Strategies for designing and optimizing new generation vaccines.

Although the field of immunology developed in part from the early vaccine studies of Edward Jenner, Louis Pasteur and others, vaccine development had largely become the province of virologists and other microbiologists, because the model for classic vaccines was to isolate the pathogen and prepare a killed or attenuated pathogen vaccine. Only recently has vaccinology returned to the realm of immunology, because a new understanding of immune mechanisms has allowed translation of basic discoveries into vaccine strategies.

Mucosal AIDS vaccine reduces disease and viral load in gut reservoir and blood after mucosal infection of macaques.

Given the mucosal transmission of HIV-1, we compared whether a mucosal vaccine could induce mucosal cytotoxic T lymphocytes (CTLs) and protect rhesus macaques against mucosal infection with simian/human immunodeficiency virus (SHIV) more effectively than the same vaccine given subcutaneously. Here we show that mucosal CTLs specific for simian immunodeficiency virus can be induced by intrarectal immunization of macaques with a synthetic-peptide vaccine incorporating the LT(R192G) adjuvant. This response correlated with the level of T-helper response. After intrarectal challenge with pathogenic SHIV-Ku2, viral titers were eliminated more completely (to undetectable levels) both in blood and intestine, a major reservoir for virus replication, in intrarectally immunized animals than in subcutaneously immunized or control macaques. Moreover, CD4+ T cells were better preserved. Thus, induction of CTLs in the intestinal mucosa, a key site of virus replication, with a mucosal AIDS vaccine ameliorates infection by SHIV in non-human primates.

[Treatment options for cartilage damage in the knee joint]. / Die Behandlung von Knorpelschäden am Kniegelenk (I).

The complex structure of articular cartilage is essential for understanding biology and function. Despite performing with a relatively low metabolic activity, healthy articular cartilage has amazing capacity to sustain itself and carry out its function. Chondrocytes are active in maintaining the tissue's matrix, showing differences in the anatomic structure. Articular cartilage has extraordinary mechanical properties and lasting durability. It is only a few millimetres thick. The cartilage wears resistance while bearing large loads throughout a person's lifetime. Otherwise we see cartilage destructions in young patients without a trauma. It is important to know that the articular cartilage decreases with age. The ability of the MRI to obtain reproducible, accurate images of cartilage has enabled early detection of cartilage lesions and provides clinically relevant information when planning cartilage repair. MRI depicts the subchondral bone and all other anatomic structures of the knee. This information is vital when planning for complex techniques that require careful size delineation of the cartilage lesion and evaluation of the surrounding subchondral bone. In addition to aiding in preoperative planning, the MRI offer an important objective evaluation of cartilage repair to be correlated with the more subjective clinical outcome instruments and provide insight into the biology of the repair process. The repair strategies are currently available for clinical use when treating articular cartilage lesions. These strategies fall into one of the following categories: 1. Palliative-conservative treatment (local or systemic application), 2. Intrinsic-repair enhancement/marrow stimulation (drilling, microfracture, arthroplasty), 3. Cell-based repair (collagen associated chondrocyte transplantation or microfracture with collagen). The use of biodegradable scaffolds can be used either alone or as delivery vehicles for growths factors. This technique could be a new way for winning structurally and biomechanically appropriate tissue.

High-affinity T helper epitope induces complementary helper and APC polarization, increased CTL, and protection against viral infection.

Natural viral proteins do not always make optimal vaccines. We have found that sequence modification to increase epitope affinity for class II MHC molecules (epitope enhancement) can improve immunogenicity. Here we show first that a higher-affinity helper epitope-enhanced HIV vaccine not only induces more cytotoxic T lymphocytes (CTLs), but also skews helper cells toward Th1 cytokine production and protects against HIV-1 recombinant vaccinia viral challenge. Furthermore, we elucidate a novel mechanism in which the higher-affinity vaccine induces dramatically more effective helper cells with a higher level of CD40L per helper cell and more positive cells, which in turn more effectively conditions dendritic cells (DCs) for CTL activation in a second culture. The improved helper cells also induce much greater IL-12 production by DCs, accounting for the reciprocal T helper polarization to Th1, and increase costimulatory molecule expression. Thus, increasing affinity for class II MHC results in a complementary interaction in which T helper and antigen-presenting cells polarize each other, as well as increase CTL, and provide greater vaccine efficacy against viral infection.

