Journal of Investigative Medicine

Proinflammatory mediators that include tumor necrosis factor-a (TNF-a) and macrophage inflammatory protein-2 (MIP-2) and anti-inflammatory mediators such as interleukin-10 (IL-10) modulate the immune response to endotoxemia. IL-10 downregulates the production of TNF-a and MIP-2. Acute lung injury may occur secondary to neutrophil chemotaxis mediated by chemokine MIP-2. We studied the temporal relationship of TNF-a, MIP-2, and IL-10 in rat endotoxemia and correlation of MIP-2 concentrations with acute lung injury.

Ten ventilated rats were randomized to receive an intravenous infusion of 2 mg/kg Escherichia coli lipopolysaccharide (n = 6) or saline placebo (n = 4). Blood pressure was continuously monitored and arterial blood was obtained for lactate, blood gas, TNF-a, IL-10, and MIP-2 measurements at baseline, 2, 4, and 5.5 hours after LPS or saline infusion.

Endotoxemia resulted in hypotension, lactic acidemia, and increased alveolar-arterial oxygen gradient (A-a O₂ gradient) compared with the placebo group. TNF-α, MIP-2, and IL-10 levels were increased 2 hours after endotoxemia. Subsequently, TNF-a levels declined while IL-10 and MIP-2 levels remained elevated. Control rats had no significant increase in cytokine production at any time point. MIP-2 concentrations correlated with A-a O₂ gradient, an indicator of lung injury (r = 0.56, p < 0.001).

MIP-2, possibly released by TNF-a stimulation of macrophages, is associated with acute lung injury possibly by inducing neutrophil chemotaxis. IL-10 may exert its counter-inflammatory response by inhibiting the release of TNF-a in endotoxemia.

Left ventricular assist devices (VAD) have improved survival in patients with end-stage heart failure. Past studies have shown that interactions between blood and synthetic surfaces promote initial bleeding and later thromboembolism. The exact mechanism of blood activation during VAD circulation remains unclear. The purpose of this study was to test the hypothesis that platelet glycoprotein (GP) IIb/IIIa receptor degradation occurs during clinical use of ventricular assist devices.

Five in vitro nonpulsatile centrifugal VAD circuits were simulated for 4 days using 450 mL of fresh human whole blood. Temperature, activated clotting time, pH, PCO₂, PO₂, Ca++, and glucose were maintained at physiologic values. Flow was maintained at a constant 2.0 L/min/mβ. We examined whole blood platelet aggregation induced by ristocetin, collagen, and adenosine diphosphate (ATP). We also examined whole blood platelet degranulation induced by collagen and ADP.

Platelet aggregation in response to ristocetin, collagen, and ADP irreversibly and progressively declined with prolonged circulation in the VAD. While ADP-induced aggregation declined within the first hour, ristocetin and collagen-induced aggregation declined after 10 hours. Collagen-induced platelet degranulation decreased similar to aggregation, whereas ADP-induced degranulation continued and was preserved throughout the experiment.

These results suggest prolonged circulation of human blood in a VAD circuit irreversibly impair platelet aggregation. The response of circulating platelets to individual agonists suggests that this platelet degradation is partially receptor specific. In our VAD system, ADP-stimulated platelet aggregation is more rapidly degraded with circulation. These results offer preliminary evidence that circulation of human blood in a VAD circuit leads to early degradation of the platelet GP IIb/IIIa complex. GP IIb/IIIa complex degradation is likely to be the mechanism of early VAD associated bleeding.

In the model of aminonucleoside of Puromycin (PAN)-induced nephrotic syndrome we assessed changes in glomerular expression of three proteins that regulate cell adhesion to extracellular matrix: paxillin, focal adhesion kinase (FAK), and Rho.

Following a single intravenous injection of PAN in Sprague-Dawley rats, proteinuria ensued and glomeruli were isolated at three stages: prior to onset of proteinuria (days 1 and 2), and when proteinuria peaked (day 9), subsided (day 29) or resolved (day 35). Glomerular protein lysates were analyzed by Western blot for expression of paxillin, FAK, and Rho.

