Shedding light on the effects of blood on meniscus tissue: the role of mononuclear leukocytes in mediating meniscus catabolism.

OBJECTIVE: Traumatic meniscal injuries can cause acute pain, hemarthrosis (bleeding into the joint), joint immobility, and post-traumatic osteoarthritis (PTOA). However, the exact mechanism(s) by which PTOA develops following meniscal injuries is unknown. Since meniscus tears commonly coincide with hemarthrosis, investigating the direct effects of blood and its constituents on meniscus tissue is warranted. The goal of this study was to determine the direct effects of blood and blood components on meniscus tissue catabolism. METHODS: Porcine meniscus explants or primary meniscus cells were exposed to whole blood or various fractions of blood for 3 days to simulate blood exposure following injury. Explants were then washed and cultured for an additional 3 days prior to collection for biochemical analyses. RESULTS: Whole blood increased matrix metalloproteinase (MMP) activity. Fractionation experiments revealed blood-derived red blood cells did not affect meniscus catabolism. Conversely, viable mononuclear leukocytes induced MMP activity, nitric oxide (NO) production, and loss of tissue sulfated glycosaminoglycan (sGAG) content, suggesting that these cells are mediating meniscus catabolism. CONCLUSIONS: These findings highlight the potential challenges of meniscus healing in the presence of hemarthrosis and the need for further research to elucidate the in vivo effects of blood and blood-derived mononuclear leukocytes due to both hemarthrosis and blood-derived therapeutics.

Natural history of monoclonal B-cell lymphocytosis among relatives in CLL families.

Chronic lymphocytic lymphoma (CLL) has one of the highest familial risks among cancers. Monoclonal B-cell lymphocytosis (MBL), the precursor to CLL, has a higher prevalence (13%-18%) in families with 2 or more members with CLL compared with the general population (5%-12%). Although, the rate of progression to CLL for high-count MBLs (clonal B-cell count ≥500/µL) is ∼1% to 5%/y, no low-count MBLs have been reported to progress to date. We report the incidence and natural history of MBL in relatives from CLL families. In 310 CLL families, we screened 1045 relatives for MBL using highly sensitive flow cytometry and prospectively followed 449 of them. MBL incidence was directly age- and sex-adjusted to the 2010 US population. CLL cumulative incidence was estimated using Kaplan-Meier survival curves. At baseline, the prevalence of MBL was 22% (235/1045 relatives). After a median follow-up of 8.1 years among 449 relatives, 12 individuals progressed to CLL with a 5-year cumulative incidence of 1.8%. When considering just the 139 relatives with low-count MBL, the 5-year cumulative incidence increased to 5.7%. Finally, 264 had no MBL at baseline, of whom 60 individuals subsequently developed MBL (2 high-count and 58 low-count MBLs) with an age- and sex-adjusted incidence of 3.5% after a median of 6 years of follow-up. In a screening cohort of relatives from CLL families, we reported progression from normal-count to low-count MBL to high-count MBL to CLL, demonstrating that low-count MBL precedes progression to CLL. We estimated a 1.1% annual rate of progression from low-count MBL, which is in excess of that in the general population.

Cerebrospinal Fluid Pterins, Pterin-Dependent Neurotransmitters, and Mortality in Pediatric Cerebral Malaria.

BACKGROUND: Cerebral malaria (CM) pathogenesis remains incompletely understood. Having shown low systemic levels of tetrahydrobiopterin (BH4), an enzymatic cofactor for neurotransmitter synthesis, we hypothesized that BH4 and BH4-dependent neurotransmitters would likewise be low in cerebrospinal fluid (CSF) in CM. METHODS: We prospectively enrolled Tanzanian children with CM and children with nonmalaria central nervous system conditions (NMCs). We measured CSF levels of BH4, neopterin, and BH4-dependent neurotransmitter metabolites, 3-O-methyldopa, homovanillic acid, and 5-hydroxyindoleacetate, and we derived age-adjusted z-scores using published reference ranges. RESULTS: Cerebrospinal fluid BH4 was elevated in CM (n = 49) compared with NMC (n = 51) (z-score 0.75 vs -0.08; P < .001). Neopterin was increased in CM (z-score 4.05 vs 0.09; P < .001), and a cutoff at the upper limit of normal (60 nmol/L) was 100% sensitive for CM. Neurotransmitter metabolite levels were overall preserved. A higher CSF BH4/BH2 ratio was associated with increased odds of survival (odds ratio, 2.94; 95% confidence interval, 1.03-8.33; P = .043). CONCLUSION: Despite low systemic BH4, CSF BH4 was elevated and associated with increased odds of survival in CM. Coma in malaria is not explained by deficiency of BH4-dependent neurotransmitters. Elevated CSF neopterin was 100% sensitive for CM diagnosis and warrants further assessment of its clinical utility for ruling out CM in malaria-endemic areas.

