Automatic assay of blood sugar by Trinder's method]

An automated method for determining blood "true glucose" has been adapted to the single channel "AutoAnalyzer". The action of the glucose-oxidase peroxidase system is directly coupled with an oxygen acceptor chromogen. The validity for accuracy, contamination and the absence of interferences by certain compounds have been tested. Experimenters proceeded then with a thorough examination of the relation existing between the "true glucose" determined by the present method combined with the Hexokinase method and the "true guclose" determined with the Beckman "Glucose Analyzer".     

Epstein-Barr virus (EBV) reactivation in allogeneic stem-cell transplantation: relationship between viral load, EBV-specific T-cell reconstitution and rituximab therapy

BACKGROUND: Monitoring of Epstein-Barr virus (EBV) reactivation after allogeneic hematopoietic stem-cell transplantation markedly improved with quantitative real-time polymerase chain reaction amplification of EBV DNA and visualization of EBV-specific CD8+ T cells with peptide-human leukocyte antigen (HLA) class I tetramers. We decided to combine these methods to evaluate posttransplant EBV reactivation and rituximab therapy. METHODS: We followed 56 patients treated with an HLA-genoidentical sibling (n=32), an HLA-matched unrelated donor (MUD, n=19), or an unrelated cord-blood transplant (n=5). EBV DNA was quantified in plasma and in peripheral blood mononuclear cells (PBMC). Patient CD8+ T cells were stained with a panel of eight tetramers. RESULTS: EBV DNA was detected in half of the patients, mainly in the MUD group (17/19). In 19 patients, viral DNA was detected only in the cellular compartment. All patients who controlled reactivation without rituximab and despite a viral load of greater than 500 genome equivalents (gEq)/150,000 PBMC mounted an EBV-specific CD8+ T-cell response in greater than 1.4% of CD3+CD8+ T cells. Plasmatic EBV genome was found in nine patients preceded by a high cellular viral load. Three of these patients controlled the reactivation before or without the introduction of rituximab, and they all developed a significant and increasing EBV-specific T-cell response. Patients with EBV-specific T cells at the onset of reactivation controlled viral reactivation without rituximab. CONCLUSION: This study emphasizes the benefit of an early and close monitoring of EBV reactivation and CD8+-specific immune responses to initiate rituximab only when necessary and before the immune response becomes overwhelmed by the viral burden.     

2-Amino metabolites are key mediators of CB 1954 and SN 23862 bystander effects in nitroreductase GDEPT

An important feature of gene-directed enzyme-prodrug therapy is that prodrug activation can provide diffusible cytotoxic metabolites capable of generating a local bystander effect in tumours. Activation of the aziridinyl dinitrobenzamide CB 1954 by E. coli nitroreductase (NTR) provides a bystander effect assumed to be due to the potently cytotoxic 4-hydroxylamine metabolite. We show that there are four cytotoxic extracellular metabolites of CB 1954 in cultures of NTR-expressing tumour cells (the 2- and 4-hydroxylamines and their corresponding amines). The 4-hydroxylamine is the most cytotoxic in DNA crosslink repair defective cells, but the 2-amino derivative (CB 10-236) is of similar potency to the 4-hydroxylamine in human tumour cell lines. Importantly, CB 10-236 has much superior diffusion properties to the 4-hydroxylamine in multicellular layers grown from the SiHa human cervical carcinoma cell line. These results suggest that the 2-amine, not the 4-hydroxylamine, is the major bystander metabolite when CB 1954 is activated by NTR in tumours. The corresponding dinitrobenzamide nitrogen mustard SN 23862 is reduced by NTR to form a single extracellular metabolite (also the 2-amine), which has superior cytotoxic potency and diffusion properties to the CB 1954 metabolites. These results are consistent with the reported high bystander efficiency of SN 23862 as an NTR prodrug in multicellular layers and tumour xenografts.     

Synthesis and evaluation of nitroheterocyclic carbamate prodrugs for use with nitroreductase-mediated gene-directed enzyme prodrug therapy

