinely evaluate the levels of these cytokines would be invaluable in determining the role platelet-derived chemokines play in febrile nonhemolytic transfusion reactions (FNHTRs). Such an assay would allow one to evaluate platelet activation and chemokine release in stored concentrates before transfusion. To develop such an assay, the authors looked for immunoreagents that were commercially available and relatively inexpensive. They then sought to optimize assay conditions so quantification of PF4 was sensitive and linear over a range of concentrations. In the past, direct enzyme-linked immunosorbent assays (ELISAs) for PF4 were unreliable because undiluted samples containing PF4 were directly adsorbed to the plate wells. The capriciousness of these assays stemmed from the lack of antibody-binding site exposure, interference from the matrix, or inconsistent antigen binding due to the large positive charge. For these reasons, the authors developed an indirect sandwich ELISA to measure PF4. The sandwich ELISA captures soluble PF4 through its interaction with an antibody preadsorbed to the plate. The authors developed an ELISA for measuring PF4 from whole human platelets or secreted from activated platelets. They determined optimal concentrations of capture antibody, detection antibody, and enzyme conjugate with serial twofold dilutions of recombinant PF4. This assay was used to determine the ideal sample dilutions needed to reliably quantitate PF4 in releasates or from whole platelet extracts. Serial dilutions of recombinant PF4 resulted in a sigmoid titration curve with a maximal sensitivity of 10 pg and a dynamic quantitative range from 100 to 2,500 pg. This ELISA was used to measure secretion from permeabilized platelets stimulated with free calcium. In a secretion experiment with 2.5 ¥ 108 platelets per mL, samples required a 1:10-fold dilution to reliably evaluate a-granule release. The authors concluded that the parameters described yield an ELISA method with low background and high sensitivity over a range of PF4 concentrations. Using the commercial reagents described makes this assay cost effective and therefore suitable for analyzing multiple samples in the research setting.
Schraw T, Whiteheart S. The development of a quantitative
enzyme-linked immunosorbent assay to detect human platelet factor 4. Transfusion.
Reprints: Dr. S. W. Whiteheart, Dept. of Molecular
and Cellular Biochemistry, University of Kentucky College of Medicine,
800 Rose St., Lexington, KY 40536; firstname.lastname@example.org
Human histocompatibility leukocyte Ag class I molecules play a pivotal role in defending against intracellular pathogens. The activation of CD8+ T cells is initiated by recognizing pathogen-derived peptides bound in the HLA class I molecules of APCs, and this interaction requires at least partial MHC identification of T cells and APCs. In the case of MHC disparity, however, HLA molecules themselves are recognized as foreign, resulting in an adaptive alloimmune response to HLA class I. After blood transfusion, or transplantation of an incompatible graft, differences in the HLA type of donor and recipient can cause formation of HLA-specific Abs and T cell activation. As a clinical consequence, preformed HLA Abs, detectable in lymphocyte crossmatch, are a contraindication to transplantation with an organ bearing the HLA Ags that are detected by these Abs. Peptide embedded in alloantigen is a critical factor that determines the recognition of alloreactive MHC class I-specific T cells. Whether serum HLA Abs are equally sensitive to peptide bound in the HLA class I groove has not been documented before. To determine whether peptide-induced changes of HLA class I conformation affect Ab binding in humans, the authors undertook a study in which a series of recombinant HLA-A2 molecules, each containing a different peptide, served as ligands for human HLA mAbs. Differential binding characteristics indicated that the nature of the peptide bound in HLA class I is decisive for binding of HLA-A2 Abs and explained the limited binding patterns of some human mAbs to cell surface-expressed HLA-A2. The solid-phase assay of a series (n=11) of HLA-A2-reactive, pregnancy-induced, human mAbs on a panel (n=12) of recombinant monomeric HLA-A2 molecules, each containing a single peptide, revealed peptide selectivity of the mAbs. The flow cytometry membrane staining intensities on the HLA-A2 transduced cell line K562, caused by these mAbs, correlated with the number of monomer species detected by the mAbs. Flow cytometry staining on HLA-A2-bearing cell lines of a variety of lineages was indicative of tissue selectivity of these HLA-A2 mAbs. This tissue selectivity suggests that the deleterious effect on allografts is confined to alloantibodies recognizing only HLA class I loaded with peptides that are derived from tissue-specific and household proteins. Since Abs that are only reactive with HLA loaded with irrelevant peptides are expected to be harmless toward allografts, the practice of HLA Ab determination on lymphocyte-derived HLA deserves reconsideration.
