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Biomedical subjects

Dieter Klein

Publications and source records attributed to Dieter Klein.

14 recordsLinked to original sources
PubMed2007

Lactoferrin, a glycoprotein with immunomodulatory and mast cell stabilising properties, in skin of horses suffering from Culicoides hypersensitivity.

Lactoferrin (LF), a glycogen of the transferrin family with anti-bacterial and immunomodulatory properties, is expressed in various secretions and tissues. Cutaneous LF serves as a mast cell stabilising compound, modulates T cell activity and is found during IgE-mediated late phase reactions at allergen challenged sites. Culicoides hypersensitivity (CHS) in horses is a common IgE-mediated allergic dermatitis, characterised by an early and late phase cutaneous reaction upon allergen challenge. The aim of the study presented here was to examine whether LF mRNA expression in skin biopsies from horses affected by CHS prior to and 4h following intradermal challenge with a commercial C. nubeculosus extract is modified in comparison to skin biopsies from non-affected horses. In order to obtain reliable data, real time PCR was performed and genes of interest were normalized using three different housekeeping genes, beta-actin, GAPDH, beta-2-microglobulin. In comparison to non-affected horses, higher variation in LF mRNA levels both prior to and post-intradermal challenge with C. nubeculosus extract was seen in horses affected by CHS. However, the statistical analysis demonstrated that LF mRNA expression was not significantly different between CHS affected and non-affected horses prior to intradermal challenge with C. nubeculosus extract. Intradermal injection of C. nubeculosus extract did not result in local upregulation of LF mRNA at 4h post-injection. LF mRNA expression was therefore not significantly different pre- or post-intradermal challenge with C. nubeculosus extract in either group. Our data indicate that clinically normal skin of horses affected by CHS is not characterized by modified maintenance levels of LF mRNA. In contrast to human skin allergen challenged sites, LF mRNA levels in horses affected by CHS are not significantly different to that of control sites at 4h post-injection of C. nubeculosus extract.

Animals↗
PubMed2006

Lactoferrin expression in the horse endometrium: relevance in persisting mating-induced endometritis.

Lactoferrin (LF) is an estrogen-regulated glycoprotein with well-described antibacterial and immunomodulatory properties. The present study is the first report on LF expression in horse endometrial specimens. Mares chosen for the study were either resistant or susceptible for persisting mating-induced endometritis (PMIE) during the natural ovulatory cycle and in early pregnancy. Our investigations included immunostaining for LF protein and CD18, a leukocyte marker, as neutrophils are a possible source for LF in the endometrium. Quantification of LF mRNA was performed by use of real-time RT-PCR. This study demonstrated that LF protein in equine endometrium was expressed in glandular and luminal epithelium and in neutrophils. Similar to other mammalian species, the level of endometrial LF transcription in the mare was modulated according to the stage of the estrus cycle and was 5500-fold higher during estrus compared with diestrus and early pregnancy. The endometria from mares susceptible for PMIE and delayed uterine clearance exhibited an increased LF transcription during all stages of the estrus cycle that reached statistical significance in proestrus. In the endometria of mares susceptible for PMIE the upregulated LF mRNA expression was not associated with a higher number of CD18 positive leukocytes but correlated with the number of uterine glands. Enhanced LF transcription within the endometrial epithelium might therefore be a response to recurrent persisting inflammation following insemination in mares with delayed uterine clearance.

Animals↗
PubMed2006

Prime-boost vaccination using DNA and whole inactivated virus vaccines provides limited protection against virulent feline immunodeficiency virus.

Protection against feline immunodeficiency virus (FIV) has been achieved using a variety of vaccines notably whole inactivated virus (WIV) and DNA. However protection against more virulent isolates, typical of those encountered in natural infections, has been difficult to achieve. In an attempt to improve protection against virulent FIV(GL8), we combined both DNA and WIV vaccines in a "prime-boost" approach. Thirty cats were divided into four groups receiving vaccinations and one unvaccinated control group. Following viral challenge, two vaccinated animals, one receiving DNA alone and one the prime-boost vaccine remained free of viraemia, whilst all controls became viraemic. Animals vaccinated with WIV showed apparent early enhancement of infection at 2 weeks post challenge (pc) with higher plasma viral RNA loads than control animals or cats immunised with DNA alone. Despite this, animals vaccinated with WIV or DNA alone showed significantly lower proviral loads in peripheral blood mononuclear cells and mesenteric lymph node cells, whilst those receiving the DNA-WIV prime-boost vaccine showed significantly lower proviral loads in PBMC, than control animals, at 35 weeks pc. Therefore both DNA and WIV vaccines conferred limited protection against viral challenge but the combination of WIV and DNA in a prime-boost approach appeared to offer no significant advantage over either vaccine alone.

