Analysis of coenzyme Q10 content in human plasma and other biological samples.
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Biomedical subjects
Publications and source records attributed to S Graves.
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A 48-yr-old man with stage IV non-Hodgkin's lymphoma, became neutropenic following chemotherapy and developed a fever. His blood cultures were processed to enhance the yield of fastidious bacteria. A slow-growing, capnophilic Gram-negative rod was isolated. The febrile episode was treated with cefotaxime, imipenem and vancomycin and resolved. The bacterial isolate was identified as Bartonella (Rochalimaea) quintana by 16S-rDNA gene sequencing. The isolate showed 99.8% sequence homology with the type strain. This is the first isolation of Bartonella (Rochalimaea) quintana from a bacteremic patient in Australia. This bacterium is a fastidious Gram-negative rod requiring prolonged culture for its isolation. Patients with culture-negative pyrexia, especially immunocompromised patients, may need to be investigated for infection with this agent.
Five fresh-frozen cadaver feet obtained from traumatic amputations were tested during hyperdorsiflexion stress of the first metatarsophalangeal joint. Three different types of injury were observed: (1) rupture of the capsule proximal to the sesamoids, (2) rupture of the plantar plate distal to the sesamoids, and (3) rupture of the capsular structures medially, allowing a lateral swing of the sesamoids around the metatarsal head. Incomplete dislocation can be associated with significant damage to the plantar plate and other soft tissues of the foot.
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Forty-three patients exhibiting psychogenic seizures with onset before the age of 16 years were studied. All patients underwent intensive electroencephalography and video-electroencephalography monitoring. Thirty-two were female and 11 male. Mean age of the population at seizure onset was 12.4 years (range, 5 to 16 years). Twenty-one patients (48.8%) were taking anticonvulsants. Neurologic past history was abnormal in nine cases. Family history of epilepsy was found in 15 cases (34.9%). Median seizure frequency was one seizure every 5 days. Clinical characteristics of the seizures varied. However, unresponsiveness with generalized violent and uncoordinated movements involving the whole body (n = 19) or with generalized trembling (n = 11) were the most common features. Neuropsychological testing, carried out in 22 cases, failed to show major abnormalities in most of the cases. Significant personal and family distress was found in most of the cases. An important impact on patient's quality of life was evident when the seizures were present as compared to the seizure-free periods. There were no statistically significant predictors of clinical outcome.
In conclusion, monoclonal antibody STRO-1 has proven to be an extremely valuable reagent for the identification, isolation and functional characterization of human bone marrow stromal cell precursors. We were able to isolate CFU-F free of contaminating hemopoietic progenitors and are beginning to construct a detailed picture of their cell surface phenotype and have developed assays to examine the conditions required for their growth and differentiation. In the future, we hope to determine whether multipotential stromal stem cells exist in human bone marrow and to establish culture conditions that selectively promote self-renewal or development along any of a number of stromal cell lineages. Such cells would be an ideal target for gene therapy and may provide a means of treating disorders of the hemopoietic system with a suspected stromal etiology, such as aplastic anemia. We have recently succeeded in generating bone cells from STRO-1+ marrow cells (Gronthos et al, in preparation) and thus marrow stromal cell precursors may also find application in the treatment of other disorders such as those of the osteogenic system. For the present, these must remain tantalizing possibilities.
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Smooth muscle responses to Na+ pump inhibition are thought to reflect two elements: a neurogenic contribution, involving catecholamine release from nerve terminals, and a myogenic response, attributed to relations between pump activity, [Na+]i, and [Ca2+]i. In the present study, we describe the time course and magnitude of cell Na+ changes, assessed by two methods, atomic absorption and nuclear magnetic resonance spectroscopy during the myogenic contractile response of rabbit aorta strips to ouabain. A threshold concentration of 3 x 10(-7) mol/L induced a gradual rise in [Na+]i. Both methods showed an essentially identical monotonic rise over 4 to 8 hours from a baseline level of 8 to 10 mmol/L water to a peak, which was approximately fivefold higher. The neurogenic (rapid) and myogenic (delayed and gradual) contractile responses were temporally distinct. Ouabain at 10(-7) mol/L, a concentration 10- to 100-fold lower than the threshold for catecholamine-dependent rapid-onset responses, induced only a delayed and gradual contractile response, which reached a maximum at 6 to 8 hours. With 10(-6) mol/L ouabain, the delayed response of 1.6 +/- 0.2 g peaked at 7.3 +/- 1.1 hours and was sustained for 16 hours. The time course was similar to that for change in [Na+] but somewhat later. Ouabain at 10(-5) and 10(-4) mol/L induced a delayed response that was identical in magnitude but also induced an early rapid contractile response, which was prevented by reserpine or phentolamine pretreatment. These agents did not influence the delayed response.(ABSTRACT TRUNCATED AT 250 WORDS)
Several assay systems were compared for measuring the concentration of viable Rickettsia australis, including embryonated eggs, tissue cultures, and mouse inoculation. Direct rickettsial counts that included the enumeration of both viable and nonviable rickettsiae were used to obtain baseline values. Assays were conducted in parallel using serially diluted R. australis preparations to establish which assay displayed the greatest sensitivity and reproducibility. Overall, the plaque assay using buffalo green monkey kidney cells with centrifugation of the rickettsiae onto the monolayers was the most sensitive assay for detecting R. australis, while the embryonated egg assay and mouse lethality titrations were the least sensitive.
