Biomedical subjects
Publications and source records attributed to J Novotny.
Explore the source record for details and available documents.
Non-infectious lung complications (NILC) are frequent, influencing morbidity and mortality of patients after allogeneic BMT. Although the term NILC encompasses a number of different entities, an association with GVHD has been noted for almost all of them. Our study was directed towards assessing the incidence and risk factors for developing NILC, as well as the response to treatment and long-term outcome. Forty (14.7%) out of 272 patients surviving for more than 3 months after allogeneic BMT, developed lung complications fulfilling the criteria for NILC. The evaluation was based on clinical investigation, radiologic imaging, lung function tests, broncho-alveolar lavage and biopsies. Risk factors were assessed by univariate and multiple statistical regression models, where chronic GVHD proved to be the only significant risk factor for the development of NILC (P = 0.011). In three patients NILC developed in direct association with donor lymphocyte infusions. The majority of patients responded well to treatment with corticosteroids and immunosuppressive drugs. NILC had no adverse effect on survival. The frequency of NILC was low in autologous (5%) as compared with allogeneic transplants (14.7%) but this difference was not statistically significant. Bone Marrow Transplantation (2000) 25, 1263-1268.
We compared the biological effects of the CXC chemokine SDF-1alpha on immunomagnetically purified CD34+ cells isolated from human normal bone marrow (NBM), leukapheresis products (LP) and patients with chronic myeloid leukaemia (CML). LP CD34+ cells showed a significantly stronger migration response to SDF-1alpha (100 ng/ml) than CD34+ cells isolated from the peripheral blood (PB) of CML patients (P < 0.05). The chemotactic response to SDF-1alpha was also reduced in CML BM CD34+ cells in comparison to NBM CD34+ cells but the observed differences were not statistically significant. In analogy to normal CD34+ cells circulating CML PB CD34+ cells were less responsive to SDF-1alpha than their BM counterparts (P < 0.05). Furthermore, SDF-1alpha elicited similar concentration-dependent growth suppressive effects on normal and CML CD34+ cells (P > 0.05) in colony-forming cell assays. We then demonstrated that SDF-1alpha triggers intracellular calcium increases in CD34+ cells and there were no differences in the time course and dose response characteristics of normal and CML CD34+ cells. The reduced migration response to SDF-1alpha in CML CD34+ cells was not due to a down-regulation of the SDF-1alpha receptor CXCR-4 as flow cytometric analysis revealed similar CXCR-4 expression levels on NBM, LP, CML PB and CML BM CD34+ cells (P > 0.05). Finally, no differences in the modulation of CXCR-4 levels in response to SDF-1alpha and serum were observed in CML and normal CD34+ cells. Our data suggest that the impaired chemotactic response of CML CD34+ cells to SDF-1alpha is not caused by a lack or complete uncoupling of CXCR-4, but may be due to an intracellular signalling defect downstream of the receptor.
Adherence and phagocytosis of invasive and noninvasive Neisseria meningitidis strains was investigated using light, fluorescence and electron microscopy. Invasive strains were isolated from the cerebrospinal fluid and/or blood of the patients with invasive meningococcal disease and noninvasive strains from the nasopharynx and/or larynx of healthy carriers. Adherence/endocytosis was studied on monkey kidney cells (the LLC-MK2 cell line) and phagocytosis on mouse monocytes and human macrophages (the P388D1 and U-937 cell lines, respectively). Although invasive and noninvasive meningococci isolated in the same cluster showed identical genotype and phenotype markers, they were found to interact differently with epithelial cells as well as with monocytes/macrophages. Invasive isolates displayed higher adherence to the surface of LLC-MK2 cells compared to noninvasive ones. Phagocytosis by P388D1 cells of noninvasive strains was effective and the bacteria were damaged by cytolysis. In contrast, invasive bacteria frequently persisted in "coiling" vacuoles and in effect could destroy the host cell. This is the first demonstration of coiling phagocytosis induced by meningococci. Efficiency of phagocytosis by U-937 cells was significantly higher for the noninvasive than invasive strains. Different behaviour of invasive and noninvasive strains of N. meningitidis observed during 4 hours of interactions with epithelial cells and monocytes/macrophages reflects well the higher pathogenic potential of invasive bacteria.
