Ilse Beckmann

Treatment of Vulvar Intraepithelial Neoplasia with Topical Imiquimod

BACKGROUND Alternatives to surgery are needed for the treatment of vulvar intraepithelial neoplasia. We investigated the effectiveness of imiquimod 5% cream, a topical immune-response modulator, for the treatment of this condition. METHODS Fifty-two patients with grade 2 or 3 vulvar intraepithelial neoplasia were randomly assigned to receive either imiquimod or placebo, applied twice weekly for 16 weeks. The primary outcome was a reduction of more than 25% in lesion size at 20 weeks. Secondary outcomes were histologic regression, clearance of human papillomavirus (HPV) from the lesion, changes in immune cells in the epidermis and dermis of the vulva, relief of symptoms, improvement of quality of life, and durability of response. Reduction in lesion size was classified as complete response (elimination), strong partial response (76 to 99% reduction), weak partial response (26 to 75% reduction), or no response (< or =25% reduction). The follow-up period was 12 months. RESULTS Lesion size was reduced by more than 25% at 20 weeks in 21 of the 26 patients (81%) treated with imiquimod and in none of those treated with placebo (P<0.001). Histologic regression was significantly greater in the imiquimod group than in the placebo group (P<0.001). At baseline, 50 patients (96%) tested positive for HPV DNA. HPV cleared from the lesion in 15 patients in the imiquimod group (58%), as compared with 2 in the placebo group (8%) (P<0.001). The number of immune epidermal cells increased significantly and the number of immune dermal cells decreased significantly with imiquimod as compared with placebo. Imiquimod reduced pruritus and pain at 20 weeks (P=0.008 and P=0.004, respectively) and at 12 months (P=0.04 and P=0.02, respectively). The lesion progressed to invasion (to a depth of <1 mm) in 3 of 49 patients (6%) followed for 12 months (2 in the placebo group and 1 in the imiquimod group). Nine patients, all treated with imiquimod, had a complete response at 20 weeks and remained free from disease at 12 months. CONCLUSIONS Imiquimod is effective in the treatment of vulvar intraepithelial neoplasia. (Current Controlled Trials number, ISRCTN11290871 [controlled-trials.com].).

Bioactive tumour necrosis factor α in pre‐eclamptic patients with and without the HELLP syndrome

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Tumor necrosis factor-α in whole blood cultures of preeclamptic patients and healthy pregnant and nonpregnant women

Objectives: Tumor necrosis factor‐α (TNF‐α) is recognized as a likely mediator of the excessive endothelial activation and injury that is a key pathogenetic mechanism of preeclampsia. We used whole blood cell cultures from 12 patients with severe preeclampsia and from 12 healthy pregnant and nonpregnant women to determine the release of TNF‐α by unstimulated leukocytes as a measure of their state of activation, and their response to stimulation with lipopolysaccharide (LPS) as an indicator of their state of priming. Methods: Blood was cultivated without and with LPS, and TNF‐α release was measured after six and 24 hours of cultivation by enzyme‐linked immunoassays. Differential leukocyte counts were performed, and TNF‐α values calculated per 105 monocytes. Results: In unstimulated whole blood cultures, TNF‐α release after six hours of cultivation was similar in all three groups; but after 24 hours, TNF‐α concentrations in culture supernatants from preeclamptic patients were significantly higher than were values obtained in blood from normotensive pregnant women. In LPS‐stimulated blood cultures with a maximum of TNF‐α release at six hours cultivation time, TNF‐α concentrations were significantly lower in preeclamptic women than they were in both control groups. We showed in an additional experiment that a strong LPS challenge following preactivation with high doses of LPS resulted in reduced release of TNF‐α compared with release of TNF‐α following preactivation with low doses of LPS. Conclusions: The observed high capacity for spontaneous TNF‐α release by leukocytes in preeclampsia indicates activation of TNF‐α producing leukocytes by the disease process. Preactivation and exhaustion of leukocytes by leakage of TNF‐α could lead to the reduced response to TNF‐α inducer LPS as observed in blood cultures from preeclamptic patients.

Soluble tumor necrosis factor receptor II and soluble cell adhesion molecule 1 as markers of tumor necrosis factor‐α release in preeclampsia

Background.  The purpose of this case‐controlled study was to investigate whether plasma concentrations of TNF‐receptors I and II and tumor necrosis factor‐α‐induced cell adhesion molecule 1 VCAM‐1 could serve as more sensitive markers of tumor necrosis factor‐α release in preeclamptic women than a direct measurement of circulating tumor necrosis factor‐α.

Cytokine release in HR-HPV(+) women without and with cervical dysplasia (CIN II and III) or carcinoma, compared with HR-HPV(-) controls

Aims. We investigated the effect of HR-HPV infection on the capacity of the cytokine network in whole blood cultures during carcinogenesis of cervical carcinoma. Methods. Thirty-nine women with moderate dysplasia, severe dysplasia, cervical carcinoma, or without dysplasia formed the study group. The control group consisted of 10 HR-HPV-negative women without CIN. Whole blood cultures were stimulated with phytohemagglutinin (PHA) and concentrations of tumour necrosis factor α (TNFα), interferon γ (IFNγ), interleukin 2 (IL-2), interleukin 12 (IL-12), interleukin 4 (IL-4), and interleukin 10 (IL-10) were determined by ELISAs. Results. A significant increase in cytokine release was detected in HR-HPV-positive women without dysplasia. In women with cervical cancer, release of IFNγ and IL-12 was of the same magnitude as in HR-HPV-positive women without clinical manifestations. Most Th1-type/Th2-type ratios decreased form CIN II to CIN III, and increased from CIN III to invasive carcinoma. Conclusions. (1) Infection with HR-HPV without expression of cervical dysplasia induces activation of the cytokine network. (2) Increases in ratios of Th1-type to Th2-type cytokines at the stage of cervical carcinoma were found by comparison with stage CIN III. (3) Significant changes in the kinetics of cytokine release to a Th2-type immune response in blood of women with cervical dysplasia occurred progressively from CIN II to CIN III.

