Biomedical subjects
T Baldwin
Publications and source records attributed to T Baldwin.
Emergence of a new Vibrio parahaemolyticus serotype in raw oysters: A prevention quandary.
CONTEXT: In May and June 1998, reported Vibrio parahaemolyticus infections increased sharply in Texas. OBJECTIVE: To determine factors that contributed to the increase in V parahaemolyticus infections. DESIGN, SETTING, AND PARTICIPANTS: Cross-sectional survey of persons reporting gastroenteritis after eating seafood in Texas; survey of environmental conditions in Galveston Bay. MAIN OUTCOME MEASURES: Traceback of oysters, water quality measures in harvest areas, presence of V parahaemolyticus in stool cultures; comparison of median values for environmental conditions before and during the outbreak compared with during the previous 5 years. RESULTS: Between May 31 and July 10, 1998, 416 persons in 13 states reported having gastroenteritis after eating oysters harvested from Galveston Bay. All 28 available stool specimens from affected persons yielded V parahaemolyticus serotype O3:K6 isolates. Oyster beds met current bacteriologic standards during harvest and fecal coliform counts in water samples were within acceptable limits. Median water temperature and salinity during May and June 1998 were 30.0 degrees C and 29.6 parts per thousand (ppt) compared with 28.9 degrees C and 15.6 ppt for the previous 5 years (P<.001). CONCLUSIONS: This is the first reported outbreak of V parahaemolyticus serotype O3:K6 infection in the United States. The emergence of a virulent serotype and elevated seawater temperatures and salinity levels may have contributed to this large multistate outbreak of V parahaemolyticus. Bacteriologic monitoring at harvest sites did not prevent this outbreak, suggesting that current policy and regulations regarding the safety of raw oysters require reevaluation. Consumers and physicians should understand that raw or undercooked oysters can cause illness even if harvested from monitored beds. In patients who develop acute gastroenteritis within 4 days of consuming raw or undercooked oysters, a stool specimen should be tested for Vibrio species using specific media. JAMA. 2000;284:1541-1545.
Therapy of murine cutaneous leishmaniasis by DNA vaccination.
Prophylactic DNA vaccination protects mice against infection with Leishmania major by inducing an exclusive Th1 immune response dominated by the production of IFN-gamma. Here we show that DNA vaccines, initially designed to prevent infection, can also have a significant therapeutic effect. In L. major infected mice, vaccination with DNA encoding the Parasite Surface Antigen/gp46/M2 causes reduction in lesion size and promotes healing in both genetically resistant C3H/He mice and susceptible BALB/c mice. The therapeutic effect is underpinned by a shift in the T cell-derived cytokine environment with an increase in the IFN-gamma producing Th1 type cells. Application of such immunotherapy in conjunction with antiparasite drugs may result in faster or more certain cure of the disease in humans.
Mice unresponsive to GM-CSF are unexpectedly resistant to cutaneous Leishmania major infection.
Granulocyte-macrophage colony-stimulating factor (GM-CSF) has been shown to play a protective role in leishmanial infection. Mice with a null mutation in the gene for the beta common (beta c) chain of the receptors for GM-CSF, interleukin(IL)-3 and IL-5 (beta c-null mice) display normal steady state hemopoiesis and develop lung disease similar to the human condition, alveolar proteinosis, due to a lack of signaling by GM-CSF. We therefore expected to observe a heightened sensitivity to Leishmania major in the beta c-null mice. Surprisingly, the beta c-null mice were more resistant to cutaneous infection than wild-type (wt) mice. Upon intradermal injection of L. major promastigotes, fewer beta c-null mice developed cutaneous lesions than wt mice and these lesions were smaller and healed more rapidly than in wt mice. This resistance to disease was associated with a reduced percentage of in vitro infected beta c-null macrophages. Macrophages from beta c-null mice displayed a more activated phenotype and produced increased amounts of nitric oxide following infection with L. major, both in vivo and in vitro. Paradoxically, however, the parasite burden in the draining lymph nodes was similar in both beta c-null and wt mice, suggesting that at least a subpopulation of cells was susceptible to the parasite. The mechanism preventing normal lesion development remains to be elucidated.
Glycosylphosphatidylinositol toxin of Plasmodium induces nitric oxide synthase expression in macrophages and vascular endothelial cells by a protein tyrosine kinase-dependent and protein kinase C-dependent signaling pathway.
