Kati Hakkarainen

DNA vaccination in mice using HIV-1 nef, rev and tat genes in self-replicating pBN-vector.

The immunogenicity of a self-replicating DNA-vector containing HIV-1 nef gene (pBN-Nef) was characterized using various DNA delivery methods. In addition, gene gun immunisation was used for assessing immunogenicity of two other HIV-1 genes (rev and tat) given in the same vector. The pBN-Nef was the most immunogenic raising both humoral and cell-mediated immune responses in mice; these responses lasted for up to six months. The pBN-Nef vector was immunogenic also when given intramuscularly or intradermally. The pBN-Rev construct did not elicit humoral responses but did elicit proliferative as well as CTL-response against the corresponding protein. The pBN-Tat was a poor immunogen in all respects. The antibodies elicited with various DNA delivery methods belonged to different antibody subclasses; however, two main epitopes in Nef were frequently recognized by all of them.

Humoral and cellular immune responses to HIV-1 nef in mice DNA-immunised with non-replicating or self-replicating expression vectors.

OBJECTIVE HIV accessory protein Nef is expressed early in the infectious cycle of the virus and has been shown to be an effective immunogen in humoral and cellular immune responses. We have used two different self-replicating pBN vectors and one non-replicating pCGal2 derived (pCG) vector expressing HIV-1 Nef in DNA immunisation of mice in order to determine their efficiency in raising humoral and cellular immune responses. DESIGN AND METHODS The expression of Nef by the three plasmids was tested by transfections into COS-1 cells. Balb/c mice were immunised with the pBN-NEF and pCGE2-NEF constructs using gold particle bombardment. Immunoblotting and immunocytochemistry were used to detect in vitro expression of Nef. 51Cr release assay, ELISA and immunoblotting were used to detect cellular and humoral immune responses in immunised mice. RESULTS Efficient in vitro expression of Nef was detected in pBN and pCGE2-NEF transfected cells, in pBN-NEF transfected cells the expression lasting up to three weeks. Anti-Nef antibodies in sera of 13 of 16 pBN-NEF immunised mice were detected within four weeks after the last immunisation, whereas only 2 of 12 pCGE2-NEF immunised mice had very weak anti-Nef antibodies. Twelve of the pBN-NEF immunised mice (75%) and 6 the pCGE2-NEF immunised mice (50%) showed Nef-specific cytotoxic T lymphocyte (CTL) responses within four weeks. CONCLUSIONS We conclude that the three eukaryotic expression vectors tested are capable of inducing a cell mediated immune response towards HIV-1 Nef and should be considered as part of a genetic HIV vaccine.

Serological responses to mycoplasmas in Hiv-infected and non-infected individuals

ObjectiveTo assess the frequency of mycoplasma infections in HIV-antibody-positive and -negative individuals by studying the serological responses against mycoplasmas, especially Mycoplasma fermentans and M. pirum. DesignAn enzyme-linked immunosorbent assay (ELISA) was used to measure immunoglobulin G (IgG) class antibody concentrations against six mycoplasma species in sera of HIV-positive and HIV-negative individuals. MethodsSerum samples were obtained from 30 HIV-positive individuals (10 asymptomatics, 10 with lymphadenopathy syndrome and 10 with AIDS), 10 HIV-negative partners of HIV-positive individuals and 40 HIV-negative blood donors. Antibodies to M. fermentans strains incognitus and PG18, M. pirum, M. genitalium, M. pneumoniae and M. hominis were assessed by immunoblot or ELISA. Absorbance values were taken as a semiquantitative measurement for antibody concentration and an arbitrary cut-off value (0.8) was set to establish seroprevalence. ResultsThere was no significant difference in the mean IgG concentrations of any of the six mycoplasmas between HIV-positive and HIV-negative groups. Antibody concentrations were also similar in different clinical phases of HIV infection. Antibody concentrations to different mycoplasma strains were compared with each other to reveal eventual cross-reactions caused by shared antigens; the strongest correlation (r = 0.836) was found between M. fermentans strains incognitus and M. pirum antibody concentrations. The correlation between M. fermentans strains incognitus and PG18 was also significant but weaker (r = 0.522). No shared antigens between M. fermentans strain incognitus and M. pirum were demonstrated by immunoblot. ConclusionsAntibodies against M. fermentans type strain PG18, strain incognitus and against M. pirum are detected infrequently and their presence does not correlate with HIV infection per se or with the clinical stage of HIV infection.

