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Biomedical subjects

G J Buffone

Publications and source records attributed to G J Buffone.

At least 37 records · Page 2Linked to original sources
PubMed1993

The diagnosis of CMV pneumonitis in lung and heart/lung transplant patients by PCR compared with traditional laboratory criteria.

Polymerase chain reaction (PCR) amplification of CMV DNA recovered from bronchial alveolar lavage (BAL) and peripheral blood samples was compared with tissue culture, cytology, and/or histology for the earlier detection of CMV pneumonitis in 12 recipients of single-lung or heart/lung transplants. In patients with confirmed CMV pneumonitis, cytological evidence of CMV disease in BAL samples was detected 38 +/- 14 days posttransplantation, while tissue culture and PCR-positive results were noted as early as 30 +/- 4.0 days and 18 +/- 4.6 days, respectively. While PCR was positive earlier than culture in a number of cases, culture-positive results were subsequently obtained in each case, consistent with earlier detection of viral replication by PCR as opposed to detection of latent virus. CMV was detected by PCR in 6 of 24 blood samples from patients with confirmed or suspected CMV pneumonitis, while results of all 24 blood samples were negative when assayed by tissue culture. PCR-based testing was more sensitive than traditional tests, allowing detection of viral replication earlier than tissue culture in the posttransplant period. PCR could provide a powerful means of monitoring the immunocompromised patients in whom preemptive therapeutic intervention for CMV disease is desirable.

Biopsy↗
PubMed1992

Rapid epidemiologic analysis of cytomegalovirus by using polymerase chain reaction amplification of the L-S junction region.

A technique based on polymerase chain reaction (PCR) amplification was developed to facilitate the study of the epidemiology of cytomegalovirus (CMV). Consensus oligonucleotide primers from repetitive DNA sequences were designed to amplify interspersed repetitive sequences in an area of heterogeneity within the L-S junction region of the CMV genome, and PCR products were detected by gel electrophoresis. Purified CMV DNAs from 25 CMV isolates, 13 from members of five families in which person-to-person transmission was documented, 9 random clinical isolates of CMV, and 3 laboratory reference strains of CMV (Towne, Davis, and AD169), were analyzed. The gel electrophoretic patterns of DNA bands, or PCR profiles, produced by amplification with the L-S primers were unique for epidemiologically unrelated strains and laboratory reference strains, yet similar patterns were observed for epidemiologically related strains isolated from members of the same family. This method of rapid fingerprinting of CMV DNA within the hypervariable L-S junction region by PCR to produce strain-specific, variably sized PCR products should simplify the molecular epidemiologic analysis of CMV.

Base Sequence↗
PubMed1992

Heart allograft involvement with Epstein-Barr virus-associated posttransplant lymphoproliferative disorder.

We describe a 60-year-old man who underwent heart transplant and died 5 months later. At autopsy, the patient was found to have posttransplant lymphoproliferative disorder (PTLD), which was not suspected ante mortem. The PTLD involved the lung, lymph nodes, spleen, and kidney and the intima of right coronary artery of the transplanted heart. Epstein-Barr virus infection was demonstrated on direct gel and dot blot after polymerase chain reaction amplification. In situ hybridization for Epstein-Barr virus DNA confirmed the presence of Epstein-Barr virus-infected lymphocytes in the intima of the right coronary artery of the cardiac allograft. To our knowledge, this case represents the first report of involvement of a heart allograft by PTLD in which Epstein-Barr virus infection of the atypical cells is documented. Although primary allograft involvement by PTLD has rarely been reported in heart transplant recipients, recognition that this may occur is important, since the lymphoid infiltrate of PTLD must be distinguished from the lymphoid infiltrate of acute rejection to avoid inappropriate therapy.

Base Sequence↗
PubMed1991

Improved amplification of cytomegalovirus DNA from urine after purification of DNA with glass beads.

