Reversible Increase In Permeability Research Articles

Different effects of temperature and salinity on permeability reduction by fines migration in Berea sandstone

Hot water injection into geothermal aquifers is considered in order to store energy seasonally. Berea sandstone is often used as a reference formation to study mechanisms that affect permeability in reservoir sandstones. Both heating of the pore fluid and reduction of the pore fluid salinity can reduce permeability in Berea sandstone. These effects could be caused by mobilisation of fines by increasing the repulsive electrical double layer forces among sandstone grains and the fines. We investigated the reversibility and the dependence on flow velocity and flow direction of the permeability change by means of flow through experiments and examined thin sections of samples prior to and after tests. A permeability reduction at 20°C with decreasing salinity was not reversed by restoring the salinity, whereas a permeability reduction due to heating to 80°C was reversible by restoring the temperature to 20°C. A reversible permeability increase with increasing flow rate was observed at 80°C but not at 20°C. We observed no difference in the distribution of kaolinite clay minerals in thin sections of untested and tested samples. Dissolution of iron bearing carbonates and precipitation of iron hydroxides was observed but no effect on permeability was found. The experimental results suggest that different mechanisms are responsible for permeability reduction depending on temperature and salinity.

Neutrophils block permeability increases induced by oxygen glucose deprivation in a culture model of the human blood–brain barrier

Experimentally, oxygen glucose deprivation (OGD) has been widely used to mimic the environmental conditions present during cerebral ischemia–reperfusion (IR) injury. OGD is known to increase permeability across cultured cerebral endothelial cells, which models the effect of IR on permeability across the blood–brain barrier (BBB); however, studies have yet to be performed in a human model. The effect of neutrophils on the increase in BBB permeability associated with IR injury has yet to be modeled in vitro. To address these questions, the human brain endothelial cell line hCMEC/D3 was exposed to OGD with reoxygenation, and permeability was measured for a range of OGD exposure times (1–24 h). One hour of exposure to OGD induced a reversible increase in permeability, unassociated with cytotoxicity (assessed from lactate dehydrogenase release). However, 12–24 h OGD exposures induced sustained increases in permeability associated with cytotoxicity. The 1 h permeability increase was inhibited with the nitric oxide synthase inhibitors l-NAME (10 − 7 mol/l) and 1400W (10 − 7 mol/l). Neutrophils (5 × 10 6/ml) blocked the permeability increase associated with 1 h OGD, whether applied during or after OGD exposure. Permeability remained low if neutrophils were activated with leukotriene (Lt)B 4 (10 − 7 mol/l) or exposed to a transendothelial gradient of LtB 4, while neutrophil activation with phorbyl myristate acetate (4 × 10 − 8 mol/l) induced a small increase. Neutrophils had no effect on the permeability increase induced by 12 h OGD exposure. This study finds that OGD induces reversible increases in permeability linked to nitric oxide synthesis in a human culture model of the BBB and shows that neutrophils mitigate permeability increases in this context.

3625: Sonoporating cells for drug delivery: Effect of pulse bandwidth and duty-cycle

The combination of ultrasound and microbubbles is currently being exploited to enhance local drug delivery and uptake. This work investigates the effect of ultrasound parameters on sonoporation, the reversible increase in permeability of biological membranes. Suspensions of KHT-C cells were exposed to ultrasound of varying pressures, pulse lengths and duty cycles, in the presence of contrast agent microbubbles (Definity). Fluorescently labeled dextran (70 kDa) was used as a permeability marker. Flow cytometry and microscopy were used to quantify permeability and viability.

Augmentation of endothelial cell monolayer permeability by hyperthermia but not tumor necrosis factor: Evidence for disruption of vascular integrity via VE-cadherin down-regulation

