Search PubMed⌕ Search

Biomedical subjects

H Katinger

Publications and source records attributed to H Katinger.

At least 19 recordsLinked to original sources
PubMed2006

Nebulization of liposomal rh-Cu/Zn-SOD with a novel vibrating membrane nebulizer.

Liposomes are potential drug carriers for pulmonary drug delivery: They can be prepared from phospholipids, which are endogenous to the respiratory tract as a component of pulmonary surfactant, and at an appropriate dose liposomes do not pose a toxicological risk to this organ. Among the various categories of drug that benefit from liposomal entrapment is the anti-inflammatory enzyme superoxide dismutase, thus prolonging its biological half-life. The delivery of liposomes by nebulization is hampered by stability problems, like physical and chemical changes that may lead to chemical degradation and leakage of the encapsulated drug. Here we present data of liposomes aerosolized with a novel electronic nebulizer based on a vibrating membrane technology (PARI eFlow), which amends drawbacks like liposomes degradation and product release. The data acquisition included aerosol properties such as aerodynamic particle size, nebulization efficiency, and liposome leakage upon nebulization. In conclusion, this study shows the ability of the PARI eFlow to nebulize high amounts of liposomal recombinant human superoxide dismutase with reduced vesicle disruption tested in an enclosing experimental protocol.

Aerosols↗
PubMed2005

Establishment of human fibroma cell lines from a MEN1 patient by introduction of either hTERT or SV40 early region.

Establishment of tumor cell lines as model systems for studying tumor biology or as a part of immunotherapeutic anti-cancer strategies is of high importance, whereby the highest possible preservation of the original tumor cell phenotype is a prerequisite for these aims. Since overexpression of the catalytic subunit of human telomerase (hTERT) is known to minimally alter the cellular phenotype, we focused on the establishment of cell lines derived from human fibroma from a MEN1 patient by ectopic expression of hTERT. Additionally, a cell line was generated by introduction of the early region of SV40 (SV40 ER). Both approaches resulted in continuous cell lines, and neither T1-LOHG (hTERT) nor SV1-LOHG (SV40 ER) showed a transformed phenotype. While SV40 ER-transfected cells underwent dramatic changes in morphology and growth characteristics, hTERT-expressing cells indeed retained a phenotype highly similar to the parental cells. Nevertheless, hTERT overexpression resulted in increased growth rates after about 70 population doublings (PD) and alterations of mRNA levels of genes associated with tumor pathogenesis. Thus, our data suggest that ectopic hTERT expression leads to immortalization of LOHG-F, sustaining many characteristics of the non-transfected counterparts, but continuous growth in vitro is associated with changes of the cellular phenotype.

Antigens, Polyomavirus Transforming↗
PubMed2002

Postnatal pre- and postexposure passive immunization strategies: protection of neonatal macaques against oral simian-human immunodeficiency virus challenge.

Simian-human immunodeficiency viruses (SHIV) allow the evaluation of antiviral strategies that target the envelope glycoproteins of the human immunodeficiency virus 1 (HIV-1) in macaques. We previously protected neonates from oral challenge with cell-free SHIV-vpu+ by passive immunization with synergistic human neutralizing monoclonal antibodies (mAbs) (Baba et al., Nat Med 6:200-206, 2000). mAbs were administered prenatally to pregnant dams and postnatally to the neonates. Here, we used solely postnatal or postexposure mAb treatment, thus significantly reducing the amount of mAbs necessary. All neonatal monkeys were also protected with these abbreviated mAb regimens. Our results are directly relevant for humans because we used mAbs that target HIV-1 envelope glycoproteins. Thus, the large-scale use of passive immunization with neutralizing mAbs may be feasible in human neonates. The mAbs, being natural human proteins, can be expected to have low toxicity. Passive immunization has promise to prevent intrapartum as well as milk-borne virus transmission from HIV-1-infected women to their infants.

Administration, Oral↗
PubMed2001

A potent cross-clade neutralizing human monoclonal antibody against a novel epitope on gp41 of human immunodeficiency virus type 1.

