Garth D. Ehrlich

Detection of Human T-Cell Lymphoma/Leukemia Virus Type I DNA and Antigen in Spinal Fluid and Blood of Patients with Chronic Progressive Myelopathy

The presence of antibodies to human T-cell lymphoma/leukemia virus Type I (HTLV-I) has been associated with chronic progressive myelopathy. We attempted to isolate the virus from the blood and spinal fluid of patients with chronic progressive myelopathy and to define the clinical, radiologic, and electrophysiologic features of this disease. Ten of 13 patients from tropical countries and 2 of 8 from the United States had serum antibodies to HTLV-I. The virus was detected in cultures of peripheral-blood lymphocytes from three of seven patients by means of Southern blot hybridization. Using a sensitive in vitro enzymatic gene-amplification technique, we detected HTLV-I sequences in fresh peripheral-blood mononuclear cells of all of 11 patients tested who were positive for the antibody, and in cell cultures of the spinal fluid from 3 of the 11 tested. Magnetic resonance imaging of the cranium revealed periventricular lesions in the white matter of 3 of the 12 antibody-positive patients. Five of these patients had mild axonal sensorimotor polyneuropathy, and one had bilateral lumbar radiculopathy. Visual evoked potentials were abnormal in three seropositive patients, and brain-stem evoked responses were abnormal in two. The detection of the DNA and proteins of HTLV-I strengthens the proposition that this virus is involved in the pathogenesis of a subset of cases of chronic progressive myelopathy.

Linkage and Association between Inflammatory Bowel Disease and a Locus on Chromosome 12

Genetic epidemiological studies have shown that genetic factors are important in the pathogenesis of the idiopathic inflammatory bowel diseases (IBD), Crohn disease (CD), and ulcerative colitis (UC). A genome screen in the United Kingdom found linkage of IBD to a 41-cM region of chromosome 12, surrounding D12S83. We aimed to replicate this linkage and to narrow the region of interest. Nonparametric linkage analyses at microsatellites surrounding D12S83 were performed in 122 North American Caucasian families containing 208 genotyped IBD-affected relative pairs. Transmission/disequilibrium tests (TDTs) were also performed. We confirmed that IBD is linked to chromosome 12 (peak GENEHUNTER-PLUS LOD* score 2.76 [P = .00016] between D12S1724 and D12S90). The evidence for linkage is contributed by both the group of CD-affected relative pairs (peak GENEHUNTER-PLUS LOD* score 1.79 [P = .0021] between D12S1724 and D12S90) and the group of UC-affected relative pairs (peak GENEHUNTER-PLUS LOD* score 1.82 [P = .0019] at D12S335). The TDT is positive at the D12S83 locus (global chi2 = 16.41, 6 df, P = .012). In conclusion, we have independently confirmed linkage of IBD to the chromosome 12 region that we investigated. A positive TDT at D12S83 suggests that we have greatly narrowed the chromosome 12 region that contains an IBD locus.

Pediatric Respiratory Papillomatosis: Prognostic Role of Viral Typing and Cofactors

Children with recurrent respiratory papillomatosis vary greatly in their clinical disease course. Many have mild disease with eventual remission while others present with an early aggressive airway obstructive course. This study consisted of 24 pediatric patients whose specimens underwent polymerase chain reaction analysis for cytomegalovirus (CMV), herpes simplex virus (HSV), and human papillomavirus (HPV) type. Nineteen of 24 specimens contained enough DNA for this study. None of the specimens were found to contain DNA from HPV‐16, ‐18, ‐31, ‐33; CMV; or HSV, which contrasts with our previous findings in adults. Ten patients were infected by HPV‐11 and seven of these underwent tracheotomy because of an aggressive tumorigenic clinical course. Nine patients were infected by HPV‐6 alone of whom only two required a tracheotomy (P = 0.05, Fishers Exact Test). The early airway obstructive course associated with HPV‐11, however, had no bearing on achieving eventual disease remission, with decannulation achieved in eight of nine children.

Multiple sclerosis, retroviruses, and PCR

Previously reported serologic and polymerase chain reaction (PCR)-based findings have suggested an association between the human retrovirus, HTLV-I, and multiple sclerosis (MS). Due to the inherent ability of PCR to produce false-positive results, we developed a set of physical and procedural safeguards to minimize the possibility of molecular carryover. These were applied as part of a blinded, large-scale, multipopulation, multiplex PCR-based study designed to examine this issue of association. Our results do not support the hypothesis that HTLV-I, which plays a role in the pathogenesis of an encephalomyeloneuropathy, HTLV-II, or closely related agents are associated with MS. A concomitant review of the current literature supports this view.