Blood in the joint: effects of hemarthrosis on meniscus health and repair techniques.

Injury to the meniscus is common and frequently leads to the development of post-traumatic osteoarthritis (PTOA). Many times meniscus injuries occur coincident with anterior cruciate ligament (ACL) injuries and lead to a bloody joint effusion. Hemarthrosis, or bleeding into the joint, has been implicated in degeneration of joint tissues. The goal of this review paper is to understand the pathophysiology of blood-induced joint damage, the possible effects of blood on meniscus tissue, and the implications for current meniscus repair techniques that involve the introduction of blood-derived products into the joint. In this review, we illustrate the similarities in the pathophysiology of joint damage due to hemophilic arthropathy (HA) and osteoarthritis (OA). Although numerous studies have revealed the harmful effects of blood on cartilage and synovium, there is currently a gap in knowledge regarding the effects of hemarthrosis on meniscus tissue homeostasis, healing, and the development of PTOA following meniscus injury. Given that many meniscus repair techniques utilize blood-derived and marrow-derived products, it is essential to understand the effects of these factors on meniscus tissue and the whole joint organ to develop improved strategies to promote meniscus tissue repair and prevent PTOA development.

Early Endothelial Activation Precedes Glycocalyx Degradation and Microvascular Dysfunction in Experimentally Induced Plasmodium falciparum and Plasmodium vivax Infection.

Endothelial activation and microvascular dysfunction are key pathogenic processes in severe malaria. We evaluated the early role of these processes in experimentally induced Plasmodium falciparum and P. vivax infection. Participants were enrolled in induced blood-stage malaria clinical trials. Plasma osteoprotegerin, angiopoietin-2, and von Willebrand Factor (vWF) levels were measured as biomarkers of endothelial activation. Microvascular function was assessed using peripheral arterial tonometry and near-infrared spectroscopy, and the endothelial glycocalyx was assessed by sublingual videomicroscopy and measurement of biomarkers of degradation. Forty-five healthy, malaria-naive participants were recruited from 5 studies. Osteoprotegerin and vWF levels increased in participants following inoculation with P. vivax (n = 16) or P. falciparum (n = 15), with the angiopoietin-2 level also increasing in participants following inoculation with P. falciparum For both species, the most pronounced increase was seen in osteoprotegerin. This was particularly marked in participants inoculated with P. vivax, where the osteoprotegerin level correlated with the levels of parasitemia and the malaria clinical score. There were no changes in measures of endothelial glycocalyx or microvascular function. Plasma biomarkers of endothelial activation increased in early P. falciparum and P. vivax infection and preceded changes in the endothelial glycocalyx or microvascular function. The more pronounced increase in osteoprotegerin suggests that this biomarker may play a role in disease pathogenesis.

Association of polygenic risk score with the risk of chronic lymphocytic leukemia and monoclonal B-cell lymphocytosis.

Inherited loci have been found to be associated with risk of chronic lymphocytic leukemia (CLL). A combined polygenic risk score (PRS) of representative single nucleotide polymorphisms (SNPs) from these loci may improve risk prediction over individual SNPs. Herein, we evaluated the association of a PRS with CLL risk and its precursor, monoclonal B-cell lymphocytosis (MBL). We assessed its validity and discriminative ability in an independent sample and evaluated effect modification and confounding by family history (FH) of hematological cancers. For discovery, we pooled genotype data on 41 representative SNPs from 1499 CLL and 2459 controls from the InterLymph Consortium. For validation, we used data from 1267 controls from Mayo Clinic and 201 CLL, 95 MBL, and 144 controls with a FH of CLL from the Genetic Epidemiology of CLL Consortium. We used odds ratios (ORs) to estimate disease associations with PRS and c-statistics to assess discriminatory accuracy. In InterLymph, the continuous PRS was strongly associated with CLL risk (OR, 2.49; P = 4.4 × 10-94). We replicated these findings in the Genetic Epidemiology of CLL Consortium and Mayo controls (OR, 3.02; P = 7.8 × 10-30) and observed high discrimination (c-statistic = 0.78). When jointly modeled with FH, PRS retained its significance, along with FH status. Finally, we found a highly significant association of the continuous PRS with MBL risk (OR, 2.81; P = 9.8 × 10-16). In conclusion, our validated PRS was strongly associated with CLL risk, adding information beyond FH. The PRS provides a means of identifying those individuals at greater risk for CLL as well as those at increased risk of MBL, a condition that has potential clinical impact beyond CLL.

