Wafer-scale epitaxial modulation of quantum dot density.

Precise control of the properties of semiconductor quantum dots (QDs) is vital for creating novel devices for quantum photonics and advanced opto-electronics. Suitable low QD-densities for single QD devices and experiments are challenging to control during epitaxy and are typically found only in limited regions of the wafer. Here, we demonstrate how conventional molecular beam epitaxy (MBE) can be used to modulate the density of optically active QDs in one- and two- dimensional patterns, while still retaining excellent quality. We find that material thickness gradients during layer-by-layer growth result in surface roughness modulations across the whole wafer. Growth on such templates strongly influences the QD nucleation probability. We obtain density modulations between 1 and 10 QDs/µm2 and periods ranging from several millimeters down to at least a few hundred microns. This method is universal and expected to be applicable to a wide variety of different semiconductor material systems. We apply the method to enable growth of ultra-low noise QDs across an entire 3-inch semiconductor wafer.

Dual role of tumour-infiltrating T helper 17 cells in human colorectal cancer.

BACKGROUND: The immune contexture predicts prognosis in human colorectal cancer (CRC). Whereas tumour-infiltrating CD8+ T cells and myeloid CD16+ myeloperoxidase (MPO)+ cells are associated with favourable clinical outcome, interleukin (IL)-17-producing cells have been reported to correlate with severe prognosis. However, their phenotypes and functions continue to be debated. OBJECTIVE: To investigate clinical relevance, phenotypes and functional features of CRC-infiltrating, IL-17-producing cells. METHODS: IL-17 staining was performed by immunohistochemistry on a tissue microarray including 1148 CRCs. Phenotypes of IL-17-producing cells were evaluated by flow cytometry on cell suspensions obtained by enzymatic digestion of clinical specimens. Functions of CRC-isolated, IL-17-producing cells were assessed by in vitro and in vivo experiments. RESULTS: IL-17+ infiltrates were not themselves predictive of an unfavourable clinical outcome, but correlated with infiltration by CD8+ T cells and CD16+ MPO+ neutrophils. Ex vivo analysis showed that tumour-infiltrating IL-17+ cells mostly consist of CD4+ T helper 17 (Th17) cells with multifaceted properties. Indeed, owing to IL-17 secretion, CRC-derived Th17 triggered the release of protumorigenic factors by tumour and tumour-associated stroma. However, on the other hand, they favoured recruitment of beneficial neutrophils through IL-8 secretion and, most importantly, they drove highly cytotoxic CCR5+CCR6+CD8+ T cells into tumour tissue, through CCL5 and CCL20 release. Consistent with these findings, the presence of intraepithelial, but not of stromal Th17 cells, positively correlated with improved survival. CONCLUSIONS: Our study shows the dual role played by tumour-infiltrating Th17 in CRC, thus advising caution when developing new IL-17/Th17 targeted treatments.

Evaluation of platelet function using PFA-100® in patients treated with Acetylsalicylic acid and qualified for Trauma and Orthopedic surgery procedures.

The phenomenon of high on-acetylsalicylic acid (ASA) treatment platelet (PLT) reactivity - HATPR - and its clinical implications have not been fully understood. Little data is available on assessing PLT activity based on the severity of intra- and postoperative bleeding in a population of orthopedic patients with normal closure time (CT) measured by a PLT function analyzer PFA-100®, despite being given long-term ASA therapy. The aim is to assess PLT function using PFA-100® in patients with ASA therapy and qualified for trauma and orthopedic surgery procedures. The retrospective analysis covered 384 patients whose PLT reactivity was assessed using PFA-100®. Out of those, 198 had been taking ASA with a 75 mg dose until hospital admission. In addition, a group of 70 patients with a proximal femoral fracture surgically treated using the dynamic hip screw (DHS) was selected, in whom severity of bleeding was assessed by HIP ASA (+). The reference group comprised 52 patients (without ASA therapy) who were operated on due to the same indications. Normal CT was found in 37% of ASA-receiving patients. Patients with normal CT, despite ASA therapy, exhibited significantly more intense bleeding after DHS surgery. A similar number of patients required red blood cells (RBCs) transfusion in HIP ASA (+) and HIP ASA (-). Increased risk of complications in HIP ASA (+) group was not found. CONCLUSIONS: Normal PLT function assessed using PFA-100® is a common phenomenon in patients with long-term ASA treatment and who are qualified for trauma and orthopedic surgery procedures. In many cases, it seems that inadequate response to ASA is only a laboratory phenomenon.

