Andrological findings in infertile men with two (biallelic) CFTR mutations: results of a multicentre study in Germany and Austria comprising 71 patients.

STUDY QUESTION: When should cystic fibrosis transmembrane conductance regulator (CFTR) mutation analysis be recommended in infertile men based on andrological findings? SUMMARY ANSWER: CFTR mutation analysis is recommended in all men with unexplained azoospermia in the presence of normal gonadotropin levels. WHAT IS KNOWN ALREADY: While 80-97% of men with congenital bilateral absence of the vas deferens (CBAVD) are thought to carry CFTR mutations, there is uncertainty about the spectrum of clinical and andrological abnormalities in infertile men with bilallelic CFTR mutations. This information is relevant for evidence-based recommendations to couples requesting assisted reproduction. STUDY DESIGN, SIZE, DURATION: We studied the andrological findings of patients with two CFTR mutations who were examined in one of the cooperating fertility centres in Germany and Austria. In the period of January till July 2019, the completed and anonymized data sheets of 78 adult male patients were returned to and analysed by the project leader at the Institute of Human Genetics in Innsbruck, Austria. PARTICIPANTS/MATERIALS, SETTING, METHODS: Minimum study entry criteria were the presence of two (biallelic) CFTR mutations and results of at least one semen analysis. Andrological assessments were undertaken by standardized data sheets and compared with normal reference values. Seventy-one patients were eligible for the study (n = 30, 42% from Germany, n = 26, 37% from Austria, n = 15, 21% other nations). MAIN RESULTS AND THE ROLE OF CHANCE: Gonadotropin levels (FSH, LH) were normal, 22% of patients had reduced testosterone values. Mean right testis volume was 23.38 ml (SD 8.77), mean left testis volume was 22.59 ml (SD 8.68) and thereby statistically increased compared to normal (P < 0.01). although the means remained in the reference range of 12-25 ml. Semen analysis revealed azoospermia in 70 of 71 (99%) patients and severe oligozoospermia <0.1 × 106/ml in one patient. Four semen parameters, i.e. ejaculate volume, pH, α-glucosidase and fructose values, were significantly reduced (P < 0.01). Only 18% of patients had a palpatory and sonographically diagnosed CBAVD, while in 31% the diagnosis of CBAVD was uncertain, in 12% patients, the vas deferens was present but hypoplastic, and in 39% the vas deferens was normally present bilaterally. Seminal vesicles were not detectable in 37% and only unilaterally present in 37% of patients. Apart from total testes volume, clinical findings were similar in patients with two confirmed pathogenic CFTR mutations (Group I) compared with patients who carried one pathogenic mutation and one CFTR variant of unknown significance (Group II). LIMITATIONS, REASONS FOR CAUTION: We could not formally confirm the in trans position of genetic variants in most patients as no family members were available for segregation studies. Nonetheless, considering that most mutations in our study have been previously described without other rare variants in cis, and in view of the compatible andrological phenotype, it is reasonable to assume that the biallelic genotypes are correct. WIDER IMPLICATIONS OF THE FINDINGS: Our study reveals that CFTR mutation analysis has a broader indication than just the absence of the vas deferens. We recommend to completely sequence the CFTR gene if there is a suspicion of obstructive azoospermia, and to extend this analysis to all patients with unexplained azoospermia in the presence of normal gonadotropin levels. STUDY FUNDING/COMPETING INTEREST(S): German Research Foundation Clinical Research Unit 'Male Germ Cells: from Genes to Function' (DFG CRU326, grants to F.T.). There are no conflicts of interest to declare. TRIAL REGISTRATION NUMBER: N/A.

Relationship between follicular volume and oocyte competence, blastocyst development and live-birth rate: optimal follicle size for oocyte retrieval.

