The Rogosin Institute

BACKGROUND: The interrogation of proteomes ("proteomics") in a highly multiplexed and efficient manner remains a coveted and challenging goal in biology and medicine. METHODOLOGY/PRINCIPAL FINDINGS: We present a new aptamer-based proteomic technology for biomarker discovery capable of simultaneously measuring thousands of proteins from small sample volumes (15 µL of serum or plasma). Our current assay measures 813 proteins with low limits of detection (1 pM median), 7 logs of overall dynamic range (~100 fM-1 µM), and 5% median coefficient of variation. This technology is enabled by a new generation of aptamers that contain chemically modified nucleotides, which greatly expand the physicochemical diversity of the large randomized nucleic acid libraries from which the aptamers are selected. Proteins in complex matrices such as plasma are measured with a process that transforms a signature of protein concentrations into a corresponding signature of DNA aptamer concentrations, which is quantified on a DNA microarray. Our assay takes advantage of the dual nature of aptamers as both folded protein-binding entities with defined shapes and unique nucleotide sequences recognizable by specific hybridization probes. To demonstrate the utility of our proteomics biomarker discovery technology, we applied it to a clinical study of chronic kidney disease (CKD). We identified two well known CKD biomarkers as well as an additional 58 potential CKD biomarkers. These results demonstrate the potential utility of our technology to rapidly discover unique protein signatures characteristic of various disease states. CONCLUSIONS/SIGNIFICANCE: We describe a versatile and powerful tool that allows large-scale comparison of proteome profiles among discrete populations. This unbiased and highly multiplexed search engine will enable the discovery of novel biomarkers in a manner that is unencumbered by our incomplete knowledge of biology, thereby helping to advance the next generation of evidence-based medicine.

Leptin elicits a metabolic response that cannot be explained by its anorectic effects alone. To examine the mechanism underlying leptin's metabolic actions, we used transcription profiling to identify leptin-regulated genes in ob/ob liver. Leptin was found to specifically repress RNA levels and enzymatic activity of hepatic stearoyl-CoA desaturase-1 (SCD-1), which catalyzes the biosynthesis of monounsaturated fatty acids. Mice lacking SCD-1 were lean and hypermetabolic. ob/ob mice with mutations in SCD-1 were significantly less obese than ob/ob controls and had markedly increased energy expenditure. ob/ob mice with mutations in SCD-1 had histologically normal livers with significantly reduced triglyceride storage and VLDL (very low density lipoprotein) production. These findings suggest that down-regulation of SCD-1 is an important component of leptin's metabolic actions.

Low serum albumin and low serum cholesterol levels are among the most consistent predictors of mortality in patients with end-stage renal disease (ESRD) undergoing hemodialysis. Hypoalbuminemia is often interpreted as a marker of poor nutrition, but serum albumin and cholesterol levels can also be low as part of a cytokine-mediated acute-phase reaction to acute or chronic inflammation. Here we report the results from a 900-day prospective study designed to determine whether tumor necrosis factor-alfa (TNF-alpha) and interleukin-6 (IL-6) predict serum albumin and cholesterol levels and mortality in a group of 90 ambulatory, adult hemodialysis patients with no acute infection, hospitalization or surgery, and no known acquired immunodeficiency syndrome (AIDS), malignancy, or liver disease. Measurable levels of TNF-alpha and/or IL-6 were found in 89 of 90 patients. Significant relationships were found between TNF-alpha and IL-6 and the degree of hypoalbuminemia and dyslipoproteinemia. IL-6 was the strongest predictor of mortality in univariate and multivariate analysis, followed by age, albumin level, and body mass index (BMI). Although the cause of hypercytokinemia was not addressed in this study, the data support the view that hypoalbuminemia and hypocholesterolemia are negative acute-phase responses to inflammatory stimuli. These results suggest that efforts to identify the nature of the stimuli for cytokine production and to lower cytokine levels in hemodialysis patients might be effective in improving the survival of patients undergoing hemodialysis.

