Immunosuppressive Substance Research Articles

Supplementation of rapamycin during in vitro maturation promotes oocyte quality and subsequent embryo development in bovine

BackgroundThe developmental competence of oocyte derived from in vitro maturation (IVM) is significantly lower than those of oocyte matured in vivo. Rapamycin is an immunosuppressive substance and widely used to prolong the survival and preserve the cells culture. The objective of this research was to explore whether rapamycin supplementation during IVM enhances meiotic maturation, oocyte quality, and subsequent embryonic development.MethodsDepending on the purpose, bovine cumulus oocyte complexes were matured without (control) or with rapamycin at different concentrations (0, 1, 10, and 100 nM) for 22 h (h) followed by 6 h in vitro fertilization, and zygotes were cultured for 8 days. Following treatment, oocytes developmental competence was assessed by meiotic progression, intra-oocyte GSH synthesis, reactive oxygen species (ROS) levels, and subsequent embryonic development.ResultsThe results showed that addition of 1 nM rapamycin to IVM medium significantly increased the nuclear maturation (90.1 ± 1.5 vs. 81.2 ± 2.5; P < 0.01) and subsequent embryonic development (47.6 ± 2.2 vs. 41.5 ± 1.9; P < 0.05) than that observed in the control group. Consequently, compared to the control group, the relative fluorescence’s intensity of ROS levels in oocyte significantly reduced by rapamycin treatment. More importantly, rapamycin supplementation during maturation significantly increased the reduced glutathione synthesis levels in oocyte compared to that observed in control, indicating the bidirectional communication between cumulus cells and oocyte becomes easy via rapamycin treatment.ConclusionsThis study suggests that the presence of rapamycin during maturation can afford to enhance reduced glutathione synthesized by the oocyte, which is an internal oocyte antioxidant defense and contributes to improve subsequent high-quality embryo production.

Incomplete reminder cues trigger memory reconsolidation and sustain learned immune responses

Peripheral immune responses can be modulated by taste-immune associative learning where the presentation of a sweet taste as conditioned stimulus (CS) is paired with the injection of an immunosuppressive substance as unconditioned stimulus (US). Previous findings demonstrate conditioned immunopharmacological properties of the mechanistic target of rapamycin (mTOR)-inhibitor rapamycin, a drug used to ameliorate neurological diseases and for the prevention of graft rejection. However, conditioned responses gradually weaken over time and eventually disappear following repeated exposure to the CS in the absence of the US. Thus, in order to employ learning paradigms in clinical conditions as supportive immunopharmacological therapy it is important to understand the central and peripheral mechanisms of how learned immune responses can be protected from extinction. Against this background, the present study used a taste-immune learning paradigm with rapamycin as US (5 mg/kg). By applying only 10% (0.5 mg/kg) of the therapeutic dose rapamycin together with the CS (taste stimulus) during eight retrieval trials, conditioned animals still displayed suppressed interleukin-10 production and T cell proliferation in splenocytes as well as diminished activity of the mTOR target protein p70s6k in amygdala tissue samples. Together, these findings indicate that reminder cues in form of only 10% (0.5 mg/kg) of the therapeutic dose rapamycin together with the CS (taste stimulus) at retrieval preserved the memory of conditioned properties of rapamycin, characterizing this approach as a potential supportive tool in peripheral and central pharmacotherapy with the aim to maximize the therapeutic outcome for the patient’s benefit.

Allatostatin C modulates nociception and immunity in Drosophila

Bacterial induced inflammatory responses cause pain through direct activation of nociceptive neurons, and the ablation of these neurons leads to increased immune infiltration. In this study, we investigated nociceptive-immune interactions in Drosophila and the role these interactions play during pathogenic bacterial infection. After bacterial infection, we found robust upregulation of ligand-gated ion channels and allatostatin receptors involved in nociception, which potentially leads to hyperalgesia. We further found that Allatostatin-C Receptor 2 (AstC-R2) plays a crucial role in host survival during infection with the pathogenic bacterium Photorhabdus luminescens. Upon examination of immune signaling in AstC-R2 deficient mutants, we demonstrated that Allatostatin-C Receptor 2 specifically inhibits the Immune deficiency pathway, and knockdown of AstC-R2 leads to overproduction of antimicrobial peptides related to this pathway and decreased host survival. This study provides mechanistic insights into the importance of microbe-nociceptor interactions during bacterial challenge. We posit that Allatostatin C is an immunosuppressive substance released by nociceptors or Drosophila hemocytes that dampens IMD signaling in order to either prevent immunopathology or to reduce unnecessary metabolic cost after microbial stimulation. AstC-R2 also acts to dampen thermal nociception in the absence of infection, suggesting an intrinsic neuronal role in mediating these processes during homeostatic conditions. Further examination into the signaling mechanisms by which Allatostatin-C alters immunity and nociception in Drosophila may reveal conserved pathways which can be utilized towards therapeutically targeting inflammatory pain and chronic inflammation.