Animals↗
PubMed2006

Increased interleukin-1beta mRNA expression in skin biopsies of horses with Culicoides hypersensitivity following challenge with Culicoides nubeculosus extract.

Interleukin-1beta (IL-1beta) is a primary cytokine of the skin that has a pivotal role in keratinocyte differentiation, epidermal wound healing and host defense. Pathological increase of cutaneous IL-1beta is associated with edema formation, epidermal hyperproliferation and atopic dermatitis in humans. However, in horses the role of cutaneous IL-1beta in edema formation and allergic skin disease has not been characterised so far. Particularly in Culicoides hypersensitivity (CHS), intradermal injection of Culicoides extract may be associated with enhanced transcription of local IL-1beta. To examine the mRNA expression of IL-1beta and its receptor antagonist IL-1RA in the skin of horses, biopsy specimens of horses affected and non-affected by CHS prior and following intradermal challenge with a commercial C. nubeculosus extract were examined. Our hypothesis was that cutaneous IL-1beta mRNA was significantly upregulated in horses with CHS in response to Culicoides allergen. Biopsies were taken from sites prior to and 4 h following intradermal challenge with C. nubeculosus extract. In order to obtain reliable data, real time PCR was performed and genes of interest were normalized using three different housekeeping genes, beta-actin, GAPDH, beta-2-microglobulin. No significant difference was detected in non-challenged cutaneous IL-1beta mRNA and IL-1RA mRNA levels between CHS affected and non-affected horses. Intradermal injection of C. nubeculosus extract resulted in local upregulation of IL-1beta mRNA both in horses with typical history, characteristic clinical signs for CHS and a positive intradermal skin test (IDT), and non-affected horses with a negative IDT. However, the difference in prior and post challenged site IL-1beta mRNA levels only reached statistical significance in the affected horses (p=0.01 versus 0.7). In contrast, IL-1RA mRNA levels did not demonstrate any modification following intradermal injection with C. nubeculosus in either group. In contrast to human atopic dermatitis, clinically normal skin of horses affected by CHS is not characterized by increased maintenance levels of IL-1beta mRNA. C. nubeculosus stimulates local IL-1beta transcription in all horses independent from disease, but the extent of upregulation from basal levels only reaches statistical significance in horses affected by CHS and active stage of disease.

Animals↗
PubMed2005

Effects of semen extender and semen processing on motility and viability of frozen-thawed dog spermatozoa.

The aims of the present study were, to assess the effects of semen centrifugation, two different diluents and two different freezing methods on post-thaw semen quality in canine semen, and to elucidate the interdependence of these parameters. For this purpose, the sperm-rich fractions of ejaculates from 12 healthy male beagles were divided into four aliquots. Two aliquots were centrifuged and resuspended with two TRIS-egg yolk based extenders: with Uppsala and Gill extender (Gill). The diluents differed in the concentration of glycerol and in the admixture of Equex STM paste (Nova Chemical Sales Inc., Scituate, MA, USA). Diluted semen was frozen either in a styrofoam box or with a computerized freezing machine and an optimized freezing curve (IceCube 1,810; Sy-Lab, Purkersdorf, A). The change in temperature inside the straws was measured during the freezing procedure. Thawed semen samples were assessed for motility and viability (SYBR-14/PI) using the computer assisted sperm analyzer SpermVision (Minitüb, G) and a modified triple staining technique (flow cytometry). Deep freezing in the machine resulted in better motility and viability than in the box. The combination centrifugation-Uppsala extender-machine was superior to all other combinations, which was most evident after storage at +5 degrees C for 7 h (motility: 53.1%, viability: 64.9%). Post-thaw longevity and progressive motility were significantly improved by the use of the here introduced freezing curve. This was shown to be partly caused by less pronounced fluctuations of temperature inside the straws when compared to box-freezing.