Construction of peptides encompassing multideterminant clusters of human immunodeficiency virus envelope to induce in vitro T cell responses in mice and humans of multiple MHC types.

To make synthetic peptide vaccines effective in a broad population of outbred humans, one would have to incorporate enough antigenic determinants to elicit recognition by T cells of most HLA types. We have previously defined multideterminant regions of the human immunodeficiency virus (HIV) envelope that include overlapping determinants seen by proliferating T cells of three or four haplotypes of mice. We have now tested the hypothesis that synthetic peptides encompassing such multideterminant regions will be recognized by T cells of multiple murine MHC types as well as by human T cells representing multiple HLA types. Six such peptides of 20-33 residues in length were prepared, and tested for their ability to stimulate T cells from mice of four distinct MHC types immunized with recombinant envelope protein rgp 160, as well as from 42 HIV-infected humans of different HLA types. Results identify several such peptides that are broadly recognized by mice of four H-2 types and by 52-73% of infected humans who still retain IL-2 productive responses to control recall antigens such as influenza A virus or tetanus toxoid. 86% of such infected donors tested against at least three peptides respond to at least one of the six peptides, and 77% of an additional group of seropositives respond to a mixture of the peptides. Moreover, the peptides can be used to immunize mice to elicit T cells reactive with the intact HIV envelope protein. These peptides therefore may be useful for both vaccine development in the broad human population, and diagnostic or prognostic use.

The importance of local mucosal HIV-specific CD8(+) cytotoxic T lymphocytes for resistance to mucosal viral transmission in mice and enhancement of resistance by local administration of IL-12.

Although crucial to mucosal vaccine development, the mechanisms of defense against mucosal viral infection are still poorly understood. Protection, cytotoxic T lymphocytes (CTL), and neutralizing antibodies have all been observed, but cause and effect have been difficult to determine. The ability of CTL in the mucosa to mediate protection against mucosal viral transmission has never been proven. Here, we use an HIV peptide immunogen and an HIV-1 gp160-expressing recombinant vaccinia viral intrarectal murine challenge system, in which neutralizing antibodies do not play a role, to demonstrate for the first time that long-lasting immune resistance to mucosal viral transmission can be accomplished by CD8(+) CTL that must be present in the mucosal site of exposure. The resistance is ablated by depleting CD8(+) cells in vivo and requires CTL in the mucosa, whereas systemic (splenic) CTL are shown to be unable to protect against mucosal challenge. Furthermore, the resistance as well as the CTL response can be increased by local mucosal delivery of IL-12 with the vaccine. These results imply that induction of local mucosal CTL may be critical for success of a vaccine against viruses transmitted through a mucosal route, such as HIV.

Temperature effects on kinetic properties of plasma membrane ATPase from the yeast Saccharomyces cerevisiae.

The reaction of plasma membrane ATPase from yeast with Mg2+ and Mg X ATP was studied in a temperature range of 10-30 degrees C. The random mechanism of activation by Mg2+ and the pseudocompetitive inhibition at higher concentrations was not altered when the temperature was varied, nor were the kinetic constants representing substrate binding. However, at low temperature, the affinity of the enzyme for Mg2+ is greatly reduced. The Arrhenius plot of log V vs. 1/T shows straight lines with an inflection point at 24 degrees C, which disappears in the presence of detergent. Calorimetric studies of the plasma membranes show a transition point at the same temperature. From these findings we suppose that Mg2+ is bound at a regulatory site of the ATPase, which is influenced by surrounding phospholipids.

The influence of tetraphenylborates (hydrophobic anions) on yeast cell electro-rotation.