Mulder A, Eijsink C, Kester MGD, et al. Impact of peptides
on the recognition of HLA class I molecules by human HLA antibodies. J
Reprints: Dr. Arend Mulder, Dept. of Immunohematology
and Blood Transfusion, Leiden University Medical Center, Building 1, Mail
stop E3Q, P.O. Box 9600, 2300 RC Leiden, Netherlands; email@example.com
Beta thalassemia is a highly heterogeneous inherited disorder distributed worldwide, with more than 200 different mutations reported to date and each ethnic population having its own cluster of common mutations. Most b thalassemias are caused by a point mutation, minor deletion, or insertion in the b globin gene, which result in reduced synthesis or lack of synthesis of the hemoglobin b globin chain. In southern China, b thalassemia is a common disease with high carrier prevalence. For instance, Guangdong Province, located in the southernmost part of mainland China, has a carrier prevalence of 2.54 percent. Searching for and identifying new mutations is a constant priority in population screening, genetic counseling, and prenatal diagnosis of thalassemia. Frameshift mutations of the b globin gene frequently generate a new premature termination codon (PTC), which makes it impossible to synthesize normal functional protein and results in b0 thalassemia. To assess the functional effect of this PTC mutation, the authors quantitatively analyzed b globin mRNA for all heterozygous members of a Chinese family by means of a real-time, one-step, reverse-transcription polymerase chain reaction (RT-PCR) assay. In addition, for the purposes of prenatal diagnosis, an extended reverse dot blot (RDB) method was developed to detect the 23 common mutations of b thalassemia found in Chinese populations. The authors found a novel frameshift mutation—an insertion of G between codons 15 and 16 in a homonucleotide run of four guanines—which generates a new premature chain terminator at the 22nd codon. Relative quantitative analysis of the b globin mRNA in heterozygous subjects demonstrated a 39.83 percent reduction compared to that in normal controls. The authors concluded that the significantly lower amounts of b globin mRNA found in mutation carriers is probably caused by the rapid nonsense-mediated degradation of the mutant mRNA. These data, combined with hematological analysis, suggest that this novel mutation of CDs 15/16 (+G) results in a b0 thalassemia phenotype.
Mo Q-H, Li X-R, Li C-F, et al. A novel frameshift mutation
(+G) at codons 15/16 in a b0 thalassaemia gene results in a
significant reduction of b globin mRNA values. J
Clin Pathol. 2005;58:923-926.
Reprints: Dr. X-M Xu, Dept. of Medical Genetics, Southern
Medical University, Tonghe 510515, Guanzhou, Guangdong, P.R. China; firstname.lastname@example.org
Antibodies play a major role in the adaptive immune response due to high-affinity binding to specific epitopes on target antigens. Human sera contain approximately 10 million different antibodies with activity against a wide range of potential pathogens. Patients' sera are frequently analyzed for the presence or absence of a few specific antibodies as a guide to diagnosis and therapy. More recently, it has been suggested that high-throughput antibody screening might have additional uses in the clinic and laboratory. For example, detecting autoantibodies that recognize tumor antigens may become an effective screening tool for cancer. In this approach, patient sera would be tested for any one of a relatively large panel of antibodies against unique antigens expressed by neoplastic cells. Applied successfully, it would allow physicians to screen whole populations, or specific at-risk populations, for the presence or recurrence of a tumor as an adjunctive tool to current diagnostic techniques. In the laboratory, multiplex antibody screening may facilitate research efforts—for example, by allowing investigators to rapidly and inexpensively identify hybridoma clones that produce antibodies with a well-characterized antigen-binding profile. In the current genomic era, high-throughput analysis tools have found widespread popularity and have facilitated a number of laboratory operations, ranging from large-scale DNA sequencing strategies, to high-density expression microarrays, to production and analysis of complex proteomic data sets. In each case, the work was made possible because highly parallel analyses could be performed at relatively low cost. Such new technology is needed to advance the field of antibody screening. The authors developed a new technique, termed layered peptide array (LPA), as a screening tool to detect antibodies in a highly multiplexed format. They demonstrated that a prototype LPA could produce approximately 5,000 measurements per experiment and appeared to be scalable to higher throughput levels. Sera and saliva from Sjögren's syndrome patients served as a test set to examine antibody titers in clinical samples. The LPA platform exhibited a high sensitivity (100%) and high specificity (94%) for identifying SSB antigen-positive samples. The multiplex capability of the platform was also confirmed when serum and saliva samples were analyzed for antibody reactivity to several peptides, including Sjögren's syndrome antigens A and B. The authors concluded that LPA analysis will be a useful method for a number of screening applications.
