Efficient fluorescence-based imaging methods for quantitating infectivity and oncolytic efficacy of Newcastle disease virus.

A high throughput quantitation protocol is desired to determine the replication of various recombinant oncolytic viruses in vitro. Plaque assay is the classic method for viral infectivity quantitation but is laborious and time consuming; moreover it does not report the oncolytic efficacy of a virus. In this paper, three new imaging methods for quantitating viral infectivity are derived and evaluated: fluorescence intensity, infection counts, and infection degree. Infection of oncolytic Newcastle disease virus in human tumor and normal cells was followed over a time course by plaque assay and the imaging methods. For the latter, brightfield and green channel images were acquired at various fixed locations in the cell culture, and later analyzed. One of the imaging methods was found to be highly correlated with viral titer; the other methods are complementary to plaque assay and provide additional information like oncolytic efficacy, syncytium formation etc. The new methods significantly reduce the time and material costs required by plaque assay, and provide an efficient system for quantitating and characterizing infectivity and efficacy of oncolytic viruses.

New image compression techniques using multiwavelets and multiwavelet packets.

Advances in wavelet transforms and quantization methods have produced algorithms capable of surpassing the existing image compression standards like the Joint Photographic Experts Group (JPEG) algorithm. For best performance in image compression, wavelet transforms require filters that combine a number of desirable properties, such as orthogonality and symmetry. However, the design possibilities for wavelets are limited because they cannot simultaneously possess all of the desirable properties. The relatively new field of multiwavelets shows promise in obviating some of the limitations of wavelets. Multiwavelets offer more design options and are able to combine several desirable transform features. The few previously published results of multiwavelet-based image compression have mostly fallen short of the performance enjoyed by the current wavelet algorithms. This paper presents new multiwavelet transform and quantization methods and introduces multiwavelet packets. Extensive experimental results demonstrate that our techniques exhibit performance equal to, or in several cases superior to, the current wavelet filters.

Standardization of lymphocyte antibody binding capacity - a multi-centre study.

As quantitative flow cytometry is being increasingly used to characterize non-malignant and malignant disorders, interlaboratory standardization becomes an important issue. However, the lack of standardized methods and process controls with predefined antibody binding capacity values, limits direct interlaboratory comparison. The present study has addressed these issues using a stable whole blood product and a standardized antigen quantification protocol. It was demonstrated that: (i) a standard technical protocol can result in a high degree of interlaboratory concordance; (ii) interlaboratory variation of less than 12% can be achieved for CD4 antibody binding capacity values; and (iii) stable whole blood can be used as a process control with predefined antibody binding capacity values. Furthermore, using such an approach, a normal range was established for CD3, CD4 CD8 and CD19. These antigens appear to be expressed in a hierarchical manner, a factor that could be used as a procedural quality control measure.

Evaluation of the flow cytometric crossmatch. Preliminary results of a multicenter study.

The flow cytometric crossmatch is a technique that is increasingly being used by clinical transplant laboratories. In this multicenter study by the British Society for Histocompatibility and Immunogenetics Flow Cytometry Group, a series of crossmatches were carried out to determine whether different centers obtained same results when performing the same crossmatch. There was greater than 80% agreement among participating laboratories on the results of 35/54 tests. There was no clear agreement in the remaining 20 cases. Quantitative analysis, estimating the number of cell-bound fluorescein molecules, demonstrated that differences in the criteria used by each center to define a positive crossmatch were responsible for some discordant results. When applied, definition of positivity based on the molecules of fluorescein increased concordance from 57.5% to 81.4%.l. These results suggest that a criterion for the interpretation of results based on quantitative analysis of bound antibody may be more reliable than methods in current routine use.

Sequence analysis of a mammalian phospholipid-binding protein from testis and epididymis and its distribution between spermatozoa and extracellular secretions.

