In vivo and in vitro effects of cord blood hematopoietic stem and progenitor cell (HSPC) expansion using valproic acid and/or nicotinamide.

BACKGROUND: High self-renewal capacity and most permissive nature of umbilical cord blood (CB) results with successful transplant outcomes but low hematopoietic stem and progenitor cell (HSPC) counts limits wider use. In order to overcome this problem ex vivo expansion with small molecules such as Valproic acid (VPA) or Nicotinamide (NAM) have been shown to be effective. To the best of our knowledge, the combinatory effects of VPA and NAM on HSPC expansion has not been studied earlier. The aim of this study was to analyze ex vivo and in vivo efficacy of VPA and NAM either alone or in combination in terms of expansion and engraftment. METHODS: A total of 44 CB units were included in this study. To determine the ex vivo and in vivo efficacy, human CB CD34+ cells were expanded with VPA and/or NAM and colony forming unit (CFU) assay was performed on expanded HSPC. Xenotransplantation was performed simultaneously by intravenous injection of expanded HSPC to NOD-SCID gamma (NSG) mice (n = 22). Significance of the difference between the expansion groups or xenotransplantation models was analyzed using t-test, Mann-Whitney, ANOVA or Kruskal-Wallis tests as appropriate considering the normality of distributions and the number of groups analyzed. RESULTS: In vitro CD34+ HSPC expansion fold relative to cytokines-only was significantly higher with VPA compared to NAM [2.23 (1.07-5.59) vs 1.48 (1.00-4.40); p < 0.05]. Synergistic effect of VPA+NAM has achieved a maximum relative expansion fold at 21 days (D21) of incubation [2.95 (1.00-11.94)]. There was no significant difference between VPA and VPA+NAM D21 (p = 0.44). Fold number of colony-forming unit granulocyte-macrophage (CFU-GM) colonies relative to the cytokine-only group was in favor of NAM compared to VPA [1.87 (1.00-3.59) vs 1.00 (1.00-1.81); p < 0.01]. VPA+NAM D21 [1.62 (1.00-2.77)] was also superior against VPA (p < 0.05). There was no significant difference between NAM and VPA+NAM D21. Following human CB34+ CB transplantation (CBT) in the mouse model, fastest in vivo leukocyte recovery was observed with VPA+NAM expanded cells (6 ± 2 days) and the highest levels of human CD45 chimerism was detectable with VPA-expanded CBT (VPA: 5.42 % at day 28; NAM: 2.45 % at day 31; VPA+NAM 1.8 % at day 31). CONCLUSION: Our study results suggest using VPA alone, rather than in combination with NAM or NAM alone, to achieve better and faster expansion and engraftment of CB HSPC.

Fetal Cell Microchimerism; Normal and Immunocompromised Gestations in Mice.

Objective: To compare fetal cell microchimerism in normal and immunocompromised gestations. Materials and methods: The study consists of two groups of mature female mice. In the control group and the immunocompromised study group, 5 mg of saline and cyclosporine were injected intraperitoneally, respectively. In the second step, all female mice were mated with "Actine-Luc (+) green fluorescent protein (GFP)" transgenic male mice. Immunohistochemical studies (ALPL-antiluciferase, cytokeratin-antiluciferase, and CD 105-antiluciferase) were carried out on maternal liver, skin, and lung tissues at 6-7th and 14-15th gestational days, and postpartum 3-4th, 12th, and 18-24 months. Results: GFP (+) cells were detected in maternal liver and skin but not in lung tissue. Liver was the most affected tissue. GFP was found to be more intense in the immunocompromised group. Conclusion: Fetal microchimerism was demonstrated in maternal liver and skin and found to be more intensive in the immunocompromised group.

Antenatal hydronephrosis and fetal urine sampling.

