Use of coal mining waste for the removal of acidity and metal ions Al (III), Fe (III) and Mn (II) in acid mine drainage.

The coal industry may generate acid mine drainage (AMD) and mining wastes, which may adversely affect the quality of the environment. In this study we propose the use of this waste in the removal of acidity and metal ions, as well as in the reduction of the toxicity of AMD. A physico-chemical analysis of the waste shows the presence of mainly SiO2, Al2O3 and Fe2O3 and a superficial area of 4.316 m2 g(-1). The treatment of AMD with the waste resulted in an increase in pH from 2.6 to 7.8 and removed 100% of the Al (III), 100% of the Fe (III) and 89% of the Mn (II). We also observed that the high toxicity of the AMD towards Daphnia magna (LC50 = 3.68%) and Artemia sp. (LC50 = 4.97%) was completely eliminated after treatment with the waste. The data obtained allow us to propose that the waste can be used in the treatment of AMD, providing an economic use for the waste.

Enhanced major histocompatibility complex class I-dependent presentation of antigens modified with cationic and fusogenic peptides.

Soluble extracellular protein antigens are notoriously poor stimulators of CD8+ cytotoxic T-lymphocyte (CTL) responses, largely because these antigens have inefficient access to an endogenous cytosolic pathway of the major histocompatibility complex (MHC) class I-dependent antigen presentation. Here, we present a strategy that facilitates antigen penetration into the cytosol of antigen-presenting cells (APC) by addition to the antigen of charge-modifying peptide sequences. As a result of this intervention, the charge modification enhances antigen uptake into APC by counteracting the repulsive cell surface charge, and then endosomal membranes are disrupted with a subsequent release of antigen into the cytosol. This technology significantly improves MHC class I-dependent antigen presentation to CTL, enabling a more efficient generation of specific CTL immunity in vivo. The strategy described here has potential for use in developing efficient vaccines for antigen-specific immunotherapy of human malignancies.

Trp-p8, a novel prostate-specific gene, is up-regulated in prostate cancer and other malignancies and shares high homology with transient receptor potential calcium channel proteins.

We have identified and cloned a novel gene, trp-p8, by screening a prostate-specific subtracted cDNA library. The 5694-bp cDNA has a 3312-bp open reading frame, which codes for a 1104 amino acid putative protein with seven transmembrane domains. The predicted protein revealed significant homology with the transient receptor potential (trp) family of Ca(2+) channel proteins. Northern blot analysis indicated that trp-p8 expression within normal human tissues is mostly restricted to prostate epithelial cells. In situ hybridization analysis showed that trp-p8 mRNA expression was at moderate levels in normal prostate tissue and appears to be elevated in prostate cancer. Notably, trp-p8 mRNA was also expressed in a number of nonprostatic primary tumors of breast, colon, lung, and skin origin, whereas transcripts encoding trp-p8 were hardly detected or not detected in the corresponding normal human tissues.

Immunotherapy of hormone-refractory prostate cancer with antigen-loaded dendritic cells.

PURPOSE: Provenge (Dendreon Corp, Seattle, WA) is an immunotherapy product consisting of autologous dendritic cells loaded ex vivo with a recombinant fusion protein consisting of prostatic acid phosphatase (PAP) linked to granulocyte-macrophage colony-stimulating factor. Sequential phase I and phase II trials were performed to determine the safety and efficacy of Provenge and to assess its capacity to break immune tolerance to the normal tissue antigen PAP. PATIENTS AND METHODS: All patients had hormone-refractory prostate cancer. Dendritic-cell precursors were harvested by leukapheresis in weeks 0, 4, 8, and 24, loaded ex vivo with antigen for 2 days, and then infused intravenously over 30 minutes. Phase I patients received increasing doses of Provenge, and phase II patients received all the Provenge that could be prepared from a leukapheresis product. RESULTS: Patients tolerated treatment well. Fever, the most common adverse event, occurred after 15 infusions (14.7%). All patients developed immune responses to the recombinant fusion protein used to prepare Provenge, and 38% developed immune responses to PAP. Three patients had a more than 50% decline in prostate-specific antigen (PSA) level, and another three patients had 25% to 49% decreases in PSA. The time to disease progression correlated with development of an immune response to PAP and with the dose of dendritic cells received. CONCLUSION: Provenge is a novel immunotherapy agent that is safe and breaks tolerance to the tissue antigen PAP. Preliminary evidence for clinical efficacy warrants further exploration.

