Placenta microRNA profile of patient with Obstetric Antiphospholipid Syndrome.

The onset of obstetric antiphospholipid syndrome (APS) occurs when antiphospholipid antibodies act upon the placenta. During pregnancy, APS exhibits traits such as vascular thrombosis, inflammation, and hindered trophoblast implantation. The involvement of microRNA expression has been proposed as a genetic factor contributing to the syndrome's development. MicroRNAs play a role in regulating gene expression in various cellular processes, including the formation of placental tissue. Therefore, additional research is needed to explore the control of placental miRNA in APS. In this study, we aimed to profile miRNA expressions from placenta tissue of patients with APS. Differentially expressed miRNAs were determined for its targeted genes and pathways. Agilent microarray platform was used to measure placental microRNA expressions between normal placental tissue and those obtained from patients with APS. Differentially expressed miRNAs were detected using GeneSpring GX software 14.2 and sequences were mapped using TargetScan software to generate the predicted target genes. Pathway analysis for the genes was then performed on PANTHER and REACTOME software. Selected miRNAs and their associated genes of interest were validated using qPCR. Microarray findings revealed, 9 downregulated and 21 upregulated miRNAs expressed in placenta of patients with APS. Quantitative expressions of 3 selected miRNAs were in agreement with the microarray findings, however only miR-525-5p expression was statistically significant. Pathway analysis revealed that the targeted genes of differentially expressed miRNAs were involved in several hypothesised signalling pathways such as the vascular endothelial (VE) growth factor (VEGF) and inflammatory pathways. VE-cadherin, ras homolog member A (RHOA) and tyrosine kinase receptor (KIT) showed significant downregulation while Retinoblastoma gene (RET), Dual specificity protein phosphatase 10 (DUSP10) and B-lymphocyte kinase (BLK) genes were significantly upregulated. These preliminary findings suggest the involvement of miRNAs and identified novel associated genes involvement in the mechanism of obstetric APS, particularly through the alteration of vascular-associated regulators and the inflammatory signalling cascade.

First report and molecular characterization of Cryptosporidium spp. in humans and animals in Khartoum state, Sudan.

BACKGROUND AND AIM: Cryptosporidium is recognized to infect several mammalian species as well as humans, causing substantial economic losses and serious public health concern. Infected animals can be a source of environmental contamination and human infections. In general, the occurrence of Cryptosporidium species in animals and human in Sudan and zoonotic importance is not well documented. This study aimed to identify Cryptosporidium spp. infecting different animal species and humans and to compare between different isolates obtained. MATERIALS AND METHODS: To provide molecular information about Cryptosporidium in animals and humans, both modified Ziehl-Neelsen (MZN) specific stain and molecular assay were used. Concentration techniques followed by three protocols of DNA extraction were carried out. After microscopic screening of 263 fecal samples (goats [n=197], cattle [n=12], sheep [n=12], and human [n=42]), 61 positive and 30 negative, randomly selected samples were used in nested polymerase chain reaction (PCR) targeting part of the 18S RNA. RESULTS: Nested PCR amplification confirmed 91.8% (56/61) of microscopic-positive samples. 8.2% (5/61) of negative samples by PCR (positive by microscopy) were considered false negatives. Sequencing followed by alignment of the 14 isolates indicated that all samples were identical (100%) and belonged to Cryptosporidium parvum. CONCLUSION: MZN staining procedure is reliable for the routine diagnosis of Cryptosporidium; cetyltrimethylammonium bromide extraction buffer and nested PCR targeting 18S rRNA gene are reliable and useful in epidemiological studies of this parasite.

Bioavailability of a new generic formulation of mycophenolate mofetil MMF 500 versus CellCept in healthy adult volunteers.

