Cancer immunotherapy.

Important contributions that stimulated studies in cancer immunotherapy included: (1) the discovery of tumour-associated antigens; (2) the observation that infection with bacille Calmette-Guérin (BCG) in animals was protective against tumour challenge; and (3) the observation that immunodepression due either to malignant disease or to treatment of the disease, was, in some instances, related to prognosis. Immunotherapy trials with microbial agents have involved attempts to obtain a local effect by injecting the agent into the tumour or into the region of the tumour and to obtain a "systemic" effect distant from the site of injection. Trials with active specific immunotherapy involving tumour cells or tumour cell extracts have frequently involved the combination of these specific agents with a nonspecific adjuvant such as BCG. Recent studies with thymosin and levamisole in patients with lung cancer and other types of malignant disease have shown prolonged survival in the groups receiving immunotherapy.     

Microtubules and cyclic amp in human leukocytes: on the order of things

We have shown previously that the β-adrenergic agonist isoproterenol (2μM) and the phosphodiesterase inhibitor isobutylmethylxanthine (1 mM) produce a much greater increase in cyclic AMP in human leukocytes that have been pretreated with colchicine (or with other agents that affect microtubule assembly) than in control leukocytes. The effects of colchicines were both time- and dose-dependant. These and other data suggested that the generation of cyclic AMP is normally restricted by an intact system of cytoplasmic microtubules. If so, then the same time and dose dependencies might apply to other colchicines-induced changes in leukocyte function. We have now assayed the distribution of concanavalin A (Con A)-receptor complexes on the leukocyte membrane, taking into account that leukocytes competent to assemble microtubules show a uniform distribution of surface- bound Con A whereas microtubule-deficient cells accumulate Con A in surface caps. We have found that the effect of colchicine on capping is also both time- and dose dependent, and that the dose-response relationships conform to those required to increase cyclic AMP levels. These findings provide further evidence that both colchicine-induced Con-A capping and colchicine- induced cyclic AMP generation depend upon the relaxation of constraints normally imposed by cytoplasmic microtubules upon the plasma membrane, which limit, respectively, lateral mobility of the lectin-receptor complexes, and expression of hormone-sensitive adenylate cyclase. Moreover, colchicine-induced Con-A cap formation is not affected even by very large changes in leukocyte cyclic AMP levels. Thus, elevated cyclic AMP levels do not appear to promote the dissolution of microtubules; rather, the dissolution of microtubules permits the generation of increased amounts of cyclic AMP.     

A procedure for total protein determination with special application to allergenic extract standardization

A method for total protein determination of allergenic extracts has been developed and evaluated. Samples were hydrolyzed with 5 M NaOH followed by colorimetric determination with ninhydrin of the released amino acids using bovine serum albumin as the standard. The entire procedure was carried out in disposable plastic tubes. Substances (glycerol, phenol and mannitol) commonly present in allergenic extracts manufactured for human use did not affect the assay results. Analyses of four different pollen extracts by the method gave good agreement with amino acid analyses. Other methods of analysis (total N, protein N unit assay, Lowry) gave more variable results compared with amino acid analysis. Analysis of the total protein content of 53 different lots of allergenic extracts gave narrow ranges of values for each species. Standardized mite extracts analyzed for total protein by US FDA-licensed manufacturers using this assay showed a good correlation of biological activity with total protein.     

Locally released retinoic acid repatterns the first branchial arch cartilages in vivo

The fates of cranial neural crest cells are unique compared to trunk neural crest. Cranial neural crest cells form bone and cartilage and ultimately these cells make up the entire facial skeleton. Previous studies had established that exogenous retinoic acid has effects on neurogenic derivatives of cranial neural crest cells and on segmentation of the hindbrain. In the present study we investigated the role of retinoic acid on the skeletal derivatives of migrating cranial neural crest cells. We wanted to test whether low doses of locally applied retinoic acid could respecify the neural crest-derived, skeletal components of the beak in a reproducible manner. Retinoic acid-soaked beads were positioned at the presumptive mid-hindbrain junction in stage 9 chicken embryos. Two ectopic cartilage elements were induced, the first a sheet of cartilage ventral and lateral to the quadrate and the second an accessory cartilage rod branching from Meckel's cartilage. The accessory rod resembled a retroarticular process that had formed within the first branchial arch domain. In addition the quadrate was often displaced laterally and fused to the retroarticular process. The next day following bead implantation, expression domains of Hoxa2 and Hoxb1 were shifted in an anterior direction up to the mesencephalon and Msx-2 was slightly down-regulated in the hindbrain. Despite down-regulation in neural crest cells, the onset of Msx-2 expression in the facial prominences at stage 18-20 was normal. This correlates with normal distal beak morphology. Focal labeling of neural crest with DiI showed that instead of migrating in a neat group toward the second branchial arch, a cohort of labeled cells from r4 spread anteriorly toward the proximal first arch region. AP-2 expression data confirmed the uninterrupted presence of AP-2-expressing cells from the anterior mesencephalon to r4. The morphological changes can be explained by mismigration of r4 neural crest into the first arch, but at the same time maintenance of their identity. Up-regulation of the Hoxa2 gene in the first branchial arch may have encouraged r4 cells to move in the anterior direction. This combination of events leads to the first branchial arch assuming some of the characteristics of the second branchial arch.     

