Antibodies to 70 kD and 90 kD heat shock proteins are associated with graft-versus-host disease in peripheral blood stem cell transplant recipients.

The development of graft-versus-host disease (GVHD) can be modified by non-MHC factors. Based on our previous studies that showed an involvement of 70kD heat shock protein (hsp70) in the pathology of acute GVHD in a rat model, we determined serum levels of antibodies to hsp70, hsp90 and hsp60 in human recipients after allogeneic peripheral blood stem cell transplantation (PBSCT). Serum levels of these antibodies were correlated with GVHD status in the recipients. Twenty-nine recipients with high-risk haematological malignances, who received G-CSF mobilized allogeneic PBSCT from HLA matched family donors, were evaluated between 30 and 960 days after transplantation. Two recipients had no GVHD, 18 developed acute followed by chronic GVHD and nine developed only chronic GVHD. Patients with acute GVHD had a significant increase in IgM anti-hsp70 and/or anti-hsp90 early (30-90 days) after transplantation. In addition, an increase in IgM anti-hsp70 and/or anti-hsp90 antibodies preceded or accompanied chronic GVHD. Antibody levels returned to normal within the next 400 days in the majority of patients. Anti-hsp60 antibody levels were not different from control levels regardless of GVHD status. This study implies that the development of acute and/or chronic GVHD in humans is accompanied by an increase in anti-hsp70 and anti-hsp90 antibodies. Monitoring levels of anti-hsp70 and anti-hsp90 antibodies in stem cell transplant recipients may serve as a diagnostic tool and help to predict the onset of GVHD.

Reduced levels of Hsp70 result in a therapeutic effect of 15-deoxyspergualin on acute graft-versus-host disease in (DA x LEW)F1 rats.

We have shown previously that increased levels of hsp70, and antibodies reactive with hsp70 parallel the onset and severity of graft-versus-host disease (GVHD) in a parent --> (DA x LEW)F1 rat model. In this study we have assessed the effect of reducing the levels of the 70 kDa heat shock protein (hsp70), on the morbidity and mortality of acute GVHD in (DA x LEW)F1 rats. The reduction was accomplished by the administration of 15-deoxyspergualin (DSG), an immunosuppressive agent which binds to a constitutively expressed member of the 70 kDa heat shock protein family. DSG administered via three different protocols reduced GVHD-associated morbidity. One of the regimens, which consisted of intermittent DSG administration, also significantly reduced GVHD associated mortality. This DSG treatment reduced hsp70 levels in spleen and lymph nodes, inhibited anti-hsp70 antibody production, and diminished the serum levels of IL-2, IFN-gamma, TNF-alpha, and IL-10. IL-4 levels in the serum did not change during GVHD and were not effected by DSG. These results show that the mechanism of DSG immunosuppressive effect in rat GVHD may involve DSG's capacity to bind to hsp70, which in turn may lead to a decrease in levels of circulating anti-hsp70 antibodies, and reduced production of cytokines.

[Selected markers of bone metabolism in boys with constitutional delay of growth and puberty]. / Wybrane wskazniki metabolizmu kostnego u chlopców z konstytucjonalnie wolnym przebiegiem wzrastania i rozwoju.

The aim of the study was to determine serum concentration and urinary excretion of selected parameters of bone metabolism in boys with constitutional delay of growth and puberty (CDGP) in the relation to pubertal stages. The study group consisted of 41 boys (aged 8 to 18 yrs) with CDGP. Patients were divided on the basis of Tanner's criteria into 4 groups according to the pubertal stage. Serum alkaline phosphatase (AP) activity and serum osteocalcin (OC) concentration as markers of bone growth, as well as urinary deoxypyridinoline (DPD) as a marker of bone resorption were determined. Serum AP activity in 31 (75.6%) boys was within normal age range. Mean values of serum AP activity increased insignificantly from I to IV stage of puberty. Serum OC concentrations were normal in 33 (80.5%) patients. Eight (19.5%) boys showed decreased values of serum OC concentration. Mean OC concentration insignificantly increased with the pubertal stages. DPD urinary excretion was within normal age range in all patients and was the highest in stage III of pubertal stage. No significant abnormalities of bone metabolism parameters in boys with constitutional delay of growth and puberty were found.

hsp70, hsp32, and grp78 are increased in thermally injured skin with and without antithrombin(human) concentrate infusion.

