Modulation of neutrophil oxidative responses to soluble stimuli by platelet-activating factor

The role of platelet activating factor (PAF) as a regulator of human neutrophil superoxide (O2-) generation in response to soluble and particulate stimuli was examined. At concentrations greater than 10(-7) mol/L, PAF alone induced a brief burst of O2- production. When cells were exposed to PAF and either the chemotactic peptide n-formyl- methionyl-leucyl-phenylalanine (FMLP 10(-7) mol/L) or the tumor promoter phorbol myristate acetate (PMA 10 ng/mL), a marked synergistic augmentation of O2- release was noted when compared to control cells stimulated with FMLP or PMA alone. Mean percentage of enhancement by 10(-5) mol/L of PAF was 297% +/- 35% (n = 9) of control responses to FMLP and 185% +/- 16% (n = 3) of control responses to PMA. Consistent enhancement occurred with PAF concentrations of as low as 10(-9) mol/L. Enhancement could be demonstrated when neutrophils were exposed to PAF either at the same time as, or up to 60 minutes prior to, the second stimulus, and was neither reversed by removal of PAF from the medium prior to addition of FMLP or PMA nor dependent on the presence of extracellular divalent cations. Continuous recordings revealed that the enhancement was due to an increased maximal rate of O2- production. In contrast, PAF concentrations up to 10(-5) mol/L had only a minimal effect on the response to neutrophils to opsonized zymosan. Analysis of the enhancing properties of lipids structurally related to PAF revealed that the critical moiety was the saturated fatty acid at position 1. These results indicate the presence of a PAF-mediated positive feedback loop whereby the oxidative burst induced by some soluble stimuli is augmented. Modulation of neutrophil O2- production by PAF may serve to amplify neutrophil oxidative responses at sites of inflammation.     

Contemporary surgical treatment of primary hyperparathyroidism without intraoperative parathyroid hormone measurement

Introduction

Primary hyperparathyroidism (pHPT) is usually the result of a single adenoma that can often be accurately located preoperatively and excised by a focused operation. Intraoperative parathyroid hormone (IOPTH) measurement is used occasionally to detect additional abnormal glands. However, it remains controversial as to whether IOPTH monitoring is necessary. This study presents the results of a large series of focused parathyroidectomy without IOPTH measurement.

Methods

Data from 2003 to 2014 were collected on 180 consecutive patients who underwent surgical treatment for pHPT by a single surgeon. Preoperative ultrasonography and sestamibi imaging was performed routinely, with computed tomography (CT) and/or selective venous sampling in selected cases. The preferred procedure for single gland disease was a focused lateral approach guided by on-table surgeon performed ultrasonography. Frozen section was used selectively and surgical cure was defined as normocalcaemia at the six-month follow-up appointment.

Results

Focused surgery was undertaken in 146 patients (81%) and 97% of these cases had concordant results with two imaging modalities. In all cases, an abnormal gland was discovered at the predetermined site. Of the 146 patients, 132 underwent a focused lateral approach (11 of which were converted to a collar incision), 10 required a collar incision and 4 underwent a mini-sternotomy. At 6 months following surgery, 142 patients were normocalcaemic (97% primary cure rate). Three of the four treatment failures had subsequent surgery and are now biochemically cured. There were no complications or cases of persistent hypocalcaemia.

Conclusions

This study provides further evidence that in the presence of concordant preoperative imaging, IOPTH measurement can be safely omitted when performing focused parathyroidectomy for most cases of pHPT.     

