Refractory mixed proliferative and membranous lupus nephritis treated with the topoisomerase I inhibitor irinotecan as add-on therapy.

OBJECTIVE: To evaluate the safety and effects of irinotecan, an inhibitor of topoisomerase I, on refractory lupus nephritis. METHOD: A patient with refractory lupus nephritis under medication with mycophenolic acid, prednisolone, and hydroxychloroquine was treated with add-on low-dose irinotecan. Irinotecan was applied every fourth week at a dose of 50 mg/m2 for four cycles followed by 100 mg/m2 for another eight cycles. Renal function and anti-double-stranded DNA antibodies as well as blood count for evaluation of side effects were assessed during the treatment with irinotecan. RESULTS: Before starting the treatment with irinotecan, a urine protein/creatinine ratio of 1298 mg/g was determined. This declined to 613 mg/g after four cycles with 50 mg/m2 irinotecan and was further reduced to 198 mg/g when using the higher dose of irinotecan. Kidney function remained stable, with creatinine levels of 1.66 mg/dL at the beginning and 1.76 mg/dL at the end of treatment with irinotecan. Importantly, no side effects, such as diarrhoea or neutropenia, were observed during the entire course of treatment. CONCLUSION: Administration of low-dose irinotecan as add-on medication for the treatment of refractory lupus nephritis was shown to be safe. Clinical trials are needed to determine whether irinotecan can improve kidney function and the outcome of patients with refractory lupus nephritis.

Characterization of porcine milk oligosaccharides during early lactation and their relation to the fecal microbiome.

The composition of porcine milk oligosaccharides (PMO) was analyzed during early lactation and their relation to piglet gut microbiome was investigated. Pigs are considered ideal intestinal models to simulate humans because of the striking similarity in intestinal physiopathology to humans. The evolution of PMO was investigated in the milk from 3 healthy sows at prefarrowing, farrowing, and d 7 and 14 postpartum by Nano-LC Chip Quadrupole-Time-of-Flight mass spectrometer (Agilent Technologies, Santa Clara, CA). Previously sequenced metagenome libraries were reanalyzed to examine changes with specific gut bacterial populations. Over 30 oligosaccharides (OS) were identified in the milk, with 3'-sialyllactose, lacto-N-tetraose, α1-3,ß1-4-d-galactotriose, 2'-fucosyllactose, and 6'-sialyllactose being the most abundant species (accounting for ~70% of the total OS). Porcine milk had lower OS diversity (number of unique structures) than human milk, and appeared closer to bovine and caprine milk. In agreement with previous studies, only 3 fucosylated OS were identified. Surprisingly, their contribution to total OS abundance was greater than in bovine milk (9 vs. 1%). Indeed, fucosylated PMO increased during lactation, mirroring a similar trend observed for neutral and type I OS content during early lactation. Taken together, these results suggest that, in terms of abundance, PMO are closer to human milk than other domestic species, such as bovine and caprine milks. Metagenomic sequencing revealed that fucose-consuming bacterial taxa in the gut microbiota of piglets were qualitatively but not quantitatively different between nursing and weaning stages, suggesting that both the composition and structure of dietary glycans may play a critical role in shaping the distal gut microbiome. The similarity of both intestinal physiopathology and milk OS composition in human and porcine species suggests similar effects on gastrointestinal development of early nutrition, reinforcing the use of the pig intestinal model to simulate human intestinal models in the clinical setting.

Influence of glycemic status and physical fitness on oxidative stress and the peroxiredoxin system in the erythrocytes of non-insulin-dependent type 2 diabetic men.