Following isolated limb perfusion (ILP) with hyperthermia (H T), TNF and melphalan, there is immediate tumor softening secondary to augmentation of capillary leak in the tumor neovasculature. TNF can induce vascular permeability but is always used with HT during ILP and the contribution of the latter on permeability is not known. This study characterizes the effects of HT and TNF on vascular permeability in vitro. Permeability across confluent human umbilical vein endothelial cells exposed to HT (40 degrees C) with or without 0.1-1000 ng/ml TNF was assessed by quantitating flux of albumin bound Evan's Blue dye from the upper to lower chamber. Immunofluorescent staining for VE-cadherin and F-actin was performed after human umbilical vein endothelial cells (hUVECs) were exposed to these conditions. HT induced a significant and reversible increase in permeability compared to untreated hUVECs (p<0.001) whereas barrier function was not altered by TNF. Untreated hUVECs had uniform cell surface staining for VE-cadherin, the primary endothelial intercellular adhesion molecule, with colocalization of F-actin cytoskeletal elements. HT resulted in a marked decrease in VE-cadherin staining and contraction of F-actin at sites of endothelial cell-cell separation. These data demonstrate that under conditions relevant to those used in ILP, HT but not TNF contributes to a rapid and reversible change in endothelial cell permeability in association with a down regulation of VE-cadherin. These data support the use of HT in isolation perfusion and demonstrate a novel mechanism for alterations in microvascular permeability by HT.

An improved endothelial barrier model to investigate dengue haemorrhagic fever

A cell culture model suitable for studies of dengue haemorrhagic fever was developed, based on culture of primary human umbilical vein endothelial cells (HUVECs) on a permeable membrane. By electron microscopy, cultured HUVECs at day 11 resembled morphologically microvascular endothelium. Endothelial barrier function was assessed by measuring transendothelial flux of albumin. Instead of using a labelled tracer molecule, an enzyme-linked immunosorbent assay (ELISA) was developed to measure concentrations of native human albumin. The permeability characteristics of the HUVEC monolayer were found to be improved significantly (approximately 1 log reduction in permeability coefficient for albumin) by culturing HUVECs in human serum rather than fetal calf serum. Permeability coefficients for albumin in the range 1–4×10 −7 cm/s were achieved, which is an improvement on previous in vitro models of the endothelial barrier. Comparison of transendothelial flux of albumin and urea provided evidence of molecular sieving by the HUVEC monolayer. Moreover, tumour necrosis factor-α induced a dose-dependent, reversible increase in permeability of the HUVEC monolayer. This endothelial barrier model thus has many important characteristics that resembled human microvascular endothelium and is an improvement on the previous model proposed for studies of dengue haemorrhagic fever.

Dehydration response of sickle cells to sickling-induced Ca++ permeabilization

Interaction of hemoglobin S polymers with the red blood cell (RBC) membrane induces a reversible increase in permeability ("P(sickle)") to (at least) Na(+), K(+), Ca(2+), and Mg(2+). Resulting changes in [Ca(2+)] and [H(+)] in susceptible cells activate 2 transporters involved in sickle cell dehydration, the Ca(2+)-sensitive K(+) ("Gardos") channel (K(Ca)) and the acid- and volume-sensitive K:Cl cotransport. We investigated the distribution of P(sickle) expression among deoxygenated sickle cell anemia (SS) RBCs using new experimental designs in which the RBC Ca(2+) pumps were partially inhibited by vanadate, and the cells' dehydration rates were detected as progressive changes in the profiles of osmotic fragility curves and correlated with flow cytometric measurements. The results exposed marked variations in (sickling plus Ca(2+))-induced dehydration rates within populations of deoxygenated SS cells, with complex distributions, reflecting a broad heterogeneity of their P(sickle) values. P(sickle)-mediated dehydration was inhibited by clotrimazole, verifying the role of K(Ca), and also by elevated [Ca(2+)](o), above 2 mM. Very high P(sickle) values occurred with some SS discocytes, which had a wide initial density (osmotic resistance) distribution. Together with its previously shown stochastic nature, the irregular distribution of P(sickle) documented here in discocytes is consistent with a mechanism involving low-probability, reversible interactions between sickle polymers and membrane or cytoskeletal components, affecting only a fraction of the RBCs during each deoxygenation event and a small number of activated pathways per RBC. A higher participation of SS reticulocytes in P(sickle)-triggered dehydration suggests that they form these pathways more efficiently than discocytes despite their lower cell hemoglobin concentrations.

The role of guanylyl cyclases in the permeability response to inflammatory mediators in pial venular capillaries in the rat.