The action of a series of tetraphenylborate ion (TPB) derivatives on yeast cells was studied by electro-rotation of the pre-treated cells. TPB derivatives in which all four phenyl groups were substituted with fluorine, chlorine or trifluoromethyl were much more toxic than the unsubstituted compound, the effect increasing dramatically with increasing size of substituents. These observations suggest that the toxicity of these hydrophobic ions is determined mainly by their size and possibly also by the chemical inductivity of their substituent groups. The order of the toxicities of these ions was in fair agreement with literature values for their translocation rates across artificial bilayers. Incubation times of 3 h were used as standard, longer incubations (up to 48 h) showed that the number of cells affected by low doses of TPB increased with the logarithm of time after the first hour of incubation. Although measurements of the percentage of cells showing co-field rotation showed that controls were not adversely affected by incubations as long as 9 h, rotation spectra showed that some cells suffer loss of internal conductivity during extended incubations. Decrease of the pH of the incubation medium, or inclusion of high concentrations of NaCl or KCl, potentiated the effects of these hydrophobic ions. The toxicity developed slowly, and the sensitivity of the assay was only very weakly dependent on the cell suspension density.

The comparative influence of substituted phenols (especially chlorophenols) on yeast cells assayed by electro-rotation and other methods.

The toxicity of 31 phenols was studied by electro-rotation of yeast cells. Control yeast cells show both anti-field and co-field rotation, depending upon the field frequency applied. After treatment with supra-threshold amounts of phenols the anti-field rotation is weakened or abolished and a stronger co-field rotation can be seen. The proportion of cells showing the co-field rotation was found to be a sensitive measure of toxicity. Doses of 2.2 mumol/l of pentachlorophenol, or of 0.3 mumol/l of pentabromophenol were detectable after 3 h incubation at pH 4.0. At a given pH, the toxicity of the chlorophenols correlated extremely well with their octanol:water partition coefficients (Pow). The complete set of phenols showed fair overall correlation with Pow, but less good correlation with their acidity constants (pKa). In particular the toxicity of a given phenol was less than predicted from its pKa if the incubation pH was higher than the pKa. Biochemical assays on 23 of the phenols showed that the rotational sensitivity runs closely parallel to the sensitivities of cell growth rate and of the plasmamembrane ATPase, but less closely to the inhibition of purine incorporation. It appears that the electro-rotation method provides a useful and rapid test for the presence of organic ecotoxins. The test enables us to distinguish differences between single cells, and is comparable in sensitivity to biochemical tests that use vesicles or homogenates derived from a cell population.

HIV-specific immunity following immunization with HIV synthetic envelope peptides in asymptomatic HIV-infected patients.

OBJECTIVE: A phase I trial was conducted to evaluate the safety and immunogenicity of an HIV synthetic peptide vaccine in HIV-seropositive individuals. The immunogens used in this study were PCLUS 3-18MN and PCLUS 6.1-18MN envelope peptides. METHODS: Eight HIV-infected patients received six subcutaneous injections of 160 microg PCLUS 3-18MN in Montanide ISA 51 and were followed longitudinally for a year after the first immunization. Peripheral blood mononuclear cells (PBMC) were tested for peptide-specific T helper and cytotoxic T cell (CTL) responses, HIV-1MN neutralizing antibodies and antibodies against HIV PCLUS 3 and P18 MN peptides. RESULTS: PCLUS 3-1 8MN-specific T helper responses were significantly increased at 36 weeks (P < 0.05, after adjustment for multiple comparisons) following initial immunization with PCLUS 3-18MN. A P18MN-specific CTL response, not present prior to vaccination, was observed after immunization in one patient. Serum HIV-1 MN-neutralizing antibody titers increased in each of the three patients who had low titers prior to immunization. Plasma HIV RNA levels and CD4 cell counts did not change appreciably during the study period. CONCLUSIONS: This trial demonstrates that both peptides can be safely administered to HIV-infected individuals and that PCLUS 3-18MN induces increases in HIV peptide-specific immune responses.

Candidate HIV type 1 multideterminant cluster peptide-P18MN vaccine constructs elicit type 1 helper T cells, cytotoxic T cells, and neutralizing antibody, all using the same adjuvant immunization.