The aim of this study is to evaluate the significance of renal pelvis aspiration (RPA) in the management of antenatal hydronephrosis (AHN). This study enrolled 15 AHN cases (one twin pregnancy) that necessitated RPA for AHN. Chromosomal abnormalities, gene disorders, and additional life-threatening congenital abnormalities were eliminated prior to intrauterine interventions. Urine analysis were performed for the evaluation of renal function. Normal renal function was observed in six neonates/infants (40%) (group 1), whereas impaired renal function and various type of urinary system anomalies were observed in 9 neonates/infants (60%) (group 2) during the short-term and longitudinal follow-up periods. There were statistically significant differences in the oligohydroamniosis rate, mean fetal urine sodium value, mean fetal urine ß2-microglobulin, mean gestational week at birth, and mean birthweight values between the groups (P = 0.007, P < 0.001, P = 0.035, P < 0.001, and P = 0.001, respectively). Renal pelvis aspiration and urine analysis were substantial for the management of AHN in necessary cases. ß2-microglobulin and sodium are clinically useful markers to detect the presence of severe renal damage due to obstructive uropathy and thus, important adjuvants in the proper selection of fetuses for further antenatal interventions.

Neural and cardiac injury markers in fetal growth restriction and their relation to perinatal outcomes.

OBJECTIVE: To compare the levels of umbilical cord blood Neuron-Specific Enolase (NSE) and troponin T and venous blood gas samples between healthy newborns and growth-retarded fetuses with impaired Doppler velocity or low APGAR scores. MATERIALS AND METHODS: This study was a prospective cohort study. The study group comprised 26 patients with intrauterine growth restriction and pathologic Doppler symptoms, and the control group included 24 healthy fetuses. Umbilical cord blood and blood gas samples were taken from all patients. The blood samples were centrifuged and sent to a laboratory to study NSE and troponin T Perinatal outcomes were evaluated from the medical records of the newborns. RESULTS: Both groups were similar in terms of demographic characteristics. Fetuses with fetal growth restriction (FGR) were born earlier and had lower APGAR scores than the study group. Chronic hypoxemic fetuses in the study group had lower cord pH and HCO3 levels. Further, troponin T levels were higher in the study group than in the control group. There were no major differences in Doppler velocity measurements. CONCLUSION: It has been understood that cardiac and neuronal injury detection on fetuses with FGR, troponin T, and NSE are indicators that can be used. In the literature there are studies with heterogeneous paradigms using different indicators to find neuronal injury. As a result of this study, it is clear that to assess neonatal prognosis, wider-scoped and comparative studies will provide more information about the subject.

Evaluation of trace elements associated with antioxidant enzymes in blood of primary epithelial ovarian cancer patients.

Epithelial ovarian cancer (EOC) has been associated with oxidative stress (OS) due to epithelial inflammation which makes ovaries more vulnerable to the deleterious effects of reactive oxygen species (ROS). However, antioxidant enzymes (AOEs) such as manganese-superoxide dismutase (Mn-SOD), copper,zinc-superoxide dismutase (Cu,Zn-SOD) and glutathione peroxidase (GPx1) protect cells against the biological damage of ROS-induced OS and support cancer prevention by maintaining normal cell cycle progression, inhibiting proliferation, tumor invasion, angiogenesis, inflammation or inducing apoptosis. In the present study, we aimed to measure the levels of trace elements [manganese (Mn), copper (Cu), zinc (Zn) and selenium (Se)] which are structurally and/or functionally associated with the AOEs by inductively coupled plasma/mass-spectrometry (ICP/MS) in blood samples of patients with EOC (M, n = 26) and compare the data with healthy subjects (C, n = 46). Serous EOC (M1, n = 18) data were also evaluated according to the tumor grading [well or moderately well differentiated (G 1-2) vs. poorly differentiated or undifferentiated (G3)] and staging of disease [stage I-II (SI-II) vs. stage III (SIII)]. We obtained; i) The Mn and Se levels of M were significantly lower than C, ii) only Mn levels were changed [(G3(Mn) < G 1-2 (Mn)] in M1, iii) significant correlations were observed between [Cu and Zn levels (r = 0.701, p = 0.036) in G 1-2 and (r = 0.686, p = 0.041) in G3; Cu and Se levels (r = 0.960, p = 0.000) in G3; Mn levels and Mn-SOD expression (r = 0.551, p = 0.006) in M, (r = 0.857, p = 0.007) in G 1-2 and (r = 0.690, p = 0.056) in G3; Se levels and erythrocyte GPx1 activity (r = 0.660, p = 0.053) in G 1-2 ; Se levels and erythrocyte Cu,Zn-SOD activity (r = 0.693, p = 0.038) in G3]. The study revealed that trace elements, particularly low Mn and Se levels along with high Cu/Se ratios might be of value in all histologic subtypes of EOC. Although Mn level was important in terms of discriminating tumor grades, positive correlation between Cu-Se levels was also remarkable in patients with G 1-2 tumors of M1. Moreover, high erythrocyte Cu/Se ratios might be a favourable marker for EOC.