In this study, we demonstrate that glycosylphosphatidylinositol (GPI) is a major toxin of Plasmodium falciparum origin responsible for nitric oxide (NO) production in host cells. Purified malarial GPI is sufficient to induce NO release in a time- and dose-dependent manner in macrophages and vascular endothelial cells, and regulates inducible NO synthase expression in macrophages. GPI-induced NO production was blocked by the NO synthase-specific inhibitor L-N-monomethylarginine. GPI also synergizes with IFN-gamma in regulating NO production. The structurally related molecules dipalmitoylphosphatidylinositol and iM4 glycoinositolphospholipid from Leishmania mexicana had no such activity, and the latter antagonized IFN-gamma-induced NO output. GPI activates macrophages by initiating an early onset tyrosine kinase-mediated signaling process, similar to that induced by total parasite extracts. The tyrosine kinase antagonists tyrphostin and genistein inhibited the release of NO by parasite extracts and by GPI, alone or in combination with IFN-gamma, demonstrating the involvement of one or more tyrosine kinases in the signaling cascade. GPI-induced NO release was also blocked by the protein kinase C inhibitor calphostin C, demonstrating a role for protein kinase C in GPI-mediated cell signaling, and by pyrrolidine dithiocarbamate, indicating the involvement of the NF-kappa B/c-rel family of transcription factors in cell activation. A neutralizing mAb to malarial GPI inhibited NO production induced by GPI and total malarial parasite extracts in human vascular endothelial cells and murine macrophages, indicating that GPI is a necessary agent of parasite origin in parasite-induced NO output. Thus, in contrast to dipalmitoylphosphatidylinositol and glycoinositolphospholipids of Leishmania, malarial GPI initiates a protein tyrosine kinase- and protein kinase C-mediated signal transduction pathway, regulating inducible NO synthase expression with the participation of NF-kappa B/c-rel, which leads to macrophage and vascular endothelial cell activation and downstream production of NO. These events may play a role in the etiology of severe malaria.
Regulation and characterization of the interferon-alpha present in patients with advanced human immunodeficiency virus type 1 disease.
To examine a possible association between plasma viremia and interferon-alpha (IFN-alpha) in patients with the acquired immunodeficiency syndrome (AIDS), we performed IFN plasma immunoadsorption by apheresis (IFN-alpha apheresis) in four volunteers with AIDS who had sustained levels of endogenous plasma IFN-alpha. IFN-alpha apheresis with two plasma volume exchanges was performed daily for 5 days. Clinical signs and symptoms and hematologic, virologic, and immunologic parameters were monitored. Two subjects developed anemia from phlebotomy, and one had a catheter++-associated bacteremia. The IFN-alpha apheresis was effective only in transiently removing IFN-alpha: depletion of IFN-alpha led only to its rapid reconstitution. Cell-associated HIV-1 was unchanged, but three of four subjects had a modest decrease in culturable plasma virus burden following the procedures. The recovery of in vivo HIV-1-related IFN-alpha by apheresis allowed its biologic and biochemical characterization. The HIV-1 IFN-alpha showed characteristics on ELISA, western blot, and biologic assays similar to two subspecies of the natural protein. The natural, recombinant, and HIV-1-induced IFN-alpha s demonstrated nearly identical antiviral activities. The HIV-1 IFN-alpha eluted from the column was not acid labile. The inability of large amounts of plasma IFN-alpha found in some patients with AIDS to affect viral burden likely reflects properties of the virus or of host factors independent of IFN-alpha.
Human immunodeficiency virus encephalitis in SCID mice.
The human immunodeficiency virus (HIV) is neuroinvasive and commonly causes cognitive and motor deficits during the later stages of viral infection. (referred to as HIV dementia). The mechanism(s) for disease revolves around secretory products produced from immune-activated brain macrophages/microglia. Recently, we developed an animal model system for HIV dementia that contains xenografts of HIV-1-infected cells inoculated into brains of mice with severe combined immunodeficiency (SCID). This animal system was used to quantitatively evaluate HIV-induced neuropathology. Xenografts of HIV-1-infected human monocytes (placed into the putamen and cortex of SCID mice) remained viable for 5 weeks. HIV-1 p24 antigen expression in mouse brain was persistent. Progressive inflammatory responses (including astrogliosis and cytokine production), which began at 3 days, peaked at day 12. The range of astrocyte proliferative reactions exceeded the inoculation site by > 1000 microns. Brains with virus-infected monocytes showed a > or = 1.6-fold increase in glial fibrillary acidic protein (staining distribution and intensity) as compared with similarly inoculated brains with uninfected control monocytes. These findings paralleled the accumulation and activation of murine microglia (increased branching of cell processes, formation of microglial nodules, interleukin (IL)-1 beta and IL-6 expression). An inflammatory reaction of human monocytes (as defined by HLA-DR, IL-1 beta, IL-6, and tumor necrosis factor-alpha expression) and neuronal injury (apoptosis) also developed after virus-infected monocyte xenograft placement into mouse brain tissue. These data, taken together, demonstrate that this SCID mouse model of HIV-1 neuropathogenesis can reproduce key aspects of disease (virus-infected macrophages, astrocytosis, microglial activation, and neuronal damage). This model may serve as an important means for therapeutic development directed toward improving mental function in HIV-infected subjects with cognitive and motor dysfunction.
