Some generalized stochastic compartment models for digesta flow

Digesta flow models have been based on linear compartment theory that assumes exponential retention times, and on a generalized theory that incorporates nonexponential (Erlang) retention times (Matis, 1987, Journal of Theoretical Biology 124, 371-376). This paper develops a new family of passage models for heterogeneous digesta by mixing the previous models with assumed parametric, usually gamma, mixing distributions. The utility of the resulting models is demonstrated with experimental data on two treatments, namely a chopped and a ground straw, given to each of four cows. Treatment differences are apparent in the preferred model form and in the means of the estimated mean residence times. The models are relatively easy to fit to data using standard estimation procedures, and they should have broad application to other compartment modeling problems with "heterogeneous particles."     

Development of a sensitive quantitative focal assay for human immunodeficiency virus infectivity.

Accurate and sensitive quantitation of infectious human immunodeficiency virus (HIV) has been difficult to achieve. In this report, a quantitative focal immunoassay (FIA) for HIV was developed using human HeLa cells rendered susceptible to HIV infection by introduction of the CD4 gene via a retrovirus vector. Infected cells were identified by using human anti-HIV antibodies or mouse monoclonal antibodies specific for HIV together with secondary fluorescein- or peroxidase-conjugated antibody specific for mouse or human immunoglobulins. The assay identified cells infected with either wild-type or culture-adapted HIV isolates and was capable of detecting 1 positive cell in 10(6) cells. The FIA was also effective at detecting cell-free HIV, and in contrast to assays using A3.01, CEM, and other human leukemia cells, the FIA detected most wild-type HIV isolates. HIV neutralization could be determined by using the FIA, and two monoclonal antibodies reactive with HIV gp120 were found to neutralize only the LAV-IIIB strain of HIV. These monoclonal antibodies, as well as antibodies in serum samples from patients with acquired immune deficiency syndrome, were able to inhibit the spread of HIV infection in human lymphocyte suspension cultures but not in CD4-positive HeLa cells growing attached to plastic dishes.     

Failure of human immunodeficiency virus entry and infection in CD4-positive human brain and skin cells.

CD4 molecules on human cells function as a major receptor for human immunodeficiency virus (HIV); however, certain CD4-negative cell types may also be susceptible to infection. Therefore, we attempted to quantitate the relationship between HIV infection and CD4 expression on human cell lines before and after introduction of the CD4 gene by using a retrovirus vector. Prior to introduction of the CD4 expression vector, low levels of HIV infection were detected by a sensitive focal immunoassay on all three cell types studied. With several HIV strains in clones of human cervical carcinoma (HeLa) cells expressing different levels of CD4, HIV titer increased with increasing CD4 expression. In contrast, in squamous cell carcinoma cells (SCL1) and astroglial cells (U87MG), even high levels of CD4 expression failed to augment HIV infection. The CD4 protein expressed in these two cell lines had the expected molecular weight and was capable of binding HIV virions. However, in contrast to CD4-positive HeLa cells, CD4-positive U87MG and SCL1 cells were unable to form syncytia when cultured with cells expressing HIV envelope protein. Thus, the inability of HIV to infect these cells appeared to be due to lack of fusion between HIV virion envelope proteins and CD4-positive cell membranes. This block is infectivity was overcome when cells were infected with HIV which was pseudotyped with the envelope protein of amphotropic murine leukemia virus. Thus, in addition to CD4, other cell surface molecules appear to be required for successful HIV entry into and infection of these two human cell lines.     

Sequence variation in the outer-surface-protein genes of Borrelia burgdorferi

Borrelia burgdorferi is a spirochete pathogen transmitted among warm- blooded hosts by ixodid ticks. Frequency-dependent selection for variant outer-surface proteins might be expected to arise in this species, since rare variants are more likely to avoid immune surveillance in previously infected hosts. We sequenced the OspA and OspB genes of nine North American strains and compared them with nine strains previously described. For each gene, the mean number of synonymous substitutions per synonymous site and the mean number of nonsynonymous substitutions per nonsynonymous site show only a twofold excess of silent mutations. Synonymous rates vary widely along the OspB protein. Some regions show a significant excess of silent substitutions, while divergence in other regions is constrained by biased base composition or selection. The presence, in antigenically important regions of the protein, of significant variation among strains, as well as evidence for recombination among strains, should be considered in attempts to develop vaccines against this disease.      