Assessment of oxidant-antioxidant status alterations with tumor biomarkers and reproductive system hormones in uterine MYOMAS.

OBJECTIVES: Uterine myomas (UM) are responsible for significant morbidity and have adverse effects on quality of life in women. Reactive oxygen species (ROS) and antioxidant enzymes (AOE), as well as sex steroids play important roles in the reproductive physiology processes. Thus, we aimed to investigate the role of oxidant-antioxidant status in UM by measuring the AOE activities and lipid peroxidation (LPO) levels. This is the first study assessing these parameters together in UM based on also menopausal status and evaluating possible correlations between AOE activities, LPO markers, tumor biomarkers, female reproductive system hormone levels, comprehensively. STUDY DESIGN: The study group consisted of patients who have undergone surgical resection with confirmed pathology of uterine myoma (UM, n = 25) and divided into subgroups; premenopausal (UMpre) and postmenopausal (UMpost). Erythrocyte copper-zinc superoxide dismutase (Cu,Zn-SOD), catalase (CAT), glutathione peroxidase (GPx1) activities were measured along with plasma malondialdehyde (MDA) and urinary 8-epi-prostaglandin F2α (8-epi-PGF2α) levels in patients with UM. The obtained data were compared to the data of healthy individuals (C, n = 25) and its subgroups; premenopausal (Cpre) and postmenopausal (Cpost). RESULTS: All AOE activities were higher (∼40% for Cu,Zn-SOD, p = 0.003; ∼55% for CAT, p = 0.001; ∼15% for GPx1, p = 0.169) and the LPO levels were lower (∼60% for MDA, p = 0.011 and ∼45% for 8-epi-PGF2α, p = 0.055) in patients with UM vs control. Approximately similar alterations were observed in UMpre vs Cpre and in UMpost vs Cpost. A significant negative correlation between erythrocyte Cu,Zn-SOD activity and plasma MDA levels (r = -0.431, p = 0.005) was reported. CONCLUSION: Decreased LPO levels might be the consequence of compensator high antioxidant enzyme activities against mild oxidative stress in the circulation of patients with UM. The marked negative correlation between erythrocyte Cu,Zn-SOD activity and plasma MDA levels also supported this finding.

Impaired antioxidant enzyme functions with increased lipid peroxidation in epithelial ovarian cancer.

