Gas exchange measurement as a non-destructive viability assay for frozen-thawed, winter-dormant apple buds.

Low temperature studies with winter-dormant buds are severely limited by the lack of a rapid, non-destructive assay for their viability. Investigations involving the winter harvest of ecodormant buds of woody subjects, including cryopreservation, are restricted if viability cannot be assessed until dormancy is broken. If post-treatment grafting indicates low survival of the harvested population then further collection and study has to be delayed until the next winter season. This study trials the use of a portable gas exchange system able to discriminate between live and dead buds rapidly, with the assay confirmed as non-destructive by subsequent micropropagation. Active respiration was recorded for 85% of a winter-dormant Malus domestica buds population that showed 91% viability when grafted (n = 45). Lethally stressed material gave no false positive results. When micropropagated after respiratory measurement, a population viability of 76% was recorded. There was a significant, positive correlation between respiration and fresh weight for buds of mass >10 mg, from a population with a mean fresh weight of 17 mg.

Avidity of IgG antibodies against meningococcal serogroup a polysaccharide and correlations with bactericidal activity in sera from meningitis patients and controls from Ethiopia.

Meningococcal meningitis is a significant global health challenge, especially for sub-Saharan area: the African meningitis belt. Neisseria meningitidis of serogroup A (MenA) is responsible for the large number of epidemics that have been recorded in these countries. To determine the level of antibodies against meningococcal A polysaccharide (APS) that correlates with protection against MenA disease in the African meningitis belt, it may be important to consider antibody avidity along with quantity. In this study, two ELISA methods using the chaotropic agent ammonium thiocyanate were compared and employed to measure avidity indexes (AI) of IgG antibodies against APS in controls and in acute and convalescent sera from Ethiopian meningococcal patients. High statistical correlations between the AIs determined by the two methods were observed. The geometric mean AI (GMAI) increased with time from acute to convalescent sera indicating affinity maturation. GMAI was significantly higher in convalescent sera from the MenA patients and in sera from the controls than in acute sera from patients with meningococcal disease. A significant correlation between serum bactericidal activity titres (SBA) and concentration of IgG antibodies against APS was observed; however, our results did not indicate that determination of antibody avidities by the thiocyanate elution method gave a better correlation with SBA than anti-APS IgG concentrations determined by the standard ELISA method.

An outer membrane vesicle vaccine for prevention of serogroup A and W-135 meningococcal disease in the African meningitis belt.

The bacterium Neisseria meningitidis of serogroups A and W-135 has in the recent decade caused most of the cases of meningococcal meningitis in the African meningitis belt, and there is currently no efficient and affordable vaccine available demonstrated to protect against both these serogroups. Previously, deoxycholate-extracted outer membrane vesicle (OMV) vaccines against serogroup B meningococci have been shown to be safe and induce protection in humans in clonal outbreaks. The serogroup A and W-135 strains isolated from meningitis belt epidemics demonstrate strikingly limited variation in major surface-exposed protein structures. We have here investigated whether the OMV vaccine strategy also can be applied to prevent both serogroups A and W-135 meningococcal disease. A novel vaccine combining OMV extracted from recent African serogroup A and W-135 strains and adsorbed to aluminium hydroxide was developed and its antigenic characteristics and immunogenicity were studied in mice. The specificity of the antibody responses was analysed by immunoblotting and serum bactericidal activity (SBA) assays. Moreover, the bivalent A+W-135 vaccine was compared with monovalent A and W-135 OMV vaccines. The bivalent OMV vaccine was able to induce similar SBA titres as the monovalent A or W-135 OMV towards both serogroups. High SBA titres were also observed against a meningococcal serogroup C strain. These results show that subcapsular antigens may be of importance when developing broadly protective and affordable vaccines for the meningitis belt.

Avidity of serogroup A meningococcal IgG antibodies after immunization with different doses of a tetravalent A/C/Y/W135 polysaccharide vaccine.