Macrophage-tropic human immunodeficiency virus isolates from different patients exhibit unusual V3 envelope sequence homogeneity in comparison with T-cell-tropic isolates: definition of critical amino acids involved in cell tropism.

Previous experiments indicate that the V3 hypervariable region of the human immunodeficiency virus (HIV) envelope protein influences cell tropism of infection; however, so far no consistent V3 sequence can account for macrophage or T-cell tropism. In these experiments, we studied infectious recombinant HIV clones constructed by using V3 region sequences of HIV isolates from 16 patients to search for sequences associated with cell tropism. Remarkable homology was seen among V3 sequences from macrophage-tropic clones from different patients, and a consensus V3 region sequence for patient-derived macrophage-tropic viruses was identified. In contrast, V3 sequences of T-cell-tropic clones from different patients were highly heterogeneous, and the results suggested that sequence diversity leading to T-cell tropism might be generated independently in each patient. Site-specific mutations identified amino acids at several positions on each side of the GPGR motif at the tip of the V3 loop as important determinants of tropism for T cells and macrophages. However, a wide variety of mutant V3 sequences induced macrophage tropism, as detected in vitro. Therefore, the homogeneity of macrophage-tropic patient isolates appeared to be the result of selection based on a biological advantage in vivo.     

An approach to the residence time distribution for stochastic multi-compartment models

Stochastic compartmental models are widely used in modeling processes such as drug kinetics in biological systems. This paper considers the distribution of the residence times for stochastic multi-compartment models, especially systems with non-exponential lifetime distributions. The paper first derives the moment generating function of the bivariate residence time distribution for the two-compartment model with general lifetimes and approximates the density of the residence time using the saddlepoint approximation. Then, it extends the distributional approach to the residence time for multi-compartment semi-Markov models combining the cofactor rule for a single destination and the analytic approach to the two-compartment model. This approach provides a complete specification of the residence time distribution based on the moment generating function and thus facilitates an easier calculation of high-order moments than the approach using the coefficient matrix. Applications to drug kinetics demonstrate the simplicity and usefulness of this approach.     

Low dose vaccination with attenuated Francisella tularensis strain SchuS4 mutants protects against tularemia independent of the route of vaccination

Tularemia, caused by the gram-negative bacterium Francisella tularensis, is a severe, sometimes fatal disease. Interest in tularemia has increased over the last decade due to its history as a biological weapon. In particular, development of novel vaccines directed at protecting against pneumonic tularemia has been an important goal. Previous work has demonstrated that, when delivered at very high inoculums, administration of live, highly attenuated strains of virulent F. tularensis can protect against tularemia. However, lower vaccinating inoculums did not offer similar immunity. One concern of using live vaccines is that the host may develop mild tularemia in response to infection and use of high inoculums may contribute to this issue. Thus, generation of a live vaccine that can efficiently protect against tularemia when delivered in low numbers, e.g. <100 organisms, may address this concern. Herein we describe the ability of three defined, attenuated mutants of F. tularensis SchuS4, deleted for FTT0369c, FTT1676, or FTT0369c and FTT1676, respectively, to engender protective immunity against tularemia when delivered at concentrations of approximately 50 or fewer bacteria. Attenuated strains for use as vaccines were selected by their inability to efficiently replicate in macrophages in vitro and impaired replication and dissemination in vivo. Although all strains were defective for replication in vitro within macrophages, protective efficacy of each attenuated mutant was correlated with their ability to modestly replicate and disseminate in the host. Finally, we demonstrate the parenteral vaccination with these strains offered superior protection against pneumonic tularemia than intranasal vaccination. Together our data provides proof of principle that low dose attenuated vaccines may be a viable goal in development of novel vaccines directed against tularemia.     

Thf complete amino acid sequence of C-I (apoLp-Ser), an apolipoprotein from human very low density lipoproteins.

C-I was prepared from very low density lipoproteins of patients with familial type V hyperliporproteinemia. Peptides from tryptic digests of unmodified and succinylated C-I, chymotryptic peptides, and the products of cayanogen bromide cleavage were isolated and characterized. Sequence analysis of tryptic peptides was performed by the dansyl (5-dimethylaminonaphthalene-1-sulfonyl) technique and hydrolytic regeneration of the amino acid residues from the phenylthiocarbamyl derivatives. Alignment of the tryptic fragments within the cyanogen bromide and succinyl-tryptic peptides was confirmed by the overlap chymotryptic peptides. The complete amino acid sequence of C-I, 57 residues in length, does not reveal any obvious basis for its lipophilic properties.