Animals↗
PubMed2005

Genetic variability of PRRS virus in Austria: consequences for molecular diagnostics and viral quantification.

Porcine reproductive and respiratory syndrome virus is a virus with a relatively high level of sequence variation, especially between the European and the American-type strains. This high degree of sequence variations will influence the accuracy of results obtained by molecular diagnostic methods, especially if mutations are located in the primer or probe binding regions of PCR or real-time PCR based assays. In order to provide the basis for the development of an accurate, region-tailored assay we have analysed samples obtained from several Austrian pig herds using a series of RT-PCR assays. Subsequent sequencing of the PCR products from ORF5 and phylogenetic analysis revealed the occurrence of both the European and the American-type of porcine reproductive and respiratory syndrome virus in Austria. Since vaccination with the American-type vaccine is not authorized in Austria the import of vaccinated animals is the most likely explanation for the occurrence of these strains. The study highlights the importance of a detailed study on the sequence variations occurring in the region of interest before the development of a reliable PCR-based assay.

Animals↗
PubMed2005

Vaccination with an inactivated virulent feline immunodeficiency virus engineered to express high levels of Env.

An inactivated virus vaccine was prepared from a pathogenic isolate of feline immunodeficiency virus containing a mutation that eliminated an endocytic sorting signal in the envelope glycoprotein, increasing its expression on virions. Cats immunized with inactivated preparations of this modified virus exhibited strong titers of antibody to Env by enzyme-linked immunosorbent assay. Evidence of protection following challenge demonstrated the potential of this approach to lentiviral vaccination.

Amino Acid Sequence↗
PubMed2004

Multiple modifications allow high-titer production of retroviral vectors carrying heterologous regulatory elements.

Tumor-specific expression of therapeutic genes is a prerequisite in many approaches to retrovirus-mediated cancer gene therapy. However, tissue specificity is often associated with a reduction in viral titer. To overcome this problem, we constructed a series of murine leukemia virus (MLV)-based retroviral promoter conversion (ProCon) vectors carrying either the simian virus 40 poly(A) signal trimer (3pA) inserted in the 3' long terminal repeat (LTR) of these vectors or the human cytomegalovirus enhancer region (CMVe) inserted 5' and 3' of the retroviral LTRs. Furthermore, an extended AT stretch/attachment site (AT/att) of wild-type MLV was introduced into the vector. In the vector-producing cells, insertion of the CMVe and/or the 3pA resulted in a three- to fourfold-enhanced marker gene expression compared to the parental vector, whereas insertion of the AT/att gave a slight decrease in expression. The combination of all three modifications had no additional effects. In contrast, however, neomycin selection of infected cells revealed only a slight increase in virus titer with vectors carrying the 3pA modification; the titer was increased by 1 with vectors containing the extended AT/att, although the viral DNA copy numbers in infected cells were similar with both types of vectors. Thus, insufficient integration rather than insufficient reverse transcription and/or production of virus RNA is the major cause for the low titer obtained with the ProCon vectors. The combination of all three modifications resulted in a 2- to 3-log increase in the virus titer. These modifications result in expression targeted ProCon vectors with titers similar to those of nonmodified MLV-based vectors.

Animals↗
PubMed2003

Phylogenetic analysis of feline immunodeficiency virus in Central Europe: a prerequisite for vaccination and molecular diagnostics.

Feline immunodeficiency virus (FIV) is a worldwide-occurring lentivirus that severely impairs the immune function of infected domestic cats. Due to structural and biological similarities, FIV represents a promising model for human immunodeficiency virus (HIV) and AIDS. A major obstacle in developing vaccines against lentiviruses is their high mutation rate. Furthermore, mutations in target sequences provide a pitfall for molecular diagnostics. It is therefore important to determine the genetic diversity of lentiviruses in any region where vaccination or implementation of new diagnostic techniques are planned. This study presents a phylogenetic analysis of 30 FIV strains derived from Central Europe. In order to improve the reliability of genotyping, DNA from two different proviral genes was amplified and comparative phylogenetic trees were inferred. The highly coincident results point to the existence of extensive virus variation with the presence of at least two highly divergent subtypes of FIV in Austria and Germany.

Animals↗
PubMed2002

Quantification using real-time PCR technology: applications and limitations.

