Chemically synthesized protein as tumour-specific vaccine: immunogenicity and efficacy of synthetic HPV16 E7 in the TC-1 mouse tumour model

Many successful candidate vaccines capable of combating tumours in animal models come to an untimely end because of the costs associated with the approval and production of the GMP-grade materials, which are usually of biological origin, for use in humans. We have used a GMP-compatible method to chemically synthesize a pure synthetic E7 protein of the human papillomavirus type 16 (HPV16-E7). This oncogen-derived protein is constitutively expressed in cervical cancer and its precursors and is thus considered as an excellent target for tumour-specific immunity. Injection of a mixture of the synthetic HPV16-E7 protein and the synthetic adjuvant CpG in mice resulted in strong functional HPV16-specific cytotoxic T-lymphocyte responses as measured by CD8+ MHC class I-tetramer staining, the detection of antigen-specific intracellular IFNgamma production and the ability to protect mice against a challenge with HPV16-E7+ TC-1 tumour cells in both prophylactic and therapeutic vaccination regimens. Our results demonstrate the potential use of pure synthetic vaccines that can be efficiently produced under GMP at low cost, which will stimulate the translation of new vaccination strategies into phase I/II clinical trials.     

Cellular response to pulsed low-dose rate irradiation in X-ray sensitive hamster mutant cell lines

The role of DNA repair mechanisms in the cellular response to low dose rate (LDR) irradiation was studied with the aim to gain insight in the process of sublethal damage (SLD) repair. Chinese hamster cell lines mutated in either DNA single strand break (ssb) repair or DNA double strand break (dsb) repair by non homologous end joining (NHEJ) and homologous recombination (HR), or showing an AT-like phenotype, were irradiated in plateau-phase either at high dose rate (HDR, 3.3 Gy/min) or at pulsed low dose rate (p-LDR, average 1 Gy/h). Cell survival after irradiation was assessed using the clonogenic assay. A change in sensitivity when the dose rate was decreased was observed for all parental cell lines and the DNA ssb repair mutant. No difference in cell survival after p-LDR versus. HDR irradiation was observed for the two NHEJ mutants, the AT-like mutant and the HR mutant. Based on these results we conclude that single strand break repair does not play a role in the dose rate effect. The AT like protein, functional NHEJ and XRCC3 are required for the dose rate effect.     

Relationship between zona pellucida-induced acrosome reaction,sperm morphology,sperm-zona pellucida binding,and in vitro fertilization

OBJECTIVE: To evaluate the possible relationships between sperm morphology, acrosome responsiveness to solubilized human zona pellucida, and sperm-zona binding potential among [1] consecutive andrology referrals and [2] randomly selected in vitro fertilization (IVF) cases. DESIGN: Prospective analytical study. SETTING: Academic training hospital.Randomly selected couples consulting for infertility. INTERVENTION(S): Acrosome reaction response to solubilized human zona pellucida was recorded. MAIN OUTCOME MEASURE(S): We determined the difference in the percentage of sperm that acrosome reacted after exposure to solubilized zona pellucida and spontaneous acrosome reaction. The results were expressed as percentage zona induced acrosome reaction (ZIAR). RESULT(S): Data were analyzed using correlation coefficients (r) and receiver operator characteristics (ROC curve analyses). The ROC curve analyses indicated ZIAR to be a sensitive indicator for fertilization failure during IVF therapy, with sensitivity and specificity of 81% and 75%, respectively. For andrology referrals, a positive and statistically significant correlation existed between ZIAR data and sperm morphology (r = 0.65) and sperm-zona binding (r = 0.57). CONCLUSION(S): ZIAR results provide further information regarding dysfunctional sperm and can be used as an additional diagnostic test. Our results predicted fertilization failure during IVF treatment.     

First-trimester diagnosis of late-infantile neuronal ceroid lipofuscinosis (LINCL) by tripeptidyl peptidase I assay and CLN2 mutation analysis

Late-infantile neuronal ceroid lipofuscinosis (LINCL) is a progressive neurodegenerative disorder caused by the deficiency of lysosomal tripeptidyl peptidase I (TPP-I) encoded by the CLN2 gene. We report the first case of early prenatal diagnosis of LINCL by combined enzyme and mutation analysis. TPP-I activity in chorionic villi (CV) was less than 2% of the mean normal control level and g.1946A > G and g.3670C > T mutations were demonstrated, as in the two previously affected children. After termination of pregnancy, TPP-I deficiency was confirmed in cultured CV cells and in the fetal skin fibroblasts. The expression of unequivocal TPP-I deficiency in CV demonstrates that enzyme assay is a reliable option for prenatal diagnosis of LINCL.     