The cellular origin of a soluble phospholipid-binding protein (PBP) in rat testicular and epididymal secretions has been investigated genetically and immunologically. PBP is ubiquitous in tissue cytosols but is not present in blood serum, lymph or milk. The relatively large amounts present in cauda epididymal plasma (CEP) and rete testis fluid suggested therefore that it may be secreted specifically by these tissues. However, when PBP cDNAs from testis and epididymis were cloned and sequenced, they did not contain a signal peptide and only one size of transcript was obtained on Northern blots of RNAs from liver, brain, placenta, testis and epididymis. Moreover, PBP could not be detected in sperm-free CEP from castrated, androgen-stimulated animals or in medium from Sertoli cell cultures. Spermatozoa, on the other hand, contained significant amounts of PBP that could be solubilized by washing cells in dissociating reagents or high-salt solutions. These results indicate that, contrary to previous interpretations, PBP is not secreted by classical pathways in either the testis or epididymis but that its presence in CEP and rete testis fluid is attributable largely to release from spermatozoa. Thus, spermatozoa have a significant influence on the composition of CEP as well as on the secretory and absorptive activity of the epididymal epithelium. A possible role for PBP in membrane biogenesis and maintenance of antigen segregation in spermatozoa is discussed.

The value of flow cytometric crossmatch in cardiac transplantation.

One of the major clinical problems in cardiac transplantation is that of moderate rejection of the graft, and over the past few years there is increasing evidence that humoral antibody may be important in graft prognosis. The sensitivity of the conventional cytotoxic crossmatch has been questioned, and an increased significance of there of the flow cytometric crossmatch (FCXM) to detect the presence of antibodies before transplantation has been reported. In this study we have examined the sera of 138 cardiac transplants (1988-1992) for the presence of donor-directed IgG and IgM antibodies using FCXM. Sera were collected immediately before transplantation and before the institution of immunosuppressive therapy. All pretransplant cytotoxic crossmatches were negative. After a minimum follow-up period of 3 months, the performance of the transplants was graded by endomyocardial biopsy: 1, no or mild evidence of rejection; 2, patients showing moderate rejection requiring increased immunosuppression. Of the 138 patients studied, 10 patients were excluded as they died within the first week of transplantation. Eight children were excluded since they were given prophylactic ATG (Merieux). A positive FCXM result was defined as showing values in excess of that found for the AB control sera. A significant association was found between the presence of both IgG to T and B cells and IgM to T cells and graft performance (P = 0.02 and 0.93, respectively). Indeed, IgM-directed T-cell antibodies were only found in patients with moderate rejecton. These two groups were mutually exclusive, so that the FCXM was able to identify the presence of moderate rejection in 55% of the patients. In conclusion, results show that pretransplant FCXM in cardiac transplantation provides a more sensitive assay of antibody status in recipients and has proved to be of prognostic value.

Mitral valve replacement with and without chordal preservation in patients with chronic mitral regurgitation. Mechanisms for differences in postoperative ejection performance.

BACKGROUND: Standard mitral valve replacement (MVR) in patients with chronic mitral regurgitation consistently results in a decrease in postoperative left ventricular (LV) ejection performance. This fall in ejection performance has been attributed, at least in part, to unfavorable loading conditions imposed by the elimination of the low-impedance pathway for LV emptying into the left atrium. In contrast to standard MVR in which the chordae tendineae are severed, however, MVR with chordal preservation (MVR-CP) does not usually decrease LV ejection performance despite similar removal of the low-impedance pathway. The purpose of the present study was to define the mechanisms responsible for this discordance in postoperative ejection performance between MVR with and without chordal preservation. METHODS AND RESULTS: Echocardiography and sphygmomanometer blood pressures were obtained in 15 patients with pure chronic mitral regurgitation before and 7-10 days after mitral valve surgery. These measurements were used to calculate ventricular volume, wall stress, and ejection fraction. Seven patients underwent MVR with chordal transection (MVR-CT), and eight patients underwent MVR-CP. MVR-CT resulted in no postoperative change in LV end-diastolic volume, a significant increase in LV end-systolic volume, a significant increase in end-systolic stress, from 89 +/- 9 to 111 +/- 12 g/cm2 (p < 0.05), and a significant decrease in ejection fraction, from 0.60 +/- 0.02 to 36 +/- 0.02 (p < 0.05). In contrast, patients who underwent MVR-CP had a significant decrease in LV end-diastolic and end-systolic volumes. End-systolic wall stress actually fell from 95 +/- 6 to 66 +/- 6 g/cm2 (p < 0.05), and ejection fraction was unchanged (0.63 +/- 0.01 before and 0.61 +/- 0.02 after mitral valve surgery) instead of reduced. CONCLUSIONS: MVR-CT resulted in a decrease in ejection performance caused in part by an increase in end-systolic stress, which in turn increased end-systolic volume. Conversely, MVR-CP resulted in a smaller LV size, allowing a reduced end-systolic stress and preservation of ejection performance despite closure of the low-impedance left atrial ejection pathway.

