Adenovirus 5 vector genetically re-targeted by an Affibody molecule with specificity for tumor antigen HER2/neu.

In order to use adenovirus (Ad) type 5 (Ad5) for cancer gene therapy, Ad needs to be de-targeted from its native receptors and re-targeted to a tumor antigen. A limiting factor for this has been to find a ligand that (i) binds a relevant target, (ii) is able to fold correctly in the reducing environment of the cytoplasm and (iii) when incorporated at an optimal position on the virion results in a virus with a low physical particle to plaque-forming units ratio to diminish the viral load to be administered to a future patient. Here, we present a solution to these problems by producing a genetically re-targeted Ad with a tandem repeat of the HER2/neu reactive Affibody molecule (ZH) in the HI-loop of a Coxsackie B virus and Ad receptor (CAR) binding ablated fiber genetically modified to contain sequences for flexible linkers between the ZH and the knob sequences. ZH is an Affibody molecule specific for the extracellular domain of human epidermal growth factor receptor 2 (HER2/neu) that is overexpressed in inter alia breast and ovarian carcinomas. The virus presented here exhibits near wild-type growth characteristics, infects cells via HER2/neu instead of CAR and represents an important step toward the development of genetically re-targeted adenoviruses with clinical relevance.

Selection and characterization of HER2/neu-binding affibody ligands.

Affibody (affibody) ligands that are specific for the extracellular domain of human epidermal growth factor receptor 2 (HER2/neu) have been selected by phage display technology from a combinatorial protein library based on the 58 amino acid residue staphylococcal protein A-derived Z domain. The predominant variants from the phage selection were produced in Escherichia coli, purified by affinity chromatography, and characterized by biosensor analyses. Two affibody variants were shown to selectively bind to the extracellular domain of HER2/neu (HER2-ECD), but not to control proteins. One of the variants, denoted His6-ZHER2/neu:4, was demonstrated to bind with nanomolar affinity (approximately 50 nM) to the HER2-ECD molecule at a different site than the monoclonal antibody trastuzumab. Furthermore, radiolabeled His6-ZHER2/neu:4 affibody showed specific binding to native HER2/neu, overexpressed on the SKBR-3 tumor cell line. Such affibody ligands might be considered in tumor targeting applications for radionuclide diagnostics and therapy of adenocarcinomas such as breast and ovarian cancers.

In vivo and in vitro lipidation of recombinant immunogens for direct iscom incorporation.

We have previously reported strategies for Escherichia coli production of recombinant immunogens fused to hydrophobic tags to improve their capacity to be incorporated into an adjuvant formulation (J. Immunol. Methods 222 (1999) 171; 238 (2000) 181). Here, we have explored the possibility to use in vivo or in vitro lipidation of recombinant immunogens as means to achieve iscom incorporation through hydrophobic interaction. For the in vivo lipidation strategy, a general expression vector was constructed encoding a composite tag consisting of a sequence (lpp) of the major lipoprotein of E. coli, fused to a dual affinity fusion tag to allow efficient recovery by affinity chromatography. Upon expression in E. coli, fatty acids would be linked to the produced gene products. To achieve in vitro lipidation, the target immunogen would be expressed in frame with an N-terminal His6-ABP affinity tag, in which the hexahistidyl tag was utilized to obtain lipidation via a Cu2+-chelating lipid. A 238 amino acid segment DeltaSAG1, from the central region of the major surface antigen SAG1 of Toxoplasma gondii, served as model immunogen in this study. The two generated fusion proteins, lpp-His6-ABP-DeltaSAG1 and His6-ABP-DeltaSAG1, both expressed at high levels (approximately 5 and 100 mg/l, respectively), could be recovered to high purity by ABP-mediated affinity chromatography, and were evaluated in iscom-incorporation experiments. The His6-ABP-DeltaSAG1 fusion protein was associated to iscom matrix with pre-incorporated chelating lipid. Both fusion proteins were found in the iscom fractions after analytical ultracentrifugation in a sucrose gradient, indicating successful iscom incorporation/association. Iscom formation was further supported by electron microscopy analysis. In addition, these iscom preparations were demonstrated to induce high-titer antigen-specific antibody responses upon immunization of mice. For this particular target immunogen, DeltaSAG1, the induced antibodies demonstrated poor reactivity to the native antigen, although slightly better for the preparation employing the in vitro lipidation strategy, indicating that DeltaSAG1 was suboptimally folded or presented. Nevertheless, we believe that the presented strategies offer convenient alternative ways to achieve efficient adjuvant incorporation for recombinant immunogens.