Expression of programmed death ligand 1 (PD-L1) is associated with poor prognosis in human breast cancer.

Recent studies in multiple epithelial cancers have shown that the inhibitory receptor programmed cell death 1 (PD-1) is expressed on tumor-infiltrating lymphocytes and/or programmed death ligand 1 (PD-L1) is expressed on tumor cells, suggesting that antitumor immunity may be modulated by the PD-1/PD-L1 signaling pathway. In addition, phase 1 clinical trials with monoclonal antibodies targeting PD-1 or PD-L1 have shown promising results in several human cancers. The purpose of this study was to investigate the impact of PD-L1 expression in human breast cancer specimens. We conducted an immunohistochemistry study using a tissue microarray encompassing 650 evaluable formalin-fixed breast cancer cases with detailed clinical annotation and outcomes data. PD-L1 was expressed in 152 (23.4 %) of the 650 breast cancer specimens. Expression was significantly associated with age, tumor size, AJCC primary tumor classification, tumor grade, lymph node status, absence of ER expression, and high Ki-67 expression. In univariate analysis, PD-L1 expression was associated with a significantly worse OS. In multivariate analysis, PD-L1 expression remained an independent negative prognostic factor for OS. In subset analyses, expression of PD-L1 was associated with significantly worse OS in the luminal B HER2(-) subtype, the luminal B HER2(+) subtype, the HER2 subtype, and the basal-like subtype. This is the first study to demonstrate that PD-L1 expression is an independent negative prognostic factor in human breast cancer. This finding has important implications for the application of antibody therapies targeting the PD-1/PD-L1 signaling pathway in this disease.

Multiple mechanisms underlie defective recognition of melanoma cells cultured in three-dimensional architectures by antigen-specific cytotoxic T lymphocytes.

Cancer cells' growth in three-dimensional (3D) architectures promotes resistance to drugs, cytokines, or irradiation. We investigated effects of 3D culture as compared to monolayers (2D) on melanoma cells' recognition by tumour-associated antigen (TAA)-specific HLA-A(*)0201-restricted cytotoxic T-lymphocytes (CTL). Culture of HBL, D10 (both HLA-A(*)0201+, TAA+) and NA8 (HLA-A(*)0201+, TAA-) melanoma cells on polyHEMA-coated plates, resulted in generation of 3D multicellular tumour spheroids (MCTS). Interferon-gamma (IFN-gamma) production by HLA-A(*)0201-restricted Melan-A/MART-1(27-35) or gp 100(280-288)-specific CTL clones served as immunorecognition marker. Co-culture with melanoma MCTS, resulted in defective TAA recognition by CTL as compared to 2D as witnessed by decreased IFN-gamma production and decreased Fas Ligand, perforin and granzyme B gene expression. A multiplicity of mechanisms were potentially involved. First, MCTS per se limit CTL capacity of recognising HLA class I restricted antigens by reducing exposed cell surfaces. Second, expression of melanoma differentiation antigens is downregulated in MCTS. Third, expression of HLA class I molecules can be downregulated in melanoma MCTS, possibly due to decreased interferon-regulating factor-1 gene expression. Fourth, lactic acid production is increased in MCTS, as compared to 2D. These data suggest that melanoma cells growing in 3D, even in the absence of immune selection, feature characteristics capable of dramatically inhibiting TAA recognition by specific CTL.

