A phase I vaccination study with tyrosinase in patients with stage II melanoma using recombinant modified vaccinia virus Ankara (MVA-hTyr)

A significant percentage of patients with stage II melanomas suffer a relapse after surgery and therefore need the development of adjuvant therapies. In the study reported here, safety and immunological response were analyzed after vaccination in an adjuvant setting with recombinant modified vaccinia virus Ankara carrying the cDNA for human tyrosinase (MVA-hTyr). A total of 20 patients were included and vaccinated three times at 4-week intervals with 5×10⁸ IU of MVA-hTyr each time. The responses to the viral vector, to known HLA class I–restricted tyrosinase peptides, and to dendritic cells transfected with tyrosinase mRNA, were investigated by ELISpot assay on both ex vivo T cells and on T cells stimulated in vitro prior to testing. The delivery of MVA-hTyr was safe and did not cause any side effects above grade 2. A strong response to the viral vector was achieved, indicated by an increase in the frequency of MVA-specific CD4⁺ and CD8⁺ T cells and an increase in virus-specific antibody titers. However, no tyrosinase-specific T-cell or antibody response was observed with MVA-hTyr in any of the vaccinated patients. Although MVA-hTyr provides a safe and effective antigen-delivery system, it does not elicit a measurable immune response to its transgene product in patients with stage II melanoma after repeated combined intradermal and subcutaneous vaccination. We presume that modification of the antigen and/or prime-boost vaccination applying different approaches to antigen delivery may be required to induce an effective tyrosinase-specific immune response.     

A Novel Triplex Quantitative PCR Strategy for Quantification of Toxigenic and Nontoxigenic Vibrio cholerae in Aquatic Environments

Vibrio cholerae is a severe human pathogen and a frequent member of aquatic ecosystems. Quantification of V. cholerae in environmental water samples is therefore fundamental for ecological studies and health risk assessment. Beside time-consuming cultivation techniques, quantitative PCR (qPCR) has the potential to provide reliable quantitative data and offers the opportunity to quantify multiple targets simultaneously. A novel triplex qPCR strategy was developed in order to simultaneously quantify toxigenic and nontoxigenic V. cholerae in environmental water samples. To obtain quality-controlled PCR results, an internal amplification control was included. The qPCR assay was specific, highly sensitive, and quantitative across the tested 5-log dynamic range down to a method detection limit of 5 copies per reaction. Repeatability and reproducibility were high for all three tested target genes. For environmental application, global DNA recovery (GR) rates were assessed for drinking water, river water, and water from different lakes. GR rates ranged from 1.6% to 76.4% and were dependent on the environmental background. Uncorrected and GR-corrected V. cholerae abundances were determined in two lakes with extremely high turbidity. Uncorrected abundances ranged from 4.6 × 10² to 2.3 × 10⁴ cell equivalents liter⁻¹, whereas GR-corrected abundances ranged from 4.7 × 10³ to 1.6 × 10⁶ cell equivalents liter⁻¹. GR-corrected qPCR results were in good agreement with an independent cell-based direct detection method but were up to 1.6 log higher than cultivation-based abundances. We recommend the newly developed triplex qPCR strategy as a powerful tool to simultaneously quantify toxigenic and nontoxigenic V. cholerae in various aquatic environments for ecological studies as well as for risk assessment programs.     

Theodor and Marcella Boveri: chromosomes and cytoplasm in heredity and development

The chromosome theory of heredity, developed in 1902-1904, became one of the foundation stones of twentieth-century genetics. It is usually referred to as the Sutton-Boveri theory after Walter Sutton and Theodor Boveri. However, the contributions of Theodor Boveri and his co-worker, Marcella O'Grady Boveri (also his wife), to the understanding of heredity and development go beyond the localization of the Mendelian hereditary factors onto the chromosomes. They investigated the interaction of cytoplasm and chromosomes, and demonstrated its relevance in heredity and development.     