In the absence of an affordable conjugate meningococcal vaccine, mass vaccination campaigns with polysaccharide vaccines are the means to control meningitis epidemics in sub-Saharan Africa. Facing global vaccine shortage, the use of reduced doses, which have been shown to be protective by serum bactericidal activity, can save many lives. In this study, we investigated the antibody responses and avidity of IgG antibodies evoked against the serogroup A capsule of Neisseria meningitidis by different doses of an A/C/Y/W135 polysaccharide vaccine. Volunteers in Uganda were vaccinated with 1/10, 1/5 or a full dose (50 µg) and revaccinated with a full dose after 1 year. Specific IgG geometric mean concentrations and geometric mean avidity indices (GMAI) were determined by a modified enzyme-linked immunosorbent assay (ELISA) using thiocyanate as a chaotropic agent. After vaccination with 1/10 or 1/5 doses, the GMAI increased from 1 month to 1 year. One year following the initial dose, the GMAI levels were higher in the arm receiving reduced doses than for the arm receiving a full dose. Following the second full dose, avidity indices equalized at approximately the same level in the three arms. Although there are practical challenges to the use of reduced doses in the field, our findings suggest that reduced doses of polysaccharide vaccine are able to elicit antibodies of as good avidity against serogroup A polysaccharide as a full dose.

Leaf physiological versus morphological acclimation to high-light exposure at different stages of foliar development in oak.

We investigated light acclimation in seedlings of the temperate oak Quercus petraea (Matt.) Liebl. and the co-occurring sub-Mediterranean oak Quercus pyrenaica Willd. Seedlings were raised in a greenhouse for 1 year in either 70 (HL) or 5.3% (LL) of ambient irradiance of full sunlight, and, in the following year, subsets of the LL-grown seedlings were transferred to HL either before leaf flushing (LL-HLBF plants) or after full leaf expansion (LL-HLAF plants). Gas exchange, chlorophyll a fluorescence, nitrogen fractions in photosynthetic components and leaf anatomy were examined in leaves of all seedlings 5 months after plants were moved from LL to HL. Differences between species in the acclimation of LL-grown plants to HL were minor. For LL-grown plants in HL, area-based photosynthetic capacity, maximum rate of carboxylation, maximum rate of electron transport and the effective photochemical quantum yield of photosystem II were comparable to those for plants grown solely in HL. A rapid change in nitrogen distribution among photosynthetic components was observed in LL-HLAF plants, which had the highest photosynthetic nitrogen-use efficiency. Increases in mesophyll thickness and dry mass per unit area governed leaf acclimation in LL-HLBF plants, which tended to have less nitrogen in photosynthetic components and a lower assimilation potential per unit of leaf mass or nitrogen than LL-HLAF plants. The data indicate that the phenological state of seedlings modified the acclimatory response of leaf attributes to increased irradiance. Morphological adaptation of leaves of LL-HLBF plants enhanced photosynthetic capacity per unit leaf area, but not per unit leaf dry mass, whereas substantial redistribution of nitrogen among photosynthetic components in leaves of LL-HLAF plants enhanced both mass- and area-based photosynthetic capacity.

Effect of streptolysin O and digitonin on egg lecithin/cholesterol vesicles.

Artificial membrane vesicles (liposomes) have been used to study the lytic mechanism of the bacterial toxin, streptolysin O, compared to that of the well-known plant glycoside, digitonin. Two types of vesicle were prepared: large unilamellar vesicles and multilamellar liposomes. The vesicles were prepared with varying molar ratios of egg lecithin and cholesterol and loaded with the water-soluble spin label, TEMPO-choline chloride. Lysis of the vesicles was registered as release of spin label and monitored by change in the electron spin resonance (ESR) spectrum. In this system digitonin was able to lyse both large unilamellar vesicles and multilamellar liposomes. The effectiveness of lysis increased by increasing the percentage of cholesterol, but even at 0% cholesterol a significant level of lysis was observed by addition of a large enough concentration of digitonin. In contrast, no lysis was detected from multilamellar liposomes after exposure to streptolysin O, even when they consisted of 50 mol% cholesterol. On the other hand, large unilamellar vesicles could be lysed by streptolysin O, provided the cholesterol content was greater than 33%. At 67 mol% cholesterol in the membranes, the degree of lysis was diminished compared to 50%, which appeared to be optimal. This is the first demonstration of liposome lysis by streptolysin O and demonstrates the cholesterol specificity which has previously been shown by inhibition studies.

