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The origins of plasmacytoid dendritic cells (pDCs) have long been controversial and progenitors exclusively committed to this lineage have not been described. We show here that the fate of hematopoietic progenitors is determined in part by their surface levels of 9-O-acetyl sialic acid. Pro-pDCs were identified as lineage negative 9-O-acetyl sialic acid low progenitors that lack myeloid and lymphoid potential but differentiate into pre-pDCs. The latter cells are also lineage negative, 9-O-acetyl sialic acid low cells but are exclusively committed to the pDC lineage. Levels of 9-O-acetyl sialic acid provide a distinct way to define progenitors and thus facilitate the study of hematopoietic differentiation.

9-O-acetylation of sialic acid is a common modification that plays important roles in host-pathogen interactions. CASD1 has been described as a sialate-O-acetyltransferase and has been shown to be essential for 9-O-acetylation of sialic acid in some cell lines in vitro. In this study, we used knockout mice to confirm that CASD1 is indeed responsible for 9-O-acetylation of sialic acids in vivo. We observed a complete loss of 9-O-acetylation of sialic acids on the surface of myeloid, erythroid and CD4+ T cells in Casd1-deficient mice. Although 9-O-acetylation of sialic acids on multiple hematopoietic lineages was lost, there were no obvious defects in hematopoiesis. Interestingly, red blood cells from Casd1-deficient mice also lost reactivity to TER-119, a rat monoclonal antibody that is widely used to mark the murine erythroid lineage. The sialic acid glyco-epitope recognized by TER-119 on red blood cells was sensitive to the sialic acid O-acetyl esterase activity of the hemagglutinin esterase from bovine coronavirus but not to the corresponding enzyme from the influenza C virus. During erythrocyte development TER-119+ Ery-A and Ery-B cells could be stained by catalytically inactive bovine coronavirus hemaggutinin-esterase but not by the inactive influenza C hemagglutinin esterase, while TER-119+ Ery-C stage cells and mature erythrocytes were recognized by both virolectins. These results suggest that throughout murine erythrocyte development, cells of the erythroid lineage express a glycoconjugate bearing a modified 7,9-di-O-acetyl form of sialic acid, that is recognized specifically by the bovine coronavirus lectin and not by the influenza C hemagglutinin, and this modified sialic acid moiety is a component of the TER-119 epitope. As erythrocytes mature, the surface of Ery-C cells and mature erythrocytes also acquires a distinct CASD1-dependent 9-O-acetyl sialic acid moiety that can be recognized by virolectins from both influenza C and bovine coronavirus that are specific for 9-O-acetyl sialic acid.

The enzyme-linked immunosorbent assay (ELISA) has evolved from other types of immunoassays in the early 1970s and is now one of the most widely used laboratory techniques in clinical, translational, and basic sciences as well as clinical medicine. In this chapter, the basic concepts of ELISA and its most commonly used forms, such as sandwich, indirect, and competitive assays, are discussed, as well as their applications and the considerations involved in designing a new ELISA assay. The focus of this chapter is on the use of ELISA in clinical and translational research, aiming to provide the reader with the necessary tools to apply ELISA in their own research context. An example of how ELISA can be employed in a translational research scenario is provided, as well as a detailed protocol describing how to do so in that example.

Upon arriving in the spleen, newly minted transitional B cells differentiate into either follicular or marginal zone (MZ) B cells. This decision depends on integration of signals from the B cell antigen receptor, B cell activating factor (BAFF)-dependent activation of the canonical NF-κB pathway, Notch2-driven gene expression as well as integrin-mediated retention. Whether such signals are received simultaneously or in a sequential manner is currently unknown. It is also unclear whether integrin-mediated retention is a pre- or postlineage commitment event. The exact anatomic location in the spleen where MZ precursors receive these signals is also not known. It is also unclear as to how much of what has been learned about mouse MZ B cell development can be directly applied to human MZ B cells.

