The FDA-approved compound, pramipexole and the clinical-stage investigational drug, dexpramipexole, reverse chronic allodynia from sciatic nerve damage in mice, and alter IL-1ß and IL-10 expression from immune cell culture.

During the onset of neuropathic pain from a variety of etiologies, nociceptors become hypersensitized, releasing neurotransmitters and other factors from centrally-projecting nerve terminals within the dorsal spinal cord. Consequently, glial cells (astrocytes and microglia) in the spinal cord are activated and mediate the release of proinflammatory cytokines that act to enhance pain transmission and sensitize mechanical non-nociceptive fibers which ultimately results in light touch hypersensitivity, clinically observed as allodynia. Pramipexole, a D2/D3 preferring agonist, is FDA-approved for the treatment of Parkinson's disease and demonstrates efficacy in animal models of inflammatory pain. The clinical-stage investigational drug, R(+) enantiomer of pramipexole, dexpramipexole, is virtually devoid of D2/D3 agonist actions and is efficacious in animal models of inflammatory and neuropathic pain. The current experiments focus on the application of a mouse model of sciatic nerve neuropathy, chronic constriction injury (CCI), that leads to allodynia and is previously characterized to generate spinal glial activation with consequent release IL-1ß. We hypothesized that both pramipexole and dexpramipexole reverse CCI-induced chronic neuropathy in mice, and in human monocyte cell culture studies (THP-1 cells), pramipexole prevents IL-1ß production. Additionally, we hypothesized that in rat primary splenocyte culture, dexpramixole increases mRNA for the anti-inflammatory and pleiotropic cytokine, interleukin-10 (IL-10). Results show that following intravenous pramipexole or dexpramipexole, a profound decrease in allodynia was observed by 1 hr, with allodynia returning 24 hr post-injection. Pramipexole significantly blunted IL-1ß protein production from stimulated human monocytes and dexpramipexole induced elevated IL-10 mRNA expression from rat splenocytes. The data support that clinically-approved compounds like pramipexole and dexpramipexole support their application as anti-inflammatory agents to mitigate chronic neuropathy, and provide a blueprint for future, multifaceted approaches for opioid-independent neuropathic pain treatment.

Phenotypic analysis of adhesion molecules in first-trimester decidual tissue from chorion villus samples.

PROBLEM: First-trimester decidua contain CD56bright/CD16neg lymphocytes that phenotypically resemble a subset of peripheral blood natural killer (NK) cells. Factor controlling their localization to decidua are unknown, but they may relate to trophoblast invasion, endometrial decidualization, and adhesion molecule expression. METHOD OF STUDY: Ninety-one chorion villous samples (CVS) were screened for the presence of decidua, and selected specimens were analyzed for the expression of adhesion molecule pairs using a panel of monoclonal antibodies. RESULTS: Lymphoid cells in CVS-derived decidua expressed CD56, PECAM, intracellular adhesion molecule-1 (ICAM-1), and the integrins LFA-1, alpha E beta 7, and alpha 4 beta 1, and co-receptors for these molecules were expressed by decidual stromal cells, invasive trophoblasts, and venule endothelium. Some endothelium expressed a cuboidal phenotype. CONCLUSIONS: The expression of complimentary pairs of adhesion molecules by maternal lymphoid cells and by either extravillous cytotrophoblast or decidual stromal cells supports the hypothesis that these cells interact within decidua. Also, both LFA-1 and alpha 4 beta 1 may contribute to decidual localization of CD56bright NK cells.

Prenatal diagnosis using fetal cells isolated from maternal peripheral blood: a review.

