Immunisation with recombinant AMA-1 protects mice against infection with Plasmodium chabaudi

The Plasmodium merozoite surface antigen apical membrane antigen-1 (AMA-1) has previously been shown to provide partial protection to Saimiri and rhesus monkeys immunised with recombinant Plasmodium fragile or parasite-derived Plasmodium knowlesi AMA-1, respectively. In the study reported here we have used the Plasmodium chabaudi/mouse model system to extend our pre-clinical assessment of an AMA-1 vaccine. We describe here the expression of the full-length Plasmodium chabaudi adami AMA-1 and the P. chabaudi adami AMA-1 ectodomain using both baculovirus and Escherichia coli. The ectodomain expressed in E. coli, which contained an N-terminal hexa-his tag, was purified by Ni-chelate chromatography and refolded in vitro in the presence of oxidised and reduced glutathione to generate intramolecular disulphide bonds. In a series of vaccine trials, in both inbred and outbred mice, highly significant protection was obtained by immunising with the refolded AMA-1 ectodomain. Protection was shown to correlate with antibody response and was dependent on intact disulphide bonds. Passive transfer of antibodies raised in rabbits against the refolded AMA-1 ectodomain was also protective. In view of this demonstration that E. coli expression of a soluble P. chabaudi AMA-1 domain can generate a vaccine that is effective in mice, we are pursuing a similar approach to generating a vaccine against P. falciparum for testing in human volunteers.     

Influence of absorbable dusting powders on wound infection

OBJECTIVE: To investigate the lymphocyte subpopulations (T4, T8 and macrophages) and major histocompatibility (MHC) II antigens in patients with superficial bladder cancer before and after intravesical instillations of recombinant interferon-gamma (IFN-gamma). PATIENTS AND METHODS: Four intravesical weekly instillations of either 1.3 mg (20 patients, group A) or 0.7 mg (11 patients, group B) IFN-gamma were administered in 31 evaluable patients (28 men and three women, mean age 68.5 years). The CD4+, CD8+, CD68+ and HLA-DR antigens were detected immunohistochemically in tumours and a marker tumour before and after intravesical instillations. RESULTS: The median number of T4 lymphocytes increased from 15 per high-power field (HPF) to 27.5 in group A (P = 0.0029) and to 45 in group B (P = 0.0117). Macrophages increased from 6 cells/HPF to 15 cells/HPF in group A (P = 0.0029) and from 2 to 8.75 cells/HPF in group B (P = 0.0117). The T8 lymphocyte subpopulation decreased from 4 to 3 cells/HPF (P = 0.0231) in group A and from 5 to 2 cells/HPF (P = 0.0759) in group B. The median percentage of HLA-DR antigens increased from 1.5% to 18% in general, (P < 0.001), from 2.5% to 15% in group A (P = 0.0064) and from 0% to 20% in group B (P = 0.0077). The induction of HLA-DR antigens was statistically significant in those receiving the lower dose (from 0% before instillation to 20% afterward, P = 0.0277), while it was not with the higher dose (from 0% to 5%, P = 0.068). Irrespective of the dose of IFN used. T4 lymphocytes and macrophages increased significantly after treatment in patients in whom the tumour HLA-DR antigens were either up-regulated or remained stable. The median net increase in T4 cells was 17.5 and 30 cells/HPF for groups A and B, respectively (P = 0.0429). CONCLUSION: T4 lymphocytes, macrophages and HLA-DR antigens increased after intravesical IFN-gamma in patients with superficial bladder cancer, but T8 lymphocytes decreased. Irrespective of the drug dose used, patients with either upregulated or stable HLA-DR antigens after treatment showed the same pattern of changes in the lymphocyte subpopulations. The two doses generally had the same effect on the immunological variables assessed but the lower dose was more effective in inducing HLA-DR antigens and in increasing the number of T4 lymphocytes in the tumours.     