Priming tissue-specific cellular immunity in a phase I trial of autologous dendritic cells for prostate cancer.

We attempted to induce therapeutic immunity against prostate-derived tissues in patients suffering from progressive hormone-refractory metastatic prostate carcinoma. Thirteen patients were treated with two infusions, 1 month apart, of autologous dendritic cells (APC8015) preexposed ex vivo to PA2024, a fusion protein consisting of human granulocyte/macrophage-colony stimulating factor (GM-CSF) and human prostatic acid phosphatase (PAP). The infusions were followed by three s.c. monthly doses of PA2024 without cells. Three groups of patients each received PA2024 at 0.3, 0.6, or 1.0 mg/injection. All Ps were two-sided. Treatment was well tolerated. After infusions of APC8015, patients experienced only mild (grade 1-2) short-lived fever and/or chills, myalgia, pain, and fatigue. One patient developed grade 3 fatigue. Four patients developed mild local reactions to s.c. PA2024. Twelve patients were evaluable for response to treatment. Circulating prostate-specific antigen levels dropped in three patients. T cells, drawn from patients after infusions of APC8015, but not before, could be stimulated in vitro by GM-CSF (P = 0.0004) and PAP (P = 0.0001), demonstrating broken immune tolerance against these two normal proteins. Injections of PA2024 did not influence the reactivity of T cells against PAP and GM-CSF. However, antibodies to GM-CSF and, to a much lesser extent, to PAP reached maximum titers only after two or even three injections of PA2024, showing that directly injected PA2024 was involved in stimulation of humoral immunity. Dendritic cells exposed to antigen ex vivo can induce antigen-specific cellular immunity in prostate cancer patients, warranting further studies of this mode of immunotherapy.

New parental cell lines for generating human hybridomas.

In contrast to the success achieved with the production of hybridomas in the mouse system, creating human hybridomas is problematic. The reason is believed to be a lack of suitable malignant human cell lines. The work presented here demonstrates the establishment of three human parent cell lines--two of which are of T cell origin--by installing hypoxanthine guanosine phosphoribosyl transferase (HGPRT) and/or thimidine kinase (TK) deficiencies into the leukemic cell lines REH, 1301 and SKW-3. In order to isolate true hybridomas, selection procedures must guarantee complete death of the enzyme-deficient malignant parent cells. In this respect sublines with a combined HGPRT and TK deficiency proved to be superior to those with only one enzyme deficiency, especially in combination with the newly developed hypoxanthine/aminopterine/thymidine/azaserine (HATA) selection medium. However, selection media should be of low nonspecific toxicity. This was shown to be a particular property of the thymidine-free azaserine/hypoxanthine (AH) selection medium. Preliminary data show an extraordinary ability of one subclone of the hypoxanthine guanosine phosphoribosyl transferase-negative T cell line SKW-3 to generate human T-T hybridomas. They are of a stable, nearly tetraploid karyotype and express new surface antigens, thus providing new possibilities for the investigation of human T lymphocyte function by means of hybridoma technology.

In vivo persistence of donor cells following adoptive transfer of allogeneic dendritic cells in HIV-infected patients.

Peripheral blood samples from HIV-seropositive individuals enrolled in a pilot clinical trial investigating the use of allogeneic dendritic cell therapy were evaluated for mixed chimerism. In this study, dendritic cells from HLA-identical, HIV-seronegative siblings were used. Patients received an infusion of dendritic cells pulsed with HIV MN gp160 protein or with peptides from HLA-A2 restricted epitopes of env, gag, and pol proteins every month for 6-9 months. Of the five allogeneic dendritic cell recipients, two showed increases in HIV antigen-specific immune responses. Allele-specific polymorphisms were identified in three sib-pairs that allowed infused donor cells to be detected using sensitive PCR-based molecular methods. Analysis of blood samples from patients showed similar patterns of donor cell persistence after the first infusion, in that cells were detectable for at least 1 week. Also, differences were observed in the kinetics of cell survival between the first and subsequent infusion cycles in all three patients. This suggests variation in HIV-specific immune responses detected among these three patients was not due to differences in persistence of infused donor cells.