We have established a panel of human monoclonal antibodies against human immunodeficiency virus type 1 (HIV-1). The antibodies 2F5 and 2G12 have been identified to be two of the most potently in vitro neutralizing antibodies against HIV-1. Here we report on a further antibody, 4E10, of similar in vitro neutralizing potency. 4E10 binds to a novel epitope C terminal of the ELDKWA sequence recognized by 2F5, which has been so far the only described broadly neutralizing anti-gp41 antibody. Both 4E10 and 2F5 bind only weakly to infected cells compared with gp120-specific 2G12 and polyclonal anti-HIV-1 immunoglobulin (HIVIG), but show potent in vitro neutralizing properties. 4E10 neutralizes potently not only tissue culture-adapted strains but also primary isolates of different clades, including A, B, C, D, and E. Viruses that were found to be resistant to 2F5 were neutralized by 4E10 and vice versa; none of the tested isolates was resistant to both anti-gp41 antibodies. This confirms that the region recognized by 2F5 and 4E10 is essential for viral infectivity and may be important for vaccine design. Moreover, our results suggest that 4E10 should be further investigated for passive anti-HIV immunotherapy.

Amino Acid Sequence↗
PubMed2001

Analysis of alterations in gene expression after amplification of recombinant genes in CHO cells.

Dihydrofolate reductase (DHFR) based amplification of recombinant genes using increasing concentrations of methotrexate (MTX) is a common method to establish CHO cell lines producing high amounts of the desired protein. Once, cell lines with highly amplified target genes and good expression rates are isolated, further characterization of their transcriptional pattern is intended to clarify the question what other factors are elevated, as a prerequisite or consequence of recombinant protein production. In order to define genes which are upregulated in a cell line that shows high production rates, we have investigated alterations in gene expression which occur beside amplification of the recombinant genes. For this purpose, the suppression subtractive hybridization method was used to create a cDNA library enriched for differentially expressed sequences in the recombinant antibody producing CHO cell line versus the original counterpart. Differential expression was confirmed by Northern blotting and Northern ELISA. In addition to the expected recombinant genes, we have identified 5 transcripts which are upregulated in the recombinant cell line. One sequence has not been found in existing data bases, the others revealed to be genes involved in protein synthesis and regulation of transcription. Furthermore, an alternatively spliced, non-functional form of the DHFR mRNA was detected, suggesting a dramatic increase of the selection pressure exerted by MTX.

Animals↗
PubMed2001

Topically applied liposome encapsulated superoxide dismutase reduces postburn wound size and edema formation.

The overproduction of biochemical mediators, and activation of leukocytes and endothelial cells, generated in thermally injured tissue, gives rise to both local and distant effects. The formation of short-lived, highly reactive metabolites, such as oxygen free radicals, increases with increasing tissue ischemia, and causes further cell damage. Human recombinant Cu/Zn-superoxide dismutase (rh-Cu/Zn-SOD), an enzyme which captures these radicals, may have a beneficial effect on the postburn inflammation processes. In this study, the influence of rh-Cu/Zn-SOD application to thermally injured tissue of rabbit backskin was examined. Three different delivery strategies were compared, pure or liposomally encapsulated enzyme, or intralesionally injected rh-Cu/Zn-SOD. For control, one animal group was treated with plain gel and another group was kept untreated. At 24 h following trauma a statistically significant difference in lesion sizes between the enzyme treated and control groups was observed. After 72 h tissue swelling had diminished significantly more in the rh-Cu/Zn-SOD treated groups as compared to the control animals. The best results were achieved by spreading liposomes encapsulating the enzyme onto the wounds. Our results suggest that local treatment of burn wounds with enzymatic radical scavengers such as rh-Cu/Zn-SOD has a beneficial effect on the extent of the postburn damage.

Animals↗
PubMed2001

Protection of neonatal macaques against experimental SHIV infection by human neutralizing monoclonal antibodies.