Pitfalls and outcomes from accelerated wear testing of mechanical heart valves.
In 1990 Sorin Biomedica introduced a new bileaflet heart valve called the Bicarbon valve. This design was reported to eliminate wear in the hinge mechanism. Clinical quality Sorin Bicarbon, CarboMedics, St. Jude Medical, Duromedics and Jyros valves were obtained to test this claim and to compare the wear in the pivot of this new valve to other available heart valves. The valves were visually inspected then subjected to 4,000 cycles at a physiological beat rate in vitro. The valves were re-inspected then subjected to 400 million cycles in a Reul type accelerated wear tester. Scanning electron microscope photographs were taken of all contact areas at 40, 80, 120, 160, 200, 240, 280 and 400 million cycles. Wear marks on the inflow side of the Sorin, CarboMedics and St. Jude leaflets were measured and compared. Orifice wear was not quantified because of difficulty with measuring inside complex depressions. After 4,000 cycles of testing at a physiological beat rate the CarboFilmTM coating on the Sorin orifice showed signs of erosion. The other valve components only exhibited minor burnishing after 4,000 cycles. Following completion of 400 million cycles in an accelerated wear tester, approximately ten years in vivo, all valves showed significant wear. The inflow face of the pivot on the Sorin Bicarbon leaflets exhibited the deepest wear marks. The CarboFilm coating on the Sorin Bicarbon orifices was removed from most areas of leaflet contact. The transition between the remaining coating and the eroded areas created a rough edge. The tips of the Sorin leaflets contacted the bottom of the orifice pivot, in contrast to the St. Jude Medical and CarboMedics designs, which had minimal contact between the leaflet and the orifice.
Carbon dioxide regulated secretion of the EaeB protein of enteropathogenic Escherichia coli.
An eaeA mutant (intimin deficient) of enteropathogenic Escherichia coli stimulated phosphorylation of several host cell proteins, showing that intimate adherence is not required to activate signal transduction pathways in enteropathogenic E. coli-infected cells. Growth of enteropathogenic E. coli in tissue culture medium in 5% CO2, in the presence or absence of cultured cells, resulted in the secretion of several bacterial proteins. Two of these, 36 kDa and 20 kDa in size, were expressed at significantly lower levels in air. N-terminal sequencing and analysis of secreted proteins of an eaeB mutant indicated that the 36 kDa secreted protein was EaeB, previously implicated in the stimulation of signalling pathways in enteropathogenic E. coli-infected cells.
A regulatory role for astrocytes in HIV-1 encephalitis. An overexpression of eicosanoids, platelet-activating factor, and tumor necrosis factor-alpha by activated HIV-1-infected monocytes is attenuated by primary human astrocytes.
HIV-1-infected brain macrophages participate in neurologic dysfunction through their continual secretion of neurotoxins. We previously demonstrated that astroglial cells activate HIV-1-infected monocytes to produce such neurotoxic activities. In this study, the mechanism underlying these monocyte secretory activities was unraveled and found dependent on HIV-1's ability to prime monocytes for activation. LPS stimulation of HIV-1-infected monocytes resulted in an overexpression of eicosanoids, platelet-activating factor (PAF), and TNF-alpha. This was dependent on the level of HIV-1 infection and monocyte stimulation. Cell to cell interactions between activated virus-infected monocytes and primary human astrocytes reduced monocyte secretions. The capacity of astrocytes to deactivate monocytes was, notably, TGF-beta independent. Although astrocytes constitutively produced latent TGF-beta 2, HIV-1-infected monocytes neither affected TGF-beta 2 production nor converted it into a bioactive molecule. Furthermore, addition of rTGF-beta 1 or rTGF-beta 2 or its Abs to LPS-stimulated monocyte-astrocyte mixtures had no effect on monokine production. In contrast, addition of rIL-10 to LPS-stimulated monocytes produced a dose-dependent decrease in TNF-alpha. IL-10 mRNAs were detected in monocytes, but not astrocytes, following LPS treatment. These results suggest that macrophage activation, a major component of HIV-1 infection in the brain, precipitates neuronal injury by causing virus-infected cells to synthesize neurotoxins. The neurotoxins produced by monocytes are then regulated by astrocytes. Astrocytes therefore, can play either positive or negative roles for disease depending on prior macrophage activation. These findings begin to unravel the cellular control mechanisms that influence cognitive and motor dysfunctions in HIV-1-infected individuals.
