PubMed Health⌕ Search

Biomedical subjects

G J Nuovo

Publications and source records attributed to G J Nuovo.

At least 19 recordsLinked to original sources
PubMed2006

In situ detection of SOCS and cytokine expression in the uterine cervix from HIV/HPV coinfected women.

The purpose of this study was to look for associations between a newly described class of suppressors of cytokine signaling (SSI/SOCS) and cytokine expression in the uterine cervix from HIV/HPV coinfected women. We examined the pro-inflammatory cytokines TNF-alpha and IL-6 since their expressions are linked and responsible for many aspects of both localized and systemic inflammatory responses. Further, expression of SSI/SOCS has been implicated in the negative feedback regulation of cytokine receptor signaling. PCR-amplified HIV-1 cDNA was noted mainly in the stroma, showing a perivascular distribution, and most of the infected cells colabeled with the macrophage marker CD68. The distribution of IL-6 and TNF-alpha was in the same area to HIV-1 and much greater than normal cervices from women with no evidence of viral infection. SOCS/SSI-1 and -3 mRNA positive cells in the uterine cervix were commonly detected in these noninfected cervical tissues; however, very few cells that contained SOCS were evident in areas where HIV-1, TNF-alpha, and IL-6 expressing cells were found. This suggests that viral-related suppression of SOCS/SSI-1-3 expression may be a factor in the marked local enhancement of TNF-alpha and IL-6 production which, in turn, may help facilitate viral spread; however, further studies should be done in order to elucidate the exact mechanisms of SOCS in the cervix.

Adult↗
PubMed2001

Increased incidence of atypical Papanicolaou tests from ThinPreps of postmenopausal women receiving hormone replacement therapy.

BACKGROUND: The authors noticed a significant increase in the rate of atypical squamous cells of undetermined significance (ASCUS) diagnoses in postmenopausal women receiving hormone replacement therapy with ThinPrep (monolayer) Papanicolaou (Pap) tests (8.3%) as compared with conventional Pap tests (2.3%; P = 0.025). The purpose of this study was to correlate these morphologic changes with human papillomavirus (HPV) DNA detection by in situ hybridization and clinical outcome. METHODS: Twenty-five monolayer Pap tests diagnosed as ASCUS in postmenopausal women receiving hormone replacement, as well as 2 cases of low-grade squamous intraepithelial lesion (SIL) from the same group, were destained in acid alcohol. In situ HPV hybridization was performed with a consensus probe that can detect greater than 90% of the HPV types that occur in cervical SIL. In addition, 11 of the ASCUS tests were analyzed with the periodic acid-Schiff (PAS) stain. RESULTS: All 25 ASCUS tests were negative for HPV DNA as compared with a detection rate of 15 of 17(88%) for low-grade SIL. Glycogen that was PAS positive was present in the atypical squamous cells of the postmenopausal women for whom HPV was not detected. Clinical follow-up demonstrated that only 2 of the 25 women with ASCUS had a subsequent biopsy proven SIL. CONCLUSIONS: The ThinPrep preparation can produce artifactual HPV-like changes in postmenopausal women receiving hormone replacement therapy that may be caused by glycogenization and, because of the high incidence of HPV negativity and negative clinical outcome, are best considered as benign cellular changes.

Aged↗
PubMed2001

Age-related hypermethylation of the 5' region of MLH1 in normal colonic mucosa is associated with microsatellite-unstable colorectal cancer development.

Hypermethylation of the MLH1 promoter underlies most sporadic colorectal cancers with microsatellite instability (MSI). To investigate the role of hypermethylation in the normal colonic mucosa as a possible precursor lesion, we studied 700 bp upstream of MLH1 covering 51 CpG sites. We found partially methylated alleles in 15 of 34 (44%) patients <60 years of age and 20 of 24 (83%) patients > or =80 years of age (P = 0.0026). Fully methylated alleles were present in 18 of 33 (55%) patients with MSI+ tumors but in only 18 of 90 (20%) patients with MSI- tumors (P = 0.00019). By in situ analysis, methylation was patchy and located mainly in the cryptal regions close to the lumen. We conclude that the spread of methylation in the MLH1 promoter in the normal colonic mucosa is closely associated with age and the development of sporadic MSI+ colorectal cancers.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Coxsackie virus infection of the placenta associated with neurodevelopmental delays in the newborn.