Cytomegalovirus can be detected in a variety of specimens including leukocytes, urine, saliva, feces, and various tissues by polymerase chain reaction (PCR) amplification of viral DNA. Although methods for amplification are fairly standard, sample preparation is not well characterized, especially for tissue. Typically, preparation of samples for PCR amplification ranges from simple boiling to phenol/chloroform extraction and quantification before testing. Several reports have described inhibition of the PCR in some samples types. Here we show that reliable detection of cytomegalovirus DNA in urine is obtained only after some degree of DNA purification, presumably because of PCR inhibition by a yet unidentified component present in a few of the urine samples tested. Glass, in the form of fine beads, was used to adsorb DNA such that protein and other substances could be selectively eluted before the recovery of DNA for PCR amplification. Urine samples prepared by this method did not show inhibition, and results correlated well with those by tissue culture for detection of cytomegalovirus.

Acquired Immunodeficiency Syndrome↗
PubMed1990

Demonstration of Epstein-Barr virus in primary central nervous system lymphomas by the polymerase chain reaction and in situ hybridization.

Primary lymphomas of the central nervous system (CNS) account for 0.3% to 1.5% of all intracranial neoplasms. Several reports have noted a coincidence between this neoplasm and serologic evidence of Epstein-Barr virus (EBV) infection, but in only a few instances has the EBV genome been demonstrated in these tumors. To further evaluate the frequency of this occurrence, we analyzed primary CNS lymphomas using nucleic acid hybridization methods and the polymerase chain reaction (PCR). In situ hybridization was used in selected cases. Sequences of EBV were found in two of nine cases by PCR and in situ hybridization. Southern blot hybridization of genomic DNA from these samples was negative for EBV. Both tumors arose in patients with conditions shown to produce secondary immunodeficiency, namely, chronic alcohol abuse and diabetes mellitus. We conclude that the association of EBV and CNS lymphoma is not restricted to patients with severe primary immune deficiency, and that PCR can be applied successfully to paraffin-embedded tissue for the detection of low-abundance viral sequences.

Central Nervous System Diseases↗
PubMed1990

Detection of mRNA from the immediate early gene of human cytomegalovirus in infected cells by in vitro amplification.

It would be desirable to have a facile means of detecting the presence of viral DNA or mRNA in any given biological sample, especially in view of the growing immunocompromised population in which the diagnosis of viral disease is a common problem. In vitro amplification of DNA using methods such as the polymerase chain reaction, offers a sensitive means of detecting both DNA and mRNA. We have used the polymerase chain reaction to detect DNA and mRNA from human cytomegalovirus infected human fibroblasts. Viral mRNA was differentiated from DNA using primers which flank a splice junction, resulting in a smaller product for the mRNA template. A cDNA was prepared from total RNA using a primer specific for the gene of interest, in this case the major immediate early transcript of human cytomegalovirus. The cDNA was then amplified using a modified polymerase chain reaction protocol. mRNA from the major immediate early gene was detected in cultured fibroblasts as early as 6 h after infection, and continued to be expressed for at least 96 h post infection. Sensitive and facile detection of viral mRNA should facilitate diagnostic and basic studies of viral pathogenesis.

Amino Acid Sequence↗
PubMed1990

Analysis of DNA in fresh and fixed tissue by the polymerase chain reaction.

The polymerase chain reaction (PCR) was used to amplify viral or oncogene sequences from frozen or formalin-fixed, paraffin-embedded tissue sections. Methods for preparing fixed, embedded colonic tissue for PCR amplification of c-K-ras sequences from genomic DNA and for amplification of viral DNA from other tissues, including brain, lung, and liver, were evaluated. The effect of formalin fixation on the efficiency of amplification was also determined. While there seemed to be only a modest variation in the efficiency of the PCR for amplification of single-copy human genes, regardless of the methods used for tissue preparation, amplification of viral DNA sequences against a human genomic DNA background was more efficient when the DNA was purified to some degree before amplification of the tissue. We used the PCR to examine frozen and fixed embedded tissue sections for the presence of Epstein-Barr virus (EBV) and cytomegalovirus (CMV) DNA. One patient with a heart-lung transplant succumbed to a lymphoproliferative disorder, and EBV genome was present in tissues with abnormal lymphoid infiltrates. CMV was also present in bronchial lavages from the same patient, where cytologic diagnosis was not apparent. Another patient with a liver transplant showed CMV genome in multiple liver biopsies, with negative histologic results for CMV. In vitro DNA amplification with the PCR demonstrated sensitivity superior to that of histology in detecting CMV and EBV in the cases examined.