BACKGROUND: The outcome of renal transplantation after an episode of acute rejection is difficult to predict, even with an allograft biopsy. METHODS: We studied urine specimens from 36 subjects with acute rejection, 18 subjects with chronic allograft nephropathy, and 29 subjects with normal biopsy results. Levels of messenger RNA (mRNA) for FOXP3, a specification and functional factor for regulatory T lymphocytes, and mRNA for CD25, CD3epsilon, perforin, and 18S ribosomal RNA (rRNA) were measured with a kinetic, quantitative polymerase-chain-reaction assay. We examined associations of mRNA levels with acute rejection, rejection reversal, and graft failure. RESULTS: The log-transformed mean (+/-SE) ratio of FOXP3 mRNA copies to 18S ribosomal RNA copies was higher in urine from the group with acute rejection (3.8+/-0.5) than in the group with chronic allograft nephropathy (1.3+/-0.7) or the group with normal biopsy results (1.6+/-0.4) (P<0.001 by the Kruskal-Wallis test). FOXP3 mRNA levels were inversely correlated with serum creatinine levels measured at the time of biopsy in the acute-rejection group (Spearman's correlation coefficient = -0.38, P=0.02) but not in the group with chronic allograft nephropathy or the group with normal biopsy results. Analyses of receiver-operating-characteristic curves demonstrated that reversal of acute rejection can be predicted with 90 percent sensitivity and 73 percent specificity with use of the optimal identified cutoff for FOXP3 mRNA of 3.46 (P=0.001). FOXP3 mRNA levels identified subjects at risk for graft failure within six months after the incident episode of acute rejection (relative risk for the lowest third of FOXP3 mRNA levels, 6; P=0.02). None of the other mRNA levels were predictive of reversal of acute rejection or graft failure. CONCLUSIONS: Measurement of FOXP3 mRNA in urine may offer a noninvasive means of improving the prediction of outcome of acute rejection of renal transplants.

Overwhelming bacterial infection is accompanied by fever, hypotension, disseminated intravascular coagulation, and multiple organ failure leading to death in 30-80% of cases. These classical symptoms of septic shock are caused by potent cytokines that are produced in response to endotoxin released from Gram-negative bacteria. Treatments with antibodies and receptor antagonists to block endotoxin or cytokine mediators have given mixed results in clinical trials. High density lipoprotein (HDL) is a natural component of plasma that is known to neutralize endotoxin in vitro. We report here that raising the plasma HDL concentration protects mice against endotoxin in vivo. Transgenic mice with 2-fold-elevated plasma HDL levels had more endotoxin bound to HDL, lower plasma cytokine levels, and improved survival rates compared with low-HDL mice. Intravenous infusion of HDL also protected mice, but only when given as reconstituted HDL prepared from phospholipid and either HDL apoprotein or an 18-amino acid peptide synthesized to mimic the structure of apolipoprotein A-I of HDL. Intact plasma HDL was mildly toxic, and HDL apoprotein was ineffective. The effectiveness of the reconstituted peptide renders very unlikely any significant contribution to protection by trace proteins in apo-HDL. These data suggest a simple leaflet insertion model for binding and neutralization of lipopolysaccharide by phospholipid on the surface of HDL. Plasma HDL may normally act to protect against endotoxin; this protection may be augmented by administration of reconstituted HDL or reconstituted peptides.

Renal transplantation is the treatment of choice for patients with end-stage renal disease. Better comprehension of the rejection response, improved preservation of organs, judicious use of cyclosporine, the application of antilymphocyte agents for the prevention and treatment of rejection, and specific protocols for the prevention and treatment of infection have all contributed to the recent improvement in outcome after renal transplantation.Immunobiology of Renal TransplantationThe Anti-Allograft (Rejection) ResponseRenal-allograft rejection depends on the coordinated activation of alloreactive T cells and antigen-presenting cells (e.g., monocyte-macrophages, dendritic cells, and B cells). Whereas acute rejection is a T-cell-dependent process, a broad array . . .

