Timothy C. Hunter

In vivo ionizing irradiations produce deletions in the hprt gene of human T-lymphocytes

The hprt T-lymphocyte cloning assay, which detects mutations occurring in vivo in humans, has been used to examine mutants induced in patients receiving radioimmunoglobulin therapy (RIT) for cancer. Samples from 13 patients before treatment (controls) and 15 samples from 12 patients after treatment were studied for both mutant frequencies and molecular changes in the hprt mutant T-cell clones. Patients were studied up to 48 months after treatment. Post-RIT patients showed increased mutant frequencies as compared to pre-treatment values. T-cell receptor (TCR) gene analysis of mutant T-cell clones demonstrated that 84% arose independently, both pre- and post-treatment, which is the same proportion as seen in normal individuals. However, several individuals did show large sets of mutants with the same TCR gene rearrangement patterns. Molecular analysis of mutants demonstrated a greater proportion of mutations with hprt gene changes on Southern blots after RIT treatment than before (40% versus 20%). RIT increases the proportion of mutations with total rather than partial gene deletions or other gross structural changes compared to normal individuals or pre-treatment patients. These studies are defining the spectrum for radiation-induced hprt gene mutations in vivo in human T-lymphocytes.

Southern-blot analyses of human T-lymphocyte mutants induced in vitro by γ-irradiation

Abstract G0 phase cultures of human peripheral blood T-lymphocytes from a single individual were exposed to 300 rad of γ-irradiation from a 137Cs source and cultured in vitro for 8 days to allow phenotypic expression. Thioguanine-resistant (TGr) mutants were isolated by a cell cloning assay in microtiter plates. These mutants were studied by Southern blot analysis to define the gross structural alterations in the hypoxanthine-guanine phosphoribosyl transferase (hprt) gene by use of an hprt cDNA probe. A similar analysis of the T-cell receptor (TCR) gene rearrangement patterns was employed to define the independent nature of each mutant colony by use of TCR β and γ cDNA probes. 74 mutants were isolated in 5 separate experiments. TCR gene rearrangement analysis showed these to represent 24 independent mutations, of which 18 contained hprt structural alterations. These alterations included simple deletions ( 10 18 ) as well as more complex rearrangements resulting in molecular weight changes of restriction fragments representing both the 5′ and 3′ regions of the hprt gene ( 4 18 and 4 18 , respectively). These results demonstrate that γ-irradiation primarily induces TGr mutations through gross structural alterations in the hprt gene and that these alterations are randomly distributed across the gene. This approach to mutation analysis will provide information on the types of alterations induced by this irradiation, especially the extent of deletions involving the hprt gene. These results also demonstrate the feasibility of employing in vitro exposure of human T-lymphocytes to a single mutagenic agent as an aid to understanding the mechanisms of mutations occurring in vivo in humans.

Pulsed field analysis of hprt T-cell large deletions : telomeric region breakpoint spectrum

In order to determine a large deletion breakpoint spectrum, 25 independent hprt T-lymphocyte mutants with deletions extending from hprt into the telomeric or centromeric flanking chromosomal region were analyzed by pulsed field gel electrophoresis (PFGE). PFGE was used to determine deletion sizes which allowed localization of breakpoints external to hprt to specific chromosomal positions in mutants containing an intra-hprt breakpoint. A breakpoint spectrum based on 19 large deletion mutants is reported for the Xq26 chromosomal region telomeric to hprt. A potential cluster of breakpoints (4/19) was observed approximately 60 kb from hprt. In addition, maximum recoverable deletion size was at least 3.5 Mb. Three of the 25 mutants analyzed appeared to be complex deletion events.

Mammary gland morphological and gene expression changes underlying pregnancy protection of breast cancer tumorigenesis

