Recombinant DNA technology for melanoma immunotherapy: anti-Id DNA vaccines targeting high molecular weight melanoma-associated antigen.

Anti-idiotypic MK2-23 monoclonal antibody (anti-Id MK2-23 mAb), which mimics the high molecular weight melanoma-associated antigen (HMW-MAA), has been used to implement active immunotherapy against melanoma. However, due to safety and standardization issues, this approach never entered extensive clinical trials. In the present study, we investigated the usage of DNA vaccines as an alternative to MK2-23 mAb immunization. MK2-23 DNA plasmids coding for single chain (scFv) MK2-23 antibody were constructed via the insertion of variable heavy (V H) and light (V L) chains of MK2-23 into the pVAC-1mcs plasmids. Two alternative MK2-23 plasmids format V H/V L, and V L/V H were assembled. We demonstrate that both polypeptides expressed by scFv plasmids in vitro retained the ability to mimic HMW-MAA antigen, and to elicit specific anti-HMW-MAA humoral and cellular immunoresponses in immunized mice. Notably, MK2-23 scFv DNA vaccines impaired the onset and growth of transplantable B16 melanoma cells not engineered to express HMW-MAA. This pilot study suggests that optimized MK2-23 scFv DNA vaccines could potentially provide a safer and cost-effective alternative to anti-Id antibody immunization, for melanoma immunotherapy.

Co-expression of Flt-3 ligand gene ablates tumor immunity elicited by HER-2/neu DNA vaccine in transgenic mice.

Fms-like tyrosine-kinase 3 ligand (Flt-3L), is a powerful hematopoyetic growth factor, known to modulate the immune response against delivered antigens by acting either as an adjuvant or tolerogenic stimulus. In this study we evaluated the use of murine Flt-3 ligand plasmid (pFl) in combination with a DNA vaccine encoding rat-p185 oncoprotein extra cellular domain (pECD) in the prevention of mammary carcinogenesis in rat-neu HER-2 mutated (neuT) transgenic mice. We demonstrate that intramuscular (i.m.) co-immunization of pFl inhibits the production of anti-HER-2 antibody elicited by pECD vaccine, resulting in the development of spontaneous carcinomas in all co-immunized mice. The inhibitory effect on antibody production by mFlt3 gene appeared to be: dose-dependent, linked to the injection site and timing, and transient in nature. Additionally, we show that co-administration of pFI and pECD plasmids was unable to trigger cytotoxic T-cell immune response in neuT mice. On the other hand, we found that the combination of pFl with pECD had no impact on the ability of pECD to reject HER-2+ transplantable tumors in parental mice. In summary our results demonstrate that, depending on tumor model, co-administration of pFl gene can produce untoward effects to immune response, and thus its application as a vaccine adjuvant should be carefully evaluated.

Tumor endothelial marker 8 enhances tumor immunity in conjunction with immnization against differentiation Ag.

BACKGROUND: We have previously shown that xenogenic DNA vaccines encoding rat neu and melanosomal differentiation Ag induce tumor immunity. Others have developed vaccines targeting tumor neovasculature. Tumor endothelial marker 8 (TEM8) is expressed in the neovasculature of human tumors, and in the mouse melanoma B16, but its expression is limited in normal adult tissues. We describe a DNA vaccine combining xenogeneic tumor Ag and TEM8. METHODS: In-situ hybridization was used to detect TEM8 RNA in mouse tumors. Mice transgenic for the rat neu proto-oncogene were immunized with DNA vaccines encoding TEM8 and the extracellular domain of rat neu and challenged with the 233-VSGA1 breast cancer cell line. In parallel experiments, C57BL/6 mice were immunized with TEM8 and human tyrosinase-related protein 1 (hTYRP1/hgp75) and challenged with B16F10 melanoma. RESULTS: TEM8 was expressed in the stroma of transplantable mouse breast and melanoma tumors. In both model systems, TEM8 DNA had no activity as a single agent but significantly enhanced the anit-tumor immunity of neu and hTYRP1/hgp75 DNA vaccines when given in concert. The observed synergy was dependent upon CD8+ T cells, as depletion of this cell population just prior to tumor challenge obviated the effect of the TEM8 vaccine in both tumor models. DISCUSSION: A local immune responses to TEM8 may increase inflammation or tumor necrosis within the tumor, resulting in improved Ag presentation of HER2/neu and hTYRP1/hgp75. Alternatively, TEM8 expression in host APC may act more as an adjuvant than an immunologic target.

Tumor endothelial marker 8 enhances tumor immunity in conjunction with immunization against differentiation Ag.