Regulation of proliferation of normal human T lymphocytes (T cells) by glutathione (GSH) was explored with T-cell activation models that do not require accessory cell signals. L-Buthionine-(S,R)-sulfoximine (BSO), which inactivates gamma-glutamylcysteine synthetase and therefore inhibits GSH synthesis, inhibited proliferation elicited by monoclonal antibodies directed at cluster designation 2 (CD2) and CD3 antigens, or by sn-1,2-dioctanoylglycerol and ionomycin. L-Buthionine-(R)-sulfoximine, which does not inactivate gamma-glutamylcysteine synthetase, did not affect proliferation. BSO-induced inhibition of accessory cell-independent T-cell proliferation was not reversed by recombinant human interleukin 2, despite activation-dependent expression of interleukin 2 receptor alpha by T cells treated with BSO. However, BSO-associated inhibition of T-cell proliferation was reversed by GSH or GSH ester. These studies, which show that GSH can directly modulate proliferation of highly purified T cells, suggest that GSH is essential for steps close to or at DNA synthesis. The availability of methods for decreasing and for increasing GSH levels suggest therapies to produce (i) immunosuppression (of value in organ transplantation), and (ii) immunopotentiation (of potential value in treatment of immunodeficiency states such as AIDS).

Abstract Interrogation of the human proteome in a highly multiplexed and efficient manner remains a coveted and challenging goal in biology. We present a new aptamer-based proteomic technology for biomarker discovery capable of simultaneously measuring thousands of proteins from small sample volumes (15 [mu]L of serum or plasma). Our current assay allows us to measure ~800 proteins with very low limits of detection (1 pM average), 7 logs of overall dynamic range, and 5% average coefficient of variation. This technology is enabled by a new generation of aptamers that contain chemically modified nucleotides, which greatly expand the physicochemical diversity of the large randomized nucleic acid libraries from which the aptamers are selected. Proteins in complex matrices such as plasma are measured with a process that transforms a signature of protein concentrations into a corresponding DNA aptamer concentration signature, which is then quantified with a DNA microarray. In essence, our assay takes advantage of the dual nature of aptamers as both folded binding entities with defined shapes and unique sequences recognizable by specific hybridization probes. To demonstrate the utility of our proteomics biomarker discovery technology, we applied it to a clinical study of chronic kidney disease (CKD). We identified two well known CKD biomarkers as well as an additional 58 potential CKD biomarkers. These results demonstrate the potential utility of our technology to discover unique protein signatures characteristic of various disease states. More generally, we describe a versatile and powerful tool that allows large-scale comparison of proteome profiles among discrete populations. This unbiased and highly multiplexed search engine will enable the discovery of novel biomarkers in a manner that is unencumbered by our incomplete knowledge of biology, thereby helping to advance the next generation of evidence-based medicine.

Recent work in this laboratory has identified immune-stimulatory properties of the oxidant hemin. In this study, we examined whether the nitrogen-based oxidant nitric oxide (NO) had inductive effects on human lymphocytes. We found that the NO-generating compounds sodium nitroprusside and S-nitroso-N-acetylpenicillamine rapidly enhanced the rate of glucose transport in resting human PBMC. In addition, NF-kappa B binding activity was induced by these agents as was the secretion of TNF-alpha. The data suggest that a cGMP-independent mechanism is involved as the cell permeant cGMP analogue, 8-Br-cGMP, had no effect in eliciting these inductive events. Activation of lymphocytes by these NO-generating compounds may be mediated through the protein tyrosine phosphorylation signal transduction pathway. We found that membrane-associated protein tyrosine phosphatase activity was enhanced in PBMC treated with sodium nitroprusside or S-nitroso-N-acetylpenicillamine and that the src family protein tyrosine kinase p56lck was activated in these cells. Inasmuch as p56lck activity is negatively controlled by tyrosine phosphorylation, its activation may be related to the enhancement of protein tyrosine phosphatase activity. 8-Br-cGMP had no effect on these enzymes. Taken together, these data suggest that NO may have immune-stimulatory properties and may signal through a hitherto undescribed cGMP-independent pathway.