OBJECTIVE: To analyze oocyte competence in gonadotropin-releasing hormone agonist (GnRHa) stimulation cycles with regard to maturity, fertilization and blastocyst rate, as well as clinical outcome (pregnancy and live-birth rate), in relation to follicular volume, measured by three-dimensional transvaginal sonography (3D-TVS), and follicular fluid composition. METHODS: This was a prospective single-center study conducted between June 2012 and June 2014, including 118 ovum pick-ups with subsequent embryo transfer. Ovarian stimulation was performed using the GnRHa long protocol. Of 1493 follicles aspirated individually, follicular volume was evaluated successfully in 1236 using automated 3D-TVS during oocyte retrieval. Oocyte maturity and blastocyst development were tracked according to follicular volume. Intrafollicular concentrations of estradiol, testosterone, progesterone, luteinizing hormone, follicle-stimulating hormone and granulocyte-colony stimulating factor were quantified by immunoassay. Clinical outcome, in terms of implantation rate, (clinical) pregnancy rate, miscarriage and live-birth rate (LBR), was evaluated. RESULTS: Follicles were categorized, according to their volume, into three arbitrary groups, which included 196 small (8-12 mm/0.3-0.9 mL), 772 medium (13-23 mm/1-6 mL) and 268 large (≥ 24 mm/> 6 mL) follicles. Although oocyte recovery rate was significantly lower in small follicles compared with medium and large ones (63.8% vs 76.6% and 81.3%, respectively; P < 0.001), similar fertilization rates (85.1% vs 75.3% and 81.4%, respectively) and blastocyst rates (40.5% vs 40.6% and 37.2%, respectively) per mature metaphase II oocyte were observed. A trend towards higher LBR after transfer of blastocysts derived from small (< 1 mL) follicles compared with medium (1-6 mL) or large (> 6 mL) follicles (54.5% vs 42.0%, and 41.7%, respectively) was observed. No predictive value of follicular fluid biomarkers was identified. CONCLUSIONS: Our data indicate that the optimal follicular volume for a high yield of good quality blastocysts with good potential to lead to a live birth is 13-23 mm/1-6 mL. However, oocytes derived from small follicles (8-12 mm/0.3-0.9 mL) still have the capacity for normal development and subsequent delivery of healthy children, suggesting that aspiration of these follicles should be encouraged as this would increase the total number of blastocysts retrieved per stimulation. Copyright © 2017 ISUOG. Published by John Wiley & Sons Ltd.

Pregnancy and birth outcomes following fresh or vitrified embryo transfer according to blastocyst morphology and expansion stage, and culturing strategy for delayed development.

STUDY QUESTION: How do live birth rates (LBRs), following fresh and vitrified/warmed embryo transfer, compare according to morphological grade, developmental stage and culturing strategy of human blastocysts in vitro? SUMMARY ANSWER: Equivalent LBRs were obtained after fresh embryo transfer and after vitrified/warmed embryo transfer of blastocysts of top or non-top quality, while vitrification after prolonged embryo culture of blastocysts with delayed development had a positive impact on LBR. WHAT IS KNOWN ALREADY: Blastocyst morphology correlates with clinical outcome; however, few data are available on vitrified/warmed embryo transfer using non-top quality blastocysts. The aim of this study was to determine clinical outcomes of non-top quality blastocysts and blastocysts with delayed development that underwent vitrified/warmed embryo transfer. STUDY DESIGN, SIZE, DURATION: This retrospective, single-centre study (conducted January 2009 to June 2013) compared 1010 fresh embryo transfer and 1270 vitrified/warmed embryo transfer of blastocysts originating from the same stimulation cycle. Furthermore, 636 fresh embryo transfers and 304 vitrified/warmed embryo transfer after delayed expansion or blastulation in the same period were also analysed. PARTICIPANTS/MATERIALS, SETTING, METHODS: Clinical outcomes after fresh and vitrified/warmed embryo transfer according to blastocyst morphology were compared in both groups. MAIN RESULTS AND THE ROLE OF CHANCE: Similar LBRs after fresh embryo transfer or after vitrified/warmed embryo transfer of top or non-top quality blastocysts were observed. A statistically significant improvement in clinical outcomes was obtained after vitrified/warmed embryo transfer of Day 5 embryos with delayed expansion or blastulation when applying prolonged culture. Our study suggests that vitrification of non-top quality blastocysts as well as delayed cavitating and blastulating Day 5 embryos should be considered in autologous IVF cycles. LIMITATIONS AND REASONS FOR CAUTION: Given that the present retrospective study used aseptic vitrification of blastocysts, the results, particularly the survival rates, may not be fully applicable to other vitrification protocols. The retrospective nature of the study has to be mentioned. WIDER IMPLICATIONS OF THE FINDINGS: Restriction of vitrification to top quality blastocysts may result in discarding potentially viable embryos. STUDY FUNDING AND COMPETING INTERESTS: This study was not externally funded. There are no conflicts of interest to declare.

