Association of genetic polymorphisms of macrophage inhibitory factor (MIF) and B-cell activating factor (BAFF) with the detection of donor specific antibodies in kidney allograft recipients

The posttransplant development of donor specific antibodies (DSA) initiates the antibody mediated rejection (AMR), which is associated with the increased rate of graft loss. One of the characteristics of AMR is the infiltration of innate immune system including macrophages, monocytes, neutrophils or NK cells. Macrophage inhibitory factor (MIF) and B-cell activating factor (BAFF) are well known cytokines that are associated with the activation of the innate immune system which can damage kidney allograft. In this article, the association of the genetic polymorphisms of MIF and BAFF with the development of DSA including Class I and II in kidney transplant patients is investigated. A total of 231 renal transplant patients between 2008 and 2012 at St. Vincent Medical Center, CA were studied in a retrospective study design. DSA were determined by Luminex technology, and single nucleotide polymorphisms (SNP) of MIF and BAFF were determined by the real time PCR based on 5′ nuclease allelic discrimination assay. The genetic polymorphisms of MIF rs1007888 (C/T) was associated with increased risk of positive DSA detection (p = 0.04) after transplantation, and consistently significant after 1 year (p = 0.016). Furthermore, the presence of C allele were associated with the increased risk of Class I DSA detection (OR 1.816, CI 1.141–2.889, p = 0.011). Also, genetic polymorphisms of BAFF rs12583006 were associated with the increased risk of Class II DSA detection (p = 0.033). In conclusion, the genetic polymorphisms of MIF and BAFF may increase the risk of posttransplant development of DSA. This result suggests the association between the development of posttransplant DSA and the activation of innate immune system.     

Classification of sensitizing and irritative potential in a combined in-vitro assay

We have developed a coculture system which in parallel indicates the sensitizing and irritative potential of xenobiotics. The assay is named loose-fit coculture-based sensitization assay (LCSA) and may be performed within 5 days. The system is composed of human monocytes that differentiate to a kind of dendritic cells by 2-day culturing in the presence of allogenic keratinocytes. The culture medium is enriched by a cocktail of recombinant cytokines. On day 3, concentration series of probes are added. On day 5, cells are harvested and analyzed for expression range of CD86 as a marker of sensitizing potential and for uptake of the viability stain 7-AAD as a marker of irritative potential. Estimation of the concentration required to cause a half-maximal increase in CD86 expression allowed quantification of sensitizing potential, and estimation of the concentration required to reduce viability to 50% allowed quantification of irritative potential. Examination of substances with known potential resulted in categorization of test scores. To evaluate our data, we have compared results with those of the validated animal-based sensitization test, the murine local lymph node assay (LLNA, OECD TG 429). To a large extent, results from LCSA and from LLNA achieved analogous grouping of allergens into categories like weak-moderate-strong. However, the new assay showed an improved capacity to distinguish sensitizers from non-sensitizers and irritants. In conclusion, the LCSA contains potential to fulfil the requirements of the EU's programme for the safety of chemicals “Registration, Evaluation, Authorisation and Restriction of chemical substances” (REACH, 2006) to replace animal models.     

Microparticles and their emerging role in cancer multidrug resistance

Drug resistance is a major obstacle to the successful treatment of cancer as tumor cells either fail to reduce in size following chemotherapy or the cancer recurs after an initial response. The phenomenon of multidrug resistance (MDR) is particularly problematic as it involves the simultaneous resistance to numerous chemotherapeutics of different classes. MDR is predominantly attributed to the overexpression of efflux transporters such as P-glycoprotein (P-gp) and the Multidrug Resistance-Associated Protein 1 (MRP1). P-gp and MRP1 are members of the ATP Binding Cassette (ABC) superfamily of transporters and are capable of effluxing many chemotherapeutics out of cancer cells, allowing them to survive the toxic insult. Numerous strategies have been developed over the years to circumvent MDR. Of these, the discovery and implementation of P-gp and MRP1 inhibitors have been most extensively studied. However, these inhibitors have not been able to be used clinically. While research continues in this area, it must also be acknowledged that other avenues must be explored. Recently, the novel 'non-genetic' acquisition of P-gp-mediated MDR by microparticles (MPs) has been reported. MPs are vesicles 0.1-1μm in diameter that are released via plasma membrane blebbing. They are important mediators of inflammation, coagulation and vascular homeostasis. In addition to surface P-gp protein, MPs also carry various nucleic acid species as cargo. This 'non-genetic' intercellular transfer provides an alternative pathway for the cellular acquisition and dissemination of traits and implicates MPs as important mediators in the spread of MDR and provides a novel pathway for the circumvention of MDR.     