Encapsulation into sterically stabilised liposomes enhances the immunogenicity of melanoma-associated Melan-A/MART-1 epitopes.

Tumour-associated antigens (TAA)-specific vaccination requires highly immunogenic reagents capable of inducing cytotoxic T cells (CTL). Soluble peptides are currently used in clinical applications despite an acknowledged poor immunogenicity. Encapsulation into liposomes has been suggested to improve the immunogenicity of discrete antigen formulations. We comparatively evaluated the capacity of HLA-A2.1 restricted Melan-A/MART-1 epitopes in soluble form (S) or following inclusion into sterically stabilised liposomes (SSL) to be recognised by specific CTL, to stimulate their proliferation and to induce them in healthy donors' peripheral blood mononuclear cells (PBMC), as well as in melanoma-derived tumour-infiltrating lymphocytes (TIL). HLA-A2.1(+), Melan-A/MART-1-NA-8 melanoma cells served as targets of specific CTL in 51Cr release assays upon pulsing by untreated or human plasma-treated soluble or SSL-encapsulated Melan-A/MART-1 27-35 (M27-35) or 26-35 (M26-35) epitopes. These reagents were also used to stimulate CTL proliferation, measured as 3H-thymidine incorporation, in the presence of immature dendritic cells (iDC), as antigen-presenting cells (APC). Induction of specific CTL upon stimulation with soluble or SSL-encapsulated peptides was attempted in healthy donors' PBMC or melanoma-derived TIL, and monitored by 51Cr release assays and tetramer staining. Na-8 cells pulsing with SSL M27-35 resulted in a five-fold more effective killing by specific CTL as compared with equal amounts of S M27-35. Encapsulation into SSL also provided a partial (50%) protection of M27-35 from plasma hydrolysis. No specific advantages regarding M26-35 were detectable in these assays. However, at low epitope concentrations (

Phase I/II clinical trial of a nonreplicative vaccinia virus expressing multiple HLA-A0201-restricted tumor-associated epitopes and costimulatory molecules in metastatic melanoma patients.

We performed a phase I/II clinical trial in metastatic melanoma patients with an ultraviolet (UV)-inactivated nonreplicating recombinant vaccinia virus enabling the expression, from a single construct, of endoplasmic reticulum-targeted HLA-A0201-restricted Melan-A/MART-1(27-35), gp100(280-288), and tyrosinase(1-9) epitopes, together with CD80 and CD86 costimulatory proteins. Corresponding soluble peptides were used to boost responses and granulocyte-macrophage colony-stimulating factor was used as systemic adjuvant. Safety and immunogenicity, as monitored with in vitro-restimulated peripheral blood mononuclear cells by cytotoxic T lymphocyte precursor (CTLp) frequency analysis and tetramer staining, were specifically addressed. Of 20 patients entering the protocol, 2 had to withdraw because of rapidly progressing disease. Immune responses were evaluated in 18 patients (stage III, n = 5; stage IV, n = 13) and increases in specific CTLp frequencies were observed in 15. In 16 patients responsiveness against all 3 antigens could be analyzed: 7 (43%), including all stage III cases, showed evidence of induction of CTLs specific for the three epitopes, and 2 (12%) and 4 (25%), respectively, showed reactivity against two or one tumor-associated antigen. In three stage IV patients no specific CTL reactivity could be induced. Increases in CTLp frequency were detected mostly after viral vaccine injections. However, in a majority of patients final CTLp levels were comparable to initial levels. Tetramer characterization of Melan-A/MART-1(27-35)-specific CTLs during the protocol also suggested preferential expansion after recombinant virus administration. Vector-specific humoral responses, frequently undetectable in stage IV patients, did not appear to prevent tumor-associated antigen-specific CTL induction. Aside from a single occurrence of transient grade 3 leukopenia, no major clinical toxicity was reported. Seventeen of 18 patients completed the 3-month trial (one patient died before the last delayed-type hypersensitivity test). Three displayed regression of individual metastases, seven had stable disease, and progressive disease was observed in seven patients. This is the first report on the administration of a UV-inactivated recombinant vaccinia virus coexpressing five transgenes in cancer patients. The results described here, in terms of safety and immunogenicity, support the use of this reagent in active specific immunotherapy.

