Biomedical subjects
Publications and source records attributed to E Wilander.
Swedish and Italian women with HPV 16-positive cervical disease were checked for codon 72 polymorphisms of p53. In both groups, arginine homozygotes were enriched in cancer compared with controls and precursor lesions.
High risk types of human papillomavirus (HPV) are agents in the aetiology of cervical carcinoma. The products of two early genes, E6 and E7, appear to be the principal transforming proteins. Studies of various monolayer cell culture systems have shown that the E7 oncoprotein of human papillomavirus type 16 is able to neutralize or bypass the inhibitory effect of the cell cycle-dependent kinase (CDK) inhibitors (CKIs) p21WAF1/CIP1 and p27KIP1. To understand whether the p21WAF1/CIP1 or p27KIP1 neutralization also plays a role in vivo, we performed studies on clinical specimens. Forty-five cervical biopsies, including HPV-negative mucosa, HPV 16-positive preinvasive (low and high grade lesions) and invasive neoplasia as well as HPV 6-positive condyloma acuminatum were analysed by single and double immunohistology. We examined the positive cell cycle regulator cyclin A and the universal cell cycle marker Ki67 as well as the negative cell cycle regulators p21WAF1/CIP1 and p27KIP1. Here, we show that in a significant fraction of cells the G1 block can be overcome despite high levels of CKIs in HPV lesions. This phenomenon, which was more evident for p21WAF1/CIP1 than for p27KIP1 was most marked in low grade lesions and in condylomata acuminata, in which a high viral productivity is expected. These results indicate that the overriding of CKI inactivation by viral oncoproteins appears to be a conserved property between low and high risk HPV types. We conclude that the CKI neutralization by HPVs is likely to be required for viral DNA replication rather than for malignant transformation of the host cell.
Anal warts are, from an aetiological point of view, a diverse category of lesions including condylomata acuminata, fibroepithelial polyps and seborrhoeic keratosis. Human papillomavirus induced anal warts, in contrast to other types of warts, are contagious and not infrequently sexually transmitted, they therefore need to be accurately identified. A total of 24 anal warts were randomly collected and the histopathological diagnoses based on microscopy, alone or in combination with a sensitive PCR-based human papillomavirus test, were compared using the SHARP Signal system for detection. Three lesions were identified as condyloma acuminatum by morphology alone due to the obvious presence of koiloytotic atypia; 11 warts without koilocytes were identified only after a positive test for anogenital human papillomavirus. One additional lesion contained human papillomavirus DNA of cutaneous type and 9 papillomas were human papillomavirus-negative and tentatively diagnosed as fibroepithelial polyps or seborrhoeic keratosis. All 14 condylomas contained human papillomavirus of low-risk type. Of these, 12 warts showed a positive human papillomavirus reaction with in situ hybridization. Morphology alone cannot reveal the true nature of most anal papillomas, even when koilocytotic atypia is considered as a diagnostic hallmark. An optimal diagnosis of anal warts requires a sensitive PCR-based human papillomavirus DNA test. A test for identification of cutaneous human papillomavirus DNA is also worthwhile.
A variant of human papillomavirus (HPV) 16 has been shown recently to be more prevalent in invasive cervical carcinoma than in preinvasive lesions. This HPV 16 variant possesses a common mutation (T to G) in nucleotide 350 (codon 83) of the E6 gene, resulting in an amino acid shift, L83V, in the E6 protein. This mutation was believed to signify preinvasive cervical lesions with a high probability of progression to invasive carcinoma. The purpose of the present investigation is to describe a rapid method for the detection of this variant HPV 16, E6 (L83V). Paraffin blocks of 18 gynecologic biopsy specimens were collected, all displaying the morphology of cervical intraepithelial neoplasia (CIN I-III) and a positive HPV 16 test. Sections from these blocks were used for DNA extraction. A DNA sequence of the E6 gene containing 176 bases (including codon 83) was amplified by polymerase chain reaction (PCR) and analyzed by non-radioactive single-strand conformational polymorphism (SSCP) analysis. A divergent SSCP pattern was observed in 7 of the HPV 16 positive biopsy specimens. A DNA sequence analysis of the PCR products revealed the conversion of Leu to Val in codon 83 of the E6 gene, correlating to the divergent band pattern. This PCR-SSCP method can be used to test for HPV 16 in women who are at serious risk of developing invasive cervical carcinoma.
