Complement deficiencies limit CD20 monoclonal antibody treatment efficacy in CLL.

Monoclonal antibodies (MAbs) form a central part of chronic lymphocytic leukaemia (CLL) treatment. We therefore evaluated whether complement defects in CLL patients reduced the induction of complement-dependent cytotoxicity (CDC) by using anti-CD20 MAbs rituximab (RTX) and ofatumumab (OFA). Ofatumumab elicited higher CDC levels than RTX in all CLL samples examined, particularly in poor prognosis cohorts (11q- and 17p-). Serum sample analyses revealed that 38.1% of patients were deficient in one or more complement components, correlating with reduced CDC responses. Although a proportion of patients with deficient complement levels initially induced high levels of CDC, on secondary challenge CDC activity in sera was significantly reduced, compared with that in normal human serum (NHS; P<0.01; n=52). In addition, a high CLL cell number contributed to rapid complement exhaustion. Supplementing CLL serum with NHS or individual complement components, particularly C2, restored CDC on secondary challenge to NHS levels (P<0.0001; n=9). In vivo studies revealed that complement components were exhausted in CLL patient sera post RTX treatment, correlating with an inability to elicit CDC. Supplementing MAb treatment with fresh-frozen plasma may therefore maintain CDC levels in CLL patients with a complement deficiency or high white blood cell count. This study has important implications for CLL patients receiving anti-CD20 MAb therapy.

Electrogenic L-histidine transport in neutral and basic amino acid transporter (NBAT)-expressing Xenopus laevis oocytes. Evidence for two functionally distinct transport mechanisms induced by NBAT expression.

We have investigated the neutral and basic amino acid transporter (NBAT)-induced transport of L-histidine in Xenopus laevis oocytes. Transport of L-histidine (pH 7.5) was electrogenic and Na+-dependent with a 14-fold increase in L-histidine- (1 mM) evoked current (I(His) = -14.7 +/- 1.5 nA) in NBAT-expressing oocytes compared with native (water-injected or uninjected) oocytes (-1.0 +/- 0.2 nA); the Na+-dependent histidine transport showed a stoichiometry of 1:1 (histidine:sodium). I(His) was stereospecific at pH 7.5 and saturable in both NaCl and tetramethylammonium chloride media. L-Histidine (1 mM) at pH 8.5, at which histidine is uncharged, evoked an Na+-independent outward current (11 +/- 1.2 nA) in NBAT-expressing oocytes. The total inward 0.1 mM I(His) increased from -9 +/- 0.8 nA at pH 7.5 to -19 +/- 2.6 nA at pH 6.5, at which histidine is predominantly cationic. The increase in I(His) from pH 7.5 to 6.5 was found to be almost entirely due to the Na+-independent component. At pH 7.5, L-histidine weakly inhibited the Na+-independent L-arginine uptake; however, this inhibition was much stronger (>90%) at pH 6.5. L-Histidine transport, at pH 7.5, is stimulated by NBAT expression, but unlike L-phenylalanine or L-arginine transport, L-histidine transport is Na+-dependent and stereoselective. The induction of Na+-dependent L-histidine transport in NBAT-expressing oocytes provides new evidence that NBAT stimulates functionally distinct amino acid transporters including Na+-dependent L-histidine and Na+-independent L-arginine and L-phenylalanine transporters. The parallel induction of two different mechanisms argues that NBAT is not an amino acid transporter itself but, instead, is a transport-activating protein for a range of amino acid translocases.

Angiographic features and outcome of questionable recurrent choroidal neovascularization.

PURPOSE: We sought to identify specific fluorescein angiographic patterns that may have led to the diagnosis of questionable recurrent choroidal neovascularization. We evaluated follow-up information to determine whether any specific angiographic patterns could be used to identify patients at high risk for definite recurrence. METHODS: We identified fluorescein angiograms graded as questionable for recurrent choroidal neovascularization that were taken from a previous prospective study involving 401 consecutive follow-up visits of patients treated with photocoagulation for choroidal neovascularization. We reviewed these angiograms to identify specific angiographic patterns that might have led to the classification of questionable recurrent choroidal neovascularization. Angiograms from visits subsequent to a questionable recurrence were reviewed to determine which patterns, if any, were associated with an increased risk for a definite recurrence to develop later. RESULTS: Forty-four eyes (44 patients) with questionable recurrences (of which 40 had at least four months of follow-up) were categorized into six angiographic patterns. The three most common patterns included the following: (1) focal staining along the edge of the laser lesion (20 cases, 15 subsequently recurred); (2) blocked fluorescence from subretinal hemorrhage not documented at the previous visit (eight cases, five subsequently recurred); (3) speckled hyperfluorescence noted beyond the edge of the laser lesion (eight cases, six subsequently recurred). CONCLUSIONS: Questionable recurrent choroidal neovascularization may be identified by specific angiographic patterns. Focal staining along the edge of the laser lesion and speckled hyperfluorescence were the patterns that were most likely to progress to definite recurrence.

