Adenosine A2A receptors in diffuse dermal fibrosis: pathogenic role in human dermal fibroblasts and in a murine model of scleroderma.

OBJECTIVE: Adenosine regulates inflammation and tissue repair, and adenosine A2A receptors promote wound healing by stimulating collagen matrix production. We therefore examined whether adenosine A2A receptors contribute to the pathogenesis of dermal fibrosis. METHODS: Collagen production by primary human dermal fibroblasts was analyzed by real-time polymerase chain reaction, 14C-proline incorporation, and Sircol assay. Intracellular signaling for dermal collagen production was investigated using inhibitors of MEK-1 and by demonstration of ERK phosphorylation. In vivo effects were studied in a bleomycin-induced dermal fibrosis model using adenosine A2A receptor-deficient wild-type littermate mice, C57BL/6 mice, and mice treated with adenosine A2A receptor antagonist. Morphometric features and levels of hydroxyproline were determined as measures of dermal fibrosis. RESULTS: Adenosine A2A receptor occupancy promoted collagen production by primary human dermal fibroblasts, which was blocked by adenosine A2A, but not A1 or A2B, receptor antagonism. Adenosine A2A receptor ligation stimulated ERK phosphorylation, and A2A receptor-mediated collagen production by dermal fibroblasts was blocked by MEK-1 inhibitors. Adenosine A2A receptor-deficient and A2A receptor antagonist-treated mice were protected from developing bleomycin-induced dermal fibrosis. CONCLUSION: These results demonstrate that adenosine A2A receptors play an active role in the pathogenesis of dermal fibrosis and suggest a novel therapeutic target in the treatment and prevention of dermal fibrosis in diseases such as scleroderma.

Persistence of intestinal antibody response to heterologous rotavirus infection in a murine model beyond 1 year.

We used an ELISPOT (enzyme-linked immunosorbent spot) assay to quantitate the long-term rotavirus-specific intestinal antibody response in a murine model. The frequency of murine intestinal antibody-secreting cells (ASCs) was followed for a period of 1 year after a single dose of rhesus rotavirus (10(6) PFU) was administered at 10 days of age. Some animals were boosted at that time with a second dose. One year after infection, virus-specific ASCs declined from acute-phase levels, but they were still present at significant levels (1.32 x 10(4) virus-specific ASCs per 10(6) intestinal mononuclear cells; approximately 17% of the previously reported response at 1 month after infection). A booster dose 1 year after the primary infection produced a 100% increase in virus-specific ASCs but did not restore the response to that of the primary infection.

Recombinant baculovirus-expressed rotavirus protein (VP4) in an ELISPOT assay of antibody secretion.

Studies on the protein specificity of the intestinal antibody response to rotavirus infection have been hampered by lack of antigenically conserved isolated proteins to serve as antigens in immunochemical assays. In this report, the use of an antigenically conserved baculovirus-expressed rotavirus protein (VP4) as a capture antigen in the ELISPOT assay is described. Anti-VP4 antibody-secreting hybridoma cells are used as a test population to show that expressed VP4 as the capture antigen detects numbers of antibody secreting cells comparable to intact rotavirus particles. Hybridoma cells specific for other rotavirus proteins are used to ensure the specificity of the expressed VP4 in the assay. The flexibility and ease of use of a recombinant expressed protein product as a capture antigen in this assay dramatically enhances the ability to quantitate intestinal antibody responses to specific viral proteins.

Murine intestinal antibody response to heterologous rotavirus infection.

Rotavirus is the most important worldwide cause of severe gastroenteritis. Extensive efforts have been devoted to the design of a vaccine that will prevent disease, but development of a more effective vaccine strategy may require progress in the understanding of the mucosal immune response to replicating viral antigens. In this article, we report the characterization of the intestinal antibody response of a murine model to heterologous infection with the rhesus rotavirus vaccine strain. We have adapted the enzyme-linked immunospot assay to measure this response without the difficulties associated with measurement of antibodies in intestinal contents or the artifacts associated with culturing of lymphocytes. The predominant response in terms of antibody-secreting cells (ASC) is seen in the small intestine lamina propria, which can be measured within 4 days of infection, peaks 3 weeks after infection, and remains near that level for longer than 8 weeks. The magnitude of the immunoglobulin A (IgA) cell response is approximately 10 times greater than the intestinal IgG cell response, and IgM cells are rare. Virus-specific ASC constitute approximately 50% of all ASC in the gut at the peak of the virus-specific response. This response is considerably greater than responses to nonreplicating mucosal antigens measured by similar techniques. Enteral infection engenders minimal virus-specific ASC response in the spleen. Rhesus rotavirus-specific enzyme-linked immunosorbent assay and neutralization assays of serum and intestinal contents did not correlate with virus-specific ASC response.