Several studies have revealed a decreased incidence of early graft rejection with the use of mycophenate mofetil (MMF). The cost of the drug is, however, prohibitive especially in developing countries with limited resources. We compared the pharmacokinetic profile of a new MMF generic formulation (MMF 500 batch number: 06T3001; Medis Tunis) with those of Cellcept, (batch number: M1427; Hoffmann La Roche, Switzerland) in healthy volunteers. The study was double-blinded to investigator and volunteers. It had a balanced randomized, two-treatment, two-period, two-sequence, single-dose, crossover, comparative oral bioavailability design in adult healthy human volunteers. The study was designed, performed, and monitored by CRO Transmedical s.a.l International (Beirut, Lebanon) in accordance with the Basic Principals defined in the US 21 CFR Part 312.20, and the principals enunciated in the World Medical Association Declaration of Helsinki. We included nonsmoking healthy volunteers between the ages of 22 and 45 years. The subjects were admitted to the hospital one night prior to blood sampling. After volunteers received the same dinner, they were fasted overnight and for 2 hours postdosing. At 8 am each person received a single oral dose of 500 mg of either formulation. Blood samples were collected to construct the pharmacokinetic profiles as follows: 0, 0.15, 0.30, 0.45 minutes and 1, 1.15, 1.30, 2, 4, 6, 10, 12, and 24 hours. Water and food intake were the same for all volunteers during the whole study period. Following an 8-day washout period, the subjects were crossed over. Plasma mycophenolic acid concentrations were determined using a high-performance liquid chromatography validated enzyme-linked immunosorbent assay-based method (TransMedical, Beirut Lebanon). Physical examinations, hematology, urinanalysis, serum chemistry tests, and liver enzymes were performed at screening and at the end of each period. Subjects were monitored for safety and adverse events throughout the study by two physicians (one from the hospital and one from TransMedical). The Cmax, Tmax, and AUC for MMF 500 were 10.14 ng/mL, 51.82 minutes, and 18.33 ng/mL/h vs 10.94 ng/mL, 49.09 minutes, and 17.46 ng/mL/h for CellCept, respectively. The 90% confidence intervals (LSM) of Cmax, Tmax, and AUC for MMF 500 were 92.7%, 105.6%, and 105%, respectively, which is within the Food and Drug Administration (FDA)-assigned range for immunosuppressive drugs (90% to 111%). These results indicated that the products are equivalent and switchable according to FDA rulings.

Calcineurin inhibitor-free protocols: risks and benefits.

The nephrotoxic and extra-renal adverse effects associated with calcineurin inhibitor (CNI) therapies appear to have a negative impact on long-term graft survival. Several CNI minimization protocols have been recently studied. These protocols involve either early CNI avoidance or CNI withdrawal. CNI withdrawal strategies are associated with a significant improvement in renal function and graft survival on both a short and long-term basis. Delayed and progressive withdrawal appears to be safer. Maintaining a high mycophenolate mofetil (MMF) or sirolimus (SIR) exposure minimizes the risk of acute rejection. CNI avoidance regimens using maintenance mono-therapy or combination therapies without induction appear to be immunologically risky and unsafe. In contrast, the combination of SIR + MMF with induction therapy reduces markedly the incidence of acute rejection and chronic allograft nephropathy (CAN). Two year patient and graft survival levels were comparable. CAN as well as the incidence and the risk for cancer in addition to blood pressure profiles and uric acid levels were overall lower in the SIR-based treatment. In contrast, hyperlipidemia, delayed wound healing, lymphocele, arthralgias, thrombocytopenia and study protocol deviations were reported more frequently in the SIR-maintenance protocols. Longer-term follow-ups are definitely needed to determine whether these avoidance strategies will result in a significant improvement in long-term patient and graft survival. Outcome differences among various protocols within the same CNI elimination strategy are probably related to study design, patient selection criteria, immunosuppression monitoring methods, indications for graft biopsies, environmental, and both genetic and ethnic factors. All monitoring techniques are unreliable short of a graft biopsy. Preliminary results on drug lymphocyte binding may offer new guidelines for tailoring immunosuppression. Whether these protocols based on SIR or SIR + MMF can also be extended to high risk patients is currently unknown. These encouraging results allow speculation but with caution that the use of the combination of non-nephrotoxic immunosuppression such as SIR and MMF, might change dramatically the natural course of CAN and may influence long-term patient survival.

Cyclosporine lymphocyte maximum level monitoring in de novo kidney transplant patients: a prospective study.