As a result of motor vehicle accident soft-tissue injury, temporomandibular joint articular disc derangement may develop and persist despite symptomatic treatment and medication. This study reports the effectiveness of management directed at controlling the TMJ and masticatory neuromuscular pain dysfunction with a TMJ/interocclusal stabilization appliance, specific biofeedback and ultrasound therapy. Following these conservative measures residual articular disc derangement was present in some subjects who were offered arthroscopic surgery and infrared midlaser with TMJ/occlusal stabilization. Twenty subjects with residual disc derangement were randomly selected into two groups with and without arthroscopic surgery, and analyses of variance made before treatment, 12 months after conservative procedures, 3 months following arthroscopic surgery and midlaser therapy and 3 years since commencement of management. Dependent variables compared were pain-discomfort, Clinical Dysfunction Index, articular disc derangement and maximal voluntary jaw opening. Conservative management alone provided significant reduction of pain-discomfort and clinical dysfunction, while arthroscopic surgery resulted in significant reduction in articular disc derangement. The midlaser with TMJ/occlusal stabilization maintained significant improvement in the variables (p < 0.01) for both groups. The common articular deviations in form found at arthroscopy were soft tissue alteration with hyperaemia, synovitis, synovial membrane and posterior attachment folding with connective tissue hyperplasia, and disc displacement with fibrous adhesions. The Global Status Score of pain behaviour compared with residual function, confirmed the presence of greater pain before treatment commenced.

An 11-year-old patient with a history of oligodontia and hypohidrotic ectodermal dysplasia had implants placed in the anterior and posterior mandible as part of his prosthetic rehabilitation. The maxilla was restored by using traditional prosthodontic methods. The long-term follow-up of the treatment is presented, and the clinical implications of placing implants in an actively growing child are discussed.

A patient with juvenile chronic arthritis presented with a malocclusion characterized by mandibular hypoplasia, symphysial deficiency, and an increased mandibular occlusal plane angle. Correction of the mandibular defect required both the horizontal advancement of the mandible and a counterclockwise rotation of the proximal segment to reduce the mandibular occlusal plane angle. This was achieved by a combination of distraction osteogenesis to horizontally advance the mandible (14 mm), followed by manipulation of the postdistraction regenerate to reduce the mandibular occlusal plane and increase the symphysial projection. The counterclockwise rotation of the mandibular body resulted in the creation of a posterior open bite. After a three-month period to allow consolidation of the mandibular distraction osteogenesis, secondary maxillary surgery at the Le Fort 1 level was performed to reestablish maxillary occlusal contact at the new mandibular occlusal plane. A genioplasty was also performed to improve chin projection.

The developmentally regulated expression of Trypanosoma cruzi neuraminidase in culture cells was monitored by immunofluorescence with a monoclonal antibody (TCN-2) against the enzyme. The results showed that TCN-2 reacted with all intracellular trypomastigote forms (NA+) but not with amastigotes. Immunoprecipitation of radiolabeled neuraminidase confirmed TCN-2 reactivity with trypomastigotes and the specificity of the antibody binding. During exiting from the host cells, all trypomastigotes were still NA+. However, when free in the extracellular environment, the relative proportion of NA+ parasites declined from 100% to about 20%, thereby establishing a subpopulation of trypomastigotes which did not express enzyme (NA-). The expression of neuraminidase in all intracellular trypomastigotes and in only a subpopulation of the extracellular counterpart suggests that the enzyme may play a role in parasite exiting from infected cells.

Ninety patients underwent arthroscopic temporomandibular joint surgery to 124 joints for arthropathy which had failed to respond to at least six months of non-surgical treatment. They were surveyed at between 6 months and 5 years (mean 2.5 years) after surgery and 63 per cent responded to the survey. They reported an 82 per cent improvement for pain (50 to 100 per cent better), 80 per cent for clicking and 82 per cent for locking. There was no morbidity following the treatment. Arthroscopic surgery should be considered for advanced temporomandibular joint arthropathy which is refractory to non-surgical treatment.

A retrospective study was conducted to determine the effect of intraoperative Haemonetics Cell Saver® (HCS) usage on postoperative homologous blood product requirements in CABG patients. From 1 January to 31 December 1993, 516 patients without renal disease or postoperative surgical or gastrointestinal haemorrhage had elective, first-time CABG surgery. The HCS was utilized in 435 of these patients (Group CS) and in 81 patients the HCS was excluded (Group NCS). Preoperative patient variables were similar in the group. We evaluated the HCS effect on blood product transfusion by comparing x units of red blood cells (RBC), fresh frozen plasma (FFP) and platelets (PLTS) transfused per patient between groups CS and NCS. There were no differences in the x units of RBC (1.9 ± 2.7 CS vs 1.8 ± 1.5 NCS) or in the RBC transfusion rate (48% CS vs 50% NCS). There were also no significant differences between the groups in x units of FFP (0.9 ± 0.8 CS vs 0.4 ± 0.9 NCS) or PLTS (0.7 ± 3.1 CS vs 0.4 ± 2.5 NCS), or in the percentage of patients receiving these products (12% CS vs 8% NCS). These data provide no evidence that the use of the HCS decreases the amount of homologous blood bank products required postoperatively in patients having routine first-time CABG surgery. The current era of aggressive blood conservation may have limited the role of the HCS in routine CABG surgery.