This 2023 statement updates clinical guidance for homozygous familial hypercholesterolaemia (HoFH), explains the genetic complexity, and provides pragmatic recommendations to address inequities in HoFH care worldwide. Key strengths include updated criteria for the clinical diagnosis of HoFH and the recommendation to prioritize phenotypic features over genotype. Thus, a low-density lipoprotein cholesterol (LDL-C) >10 mmol/L (>400 mg/dL) is suggestive of HoFH and warrants further evaluation. The statement also provides state-of-the art discussion and guidance to clinicians for interpreting the results of genetic testing and for family planning and pregnancy. Therapeutic decisions are based on the LDL-C level. Combination LDL-C-lowering therapy-both pharmacologic intervention and lipoprotein apheresis (LA)-is foundational. Addition of novel, efficacious therapies (i.e. inhibitors of proprotein convertase subtilisin/kexin type 9, followed by evinacumab and/or lomitapide) offers potential to attain LDL-C goal or reduce the need for LA. To improve HoFH care around the world, the statement recommends the creation of national screening programmes, education to improve awareness, and management guidelines that account for the local realities of care, including access to specialist centres, treatments, and cost. This updated statement provides guidance that is crucial to early diagnosis, better care, and improved cardiovascular health for patients with HoFH worldwide.

Chronic inflammatory diseases are associated with premature atherosclerosis; however, it is unknown whether arterial stiffness is increased in this setting, possibly as a manifestation of vascular disease preceding and/or independent of atherosclerosis. Carotid ultrasonography and radial applanation tonometry were performed in 101 patients with systemic lupus erythematosus, 80 patients with rheumatoid arthritis, and 105 healthy control subjects. The 3 groups were comparable in age, gender, and carotid artery absolute and relative wall thickness. Atherosclerotic plaque was more common in lupus (46%) and rheumatoid arthritis (38%) patients than in controls (23%) (P<0.003). Although control subjects had higher central and peripheral blood pressures, arterial stiffness was increased in patient groups compared with controls (lupus, rheumatoid arthritis, controls, respectively: beta: 3.36 versus 3.22 versus 2.60, P<0.001; Young's modulus: 441 versus 452 versus 366 mm Hg/cm, P=0.004; Peterson's elastic modulus: 278 versus 273 versus 216 mm Hg, P<0.001) after adjustment for differences in mean brachial pressure. In multivariate analysis involving the entire population, arterial stiffness was independently related to age, serum glucose, and the presence of chronic inflammatory disease. In multivariate analysis restricted to the patients, arterial stiffness was independently related to age at diagnosis, disease duration, serum cholesterol, and C-reactive protein (and IL-6, when substituted for C-reactive protein). When analyses were repeated in the 186 study subjects without carotid plaque, arterial stiffness remained significantly elevated in patient groups after adjustment for differences in age and mean brachial pressure. In conclusion, arterial stiffness is increased in chronic inflammatory disorders independent of the presence of atherosclerosis and is related to disease duration, cholesterol, and the inflammatory mediator C-reactive protein and the cytokine that stimulates its production, IL-6.

