Peter L. Rady

Herpesvirus-like DNA sequences in non-Kaposi's sarcoma skin lesions of transplant patients

Herpesvirus-like DNA sequences (KSHV) have been reported to be associated with various forms of Kaposis sarcoma (KS). To determine if KSHV was associated with other proliferative skin lesions from non-AIDS immunocompromised patients, 33 skin lesions (basal cell carcinomas, squamous cell carcinomas, actinic keratoses, verruca vulgaris, atypical squamous proliferations, and seborrhoeic keratosis) from 4 organ-transplant patients receiving immunosuppressive therapy were tested for KSHV by PCR. KSHV sequences were detected in 82% of these skin lesions. Our results suggest that KSHV is associated with lesions other than KS in non-AIDS immunocompromised patients, and may also be involved in the pathogenesis of the various forms of proliferative skin lesions from organ-transplant patients.

Knock‐out mouse for Canavan disease: a model for gene transfer to the central nervous system

Canavan disease (CD) is an autosomal recessive leukodystrophy characterized by deficiency of aspartoacylase (ASPA) and increased levels of N‐acetylaspartic acid (NAA) in brain and body fluids, severe mental retardation and early death. Gene therapy has been attempted in a number of children with CD. The lack of an animal model has been a limiting factor in developing vectors for the treatment of CD. This paper reports the successful creation of a knock‐out mouse for Canavan disease that can be used for gene transfer.

Interleukin-10 messenger ribonucleic acid in human placenta: implications of a role for interleukin-10 in fetal allograft protection.

OBJECTIVE Our purpose was to determine whether interleukin-10 is expressed in human placental tissue, which might imply a role for it in fetal allograft protection. STUDY DESIGN Detection of interleukin-10 messenger ribonucleic acid in human placental tissue and in human placental JAR cells by reverse transcription-coupled polymerase chain reaction was studied. RESULTS Interleukin-10 messenger ribonucleic acid was detected in human placental tissue from term mothers and in human placental JAR cells. Sequence analysis of the expected interleukin-10 complementary deoxyribonucleic acid fragment revealed 100% homology to authentic interleukin-10 complementary deoxyribonucleic acid. CONCLUSION Our results indicated that human placental tissue from term mothers expressed high levels of interleukin-10 messenger ribonucleic acid, suggesting that cells that produce interleukin-10 and that are associated with the placenta may play a role in preventing rejection of the fetal allograft by the mother.

Clinical aspects of epidermodysplasia verruciformis

Thirteen patients with epidermodysplasia verruciformis (EV) were studied over a period of 7 years. EV is a rare genodermatosis characterized by a generalized infection with a specific group of human papilloma virus (HPV) and a propensity for developing skin malignant tumours in 30%−50% of patients. The diagnosis of EV was confirmed by histopathological and immunohistochemical findings. Three of our patients had the benign form of EV, which is characterized by monomorphous lesions and no malignant changes, whereas 10 had the malignant form, which is characterized by polymorphic lesions and development of cutaneous malignant tumours. All EV patients with the malignant form developed multiple skin tumours (77%). They started to appear at age 20, predominantly on the forehead (50%). Most were squamous cell carcinoma, extremely aggressive and invasive, which provoked metastasis and death in two patients.

Methylenetetrahydrofolate reductase (MTHFR): The incidence of mutations C677T and A1298C in the Ashkenazi Jewish population

The polymorphic mutation C677T in the gene of MTHFR is considered a risk mutation for spina bifida and vascular disease. Another common mutation on the MTHFR gene, A1298C, has also been described as another risk mutation. We studied the frequencies of these two mutations on DNA samples from healthy Jewish individuals and compared them to the frequency of these mutations in DNA samples obtained from healthy individuals in South Texas. The presence of the C677T allele was determined by PCR and Hinf I digestion, and mutation A1298C by PCR and Mbo II digestion. A total of 310 alleles was examined for C677T in the Ashkenazi samples and 400 alleles in the non-Jewish samples. The rate of C677T among the Ashkenazi Jewish alleles was 47.7% as compared to 28.7% among the alleles from the non-Jewish population. The difference is statistically significant, P < 0.0005. Mutation A1298C was examined in 298 alleles of Jewish individuals and 374 alleles of non-Jewish counterparts from Texas. The rate of the A1298C mutation in the Jewish samples was 27.2% whereas in the non-Jewish was 35%. This was also statistically significant, P < 0.031. No individuals were homozygous for both mutations or were found to be homozygous for one mutation with heterozygosity of the other mutation, and that the C677T and the A1298C alleles did not occur in cis position. This study shows a unique distribution of C677T and the A1298C alleles among the Ashkenazi Jews. In spite of high frequency of C677T mutation, spina bifida is less common among Ashkenazi Jews. Further studies are needed to establish whether the C677T and the A1298C mutations have an impact on vascular disease in the Ashkenazi Jewish population.

Adeno-associated virus-mediated aspartoacylase gene transfer to the brain of knockout mouse for canavan disease.