Neonatal macaques were completely protected against oral challenge with SHIV-vpu+, a simian-human immunodeficiency virus that encodes the envelope gene of a laboratory-adapted HIV strain, by pre- and post-natal treatment with a triple combination of human neutralizing monoclonal antibodies (mAbs). The mAbs were directed either against the CD4 binding site, a glycosylation-dependent gp120 epitope, or against a linear epitope on gp41. This triple combination was highly synergistic in vitro and neutralized primary HIV completely. Subsequently, oral challenge was performed with pathogenic SHIV89.6P, an animal-passaged variant of a chimeric virus that encodes the envelope gene of the primary, dual-tropic HIV89.6. Only post-natal treatment with a similar triple mAb combination was used. One out of 4 mAb-treated infants was completely protected from infection. In the other 3 treated animals, there was a tendency towards lower peak viral RNA loads compared with untreated controls. Two out of 4 mAb-treated infants maintained normal CD4+ T-cell numbers, in contrast to all controls that had steep declines at 2 weeks post-challenge. We conclude that the triple mAb combination significantly protected the neonates, even against mucosal challenge with pathogenic SHIV89.6P. Passively administered synergistic human mAbs may play a role in preventing mother-infant transmission of HIV, both against intrapartum transmission as well as against infection through breast milk. As passive immunization is a tool to assess correlates of immune protection, we conclude that the epitopes recognized by the mAbs in our combinations are important for AIDS vaccine development. Future passive immunization studies may reveal other important conserved epitopes.

AIDS Vaccines↗
PubMed2001

Passive immunization against oral AIDS virus transmission: an approach to prevent mother-to-infant HIV-1 transmission?

To develop immunoprophylaxis regimens against mother-to-child human immunodeficiency virus type 1 (HIV-1) transmission, we established a simian-human immunodeficiency virus (SHIV) model in neonatal macaques that mimics intrapartum mucosal virus exposure (T.W. Baba, J. Koch, E.S. Mittler et al: AIDS Res Hum Retroviruses 10:351-357, 1994). We protected four neonates from oral SHIV-vpu+ challenge by ante- and postpartum treatment with a synergistic triple combination of immunoglobulin (Ig) G1 human anti-HIV-1 neutralizing monoclonal antibodies (mAbs) (T.W. Baba, V. Liska, R. Hofmann-Lehmann et al: Nature Med 6:200-206, 2000), which recognize the CD4-binding site of Env, a glycosylation-dependent gp120, or a linear gp41 epitope. Two neonates that received only postpartum mAbs were also protected from oral SHIV-vpu+ challenge, indicating that postpartum treatment alone is sufficient. Next, we evaluated a similar mAb combination against SHIV89.6P, which encodes env of primary HIV89.6. One of four mAb-treated neonates was protected from infection and two maintained normal CD4+ T-cell counts. We conclude that the epitopes recognized by the three mAbs are important determinants for achieving protection. Combination immunoprophylaxis with synergistic mAbs seems promising to prevent maternal HIV-1 transmission in humans.

Acquired Immunodeficiency Syndrome↗
PubMed2001

Postnatal passive immunization of neonatal macaques with a triple combination of human monoclonal antibodies against oral simian-human immunodeficiency virus challenge.

To develop prophylaxis against mother-to-child human immunodeficiency virus (HIV) transmission, we established a simian-human immunodeficiency virus (SHIV) infection model in neonatal macaques that mimics intrapartum mucosal virus exposure (T. W. Baba et al., AIDS Res. Hum. Retroviruses 10:351-357, 1994). Using this model, neonates were protected from mucosal SHIV-vpu(+) challenge by pre- and postnatal treatment with a combination of three human neutralizing monoclonal antibodies (MAbs), F105, 2G12, and 2F5 (Baba et al., Nat. Med. 6:200-206, 2000). In the present study, we used this MAb combination only postnatally, thereby significantly reducing the quantity of antibodies necessary and rendering their potential use in humans more practical. We protected two neonates with this regimen against oral SHIV-vpu(+) challenge, while four untreated control animals became persistently infected. Thus, synergistic MAbs protect when used as immunoprophylaxis without the prenatal dose. We then determined in vitro the optimal MAb combination against the more pathogenic SHIV89.6P, a chimeric virus encoding env of the primary HIV89.6. Remarkably, the most potent combination included IgG1b12, which alone does not neutralize SHIV89.6P. We administered the combination of MAbs IgG1b12, 2F5, and 2G12 postnatally to four neonates. One of the four infants remained uninfected after oral challenge with SHIV89.6P, and two infants had no or a delayed CD4(+) T-cell decline. In contrast, all control animals had dramatic drops in their CD4(+) T cells by 2 weeks postexposure. We conclude that our triple MAb combination partially protected against mucosal challenge with the highly pathogenic SHIV89.6P. Thus, combination immunoprophylaxis with passively administered synergistic human MAbs may play a role in the clinical prevention of mother-to-infant transmission of HIV type 1.