BACKGROUND: We have previously shown that xenogeneic DNA vaccines encoding rat neu and melanosomal differentiation Ag induce tumor immunity. Others have developed vaccines targeting tumor neovasculature. Tumor endothelial marker 8 (TEM8) is expressed in the neovasculature of human tumors, and in the mouse melanoma B16, but its expression is limited in normal adult tissues. We describe a DNA vaccine combining xenogeneic tumor Ag and TEM8. METHODS: In-situ hybridization was used to detect TEM8 RNA in mouse tumors. Mice transgenic for the rat neu proto-oncogene were immunized with DNA vaccines encoding TEM8 and the extracellular domain of rat neu and challenged with the 233-VSGA1 breast cancer cell line. In parallel experiments, C57BL/6 mice were immunized with TEM8 and human tyrosinase-related protein 1 (hTYRP1/hgp75) and challenged with B16F10 melanoma. RESULTS: TEM8 was expressed in the stroma of transplantable mouse breast and melanoma tumors. In both model systems, TEM8 DNA had no activity as a single agent but significantly enhanced the anti-tumor immunity of neu and hTYRP1/hgp75 DNA vaccines when given in concert. The observed synergy was dependent upon CD8+ T cells, as depletion of this cell population just prior to tumor challenge obviated the effect of the TEM8 vaccine in both tumor models. DISCUSSION: A local immune response to TEM8 may increase inflammation or tumor necrosis within the tumor, resulting in improved Ag presentation of HER2/neu and hTYRP1/hgp75. Alternatively, TEM8 expression in host APC may alter T-cell interactions or homing. In this way, TEM8 may act more as an adjuvant than an immunologic target.

Adaptabilidad de granos de híbridos de maíz super dulce sh2 al procesamiento industrial. II Enlatado / Super sweet corn hybrid sh2 adaptability for industrial canning process

The super sweet corns Krispy king, Victor and 324 (sh2 hybrids) were evaluated to determine their adaptabilities to the industrial canning process as whole kernels. All these hybrids and Bonanza (control) were sown in San Joaquín (Carabobo, Venezuela), harvested and canned. After 110 days storage at room temperature they were analyzed to be compared physically, chemically and sensorially with Bonanza hybrid. Results did not show significant differences among most of the physical characteristics, except for percentage of broken kernels which was higher in 324 hybrid. Chemical parameters showed significant differences (P < 0.05) comparing each super sweet hybrid with Bonanza. The super sweet hybrids presented a higher sugar content and soluble solid of the brine than Bonanza, also a lower pH. The super sweet whole kernel presented a lower soluble solids content than Bonanza but they were not significant (Krispy king and 324). Appearance, odor and overall quality were the same for super sweet hybrids and Bonanza (su). Color, flavor and sweetness were better for 324 than all the other hybrids. Super sweet hybrids presented a very good adaptation to the canning process, having as an advantage that doesn't require sugar addition in the brine and a very good texture (firm and crispy).

[Suitability study for super sweet corn on the ears hybrids under refrigerated conditions evaluating chemicals microbiological and sensorial characteristics]. / Estudio de la estabilidad de las características químicas, microbiológicas y sensoriales de mazorcas refrigeradas de híbridos de maíz super dulce.

Krispy King, Víctor and 324, super sweet hybrids (sh2) were cultivated in San Joaquín, estado Carabobo, Venezuela. The scheme was stablished to produce refrigerated fresh ears to be commercialized. The chemistry, microbiology and sensorial characteristics were evaluated at 0; 7; 14; 21 and 28 days of storage. One hundred ears of each hybrid were picked at the ripe fresh stage and packed in polystyrene trays covered with polyethylene. The storage temperature was 4 degrees C +/- 1 degree C. The scheme used was well adapted, allowing a good stability of the ears until 28 days of storing. The plastic cover avoid the lost of humidity. The soluble solids, total sugars and pH went down during the storage. The acidity and the microorganisms increased as expected. The sensorial variables kept the same for Krispy king and Víctor, while the hybrid 324 shown the lowest humidity content, the highest count of microorganisms and the poorest sensorial quality.

Prognostic relevance of altered Fas (CD95)-system in human breast cancer.

The Fas ligand (FasL) and its receptor Fas (APO-1 or CD95) are members, respectively, of the tumor necrosis factor family that, upon interaction with each other, play a key role in the initiation of one apoptotic pathway. Faulty regulation of the Fas system has been described in a variety of human tumors with different histogenetic origin. Here, we describe the expression and distribution of Fas receptor and ligand pair antigens in surgical samples collected from a cohort of 186 patients bearing breast neoplasms (45 benign and 141 malignant lesions). Immunoperoxidase staining of formalin-fixed tissues showed that 91.1% of benign lesions expressed Fas, which was present in only 56.7% of malignant tumors. On the other hand, FasL was found positive in 22.2% of benign neoplasms and up-regulated in in situ as well as invasive carcinomas (53.9%). Moreover, in malignant tumors, the expression of receptor and ligand antigens appeared to be inversely related. When these findings were correlated with pathological parameters of prognostic relevance, a significant association was observed between FasL and the presence of metastatic lymph nodes and larger tumor size while Fas expression correlated to node-negative status and smaller tumor size. Patients with Fas positive tumors exhibited longer disease-free survival than those with Fas-negative carcinoma while FasL did not influence patient outcome. These relationships indicate that benign and malignant mammary lesions are characterized by differential cellular expression of Fas and FasL and suggest that a neoplastic Fas negative/FasL positive phenotype may be linked to breast cancer progression.