Inflammatory mediators have a role in the formation of cerebral oedema and there is evidence that cGMP is an important signal in vascular permeability increase. We have investigated the role and the source of cGMP in mediating the permeability response to acutely applied bradykinin and the histamine H(2) agonist dimaprit on single cerebral venular capillaries, by using the single vessel occlusion technique. We found that 8-bromo-cGMP applied acutely resulted in a small and reversible permeability increase with a log EC(50) -7.2 +/- 0.15 M. KT 5823, the inhibitor of cGMP-dependent protein kinase, abolished the permeability responses to both bradykinin and dimaprit, while zaprinast, an inhibitor of type 5 phosphodiesterase, potentiated the response to bradykinin. On the other hand, L-NMMA blocked the response to dimaprit, but not that to bradykinin. Inhibitors of soluble guanylyl cyclase, LY 85353 and methylene blue, also inhibited the permeability response to dimaprit, but not bradykinin. The permeability responses to the natriuretic peptides ANP and CNP were of similar magnitude to that of bradykinin with log EC(50) -10.0 +/- 0.33 M and -8.7 +/- 0.23 M, respectively. The natriuretic peptide receptor antagonist HS-142-1 blocked permeability responses to bradykinin as well as to ANP, and leukotriene D(4) blocked the responses to CNP and bradykinin, but not to dimaprit. In conclusion, the histamine H(2) receptor appears to signal via cGMP that is generated by a NO and soluble guanylyl cyclase, while bradykinin B(2) receptor also signals via cGMP but through particulate guanylyl cyclase.

Increased permeability of primary cultured brain microvessel endothelial cell monolayers following TNF-α exposure

TNF-α is a cytokine that produces increased permeability in the peripheral vasculature; however, little is known about the effects of TNF-α on the bloodbrain barrier (BBB). Using primary cultured bovine brain microvessel endothelial cells (BBMEC) as an in vitro model of the BBB, this study shows that TNF-α produces a reversible increase in the permeability of the brain microvessel endothelial cells. The BBMEC monolayers were pre-treated with 100 ng ml of TNF-α for periods ranging from 2 to 12 hours. Permeability was assessed using three molecular weight markers, fluorescein (376 MW), fluorescein-dextran (FDX-4400; 4400 MW), and FDX-70000 (MW 70000). The permeability of BBMEC monolayers to all three fluorescent markers was increased two-fold or greater in the TNF-α treatment group compared to control monolayers receiving no TNF-α. Significant changes in permeability were also observed with TNF-α concentrations as low as 1 ng ml . These results suggest that TNF-α acts directly on the brain microvessel endothelial cells in a dynamic manner to produce a reversible increase in permeability. Exposure of either the lumenal or ablumenal side of BBMEC monolayers to TNF-α resulted in similar increases in permeability to small macromolecules, e.g. fluorescein. However, when a higher molecular weight marker was used (e.g. FDX-3000), there was a greater response following lumenal exposure to TNF-α. Together, these studies demonstrate a reversible and time dependent increase in brain microvessel endothelial cell permeability following exposure to TNF-α. Such results appear to be due to TNF's direct interaction with the brain microvessel endothelial cell.

Electrical resistance and macromolecular permeability of retinal capillary endothelial cells in vitro.

This study was undertaken to examine the ability of retinal capillary endothelial cells to retain blood-retinal barrier properties in vitro. Second passage bovine retinal capillary endothelial cells were grown to confluence on polycarbonate filters in two chamber systems coated with laminin, fibronectin and type IV collagen. The electrical resistances, permeability of 3H-insulin and expression of blood-brain barrier related enzymes by retinal cells was observed and compared with bovine aortic endothelial cells and bovine fibroblasts. The electrical resistance of retinal cells rose over the first week of culture, peaking after 5-9 days in culture. In eleven separate experiments (n = 5 for each experiment) the average peak resistance of retinal endothelial cells ranged from 89.3-186.6 with a mean average of 129.0 ohm.cm2. In one of these experiments, the peak electrical resistance of retinal cells was 149.0 +/- 10.3 compared with 34.8 +/- 6.8 for aortic cells and 37.8 +/- 3.8 ohm.cm2 for fibroblasts. The permeability coefficients of inulin were: retinal cells 0.17 +/- 0.09, aortic cells 3.47 +/- 1.58 (p = 0.015), fibroblasts 3.93 +/- 0.78 (p = 0.002) x 10(-6) cm/sec. Retinal cells expressed significantly higher activities of gamma-glutamyl transpeptidase and alkaline phosphatase than the other cell types. Treatment of the monolayers with the calcium ionophore, A23187, resulted in a reversible increase in permeability as has been described for peripheral vascular endothelium. We conclude that BRCEC retain at least some of their specialised barrier properties in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Electroporation of the photosynthetic membrane: structural changes in protein and lipid-protein domains