Non-additive gene effects in populations under different methods of selection.

The genetic parameters of two quantitative traits, 13-day larval weight and pupal weight, in Tribolium populations developed by reciprocal recurrent selection (RRS) and by within-line purebred selection (WLS) were compared each with the other and also with the parameters of the unselected base populations using the genetic model of Carbonell, Nyquist and Bell. The variability for two and three-way crosses of inbred lines derived from "companion" populations (two strains, breeds, or varieties used for a terminal cross or hybrid) was analyzed into genetic effects: autosomal additivity ((*) g), autosomal heterosis ((*) s), sex-linked additivity (L), sex-linked heterosis (LL), general maternal (m), specific maternal or reciprocal (r), additive by additive epistasis (aa), and deviations from the model due, among other causes, to higher order epistasis (dev). One series of crosses involved companion populations with diverse origins. For contrast, a second series of crosses involved companion populations originating from a common heterogenous base population. For the heterotic trait larval weight, (*) g and (*) s effects were equally important and accounted for over 50% of the total variation. The aa epistasis contributed another 20% and was followed in importance by higher order epistasis and general maternal effects. For the more highly heritable trait, pupal weight, (*) g effects were most important with (*) s, aa, and m effects having smaller but significant influences. Sex-linked and reciprocal effects were statistically significant for many crosses, but they were relatively unimportant overall. In general, the unselected base populations showed higher (*) g variation than either RRS or WLS populations with the reverse true for (*) s effects. In agreement with theoretical expectations, RRS was more effective than WLS in exploiting (*) s effects. The aa epistatic effects for larval weight were of major importance in the unselected populations, but RRS and WLS did not differ significantly for exploiting superior aa gene combinations. Companion populations with diverse origins revealed significantly larger variation due to (*) g and (*) s effects in crosses than did populations initiated from a common heterogeneous base.

Multiple vital functions of the daughterless (da) gene in Drosophila melanogaster and factors influencing its expression.

Multiple sub-vital effects, here designated as reductions in fitness S1-S5, for the second chromosome gene 'daughterless' (da) of Drosophila melanogaster were described as (S1) a recessive maternal lethality for daughters, (S2) a reduced fertility of da/da females, (S3) a recessive sub-vital zygotic effect, (S4) a recessive female-specific zygotic effect and (S5) a recessive maternal interaction effect on sons. (For S1-3 see also: Bell, 1954; Sandler, 1972; Cline, 1976). These five distinct effects were initially quantified from estimates of viability in single generation crossing experiments. Dynamic estimates of these fitness parameters were obtained by fitting the elimination rate of da from a series of large random mating cage populations to a recurrence equation by the method of minimum chi-square. The stability of these estimates discerns those effects which are truly pleiotropic versus those due to linked genes. The dynamic estimates supported only S1 and S4 effects. Evidence for S2 and S5 was indeterminate, but the S3 effect was rejected (P less than 0.01). The observed reduction in fitness, supposedly due to this recessive zygotic effect for da, was most likely the result of linked deleterious genes. These results indicate that pleiotropic vital effects observed in single generation test-cross matings may be caused by linked genes rather than the specific mutant per se. This problem is of particular importance when the mutant allele has been maintained with a balancer chromosome. Experiments on the rescue of daughters from da/da mothers with low temperatures during embryogenesis and with dechorionation of eggs were described in which the findings failed to confirm previously reported actions of the da gene. Modifying genes rather than environmental variables were cited as the probable cause for these conflicting results.

Properties of gastric and duodenal mucus: effect of proteolysis, disulfide reduction, bile, acid, ethanol, and hypertonicity on mucus gel structure.

