Ex vivo cytotoxic drug evaluation by DiSC assay to expedite identification of clinical targets: results with 8-chloro-cAMP

There is a pressing need to reduce the time and cost of developing new cytotoxic agents and to accurately identify clinically active agents at an early stage. In this study, the differential staining cytotoxicity (DiSC) assay was used to assess the efficacy of the novel antitumour cAMP analogue, 8-chloro-cAMP (8-Cl-cAMP) (and its metabolite 8-Cl-adenosine) against 107 fresh specimens of human neoplastic and normal cells. Diagnoses included chronic and acute leukaemias, myeloma, non-Hodgkin's lymphoma (NHL) and miscellaneous solid tumours. The aim was to identify targets for subsequent phase I, II and III trials. 8-Cl-cAMP was tested at 4-985 microM, along with standard chemotherapeutic drugs. 8-Cl-cAMP and its metabolite caused no morphologically observable cell differentiation but induced dose-dependent cytotoxicity. Compared with untreated patients, previously treated chronic lymphocytic leukaemia (CLL) patients showed no increase in ex vivo resistance to 8-Cl-cAMP (P = 0.878); minimal cross-resistance with other cytotoxic drugs was detected. Compared with normal cells (mean LC90 = 1803 microM), 8-Cl-cAMP showed significant ex vivo activity against CLL (117.0 microM; P < 0.0001) and NHL (140.0 microM; P < 0.0001), of which eight were mantle cell NHL (84.7 microM), and greatest activity against cells from patients with acute myeloid leukaemia (AML; mean LC90 = 24.3 microM; in vitro therapeutic index 74-fold, P < 0.0001). Solid tumour specimens were comparatively resistant to 8-Cl-cAMP. The results highlight the clinical potential of 8-Cl-cAMP, point to several new phase I, II and III trial possibilities and provide a rationale for the inclusion of ex vivo cytotoxic drug evaluation in the drug development process.     

Fluorescent labelled analogues of neuropeptide Y for the characterization of cells expressing NPY receptor subtypes

Porcine neuropeptide Y (NPY), a 36 amino acid hormone of the pancreatic polypeptide family, and subtype selective analogues have been synthesized by solid phase peptide synthesis. The peptides were labelled with Cy3, a commercially available fluorescent marker based on a cyanine dye, by solid phase strategy. During the cleavage a partial fragmentation of the fluorescent marker occurred. This has been investigated by means of HPLC and electrospray mass spectrometry. The labelled analogues of NPY showed high affinity to the NPY receptor subtypes Y1 and Y2. Thus, Cy3-NPY, Y1-selective Cy3-[Pro34] NPY and Y2 selective Cy3-[Ahx5-24] NPY were used to label SK-N-MC- and SMS-KAN-cells, which are stably expressing the Y1-(SK-N-MC) and the Y2-receptor subtype (SMS-KAN). The binding of the labelled analogues to the receptors was reversible and specific. The photoactivatable analogue, [(Tmd)Phe27] NPY, which showed high affinity to both receptor subtypes was labelled with Cy3 in solution. Whereas the fluorescent labelling of the cells with analogues without photoactivatable amino acid was reversible, successful photocrosslinking could be investigated by the irreversible staining of the cells using Cy3-[(Tmd)Phe27] NPY. These subtype selective analogues are exciting tools to trace receptors in tissues and to identify the pharmacologically characterized subtypes without radioactivity.     

DNA- and RNA-hydrolyzing antibodies from the blood of patients with various forms of viral hepatitis

Antibodies (Abs) hydrolyzing proteins, DNA, and RNA are detected in the blood of patients with various autoimmune diseases. In the present work, homogeneous preparations of IgG Abs from the blood of the healthy donors as well as patients with A, B, C, and delta types of viral hepatitis, influenza, pneumonia, tuberculosis, tonsillitis, duodenal ulcer, and some types of cancer were purified. For the first time, the fraction of IgG and its Fab fragments of patients with viral hepatitis were shown to have high DNA- and RNA-hydrolyzing activity. In case of Abs from the healthy donors and patients with other diseases, high activity of Abs was not detected. The data obtained by various methods indicate that the activity of hepatitis Abs is an intrinsic property of the immunoglobulins. The relative rates of hydrolysis of cCMP, poly(U), poly(A), poly(C), and tRNA(Phe) by hepatitis Abs were compared with those of RNase A and other RNases from human blood. Significant differences in activities of Abs and nucleases in hydrolysis of model substrates were demonstrated. Thus, catalytically active Abs can appear in the blood of patients not only with autoimmune disorders, but with viral diseases as well.     