Administration, Oral↗
PubMed2001

Hyperattenuated recombinant influenza A virus nonstructural-protein-encoding vectors induce human immunodeficiency virus type 1 Nef-specific systemic and mucosal immune responses in mice.

We have generated recombinant influenza A viruses belonging to the H1N1 and H3N2 virus subtypes containing an insertion of the 137 C-terminal amino acid residues of the human immunodeficiency virus type 1 (HIV-1) Nef protein into the influenza A virus nonstructural-protein (NS1) reading frame. These viral vectors were found to be genetically stable and capable of growing efficiently in embryonated chicken eggs and tissue culture cells but did not replicate in the murine respiratory tract. Despite the hyperattenuated phenotype of influenza/NS-Nef viruses, a Nef and influenza virus (nucleoprotein)-specific CD8(+)-T-cell response was detected in spleens and the lymph nodes draining the respiratory tract after a single intranasal immunization of mice. Compared to the primary response, a marked enhancement of the CD8(+)-T-cell response was detected in the systemic and mucosal compartments, including mouse urogenital tracts, if mice were primed with the H1N1 subtype vector and subsequently boosted with the H3N2 subtype vector. In addition, Nef-specific serum IgG was detected in mice which were immunized twice with the recombinant H1N1 and then boosted with the recombinant H3N2 subtype virus. These findings may contribute to the development of alternative immunization strategies utilizing hyperattenuated live recombinant influenza virus vectors to prevent or control infectious diseases, e.g., HIV-1 infection.

AIDS Vaccines↗
PubMed2001

Broadly neutralizing antibodies targeted to the membrane-proximal external region of human immunodeficiency virus type 1 glycoprotein gp41.

The identification and epitope mapping of broadly neutralizing anti-human immunodeficiency virus type 1 (HIV-1) antibodies (Abs) is important for vaccine design, but, despite much effort, very few such Abs have been forthcoming. Only one broadly neutralizing anti-gp41 monoclonal Ab (MAb), 2F5, has been described. Here we report on two MAbs that recognize a region immediately C-terminal of the 2F5 epitope. Both MAbs were generated from HIV-1-seropositive donors, one (Z13) from an antibody phage display library, and one (4E10) as a hybridoma. Both MAbs recognize a predominantly linear and relatively conserved epitope, compete with each other for binding to synthetic peptide derived from gp41, and bind to HIV-1(MN) virions. By flow cytometry, these MAbs appear to bind relatively weakly to infected cells and this binding is not perturbed by pretreatment of the infected cells with soluble CD4. Despite the apparent linear nature of the epitopes of Z13 and 4E10, denaturation of recombinant envelope protein reduces the binding of these MAbs, suggesting some conformational requirements for full epitope expression. Most significantly, Z13 and 4E10 are able to neutralize selected primary isolates from diverse subtypes of HIV-1 (e.g., subtypes B, C, and E). The results suggest that a rather extensive region of gp41 close to the transmembrane domain is accessible to neutralizing Abs and could form a useful target for vaccine design.

Amino Acid Motifs↗
PubMed2001

Fine definition of the epitope on the gp41 glycoprotein of human immunodeficiency virus type 1 for the neutralizing monoclonal antibody 2F5.

Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS), in combination with proteolytic protection assays, has been used to identify the functional epitope on human immunodeficiency virus envelope glycoprotein gp41 for the broadly neutralizing anti-gp41 human monoclonal antibody 2F5. In this protection assay-based procedure, a soluble gp140 protein with a stabilizing intermolecular disulfide bond between the gp120 and gp41 subunits (SOS gp140) was affinity bound to immobilized 2F5 under physiological conditions. A combination of proteolytic enzymatic cleavages was then performed to remove unprotected residues. Residues of SOS gp140 protected by their binding to 2F5 were then identified based on their molecular weights as determined by direct MALDI-MS of the immobilized antibody beads. The epitope, NEQELLELDKWASLWN, determined by this MALDI-MS protection assay approach consists of 16 amino acid residues near the C terminus of gp41. It is significantly longer than the ELDKWA core epitope previously determined for 2F5 by peptide enzyme-linked immunosorbent assay. This new knowledge of the structure of the 2F5 epitope may facilitate the design of vaccine antigens intended to induce antibodies with the breadth and potency of action of the 2F5 monoclonal antibody.

AIDS Vaccines↗
PubMed2001

Neutralization synergy of human immunodeficiency virus type 1 primary isolates by cocktails of broadly neutralizing antibodies.

Several reports have described the existence of synergy between neutralizing monoclonal antibodies (MAbs) against human immunodeficiency virus type 1 (HIV-1). Synergy between human MAbs b12, 2G12, 2F5, and 4E10 in neutralization of primary isolates is of particular interest. Neutralization synergy of these MAbs, however, has not been studied extensively, and the mechanism of synergy remains unclear. We investigated neutralization synergy among this human antibody set by using the classical approach of titrating antibodies mixed at a fixed ratio as well as by an alternative, variable ratio approach in which the neutralization curve of one MAb is assessed in the presence and absence of a fixed, weakly neutralizing concentration of a second antibody. The advantage of this second approach is that it does not require mathematical analysis to establish synergy. No neutralization enhancement of any of the MAb combinations tested was detected for the T-cell-line-adapted molecular HIV-1 clone HxB2 using both assay formats. Studies of primary isolates (89.6, SF162, and JR-CSF) showed neutralization synergy which was relatively weak, with a maximum of two- to fourfold enhancement between antibody pairs, thereby increasing neutralization titers about 10-fold in triple and quadruple antibody combinations. Analysis of b12 and 2G12 binding to oligomeric envelope glycoprotein by using flow cytometry failed to demonstrate cooperativity in binding between these two antibodies. The mechanism by which these antibodies synergize is, therefore, not yet understood. The results lend some support to the notion that an HIV-1 vaccine that elicits moderate neutralizing antibodies to multiple epitopes may be more effective than hereto supposed, although considerable caution in extrapolating to a vaccine situation is required.

AIDS Vaccines↗
PubMed2000

Determination of liposome size distribution by flow cytometry.

BACKGROUND: An essential parameter that describes the quality of liposome suspensions is the mean size, respectively the size distribution. Currently several analytical methods including laser light scattering techniques (LLST) are being employed. METHODS: Here we present an alternative technique using flow cytometry (FCM) to characterize uni- and polydisperse suspensions. As model liposomes preparations containing dipalmitoylphosphatidylcholine (DPPC) were used. A constant number of particles (1,500/s) in the fluid stream and a representative number of 10,000 particles of each sample was measured. Fluorescence-labeled latex beads were measured identically, and their side scatter signals were calibrated and correlated to the results obtained with liposome vesicles. RESULTS: Evaluation of the measurement and validation of the FCM results in comparison to LLST confirm the reliability of results obtained with our method. Latex beads in the range of 100-1000 nm were used for calibration to classify liposomes. Although measurement characteristics and calculation in both methods are basically different, very good agreement of the results was achieved. CONCLUSIONS: Demonstration of stability, reproducibility, and reliability of results make the employment of this method acceptable for an adequate routine analysis technique.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed2000

Stable recombinant expression of the anti HIV-1 monoclonal antibody 2F5 after IgG3/IgG1 subclass switch in CHO cells.