Characterisation of species differences in the platelet ADP and thrombin response

INTRODUCTION: A number of animal models are used to study platelet-dependent diseases. In the present investigation, we have used a simple flow cytometry assay to evaluate platelet function in man, rat, mouse, guinea pig and dog. MATERIALS AND METHODS: Platelet activation was evaluated in diluted whole blood by measuring fibrinogen binding to activated platelets using a polyclonal anti-human fibrinogen antibody that cross-reacts with fibrinogen from all species tested. The assay was used to evaluate platelet function with respect to ADP and thrombin sensitivity. The relative importance of the two platelet ADP receptors and total ADP in the thrombin response was also studied by using receptor-specific antagonists and apyrase, respectively. RESULTS: Mouse platelets were most sensitive to both agonists. Unlike in man and dog the maximal response to ADP was greater than to thrombin in mouse, rat and guinea pig. P2Y(12) blockade was in all species equally effective as ADP removal in inhibiting thrombin-induced platelet activation whereas P2Y(1) blockade was almost ineffective. CONCLUSION: The present study describes a simple platelet function test that can be used to evaluate platelet function in man, rat, mouse, guinea pig and dog. Platelets from the tested species differed in their sensitivity to ADP and thrombin. In contrast to human and canine platelets, mouse, rat and guinea pig platelets displayed a stronger maximal response to ADP than to thrombin. In terms of the relative contribution of P2Y(1) and P2Y(12) in the thrombin response, the P2Y(12) receptor was the key receptor in all species and its blockade gave equal effect as total removal of ADP.     

Dendritic cells maturation promoted by M1 and M4, end products of steroidal ginseng saponins metabolized in digestive tracts, drive a potent Th1 polarization

Ginseng is a medicinal herb widely used in Asian countries, and many of its pharmacological actions are attributed to the ginsenosides. Dendritic cells (DCs) play a pivotal in the initiation of T-cell-mediated immune responses, making them an attractive cellular adjuvant for use in cancer vaccines. In this study, we investigated whether M1 and M4, end products of steroidal ginseng saponins metabolized in digestive tracts, can drive DCs maturation from human monocytes in vitro. Human monocytes were cultured with GM-CSF and IL-4 for 6 days, followed by another 2 days in the presence of M1, M4 or TNF-alpha as a maturation stimulus. Stimulation with 20 microM of M1 or M4 increased expression level of CD80, CD83 and CD86 as expressed by mean fluorescence intensity (MFI) and decreased endocytic activity. M4-primed mature DCs also displayed enhanced T cells stimulatory capacity in a MLR, as measured by T cell proliferation. Mature DCs differentiated with M1 or M4 induced the differentiation of na?ve T cells towards a helper T cell type 1 (Th1) response at DC/T (1:5) cells ratio depending on IL-12 secretion. In CTL assay, the production of IFN-gamma and 51Cr release on M4-primed mature DCs was more augmented than of immature DCs or TNF-alpha-primed mature DCs. These results suggest that M4 may be used on DC-based vaccines for cancer immunotherapy.     

Surface and cytoplasmic immunoglobulin expression in B-cell chronic lymphocytic leukemia (CLL)

Flow cytometric analysis of abnormal lymphocyte populations in chronic lymphocytic leukemia (CLL) has been widely reported to show weak expression of surface immunoglobulin (sIg). The international scoring system to help discriminate between CLL and other B-cell lymphoproliferative disorders lists this as the first of 5 criteria worth 1 point each. In the present study, 30 cases of CLL were studied for surface and cytoplasmic Ig expression. 23 of these 30 (76.7%) cases were positive for sIg. Of these 23, 14 cases (60.9%) showed moderate to bright sIg expression. All of these 23 cases were positive for CD5 and CD23; all were negative for CD10 and only 6 (26.1%) were positive for FMC7. 27 of 30 (90.0%) cases expressed cytoplasmic immunoglobulin (cIg) compared to 5% reported by others. This shows that cytoplasmic Ig occurs in a much greater percentage of cases than reported previously. 23 of 30 (76.7%) cases showed positivity for both surface and cytoplasmic Ig, with 15 showing kappa light chain restriction and 8 showing lambda light chain restriction. Six expressed cytoplasmic Ig only, with 4 showing kappa light chain restriction and 2 showing lambda light chain restriction. One case expressed neither cytoplasmic nor surface Ig. CD38 positivity portends a worse prognosis. Of the 29 cases tested for CD38, 13 (44.8%) were positive. Of these 13 cases, 12 were in the surface Ig/cytoplasmic Ig+ group and 1 in the cytoplasmic Ig+ group only. Also, of the 23 cases tested for CD22 expression, 16 (69.6%) were positive. These data question the use of both "weak" surface Ig expression and lack of CD22 expression as valid scoring criteria for CLL.     