Cell fusion: an approach to generating constitutively proliferating human tumor antigen-presenting cells.

Somatic cell hybrids of HLA-A2(+) EBV-transformed B- or dendritic cells (DC) and allogeneic HLA-A2(-) melanoma cell line Me15 were obtained by in vitro electrofusion using an electroporator. Before fusion, melanoma cells were stably transfected with green fluorescent marker protein (GFP) and neomycin resistance gene (neo(+)). Stably growing hybrid antigen-presenting cells (HAPC) expressing HLA-DR and HLA-A2 (or HLA-A30/31), and melanoma-associated antigens (MART-1, gp100) were selected by a double strategy of immunomagnetic MACS and neomycin selection. Fusion efficiency ranged between 3% and 18% (mean: 8.0+/-4.7%) as defined by simultaneous GFP and HLA-A2 detection. Expression of melanoma-associated antigens (MART-1, gp100) in hybrid cells was determined by reverse transcription-polymerase chain reaction (RT-PCR). HLA-restricted antigen-specific presentation of melanoma antigens was demonstrated by killing of semi-allogenic HAPC by HLA-A2-restricted MART-1 or gp100-specific cytotoxic T lymphocyte (CTL) clones. HLA restriction and antigen specificity were confirmed by inhibition of specific cytotoxicity by anti-HLA antibodies and cold target inhibition. During long-term (42-70 days) neomycin selection of HAPC, a drastic loss of antigen-presenting cell (APC)-derived determinants (e.g. HLA-DR, HLA-A2) was observed which, however, could be "reversed" by repeated MACSorting (days 10, 21 and 49). Our method allows the generation of semi-allogenic HAPC that constitutively proliferate in vitro. This opens the possibility of establishing a number of tumor-APC hybrids expressing defined HLA haplotypes and tumor antigens, of investigating their specific properties (e.g. antigen processing), and testing their diagnostic or therapeutic potential.

Immunogenicity of nonreplicating recombinant vaccinia expressing HLA-A201 targeted or complete MART-1/Melan-A antigen.

The effect on immunogenicity of different tumor T cell epitope formulations was evaluated in vitro using nonreplicating recombinant vaccinia vector expressing two forms of the melanoma-associated MART-1/Melan-A antigen. The first recombinant virus expressed a minigene encoding a fusion product between an endoplasmic reticulum (ER)-targeting signal and the HLA-A201 binding 27-35 peptide. The second viral construct encoded the complete MART-1/Melan-A protein. The capacity of HLA-A201 cells infected with either viral construct to generate and to stimulate MART-1/Melan-A 27-35 specific cytotoxic T-lymphocytes (CTL), was comparatively characterized. The results obtained here with a tumor antigen confirmed the capacity of vaccinia virus-encoded ER-minigene to generate a very strong antigenic signal. In cytotoxicity assays, recognition of target cells infected with high amounts of both recombinant viruses with activated specific CTL clones, resulted in similar lytic activity. With regard to calcium mobilization, TCR down-regulation, IFN-gamma release, and T cell proliferation assays, the targeted epitope elicited 10- to 1000-fold stronger responses. Remarkably, the immunogenic difference between the two formulations, in their respective capacity to generate CTL from naive HLA-A2 peripheral blood mononuclear cells in vitro as measured by tetramer detection, was lower (2- to 3-fold). Recombinant vectors expressing complete antigens have demonstrated their capacity to generate specific responses and such vaccines might take advantage of a broader potential of presentation. However, as demonstrated here for the HLA-A201-restricted MART-1/Melan-A immunodominant epitope, nonreplicative vaccinia virus expressing ER-targeted minigenes appear to represent a significantly more immunogenic epitope vaccine formulation.