Use of chitosan microspheres as remedial material for acidity and iron (III) contents of coal mining wastewaters.

Chitosan microspheres are highly effective in neutralizing the acidity of wastewaters from coal mining. The saturation capacity for the formation of a superficial monolayer on the adsorbent was interpreted using Langmuir isotherm and considering the amino groups as the adsorption sites for hydronium ions. The saturation capacity of the surface of the static system was 0.428 mol kg(-1), higher than that of the dynamic one. This value corresponds to the neutralization of 135 liters of wastewater per kilogram of microspheres. One gram of chitosan microspheres was capable of increasing wastewater pH from 2.5 to 4.0 and removing approximately 100% of its iron (III) contents.

Biometrics of hoof balance in equids / Biometria do equilíbrio podal de equídeos

Differences in hoof balance between horses, mules and donkeys were identified in order to form more specific considerations for proper management of the animals. Measurements of the natural dimensions of hooves in sixty animals were used: 20 horses from the Crioulo breed, 20 mules and 20 donkeys from the Pêga breed. Liveweight was estimated using the correlation equations in each species by heart girth. Using a caliper rule, tape measure and hoof gauge, measurements of the length and width of the frog, hoof height, angle of heel, medial and lateral dorsal length, angle of the toe and crown circumference of the hooves of forelimbs and hindlimb were taken. Within each group the hooves of the hindlimbs exhibited narrower measurements than the hooves of the forelimbs and no difference was observed between the hoof angle of both members of groups. The conformation of the hooves of donkeys is shown to be substantially different from that observed in horses, the mules being in an intermediate condition, being smaller, angled and robust frog and proportionally more developed. Similarly, the hooves of donkeys provide greater support area compared to mules and horses, in descending order, even being dimensionally smaller. We conclude that the hooves of horses, mules and donkeys, have specific patterns of geometric balance that must be taken into consideration at the time of trimming and imbalance inferences.(AU)

O objetivo deste trabalho foi determinar o equilíbrio dos cascos de equídeos. Foram utilizados 60 animais, sendo estes: 20 equinos da raça Crioula, 20 muares e 20 asininos da raça Pêga. O peso vivo foi estimado por meio de equações de correlação com o perímetro torácico específico a cada espécie. Utilizando-se paquímetro, fita métrica e podogoniômetro, foram mensurados comprimento e largura da ranilha e do casco, altura e ângulo dos talões medial e lateral, comprimento dorsal e ângulo da pinça e perímetro da banda coronária dos cascos dos membros torácicos e pélvicos. Dentro de cada grupo, observou-se que os cascos dos membros pélvicos exibem-se mais estreitos que os cascos dos membros torácicos, e não houve diferença entre o ângulo das pinças de ambos os grupos de membros. A conformação dos cascos dos asininos mostra-se substancialmente divergente do observado nos equinos, estando os muares numa condição intermediária, sendo aqueles menores, mais angulados e com ranilha robusta e proporcionalmente mais desenvolvida. Da mesma forma, os cascos dos asininos proporcionam maior área de apoio em relação aos muares e equinos, em ordem decrescente, mesmo sendo dimensionalmente menores. Conclui-se que os cascos de equinos, muares e asininos apresentam padrões de equilíbrio geométrico específicos, que devem ser levados em consideração no momento do casqueamento e na inferência de desequilíbrios.(AU)

Evaluation of remediation of coal mining wastewater by chitosan microspheres using biomarkers.