Canavan disease (CD) is an autosomal recessive leukodystrophy caused by deficiency of aspartoacylase (ASPA). Deficiency of ASPA leads to elevation of N-acetyl-L-aspartic acid (NAA) in the brain and urine. To explore the feasibility of gene transfer to replace ASPA in CD, we generated a knockout mouse and constructed an AAV vector that encodes human ASPA cDNA (hASPA) followed by green fluorescent protein (GFP) after an intraribosomal entry site. We injected CD mice with rAAV-hASPA-GFP in the striatum and thalamus or injected rAAV-GFP identically into control animals. Three to five months after the injection, we determined the presence of ASPA in the CD mouse brain by ASPA activity assay, GFP expression, and Western blot analysis. While rAAV-GFP-injected animals displayed undetectable levels of ASPA, all detection methods revealed significant ASPA levels in rAAV-hASPA-GFP-injected CD mice. We evaluated the functional effects of rAAV-hASPA-GFP-mediated ASPA expression by standard histological methods, magnetic resonance spectroscopy (MRS) for in vivo NAA levels, and magnetic resonance imaging of CD mice. rAAV-hASPA-injected animals displayed a remarkable lack of spongiform degeneration in the thalamus. However, pathology in sites unrelated to the injected areas showed no improvement in histopathology. The improvement in thalamic neuropathology was also detectable via in vivo MRI. MRS revealed that in vivo NAA levels were also reduced. These data indicate that rAAV-mediated ASPA delivery may be an interesting avenue for the treatment of CD.

Detection of human herpesvirus-8 DNA in Kaposi’s sarcomas from iatrogenically immunosuppressed patients

BACKGROUND Kaposis sarcoma (KS) accounts for more than 5% of malignancies in immunosuppressed organ transplant patients (OKS). A new herpesvirus (HHV-8) was identified with high prevalence in biopsy specimens of AIDS-KS, endemic KS, and classic KS and in OKS. KS has also been associated with other underlying diseases in patients treated with corticosteroids, but this subset of KS has been reported to contain HHV-8 in only a few case reports. OBJECTIVE In this larger study, we determined the prevalence of HHV-8 in seven patients of Jewish origin in whom KS developed during immunosuppressive therapy for different primary diseases (ISKS). METHODS The study included HHV-8 DNA detection by polymerase chain reaction (PCR) coupled with Southern blot and sequence analysis as well as by in situ hybridization. RESULTS HHV-8 sequences were detected by PCR with confirmation by Southern blot and sequence analysis in 100% of the ISKS samples. Direct sequencing revealed several previously unknown base changes within the 208 bp region from open reading frame 26 (ORF26[208]) of HHV-8 in ISKS. CONCLUSION Ours is the largest known study describing the presence of HHV-8 in iatrogenic KS from immunosuppressed nontransplant patients and provides data of previously unknown sequence variations within the ORF26 of HHV-8 DNA.

Gastrinomas demonstrate amplification of the HER-2/neu proto-oncogene.

ObjectiveThis study determined whether genomic amplification of HER-2/neu or mutations of the p53 and ras genes were present in gastrinomas. Summary Background DataAmplification of HER-2/neu, a proto-oncogene related to the epidermal growth factor receptor, and mutation of the ras proto-oncogene and p53 tumor suppressor gene appear to play a role in the pathogenesis of some human cancers. Little is known about possible molecular alterations in gastrinomas, tumors that may be particularly virulent because of gastrin overproduction, resulting in the severe ulcer diathesis, the Zollinger-Ellison syndrome. MethodsThe differential polymerase chain reaction (PCR) procedure was used to detect amplification of the HER-2/neu gene in DNA samples from the novel human gastrinoma cell line (PT) and from paraffin-embedded samples of gastrinomas. Sequencing techniques were used to determine whether mutations of the p53 or ras (Ha-ras, N-ras, Ki-ras) genes were present. ResultsAmplification (> twofold) occurred in all gastrinoma tumor samples. Compared with normal pancreas or ileum, a 4− to 12-fold amplification of HER-2/neu was found in 3 gastrinomas, 3 to 3.3-fold in four samples and 2.1− to 2.4-fold in the remaining five tumors. A heterozygous point mutation in the p53 gene (codon 273) was found in a single sample; none of the gastrinomas contained a mutation of the ras genes. ConclusionsAmplification of the HER-2/neu gene, but not alterations of either p53 or ras, may be involved in the pathogenesis of gastrinomas. The unique PT cell line will be a useful model to further elucidate the molecular mechanisms that contribute to gastrinoma formation and growth.

Human papillomavirus (HPV) viral load and HPV type in the clinical outcome of HIV-positive patients treated with imiquimod for anogenital warts and anal intraepithelial neoplasia

Objective  To evaluate the efficacy of 5% imiquimod in HIV‐positive male patients with anogenital warts or anal intraepithelial neoplasia (AIN), and to elucidate whether human papillomavirus (HPV) type and viral load were important for clinical outcome and recurrences.

Serologic and molecular evidence of human herpesvirus 8 activation in renal transplant recipients.

This study was designed to determine whether there is serologic or molecular evidence of human herpesvirus 8 (HHV-8) activation in renal transplant patients, an immunocompromised population at risk for development of Kaposis sarcoma. Indirect immunofluorescence for detection of HHV-8 serum antibody and Southern blot polymerase chain reaction (PCR) for detection of viral DNA in whole blood were used. Seroprevalence and geometric mean titer (GMT) were significantly increased in the transplant group compared with healthy adults and were comparable to those in human immunodeficiency virus (HIV)-positive adults (transplant patients, 50% [GMT 1:210]; healthy adults, 7% [GMT 1:44]; HIV-positive patients, 73% [GMT 1:172]). Viral DNA was present in the blood of some renal transplant patients (3/33 PCR-positive) but in none of 20 healthy adults. Thus, there is both serologic and molecular evidence of HHV-8 activation in the renal transplant population compared with healthy adults (P<.01). The serologic results approximate those obtained for HIV-positive adults.