The aim of this study was to investigate the permeability of the adherent mucus gel layer in rat duodenum in vivo to macromolecules applied in the lumen. Rats were anesthetized with thiobarbiturate, and the duodenum was perfused with isotonic NaCl solution containing large-molecular-size secretagogues. Effects on mucosal HCO(-)(3) secretion and blood-to-lumen (51)chromium-labeled EDTA clearance were used as indexes that compounds had migrated across the mucus layer. Exposure to a low concentration of papain (10 U/100 ml) for 30 min removed the mucus layer without damage to the epithelium and induced or markedly enhanced HCO(-)(3) secretory responses to cholera toxin (molecular mass of 85 kDa) or glucagon (3.5 kDa). Water extracts from a VacA cytotoxin (89 kDa) producing Helicobacter pylori strain, but not from a toxin-negative isogenic mutant, caused a small increase in HCO(-)(3) secretion but only after the mucus layer had been removed by papain. The duodenal surface mucus gel thus significantly restricts migration of macromolecules to the duodenal surface. Release of bacterial toxins at the cell-mucus interface may enhance or be a prerequisite for their effects on the gastrointestinal mucosa.
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High-risk human papillomavirus (HPV) is a known risk factor in the etiology of cervical intraepithelial neoplasia (CIN) I-III and invasive cervical carcinoma (ICC). The most severe preinvasive lesion is CIN III, and it is still not entirely understood why some cases progress to invasion, whereas others do not. Our hypothesis that this could be predicted by intratype variation of the immortalizing and transforming early proteins E6 and E7 was tested. Because HPV16 is frequently detected in cervical neoplastic lesions, 25 CIN III and 17 ICC cases from Swedish women, all positive for this genotype, were selected to investigate the E6 and E7 genes for mutations. PCR-amplified products were sequenced by the fluorescent dideoxy termination method. ICC harbored almost exclusively HPV16 E6 variants (94%) and rarely harbored the prototype (6%), whereas CIN III demonstrated a more uniform distribution of variants (56%) and prototype (44%; P = 0.013). All variants contained variations that were identified in areas likely to be important for protein-protein interaction with p53 or in areas of immunological significance. The most frequent E6 variation was seen at residue 83. This polymorphism was detected alone or in combination with others in 88% of ICC and 44% of CIN III cases. E7 variations were extremely rare and were only detected together with E6 variations in 4% of CIN III and in 6% of ICC cases, suggesting that the HPV16 E7 but not the HPV16 E6 oncoprotein is highly conserved in vivo. This indicates that HPV16 E6 variants, specifically those containing the substitution at residue 83, may be more oncogenic than the prototype and thus carry a higher risk for the development of invasive cervical disease. This may be due to subtle differences in the type of transformation produced or to evasion of host immune defenses. These results might have implications for future in vitro studies, diagnostics, treatment, and vaccine design.
BACKGROUND: Type 1 gastric carcinoids are associated with hypergastrinaemia and chronic atrophic gastritis, type 2 occur in patients with multiple endocrine neoplasia type 1 combined with Zollinger-Ellison syndrome, and type 3 lack any relation to hypergastrinaemia. Type 1 tumours are usually benign whereas type 3 are highly malignant. AIMS: To identify possible tumour markers in patients with gastric carcinoids. PATIENTS/METHOD: Nine patients with type 1, one with type 2, and five with type 3 were evaluated with regard to symptoms, hormone profile, and prognosis. RESULTS: Plasma chromogranin A was increased in all patients but was higher (p < 0.01) in those with type 3 than those with type 1 carcinoids. All patients with type 3 carcinoids died from metastatic disease, but none of the type 1 patients died as a result of their tumours. One type 1 patient with a solitary liver metastasis received interferon alpha and octreotide treatment. Nine months later, the metastasis was no longer detectable. She is still alive eight years after diagnosis, without recurrent disease. This represents the only reported case of foregut carcinoid with an unresectable liver metastasis that seems to be have been cured by biotherapy. CONCLUSIONS: Plasma chromogranin A appears to be a valuable tumour marker for all types of gastric carcinoid. Combination therapy with interferon alpha and octreotide may be beneficial in patients with metastasising type 1 gastric carcinoids.