Glycocalyx breakdown is increased in African children with cerebral and uncomplicated falciparum malaria.

Cerebral malaria (CM) from Plasmodium falciparum infection is associated with endothelial dysfunction and parasite sequestration. The glycocalyx (GCX), a carbohydrate-rich layer lining the endothelium, is crucial in vascular homeostasis. To evaluate the role of its loss in the pathogenesis of pediatric CM, we measured GCX degradation in Tanzanian children with World Health Organization-defined CM (n = 55), uncomplicated malaria (UM; n = 20), and healthy controls (HCs; n = 25). Urine GCX breakdown products [glycosaminoglycans (GAGs)] were quantified using dimethylmethylene blue (DMMB) and liquid chromatography-tandem mass spectrometry assays. DMMB-GAG and mass spectrometry (MS)-GAG (g/mol creatinine) were increased in CM and UM compared with HCs (P < 0.001), with no differences in DMMB-GAG and MS-GAG between CM and UM children or between those with and without a fatal outcome. In CM survivors, urinary GCX DMMB-GAG normalized by d 3. After adjusting for disease severity, DMMB-GAG was significantly associated with parasitemia [partial correlation coefficient (Pcorr) = 0.34; P = 0.01] and plasma TNF (Pcorr = 0.26; P = 0.04) and inversely with plasma and urine NO oxidation products [Pcorr = -0.31 (P = 0.01) and Pcorr = -0.26 (P = 0.03), respectively]. GCX breakdown is increased in children with falciparum malaria, with similar elevations in CM and UM. Endothelial GCX degradation may impair endothelial NO production, exacerbate adhesion-molecule expression, exposure, and parasite sequestration, and contribute to malaria pathogenesis.-Yeo, T. W., Bush, P. A., Chen, Y., Young, S. P., Zhang, H., Millington, D. S., Granger, D. L., Mwaikambo, E. D., Anstey, N. M., Weinberg, J. B. Glycocalyx breakdown is increased in African children with cerebral and uncomplicated falciparum malaria.

Evaluation of culture conditions for in vitro meniscus repair model systems using bone marrow-derived mesenchymal stem cells.

Purpose: Meniscal injury and loss of meniscus tissue lead to osteoarthritis development. Therefore, novel biologic strategies are needed to enhance meniscus tissue repair. The purpose of this study was to identify a favorable culture medium for both bone marrow-derived mesenchymal stem cells (MSCs) and meniscal tissue, and to establish a novel meniscus tissue defect model that could be utilized for in vitro screening of biologics to promote meniscus repair.Materials and Methods: In parallel, we analyzed the biochemical properties of MSC - seeded meniscus-derived matrix (MDM) scaffolds and meniscus repair model explants cultured in different combinations of serum, dexamethasone (Dex), and TGF-ß. Next, we combined meniscus tissue and MSC-seeded MDM scaffolds into a novel meniscus tissue defect model to evaluate the effects of chondrogenic and meniscal media on the tissue biochemical properties and repair strength.Results: Serum-free medium containing TGF-ß and Dex was the most promising formulation for experiments with MSC-seeded scaffolds, whereas serum-containing medium was the most effective for meniscus tissue composition and integrative repair. When meniscus tissue and MSC-seeded MDM scaffolds were combined into a defect model, the chondrogenic medium (serum-free with TGF-ß and Dex) enhanced the production of proteoglycans and promoted integrative repair of meniscus tissue. As well, cross-linked scaffolds improved repair over the MDM slurry.Conclusions: The meniscal tissue defect model established in this paper can be used to perform in vitro screening to identify and optimize biological treatments to enhance meniscus tissue repair prior to conducting preclinical animal studies.