UNLABELLED: Oxidative stress plays a leading role in the progression of diabetic secondary complications, e.g., of cardio-vascular illnesses. Physical activity has been shown to delay and even prevent the progression of type 2 diabetes by improving the antioxidative capacity and thereby decreasing systemic oxidative stress. Peroxiredoxins (PRDX) are important antioxidative components that are highly abundant in erythrocytes. The present study examines the influence of glycemic control and physical fitness on oxidative stress and the peroxiredoxin system in the erythrocytes of non-insulin-dependent type 2 diabetic men ( N=22, years=61 ± 10) at rest. Oxidative stress was measured by immunohistochemical stainings for 8-iso-prostaglandin-F2α (8-Iso-PGF) and the overoxidized form of peroxiredoxins (PRDX-SO (2-3)). Peroxiredoxin isoforms PRDX1 and PRDX2 were also quantified immunohistochemically. Physical fitness was determined during the WHO-step test. Regression analyses showed a positive relationship between 8-Iso-PGF plotted against HbA (1c) (hyperbolic curve (y=a+b/x), R (2)=0.346, P=0.013), a positive relationship between 8-Iso-PGF plotted against fasting glucose (hyperbolic curve (y=a+b/x), R (2)=0.440, P=0.003), as well as positive relationships between PRDX2 plotted against VO (2 peak) (S-curve (y=e(a+b/x)), R(2)=0.259, P=0.018) and between PRDX2 plotted against the workload corresponding to the 4 mmol/l blood lactate concentration (hyperbolic curve (y=a+b/x), R(2)=0.203, P=0.041). Further significant relationships were not found. CONCLUSIONS: Poor glycemic control may increase oxidative stress in the erythrocytes of type 2 diabetic men. Good physical fitness seems to be associated with increased peroxiredoxin contents. Therefore, it can be speculated that physical training can contribute to the improvement of the erythrocyte peroxiredoxin system to counteract free radicals in type 2 diabetic patients.

Myosin heavy chain expression pattern as a marker for anabolic potency: desoxymethyltestosterone (madol), norandrostenedione and testosterone repress MHC-IIb expression and stimulate MHC-IId/x expression in orchiectomized rat gastrocnemius muscle.

Both 19-norandrostenedione (estr-4-ene-3,17-dione, NOR) and desoxymethyltestosterone (17alpha-methyl-5alpha-androst-2-en-17beta-ol, DMT or "madol") are 'designer steroids' misused for doping purposes in the bodybuilding scene. We have previously characterized the pharmacological profile of madol and identified potential adverse side effects. The aim of this study was to investigate the anabolic potency of NOR, madol and the reference substance testosterone propionate (TP). Besides wet weight of the M.levator ani (LA), we examined the effects on muscle fiber type composition and myosin heavy chain (MHC) expression in the M.gastrocnemius (Gas) muscle as additional markers for anabolic potency. A Hershberger assay was performed, where orchiectomized (orchi) male Wistar rats were treated subcutaneously with NOR, madol, TP or vehicle control (all 1 mg/kg BW/day) for 12 days. Wet weights of the Gas, LA, prostate and seminal vesicle were examined to determine anabolic and androgenic effects. Fiber type composition of the Gas muscle was analyzed using ATPase staining, and MHC protein profiles were determined by silver stain and Western blot analysis. NOR and madol exhibited strong anabolic and weak androgenic potency by stimulating growth of the LA but not the prostate and seminal vesicle. Skeletal muscle fiber type composition characterized by ATPase staining was not significantly altered between the treatment groups, although there was a tendency toward lower levels of type IIB and increased type IIA fibers in all treatment groups relative to orchi. MHC protein expression determined by Western blot and silver stain analysis revealed that MHC IId/x was significantly up-regulated, while MHC IIb was significantly down-regulated in NOR, madol and TP groups relative to orchi. There were no significant differences for MHC IIa and MHC I expression between groups. Results suggest that the observed MHC expression shift could serve as a molecular marker to determine anabolic activity of anabolic steroids at least in skeletal muscle of orchi rats. The molecular mechanisms as well as the androgen-dependent regulation of MHC expression in intact skeletal muscle remain to be further investigated.

Synergistic induction of cell death in liver tumor cells by TRAIL and chemotherapeutic drugs via the BH3-only proteins Bim and Bid.

Although death receptors and chemotherapeutic drugs activate distinct apoptosis signaling cascades, crosstalk between the extrinsic and intrinsic apoptosis pathway has been recognized as an important amplification mechanism. Best known in this regard is the amplification of the Fas (CD95) signal in hepatocytes via caspase 8-mediated cleavage of Bid and activation of the mitochondrial apoptosis pathway. Recent evidence, however, indicates that activation of other BH3-only proteins may also be critical for the crosstalk between death receptors and mitochondrial triggers. In this study, we show that TNF-related apoptosis-inducing ligand (TRAIL) and chemotherapeutic drugs synergistically induce apoptosis in various transformed and untransformed liver-derived cell lines, as well as in primary human hepatocytes. Both, preincubation with TRAIL as well as chemotherapeutic drugs could sensitize cells for apoptosis induction by the other respective trigger. TRAIL induced a strong and long lasting activation of Jun kinase, and activation of the BH3-only protein Bim. Consequently, synergistic induction of apoptosis by TRAIL and chemotherapeutic drugs was dependent on Jun kinase activity, and expression of Bim and Bid. These findings confirm a previously defined role of TRAIL and Bim in the regulation of hepatocyte apoptosis, and demonstrate that the TRAIL-Jun kinase-Bim axis is a major and important apoptosis amplification pathway in primary hepatocytes and liver tumor cells.