Exposure to Mycophenolate and Fatherhood

BackgroundMycophenolic acid (MPA) is the active immunosuppressive substance in both mycophenolate mofetil and mycophenolate sodium, and it is widely used after organ transplantation. In women, taking MPA is teratogenic and may also influence spermatogenesis. There is a lack of knowledge regarding outcome of pregnancies fathered by men exposed to MPA.MethodsWe compared outcomes in pregnancies fathered by renal transplant men per whether they had been exposed to MPA or not at time of conception. A nationwide population-based retrospective cohort study was performed. Data from the Norwegian Renal Registry with all renal transplanted men alive between January 1, 1995 and December 31, 2015 were included, and relevant outcome data were extracted from the Medical Birth Registry of Norway.ResultsDuring the given time, 230 immunosuppressed renal transplanted men fathered 350 children (155 on MPA/195 not on MPA). There were no significant increased risks of malformation (3.9% vs. 2.6%, P = 0.49) in MPA exposed versus unexposed cohorts of children. The average dose (±SD) of mycophenolate was 1.42 ± 0.3 g/day and the individual median MPA trough concentration in the time period of anticipated conception and pregnancy was 2.8 ± 1.6 mg/L. Birth weight was similar in exposed and unexposed cohorts of children; 3381 ± 681 g vs. 3429 ± 714 g (P = 0.53).ConclusionsPaternal exposure to MPA did not increase the risk of adverse birth outcomes in children fathered by male kidney transplanted patients. These results are reassuring and support the continuation of paternal MPA treatment before, during, and after conception.

Cancer risk of immunosuppressants in manufacturing

During the chemical and pharmaceutical production of active pharmaceutical substances which are intended for immunosuppressive therapy, the employees may be exposed to these substances via inhalation. Immunosuppressants are linked to development of certain types of cancers e.g., lymphoma or skin cancer in transplant patients. The development of these cancers in patients is linked to the level of immunosuppression needed for transplantation in order to avoid organ rejection. Below these levels, with the immune system functioning uninhibited, cancer is unlikely to develop.An internal workshop was conducted to compare several pharmaceutical substances with the intrinsic property to cause immunosuppression, with the attempt to define the risk of healthy employees to develop cancer due to exposure to immunosuppressive substance at work and to determine the appropriate hazard classification for regulatory purposes. Data are discussed with emphasis on cyclosporine to reason the dose–response relationship and the safe level for occupational exposure. Our review indicates that if the exposure to cyclosporine at the workplace is below the threshold necessary to induce immunosuppression, the risk to develop cancer is negligible. Non-mutagenic immunosuppressants do not contribute to malignancies in occupational setting if their air concentrations do not exceed the immunosuppressive threshold limited with occupational exposure limits (OELs), which is for cyclosporine 17.5μg/m3.

236: Immunosuppressive Agents Differently Suppress T Cell Subpopulations

The recent discovery of a CD4+ T cell subtype characterized by the secretion of interleukin IL-17 (Th-17 cells) had a major impact on our understanding of immune processes not readily explained by the Th-1/Th-2 paradigm. We investigated the efficacy of each member of the main immunosuppressive substance classes to specifically suppress activated lymphocyte subpopulations.