Gannot G, Tangrea MA, Gillespie JW, et al. Layered
peptide arrays: high-throughput antibody screening of clinical samples.
Mol Diagn. 2005;7:427-436.
Reprints: Michael R. Emmert-Buck, Pathogenetics Unit,
Advanced Technology Center, Laboratory of Pathology and Urologic Oncology
Branch, Center for Cancer Research, National Cancer Institute, 8717 Grovemont
Circle, Bethesda, MD 20892-4605; email@example.com
Necrotizing enteritis is an often fatal syndrome characterized by hemorrhagic, inflammatory, or ischemic necrosis of the jejunum. The syndrome was first described in the 1940s in Germany in chronically starved people who had eaten a large meal, possibly including poorly cooked meat. A previously unknown strain of Clostridium welchii was isolated and subsequently renamed Clostridium perfringens type C. The syndrome was called Darmbrand (burnt intestine), and cases ceased when standards of living and nutrition improved after World War II. A similar syndrome in children and young adults was recognized as a surprisingly common condition in the early 1960s in Papua, New Guinea, where it was associated with protein-deficient people consuming poorly cooked pork in ceremonial feasts. Additional investigation identified C. perfringens type C as the causative organism and the bacteria's b-toxin as the direct mediator of intestinal necrosis. Necrotizing enteritis, locally referred to as pigbel, was the leading cause of childhood mortality in Papua, New Guinea, until a program of vaccination against C. perfringens type C b-toxin dramatically reduced disease incidence. In developed countries, a handful of adult necrotizing enterocolitis cases caused by C. perfringens type C or undetermined type have been described, principally in diabetics. The authors conducted a study that included a case series and literature review. They reviewed charts and autopsy reports from four patients with adult necrotizing enterocolitis (ANEC). They subtyped C. perfringens isolates by mouse bioassay and pulsed-field gel electrophoresis. Then the authors tested fixed tissue specimens with an anticlostridial antibody using an immunohistochemical assay. The authors found that between 2000 and 2003, ANEC developed in four previously healthy men; three died. The small bowel was affected in three patients and the colon in two patients. Portal or mesenteric vein thrombosis occurred in three patients. C. perfringens type A was isolated from three patients, and immunohistochemical assay demonstrated clostridial antigens limited to affected areas of the intestine of all four. The nonculture-positive patient had a strong epidemiologic link to one of the others and a compatible clinical course. C. perfringens of the same pulsed-field gel electrophoresis-defined molecular subtype was isolated from stool samples of one patient, his wife, and food from a restaurant they patronized. The authors concluded that ANEC associated with C. perfringens type A infection occurred in four North American adults. Culture for C. perfringens type A should be performed in cases of ANEC. Alternative tests, such as immunohistochemical assay, were diagnostically useful. Additional research might uncover virulence factors, host factors, and the burden of disease in the population.
Sobel J, Mixter CG, Kolhe P, et al. Necrotizing enterocolitis
associated with Clostridium perfringens type A in previously
healthy North American adults. J
Am Coll Surg. 2005;201:48-56.
