Pilot study of an HLA-A2 peptide vaccine using flt3 ligand as a systemic vaccine adjuvant

A pilot vaccine study was conducted to test the safety and immunological efficacy of four monthly immunizations of an MHC class I peptide vaccine, the E75 HLA-A2 epitope from HER-2/neu, using flt3 ligand as a systemic vaccine adjuvant. Twenty HLA-A2-expressing subjects with advanced stage prostate cancer were randomly assigned to one of four immunization or treatment schedules: (a) Flt3 ligand (20 g/kg per day) administered subcutaneously daily for 14 days on a 28-day cycle, monthly for four months; (b) flt3 ligand course as above with the E75 peptide vaccine administered on day 7 of each flt3 ligand cycle; (c) flt3 ligand course as above with the E75 peptide vaccine administered on day 14 of each flt3 ligand cycle; or (d) E75 peptide admixed with granulocyte–macrophage colony-stimulating factor and administered intradermally once every 28 days, as has previously been reported. The primary endpoints of the study were the determination of safety and immunological efficacy in generating E75-specific T cells as determined by peptide-specific interferon-gamma ELIspot. Adverse events included one grade 3 skin reaction and the development of grade 2 autoimmune hypothyroidism in two subjects with preexisting subclinical autoimmune hypothyroidism. Dendritic cells were markedly increased in the peripheral blood of subjects receiving flt3 ligand with each repetitive cycle, but augmentation of antigen-presenting cells within the dermis was not observed. Apart from a single subject, no significant peptide-specific T-cell responses were detected by ELIspot, whereas delayed-type hypersensitivity responses were detectable in control subjects and in subjects receiving peptide vaccine early in the course of flt3 ligand administration. The absence of robust peripheral immune responses in the current study may be attributable to the small numbers of subjects or differences in the subject population. In addition, the inability of flt3 ligand to augment the number of peripheral skin antigen-presenting cells may have contributed to the absence of robust peptide-specific immunity detectable in the peripheral blood of immunized subjects treated with flt3 ligand.     

Changes in retinoblastoma gene expression during cervical cancer progression

The role of tumour suppressor genes in the development of human cancers has been studied extensively. In viral carcinogenesis, the inactivation of suppressor proteins such as retinoblastoma (pRb) and p53, and cellular oncogenes overexpression, such as c-myc, has been the subject of a number of investigations. In uterine-cervix carcinomas, where high-risk human papillomavirus (HPV) plays an important role, pRb and p53 are inactivated by E7 and E6 viral oncoproteins, respectively. However, little is known about the in situ expression of some of these proteins in pre-malignant and malignant cervical tissues. On the other hand, it has also been demonstrated that c-myc is involved in cervical carcinogenesis, and that pRb participates in the control of c-myc gene expression. By using immunostaining techniques, we investigated pRb immunodetection pattern in normal tissues, squamous intraepithelial lesions (SILs) and invasive carcinomas from the uterine cervix. Our data show low pRb detection in both normal cervical tissue and invasive lesions, but a higher expression in SILs. C-Myc protein was observed in most of the cellular nuclei of the invasive lesions, while in SILs was low. These findings indicate a heterogeneous pRb immunostaining during the different stages of cervical carcinogenesis, and suggest that this staining pattern could be a common feature implicated in the pathogenesis of uterine-cervix carcinoma.     

阴道炎患者HPV6/11-DNA定量分析

目的：了解女性阴道炎患者人乳头瘤病毒6／11型（HPV6／11）的感染情况。方法：荧光定量聚合酶链反应（FQ－PCR）技术检测258例门诊患者阴道分泌物中HPV6／11的病毒拷贝数。结果：共检出HPV6／11阳性者94例,阳性率36．43％,拷贝数在10^5以上者78例,占阳性者中82．98％,40岁以上年龄组阳性率高且病毒复制量均在10^5以上。结论：（1）中老年阴道炎患者就诊晚,感染重。（2）FQ－PCR检测HPV敏感,快速,准确,特别是其定量特点对临床很有意义。     

Dose and Load Studies for Subcutaneous and Oral Delivery of Poly(lactide-co-glycolide) Microspheres Containing Ovalbumin

Poly(lactide-co-glycolide) microspheres containing different loads of OVA (0.05, 0.1, 0.5 and 1.0% w/w) were manufactured by a w/o/w emulsion/solvent evaporation method. Low load efficiencies of less than 20% were observed. Normal size distributions with mean volume diameters ranging from 3.7 to 4.7 µm were obtained for different batches. The in vitro release of OVA from different loaded microspheres showed an expected burst release with all batches. The in vivo dose study (1, 10, 25, 50 µg of OVA) was performed by subcutaneous and oral inoculation in mice by single (0 week) or double (0 and 3 weeks) administration of PLGA 50/50 microspheres containing 0.1% OVA. Subcutaneous administration showed an immune response (serum Ig levels by ELISA) statistically (Fishers paired t-test; P < 0.05) above OVA saline negative controls at 3, 6 and 12 weeks after administration. Oral administration of microspheres produced statistically higher systemic immune responses at the higher doses. Single and double inoculation orally and subcutaneously produced similar serum antibody levels. The in vivo load study was performed by subcutaneous and oral administration to mice of 25 µg OVA contained in various loaded (0.05, 0.1, 0.5 and 1.0% w/w) microspheres. Serum immune responses at 3, 6, and 12 weeks after inoculation were statistically above OVA saline controls and were inversely proportional to the OVA load using either route. This observation suggested a relationship between the number of microspheres delivered and the in vivo serum response. Single subcutaneous administration of 0.05 or 0.1% OVA loaded PLGA 50/50 microspheres induced larger immune responses compared with complete Freunds adjuvant.     

