A novel ELISpot method for adherent cells

The purpose of this study was to develop an enzyme-linked immunospot assay (ELISpot assay) that can be used with human adherent cells. While standard enzyme-linked immunosorbent assays (ELISAs) are available and widely used and ELISpot assays are used for nonadherent lymphocytes, no ELISpot assay has been developed for adherent cells. We used primary human fibroblasts from four different tissues (myometrium, lung, gingiva, and orbit), either unstimulated or interleukin (IL)-1beta-activated, to evaluate an ELISpot assay. Antibody pairs for IL-6 and IL-8 were used and results were compared to a standard ELISA. We found that we could reliably detect IL-6 and IL-8 spots with as few as 10 fibroblasts. Optimal cell numbers were 50 cells per well incubated for 8 h, although spots appeared as early as 2 h after incubation. Spots were absent when cells, primary, or secondary anti-cytokine antibodies were omitted from the protocol. Spot number and size can be ascertained using current automated ELISpot reader technology. The frequency of IL-6 and IL-8-producing human fibroblasts could also be determined. For example, 60% of the lung fibroblasts express IL-6, but IL-8 can be detected from only 40% of the cells. Approximately 80% of the human orbital fibroblasts make IL-6, whereas approximately 50% generate IL-8 following IL-1beta stimulation. These new findings show that fibroblasts from different human tissues display different frequencies of cytokine production and this further supports the concept of fibroblast diversity. The sensitivity of this new ELISpot assay is adequate for cytokine detection in just a few cells, unlike the standard ELISA. It should permit ascertaining the frequency of fibroblasts and other adherent cells that produce cytokines and, if desired, can be used in tandem with a standard ELISA to determine total cytokine produced. Moreover, the assay is suitable for normal human adherent cells that are often short-lived and difficult to cultivate.     

Cyclooxygenase-2 deficiency results in a loss of the anti-proliferative response to transforming growth factor-beta in human fibrotic lung fibroblasts and promotes bleomycin-induced pulmonary fibrosis in mice

Prostaglandin E(2) (PGE(2)) inhibits fibroblast proliferation and collagen production. Its synthesis by fibroblasts is induced by profibrotic mediators including transforming growth factor (TGF)-beta(1). However, in patients with pulmonary fibrosis, PGE(2) levels are decreased. In this study we examined the effect of TGF-beta(1) on PGE(2) synthesis, proliferation, collagen production, and cyclooxygenase (COX) mRNA levels in fibroblasts derived from fibrotic and nonfibrotic human lung. In addition, we examined the effect of bleomycin-induced pulmonary fibrosis in COX-2-deficient mice. We demonstrate that basal and TGF-beta(1)-induced PGE(2) synthesis is limited in fibroblasts from fibrotic lung. Functionally, this correlates with a loss of the anti-proliferative response to TGF-beta(1). This failure to induce PGE(2) synthesis is because of an inability to up-regulate COX-2 mRNA levels in these fibroblasts. Furthermore, mice deficient in COX-2 exhibit an enhanced response to bleomycin. We conclude that a decreased capacity to up-regulate COX-2 expression and COX-2-derived PGE(2) synthesis in the presence of increasing levels of profibrotic mediators such as TGF-beta(1) may lead to unopposed fibroblast proliferation and collagen synthesis and contribute to the pathogenesis of pulmonary fibrosis.     

Identification of an antigenic subtype of eastern equine encephalitis virus isolated from a human.

Eastern equine encephalitis (EEE) virus was isolated from the cerebrospinal fluid of a 6-year-old male who had clinically diagnosed aseptic meningitis and subsequently died. Several standard serologic tests that use polyclonal antibody and indirect immunofluorescence and hemagglutination inhibition tests that use monoclonal antibody provided evidence that the isolate was an antigenic subtype of prototype North American EEE virus. We believe that this is the first evidence of an antigenic subtype of EEE virus.     

Immunohistochemical staining of colorectal tissues with monoclonal antibodies to ras oncogene p21 product and carbohydrate determinant antigen 19-9.