An acquired deficiency of antithrombin (AT), an anti-inflammatory protein, develops in patients with thermal injuries. Skin thermotolerance is regulated by heat shock protein (hsp) genes. hsp70, hsp32, hsp27, and glucose-regulated protein78 (grp78) were studied in burned and unburned human skin to determine whether correction of the AT deficiency modulated the intensity of expression of these proteins. Fifty-four human skin samples were prepared by Western blot analysis: 11 unburned and 22 burned control skin samples and 7 unburned and 14 burned skin samples from patients treated with AT(Human), or AT(H). The intensity of hsp32 expression in burned AT(H)-treated skin (P < .001) and in burned control skin (P < .01) was significantly increased compared with unburned control skin. The intensity of expression of hsp70 was statistically significant in burned AT(H)-treated skin compared with unburned control skin (P < .02), as was that of grp78 (P < .01). Thermally injured skin with or without AT(H) treatment had an increased expression of hsp70, hsp32, and grp78 compared with unburned control skin.

Expression of 70-kDa heat-shock protein during acute graft-versus-host disease.

Acute lethal graft-versus-host disease (GVHD) was induced in unirradiated (LEW x DA)F1 rats by injection of DA parental lymphoid cells. Control rats received the same dose of (LEW x DA)F1 cells. The level of hsp70 at different stages of GVHD was examined in the spleen, lymph nodes, thymus, liver, lung, kidney, heart, and skeletal muscles of GVHD and control animals. Hsp70 was present in all organs analyzed in both groups and, with the exception of the spleen and lymph nodes, detectable levels remained constant during the disease. However, the spleen and lymph nodes of GVHD rats showed distinct changes in the hsp70 levels throughout the course of the disease. Expression of hsp70 in spleen and lymph nodes was augmented 7 and 14 days after induction of the disease. During the last stages of GVHD, 18-21 days after the induction, expression of hsp70 diminished to a level similar to that in untreated and control animals. Elevated levels of hsp70 in lymphoid organs during GVHD suggest an involvement of hsp70 in the pathology of the disease.

Paediatric preoperative teaching: effects at induction and postoperatively.

In a randomized controlled trial at Duke University and University of North Carolina Hospitals, 143 two to six year old children undergoing elective ambulatory surgery were randomized into a control group who received routine preoperative teaching or an intervention group who received an interactive teaching book. Changes in behaviour were measured by a Vernon behavioural questionnaire preoperatively and again two weeks postoperatively. A Global Mood Score was assessed in the preoperative holding area, at arrival into the operating room, and at the time of mask induction. The children in the control group were significantly more aggressive postoperatively than the intervention group (P < 0.05). Children who received the interactive teaching book exhibited higher levels of anxiety on the day of surgery, but fewer behavioural changes two weeks following surgery. Parents in the intervention group reported that preoperative teaching helped their child (87%) and themselves (83%).

Effect of d-fenfluramine on the lymphocyte response of HIV+ humans.

The objective of this study was to analyse the effect of d-dexfenfluramine (d-FEN) on the human lymphocyte response, in vitro. Experiments were designed to determine whether d-FEN augments specific human immune parameters associated with protection from opportunistic microbial pathogens and particularly focuses on d-FEN as a means by which to augment the function of CD8+ and CD4+ lymphocytes. Lymphocytes were examined for three reasons: (1) for their ability to inhibit the growth of Candida albicans; (2) for their ability to proliferate in response to a mitogen; and, (3) their cytokine profile (vis., production of IL-2, IFN-gamma and TNF-alpha). Peripheral blood mononuclear cells (PBMC) were obtained from 20 HIV+ patients. The patients were diagnosed as HIV+ within the past 0.5-9 years. d-FEN was found to augment the capacity of CD8+ lymphocytes to inhibit the growth of the opportunistic microbial pathogen, C. albicans. d-FEN enhanced the capacity of CD4+ lymphocytes to proliferate in response to the mitogen, Concanavalin A, and to increase the amount of IL-2 produced by CD4+ and CD8+ lymphocytes from AIDS patients. d-FEN increased the number of CD4+ and CD8+ lymphocytes that produced IFN-gamma from either non-AIDS or AIDS patients and increased the number of AIDS patient's CD8+ lymphocytes that produce TNF-alpha. These in vitro data suggest that d-FEN may be effective in enhancing immune function in immunocompromised individuals.