BACKGROUND: Rheumatoid arthritis is associated with increased morbidity and mortality because of cardiovascular disease, independent of traditional risk factors. OBJECTIVE: To determine the prevalence of preclinical atherosclerosis in patients with rheumatoid arthritis and to identify clinical and biological markers for atherosclerotic disease in this patient population. DESIGN: Matched, cross-sectional study. SETTING: Hospital for Special Surgery in New York City. PATIENTS: 98 consecutive outpatients with rheumatoid arthritis who were followed by rheumatologists and 98 controls matched on age, sex, and ethnicity. MEASUREMENTS: Cardiovascular risk factor ascertainment and carotid ultrasonography in all participants; disease severity, disease treatment, and inflammatory markers in patients with rheumatoid arthritis. RESULTS: Despite a more favorable risk factor profile, patients with rheumatoid arthritis had a 3-fold increase in carotid atherosclerotic plaque (44% vs. 15%; P < 0.001). The relationship between rheumatoid arthritis and carotid atherosclerotic plaque remained after accounting for age, serum cholesterol levels, smoking history, and hypertensive status; adjusted predicted prevalence was 7.4% (95% CI, 3.4% to 15.2%) for the control group and 38.5% (CI, 25.4% to 53.5%) for patients with rheumatoid arthritis. Age (P < 0.001) and current cigarette use (P < 0.014) were also significantly associated with carotid atherosclerotic plaque. Among patients with rheumatoid arthritis, atherosclerosis was related to age, hypertension status, and use of tumor necrosis factor-alpha inhibitors (a possible marker of disease severity). LIMITATIONS: The study had a cross-sectional design, and inflammatory markers were determined only once. CONCLUSIONS: Patients with rheumatoid arthritis have a high prevalence of preclinical atherosclerosis independent of traditional risk factors, suggesting that chronic inflammation and, possibly, disease severity are atherogenic in this population.

Lipoprotein (a) [Lp(a)] is a plasma component whose concentration is related to the development of atherosclerosis, although the underlying mechanisms are not known. Lp(a) contains a unique structure, apolipoprotein (a), that shares partial homology with plasminogen. We now report that plasmin catalyzes the binding of Lp(a) to both immobilized fibrinogen and fibrin in a manner analogous to our previously reported studies with plasminogen. Plasmin treatment of immobilized fibrinogen induces a 3.7-fold increase in Lp(a) binding. Low density lipoprotein, molecules similar to Lp(a) but lacking apolipoprotein (a), bind poorly to immobilized fibrinogen and binding is not increased by plasmin. Trypsin but not neutrophil elastase also increases the binding of Lp(a) to fibrinogen. Lp(a) also complexes to plasmin-fibrinogen digests, and binding increases in proportion to the time of plasmin-induced fibrinogen degradation. Lp(a) binding is lysine-binding site dependent as it is inhibited by epsilon-aminocaproic acid. Lp(a) inhibits the binding of plasminogen to plasmin-modified immobilized fibrinogen, indicating that both molecules compete for similar lysine-binding sites. These findings demonstrate an affinity between Lp(a) and protease-modified fibrinogen or fibrin and thereby provide a potential mechanism to explain the association between thrombosis, coronary atherosclerosis, and increased blood concentrations of Lp(a).

From The Department of Medicine and The Rogosin Kidney Center, The New York Hospital, 525 East 68th Street, New York, New York 10021

Because the pulmonary alveolar epithelium separates air spaces from a fluid-filled compartment, it is expected that this barrier would be highly resistant to the flow of solutes and water. Investigation of alveolar epithelial resistance has been limited due to the complex anatomy of adult mammalian lung. Previous efforts to study isolated alveolar epithelium cultured on porous substrata yielded leaky monolayers. In this study, alveolar epithelial cells isolated from rat lungs and grown on tissue culture-treated Nucleopore filters resulted in tight monolayers with transepithelial resistance greater than 2,000 omega.cm2. Changes in bioelectric properties of these alveolar epithelial monolayers in response to ouabain, amiloride, and terbutaline are consistent with active sodium transport across a polarized barrier. 22Na flux measurements under short-circuit conditions directly confirm net transepithelial absorption of sodium by alveolar epithelial cells in the apical to basolateral direction, comparable to the observed short-circuit current (4.37 microA/cm2). The transport properties of these tight monolayers may be representative of the characteristics of the mammalian alveolar epithelial barrier in vivo.