The solution shapes of IgG3 immunoglobulin and its Fch and Fc fragments. A small-angle X-ray scattering study.

A homogeneous IgG3 protein and its corresponding Fch and Fc fragments have been studied in solution using the small-angle X-ray scattering method. The Fch and Fc fragments were produced by short digestion of IgG3 with papain and trypsin. The results indicate that the overall shape of the IgG3 molecule in solution can best be described as an elliptical cylinder with a total length of 29 nm and with a cross-section having the semiaxes 3.8 and 0.9 nm. Thus, the overall shape of IgG3 is considerably different from the Y-shape normally adopted for the IgG1 molecule. The analysis of the data obtained for the Fch and Fc fragments also yields elliptical cylinders with almost the same dimensions of the cross-section but with shorter total lengths, 11.4 and 6.7 nm, respectively. The molecular weights of the IgG3 protein and the Fch and Fc fragments were determined to be 1.8 x 10(5), 0.61 x 10(5), and 0.50 x 10(5), respectively.

Thermal properties of human IgG.

Dynamic light scattering experiments have been performed to study the aggregation kinetics of human immunoglobulin G (IgG). Aggregation and irreversible cluster growth results when IgG solutions (2-15 mg/ml) are heated above 50 degrees C. The measured scattering intensity I and effective hydrodynamic radius (R) can be described consistently by a Smoluchowski aggregation process. The number of clusters ni(t) containing i monomers at time t are computed. The radius of an i cluster is assumed to be Ri = R0 i beta, where beta is the cluster exponent. This kinetic process results in the following characteristic power law behavior: (R)/R0 = (1 + gamma R (T, C, c)t) alpha R and (I)/I0 = (1 + gamma 1 (T, C, c)t) alpha I. Here R0 = 5.51 nm, is the monomer hydrodynamic radius, and I0 the scattered intensity from the monomer solution at temperature T and concn C. A fraction, c approximately 0.48 of the IgG monomers are heat stable up to 63 degrees C and do not participate in the aggregation process. The power-law behavior of mean value of R/R0 and mean value of I/I0 indicates scaling, and indeed a very satisfactory data collapse results from our data. The best non-linear fit of the power-law forms gives alpha R = 0.48 +/- 0.05, alpha I = 1.00 +/- 0.01 and beta = 0.39 +/- 0.04. We also find that the heat aggregation of IgG is an activated process. Fits of the experimental data Gibbs free energy for the activated complex delta G* = 13.8 +/- 0.1 kcal/mole at 56 degrees C. The temp dependence of the growth rates exhibits an Arrhenius behavior with an enthalpy of activation delta H* = 120 +/- 5 kcal/mole.

A comparison of human and murine monoclonal IgGs specific for the P1.7 PorA protein of Neisseria meningitidis.

Monoclonal human IgG SS269 reacts with Neisseria meningitidis expressing the P1.7 PorA protein and with linear peptides containing NGGAS, which accounts for the P1.7 specificity. Murine monoclonal antibody to P1.7 reacts with peptides containing the overlapping epitope, ASGQ. The human and murine antibodies have similar affinities. The low avidity human antibody is very inefficient at stimulating complement-mediated bactericidal killing while the high avidity murine antibody efficiently kills bacteria. However, efficient opsonophagocytosis was mediated even at low concentrations of the human antibody and in the absence of complement, suggesting that low avidity antibodies might be protective against disease.

Cellular activating properties and morphology of membrane-bound and purified meningococcal lipopolysaccharide.