Long-term immunologic effects of thymectomy in patients with myasthenia gravis.

BACKGROUND: Thymectomy (Tx) is a common therapeutic option to treat myasthenia gravis (MG), but its effects on the immune system are still obscure in humans. OBJECTIVE: We sought to evaluate long-term immunologic effects of therapeutic Tx in patients with MG. METHODS: T- and B-cell subsets and T-cell repertoire were analyzed in 35 patients with MG, 16 with previous Tx (at least 8 years before), 6 with recent (<1 year) Tx, and 13 without Tx, as well as in 32 healthy subjects used as normal control subjects. Serum immunoglobulins and a variety of autoantibodies were also measured. A subsequent 3-year clinical follow-up was performed to verify the possible appearance of systemic autoimmune diseases. RESULTS: The long-term thymectomized (Txd) patients had mild T-cell lymphopenia and an expansion of some Vbeta families among circulating CD4+ and CD8+ T cells. They displayed a normal number of total B and CD5+ B-circulating lymphocytes, but they also displayed a polyclonal increase in serum IgM and IgG associated with the presence of high levels of a variety of organ- and nonorgan-specific autoantibodies, including anti-dsDNA and anticardiolipin, without clinical evidence of autoimmune disease. These serologic abnormalities were not detectable in both non-Txd and recently Txd patients. After 3 years, 2 long-term Txd patients had systemic lupus erythematosus and an undifferentiated connective tissue disease. CONCLUSIONS: The association between MG and laboratory findings of systemic autoimmune disease may be in part related to Tx rather than to MG. Tx may represent a risk for the development of systemic autoimmune disorders over years in patients with MG.

Binding of feline immunodeficiency viral gag-p24 polypeptide to nonimmune Igs.

Available data on the existence of lentivirus proteins with properties of unconventional Ag for B cells, have been so far restricted to human immunodeficiency virus (i.e. gp-120 of HIV-I). By using biotinylated-MAbs-anti-biotin IgG as readout system, we now report that gag-p24 antigen, either assembled in feline immunodeficiency virus (FIV) particles or expressed as recombinant polypeptide (rec.p24) may bind to nonimmune IgGs purified from mouse or cat sera. Moreover, FACS scanning experiments are consistent with the possibility that rec.p24 interacts with surface-Ig in a sub-population (5-6%) of rodent B cells. We hypothesize that gag-p24 peptide encoded regions may bind to unconventional Ig sites or, alternatively, that they may represent 'public' epitopes for natural polyreactive antibody.

Genetic immunization against neu/erbB2 transgenic breast cancer.

erbB2/neu, an overexpressed oncogene product, has been proposed as a human cancer vaccine target. In the present study, transgenic (rat neuNT oncogene) FVB/neu mice, developing metastasizable mammary carcinoma, were immunized with a plasmid DNA encoding are not tolerant to the self antigen and sequences. We report that transgenic tumour-bearing mice, like some breast cancer patients erbB2 + X, develop anti-neu autoimmune responses, which can be boosted and skewed to a Th1 phenotype by DNA immunization. Intramuscular injections of neuNT plasmid drastically reduced (or even prevented in a small number of treated mice) the outgrowth of mammary neoplasms as well as their metastatic penetrance. Furthermore, DNA immunization caused haemorrhagic necrosis of established cancer nests, leaving a greatly reduced portion of the tumour burden for the host to cope with. The antitumour activities we obtained, in this very challenging model for cancer immunotherapy, lay the foundation for DNA-based immunization to control erbB2/neu-overexpressing neoplasms.

Autoantibody to p185erbB2/neu oncoprotein by vaccination with xenogenic DNA.