Many questions remain about the feasibility of using fetal cells from maternal blood for prenatal diagnosis. Although recently there has been more focus on clinically relevant methods, many studies have been performed using blood drawn after invasive procedures, and over a wide range of gestational ages. For methods to be applicable to clinical use, more work is needed on isolating cells early in pregnancy, when termination is still an option for parents who are found to have an affected pregnancy. It is generally agreed that fetal nucleated erythrocytes are the most efficacious cell type for prenatal diagnosis, but it has not yet been shown definitively whether there is an ideal gestational age for sampling, whether ABO incompatibility might limit availability of fetal cells, or whether the number of cells present might be different in normal versus abnormal pregnancies. PCR has been shown to be a powerful tool in allowing amplification and identification of very small amounts of fetal DNA. However, this is limited to cases in which a specific and unique gene from the father is sought. This means that there is the potential to diagnose many paternally inherited autosomal dominant diseases and some autosomal recessive diseases, in which the parents have different and identifiable mutations. However, when parents are both carriers of the same autosomal recessive mutations, or when the disease is X linked, PCR will not aid in prenatal diagnosis. Cytogenetic analysis of fetal cells by FISH after cell sorting is another potentially useful method of prenatal diagnosis, but requires relatively pure samples of fetal cells or an independent marker that allows easy microscopic identification. The latter might be accomplished by identifying fetal cells through their expression of embryonic hemoglobins or because they contain HLA-G mRNA. In addition, current techniques of cell sorting must be improved so that a higher percentage of fetal cells can be isolated. Currently, the best cell sorting techniques usually produce a maximum purity of 10% fetal cells. Commonly, in normal pregnancies, fewer than 0.1% of the cells isolated after sorting are fetal in origin. Improving the concentration and quantity of fetal cells will improve the accuracy of FISH. Methods such as immunophenotyping that allow the selective identification of fetal cells by microscopy, and can be used in conjunction with FISH, may be extremely valuable because they may allow the genetic analysis of only the few fetal cells within a background preponderance of maternal cells. Although the retrieval of fetal cells from maternal blood is an attractive concept, it must be clearly stated that presently it is only in the investigational phase because of the low sensitivity and specificity. There is no current application for these methods in clinical practice. It remains to be determined whether testing maternal blood for fetal cells or DNA will be used as a screening tool, similar to the maternal serum screening currently in use, or whether the accuracy can be improved to a level such that the techniques can be used diagnostically. Although there are many questions that remain unanswered at this time, the outlook for noninvasive prenatal genetic testing in the future is optimistic.

Isolation of decidual lymphocytes from chorionic villus samples: phenotypic analysis and growth in vitro.

PROBLEM: Giemsa stained cell isolates prepared from chorionic villus samples (CVS) contain granulated cells morphologically similar to large granular lymphocytes. METHOD: Phenotypic characterization of these cellular isolates by two-color immunofluorescence and subsequent in vitro culture in the presence of recombinant interleukin-2 (rIL-2) were done in order to determine whether CVS could serve as a source of decidual lymphocytes. RESULTS: A major fraction of the CVS-derived lymphocytes were characterized as decidual NK cells, exhibiting high levels of CD56 expression (CD56+bright), without concomitant expression of CD16. The T cell population present in CVS-derived lymphocytes contained both CD4+ and CD8+ cells in a ratio somewhat reduced compared to that found in peripheral blood. While both T cells and CD56+bright cells from CVS proliferate in vitro in response to rIL-2 alone, preferential growth of CD56+bright cells was accomplished using a selective culture technique wherein co-culture with an irradiated, B lymphoblastoid cell line promoted the growth of CD56+ cells. CONCLUSION: CVS contains decidual NK cells and T cells that proliferate in response to rIL-2 and/or third party stimulator cells. These culture techniques will allow investigations into the maturation and/or activation of decidual NK cells and T cells.

Alloantibody responses to antigens recognized by rabbit antitrophoblast antisera in trophoblast and mononuclear cell (MNC) membranes by primary aborting women before and after paternal leukocyte immunization.