Cytotoxic T lymphocytes and Th1 cells have been suggested to play a critical role in the control of HIV infection. It is therefore considered that a vaccine that induces a strong Th1 response and CTL response would be more efficacious than one that does not in providing protection against infection and progression toward AIDS. In this study we show that immunization with vaccine constructs consisting of multideterminant cluster peptides containing Th epitopes from the HIV-1IIIB envelope colinearly synthesized to peptide 18MN, is capable of inducing a Th1 response in mice and, dependent on this help, both cytotoxic T cell responses and neutralizing antibody toward the homologous strain of HIV. Moreover, the cytotoxic T cell response elicited by immunization with a mixture of cluster peptide-P18MN vaccine constructs was at least as cross-reactive against known viral variant P18 target sequences as a CTL line produced by immunization with a vaccinia construct expressing recombinant gp160 MN. Four adjuvants were compared to optimize both CTL and antibody responses. A single adjuvant formulation of peptide in ISA 51 could elicit all three: Th1 cells, CTLs, and neutralizing antibody. Thus, immunization directed toward the development of a cytotoxic T cell response does not preclude the development of neutralizing antibody and vice versa, i.e., the responses are not mutually exclusive. The immunization protocol described here should be directly applicable for study in clinical trials aimed at HIV-1 immunotherapy or prophylaxis.

Correlation between the lipophilicity of substituted phenols and their inhibition of the Na+/K+-ATPase of Chinese hamster ovary cells.

The Na+/K+-ATPase of Chinese Hamster Ovary (CHO) cells, a plasma membrane bound protein was used as a test system to evaluate the toxicity of several phenol derivatives on membranes. Taking only 2 physico-chemical parameters into consideration, viz., the logarithm of the octanol/water partition coefficient as an indicator for the lipophilicity and the sigma-Hammett constant as a measure for the polarity of the phenol substitutes, it was possible to predict the toxicity with high significance. A multivariate regression analysis calculated a correlation coefficient of 0.99. The results confirm studies performed in our laboratory on cytotoxicity and on functional membrane proteins of fungal and mammalian cells [1,2], suggesting a common mechanism of toxicity by the action of hydrophobic xenobiotics on biomembranes. Taking into account the different sensitivities of the test systems, Quantitative Structure-Activity Relationship (QSAR) analyses could help to explain the basic toxicity of several classes of environmental chemicals.

Influence of cations on the mode of action of miconazole on yeast cells.

The influence of the antifungal agent miconazole nitrate on yeast plasma membranes was studied in a concentration range 0-100 microM. The reaction of 100 microM miconazole with the plasma membranes lead to a rapid breakdown of the transmembrane pH gradient and to an efflux of metabolites from the cytoplasm of the cells. This effect of miconazole could be reversed by mono-, di- and most effectively by trivalent cations due to the formation of miconazole-cation complexes. At a ratio of trivalent cation/miconazole (1:3) the effect was completely reversed. X-Ray diffraction studies indicated a crystalline structure of the aluminium-miconazole complex.

[The anatomy, radiology and clinical features of fractures of the sacrum (author's transl)]. / Anatomie, Röntgenologie und Klinik der Sakrumfrakturen.

In the presence of multiple injuries, 30% of fractures of the sacrum are missed on the first examination. The clinical features are non-specific and are often obscured by other injuries. Interruption or displacement of contrast lines on the A. P. view, if bilateral, are definite evidence of a fracture. In evaluating films of the sacrum it is therefore recommended that these lines be examined carefully. Such changes were found in two thirds of longitudinal and one third of horizontal fractures. Special attention should also be paid to the lateral and cranial articular processes, since these are areas of weakness in the pelvis and are predisposed to fracture.

[Shoulder luxation. A late clinico-radiologic study]. / Die Schulterluxation. Eine klinisch-radiologische Spätuntersuchung.

There are various opinions regarding the aetiology and pathogenesis of dislocation of the shoulder. Various aspects of these theories and their clinical significance have been examined in the course of a clinical and radiological follow-up. Crucial to the development of subluxation is the occurrence of trauma. The age of the patient is important in the prognosis: more than 60% of patients under the age of 30 develop recurrent or persistent dislocation. Apart from a congenital disposition and inadequate treatment, accompanying injuries are primarily responsible for the development of persistent dislocation. Late follow-up has shown that radiologically recognisable post-traumatic changes are apparent in at least one-third of the patients. Persistent symptoms are much more common and consist of pain, limitation of movement, weakness and paraesthesia. In evaluating the late results, one should distinguish between objective functional limitation of movement and subjectively felt reduction in function.