Reprints: Dr. Jeremy Sobel, Foodborne and Diarrheal Diseases Branch, Centers for Disease Control and Prevention, 1600 Clifton Rd., MS-A38, Atlanta, GA 30333
Atherosclerosis is a complex inflammatory process that is characterized by monocytes/macrophages and T lymphocytes in the atheroma. More advanced atherosclerotic lesions, such as fibro-fatty plaques, are the result of monocyte recruitment together with smooth muscle cell migration and proliferation. Macrophage density is greater in the shoulder region of plaques that rupture. Monocytes/macrophages are pivotal in atherosclerosis and are present at all stages of atherosclerosis, from nascent fatty streak lesions to culmination in acute coronary syndromes. Macrophages promote atherosclerosis via production of various key biomediators, including cytokines such as interleukin (IL)-1b, tumor necrosis factor-a (TNF-a), and IL-6; chemokines such as IL-8 and monocyte chemoattractant protein-1 (MCP-1); matrix metalloproteinases; and integrins (CDllb and VLA4). Therapies shown to reduce cardiovascular events appear to have anti-inflammatory properties. This has been well documented with the hydroxymethylglutaryl-CoA (HMG-CoA) reductase inhibitors (statins). Because newer, specific anti-inflammatory therapies are also being developed, it is essential to have a simple in vitro screening assay to test their anti-inflammatory effects. The authors developed an in vitro model system of monocytic cells to test the anti-inflammatory properties of dietary supplements and pharmacologic agents. They stimulated THP-1 cells (human monocytic cell line) with different concentrations of lipopolysaccharide (0 to 1,000 μg/L) for different times (4, 12, and 24 hours) and assessed the secretion of proinflammatory cytokines (IL-1, IL-6, and TNF-a). They found that TNF-a secretion was maximum at the lowest lipopolysaccharide concentration (100 μg/L) and at the shortest duration of incubation (four hours). Maximum secretion of IL-1b and IL-6 was achieved at 24 hours with higher doses of lipopolysaccharides. Treatment of THP-1 with dietary supplements (a-tocopherol, N-acetylcysteine, catechin, and epigallocatechin gallate) and pharmacologic agents (statins, peroxisome proliferator-activated receptor-g agonists, and an angiotensin II receptor blocker) significantly inhibited lipopolysaccharide-stimulated TNF-a release. The authors concluded that the release of TNF-a after stimulation of THP-1 cells with
lipopolysaccharides is a valid model system to test novel compounds for potential
Singh U, Tabibian J, Venugopal SK, et al. Development
of an in vitro screening assay to test the antiinflammatory properties
of dietary supplements and pharmacologic agents. Clin
Reprints: Ishwarlal Jialal, Laboratory for Atherosclerosis
and Metabolic Research, Dept. of Pathology & Laboratory Medicine, University
of California, Davis Medical Center, 4635 2nd Ave., Research Bldg. 1,
Room 3000, Sacramento, CA 95817; firstname.lastname@example.org
H antigen is the precursor of A and B antigens.The
ABO locus determines the A and B antigens, whereas a-(1,2)-fucosyltransferase
genes FUT1 and FUT2 determine the H antigen. FUT1
is responsible for H antigen expression on red blood cells (RBCs), and
FUT2 encodes the enzyme that determines H expression in secretions.
In the para-Bombay phenotype, ABH antigens are not expressed on RBCs,
but ABH substances are present in saliva. The genetic polymorphism of
human FUT1 was first recognized in a person in Bombay, India,
whose RBCs lacked H antigen and whose plasma agglutinated RBCs of all
normal ABO phenotypes. The authors described FUT1 mutations in
two Chinese people with the para-Bombay phenotype. RBC phenotypes were
characterized by conventional serologic methods. Exons 6 and 7 of the
ABO gene, as well as the entire coding region for FUT1 and FUT2,
were amplified with four independent polymerase chain reactions (PCRs)
from genomic DNA. PCR products were excised, purified from agarose gels,
and sequenced directly. Mutations of FUT1 were identified by
TOPO cloning sequencing. RBC ABO genotypes correlated with ABH substances
in both subjects' saliva. One patient had two heterozygous mutations of
FUT1 by direct DNA sequencing, namely a CÆT heterozygous
mutation at position 293(C293T) and AG heterozygous deletion (CAGAGAGÆCAGAG)
at position 547 to 552. These two mutations were confirmed to be compound
heterozygotes; that is, each mutation was determined to be on a separate
homologous chromosome by TOPO cloning sequencing. The FUT2 genotype
The other individual, a blood donor, had an AG deletion at position 547
to 552 homozygous allele in FUT1. The FUT2 genotype
357. C293T mutation can cause Thr/Met
at amino acid position 98. AG deletion at position 547 to 552 caused a
reading frameshift and a premature stop codon. The authors concluded that
a novel nonfunctional FUT1 allele C293T was identified in a person
with the para-Bombay phenotype. This rare H-deficient phenotype may result
from different nonfunctional alleles.
Yan L, Zhu F, Xu X, et al. Molecular basis for para-Bombay
phenotypes in Chinese persons, including a novel nonfunctional FUT1 allele.
Reprints: Dr. Lixing Yan, Blood Center of Zhejiang
Province, Hangzhou, Zhejiang, 310006 People's Republic of China; email@example.com
Dr. Bissell is Professor and Director of Clinical
Services and Vice Chair, Department of Pathology, Ohio State University
Medical Center, Columbus.