Modulation of dendritic cell phenotype and mobility by tumor cells in vitro.

To gain new insights into the functional interaction between DC and neoplastic cells, we have analyzed the effects of melanoma and colorectal cancer lines on the chemotaxis and the phenotype of monocyte-derived DC in vitro. Both types of tumor cells displayed effective chemoattractive capacity towards immature, but not mature DC. Furthermore, conditioned medium of discrete melanoma lines induced upregulation of CD80, CD86, MHC class I, and MHC class II molecules on immature DC. However, de novo expression of E-cadherin and strong upregulation of CD15 could also be detected in the absence of CD83 expression. Melanoma-conditioned DC exhibited an increased adhesion capacity to a melanoma cell line in vitro and did not migrate in response to SLC chemokine. Tumor-infiltrating CD15(+) cells displaying DC morphology could also be detected by immunohistochemistry in the original tumor specimens from which discrete melanoma cell lines under investigation were derived. Colorectal cancer cell lines, although able to chemoattract immature DC, were apparently unable to modulate their phenotype. Altogether our results suggest that tumor cells can attract immature DC in vitro and, eventually, modulate their phenotype. As a result, DC mobility could be severely impaired.

[The role of arginine vasopressin (AVP) in pain transmission and perception]. / Rola wazopresyny argininowej (AVP) w procesach transmisji i percepcji bólu.

In mammalian organisms were found composed systems controlling the transmission and perception of nociceptive impulses. The present review focuses on clinical and laboratory studies that are aimed at identifying the role of AVP, known antidiuretic hormone in the mechanism of pain phenomenon.

NY-ESO-1 tumour associated antigen is a cytoplasmic protein detectable by specific monoclonal antibodies in cell lines and clinical specimens.

NY-ESO-1 gene encodes a novel member of the cancer/testis (CT) family of human tumour-associated antigens (TAA). Specific monoclonal antibodies (mAb) have identified the corresponding gene product in lysates of tumour cell lines as a 22 kDa protein but no data are available concerning its intracellular location or distribution within neoplastic tissues. We have generated NY-ESO-1 specific mAbs recognizing the target molecule in cytospin preparations and in sections from clinical tumour specimens. These reagents identify NY-ESO-1 TAA in melanoma cell lines expressing the specific gene as a cytoplasmic protein, sharing the intracellular location of most MAGE TAA. In a series of 12 melanoma specimens, specific staining, limited to neoplastic cells, was detectable in the five cases where NY-ESO-1 gene expression was observed. In two of them over 90% of tumour cells showed evidence of positive staining. Lower percentages of positive neoplastic cells ranging between single cells and 50% were observed in the remaining tumours. These data suggest that active specific immunotherapies targeting NY-ESO-1, alone or in combination with other TAA could be of high clinical relevance in sizeable subgroups of melanoma patients.

Naturally processed and concealed HLA-A2.1-restricted epitopes from tumor-associated antigen tyrosinase-related protein-2.

