Association of tumor necrosis factor-alpha gene expression and apoptotic cell death with regression of Shope papillomas.

The objective of this study was to test the hypothesis that spontaneous regression of Shope papillomas involves tumor necrosis factor-alpha and apoptotic cell death of the papilloma cells. In situ hybridization using RNA probes of rabbit tumor necrosis factor-alpha revealed tumor necrosis factor-alpha mRNA in most of the numerous mononuclear cells infiltrating the upper dermis of regressing papillomas and at the dermoepidermal junction. Such cells in progressing papillomas were much fewer in number and were located in the deeper dermis. In situ terminal deoxynucleotidyl transferase assay demonstrated DNA strand breaks in many scattered epidermal keratinocytes of regressing papillomas but in only a few thin layers just beneath the horny layer in progressing papillomas. Electron microscopy demonstrated that regressing papillomas contained many apoptotic bodies and keratinocytes showing apoptotic changes such as chromatin condensation, degradation of condensed nuclei, surface protuberances, and a filamentous degeneration, as well as infiltrating lymphocytes and macrophages. We propose that tumor necrosis factor-alpha produced by infiltrating mononuclear cells probably plays a role in the papilloma regression.

Podofilox-induced regression of Shope papillomas may be independent of host immunity.

We tested the hypothesis that infiltrating leukocytes might contribute to papilloma destruction following podofilox treatment. New Zealand White (NZW) rabbits were inoculated with cottontail rabbit papillomavirus (CRPV) onto abraded areas of the dorsal skin. At 21 d after viral inoculation, 5.0% podofilox solution was applied to some papillomas, whereas others were used as controls. Three rabbits were sacrificed at each of three different periods after treatment initiation (1, 4, and 7 d). Four monoclonal antibodies (MoAbs), RG-16 (for B cells), L11/135 (specific for T cells), 2C4 (specific for class II antigen), and Ki67 (specific for proliferating cells), were used in an immunohistochemical study. All positive cells and total cells in the field were counted with an ocular grid. After 1 d of treatment, proliferation of papilloma cells was strongly suppressed in treated papillomas, but leukocytic infiltration was not altered. At 4 d and 7 d of treatment, there were substantial increases (about two to three times) in the numbers of B and T cells and class II-expressing leukocytes. The upper layers of the papillomas were highly necrotic and cell proliferation was absent in all layers. These data support the view that podofilox has a direct toxic effect on papilloma tissue. Leukocyte infiltration is not strongly associated with papilloma tissue and may not contribute to papilloma destruction.

Endocervical and endometrial adenocarcinoma: an immunoperoxidase and histochemical study.

Carcinoembryonic antigen (CEA) has been demonstrated in endocervical but not in endometrial adenocarcinomas, suggesting a valuable differentiating feature. Using an immunoperoxidase technique for CEA, alcian blue (AB)-periodic acid-Schiff stain for acidic and neutral mucosubstances, respectively, and Mayer's mucicarmine for epithelial mucosubstances, 13 endocervical and 21 endometrial adenocarcinomas were studied to delineate differentiating criteria. CEA was present in all endocervical adenocarcinomas with strongly positive immunostaining in 69%, diffusely throughout the tumor in 77%, intracellularly in 77%, in luminal secretions in 39%, and at the cell surface in 31%. Only 11 (52%) endometrial adenocarcinomas contained CEA, 82% with weak immunostaining, 64% focally, 82% at the cell surface and 36% intracellularly. Mucin was present in 12 (92%) endocervical lesions with AB-positive intracellular and luminal secretion in 83%, strong positivity in 42%, and a diffuse distribution in 60% with all three stains. Moderate or small amounts of mucin were present in 19 (91%) endometrial lesions, focally in 80%, in luminal secretions in 94%, and intracellularly in 42%. Thus, the majority of endocervical adenocarcinomas showed abundant diffusely distributed intracellular CEA and mucin, and mucinous luminal secretions. Half of the endometrial adenocarcinomas contained focal small amounts of apical surface CEA. Mucin was present focally particularly in luminal secretions.

Viral diagnosis by in situ hybridization. Description of a rapid simplified colorimetric method.

An improved method of colorimetric in situ hybridization for the diagnosis of viral infections in standard formalin-fixed, paraffin-embedded tissue sections has been developed. This method employs a 2-hour hybridization with biotin-labeled DNA probes followed by direct colorimetric detection with avidin-alkaline phosphatase complexes. Visual results are obtained within 8 h of cutting the tissue section. Specific histologic localization of cytomegalovirus and adenovirus genetic information has been achieved in infected lung tissues from autopsy or biopsy. Simultaneous denaturation of tissue and probe DNA at elevated temperature (100-105 degrees C) resulted in increased signal. It is our suggestion that these denaturing conditions may be required to denature more fully formalin cross-linked tissue DNA and favor penetrance of probe into the tissues. Comparison of the results of hybridization and viral culture for the diagnosis of CMV infections suggest that in clinical situations hybridization will allow specific diagnosis of productive viral infection more rapidly than viral culture with some loss in sensitivity. Colorimetric in situ DNA hybridization offers the surgical pathologist a powerful new technique that provides an alternative to immunocytochemistry and electron microscopy in the diagnosis of viral pathogens.