Adult↗
PubMed1989

New methodologies. Their role in pediatric pathology.

Recombinant DNA technology is providing the means for early and specific etiologic diagnoses of infectious and immunologic diseases, replacing or complementing older methodologies. The new tools that have been so useful in detecting gene rearrangements in leukemias and lymphomas are being applied to the unresolved questions of embryogenesis and disorderly cell differentiation and are being used to completely re-map the nervous system. Flow cytometry and cell sorting are becoming standard features of clinical laboratories and are instrumental not only in defining alterations in lymphoid cell populations but in examining cellular functions as well as surface markers. Bone marrow and organ transplantation for genetic, metabolic, and neoplastic diseases will be performed much more effectively as these newer technologies are applied to the selection of compatible donors and to the follow-up of rejection and infectious complications.

Adolescent↗
PubMed1989

Factors contributing to mortality in lung transplant recipients: an autopsy study.

Despite improved surgical techniques and advances in immunosuppressive therapy, posttransplant mortality rates remain significantly high in lung transplant patients. Since 1985, 3 of 6 single lung recipients, 3 of 3 double lung recipients, and 4 of 7 heart-lung recipients have died and undergone autopsy. We reviewed the autopsy findings in these patients to determine the type and frequency of pathologic processes associated with mortality. One or more infectious processes was found in every patient at autopsy. Gram-negative bacterial pneumonia and sepsis, found in 7 and 8 of 10 autopsy cases, respectively, were by far the most frequent contributing factors to mortality. Epstein-Barr virus infection was demonstrated in one patient using polymerase chain reaction amplification. Acute transplant rejection was found in only 2 patients and therefore is a much less common factor in the death of lung transplant recipients. Diffuse alveolar damage occurred in 6 patients and bronchiolitis obliterans occurred in 3 patients. These latter two processes may have different etiologies in different patients.

Adolescent↗
PubMed1989

Linkage disequilibrium, cystic fibrosis, and genetic counseling.

Strong linkage disequilibrium occurs between the cystic fibrosis (CF) locus and polymorphisms detected with the DNA probes XV-2c and KM-19. In a North American population, 86% of CF chromosomes occur with a haplotype which occurs on only 14% of normal chromosomes. An individual homozygous for the highest-risk haplotype has an 81-fold greater probability of carrying a CF allele than does an individual homozygous for the lowest-risk haplotype. The linkage-disequilibrium data can be used for prenatal diagnosis and genetic counseling in CF families. The data are useful in 1-in-4-risk pregnancies when DNA is not available from the propositus and in counseling close relatives of CF families. Serious problems arise with some pregnancies which remain at intermediate risks after analysis, and families are left with difficult decisions. It is not clear that genetic testing for couples at less than 1-in-4 risk is cost-effective or standard care, but use of linkage-disequilibrium data will provide more accurate risk probabilities in a substantial proportion of cases if such testing is carried out. Our results emphasize the need for a specific biological or molecular carrier test. This experience in using linkage-disequilibrium and linkage data in combination for genetic counseling provides a model system for the diagnosis of other disorders.

Cystic Fibrosis↗
PubMed1988

Detection of cytomegalovirus in urine from newborns by using polymerase chain reaction DNA amplification.

Polymerase chain reaction (PCR) amplification was used to detect cytomegalovirus (CMV) in tissue culture and in urine specimens from newborns. Synthetic oligonucleotide primer pairs were used to amplify DNA from the major immediate-early and the late antigen genes of CMV. Amplified products were detected by gel electrophoresis and by dot-blot hybridization with oligonucleotide probes. Using one or both of the primer pairs and associated probes, we found 46 different tissue culture isolates of CMV that were positive; no reaction products were detected when the same primers and probes were used to amplify other herpes family viruses or human genomic DNA. Urine samples from 44 congenitally infected infants were positive when tested with one or both primer pairs and probes. When compared with tissue culture, detection by gel electrophoresis provided a sensitivity of 93%, a specificity of 100%, and a predictive value of a positive result of 100%. Dot-blot analysis raised the sensitivity to 100%. We conclude that PCR amplification may be a valuable tool for diagnosing congenital CMV infection.