Acidic mine waters have a marked influence on the surrounding environment and pose a serious threat through long-term environmental degradation. Therefore, it is important to improve and monitor water quality with the aim of decreasing the hazard presented by this effluent emission. The aim of this work was to evaluate the remediation of mining wastewater effluents by chitosan microspheres using biomarkers of exposure and effect. DNA damage (Comet assay) and several biomarkers of oxidative stress, such as lipoperoxidation levels (TBARS), superoxide dismutase (SOD), catalase (CAT), and glutathione S-transferase (GST) activities, and contents of reduced glutathione (GSH), were measured in blood and liver of tilapia (Oreochromis niloticus) exposed for 7, 15, and 30 days to dechlorinated tap water, 10% coal mining wastewater (CMW), and coal mining wastewater treated with chitosan microspheres (RCM). The results indicate that hepatic TBARS levels were significantly higher in fish exposed to CMW after 7, 15, and 30 days (100%, 86%, and 63%, respectively), and after remediation there was no significant difference in relation to the control group. Hepatic GSH concentrations were lower than control values for CMW after 7 and 15 days of exposure (34% decrease at both times), and this concentration was normalized by treatment with chitosan. SOD showed increased activity in liver after 15 and 30 days of exposure, 30% and 36%, respectively, and in fish exposed to RCM there was no change in this activity compared with the control group. Increased CAT activity in liver was observed during all experimental periods in fish exposed to CMW (46%, 50%, and 56% at 7, 15, and 30 days, respectively) compared with the control or treated-water groups. The highest increase in hepatic GST activity (106%) was observed only in fish exposed to CMW for 30 days. There was an increase in DNA damage in liver (50% at 7 and 15 days) and blood (79%, 77%, and 48% at 7, 15, and 30 days, respectively) after exposure to CMW. In contrast, the fish exposed to wastewater treated with chitosan microspheres exhibited DNA fragmentation indexes similar to the control group. The results obtained indicate the use of oxidative stress biomarkers as useful tools for the toxicity evaluation of coal mining effluents and also suggest that chitosan microspheres may be used as an alternative approach for remediation of coal mining wastewaters.

Carbohydrate-specific human heterophile antibodies in normal human sera that react with xenogeneic cells.

Human heterophile antibodies (HHA) that are present in normal human sera (NHS) play an important role in hyperacute xenograft rejection. The aim of this study was to analyze the occurrence, mode of action and molecular specificity of HHA in NHS that are directed against xenogeneic lymphocytes (isolated from mouse, rat, guinea pig, rabbit, cattle and pig) and isolated rat pancreatic islets. All sera contained variable amounts of HHA that killed the target cells via the classical complement pathway. The cytotoxic activity of these HHA was specifically inhibited by certain carbohydrates (alpha-D-melibiose, beta-lactose, beta-gentiobiose, beta-cellobiose, D-mannose, N-acetyl-beta-D-mannosamine and alpha-D-rhamnose) and by rat IgM. By means of affinity chromatography with immobilized inhibitors we obtained an antibody preparation of mainly IgG type from NHS (up to 3.5 mg/10 ml serum) that reacted strongly with rat lymphocytes and isolated rat pancreatic islets. Though thus far residual xenospecific antibody activity has remained in the sera even after multiple affinity chromatography, these data suggest that specific elimination of HHA is feasible and that it may be thus possible to overcome a major obstacle to xenotransplantation.

Alloantigen-specific cytotoxic clones bearing the alpha,beta T cell antigen receptor but not CD4 or CD8 molecules.

The vast majority of circulating lymphocytes that express the alpha,beta TCR in association with CD3 also express either CD4 or CD8 molecules, which are thought to act as important accessory structures in HLA class II- and I-restricted T cell functions, respectively. In the current study alpha,beta TCR+ clones devoid of detectable CD4 or CD8 were generated by repeated stimulation of fresh CD3+,CD4-,CD8- cells with an allogeneic lymphoblastoid cell line in the presence of conditioned medium containing IL-2. Except for the absence of CD4 and CD8, which was associated with undetectable levels of CD4 and CD8 mRNA, the clones were phenotypically indistinguishable from classical CD3+,alpha,beta TCR+ cells. Furthermore, they mediated potent cytolysis of their specific stimulator line but did not kill irrelevant LCL or NK-sensitive targets. mAb to CD3 and the alpha,beta TCR inhibited cytolysis, suggesting that the clones use the TCR/CD3 complex to recognize and respond to their targets. mAbs to CD2 and CD11a also inhibited cytolysis, indicating that the clones use these accessory molecules to interact with their targets. Finally, cytolysis was inhibited by an HLA-A,B,C framework-specific mAb (W6/32) as well as a mAb (MA2.1) specific for an HLA-A2 epitope. These results demonstrate that CD3+,alpha,beta TCR+,CD4-,CD8- cytotoxic clones can be generated from the peripheral blood of healthy adults, and use their TCR/CD3 complexes to function in an HLA class I-restricted manner.

