A potential tolerogenic immune mechanism in a trophoblast cell line through the activation of chemokine-induced T cell death and regulatory T cell modulation

BACKGROUND: Successful implantation is followed by a local pro-inflammatoryand Th1 response, subsequently controlled by Th2. Regulatedupon activation, normal T cell expressed and secreted (RANTES)promotes a Th1 response and is implicated as a physiologic tolerogenicfactor; therefore, we studied its potential role in the trophoblast–maternalleukocyte dialog. METHODS: We performed co-cultures of immortalized trophoblast cell line(Swan 71) and peripheral blood mononuclear cells (PBMCs) fromfertile women (n = 23) or with recurrent spontaneous abortions(n = 18, RSA). After 24 and 48 h, supernatant and cells wereanalyzed by enzyme-linked immunosorbent assay, fluorescence-activatedcell sorting, Western blot and apoptosis assay. To investigatethe physiological effects at peripheral level, we co-culturedmaternal and paternal PBMCs with conditioned media from Swancells and progesterone. RESULTS: Following interaction of maternal PBMCs and trophoblast cells,RANTES production increased (P < 0.05) and was accompaniedby low levels of interferon , interleukin-12 p70 and high levelsof tumor necrosis factor-, nitrites and leukemia-inhibitoryfactor. RANTES production resulted in elevated apoptosis ofpotentially deleterious maternal CD3+ lymphocytes, accompaniedby a decrease in the proliferative maternal response. Duringfetal–maternal dialog, the anti-RANTES antibody significantlyreduced the frequency of CD4+CD25+Foxp3+ cells (P < 0.05)and was associated with trophoblast cell survival. However,co-cultures of Swan cells and RSA-PBMCs displayed a differentialRANTES kinetics, lower levels of regulatory T cells (Tregs)and CD3+annexin-V+cells, accompanied by higher levels of apoptotictrophoblast cells. CONCLUSIONS: RANTES promotes an adequate pro-implantatory microenvironmentthat influences trophoblast cell survival and modulates thebalance of maternal Treg/T effector lymphocytes in favor ofmaternal tolerance.     

Development and Validation of a Fluorescent Microsphere Immunoassay for Soluble CD30 Testing

Testing for soluble CD30 (sCD30), an indicator of Th2 immune response, is a useful prognostic marker in solid organ transplantation, lymphoproliferative disorders, autoimmunity, and various parasitic diseases. In this study we report the development and validation of a fluorescent microsphere immunoassay for the detection of sCD30 in serum, plasma, and culture supernatants. The dynamic range of this assay is 1 to 400 ng/ml, and the rate of recovery of various concentrations of recombinant sCD30 ranges from 97 to 116% (average recovery, 105%). The test showed a high degree of precision in both intra-assay and interassay studies (coefficients of variation, as high as 7% and 8%, respectively), with a sensitivity of 1 ng/ml. The normal reference range calculated for a cohort of 151 healthy individuals was 1 to 29 ng/ml. The clinical usefulness of the sCD30 fluorescent microsphere immunoassay was demonstrated by showing that levels of sCD30 have a positive correlation with specimens containing high titers of anti-double-stranded DNA antibodies and high titers of immunoglobulin G against Leishmania species. Given the multiplexing potential of the sCD30 fluorescent microsphere immunoassay reported in this study, it is expected that testing of sCD30 concentrations along with those of other cytokines will become an important diagnostic tool for selected immunological and inflammatory diseases where Th2-type cytokine responses have been reported.CD30 (TNFRSF 8) is a transmembrane protein, a member of the tumor necrosis factor (TNF) receptor superfamily. It was originally described as a marker for Reed-Sternberg cells (“Ki-1 antigen”) in Hodgkin''s disease (12, 18, 20). CD30 is expressed on CD4⁺ and CD8⁺ T cells that secrete Th-2 type cytokines (8, 17). Signaling through CD30 plays important roles in T- and B-cell growth, differentiation, and function. The soluble form of CD30 (sCD30) is produced after proteolytic cleavage of the membrane-bound CD30 ectodomain by the TNF-α-converting enzyme (9).Numerous studies have reported that circulating levels of sCD30 may represent a biomarker for outcomes in solid-organ transplantation (16, 21). In addition, other studies have reported that levels of sCD30 have important prognostic value for various lymphoproliferative disorders (4, 15, 22), systemic lupus erythematosus (SLE) (5, 7), and leishmaniasis (1, 2). The current method for quantitation of sCD30 is the enzyme-linked immunosorbent assay (ELISA), which has good sensitivity and specificity. However sCD30 production differs greatly between patients, and the dynamic range of ELISAs requires that many samples be diluted and retested. Moreover, ELISA measures only 1 analyte per well, which precludes the testing of multiple analytes in the same test. In this study, we report the development and validation of a fluorescent microsphere immunoassay suitable for multiplexed determination of sCD30 levels, along with those of other cytokines, in serum and plasma specimens and in tissue culture supernatants. We present data showing the positive correlation of sCD30 levels with titers of anti-double-stranded DNA (anti-dsDNA) antibodies in SLE and with immunoglobulin G (IgG) levels in patients with leishmaniasis.     