Agarose-embedded tissue arrays for histologic and genetic analysis.

To facilitate the histologic analysis of large numbers of 7-day-old zebrafish (Danio rerio), a method has been developed to process them in agarose-embedded arrays. Using thin tissue sections, the morphology of cells and tissues can be examined microscopically to investigate a variety of biologic processes. Because of their small size, precise arrangement of the larvae is necessary to section them simultaneously. A technique was designed to embed groups of zebrafish larvae in a single plane in agarose before sectioning. Stained tissue sections of thousands of larvae can be examined efficiently using this embedding method. In addition to histologic analysis, PCR-based genotypic analysis of DNA from individual larval sections is also possible. This technique can be modified to accommodate any study that requires the histologic examination of many pieces of tissue.

In vivo anti-papillomavirus activity of nucleoside analogues including cidofovir on CRPV-induced rabbit papillomas.

A series of nucleoside analogues were tested for in vivo anti-papillomavirus activity using the cottontail rabbit papillomavirus (CRPV) domestic rabbit model. Compounds were delivered either topically, injected into growing papillomas, or delivered subcutaneously at a site remote from the papillomas. Compounds tested included cidofovir [(S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine] (HPMPC); cyclic HPMPC (cHPMPC); cyclopentenylcytosine (CPE-C); lobucavir [1R(1alpha,2beta,3alpha)]-9-[2, 3-bis(hydroxymethyl)cyclobutyl]guanine; 9-((2-phosphonylmethoxy)propyl)adenine (PMPA); adefovir 9-((2-phosphonylmethoxy)ethyl)adenine(PMEA) and cyclopropyl 9-(2-phosphonylmethoxyethyl)-2,6-diaminopurine (cyclopropylPMEDAP). Dose response curves and time-course treatments were included for most compounds tested. Strong anti-viral activity was detected using cidofovir and cHPMPC when delivered either topically or by the intralesional route. Complete cures were obtained using 1% (w/v) topical cidofovir at dosing schedules of twice daily for 8 weeks beginning at 4 weeks after CRPV infection, which represents a time when papillomas were clearly visible. Complete cures of large established papillomas were obtained by intralesional injection of 1% cidofovir three times per week for 8 weeks. Topical treatments with adefovir had strong anti-viral activity, cyclopropyl PMEDAP had moderate anti-viral activity, and CPE-C, PMPA and lobucavir showed no effects. These data indicate that certain nucleoside analogues have strong in vivo anti-papillomavirus activity and that the CRPV/rabbit model is a good model for assessing clinical responses of anti-viral treatments for patients with HPV disease.

Commercial immunoperoxidase kits in the study of 13 pancreatic islet-cell tumors.

Use of commercial immunoperoxidase kits for the study of gastrin, glucagon, insulin, and somatostatin production in 13 islet-cell tumors was assessed. Kits save time in preparation and technic, are convenient and easy to use, and are economical for occasional tests. Results are reproducible, with crisp immunostain, minimal background staining, and high sensitivity so that interpretation is easy. The relevant hormone was specifically identified in seven symptomatic tumors. Of six asymptomatic tumors, five showed strongly positive glucagon immunostain and one somatostatin. Nine tumors, in addition, contained somatostatin. Three tumors produced a third hormone. Use of kits can prove expensive for large numbers of tests. Reagents supplied were of unknown dilution and usually insufficient to run negative controls concurrently. Fading of immunostain was noticed after six months of storage.

Evaluation of commercial immunoperoxidase kits in diagnosis of prostate carcinoma.

Comparison of one commercial immunoperoxidase kit for prostate specific acid phosphatase (PSAP) and two for prostate specific antigen (PA) with an unlabelled antibody peroxidase-antiperoxidase (PAP) technique using rabbit antihuman PSAP revealed generally good reproducibility of results on formalin-fixed, paraffin-embedded sections of primary and metastatic prostatic carcinoma. Frequency of positivity was 92 per cent with both our noncommercial PAP and the kit method for PSAP, and 82 per cent and 89 per cent with kits for PA. Thus, sensitivity with the PA kits was less than with our noncommercial PAP and the kit PSAP, with less intense immunostain and occasional false negatives. Specificity was good with all three kits, and no false positive results were obtained. Commercial immunoperoxidase kits provide a relatively easy and inexpensive means for practicing pathologists in a service-oriented setting to make more precise diagnoses in cases of poorly differentiated or metastatic carcinoma of prostatic origin.