Influence of age and human papillomavirus-infection on reliability of cervical cytopathology

The aim of the present study was to evaluate the effect of age and human papillomavirus (HPV) infection associated cellular changes on the predictive value of cervical cytology. In a group of 671 women with Papanicolaou smears suggesting low grade squamous intraepithelial lesion (LSIL), high grade squamous intraepithelial lesion (HSIL) or invasive cervical cancer, cervical cytology was correlated with the histological finding. Predictive values were calculated and related to severity of the lesion, age and HPV associated changes. The predictive values of Papanicolaou (cervical) smears suggesting LSIL, HSIL and invasive carcinoma were 40%, 86%, and 78%, respectively. A poor predictive value of smears suggesting LSIL was found among older women. HPV associated changes were diagnosed in 80% of women 25 years of age, 66% in the age group 26 to 35 years, 51% in the age group 36 to 45 years and 38% in women aged 46 years (P = 0.03). The presence of HPV associated cellular changes led to a significantly higher number of overdiagnoses (9% with HPV infection compared to 4% without HPV infection) and HPV negative cases were more frequently associated with underdiagnosis (15% without HPV infection compared to 8% with HPV infection,P = 0.0011). This result remained significant after adjustment for age (P = 0.004). Cellular changes associated with HPV infection most frequently occurred in young women. HPV infection should therefore be acknowledged as source of overdiagnosis in the cytological evaluation of SIL especially in woung women.Supported by the Research Grant of the Mayor of Vienna (no. 1045 [to Dr. Kainz])     

Malaria vaccines

Significant progress has been made in the development of the malaria vaccine during the last 20 years. Ninety percent of the 300–500 million clinical cases of malaria per year worldwide occur in Africa. Thus, research must be directed toward the 1 million African children under 5 years of age who die every year of malaria. An asexual blood-stage vaccine, capable of reducing severe and complicated malaria and malaria-related mortality, is therefore an important public health tool in these countries. Although knowledge of the parasite's biology is incomplete, research has allowed insight into some of the mechanisms that the parasite uses to evade host immunity. This is the basis for adopting an antigenic cocktail approach toward obtaining a synthetic or recombinant subunit vaccine such as the synthetic Colombian Malaria vaccine SPf 66. During the development of Spf66, field trials under both low and high malaria endemicity areas in Latin America and Africa have been carried out. The results from these studies showed a protective efficacy ranging between 38.8 and 60.2% againstPlasmodium falciparum malaria. Given the characteristics of the normal immune response to malaria (relatively short-lived and not completely effective), it is understandable that the main goal is to try to increase the host's natural immunity. The best candidates for designing a malaria vaccine are the proteins required for parasite survival, those with low mutation rates and conserved epitopes. Because these proteins play an important role in multiple or alternative steps during the invasion process, they should be the targets against which a protective immune response should be elicited. The interaction between the malaria parasite and its host is complex. It is therefore crucial to define new ways of improving the immune response—such as directly modifying the chemical structure of epitopes or using new adjuvants or DNA immunization techniques—to produce novel vaccines against this disease.     

Production of 5–15 µm Diameter Alginate-Polylysine Microcapsules by an Air-Atomization Technique

A novel method of preparing small-sized microcapsules using a Turbotak air-atomizer is reported. Alginate-polylysine microcapsules containing Bacillus Calmette Guérin vaccine have been prepared by an adaptation of the method of Lim (1) which allows the manufacture of small-sized microcapsules. A Turbotak is used to spray sodium alginate solution into calcium chloride solution to form temporary calcium alginate microgel capsules. These temporary microgel droplets are subsequently cross-linked with polylysine to form permanent membranes. Microcapules in the size range of 5–15 µm have been produced which can be compared to an average diameter of 300 µm obtained by the method reported by Lim. The microcapsule size is dependent on the conditions of operation of the Turbotak and the concentration of the sodium alginate solution. Particles within the size range 5–15 µm can be reproducibly manufactured using the conditions of operation reported here. Other size ranges below the minimum of 300 µm reported by Lim are also feasible using this technique.     

A residual cystic lesion in acute disseminated encephalomyelitis

We report a case of acute disseminated encephalomyelitis (ADEM) with a residual cystic lesion on MRI. This seemed to be induced by Japanese encephalitis vaccination. Despite complete clinical improvement with high-dose steroid therapy, the cystic lesion has persisted for 3 years on MRI. There have been no previous reports of residual cystic lesions in ADEM. Received: 20 August 1999/Accepted: 11 November 1999