A variety of nitroheterocyclic carbamate prodrugs of phenylenediamine mustard and 5-amino-1-(chloromethyl)-3-[(5,6,7-trimethoxyindol-2-yl)carbonyl]-1,2-dihydro-3H-benz[e]indoline (amino-seco-CBI-TMI), covering a wide range of reduction potential, were prepared and evaluated for use in gene-directed enzyme prodrug therapy (GDEPT) using a two-electron nitroreductase (NTR) from Escherichia coli B. The carbamate prodrugs and corresponding amine effectors were tested in a cell line panel comprising parental and NTR-transfected human (SKOV3/SKOV3-NTR(neo), WiDr/WiDr-NTR(neo)), Chinese hamster (V79(puro)/V79-NTR(puro)), and murine (EMT6/EMT6-NTR(puro)) cell line pairs and were compared with the established NTR substrates CB1954 (an aziridinyl dinitrobenzamide) and the analogous dibromomustard. The 1-methyl-2-nitroimidazol-5-ylmethyl carbamate of phenylenediamine mustard was metabolized rapidly by EMT6-NTR(neo) but not EMT6 cells, demonstrating that it is an efficient substrates for NTR. Despite this, the carbamates of phenylenediamine mustards show relatively low differential cytotoxicity for NTR+ve cells in IC(50) assays, apparently because they retain sufficient alkylating reactivity that most of the prodrug reacts with nucleophiles during the drug exposure period. In contrast, the corresponding amino-seco-CBI-TMI prodrugs were less efficient NTR substrates but had greater chemical stability, were more potent, and showed substantial NTR-ve/NTR+ve ratios in the cell line panel, with ratios of 15-100-fold for the 1-methyl-2-nitro-1H-imidazol-5-ylmethyl and 1-methyl-5-nitro-1H-imidazol-2-ylmethyl carbamates of amino-seco-CBI-TMI. The activity of these two prodrugs was evaluated against NTR-expressing EMT6 tumors comprising ca. 10% NTR+ve cells. Small but not statistically significant killing of NTR+ve cells was observed, with no effect against NTR-ve target cells. The lack of activity against NTR+ve cells in tumors, despite potent and selective activity in culture, indicates that pharmacokinetic optimization will be required if in vivo efficacy against solid tumors is to be achieved with this new class of NTR prodrugs.     

Recovery of slow skeletal muscle after injury in the senescent rat

We studied the contractile, histological and biochemical characteristics of regenerating slow (soleus) muscles of aged rats and the effect of IGF-1 treatment on these parameters. Regenerating soleus muscles were studied 21 days after myotoxic injury. In senescent rats (24 month old), the in situ isometric maximal tetanic force (P0), resistance to fatigue (T20%P0) and shortening speed with an afterload of 20%P0 (SS20%P0) were lower (p<0.05) in regenerating soleus muscles as compared to uninjured controlateral soleus muscles. Moreover, the expression of type 1 myosin heavy chain (MHC-1) was decreased by injury in the soleus muscles of senescent rats (p<0.05). Furthermore, a single injection of IGF-1 (3 microg) into the soleus of senescent rats only slightly increased the level of sarcoplasmic reticulum type 2 Ca(2+)-ATPase in regenerating soleus muscles (p<0.01). Contrary to senescent animals, regenerating soleus of adult rats (10 month old) did not present significantly lower P0 and MHC-1 expression than uninjured controlateral muscles (p>0.05). In conclusion, the regeneration of a slow muscle is more uncompleted 3 weeks after myotoxic injury in senescent rats than in adult rats. It cannot be made more effective by a single injection of IGF-1 into the senescent slow muscle.     

Dimeric architecture of the human bumetanide-sensitive Na-K-Cl Co-transporter

The primary mediator of NaCl reabsorption in the renal distal tubule is the human bumetanide-sensitive Na(+)-K(+)-2Cl(-) co-transporter (hNKCC2), located at the apical membrane of the thick ascending limb of Henle's loop. The physiologic importance of this transporter is emphasized by the tubular disorder Bartter syndrome type I, which arises from the functional impairment of hNKCC2 as a result of mutations in the SLC12A1 gene. The aim of the present study was to investigate the oligomeric state of hNKCC2 to understand further its operational mechanism. To this end, hNKCC2 was heterologously expressed in Xenopus laevis oocytes. Chemical cross-linking with dimethyl-3,3-dithio-bis-propionamidate indicated that hNKCC2 subunits can reversibly form high molecular weight complexes. Co-immunoprecipitation of tagged hNKCC2 subunits further substantiated a physical interaction between individual hNKCC2 subunits. The size of the hNKCC2 multimers was determined by sucrose gradient centrifugation, and a preference for dimeric complexes (approximately 320 kD) was demonstrated. Finally, concatemeric constructs consisting of two wild-type subunits or a wild-type and a functionally impaired hNKCC2 subunit (G319R) were expressed in oocytes. Subsequently, the concatemers were functionally characterized, resulting in a significant bumetanide-sensitive (22)Na(+) uptake of 2.5 +/- 0.2 nmol/oocyte per 30 min for the wild-type-wild-type concatemer, which was reduced to 1.3 +/- 0.1 nmol/oocyte per 30 min for the wild-type-G319R concatemer. In conclusion, this study suggests that hNKCC2 forms at least functional dimers when expressed in Xenopus laevis oocytes of which the individual subunits transport Na(+) independently.