A biological membrane undergoes a reversible permeability increase through structural changes in the lipid domain when exposed to high external electric fields. The present study shows the occurrence of electric field-induced changes in the conductance of the proton channel of the H(+)-ATPase as well as electric field-induced structural changes in the lipid-protein domain of photosystem (PS) II in the photosynthetic membrane. The study was carried out by analyzing the electric field-stimulated delayed luminescence (EPL), which originates from charge recombination in the protein complexes of PS I and II of photosynthetic vesicles. We established that a small fraction of the total electric field-induced conductance change was abolished by N,N'-dicyclohexylcarbodiimide (DCCD), an inhibitor of the H(+)-ATPase. This reversible electric field-induced conductance change has characteristics of a small channel and possesses a lifetime < or = 1 ms. To detect electric field-induced changes in the lipid-protein domains of PS II, we examined the effects of phospholipase A2 (PLA2) on EPL. Higher values of EPL were observed from vesicles that were exposed in the presence of PLA2 to an electroporating electric field than to a nonelectroporating electric field. The effect of the electroporating field was a long-lived one, lasting for a period > or = 2 min. This effect was attributed to long-lived electric field-induced structural changes in the lipid-protein domains of PS II.

Superoxide dismutase as an inhibitor of postischemic microvascular permeability increase in the hamster

The purpose was to elucidate the involvement od superoxide radical (O 2 −·) in the postischemic increase in the vascular permeability in the hamster cheek pouch. Cheek pouches of anesthetized hamsters were everted, prepared for intravital microscopy, and superfused with a bicarbonate buffered saline solution. Local ischemia for 30 min was obtained using a cuff placed around the proximal part of the cheek pouch. The vascular permeability in the postcapillary venules was quantified as leakage of intravenously injected fluorescein labeled dextran (FITC-dextran, Mw 150 000), using intravital microscopy and fluorimetry. There was a significant and reversible permeability increase after the reperfusion started. In the first series of experiments, combined intravenous infusion and topical application of human recombinant extracellular superoxide dismutase C (EC-SOD C) reduced the postischemic permeability response by 80%. Bovine CuZn-SOD given in exactly the same way reduced the response by 60%. In the second series of experiments, inactivated EC-SOD C was given to the control animals and active EC-SOD C was given to the treated animals. The topical treatment was excluded. Only active EC-SOD C reduced significantly the postischemic permeability increase when present during the ischemic period. Treatment with mannitol (i.v.) did not alter the postischemic response. Since active EC-SOD C and CuZn-SOD but not inactivated EC-SOD C effectively inhibited the response, we suggest that the superoxide anion is involved in the mediation of the postischemic permeability increase in the hamster.

Terbutaline and budesonide as inhibitors of postischaemic permeability increase.

A temporary ischaemia with total circulatory arrest of the hamster cheek pouch was obtained by clamping the neck of the everted cheek pouch. The macromolecular permeability increase in postcapillary venules was quantified as the leakage of fluorescein-labelled dextran using intravital microscopy and a fluorometer simultaneously. At reperfusion after 30 min ischaemia, there was a significant and reversible permeability increase. This response could be totally prevented by topical administration of either terbutaline, a selective beta 2-receptor agonist, or budesonide, a glucocorticoid, but it was not significantly impeded by the antihistamine mepyramine. The study shows that, in conformity with the situation in inflammation, the postischaemic permeability increase at reperfusion after ischaemia can be blocked by either beta 2-stimulation or glucocorticoids. Furthermore, it indicates that histamine, a common inflammatory mediator, is not responsible for the postischaemic permeability increase.

A calcium-dependent reversible permeability increase in microvessels in frog brain, induced by serotonin.

