Detection of Serum Antibodies to Ovine Progressive Pneumonia Virus in Sheep by Using a Caprine Arthritis-Encephalitis Virus Competitive-Inhibition Enzyme-Linked Immunosorbent Assay

A competitive-inhibition enzyme-linked immunosorbent assay (cELISA) for detection of antibodies to the surface envelope (SU) of caprine arthritis-encephalitis virus (CAEV) was recently reported (L. M. Herrmann, W. P. Cheevers, T. C. McGuire, D. Scott Adams, M. M. Hutton, W. G. Gavin, and D. P. Knowles, Clin. Diagn. Lab. Immunol. 10:267-271, 2003). The cELISA utilizes CAEV-63 SU captured on microtiter plates using the monoclonal antibody (MAb) F7-299 and measures competitive displacement of binding of the anti-CAEV MAb GPB 74A by goat serum. The present study evaluated the CAEV cELISA for detection of antibodies to ovine progressive pneumonia virus (OPPV) in sheep. Three hundred thirty-two sera were randomly selected from 21,373 sheep sera collected throughout the United States to determine the sensitivity and specificity of cELISA and agar gel immunodiffusion (AGID) based on immunoprecipitation (IP) of [³⁵S]methionine-labeled OPPV antigens as a standard of comparison. A positive cELISA test was defined as >20.9 percent inhibition (% I) of MAb 74A binding based on two standard deviations above the mean % I of 191 IP-negative sheep sera. At this cutoff, there were 2 of 141 false-negative sera (98.6% sensitivity) and 6 of 191 false-positive sera (96.9% specificity). Sensitivity and specificity values for IP-monitored AGID were comparable to those for cELISA for 314 of 332 sera with unambiguous AGID results. Concordant results by cELISA and IP resolved 16 of the 18 sera that were indeterminate by AGID. Additional studies evaluated cELISA by using 539 sera from a single OPPV-positive flock. Based on IP of 36 of these sera, there was one false-negative by cELISA among 21 IP-positive sera (95.5% sensitivity) and 0 of 15 false-positives (100% specificity). We conclude that the CAEV cELISA can be applied to detection of OPPV antibodies in sheep with high sensitivity and specificity.     

Expression of cytokine mRNA in lentivirus-induced arthritis.

Infection of goats with the lentivirus caprine arthritis encephalitis virus (CAEV) leads to persistent infection and development of chronic arthritis. We analyzed the expression of cytokines and viral RNA in the joints of goats at early time points after experimental infection with CAEV and in those of animals suffering from chronic arthritis as a result of natural infection. In situ hybridization experiments showed that the pattern of cytokine expression in caprine arthritis was similar to that found in rheumatoid arthritis (RA), with a few cells expressing the lymphocyte-derived cytokines interferon (IFN)-gamma and interleukin (IL)-2 and rather more cells expressing monocyte chemoattractant protein (MCP)-1, IL-6, and tumor necrosis factor (TNF)-alpha. IFN-gamma mRNA expression in experimentally infected joints peaked at day 12 and was mostly detected in areas containing viral RNA. At later time points, no IFN-gamma- or virus-expressing cells were found in inflamed joints but both were again detected in goats with severe arthritis. Interestingly, at the clinical stage of arthritis reflecting the chronic stage of infection, the inflammatory lesion was found to be immunologically compartmentalized. Humoral immune responses and cell-mediated immune responses appeared to concurrently occur in distinct areas of the synovial membrane.     

Evaluation of agar gel immunodiffusion serology using caprine and ovine lentiviral antigens for detection of antibody to caprine arthritis-encephalitis virus.

The sensitivity of the agar gel immunodiffusion (AGID) test for the detection of antibody to caprine arthritis-encephalitis virus (CAEV) was investigated with CAEV or ovine progressive pneumonia virus (OPPV) as the source of antigen. A total of 218 goat serum specimens were tested for anti-CAEV antibody by AGID and immunoprecipitation of [35S]methionine-labeled CAEV. In comparison with that of immunoprecipitation, the sensitivity of the CAEV AGID test was 0.91, and that of the OPPV AGID test was 0.56. The AGID test with either antigen was 100% specific. The lower sensitivity of the OPPV AGID test in detecting caprine antibody to CAEV indicates that OPPV antigen is of limited value for use in CAEV diagnosis and control programs.     

Systemic hematologic effects of PEG-rHuMGDF-induced megakaryocyte hyperplasia in mice

PEG-rHuMGDF injected daily in normal mice causes a rapid dose-dependent increase in megakaryocytes and platelets. At the same time that platelet numbers are increased, the mean platelet volume (MPV) and platelet distribution width (PDW) can be either decreased, normal, or increased depending on the dose and time after administration. Thus, PEG-rHuMGDF at a low dose causes decreases in MPV and PDW, MGDF at an intermediate dose causes an initial increase followed by a decrease in MPV and PDW, and PEG-rHuMGDF at higher doses causes an increase in MPV and PDW followed by a gradual normalization of these platelet indices. In addition to the expected thrombocytosis after 7 to 10 days of daily injection of high doses of PEG-rHuMGDF, a transient decrease in peripheral red blood cell numbers and hemoglobin is noted accompanied in the bone marrow by megakaryocytic hyperplasia, myeloid hyperplasia, erythroid and lymphoid hypoplasia, and deposition of a fine network of reticulin fibers. Splenomegaly, an increase in splenic megakaryocytes, and extramedullary hematopoiesis accompany the hematologic changes in the peripheral blood and marrow to complete a spectrum of pathologic features similar to those reported in patients with myelofibrosis and megakaryocyte hyperplasia. However, all the PEG-rHuMGDF-initiated hematopathology including the increase in marrow reticulin is completely and rapidly reversible upon the cessation of administration of PEG-rHuMGDF. Thus, transient hyperplastic proliferation of megakaryocytes does not cause irreversible tissue injury. Furthermore, PEG-rHuMGDF completely ameliorates carboplatin-induced thrombocytopenia at a low-dose that does not cause the hematopathology associated with myelofibrosis.     

