No abstract

The various granule subtypes of the human neutrophil differ in propensity for exocytosis. As a rule, granules formed at late stages of myelopoiesis have a higher secretory potential than granules formed in more immature myeloid cells. Neutrophils contain four closely related alpha-defensins, which are stored in a subset of azurophil granules. These defensin-rich azurophil granules (DRG) are formed later than defensin-poor azurophil granules, near the promyelocyte/myelocyte transition. In order to characterize the secretory properties of DRG, we developed a sensitive and accurate ELISA for detection of the neutrophil alpha-defensins HNP 1-3. This allowed us to quantify the exocytosis of alpha-defensins and markers of azurophil (myeloperoxidase), specific (lactoferrin) and gelatinase (gelatinase) granules from neutrophils stimulated with different secretagogues. The release pattern of alpha-defensins correlated perfectly with the release of myeloperoxidase and showed no resemblance to the exocytosis of lactoferrin or gelatinase. This finding was substantiated through subcellular fractionation experiments. In conclusion, despite a distinct profile of biosynthesis, DRG are indistinguishable from defensin-poor azurophil granules with respect to exocytosis. Thus, in contrast to peroxidase-negative granules, azurophil granules display homogeneity in their availability for extracellular release.

No abstract

The clinical utility and medico-economic value of several personalized diagnostic tests has been well described in the literature. Development of such tests, including generation of the necessary supportive clinical validation data, is a complex and expensive endeavor. In general, sponsors of such tests lack sufficient clarity on appropriate reimbursement and regulatory pathways to provide the clear development framework necessary to incentivize the required level of investment. In the USA, an imperfect reimbursement paradigm has evolved to accommodate a small number of 'value-priced' laboratory-developed tests, although major structural barriers remain to broader implementation. In Europe, by contrast, there is virtually no precedent for value-based public sector pricing, and even such procedurally based pricing as currently exists is administered by a complex network of largely decentralized bodies. As a consequence, patient access is limited and health-economic savings are not realized. This article explores some of the European market entry barriers, with a focus on reimbursement challenges, and highlights some collaborative proposals to address such.

Serglycin is a major proteoglycan of hematopoietic cells. It is thought to play a role in the packaging of granule proteins in human neutrophil granulocytes. The presence of serglycin in myeloid cells has been demonstrated only at the transcriptional level. We generated a polyclonal antibody against recombinant human serglycin. Here, we show the localization of serglycin in humans during neutrophil differentiation. Immunocytochemistry revealed serglycin immunoreactivity in the Golgi area of promyelocytes (PM) and myelocytes (MC), as well as in a few band cells and mature neutrophil granulocytes. Granular staining was detected near the Golgi apparatus in some of the PM, and the major part of the cytoplasm was negative. Immunoelectron microscopy showed serglycin immunoreactivity located to the Golgi apparatus and a few immature granules of PM and MC. The decreasing level of serglycin protein during myeloid differentiation coincided with a decrease of mRNA expression, as evaluated by Northern blotting. Subcellular fractions of neutrophil granulocytes were obtained. Serglycin immunoreactivity was detected in the fraction containing Golgi apparatus, plasma membrane, and secretory vesicles by Western blotting and enzyme-linked immunosorbent assay. Serglycin was not detected in subcellular fractions containing primary, secondary, or tertiary granules. Together, these findings indicate that serglycin is located to the Golgi apparatus and a few immature granules during neutrophil differentiation. This is consistent with a function for serglycin in formation of granules in neutrophil granulocytes. Our findings contrast the view that native serglycin is present in mature granules and plays a role in packaging and regulating the activity of proteolytic enzymes there.