There was a progressive increase in glomerular paxillin level that peaked concomitantly with heavy proteinuria (day 9). Paxillin remained increased during the recovery phase of PAN-induced injury and when proteinuria resolved. Expression of FAK and Rho remained unchanged at all time points. To explore whether the increase in paxillin expression following administration of PAN was due to a direct effect on glomerular epithelial cells (GEC), cultured rat GECs were incubated with PAN for 3, 6, and 24 hours, and expression of paxillin was assessed in GEC lysates by Western blot analysis. No change in paxillin levels was observed.

In PAN-induced nephrotic syndrome there is a preferential increase in paxillin expression that cannot be accouted for by an effect of PAN on GEC paxillin synthesis. We propose that the enhanced paxillin synthesis in the course of PAN-induced GEC injury reflects perturbations in contact between GEC and the GBM and may play a role in regulating adherence of GEC to the GBM. (Journal Investig Med 1998;46:284-289)

Nonsteroidal anti-inflammatory drugs (NSAIDs) can block renin release via inhibition of cyclooxygenase (COX). The responsible COX-isoenzyme in man is unknown. Therefore, we assessed the effects of meloxicam, a selective inhibitor of COX-2, and indomethacin, an unselective inhibitor of COX-1 and COX-2, on furosemide stimulated plasma renin activity (PRA).

In a randomized cross-over design 15 healthy female volunteers received no NSAID or meloxicam 7.5 mg/d for 6 days or indomethacin 25 mg tid for 2 days and 25 mg on the 3rd day. On the control day and on the last day of each treatment the following parameters were evaluated before and after furosemide 20 mg IV: PRA measured by RIA, urinary excretion of prostaglandin E₂ (PGE₂) assessed by gas chromatography-tandem mass spectrometry, urine volume and urinary excretion of sodium, potassium, and creatinine, and serum concentrations of sodium, potassium, and creatinine.

Furosemide led to a more than two-fold rise of PRA. Indomethacin as well as meloxicam had no significant effect on basal PRA but inhibited the furosemide-stimulated PRA increase. PGE₂ excretion on the control day rose twofold after furosemide. Meloxicam had no effect on basal PGE₂ excretion, whereas indomethacin reduced this parameter by 30%. Both drugs inhibited the increase of urinary PGE₂ after furosemide. No drug effects on urine flow nor on electrolytes and creatinine in serum and urine could be observed.

Meloxicam inhibited furosemide stimulated renin release, suggesting that in man COX-2 is responsible for prostaglandin synthesis mediating renin release.

Mice, transgenic for HIV-1 genes, have been demonstrated to develop renal lesions mimicking HIV-associated nephropathy. Focal glomerulosclerosis (FGS) has been reported to be the predominant glomerular lesion in these animals. In the other models of FGS, the accumulation of mesangial matrix and mesangial cell proliferation have been shown to be the preceding abnormalities. We evaluated the proliferation, apoptosis, and matrix accumulation by mesangial cells derived from mice transgenic for HIV-1 genes as well as from nontransgenic mice.

Mesangial cells were cultured from mice transgenic for HIV-1 genes (HTrMC) and nontransgenic mice (NTrMC) of the same age and sex. The growth rate of HTrMC and NTrMC was determined under identical conditions. Morphologic evaluation of apoptosis was performed by staining cells with Hoechst (H)-33342 and propidium iodide. Accumulation of mesangial cell collagen type IV, laminin, and fibronectin was measured by the dot blot assay. Total RNA was extracted from HTrMC and NTrMC and Northern blots were generated. These blots were probed with specific probes for TGF-ß, proteoglycan (P16), and GAPDH.

Mesangial cells (HTrMC) derived from transgenic mice had greater (P < 0.004) proliferation when compared to mesangial cells (NTrMCs) from nontransgenic mice (HTrMCs, 4.2 ± 0.3 vs NTrMCs, 3.0 ± 0.2×10 ⁴ cells/well). HTrMCs also showed enhanced (P < 0.0001) apoptosis compared to NTrMCs (HTrMCs, 13.2 ± 1.5% vs NTrMCs, 3.1 ± 0.5% apoptotic cells/field). HTrMCs accumulated an increased (P < 0.02) amount of collagen type IV (HTrMCs, 5659.7 ± 472.8 vs NTrMCs, 3882.2 ± 339.7 ng/well); whereas NTrMCs accumulated a greater amount of laminin when compared to HTrMCs (HTrMCs, 12.8 vs NTrMCs, 29.6 ± 2.9 ng/well). HTrMCs also showed an enhanced mRNA expression of TGF-ß and an attenuated expression of proteoglycan (P16).