Squamous carcinoma of the uterine cervix accounts for a considerable mortality among gynecological malignancies, although both the incidence of and mortality from cervical cancer have decreased in the Nordic countries since 1970. The reduction is a result of the cytologic screening program, through which precursor lesions can be detected and removed. Our aim was to determine why women in the county of Uppsala get cervical cancer despite extensive gynecological screening. A retrospective study of all women (43 cases) who developed histologically verified cervical squamous carcinoma in the county of Uppsala during the years 1991 to 1994 was undertaken. A central computer database covering all histopathological and cytological material made it possible to compare each woman's previous smears or lack of smears with her cancer diagnosis. Twenty women (47%, mean age at diagnosis 64.4 years) had not undergone cytological screening. Twenty-three women (53%) had been screened at least once within 6 years before tumor diagnosis. Of these, 11 (mean age 47.5 years) had had normal smears for the last 6 years, 8 (mean age 44.3 years) had shown abnormal cytology for less than one year and 4 (mean age 39.0 years) had shown abnormal cytology for more than one year and up to 6 years before their cervical cancer diagnosis. Women with cervical squamous carcinoma, who are not covered by the gynecological screening program (47%) are at increased risk of developing cervical cancer compared with other women. Consequently the average age of non-screened women developing cancer is considerably higher (64.4 years) than that of women with cancer screened previously (44.9 years). In 26% of the total group of women with cervical squamous carcinoma previous cytology displayed normal features.
Clonality of invasive human cervical cancer was assessed in microdissected archival formaldehyde-fixed, paraffin-embedded tissues by PCR-based analysis of X-chromosome inactivation of the androgen receptor gene. In 18 informative cases, cancers from 12 cases including 2 early-invasive cancers were scored as monoclonal. Among them, 8 cases had the combination of random X-chromosome inactivation in normal cervical tissue and non-random in the cancer and in other 4 cases normal and cancer tissue were both non-random but had discordant X-chromosome inactivation. Six cases showed that the non-random inactivation affected the same allele in cancer and normal tissue and thus were considered to be inconclusive. The results suggest the monoclonal origin of cervical cancer and indicate that genetic events are critical in transition of pre-malignant epithelia to invasive cancer in cervical carcinogenesis. Finding of monoclonal early invasive cancer also argues that monoclonality of human tumors is not a late event due to clonal competition or selection. X-chromosome inactivation patterns in normal cervical epithelia and tissues were analyzed in 21 informative cases. When a mixture of normal stroma and epithelium was analyzed, 38% (8/21) of cases showed non-random inactivation pattern, whereas using pure epithelium it was 64% (11/17). The X-chromosome inactivation pattern between mixed epithelium/stroma and matched epithelium differed in 2/8 cases. These results point to a certain amount of overlooked source of error in X-inactivation-based tumor clonality analysis in which peripheral blood monocytes were chosen as control and strongly recommend that control tissues which share close histological origin with analyzed tumors should be selected in any clonality study of human neoplasm. Post-embryological mechanisms of clonal patch in normal cervical epithelia are also discussed.
Genetic alteration of the RET proto-oncogene is associated with multiple endocrine neoplasia type 2A and 2B (MEN 2A and MEN 2B), familial medullary thyroid carcinoma (FMTC) and Hirschprung's disease. Oncogenically activated RET has also been demonstrated in sporadic medullary thyroid tumors, which in some cases show somatic missense mutations. We have recently described a complex 9 bp deletion in RET exon 11 in a single case of sporadic MTC. In order to determine the prevalence of this mutation among sporadic MTC tumors, we have now analysed 15 cases and five normal controls by PCR-based nonradioactive single-strand conformational polymorphism analysis (PCR-SSCP) and fragment size analysis of exon 11. DNA was extracted from microdissected tumor tissue or normal cells and subjected to nested PCR prior to analysis. A markedly divergent SSCP pattern and a PCR fragment 9 bp shorter than normal were demonstrated in 14 of the 15 MTC tumors. Sequencing revealed the deletion of nine bases encompassing a key cysteine at codon 634, often altered in MEN 2A. Four lymphocyte controls and normal thyroid tissue from one patient failed to show the deletion. Several factors in the DNA sequence environment immediately surrounding the deletions, including an extended inverted repeat, several direct repeats and a so-called symmetric element suggest that the deletional events may be non-random.
The prevalence of human papillomavirus (HPV) DNA sequences in 45 cervical cancer biopsies was examined with the hot-start polymerase chain reaction (PCR), employing HPV consensus primers from the L1 region. The cases comprised 38 squamous cell carcinomas, three adenosquamous carcinomas, and four adenocarcinomas. PCR products were typed with single-strand conformation polymorphism (SSCP) and the HPV types detected were correlated with tumour type. Forty-three biopsies were HPV-positive, HPV16 being the most prevalent type. HPV18/33/45/58 were also detected, but no low-risk or multiple types. Keratinizing squamous cell carcinoma was invariably associated with HPV16 and adenosquamous carcinoma and adenocarcinoma with HPVs 18/45. Non-keratinizing squamous cell carcinomas harboured all five detected types. Our data corroborate the view that malignant cervical tumours are almost invariably associated with high-risk HPV and that certain malignant cervical tumour phenotypes correlate with specific HPV types.