Neisseria meningitidis, the cause of epidemic meningitis and acute lethal sepsis, synthesizes surplus lipopolysaccharides (LPSs) during growth, which are released as outer membrane vesicles (OMV) or "blebs". Meningococcal disease severity is related to plasma LPS levels. We have compared the biological activities of native outer membrane vesicles (nOMV) to those of purified Nm-LPS (Nm-LPS) and LPS-depleted OMV (dOMV) prepared from N. meningitidis. The LPS content of nOMV was determined spectrophotometrically by quantifying KDO and by silver-stained SDS-PAGE gels. The morphology of the preparations was studied by transmission electron microscopy. The Limulus amoebocyte lysate (LAL) assay was used to quantify LPS in the plasma solutions. The preparations were diluted in endotoxin-free heparin plasma to equal amounts of LPS (w/w) in the range 50-5000 pg/ml. The biological reactivity was tested by: (i) a monocyte target-assay (monocyte purity > or =96%); and (ii) a whole blood model, measuring the secretion of TNF-alpha and IL-6 induction of procoagulant activity in monocytes (PCA). In both models, nOMV induced dose-dependent cell responses (TNF-alpha, IL-6, PCA) similar to purified Nm-LPS, whereas dOMV induced minimal responses. However, LAL activity was significantly higher for nOMV than for purified Nm-LPS and dOMV. The cellular responses of purified Nm-LPS and nOMV were reduced (>95%) by a specific anti-CD14-antibody.

Immune lysis of spin label loaded liposomes incorporating cardiolipin; a new sensitive method for detecting anticardiolipin antibodies in syphilis serology.

Liposomes prepared from a mixture of the pure lipids cholesterol, lecithin and cardiolipin (molar ratio 50/45/5), are able to bind antibodies directed against Treponema pallidum. When the liposomes are loaded with the water soluble spin label tempocholine chloride, the release of spin label from the liposomes can be monitored directly by observing changes in the paramagnetic resonance (ESR) spectrum from the spin label. The method offers a convenient technique for monitoring the complement-mediated lysis of liposomes, and may be applied in the serological diagnosis of syphilis, and a method for quantitative measurement of complement.

Quantitation of IgG subclass antibody responses after immunization with a group B meningococcal outer membrane vesicle vaccine, using monoclonal mouse-human chimeric antibodies as standards.

An ELISA method was developed to quantitate gravimetrically (microgram/ml) the IgG subclass response against a Norwegian vaccine composed of outer membrane vesicles (OMV) isolated from a Neisseria meningitidis B:15:P1.7,16 epidemic strain. Chimeric mouse-human anti-hapten NIP (5-iodo-4-hydroxy-3-nitrophenacetyl) antibodies of each subclass were used for calibration purposes. Before vaccination, low amounts of IgG1 and IgG2 antibodies against OMV were detectable in all vaccinees, whereas IgG3 was only detectable in one of the 21 vaccinees. After vaccination, IgG1 antibodies dominated the response followed by IgG3 and low to moderate levels of IgG2 antibodies. IgG4 was only detectable at very low levels in a few vaccinees. All sera showed close to parallel dose-response curves to each other for IgG1 and IgG3, whereas the IgG2 curves were not parallel to chimeric IgG2 and could thus not be quantitated gravimetrically. For IgG3, 1/3 of the vaccinee sera showed non-parallel dose-response curves to the rest of the vaccinee sera and to chimeric IgG3 and could not be gravimetrically quantitated. The rest of the sera showed parallel dose-response curves with the chimeric IgG3 and gravimetric quantitation was possible. This study illustrates that chimeric antibodies can be used as calibrators to quantitate IgG subclass antibody responses against OMV in gravimetric units and that the vaccine mainly induces IgG1 and IgG3 antibodies in humans.

Light acclimation maintains the redox state of the PS II electron acceptor Q(A) within a narrow range over a broad range of light intensities.

Chrysanthemum inducum-hybrid 'Coral Charm', Hibiscus rosa-sinensis L. 'Cairo Red' and Spathiphyllum wallisii Regel 'Petit' were grown in natural light in a greenhouse at three levels of irradiance using permanent shade screens. Light acclimation of photosynthesis was characterized using modulated chlorophyll a fluorescence of intact leaves. A close correlation was found between the degree of reduction of the primary electron acceptor Q(A) of Photosystem II (PS II) approximated as the fluorescence parameter 1-q(P), and light acclimation. The action range of 1-q(P) was 0-0.4 from darkness to full irradiance around noon, within the respective light treatments in the greenhouse, indicating that most PS II reaction centres were kept open. In general, the index for electron transport (ETR) measured by chlorophyll fluorescence was higher for high-light (HL) than intermediate-(IL) and low-light (LL) grown plants. However, HL Chrysanthemum showed 40% higher ETR than HL Hibiscus at light saturation, despite identical redox states of Q(A). The light acclimation of the non-radiative dissipation of excess energy in the antenna, NPQ, varied considerably between the species. However, when normalized against q(P), a strong negative correlation was found between thermal dissipation and ETR measured by chlorophyll fluorescence. To be able to accommodate a high flux of electrons through PS II, the plants with the highest light-saturated ETR had the lowest NPQ/q(P). The possibility of using chlorophyll fluorescence for quantification of the energy balance between energy input and utilization in PS II in intact leaves is discussed.