Two monoclonal antibodies were applied to benign, dysplastic, and malignant human colorectal tissues using immunohistochemical techniques on formalin fixed paraffin embedded material. RAP-5 antibody is directed against a synthetic peptide, reflecting an amino acid sequence of the ras oncogene p21 protein product. Despite using several different techniques and antibody dilutions differential staining between the various epithelial populations was not obtained. RAP-5 also showed other tissue components such as plasma cells, histiocytes, fibroblasts, smooth muscle and vascular endothelium. CA19-9 antibody recognizes an epithelial surface carbohydrate antigen originally derived from a human colorectal carcinoma cell line: it did not stain normal colorectal mucosa or adenomatous polyps, but showed focal expression of variable strength in regenerative, dysplastic, and cancerous mucosa in ulcerative colitis, and in non-colitic colorectal carcinoma. Neither antibody was found to be a reliable marker of the evolution of malignant mucosal changes, although CA19-9 may be of limited use in confirming adenocarcinoma of gastrointestinal origin.     

Analysis of the targeting of the hypermutational machinery and the impact of subsequent selection on the distribution of nucleotide changes in human V rearrangements

Summary: B cells are unique in that they generate and tolerate a high rate of mutations in their antigen receptor genes and employ these mutations as a basis of avidity maturation. The precise role of the mutational machinery versus subsequent selection in determining the frequency and distribution of mutations has not been fully analyzed. To address these issues, the influence of the intrinsic mutational machinery and subsequent selection on the frequency and distribution of mutations in the expressed human immunoglobulin repertoire was analyzed. Analysis of non-productively rearranged v_H genes from individual human B cells provided an opportunity to examine the immediate impact of somatic hypermtitation without superimposed selective influences. Comparison with the frequency and distribution of mutations in the productively rearranged human V_H genes permitted an estimate of the influences of subsequent selection.     

Molecular cloning and nucleotide sequence of a pestivirus genome, noncytopathic bovine viral diarrhea virus strain SD-1.

Genomic RNA of noncytopathic (NCP) bovine viral diarrhea virus (BVDV) strain SD-1 was extracted directly from serum obtained from a persistently infected animal. cDNA was synthesized and amplified by polymerase chain reaction (PCR) before cloning. The complete genomic nucleotide sequence was determined by sequencing at least two different clones from independent PCR reactions. The 5' and 3' end sequences of the SD-1 genome was determined from 5'-3' ligation clones. The complete genome sequence was comprised of 12,308 nucleotides containing one large open reading frame which encodes an amino acid sequence of 3898 residues with a calculated molecular weight of 438 kDa. In contrast to cytopathic (CP) BVDV strain NADL, which contains a cellular RNA insert of 270 nucleotides and CP BVDV strain Osloss, which has an inserted ubiquitin RNA sequence of 228 nucleotides, the NCP strain SD-1 had no insertion along the genome. Sequence comparison with other pestiviruses revealed that the overall nucleotide sequence homologies of SD-1 are 88.6% with NADL, 78.3% with Osloss, 67.1% with HoCV Alfort, and 67.2% with HoCV Brescia. The overall deduced amino acid sequence homologies of SD-1 are 92.7% with NADL, 86.2% with Osloss, 72.5% with HoCV Alfort, and 71.2% with HoCV Brescia. The most conserved nucleotide and amino acid sequences are located in the 5' untranslated region (5'UTR) and nonstructural protein p80 region, respectively. The viral glycoproteins, particularly gp53, and nonstructural proteins p54 and p58 have the lowest homology comparing both nucleotide and amino acid sequences between SD-1 and other pestiviruses. Extensive analyses of amino acid sequences for the viral structural proteins and nonstructural protein p54 regions from five pestiviruses led to the identification of four conserved domains (designated as C1, C2, C3, C4) and three highly variable domains (designated as V1, V2, V3) within this region. The C1, C2, and C3 domains are located in the capsid protein p14, glycoprotein gp48, and gp25, respectively. The C4 domain is located in the junction between gp53 and p54. Interestingly, out of three variable domains, two (V1, V2) are located in the same glycoprotein gp53. The third variable domain is located in the nonstructural protein p54.     