We have tested hypotheses relating lipoprotein structure to function as measured by the relative ability to neutralize endotoxin by comparing natural human lipoproteins, a chemically defined form of reconstituted high-density lipoprotein (R-HDL), and a lipid emulsion (Intralipid). The human whole-blood system was used as an in vitro model of lipopolysaccharide (LPS) binding protein and CD14-dependent activation of cytokine production. When lipoproteins were compared on the basis of protein content, R-HDL was most effective in reducing tumor necrosis factor alpha (TNF-alpha) production followed in order by very low density lipoprotein, low-density lipoprotein, Intralipid, and natural HDL. However, when these particles were compared by protein, phospholipid, cholesterol, or triglyceride content by stepwise linear regression analysis, only phospholipid was correlated to effectiveness (r2 = 0.873; P < 0.0001). Anti-CD14 monoclonal antibodies MY4 and 3C10 inhibited LPS binding protein and CD14-dependent activation of TNF-alpha production by LPS at LPS concentrations up to approximately 1.0 ng/ml. R-HDL (2 mg of protein per ml) blocked TNF-alpha production by LPS from both smooth- and rough-type gram-negative bacteria at concentrations up to 100 ng of LPS per ml but had little effect on heat-killed gram-positive Staphylococcus aureus and no effect on other LPS-independent stimuli tested. These results support our hypothesis that LPS is neutralized by binding to phospholipid on the surface of R-HDL and demonstrate that R-HDL is a potent inhibitor of the induction of TNF-alpha by LPS from both rough- and smooth-form gram-negative bacteria in whole human blood.

OBJECTIVE: To determine the relationship of hypolipidemia to cytokine concentrations and clinical outcomes in critically ill surgical patients. DESIGN: Consecutive, prospective case series. SETTING: Surgical intensive care unit of an urban university hospital. PATIENTS: Subjects were 111 patients with a variety of critical illnesses, for whom serum lipid, lipoprotein, and cytokine concentrations were determined within 24 hrs of admission to a surgical intensive care unit. Controls were 32 healthy men and women for whom serum lipid, lipoprotein, and cytokine concentrations were determined. INTERVENTIONS: Blood samples were drawn on admission to the intensive care unit. Predetermined clinical outcomes including death, infection subsequent to intensive care unit admission, length of intensive care unit stay, and magnitude of organ dysfunction were monitored prospectively. MEASUREMENTS AND MAIN RESULTS: Measurements included total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, apolipoproteins A-I and B, phospholipid, triglyceride, interleukin-6, interleukin-10, soluble interleukin-2 receptor, tumor necrosis factor-alpha, and soluble tumor necrosis factor receptors p55 and p75. Mean serum lipid concentrations were extremely low: total cholesterol, 127 +/- 52 mg/dL; low-density lipoprotein cholesterol, 75 +/- 41 mg/dL; high-density lipoprotein cholesterol, 29 +/- 15 mg/dL. Total, low-density lipoprotein, and high-density lipoprotein cholesterol concentrations and apolipoprotein concentrations inversely correlated with interleukin-6, soluble interleukin-2 receptor, and interleukin-10 concentrations, whereas the triglyceride concentration correlated positively with tumor necrosis factor soluble receptors p55 and p75. Clinical outcomes were related to whether the admission cholesterol concentration was above (n = 56) or below (n = 55) the median concentration of 120 mg/dL. Each of the clinical end points occurred between 1.9- and 3.5-fold more frequently in the very low cholesterol (<120 mg/dL) group. Nine patients (8%) died during the hospitalization. Seven of the nine patients who died had total cholesterol concentrations below the median concentration of 120 mg/dL. CONCLUSIONS: Low cholesterol and lipoprotein concentrations found in critically ill surgical patients correlate with interleukin-6, soluble interleukin-2 receptor, and interleukin-10 concentrations and predict clinical outcomes.

The regulation of mRNA encoding transforming growth factor beta (TGF-beta) and interleukin 2 (IL-2) in normal human T cells was explored using novel competitor DNA constructs in the quantitative polymerase chain reaction and accessory cell-independent T cell activation models. Our experimental design revealed the following: (a) TGF-beta mRNA and IL-2 mRNA are regulated differentially in normal human T cells, quiescent or signaled with the synergistic combinations of: sn-1,2-dioctanoylglycerol and ionomycin or anti-CD3 monoclonal antibody (mAb) and anti-CD2 mAb; (b) the steady-state level of TGF-beta mRNA in the stimulated T cells, in contrast to that of IL-2 mRNA, is increased by the immunosuppressant cyclosporine (CsA); and (c) the paradoxical effect of CsA on TGF-beta mRNA levels is also appreciable at the level of production of functionally active TGF-beta protein. Our findings, in addition to demonstrating the utility of the competitor DNA constructs for the precise quantification of immunoregulatory cytokines, suggest a novel and unifying mechanistic basis for the immunosuppression and some of the complications (e.g., renal fibrosis) associated with CsA usage.