The effect of serotonin on brain microvascular permeability was studied by measurement of changes in the electrical resistance of the venular vascular wall, induced by this substance. Intravenous administration of serotonin decreased the electrical resistance in a dose-dependent manner with Kd congruent to 8.2 microM. The maximal decrease in electrical resistance was about 33%. The electrical resistance fell within seconds following the application and returned to the control value after 1-5 min. Serotonin applied to the outside of the brain vessels had no effect on electrical resistance. Pre-treatment with the 5-HT2 receptor antagonist Ketanserin blocked the serotonin response completely. The serotonin response was strongly inhibited by pre-treatment with the calcium-entry blocker verapamil (Isoptin). The findings demonstrate that serotonin reversibly increases blood-brain barrier permeability. The effect is mediated via 5-HT2 receptors located at the luminal surface of the cerebrovascular endothelium and is dependent on mobilization of extracellular Ca2+.

Antidepressants alter cerebrovascular permeability and metabolic rate in primates.

External detection of the annihilation radiation produced by water labeled with oxygen-15 was used to measure cerebrovascular permeability and cerebral blood flow in six rhesus monkeys. Use of oxygen-15 also permitted assessment of cerebral metabolic rate in two of the monkeys. Amitriptyline produced a dose-dependent, reversible increase in permeability at plasma drug concentrations which are therapeutic for depressed patients. At the same concentrations the drug also produced a 20 to 30 percent reduction in cerebral metabolic rate. At higher doses normal autoregulation of cerebral blood flow was suspended, but responsivity to arterial carbon dioxide was normal.

363 - Development of drug carrier systems: Electrical field induced effects in cell membranes

Mouse thymocytes and erythrocytes are loaded electrically with drugs and dyes in isotonic solution. The loaded cells are used for targeting the drugs to specific sites in the organism in order to achieve a controlled drug release in time and space. Erythrocytes are directed to the liver by changing the volume and the shape. Using erythrocytes as drug carrier systems, methotrexate, the most widely studied agent in chemotherapeutic cancer treatment, could be directed exclusively to the liver. Directing to other organs is obtained by either using electrically fused cells or by loading cells with magnetic particles (about 10 nm in diameter) and the drug simultaneously and by guiding the cells to any selected site of the organism. The field technique used for the loading of the cells is based on the electrical breakdown of the cell membrane which is observed when cell suspensions are subjected to external field pulses of 2 to 20 kV/cm for short time intervals (ns to ms). When an apparent membrane potential of about 1 V (pulse length in the range of μs) is reached in response to the external field the membrane breaks down reversibly. The breakdown of the membrane is associated with a remarkable and reversible permeability increase of the cell membrane. The increase in permeability depends on the strength and the duration of the field pulse. The duration of the high conductance state of the cell membrane induced by the electric field is sufficiently long to entrap large quantities of drugs or dyes inside the cells. Electron-micrographs of thymocytes subjected to field pulses of various strengths and durations show that the pulse length is a critical factor for the reversibility of the field induced effects in the cell. When loading thymocytes the field pulse length has to be less than 1 μs to avoid irreversible changes in the ultrastructure of the cell. On the other hand, erythrocytes can be subjected to field pulses of 40 μs duration without any irreversible changes in the membrane structure. The life-span of the loaded erythrocytes in the blood circulation can be considerably prolonged if loss of haemoglobin and intracellular enzymes is avoided during field application and the resealing process. The loss of intracellular proteins can be minimized if the erythrocytes are immobilized in a polymeric network of calcium-alginate during the field application. The matrix is permeable to low molecular weight compounds but impermeable towards larger molecules such as proteins. Chelation of the Ca 2+-ions in the alginate network by addition of sodium citrate leads to the release of the loaded erythrocytes from the calcium-alginate matrix.

Protein synthesis in transformed 3T3 cells permeabilized by exogenous ATP.

Exogenous ATP has been shown to cause a rapid and reversible increase in permeability in transformed 3T3 cells (3T6 and SV3T3) but not in untransformed 3T3 cells. The cells remain viable, but lose intracellular acid-soluble pools. Treatment of transformed cells with ATP greatly reduces incorporation of 14C-leucine into protein, which is restored by the incubation of the cells with Dulbecco's modified Eagle's medium or by the external additions of certain ions and energy sources. tRNA is not required for the restoration of protein synthesis. In the permeabilized cells the energy for protein synthesis can be provided by glycolysis, oxidative phosphorylation, or direct addition of ATP. These studies demonstrate the usefulness of this method for studying the control of metabolism and macromolecular synthesis in monolayer cultures of transformed mammalian cells.