Isolated calcium-binding loops of EF-hand proteins can dimerize to form a native-like structure.

Helix-loop-helix fragments of EF-hand proteins are known to dimerize in solution, re-producing the characteristic structure of native protein domains [Shaw, G.S., Hodges, R.S., & Sykes, B. D. (1990) Science 249, 280-283]. In this paper we present evidence that isolated calcium-binding loops can also dimerize, when saturated with lanthanide ions, interacting with each other in a similar way as do loops in intact proteins. A synthetic analogue of calcium binding loop III of calmodulin, AcDKDGDGYISAAE-NH2, has been studied by 1H NMR spectroscopy. For the La(3+)-saturated peptide, concentration dependent broadenings and shifts of certain signals have been observed indicating dimerization process of intermediate rate on the NMR time scale. Analysis of signal shape and position of the Tyr7 ring protons as a function of concentration makes it possible to determine the association and dissociation rate constants of the process for various temperatures within the range of 10-80 degrees C. The dimerization constant changes according to van't Hoff relationship with delta S = 233 J/mol.K and delta H = 62 kJ/mol. A distance of 11.4 +/- 0.4 A between the ions coordinated by dimer molecules has been determined by measurements of Tb(3+)-->Ho3+ luminescence energy transfer. This value suggests that the dimer structure is similar to that of two-loop structural elements in native EF-hand proteins. From a thermodynamic cycle it can be shown that La3+ ion binding to the peptide dimers must be highly cooperative. Therefore, cooperativity of ion binding to domains of EF-hand proteins is, at least partly, due to local interactions between binding loops.

[Correlations between left atrial size and parameters of averaged atrial signal in patients with paroxysmal atrial fibrillation in the course of ischemic heart diseases]. / Korelacje miedzy wielkoscia lewego przedsionka a parametrami usrednionego sygnalu przedsionkowego u chorych z napadowym migotaniem przedsionków w przebiegu choroby niedokrwiennej serca.

The aim of this study was to assess the correlation between left atrial size and atrial signal-averaged electrocardiogram (ALP) in time-domain and frequency analysis in patients with paroxysmal atrial fibrillation (PAF) during ischaemic heart disease (IHD). The study population consisted of 75 patients with PAF during IHD who were divided into two groups; gr. I (left atrial dimension 40 mm) and gr. II (left atrial dimension 40 mm). The control group (gr. III) consisted of 50 healthy subjects. Recording of ALP was carried out by HIPEC-200HA. The high gain ECG was recorded during basic rhythm with orthogonal Frank leads X,Y,Z. These signals were amplified, averaged and filters. The filtered signals were combined into a vector magnitude and time-domain and frequency analysis was done. These findings suggest that the size of the left atrium does not change time-domain and frequency parameters of ALP in patients with PAF and IHD. We noticed significant influence of IHD on these parameters. On the other hand frequency analysis of ALP did not show any uselessness in diagnostic identification of patients with PAF during IHD and healthy subjects.

[The effect of intravenously given amiodarone on maintenance of ventricular signal averaged electrocardiogram in patients with ischemic heart disease]. / Wplyw dozylnie podanego amiodaronu na zachowanie sie usrednionego sygnalu komorowego u chorych na chorobe niedokrwienna serca.