We aimed to identify the possible role of oxidant-antioxidant status in epithelial ovarian cancer (EOC) by measuring (a) antioxidant enzyme (AOE) activities [total superoxide dismutase (SODtotal ), manganese-SOD (Mn-SOD), copper,zinc-SOD (Cu,Zn-SOD), catalase (CAT) and glutathione peroxidase (GPx1)], (b) Mn-SOD protein expression, (c) lipid peroxidation markers [malondialdehyde (MDA), 8-epi-prostaglandin-F2α (8-epi-PGF2α)] and by evaluating the possible correlations between tumor biomarkers, reproductive hormone levels and all measured parameters, comprehensively. The data obtained from the patients with EOC (M, n = 26) evaluated according to the histopathological/clinical characteristics of tumors and compared with data of healthy controls [Ctissue (C1) and Cblood/urine (C2), n = 30, respectively). Significantly, low activities of tumor SODtotal (52%), Mn-SOD (42%), Cu,Zn-SOD (55%); high activities of tumor and erythrocyte CAT (66%, 33% respectively) and tumor GPx1 (60%); high levels of tumor Mn-SOD protein expression; tumor MDA (193%) and urinary 8-epi-PGF2α (179%) were observed in serous EOC tumors (M1, n = 18) compared with controls (P < 0.05). However, higher levels of tumor MDA, Mn-SOD protein expression and urinary 8-epi-PGF2α were observed along with lower tumor CAT activity in poorly differentiated or undifferentiated (grade 3, G 3) versus well or moderately well differentiated (grade 1-2, G 1-2) serous EOC tumors. Obtained data indicate the presence of a severe redox imbalance in EOC and draw attention to the criticial role of AOEs in the pathogenesis of the disease. © 2017 IUBMB Life, 69(10):802-813, 2017.

Placental, hepatic, and supraclavicular lymph node metastasis in pancreatic adenocarcinoma during pregnancy: A case report.

The occurrence of coexisting cancer in pregnant women is not a common phenomenon. It complicates approximately 1 in 1000 to 1500 pregnancies. We present a multiparous woman aged 27 years in her 28th week of pregnancy who was admitted to our clinic with right upper quadrant pain and was finally revealed to have multiple metastatic pancreatic adenocarcinoma. To the best of our knowledge, this is the first documented case of pancreatic adenocarcinoma to metastasize both to the placenta and multiple maternal sites (liver, supraclavicular, para-aortic lymph nodes) in a pregnant patient. Unpredictable metastases to the placenta may be encountered and may even lead to definitive diagnosis, as in our case. Therefore, the placenta in any patient with known malignancy should be sent for pathologic evaluation.

Effects of in utero cord blood collection on post-cesarean hemoglobin levels.

OBJECTIVE: To assess effects of in utero cord blood collection on postoperative hemoglobin, hematocrit levels. STUDY DESIGN: Elective cesarean deliveries in which cord blood was collected were compared with match paired elective cesarean deliveries without cord blood collection. Pre-post-operative hemoglobin and hematocrit level differences were compared between study groups with Student's t test. Multivariate regression models were used to address confounders. Correlation between volume of collected UCB and mean decrease in blood count parameters was analyzed. RESULTS: A total of 399 cesarean deliveries during a 12 months period were included in the analysis. Mean decrease in hemoglobin levels was 1.08g/dL (SD=1.0) in UCB collected group compared to 0.84g/dL (SD=1.0) in control group (p=0.002). Mean decrease in hematocrit levels was 3.1% (SD=3.4) in cord blood collected cesarean delivery group compared to 1.9% (SD=2.4) in control group (p=0.002). Univariate analysis has shown the collected UCB volume to be uncorrelated with the change in hemoglobin levels (r=0.013). Multivariate regression models, after adjusting for birth weight, age and number of prior cesarean, have shown the UCB collection to be significantly associated with the mean decrease in blood count parameters (estimate=0.23g/dL, t=-2.23, p=0.02). CONCLUSION: In utero UCB collection is associated with a small increase in bleeding of little clinical importance. Amount of UCB is not associated with amount of change in hemoglobin and hematocrit levels. In utero UCB collection seems to be safe for expectant mothers scheduled for low-risk cesarean delivery.

Maternal blood and amniotic fluid levels of moxifloxacin, levofloxacin and cefixime.

