Nonnucleoside reverse transcriptase inhibitors (NNRTIs) are potent inhibitors of human immunodeficiency virus type 1 (HIV-1); however, currently marketed NNRTIs rapidly select resistant virus, and cross-resistance within the class is extensive. A parallel screening strategy was applied to test candidates from a series of diarylpyrimidines against wild-type and resistant HIV strains carrying clinically relevant mutations. Serum protein binding and metabolic stability were addressed early in the selection process. The emerging clinical candidate, TMC125, was highly active against wild-type HIV-1 (50% effective concentration [EC50] = 1.4 to 4.8 nM) and showed some activity against HIV-2 (EC50 = 3.5 microM). TMC125 also inhibited a series of HIV-1 group M subtypes and circulating recombinant forms and a group O virus. Incubation of TMC125 with human liver microsomal fractions suggested good metabolic stability (15% decrease in drug concentration and 7% decrease in antiviral activity after 120 min). Although TMC125 is highly protein bound, its antiviral effect was not reduced by the presence of 45 mg of human serum albumin/ml, 1 mg of alpha1-acid glycoprotein/ml, or 50% human serum. In an initial screen for activity against a panel of 25 viruses carrying single and double reverse transcriptase amino acid substitutions associated with NNRTI resistance, the EC50 of TMC125 was <5 nM for 19 viruses, including the double mutants K101E+K103N and K103N+Y181C. TMC125 also retained activity (EC50 < 100 nM) against 97% of 1,081 recent clinically derived recombinant viruses resistant to at least one of the currently marketed NNRTIs. TMC125 is a potent next generation NNRTI, with the potential for use in individuals infected with NNRTI-resistant virus.

Type 2 diabetes is frequently associated with an inflammatory status; the relationships between low-grade inflammation and diabetic nephropathy are still unclear. The aim of this study was to evaluate the relationships between acute-phase markers of inflammation, glomerular structure, and albumin excretion rate (AER) in type 2 diabetes. In 74 patients with type 2 diabetes (23 normoalbuminuric, 30 microalbuminuric, and 21 proteinuric) fibrinogen, serum amyloid A protein (SAA), C-reactive protein (CRP), and IL-6 were determined. AER was measured on three 24-h urine collections; GFR was measured by 51Cr EDTA plasma clearance. A kidney biopsy was performed, and mesangial fractional volume [Vv(mes/glom)] and glomerular basement membrane (GBM) width were estimated by electron microscopic morphometric analysis. CRP, fibrinogen, SAA, and IL-6 differed among groups, with proteinuric patients having the highest levels. SAA and fibrinogen correlated with AER (P < 0.03 and P < 0.001, respectively). GBM width and Vv(mes/glom) increased from normoalbuminuric to proteinuric patients [P < 0.005 normoalbuminuric and microalbuminuric versus proteinuric for GBM, P < 0.01 normoalbuminuric versus proteinuric for Vv(mes/glom)]. In patients with increased GBM width >> 396 nm), CRP, SAA, and IL-6 were higher than in patients with normal GBM width (P < 0.003, P < 0.004, and P < 0.0004, respectively). GBM width was directly correlated with fibrinogen (r = 0.33, P < 0.002) and IL-6 (r = 0.25 P < 0.05). In conclusion, this study demonstrates that acute-phase markers of inflammation are associated with nephropathy status and GBM thickening, suggesting a role for inflammation in the pathogenesis of diabetic glomerulopathy.

Glycerophospholipid and sphingolipid species and their bioactive metabolites are important regulators of lipoprotein and cell function. The aim of the study was to develop a method for lipid species profiling of separated lipoprotein classes. Human serum lipoproteins VLDL, LDL, and HDL of 21 healthy fasting blood donors were separated by fast performance liquid chromatography (FPLC) from 50 microl serum. Subsequently, phosphatidylcholine (PC), lysophosphatidylcholine, sphingomyelin (SM), ceramide (CER), phosphatidylethanolamine (PE), PE-based plasmalogen (PE-pl), cholesterol, and cholesteryl ester (CE) content of the separated lipoproteins was quantified by electrospray ionization tandem mass spectrometry (ESI-MS/MS). Analysis of FPLC fractions with PAGE demonstrated that albumin partially coelutes with HDL fractions. However, analysis of an HDL deficient serum (Tangier disease) showed that only lysophosphatidylcholine, but none of the other lipids analyzed, exhibited a significant coelution with the albumin containing fractions. Approximately 60% of lipoprotein CER were found in LDL fractions and 60% of PC, PE, and plasmalogens in HDL fractions. VLDL, LDL, and HDL displayed characteristic lipid class and species pattern. The developed method provides a detailed lipid class and species composition of lipoprotein fractions and may serve as a valuable tool to identify alterations of lipoprotein lipid species profiles in disease with a reasonable experimental effort.