Functional genomics uncovers three glucosyltransferases involved in the synthesis of the major sweet glucosides of Stevia rebaudiana

Stevia rebaudiana leaves accumulate a mixture of at least eight different steviol glycosides. The pattern of glycosylation heavily influences the taste perception of these intensely sweet compounds. The majority of the glycosides are formed by four glucosylation reactions that start with steviol and end with rebaudioside A. The steps involve the addition of glucose to the C-13 hydroxyl of steviol, the transfer of glucose to the C-2' and C-3' of the 13-O-glucose and the addition of glucose to the hydroxyl of the C-4 carboxyl group. We used our collection of ESTs, an UDP-glucosyltransferase (UGT)-specific electronic probe and key word searches to identify candidate genes resident in our collection. Fifty-four expressed sequence tags (ESTs) belonging to 17 clusters were found using this procedure. We isolated full length cDNAs for 12 of the UGTs, cloned them into an expression vector, and produced recombinant enzymes in Escherichia coli. An in vitro glucosyltransferase activity enzyme assay was conducted using quercetin, kaempferol, steviol, steviolmonoside, steviolbioside, and stevioside as sugar acceptors, and (14)C-UDP-glucose as the donor. Thin layer chromatography was used to separate the products and three of the recombinant enzymes produced labelled products that co-migrated with known standards. HPLC and LC-ES/MS were then used to further define those reaction products. We determined that steviol UGTs behave in a regioselective manner and propose a modified pathway for the synthesis of rebaudioside A from steviol.     

Human immunodeficiency virus type 1 clade B superinfection: evidence for differential immune containment of distinct clade B strains

Sequential infection with different strains of human immunodeficiency virus type 1 (HIV-1) is a rarely identified phenomenon with important implications for immunopathogenesis and vaccine development. Here, we identify an individual whose good initial control of viremia was lost in association with reduced containment of a superinfecting strain. Subject 2030 presented with acute symptoms of HIV-1 infection with high viremia and an incomplete seroconversion as shown by Western blotting. A low set point of viremia (approximately 1,000 HIV-1 copies/ml) was initially established without drug therapy, but a new higher set point (approximately 40,000 HIV-1 copies/ml) manifested about 5 months after infection. Drug susceptibility testing demonstrated a multidrug-resistant virus initially but a fully sensitive virus after 5 months, and an analysis of pol genotypes showed that these were two phylogenetically distinct strains of virus (strains A and B). Replication capacity assays suggested that the outgrowth of strain B was not due to higher fitness conferred by pol, and env sequences indicated that the two strains had the same R5 coreceptor phenotype. Delineation of CD8+-T-lymphocyte responses against HIV-1 showed a striking pattern of decay of the initial cellular immune responses after superinfection, followed by some adaptation of targeting to new epitopes. An examination of targeted sequences suggested that differences in the recognized epitopes contributed to the poor immune containment of strain B. In conclusion, the rapid overgrowth of a superinfecting strain of HIV-1 of the same subtype raises major concerns for effective vaccine development.     

Semen-specific genetic characteristics of human immunodeficiency virus type 1 env

Human immunodeficiency virus type 1 (HIV-1) in the male genital tract may comprise virus produced locally in addition to virus transported from the circulation. Virus produced in the male genital tract may be genetically distinct, due to tissue-specific cellular characteristics and immunological pressures. HIV-1 env sequences derived from paired blood and semen samples from the Los Alamos HIV Sequence Database were analyzed to ascertain a male genital tract-specific viral signature. Machine learning algorithms could predict seminal tropism based on env sequences with accuracies exceeding 90%, suggesting that a strong genetic signature does exist for virus replicating in the male genital tract. Additionally, semen-derived viral populations exhibited constrained diversity (P < 0.05), decreased levels of positive selection (P < 0.025), decreased CXCR4 coreceptor utilization, and altered glycosylation patterns. Our analysis suggests that the male genital tract represents a distinct selective environment that contributes to the apparent genetic bottlenecks associated with the sexual transmission of HIV-1.     

Characterization of human immunodeficiency virus type 1 (HIV-1) envelope variation and neutralizing antibody responses during transmission of HIV-1 subtype B

We analyzed neutralization sensitivity and genetic variation of transmitted subtype B human immunodeficiency virus type 1 (HIV-1) in eight recently infected men who have sex with men and the virus from the six subjects who infected them. In contrast to reports of heterosexual transmission of subtype C HIV-1, in which the transmitted virus appears to be more neutralization sensitive, we demonstrate that in our study population, relatively few phenotypic changes in neutralization sensitivity or genotypic changes in envelope occurred during transmission of subtype B HIV-1. We suggest that limited genetic variation within the infecting host reduces the likelihood of selective transmission of neutralization-sensitive HIV.