The time aspect in storing vitrified blastocysts: its impact on survival rate, implantation potential and babies born.

STUDY QUESTION: Does the storage time of vitrified human blastocysts negatively impact their survival, the implantation potential of embryos or the malformation rate of babies born? SUMMARY ANSWER: There was no evidence that storage times of up to 6 years after vitrification (VIT) had a negative impact on blastocyst survival, the implantation potential of embryos or the malformation rate of babies born. WHAT IS KNOWN ALREADY: Although several thousand children have been born after blastocyst VIT, many aspects of this technique remain to be elucidated. New applications, such as fertility preservation, lead to long storage times of vitrified gametes or embryos but it remains to be determined if these vitrified embryos are stable over time. STUDY DESIGN, SIZE, DURATION: A retrospective study including 603 transfers was conducted between January 2009 and April 2012. Blastocysts were vitrified using a closed system. PARTICIPANTS/MATERIALS, SETTING, METHODS: All patients underwent the transfer of aseptically vitrified/warmed blastocysts in a cryo-cycle. A total of 1077 blastocysts were transferred. Survival rates (SRs), implantation potential, birth rates and characteristics of the children born were evaluated. MAIN RESULTS AND THE ROLE OF CHANCE: We found that the storage of vitrified blastocysts in aseptic conditions neither impaired blastocyst viability (SR after warming during the first year of storage was 83.0% compared with 83.1% after 5-6 years of storage: NS) nor decreased pregnancy rates (clinical pregnancy rate after 1 year of storage was 40.0 versus 38.5% after 6 years: NS). In addition, no increase in the malformation rate over time was observed. LIMITATIONS, REASONS FOR CAUTION: Our study only included the transfer of blastocysts which had been vitrified aseptically (i.e. using a closed system). Therefore, our results might not be applicable to 'open' VIT systems. The long-term follow-up of children born will be necessary to confirm our findings. WIDER IMPLICATIONS OF THE FINDINGS: The results suggest that vitrified human blastocysts can be stored for long periods of time without significant negative consequences for the offspring. Therefore, the method should be of benefit to those patients who need to consider taking measures for fertility preservation. STUDY FUNDING/COMPETING INTEREST(S): No external funding was sought for this study and the authors have no conflict of interest to declare.

Lower intracellular concentration of cryoprotectants after vitrification than after slow freezing despite exposure to higher concentration of cryoprotectant solutions.

STUDY QUESTION: What is the intracellular concentration of cryoprotectant (ICCP) in mouse zygotes during vitrification (VIT) and slow-freezing (SLF) cryopreservation procedures? SUMMARY ANSWER: Contrary to common beliefs, it was observed that the ICCP in vitrified zygotes is lower than after SLF, although the solutions used in VIT contain higher concentrations of cryoprotectants (CPs). WHAT IS KNOWN ALREADY: To reduce the likelihood of intracellular ice crystal formation, which has detrimental effects on cell organelles and membranes, VIT was introduced as an alternative to SLF to cryopreserve embryos and gametes. Combined with high cooling and warming rates, the use of high concentrations of CPs favours an intracellular environment that supports and maintains the transition from a liquid to a solid glass-like state devoid of crystals. Although the up-to-date publications are reassuring in terms of obstetric and perinatal outcomes after VIT, a fear about exposing gametes and embryos to high amounts of CPs that exceed 3-4-fold those found in SLF was central to a debate initiated by advocates of SLF procedures. STUDY DESIGN, SIZE, DURATION: Two experimental set-ups were applied. The objective of a first study was to determine the ICCP at the end of the exposure steps to the CP solutions with our VIT protocol (n = 31). The goal of the second investigation was to compare the ICCP between VIT (n = 30) and SLF (n = 30). All experiments were performed in triplicates using mouse zygotes. The study took place at the GIGA-Research Institute of the University of Liège. PARTICIPANTS/MATERIALS, SETTING, METHODS: Cell volume is modified by changes in extracellular osmolarity. Hence, we estimated the final ICCP after the incubation steps in the VIT solutions by exposing the cells to sucrose (SUC) solutions with defined molarities. The ICCP was calculated from the SUC concentration that produced no change in cell volume, i.e. when intra- and extracellular osmolarities were equivalent. Cell volume was monitored by microscopic cinematography. ICCP was compared between SLF and VIT based on the principle that a high ICCP lowers the probability of (re)crystallization during warming but increases the probability of over-swelling of the cell due to fast inflow of water. The survival rates of mouse zygotes after SLF or VIT were compared using either (i) various warming rates or (ii) various concentrations of SUC in the warming dilution medium. MAIN RESULTS AND THE ROLE OF CHANCE: The ICCP in mouse zygotes during the VIT procedure prior to plunging them in liquid nitrogen was ∼2.14 M, i.e. one-third of the concentration in the VIT solution. After SLF, the warming rate did not affect the zygote survival rate. In contrast, only 3/30 vitrified zygotes survived when warmed slowly but as many as 30/30 zygotes survived when warming was fast (>20 000°C/min). Vitrified zygotes showed significantly higher survival rates than slow-frozen zygotes when they were placed directly in the culture medium or in solutions containing low concentrations of SUC (P < 0.01). These two experiments demonstrate a lower ICCP after VIT than after SLF. LIMITATIONS, REASONS FOR CAUTION: The results should not be directly extrapolated to other stages of development or to other species due to possible differences in membrane permeability to water and CPs. WIDER IMPLICATIONS OF THE FINDINGS: The low ICCP we observed after VIT removes the concern about high ICCP after VIT, at least in murine zygotes and helps to explain the observed efficiency and lack of toxicity of VIT. STUDY FUNDING / COMPETING INTEREST(S): The study was funded by the FNRS (National Funds for Scientific Research). The authors declare that they have no competing interests.