A full-term pregnancy early in life reduces lifetime risk of developing breast cancer, and the effect can be mimicked in rodents by full-term pregnancy or short-term treatment with exogenous estrogen and progesterone. To gain insight into the protective mechanism, 15 3-mo-old postpubertal virgin Lewis rats were randomly assigned to three groups: control (C), pregnancy (P), or hormone (H). The P group animals underwent a full-term pregnancy, and H group animals were implanted subcutaneously with silastic capsules filled with ethynyl estradiol and megesterol acetate for 21 days. C and P animals were implanted with sham capsules. On day 21 capsules were removed, which was followed by a 49-day involution period, euthanasia, and mammary tissue collection. Global gene expression was measured using Rat Genome 230.2 Arrays. Histological analysis revealed that P and H treatments induced sustained morphological changes in the mammary gland with significantly increased percentages of mammary parenchyma and stromal tissues and higher ratio of stroma to parenchyma. Transcriptome analysis showed that P and H treatments induced sustained global changes in gene expression in the mammary gland. Analysis of commonly up- and downregulated genes in P and H relative to C treatment showed increased expression of three matrix metallopeptidases (Mmp3, 8, and 12), more differentiated mammary phenotype, enhanced innate and adaptive immunity, and reduced cell proliferation and angiogenic signatures. The sustained morphological and global gene expression changes in mammary tissue after pregnancy and hormone treatment may function together to provide the protective effect against breast cancer.

Analysis of T cell receptor β and γ genes from peripheral blood, regional lymph node and tumor-infiltrating lymphocyte clones from melanoma patients

SummaryA total of 199 T cell clones from two melanoma patients were derived from progenitor T cells from recurrent melanoma, regional lymph nodes (either involved or uninvolved with malignancy) and peripheral blood by inoculating single cells directly into the wells of microtiter plates before in vitro expansion. The surface marker phenotype of most clones was CD4+CD8−, although some were CD4−CD8+. Genomic DNA prepared from all clones was analyzed by Southern blot hybridization using T cell receptor (TCR) β and γ gene probes, seeking clones with identical TCR gene rearrangement patterns as direct evidence for in vivo progenitor T cell clonal amplification. ProbingHindIII-digested DNA with TCRβ and TCRγ probes revealed several clones with identical TCR gene rearrangement patterns. These clones had subsequent probing ofBamHI-digested DNA with TCRβ and TCRγ probes, which showed all but 2 clones to have distinct rearrangement patterns. These analyses provide clear molecular evidence for in vivo polyclonal CD4+ T cell populations in each of several separate immune compartments in these patients.

Regional Expression of Aquaporin 1, 4, and 9 in the Brain During Pregnancy

Pregnancy is a state of physiologic adaptation, with significant changes in cardiovascular, renal, and hemodynamic systems. Aquaporins (AQPs) may play a role in facilitating these changes. While AQP expression has been assessed in several organs during pregnancy, little is known about its expression in the brain during pregnancy. Therefore, this study assesses the regional expression of AQP1, 4, and 9 during pregnancy and the postpartum period using real-time quantitative polymerase chain reaction. The authors show that AQP1, 4, and 9 are expressed in the anterior and posterior cerebrum, cerebellum, and brainstem of nonpregnant, midpregnant, late pregnant, and postpartum rats. The regional distribution pattern of AQP4 and 9 remained similar during gestation, whereas this pattern changed for AQP1. The expression levels of AQP1, 4, and 9 in the brainstem did not change with gestation, whereas changes were found in the anterior cerebrum for AQP4 and in the posterior cerebrum and cerebellum for all AQPs.

Coxsackievirus B detection in cases of myocarditis, myopericarditis, pericarditis and dilated cardiomyopathy in hospitalized patients

Coxsackieviruses B (CV-B) are known as the most common viral cause of human heart infections. The aim of the present study was to assess the potential role of CV-B in the etiology of infectious heart disease in hospitalized patients. The present study is based on blood, pericardial fluid and heart biopsies from 102 patients and 100 control subjects. All of the samples were examined for the detection of specific enteroviral genome using the reverse transcription polymerase chain reaction (RT-PCR) and sequence analysis. Immunohistochemical investigations for the detection of the enteroviral capsid protein, VP1, from the biopsies were performed. The samples were cultured on confluent KB monolayer cell line for possible virus isolation. The epidemiological data were also collected. CV-B was detected in 28 of the 102 patients. The sequence analysis demonstrated that 27 strains were identical to CV-B3 and only one strain was identical to CV-B1. Furthermore, VP1 in the heart biopsies was detected in enterovirus-positive cases, as revealed by RT-PCR. Pericarditis infection was more frequent than myocarditis (P<0.05) or myopericarditis (P=0.05). The epidemiological data demonstrate that CV-B heart infections occur mainly during autumn and winter, and young male adults are more susceptible than adolescents or adults (P<0.5). The present findings demonstrate a higher prevalence of viral heart infections, suggesting that CV-B may significantly contribute to heart infections.