Monoclonal antibodies to the human mammary gland: III. Monoclonal antibody LICR-LON-M18 identifies impaired expression and excess sialylation of the I(Ma) cell-surface antigen by primary breast carcinoma cells

Monoclonal antibody LICR -LON- M18 identifies the immunodominant oligosaccharide sequence of the I(Ma) blood-group antigen: Gal beta 1----4GlcNAc beta 1----6--. In primary breast cancers this structure is almost totally cryptic, due to "masking" by sialic acid, but can be revealed by digestion with the specific glycosidase neuraminidase. Following desialylation, light microscopic immunohistochemical examination has revealed the epitope identified by LICR -LON- M18 to be heterogeneously distributed throughout the population of breast carcinoma cells. These tumor cells express the antigen as both a cytoplasmic and a surface membrane determinant. In the normal human breast, this structure is expressed exclusively along the luminal plasma membranes of the duct and alveolar littoral epithelial cells. Desialylation of tissue sections of normal resting and lactating breast epithelium with neuraminidase virtually abolishes the heterogeneous intercellular distribution of the I(Ma) determinant. In desialylated nonneoplastic breast tissues, the expression of this antigen is observed within the cytoplasm of some myoepithelial cells, but not in the littoral epithelial cells. The expression of the I(Ma) antigen by neoplastic and normal breast epithelial cells has also been compared with that of the oligosaccharide sequence Gal beta 1----3GalNAc. This structure, recognized by peanut agglutinin, forms the dominant portion of the Thomsen-Friedenreich antigen. With respect to normal and lactating breast epithelial cells, both oligosaccharide structures are sialylated and appear to be similarly misprocessed by breast carcinomas. The masking of surface carbohydrate determinants and the faulty processing of structures usually expressed on the surface of non-neoplastic breast epithelial cells may be important phenomena in the pathobiology of breast carcinomas.     

Ex vivo expansion and prophylactic infusion of CMV-pp65 peptide-specific cytotoxic T-lymphocytes following allogeneic hematopoietic stem cell transplantation.

Cytomegalovirus reactivation and infection post-allogeneic hematopoietic stem cell transplant continue to cause morbidity and mortality. Current pharmacologic therapies are limited by side effects. Adoptive transfer of ex vivo generated cytomegalovirus-specific T cells has the potential to restore immunity, prevent cytomegalovirus, and circumvent the need for pharmacologic therapies. We have generated donor-derived cytomegalovirus-specific cytotoxic T cells using dendritic cells pulsed with the HLA-A2 restricted nonapeptide NLVPMVATV (NLV) derived from the cytomegalovirus-pp65 protein. These cytotoxic T cells have been given prophylactically to 9 recipients aged 4 to 65 years on or after day 28 post-allogeneic hematopoietic stem cell transplant. Only 2 of 9 recipients received T cell depletion in vivo or in vitro. There were no immediate adverse reactions to the infusions. During 97-798 days of follow-up, 2 recipients developed cytomegalovirus reactivation; neither developed cytomegalovirus disease or required pharmacotherapy. Three recipients developed acute graft versus host disease after infusion. Two recipients died, 1 from thrombotic thrombocytopenia purpura secondary to cyclosporine, 1 from complications of graft versus host disease. A transient increase in numbers of cytomegalovirus-specific T cells demonstrated by NLV-tetramer binding was seen in 6 recipients. Prophylactic adoptive transfer of NLV-specific T cells is safe and may be effective in preventing cytomegalovirus reactivation.     

Increase in radiosensitivity of lung micrometastases by hyperbaric oxygen

Four-day-old artificial pulmonary micrometastases of two murine fibrosarcomas, designated FSA and NFSA, showed increased sensitivity to ionizing radiation by a factor of 1·13 when animals were exposed to hyperbaric oxygen breathing before and during irradiation, implying the presence of hypoxia in the micrometastases. At the time of irradiation the diameter of FSA and NFSA metastases was smaller than 200 and 100m, respectively, which, on the basis of oxygen diffusion, could not be responsible for hypoxia. It is assumed that hypoxia of micrometastases is passive, reflecting the radiobiological hypoxia of lung tissue that could exist under normal breathing conditions.