Recording of ventricular signal-averaged ECG (VSAE) were carried out in 19 patients suffering from ischemic heart disease before and after intravenously administration of amiodarone using a commercially available machine HIPEC-200 HA device of Aerotel's. The high gain ECG was recorded during basic rhythm with orthogonal Frank leads X, Y, Z. These combined into a vector magnitude and the QRS duration (QRS), the duration of low amplitude signals < 40 microV (LPD) and the root mean square voltage of the signals in the last 40 and 50 ms of the filtered QRS (RMS 40,50) were calculated for VSAE. It was time-domain analysis of VSAE. In frequency domain analysis were calculated the following parameters in range from 40 Hz to 400 Hz in linear scale: area ratio 20-50 Hz/0-20 Hz, peak and magnitude ratio and in logarithmic scale: energy spectrum > -60 dB and decibel drop at 40 Hz. Subsequently amiodarone hydrochloride was given intravenously 5 mg/kg c.c. and again time-domain and frequency domain analysis were done. Data presented are mean +/- standard deviation. Statistical comparisons were performed with paired t-Student test. Significance refers to a p values < 0.05. Time-domain analysis of VSAE didn't show any significant differences after amiodarone injection. During frequency-domain analysis of VSAE statistically significant differences were displayed in Frank lead X and Y for energy spectrum > -60 dB. In Frank lead X decrease of this parameter after amiodarone (from 3322 +/- 1057 dB-Hz to 2628 +/- 733) and in lead Y increase (from 2793 +/- 500 dB-Hz to 3199 +/-938 dB-Hz) was noticed. Our observation have indicated that intravenously application of amiodarone hydrochloride didn't change parameters in time-domain analysis. Statistically significant differences were noticed only during frequency analysis VSAE in logarithmic scale -energy spectrum > -60 dB but only in two leads X and Y.

Antibodies specific for the 70-kDa heat-shock protein parallel the development of acute graft-versus-host disease in (DA x LEW)F1 rats.

Acute GVHD was induced in unirradiated (DA x LEW)F1 rats by the injection of parental DA lymphoid cells. Serum sequentially derived from such animals was found to contain increased levels of antibodies reactive with 70-kDa heat-shock protein (hsp70). Elevated levels of antibody to hsp70 were detected as early as 7 days and were maximal 14 days after the induction of GVHD. These increased levels of antibodies reactive with hsp70 paralleled the onset of the signs and symptoms associated with the development of GVHD. Increased anti-hsp70 reactivity in GVHD sera was associated statistically with increased levels of hsp70-reactive IgM, IgG2a, and IgG2b. In addition, GVHD sera reacted with a 70-kDa protein found within (DA x LEW)F1 lymphoid tissue. These results demonstrate that increased levels of antibodies to hsp70 parallel the onset and development of GVHD in an acute model and are the first to show that antibodies reactive with hsp70 are associated with an ongoing disease process.

Luminescence of peptide-bound terbium ions. Determination of binding constants.

Luminescence of Tb3+ ions bound to a calmodulin fragment has been studied. It is shown that during their lifetime excited ions dissociate from the peptide. If concentration of free peptide is high enough they can be coordinated again. As a consequence, observed terbium luminescence lifetime and intensity depends not only on binding equilibrium, but also on concentration of free peptide molecules. In such a system terbium binding constant cannot be correctly determined by simple steady-state measurements of luminescence intensities. Instead, terbium luminescence decay curves measured at various peptide concentrations must be analysed. Such an analysis has been made for a fragment of the IIIrd calcium binding domain of rat testis calmodulin. Rate constant of terbium association and the equilibrium binding constant corresponding to the best fit of theoretical functions to experimental points have been determined.

Neurochemical, endocrine and immunological responses to stress in young and old Fischer 344 male rats.

