Andrzej Loesch

Antibodies to human serum amyloid P component eliminate visceral amyloid deposits

Accumulation of amyloid fibrils in the viscera and connective tissues causes systemic amyloidosis, which is responsible for about one in a thousand deaths in developed countries. Localized amyloid can also have serious consequences; for example, cerebral amyloid angiopathy is an important cause of haemorrhagic stroke. The clinical presentations of amyloidosis are extremely diverse and the diagnosis is rarely made before significant organ damage is present. There is therefore a major unmet need for therapy that safely promotes the clearance of established amyloid deposits. Over 20 different amyloid fibril proteins are responsible for different forms of clinically significant amyloidosis and treatments that substantially reduce the abundance of the respective amyloid fibril precursor proteins can arrest amyloid accumulation. Unfortunately, control of fibril-protein production is not possible in some forms of amyloidosis and in others it is often slow and hazardous. There is no therapy that directly targets amyloid deposits for enhanced clearance. However, all amyloid deposits contain the normal, non-fibrillar plasma glycoprotein, serum amyloid P component (SAP). Here we show that administration of anti-human-SAP antibodies to mice with amyloid deposits containing human SAP triggers a potent, complement-dependent, macrophage-derived giant cell reaction that swiftly removes massive visceral amyloid deposits without adverse effects. Anti-SAP-antibody treatment is clinically feasible because circulating human SAP can be depleted in patients by the bis-d-proline compound CPHPC, thereby enabling injected anti-SAP antibodies to reach residual SAP in the amyloid deposits. The unprecedented capacity of this novel combined therapy to eliminate amyloid deposits should be applicable to all forms of systemic and local amyloidosis.

Electron-immunocytochemical localization of P2X1 receptors in the rat cerebellum.

Abstract The distribution of the P2X1 subtype of purinoceptors associated with the extracellular activities of ATP was studied in the rat cerebellum at the electron-microscope level. Receptors were labelled with peroxidase-antiperoxidase and the avidin-biotin-peroxidase complex for immunocytochemistry. Immunoreactivity to P2X1 receptors was localized in subpopulations of synapses between varicosities of parallel fibres of granule cells and dendritic spines of Purkinje cells. Unlabelled varicosities of parallel fibres formed asymmetric synapses with labelled dendritic spines, whereas labelled varicosities of parallel fibres formed asymmetric synapses with unlabelled dendritic spines. P2X1 immunoreactivity was also localized in some astrocyte processes. The functional significance of these findings is discussed.

Localization of vasopressin, serotonin and angiotensin II in endothelial cells of the renal and mesenteric arteries of the rat.

SummaryThe localization of vasopressin, serotonin and angiotensin II in the endothelial cells of renal and mesenteric arteries was investigated using the pre-embedding peroxidase-antiperoxidase technique for electron microscopy. Vasopressin-and serotonin-positive endothelial cells were present in both renal and mesenteric arteries while angiotensin II-positive cells were observed in the mesenteric artery exclusively. Both arteries showed less than 10% immunoreactive cells. The lack of angiotensin II in the endothelial cells of the renal artery suggests that there may be subtle physiological differences between the renal and mesenteric arteries with respect to the local control of blood flow.

Ultrastructural localisation of ATP-gated P2X2 receptor immunoreactivity in the rat hypothalamo-neurohypophysial system

The distribution of the P2X2 subtype of the purine receptor associated with the extracellular signalling activities of ATP was studied in the rat hypothalamo-neurohypophysial system at the electron microscope level. Receptors were labelled with ExtrAvidin-horseradish peroxidase preembedding immunocytochemistry using a polyclonal antibody against a fragment of an intracellular domain of the receptor. Immunoreactivity to P2X2 receptors was localised in: (i) paraventricular and supraoptic nuclei—in subpopulations of endocrine neurones, neurosecretory and non-neurosecretory axons and dendrites; and (ii) the neurohypophysis—in pituicytes and subpopulation of neurosecretory axons. In both the hypothalamic nuclei examined, labelled asymmetric axo-dendritic synapses were commonly observed. These synapses involved either P2X2-labelled axon terminals (synaptic buttons) and unlabelled dendrites or labelled dendrites and unlabelled axon terminals. Axo-somatic synapses established by P2X2-positive axons on P2X2-positive endocrine cell bodies as well as on P2X2-negative somata were also observed. The functional significance of these findings is discussed.

Hypothesis: A potential role for the vasa vasorum in the maintenance of vein graft patency

Autologous saphenous vein is the most commonly used conduit for coronary artery bypass surgery with more than 50% grafts occluding within 10 years. In conventional preparation the vein undergoes considerable surgical trauma with damage to the outer layers during harvesting. Within these regions are situated the vasa vasorum and small vessels providing oxygen and nutrients to the vessel wall. Certain vasa vasorum terminate in the vessel lumen where it is suggested that they have a physiological role. Preservation of the vasa vasorum of saphenous veins used as bypass conduits may play an important role in the maintenance of graft patency.

Loss of phosphatidylinositol 4-kinase 2α activity causes late onset degeneration of spinal cord axons

Phosphoinositide (PI) lipids are intracellular membrane signaling intermediates and effectors produced by localized PI kinase and phosphatase activities. Although many signaling roles of PI kinases have been identified in cultured cell lines, transgenic animal studies have produced unexpected insight into the in vivo functions of specific PI 3- and 5-kinases, but no mammalian PI 4-kinase (PI4K) knockout has previously been reported. Prior studies using cultured cells implicated the PI4K2α isozyme in diverse functions, including receptor signaling, ion channel regulation, endosomal trafficking, and regulated secretion. We now show that despite these important functions, mice lacking PI4K2α kinase activity initially appear normal. However, adult Pi4k2aGT/GT animals develop a progressive neurological disease characterized by tremor, limb weakness, urinary incontinence, and premature mortality. Histological analysis of aged Pi4k2aGT/GT animals revealed lipofuscin-like deposition and gliosis in the cerebellum, and loss of Purkinje cells. Peripheral nerves are essentially normal, but massive axonal degeneration was found in the spinal cord in both ascending and descending tracts. These results reveal a previously undescribed role for aberrant PI signaling in neurological disease that resembles autosomal recessive hereditary spastic paraplegia.