Increased levels of a circulating digoxin-like factor (DLF) occur in a number of physiologic states in which sodium homeostasis is altered, and may contribute to the pathogenesis of hypertension. We exploited the different affinities for DLF of seven antisera directed at digoxin to develop an immunochemical profile, and then employed this index to address two questions: does the same DLF species exist in several conditions associated with increased DLF levels, including pregnancy, renal failure, hepatic failure, and neonatal cord blood? Will this approach prove useful in assessing candidates proposed to be DLF? An identical profile was identified in serum from pregnant women and patients with renal or hepatic failure, and a highly significant correlation existed between DLF levels measured with antisera of high and intermediate affinity in 42 subjects with increased levels (r = 0.93; P less than .001). In patients with renal failure, when endogenous DLF levels were too low to assess the profile, concentration of the serum resulted in measurable DLF levels that had an identical profile. The profile was somewhat altered in umbilical cord blood, perhaps reflecting an influence of increased steroid hormone levels. Among agents suggested as candidates for DLF, neither lysophosphatidylcholine nor ouabain showed a profile resembling DLF. Progesterone, 17-OH-progesterone, and bufalin, on the other hand, did show substantial similarity, perhaps providing a clue to the structure of DLF. The normal plasma levels of progesterone and 17-OH-progesterone are 100- to 1000-fold too low to be candidates for DLF and bufalin was sufficiently dissimilar not to be a candidate. DLF in at least three different patient populations probably represents identical chemical species.(ABSTRACT TRUNCATED AT 250 WORDS)
Recent epidemiologic data suggests that Rickettsia australis, the cause of Queensland tick typhus, is present in southeastern Australia. In order to further confirm this observation, a canine serosurvey was undertaken to determine if naturally occurring antibodies were present in pet and farm dogs from this newly-recognized endemic area. Thirty-five of 312 surveyed dogs (11.2%) had indirect immunofluorescent antibody titers of 1:64 or greater against R. australis antigen. Positive control sera were obtained from two dogs experimentally inoculated with R. australis. One of these dogs was serially sampled and a rickettsemia could not be documented. None of 26 control sera obtained from dogs from South Australia, New Zealand, western Victoria, or North Carolina had antibody titers greater than or equal to 1:64. These results suggest that spotted fever group rickettsiae are present in Southeastern Australia.
New endemic areas of spotted fever-like rickettsial disease have been found in south-eastern Australia (Gippsland, Victoria and Flinders Island, Tasmania). The rickettsia responsible is currently unknown although it may be Rickettsia australis. To investigate serological evidence of rickettsial exposure in various wild animal species, a competitive ELISA was developed which detected antibodies to R. australis. It was based on inhibition of an indirect ELISA detecting antibody to R. australis in guinea pig sera. Pre- and post-infection sera from 2 dogs, 2 rabbits, 5 mice and 6 rats, experimentally infected with R. australis, were tested by competitive ELISA. The results showed that all pre-infection sera were negative and all post-infection sera positive for antibody to R. australis. To test the utility of the competitive ELISA for detecting natural rickettsial infection in non-laboratory animals, 51 dog sera, negative for rickettsial antibody by immunofluorescence (IF) and 20 IF positive dog sera (collected from various locations on the east coast of Australia) were tested. Compared to the IF test the competitive ELISA was 90% sensitive and 96% specific. This new test has potential for detecting antibody to R. australis in the sera of different wild animal species.
Diphtheria toxin (DTx) is an extremely potent inhibitor of protein synthesis. Cell death has been generally accepted as a straightforward effect of translation inhibition. Using human U937 cells, we found that DTx intoxication leads to cytolysis; indeed, release of 51Cr- and 75Se-labeled proteins could be detected within 7 h. However, little or no cell lysis was observed over a 20-50-h period when human U937 cells were exposed to cycloheximide, amino acid-deficient medium, or metabolic poisons even though protein synthesis was rapidly inhibited to levels observed with DTx. Likewise, investigations with human K562 cells revealed full resistance to the cytolytic action of DTx over a 50-h period despite a severe reduction in translation activity. These observations establish that inhibition of protein synthesis per se is not sufficient to provoke cell lysis. A characterization of DTx-induced cytolysis revealed a long lag period (6-7 h) which could be shortened considerably by a short exposure to low pH. NH4Cl and metabolic poisons blocked the cytolytic action of DTx, indicating that endocytic uptake of toxin is required for lytic activity. Surprisingly, DTx also induced extensive internucleosomal degradation of cellular DNA, a characteristic feature of apoptosis or programmed cell death. DNA-fragmentation preceded cell lysis and did not occur in DTx-treated K562 cells or in U937 cells that were treated with the other protein synthesis inhibitors. From these observations, we conclude that DTx-mediated cytolysis is not a simple consequence of translation inhibition and that internucleosomal DNA fragmentation is a newly identified and relatively early step in the cytolytic pathway of DTx.