Fingolimod – ein neuer Immunmodulator

Fingolimod is a derivative of the naturally occurring immunosuppressive substance myriocin, with structural similarity to sphingosine, a ubiquitous sphingolipid. It acts as an immunomodulator interfering with the egress of T and B lymphocytes from secondary lymph organs, which results in lymphopenia. A phase II study in patients with multiple scerosis showed a significant reduction in annual relapse rate and lesions in magnetic resonance imaging with an acceptable rate of side effects. An advantage of fingolimod compared to interferon beta, glatirameracetate and natalizumab is its oral availability. Definitive assessment of the compound has to await the results of trials currently being performed.

Oscillation between BK Virus Nephropathy and Rejection – the Frustrating Course of a Living Donor Transplantation

A 56-year-old male patient with terminal renal insufficiency received a living donor kidney transplant from his wife in June 2007. Initial immunosuppression consisted of prednisolone, tacrolimus, and mycophenolate mofetil (MMF). 4 months after transplantation, the serum creatinine increased to 192 micromol/l and urinary analysis revealed the presence of decoy cells. A biopsy showed a focal interstitial nephritis and SV40 antigen was detected in tubular nuclei. Polymerase chain reaction (PCR) in serum and urine showed high titers of BK virus. Tacrolimus was stopped, MMF was reduced, and leflunomide (20 mg/day) was started. The patient was readmitted because the serum creatinine further increased to 262 micromol/l. Leflunomide concentrations were in the target range, but renal biopsy still revealed the presence of BK virus nephropathy. MMF was stopped. Serum creatinine stabilized at 233 micromol/l and PCR for BK virus in serum was negative. In April 2008, a deterioration of renal function occurred (serum creatinine 308 micromol/l) and renal biopsy revealed signs of acute interstitial rejection without the presence of SV40 antigen. A methylprednisolone pulse therapy for 5 days was performed and cyclosporine was added. After a few weeks serum creatinine increased to 444 micromol/l and a new biopsy revealed the reoccurrence of BK virus nephropathy. Since the tubulointerstitial injury was > 80%, no further therapy was performed and soon after dialysis therapy was initiated. BK virus nephropathy is a still rare, but increasing complication of renal transplantation, presumably mediated by intensive immunosuppression. The disease can induce graft dysfunction and may ultimately lead to graft failure. BK virus nephropathy can trigger acute rejection. Unfortunately, therapy of BK virus nephropathy and acute rejection is just the opposite: BK virus nephropathy requires a reduction of immunosuppression whereas acute rejection calls for intensification. A potential therapeutic approach may be leflunomide, an immunosuppressive substance with antiviral properties, but potential severe side effects. The described case demonstrates the frustrating course of a graft from a living donor despite initial successful therapy with leflunomide and illustrates the problems choosing between intensive and moderate immunosuppression.

Human Early Pregnancy Factor and Early Pregnancy Associated Protein before and after Therapeutic Abortion in Comparison with ß-hCG, Estradiol, Progesterone and 17-Hydroxyprogesterone*)

Serum activities and concentrations, respectively, of early pregnancy factor (EPF), early pregnancy associated protein (EPAP), beta-hCG, estradiol, progesterone and 17-hydroxyprogesterone were estimated in 20 healthy primigravidae within the tenth to twelfth completed gestational week before and after therapeutic abortion. EPF, beta-hCG and estradiol markedly decreased after termination of pregnancy, whereas progesterone and 17-hydroxyprogesterone concentrations moderately fell. EPAP showed no significant alterations in a 24 hours period. Because of its possible role as immunosuppressive substance and its short half-life EPF may be a promising immunobiomarker for disturbances in early pregnancy.

Does cyclosporin A influence sex hormone level?

Cyclosporin A is an effective immunosuppressive substance which is extensively applied in various conditions. A well known side effect of high dose Cyclosporin A treatment is the occurrence of hypertrichosis, often referred to as hirsutism. The purpose of the study was therefore to investigate ovarian, adrenal and pituitary hormones as possible mediators of increased hair growth. In 5 female and 11 male patients who had developed hair overgrowth during Cyclosporin A treatment serum androgens (testosterone, dehydroepiandrosterone-sulfate, androstendione), sex hormone binding globuline (SHBG), prolactin (HPRL), 17-hydroxyprogesterone (17-OHP), 17 beta-estradiol (17 beta-E) and cortisol (F) were determined by standard radioimmunoassay methods. The same number of age matched patients with Azathioprine treatment served as control. Both patient groups received additive cortisone treatment of the same dosage. No significant differences of serum androgens were noted between Cyclosporin A treated patients and controls. In addition, normal prolactin levels were defected. 17-OHP was in the low normal range and cortisol below normal due to the additive cortisone treatment. SHBG was within normal range. The findings thus indicate that increased hair growth caused by Cyclosporin A seems not to be caused by an action of the substance on the hormonal level.

