Tumor immunotherapy by epicutaneous immunization requires langerhans cells.

A role for Langerhans cells (LC) in the induction of immune responses in the skin has yet to be conclusively demonstrated. We used skin immunization with OVA protein to induce immune responses against OVA-expressing melanoma cells. Mice injected with OVA-specific CD8(+) T cells and immunized with OVA onto barrier-disrupted skin had increased numbers of CD8(+) T cells in the blood that produced IFN-gamma and killed target cells. These mice generated accelerated cytotoxic responses after secondary immunization with OVA. Prophylactic or therapeutic immunization with OVA onto barrier-disrupted skin inhibited the growth of B16.OVA tumors. LC played a critical role in the immunization process because depletion of LC at the time of skin immunization dramatically reduced the tumor-protective effect. The topically applied Ag was presented by skin-derived LC in draining lymph nodes to CD8(+) T cells. Thus, targeting of tumor Ags to LC in vivo is an effective strategy for tumor immunotherapy.

Recombinant cathepsin S propeptide attenuates cell invasion by inhibition of cathepsin L-like proteases in tumor microenvironment

Human cathepsin L along with cathepsin S, K, and V are collectively known as cathepsin L-like proteases due to their high homology. The overexpression and aberrant activity of each of these proteases has been implicated in tumorigenesis. These proteases contain propeptide domains that can potently inhibit both their cognate protease and other proteases within the cathepsin L-like subfamily. In this investigation, we have produced the cathepsin S propeptide recombinantly and have shown that it is a potent inhibitor of the peptidolytic, elastinolytic, and gelatinolytic activities of the cathepsin L-like proteases. In addition, we show that this peptide is capable of significantly attenuating tumor cell invasion in a panel of human cancer cell lines. Furthermore, fusion of an IgG Fc-domain to the COOH terminus of the propeptide resulted in a chimeric protein with significantly enhanced ability to block tumor cell invasion. This Fc fusion protein exhibited enhanced stability in cell-based assays in comparison with the unmodified propeptide species. This approach for the combined inhibition of the cathepsin L-like proteases may prove useful for the further study in cancer and other conditions where their aberrant activity has been implicated. Furthermore, this strategy for simultaneous inhibition of multiple cysteine cathepsins may represent the basis for novel therapeutics to attenuate tumorigenesis.

An optimized method for the generation of AFLP markers in a stingless bee (Melipona quadrifasciata) reveals a high degree of intracolonial genetic polymorphism

This study describes an optimized protocol for the generation of Amplified Fragment Length Polymorphism (AFLP) markers in a stingless bee. Essential modifications to standard protocols are a restriction enzyme digestion (EcoRI and Tru1I) in a two-step procedure, combined with a touchdown program in the selective PCR amplification step and product labelling by incorporation of alpha[P-33]dATP. In an analysis of 75 workers collected from three colonies of Melipona quadrifasciata we obtained 719 markers. Analysis of genetic variability revealed that on average 32% of the markers were polymorphic within a colony. Compared to the overall percentage of polymorphism (44% of the markers detected in our bee samples), the observed rates of within-colony polymorphism are remarkably high, considering that the workers of each colony were all of spring of a singly mated queen.

Studying genetic relationships among coconut varieties/populations using microsatellite markers

The extent of genetic diversity and the genetic relationships among 94 coconut varieties/populations (51 Talls and 43 Dwarfs) representing the entire geographic range of cultivation/distribution of the coconut was assessed using 12 pairs of coconut microsatellite primers. A high level of genetic diversity was observed in the collection with the mean gene diversity of 0.647+/-0.139, with that of the mean gene diversity of Talls 0.703+/-0.125 and 0.374+/-0.204 of Dwarfs. A phenetic tree based on DAD genetic distances clustered all the 94 varieties/ populations into two main groups, with one group composed of all the Talls from southeast Asia, the Pacific, west coast of Panama, and all Dwarfs and the other of all Talls from south Asia, Africa, and the Indian Ocean coast of Thailand. The allele distribution of Dwarfs highlighted a unique position of Dwarf palms from the Philippines exhibiting as much variation as that in the Tall group. The grouping of all Dwarfs representing the entire geographic distribution of the crop with Talls from southeast Asia and the Pacific and the allele distribution between the Tall and Dwarf suggest that the Dwarfs originated from the Tall forms and that too from the Talls of southeast Asia and the Pacific. Talls from Pacific Islands recorded the highest level of genetic diversity (0.6+/-0.26) with the highest number of alleles (51) among all the regions.

Levels and distribution of genetic diversity of coconut (Cocos nucifera L., var. Typica form typica) from Sri Lanka assessed by microsatellite markers

The coconut variety Typica, form typica, commonly known as Sri Lanka tall coconuts is the most widely exploited and grown variety in Sri Lanka. Under the coconut bio-diversity conservation programme, several Typica populations have been collected by island-wide surveys and planted ex situ. Thirty-three coconut populations were subjected to microsatellite assay with eight coconut-specific microsatellite primer pairs in order to study the levels and distribution of genetic variation of the collected materials for formulating future collection strategies and selecting parents for the breeding programme. A total of 56 alleles were detected ranging from 3 to 10 alleles per primer pair with an average of 7 alleles per locus. Overall a very high level of genetic diversity was detected (0.999) for all the populations studied ranging from 0.526 for population Debarayaya to 0.683 for population Dickwella. Only four introduced coconut populations, i.e. Clovis, Margeret, Dickwella, Mirishena and an embryo-cultured population were clearly separated from the resulting dendrogram. A very high level of within population variation (99%) accounted for native populations suggests a common history and a restricted genetic base for native Sri Lankan tall coconuts. Categorization of alleles into different classes according to their frequency and distribution confirmed the results of the dedrogram and concluded the adequacy of single large collection from the entire target area to represent the total genetic diversity in Sri Lanka. This study discusses useful information regarding conservation and breeding of coconut in Sri Lanka.

Genetic variation of the razor clam Ensis siliqua (Jeffreys, 1875) along the European coast based on random amplified polymorphic DNA markers

Ensis siliqua is regarded as an increasingly valuable fishery resource with potential for commercial aquaculture in many European countries. The genetic variation of this razor clam was analysed by randomly amplified polymorphic DNA (RAPD) in six populations from Spain, Portugal and Ireland. Out of the 40 primers tested, five were chosen to assess genetic variation. A total of 61 RAPD loci were developed ranging in size from 400 to 2000 bp. The percentages of polymorphic loci, the allele effective number and the genetic diversity were comparable among populations, and demonstrated a high level of genetic variability. The values of Nei's genetic distance were small among the Spanish and Portuguese populations (0.051-0.065), and high between these and the Irish populations. Cluster and principal coordinate analyses supported these findings. A mantel test performed between geographic and genetic distance matrices showed a significant correlation (r=0.84, P