OBJECTIVES: To determine prospectively the temporal variations of cyclosporine-A lymphocyte maximum level, whole blood maximum concentration, and total lymphocyte count in patients with de novo kidney transplantation. MATERIALS AND METHODS: Lymphocyte maximum level, whole blood maximum concentration, and total lymphocyte count were prospectively measured in 35 patients at 1, 2, and 3 months after kidney transplantation. Two groups--a biopsy-proven acute rejection group (REJ+) and a rejection-free group (REJ-)--were compared. RESULTS: Both groups had similar lymphocyte maximum levels, whole blood maximum concentrations, and total lymphocyte counts at the first month after transplantation. REJ+ patients had significantly lower lymphocyte maximum levels at 2 and 3 months (59+/-34 and 33+/-9 pg/Lc) and higher total lymphocyte counts (0.00204+/-0.00078x10(9)/L and 0.00203+/-0.00022x10(9)/L) when compared with their REJ- counterparts (87+/-56 and 63+/-30 pg/Lc, P<.05 and P<.007) and (0.00137+/-0.00074x10(9)/L and 0.0015+/-0.0006x10(9)/L, P<.02 and P<.003) respectively. Whole blood maximum concentrations were significantly higher in patients in the REJ+ group (2050+/-623 vs 1414+/-536 ng/mL, P<.02) at 2 months. At 3 months, the 2 groups were comparable (1158+/-340 vs 1365+/-525 ng/mL, P=NS). CONCLUSIONS: These results suggest that acute rejection is associated with a relatively low cyclosporine- A lymphocyte maximum level and high total lymphocyte count in the early posttransplant period. Cyclosporine-A whole blood maximum concentration failed to correlate with clinical outcome. Cyclosporine-A lymphocyte maximum level seems to offer a more reliable alternative than does whole blood maximum concentration for cyclosporine-A monitoring in patients with kidney transplantation.

Switchability of neoral and equoral according to Food and Drug Administration rules and regulations.

According to the US Food and Drug Administration (FDA), if a drug product contains a drug substance that is chemically identical and is delivered to the site of action at the same rate and extent as another drug product, then it is equivalent and can be substituted (switchable) for that drug product. Methods used to define bioequivalence as stated by the FDA rules (FDA 21 CFR 320, 24) are (1) pharmacokinetic (PK) studies in healthy volunteers, (2) comparative clinical trials, and (3) pharmacodynamic (PD) studies (bioactivity). We evaluated the switchability of Equoral (IVAX-USA) with Neoral (Novartis Switzerland using all FDA rules. In a single oral dose, we undertook a comparative bioavailability study of Equoral (IVAX, USA) Neoral (Novartis, USA), and Neoral (Novartis UK). The pharmacokinetics of Equoral and Neoral were determined with blood levels at 0, 0.25, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.5, 3, 3.5, 4, 5, 6, 8, 10, 12, 16, 24, 30, 36, 42, and 48 hours. The area under curve (AUC), AUC extrapolated to infinity (AUC0-inf), rate of absorption (Tmax), extent of absorption (Cmax), half time (t1/2) of Equoral and Neoral were all within the 90% confidence interval of 80% to 125% boundaries. A comparative multinational multicenter clinical trial in stable renal transplant patients included 70 patients (22 women and 48 men) of mean age of 33 years (range, 26 to 43) was performed in Turkey, Lebanon, and Pakistan. In this study the ratios of LSM and the 90% confidence intervals for the Nontransformed/Parameters (AUC0-t, AUCinf, Tmax, and Cmax) of Equoral and Neoral SGC were 98% and 95%, respectively, which are within the 80% to 125% FDA acceptance range. For immunosuppressive drugs, the site of action is the lymphocyte and the measurable response is the decrease in lymphocyte count caused by the relative concentration of the drug in the lymphocyte. In a controlled switch, fixed-dose study, both Equoral and Neoral achieved the same concentration in the lymphocytes and caused the same degree of lymphocyte count reduction. The results of the testing (bioavailability-bioequivalence, clinical studies, and pharmacodynamic-bioactivity) required by FDA for interchangeability ("switchability") of immunosuppressive agents suggests that Neoral and Equoral are switchable.

Mycophenolic acid plasma trough level: correlation with clinical outcome.

OBJECTIVES: Assess the relationship between clinical diagnosis, state of immunosuppression, mycophenolic acid (MPA) plasma trough levels (MPACmin), and mycophenolate mofetil (MMF) dosage in renal transplant recipients. MATERIALS AND METHODS: MPACmin were determined in 30 kidney transplant patients, of whom 7 exhibited biopsy-proven acute rejection. The remaining 23 had normal graft function. Graft outcome, defined by clinical diagnosis and serum creatinine level, was compared according to MPACmin, MMF dosage, and total lymphocyte count (LC). RESULTS: Patients with acute rejection had similar MPACmin (2.4 +/- 1.7 microg/mL), MMF dosages (1.7 +/- 0.5 g), and LCs (0.001165 +/- 0.0040 x 10(9)/L) when compared with normal patients (2.2 +/- 0.7 microg/mL, 1.7 +/- 0.4 g and 0.001160 +/- 0.00527 x 10(9)/L) respectively. Rejection rates were comparable irrespective of MPACmin)ranges and higher in those receiving the 1-g dose (30%) when compared with those receiving 1.5-g and 2-g doses (12.5% and 11.7%). No relationship was observed between MPACmin and MMF doses, and neither parameter correlated with LC. CONCLUSIONS: These results suggest that MPACmin is a poor correlate of clinical outcome and state of immunosuppression. Although the usually recommended dosage of MMF (2 g) may be associated with acute rejection, low-dose MMF (1 g) seems to constitute a higher risk.