A cyclopentanediol analogue selectively suppresses the conserved innate immunity pathways, Drosophila IMD and TNF-α pathways

Innate immunity comprises evolutionarily conserved self-defense mechanisms against microbial infections. In mammals, innate immunity interacts with adaptive immunity and has a key role in the regulated immune response. Therefore, innate immunity is a pharmaceutical target for the development of immune regulators. Using Drosophila ex vivo culture systems, we isolated a cyclopentanediol analogue from Aspergillus sp. as an immunosuppressive substance. This compound selectively suppressed activation of the IMD pathway in Drosophila in vivo and the target molecules of the compound lie between the Imd adaptor protein and dTAK1 kinase in the IMD pathway. In human cells, the compound suppressed TNF-α, but not IL-1β, stimulation-induced activation of NF-κB, suggesting that its target molecules are upstream of TAK1 in mammalian innate immunity.

A novel immunotherapeutic modality with direct hemoperfusion targeting transforming growth factor-β prolongs the survival of tumor-bearing rats

Immune responses are frequently depressed in patients with cancer. One of the reasons for a poor immune response is the presence of increased levels of immunosuppressive substances associated with tumor growth. Transforming growth factor-beta (TGF-beta), a representative immunosuppressive cytokine, plays various roles in the progression of cancer. To remove immunosuppressive substances from tumor-bearing hosts, we developed an immunosuppressive substance adsorption (ISA) column for direct hemoperfusion (DHP) treatment. It is filled with extra-fine fibers that can adsorb TGF-beta. In this study, we investigated the effects of this DHP treatment on serum levels and activities of TGF-beta, cellular immune responses, and anti-tumor effects in KDH-8 (TGF-beta-producing hepatocellular carcinoma cell line)-bearing rats. We further studied the ability of ISA fibers to adsorb tumor-associated immunosuppressive cytokines [TGF-beta, interleukin (IL)-6 and vascular endothelial growth factor (VEGF)] in samples of body fluids obtained from patients with metastatic cancer. DHP treatment decreased serum levels and activities of TGF-beta in tumor-bearing rats and restored T lymphocyte response to mitogen. Tumor growth in rats treated by DHP was significantly slower than that in untreated rats. The survival time of treated rats was significantly longer than that of untreated rats. The concentrations of TGF-beta, IL-6, and VEGF in the samples of human body fluids were decreased markedly by in vitro treatment with ISA fibers. These results suggest that DHP treatment with an ISA column, which removes TGF-beta and other immunosuppressive substances from the sera of tumor-bearing hosts, is potentially a new immunotherapeutic strategy for cancer.

'Vegetable wasps and plant worms' and G-protein-coupled receptors

Following the discovery of an immunosuppressive substance isolated from one of the entomopathogenic fungi 'vegetable wasps and plant worms', FTY720, whose immunosuppressive action is more potent than that of cyclosporin, was developed. In contrast to classical immunosuppressants such as cyclosporin, FTY720 does not affect the growth, maturation and activation of lymphocytes, but causes a reduction in the number of circulating lymphocytes as a result of enhanced homing of lymphocytes to the secondary lymphoid organs. It has recently been reported that FTY720 is phosphorylated in vivo by sphingosine kinase, and the phosphorylated FTY720 is a ligand of the receptors of sphingosine 1-phosphate (S1P), which are G-protein-coupled receptors. S1P has been recognized most prominently as a platelet-derived lipid mediator which regulates several functions of endothelial and smooth muscle cells. In addition, recent studies have suggested the crucial roles for S1P in immunological, and inflammatory modulation.

Therapeutic potential of 4-1BB (CD137) as a regulator for effector CD8(+) T cells.