Kinetic and Cross-Sectional Studies on the Genesis of Hypoargininemia in Severe Pediatric Plasmodium falciparum Malaria.

The low bioavailability of nitric oxide (NO) and its precursor, arginine, contributes to the microvascular pathophysiology of severe falciparum malaria. To better characterize the mechanisms underlying hypoargininemia in severe malaria, we measured the plasma concentrations of amino acids involved in de novo arginine synthesis in children with uncomplicated falciparum malaria (UM; n = 61), children with cerebral falciparum malaria (CM; n = 45), and healthy children (HC; n = 109). We also administered primed infusions of l-arginine uniformly labeled with 13C6 and 15N4 to 8 children with severe falciparum malaria (SM; age range, 4 to 9 years) and 7 healthy children (HC; age range, 4 to 8 years) to measure the metabolic flux of arginine, hypothesizing that arginine flux is increased in SM. Using two different tandem mass spectrometric methods, we measured the isotopic enrichment of arginine in plasma obtained at 0, 60, 90, 120, 150, and 180 min during the infusion. The plasma concentrations of glutamine, glutamate, proline, ornithine, citrulline, and arginine were significantly lower in UM and CM than in HC (P ≤ 0.04 for all pairwise comparisons). Of these, glutamine concentrations were the most markedly decreased: median, 457 µM (interquartile range [IQR], 400 to 508 µM) in HC, 300 µM (IQR, 256 to 365 µM) in UM, and 257 µM (IQR, 195 to 320 µM) in CM. Arginine flux during steady state was not significantly different in SM than in HC by the respective mass spectrometric methods: 93.2 µmol/h/kg of body weight (IQR, 84.4 to 129.3 µmol/h/kg) versus 88.0 µmol/h/kg (IQR, 73.0 to 102.2 µmol/h/kg) (P = 0.247) by the two mass spectrometric methods in SM and 93.7 µmol/h/kg (IQR, 79.1 to 117.8 µmol/h/kg) versus 81.0 µmol/h/kg (IQR, 75.9 to 88.6 µmol/h/kg) (P = 0.165) by the two mass spectrometric methods in HC. A limited supply of amino acid precursors for arginine synthesis likely contributes to the hypoargininemia and NO insufficiency in falciparum malaria in children.

Glycocalyx Breakdown Is Associated With Severe Disease and Fatal Outcome in Plasmodium falciparum Malaria.

BACKGROUND: Interactions between the endothelium and infected erythrocytes play a major role in the pathogenesis of falciparum malaria, with microvascular dysfunction and parasite sequestration associated with worsening outcomes. The glycocalyx is a carbohydrate-rich layer that lines the endothelium, with multiple roles in vascular homeostasis. The role of the glycocalyx in falciparum malaria and the association with disease severity has not been investigated. METHODS: We prospectively enrolled Indonesian inpatients (aged ≥18 years) with severe (SM) or moderately severe (MSM) falciparum malaria, as defined by World Health Organization criteria, and healthy controls (HCs). On enrollment, blood and urine samples were collected concurrently with measurements of vascular nitric oxide (NO) bioavailability. Urine was assayed for glycocalyx breakdown products (glycosaminoglycans) using a dimethylmethylene blue (GAG-DMMB) and liquid chromatography-tandem mass spectrometry (GAG-MS) assay. RESULTS: A total of 129 patients (SM = 43, MSM = 57, HC=29) were recruited. GAG-DMMB and GAG-MS (g/mol creatinine) were increased in SM (mean, 95% confidence interval: 3.98, 2.44-5.53 and 6.82, 5.19-8.44) compared to MSM patients (1.78, 1.27-2.29 and 4.87, 4.27-5.46) and HCs (0.22, 0.06-0.37 and 1.24, 0.89-1.59; P < 0.001). In SM patients, GAG-DMMB and GAG-MS were increased in those with a fatal outcome (n = 3; median, interquartile range: 6.72, 3.80-27.87 and 12.15, 7.88-17.20) compared to survivors (n = 39; 3.10, 0.46-4.5 and 4.64, 2.02-15.20; P = 0.03). Glycocalyx degradation was significantly associated with parasite biomass in both MSM (r = 0.48, GAG-DMMB and r = 0.43, GAG-MS; P < 0.001) and SM patients (r = 0.47, P = 0.002 and r = 0.33, P = 0.04) and inversely associated with endothelial NO bioavailability. CONCLUSIONS: Increased endothelial glycocalyx breakdown is associated with severe disease and a fatal outcome in adults with falciparum malaria.