Vulvar vestibulitis: a multi-factorial condition.

OBJECTIVE: To study differences in somatic symptoms and personality dimensions between women with vulvar vestibulitis and a non-symptomatic control group. DESIGN: A case-control study conducted in 1998. SETTING: Two clinics in northern Sweden. SAMPLE: Thirty-eight women, 18-25 years of age, suffering from vulvar vestibulitis, and 70 healthy controls. METHODS: The women completed two questionnaires: the temperament and character inventory to study personality aspects, and the Giessen subjective complaints list, which is a checklist of subjective bodily complaints. RESULTS: Regarding personality aspects the women with vulvar vestibulitis scored significantly higher than the control group, on exclusively one out of seven subscales of the temperament and character inventory (i.e. harm avoidance). This trait is considered to be partly inherited and stable throughout life, and to give the person a tendency to react to problems with pessimistic thoughts, increased anxiety and fatigue. On the Giessen subjective complaints list the women with vestibulitis reported a significantly higher number of somatic complaints in several areas. CONCLUSIONS: The findings that women suffering from vulvar vestibulitis have more bodily complaints than the controls is interpreted as an indication of a psychosomatic element in their illness, which could be primary or secondary. Furthermore, these women are characterised by a particular personality trait, and it is suggested that this trait might influence their experience and management of pain and stress. A multi-factorial origin of vulvar vestibulitis is advocated and a multimodal interdisciplinary treatment approach is suggested. To elucidate further the mechanisms behind this health problem, prospective controlled studies are urgently needed.

Somalis giving birth in Sweden: a challenge to culture and gender specific values and behaviours.

OBJECTIVES: Giving birth in a foreign country implies going through a life event with little or no access to your own traditions and social support. The aim of this study was to study the childbirth experiences of Somali women and men in Sweden. DESIGN: Qualitative. Nine women and seven men were interviewed. Data collection was characterised by an openness to new ideas during the interview and the interviews were analysed according to the grounded theory technique. FINDINGS: The meeting of Somalis with Swedish antenatal and delivery care was a multicultural event. It revealed social, medical, cultural and gender factors advocating space in the arena of childbirth. The Somalis constituted a homogeneous group with regard to their cultural belonging and motives for exile. The subjects were heterogeneous in that they represented a great variety in social and demographic background as well as in experiences, feelings and modes of expression. One striking finding was the Somali man's dramatic entrance into childbirth, which seemed to have a strong impact on the Somali woman's well-being during delivery. The study showed difficulties in getting used to the Swedish model of parenthood and in finding new role divisions in the couple relationship. Some of the subjects had experienced a strengthening of their marriage and an increased understanding of each other. Others commented that various aspects of traditional womanhood and manhood were lost as a result of the unfamiliar gender structures in Sweden. CONCLUSION: The Somalis' experiences of childbirth in Sweden can be understood by using the theoretical concept of gender, rather than culture. Our own and other studies show that women and men may have different frames of reference in childbirth, where the women mainly focus on biological circumstances and the men on the social and cultural aspects of birth. The Somali couple were found to be vulnerably positioned, with the professionals having the important role of supporting and empowering Somali parents.

Vulvar vestibulitis: medical, psychosexual and psychosocial aspects, a case-control study.