AIM: Moxifloxacin and levofloxacin are wide spectrum quinolones and cefixime is a third-generation cephalosporin with a wider spectrum of activity against gram-positive and gram-negative bacteria and anaerobics. Although they are widely used, little is known about the amniotic fluid levels of these antibiotics. The aim of the present investigation was to study and compare the maternal blood and amniotic fluid levels of these antibiotics in second trimester pregnancy. METHODS: To assess the amniotic fluid levels of these antibiotics, 10 pregnant women were given moxifloxacin, 10 were given levofloxacin and 6 were given cefixime orally 2 h before amniocentesis as a single dose for prophylaxis. During amniocentesis, an extra 2 mL amniotic fluid sample and 2 mL maternal venous blood were drawn. The levels of these agents in samples were analyzed using high performance liquid chromatography. RESULTS: The amniotic fluid levels of moxifloxacin and levofloxacin were 0.27 +/- 0.21 microg/mL and 0.60 +/- 0.41 microg/mL, respectively. The maternal blood levels were 3.53 +/- 0.65 microg/mL and 3.95 +/- 0.77 microg/mL in the moxifloxacin and levofloxacin groups, respectively. The maternal blood level of cefixime was 2.59 +/- 1.10 microg/mL and the amniotic fluid level was 0.85 +/- 0.42 microg/mL. The amniotic fluid passage rates were 7.83% for moxifloxacin, 15.67% for levofloxacin and 37.55% for cefixime. CONCLUSION: Of these three antibiotics, cefixime has the highest transplacental passage rate and, therefore, can be used as a therapeutic agent in infectious conditions in which membranes and the placenta are involved. Moxifloxacin and levofloxacin have low passage rates, which should be considered when using as a therapeutic agent.

Maternal and fetal blood levels of moxifloxacin, levofloxacin, cefepime and cefoperazone.

Wide-spectrum quinolones such as moxifloxacin and levofloxacin as well as high-order cephalosporins such as cefoperazone and cefepime have increased antimicrobial activity. However, little is known about their distribution in fetal blood. Therefore, the aim of this study was to measure and compare maternal and fetal blood levels of these agents. For the measurement of blood levels, 9 pregnant women received cefepime hydrochloride, 10 received cefoperazone, 10 received moxifloxacin and 12 received levofloxacin intravenously. Maternal and umbilical cord blood samples were drawn during delivery. Antibiotic levels were analysed by high-performance liquid chromatography. Mean transplacental passage rates of moxifloxacin, levofloxacin, cefepime and cefoperazone were 74.84%, 66.53%, 23.21% and 12.68%, respectively, and mean transfetal passage rates were 90.78%, 84.22%, 79.17% and 79.78%, respectively. The transplacental passage rate for either quinolone was significantly higher than that of either cephalosporin, and the transplacental passage rate of cefoperazone was lower than that of cefepime. In conclusion, both quinolones have high transplacental passage rates. Cefepime and cefoperazone have a lower transplacental passage rate and thus may be used as prophylaxis in situations where transplacental passage is undesirable.

Comparison of pharmacokinetic profiles of moxifloxacin in Caesarean versus non-pregnant sectioned women by fully validated HPLC with fluorescence detection.

In this study, a simple, rapid, cost-effective, and sensitive reversed-phase high-performance liquid chromatographic method has been developed and validated for the analysis of moxifloxacin in plasma. The chromatographic separation was achieved on a Zorbax Eclipse XDB-C18 column (150 mm x 4.6 mm i.d.) connected to a Phenomenex C(18) column (4 mm x 3.0 mm i.d.) using a mixture of acetonitrile: 15 mM citrate buffer (pH 3) (23:77, v/v) as the mobile phase with isocratic system at a flow rate of 1 mL/min. Fluorescence detection was employed with excitation at 290 nm and emission at 500 nm. Lomefloxacin was used as internal standard. Plasma samples were prepared with addition of acetonitrile only. The method was fully validated according to the International Conference on Harmonization (ICH) guidelines. The results of the validation parameters were: linearity range, 3-6000 ng/mL (R(2) = 0.9994); mean recovery, 100.48 %; limit of quantification, 5 ng/mL; limit of detection, 1 ng/mL; and intra- and inter-day precision less than 3.2% and 5.1%, respectively. The robustness of the method was evaluated and confirmed with fractional factorial design. After validation studies, the method was applied in order to conclude the effects of pregnancy on postoperative pharmacokinetic profiles of moxifloxacin. For this aim, moxifloxacin was given to non-pregnant women (n=9) and caesarean-sectioned women (n=6) as a single intravenous dose (400 mg Avelox(R) infusion). Plasma samples were analyzed in order to compare pharmacokinetic profiles of pregnants and non-pregnants. Peak serum concentrations of non-pregnant and caesarean-sectioned women at the arterial port after the infusion were 4.95 +/- 1.50 and 1.56 +/- 0.16 microg/mL, respectively. The mean elimination half-life, volume of distribution and calculated area under the concentration-time curve (AUC)(0-infinity) were 5.54 +/- 0.73 h, 65.58 +/- 6.30 L and 49.95 +/- 6.30 microg.h/mL for non-pregnant women and 3.50 +/- 0.37 h, 215.85 +/- 24.87 L and 10.53 +/- 0.66 microg.h/mL for caesarean-sectioned women, respectively. These results indicated that pregnancy has a significant effect on the pharmacokinetics of moxifloxacin.