Murine monoclonal antibodies (MAB) of the IgG3 subclass generated to colorectal tumor-associated glycolipids were assessed for enhancement of antibody-dependent cell-mediated cytotoxicity (ADCC) by interleukin 2 (IL-2). Mononuclear cell preparations containing large granular lymphocyte effectors required only low doses of IL-2 (0.1-10 units/ml) and short exposure (3 h) for maximal enhancement of ADCC. Exposure of mononuclear cells for longer periods (1-6 days) to higher levels of IL-2 (100-1000 units/ml) resulted in the generation of lymphokine-activated killer N cells which were lytically active without antibody. A non-ADCC MAB of the IgG1 subclass showed no ADCC even after stimulation of effector cells with IL-2. Effector cells pretreated with IL-2 showed an enhanced rate of cytolysis of target cells in the presence of antibody. Pretreatment of effector cells with IgG3 MAB resulted in lower ADCC, but pretreatment of target cells or simultaneous addition of MAB and effectors to target cells gave higher levels. These results indicated that ADCC with murine IgG3 is enhanced by levels of IL-2 achievable in current patient trials. The combination of antibody and IL-2-boosted effector cells gives comparable levels of killing to lymphokine-activated killer cells but should not suffer from similar toxicity. The NR-Co-04 antibody in combination with lymphokine may prove effective in treating colon cancer, a disease for which both chemotherapeutic and current biological therapies suggest a need for improved forms of therapies.
Studies with human U937 cells as targets established that a 15-min exposure to rTNF at pH 5.3 caused a significant increase in TNF-mediated cytolysis when compared to cells exposed to TNF at pH 7.4. A detailed examination of TNF-membrane interactions revealed that although TNF bound avidly to model membrane targets, no damage was generated under any condition tested. Binding of TNF, monitored with 125I-labeled as well as unlabeled protein, was enhanced at low pH. In the pH range tested (i.e., 4 to 8), target membrane permeability actually decreased in the presence of TNF. This membrane stabilization may be a consequence of TNF insertion into the target bilayer, a process we detected through use of an intramembranous photolabeling assay; interestingly, the efficiency of TNF insertion into membranes increased dramatically with decreasing pH. We conclude that native TNF does not cause pore formation directly and that its ability to induce cell lysis, as monitored by 51Cr release, is a consequence of some as yet obscure signaling event or intracellular activity. Parallel studies were carried out with diphtheria toxin, a protein with a more thoroughly characterized pH-dependent intoxification pathway. This toxin displayed acid-enhanced activities with both biologic and artificial targets.
Transferrin and, to a lesser extent, insulin were two components of serum that enhanced the survival of Treponema pallidum in microaerophilic, acellular medium. These two components also enhanced the attachment of T pallidum to baby rabbit genital organ (BRGO) cells in both aerobic and microaerophilic tissue culture systems. Growth hormone and epidermal growth factor marginally enhanced the attachment of T pallidum to BRGO cells.
We conducted a retrospective chart review on 50 patients under age 65 (average age 52.9 years) and 55 patients over 65 (average age 75.6 years). The older patients were much more likely to have atypical pain or no pain (38% vs 4%, P less than .0001). They were less likely to have electrocardiographic QRS changes (47% vs 72%), but more likely to have congestive heart failure (44% vs 16%, P less than .01). In 25% of the older patients, no diagnosis was made in the first 24 hours, as compared to 8% of the younger group. The increased mortality in the older group (16% vs 4%) approached statistical significance (P = .08). We conclude that the manifestations of acute myocardial infarction are more subtle in the elderly, with a higher proportion of atypical chest pain and nondiagnostic electrocardiograms, but the elderly are more likely to have congestive heart failure.
Rabbits infected intradermally with 10(3) Treponema pallidum (Melbourne 1) cells were examined for their susceptibility to reinfection with 10(2) T. pallidum cells (homologous or heterologous strains) at various intervals after the initial infection. At 2.5 weeks after infection, the rabbits were extremely sensitive to reinfection and developed syphilitic lesions significantly faster (i.e., shorter latent periods) than control rabbits that had not received the initial infection. This phenomenon may represent a state of immunosuppression or hypersensitivity in the infected rabbits. Whatever its etiology (at present unknown), it was a transient state since at 5 weeks after infection the rabbits were no longer different from control rabbits in their susceptibility to reinfection. They showed neither immunity (i.e., longer latent periods) nor immunosuppression or hypersensitivity (shorter latent periods) upon reinfection. At 6.5 weeks after infection, two of the three experimental rabbits were fully immune (no lesions upon reinfection), whereas the other rabbit exhibited immunosuppression or hypersensitivity upon reinfection. At 7.5 and 10 weeks after infection, all of the experimental rabbits were immune to reinfection. We conclude that syphilitic rabbits show a biphasic response to reinfection, consisting of an early phase of enhanced sensitivity to T. pallidum and a later phase of immunity to T. pallidum.