OBJECTIVE: To determine if viral infection of the placenta was associated with long-term neurodevelopmental delays in the newborn. METHODS: Placental tissue from seven newborn infants with severe respiratory failure and subsequent neurodevelopmental abnormalities as well as ten normal controls and five cases of known placental infection (cytomegalovirus, herpes simplex virus, and parvovirus) were tested by in situ hybridization or reverse transcriptase in situ polymerase chain reaction (PCR) for adenovirus, coxsackie virus, cytomegalovirus, Epstein Barr virus, herpes simplex virus, influenza A virus, picornavirus, polyoma virus, parvovirus, respiratory syncytial virus, rotavirus, and varicella zoster virus. RESULTS: Coxsackie virus RNA was detected in six of the seven cases, and in none of the ten normal controls or five cases with known viral infection. Viral RNA localized primarily to the Hofbauer cells and trophoblasts of the terminal villi. Immunohistochemical analysis for the coxsackie virus antigen VP1 yielded equivalent results. CONCLUSIONS: In utero coxsackie virus of the placenta is associated with the development of severe respiratory failure and central nervous system sequelae in the newborn. This underscores the importance of detailed pathologic and viral examination of the placenta in cases of systemic illness in the newborn.

Adolescent↗
PubMed2001

Histologic distribution of fatal rotaviral infection: an immunohistochemical and reverse transcriptase in situ polymerase chain reaction analysis.

Rotaviral infection is the most common cause of gastroenteritis in young children. Although rotavirus infection has a high morbidity and mortality rate in animals, in most cases in the United States the disease appears to be self-limited in humans. We report on 2 cases of fatal rotaviral infection in a 1 year old and a 4 year old. In each case, the illness showed a rapid systemic course dominated by cardiac and central nervous system involvement; in one case, rotaviral infection was documented by stool culture. Viral RNA was localized by reverse transcriptase in situ polymerase chain reaction to the mucosal cells of the small and large intestine and to many other tissues, including the heart and central nervous system, where it was noted in the endothelial cells of the microvasculature. Immunohistochemical analysis for the virus showed an identical histologic distribution in the intestinal epithelial cells and the systemic microvasculature. It is concluded that rotaviral infection can lead to a fatal disseminated infection in humans and the mechanism of this complication is based on a diffuse endothelialitis and concomitant tissue damage.

Child, Preschool↗
PubMed2001

Highly differentiated keratinizing squamous cell cancer of the cervix: a rare, locally aggressive tumor not associated with human papillomavirus or squamous intraepithelial lesions.

The purpose of this study is to report an unusual variant of cervical squamous cell carcinoma, not associated with either human papillomavirus infection or antecedent squamous intraepithelial lesions. Five women had a diagnosis of invasive cervical cancer discovered at hysterectomy performed for prolapse (two cases), leiomyoma (one case), or a vaginal fistula (two cases). The women ranged in age from 47 to 78 years (mean 59 years). Four of the five had a history of normal Papanicolaou (Pap) smears; the other had a Pap smear diagnosis of atypical squamous cells of undetermined significance (ASCUS). All had large cervical tumors (two with parametrial involvement and one with vaginal involvement) that showed extensive keratin formation, an inverted pattern of growth, and, except for one case, minimal cytologic atypia. There was extensive hyperkeratosis and parakeratosis adjacent to each tumor; none had evidence of squamous intraepithelial lesion. Human papillomavirus testing by polymerase chain reaction in situ hybridization and reverse-transcribed polymerase chain reaction in situ was negative in each case, compared with a detection rate of 107 of 108 (99%) for squamous intraepithelial lesion-associated cervical squamous cell and adenocarcinomas. Two of the women died of extensive local recurrence; two other women were recently diagnosed. We conclude that highly differentiated keratinizing squamous cell carcinoma of the cervix is a rare entity not associated with human papillomavirus infection or squamous intraepithelial lesion and thus difficult to detect on routine cervical cancer screening.

Adult↗
PubMed2001

In situ determination of B-cell heavy chain and kappa/lambda light chain expression patterns: methodology and clinical utility.