Oral mucosal soft tissue necrosis caused by superinfection : report of three cases

Three patients in whom flap necrosis developed after oral surgeries and antibiotic therapies were studied microbiologically by routine aerobic and anaerobic methods. In all cases the bacteria Enterobacter cloacae, Klebsiella oxytoca, Escherichia coli, and coagulase-negative penicillinase-producing staphylococci were resistant to the antibiotics used. These bacteria are frequent microorganisms of superinfection and were not found after the necrotic tissues had repaired.

Naturally occurring human autoantibodies recognize a fetal brain antigen identified as microtubulus associated Protein 1B

We have recently reported naturally occurring autoantibodies against a large fetal brain antigen (FBA). Now we describe the process of purification and identification of this particular FBA. The brains of newborn rabbits were solubilized and purified with preparative gel electrophoresis. The protein fractions were concentrated and desalted and the fractions were tested by a known positive serum. On membrane digestion of the FBA-band gave a twelve amino acid sequence that resulted in best identity score for mouse, rat and human microtubule-associated protein (MAP) 1B: a member of the microtubule-associated protein family. Monoclonal anti-MAP1B recognized a band in immunoblots of the brain homogenate and of the partially purified fractions with the same electrophoretic mobility as that recognized by a known anti-FBA positive serum. When adult rabbit brain was used as an antigen, the anti-MAP1B failed to recognize any bands on immunoblots. MAP lB has not been previously known as an autoantigen, even though many structural proteins of the neuronal cytoskeleton are known to be targets of naturally occurring autoantibodies. MAP 1B is a functionally important regulatory protein in the developing brain; thus autoantibodies against MAP1B may affect the normal development.

Undergraduate curriculum in palliative medicine at Tampere University increases students' knowledge

BackgroundEducation in palliative medicine (PM) at medical schools reveals wide variation despite the increasing importance of palliative care. Many universities present poor description of the benefits and detailed content of the total curriculum in PM. Using the recommendations of European Association for Palliative Care (EAPC) as a reference, we evaluated the content and outcomes of the curriculum in PM at the University of Tampere, Finland.MethodsWe searched for a PM curriculum by examining the teaching offered by every specialty and compared it to EAPC recommendations. Students’ knowledge was evaluated using a progress test over three consecutive years.ResultsWe found 53.5 teaching hours addressing PM issues, which exceeds the recommendation of the EAPC. Basics, symptom management, ethics, and communication skills were well established, while education in psychosocial/spiritual aspects, teamwork and self-reflection failed to reach the recommendations. Out of the maximum of 4.0, the progress test mean scores in PM among the third, fourth, fifth and sixth year students were 0.1 (SD 0.71), 0.69 (SD 1.28), 1.38 (SD 1.46) and 2.53 (SD 1.26), respectively (p < 0.001). This growing knowledge was associated with the timely increase in teaching provided through the PM discipline. In addition, the students who completed the optional PM course achieved better mean scores (2.66; SD 1.27) than the others (1.33; SD 1.43) (p < 0.001).ConclusionsThe curriculum in PM at the University of Tampere is integrated into the teaching of many disciplines and complied well with the EAPC recommendations. This education led to increasing knowledge in PM among medical students.

Simulated Encounters With Vaccine-Hesitant Parents: Arts-Based Video Scenario and a Writing Exercise

Vaccine hesitancy is an increasing and urgent global public health challenge. Medical students’ encounters with vaccine-hesitant parents, however, remain incidental and unexplored. During pre-clinical training, the vaccine-hesitant parents are typically represented through impersonal text-based cases, lists of their concerns, and sometimes a virtual patient. However, in reality, vaccine-hesitant parents have many health beliefs and arguments that are accompanied with intense emotions, and students remain unaware and unprepared for them. This study is an experimental pilot test in stimulating the medical students’ understanding of, and ability to respond to, vaccine-hesitant parents’ beliefs and questions. An arts-based video scenario and a writing exercise are used to demonstrate a rich case of vaccine hesitancy, including a simulated dialogue between a parent and a student. The study invites vaccine-hesitant parents to ask questions to medical students, then it incorporates these questions in a video scenario and subsequently invites the students to answer these questions as junior doctors. The study examines how the peer group discussion after the video viewing resembles a hospital breakroom conversation and how the written dialogue with a vaccine-hesitant parent simulates a consultation-room encounter.

Circulating autoantibodies to a 240 KD fetal brain protein

Antibodies (IgG and IgM) recognizing a 240 kD antigen of the cat fetal brain were found in sera of healthy people and in sera (IgG) obtained at uncomplicated delivery from the umbilical cord of the newborn infant. The method applied was immunoblotting. Using the same method, the 240 kD antigen could not be detected in the adult brain or other fetal tissues. It seems that the antigen is specific for the fetal brain. The role of the antigen and the origin of generation and significance of function of the antibodies in the circulation are the objects of our further studies.