The rat thyrotropin-releasing hormone receptor-1 (TRHR-1) was modified by the addition of green fluorescent protein (GFP) and expressed stably in HEK293 cells. Extensive overlap of plasma membrane distribution of autofluorescent TRHR-1-GFP with that of the phosphoinositidase C-linked G-proteins Gqalpha/G11alpha, identified by indirect immunofluorescence, was monitored concurrently. Addition of thyrotropin-releasing hormone resulted in rapid separation of TRHR-1-GFP and Gqalpha/G11alpha signals as the receptor was internalized. This situation persisted for more than an hour. At longer time periods a fraction of the cellular Gqalpha/G11alpha was also internalized, although much of the Gqalpha/G11alpha immunoreactivity remained associated with the plasma membrane. Parallel experiments, in which the cellular distribution of TRHR-1-GFP and Gqalpha/G11alpha immunoreactivity were monitored in sucrose-gradient fractions following cell disruption, also demonstrated a rapid, agonist-induced movement of TRHR-1-GFP away from the plasma membrane to low-density vesicular fractions. At later time points, a fraction of the cellular Gqalpha/G11alpha immunoreactivity was also redistributed to overlapping, but non-identical, low-density-vesicle-containing fractions. Pretreatment of the cells with cytochalasin D or nocodazole prevented agonist-induced redistribution of G-protein but not TRHR-1-GFP, further indicating resolution of the mechanics of these two processes. The combination of a GFP-modified receptor and immunostaining of the G-proteins activated by that receptor allows, for the first time, concurrent analysis of the varying dynamics and bases of internalization and redistribution of two elements of the same signal-transduction cascade.
Thyroid hormones influence a wide range of physiological responses and the heart is considered a major target organ for triiodothyronine action. In the present study we examined closely the presumed relationship between altered thyroid status in the newborn rat and maturation of the regulatory components of the myocardial hormone-sensitive adenylyl cyclase signaling system. Beta -adrenoceptors and the alpha subunits of the stimulatory (Gs) as well as inhibitory (Gi) G proteins were determined in ventricular myocardium of immature (21-day-old) hypo- or hyperthyroid rats and in adult (84-day-old) previously hypo- or hyperthyroid rats. The changes in receptor and G protein levels were correlated with basic parameters of cardiac function and inotropic responsiveness to isoproterenol. All results were compared with those obtained in age-matched controls. Hypothyroidism in immature rats was associated with markedly reduced myocardial beta-adrenoceptor density, lower content of the long isoform (Gsalpha-L) of the stimulatory G protein and increased amounts of Gialpha2 and Gialpha3. These changes were accompanied by substantially diminished sensitivity to the inotropic effect of isoproterenol. On the other hand, no change in beta-adrenoceptor number, an increased level of Gsalpha-L and decreased levels of Gialpha2 were found in hyperthyroid myocardium. Cardiac inotropic responsiveness to isoproterenol in immature hyperthyroid rats was not significantly altered. In adult hearts of previously hyper- or hypothyroid rats, beta-adrenoceptor density was decreased but G protein levels as well as functional response were comparable to those determined in control animals. It may be concluded that physiological level of thyroid hormones is an important modulator of the normal maturation of the beta-adrenergic system in the developing rat ventricular myocardium. In adult rats previously affected by altered thyroid status, the function of their myocardial beta-adrenergic signaling appears to be compensated despite a lower number of the receptors.
Forty-five cases of fungaemia due non-albicans Candida spp. (NAC) in a single National Cancer Institution within 10 years were analysed for aetiology, risk factors and outcome. There had been 12 cases of fungaemia that were due to C. krusei, 14 due to C. parapsilosis, 7 due to C. (T.) glabrata, 6 to C. tropicalis, 2 to C. guillermondii, 2 to C. lusitaniae, 1 to C. stellatoidea, and 1 to C. rugosa. Comparison of 45 NAC fungaemia with 75 episodes of C. albicans fungaemia revealed differences only in two risk factors: previous empiric therapy with amphotericin B (16.0 vs 2.2%, P<0.01) appeared more frequently in cases of C. albicans fungaemia, and prior prophylaxis with fluconazole (8.9 vs 0%, P<0.02) was conversely more frequently observed with NAC. The incidence of other risk factors, such as underlying disease, chemotherapy, antibiotic prophylaxis or therapy, treatment with corticosteroids, catheter insertion, mucositis, cytotoxic chemotherapy, and neutropenia, was similar in both groups. There was no difference either in attributable or in overall mortality between NAC and C. albicans fungaemia in our cancer patients.