The authors describe a reverse transcriptase in situ polymerase chain reaction (RT in situ PCR) method that allows the determination of B-cell clonality as defined by expression of kappa and lambda mRNA as well as the different VH families that comprise the entire heavy chain (IgH) sequence using paraffin-embedded, formalin-fixed tissue. Polyclonal expression of B-cell light and heavy chains was documented in two reactive lymph nodes and a spleen in the same histologic distribution as the B-cell marker CD20. Monoclonal expression of kappa versus lambda mRNA was documented in 11 cases of B-cell lymphoma and was corroborated in 5 cases by flow cytometry; each case showed monoclonal expression of IgH. The authors analyzed seven additional tissues where a definitive diagnosis of B-cell lymphoma could not be rendered based on histologic, immunohistologic. and clinical analysis. RT in situ PCR for IgH and kappa versus lambda expression differentiated the B-cell lymphomas (n = 2) from reactive B-cell processes (n = 3) and from two cases of B-cell-rich T-cell lymphoma. The described RT in situ PCR methodology allows routine determination of the monoclonal versus multiclonal expression patterns of B cells, which will facilitate the diagnosis of B-cell lymphomas and aid in understanding the pathogenesis of B-cell malignancies using archival, paraffin-embedded tissues.

Antigens, CD20↗
PubMed2001

In situ determination of T-cell receptor beta expression patterns.

A definitive diagnosis of T-cell lymphoma may be contingent on the rearrangement profile of the T-cell receptor. This is most accurately done by molecular analysis of the beta-chain of the T-cell receptor (TCR beta) by Southern blotting hybridization that requires unfixed tissue. We describe a reverse transcriptase in situ PCR (RT in situ PCR) method that permits the target-specific direct incorporation of the reporter nucleotide into the different transcripts that comprise the TCR beta, using paraffin-embedded, formalin-fixed tissue. Each of the 25 possible V beta segment rearrangements was documented in three lymph nodes with nonspecific lymphadenitis, with clonal expansion evident in a case of metastatic melanoma. Monoclonal expression was documented in seven tissues diagnostic of a T-cell lymphoma. We analyzed five additional tissues for which a definitive diagnosis of T-cell vs B-cell lymphoma could not be rendered on the basis of histological, immunohistological, and flow cytometric analysis. RT in situ PCR for TCR beta expression with CD3 co-labeling demonstrated which of these lesions was a B-cell-rich T-cell lymphoma. We conclude that the RT in situ PCR methodology will allow the routine determination of monoclonal vs multiclonal expression patterns of the TCR beta using archival paraffin-embedded tissues.(J Histochem Cytochem 49:139-145, 2001)

Diagnosis, Differential↗
PubMed2001

Co-labeling using in situ PCR: a review.

In situ amplification permits the histological localization of low-copy DNA and RNA targets. However, in many instances it would be useful to know the specific phenotype of the target-containing cell or to ascertain the distribution of a different nucleic acid sequence in the same tissue section. This review describes a methodology that allows co-in situ localization of two nucleic acid targets or a DNA/RNA sequence and a protein in paraffin-embedded, formalin-fixed tissue. The key variable for detection of low-copy RNA targets by RT in situ PCR is optimal protease digestion to permit cDNA target-specific incorporation of the reporter nucleotide. This is achieved via inactivation of nonspecific DNA synthesis by overnight DNase digestion. The key variable for immunohistochemical localization of proteins is to determine the effect of protease digestion on the antigen-based signal intensity. Background for DNA targets by in situ hybridization or, for targets present in 1-10 copies per cell, PCR ISH is dependent primarily on probe concentration and the stringency of the post-hybridization wash. Radioactive 3H-labeled nucleotides permit an excellent distinction with colorimetric signals for co-localization, although two distinct chromogens can in many instances allow successful localization of two different targets.

DNA↗
PubMed2001

Longterm efficacy of interferon-alpha for extrahepatic disease associated with hepatitis C virus infection.