In this study, a computer-assisted reverse immunology approach was utilized in order to identify potentially antigenic peptides derived from the differentiation antigen TRP-2, a melanosomal protein frequently expressed in melanoma. Among the seven peptides complying with HLA-A2.1-binding motifs, two induced specific CD8(+) cytotoxic T lymphocytes. HLA-A2.1(+) melanoma cells expressing TRP-2 were lysed by clones specific for TRP-2(360-368) (TLDSQVMSL) peptide, thus identifying it as a naturally processed epitope. Other T-cell clones directed against TRP-2(476-484) (VMGTLVALV) were unable to lyse HLA-matched TRP-2(+) cell lines. The role of intracellular proteolytic processing in the generation of this epitope was investigated by transfecting mini-genes encoding the TRP-2(476-484) peptide alone or carrying N- or C-terminal extensions. Specific T-cell clones recognized target cells expressing the cytotoxic T-lymphocyte (CTL)-defined epitope or its C-terminally extended precursor, but failed to recognize cells expressing the N-terminally extended TRP-2(476-484) peptide, suggesting the presence of a negative processing signal (NPS). Regarding C-terminus-flanking regions, mutational analysis indicates that the GLY485 residue plays a key role in the processing of the TRP-2(476-484) epitope. Interestingly, proteasome inhibitors preventing the generation of the MART-1/Melan-A(27-35) immunodominant melanoma tumor-associated antigen (TAA) promoted detectable presentation of TRP-2(476-484) epitope in HLA-A2.1(+) and TRP-2(+) tumor lines, as witnessed by cytokine release by specific T-cell clones.

FLT3 ligand gene expression and protein production in human colorectal cancer cell lines and clinical tumor specimens.

Dendritic cells (DC) are professional antigen presenting cells (APC) whose proliferation and functional differentiation can be induced by hematopoietic growth factors including GM-CSF and FLT3 ligand (FL). Colorectal cancers are known to be infiltrated by dendritic cells (DC) and neoplastic cells have been shown to produce GM-CSF. In this work we investigated FLT3 ligand (FL) gene expression and protein production in human colorectal cancer cell lines and clinical tumor specimens. Using reverse transcription polymerase chain reaction (RT-PCR), 6 out of 6 established tumor lines were found to express to variable extents FL gene. In 1 of them, SW480, FL immunoreactivity could be observed by taking advantage of specific antibodies. In contrast, soluble FL could not be detected in any culture supernatant. FLT3 receptor (FR) gene was not expressed and exogenous addition to the cultures of recombinant FL (rFL) did not affect the proliferation of the tumor lines. FL gene expression was investigated using a densitometry-assisted, semiquantitative RT-PCR in clinical tumor specimens. Specific FL gene transcripts were amplified from 12 of 12 surgical samples. In these cases, FL gene expression of significantly lower intensity was also detected in healthy mucosa sampled in the vicinity (2 cm) or at a distance (10 cm) from neoplastic outgrowth. Immunohistochemical studies identified FL-positive cancer cells in 5 of 5 cases tested. No positivity was detected in healthy mucosa epithelia at a distance from the tumor or in stromal cells. FL content in preoperative sera from colorectal cancer patients (n = 13) did not exceed the levels detected in healthy donors (

Chemical and physical properties of processed newspaper compared to wheat straw and wood shavings as animal bedding.

Because of continuing concerns about the safety and the suitability of recycled newspaper as an animal bedding material, municipal curbside-collected newspaper was processed into chopped and pelleted forms for comparison studies with wheat straw and kiln-dried pinewood shavings. Measurements included nutrient, heavy metal, dioxin and furan content, particle size distribution, density, combustion potential, and water-holding capacity. Recycled newspaper, straw, and wood shavings tested below or equivalent to National Research Council dietary tolerance levels and US Environmental Protection Agency toxic equivalent levels. Small particle size distribution was shavings > straw > all forms of newspaper. The density of pelleted newspaper was 50-fold greater than that of chopped newspaper and straw and 15-fold greater than shavings. In simulated flash burns, chopped newspaper, straw, and shavings ignited, and flames spread rapidly in newspaper and shavings and lasted the longest in shavings. Pelleted newspaper did not ignite. Chopped and pelleted forms of newspaper and wood shavings had higher water holding capacities (>400%) than did straw (200%). Animal industries can, in confidence, utilize recycled newspaper as an animal bedding material, providing that sources of low toxicity are identified, and suitable processed forms are produced.

Induction of potent antitumor CTL responses by recombinant vaccinia encoding a melan-A peptide analogue.

