Effect of mast cell chymase on the morphology of thyroid cells in vitro.

We studied the effect of mast cell chymase on the thyroid cells in culture. Rat serosal mast cells, similar functionally to connective tissue mast cells, were obtained after lavage of the peritoneal cavity and lyzed by freezing. The resulting lysate was used as crude enzyme preparation. Mast cell chymase was purified from the crude preparation by anion exchange chromatography. Crude and purified chymase incubated with thyroid cells induced cellular retraction, the appearance of long processes and gradual cell detachment from the substratum. The effect of the enzyme was not cytotoxic. The immunofluorescence studies of thyroid cells showed a decreased amount of polymerized actin and tubulin after incubation with chymase. Neutral protease inhibitor abolished the effect of crude and purified chymase on thyroid cell morphology. The above findings suggest that mast cell chymase may have a function in the control of cell morphology and cell-matrix interaction.     

Immunopathogenesis of infection with the visceralizing Leishmania species

Human leishmaniasis is a spectral disease that includes asymptomatic self-resolving infection, localized skin lesions, and progressive visceral leishmaniasis. With some overlap, visceral and cutaneous leishmaniasis are usually caused by different species of Leishmania. This review focuses on host responses to infection with the species that cause visceral leishmaniasis, as they contrast with species causing localized cutaneous leishmaniasis. Data from experimental models document significant differences between host responses to organisms causing these diverse syndromes. The visceralizing Leishmania spp. cause localized organ-specific immune responses that are important determinants of disease outcome. Both the Leishmania species causing cutaneous and those causing visceral leishmaniasis require a Type 1 immune response to undergo cure in mouse models. However, during progressive murine infection with the visceralizing Leishmania sp., the Type 1 response is suppressed at least in part by TGF-beta and IL-10 without type 2 cytokine production. This contrasts with the cutaneous species L. major, in which a Type 2 response suppresses type 1 cytokines and leads to murine disease progression. Population and family studies are beginning to elucidate human genetic determinants predisposing to different outcomes of Leishmania infection. These studies should eventually result in a better understanding of the immunopathogenesis and the spectrum of human leishmaniasis.     

Lactoperoxidase and human airway host defense

The lactoperoxidase (LPO) antibiotic system is a well-characterized component of mammary and salivary gland secretions. Because LPO has been shown to function in ovine airways, human airway tissue and secretions were examined for the presence of LPO and its substrate, the anion thiocyanate (SCN-). In addition, human airway secretions were tested for LPO-mediated antibacterial activity, and LPO's activity was assessed against some human airway pathogens. The data showed that normal human airway secretions contained LPO enzyme activity (0.65 +/- 0.09 microg/mg secreted protein; n = 17), and Western blots of secretions demonstrated bands of the expected sizes for LPO. LPO mRNA was detected in trachea by sequencing PCR-amplified cDNA. SCN-, LPO's substrate, was present in undiluted airway secretions at concentrations sufficient for LPO catalysis (0.46 +/- 0.19 mM; n = 8), and diluted secretions contained antibacterial activity with LPO-like properties. Immunocytochemistry localized LPO to submucosal glands in human bronchi. Finally, as expected based on the known antibacterial spectrum of the LPO system, airway secretions showed LPO-dependent activity against Pseudomonas aeruginosa. In addition, the airway LPO system was shown to be effective against Burkholderia cepacia and Haemophilus influenzae. Thus, a functional LPO system exists in human airways and may contribute to airway host defense against infection.     

Cyclooxygenase-2 in normal,hyperplastic and neoplastic follicular cells of the human thyroid gland

This study was undertaken to investigate cyclooxygenase-2 (COX-2) expression in follicular cells of the human thyroid. COX-2 expression was studied immunohistochemically in a total of 174 samples. COX-2 immunoreactivity was confined to the cell cytoplasm with the nuclei remaining unlabelled. COX-2 expression was observed in five cases (17.2%) of normal follicular cells and in one case (16.6%) of solid cell nests. Follicular carcinoma expressed COX-2 more frequently than follicular adenoma (93.4% vs 21.1%) (p0.001). A higher percentage of cases of papillary microcarcinomas up-regulated COX-2 in comparison with all papillary carcinomas (p0.05). However, we could not establish any relationships among COX-2, patients ages or lymph node metastases in papillary carcinomas. COX-2 expression was found in 12 (92.3%) poorly differentiated carcinomas and in 13 (92.8%) undifferentiated carcinomas. We found that COX-2 is not always useful as a marker of malignancy. Our results suggest that COX-2 plays a role in progression of all thyroid carcinomas, but in papillary carcinomas, seems more important only in the early stages. COX-2 expression in the undifferentiated carcinoma deserves special consideration due to its prognosis and to the fact that selective COX-2 inhibitors were found to enhance tumour response to radiation in some studies.     

Neural cell adhesion molecule immunoreactivity in Merkel cells and Merkel cell tumours

We have analysed the expression of the neural cell adhesion molecule (NCAM) in normal Merkel cells of pig and human skin, and in nine neuroendocrine carcinomas of the skin (Merkel cell carcinomas). NCAM immunoreactivity was observed in virtually all Merkel cells, both in epidermis and vibrissae of pig snout skin and in human epidermis. Immunostaining surrounded the entire surface of Merkel cells and was not restricted to the contact areas between Merkel cells and nerve terminals. All Merkel cell carcinomas studied were also positive for NCAM. The immunostaining pattern of the tumour cells was similar to that observed in normal Merkel cells; the immunoreactivity was confined to the cell membranes. These results suggest that NCAM may be used as an immunohistochemical marker for both Merkel cells and Merkel cell tumours.     