Fine mapping of the split-hand/split-foot locus (SHFM3) at 10q24: evidence for anticipation and segregation distortion.

Split-hand/split-foot malformation (SHFM, ectrodactyly, or lobster-claw deformity) is a human limb malformation characterized by aberrant development of central digital rays with absence of fingers and toes, a deep median cleft, and fusion of remaining digits. SHFM is clinically heterogeneous, presenting both in an isolated form and in combination with additional abnormalities affecting the tibia and/or other organ systems, including the genitourinary, craniofacial, and ectodermal structures. Three SHFM disease loci have been genetically mapped to chromosomes 7q21 (SHFM1), Xq26 (SHFM2), and 10q24 (SHFM3). We mapped data from a large Turkish family with isolated SHFM to chromosome 10q24 and have narrowed the SHFM3 region from 9 cM to an approximately 2-cM critical interval between genetic markers D10S1147 and D10S1240. In several instances we found evidence for a more severe phenotype in offspring of a mildly affected parent, suggesting anticipation. Finally, data from this family, combined with those from six other pedigrees, mapped to 10q24, demonstrate biased transmission of SHFM3 alleles from affected fathers to offspring. The degree of this segregation distortion is obvious in male offspring and is possibly of the same magnitude for female offspring.

DNA sequence analysis of the gene encoding the HTLV-I p21e transmembrane protein reveals inter- and intraisolate genetic heterogeneity.

DNA sequence analysis was performed on a 235-bp region of the p21 e transmembrane protein gene of the human T-cell lymphoma/leukemia virus type I (HTLV-I) which encompassess the putative immunosuppressive peptide. Polymerase chain reaction-based amplification was used to generate multiple molecular clones from isolates derived from fresh or cultured cells from 19 individuals. A dendrogram was constructed using the p21e DNA sequence information to compare the sequences among isolates in the current study and other previously published HTLV-I isolates including strains from Africa and Papua New Guinea. Examination of multiple clones from individual isolates revealed the presence of multiple genotypes in patients with tropical spastic paraparesis/HTLV-I-associated myelopathy and adult T-cell leukemia/lymphoma. These findings suggest that HTLV-I, like HIV, may be present as a quasispecies in vivo. Our studies, however, failed to identify specific sequence motifs that segregated exclusively with the lymphoproliferative or neurological forms of the disease.

Use of DNA amplification for diagnosis of cytomegalovirus enteritis after intestinal transplantation

BACKGROUND & AIMS Intestinal transplantation is a feasible therapy for patients with short-bowel syndrome. However, cytomegalovirus (CMV) enteritis can cause complications. The aim of this study was to investigate the value of polymerase chain reaction (PCR)-based detection methods for CMV in the management of patients with small bowel transplants. METHODS Comparative evaluation of PCR with histopathology, shell-vial assay, and tube culture of intestinal biopsy specimens was used for the diagnosis of CMV enteritis in 21 patients. RESULTS Ten patients experienced 21 episodes of CMV enteritis, diagnosed by histopathology, virology, or both. PCR had a sensitivity and specificity of 96% and 69%, respectively, compared with traditional methods, whereas the positive and negative predictive values were 35% and 99%, respectively. Three+ and 4+ signals corresponded to a specificity of 91% and positive predictive value of 59%, respectively. CMV was detected by PCR a median of 11 days (range, 0-32) earlier than other methods and lasted a median of 40 days (range, 21-80) in the 13 episodes, which became PCR-negative and in those patients who developed asymptomatic infection. In 8 episodes, CMV by PCR never became negative and was associated with a relapse of disease confirmed by other methods. CONCLUSIONS PCR is a sensitive method for the early detection of CMV in intestinal biopsy specimens and can be used for preemptive therapy after intestinal transplantation.

RNA differential display of scarless wound healing in fetal rabbit indicates downregulation of a CCT chaperonin subunit and upregulation of a glycophorin-like gene transcript.

