Activation of host phospholipases C and D in macrophages after infection with Listeria monocytogenes

Infection of the J774 murine macrophage-derived cell line with Listeria monocytogenes results in several elevations of intracellular calcium during the first 15 min of infection. These appear to result from the actions of secreted bacterial proteins, including phosphatidylinositol-specific phospholipase C (PI-PLC), a broad-range phospholipase C, and listeriolysin O (LLO) (S. J. Wadsworth and H. Goldfine, Infect. Immun. 67:1770-1778, 1999). We have measured hydrolysis of host PI and the activation of host polyphosphoinositide-specific PLC and host phospholipase D (PLD) during infection with wild-type and mutant L. monocytogenes. Elevated hydrolysis of host PI occurred within the first 10 min of infection and was dependent on both bacterial PI-PLC and LLO, both of which were required for the earliest elevations of intracellular calcium in the host cell. A more rapid hydrolysis of host PI was observed at 30 min after infection, at the time when wild-type bacteria have been internalized. Activation of host PLC, also occurred in the first 10 min of infection but was not dependent on the presence of bacterial PI-PLC. Similar observations were made in murine bone marrow-derived macrophages. In J774 cells, activation of host PLD was observed after 20 min of infection and was dependent on bacterial LLO. Mutants in the bacterial phospholipases produced levels of PLD activation similar to those produced by the wild type. Phorbol myristate acetate (PMA) also activated host PLD, while long-term treatment with PMA resulted in loss of the ability of L. monocytogenes to activate host PLD, suggesting an involvement of protein kinase C (PKC) in the activation of PLD. Rottlerin, an inhibitor of PKC delta in J774 cells, also inhibited the activation of PLD, but hispidin, an inhibitor of PKC betaI and betaII, did not. Pretreatment of J774 cells with the PLD inhibitor, 2, 3-diphosphoglycerate partially inhibited escape of the bacteria from the primary phagocytic vacuole.     

Identification of C3d receptors on Candida albicans.

Pseudohyphal but not yeast forms of Candida albicans possess both iC3b and C3d receptors, as determined by rosetting with erythrocytes carrying iC3b (EAC3bi) or C3d (EAC3d). Rosetting with EAC3d was markedly reduced when pseudohyphae were heat killed or treated with trypsin or pronase but was not inhibited by several saccharides or aminosaccharides, including alpha-methyl-D-mannoside, or by pretreatment of pseudohyphae with concanavalin A. However, mannoproteins obtained by concanavalin A affinity chromatography of whole pseudohyphal extracts inhibited the attachment of EAC3d to C. albicans, whereas soluble (nonmannosylated) proteins were less active. Thus, although the C3d receptors appeared to be glycosylated, the oligosaccharide component of the receptor was apparently not involved in the recognition of C3d. To isolate these receptors, whole-cell extracts were separated by DEAE-Trisacryl chromatography. Fractions that inhibited rosetting were pooled and affinity purified by C3d-Thiol-Sepharose chromatography. The eluate from this affinity column inhibited attachment of C. albicans to EAC3d. Monoclonal antibodies to C. albicans were prepared, and three of these antibodies blocked rosetting. Western blotting (immunoblotting) with these antibodies indicated the presence of 62- and 70-kilodalton receptors for C3d in the extracts purified by C3d affinity chromatography and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Isolation and characterization of cell surface mutants of Candida albicans.