SET alpha and SET beta mRNA isoforms in chronic lymphocytic leukaemia.

Alteration in RNA splicing is implicated in carcinogenesis and progression. Mutations in spliceosome genes and alternative splicing of other genes have been noted in chronic lymphocytic leukaemia (CLL), a common B cell malignancy with heterogeneous outcomes. We previously demonstrated that differences in the amount of SET oncoprotein (a physiological inhibitor of the serine/threonine phosphatase, PP2A) is associated with clinical aggressiveness in patients with CLL. It is unknown if alternative splicing of gene transcripts regulating kinases and phosphatases affects disease pathobiology and CLL progression. We show here for the first time that mRNA levels of the alternatively spliced SET isoforms, SETA and SETB (SETα and SETß), significantly correlate with disease severity (overall survival and time-to-first-treatment) in CLL patients. In addition, we demonstrate that relative increase of SETA to SETB mRNA can discriminate patients with a more aggressive disease course within the otherwise favourable CLL risk classifications of IGHV mutated and favourable hierarchical fluorescence in situ hybridisation groups. We validate our finding by showing comparable relationships of SET mRNA with disease outcomes using samples from an independent CLL cohort from a separate institution. These findings indicate that alternative splicing of SET, and potentially other signalling cascade molecules, influences CLL biology and patient outcomes.

Meniscus-Derived Matrix Bioscaffolds: Effects of Concentration and Cross-Linking on Meniscus Cellular Responses and Tissue Repair.

Meniscal injuries, particularly in the avascular zone, have a low propensity for healing and are associated with the development of osteoarthritis. Current meniscal repair techniques are limited to specific tear types and have significant risk for failure. In previous work, we demonstrated the ability of meniscus-derived matrix (MDM) scaffolds to augment the integration and repair of an in vitro meniscus defect. The objective of this study was to determine the effects of percent composition and dehydrothermal (DHT) or genipin cross-linking of MDM bioscaffolds on primary meniscus cellular responses and integrative meniscus repair. In all scaffolds, the porous microenvironment allowed for exogenous cell infiltration and proliferation, as well as endogenous meniscus cell migration. The genipin cross-linked scaffolds promoted extracellular matrix (ECM) deposition and/or retention. The shear strength of integrative meniscus repair was improved with increasing percentages of MDM and genipin cross-linking. Overall, the 16% genipin cross-linked scaffolds were most effective at enhancing integrative meniscus repair. The ability of the genipin cross-linked scaffolds to attract endogenous meniscus cells, promote glycosaminoglycan and collagen deposition, and enhance integrative meniscus repair reveals that these MDM scaffolds are promising tools to augment meniscus healing.

Clinical outcomes in chronic lymphocytic leukaemia associated with expression of CD5, a negative regulator of B-cell receptor signalling.

Chronic lymphocytic leukaemia (CLL) is characterized by expression of CD5 on clonal B cells, and is partly driven by activated B-cell receptor (BCR) signalling. While CD5 is known to be a negative regulator of BCR signalling, it is unknown if variability in CD5 expression exists among patients and whether CLL cell CD5 expression affects CLL clinical outcomes. We assessed the extent to which CD5 expression is correlated with clinical outcomes, and whether this information adds to currently used prognostic markers. We evaluated CD5 expression from 1275 blood samples, established prognostic markers and time to event data from 423 CLL patients followed at the Duke University and Durham VA Medical Centers. CD5 median fluorescence intensity (MFI) was largely stable over time in individual patients, but ranged between 0·5 and 760 in the entire cohort. Lower CD5 MFI was significantly associated with a shorter time to first therapy. CD5 MFI, combined with established clinical and molecular prognostic markers, significantly improved risk-stratification. CD5 may affect disease outcomes by suppressing signalling through the BCR. Thus, a strategy to modulate CLL cell CD5 expression or function could be a therapeutic approach in CLL.

Phase III randomized study of the proposed adalimumab biosimilar GP2017 in psoriasis: impact of multiple switches.