Drug craving and addiction: integrating psychological and neuropsychopharmacological approaches

In the present review, an integrated approach to craving and addiction is discussed, which is based on recent insights from psychology and neuropsychopharmacology. An integrated model explains craving and relapse in humans by the psychological mechanism of "attentional bias" and provides neuropsychopharmacological mechanisms for this bias. According to this model, cognitive processes mediate between drug stimulus and the subject's response to this stimulus and subsequent behavioral response (e.g., drug use, relapse). According to the model, a conditioned drug stimulus produces an increase in dopamine levels in the corticostriatal circuit, in particular the anterior cingulate gyrus, amygdala, and nucleus accumbens, which in turn serves to draw the subject's attention towards a perceived drug stimulus. This process results in motor preparation and a hyperattentive state towards drug-related stimuli that, ultimately, promotes further craving and relapse. Evidence for this attentional bias hypothesis is reviewed from both the psychopharmacological and the neuroanatomical viewpoints. The attentional bias hypothesis raises several suggestions for clinical approaches and further research.     

Natural T-helper immunity against human papillomavirus type 16 (HPV16) E7-derived peptide epitopes in patients with HPV16-positive cervical lesions: identification of 3 human leukocyte antigen class II-restricted epitopes

Tumor-specific T-helper (Th) immunity was found to play a pivotal role in the natural and vaccine-induced immune defense against tumors. Since the majority of cervical cancers express human papillomavirus type 16 (HPV16) E7 oncoprotein, it is important to investigate the Th response against this target antigen in detail. By means of PBMC cultures from HLA-typed healthy donors, we identified the central part of HPV16 E7 (E7(41-72)) as the major immunogenic region within this antigen. Furthermore, we mapped 3 distinct Th epitopes within this region (DR15/E7(50-62), DR3/E7(43-77), DQ2/E7(35-50)). In a parallel approach, employing IFN-gamma ELISPOT analysis, we detected Th immunity against HPV16 E7 in subjects with HPV16+ lesions. Several of these responses matched with the 3 Th epitopes defined in our study. A number of other HPV16+ subjects did not display any E7-specific type 1 cytokine-producing T-cell immunity, indicating failure of the immune response. Our combined data argue for more extensive as well as longitudinal analysis of HPV16-specific T-cell immunity using the ELISPOT assay described, as well as for HPV-specific vaccination of individuals with HPV+ lesions.     

Sperm chromatin packaging as an indicator of in-vitro fertilization rates

The development of a sequential diagnostic schedule for patients consulting for infertility disturbances would be an ideal method of approach for clinicians in the absence of an aetiological or pathophysiological diagnosis. Since sperm morphology recorded by strict criteria has often been correlated with fertilization failure, the present study aimed to evaluate the relationship between normal morphology as well as in-vitro fertilization (IVF) rates, with chromatin staining among fertile and subfertile men. Two semen smears were prepared from each specimen obtained from 72 men to record normal morphology and chromatin packaging as recorded by chromomycin A(3) (CMA(3)) staining. Following the semen analyses, the 72 men were divided into the two morphological groups, namely <4% and >4% normal forms. Significantly different percentages of CMA(3) staining (mean +/- SE) were recorded between the two morphological groups, namely 65.9% +/- 3.5 and 44.5% +/- 1.7 (P +/- 0.001). A highly negative significant correlation existed between percentage of normal morphology as recorded by strict criteria and CMA(3) staining. A highly significant and positive correlation was recorded for normal morphology and IVF rates (r +/- 0.45, P +/- 0.0001). A significant negative correlation (r +/- -0.51, P +/- 0.0001) existed between CMA(3) values and IVF rates. The discriminating power of nuclear maturity, as recorded by CMA(3) staining, to identify abnormal morphology values and poor IVF rates was calculated with receiver operator characteristic (ROC) analyses. The areas under the ROC curves were 0.86 for sperm morphology and 0. 74 for IVF rates. The calculated threshold values for CMA(3) staining to distinguish between morphology groups were 48 and 50% for IVF. Chromatin packaging assessment is a valuable addition to the sequential diagnostic programme in an assisted reproductive arena.     

Muscle wasting after radiotherapy in young and adult rats

The impact of late muscular injury after radiotherapy on the well being of patients, particularly those treated at a relatively young age, is often underestimated. To extend the present knowledge, a detailed comparative study of the radiation response of skeletal muscles was performed. The hind limb of rats was irradiated at the age of 30-60 days (young), at approximately 200 days (adult) or at the age of >360 days (old), with single doses ranging from 15-60 Gy. Muscular wet weight, volume and tibia length was followed up to 12 months post-treatment. Radiation treatment resulted in muscular wasting, which was more severe at higher doses. The radiation response depended strongly on the age of the animal at the time of treatment. Radiation induced muscle wasting and stunted tibia growth were more prominent in young animals than in adult or in old rats. ED50 values based on normalized wet weight were 12.5 (11.5-13.6) Gy for young animals and 22.5 (21.8-23.3) Gy for adult animals. The gastrocnemius muscle of young animals appears to be more radiosensitive than the gastrocnemius muscle of adult or old animals. Furthermore, in young animals, severe wasting of the muscles may already occur after relative low radiation doses (single dose of 15 Gy).     

Congenital dysplasia of C2–6

Neural arch dysplasia of the cervical 4 vertebrae is a rare occurrence. The authors present a case with dysplasia of cervical bodies and posterior arch elements at multiple levels. The embryogenesis of these anomalies is discussed.