Major histocompatibility complex class I-restricted presentation of protein antigens without prior intracellular processing.

Proteins in their native form are incapable of stimulating antigen (Ag)-specific T cells, which can only recognize major histocompatibility complex (MHC)-bound peptides that have been generated by intracellular processing within antigen-presenting cells (APCs). Here, we show that APCs can trigger MHC class I-restricted T-cell responses after presenting proteins without conventional intracellular processing, provided the immunostimulatory MHC class I-binding peptide sequence is incorporated at the carboxy-terminal position. Such MHC-bound proteins do not stimulate T cells directly, because the contact between MHC/peptide complex and its cognate ligand is sterically hindered by the amino-terminal bulk of the protein. Removal of the latter via an extracellular Ag proteolysis by the T-cell- and/or APC-derived enzymes is required for effective T-cell stimulation. Our data challenge the established concept that only small peptides can bind to the MHC class I molecules.

B4B, a novel growth-arrest gene, is expressed by a subset of progenitor/pre-B lymphocytes negative for cytoplasmic mu-chain.

We subjected PBMC of normal adults to density fractionation to enrich for an immunoblast fraction that would include early immune lineage precursors. Differential display PCR experiments identified one transcript that is expressed specifically in this immunoblast fraction. This cDNA, designated B4B, encodes a novel gene product containing four putative transmembrane-spanning domains. B4B+ cells, detected with anti-B4B Ig, were found at very low frequency in PBMC (0.01%) and were enriched significantly in intermediate density fractions (0.1-1.0%). B4B+ cells were shown to be CD19+CD45+HLA-DR+ and negative for CD20, cytoplasmic mu-chain, CD3, CD16, CD56, CD34, and CD68 (monocyte), consistent with a progenitor/pre-B lymphocyte subset that does not express cytoplasmic mu-chain and thus may lack productive Ig rearrangement. This phenotypic description of the B4B+ subset agrees with our finding that the frequency of B4B+ cells was greatly increased in bone marrow (3-10%) as compared with PBMC (0.01%). The B4B polypeptide sequence exhibits significant homology to only one known protein, PMP-22/gas-3, a Schwann cell-specific protein that induces cell growth arrest. Transient expression of B4B specifically inhibited cellular proliferation by more than 50%. Based on its antiproliferative effect and pattern of expression restricted to a subpopulation of immature B cells, the B4B gene product may be involved in the elimination of B cells before productive VDJC rearrangement of Ig loci or, alternatively, in the growth arrest of transformed progenitor B cells.

Induction of prostate tumor-specific CD8+ cytotoxic T-lymphocytes in vitro using antigen-presenting cells pulsed with prostatic acid phosphatase peptide.

BACKGROUND: Most strategies in cancer immunotherapy are aimed at the induction of a strong cellular immune response against the tumor. Particularly, CD8+ T lymphocytes have been proven in multiple animal models to be critical for the eradication of solid tumors. METHODS: We used a population of peripheral blood-derived antigen-presenting cells (APC), containing dendritic cells (DC), to generate prostate tumor-specific CD8+ T cells. Selected peptides from prostatic acid phosphatase (PAP), a prostate tissue-specific antigen, were shown to bind HLA-A2. A high-affinity peptide was used to generate peptide-specific CD8+ cytolytic T lymphocytes (CTL) from the peripheral blood of healthy donors. RESULTS: The obtained PAP-peptide-specific CTL lysed peptide-coated target cells, vaccinia-infected target cells, and HLA-A2-positive prostate-tumor cells in vitro in an antigen-specific manner. CONCLUSIONS: Our results indicate that CTL precursors to the PAP gene product exist and could be potentially recruited to elicit an antitumor response. Thus, PAP is a suitable antigen for inclusion in prostate cancer vaccines.

Induction of tissue-specific autoimmune prostatitis with prostatic acid phosphatase immunization: implications for immunotherapy of prostate cancer.