Expression of the liver-type glucose transporter (GLUT2) in 3T3-L1 adipocytes: analysis of the effects of insulin on subcellular distribution.

We have expressed the liver-type facilitative glucose transporter, GLUT2, in the insulin-sensitive 3T3-L1 adipocyte clonal cell line in an effort to address the importance of transporter isoform and cellular environment on the ability of insulin to mediate glucose-transporter translocation. Analysis of non-differentiated fibroblastic cell clones transfected with the GLUT2 cDNA identified the presence of this isoform in several independent clones. These clones exhibited increased deoxyglucose and fructose transport rates compared with control cells. Upon differentiation, the fibroblastic clones selected for study achieved > 95% phenotypic conversion into adipocytes. Expression of the GLUT2 protein was maintained throughout the differentiation protocol. Subcellular fractionation revealed that in response to insulin, unlike the native GLUT4, GLUT2 protein did not undergo significant translocation to the plasma membrane; furthermore, the subcellular distribution of the expressed GLUT2 was quite distinct from that of the endogenous GLUT4. 3T3-L1 adipocytes expressing GLUT2 only exhibited a 2-fold increase in insulin-stimulated fructose uptake, further suggesting that GLUT2 does not undergo insulin-stimulated translocation.

Immunological analysis of glucose transporters expressed in different regions of the rat brain and central nervous system.

Glucose is the major energy source for brain tissue. It is now well established that glucose crosses the blood-brain barrier by facilitated diffusion mediated by the erythrocyte-type (GLUT 1) glucose transporter. Northern blot analysis has indicated that brain tissue expresses two different glucose transporters, the erythrocyte-type transporter and the brain-type transporter (GLUT 3). Here we use a panel of antibodies specific for four glucose transporters to examine the distribution of these transporters in various regions of rat brain and central nervous system. We show that GLUT 1 and GLUT 3 are expressed in virtually all regions of the brain examined, but that the relative levels of the proteins differ. We also demonstrate that GLUT 2 appears to be widely expressed in all brain regions, but at apparently low levels. In addition, GLUT 4 is expressed in the pituitary, the hypothalamus and the medulla. These results are discussed in the light of potential glucose-regulated processes in the brain.

Analysis of the glucose transporter content of islet cell lines: implications for glucose-stimulated insulin release.

Glucose transport across the plasma membrane of mammalian cells is mediated by a family of homologous proteins. Each glucose transporter isoform has a specific tissue distribution which relates to that tissue's demand for glucose. The beta-cells of pancreatic islets are known to express a distinct glucose transporter isoform, termed GLUT 2, which has a high Km for glucose. In this study, we examined the glucose transporter content of normal rat islets and three beta cell lines, beta-TC, HIT and RIN cells. We show that at the protein level, GLUT 2 is the only detectable transporter isoform in normal islets, and that all three cell lines also express detectable GLUT 2. In contrast, all three cell lines expressed high levels of GLUT 1, but this isoform was not detected in normal islets. Neither the native islets nor any of the cell lines expressed GLUT 3. The insulin-responsive glucose transporter GLUT 4 was detected at very low levels in beta-TC cells; to our knowledge, this is the only non-muscle or adipose cell line which expresses this isoform. We propose that the elevated level of GLUT 1 expression, together with a reduced expression of the high Km transporter GLUT 2, may account for the characteristic aberrant patterns of glucose-stimulated insulin release in cell lines derived from beta-cells.

Expression of the brain-type glucose transporter is restricted to brain and neuronal cells in mice.