BACKGROUND: Adalimumab is used to treat several inflammatory diseases, including plaque psoriasis. GP2017 is a proposed adalimumab biosimilar. OBJECTIVES: To assess the impact of multiple switches between GP2017 and reference adalimumab (ref-ADMB) following the demonstration of equivalent efficacy and similar safety and immunogenicity, in adult patients with active, clinically stable, moderate-to-severe plaque psoriasis. METHODS: This 51-week double-blinded, phase III study randomly assigned patients to GP2017 (n = 231) or ref-ADMB (n = 234) 80 mg subcutaneously at week 0, then 40 mg biweekly from week 1. At week 17, patients were rerandomized to switch (n = 126) or continue (n = 253) treatment. The primary end point was patients achieving ≥ 75% improvement in Psoriasis Area and Severity Index (PASI 75) at week 16, with equivalence confirmed if the 95% confidence interval (CI) for the difference in PASI 75 between treatments was ± 18%. The key secondary end point was the change from baseline to week 16 in continuous PASI. Other end points were PASI over time; PASI 50, 75, 90 and100; pharmacokinetics; safety; tolerability and immunogenicity for the switched and continued treatment groups. RESULTS: Equivalent efficacy between GP2017 and ref-ADMB was confirmed for the primary (66·8% and 65·0%, respectively; 95% CI -7·46 to 11·15) and key secondary end points (-60·7% and -61·5%, respectively; 95% CI -3·15 to 4·84). PASI improved over time and was similar between treatment groups at week 16, and the switched and continued groups from weeks 17 to 51. There were no relevant safety or immunogenicity differences between GP2017 and ref-ADMB at week 16, or the switched and continued groups from weeks 17 to 51. No hypersensitivity to adalimumab was reported upon switching. CONCLUSIONS: Following the demonstration of GP2017 biosimilarity to ref-ADMB, switching up to four times between GP2017 and ref-ADMB had no detectable impact on efficacy, safety or immunogenicity.

Suppression of Glut1 and Glucose Metabolism by Decreased Akt/mTORC1 Signaling Drives T Cell Impairment in B Cell Leukemia.

Leukemia can promote T cell dysfunction and exhaustion that contributes to increased susceptibility to infection and mortality. The treatment-independent mechanisms that mediate leukemia-associated T cell impairments are poorly understood, but metabolism tightly regulates T cell function and may contribute. In this study, we show that B cell leukemia causes T cells to become activated and hyporesponsive with increased PD-1 and TIM3 expression similar to exhausted T cells and that T cells from leukemic hosts become metabolically impaired. Metabolic defects included reduced Akt/mammalian target of rapamycin complex 1 (mTORC1) signaling, decreased expression of the glucose transporter Glut1 and hexokinase 2, and reduced glucose uptake. These metabolic changes correlated with increased regulatory T cell frequency and expression of PD-L1 and Gal-9 on both leukemic and stromal cells in the leukemic microenvironment. PD-1, however, was not sufficient to drive T cell impairment, as in vivo and in vitro anti-PD-1 blockade on its own only modestly improved T cell function. Importantly, impaired T cell metabolism directly contributed to dysfunction, as a rescue of T cell metabolism by genetically increasing Akt/mTORC1 signaling or expression of Glut1 partially restored T cell function. Enforced Akt/mTORC1 signaling also decreased expression of inhibitory receptors TIM3 and PD-1, as well as partially improved antileukemia immunity. Similar findings were obtained in T cells from patients with acute or chronic B cell leukemia, which were also metabolically exhausted and had defective Akt/mTORC1 signaling, reduced expression of Glut1 and hexokinase 2, and decreased glucose metabolism. Thus, B cell leukemia-induced inhibition of T cell Akt/mTORC1 signaling and glucose metabolism drives T cell dysfunction.

A Steady-State Head-to-Head Pharmacokinetic Comparison of All FK-506 (Tacrolimus) Formulations (ASTCOFF): An Open-Label, Prospective, Randomized, Two-Arm, Three-Period Crossover Study.