Amino acid preferences of retroviral proteases for amino-terminal positions in a type 1 cleavage site

The specificities of the proteases of 11 retroviruses were studied using a series of oligopeptides with amino acid substitutions in the P1, P3, and P4 positions of a naturally occurring type 1 cleavage site (Val-Ser-Gln-Asn-Tyr↓Pro-Ile-Val-Gln) in human immunodeficiency virus type 1 (HIV-1). Previously, the substrate specificity of the P2 site was studied for the same representative set of retroviral proteases, which included at least one member from each of the seven genera of the family Retroviridae (P. Bagossi, T. Sperka, A. Fehér, J. Kádas, G. Zahuczky, G. Miklóssy, P. Boross, and J. Tözsér, J. Virol. 79:4213-4218, 2005). Our enzyme set comprised the proteases of HIV-1, HIV-2, equine infectious anemia virus, avian myeloblastosis virus (AMV), Mason-Pfizer monkey virus, mouse mammary tumor virus (MMTV), Moloney murine leukemia virus, human T-lymphotropic virus type 1, bovine leukemia virus, walleye dermal sarcoma virus, and human foamy virus. Molecular models were used to interpret the similarities and differences in specificity between these retroviral proteases. The results showed that the retroviral proteases had similar preferences (Phe and Tyr) for the P1 position in this sequence context, but differences were found for the P3 and P4 positions. Importantly, the sizes of the P3 and P4 residues appear to be a major contributor for specificity. The substrate specificities correlated well with the phylogenetic tree of the retroviruses. Furthermore, while the specificities of some enzymes belonging to different genera appeared to be very similar (e.g., those of AMV and MMTV), the specificities of the primate lentiviral proteases substantially differed from that observed for a nonprimate lentiviral protease.     

Efficient mRNA detection from human archival paraffin-embedded tissue: An update

We recently published an in situ hybridization protocol for archival tissue using a commercial hybridization buffer. This buffer is no more available. Therefore, we have developed an improved protocol with a defined hybridization buffer.     

Towards domestication of giant kelp (<Emphasis Type=Italic>Macrocystis pyrifera</Emphasis>) in Chile: selection of haploid parent genotypes,outbreeding, and heterosis

Macrocystis is an important marine resource in Chile, with severe problems of over-exploitation. Our study describes genetic materials and techniques for a further improvement of laboratory-based mariculture. For a systematic hybridization program we have selected one pair (cultivar) of gametophytes with favorable somatic and reproductive characteristics from each of seven localities in southern Chile. Sporophytes from all 49 crosses were grown for 10 weeks to seedling size. We report here that sporophytes from sympatric parents (intra-cultivar matings) grow to different length, depending on the locality and, importantly, that sporophytes from several inter-cultivar crossings show superior growth, suggesting heterosis with symmetric or asymmetric reciprocity. The genetic materials and techniques described here, together with our newly developed standardized seedling production protocols now available, constitute a significant step towards domestication of Macrocystis in analogy to terrestrial agriculture.     

Early events in signalling high-temperature stress in tobacco BY2 cells involve alterations in membrane fluidity and enhanced hydrogen peroxide production

Alterations in membrane fluidity are among the early events in plants that detect changes in ambient temperature. However, signal transduction downstream of the membrane-associated processes is still not well understood. We have focused here on the role of hydrogen peroxide (H(2)O(2)) in high-temperature signalling in relation to changes in membrane fluidity in cells of tobacco (Nicotiana tabacum L.) cv. Bright Yellow 2 (BY2). As final indicators of the heat-signalling cascade, we have monitored the synthesis of small heat-shock proteins (sHSPs). Elevation of temperature between 32 and 38 degrees C resulted in a fast, transient stimulation of H(2)O(2) production in the tobacco cells. A similar H(2)O(2) burst could be induced at lower temperatures (28-32 degrees C) by membrane fluidization using benzyl alcohol (BA). Diphenylene iodonium (DPI), a NADPH oxidase inhibitor, prevented both the heat- and BA-triggered H(2)O(2) rise. The synthesis of sHSPs (14.5 and 16 kDa) was shifted to lower temperatures by BA application and was suppressed by DPI treatment in the same way. The results indicate that H(2)O(2) is an early component of the heat-signalling pathway, which responds rapidly to changes in membrane fluidity and is required for the activation of sHSP synthesis.