BACKGROUND/PURPOSE Scars form as wounds heal in adult organisms. In addition to disrupting cosmetic appearance, scar tissue can cause significant morbidity, and even death if it blocks vital organ function. Previous work has established that fetal wounds, especially in early to midgestation, can heal without scarring. Because such inherent physiological mechanisms ultimately are under genetic control, a study was initiated to elucidate the differences in gene expression that produce scarless wound healing in the mammalian fetus but scarring in postnatal wounds. Reverse transcription polymerase chain reaction (RT-PCR) differential display (DD) was used to detect differentially expressed mRNA transcripts in a rabbit model of wound healing. METHODS Adult and 21-day fetal full-thickness rabbit skin specimens from wounded and unwounded sites were harvested 12 hours postwounding. RNA extracted from the tissue was used as a template in DD reactions using anchoring and random primers to generate tissue-specific gene expression fingerprints. The over 2,000 resulting amplimers (gene transcripts) were screened for differential expression among the 4 types of specimens: fetal control (unwounded), fetal wound, adult control, and adult wound. Selected bands distinctly upregulated or downregulated in fetal wound lanes on the DD gels were excised, and the cDNA was extracted, reamplified, cloned into vectors, and sequenced. DD results were confirmed by limiting-dilution RT-PCR using sequence-specific primers. RESULTS Differential display (DD) showed 22 amplimers that were significantly upregulated in all fetal wound samples as compared with little or no expression in fetal control, adult control, or adult wound tissues. Conversely, 5 transcripts were downregulated in the fetal wound specimens but highly expressed in the 3 comparison tissues. Reamplification of selected transcripts by PCR, followed by cloning and DNA sequencing, yielded 7 distinct sequences, each representing a gene expressed differently in fetal wound than in the other 3 tissues. A transcript that was downregulated in fetal wound showed very high sequence homology to part of the human gene for the eta subunit of the hetero-oligomeric particle CCT (the chaperonin containing T-complex polypeptide 1 or TCP-1). An upregulated amplimer showed significant DNA sequence homology to glycophorins A and B. One sequence was identified as 28S rRNA. The remaining 4 candidate sequences showed no significant homology to known genes, but 1 had high homology to expressed sequence tags of unknown function. CONCLUSIONS With careful experimental design and proper controls and verifications, differential display of RNA expression is a potentially powerful method of finding genes that specifically regulate a particular physiological process such as fetal wound healing. No a priori knowledge of what genes might be involved, or why, is necessary. This study indicates that downregulation of a gene that codes for a chaperonin subunit and upregulation of several other genes may be involved in the striking scarless character of wound healing in the mammalian fetus. Results suggest the hypothesis that downregulation of the CCT chaperonin in fetal wound may inhibit the formation of myofibroblasts, a cell type that correlates highly with scarring in postnatal wound healing, by preventing the folding of sufficient alpha-smooth muscle actin to form the stress fibers characteristic of these cells.

Development of a simplex polymerase chain reaction-based assay for the detection of Neisseria meningitidis

BACKGROUND This study was undertaken to develop a sensitive and specific polymerase chain reaction (PCR)-based assay for Neisseria meningitidis and Neisseria gonorrhoeae that could ultimately be incorporated into a multiplex assay designed to screen cerebrospinal fluid (CSF) for a panel of pyogenic bacterial species associated with bacterial meningitis. METHODS N.meningitidis-specific primers were designed from porA gene sequences with the aid of a commercial software program and were used to develop and validate a clinical assay using a liquid hybridization-gel retardation detection system. The analytic sensitivity of the assay was determined using CSF spiked with N. meningitidis and comparing the PCR-based results with culture. RESULTS Analytic sensitivity experiments showed the assays limit of detection to be 100 fg purified input target DNA and 0.0125 colony-forming units of N. meningitidis spiked into CSF. Specificity experiments showed the assay could detect all strains of N. meningitidis and N. gonorrhoeae tested, but did not support amplification of the commensal neisserial species or a panel of other human bacterial pathogens. CONCLUSIONS This PCR-based assay for pathogenic neisserial species is sensitive and specific and suitable for incorporation into a multiplex assay for the clinical differentiation of aseptic and septic meningitis.

Culture-negative infections in orthopedic surgery

Laboratory cultures are the main scientific input into the decision-making process that determines the course of treatment for suspected orthopedic infections, just as they constitute the mainstay of the diagnosis of infections in other medical specialties. This situation is archaic because culture techniques were virtually abandoned in Environmental Microbiology (Hugenholtz et al. 1998) many years ago, following the conclusion that <1% of the bacteria in any natural ecosystem can be recovered by standard cultural methods. Medical Microbiology has clung to culture techniques because they detect the bacteria that cause acute infections, with reasonable sensitivity and accuracy, but the time has come to examine both their sensitivity and their accuracy for the detection and identification of bacteria in chronic biofilm infections (Costerton et al. 1999).