Interactive effects of subanesthetic ketamine and subhypnotic lorazepam in humans

Ketamine is an N-methyl-D-aspartate (NMDA) receptor antagonist with psychotogenic and dissociative effects in healthy humans. These cognitive and perceptual effects in humans are reportedly reduced by benzodiazepine premedication. This study assessed the interactive effects of a ketamine (i.v. bolus of 0.26 mg/kg followed by an infusion of 0.65 mg/kg per hour) and lorazepam 2 mg., PO, in humans. Twenty-three healthy subjects completed 4 test days involving the oral administration of lorazepam or matched placebo 2 h prior to the i.v. infusion of ketamine or placebo. Ketamine: 1) produced behaviors similar to the positive and negative symptoms of schizophrenia as assessed by the Brief Psychiatric Rating Scale (BPRS); 2) evoked perceptual alterations as measured by the Clinician-Administered Dissociative States Scale (CADSS); 3) impaired performance on the Wisconsin Card Sorting Test (WCST) and other tests sensitive to frontal cortical impairment; and 4) had amnestic effects. Lorazepam produced attention impairments, concrete proverb interpretations, and recall impairments. Lorazepam reduced ketamine-associated emotional distress and there was a non-significant trend for it to decrease perceptual alterations produced by ketamine. However, it failed to reduce many cognitive and behavioral effects of ketamine, including psychosis. Further, lorazepam exacerbated the sedative, attention-impairing, and amnestic effects of ketamine. There was no evidence of pharmacokinetic interaction between these medications. These data suggest that subhypnotic lorazepam and ketamine show a spectrum of interactive effects, ranging from antagonism to potentiation.     

Highly sensitive thromboplastins do not improve INR precision

OBJECTIVE: Severe obesity (ie, at least 100% overweight or body mass index > or =40 kg/m2) is associated with significant morbidity and increased mortality. It is apparently becoming more common in this country. Conventional weight-loss treatments are usually ineffective for severe obesity and bariatric surgery is recommended as a treatment option. However, longitudinal data on the long-term outcome of bariatric surgery are sparse. Available data indicate that the outcome of bariatric surgery, although usually favorable in the short term, is variable and weight regain sometimes occurs at 2 years after surgery. The objective of this study is to present a review of the outcome of bariatric surgery in three areas: weight loss and improvement in health status, changes in eating behavior, and psychosocial adjustment. The study will also review how eating behavior, energy metabolism, and psychosocial functioning may affect the outcome of bariatric surgery. Suggestions for additional research in these areas are made. METHOD: Literature review. RESULTS: On average, most patients lose 60% of excess weight after gastric bypass and 40% after vertical banded gastroplasty. In about 30% of patients, weight regain occurs at 18 months to 2 years after surgery. Binge eating behavior, which is common among the morbidly obese, may recur after surgery and is associated with weight regain. Energy metabolism may affect the outcome of bariatric surgery, but it has not been systematically studied in this population. Presurgery psychosocial functioning does not seem to affect the outcome of surgery, and psychosocial outcome is generally encouraging over the short term, but there are reports of poor adjustment after weight loss, including alcohol abuse and suicide. CONCLUSIONS: Factors leading to poor outcome of bariatric surgery, such as binge eating and lowered energy metabolism, should be studied to improve patient selection and outcome. Long-term outcome data on psychosocial functioning are lacking. Longitudinal studies to examine the long-term outcome of bariatric surgery and the prognostic indicators are needed.     