A fundamental problem of antitumor immunity is tumor-induced immunosuppression. Tumor cells often down-regulate expression of co-stimulatory molecules, tumor antigens, and major histocompatibility complex (MHC) molecules on tumor cells, secrete immunosuppressive substance such as transforming growth factor-beta (TGF-beta) or interleukin-4 (IL-4), and induce apoptosis of effector T cells to escape surveillance. A major goal of antitumor or antivirus immunotherapy is to generate long-lived protective T cells that enable killing of target cells. In this review, we discuss the importance of 4-1BB for development or survival of functionally active effector CD8(+) T cells against tumors, virus infection, and allogeneic immune responses and for potential therapeutic application.

Suppression of immunoglobulin secretion by soluble annexin II.

Annexin II (AII) belongs to a family of glycoproteins that bind negatively charged phospholipids in the presence of calcium. The annexins exert various biological functions. We have previously shown that soluble AII suppresses mitogen-induced lymphoproliferation in vitro. In this study we address the question of whether soluble AII may also affect immunoglobulin secretion. Mononuclear cells from peripheral blood were stimulated with pokeweed mitogen in vitro and immunoglobulin-secreting cells were quantified using an ELISPOT assay. Retroplacental serum and soluble AII significantly inhibited secretion of IgG and IgM when added at concentrations that did not affect lymphoproliferation or cell viability. The inhibitory effect was dose- and time dependent. Significant suppression was observed when soluble AII was added at concentrations of 0.1 and 0.01 microg/ml. The strongest inhibition was observed when soluble AII or retroplacental serum was added initially. The data demonstrate that soluble AII can suppress immunoglobulin secretion in vitro. AII seems to be a potent immunosuppressive substance. The presence of high levels of soluble AII in retroplacental serum may indicate a possible immunomodulatory role in normal pregnancy.

Search for immunosuppressive substances produced in the culture of enteric bacteria isolated from marine fish

Culture supernatants of enteric bacteria isolated from the gastrointestinal tracts of two marine fishes were screened for the production of immunosuppressive substances. At least 50% of the 60 bacterial isolates tested were found to produce immunosuppressants that significantly inhibited the induction of cytotoxic T cells (CTL) in vitro. The strains that secreted the highest suppressive activity was identified as Aeromonas caviae. The immunosuppressive substance purified from the A. caviae culture supernatant was a polysaccharide with a molecular weight of about 70 kDa that contained mannose: glucose (4:1). This polysaccharide did not show nonspecific cytotoxicity, and was found to inhibit the proliferation and differentiation of CD8-positive CTL.

A Study of Early Pregnancy Factor Activity in Preimplantation

Early pregnancy factor (EPF), an Immunosuppressive substance, which appears in pregnant women's sera 48 h after fertilization, is a kind of pregnancy-specific protein. To determine whether the EPF activity could be a super early indicator of pregnancy, we used rosette inhibition assay to detect EPF activity in the sera, collected from 70 women 2-7 days after ovulation intending to conceive monitored by ultrasonography. Simultaneously we selected 40 non-pregnant sera and 12 early-pregnant sera as negative control and positive control, respectively. The results of this study demonstrated that EPF activity is detected in 35 women's sera out of 70 women within 2-7 days after ovulation, and 28 women out of the 35 were pregnant, which was known by follow-up, and 7 were not pregnant, possibly due to either false positive results or embryo loss because of preimplantation failure, thus causing no pregnancy. The other 35 out of 70 had no EPF activity and 34 of them were not pregnant, which was known by follow-up, but one case became pregnant, which was false negative result. Our study showed that diagnosis of the super early pregnancy could be made by detecting EPF activity in maternal serum within the time of preimplantation. The accuracy of pregnancy diagnosis by this method is 88.6%, with a false negative rate of 3.4% and a false positive rate of 17.1%. The beta-HCG level was measured from the above 70 women's sera in order to contrast EPF activity. All of the sera collected 2-6 days following ovulation indicated that there were lower beta-HCG values in very early pregnancy (> or = a5 mIU/ml). On the seventh day after ovulation, EPF activity was detected in 11 out of 15 sera with only 2 of them with a b-HCG level that reached or slightly surpassed that of the early pregnancy diagnosis (5 mIU/ml and 5.4 mIU/ml, respectively). This demonstrated that beta-HCG is not the earliest signal of pregnancy; otherwise the EPF activity is one that appears 2-6 days earlier than beta-HCG appears. We measured the progesterone level of the 48 sera from the 70 collected above within 2-7 days postovulation and found most of them reached the level of progesterone in the luteal phase (7.5-98.3 nmol/L). This indicated that ovulation had taken place in these women, which was in accordance with observations by ultrasonography. Our study showed that diagnosis (of 88.6%) of super early pregnancy could be made with an accuracy of 88.6% by detecting EPF activity in maternal serum within 2-days after ovulation. This offers a basis for pregnancy diagnosis for the women who attempt to terminate their pregnancy safely or who conceive unexpectedly, and it contributes to family-planning.