BACKGROUND: Vulvar vestibulitis is suspected to be increasingly prevalent among young women, but the etiology is still unclear. The aim of this study was to explore the differences in medical, psychosexual and psychosocial factors between women with vulvar vestibulitis and a control group. METHODS: A case-control study was made with 38 women with vulvar vestibulitis and 71 healthy age-matched controls. All the women answered a structured questionnaire about their medical and gynecological history which included psychosexual and psychosocial background factors and current aspects as well. RESULTS: Women with vulvar vestibulitis have very much the same psychosocial and sexual background factors as their controls, whereas there are many differences in their medical background factors, both gynecological and others. It is very clear that they suffer from many other somatic symptoms more often than their controls. CONCLUSIONS: There are no indications of a primary sexual disturbance in women with vulvar vestibulitis. However, the finding that women with vulvar vestibulitis have many different somatic symptoms indicates a psychosomatic strain in the illness. Regardless of whether this is primary or secondary, it should be taken into consideration when treating the patients.

[Specimen culture from all children in a day care center because of an outbreak of streptococcal infection]. / Odling på samtliga barn vid streptokockutbrott i förskola.

In an outbreak of recurrent group A streptococcal (GAS) pharyngotonsillitis between January and February 1997 at a Swedish preschool, involving 58% (11/19) of the children and several family members, clinical examination showed few of the children to manifest signs and symptoms of tonsillitis. However, throat specimens yielded GAS growth of the same T-type in 42% (8/19) of the children and in 30% (6/20) of family members, but in none of the staff. Strict hygiene, appropriate antibiotic treatment of all culture-positive individuals irrespective of symptoms, and non-attendance at the preschool during the first two days of treatment promptly terminated the outbreak and there were no cases of recurrence.

Motives for having or not having children: a comparison between questionnaire and interview methods.

This study addresses validity issues in a questionnaire constructed with the aim of exploring a private and emotionally loaded topic, namely motives for having or not having children. Themes extracted by factor analysis of data from a reference population of men and women in the fertile ages were used to structure conversational interviews with a smaller sample of women and men of the same age. Of 30 persons invited, 24 agreed to participate in the interview and they completed the questionnaire before the interview. Factor scores were estimated from data obtained by means of the written questionnaire with closed questions, and the scores were compared with results from the structured interview with open questions. Thus, the questionnaire and the factor analysis were validated and a conclusion could be drawn that this method was able to provide important informatory facts even in an area where emotional factors are predominant. The findings also supported the idea that it is important to supplement questionnaire results with validity studies. The combination of the two methods in this study was fruitful and highlighted important new areas that need further research.

Ambivalence towards parenthood among pregnant women and their men.

369 pregnant women and 345 expectant fathers visiting antenatal clinics were examined by a questionnaire concerning attitudes towards pregnancy and parenthood. Data were processed by factor analysis and the degree of agreement to important factors was estimated. Attitudes were dominated by two conflicting views: children mean existential satisfaction and children mean restriction of freedom for women as well as for men. There was a tendency to deny the items concerning the disadvantages of having children. An important factor in the women's as well as in the men's analysis was the importance of own parents as models in parenthood. The findings from this unselected population of pregnant women and their men are consistent with earlier in-depth interviews. It can be concluded that ambivalence should be regarded as a normal constituent of the adaptation to pregnancy and parenthood. Experiences of own parents play an important role in the transition to parenthood. An important task for the medical profession should be to inform about ambivalence as a normal phenomenon as it, left unaccepted, may cause unnecessary anxiety and feelings of guilt.

Attitudes toward reproduction in a nonpatient population.

OBJECTIVE: A basic question to be answered in an attempt to understand human reproductive behavior as it manifests itself in clinical practice is, why do people want to have children? STUDY DESIGN: A psychometric instrument for the assessment of reproductive attitudes was developed. A total of 746 men and women at 20, 30, and 40 years of age, who were randomly selected from a nonpatient population, answered. Data were analyzed by factor analysis. Reproductive profiles were constructed. RESULTS: The two most important factors in both sexes were "children as existential satisfaction" and "children as lack of freedom," indicating a basic conflict. The third most important factor in both sexes was "the importance of one's own parents as examples in parenthood," supporting earlier findings that reproductive conflicts are transmitted from one generation to another. Reproductive profiles were uniform in the different age groups. CONCLUSION: The element of ambivalence may be a clue to a deeper understanding of human reproductive behavior.