Several studies indicate that the neuraminidase from Trypanosoma cruzi (TCNA) [1 ] plays an important role in infection. Trypomastigotes remove sialic acid from rat myocardial and human endothelial cells in vitro [2], and from mouse [1] and human [3] erythrocytes during infection with T. cruzi in vivo. The extent of desialylation of host cells correlates with the number of parasites and TCNA activity [3]. Inhibition of enzyme activity by polyclonal [4] and monoclonal [5] antibodies and by high [6] and low-density lipoproteins [7] augment invasion of host cells by trypomastigotes suggesting that the enzyme exerts a negative control on T. cruzi infection (reviewed in ref. 8). We have previously shown that TCNA is spontaneously released into condition medium when tissue culture trypomastigotes are incubated at 4°C [4] or 37°C [5] for several hours or days, suggesting that the enzyme is anchored to the cell surface by a glycophosphatidylinositol (GPI) linkage. Release of membrane-bound proteins by PI-PLC is now regarded as convincing evidence for the involvement of GPI in membrane anchoring [9]. We therefore tested whether TCNA could be specifically removed from the surface of the Silvio X10/4

Trypanosoma cruzi exhibits a developmentally regulated neuraminidase activity that is inhibited by high-density lipoprotein (HDL). We report here that the infection of culture cells by T. cruzi trypomastigotes is enhanced by HDL in a dose-dependent manner. The enhanced infection is prevented by Vibrio cholerae neuraminidase, an enzyme whose activity is not inhibited by HDL, suggesting that sialic acid is involved in T. cruzi-host interaction. Similar enhancement of infection is also produced by low-density lipoprotein (LDL), which inhibits T. cruzi neuraminidase as well as HDL. Further evidence that the enhancement is due to lipoproteins is provided by the fact that infection of host cells in lipoprotein-deficient medium is less than in normal medium; it can be restored to the higher level by the addition of HDL, LDL or both to the lipoprotein-deficient medium. In view of these results, we propose that HDL and LDL regulate T. cruzi infection in mammalian hosts by inhibiting the parasite neuraminidase activity.

A valinomycin-mediated K+ diffusion potential across the membrane of multilamellar liposomes is stable for longer than 30 min and can be collapsed by a nonselective channel such as gramicidin. The kinetics of the potential collapse are complex but can be qualitatively broken down into a series of processes involving (1) binding of the gramicidin to the outer membrane, (2) dimerization to form a functional channel, (3) the flow of ions through the channel, (4) the establishment of a new diffusion potential on the next bilayer within the multilamellar liposome, and (5) the dissociation of gramicidin from the outer bilayer into the adjacent internal aqueous space. These processes are then repeated, in turn, for all the internal bilayers until the K+ concentration gradient (and membrane potential) is completely dissipated. Process 5 appears to be rate limiting at high gramicidin concentrations, but ion flux, process 3, becomes slower at low gramicidin concentrations where the collapse of the K+ gradient displays voltage dependence. Of course the rates of these processes can also be manipulated by changing the composition or size of the liposome and by varying the ion concentrations. Since the diffusion potential can be conveniently monitored with a voltage-sensitive fluorescent dye, 3,3'-diethylthiodicarbocyanine iodide [diS-C2-(5)], a simple method for the detection and partial characterization of membrane pores emerges from this investigation.

Entamoeba histolytica kills cells by contact dependent cytolysis. The mechanism underlying this process must be of rapid onset because target cells round up and show marked zeiosis within 15 min of contact. In earlier work, we identified a remarkable ion-channel forming protein which we named amoebapore, that may contribute to the amoeba-induced target cell killing. Within the amoeba it exists as part of a supramolecular aggregate together with other proteins of unknown function. In this work we report the purification of a solubilized form of the amoebapore. Amoebapore was found to exist as an apparent dimer of the previously reported protein whose molecular weight had been determined under denaturing conditions. Two isoforms of this dimer, with pI values of 6.8 and 5.3 present at a ratio of 7 to 1, were identified and purified. Both isoforms demonstrate ion-channel forming activity in planar lipid membranes. These channels show a unit conductance of 5–20 pS and remain open for < 1 s. Upon lateral aggregation, opening becomes concerted to a greater degree with channel conductance appearing in the 500 pS range (270 mM KCl). Aggregation seems to be highly cooperative because no intermediate single channel conductances are observed. The isolated particulate form of amoebapore depolarizes cells.