[MR tomography of injuries of the medial collateral ligaments of the knee joint]. / MR-Tomographie bei Innenbandverletzungen des Kniegelenkes.

Frequency and characteristics of lesions of the medial collateral ligaments (MCL) were studied by MRI in 155 patients with trauma to the knee. There were abnormalities of the MCL in 38% of cases with ligamentous injuries and in 27% these were combined with meniscal tears. 11% of these patients showed isolated rupture of MCL and as a result of the MRI findings were treated conservatively. By means of T2*-weighted images the individual lesions could be accurately localised. Characteristic findings have been defined.

Assessment of structurally related chemicals: toxicity and ecotoxicity of acrylic acid and acrylic acid alkyl esters (acrylates), methacrylic acid and methacrylic acid alkyl esters (methacrylates).

BUA compiled the available data on toxicity and ecotoxicity for several acrylic and methacrylic acid esters and their corresponding acids. A comparison of these data revealed a qualitative similarity in the toxicological and ecotoxicological properties of the compounds considered. The data indicate that methacrylates are less reactive than the corresponding acrylates.

[Cervical thymectomy as an expanded surgical method in primary and secondary hyperparathyroidism]. / Die cervicale Thymektomie als erweitertes Operationsverfahren beim primären und sekundären Hyperparathyreoidismus.

From 1964 to 1979 neck explorations were performed on 135 patients with primary and 30 patients with secondary hyperparathyroidism. In 28 of 165 patients (16,9%) a cervical (23 patients) or a direct (5 patients) thymectomy after sternum splitting was carried out. In 22 of 28 patients, parathyroid tissue was found within or adjacent to the thymus (13,3%). Cervical thymectomy is a safe and effective procedure when an exact indication is present and if certain rules of operative technique are followed.

Application of the criteria for classification of existing chemicals as dangerous for the environment.

The criteria for classification and labelling of substances as "dangerous for the environment" agreed upon within the European Union (EU) were applied to two sets of existing chemicals. One set (sample A) consisted of 41 randomly selected compounds listed in the European Inventory of Existing Chemical Substances (EINECS). The other set (sample B) comprised 115 substances listed in Annex I of Directive 67/548/EEC which were classified by the EU Working Group on Classification and Labelling of Existing Chemicals. The aquatic toxicity (fish mortality,Daphnia immobilisation, algal growth inhibition), ready biodegradability and n-octanol/water partition coefficient were measured for sample A by one and the same laboratory. For sample B, the available ecotoxicological data originated from many different sources and therefore was rather heterogeneous. In both samples, algal toxicity was the most sensitive effect parameter for most substances. Furthermore, it was found that, classification based on a single aquatic test result differs in many cases from classification based on a complete data set, although a correlation exists between the biological end-points of the aquatic toxicity test systems.

Environmental risk assessment of existing chemicals.

Most of the existing chemicals of high priority have been released into the environment for many years. Risk assessments for existing chemicals are now conducted within the framework of the German Existing Chemicals Program and by the EC Regulation on Existing Substances. The environmental assessment of a chemical involves: a) exposure assessment leading to the derivation of a predicted environmental concentration (PEC) of a chemical from releases due to its production, processing, use, and disposal. The calculation of a PEC takes into account the dispersion of a chemical into different environmental compartments, elimination and dilution processes, as well as degradation. Monitoring data are also considered. b) effects assessment. Data obtained from acute or long-term toxicity tests are used for extrapolation on environmental conditions. In order to calculate the concentration with expectedly no adverse effect on organisms (Predicted No Effect Concentration, PNEC) the effect values are divided by an assessment factor. This assessment factor depends on the quantity and quality of toxicity data available. In the last step of the initial risk assessment, the measured or estimated PEC is compared with the PNEC. This "risk characterization" is conducted for each compartment separately (water, sediment, soil, and atmosphere). In case PEC > PNEC an attempt should be made to revise data of exposure and/or effects to conduct a refined risk characterization. In case PEC is again larger than PNEC risk reduction measures have to be considered.