Mutant strains of Candida albicans were obtained by selecting for cells that escaped agglutination by a polyclonal antiserum raised against standard C. albicans serotype A isolate B311. Mutants were obtained from strains B311 and B792 and from four strains isolated from patients with acquired immunodeficiency syndrome. All 15 tested mutants retained characteristic sugar assimilation patterns. All but one of the mutants retained the ability to form germ tubes and chlamydospores. Two mutants from an acquired immunodeficiency syndrome-derived isolate were deficient in binding complement ligands iC3b and C3d, whereas another mutant was deficient in binding ligand iC3b but not C3d. The hyphae of these three mutants lacked antigens when examined by Western immunoblotting with monoclonal antibody Ca-A, which detects several glycoproteins, including C3d-binding proteins. One of the complement-binding-deficient mutants was tested for its ability to colonize the gastrointestinal tract of rabbits but did not differ from the wild-type parent in site or degree of colonization. The proton magnetic resonance spectra of bulk mannan carbohydrate extracted from tested mutants showed the loss of a signal characteristic of the mannosyl alpha-PO4 linkage; each mutant also had a distinct pattern of other changes.     

A critical analysis of commercially available latex particle reagents for C-reactive protein (CRP) slide agglutination tests

C-reactive protein (CRP) was assessed in pediatric serum samples using different commercial latex reagents, which were analyzed for species origin of the coating antibodies, homogeneity and density of the latex particles, and prozone agglutinating capacity. All reagents correctly agglutinated the positive and negative control sera. The antibodies coating the particles differed with regard to species origin: one was coated with rabbit, one with horse and goat, one with horse, goat, rabbit and swine, while the reference reagent had horse, goat and rabbit antibodies.Only the monospecies specific antibody-coated latex showed obvious prozoning; this reagent also had the smallest and most homogenous latex particles and showed the most clear-cut reactions. False agglutination was observed at 7–26% according to quantitation with the spot immunoprecipitate assay, which compared favorably with radial immunodiffusion measurements. The lowest percentage of false readings was noted for the rabbit antibody-coated particles; the highest for the reagent with particles coated using antibodies from 4 different species.No reagent had satisfactory precision for the low positive sera between 10 and 40 mg CRP/1.     

1040 Prophylactic infusions with an unmodified intravenous immunoglobulin product causing few side-effects in patients with antibody deficiency syndromes

Summary Thirty-two patients with common variable immunodeficiency (CVID) and two patients with IgA and IgG subclass deficiency received a total of 1,040 intravenous (i.v.) infusions during 60 patient years with 7,575 g of a new immunoglobulin (Ig) preparation. The content of prekallikrein activators and the anti-complementary activity in the tested Ig preparation was low and, in comparison to seven other commercial i.v. Igs, so was the proportion of IgG polymers and fragments. The IgA content was always 0.02 g/l, often <0.004 g/l, and it was possible to continuously give the Ig prophylactically to four patients with anti-IgA antibodies, i.e. three with CVID and one with combined IgA-IgG2 deficiency. Adverse reactions were only noted in 4.7% of the 1,040 infusions and in 12 out of the 34 patients. None of the reactions were of the anaphylactic type, but two patients had moderate reactions and one had anuria, probably not caused by the Ig. A simultaneous infection seemed to increase the risk of phlogistic reactions, as five out of six patients who reacted with temperature rise and chills had a simultaneous upper respiratory tract infection. A substudy of various dosage schedules was performed with 11 patients receiving 203 infusions over 10.8 patient years. On 25 mg/kg/week of Ig given i.v. every five weeks, a mean increase in the preinfusion serum IgG level of 0.3 g/l was observed, as compared to earlier i.m. prophylaxis with the same dose. Only 1/4 of the patients on 25 mg/kg/week every five or three weeks reached a preinfusion IgG level 3 g/l. On 50 mg/kg/week every two weeks, 4/4 CVID patients had preinfusion levels above 3 g/l with a mean preinfusion increase of 1.5 g/l over the start level. Finally, 100 mg/kg/week every three weeks gave 5/5 patients a preinfusion serum IgG level of >4 g/l with a mean rise of 3.6 g/l, as compared with the levels before the study. An association between decreasing preinfusion IgG serum levels and the presence of infection was noted on 13/17 occasions, while increasing IgG was seen in healthy periods on 14/14 observations.