Expression of constitutively active Raf-1 in the mitochondria restores antiapoptotic and leukemogenic potential of a transformation-deficient BCR/ABL mutant

The oncogenic BCR/ABL protein protects hematopoietic cells from apoptosis induced by growth factor deprivation, but the mechanisms are only partially understood. A BCR/ABL mutant lacking amino acids 176-426 in the BCR domain (p185DeltaBCR) failed to protect interleukin 3-deprived 32Dcl3 myeloid precursor cells from apoptosis, although it possessed tyrosine kinase activity and was capable of activating the Ras-Raf-MAP kinase pathway. Compared to p185 wild-type transfectants, p185DeltaBCR-transfected cells showed markedly reduced levels of Bcl-2 and expressed the hypophosphorylated, proapoptotic form of BAD. Bcl-2 expression in the mitochondrial fraction of p185DeltaBCR cells was also markedly diminished and mitochondrial RAF was undetectable. In p185DeltaBCR cells transfected with a mitochondria-targeted, constitutively active RAF (M-Raf) BAD was expressed in the hyperphosphorylated form and released from the mitochondria into the cytosol. p185DeltaBCR/M-Raf-transfected cells were completely resistant to apoptosis induced by growth factor deprivation in vitro. Moreover, constitutive expression of dominant-negative M-Raf (K375W) enhanced the susceptibility of 32Dcl3 cells expressing wild-type BCR/ABL to apoptosis. In severe combined immunodeficiency (SCID) mice, p185DeltaBCR/M-Raf double transfectants were leukemogenic, whereas cells expressing only p185DeltaBCR showed no leukemogenic potential. Together, these data support the existence of a BCR/ABL-dependent pathway that leads to expression of an active RAF in the mitochondria and promotes antiapoptotic and leukemia-inducing effects of BCR/ABL.     

More than 10 years of unrecognized nosocomial transmission of legionnaires' disease among transplant patients

OBJECTIVE: To investigate a cluster of cases of legionnaires' disease among patients at a hospital. SETTING: A university hospital that is a regional transplant center. DESIGN: Retrospective review of microbiology and serology data from the hospital laboratories and prospective surveillance via the radiology department; a case-control study and environmental sampling within the hospital and from nearby cooling towers. RESULTS: Diagnosis of seven cases of legionnaires' disease in the first 9 months of 1996 led to recognition of a nosocomial outbreak that may have begun as early as 1979. Review of charts from 1987 through September 1996 identified 25 culture-confirmed cases of nosocomial or possibly nosocomial legionnaires' disease, including 18 in bone marrow and heart transplant patients. Twelve patients (48%) died. During the first 9 months of 1996, the attack rate was 6% among cardiac and bone marrow transplant patients. For cases that occurred before 1996, intubation was associated with increased risk for disease. High-dose corticosteroid medication was strongly associated with the risk for disease, but other immunosuppressive therapy or cancer chemotherapy was not. Several species and serogroups of Legionella were isolated from numerous sites in the hospital's potable water system. Six of seven available clinical isolates were identical and were indistinguishable from environmental isolates by pulsed-field gel electrophoresis. Initial infection control measures failed to interrupt nosocomial acquisition of infection. After extensive modifications to the water system, closely monitored repeated hyperchlorinations, and reduction of patient exposures to aerosols, transmission was interrupted. No cases have been identified since September 1996. CONCLUSIONS: Legionella can colonize hospital potable water systems for long periods of time, resulting in an ongoing risk for patients, especially those who are immunocompromised. In this hospital, nosocomial transmission possibly occurred for more than 17 years and was interrupted in 1996, after a sudden increase in incidence led to its recognition. Hospitals specializing in the care of immunocompromised patients (eg, transplant centers) should prioritize surveillance for cases of legionnaires' disease. Aggressive control measures can interrupt transmission of this disease successfully.     

Venous rupture complicating hemodialysis access angioplasty: percutaneous treatment and outcomes in seven patients

OBJECTIVE: To evaluate percutaneous treatment options for preserving hemodialysis access after angioplasty-related venous rupture, we retrospectively reviewed the charts for all dialysis access angioplasties performed over a 33-month period. Seven cases of venous rupture after venous angioplasty were identified (four men and three women; mean age, 63.5 years). Treatment included observation only (n = 1), a second prolonged balloon inflation at the rupture site (n = 2), stent insertion (n = 5), and manual graft occlusion (n = 1). Treatment was successful in eliminating contrast extravasation in all patients while maintaining immediate graft function in six out of seven patients. None of the patients required emergent surgical intervention. The mean primary and secondary patency rates of the salvaged grafts after intervention were 2.3 and 9.3 months, respectively. Five of seven access sites were still patent at the most recent follow-up. CONCLUSION: Prolonged balloon inflation or placement of a stent may salvage hemodialysis access in most patients after angioplasty-related venous rupture. Primary and secondary patency have proven to be satisfactory.     