Colorectal cancer extracellular acidosis decreases immune cell killing and is partially ameliorated by pH-modulating agents that modify tumor cell cytokine profiles

Tumor cells upregulate myriad proteins that are important for pH regulation, resulting in the acidification of the extracellular tumor microenvironment (TME). Abnormal pH is known to dampen immune function, resulting in a worsened anti-tumor immune response. Understanding how extrinsic alterations in pH modulate the interactions between immune cells and tumors cells will help elucidate opportunities for new therapeutic approaches. We observed that pH impacts the function of immune cells, both natural killer (NK) and T cells, which is relevant in the context of a highly acidic TME. Decreased NK and T cell activity was correlated with decreasing pH in a co-culture immune cell-mediated tumor cell-killing assay. The addition of pH-modulating drugs cariporide, lansoprazole, and acetazolamide to the co-culture assay was able to partially mitigate this dampened immune cell function. Treatment of colorectal cancer (CRC) cells with NHE1 inhibitor cariporide increased CRC cell-secreted cytokines involved in immune cell recruitment and activation and decreased cytokines involved in epithelial-mesenchymal transition (EMT). Cariporide treatment also decreased CRC cell shed TRAIL-R2, TRAIL-R3, and PD-L1 which is relevant in the context of immunotherapy. These experiments can help inform future investigations into how the pH of the tumor microenvironment may be extrinsically modulated to improve anti-tumor immune response in solid tumors such as colorectal cancer.     

Caprine arthritis-encephalitis virus-infected goats can generate human immunodeficiency virus-gp120 cross-reactive antibodies(1)

Lentiviruses display surprisingly disparate clinical manifestations in their specific hosts, share complex genetic structures, and exhibit extensive diversity, particularly in their envelope genes. The envelope protein, gp135, of caprine arthritis-encephalitis virus (CAEV) has minimal primary sequence homology to gp120, the envelope protein of human immunodeficiency virus (HIV). Nevertheless, they bear certain similarities in that they both possess five variable regions, both are heavily glycosylated, and both share short sequence motifs. We establish a further relationship and demonstrate that some goats, infected with CAEV, possess gp135-specific antibodies which cross-react with gp120 from several HIV strains, provided the protein is expressed in insect cells. We show that, although the cross-reactivity of these immunoglobulins depends on the level of glycosylation, nevertheless, some antibodies recognize the protein epitopes on gp120, at least some of which are linear in character. Further characterization of this unexpected cross-reaction will define its potential therapeutic utility.     

Immune response to caprine arthritis-encephalitis virus surface protein induced by coimmunization with recombinant vaccinia viruses expressing the caprine arthritis-encephalitis virus envelope gene and caprine interleukin-12

The objective of this study was to determine if interleukin (IL)-12 can focus an antigen specific type 1 immune response characterized by activation of Th1 lymphocytes and production of IgG2 antibodies in vivo. Saanen goats co-immunized with recombinant vaccinia viruses expressing caprine IL-12 (rRB-IL12) and the caprine arthritis-encephalitis virus (CAEV) envelope (env) gene (rWR-63) were evaluated for development of immune responses to the CAEV env encoded surface glycoprotein (SU). Immune responses were defined by: (i) SU antibody titers; (ii) the ratio of SU IgG1 and IgG2 antibodies; (iii) interferon gamma (IFNgamma) and IL-4 gene expression and proliferative response of SU stimulated lymph node mononuclear cells (LNMC). Apart from enhancement of IFNgamma and IL-4 gene expression in SU stimulated LNMC, rRB-IL12 did not affect the immune response to rWR-63 encoded SU. Thus, localized production of exogenous species specific IL-12 at the site of immunization did not focus initial priming of antigen reactive Th lymphocytes. These results are in contrast to previous studies using inbred mice and raise questions regarding the use of cytokine adjuvants to focus immune responses in outbred animals.     

Simultaneous measurement of gallbladder emptying with cholescintigraphy and US during infusion of physiologic doses of cholecystokinin: a comparison

Both ultrasonography (US) and cholescintigraphy are used to study gallbladder dynamics. The present study was undertaken to determine whether the two methods provide the same or different information relating to gallbladder emptying. Emptying was simultaneously studied with both methods during infusion of graded physiologic doses of cholecystokinin (CCK) in six healthy subjects. Infusion of stepwise increasing doses of CCK, ranging from 0.03 to 0.5 Ivy dog units per kilogram of body weight per hour (IDU/kg.h), induced significant dose-related increases in plasma CCK, decreases in gallbladder volume assessed with US, and gallbladder emptying assessed with cholescintigraphy. The threshold dose for inducing significant gallbladder emptying was 0.13 IDU/kg.h, as determined with both techniques, indicating similar detection limits. There was a highly significant correlation between decreases in gallbladder volume and decreases in radioactive counts over the gallbladder region, with a tendency toward greater gallbladder responses at sonography during the early phase of gallbladder contraction and toward greater responses at cholescintigraphy during the later phase of gallbladder contraction. It is concluded that these methods can be used interchangeably for the quantitation of gallbladder emptying.