Subunit structure of the erythropoietin receptor analyzed by 125I-Epo cross-linking in cells expressing wild-type or mutant receptors

To analyze the structure of the murine erythropoietin receptor (EpoR), wild-type or mutant EpoR cDNAs were expressed in cell lines, and the proteins that cross-linked with 125I-labeled erythropoietin (Epo) were analyzed by immunoprecipitation using an antibody against the intracellular region of the cloned EpoR. COS-7 cell transfectants expressing the wild-type EpoR showed two major cross-linked species of 145 and 110 Kd, both of which were recognized by the antibody against the cloned EpoR after denaturation under reducing conditions. Furthermore, a reduction in sizes of both cross-linked bands was observed in COS-7 transfectants expressing a mutant receptor with an internal deletion, thus indicating that both species contain the cloned EpoR. COS-7 cells expressing mutant receptors with carboxy-terminal deletions showed cross-linked bands corresponding to the smaller species of the two observed in cells expressing the wild-type receptor. In contrast to COS-7 cell transfectants, DA3 cells expressing wild-type or mutant EpoR cDNAs showed an additional cross-like species of 130 Kd. The size of this species was not altered by deletions in EpoR, showing that it did not contain EpoR. The 130-Kd cross-linked band, which would contain a 95-Kd protein, was also observed in a murine erythroleukemia cell line, D1B. These results suggest that Epo associates with a second component of 95 Kd, which is specifically expressed in hematopoietic cells.     

Platelet IgG, IgA, IgM, and albumin: correlation of platelet and plasma concentrations in normal subjects and in patients with ITP or dysproteinemia

IgG, IgA, IgM, and albumin are primarily known as plasma proteins. Their presence in platelets is poorly understood. The total platelet content of IgG, IgA, and albumin, measured in solubilized platelets by an enzyme-linked immunosorbent-assay (ELISA) technique, was greater than 90% secreted after stimulation by thrombin, consistent with an alpha-granule location. The platelet concentrations of these proteins correlated with their plasma concentrations in normal subjects and over a wide range of abnormalities in patients with IgG or IgA myeloma or liver cirrhosis. IgM was not detectable in normal platelets but was measurable and related to the plasma IgM concentration in patients with macroglobulinemia. In patients with idiopathic thrombocytopenic purpura (ITP), the platelet concentrations of IgG, IgA, and albumin were all twofold to threefold higher than normal despite normal plasma concentrations. Platelet surface IgG, measured by 125I-monoclonal antibody binding, constituted less than 1% of the total platelet IgG, and it appeared to be a pool distinct from the alpha-granule IgG since its concentration in normal subjects and patients did not correlate with either plasma or total platelet IgG concentrations. These observations are consistent with hypotheses that megakaryocytes incorporate plasma proteins into developing alpha-granules by pinocytosis and that the increased ratio of platelet to plasma of IgG, IgA, and albumin in ITP may reflect a younger average age of these platelets.     

Human platelet surface binding of endogenous secreted factor VIII-von Willebrand factor and platelet factor 4

Washed human platelets in buffers containing either 2 mM Ca++ or 4 mM EDTA were stimulated by human alpha-thrombin to induce secretion. The binding of two endogenous secreted proteins, factor-VIII-related protein (VIII-R) (von Willebrand factor) and platelet factor 4, was measured by reacting thrombin-treated and control platelets with specific antibodies to these proteins, then quantifying antibody binding with 125I-staphylococcal protein A. Both of these granule proteins were associated with the platelet membrane surface by a calcium-dependent mechanism after thrombin-induced secretion. This ability to bind endogenous secreted proteins to the plasma membrane surface may provide a mechanism by which the platelet can concentrate and organize its secreted proteins for subsequent physiologic reactions.     

Identification of the optimal wavelengths for optical topography: a photon measurement density function analysis

A method is presented to select the optimal wavelengths for multispectral optical topography, which not only gives good separation between chromophores, absorption, and scattering, but also minimizes the differences between interrogated volumes. This method uses the sum of squared differences to compare photon measurement density functions, which were generated for wavelengths in the near-infrared (NIR) range for a suitable model of tissue optical properties. It is found that including this condition significantly influences the range of optimal wavelengths. However, for the adult human head, the differences between interrogated volumes at NIR wavelengths are very small and image reconstruction is only slightly improved when measurements with overlapping sensitivities are used.