There is considerable interest in the development of vaccination strategies that would elicit strong tumor-specific CTL responses in cancer patients. One strategy consists of using recombinant viruses encoding amino acid sequences corresponding to natural CTL-defined peptide from tumor Ags as immunogens. However, studies with synthetic tumor antigenic peptides have demonstrated that introduction of single amino acid substitutions may dramatically increase their immunogenicity. In this study we have used a well-defined human melanoma tumor Ag system to test the possibility of translating the immunological potency of synthetic tumor antigenic peptide analogues into recombinant vaccinia viruses carrying constructs with the appropriate nucleotide substitutions. Our results indicate that the use of a mutated minigene construct directing the expression of a modified melanoma tumor Ag leads to improved Ag recognition and, more importantly, to enhanced immunogenicity. Thus, recombinant vaccinia viruses containing mutated minigene sequences may lead to new strategies for the induction of strong tumor-specific CTL responses in cancer patients.

Enhanced generation of cytotoxic T lymphocytes using recombinant vaccinia virus expressing human tumor-associated antigens and B7 costimulatory molecules.

In this work, we addressed the possibility to enhance the "in vitro" generation of CTLs recognizing tumor-associated antigens (TAAs) by using an inactivated recombinant vaccinia virus encoding B7.1 and B7.2 costimulatory molecules (rVV-B7.1/2). Antigen presenting cells (APCs) infected by rVV-B7.1/2 and pulsed with MART-1/Melan-A27-35 HLA-A2.1-restricted peptide induced significantly higher specific cytotoxic activity than peptide-loaded APCs infected by wild-type VV, both in VV-sensitized and naive donors. When APCs were infected with a rVV encoding both MART-1/Melan-A27-35 and B7-1/2 (rVV-B7.1/2-M), a significantly more effective CTL generation was observed as compared with cultures stimulated by APCs infected with a rVV encoding the TAA epitope only (rVV-M). These enhancing effects were detectable irrespective of a previous VV-specific sensitization. Most importantly, fibroblasts, devoid of antigen-presenting capacity upon peptide pulsing or infection with rVV-M, could be turned into effective APCs after infection by rVV encoding TAA epitopes and costimulatory molecules. In these experiments, by using separate recombinant viral constructs, we observed a predominant role of B7-1 as compared with B7-2 in the induction of TAA-specific CTLs. Taken together, our data indicate that replication-incompetent rVV encoding TAA epitopes and costimulatory molecules are able to induce highly effective generation of tumor-specific CTLs. Therefore, these vectors could represent valuable clinical tools for immunotherapy of melanoma patients.

GM-CSF gene expression and protein production in human colorectal cancer cell lines and clinical tumor specimens.

Granulocyte-macrophage colony stimulating factor (GM-CSF) gene expression and protein production were investigated in colorectal cancer cell lines and surgical specimens. In 6 of 6 established tumor lines, expression of the GM-CSF gene was observed by reverse transcription polymerase chain reaction (RT-PCR). Furthermore, for 2 of the lines, the cytokine was detectable in > or = 100 pg/ml amounts in culture supernatants by enzyme-linked immunosorbent assay tests. Addition of recombinant GM-CSF at doses ranging between 30 pg and 30 ng/ml did not appear to affect the proliferation of colorectal cancer cell lines as measured by 3H-thymidine incorporation. GM-CSF gene expression was then examined in surgical specimens by a densitometry-assisted, semiquantitative RT-PCR technique. In the 10 samples analyzed, significantly higher expression was detectable in tumors, as compared with autologous healthy mucosa sampled in the vicinity (2 cm) or at distance (10 cm) from the neoplastic focus. Immunohistochemistry studies performed on 13 specimens led to the identification of intracytoplasmic GM-CSF in tumor cells in 9 samples. In 6 of these, positivity of stromal fibroblasts and lymphocytes adjacent to the tumor was also observed. In contrast, intracellular GM-CSF was only detectable in 2 cases in untransformed epithelial cells, close to the neoplasm, but never in healthy mucosa at distance from the tumor. Infiltration by dendritic cells (DC) was also investigated. In 5 of 8 colorectal cancers tested, DC aggregates accounted for more than 10% of stromal cells. Lower numbers were detectable in healthy mucosa. However, DC infiltration could not be correlated with the presence of GM-CSF-positive neoplastic cells in the tumor specimens. Remarkably, cultured DC were unable to exert significant cytotoxic activity against colorectal cancer cells in vitro.