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1040 Infusionen mit einem nicht modifizierten Immunglobulin-Produkt, das bei Patienten mit Antikörpermangelsyndrom wenig Nebenwirkungen hervorruft

Zusammenfassung 32 Patienten mit gewöhnlichem variablen Immunglobulinmangel (CVID) und zwei Patienten mit fehlenden IgA- und IgG-Subklassen erhielten zusammen 1040 intravenöse (i.v.) Infusionen innerhalb von 60 Patientenjahren mit 7575 g einer neuen Immunglobulinpräparation (Ig). Der Gehalt an Prä-Kallikrein-Aktivatoren und die anti-komplementäre Aktivität in der getesteten Ig-Präparation war gering, verglichen mit sieben anderen kommerziell erhältlichen i.v. Immunglobulinen. Dasselbe gilt für den Anteil an IgG-Polymeren und -Fragmenten. Der IgA-Gehalt betrug immer 0,02 g/l, oft 0,004 g/l. Das Ig konnte vier Patienten mit IgA-Antikörpern, d. h. drei mit CVID und einem mit kombiniertem IgA-IgG-Mangel kontinuierlich verabreicht werden. Nur bei 4,7% der 1040 Infusionen und bei 12 der 34 Patienten wurden Nebenwirkungen beobachtet. Keine der Nebenwirkungen war vom anaphylaktischen Typ, aber zwei Patienten hatten mittelschwere Nebenwirkungen und einer eine Anurie, die wahrscheinlich nicht durch das Ig verursacht war. Das Risiko für phlogistische Reaktionen schien durch gleichzeitige Infektionen erhöht zu werden. Dies wurde bei fünf von sechs Patienten beobachtet, die bei Infektion der oberen Atemwege auf die Infusion mit Temperaturanstieg und Schüttelfrost reagierten. Bei 11 Patienten, die über 10,8 Patientenjahre 203 Infusionen erhielten, wurde eine Sonderstudie zu verschiedenen Dosierungen durchgeführt. Bei Infusion von 25 mg/kg/Woche i.v. alle fünf Wochen wurde ein mittlerer Anstieg der Serumspiegel vor Infusion gemessen, der um 0,3 g/l höher war als bei früherer i.m. Prophylaxe mit derselben Dosis. Nur bei 1/4 Patienten, die alle fünf oder drei Wochen 25 mg/kg/Woche erhielten, wurde ein IgG-Spiegel vor Infusion von 3 g/l erreicht. Bei Gabe von 50 mg/kg/Woche alle zwei Wochen hatten 4/4 CVID-Patienten vor Infusion Spiegel über 3 g/l, dabei stiegen die Spiegel vor Infusion um 1,5 g/l höher an als vor Therapiebeginn. Bei 100 mg/kg/Woche alle drei Wochen wiesen 5/5 Patienten ein Serum IgG von >4 g/l auf; der mittlere Anstieg gegenüber den Werten vor Studienbeginn betrug 3,6 g/l. Bei 13/17 Fällen wurde eine Assoziation von abnehmenden IgG-Serumspiegeln vor Infusion mit einer Infektion beobachtet; in gesunden Phasen waren bei 14/14 Beobachtungen Anstiege der IgG-Spiegel zu beobachten.

     

Public discourse and policy change: Absence of harm from increased oversight and transparency in OPO performance

The Centers for Medicare and Medicaid Services announced changes to the Final Rule for organ procurement organizations (OPOs) in November 2020, after a 23-month period of public debate. One concern among transplant stakeholders was that public focus on OPO underperformance would harm deceased donation. Using CDC-WONDER data, we studied whether donation performance dropped during the era of public debate about OPO reform (December 2018–February 2020). Overall OPO performance as measured relative to cause, age, and location-consistent deaths rose by 12.3% in 2019, compared to a median annual change of 2.5% 2009–2019. Organ recoveries exceeded seasonally adjusted forecasts by 4.2% in the first half of 2019, by 8.1% following the Executive Order issuing a mandate for OPO metric reform, and by 14.1% between the Notice of Public Rule Making and the onset of COVID-19-related systemic disruptions. We describe changes in donor phenotype in the period of increased performance; improvement was greatest for older and donation after cardiac death (DCD) donors, and among decedents who did not have a drug-related mechanism of death. In summary, performance during an era of intense public debate and proposed regulatory changes yielded 692 additional donors over expectations, and no detriment to organ donation was observed.     