Two experiments were performed. In the first, a 20 min conditioned emotional response (CER) paradigm was used to compare the neurochemical, endocrine and immunological responses to stress of 7- and 22-month-old Fischer 344 (F344) male rats. In the second, corticosterone levels 20 min following ether stress, and regional brain type I and II corticosterone receptor densities were examined using 7- and 17.5-month-old F344 male rats. Dopamine (DA) metabolism in old nonstressed rats was significantly reduced in the medial frontal cortex, neostriatum, nucleus accumbens and hypothalamus, but not in the amygdala. The CER procedure, nevertheless, increased medial frontal cortical, nucleus accumbens and amygdaloid DA turnover in both the young and old rats. The young and old nonstressed rats did not evidence differences in norepinephrine (NE) and serotonin (5-HT) concentrations. However, stress resulted in a decrease in medial frontal cortical 5-hydroxyindoleacetic acid (5-HIAA) and hypothalamic 5-HT levels in old but not in young animals. These observations suggest age-related differences in the response of central NE and 5-HT systems to stress. Ether and the CER procedure led to exaggerated corticosterone responses in the old rats (17.5 and 22 month, respectively). Hippocampal type I but not type II corticosterone receptors were decreased by 47% in the 17.5-month-old rats. Thus, age-related changes in hippocampal corticosterone receptor types do not occur in unison, and the exacerbated corticosterone response to stress precedes the reported down-regulation of hippocampal type II corticosterone receptors in aged rats. Age-related changes were not observed in the concentrations of corticosterone receptors in other brain regions, or in the prolactin response to stress. The old rats, however, evidenced a reduction in the availability of the renin substrate, angiotensinogen, and in stress-induced renin secretion. Immune function was impaired in the old nonstressed rats, and further compromised by exposure to the CER procedure. In comparison to the young control rats, the old nonstressed rats showed an increased percentage of splenic large granular lymphocytes, reduced splenic natural killer cytotoxicity, and impaired Con-A-stimulated splenic T lymphocyte proliferation. Reductions in T splenic cell proliferation and natural killer cytotoxicity were observed in the young rats subjected to the CER paradigm, but not to the same extent as in the old rats. These observations indicate that aging male F344 rats evidence major alterations in basal central monoamine, endocrine and immune functions, and an increased sensitivity of these systems to stress.

[Effect of stimulation of adrenergic beta receptors on electrophysiologic properties of the bundle of Kent in patients with intermittent Wolff-Parkinson-White syndrome]. / Wplyw stymulacji receptorów beta-adrenergicznych na wlasciwosci elektrofizjologiczne peczka Kenta u chorych z intermitujacym zespolem Wolffa-Parkinsona-White'a.

In 15 patients with intermittent ventricular preexcitation symptoms the effect of intravenously administered dobutamine (10 mcg/kg b.w./min) on the ecg tracing, effective atrial, a-v nodal and Kent bundle refractions as well as the incidence of cardiac arrhythmias were assessed basing on results of the electrophysiologic examination. Pharmacological stimulation of the adrenergic system resulted in highly statistically significant shortening of examined refraction periods (p less than 0,02 for effective Kent bundle refraction and p less than 0,001 for the rest parameters) and improved the conduction via the accessory pathway as well as exacerbated cardiac arrhythmias.

[Effect of pharmacologic blockade of the autonomic nervous system on electrophysiologic properties of the accessory pathway and atrioventricular node in patients with Wolff-Parkinson-White syndrome]. / Wplyw blokady farmakologicznej wegetatywnego ukladu nerwowego na wlasciwosci elektrofizjologiczne drogi dodatkowej i wezla przedsionkowo-komorowego u chorych z zespolem Wolffa-Parkinsona-White'a.

In 17 patients aged 21-61 (mean 32) with WPW syndrome the transoseophageal stimulation was performed before and after intravenous administration of propranolol (0.1 mg/kg b.w.) and atropine (0.02 mg/kg b.w.). Pharmacological blockade of the autonomic nervous system resulted in statistically significant increase of heart rate (from 81 to 111/min), shortening of a-v nodal and atrial refraction (from 298 to 272 ms) as well as in shortening of stimuli cycle length revealing Wenckebach's point (from 324 to 291 ms). The Kent bundle refraction did not at the average change (333 and 324 ms), while in individuals great differences were observed. Generally, pharmacological blockade of the autonomic nervous system improves a-v nodal conduction, but in patients with WPW syndrome does not significantly effect on the accessory pathway.