Immunohistochemical acid phosphatase level and tumor grade in prostatic carcinoma.

An immunoperoxidase technique to detect prostatic-specific acid phosphatase (PSAP) was used on specimens from 98 cases of prostatic carcinoma that were graded by both the Gleason and the Mostofi systems, to see if tumor grade correlated with amount of PSAP seen in tissue. Most tumors showed strong, diffuse cytoplasmic staining; no significant difference was seen among the various grades. Other than focal, weak staining of renal tubular epithelium, the antibody to PSAP gave uniformly negative results with a variety of normal and neoplastic tissues. In light of the great sensitivity and specificity of this technique, it potential applications include diagnosis of poorly differentiated prostatic malignant neoplasms, whether primary or metastatic.

Induction of productive human papillomavirus type 11 life cycle in epithelial cells grown in organotypic raft cultures.

The study of the human papillomavirus (HPV) life cycle was hampered for more than 50 years by the lack of a conventional cell culture system for propagating HPV. Considerable progress has been made in the production of several HPV types using either organotypic rafts or human epithelial xenografts in immunocompromised mice. In this study, we demonstrated episomal maintenance of HPV-11 DNA in N-Tert cells. HPV-11 episomal DNA containing cell populations grown in raft culture showed induction of the productive viral life cycle. HPV-11 DNA amplification and viral capsid antigen synthesis were detected in differentiated layers of epithelia. The viruses generated were able to infect keratinocytes in vitro, which indicate that viruses generated were infectious. The demonstration of the productive HPV-11 life cycle in raft culture from cloned HPV-11 DNA will facilitate genetic analyses of viral gene functions that was not possible using the human xenograft athymic mouse model.

The human papillomavirus type 11 E1/\E4 protein is not essential for viral genome amplification.

An abundant human papillomavirus (HPV) protein E1/\E4 is expressed late in the virus life cycle in the terminally differentiated layers of epithelia. The expression of E1/\E4 usually coincides with the onset of viral DNA amplification. However, the function of E1/\E4 in viral life cycle is not completely understood. To examine the role of E1/\E4 in the virus life cycle, we introduced a single nucleotide change in the HPV-11 genome to result in a truncation of E1/\E4 protein without affecting the E2 amino acid sequence. This mutated HPV-11 genome was introduced into a human foreskin keratinocyte cell line immortalized by the catalytic subunit of human telomerase, deficient in p16(INK4a) expression, and previously shown to support the HPV-11 life cycle when grown in organotypic raft culture. We have demonstrated that E1/\E4 is dispensable for HPV-11 viral DNA amplification in the late stages of the viral life cycle.

Detection of Epstein-Barr virus genomes by in situ DNA hybridization with a terminally biotin-labeled synthetic oligonucleotide probe from the EBV Not I and Pst I tandem repeat regions.

We describe a case of acute, disseminated Epstein-Barr virus (EBV) infection which was analyzed for the cellular distribution of viral replication by automated, colorimetric in situ DNA hybridization using a single, synthetic, terminally biotin-labeled oligonucleotide probe composed of 23 consecutive nucleotides selected from the EBV Not I region. The GC-rich, Not I region is a 125-base pair sequence that is repeated in tandem an average of 12.6 times in the EBV genome. The synthetic sequence had 91% base homology with another EBV genomic tandem repeat, the 102-base pair Pst I region, which is also GC-rich, has an overall 70% homology with the Not I region, and is reiterated about 25 times in the viral DNA. Disseminated EBV infection was detected in nuclei of atypical lymphocytes in several organs, including lung, bronchus, trachea, spleen, liver, and stomach, with the probes. In addition, the synthetic oligomer compared favorably with a significantly more expensive, nick translated, biotinylated probe cloned from the BAM HI-V (W), large internal repeat region. This 3.0-kilobase pair (kbp) sequence is repeated an average of 11 times in EBV. Although both probes identified regions repeated multiple times in the virus, and each confirmed an identical tissue distribution for the infection, the signal obtained with the Not I/Pst I probe was more intense and confined to the nuclei of fewer lymphocytes than the general, more weakly distributed signal obtained with the probe from the large internal repeat region. Consistent positive cellular staining was obtained with the Not I/Pst I probe in both EBV-infected control tissue culture cells and in formalin-fixed, paraffin-embedded tissue sections.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunohistochemical ferritin in testicular seminoma.