BACKGROUND: Little is known about factors related to prognosis in patients with carcinoid disease. In this study we have tried to identify such factors. PATIENTS AND METHODS: We have evaluated 301 consecutive carcinoid patients (256 midgut, 39 foregut and six hindgut) referred during 15 years for medical treatment with respect to tumor distribution, hormone production, prognostic factors and survival. RESULTS: Survival was significantly shorter in midgut carcinoid patients with > or = 5 liver metastases or with high levels of urinary 5-hydroxyindoleacetic acid, plasma chromogranin A or neuropeptide K. By univariate analysis, these variables together with the presence of carcinoid syndrome were related to a higher risk of dying. In multivariate analyses, performed in the 71 patients with full information on all variables, advanced age and plasma chromogranin A > 5000 micrograms/l were independent predictors of overall survival. CONCLUSIONS: Poor prognostic factors for midgut carcinoid patients were multiple liver metastases, presence of carcinoid syndrome and high levels of the tumor markers studied. In this study the only independent predictors of bad prognosis in midgut, carcinoid patients were advanced age, which however is inherently related to overall survival, and plasma chromogranin A > 5000 micrograms/l. Thus, chromogranin A may prove to be an important prognostic marker for patients with carcinoid tumors.
The aim of the study was to determine if and by what mechanism(s) nitric oxide inhibition modulates the susceptibility of the duodenum to hydrochloric acid-induced disturbances of mucosal integrity. A second aim was to investigate whether basal permeability is a determinant of epithelial acid barrier function. Using an in situ duodenal perfusion model, mucosal permeability, alkaline secretion and morphology were investigated in anaesthetized rats. Luminal perfusion with 50 mM hydrochloric acid increased duodenal mucosal permeability in the control animals. In animals receiving the nitric oxide synthase inhibitor N-nitro-L-arginine methyl ester (L-NAME 3 mg kg(-1) and 1 mg kg(-1) h(-1)) and in those receiving vasopressin (1 IU kg(-1) h(-1)), however, the mean increase in permeability in response to acid was markedly higher. In rats treated with either hexamethonium (20 mg kg(-1)) or atropine (0.5 mg kg(-1)) L-NAME failed to augment the acid-induced increase in permeability. Perfusion with hypotonic saline (25 mM) increased basal permeability but did not influence the response to acid. Exposure of the duodenum to hydrochloric acid caused very subtle changes of duodenal morphology. It is concluded that both inhibition of endogenous nitric oxide synthesis and vasopressin treatment augment the acid-induced increase in mucosal permeability. The mechanisms involved may be related to changes of Starling forces in the microcirculatory bed. Endogenous nitric oxide may protect the duodenal mucosa by regulating vascular permeability and interstitial fluid pressure.
BACKGROUND: Knowledge about the natural course of HPV infection is still limited. In this study we investigated the presence of HPV DNA after treatment and clinical clearance of infection. METHODS: Eighty-two women treated for genital HPV infection at the STD clinic in Uppsala were consecutively selected for the study. After treatment with podophyllotoxin, and in some cases laser vaporization, a cell sample was taken at the follow-up visit 6-12 months after clinical clearance of the lesions as evaluated by colposcopy. Samples were analysed with PCR to detect HPV DNA. As a reference group, women treated for cervical intraepithelial neoplasia (CIN) with laser surgery, either with cone biopsy or vaporization, were followed-up after 6 months for the presence of HPV DNA. RESULTS: Six to 12 months after clinical clearance of HPV infection, 39 (48%) of the women showed detectable HPV DNA in cell samples from the cervix. Of these, 26 (67%) were found to harbor high risk HPV, six (15%) low risk, and seven (18%) either had more than one HPV type or HPV that could not be classified. All but three of the women treated for CIN (90%) were negative for HPV DNA. CONCLUSION: After clinical clearance of genital HPV infection half of the women had detectable HPV DNA. This does not necessarily imply that transmission to a new partner may occur, but indicates this possibility. Only 10% of the CIN treated women harbored HPV DNA in the cell samples in spite of showing high risk HPV infection before treatment.