Effect of yeast on feed intake and performance of cows fed diets based on corn silage during early lactation.

Thirty-six multiparous Holstein cows were fed a mixture of corn silage and concentrate [1:1; dry matter (DM) basis] and long hay (0.9 kg/d) through wk 18 of lactation. Beginning at 30 d prepartum through wk 4 of lactation, the total mixed rations of 18 of these cows were top-dressed daily with 10 g of Biomate Yeast Plus (Chr. Hansen's, Inc., Milwaukee, WI). The other 18 cows served as controls. At wk 5, both control and treated cows were divided into three groups and fed 0, 10, or 20 g/d of yeast. Yeast supplementation during early lactation significantly improved DM intake, milk yield, and the digestibility of crude protein and acid detergent fiber. Least squares means for DM intake, fat-corrected milk yield, crude protein digestibility, and acid detergent fiber digestibility for cows fed 0, 10, 20 g/d of yeast during wk 5 to 18 of lactation were 23.8, 24.7, and 25.0 kg/d; 37.7, 40.7, and 41.4 kg/d; 78.5, 80.8, and 79.5%; and 54.4, 60.2, and 56.8%, respectively. Although numerical responses in DM intake and milk yield were greater for cows fed 20 g/d of yeast than for cows fed 10 g/d of yeast, the response was not significant.

C-type lectin-like receptors in peptide-specific HLA class I-restricted cytotoxic T lymphocytes: differential expression and modulation of effector functions in clones sharing identical TCR structure and epitope specificity.

C-type lectin-like inhibitory receptors are heterodimers consisting of CD94 and NKG2-A-B molecules expressed on NK cells and on a subset of activated T lymphocytes. Their inhibitory effects on NK cytotoxicity and on the NK-like activity of T cell clones have been demonstrated, but no data are currently available on antigen-specific class I-restricted cytotoxic T lymphocytes (CTL). We have generated a panel of HLA-A2.1-restricted CTL clones directed against a nonapeptide derived from a melanoma-associated antigen, dopachrome tautomerase (TRP-2). All clones were CD8+ and TCR alphabeta+. About half of them expressed a CD94bright phenotype, whereas the remaining were CD94dim. Only the CD94bright CTL expressed the NKG2-A-B gene, consistent with the expression of a C-type, lectin-like, inhibitory CD94/NKG2-A-B heterodimer. Both CD94bright and CD94dim clones appeared to require similar amounts of synthetic epitope sensitizing target cells. Addition of anti-CD94 mAb resulted in a significant increase of specific killing by CD94bright, but not by CD94dim clones in the presence of suboptimal concentrations of peptide, whereas, when optimal amounts were used, the mAb did not induce a significant modulation of the cytotoxicity. Antigen-induced inward [Ca2+]i fluxes were unaffected, but an enhancement of TCR down-modulation could be observed in the presence of anti-CD94 mAb at high concentration of antigenic peptide. The analysis of the TCR-Vbeta repertoire of the CTL clones by RT-PCR and immunofluorescence revealed that all clones regardless of CD94 phenotype shared Vbeta22 expression. Most importantly, sequence analysis showed that they all expressed identical Vbeta22 TCR rearranged with Jbeta2.1 and Cbeta2. Taken together, these data indicate that different expression of functionally active lectin-like inhibitory receptors can be detected in CTL clones sharing identical TCR sequence and peptide specificity.