Isoenzyme electrophoretic patterns (zymodemes) are increasingly used to distinguish between pathogenic and non-pathogenic strains of Entamoeba histolytica. Isolates of E. histolytica from asymptomatic and symptomatic cases have been shown to differ in the electrophoretic mobility of their hexokinase and phosphoglucomutase isoenzymes. The hexokinase isoenzymes from a non-pathogenic strain and from a pathogenic strain of E. histolytica were purified by fast protein liquid chromatography in several steps, which included a separation by size, chromatofocusing, and anion exchange chromatography. The isoenzymes differed in their isoelectric points, which ranged from pH 4.8–5.4, but had very similar kinetic properties and almost identical apparent molecular weights (48 000) in sodium dodecyl sulfate polyacrylamide gels, as well as on gel filtration columns. Comparison of tryptic peptide analysis of each of the isoenzymes indicated considerable homology between the non-pathogenic and pathogenic forms. Antibodies produced against each of the two pathogenic hexokinase isoenzymes inhibited their enzymatic activity. The antibodies also inhibited the activity of the isoenzymes of the non-pathogenic strain. Our findings suggest that the isoenzymes have structural similarities, and that the pathogenic ones differ from the non-pathogenic ones in their electromobility due to post-translational modifications.

The trypanosomatid flavoprotein disulfide reductase, trypanothione reductase, is shown to catalyze one-electron reduction of suitably substituted naphthoquinone and nitrofuran derivatives. A number of such compounds have been chemically synthesized, and a structure-activity relationship has been established; the enzyme is most active with compounds that contain basic functional groups in side-chain residues. The reduced products are readily reoxidized by molecular oxygen and thus undergo classical enzyme-catalyzed redox cycling. In addition to their ability to act as substrates for trypanothione reductase, the compounds are also shown to effectively inhibit enzymatic reduction of the enzyme's physiological substrate, trypanothione disulfide. Under aerobic conditions, trypanothione reductase is not inactivated by these redox-cycling substrates, whereas under anaerobic conditions the nitrofuran compounds cause irreversible inactivation of the enzyme. When tested for biological activity against Trypanosoma cruzi trypomastigotes, many of the test compounds were trypanocidal, and this activity correlated with their relative ability to act as substrates for trypanothione reductase. The activity of the enzyme with these redox-cycling derivatives constitutes a subversion of its normal antioxidant role within the cell. For this reason these compounds may be termed "subversive" substrates for trypanothione reductase.

The production of a lymphokine, the leukocyte-migration-inhibition factor (LIF), by peripheral blood lymphocytes in response to an in vitro challenge with bovine β-lactoglobulin was assayed in infants and children suspected of having allergy to cow's milk protein. of the patients studied, 24 had cow's milk allergy, 24 were normal control subjects, 18 had recovered from milk allergy, 10 were newborns, and 10 were babies suffering from acute gastroenteritis. All patients with milk allergy demonstrated significant LIF production in response to β-lactoglobulin (23.5% ± 6.4%). In the normal control subjects, LIF was 3.1% ± 4.3% (P < .0005). Only two of the 24 control subjects and two of the ten newborns had high-normal values bordering on the positive. None of the ten babies with acute gastroenteritis gave a positive response. Most of the children who had recovered from milk allergy and were ingesting cow's milk had negative assays. This cell-mediated immune assay is shown to be a reliable test for the diagnosis of sensitivity to milk protein in infants and children, and for determining dietary treatment and when this treatment can be safely terminated. In most cases, its use should eliminate the need for the potentially dangerous and ethically questionable provocation test, as well as the need for repeated intestinal biopsies.

Separation and initial characterization of subcellular organelles from Entamoeba histolytica have been achieved by the use of self-generating Percoll gradients. Adequate resolution was obtained within one hour of amoeba homogenization. The ion-channel forming activity, amoebapore, was found associated with highly dense, small diameter particles, that were resolved from the numerous digestive vacuoles, and the plasma membrane. Following iodination of intact trophozoites with lactoperoxidase, the label was found to be incorporated into two distinct sedimentable fractions. The major component contained the near totality of the concanavalin A binding glycoproteins and the 5-iodonaphthalene-1-azide labelled intrinsic membrane proteins. It therefore was identified as the plasma membrane. The minor lactoperoxidase-iodinated component was found in the upper section of the gradient, associated with a particulate fraction nearly devoid of concanavalin A-binding glycoproteins. This fraction appears to represent particulate material on the external surface of the amoeba that is distinct from the plasma membrane. The digestive vesicles, which were identified by the presence of acid phosphatase and beta-D-glucosaminidase activities, appeared in the gradient as one main peak with a shoulder in the region of the plasma membrane. Analysis of the polypeptides and their labelling pattern in each fraction of the gradient are presented. Distinctive characteristics are discussed including the identification of a unique soluble protein highly labelled by 5-iodonaphthalene-1-azide.