Erythrocytes and lymphocytes as drug carrier systems: techniques for entrapment of drugs in living cells.

Mouse thymocytes and erythrocytes are loaded electrically with drugs in isotonic solution. The loaded cells are used for targeting the drugs to specific sites in the organism in order to achieve a controlled drug release in time and space. The field technique used for the loading of the cells is based on the dielectric breakdown of the cell membrane which is observed when cell suspensions are subjected to external field pulses of 2-20 kV/cm for short time intervals (ns to microseconds). When an apparent membrane potential of about 1 V is reached in response to the external field, the membrane breaks down reversibly. The breakdown of the membrane is associated with a remarkable and reversible permeability increase of the cell membrane. The increase in permeability depends on the strength and the duration of the field pulse.

Development of drug carrier systems: Electrical field induced effects in cell membranes

Abstract Mouse thymocytes and erythrocytes are loaded electrically with drugs and dyes in isotonic solution. The loaded cells are used for targeting the drugs to specific sites in the organism in order to achieve a controlled drug release in time and space. Erythrocytes are directed to the liver by changing the volume and the shape. Using erythrocytes as drug carrier systems, methotrexate, the most widely studied agent in chemotherapeutic cancer treatment, could be directed exclusively to the liver. Directing to other organs is obtained by either using electrically fused cells or by loading cells with magnetic particles (about 10 nm in diameter) and the drug simultaneously and by guiding the cells to any selected site of the organism. The field technique used for the loading of the cells is based on the electrical breakdown of the cell membrane which is observed when cell suspensions are subjected to external field pulses of 2 to 20 kV/cm for short time intervals (ns to ms). When an apparent membrane potential of about 1 V (pulse length in the range of μs) is reached in response to the external field the membrane breaks down reversibly. The breakdown of the membrane is associated with a remarkable and reversible permeability increase of the cell membrane. The increase in permeability depends on the strength and the duration of the field pulse. The duration of the high conductance state of the cell membrane induced by the electric field is sufficiently long to entrap large quantities of drugs or dyes inside the cells. Electron-micrographs of thymocytes subjected to field pulses of various strengths and durations show that the pulse length is a critical factor for the reversibility of the field induced effects in the cell. When loading thymocytes the field pulse length has to be less than 1 μs to avoid irreversible changes in the ultrastructure of the cell. On the other hand, erythrocytes can be subjected to field pulses of 40 μs duration without any irreversible changes in the membrane structure. The life-span of the loaded erythrocytes in the blood circulation can be considerably prolonged if loss of haemoglobin and intracellular enzymes is avoided during field application and the resealing process. The loss of intracellular proteins can be minimized if the erythrocytes are immobilized in a polymeric network of calcium-alginate during the field application. The matrix is permeable to low molecular weight compounds but impermeable towards larger molecules such as proteins. Chelation of the Ca2+-ions in the alginate network by addition of sodium citrate leads to the release of the loaded erythrocytes from the calcium-alginate matrix.

Calcium Ions Protect Beet Root Cell Membranes against Thermally Induced Changes

AbstractThe initial heat induced increase in permeability of beet (Beta vulgaris L.) root cell membranes, as measured by betacyanin efflux, is reversible. The leakage of betacyanin stopped when tissue held for 90 min at 45°C was transferred to 27°C.Stronger heat treatments, 10 min at 60°C or 150 min at 45°C, resulted in a continuous release of betacyanin indicating an irreversible change in membrane structure.Calcium ions inhibited the heat induced betacyanin efflux only if this was due to a reversible increase in permeability.

Enzymes in Blood Plasma.

This monograph reviews some 589 papers published mostly during the decade from 1951 to 1961 inclusive with regard to the possible clinical significance of plasma enzymes. It appears that plasma enzymes are either the result of cell secretion such as acid and alkaline phosphatase, amylase, pepsin, and a few others or they are lost from cells undergoing sudden disintegration or reversible increase in permeability. The recent decade has added little to our knowledge and ability to use the secreted enzymes. The bulk of recent literature deals with plasma enzymes which leak out of cells and probably have no specific purpose in plasma. Much search has been made for organ specific and disease specific plasma enzymes or combinations of enzymes. In general the plasma enzyme levels are of greatest use in situations where the site of enzyme loss is either established by other diagnostic information or at least highly suspect. It