Serogroup determination of Neisseria meningitidis by whole-cell ELISA, dot-blotting and agglutination.

Two new methods for serogrouping of meningococci, whole-cell ELISA and dot-blotting, with monoclonal antibodies against serogroups A, B, C, Y and W135 were compared with slide-agglutination applying polyclonal sera. In addition to a panel of strains with previously determined serogroups by slide-agglutination, two strain collections of meningococci were studied: 1) 50 strains isolated from patients with systemic meningococcal disease in Norway during the winter 1987-1988; 2) 133 throat strains isolated from asymptomatic carriers over the same period. For the disease strains all three methods gave identical results, whereas some carrier strains which were non-agglutinable or polyagglutinable by slide-agglutination were serogroupable by the two other methods. All the systemic strains and about half of the carrier strains were serogroupable. We find that whole-cell ELISA and dot-blotting are specific, easy to read and more sensitive compared to slide-agglutination, but the former methods are at present limited by the availability of monoclonal antibodies against only serogroups A, B, C, Y and W135.

IgG subclass antibodies to serogroup B meningococcal outer membrane antigens following infection and vaccination.

IgG and IgG subclass antibodies to the outer membrane antigens from Neisseria meningitidis (serogroup B, serotype 15:P1.16) were quantitated by an enzyme-linked immunosorbent assay (ELISA) in sera from 40 patients with group B:15:P1.16 meningococcal disease and 24 volunteers immunized with a serotype 15:P1.16 outer membrane vesicle vaccine. A second injection was given 6 weeks after the first immunization. Patient sera obtained two and six weeks after onset of the disease had significantly higher levels of total IgG, IgG1, IgG2, and IgG3 antibodies to the outer membrane antigens than acute sera, convalescent sera from patients with systemic non-meningococcal bacterial infections and sera from healthy controls. The levels of total IgG and IgG1 remained high one and three years later. Sera from the vaccinees showed high levels of total IgG and IgG1 6, 12 and 26 weeks after the first immunization and high levels of IgG3 6 weeks after the second immunization. No increase of IgG2 or IgG4 levels was observed in the postimmunization sera. Immunoblotting of three convalescent sera demonstrated individual patterns of IgG subclass binding to various outer membrane antigens with most distinct binding of IgG1 and IgG3 antibodies to the class I protein, the H.8 lipoprotein and the lipopolysaccharide. Since IgG1 and IgG3 are the most effective antibodies for complement activation and phagocytosis, group B meningococcal disease and immunization with the serotype 15:P1.16 outer membrane vesicle vaccine stimulate production of those IgG subclasses which have the strongest opsonic and bactericidal activity.

Analysis of the expression of the putatively virulence-associated neisserial protein RmpM (class 4) in commensal Neisseria and Moraxella catarrhalis strains.

The RmpM protein has been reported to be present only in pathogenic Neisseria species. In the present study we demonstrate that this protein is also present at least in N. lactamica and N. sicca strains. The N. lactamica protein reacts with a RmpM-specific monoclonal antibody (185,H-8), having a molecular mass ( approximately 31 kDa) slightly lower than that of the meningococcal RmpM, and mouse antibodies from sera against outer membrane vesicles from both N. lactamica and N. sicca strains cross-react with the meningococcal RmpM. PCR and hybridization experiments with a complete rmpM probe agree with the immunodetection experiments. Our results strongly suggest that the meningococcal RmpM should not be considered a virulence marker, and the presence of this protein in the commensal species agrees with its role as a structural protein, proposed for the RmpM, which should be considerably conserved in the Neisseria species.