P2X4 and P2X6 receptors associate with VE-cadherin in human endothelial cells

Abstract. We investigated the expression of P2X4 and P2X6 receptors on human umbilical vein endothelial cells (HUVECs) and found that both P2X receptor subtypes on plasma membranes are largely restricted to areas of cell-cell contact. Co-labelling experiments at the confocal and electron microscopy levels revealed that P2X4 and P2X6 receptors are strongly co-localised with the cell adhesion molecule VE-cadherin. The P2X4 and P2X6 receptors on plasma membranes at cellular junctions are rapidly (within 5 min) internalised specifically after decreasing extracellular [Ca2+]. Disruption of microfilaments, microtubules and integrin-mediated adhesion or stimulation of P2 receptors with ATP did not alter P2X4 and P2X6 receptor expression on HUVEC plasma membranes. Membraneous P2X4 and P2X6 receptors resisted extraction with Triton-X 100, whereas cytoplasmic P2X receptors were Triton-X 100 soluble. P2X4 receptors, but not P2X6 receptors, could be co-immunoprecipitated with VE-cadherin and vice versa. We conclude that P2X4 and P2X6 receptors are associated with VE-cadherin at HUVEC adherens junctions.

Increased Shear Stress Leads to Differential Release of Endothelin and ATP from Isolated Endothelial Cells from 4- and 12-Month-Old Male Rabbit Aorta

Freshly isolated thoracic aortic endothelial cells from 4- and 12-month-old male rabbits were exposed to two periods of increased flow rate and the levels of ATP, endothelin (ET) and arginine vasopressin (AVP) released into the perfusate were measured. The rapid, significant release of ATP and ET with increased flow from cells isolated from 12-month-old rabbits was not seen when cells isolated from 4-month-old rabbits were exposed to the same conditions. Increased flow did not stimulate AVP release from cells from either age group. Basal release with low flow also differed in endothelial cells from 4-month-old compared to 12-month-old rabbits; thus in 4-month preparations basal release of AVP was greater, that of ET was smaller, while that of ATP was unchanged relative to 12-month preparations. Immunohistochemical studies showed that almost half as many endothelial cells isolated from 4-month-old rabbits were immunoreactive to ET and AVP compared to cells isolated from 12-month-old animals. It is suggested that the endothelial contribution to the control of vascular tone in rabbit thoracic aorta changes during ageing.

Colocalization of endothelin, vasopressin and serotonin in cultured endothelial cells of rabbit aorta

The localization and colocalization of endothelin-1, arginine-vasopressin and serotonin in the endothelial cells of rabbit aorta in primary culture were investigated by preembedding and postembedding immunocytochemistry. These three substances were localized in the same population of cells, where they appeared in high proportions (greater than 60%). These findings indicate: 1) that the cell population is heterogeneous, and 2) that these substances are colocalized in some of the cultured endothelial cells. Double labeling in single cells has demonstrated the simultaneous presence of 1) endothelin and vasopressin, 2) vasopressin and serotonin, and 3) endothelin and serotonin. The immunolabeling dominated in the cytoplasmic matrix.

Immunoreactivity to P2X6 receptors in the rat hypothalamo-neurohypophysial system: an ultrastructural study with extravidin and colloidal gold-silver labelling

The distribution of the purine receptor P2X(6) subtype was studied in the rat hypothalamo-neurohypophysial system at the electron microscope level. Receptors were visualised with ExtrAvidin peroxidase conjugate and immunogold-silver pre-embedding immunocytochemistry using a polyclonal antibody against an intracellular domain of the receptor. Application of ExtrAvidin labelling revealed P2X(6) receptors in subpopulations of: (i) neurosecretory cell bodies, neurosecretory and non-neurosecretory axons and dendrites of neurones in the paraventricular and supraoptic nuclei; and (ii) pituicytes and neurosecretory axons of the neurohypophysis. Some of the neurosecretory granules observed in the supraoptic and paraventricular nuclei neurone cell bodies, dendrites and axons as well as those in neurohypophysial axons were also positive for the P2X(6) receptors. In the paraventricular nucleus, some axons and dendrites of non-neurosecretory neurones positive for P2X(6) receptors formed synapses between themselves. Using the immunogold-silver method, the electron-dense particles labelling P2X(6) receptors were found in neurosecretory cell bodies of the supraoptic and paraventricular nuclei, in relation to the cytoplasm, endoplasmic reticulum, Golgi complex and neurosecretory granules. The particles indicative of P2X(6) receptors were also located in neurosecretory and non-neurosecretory axons including axonal buttons making synapses with P2X(6)-negative dendrites. In the neurohypophysis, the electron-dense particles were localised in a subpopulation of pituicytes and neurosecretory axons. In neurohypophysial axons, particles were at times seen over the membrane of some neurosecretory granules (immunogold label) or microvesicles (immunoperoxidase label). We speculate that the P2X(6) receptors at the neurohypophysial level may be implicated not only in hormone release from the axon terminals, but also in membrane recycling of the granular vesicles and microvesicles.