Activation of transducin guanosine triphosphatase by two proteins of the RGS family

RGS proteins (regulators of G protein signaling) constitute a newly appreciated group of negative regulators of G protein signaling. Several members of this group stimulate the guanosine triphosphatase (GTPase) activity of various G protein alpha-subunits, including the photoreceptor G protein, transducin. In photoreceptor cells transducin GTPase is known to be substantially accelerated by the coordinated action of the gamma-subunit of its effector enzyme, cGMP phosphodiesterase (PDE gamma), and another yet unidentified membrane-associated protein factor. Here we test the possibility that this factor belongs to the RGS family of GTPase stimulators. We report a detailed kinetic analysis of transducin GTPase activation by two members of the RGS family, RGS4 and G alpha interacting protein (GAIP). RGS4, being at least 5-fold more potent than GAIP, stimulates the rate of transducin GTPase by 2 orders of magnitude. Neither RGS4 nor GAIP requires PDE gamma for activating transducin. Rather, PDE gamma causes a partial reversal of transducin GTPase activation by RGS proteins. The effect of PDE gamma is based on a decreased apparent affinity of RGS for the alpha-subunit of transducin. Our observations indicate that GTPase activity of transducin can be activated by at least two distinct mechanisms, one based on the action of RGS proteins alone and another involving the cooperative action of the effector enzyme and another protein.     

Effectiveness of mass neonatal BCG vaccination in the prevention of pulmonary tuberculosis: a case-control study in Nagpur, India

SETTING: Government Medical College, Nagpur, India, a tertiary care hospital. OBJECTIVE: To estimate the effectiveness of mass neonatal BCG vaccination in the prevention of pulmonary tuberculosis in Nagpur, India. DESIGN: A hospital-based pair-matched case-control study with a case of 1:3, including 144 cases of pulmonary tuberculosis and 432 controls. RESULTS: The overall vaccine effectiveness estimated in the present study was 60% (95% Confidence Interval [CI] 43%-72%). The protective effect was more in males in the age group 21-30 years. The prevented fraction was 39% (95% CI 24%-52%). CONCLUSION: The moderate effectiveness demonstrated in this study needs to be substantiated for other forms of tuberculosis by undertaking community-based case-control studies, before attempting to justify the use of mass neonatal BCG vaccination strategy as a part of the national programme.     

Usefulness of the codification of multiple causes of death in mortality statistics

BACKGROUND: The codification of multiple causes of death began in the US in 1917 and systematic publication of this data started in 1984. In Spain this began in 1988, and the data from this year have been taken as the basis for investigation. They have also been studied for regional differences. METHODS: A representative sample (595) of Spanish Standard Death Certificates (DC) was collected in Asturias for the year 1988. All were coded according to the International Classification of Diseases and a separate coding was made for each nosological entity included in the certificate (coding of multiple causes). The median, mode and the multiple cause/underlying cause ratio were also calculated. RESULTS: More than 80% of the certificates studied contained more than one cause of death. Chronic diseases are those which are accompanied by a greater number of causes and acute diseases those which appear alone. The highest ratios appear for diseases which are ill defined and also in those which are chronic. CONCLUSIONS: Our data show that information is lost in the production of the statistics of mortality and there are repercussions for the usefulness of these statistics.     

Increased accumulation of the glycoxidation product N(epsilon)-(carboxymethyl)lysine in human tissues in diabetes and aging

N(epsilon)-(Carboxymethyl)lysine (CML), a major product of oxidative modification of glycated proteins, has been suggested to represent a general marker of oxidative stress and long-term damage to proteins in aging, atherosclerosis, and diabetes. To investigate the occurrence and distribution of CML in humans an antiserum specifically recognizing protein-bound CML was generated. The oxidative formation of CML from glycated proteins was reduced by lipoic acid, aminoguanidine, superoxide dismutase, catalase, and particularly vitamin E and desferrioxamine. Immunolocalization of CML in skin, lung, heart, kidney, intestine, intervertebral discs, and particularly in arteries provided evidence for an age-dependent increase in CML accumulation in distinct locations, and acceleration of this process in diabetes. Intense staining of the arterial wall and particularly the elastic membrane was found. High levels of CML modification were observed within atherosclerotic plaques and in foam cells. The preferential location of CML immunoreactivity in lesions may indicate the contribution of glycoxidation to the processes occurring in diabetes and aging. Additionally, we found increased CML content in serum proteins in diabetic patients. The strong dependence of CML formation on oxidative conditions together with the increased occurrence of CML in diabetic serum and tissue proteins suggest a role for CML as endogenous biomarker for oxidative damage.