These results suggest that mesangial cells derived from mice transgenic for HIV-1 genes have enhanced proliferation and collagen accumulation. The enhanced expression of TGF-ß may have contributed to enhanced HTrMC proliferation and the accumulation of collagen. The present study provides the basis for a hypothesis that mesangial cells may be contributing to the development of focal glomerulosclerosis in mice transgenic for HIV-1 genes.

Quantitative assays are needed to characterize the multilineage engraftment potential of clinical hematopoietic grafts. After we observed a dose-response relationship between the number of human hematopoietic cells transplanted into nonobese diabetic-scid/scid (NOD/SCID) mice and the number of human CD45+ cells recovered in the chimeras’ marrows and spleens, we sought to develop a multiple linear regression model that allows quantitative comparisons of human cell engraftment in vivo.

We used this NOD/SCID xenotransplant model to compare the engraftment potential of cord blood vs. adult marrow or mobilized blood, after either of 2 commonly used clinical graft engineering procedures: CD34+ cell selection or T cell depletion.

The engraftment per transplanted cell was >20 fold higher for cord blood cells, as compared to hematopoietic cells from adults. However, there was no difference in engraftment per CD34+ cell transplanted between marrow and mobilized blood. Levels of human cell engraftment from all sources could be increased by administration of human hematopoietic growth factors to human/mouse chimeras after transplantation. Correlation analysis of the number of human CD13+ myeloid cells and CD19+ B lymphoid cells in the chimeras’ marrows 8 weeks after transplantation provided evidence that almost all the human myeloid and B lymphoid cells were derived from the same primitive precursor cells.

These findings and assay may be useful in the development of clinical hematopoietic cell therapies.

Despite the known potent vasoconstrictor effects of vasopressin, the role of this hormone in the maintenance of blood pressure is incompletely understood. In studies performed in animals with increased plasma vasopressin concentrations, several complex cardiovascular effects have been noted, including decreases in heart rate and cardiac output, which may account for a lack of effect on arterial pressure despite the vasopressin-induced increase in total peripheral resistance. Only a few studies have been done to assess the cardiovascular effects of vasopressin in human subjects, and most of these have been limited to measurement of heart rate and arterial pressure only. The present study was designed to identify more fully the cardiovascular effects of vasopressin when plasma vasopressin concentrations are increased by osmotic stimulation without the superimposition of major nonosmotic stimuli associated with severe volume depletion.

Studies were performed on 11 normal human subjects in supine and erect posture before and after 24 hours of fluid deprivation, and following administration of a selective V1 receptor antagonist, [d(CH2)5Tyr(ME)]AVP, after dehydration. Cardiovascular parameters were measured noninvasively by thoracic electrical bioimpedance cardiography and blood samples for measurements of plasma concentrations of vasopressin and other hormones affected by dehydration and differences in posture were collected for subsequent analysis.

After 24 hours of fluid restriction, plasma osmolality was increased from 287 ± 0.9 to 294 ± 0.7 mosm/kg H20 and plasma vasopressin concentrations (Pavp) were increased in both supine and erect posture. Mean arterial (MAP) and systolic blood pressure (SBP) were reduced by fluid restriction but were higher in erect than in supine posture both before and after fluid restriction. Heart rate (HR), diastolic blood pressure (DBP), and systemic vascular resistance (SVRI) were also higher in erect than in supine posture, while cardiac index (CI), stroke index (SI), end-diastolic index (EDI), and an index of total thoracic fluid content (TFC) were all reduced in erect posture, both before and after dehydration. Plasma renin activity (PRA) and plasma norepinephrine concentrations (Pne) were increased in erect posture, both before and after dehydration, but there was no effect of erect posture on plasma vasopressin concentrations (Pavp), either before or after dehydration. Administration of the V1 receptor antagonist after dehydration had no effect on hemodynamic parameters other than small reductions in DBP and cardiac preload.

It is concluded from these studies that small increases in Pavp associated with moderate dehydration do not play a role in the maintenance of arterial pressure in normal human subjects in either supine or erect posture.