HPRT mutations in vivo in human CD 34+ hematopoietic stem cells

The HPRT mutations in T lymphocytes are widely utilized as biomarkers of environmental exposure and effect. The HPRT gene detects a wide variety of mutation types, many of which are similar at the molecular level to those found in oncogenes in cancers. However, it remains to be determined whether the assay for mutations in T lymphocytes is reflective of mutagenic events in tissues or cells which have high frequencies of malignancy in humans. We now demonstrate that the HPRT gene can be utilized to detect mutations in myeloid stem cells, which are frequent progenitor cells of leukemias. This myeloid stem cell assay shows an age related increase in mutation at HPRT and also detects increases in mutant frequency (M-MF) in patients who have undergone chemotherapy. The myeloid mutants are confirmed to have mutations in the HPRT gene by DNA sequence analysis. Increases in M-MF are seen as expected in the clonally unstable myeloid stem cells of patients with myelodysplastic syndromes; however, unexpectedly these patients also have elevated T-lymphocyte mutant frequencies (T-MF). A good correlation is shown between M-MFs and T-MFs in the same patients. Thus, it appears that the T-lymphocyte assay, which is technically much less demanding than the myeloid assay, appears to faithfully represent the frequency of mutagenic events in the myeloid lineage.

Postmortem diagnosis of infectious heart diseases: A mystifying cause of Sudden Infant Death

Sudden infant death (SID) is an unresolved problem of high relevance and previous studies have indicated a role of viral heart infections. The diagnosis remains difficult in clinical practice using routine diagnostic tests and must be substantially improved. A prospective study based on post-mortem samples from SID victims whose heart disease was not clinically recognized was conducted for 4 years in a Tunisian University Hospital. Pediatric cases of unnatural death served as controls. Both SID victims and controls were investigated for possible coxsackievirus-B (CV-B) infection in heart tissue. During the study period, 39 cases with a male predominance (77%) were reported. There was no positive family history of coronary artery disease among the victims. In 35 cases (90%), low birth weight and/or critical development period were reported. All SID victims had complained of mild fever and insomnia for a few days preceding death, which required infectious laboratory investigations marked with an elevated white blood cell count (WBC) and C-reactive protein (CRP). The cardiac biomarkers were also elevated. The histopathological investigations of the heart tissue samples revealed signs of myocardial and pericardial inflammation. Enterovirus was detected by immunohistochemistry (IHC) and PCR from myocardial samples from 6 cases (15.3%) having myocarditis and 3 cases (7.7%) having perimyocarditis. The current study is of great interest and is aimed at urging health professionals to adopt systematically long intensive heart care in infants with underlying vulnerability as well as new diagnostic approaches including histopathology complemented with IHC and molecular pathology.

Coxsackievirus B heart infections and their putative contribution to sudden unexpected death: An 8-year review of patients and victims in the coastal region of Tunisia

Coxsackieviruses B (CV B) are known as the most common viral cause of human heart infections. Cardiac inflammations contribute to sudden unexpected death (SUD) significantly. The diagnosis remains difficult with the traditional diagnostic tests and must be substantially improved. This has prompted health professionals to seek new diagnostic procedures which may provide important clues regarding underlying etiology. The present study is based on patients with infectious heart diseases and SUD victims with no relevant pathologies. They were investigated for possible CV-B infection. Patients with coronary artery diseases and unnatural road and domestic accident victims served as controls. The samples were studied for CV-B applying PCR. Histopathology for inflammatory markers, immunohistochemistry (IHC) for immune inflammatory cells and the enteroviral VP1-capsid protein were performed. Overall, 102 patients and 87 SUD victims were studied. As controls, 100 patients and 54 SUD unnatural accident victims were enrolled. CV-B were detected in 28 patients and 15 SUD victims. The control group samples were completely virus negative. Compared to controls, IHC revealed a significant presence of T and B lymphocytes within the myocardium. Furthermore, enteroviral VP1-capsid protein were detected from samples by IHC. Applying a comprehensive combination of methods, our results demonstrate the involvement of CV-B in cases of heart infection suggesting they play a significant role in SUD. Our results emphasize the importance of opting for a combination of methods.