Specificity of antinuclear autoantibodies in diseases with systemic destruction of connective tissue]

By using the fluorescent antibody technique several patterns of antinuclear antibodies (ANA) were detected in the systemic connective tissue diseases, as well as in some other diseases and in healthy donors, depending on the character of nuclear fluorescence. Homogeneous nuclear fluorescence, homogeneous fluorescence free of nucleaolar fluorescence, ring-shaped fluorescence, lumpy fluorescence, selective nuclealar fluorescence, nuclear fluorescence in the form of long fine plexiform bands associated with fluorescence in the nuclear membrane region. The latter ANA pattern was found only in several forms of lupus erythematosus, and this fluorescence was different from the previously reported "reticular" and "filamentous" patterns.     

Male- and female-specific variants of doublesex gene products have different roles to play towards regulation of Sex combs reduced expression and sex comb morphogenesis in Drosophila

Sexually dimorphic characters have two-fold complexities in pattern formation as they have to get input from both somatic sex determination as well as the positional determining regulators. Sex comb development in Drosophila requires functions of the somatic sex-determining gene doublesex and the homeotic gene Sex combs reduced. Attempts have not been made to decipher the role of dsx in imparting sexually dimorphic expression of SCR and the differential function of sex-specific variants of dsx products in sex comb development. Our results in this study indicate that male-like pattern of SCR expression is independent of dsx function, and dsx ^F must be responsible for bringing about dimorphism in SCR expression, whereas dsx ^M function is required with Scr for the morphogenesis of sex comb.     

Acute outcome of treating patients admitted with electrical storm in a tertiary care centre

Background

Electrical storm (ES) is a life threatening emergency. There is little data available regarding acute outcome of ES.

Aims

The study aimed to analyze the acute outcome of ES, various treatment modalities used, and the factors associated with mortality.

Methods

This is a retrospective observational study involving patients admitted with ES at our centre between 1/1/2007 and 31/12/2013.

Results

41 patients (mean age 54.61 ± 12.41 years; 86.7% males; mean ejection fraction (EF) 44.51 ± 16.48%) underwent treatment for ES. Hypokalemia (14.63%) and acute coronary syndrome (ACS) (14.63%) were the commonest identifiable triggers. Only 9 (21.95%) patients already had an ICD implanted. Apart from antiarrhythmic drugs (100%), deep sedation (87.8%), mechanical ventilation (24.39%) and neuraxial modulation using left sympathetic cardiac denervation (21.95%) were the common treatment modalities used. Thirty-three (80.49%) patients could be discharged after a mean duration of 14.2 ± 2.31 days. Eight (19.5%) patients died in hospital. The mortality was significantly higher in those with EF < 35% compared to those with a higher EF (8 (42.11% vs 0 (0%), p = 0.03)). There was no significant difference in mortality between those with versus without a structural heart disease (8 (21.1% vs 0 (0%), p = 0.32)). Comparison of mortality an ACS with ES versus ES of other aetiologies (3 (50%) vs 5 (14.29) %, p = 0.076)) showed a trend towards significance.

Conclusion

With comprehensive treatment, there is reasonable acute survival rate of ES. Hypokalemia and ACS are the commonest triggers of ES. Patients with low EF and ACS have higher mortality.     

Alterations in myocardial ultrastructure and protein expression after a single injection of isoproterenol

Immunochemical and electron microscopic characterization of rat myocardium was conducted 2 h and 3 weeks after a single injection of isoproterenol in rats. The relative content of several myospecific proteins (KRP – kinase-related protein, desmin), cytoskeletal proteins (tubulin, vinculin, myosin light chain kinase – MLCK) and extracellular matrix protein fibronectin was determined by immunoblotting. Two hours after injection of 50 mg/kg isoproterenol a destruction of some cardiomyocytes, contracture of myofibrils and mild edema of intercellular space was observed. The content of all the studied proteins except KRP decreased below control levels. This situation sustained 3 weeks after injection and paralleled alterations in cardiomyocyte ultrastructure. Areas of myofibrillar contracture and lysis were noted, glycogen granules were sparse; mitochondria contained arrow-like inclusions that are characteristic for calcium overload, also huge mitochondria contacting each other by specialized intermitochondrial contacts were detected. Clumps of unripe elastic fibers in enlarged intercellular space were combined with increased deposition of collagens type I and III forming areas of fibrosis. The smaller dosage of isoproterenol (10 mg/kg) rendered no significant damage in the acute postinjection period but 3 weeks later it induced the thickening of extracellular matrix around cardiac cells and the increase in KRP and tubulin content by 26 and 32%, correspondingly. MLCK levels remained depressed throughout the experiment. The rise in KRP expression was also observed after the addition of isoproterenol to cultured chicken embryo cardiomyocytes. Obtained results indicate that even a single injection of isoproterenol creates long lasting structural alterations in cardiac muscle accompanied by the increased expression of extracellular matrix proteins and several sarcoplasmic proteins apparently involved in hypertrophic response of cardiomyocytes.     