Effects of embryo transfer quality on pregnancy and live birth delivery rates.

BACKGROUND: To analyze the effects of embryo transfer (ET) quality on clinical pregnancy (CPR) and live birth delivery rates (LBDR). METHODS: In a retrospective study at a single, private infertility center between November 2005 and December 2009 one thousand fifty-five day-3 and day-5 ETs following IVF/ICSI/IMSI were evaluated. We analyzed the impact of an atraumatic ET with a soft catheter (ET 1), after external guidance (ET 2), after probing of the cervix with a stylet (ET 3), or after grasping the portio vaginalis with a tenaculum (ET 4) on CPR and LBDR. RESULTS: The use of external guidance showed a significantly reduced LBDR as compared to an atraumatic ET (26.0% vs. 32.5%). The lowest CPR and LBDR were found in ET 4. The application of stylets in cases of difficult ETs was superior to the use of external guidance. No differences in miscarriages between ET 1-4 were noted. CONCLUSIONS: Besides embryo culture and patient history, the quality of an ET might also have an important impact on pregnancy outcome. Techniques to ensure an atraumatic ET, such as mechanic uterine cavity length measurements, before starting treatment might help identify patients at risk for a difficult ET and lead to modified treatments, such as the primary use of a stylet. Limitation of study: retrospective analysis.

Individual demands of human embryos on IVF culture medium: influence on blastocyst development and pregnancy outcome.

The elucidation of the metabolic requirements of human embryos in vivo or in vitro remains, despite being intensively investigated, a work in progress. The adoption of extended embryo culture to the blastocyst stage during the last decade has entailed new challenges. With the increased attention to culture media formulations, more evidence on the sensitivity of embryos to their early environmental conditions is accumulating which might affect phenotype and developmental potential. A retrospective study was conducted that comprised 286 IVF cycles to evaluate the effect of two different culture media on blastocyst development and pregnancy outcome. Embryos were either cultured in a one step or a sequential medium. Higher fertilization rates and augmented blastocyst rates as well as higher implantation rates were observed when embryos were cultured in one step medium (P<0.05). Interestingly, the transfer of two embryos where one embryo was cultured in either medium resulted in a significantly higher rate of twin pregnancies. Although multiple pregnancies should be avoided in assisted reproduction treatment to reduce risks for offspring and mother, this higher frequency of twin pregnancies resulting from the transfer of embryos derived from different culture media suggests that each embryo makes individual demands on its early environment.

Beer down-regulates activated peripheral blood mononuclear cells in vitro.