OBJECTIVE: To investigate longterm responsiveness to interferon-alpha (lFN-alpha) of patients with extrahepatic manifestations of hepatitis C virus (HCV) in a nonendemic area. METHODS: We prospectively evaluated 11 patients with extrahepatic manifestations of HCV infection, including 10 with Type II cryoglobulins, treated with IFN-alpha--9 had cutaneous vasculitis, 6 arthralgias, 7 neuropathy, and 4 glomerulonephritis. Liver biopsies were performed on all patients, although 6/11 had normal liver function tests. All received 3 M units IFN-alpha tiw, with total length of treatment ranging from 3 mo to 5 yrs. Periodic assessments were made of clinical activity, biochemical variables, cryoglobulin quantitation, and HCV copy number. RESULTS: Three patients were withdrawn because of toxicity. Three were nonresponders at 6, 16, and 17 mo of therapy, based on persistence of HCV RNA in blood, cryoprecipitates, and peripheral blood mononuclear cells. One patient was a partial responder at 3 yrs, with 2 major flares of cutaneous vasculitis occurring on separate attempts to withdraw IFN-alpha. Three patients (27.2%) were complete responders based on resolution of symptoms (purpura, neuropathy) and disappearance of cryoprecipitates and HCV RNA, but only one successfully tapered IFN-alpha after 3 yrs of treatment, with sustained resolution at followup 15 mo later. CONCLUSION: IFN-alpha is safely tolerated for prolonged periods in patients with extrahepatic HCV infection, and is particularly effective for treatment of cutaneous vasculitis. Careful monitoring is needed for evolution of liver pathology to cirrhosis, or for progression of renal or neurologic disease.

Adult↗
PubMed2000

Microsphere-adenoviral complexes target and transduce the glomerulus in vivo.

BACKGROUND: Developing new treatments for glomerulonephritis makes the glomerulus a logical target for gene therapy. Microspheres may lodge in the glomerulus, and replication-deficient recombinant adenoviruses are potent mediators of gene transfer. We postulated that adenoviral-microsphere complexes could result in DNA transfer (transduction) into glomerular cells in vivo. METHODS: Two adenoviruses, each one containing a luciferase or beta-galactosidase (beta-gal) transgene expression cassette, were complexed to polystyrene microspheres. To assess in vivo glomerular transduction with this tool, male Sprague-Dawley rats underwent aortic injections with adenovirus linked to 11 or 16 microm diameter microspheres. RESULTS: After 48 hours, adenoviral-microsphere complexes resulted in transduction of up to 19% of glomeruli per kidney section. Endothelial and mesangial cells were transduced with this approach, and transprotein expression persisted for 21 days. Transduction efficiency was greater in the 16 microm group. For all rats, there was a strong correlation between kidney luciferase levels and the number of beta-gal-positive glomeruli (r = 0.87), indicating that transgene expression was primarily glomerular in location. This was supported by reverse transcriptase in situ polymerase chain reaction, which demonstrated glomerular localization of the beta-gal transgene. CONCLUSIONS: The aortic injection of adenoviral-microsphere complexes transduces the glomerulus in vivo and may be a useful tool in developing approaches to gene therapy of glomerular disease.

Adenoviridae↗
PubMed2000

The role of human papillomavirus in gynecological diseases.

Papanicolaou (Pap) smears of the cervix and biopsies of the cervix, vagina, vulva, and penis are a substantial portion of the work load of any anatomic pathology laboratory. The importance of samples from these sites is highlighted by the fact that their most common disease is dysplasia, which is sexually transmitted and a recognized risk factor for carcinoma. Human papillomavirus (HPV) is an essential co-factor for the development of lower genital tract dysplasia. Molecular testing for HPV has revealed a great deal about the pathophysiology of dysplasia and its evolution to carcinoma. Importantly, it now allows diagnostic pathologists the opportunity to be more accurate in their assessment of common conditions such as Pap smears of squamous atypia and biopsies equivocal for dysplasia. This review highlights the role of HPV in the evolution of disease in the lower genital tract and stresses the utility of HPV testing to the diagnostic pathologist.

Female↗
PubMed2000

The histologic spectrum of epidermodysplasia verruciformis.

The classic histologic presentation of epidermodysplasia verruciformis is a verruca plana-type lesion with minimal hyperkeratosis and acanthotic areas where the cells contain perinuclear halos and blue-gray pallor. Whereas these lesions have a high malignant potential, it is important to elucidate the histologic spectrum of this entity and to differentiate it from its mimics. Fifteen skin biopsies from people with multiple cutaneous warts clinically suspicious for epidermodysplasia verruciformis were analyzed both histologically and for human papillomavirus (HPV) deoxyribonucleic acid (DNA) by in situ hybridization. Ten of the lesions contained HPV DNA, either type 5 (n = 6), type 8 (n = 3), or type 51 (n = 1). Only three of these lesions showed typical verruca plana. The histologic marker of HPV DNA in the other seven viral-positive cases was rare perinuclear halos in association with an irregular granular layer. The other five cases, which were also negative for viral DNA after polymerase chain reaction in situ hybridization, rarely demonstrated the abrupt variation in keratohyaline granules and concomitant perinuclear halos. The authors conclude that there is a wide spectrum of histologic changes in epidermodysplasia verruciformis and that viral testing in conjunction with the histologic and clinical findings can differentiate this premalignant entity from its mimics.