Simultaneous multiresponse optimization of an HPLC method to separate seven cephalosporins in plasma and amniotic fluid: application to validation and quantification of cefepime, cefixime and cefoperazone.

An HPLC method for the separation of seven cephalosporins [Cefepime (CEP), ceftazidime (CTA), ceftizaxime (CTI), ceftriaxone (CTR), cefotaxime (COT), cefixime (CIX) and cefoperazone (COP)] in human plasma and amniotic fluid has been developed. Optimization of the chromatographic method was performed in three steps: a series of initial experiments followed by two sets of experiments based on different experimental designs. The initial experiments were performed to decide the basic analytical requirements of the method. Then screening experiment fractional factorial design was used in order to decrease the number of parameters by eliminating parameters which having insignificant effect on responses. The parameters having significant effect were further optimized through a full factorial design. Having studied two responses (retention times and resolutions), a desirability function that assess the responses together, was used to find experimental conditions where the system generated desirable results. The desirable results were obtained with XTerra C18 (250 mm x 4.6mm, 5 microm i.d.) column, 40 mM phosphate buffer, pH 3.2, 18% MeOH, 0.85 mL min(-1) flow rate and 32 degrees C column temperature. Gradient elution with MeOH was applied. A simple and efficient solid-phase extraction was applied for the preparation of plasma and amniotic fluid samples. The validation parameters of the method were evaluated in accordance with ICH guideline. The method validated was applied to the analysis of CEP and COP in maternal venous, fetal venous and fetal arterial plasma, and to the analysis of CIX in maternal venous plasma and amniotic fluid.

Endometrial osseous metaplasia: an evolving cause of secondary infertility.

OBJECTIVE: To present an endometrial osseous metaplasia case and reemphasize that the condition is a cause of secondary infertility. DESIGN: Case report. SETTING: Department of Obstetrics and Gynecology in a university hospital in Turkey. PATIENT(S): A 33-year-old multiparous woman was admitted to our institution with secondary infertility that had lasted for 2 years. She had experienced one first-trimester and one second-trimester abortion, 3 years and 2 years ago, respectively. On transvaginal sonography, a linear curvy echogenity was observed. INTERVENTION(S): Hysteroscopic examination revealed multiple bony spicules, extending perpendicularly from the posterior uterine wall in to the uterine cavity and occupying almost two thirds of the cavity. Thereafter, a resectoscopic excision of the bony spicules was performed. MAIN OUTCOME MEASURE(S): A normal endometrium and uterine cavity. RESULT(S): Two weeks after the operation, ultrasonographic evaluation was in the normal range, and the patient currently is trying to conceive spontaneously. CONCLUSION(S): Although the role of office hysteroscopy in the evaluation of infertile couple is still under debate, clinicians should keep this rare disorder in mind, especially in patients with a history of late abortion, and should evaluate such cases by hysteroscopy when sonographic features are encountered.