Milrinone as a Rescue Therapy for Symptomatic Refractory Cerebral Vasospasm in Aneurysmal Subarachnoid Hemorrhage

Introduction  

Delayed ischemic neurological deficit associated to cerebral vasospasm is the most common cause of sequelae and death that follows the rupture of an aneurysm. The objective of this study was to evaluate the safety and efficacy of intra-arterial Milrinone in patients with symptomatic refractory cerebral vasospasm.     

Subchronic effects of cyanobacterial cells on the transcription of antioxidant enzyme genes in tilapia (<Emphasis Type="Italic">Oreochromis</Emphasis><Emphasis Type="Italic">niloticus</Emphasis>)

The increasing occurrence of toxic cyanobacterial blooms in eutrophic water bodies is nowadays of worldwide concern due to their ability to produce toxins such as microcystins (MCs). These cyanobacterial toxins have been shown to affect aquatic organisms such as fish, resulting in oxidative stress. Among the antioxidant enzymes, glutathione peroxidase (GPx) and soluble glutathione-S-transferases (sGST) play an important role in the detoxification of MCs. In the present work tilapia (Oreochromis niloticus) were orally exposed to cyanobacterial cells containing MCs and non-containing MCs for 21 days. The activity and relative mRNA expression by real-time PCR of both enzymes and the GST protein abundance by Western blot analysis were evaluated in liver and kidney. Also the induction of lipid peroxidation (LPO) was assayed. MCs containing cyanobacterial cells induced an increase of LPO products in both organs, and MCs containing and MCs non-containing cyanobacterial cells altered the activity, gene expression and protein abundance of the enzymes, indicating the importance of GPx and sGST in MCs detoxification. Moreover, liver, the main organ involved in biodegradation and biotransformation, experienced an adaptative response to the toxic insult. These results show for the first time that the subchronic exposure to cyanobacterial cells causes changes in antioxidant and detoxification enzymes and that GPx and GST gene expression are good markers of these alterations in tilapia.     

Validated liquid chromatographic method and analysis of content of tilianin on several extracts obtained from Agastache mexicana and its correlation with vasorelaxant effect

Ethnopharmacological relevance

To optimize the obtention of tilianin, an antihypertensive flavonoid isolated from Agastache mexicana (Lamiaceae), a medicinal plant used in Mexico for the treatment of hypertension. Also, a validated HPLC method to quantify tilianin from different extracts, obtained by several extraction methods, was developed.

Materials and methods

The aerial parts of Agastache mexicana were dried at different temperatures (22, 40, 50, 90, 100 and 180 °C) and the dry material was extracted with methanol by maceration to compare the content of the active constituent tilianin in the samples. Furthermore, EtOH:H₂O (7:3), infusion and decoction extracts were prepared from air-dried samples at room temperature to compare the content and composition of the different extraction methods. Moreover, an ex vivo vasorelaxant test on endothelium-intact aortic rat rings was conducted, in order to correlate the presence of tilianin with the activity of each extract.

Results

Higher concentration and amounts of tilianin were determined from chromatograms in the obtained methanolic extracts from plant material dried at 90, 50, 40 and 22 °C, followed by 100 °C; however, lower concentrations were observed in dried at 180 °C and EtOH:H₂O (7:3). It is worth to notice that methanolic extracts with higher amount of tilianin were the most potent vasorelaxant extracts, even though these extracts were less potent than carbachol, a positive control used. Finally, decoction, infusion and EtOH:H₂O (7:3) extracts did not show any vasorelaxant effect.

Conclusion

Results suggest that extracts with higher concentration of tilianin possess the best vasorelaxant activity, which allowed us to have a HPLC method for future quality control for this medicinal plant.     

Congenital leptin deficiency: diagnosis and effects of leptin replacement therapy

To describe our 10-year experience in treating leptin-deficient humans. Three adults and one boy presented with childhood-onset morbid obesity, hypogonadism and family history of obesity and early death. Serum leptin was inappropriately low. A recessive C105T leptin gene mutation was identified. Metabolic and endocrine assessments were conducted, before and while on and off leptin. The adults' body mass index decreased from 51.2 ± 2.5 to 29.5 ± 2.8 kg/m(2). Serum lipids normalized, insulin resistance decreased, and one of the initially diabetic females became normoglycemic. Hypogonadotropic hypogonadism was reversed, and other changes were observed in the adrenal, sympathetic, somatotropic and thyroid functions. Leptin replacement therapy reverses endocrine and metabolic alterations associated with leptin deficiency. Some of these results may be extrapolated to other diseases.