Small deformation oscillatory rheologic measurements have been used to investigate the structure of human and pig gastric mucus and pig duodenal mucus. All three secretions had viscoelastic properties characteristic of water-insoluble, viscoelastic gels. Mucus will flow and anneal if damaged, due to the making and breaking of its elastic structure, the measured lifetime of which was 10-120 min. Mucus reconstituted by concentration of the purified glycoprotein (pig gastric and duodenal mucus) had the same viscoelastic properties as the fresh mucus, giving evidence that the glycoprotein alone will reproduce the rheologic characteristics of the mucus. The structure of fresh mucus gel was unaffected by prolonged exposure to the following mucosal damaging agents: undiluted pig bile, 20 mM sodium taurocholate or 20 mM sodium glycocholate (all at pH 2, 6, and 8), HCl at pH 1, 2 M NaCl, and ethanol less than 40% (vol/vol). Higher concentrations of ethanol greater than 40% (vol/vol), caused dehydration and denaturation of mucus. Proteolysis by pepsin and other enzymes resulted in solubilization of the mucus gel with a complete change in the properties from an "elastic" gel to those of a "viscous" liquid. A similar collapse of mucus gel structure was observed after reduction of disulfide bonds in 0.2 M mercaptoethanol, but only after incubation for at least 50 min. This study demonstrates the stability of mucus to several mucosal damaging agents. It is proposed in vivo that although adherent gastroduodenal mucus allows penetration of these agents to the underlying mucosa, it can remain in situ and continue to protect against acid (with HCO3-) and pepsin, thus minimizing mucosal damage and maximizing repair.

Estimation of maternal, sex-linked and additive x additive epistatic gene effects for body size of Tribolium.

The genetic structure of two quantitative traits, 13-day larval weight and pupal weight, in two unselected populations of Tribolium castaneum was investigated by the genetic model of Carbonell et al. (1983). The variability among two and three-way crosses was analyzed into components due to: general and specific combining abilities, maternal, sex-linkage, specific reciprocal and additive-by-additive epistasis. Also, indirect evidence of higher order epistasis was studied. It is concluded that the heterotic trait larval weight is highly affected by sex-linked genes and by non-additive gene action with additive-by-additive as well as higher order epistasis playing major roles. Pupal weight, on the other hand, is determined mostly by additive gene action although epistasis is also a significant source for genetic variability. Both traits are significantly influenced by maternal effects.

Selected methods for the evaluation of the effects of drugs on mucus viscoelasticity and mucociliary clearance.

In order to rationally test the efficacy of mucolytic and mucospissic compounds, their effects upon mucus viscoelasticity, ciliary beat frequency (c.b.f.) and total mucociliary transport rate (t.t.r.) must all be determined. Methods are described which allow the measurement of the viscoelastic modulii of small (10 microL) quantities of mucus, while c.b.f. and t.t.r. are determined using the frog palate model.

Performance of dwarf and normal laying hens as influenced by protein level and cage density.

The performance of dwarf and normal White Leghorn laying hens with similar genotypes other than for the dwarf gene (dw) was observed over thirteen 28-day periods in a factorial arrangement involving four protein levels (20.0, 18.1, 16.4 or 14.3% protein plus supplemental methionine) and two densities (two or three birds per standard 10 X 16 in cage). The normal birds had significantly higher overall hen-day egg production, egg weight, and body weight, but the dwarf birds excelled for feed efficiency and adult viability. Significant genotype X ration interactions were observed for egg lay, egg weight, body weight, and feed efficiency, primarily due to the relatively poor performance of the dwarf birds on the 14% protein. A significant genotype X density interaction resulted for feed efficiency, due to a higher efficiency in 3-bird than in 2-bird cages for dwarf birds, while the reverse was true for normal birds. These results suggest that the potential of the dwarf gene for increasing the efficiency of commercial egg layers can be enhanced by further study as to optimal nutrition and management regimens.

Sex-linked and maternal effects in the Eberhart-Gardner general genetics model.

To deal with differences between reciprocal crosses found in many animal breeding experiments, an extension of the general model for genetic effects, given by Eberhart and Gardner (1966, Biometrics 22, 864-881), is presented. In this extension, reciprocal differences between crosses are defined in terms of several maternal and sex-linked parameters, the latter being expressed as functions of gene values and their frequencies. Models are given for several kinds of crosses. Experimental setups or designs of increasing complexity are presented for the estimation of some of the parameters in the models, particularly the sex-linked and maternal ones, as well as the interpopulation heterotic additive-by-additive epistasis. For prediction purposes, different analyses are suggested for the model with highly correlated variables. If the genetic architecture of a trait in different populations is to be compared, the analysis of variance will provide enough degrees of freedom to allow the investigation of the importance of each kind of genetic effect. An example which uses all possible two-way crosses and a partial set of three-way crosses applied to two quantitative traits in the flour beetle, Tribolium castaneum, is included merely as a guide for computations. Population means given by Eberhart and Gardner are extended to incorporate the inbreeding coefficient.