The passive transfer of antibodies and vaccination procedures against p185, the erbB2/neu oncoprotein, are approaches being explored for treatment of human breast cancer. We now report the possibility of using the erbB2/neu gene as an immunogen. This study demonstrates that intramuscular or intradermal injections of rat neuNT full-length DNA into mice generate anti-p185 autoantibodies. Anti-p185 polyclonals were also shown to bind the homologous human receptor ErbB2 and to stain specimens of breast adenocarcinoma from both neu-transgenic mice and humans. Further, in vitro assays demonstrated that anti-p185 IgG (probably dependent on CD4+ Th1) were able to inhibit human SKBR3 tumour cell growth and to mediate their lysis by natural killer cells. The continuous presence of circulating neu autoantibodies in mice did not cause any discernible toxic effects on normal tissues expressing low levels of self-antigen, even after 1 year. The experiments reported here raise the possibility that boosting anti-ErbB2 immunity by DNA vaccination will not induce harmful autoimmunity in humans.

Immunorecognition of ring skeleton of taxanes by chicken egg yolk antibodies.

Anti-10 deacetylbaccatin III (DAB) antibodies (IgY) were elicited in hens immunized with a succinyl-DAB/BSA conjugate and extracted from egg yolk. As shown by indirect competitive inhibition enzyme immunoassay (CIEIA), the addition of free-DAB competitively inhibited the binding of affinity purified anti-DAB IgY to DAB/BSA solid phase conjugated antigen. The assay enabled the detection of DAB in concentrations as low as 7.5ng/ml (13.7 nM DAB), whereas anti-DAB IgY did not react with taxol even at a concentration a thousand times higher. The structural requirements of the diterpenoid nucleus for binding to IgY were considered on the basis of the levels of cross-reaction found with 10 authentic taxanes. The results indicate that anti-DAB IgY represents the first high affinity antibody produced capable of recognizing the ring skeleton of taxol precursors.

Functional characterization of T cells bearing the gamma/delta T-cell receptor in patients with primary Sjögren's syndrome.

High percentages of gamma/delta+ T cells in the peripheral blood of a subgroup of patients with primary Sjögren's syndrome (SS) were found. This allowed us to purify and analyze them without their being previously expanded in vitro, and to investigate, therefore, the role of these cells in the pathological immune response which characterizes such systemic autoimmune disorders. The results showed poor proliferation of patient gamma/delta+ T cells in response to anti-CD3, due not to macrophage-dependent suppression but to defective interleukin 2 (IL-2) synthesis. Despite the defective proliferation patient gamma/delta+ cells, unlike those of the normal controls, provided a helper effect in inducing B cells to secrete immunoglobulins (Ig), particularly when they were preincubated with IL-2. The relative increase in a gamma/delta+ T cell subset which, although it secretes low levels of IL-2, is able to provide help for B-cell Ig synthesis, suggests that this T-cell subpopulation may be functional in vivo and may be involved in the pathological immune response encountered in pSS.

Nucleic acids in mummified plant seeds: biochemistry and molecular genetics of pre-Columbian maize.

Nucleic acids fractions were isolated from pre-Columbian maize seeds and characterized using different approaches such as polyacrylamide gel electrophoresis, anti-DNA antibody binding, HPLC fractionation, molecular hybridization with cloned genes, and DNA amplification by the polymerase chain reaction. The nucleic acids were found to be very depolymerized (less than or equal to 140 base pairs in length) and composed mainly of ribosomal RNA. Despite the very low amount and degree of polymerization of seed DNA, specific maize nuclear Mu1, Mu4, Mu8 and, possibly, Mu5 element components could be detected, thanks to the use of amplification systems as short as 90 bp. The results suggest that evaluation of the relative proportions of Mu-type element components and, possibly, other maize genomic components in single mummified kernels, may offer a new key to the study of ancient maize populations.

Escherichia coli DNA-binding protein H-NS is localized in the nucleoid.

Electron microscope localization of the 15.4-kDa DNA-binding protein H-NS was carried out in Escherichia coli cells subjected to cryosubstitution followed by immuno-labelling with the protein A/gold technique. Three types of E. coli cells were used: (1) "normal" cells growing exponentially at 37 degrees C; (2) "cold-shocked" cells two hours after the shift from 37 degrees C to 10 degrees C; and (3) cells in which an expression vector had been induced to overproduce H-NS. The results clearly indicate that in all 3 cases, the vast majority of the molecules reacting with anti-H-NS antibodies are localized within the nucleoid and at the border between the nucleoid and the ribosome-rich cytoplasm, which supports the premise that H-NS is implicated in the condensation of the nucleoid.

Residual DNA-bound proteins are a source of in vitro transcription inhibitor peptides.

Enzymatic breakdown of residual proteins occurs at mild alkaline pH (pH optimum 8.5) as monitored by using radioiodinated, purified genomic DNA from calf thymus. These DNA fibers also possess a differential ability to hydrolyze added exogenous small and linker histones. The results described argue strongly that a putative protease activity, co-purified with DNA, is the source of short chain peptides which inhibit transcription in vitro3. Therefore, we propose that RNA repressor peptides must be of higher molecular weight than previously reported.