Blotting, Southern↗
PubMed1988

DNA hybridization assay for congenital cytomegalovirus infection.

An in vitro DNA hybridization assay was used to test 281 newborns for congenital infection with cytomegalovirus. The assay utilized an abbreviated method for DNA preparation and a dot blot assay that provided good sensitivity (100%) and specificity (98.9%) when compared with standard tissue culture, yet substantially reduced the total time of analysis. This assay would be a useful adjunct to tissue culture to diagnose newborns with congenital infection with cytomegalovirus.

Cytomegalovirus Infections↗
PubMed1988

Prenatal diagnosis of cystic fibrosis: microvillar enzymes and DNA analysis compared.

We have established reference ranges for three microvillar intestinal enzymes--alkaline phosphatase (EC 3.1.3.1), gamma-glutamyltransferase (EC 2.3.2.1), and leucine aminopeptidase (EC 3.4.1.1)--measured in amniotic fluid in a reference population of 1875 women presenting for routine amniocentesis. These data were derived for use in prenatal diagnostic studies in a population at risk (1:4) for cystic fibrosis. False-positive or indeterminate results were noted for fewer than 3.5% of all low-risk cases for each enzyme evaluated. Total alkaline phosphatase and its isoenzymes and leucine amino-peptidase and gamma-glutamyltransferase were measured in amniotic fluid sampled between the 15th and 19th weeks of gestation. Restriction fragment length polymorphism analysis of DNA was also performed when possible. In 52 cases examined for cystic fibrosis thus far, 46 were diagnosed on the basis of DNA analysis and (or) by sweat testing; for the other six cases, only abnormal enzyme results were obtained before termination of pregnancy. Predictions based on microvillar enzyme results were falsely negative in three cases. In only one case was there a discrepancy between enzyme results and DNA analysis. Diagnostic accuracy was highest during the 17th and 18th week of gestation. Preliminary results suggest the false-negative rate of this diagnostic strategy may be greater than or equal to 10%.

Alkaline Phosphatase↗
PubMed1987

Prenatal diagnosis of cystic fibrosis using linked DNA markers and microvillar intestinal enzyme analysis.

Prenatal diagnosis was performed for 47 pregnancies with 1 in 4 risk of cystic fibrosis, including 7 cases analyzed with linked DNA markers, 16 cases analyzed by microvillar intestinal enzyme testing, and 24 cases where both methods of testing were attempted. DNA was obtained by chorionic villus sampling in 10 cases and by amniocentesis in 21 cases, and diagnosis was based on analysis with the tightly linked DNA markers D7S8 and met. DNA analysis using these probes was fully informative in 74.4% of 90 couples with 1 in 4 risk. In 18 cases where both DNA results and microvillar intestinal enzyme data were diagnostic, there was agreement regarding the predicted status of the fetus. No adequate diagnosis was achieved in two cases where both diagnostic tests were attempted. Outcome is known for 24 pregnancies including 10 where DNA analysis was diagnostic, and no errors in diagnosis were detected. Prenatal diagnosis of cystic fibrosis using DNA markers is highly informative and accurate, but microvillar intestinal enzyme analysis remains a valuable part of a complete diagnostic program.

Alkaline Phosphatase↗
PubMed1987

Growth and hepatospecific gene expression of human hepatoma cells in a defined medium.

The production of albumin, alpha-fetoprotein (AFP), and alpha-1 antitrypsin has been compared among human hepatoma cells cultured in medium containing serum, medium containing hormones and growth factors, and a basal medium containing selenium as the only supplement. Growth is sustained in all three media, and the expression of all three proteins was maintained for over 4 mo. in the various media. However, the quantitative production of albumin and AFP were dramatically different in the three media. Two hormones, insulin and triiodothyronine, influenced the level of secreted proteins. Triiodothyronine increases the amount of secreted albumin whereas insulin at 10 micrograms/ml reduced the level of total secreted protein.

Albumins↗