Northern blot analysis of human tissues has demonstrated the expression of the brain-type glucose transporter isoform (GLUT 3) in liver, muscle and fat, raising the possibility that this transporter isoform may play a role in the regulation of glucose disposal in these tissues in response to insulin. We have raised an anti-peptide antibody against the C-terminal 13 amino acids of the murine homologue of this transporter isoform, and determined its tissue distribution in mouse tissues and murine-derived cell lines. The antibodies recognise a glycoprotein of about 50 kilodaltons, expressed at high levels in murine brain. In contrast to human tissues, the expression of GLUT 3 in mice is restricted to the brain, and no immunoreactivity was observed in either liver, fat or muscle membranes, or in murine 3T3-L1 fibroblasts or adipocytes. In contrast, high levels of expression of this isoform were observed in the NG 108 neuroblastoma x glioma cell line, a hybrid cell derived from rat glioma and mouse neuroblastoma cells. Taken together, these data suggest that the expression of GLUT 3 in rodents is restricted to non-insulin responsive neuronal cells and hence it is likely that the factors regulating the expression of this transporter in rodents differ to those in humans.

Experimental determination and mathematical model of the transient incorporation of cholesterol in the arterial wall.

Experimental data of the radial incorporation of labeled cholesterol [14C-4] into the artery wall is regressed against a mathematical model that predicts macromolecular transport in this biological system. Data is obtained using excised canine carotid arteries that are perfused in vitro under pulsatile hemodynamic conditions for 2 hr. Vessels are exposed to either normotensive hemodynamics, hypertensive hemodynamics, or simulations in which the rate of flow or vessel compliance is deliberately altered. Several arteries are studied under normotensive conditions following balloon catheter deendothelialization. Transmural concentration profiles of [14C-4] activity are determined by microcryotomy of longitudinal sections of perfused vessels. Nonlinear Marquardt regression on 12 experimental cases yields parameter estimates of effective diffusivity, D and solute filtration velocity, V. Results of this experimental investigation support our hypothesis that hemodynamics and the endothelial lining influence wall flux in intact vessels. Exposure to altered (vs normotensive) hemodynamics is associated with increased incorporation of labeled cholesterol. A similar observation is made for deendothelialized vessels (e.g. a greater accumulation of label and a rise in convective flux). Based upon our companion measurements of vessel wall forces and endothelial cellular morphology accompanying hemodynamic simulations, we suggest that hemodynamically induced alterations to endothelial structures lead to the increased permeability, convection and incorporation that we observe in this work.

Regression of cytomegalovirus retinitis with zidovudine. A clinicopathologic correlation.

A 55-year-old man with the acquired immunodeficiency syndrome had cytomegalovirus (CMV) retinitis develop in his left eye. The patient declined treatment with ganciclovir but elected to be treated with zidovudine. Two months later his CMV retinitis regressed, and it remained inactive for a total of 9 months. The most likely explanation was zidovudine-mediated improvement in the patient's immune function with subsequent suppression of the CMV retinitis in the left eye. The patient ultimately and preterminally had CMV retinitis develop in his right eye. He subsequently died and his eyes were examined at autopsy. Active CMV retinitis lesions were noted in the right eye while inactive scars were noted in the left eye. Immunoperoxidase staining and in situ hybridization studies demonstrated the presence of CMV in the right eye but not in the left eye. While ganciclovir remains the treatment of choice for CMV retinitis, zidovudine treatment may have a beneficial effect on CMV retinitis in some patients.

Ocular manifestations in hereditary hemorrhagic telangiectasia (Rendu-Osler-Weber disease).

Although hereditary hemorrhagic telangiectasia is a cause of blood tears from the conjunctival telangiectasias, visual loss from intraocular vascular malformations is a rare complication. We examined 20 patients with hereditary hemorrhagic telangiectasia to determine the prevalence of ocular abnormalities in this disease. Seven patients (35%) had conjunctival telangiectasias and two (10%) had retinal vascular malformations.

Experimental determination of wall shear rate in canine carotid arteries perfused in vitro.

The mathematical model of Hung (Tsai and Hung, 1984) is employed to determine the wall shear rate acting on canine carotid arteries perfused in vitro. Model equations for pulsatile flow in a deformable vessel are coupled with experimental data of dynamic pressure drop, flow rate, vessel radius and radial wall motion. Derived quantities, e.g. velocity profiles and wall shear, are obtained for vessels exposed to 'normotensive' hemodynamics, 'hypertension' simulations and perfusions in which the compliance of the vessel wall is deliberately altered. Our results indicate that wall shear varies markedly as a function of the hemodynamic environment. The effects of vessel radius vs flow rate on the development of wall shear are also demonstrated. It is found that convective processes correlate with the magnitude of wall shear in the 'hypertension' simulations. The present findings and complementary published data may explain, at least in part, the variations in vessel wall transport and endothelial cell biology we observe as a function of the hemodynamic environment. For example we have documented that the exposure of canine carotids to 'hypertensive' (vs 'normotensive') hemodynamics is associated with an increased flux of lipoproteins (LDL) into the intima and luminal media. Alternations in wall compliance, on the other hand, profoundly influence endothelial shape, orientation and cytoskeletal array.