The introduction of real-time PCR technology has significantly improved and simplified the quantification of nucleic acids, and this technology has become an invaluable tool for many scientists working in different disciplines. Especially in the field of molecular diagnostics, real-time PCR-based assays have gained favour in the recent past. However, the wide use of real-time PCR methods has also highlighted some of the critical points and limitations of these assays. These aspects must be considered to increase the reliability of the obtained data.

Gene Expression Profiling↗
PubMed2002

The murine whey acidic protein promoter directs expression to human mammary tumors after retroviral transduction.

The whey acidic protein (WAP) promoter is known to be active in pregnant and lactating mammary epithelial cells as well as mammary tumors of mice. Here we show that a proximal fragment of the murine WAP promoter, including most elements postulated as being responsible for mammary-specific regulation, confers mammary-specific expression upon a marker gene in transgenic mice even though the distal promoter region, known to be important for rat WAP promoter activity, is lacking. The relatively small size of this fragment allows its insertion into a murine leukemia virus-based retroviral vector in place of the viral promoter. Infection of a number of established human mammary and nonmammary cell lines with such a retroviral vector revealed that the WAP promoter was limited in its activity to mammary tumor cell lines. Expression in tumorigenic mammary cells was even more pronounced when these cells were introduced into the mammary fat pads of mice. This is the first demonstration that the WAP promoter is active in human mammary cells and mammary tumor cells in general, and suggests that the extended proximal WAP promoter may be useful for directing therapeutic gene expression to human mammary tumors.

3T3 Cells↗
PubMed2002

Evolution of replication efficiency following infection with a molecularly cloned feline immunodeficiency virus of low virulence.

The development of an effective vaccine against human immunodeficiency virus is considered to be the most practicable means of controlling the advancing global AIDS epidemic. Studies with the domestic cat have demonstrated that vaccinal immunity to infection can be induced against feline immunodeficiency virus (FIV); however, protection is largely restricted to laboratory strains of FIV and does not extend to primary strains of the virus. We compared the pathogenicity of two prototypic vaccine challenge strains of FIV derived from molecular clones; the laboratory strain PET(F14) and the primary strain GL8(414). PET(F14) established a low viral load and had no effect on CD4(+)- or CD8(+)-lymphocyte subsets. In contrast, GL8(414) established a high viral load and induced a significant reduction in the ratio of CD4(+) to CD8(+) lymphocytes by 15 weeks postinfection, suggesting that PET(F14) may be a low-virulence-challenge virus. However, during long-term monitoring of the PET(F14)-infected cats, we observed the emergence of variant viruses in two of three cats. Concomitant with the appearance of the variant viruses, designated 627(W135) and 628(W135,) we observed an expansion of CD8(+)-lymphocyte subpopulations expressing reduced CD8 beta-chain, a phenotype consistent with activation. The variant viruses both carried mutations that reduced the net charge of the V3 loop (K409Q and K409E), giving rise to a reduced ability of the Env proteins to both induce fusion and to establish productive infection in CXCR4-expressing cells. Further, following subsequent challenge of naïve cats with the mutant viruses, the viruses established higher viral loads and induced more marked alterations in CD8(+)-lymphocyte subpopulations than did the parent F14 strain of virus, suggesting that the E409K mutation in the PET(F14) strain contributes to the attenuation of the virus.

Amino Acid Sequence↗
PubMed2000

Variations in the major envelope glycoprotein GP5 of Czech strains of porcine reproductive and respiratory syndrome virus.

The major envelope glycoprotein genes (ORF5) of seven Czech isolates of porcine reproductive and respiratory syndrome virus (PRRSV) were amplified and their nucleotide sequences were determined. ORF5 displayed nucleotide and amino acid identities of 87.5-100% and 87. 6-100%, respectively, among the isolates. In a phylogenetic tree, all European isolates were grouped in a genotype distinct from that of reference American strains (VR-2332, IAF-Klop). Among the European isolates, two different clades were identified. Two Czech isolates (V-501 and V-503) and Italian strain PRRSV 2156 fell into one clade. The remaining European strains comprised the second clade. Surprisingly, two separately clustered strains (V-501 and V-516) were isolated from the same herd. Additionally, the possible effect of in vitro cultivation on the nucleotide sequence was analysed. Nine point mutations in the ORF5 region resulted from 152 in vitro passages of the V-502 isolate in MARC-145 cells.

Amino Acid Sequence↗