Multiple P2X and P2Y receptor subtypes in mouse J774, spleen and peritoneal macrophages

We investigated P2 receptor expression and function in macrophages from mouse, and in the J774 cell line, and revealed a larger spectrum of P2 receptor subtypes than previously recognised. The nucleotides adenosine triphosphate (ATP), adenosine diphosphate, uridine triphosphate and uridine diphosphate evoked an increase in intracellular calcium and the activation of a potassium current. The sensitivity of these responses to the antagonists suramin, PPADS, MRS 2179 and Cibacron blue suggest the presence of at least three functional P2Y receptor subtypes, most probably P2Y(2), P2Y(4) and P2Y(6). ATP also activated P2X receptors, giving rise to a rapidly activating cation conductance. This response was insensitive to the antagonists suramin and Cibacron blue, was potentiated by Zn(2+) and inhibited by acidification suggesting involvement of P2X(4) receptors. In low divalent cation solution, responses to ATP became larger, and dibenzoyl-ATP became more potent than ATP, indicating the presence of P2X(7) receptors. Immunofluorescence, flow cytometry, Western blots and RT-PCR show that P2X(4) and P2X(7) receptors are the most prominent in both macrophage types, while the expression of the other P2X subunits is variable and sometimes weak or undetectable. These techniques also demonstrated the presence of mRNA for P2Y(1), P2Y(2), P2Y(4) and P2Y(6) receptors along with protein expression for the three subtypes we investigated, namely, P2Y(1), P2Y(2) and P2Y(4).     

Comparison of four different treatment conditions of extended exposure in the in vitro micronucleus assay using TK6 lymphoblastoid cells

In the OECD Guideline 487, a total of four extended exposure treatment conditions are proposed for the in vitro micronucleus (MNvit) assay in the presence and absence of a cytokinesis block and with or without a recovery period. This guideline also states that rodent cell lines and human lymphocytes can be used as shown by many validated studies but that human cell lines such as TK6 and HepG2 are not yet validated. In this present study each extended exposure condition was characterized by investigation using TK6 cells and nine chemicals known to be able to induce micronucleus (MN) in rodent cell lines. The results revealed two concerns: six chemicals did not show significant MN induction in the ‘cytokinesis block without recovery period’; two aneugens showed no dose-dependent cytotoxicity in the ‘cytokinesis block with recovery period’. Further investigation revealed that 3–4 times higher spontaneous MN frequency than that in the other conditions is a possible reason for the low sensitivity, and this high spontaneous MN frequency was not observed in Chinese hamster lung cells under the identical treatment condition. With regard to the two conditions without cytokinesis block, two negative substances were evaluated and found to be negative, suggesting the validity of the TK6 test system for these conditions. Although our findings showed a few concerns for the treatment with cytokinesis block, the TK6 cells were considered to be a reliable cell line to be used for detecting potential inducers of MN in the in vitro micronucleus assay based on the overall results.     

Characterization of human decidual mast cells and establishment of a culture system

Background

Although rodent decidual mast cells (MCs) reportedly play an important role in implantation and placenta formation, the characterization of human decidual MCs has been not well clarified. The aims of this study were to investigate the distribution and characteristics of MCs in human decidua and to establish a culture system for decidua-derived MCs.

Post-transplant increase in soluble human leukocyte antigen-G associated with non-severe cardiac allograft vasculopathy

Cardiac allograft vasculopathy (CAV) is the single most important long-term limitation to heart transplantation. This study aimed to assess the value of monitoring soluble human leukocyte antigen-G (sHLA-G) during the first year post-transplantation to predict the severity of CAV, in 21 out of 77 heart recipients assessed by intravascular ultrasound (IVUS). Serum sHLA-G concentration increased after transplant in recipients free of severe CAV, but decreased in recipients suffering from severe CAV, significant differences between these two groups were found 6 to 12 months post-transplantation. The optimal value of the change in post-transplant sHLA-G for identifying severe CAV was ?0.062%, which maximized sensitivity (80%) and specificity (100%). Importantly, increases in post-transplant sHLA-G were inversely associated with severe CAV, but directly associated with human cytomegalovirus reactivation. In addition, recipients presenting non-severe CAV or an increased sHLA-G post-transplantation, showed higher numbers of CD8⁺CD28⁻ T cells and a down-modulation of CD28 on CD4⁺ lymphocytes, which typically identifies CD8⁺ regulatory T cells and anergic/tolerogenic T helper cells, respectively. In conclusion, quantification of sHLA-G might offer a complementary non-invasive method for identifying recipients at risk of more severe CAV and who might benefit from earlier preventive therapies, although these results need to be confirmed in larger series.