Factors Affecting Tautomeric Phenomenon of a Novel Potent Immunosuppressant (FK506) on the Design for Injectable Formulation

AbstractDue to its poor solubility in water, immunosuppressive drug substance FK506 was aimed to use nonaqueous solvent such as dehydrated ethanol for designing the injectable formulation. Ethanol, however, has been known to generate the tautomeric compounds from FK506. Herein, factors affecting the tautomeric phenomenon have been studied. Polyoxyethylene hydrogenated castor oil 60 (HCO-60, nonionic surfactant), employed as a solubilizer to avoid producing precipitates in FK506 admixtures, restrained the formation of tautomeric compounds. High temperature accelerated the equilibrium among FK506 and tautomeric compounds, whereas pH and concentration of FK506 solution had no effect on the tautomeric phenomenon.

The immunosuppressive substance 2-chloro-2-deoxyadenosine modulates lipoprotein metabolism in a murine macrophage cell line (P388 cells).

A recently developed immunosuppressive substance, 2-chloro-2-deoxyadenosine (2-CdA), was reported to inhibit monocyte functions at low concentration. Because macrophages play a key role in the formation of atherosclerotic plaques, it was of interest to study the effect of 2-CdA on cellular lipid metabolism. For this purpose we have used a macrophage cell line (P388) to perform incubation studies in the presence of acetylated low density lipoprotein (Ac-LDL) and 2-CdA. The addition of 2-CdA, in concentrations ranging from 5-20 nM, induced a dose-dependent decrease in cellular cholesterol content and in the amount of extracellular [14C]oleic acid (OA) incorporated into the cholesteryl ester (CE) fraction. The effect was maximized at 20 nM 2-CdA with an 86% reduction in cholesterol esterification compared to controls (P < 0.008). To evaluate the mechanism of interaction of 2-CdA with cellular lipid metabolism, deoxycytidine (dCyt) and 3-methoxybenzamide (3-MOB), substances known to antagonize the effect of 2-CdA in different ways, were co-administered with 2-CdA. dCyt, a competitive inhibitor of dCyt kinase, which catalyzes phosphorylation to the active metabolite, antagonized the effects of 20 nM 2-CdA, producing significantly greater incorporation of extracellular [14C]OA into the CE fraction than in the presence of 2-CdA alone (P < 0.0086). Co-incubation with 2-CdA and the poly-ADP-ribose synthetase inhibitor 3-MOB, which is known to render cells resistant to 2-CdA toxicity by preventing cellular nicotinamide adenine dinucleotide (NAD)- and adenosine triphosphase-depletion, also reversed the effect of 2-CdA on lipid accumulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies on thermophile products. IX. Isofatty acid-containing phosphatidylglycerol that enhances the induction of concanavalin A-activated suppressor T cells.

A new enhancer of the induction of concanavalin A (Con A)-activated suppressor T (Ts) cells has been demonstrated in the ethanolysate of Bacillus stearothermophilus UBT8038. It was purified by successive silica gel column chromatographies and identified as phosphatidylglycerol with C14:0-C18:0 isofatty acids (Fr. 7-C). Mouse splenocytes activated with Con A and Fr. 7-C (0.01-1 microgram/ml) in vitro significantly suppressed the proliferative response of syngenic splenocytes by mitogen stimulation in a dose-dependent manner, compared to those stimulated by Con A alone. The immunosuppressive response enhanced by Fr. 7-C disappeared when the cell populations of Thy-1.2 or CD8 positive lymphocytes were depleted. The result strongly suggests that Fr. 7-C is an immunosuppressive substance which enhances the induction of Con A-activated CD8 positive Ts cells.