BACKGROUND: Transforming growth factor-beta1 (TGF-beta1) is an immunoregulatory and fibrogenic cytokine. In an earlier in vitro study, we demonstrated that cyclosporine (CsA) increases TGF-beta1 transcription rate in human T lymphocytes. Herein, we explored whether CsA augments the in vivo expression of TGF-beta1 in humans. METHODS: The inherent difficulty in studying the in vivo effect of CsA in humans was circumvented by investigating stable end-stage renal disease patients who were preconditioned with CsA before their living donor renal transplantation. Sera and peripheral blood mononuclear cells were obtained from CsA-preconditioned patients and quantified for TGF-beta1 expression at the mRNA (by competitive polymerase chain reaction) and protein (sandwich enzyme-linked immunosorbent assay) levels. RESULTS: Our studies demonstrated a significant increase in TGF-beta1 expression after CsA therapy. The stimulatory effect was unique to TGF-beta1, and CsA did not increase interleukin (IL)-10, IL-6, IL-2, or tumor necrosis factor-alpha expression. CONCLUSIONS: Our first-time demonstration of a TGF-beta1-selective in vivo stimulatory effect of CsA in humans: (1) advances a TGF-beta1-centered hypothesis for the beneficial (immunosuppression) and detrimental (fibrosis, hypertension) effects of CsA use, and (2) broadens the mechanism of immunosuppressive action of CsA to include heightened expression of an endogenous immunosuppressive cytokine.

OBJECTIVES: To determine the prevalence and clinical significance of hypolipidemia found in critically ill patients, and whether the addition of a reconstituted lipoprotein preparation could inhibit the generation of tumor necrosis factor-alpha (TNF-alpha) in acute-phase blood taken from these patients. SETTING: Surgical intensive care unit (ICU) of a large urban university hospital. DESIGN: Prospective case series. PATIENTS: A total of 32 patients with a variety of critical illnesses had lipid and lipoprotein concentrations determined. Six patients and six age- and gender-matched control subjects had whole blood in vitro studies of the effect of lipoprotein on lipopolysaccharide mediated TNF-alpha production. INTERVENTIONS: Blood samples were drawn on admission to the ICU and over a subsequent 8-day period. MEASUREMENTS AND MAIN RESULTS: Mean serum lipid and lipoprotein values obtained from patients within 24 hrs of transfer to the surgical ICU were extremely low: mean total cholesterol was 117 mg/dL (3.03 mmol/L), low-density lipoprotein cholesterol 71 mg/dL (1.84 mmol/L), and high-density lipoprotein cholesterol 25 mg/dL (0.65 mmol/L). Only the mean triglyceride concentration of 105 mg/dL (1.19 mmol/L), and the mean lipoprotein(a) concentration of 25 mg/dL (0.25 g/L) were within the normal range. During the first 8 days following surgical ICU admission, there were trends toward increasing lipid and lipoprotein concentrations that were significant for triglycerides and apolipoprotein B. Survival did not correlate with the lipid or lipoprotein concentrations, but patients with infections had significantly lower (p = .008) high-density lipoprotein cholesterol concentrations compared with noninfected patients. Lipopolysaccharide-stimulated production of TNF-alpha in patient and control blood samples was completely suppressed by the addition of 2 mg/mL of a reconstituted high-density lipoprotein preparation. CONCLUSIONS: Patients who are critically ill from a variety of causes have extremely low cholesterol and lipoprotein concentrations. Correction of the hypolipidemia by a reconstituted high-density lipoprotein preparation offers a new strategy for the prevention and treatment of endotoxemia.