The 126 kDa iron-regulated protein of Listeria monocytogenes is not a transferrin binding protein.
It has been reported that a 126 kDa protein of Listeria monocytogenes binds human transferrin. It is evident from results presented here that this is not so and that the observation of others resulted from interaction of this 126 kDa protein with streptavidin.
The attaching and effacing virulence property of enteropathogenic Escherichia coli.
Enteropathogenic Escherichia coli (EPEC) remain an important cause of infant diarrhoea in many parts of the developing world. Essential for virulence is their ability to adhere to the small intestinal mucosa and produce a striking 'attaching and effacing' (AE) lesion characterised by localised destruction of brush border microvilli, intimate attachment of bacteria to the residual apical enterocyte membrane, often in a cuplike pedestal structure, and formation of a dense plaque of actin (and other) cytoskeletal filaments beneath adherent bacteria. Fluorescence actin staining (FAS test) has turned out to be a useful diagnostic test for the AE lesion and also led to the identification of a chromosomal gene, eae, which is necessary but, by itself, not sufficient to produce the AE lesion. The 94 kDa outer membrane protein encoded by eae may be the adhesin which promotes intimate bacterial attachment. The signal transduction pathway which leads to AE lesion formation has yet to be defined although EPEC induced increased levels in intracellular calcium and phosphorylation of specific cell proteins including myosin light chain suggest that EPEC, by binding to a specific host cell receptor, may be promoting a calcium second message which would a) activate the brush border protein villin to cause microvillar breakdown and b) stimulate protein kinase activity to cause the other cytoskeletal rearrangements.
Meningitis caused by Psychrobacter immobilis in an infant.
Psychrobacter immobilis was isolated from the cerebrospinal fluid (CSF) and blood of a 2-day-old infant who appeared well except for a fever and a full anterior fontanelle. The infant was treated with antibiotics intravenously. After 48 h, he became afebrile and CSF and blood cultures were negative; he was then discharged. After 96 h of incubation, CSF and blood cultures yielded a gram-negative organism, P. immobilis. The child was readmitted to the hospital, and the same organism was again isolated from his blood and CSF.
Nonexercise cardiac stress testing.
Many patients who require evaluation for coronary artery disease are unable to undergo exercise stress testing because of physiologic or psychological limitations. Drs Vacek and Baldwin describe three alternative methods for assessment of cardiac function in these patients, all of which have high levels of diagnostic sensitivity and specificity.
Low energy level internal defibrillation during cardiopulmonary bypass.
Low energy level internal direct current shocks were used to defibrillate the hearts of 168 patients during procedures performed on cardiopulmonary bypass. In all cases, the core temperature was greater than 32 degrees C and care was taken to correct hypokalaemia and acid-base balance prior to defibrillation. In 78 patients (46%), defibrillation required 2 joules or less, and in 139 (82.7%) cases, defibrillation was effected with 4 joules or less. Only 4 patients required more than 10 joules to defibrillate the heart. This study shows that it is possible to defibrillate hearts during cardiopulmonary bypass with energy levels well below the 20-30 joule shocks commonly used.
Use of cardiovascular drugs in the elderly. What difference does age make?
Pharmacologic treatment of cardiovascular disease is both challenging and rewarding. Multiple medications and the variable nature of most cardiac problems necessitate careful monitoring of drug regimens in patients of any age. The elderly require even more attention to optimize therapy in light of variable drug absorption, effects, metabolism, and excretion. This challenge will present itself more frequently as the US population ages.
Actin accumulation at sites of bacterial adhesion to tissue culture cells: basis of a new diagnostic test for enteropathogenic and enterohemorrhagic Escherichia coli.
Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) adhere to the intestinal mucosa and produce an attaching and effacing (AE) lesion in the brush border microvillous membrane; the AE lesion is characterized by localized destruction of microvilli and intimate attachment of bacteria to the apical enterocyte membrane. A similar lesion is seen when bacteria adhere in vitro to a variety of human tissue culture cell lines. In both cases, dense concentrations of microfilaments are present in the apical cytoplasm beneath attached bacteria. Using a fluorescein-labeled phallotoxin, we have shown that these microfilaments are composed of actin. Cells infected with EPEC and EHEC strains known from electron microscopic studies to produce the AE lesion all exhibited intense spots of fluorescence which corresponded in size and position with each adherent bacterium; cells infected with adherent E. coli strains known not to produce the AE lesion did not produce this striking pattern of fluorescence and were indistinguishable from uninfected control cells. These results indicate that such site-specific concentrations of cytoskeletal actin are characteristic of the AE membrane lesion and can form the basis of a simple, highly sensitive diagnostic test for EPEC and EHEC.