Moderate consumption of alcoholic beverages is suggested to reduce cardiovascular risk. Within this context, most attention is drawn to antioxidant ingredients of wine, but also beer was found to be beneficial. Potential effects of three different types of beer including alcohol-free beer were investigated using freshly isolated human peripheral blood mononuclear cells stimulated with the mitogen phytohaemagglutinin in vitro. Neopterin production and tryptophan degradation were monitored in culture supernatants to determine effects of test substances on immunobiochemical pathways induced by interferon-gamma. In a subgroup of experiments also production of interferon-gamma was measured. Compared to unstimulated cells, phytohaemagglutinin increased production of neopterin and also triggered the degradation of tryptophan (all p < 0.01). All types of beer (2-4% dilution) were found to counteract these stimulation-induced effects and significant reduction of neopterin formation and tryptophan degradation was observed (p < 0.01). Data demonstrate that beer reduces production of neopterin and degradation of tryptophan, both these biochemical pathways are induced during cell-mediated immune response. Data suggest that the immunosuppressive capacity of beer may relate to its anti-inflammatory nature.

Two different store-operated Ca2+ entry pathways in MDCK cells.

Whole cell patch clamp experiments in conjunction with Fura-2 fluorescence microscopy were performed to study the mechanisms of 'store-operated' (capacitative) Ca2+ entry. In MDCK cells, depletion of inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ stores activates a store-operated cation current (SOCC) predominantly selective for Ca2+ than for Na+ or Mn2+ [Delles C., Haller T., Dietl P. A highly calcium-selective cation current activated by intracellular calcium release in MDCK cells. J Physiol 1995, 486: 557-569]. In the presence of extracellular Ca2+, thapsigargin (TG) stimulated both SOCC and a Ca(2+)-dependent K+ current (IK(Ca)), reflecting stimulation of store-operated Ca2+ entry. The Ca2+ entry blocker 1-[3-(4-methoxyphenyl) propoxyl]-1-(4-methoxyphenyl)-ethyl-1H-imidazole HCI (SK&F96365; 30 microM) did not inhibit SOCC. At the same concentration, it exerted a transient partial inhibition on IK(Ca) activated by TG-induced Ca2+ entry. It did, however, not directly inhibit IK(Ca). This was demonstrated by an unchanged relationship between the cytosolic Ca2+ concentration ([Ca2+]i) and IK(Ca) in experiments where [Ca2+]i was measured under whole cell patch clamp conditions and by a lacking effect of SK&F96365 on IK(Ca) prestimulated by a high 'clamped' [Ca2+]i. La3+ partially, but not directly, inhibited the TG-induced IK(Ca) at a concentration (10 microM) sufficient to entirely block SOCC. La3+ and SK&F96365 in combination exerted an additive reduction on the TG-induced whole cell conductance (G) and completely blocked IK(Ca) stimulated by TG. We conclude that two Ca2+ entry pathways with different pharmacological and biophysical properties are involved in 'store-operated' Ca2+ entry in MDCK cells.

Neopterin as a marker for immune system activation.

Increased amounts of neopterin are produced by human monocytes/macrophages upon stimulation with the cytokine interferon-y. Therefore, measurement of neopterin concentrations in body fluids like serum, cerebrospinal fluid or urine provides information about activation of T helper cell 1 derived cellular immune activation. Increased neopterin production is found in infections by viruses including human immunodeficiency virus (HIV), infections by intracellular living bacteria and parasites, autoimmune diseases, malignant tumor diseases and in allograft rejection episodes. But also in neurological and in cardiovascular diseases cellular immune activation indicated by increased neopterin production, is found. Major diagnostic applications of neopterin measurements are, e.g. monitoring of allograft recipients to recognize immunological complications early. Neopterin production provides prognostic information in patients with malignant tumor diseases and in HIV-infected individuals, high levels being associated with poorer survival expectations. Neopterin measurements are also useful to monitor therapy in patients with autoimmune disorders and in individuals with HIV infection. Screening of neopterin concentrations in blood donations allows to detect acute infections in a non-specific way and improves safety of blood transfusions. As high neopterin production is associated with increased production of reactive oxygen species and with low serum concentrations of antioxidants like alpha-tocopherol, neopterin can also be regarded as a marker of reactive oxygen species formed by the activated cellular immune system. Therefore, by neopterin measurements not only the extent of cellular immune activation but also the extent of oxidative stress can be estimated.

Hyperhomocysteinemia, pteridines and oxidative stress.