Immunohistochemical ferritin was demonstrated in all 23 (100%) testicular seminomas studied with antisera to liver ferritin (largely of basic isoferritins) and to myocardial ferritin (containing more acidic isoferritins). With liver ferritin antibody immuno-stain was strong in 74% with a diffuse distribution in 41%. The remaining six tumors showed moderate immuno-stain, of diffuse distribution in two. The myocardial ferritin antibody showed the following: strong immuno-stain in 52%, 58% of diffuse distribution; and moderate immuno-stain in 30%, 86% of focal distribution. Although ferritin is widespread in nature, the facts that both basic and acidic isoferritins are present in the majority of testicular seminomas, and that serum ferritin levels are reported to be increased in over 80% of patients with seminoma suggest that the demonstration of raised serum ferritin levels in the face of negative or low serum assays for human chorionic gonadotrophin and alpha-fetoprotein possibly may prove to be useful in the diagnosis, and evaluation of extent and recurrence of testicular seminomas.

Anatomic viral detection is automated: the application of a robotic molecular pathology system for the detection of DNA viruses in anatomic pathology substrates, using immunocytochemical and nucleic acid hybridization techniques.

This paper presents the first automated system for simultaneously detecting human papilloma, herpes simplex, adenovirus, or cytomegalovirus viral antigens and gene sequences in standard formalin-fixed, paraffin-embedded tissue substrates and tissue culture. These viruses can be detected by colorimetric in situ nucleic acid hybridization, using biotinylated DNA probes, or by indirect immunoperoxidase techniques, using polyclonal or monoclonal antibodies, in a 2.0-hour assay performed at a single automated robotic workstation.

Alpha-1-antitrypsin deficiency in Southern African hepatocellular carcinoma patients. An immunoperoxidase and histochemical study.

An increased frequency of alpha-1-antitrypsin deficiency (AATD) has been reported in patients with hepatocellular carcinoma (HCC) in Scandinavia and England. Using a specific immunoperoxidase technique for alpha-1-antitrypsin (AAT) and periodic acid-Schiff (PAS) with and without diastase predigestion, the authors examined nonneoplastic autopsy liver tissue from 58 black southern African patients with HCC and from 54 controls. No periportal PAS-positive diastase-resistant globules containing AAT (specific for AATD) were found in liver tissue from either group. A finely granular diffuse cytoplasmic AAT staining (not removed by diastase predigestion) was present in hepatocytes in a "checkerboard" pattern within the lobule in 33 (57%) HCC patients and in 20 (37%) controls (P greater than 0.1), and was particularly prominent adjacent to tumor in 11 HCC patients. Alpha-1-antitrypsin was present in neoplastic cells in 18 of the 40 HCC examined (45%). These findings suggest no major role for AATD in the etiology of HCC in southern Africa.

Liver iron stores and hepatitis B antigen status.

Higher serum iron and ferritin levels noted in hepatitis B antigen (HBAg) carriers than in noncarriers suggests that virus might actively replicate in hepatocytes, stimulate ferritin synthesis, and result in increased liver iron stores. A comparative semiquantitative study of immunohistochemical ferritin (0-12) and hemosiderin (0-9) was performed on 54 normal, 13 cirrhotic, and 70 nonneoplastic livers from patients with hepatocellular carcinoma, in each group, comparing amounts in HBAg-positive and HBAg-negative patients. Mean scores for ferritin and hemosiderin were high in all three groups, normal livers averaging 8.3 and 6, respectively, cirrhotic livers, 8.5 and 7.4, respectively, and carcinoma livers, 5.6 and 6.1, respectively. In each group, there was no significant difference in ferritin and hemosiderin mean scores in HBAg-positive and HBAg-negative patients. The large liver iron stores do not appear to be a consequence of hepatitis B virus infection alone. Their role in the development of hepatocellular carcinoma is still to be elucidated.

Tumor-associated antigens in breast carcinomas. Prognostic significance.

In an attempt to identify biologic markers that might predict prognosis in breast cancer patients, the presence or absence of seven tumor-associated antigens in 54 infiltrating breast carcinomas was correlated with tumor recurrence rates (minimum five-year follow-up), axillary lymph node metastases and tumor volume. Immunohistochemical kappa-casein was present in 30 (56%) tumors, alpha-lactalbumin in 39 (72%) tumors, secretory component of IgA in 26 (48%) tumors, carcinoembryonic antigen in 34 (63%) tumors, pregnancy-specific beta-1-glycoprotein in 7 (13%) tumors, beta subunit of human chorionic gonadotrophin in 1 (2%) tumor and human placental lactogen in 0 (0%) tumors. There was no significant correlation between the presence or absence in tumor of any of the antigens, and prognosis as assessed either by 5-year recurrence rates (P greater than 0.18) or by the presence of axillary lymph node metastases (P greater than 0.20). No significant difference was noted in mean tumor volume (cm3) +/- SEM, between tumors with or without antigen immunoreactivity (P greater than 0.05).