Adult↗
PubMed2000

In situ strand displacement amplification: an improved technique for the detection of low copy nucleic acids.

Strand displacement amplification (SDA) is an isothermal, enzymatic method for the exponential synthesis of target nucleic acids which traditionally requires two pairs of oligoprimers. One pair, described as "bumpers", serves to generate target specific single stranded DNA after heat denaturation of double stranded DNA subsequent to primer annealing and extension. We report here the in situ (IS) application of SDA (IS-SDA) using nicked DNA targets which eliminate the need for heat denaturation and bumpers. Productive nicking can be generated by restriction endonuclease activity, exposure to dry heat, paraffin embedding, or mild depurination. IS-SDA, followed by in situ hybridization (ISH) allows for the routine detection of single copy HIV-1 DNA as well as HPV DNA in both cell lines and tissue sections. Detection of hepatitis C RNA in liver biopsy specimens using reverse transcription IS-SDA (RT-IS-SDA) was performed with direct incorporation of the reporter nucleotide. The amplification of RNA targets does require bumper primers, although not an initial denaturation step. Extensive analyzes demonstrated that SDA-ISH is as sensitive as polymerase chain reaction (PCR)-ISH and that each are more sensitive than in situ hybridization coupled to catalyzed signal amplification (CSA). Moreover. SDA-ISH is technically much simpler than PCR-ISH because it is isothermal and does not require an initial denaturation.

DNA Primers↗
PubMed1999

In situ detection of the hypermethylation-induced inactivation of the p16 gene as an early event in oncogenesis.

We have developed a technique, methylation-specific PCR in situ hybridization (MSP-ISH), which allows for the methylation status of specific DNA sequences to be visualized in individual cells. We use MSP-ISH to monitor the timing and consequences of aberrant hypermethylation of the p16 tumor suppresser gene during the progression of cancers of the lung and cervix. Hypermethylation of p16 was localized only to the neoplastic cells in both in situ lesions and invasive cancers, and was associated with loss of p16 protein expression. MSP-ISH allowed us to dissect the surprising finding that p16 hypermethylation occurs in cervical carcinoma. This tumor is associated with infection of the oncogenic human papillomavirus, which expresses a protein, E7, that inactivates the retinoblastoma (Rb) protein. Thus, simultaneous Rb and p16 inactivation would not be needed to abrogate the critical cyclin D-Rb pathway. MSP-ISH reveals that p16 hypermethylation occurs heterogeneously within early cervical tumor cell populations that are separate from those expressing viral E7 transcripts. In advanced cervical cancers, the majority of cells have a hypermethylated p16, lack p16 protein, but no longer express E7. These data suggest that p16 inactivation is selected as the most effective mechanism of blocking the cyclin D-Rb pathway during the evolution of an invasive cancer from precursor lesions. These studies demonstrate that MSP-ISH is a powerful approach for studying the dynamics of aberrant methylation of critical tumor suppressor genes during tumor evolution.

Carcinoma, Squamous Cell↗
PubMed1999

Epstein-Barr virus infection of renal proximal tubule cells: possible role in chronic interstitial nephritis.

Chronic interstitial nephritis frequently accompanies renal diseases of different etiologies. Far less common is the entity of primary interstitial nephritis wherein the glomerular and vascular structures of the kidney are not the primary focus of the disease process. Using in situ hybridization and the polymerase chain reaction, we detected DNA from the Epstein-Barr Virus (EBV) exclusively in renal tissue of patients with the idiopathic variety of chronic interstitial nephritis. The EBV genome, but not that of cytomegalovirus or adenovirus, was detected primarily in renal proximal tubule cells. Furthermore, the CD21 antigen, which serves as the receptor for EBV in B lymphocytes, was detected by immunocytochemistry primarily on proximal tubule cells and was markedly upregulated in the EBV-infected tissue. Western blot analysis of primary cultures of normal proximal tubule cells identified a 140-kDa protein, confirming the expression of the CD21 antigen. Colocalization experiments using proximal and distal tubule markers confirmed that EBV DNA and the CD21 antigen are found primarily in proximal tubule cells. EBV infection of renal proximal tubular cells may participate in evoking a cellular immune response that results in a damaged renal interstitium.

Adult↗