Tetrahydrofolate is an essential cofactor for the conversion of homocysteine to methionine, and hyperhomocysteinemia is considered as a risk factor for cardiovascular and cerebrovascular diseases. In subjects with hyperhomocysteinemia usually an inverse relationship exists to folic acid levels, and supplementation with folic acid is able to lower homocysteine concentrations. The pathogenesis of most if not all diseases which are accompanied with moderate hyperhomocysteinemia involves cellular immune activation and therefore in patients very often exists also a positive correlation between homocysteine concentrations and the degree of immune activation which is indicated, e.g. by increased neopterin concentrations. Since neopterin concentrations also serves as an estimate of oxidative stress merging from immune system activation, this association suggests that cellular immune activation and oxidative stress could be involved in the development of hyperhomocysteinemia. Because tetrahydrofolate is very susceptible to oxidation, an increased oxidative degradation of tetrahydrofolates may become relevant under oxidative stress conditions. In this way folate deficiency may develop despite normal dietary intake of the vitamin. In our patients, hyperhomocysteinemia is considered as an indirect consequence of hyperconsumption of antioxidant vitamins during prolonged states of immune activation.

Neopterin and 7,8-dihydroneopterin induce apoptosis in the rat alveolar epithelial cell line L2.

The neopterin derivatives, neopterin and 7,8-dihydroneopterin, modulate the cellular oxidant-antioxidant balance as well as the expression of the inducible nitric oxide synthase (iNOS) gene. Since apoptosis can be induced by reactive oxygen intermediates and nitric oxide (NO) we investigated whether these neopterin derivatives induce apoptotic cell death. As model we selected the rat alveolar epithelial cell line L2. 24 h incubation of neopterin (1-1000 microM) as well as 7,8-dihydroneopterin (1-1000 microM) resulted in a significant increase of percent apoptotic cells (measured by FACS analysis). Coincubation of both pteridines with the cytomix (interferon-gamma plus tumor necrosis factor-alpha) led to a significantly higher apoptosis than the cytomix alone. In contrast to the cytomix, no iNOS gene expression and no NO release could be detected after incubation with neopterin or 7,8-dihydroneopterin. We conclude that neopterin and 7,8-dihydroneopterin are per se inducers of apoptosis which is not mediated by nitric oxide. This may be of importance in inflammatory pulmonary diseases associated with an activation of the cellular immune system.

Interferon-gamma-induced conversion of tryptophan: immunologic and neuropsychiatric aspects.

Tryptophan is an essential amino acid and the least abundant constituent of proteins. In parallel it represents a source for two important biochemical pathways: the generation of neurotransmitter 5-hydroxytryptamine (serotonin) by the tetrahydrobiopterin-dependent tryptophan 5-hydroxylase, and the formation of kynurenine derivatives and nicotinamide adenine dinucleotides initiated by the enzymes tryptophan pyrrolase (tryptophan 2,3-dioxygenase, TDO) and indoleamine 2,3-dioxygenase (IDO). Whereas TDO is located in the liver cells, IDO is expressed in a large variety of cells and is inducible by the cytokine interferon-gamma. Therefore, accelerated tryptophan degradation is observed in diseases and disorders concomitant with cellular immune activation, e. g. infectious, autoimmune, and malignant diseases, as well as during pregnancy. According to the cytostatic and antiproliferative properties of tryptophan-depletion on T lymphocytes, activated T-helper type 1 (Th-1) cells may down-regulate immune response via degradation of tryptophan. Especially in states of persistent immune activation availability of free serum tryptophan is diminished and as a consequence of reduced serotonin production, serotonergic functions may as well be affected. Accumulation of neuroactive kynurenine metabolites such as quinolinic acid may contribute to the development of neurologic/psychiatric disorders. Thus, IDO seems to represent a link between the immunological network and neuroendocrine functions with far reaching consequences in regard to the psychological status of patients. These observations provide a basis for the better understanding of mood disorder and related symptoms in chronic diseases.

Histamine suppresses neopterin production in the human myelomonocytoma cell line THP-1.