Forty-five consecutive diabetic patients with 46 upper extremity infections who underwent surgical debridement were retrospectively reviewed. The infections involved the skin or subcutaneous tissue in 19 patients and the fascia, tendon, muscle, or bone in 27. Twenty-three infections (50%) required a single operation and 23 required more than 1. Eighteen infections (39%) required an amputation and there were 3 deaths directly related to an infection. Six of 7 infections in which anaerobic organisms were cultured culminated in amputation. Four patients were diagnosed with necrotizing fasciitis. Twenty-one cultures (46%) were polymicrobial. An increased rate of amputation was associated with deep infections below the subcutaneous tissue, renal failure, and infections with gram-negative, anaerobic, or polymicrobial cultures. An increased rate of repeat surgery and a prolonged hospitalization were associated with deep infection and polymicrobial infections.
A distributed multimedia electronic patient record (EPR) is a central component of a medicine-telematics application that supports physicians working in rural areas of South America, and offers medical services to scientists in Antarctica. A Hyperwave server is used to maintain the patient record. As opposed to common web servers--and as a second generation web server--Hyperwave provides the capability of holding documents in a distributed web space without the problem of broken links. This enables physicians to browse through a patient's record by using a standard browser even if the patient's record is distributed over several servers. The patient record is basically implemented on the "Good European Health Record" (GEHR) architecture.
An N-glycan (N306) at the base of the V3 loop of HIV-BRU gp120 is shielding a linear neutralization epitope at the tip of the V3 loop on oligomeric Env. In contrast, this epitope is readily antigenic on monomeric gp120. Immunization with recombinant monomeric HIV-BRU gp120 may thus be expected to elicit antibodies preferentially neutralizing mutant variants of HIV-BRU lacking the N306 glycan. Therefore, two guinea pigs were immunized with monomeric wild-type HIV-BRU gp120 possessing the N306 glycan and immune sera were tested for neutralization against target viruses HIV-BRU, -A308, and -A308T321. HIV-A308 and HIV-A308T321 lack the N306 glycan; HIV-A308T321 contains an additional mutation at the tip of V3 rendering it resistant to MAb binding at this epitope. Both immune sera preferentially neutralized the two mutant virus variants lacking the N306 glycan, with a 10- to 20-fold increase in neutralization titer compared with the wild-type HIV-BRU. Thus, immunization with monomeric HIV-BRU gp120 elicited antibodies preferentially neutralizing HIV variants lacking the N306 glycan. In addition to antibodies directed against the tip of V3, other antibodies directed against epitopes shielded by the N306 glycan on the envelope oligomer were elicited by the immunization, as demonstrated by the ability of the immune sera to neutralize HIV-A308T321. One such epitope was overlapping the NEA-9284 epitope located at the amino-terminal flank of the V3 loop. Our results demonstrate that monomeric gp120 contains immunogenic structures inaccessible on the envelope oligomer. The limited ability of recombinant gp120 vaccines to induce neutralizing antibodies against primary isolates may thus not exclusively reflect genetic variation.
Using a combination of confocal immunofluorescence microscopy and subcellular fractionation, we demonstrate for the first time active internalization, trafficking, and down-regulation of a G protein alpha subunit subsequent to agonist occupation of a receptor. This proceeds on a much slower time scale than internalization of the corresponding receptor. In intact E2M11 HEK293 cells that express high levels of murine G11alpha and the rat thyrotropin-releasing hormone (TRH) receptor, the immunofluorescence signal of G11alpha was restricted almost exclusively to the plasma membrane. Exposure to TRH (10 microM) resulted first in partial relocation of G11alpha to discrete, segregated patches within the plasma membrane (10-60 min). Further exposure to TRH caused internalization of G11alpha to discrete, punctate, intracellular bodies (2-4 h) and subsequently to a virtually complete loss of G11alpha from plasma membranes and the cells (8-16 h). Short-term treatment with TRH followed by wash-out of the ligand allowed G11alpha immunofluorescence to be restored to the plasma membrane within 12 h. In subcellular membrane fractions, G11alpha was centered on plasma membranes, and this was not altered by up to 1-2 h of incubation with TRH. Further exposure to TRH (2-4 h) resulted in transfer of a significant portion of G11alpha to light-vesicular and cytosol fractions. At longer time intervals (4-16 h), an overall decrease in G11alpha content was observed.