Efficiency of non-viral gene delivery systems to rat lungs.

OBJECTIVE: Transient expression of therapeutic genes within lung allografts may modulate the pathological processes following allotransplantation. Whilst efficient gene transfer to lungs has been reported with viral vectors, their usefulness is limited on the grounds of safety. Since non-viral systems overcome many of these safety issues, our studies were designed to evaluate the efficiency of several non-viral gene delivery vectors for in vivo transfer of plasmid DNA to rat lungs via the airways. METHODS: Fischer rats (230--260 g) underwent a thoracotomy, right main bronchus occlusion and instillation of 300 microg naked or complexed DNA (pCIluci, luciferase gene/CMV promoter) to the left lung followed by ventilation for 10 min. Rats were divided into five treatment groups (n=5): (1) Glucose, (2) Naked DNA, (3) Linear polyethylenimine (PEI), (4) Branched PEI, (5) Lipid GL-67/DOPE and (6) DOTAP/cholesterol. Animals were sacrificed 24 h after gene delivery for measurement of reporter gene activity and gas exchange of the left lung. RESULTS: Linear PEI was the most efficient gene delivery vector and was significantly better than DOTAP/cholesterol (P=0.00002) and naked DNA (P=0.004). All gene delivery vectors impaired function of the transfected left lung compared with DNA alone. Of all the gene delivery vectors tested, lipid GL-67/DOPE exerted the least effect on lung function whilst DOTAP/cholesterol mediated the most adverse effect. CONCLUSION: Linear PEI was the most efficient vector for gene delivery to rat lungs in our experimental setting although it mediated a moderate impairment in lung function. Further studies are needed to evaluate whether this effect is transient.

Perinuclear localization of the protein-tyrosine phosphatase SHP-1 and inhibition of epidermal growth factor-stimulated STAT1/3 activation in A431 cells.

The SH2 domain protein-tyrosine phosphatase SHP-1 has been shown earlier to bind to the epidermal growth factor receptor and to have the capacity for receptor dephosphorylation. New bi- and tricistronic expression vectors (pNRTIS-21 and pNRTIS-33, respectively) based on the tetracycline system were constructed and employed to generate stable cell lines with inducible expression of SHP-1. Inducible overexpression of SHP-1 in A431 cells led to attenuation of epidermal growth factor (EGF) receptor autophosphorylation and of EGF-induced DNA binding of 'signal transducers and activators of transcription' (STAT) 1 and 3. SHP-1 was localized in the cytoplasm with an enrichment in the perinuclear compartment. Association of SHP-1 with perinuclear structures may form the basis for a partial cofractionation with nuclei observed in different types of transfected cells and also with endogenous SHP-1 in U-937 cells. Treatment of SHP-1-overexpressing A431 cells or of HaCaT human keratinocytes expressing SHP-1 endogenously with the Ca2+-ionophore A23187 resulted in partial nuclear accumulation of SHP-1. Thus, SHP-1 may interact with substrates or regulatory proteins in perinuclear or nuclear structures.

Selection of ventricular-like cardiomyocytes from ES cells in vitro.

Ischemic disorders of the heart can cause an irreversible loss of cardiomyocytes resulting in a substantial decrease of cardiac output. The therapy of choice is heart transplantation, a technique that is hampered by the low number of donor organs. In the present study, we describe the specific labeling, rapid but gentle purification and characterization of cardiomyocytes derived from mouse pluripotent embryonic stem (ES) cells. To isolate the subpopulation of ventricular-like cardiomyocytes, ES cells were stable transfected with the enhanced green fluorescent protein (EGFP) under transcriptional control of the ventricular-specific 2.1 kb myosin light chain-2v (MLC-2v) promoter and the 0.5 kb enhancer element of the cytomegalovirus (CMV(enh).). First fluorescent cells were detected at day 6 + 8 of differentiation within EBs. Four weeks after initiation of differentiation 25% of the cardiomyocyte population displayed fluorescence. Immunohistochemistry revealed the exclusive cardiomyogenic nature of EGFP-positive cells. This was further corroborated by electrophysiological studies where preferentially ventricular phenotypes, but no pacemaker-like cardiomyocytes, were detected among the EGFP-positive population. The enzymatic digestion of EBs, followed by Percoll gradient centrifugation and fluorescence-activated cell sorting, resulted in a 97% pure population of cardiomyocytes. Based on this study, ventricular-like cardiomyocytes can be generated in vitro from EBs and labeled using CMV(enh)./MLC-2v-driven marker genes facilitating an efficient purification. This method may become an important tool for future cell replacement therapy of ischemic cardiomyopathy especially after the proof of somatic differentiation of human ES cells in vitro.