This two-sequence, three-period crossover study is the first pharmacokinetic (PK) study to compare all three innovator formulations of tacrolimus (twice-daily immediate-release tacrolimus capsules [IR-Tac]; once-daily extended-release tacrolimus capsules [ER-Tac]; novel once-daily tacrolimus tablets [LCPT]). Stable renal transplant patients were dosed with each drug for 7 days, and blood samples were obtained over 24 h. Thirty subjects were included in the PK analysis set. A conversion factor of 1:1:0.80 for IR-Tac:ER-Tac:LCPT was used; no dose adjustments were permitted during the study. The median (interquartile range) total daily dose was 6.0 (4.0-8.0) mg for IR-Tac and ER-Tac and 4.8 (3.3-6.3) for LCPT. Significantly higher exposure on a per milligram basis, lower intraday fluctuation and prolonged time (Tmax ) to peak concentration (Cmax ) were found for LCPT versus IR-Tac or ER-Tac. ER-Tac showed no differences versus IR-Tac in exposure, Cmax , Tmax or fluctuation. The observed exposure of IR-Tac was used to normalize exposure for LCPT and ER-Tac, resulting in the following recommended total daily dose conversion rates: IR-Tac:ER-Tac, +8%; IR-Tac:LCPT, -30%; ER-Tac:LCPT, -36%. After exposure normalization, Cmax was ~17% lower for LCPT than for IR-Tac or ER-Tac; Cmin was ~6% lower for LCPT compared with IR-Tac and 3% higher compared with ER-Tac.

Impaired systemic tetrahydrobiopterin bioavailability and increased dihydrobiopterin in adult falciparum malaria: association with disease severity, impaired microvascular function and increased endothelial activation.

Tetrahydrobiopterin (BH4) is a co-factor required for catalytic activity of nitric oxide synthase (NOS) and amino acid-monooxygenases, including phenylalanine hydroxylase. BH4 is unstable: during oxidative stress it is non-enzymatically oxidized to dihydrobiopterin (BH2), which inhibits NOS. Depending on BH4 availability, NOS oscillates between NO synthase and NADPH oxidase: as the BH4/BH2 ratio decreases, NO production falls and is replaced by superoxide. In African children and Asian adults with severe malaria, NO bioavailability decreases and plasma phenylalanine increases, together suggesting possible BH4 deficiency. The primary three biopterin metabolites (BH4, BH2 and B0 [biopterin]) and their association with disease severity have not been assessed in falciparum malaria. We measured pterin metabolites in urine of adults with severe falciparum malaria (SM; n=12), moderately-severe malaria (MSM, n=17), severe sepsis (SS; n=5) and healthy subjects (HC; n=20) as controls. In SM, urinary BH4 was decreased (median 0.16 »mol/mmol creatinine) compared to MSM (median 0.27), SS (median 0.54), and HC (median 0.34)]; p<0.001. Conversely, BH2 was increased in SM (median 0.91 »mol/mmol creatinine), compared to MSM (median 0.67), SS (median 0.39), and HC (median 0.52); p<0.001, suggesting increased oxidative stress and insufficient recycling of BH2 back to BH4 in severe malaria. Overall, the median BH4/BH2 ratio was lowest in SM [0.18 (IQR: 0.04-0.32)] compared to MSM (0.45, IQR 0.27-61), SS (1.03; IQR 0.54-2.38) and controls (0.66; IQR 0.43-1.07); p<0.001. In malaria, a lower BH4/BH2 ratio correlated with decreased microvascular reactivity (r=0.41; p=0.03) and increased ICAM-1 (r=-0.52; p=0.005). Decreased BH4 and increased BH2 in severe malaria (but not in severe sepsis) uncouples NOS, leading to impaired NO bioavailability and potentially increased oxidative stress. Adjunctive therapy to regenerate BH4 may have a role in improving NO bioavailability and microvascular perfusion in severe falciparum malaria.

Impaired systemic tetrahydrobiopterin bioavailability and increased oxidized biopterins in pediatric falciparum malaria: association with disease severity.