Sixty four episodes of bacteraemia that appeared during antimicrobial prophylaxis with an oral quinolone plus an azole in neutropenic cancer patients were compared with 128 cases of bacteraemia in a cohort of controls matched for age, sex, underlying disease, neutropenia and vascular catheter in situ to assess differences in aetiology, cost of therapy and outcome. Patients who received prophylaxis had breakthrough bacteraemias of a different aetiology compared with the control group: they had significantly fewer multiply-resistant strains (21.9 vs. 51.5, P < 0.04) and a longer afebrile neutropenic period (9.55 days vs. 4.1, P < 0.001). Patients who received prophylaxis also had bacteraemias that were significantly more frequently caused by viridans streptococci (9.4%, vs. 1.7%, P < 0.01), enterococci (15.6% vs. 7.2%, P < 0.05) and Stenotrophomonas maltophilia (17.2% vs. 3.4%, P < 0.01). The cost of antimicrobial therapy per case (37401 SKK (1091 USD) vs. 31808 SKK (899 USD), P < 0.05) was also significantly higher in cases than controls; however, the number of administered antibiotics (4.18 vs. 3.21 per case, P = NS) was similar in both groups. There were no differences in outcome between both groups. However patients who received prophylaxis had significantly longer periods of afebrile neutropenia (9.55 days vs. 4.1, P < 0.001) and bacteraemia developed later than in controls. Also, the incidence of polymicrobial bacteraemia caused by multiresistant strains was lower among cases (21.9 vs. 51.5, P < 0.04).
High-dose chemotherapy with autologous transplantation of in vivo purged PBSC is a new and interesting therapeutic option for CML patients not eligible for allogeneic transplantation. We investigated the feasibility and toxicity of this approach in 57 patients with Ph-positive CML. For mobilization of Ph-negative PBSC, patients were treated either with '5 + 2/7 + 3'- type chemotherapy or with 'mini-ICE/ICE' chemotherapy followed by administration of G-CSF. Fourteen patients were in early chronic phase, 30 patients in late chronic phase and 13 patients in accelerated phase (AP) or blast crisis (BC). Cytogenetic responses in the PBSC harvests were dependent on both disease stage and type of chemotherapy: in late chronic phase and AP/BC, a complete or major cytogenetic response could be obtained in nine out of 13 patients treated with 'mini-ICE/ICE' but only in three out of 23 patients treated with '5 + 2/7 + 3' chemotherapy. However, in early chronic phase a Ph-negative autograft could be obtained in three out of eight patients upon mobilization with '5 + 2' chemotherapy. Thirty-one patients underwent PBSC transplantation and all of them successfully engrafted. Post-transplant cytogenetic analysis was available on 21 cases, of whom seven achieved a complete or major cytogenetic response, with two minor cytogenetic remissions. One patient (1/57) in blast crisis died during mobilization therapy (1.8%). Transplantation related mortality was 0%. This study demonstrates that mobilization of Ph-negative PBSC after myelosuppressive chemotherapy is feasible in CML patients and is associated with acceptable toxicity. Autologous transplantation of in vivo purged PBSC is a safe procedure with rapid and complete hematopietic recovery.
48 healthy donors underwent peripheral blood stem cell (PBSC) apheresis for allogeneic transplantation beginning on day 4 of G-CSF (2 x 5 microg/kg) mobilization. In one to four (median two) large-volume mononuclear cell aphereses, a median of 55.9 x 10(9) of lymphocytes (range 21.0-109.2 x 10[9]) were collected, an amount comparable to lymphocyte numbers removed by therapeutic lymphaphereses in autoimmune diseases. Mean peripheral lymphocyte counts decreased from premobilization values of 2.31 x 10(9)/l to 1.31 x 10(9)/l at a median of 34 d (1 month) and 1.53 x 10(9)/l at a median of 327 d (11 months). The decrease in peripheral lymphocyte counts was significantly correlated with the number of lymphocytes removed and the number of aphereses. Neutrophil and platelet counts returned to normal values after 1 month whereas monocyte counts and haemoglobin concentrations were significantly decreased at 1 month but not at 11 months.