Preparation and use of monoclonal antibodies against seal alkaline phosphatase]

Monoclonal antibodies (termed as APP.1 and related to subclass IgG1) against seal alkaline phosphatase, have been obtained. APP.1 did not influence the enzymatic activity of alkaline phosphatase. The dissociation constant for the APP.1 interaction with Greenland seal alkaline phosphatase was equal to 8.5 x 10(-10) M. It was found that APP.1 interact with intestinal isoenzymes of common and fur seal, calf and deer alkaline phosphatases. An APP.1 complex with seal alkaline phosphatase was obtained and successfully applied in immunoenzymatic analysis. The use of this complex made it possible to diminish the limit of detectability of antibodies against peptide fragments of HIV-1 and HIV-2 proteins. Moreover, this complex allowed the identification of cytokeratin-8 and vimentin in human kidney slices and embryonic fibroblast-like cells, respectively.     

Expression of fibronectin variants in vascular and visceral smooth muscle cells in development

Monoclonal antibodies recognizing extra domain A (ED-A) and extra domain B (ED-B) fibronectin (FN) sequences were used to characterize FN variants expressed in human vascular smooth muscle cells (SMC) during fetal and postnatal development and to compare spectrum of FN variants produced by vascular and visceral SMC. In 8- to 12-week-old fetuses both ED-A-containing FN (A-FN) and ED-B-containing FN (B-FN) were found in all smooth muscles studied--aorta, esophagus, stomach, and jejunum. By 20-25 weeks of gestation relative amounts of both A-FN and B-FN were reduced significantly in the aortic media (fivefold for A-FN and twofold for B-FN), while in visceral SMC only B-FN content was decreased. All the adult visceral smooth muscles examined contained A-FN rather than B-FN. Therefore, the cells from adult aortic media appear to be the only SMC so far known to produce FN that contains neither ED-A nor ED-B. Moreover, the data obtained show that, unlike other cells, medial SMC are embedded in vivo in the extracellular matrix that contains FN lacking both ED-A and ED-B. SMC from the minor intimal thickenings in the human child aorta as well as those from the atherosclerotic plaques produce A-FN rather than B-FN. We conclude that (1) vascular SMC change the spectrum of produced FN variants at least twice--during prenatal development between 12 and 20 weeks of gestation, and during the postnatal period, when they are recruited into the intimal cell population; (2) the production of FN variants in visceral SMC is also developmentally regulated; (3) all visceral SMC unlike the cells from adult aortic media produce A-FN; (4) the presence of ED-A and ED-B sequences in the FN molecule is not necessary for the extracellular matrix assembly in vivo.     

Localization of different types of collagen (I, III, IV, V) in the connective tissue of the popliteal artery and skeletal muscle of man (immunoelectronmicroscopic analysis)

By means of immunoperoxidase and immunoferritin techniques collagen of the I, III, IV and V types has been revealed in cryostat sections of the popliteal artery and in the musculus quadriceps of the femur. Areas of the vascular wall without any macroscopical signs of lesions have been investigated. They have been obtained from amputated extremities of young persons (17-22 years old), and muscle pieces have been taken during operations performed in the knee joint. After certain immunocytochemical procedures the cryostat slices are embedded in mixture of epon 812 and araldit, non-contrasted ultrathin slices are examined in the electron microscope JEM 100CX. Collagen of the I and III types is revealed in fibrills 20-80 nm thick either with or without cross striation, as well as in microfibrills. Collagen of the III type in the intercellular substance of the arterial wall occurs in nonfibrillar form. Collagen of the IV type is revealed in basal membranes of the smooth muscle cells of the arterial wall, of the muscle fibers and of endothelium of blood capillaries of the skeletal muscle. Collagen of the V type is found as accumulations having various size and form; they localize in many places of the intercellular substance of the arterial wall. A tight contact is revealed between the formations including collagen of the V type with drops of elastin and elastic fibers. A suggestion is made that collagen of the V type participates in formation of elastic fibers.     

Retroviral transfer of the p16INK4a cDNA inhibits C6 glioma formation in Wistar rats

BACKGROUND: The p16INK4A gene product halts cell proliferation by preventing phosphorylation of the Rb protein. The p16INK4a gene is often deleted in human glioblastoma multiforme, contributing to unchecked Rb phosphorylation and rapid cell division. We show here that transduction of the human p16INK4a cDNA using the pCL retroviral system is an efficient means of stopping the proliferation of the rat-derrived glioma cell line, C6, both in tissue culture and in an animal model. C6 cells were transduced with pCL retrovirus encoding the p16INK4a, p53, or Rb genes. These cells were analyzed by a colony formation assay. Expression of p16INK4a was confirmed by immunohistochemistry and Western blot analysis. The altered morphology of the p16-expressing cells was further characterized by the senescence-associated beta-galactosidase assay. C6 cells infected ex vivo were implanted by stereotaxic injection in order to assess tumor formation. RESULTS: The p16INK4a gene arrested C6 cells more efficiently than either p53 or Rb. Continued studies with the p16INK4a gene revealed that a large portion of infected cells expressed the p16INK4a protein and the morphology of these cells was altered. The enlarged, flat, and bi-polar shape indicated a senescence-like state, confirmed by the senescence-associated beta-galactosidase assay. The animal model revealed that cells infected with the pCLp16 virus did not form tumors. CONCLUSION: Our results show that retrovirus mediated transfer of p16INK4a halts glioma formation in a rat model. These results corroborate the idea that retrovirus-mediated transfer of the p16INK4a gene may be an effective means to arrest human glioma and glioblastoma.