Uterine artery estrogen receptors in the nonpregnant and pregnant guinea pig

Uterine artery, heart, and aorta or carotid specimens of nonpregnant, midpregnant, and term pregnant guinea pigs were examined for estrogen receptors by immunocytochemical methods. Estrogen receptors were found in the nuclei of cells in the endothelial, muscle, and adventitia layers of the uterine artery wall. The concentration of estrogen receptors was slightly higher in nonpregnant and term pregnant animals than in midpregnancy. No estrogen receptors were found in the heart, aorta, or carotid specimens of all animals. These results confirm the uterine artery as a target organ of estrogen action that would eventually lead to arterial functional adaptation in different biologic periods.

Immunochemical investigations of antigens isolated fromBacteroides ovatus strain ATCC 8483

A saline extract (SE) and a phenol/water extract (WL) were prepared fromBacteroides ovatus strain ATCC 8483. A fraction CS was isolated from the culture supernatant. WL was further split by ultracentrifugation into lipopolysaccharide (LPS) and supernatant (L1). Fractions SE, WL, LPS and L1 reacted serologically with homologous antiserum but did not cross-react with antisera against heterologousBacteroides serotypes. Fraction CS was inactive in haemagglutination, haemagglutination inhibition and immunoelectrophoresis tests. SE, WL, LPS and L1 proved to be serologically heterogeneous. A distinct serological specificity for SE was demonstrated. The serological reactivity in SE and WL was not altered after treatment with proteolytic enzymes yet completely destroyed in WL and partially in SE by sodium metaperiodate. SE, WL, LPS and L1 contained the sugar components rhamnose, fucose, ribose, mannose, galactose, glucose and glucosamine in different molar ratios for each fraction. Galactosamine was found in WL and LPS, uronic acid in WL and L1. Two unidentified aminohexoses were detected in WL, one of which was also detectable in L1 and SE. 2-Keto-3-deoxyaldonic acid was demonstrated in LPS and L1 after strong acid hydrolysis.

Release of tumor necrosis factor-α and prostanoids in whole blood cultures after in vivo exposure to low-dose aspirin

BACKGROUND: The preventive effect of low-dose aspirin in cardiovascular disease is generally attributed to its antiplatelet action caused by differential inhibition of platelet cyclooxygenase-1. However, there is evidence that aspirin also affects release of inflammatory cytokines, including tumor necrosis factor-alpha (TNF-alpha). It is not known whether this is caused by direct action on the cytokine pathway or indirectly through cyclooxygenase inhibition and altered prostanoid synthesis, or both. METHODS: We assessed the capacity of lipopolysaccharide-activated leukocytes in whole blood cultures of eight healthy subjects following a single oral dose of 80 mg aspirin to release TNF-alpha, prostanoid E2 (PGE2) and prostanoid I2 (PGI2), and thromboxane A2 (TXA2). TNF-alpha and prostanoids were determined by enzyme-linked immunoassays. RESULTS: In seven subjects, TNF-alpha release in blood cultures decreased 24h after intake of aspirin. The effect of aspirin on prostanoid release was assessed in three individuals: PGE2 increased in all subjects, PGI2 increased in two and remained unchanged in one, and TXA2 was reduced in two and unchanged in one individual The presence of DFU, a specific inhibitor of cyclooxygenase 2, did not affect the reduction of TNF-alpha release by aspirin, but abolished prostanoid production in all three individuals. Conclusion: The capacity of activated leukocytes to release TNF-alpha is reduced by ingestion of low-dose aspirin, independent of changes in prostanoid biosynthesis.

Immune Response to Endotoxin Isolated from Bacteroides fragilis in the Pregnant Guinea Pig

The humoral immune response to endotoxin isolated from Bacteroides fragilis was analyzed in the pregnant guinea pig by means of passive hemagglutination, passive hemolysis, a modified Coombs test, and by crossed immunoelectrophoresis. Pregnant animals were immunized with endotoxin on day 30 of gestation, and antibodies were determined on day 61 in maternal and fetal sera, and in amniotic fluid. The IgG and IgM responses in maternal sera were of the same magnitude as in sera of nonpregnant animals. Fetal sera contained IgG and sometimes IgM, and a higher percentage of incomplete antibodies against endotoxin than maternal sera. Low-titer anti-endotoxin antibodies, partially sensitive to dithiothreitol, were found in amniotic fluid. A statistically significant reduction in the growth of fetuses from endotoxin-immunized females was observed.

Detection of Bacteroides fragilis endotoxin in amniotic fluid by counterimmunoelectrophoresis

The ability of counter immunoelectrophoresis (CIE) to detectBacteroides fragilis endotoxin in amniotic fluid in small concentrations was evaluated. A method was developed which, in combination with ultrafiltration, permits detection ofB. fragilis endotoxin in amniotic fluid in a concentration of 40 ng/ml or more. The sensitivity threshold was reduced to 2 ng/ml by using a highly reactive IgG-fraction isolated from rabbit anti-B. fragilis IPL E 323 antiserum.