Prostatic acid phosphatase (PAP) is uniquely expressed in prostatic tissue and prostate cancer. In this study, the immunogenicity of PAP was investigated in a male rat model. We show that immunization with recombinant rat or human PAP in CFA leads to a significant Ab response, but does not generate CTL or result in autoimmune prostatitis. In contrast, immunization with recombinant vaccinia expressing human PAP, but not rat PAP, generates a CTL response and tissue-specific prostatitis in the absence of detectable PAP-specific Abs. These findings suggest that a cellular immune response to PAP, rather than Abs, mediates destructive autoimmune prostatitis. Thus, xenogeneic forms of PAP are a new tool for the induction of prostate-specific immunity and may prove useful for the immunotherapy of prostate cancer.

V7, a novel leukocyte surface protein that participates in T cell activation. I. Tissue distribution and functional studies.

Among a panel of mouse mAbs generated to a human T cell clone, one mAb, V7.1, inhibited T cell activation in the mixed lymphocyte reaction and was studied further. V7.1 reacted strongly with Ag-specific T cell clones, in addition to freshly isolated monocytes and granulocytes. However, the mAb reacted weakly with freshly isolated PBLs (T cells, B cells, and NK cells), T cells stimulated with phytohemagglutinin, or Con A, and did not stain the vast majority of transformed cell lines of hemopoietic origin. Stimulation of T cells with anti-CD3, or the combination of anti-CD3 and PMA, or anti-CD3, PMA and ionomycin, markedly increased V7.1 surface staining. The mAb precipitated a single polypeptide chain of approximately 135 kDa from alloactivated T cells or monocytes, which was reduced to approximately 110 kDa after treatment with N-glycanase. The proliferative response of T cells to allogeneic monocytes or B lymphoblastoid cells was inhibited by V7.1, and inhibition was maximal when the mAb was present at the initiation of culture. V7.1 also exhibited dose-dependent inhibition of the T cell response to immobilized anti-CD3 Ab in the absence of APCs, indicating that the inhibitory effect of this Ab occurs at the T cell level. Expression of CD25 (IL-2R) on anti-CD3-activated T cells and secretion of IL-2 induced with anti-CD3 and PMA were inhibited by V7.1, whereas the Ab had no effect on T cell proliferation induced by PHA or Con A or on T cell-mediated cytotoxicity. These results indicate that V7.1 recognizes a novel leukocyte surface glycoprotein, designated V7, that is up-regulated on Ag but not lectin-activated T cells, and appears to play a role in TCR/CD3-dependent T cell activation. In an accompanying study, the gene encoding the V7 Ag is described and the molecule is shown to be a novel member of the Ig superfamily.

V7, a novel leukocyte surface protein that participates in T cell activation. II. Molecular cloning and characterization of the V7 gene.

V7 is a cell surface glycoprotein expressed on Ag-activated T cells, monocytes, and granulocytes, as well as subpopulations of T cells and accessory cells present in thymic medulla and tonsil. A mAb directed against V7 inhibits the proliferative response of T cells to allogeneic cells or immobilized anti-CD3 Ab, but not lectin mitogens, suggesting that V7 plays a role in TCR/CD3-mediated T cell activation. We have used the anti-V7 Ab in eukaryotic expression cloning experiments to isolate a cDNA clone containing a 3,340-bp insert that encodes V7 when transiently expressed in simian and murine fibroblastoid cells. DNA sequence analysis revealed a novel 1,021-amino acid open reading frame the structure of which conforms to the category of type I integral membrane proteins. The protein sequence includes a 20-residue putative hydrophobic signal sequence followed by a putative extracellular domain of 934 amino acids, a prototypic hydrophobic transmembrane spanning a domain of 25 residues, and finally a short and highly charged putative cytoplasmic domain of 42 residues. The extracellular domain contains seven pairs of regularly spaced cysteine residues, suggestive of Ig-like domains. On the basis of statistical analysis of the sequences of the putative cysteine loops, all seven of the Ig-like domains belong to the variable, or V-type, category. By using fluorescence in situ hybridization, we have mapped the V7 gene to human chromosome Ip13. Thus, the V7 glycoprotein represents a novel member of the Ig superfamily that is involved in critical intracellular signals essential for immune function.