Histamine, an important inflammatory mediator in allergic diseases and asthma, was reported to have modulatory effects on T cells by down-regulating Th1-type cell cytokines like interleukin 2 (IL-2) and interferon-gamma (IFN-gamma). In this study we examined the effect of histamine and the histamine-receptor antagonists cimetidine and diphenhydramine on the production of neopterin after stimulation with IFN-gamma in the myelomonocytoma cell line THP-1. Increasing concentrations of histamine markedly suppressed IFN-gamma induced neopterin formation. Simultaneous preincubation of THP-1 cells with histamine, IFN-gamma and different concentrations of the H(2)-receptor antagonist cimetidine showed a clear antagonizing effect on neopterin formation. In contrast, the H(1)-receptor antagonist diphenhydramine was not able to abrogate the suppressive effect of histamine on neopterin production. Our results suggest, that histamine may be a potent inhibitor of effects or mechanisms induced by IFN-gamma in monocytes/macrophages. Cimetidine, and possibly other H(2)-receptor antagonists, may reverse down-regulatory actions of endogenously formed histamine on activated monocytic cells.

7,8-Dihydroneopterin-induced apoptosis in Jurkat T lymphocytes: a comparison with anti-Fas- and hydrogen peroxide-mediated cell death.

Activated cell-mediated immunity, associated for example with HIV infection, is accompanied by elevated concentrations of neopterin and 7,8-dihydroneopterin. Recent data have indicated a role of neopterin derivatives in virus activation and apoptotic cell death, processes likely to involve the action of oxygen free radicals. Because T cell death in AIDS is likely to involve the Fas/Fas ligand system and the action of oxygen free radicals and 7,8-dihydroneopterin, we compared the kinetics and sensitivity of apoptotic cell death of human leukemic Jurkat T cells to that of treatments with 7,8-dihydroneopterin, anti-Fas, and H2O2. Upon incubation with 5 mM 7,8-dihydroneopterin and 50 microM hydrogen peroxide over a period of 24 hr, bimodal kinetics were observed with peaks at 5.5 hr (7,8-dihydroneopterin, 13.1%; H2O2, 11.4%) and at 24 hr (7,8-dihydroneopterin, 11.2%; H2O2, 13.2%). In contrast, anti-Fas (20 ng/mL)-induced apoptosis increased steadily over time, peaking at 11 hr (43.2%). Interestingly, anti-Fas-induced apoptosis was suppressed upon co-incubation with 7,8-dihydroneopterin and H2O2 by 62% and 68%, respectively. We also compared the sensitivity to drug treatments of apoptosis induced by 7,8-dihydroneopterin, anti-Fas antibodies, and H2O2. 7,8-Dihydroneopterin-mediated, and similarly anti-Fas- and H2O2-mediated, apoptosis was not inhibited by a broad range of pharmacological inhibitors, such as actinomycin D, cycloheximide, cyclosporin A, and various protein kinase inhibitors. On the contrary, inhibitors with antioxidant abilities, such as pyrrolidinedithiocarbamate, significantly blocked 7,8-dihydroneopterin-, H2O2- as well as anti-Fas-mediated apoptosis. These results imply that 7,8-dihydroneopterin-, H2O2-, and anti-Fas-mediated cell death might involve related redox sensitive signal transduction pathways.

Moderate hyperhomocysteinemia and immune activation.

Moderate hyperhomocysteinemia is associated with an increased risk of atherosclerosis, thrombosis and neurodegenerative diseases. Homocysteine accumulation in the blood can be due to many underlying causes, which may interact with each other, e.g. genetic disposition and B-vitamin status. The role of the sulfur-containing amino acid homocysteine in the pathogenesis of diseases remains unclear, even if many studies suggest a causal relationship between homocysteine-mediated processes like oxidative stress, NO-inactivation and endothelial deficiency and atherogenesis. Proposed mechanisms of action of homocysteine are discussed, and the question is addressed, whether effects that are attributed to homocysteine, are not rather the consequence of folate and vitamin B12-deficiency. Deficiency of these B-vitamins in parallel with moderate hyperhomocysteinemia is often found in patients with enhanced activation of the cellular immune system, like Alzheimer's disease, rheumatoid arthritis and also vascular diseases. In patients with these diseases an association between homocysteine metabolism, oxidative stress and immune activation exists. On the one hand proliferation of immunocompetent cells having an enhanced demand for B-vitamins leads to the accumulation of homocysteine. On the other hand macrophages stimulated by TH1-type cytokine interferon-gamma form reactive oxygen species (ROS), which oxidize antioxidants, lipoproteins and oxidation-sensitive B-vitamins. Thereby Th1-type immune response could contribute importantly to the development of hyperhomocysteinemia, and may also be a major determinant of disease progression.