BACKGROUND: Studies in different species have suggested, but not established, that sex hormones influence gastric acid secretion. We studied how acid output is affected by the sex hormones estradiol or testosterone in vivo and in vitro. METHODS: In gastric fistula rats that were normal, sham-operated, neonatally gonadectomized, or treated with estradiol or testosterone, 24-h basal and pentagastrin-stimulated acid secretion was measured. The in vitro effects of estradiol and testosterone on histamine-induced aminopyrine accumulation in isolated parietal cells were also determined. RESULTS: Basal acid output was similar in the two sexes, but stimulated secretion was significantly higher (34%; P < 0.01) in males. Ovariectomy did not influence acid output, whereas orchidectomy reduced basal (18%; NS) and stimulated 24-h secretion (P < 0.01). Estradiol decreased (23%; NS) the 24-h basal output in females but not in males. Estradiol suppressed stimulated secretion in females (29%, P < 0.01) and males (42%, P < 0.01) during the day. At night the stimulated secretion increased in both females (17%, NS) and males (32%, P < 0.05). A similar pattern was found when rats were treated with testosterone. In vitro, estradiol and testosterone reduced histamine-stimulated aminopyrine accumulation in both female and male isolated parietal cells. CONCLUSIONS: Estradiol and testosterone both appear to influence gastric secretion in rats, and their action differs between day and night, between the sexes, and between basal and stimulated secretion.
The extent of apoptosis identified by in situ DNA nick end labelling (TUNEL) on tissue samples obtained from patients with neuroendocrine tumors was correlated with the clinical outcome in patients treated with high-dose somatostatin analog (lanreotide 12 mg/day), n = 8, or other biotherapy including interferon-alpha (IFN-alpha), n = 4, low-dose somatostatin analog (octreotide or lanreotide), n = 3, or a combination of both, n = 1. Biopsies were obtained before the start of treatment and/or after 6 months and 12 months. After 6 months of treatment, 5 patients receiving high-dose somatostatin analog showed a biochemical response (decrease in different neuroendocrine tumor markers) and 4 of these showed an increase in apoptotic index (AI: percentage of apoptotic cells) by 1.94 +/- 1.71%. At 12 months, AI was also increased in patients with a biochemical response (4.22 +/- 3.93%). However, none showed a decrease in tumor size on computerized tomography (CT) and none of the patients treated with low-dose somatostatin analog or IFN-alpha showed any significant increase in AI during treatment. In an experimental model, nude mice were xenografted with the neuroendocrine cell line (BON-1). From the 2nd day of tumor implantation, they received treatment with either placebo, high-dose octreotide, IFN-alpha, or a combination of both, for 28 days. In mice receiving treatment with high-dose octreotide (300 microg/kg, t.i.d) there was a threefold increase in apoptotic cells as compared to the placebo group (p = 0.0084), while the combination group had few cells with ultra-structural changes indicating apoptosis and the IFN-alpha treated group showed no significant changes. However, tumor growth inhibition was more pronounced in the combination group (p = 0.0011). This probably denotes that tumor growth inhibition could be achieved more efficiently by blocking the cell cycle than by inducing apoptosis. We concluded that treatment with high-dose somatostatin analogs may induce apoptosis in neuroendocrine tumors, while this is not found during treatment with low-dose somatostatin analogs or IFN-alpha. We also found that an increase in AI during high-dose somatostatin analog treatment was correlated with the biochemical response, but not with the tumor size as detected by CT in patients or with the tumor mass in the experimental model.
The polymerase chain reaction is being increasingly used for human papillomavirus (HPV) detection in routine diagnostics and in research. Recently, a nonisotopic, enzyme-linked immunosorbent assay-based sandwich capture hybridization assay was introduced as a commercial kit. This hybrid capture system can be used directly on extracted DNA or on polymerase chain reaction products. The latter approach, the SHARP Signal System, uses the consensus primers MY09/MY11, MY11 being biotinylated at its 5' end. We applied the SHARP Signal System to 72 neutral-buffered, formaldehyde-fixed, cervical biopsy specimens to assess the sensitivity and specificity of the kit compared with an earlier established, highly sensitive HPV detection method, GP+/GP6+ and single-stranded conformation polymorphism (SSCP). With the MY09/MY11/SHARP Signal System, 38% of the cases proved positive and with GP5+/GP6+/SSCP, 35%. Correlation of the two methods was 94% for the negative and positive cases and 100% for low- and high-risk HPV. We concluded that the MY09/MY11/ SHARP Signal System is suitable for amplified HPV DNA detection in research and for clinical purposes.