Serotyping and subtyping of Neisseria meningitidis isolates by co-agglutination, dot-blotting and ELISA.

Typing of meningococci with a panel of serotype and subtype specific monoclonal antibodies (MAbs) was compared in co-agglutination, dot-blotting and ELISA tests. Twenty reference strains, 50 case isolates and 133 throat isolates from healthy carriers were studied. The typing results with dot-blotting and ELISA were identical, whereas co-agglutination gave different results for three case and 24 carrier strains. The distribution of serotypes and subtypes among the strains is reported. The combination of the subtypes P1.1 and P1.15 in a serotype 15 patient strain was observed. With one case strain and 15 carrier strains, neither serotype nor subtype could be determined. Non-typable and non-subtypable isolates were further characterised by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Co-agglutination is useful for typing small numbers of strains with a few MAbs, but less suitable for large-scale typing than the other two methods. Dot-blotting needs less expensive equipment, smaller volumes of antibodies and fewer manipulations than ELISA.

Determination of antibody responses to meningococcal antigens by ELISA.

Enzyme-linked immunosorbent assay (ELISA, EIA) is a highly versatile and sensitive technique that can be used for quantitative as well as qualitative determination of almost any antigen or antibody. Reagents are stable, non-radioactive and, in most cases, commercially available. Owing to the simplicity and versatility of the method, ELISA represents probably one of the most used methods for studying antibody responses and antibody levels. Since Engvall and Perlman's first paper describing the ELISA in 1971 (1), almost all laboratories working in serology or immunology have designed their own assays with different protocols for coating with antigens, incubation conditions, detecting systems, and ways of reporting of the results. In most cases, there is no need for strict interlaboratory standardization of ELISAs and each laboratory will develop a system that suits their needs. However, for some ELISAs, e.g., used in diagnostic laboratories and in vaccine trials, standardization is important, and this is considered in "Meningococcal Disease" Edited by AJ Pollard and MCJ Maiden, (1a).

Outer membrane protein vesicle vaccines for meningococcal disease.

Alternative strategies exist for prevention of group B Neisseria meningitidis (meningococcal) disease through vaccination (see Chapters 5 , 8 , 13 , 14 in this volume). However, the most promising approach to date has been the use of outer-membrane vesicle (OMV) vaccines for induction of bactericidal antibodies against cell-surface outer-membrane proteins (OMPs).

Preclinical immunogenicity study of trivalent meningococcal AWX-OMV vaccines for the African meningitis belt.

In the recent decade, epidemic meningitis in the African meningitis belt has mostly been caused by Neisseria meningitidis of serogroups A, W and X (MenA, MenW and MenX, respectively). There is at present no licensed vaccine available to prevent MenX meningococcal disease. To explore a trivalent MenAWX vaccine concept, we have studied the immunogenicity in mice of MenX outer membrane vesicles (X-OMV) or MenX polysaccharide (X-PS) when combined with a bivalent A-OMV and W-OMV (AW-OMV) vaccine previously shown to be highly immunogenic in mice. The vaccine antigens were produced from three representative wild type strains of MenA (ST-7), MenW (ST-11) and MenX (ST-751) isolated from patients in the African meningitis belt. Groups of mice were immunized with two doses of X-OMV or X-PS combined with the AW-OMV vaccine or as individual components. All vaccine preparations were adsorbed to Al(OH)3. Sera from immunized mice were tested by ELISA and immunoblotting. Functional antibody responses were measured as serum bactericidal activity (SBA) and opsonophagocytic activity (OPA). Immunization of mice with X-OMV, alone or in combination with AW-OMV induced high levels of anti-X OMV IgG. Moreover, X-OMV alone or in combination with the AW-OMV vaccine induced high SBA and OPA titers against the MenX target strain. X-PS alone was not immunogenic in mice; however, addition of the AW-OMV vaccine to X-PS increased the immunogenicity of X-PS. Both AWX vaccine formulations induced high levels of IgG against A- and W-OMV and high SBA titers against the MenA and MenW vaccine strains. These results suggest that a trivalent AWX vaccine, either as a combination of OMV or OMV with X-PS, could potentially prevent the majority of meningococcal disease in the meningitis belt.