Reduced pteridine derivatives induce apoptosis in human neuronal NT2/HNT cells.

Elevated concentrations of the pteridine compound neopterin, usually accompanied by 7,8-dihydroneopterin were found in cerebrospinal fluids of patients with neurodegenerative diseases and central nervous system infections. Here, the potential of pteridines to induce apoptosis of the human neuronal cell line (NT2) was investigated. Reduced neopterin, biopterin- and folate derivatives led to a time-dependent increase of apoptosis of cells. In contrast, non-reduced pteridines did not significantly alter cell survival. After differentiation of neuronal precursor cells to neurons and astrocyte-like cells, similar effects were detected. Antioxidants partly protected NT2 from pteridines-induced apoptosis, suggesting the involvement of reactive oxygen intermediates. In vitro experiments using dichlorofluorescin-diacetate further indicated a direct formation of reactive oxygen species in cells. Results implicate that high concentrations of reduced pteridines, might contribute to the loss of neuronal cells in neurodegenerative diseases.

Neopterin production in SCID mice injected with human peripheral blood mononuclear cells.

Intraperitoneal transfer of peripheral blood mononuclear cells (PBMC) from human EBV+ donors into severe combined immunodeficiency (SCID) mice is a suitable model for studying some aspects of lymphomagenesis and immune activation. Neopterin is a soluble immune marker which was found to be a useful indicator for immune activation processes in humans, e.g. to monitor immunological complications in allograft recipients or to predict prognosis in HIV-infected individuals. In contrast, this pteridine compound is normally synthesized in murine organism in only very low amounts. The measurement of neopterin concentrations in serum and urine should be feasible in SCID mice reconstituted with human PBMC. In this study, we examined the usability of this experimental model for monitoring human T cell activation by neopterin measurements. The production of neopterin by SCID mice after injection of freshly isolated human PBMC, purified B or T cells and cultured Epstein-Barr virus (EBV)+ lymphoblastoid cells (LCL) was determined. It was found that neopterin can be detected early after injecting SCID mice with PBMC, whereas injection of purified human T or B cells did not result in neopterin production. Highest neopterin levels were detected in mice treated with LCL cells when developing lymphoma. We discuss the possible sources of neopterin along this process and its usefulness in this model.

Enhanced tryptophan degradation in systemic lupus erythematosus.

In vitro and in vivo, tryptophan degradation was found to be associated with T cell functional loss and tolerance induction. In systemic lupus erythematosus (SLE) besides the Th2-type cytokine interleukin-10, Th1-type cytokines including interferon-gamma (IFN-gamma) are expressed especially during exacerbation of the disease. IFN-gamma stimulates the enzyme indoleamine (2,3)-dioxygenase (IDO) converting tryptophan to the metabolite kynurenine which in macrophages is subsequently degraded to other, partly neurotoxic compounds like quinolinic acid, and finally to nicrotinamides. We measured kynurenine and tryptophan concentrations in the sera of 55 SLE patients. In these patients, the concentrations of tryptophan (median, interquartile range: 53.9, 45.7-64.1 microM) were lower (p < 0.0001), and the kynurenine concentrations (2.45, 1.75-3.40 microM) were increased (p < 0.0005) compared to healthy blood donors (70.0, 63.8-80.6; 1.80, 1.45-2.27 microM, respectively). Also the kynurenine per tryptophan quotients (K/T), which allow to estimate IDO activity, were significantly higher in patients than in normals (0.043, 0.033-0.062 vs. 0.027, 0.021-0.030; p < 0.0001), indicating enhanced IDO-induced tryptophan degradation in SLE. There was no significant relationship between tryptophan, kynurenine and the SLEDAI, and also the correlation of K/T with SLEDAI was rather weak (rs = 0.243, p < 0.05). Higher K/T was found in patients presenting with serositis (p = 0.01), decrease of complement (c3, c4; p < 0.01) and blood count change (anemia, leucopenia, lymphopenia; p = 0.032) than in patients without such disease manifestations. The significant correlation found between K/T and neopterin (rs = 0.808, p < 0.001), a marker of immune activation, points to a role of immune activation to be responsible for tryptophan degradation in SLE patients.