Binding of high density lipoprotein (HDL) to Trypanosoma cruzi was examined because of its ability to specifically inhibit the parasite's neuraminidase. 125I-Labeled HDL bound to live and glutaraldehyde-fixed parasites equally well either at 37 degrees C or at 4 degrees C. Binding was saturable and inhibited by unlabeled HDL but not by unrelated plasma proteins. Specificity of the T. cruzi-HDL interaction was confirmed using fluorescein labeled HDL which bound to T. cruzi but not to T. rangeli, a species whose neuraminidase is not inhibited by HDL. Binding of HDL to T. cruzi paralleled the neuraminidase activity exhibited by the parasite's different stages and strains. In agreement with this finding, Steck and Wallach analysis of the binding data showed that the number of HDL binding sites was greater in infective trypomastigotes and on strains with high neuraminidase activity. However, the association constant of the binding did not change within the various developmental forms and strains of T. cruzi, suggesting that HDL bound to the same receptor, presumably having neuraminidase activity.

When pore-forming factors insert into the hyperpolarized membranes of lipid vesicles, ion gradients are rapidly equilibrated, effecting complete depolarization. This process can be conveniently followed with a potentiometric cyanine dye. The generality of the method is demonstrated by applications to three diverse materials. The well-studied gramicidin channel is used to demonstrate that the method is sensitive down to concentrations of lo-'* M. An extract from the shark repellent skin secretion of the Red Sea flatfish displays activity in the assay and is used to demonstrate the potential of the method to elucidate some of the characteristics of the pore, including its molecularity. That membrane-active factors can be detected and assayed in crude preparations is demonstrated with an impure extract of "amoebapore" from Entamoeba histolytica. In addition, variation of the buffer composition surrounding the vesicles can provide information about the ion selectivity of the pore under investigation. Recently, we described a simple method for detection of transferable membrane pores (Loew et al., 1983). The prin- ciple of the method involved the pore-mediated collapse of a diffusion potential in lipid vesicles that could be conveniently monitored with a potential sensitive dye. In that study multilamellar vesicles (MLV's)' were employed and the rate of diffusion potential collapse was limited by the rate of bi- layer-bilayer transfer of the pore. We now report that a similar protocol with large unilamellar vesicles (LUV's) can provide sensitivities in the femtomole range and also allows more detailed characterization of the pore properties. A number of other fluorescence-based methods for studying membrane permeabilizing factors in lipid vesicles have been recently described. Together, they provide a convenient set of additions to electrical measurements on planar bilayers for

Microbiologic methods of quantitating penicillin require indicator strains resistant to other antibiotics, are influenced by non-antibiotic serum factors, and require overnight incubation; features which limit the clinical usefulness. Since aminoglycosides can be quantitated with R-factor Mediated enzymes, we examined the quantitation of penicillin with plasmid mediated β-lactamase. Osmotic shockates prepared from E. coli 1100/RKS contain β-lactamase activity which in active against penicillin G, ampicillin, methicillin, oxacillin, cloxacillin and carbenicillin; converting them to their respective penicilloic acids. Penicilloic acid will reduce arsenomolybdate in the presence of trace amounts of Hg++, yielding a blue complex which absorbs at 750 nm. We constructed a spectrophotometric assay for penicillin G based on quantitating β-lactamase dependent production of penicilloic acid. The assay reliably quantitates penicillin G in 0.2 ml serum in concentrations between 0.25 and lOOμg/ml within one hour. Non-β-lactam antibiotics at maximum therapeutic concentrations do not interfere with the assay. Mixtures of β-lactam antibiotics can not be accurately assayed. Very high concentrations of aminoglycosides (approx. 100 μg/ml) increase the blank value, but reliable (relative standard error of 5%) quantitation of penicillin G is possible. Preservative agents commonly added to blood specimens do not interfere with the assay. We conclude that plasmid mediated β-lactamase can serve as a reagent in the rapid, specific quantitation of penicillin G.