Effect of human C-reactive protein on chemokine and chemotactic factor-induced neutrophil chemotaxis and signaling

C-reactive protein (CRP) is a unique serum pentraxin and the prototype acute phase reactant. CRP is a ligand for specific receptors on phagocytic leukocytes, and mediates activation reactions of monocytes/macrophages, but inhibits the respiratory burst of neutrophils (PMN). Since CRP selectively accumulates at inflammatory sites in which IL-8 is also produced, we tested the effects of CRP on the responsiveness of PMN to IL-8 and the bacterial chemotactic peptide, FMLP-phenylalanine (FMLPP). Purified human CRP inhibited the chemotactic response of PMN to IL-8 and FMLPP. A mouse IgM mAb that was generated against the leukocyte CRP receptor (CRP-R) also inhibited the chemotactic response. Incubation of purified CRP with activated PMN generated CRP-derived peptides that also inhibited chemotaxis. A synthetic CRP peptide (residues 27-38) that binds to the CRP-R had weak chemotactic activity, whereas two other CRP synthetic peptides (residues 174-185 and 191-205) inhibited chemotaxis of PMNs to both IL-8 and FMLPP. CRP did not alter receptor-specific binding of IL-8, but exerted its effect at the level of signaling. CRP augmented both IL-8- and FMLPP-induced mitogen-activated protein kinase (extracellular signal-regulated kinase-2) activity. CRP at acute phase levels increased both agonist-induced and noninduced phosphatidylinositol-3 kinase activity. The results suggest a role for CRP as a regulator of leukocyte infiltration at inflammatory sites.     

Oxidative damage to sarcoplasmic reticulum Ca2+-ATPase AT submicromolar iron concentrations: evidence for metal-catalyzed oxidation

The sarcoplasmic reticulum (SR) calcium ATPase carries out active Ca2+ pumping at the expense of ATP hydrolysis. We have previously described the inhibition of SR ATPase by oxidative stress induced by the Fenton reaction (Fe2+ + H2O2 --> HO. + HO- + Fe3+). Inhibition was not related to peroxidation of the SR membrane nor to oxidation of ATPase thiols, and involved fragmentation of the ATPase polypeptide chain. The present study aims at further characterizing the mechanism of inhibition of the Ca2+-ATPase by oxygen reactive species at Fe2+ concentrations possibly found in pathological conditions of iron overload. ATP hydrolysis by SR vesicles was inhibited in a dose-dependent manner by micromolar concentrations of Fe2+, H2O2, and ascorbate. Measuring the rate constants of inactivation (k inact) at different Fe2+ concentrations in the presence of saturating concentrations of H2O2 and ascorbate (100 microM each) revealed a saturation profile with half-maximal inactivation rate at ca. 2 microM Fe2+. Inhibition was not affected by addition of 200 microM Ca2+ to the medium, indicating that it was not related to iron binding to the high affinity Ca2+ binding sites in the ATPase. Furthermore, inhibition was not prevented by the water-soluble hydroxyl radical scavengers mannitol or dimethylsulfoxide, nor by butylated hydroxytoluene (a lipid peroxidation blocker) or dithiothreitol (DTT). However, when Cu2+ was used instead of Fe2+ in the Fenton reaction, ATPase inhibition could be prevented by DTT. We propose that functional impairment of the Ca2+-pump may be related to oxidative protein fragmentation mediated by site-specific Fe2+ binding at submicromolar or low micromolar concentrations, which may occur in pathological conditions of iron overload.