PROBLEM: Because shared allogeneic antigens are expressed on peripheral blood lymphocytes, as well as trophoblasts, it has been proposed that lymphocyte transfusions may appropriately sensitize recurrent spontaneous aborters (RSA) to trophoblast and lead to pregnancy conservation. The degree to which these responses are affected by this treatment, however, has not been defined. METHOD: SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analyses were used to study the alloantibody responses in RSA patients both before and after immunization with paternal leukocytes (MNC) against membrane proteins isolated from peripheral blood mononuclear cells (MNC), first trimester and full term chorionic villi (CV). RESULTS: A distinct set of antigens with apparent molecular weights of 32, 36, 41, 63, 65, and 85 kDa was identified in both MNC and trophoblast membranes by rabbit anti-trophoblast antisera. In addition, a 55 kDa protein was recognized by MNC membranes. Thirty-eight percent of primary RSA sera recognized this 55 kDa protein. After paternal MNC immunization, all primary RSA samples displayed increased reactivity or produced antibodies to this 55 kDa protein when compared with preimmunization sera. The protein was identified as MCP using a rabbit polyclonal anti-MCP antisera. In contrast, reactivity to the other antigens present in the membrane preparations decreased after paternal MNC immunization. CONCLUSION: Changes in immune reactivity in RSA patients after paternal MNC immunization suggest that immunization alters serum immune reactivity to MNC and trophoblast shared antigens.

Cytokine production in first trimester chorionic villi: detection of mRNAs and protein products in situ.

Growth factor cytokines must play important roles in placental growth and differentiation. We used cDNA-mRNA in situ hybridization to examine whether human first trimester chorionic villi could produce these substances locally. IL-1 beta and IL-4 mRNA was found in both trophoblast layers, whereas CSF-1 and TNF alpha were found mostly in syncytiotrophoblast with much less localization to cytotrophoblast areas. IFN-gamma message was exclusively found in the cytotrophoblast. Immunohistochemical analysis confirmed that cytokines are produced by placental tissue, and the patterns of expression correlated well with the data obtained by in situ hybridization. These data are discussed regarding possible roles of cytokines in regulation of MHC antigen expression by trophoblasts and immunomodulation of decidual lymphocytes.

CD8+/DR+ T gamma cells inhibit the autologous mixed lymphocyte reaction.

The proliferative response of T cells during autologous mixed lymphocyte reactions (AMLR) was affected by depletion of IgG Fc receptor+ T lymphocytes (Tg). Removal of Tg cells resulted in enhanced proliferation, and EA-rosette isolated Tg cells, when added to AMLR cultures as irradiated third components, reduced the uptake of 3H-thymidine by 63-87% in a dose-dependent manner. Negative selection using an avidin-biotin affinity chromatography technique demonstrated that the suppression was mediated by DR+ Tg cells; the major proportion of which also expressed the CD8 antigen. By comparing AMLR supernatants collected from control (lacking Tg) and suppressed (containing Tg) cultures on days 2, 3, and 4, it was established that supernatants from suppressed cultures had significantly reduced levels of cytokine activity. These data indicate that the CD8+/DR+ Tg cells function as suppressor cells during an AMLR and reduce the proliferative response by inhibiting AMLR responder T cells from producing the cytokines necessary for in vitro growth.

Diverse patterns of immune and non-immune-mediated disease in EMC M-variant-infected mice.

The M variant of EMC virus exhibits unique tropism for the beta cells of the islets of Langerhans. However, the severity and character of the lesion differ in various strains of animals. This paper briefly reviews the features of these lesions and considers their pathogenesis.

Helper-inducer T-lymphocytes mediate diabetes in EMC-infected BALB/c ByJ mice.

Diabetes mellitus developing in BALB/c ByJ mice infected with the M variant of encephalomyocarditis (EMC) virus is dependent on thymic immune mechanisms. We treated mice with the anti-T-lymphocyte monoclonal anti-L3T4 and anti-Lyt2.2 antibodies before virus inoculation and monitored the infection and occurrence of diabetes thereafter. Mice depleted of L3T4+ cells exhibited a reduced incidence and severity of diabetes compared with both untreated and anti-Lyt2.2-treated animals. All mice sustained pancreatic infections, but islet lesions with beta-cell degranulation only occurred in control infected and anti-Lyt2.2-treated animals. These data support the conclusion that the induction of diabetes in EMC-infected BALB/c mice is immune mediated and controlled by the L3T4 T-lymphocyte subpopulation.