The human monoclonal antibody (humAb) 2F5 is a potent candidate for immunotherapy of HIV-1. The xenohybridoma derived humAb 2F5 is of IgG3 kappa isotype. Generally, IgG1 isotype has a longer half-life (beta-clearance) in humans than IgG3. Therefore the isotype was switched to an IgG1 by ligation of the 2F5 heavy chain variable region to an IgG1 constant region and expressed as IgG1 kappa in CHO cells. CHO clones have been established, which stably express humAb 2F5 at high levels. The specificity, affinity and in vitro function of both isotypes were identical.

Animals↗
PubMed2000

Subtractive hybridization of mRNA from early passage and senescent endothelial cells.

Regulation of cellular processes that eventually lead to a state of growth arrest is an important manifestation of in vitro cellular senescence caused and accompanied by variations of the gene expression pattern. Whereas these changes at the mRNA level have been studied mainly in fibroblast cultures, we concentrated on endothelial cells that represent an accepted model for vascular systems and may be involved in the pathogenesis of diseases related to aging. To isolate differentially expressed genes, we created a subtractive cDNA library using mRNA from senescent (35 passages) and young (five passages) human umbilical vein endothelial cells (HUVECs). Candidate clones were isolated from the cDNA library, differential expression was confirmed by Northern blot analyses and sequences were compared with a genbank data base. Because many mRNAs were below the detection limit of Northern blot analysis, we were forced to establish a more sensitive PCR based method (ATAC-PCR) to quantify and confirm altered levels of gene expression. Several mRNAs were found to be upregulated in senescent HUVECs including two components of the extracellular matrix (ECM): plasminogen activator inhibitor and fibronectin. Elevated expression of both has already been described in senescent cells. The mRNAs of TGF-beta-inducible gene H3 (beta-IG-H3; ECM protein), insulin-like growth factor binding protein (IGFBP-3), p53-inducible gene (PIG3) a protein involved in vesicular transport (SEC13R) and ribosomal protein L28 have likewise been shown to be preferentially expressed in senescent cells. Because studies support the involvement of ECM components, TGF-beta and p53 in tumor suppressing mechanisms, our data supports the hypothesis that cellular senescence and upregulation of ECM proteins may be associated with tumor preventive functions.

Cells, Cultured↗
PubMed2000

Human neutralizing monoclonal antibodies of the IgG1 subtype protect against mucosal simian-human immunodeficiency virus infection.

Although maternal human immunodeficiency virus type 1 (HIV-1) transmission occurs during gestation, intrapartum and postpartum (by breast-feeding), 50-70% of all infected children seem to acquire HIV-1 shortly before or during delivery. Epidemiological evidence indicates that mucosal exposure is an important aspect of intrapartum HIV transmission. A simian immunodeficiency virus (SIV) macaque model has been developed that mimics the mucosal exposure that can occur during intrapartum HIV-1 transmission. To develop immunoprophylaxis against intrapartum HIV-1 transmission, we used SHIV-vpu+ (refs. 5,6), a chimeric simian-human virus that encodes the env gene of HIV-IIIB. Several combinations of human monoclonal antibodies against HIV-1 have been identified that neutralize SHIV-vpu+ completely in vitro through synergistic interaction. Here, we treated four pregnant macaques with a triple combination of the human IgG1 monoclonal antibodies F105, 2G12 and 2F5. All four macaques were protected against intravenous SHIV-vpu+ challenge after delivery. The infants received monoclonal antibodies after birth and were challenged orally with SHIV-vpu+ shortly thereafter. We found no evidence of infection in any infant during 6 months of follow-up. This demonstrates that IgG1 monoclonal antibodies protect against mucosal lentivirus challenge in neonates. We conclude that epitopes recognized by the three monoclonal antibodies are important determinants for achieving substantial protection, thus providing a rational basis for AIDS vaccine development.

Animals↗