Biomechanics of the arterial wall under simulated flow conditions.

A perfusion apparatus is employed to reproduce quantifiable pulsatile hemodynamics within freshly excised canine carotid arteries. From measurements of pulsatile intraluminal and transmural pressure and the dynamic radial motion of the vessel wall, calculations are made of the vascular incremental modulus of elasticity and hoop, axial, and radial wall stresses. The results of this investigation suggest that an increase in transmural pressure from 120/80 to 240/120 mmHg produces a marked elevation in incremental modulus and arterial wall stress. These parameters are reduced when transmural pressure is lowered while maintaining intraluminal pressure at physiologic values.

Hemodynamics and the vascular endothelial cytoskeleton.

Although there is considerable evidence to suggest that hemodynamics play an important role in vascular disease processes, the exact mechanisms are unknown. With this in mind, we have designed a pulsatile perfusion apparatus which reproducibly delivers pulsatile hemodynamics upon freshly excised canine carotid arteries in vitro. Quantifiable simulations included normotension with normal or lowered flow rates (120/80 mmHg, 120 and 40 ml/min), normotension with lowered or elevated transmural pressures (40-170 mmHg), and elevated pulse pressure (120 and 80 mmHg) with normal (150 ml/min) or elevated rates of flow (300 and 270 ml/min). Arterial biomechanical stresses and cellular behaviors were characterized biochemically and morphologically under all these stimulations which continued for 2-24 h. We found that increased pulse pressure alone had little effect on the total amount of radiolabeled [4-14C]cholesterol present within the medial compartment. However, normotension when coupled with altered transmural pressure yielded a three- to fourfold increase. Combinations of increased pulse pressure and flow potentiated cholesterol uptake by a factor of 10 when compared with normotension control values. Simulations that enhanced carotid arterial cholesterol uptake also influenced the endothelial cytoskeletal array of actin. Stress fibers were not present within the carotid endothelial cells of either the sham controls or the normotension and increased pulse pressure (normal flow) simulations. Endothelial cells lining carotids exposed to elevations in flow or those present within vessels perfused as per simulation b above assembled stress fibers (x = 4 and 10 per cell, respectively) within the time course of these studies. When endothelial cells were subjected to hemodynamic conditions that potentiated maximally cholesterol transport, no diffuse or stress fiber staining could be seen, but the cortical array of actin was intact. These results suggest that those biomechanical stresses that alter endothelial permeability and intimal integrity may do so via cytoskeletal actin signaling.

Measurement in vitro of pulsatile arterial diameter using a helium-neon laser.

A noncontacting in vitro measurement of pulsatile arterial diameter using a scanning optical micrometer is described. The major component of this system is a He-Ne laser whose beam scans the pulsating artery to be measured. The laser micrometer was integrated into a pulsatile perfusion apparatus that imposed various hemodynamic conditions on excised canine vessels. The laser system reliably tracked the pulsating arterial diameter at a particular longitudinal site as well as at various increments in the presence of an experimentally created stenosis. The He-Ne laser measured the radial motion of canine arteries and various vascular substitutes anastomosed in an end-to-end fashion. From these novel measurements, calculations were made of arterial compliance and bending stress, two biomechanical parameters that are implicated as potential causes of anastomotic intimal hyperplasia and graft failure. Although this device is inherently limited to in vitro use, it is a potentially useful instrument for vascular physiology and biophysics.

Effect of variations in pressure and flow on the geometry of isolated canine carotid arteries.

A study is described in which the effects of hemodynamics on arterial geometry are investigated in vitro. A novel perfusion apparatus is employed to deliver pulsatile flow through excised canine carotid arteries under carefully controlled conditions. Data of perfused vessel diameter and arterial wall thickness are derived from the radial displacement of the pulsating vessel as measured using a scanning laser micrometer whose accuracy is determined to be 0.0125 mm (0.0005 in). The results of 30 perfusion experiments suggest that the hemodynamic variables of transmural pressure, pulse pressure and flow rate influence vessel size and radial strain. The physiologic implications of these findings are discussed.