Roux-Y Gastric Bypass: an effective anti-reflux procedure

Gastric limiting procedures have made an improvement in the lives of those patients in whom they have been successful. Not only have there been marked improvements in diabetes, hypertension, and arthritis, but there have been a number of other ‘spin-offs’, not the least of which is control of reflux esophagitis by totally eliminating the secretion of the parietal cell mass of the stomach from rising into the esophagus. We compared a group of 100 obese patients with reflux esophagitis who underwent Roux-Y gastric bypass (RYGBP) to a normalsized group of 23 patients on whom we had done Nissen fundoplications in the past. Visick gradings I-II of 100% vs 87%, respectively, may indicate a superiority of RYGBP over the Nissen procedure. Although the groups and time periods are too divergent to draw statistically significant conclusions, one can see that the RYGBP population was apparently better served considering their cure of ‘heartburn’ and other reflux symptoms as well as their achievement of sustained weight loss.     

Serum protein profiles to identify head and neck cancer.

PURPOSE: New and more consistent biomarkers of head and neck squamous cell carcinoma (HNSCC) are needed to improve early detection of disease and to monitor successful patient management. The purpose of this study was to determine whether a new proteomic technology could correctly identify protein expression profiles for cancer in patient serum samples. EXPERIMENTAL DESIGN: Surface-enhanced laser desorption/ionization-time of flight-mass spectrometry ProteinChip system was used to screen for differentially expressed proteins in serum from 99 patients with HNSCC and 102 normal controls. Protein peak clustering and classification analyses of the surface-enhanced laser desorption/ionization spectral data were performed using the Biomarker Wizard and Biomarker Patterns software (version 3.0), respectively (Ciphergen Biosystems, Fremont, CA). RESULTS: Several proteins, with masses ranging from 2778 to 20800 Da, were differentially expressed between HNSCC and the healthy controls. The serum protein expression profiles were used to develop and train a classification and regression tree algorithm, which reliably achieved a sensitivity of 83.3% and a specificity of 100% in discriminating HNSCC from normal controls. CONCLUSIONS: We propose that this technique has potential for the development of a screening test for the detection of HNSCC.     

Subjective cognitive complaints and amyloid burden in cognitively normal older individuals

Accumulating evidence suggests that subjective cognitive complaints (SCC) may indicate subtle cognitive decline characteristic of individuals with preclinical Alzheimer’s disease (AD). In this study, we sought to build upon previous studies by associating SCC and amyloid-β deposition using positron emission tomography with Pittsburgh Compound B (PiB-PET) in cognitively normal older individuals. One-hundred thirty one subjects (mean age 73.5±6) were administered three subjective cognitive questionnaires and a brief neuropsychological battery. A relationship between a subjective memory complaints composite score and cortical PiB binding was found to be significant, even after controlling for depressive symptoms. By contrast, there were no significant relationships between objective cognitive measures of memory and executive functions and cortical PiB binding. Our study suggests that SCC may be an early indicator of AD pathology detectable prior to significant objective impairment.     

Care of the Sexually Assaulted Woman

Sexual assault is very common. Unfortunately, many sexual assault survivors receive inadequate care, including lack of proper prophylaxis against sexually transmitted infections or pregnancy. This article describes the short- and long-term care consequences of female sexual assault and, using case studies, provides a guide for nurse practitioners on comprehensive and empowering care, from proper triage after sexual assault to sexually transmitted infection and pregnancy prophylaxis and follow-up and referral for the sexually assaulted woman.