VP4-specific intestinal antibody response to rotavirus in a murine model of heterotypic infection.

We have adapted a murine model of heterotypic rotavirus infection for the purpose of evaluating the intestinal antibody response to an infection that mimics human vaccination. Neonatal mice were infected with the rhesus rotavirus (RRV). The enzyme-linked immunospot assay was used in order to avoid common artifacts in the quantitation of intestinal immune responses inherent in measurements of luminal or serum immunoglobulins and to obtain easily quantifiable data in a flexible and convenient format. Functionally active lymphocytes were harvested from the spleen, small intestinal lamina propria, Peyer's patches, and mesenteric lymph nodes and processed into single-cell suspensions. Antibody-secreting cells (ASC) were quantitated from 5 to 50 days after infection for total, RRV-specific, baculovirus-expressed VP4-specific, and single-shell RRV-specific ASC secreting either immunoglobulin G (IgG), IgM, or IgA. The response to VP4 constituted less than 1.5% of the total virus-specific response, which was located almost exclusively in the gut and was 90% IgA. Intestinal ASC were directed overwhelmingly toward proteins incorporated in the single-shell particle, predominantly VP2 and VP6. We conclude that the antibody response to VP4, thought to be the site of the important neutralization sites conserved among several rotavirus serotypes, is an extremely small portion of the overall antibody response in the intestinal tract.

Specific chromosomal changes in patients with acute nonlymphocytic leukemia from India.

We analysed forty consecutive patients with acute nonlymphocytic leukemia (ANLL) using methotrexate cell synchronization and 24 h-unstimulated cultures of bone marrow cells to determine the incidence of chromosomal aberrations and the association of specific anomalies with FAB morphological subtypes, in an Indian population. All patients demonstrated an abnormal karyotypic pattern. The specific chromosomal changes viz., t(9;22), t(8;21), t(15;17), t/del(11q), 12p- were found in M 1(3/5), M2(8/15), M3(8/8), M4(1/1) M5(2/4) and M2Ba(1/1) (M2 with Basophilia) patients. Abnormalities of 11q were also noted in two M2 patients showing monocytic involvement. A translocation involving chromosomes 6 and 9 was seen in one patient with M1 and two patients with M2. An inv(16) was observed in M1 (one case), M2 (two cases) and M6 (one case). A del(16) was noted in an M4 case. Although t(9;22) is frequently associated with M1 patients, it was also detected in M2 (two patients) and M4 (one patient). Among all the FAB specific anomalies described above, t(8;21) and t(15;17) were observed only in M2 and M3 patients, respectively. Interestingly, one M2 patient had two independent clones, one with t(8;21) and t(9;11). Deletion or translocation involving 11q was found in a Ph positive M4 patient. New structural rearrangements such as t(1;7) (q32;q36) in association with t(8;21), and t(14;22) (q32;q11) in association with del(11)(q23) were detected in a M2 and a M5 patient, respectively. In conclusion, our studies have revealed that the incidence of FAB specific abnormalities viz., t(8;21); t(15;17), t(9;22), t/del(11q) and also other recurrent anomalies viz., -7/7q-, +8 is much higher in our patients, as compared with other countries. This difference may be attributed to the influence of differential environmental exposure to unknown carcinogenic agents.

Cytogenetic findings in patients with acute promyelocytic leukemia and a case of CML blast crisis with promyelocytic proliferation.

Nineteen patients with acute promyelocytic leukemia (APL) and one with promyelocytic blast crisis were studied by a methotrexate cell synchronization technique and by 24-hour short-term culture. Nineteen cases of APL included two cases of the microgranular variant (M3V). Except in one case of M3V, t(15;17) was detected in all patients. The breakpoints were determined as 15q22 and 17q12-21. A chronic myeloid leukemia (CML) blast crisis patient with high promyelocyte count also had a t(15;17) as well as a masked Ph chromosome. Other abnormalities, such as +8,del(7q) and an i(17q), were also observed in some patients. Our studies have indicated that 1) the translocation (15;17), characteristic of APL, was present in our population in almost all patients; 2) the presence of an identical abnormality in a promyelocytic CML blast crisis supported and confirmed the phenomenon of association of specific chromosome change with target cell type; and 3) the precise localization of breakpoints on chromosome 17 is still difficult to determine. The identification of as yet unknown genes at region 17q11-q21 and their subsequent translocation on chromosome 15 will help in assigning a precise position to these breakpoints.