The role of generics in transplantation: TM-MMF versus Cellcept in healthy volunteers.

Because several studies having revealed a relation between early graft rejection and long-term graft survival, potential benefits have been attributed to MMF. The cost of the drug is, however, prohibitive, which renders its long-term use in countries with limited income. We thus compared the pharmacokinetic profiles of a new MMF generic formulation (MM-Cept developed by Ivax CR) with those of Cellcept (Hoffman La Roche) in healthy volunteers. This open label, balanced randomized, two-treatment, two-period, two-sequence, single-dose, crossover, comparative oral bioavailability study was conducted in non-smoking adult male healthy volunteers between the ages of 18 and 45 years. The study was performed in accordance with the basic principles defined in the US 21 CFR Part 312.20 and the principles enunciated in the Declaration of Helsinki (World Medical Association Declaration of Helsinki). The subjects were given a single oral 1 g dose with a washout period of 10 days. Pharmacokinetic profiles included blood levels at 0, 0.25, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.5, 3, 4, 6, 10, 12, 16, 20, 24, 30, 36, and 48 hours following each dose. The formulations were MMCept 500 mg tablets and Cellcept 500 mg tablets. Subjects were fasted overnight and for 4 hours postdosing. Mycophenolic acid (MPA) concentrations were determined using HPLC. Physical examinations, hematology, urinalysis, and serum chemistry tests including liver enzymes were performed at screening and at the end of the study. Subjects were monitored for safety and adverse events throughout the study. Both products showed similar bioavailability. The LSM were within the limits for FDA approval (80 to 125), suggesting that the two products are equivalent and switchable.

The pharmacokinetics of equoral versus neoral in stable renal transplant patients: a multinational multicenter study.

We studied the pharmacokinetics (PKs) of the new generic cyclosporine formulation, Equoral capsules, after the switch from original formulation Neoral capsules in stable renal transplant patients. The study was carried out in accordance with the basic principles defined in the US 21 CFR Part 312.20 and the principles of the Declaration of Helsinki. The study included clinically stable first renal transplant patients maintained on cyclosporine with no rejection episode during the past 6 months. Hematology, biochemistry, and urine chemistry were determined on day 7, and day 21. The patients were all switched to Neoral (lot number 416MFD0601) on day 0 when the first sparse sampling PK was performed. On day 14 a 12-hour PK profile included predose, 30 minutes; 1 hour; 1 hour 30 minutes; 2 hours; 3 hours; 4 hours; 5 hours; 6 hours; 8 hours; 10-hours and 12-hour samples. Cyclosporine levels were determined using a CYA kit (Abbott TDx). On day 15 the patients were switched from Neoral capsules to Equoral capsules (lot 5T111014) at an equivalent dosage (mg/mg). The second sparse sampling PK was performed on day 21 and a 12-hour PK was performed on day 28. On the morning of day 29 patients were switched from Equoral capsules to Neoral capsules at an equivalent dosage (mg/mg). Additional concentrations were measured on days -7, 18, and 35. Safety parameters were monitored at each visit. The pharmacokinetics of both formulations were equivalent. The mean AUC for Neoral and Equoral was 2856 and 2892, respectively. The ratios of LSM and the 90% confidence intervals for the in-transformed parameters (AUC o-t, AUC inf, and Cmax) of Equoral and Neoral SGC were 98% and 95%, respectively, suggesting that Equoral and Neoral SGC are bioequivalent.

Minor role for BRCA2 (exon11) and p53 (exon 5-9) among Sudanese breast cancer patients.

A cohort of 20 breast cancer patients from the Sudan was tested for germ line and somatic mutation in their BRCA2 exon 11 as well as the main conserved area of the p53 tumor suppressor gene. The results indicate that both regions may play a limited role in the pathogenesis of breast cancer in those patients. The fact that there are no somatic mutations detected in p53 was particularly surprising as the expected rate for mutations in breast cancer is 30-50%.