A survey of the infrastructure in radiotherapy centres in three Central European countries has been performed as a first step in the development of a quality assurance network. Data concerning radiotherapy equipment, staff and number of patients treated in most of the radiotherapy centres from Czech Republic, Poland and Hungary were collected at the beginning of 1994. Equipment data have shown that 35% of 182 treatment units are conventional x-ray units, 35% 60Co units, 19% linear accelerators, 7% 137Cs units and 4% betatrons. About 47% of high energy units are older than 12 years and about 20% older than 21 years. An important number of centres still have no simulator which would constitute an important handicap to carry out adequate radiotherapy. The number of treatment planning systems has also been registered; 44% being PC-based systems with locally developed software. Large variations are observed in the number of patients treated per year, per high energy unit, but 12/47 centres treat more than 700 patients per year and unit. On the average, staffing seems adequate in numbers though there are wide variations. The main limitation of radiotherapy infrastructure in the Central European countries is the low number of linear accelerators and simulators and the advanced age of therapy units.
To determine the etiology of genital ulcers and to assess the prevalence of human immunodeficiency virus (HIV) infection in ulcer patients in 10 US cities, ulcer and serum specimens were collected from approximately 50 ulcer patients at a sexually transmitted disease clinic in each city. Ulcer specimens were tested using a multiplex polymerase chain reaction assay to detect Haemophilus ducreyi, Treponema pallidum, and herpes simplex virus (HSV); sera were tested for antibody to HIV. H. ducreyi was detected in ulcer specimens from patients in Memphis (20% of specimens) and Chicago (12%). T. pallidum was detected in ulcer specimens from every city except Los Angeles (median, 9% of specimens; range, 0%-46%). HSV was detected in >/=50% of specimens from all cities except Memphis (42%). HIV seroprevalence in ulcer patients was 6% (range by city, 0%-18%). These data suggest that chancroid is prevalent in some US cities and that persons with genital ulcers should be a focus of HIV prevention activities.
The relative proportions and tissue distribution of the long (Gs[alpha]-L) and short (Gs[alpha]-S) variants of the a subunit of the stimulatory G-protein (Gs[alpha]) change under a wide range of metabolic conditions, such as cellular differentiation, ontogenetic development, ageing and various adaptive processes. Although the two variants of Gs(alpha) are generally regarded to be functionally identical, this review summarizes recent experimental support for the non-identical behaviour of these proteins. Similarly, there is no consistent evidence for the functional meaning of these changes as far as regulation of adenylate cyclase activity is concerned. Since it is hard to believe that the complicated scheme of alternative splicing and the energy-consuming synthesis of proteins would be performed for no reason, it is suggested that Gs[alpha] variants might be involved in controlling other effector molecules and processes besides adenylate cyclase and cAMP metabolism. Such an idea is indirectly supported by specific alterations in the Gs[alpha]-L/Gs[alpha]-S ratio under various physiological and pathophysiological conditions.
Empirical free energy calculations were done for two X-ray DNA-protein complexes. Complex formation energies were estimated for the phage 434 repressor and the 434 cro protein, both in complex with wild-type versions of their respective nucleic acid binding sites. The resulting quantities were subdivided according to the relative contributions of hydrophobicity, electrostatics and entropy (including both conformational changes and those pertinent to physical association of complex components). The non-linearized finite difference Poisson-Boltzmann equation was solved over a range of salt concentrations to determine magnitudes of coulombic and desolvation effects. Though approximate at best, results confirmed (1) the importance of centrally located charged protein residues that do not make direct contact with the DNA, (2) explicit protein backbone interactions with DNA, and (3) the varied parts of the two proteins used to harness the energy of binding. Although a large difference exists between calculated and experimentally measured free energy values, these can be reconciled by assuming that approximately 1.5 e.u. (about 0.4-0.5 kcal at room temperature) of DNA vibrational and/or conformational entropy is lost for every basepair torsion in contact with the protein. Such a conclusion supports an "indirect readout" ("compound recognition") hypothesis of DNA binding specificity, wherein sequence-dependent differences in protein-DNA interaction are due to the topological peculiarities of the interactions rather than intrinsic chemical differences alone.