Looking for ferns: optimization of digestion pretreatment in fluorescence in situ hybridization (FISH) technique on paraffin-embedded tissues.

Fluorescence in situ hybridization (FISH) is a useful cytogenetic technique for the detection of chromosome aberrations. However, applying this technique routinely on paraffin-embedded tissue is hampered by technical problems. The efficiency of hybridization is influenced by formalin fixation time, and this may vary considerably between specimens. We present a simple method for improving hybridization by microscopically monitoring the time of enzymatic digestion. To establish optimal digestion time, enzymatic digestion was stopped at 3-minute intervals for biopsies and 10-minute intervals for autopsies in 24 paraffin-embedded samples. At every stop, tissue morphology was examined under light microscopy to determine if observed changes could be correlated with subsequent FISH results. The appearance of fernlike formations was found to mark the optimal digestion time that produced the strongest hybridization signals. Using this method of digestion time control, an additional 41 cases were evaluated for FISH with various types of probe. Monitoring under the microscope could be more spaced if the morphology did not change after the first visual control and could be adapted to the type of sample (in general, endoscopic samples, total digestion time of about 10 min; routine biopsies, 15 to 30 min; autopsy samples, 20 to 40 min). In every case, the appearance of the fernlike pattern correlated with proper hybridization signal. Monitoring digestion time for the appearance of fernlike structures is a useful method for improving reproducibility of FISH technique on paraffin-embedded samples. It is particularly useful when dealing with samples under heterogeneous fixation conditions (consultations, autopsies, etc.), because it eliminates the need for repetition.     

Visual inspection with acetic acid for cervical cancer screening outside of low-resource settings.

OBJECTIVES: To assess visual inspection with acetic acid (VIA) as a screening tool for use in a well-equipped health center in Peru, to evaluate VIA as an alternative or adjunct to the Papanicolaou (Pap) smear, and to determine if VIA can play a role in settings other than low-resource ones. METHODS: This was a prospective study of 1 921 asymptomatic women living in Lima, Peru, carried out in 1999 and 2000. The study was performed at a cancer center equipped with the latest-generation technology and highly trained oncologists. The women underwent a complete clinical evaluation, including a Pap smear and VIA. Participants with any positive test were referred for colposcopy and biopsy. RESULTS: More women tested positive by VIA than on the Pap smear (6.9% vs. 4.2%; P = 0.0001). There were 35 women with histologic cervical intraepithelial neoplasia grade 1 (CIN 1); of these, 15 were detected by Pap and 20 by VIA (P = 0.4). A diagnosis of CIN 2 or 3 (CIN 2-3) was confirmed in a total of 13 cases; Pap detected 5 of the cases and VIA 11 of the cases (P = 0.06). The positive predictive value for detection of CIN 2+ was 8.3% for VIA and 6.3% for Pap (P = 0.5). Most importantly, while only 2.3% of patients with a positive VIA were lost to follow-up before colposcopy, that was true for 26.3% of the women with a positive Pap smear (P < 0.0001). CONCLUSIONS: VIA is useful for detection of precursor lesions of cervical cancer not only in low-resource settings but also in well-equipped health centers and cancer centers. In these non-low-resource settings, VIA has a positive predictive value comparable to the conventional Pap smear, but it is more likely to achieve earlier diagnosis, follow-up, and treatment than cytology-based screening.     

Neither one‐time negative screening tests nor negative colposcopy provides absolute reassurance against cervical cancer

A population sample of 10,049 women living in Guanacaste, Costa Rica, was recruited into a natural history of human papillomavirus (HPV) and cervical neoplasia study in 1993–1994. At the enrollment visit, we applied multiple state‐of‐the‐art cervical cancer screening methods to detect prevalent cervical cancer and to prevent subsequent cervical cancers by the timely detection and treatment of precancerous lesions. Women were screened at enrollment with 3 kinds of cytology (often reviewed by more than one pathologist), visual inspection and cervicography. Any positive screening test led to colposcopic referral and biopsy and/or excisional treatment of CIN2 or worse. We retrospectively tested stored specimens with an early HPV test (hybrid capture tube test) and for >40 HPV genotypes using a research PCR assay. We followed women typically 5–7 years and some up to 11 years. Nonetheless, 16 cases of invasive cervical cancer were diagnosed during follow‐up. Six cancer cases were failures at enrollment to detect abnormalities by cytology screening; 3 of the 6 were also negative at enrollment by sensitive HPV DNA testing. Seven cancers represent failures of colposcopy to diagnose cancer or a precancerous lesion in screen‐positive women. Finally, 3 cases arose despite attempted excisional treatment of precancerous lesions. Based on this evidence, we suggest that no current secondary cervical cancer prevention technologies applied once in a previously under‐screened population is likely to be 100% efficacious in preventing incident diagnoses of invasive cervical cancer. © 2009 UICC