Decreased bioavailability of nitric oxide (NO) is a major contributor to the pathophysiology of severe falciparum malaria. Tetrahydrobiopterin (BH4) is an enzyme cofactor required for NO synthesis from L-arginine. We hypothesized that systemic levels of BH4 would be decreased in children with cerebral malaria, contributing to low NO bioavailability. In an observational study in Tanzania, we measured urine levels of biopterin in its various redox states (fully reduced [BH4] and the oxidized metabolites, dihydrobiopterin [BH2] and biopterin [B0]) in children with uncomplicated malaria (UM, n = 55), cerebral malaria (CM, n = 45), non-malaria central nervous system conditions (NMC, n = 48), and in 111 healthy controls (HC). Median urine BH4 concentration in CM (1.10 [IQR:0.55-2.18] µmol/mmol creatinine) was significantly lower compared to each of the other three groups - UM (2.10 [IQR:1.32-3.14];p<0.001), NMC (1.52 [IQR:1.01-2.71];p = 0.002), and HC (1.60 [IQR:1.15-2.23];p = 0.005). Oxidized biopterins were increased, and the BH4:BH2 ratio markedly decreased in CM. In a multivariate logistic regression model, each Log10-unit decrease in urine BH4 was independently associated with a 3.85-fold (95% CI:1.89-7.61) increase in odds of CM (p<0.001). Low systemic BH4 levels and increased oxidized biopterins contribute to the low NO bioavailability observed in CM. Adjunctive therapy to regenerate BH4 may have a role in improving NO bioavailability and microvascular perfusion in severe falciparum malaria.

Nitric Oxide-Dependent Endothelial Dysfunction and Reduced Arginine Bioavailability in Plasmodium vivax Malaria but No Greater Increase in Intravascular Hemolysis in Severe Disease.

BACKGROUND: Pathogenesis of severe Plasmodium vivax malaria is poorly understood. Endothelial dysfunction and reduced nitric oxide (NO) bioavailability characterize severe falciparum malaria, but have not been assessed in severe vivax malaria. METHODS: In patients with severe vivax malaria (n = 9), patients with nonsevere vivax malaria (n = 58), and healthy controls (n = 79), we measured NO-dependent endothelial function by using reactive hyperemia-peripheral arterial tonometry (RH-PAT) and assessed associations with arginine, asymmetric dimethylarginine (ADMA), and hemolysis. RESULTS: The L-arginine level and the L-arginine to ADMA ratio (a measure of L-arginine bioavailability) were reduced in patients with severe vivax malaria and those with nonsevere vivax malaria, compared with healthy controls (median L-arginine level, 65, 66, and 98 µmol/mL, respectively [P = .0001]; median L-arginine to ADMA ratio, 115, 125, and 187, respectively [P = .0001]). Endothelial function was impaired in proportion to disease severity (median RH-PAT index, 1.49, 1.73, and 1.97 in patients with severe vivax malaria, those with nonsevere vivax malaria, and healthy controls, respectively; P = .018) and was associated with the L-arginine to ADMA ratio. While the posttreatment fall in hemoglobin level was greater in severe vivax malaria as compared to nonsevere vivax malaria (2.5 vs 1 g/dL; P = .0001), markers of intravascular hemolysis were not higher in severe disease. CONCLUSIONS: Endothelial function is impaired in nonsevere and severe vivax malaria, is associated with reduced L-arginine bioavailability, and may contribute to microvascular pathogenesis. Severe disease appears to be more associated with extravascular hemolysis than with intravascular hemolysis.

Relationship of blood monocytes with chronic lymphocytic leukemia aggressiveness and outcomes: a multi-institutional study.

Monocyte-derived cells, constituents of the cancer microenvironment, support chronic lymphocytic leukemia (CLL) cell survival in vitro via direct cell-cell interaction and secreted factors. We hypothesized that circulating absolute monocyte count (AMC) reflects the monocyte-derived cells in the microenvironment, and that higher AMC is associated with increased CLL cell survival in vivo and thus inferior CLL patient outcomes. We assessed the extent to which AMC at diagnosis of CLL is correlated with clinical outcomes, and whether this information adds to currently used prognostic markers. We evaluated AMC, clinically used prognostic markers, and time to event data from 1,168 CLL patients followed at the Mayo Clinic, the Duke University Medical Center, and the Durham VA Medical Center. Elevated AMC was significantly associated with inferior clinical outcomes, including time to first therapy (TTT) and overall survival (OS). AMC combined with established clinical and molecular prognostic markers significantly improved risk-stratification of CLL patients for TTT. As an elevated AMC at diagnosis is associated with accelerated disease progression, and monocyte-derived cells in the CLL microenvironment promote CLL cell survival and proliferation, these findings suggest that monocytes and monocyte-derived cells are rational therapeutic targets in CLL. Am. J. Hematol. 91:687-691, 2016. © 2016 Wiley Periodicals, Inc.