Management of prelabor rupture of membranes at term. A randomized study.

OBJECTIVE: To compare the rate of obstetric interventions, length of labor, and maternal morbidity in pregnancies with prelabor rupture of membranes at term after either early or late induction of labor in both primiparous and pluriparous women. DESIGN: Prospective, randomized study. SUBJECTS: 362 women with singleton pregnancies, cephalic presentations, gestational age of 36 completed weeks or more were allocated at random to induction with oxytocin either 6 hours after PROM (n = 62) (early) or 24 hours (n = 62) (late). Those eligible, but not participating in the study, totalled 238 women. MAIN OBSTETRIC MEASURES: Time of spontaneous labor in the late induction group, length of labor, obstetric intervention rate, maternal morbidity, and the degree of histologic chorioamnionitis. RESULTS: The length of labor was longer in the late induction group than in the early induction group in both primiparous and pluriparous (p < 0.05). There were no overall differences in the rate of obstetric interventions or maternal morbidity, but there were marked differences between primiparous and pluriparous women. Increasing time span between the period from rupture of membranes to delivery increased the degree of histologic chorioamnionitis. CONCLUSION: If a woman wants a short labor, she will benefit from early induction. We did not find statistical differences in the rate of obstetric intervention or in the maternal morbidity, but there was a tendency towards adverse effects of late induction.

Dorsal root ganglion neurons expressing trk are selectively sensitive to NGF deprivation in utero.

In utero immune deprivation of the neurotrophic molecule nerve growth factor (NGF) results in the death of most, but not all, mammalian dorsal root ganglion (DRG) neurons. The recent identification of trk, trkB, and trkC as the putative high affinity receptors for NGF, brain-derived neurotrophic factor, and neurotrophin-3, respectively, has allowed an examination of whether their expression by DRG neurons correlates with differential sensitivity to immune deprivation of NGF. In situ hybridization demonstrates that virtually all neurons expressing trk are lost during in utero NGF deprivation. Most, if not all, neurons expressing trkB and trkC survive this treatment. In contrast, the low affinity NGF receptor, p75NGFR, is expressed in both NGF deprivation-resistant and -sensitive neurons. These experiments show that DRG neurons expressing trk require NGF for survival. Furthermore, at least some of the DRG neurons that do not require NGF express the high affinity receptor for another neurotrophin. Finally, these experiments provide evidence that trk, and not p75NGFR, is the primary effector of NGF action in vivo.

Retrospective analysis of the psychologic effects of mechanical circulatory support.

There is an increasing need to evaluate the psychologic impact of circulatory support because of the growing use of mechanical assist devices. Twenty-seven people, 12 survivors of mechanical circulatory assistance and 15 family members, were surveyed; nonsurvivors' families were not surveyed. The 12 survivors (ages 18 to 66 years; mean, 43 years) had been supported with Pierce-Donachy ventricular assist devices (nine patients) and the Novacor left ventricular assist system (one patient). One patient received a centrifugal pump, and one was supported with extracorporeal membrane oxygenation (ECMO). Duration of support ranged from 8 hours to 90 days (mean, 23 days). Ten people surveyed were spouses of these patients, and five were parents of children who had been supported with ECMO. Three patients were supported longer than 3 weeks, and four patients were awake and ambulatory during support. Financial worries were not a concern for 80% of the parents but were a concern for 80% of the spouses. Eighteen percent of the patients believed they were treated as an experiment, whereas 40% of their spouses thought they were. None of the parents thought their child was treated as an experiment. Sixty-seven percent of the patients stated that they have returned to a normal life-style, and 75% feel they have a brighter outlook on life. Six patients have returned to work, two are retired, two are disabled, and two have physical capabilities to work but choose not to. Eighty-nine percent of all those surveyed would recommend an assist device to someone who needed one. Although these procedures are often associated with severe medical and psychologic complications, 74% of the patients would agree to a second implant.