BACKGROUND

Patients with type 2 diabetes are at increased risk of macro- and microvascular disease, and the presence of albuminuria and/or reduced kidney function further enhances macrovascular risk. Angiotensin-converting-enzyme inhibitors reduce both macro- and microvascular events, yet the residual renal and cardiovascular risk still remains high. Aliskiren a novel oral direct renin inhibitor that unlike ACEi and ARBs, lowers plasma renin activity, angiotensin I and angiotensin II levels, may thereby provide greater benefit compared to ACEi or ARB alone.

METHODS

The primary objective of the ALTITUDE trial is to determine whether aliskiren 300 mg once daily, reduces cardiovascular and renal morbidity and mortality compared with placebo when added to conventional treatment (including ACEi or ARB). ALTITUDE is an international, randomized, double-blind, placebo-controlled, parallel-group study, which will include three categories of high-risk patients with type 2 diabetes (aged>> or =35 years): those with either urinary albumin/creatinine ratio (UACR)>> or =200 mg/g; microalbuminuria (UACR)>> or =20 <200 mg/g and eGFR>> or =30 <60 mL/min/1.73 m2; and thirdly, those with a history of cardiovascular disease and eGFR>> or =30 <60 mL/min/1.73 m2 with or without microalbuminuria. ALTITUDE is an event driven trial that aims to randomize 8600 patients with a planned follow-up time of 48 months. The primary outcome measure is time to first event for the composite endpoint of cardiovascular death, resuscitated death, myocardial infarction, stroke, unplanned hospitalization for heart failure, onset of end-stage renal disease or doubling of baseline serum creatinine concentration. Secondary endpoints include a composite CV endpoint and a composite renal endpoint.

CONCLUSIONS

ALTITUDE will determine whether dual RAAS blockade with the direct renin inhibitor aliskiren in combination with an ACEi or ARB will reduce major morbidity and mortality in a broad range of high-risk patients with type 2 diabetes.

Human serum albumin (HSA) nanoparticles were manufactured by desolvation. Transferrin or transferrin receptor monoclonal antibodies (OX26 or R17217) were covalently coupled to the HSA nanoparticles using the NHS-PEG-MAL-5000 crosslinker. Loperamide was used as a model drug since it normally does not cross the blood-brain barrier (BBB) and was bound to the nanoparticles by adsorption. Loperamide-loaded HSA nanoparticles with covalently bound transferrin or the OX26 or R17217 antibodies induced significant anti-nociceptive effects in the tail-flick test in ICR (CD-1) mice after intravenous injection, demonstrating that transferrin or these antibodies covalently coupled to HSA nanoparticles are able to transport loperamide and possibly other drugs across the BBB. Control loperamide-loaded HSA nanoparticles with IgG2a antibodies yielded only marginal effects.

Corneal epithelium removed from underlying extracellular matrix (ECM) extends numerous cytoplasmic processes (blebs) from the formerly smooth basal surface. If blebbing epithelia are grown on collagen gels or lens capsules in vitro, the basal surface flattens and takes on the smooth contour typical of epithelium in contact with basal lamina in situ. This study examines the effect of soluble extracellular matrix components on the basal surface. Corneal epithelia from 9- to 11-d-old chick embryos were isolated with trypsin-collagenase or ethylenediamine tetraacetic acid, then placed on Millipore filters (Millipore Corp., Bedford, Mass.), and cultured at the medium-air interface. Media were prepared with no serum, with 10% of calf serum, or with serum from which plasma fibronectin was removed. Epithelia grown on filters in this medium continue to bleb for the duration of the experiments (12-14 h). If soluble collagen, laminin, or fibronectin is added to the medium, however, blebs are withdrawn and by 2-6 h the basal surface is flat. Epithelia grown on filters in the presence of albumin, IgG, or glycosaminoglycans continue to bleb. Epithelia cultured on solid substrata, such as glass, also continue to bleb if ECM is absent from the medium. The basal cell cortex in situ contains a compact cortical mat of filaments that decorate with S-1 myosin subfragments; some, if not all, of these filaments point away from the plasmalemma. The actin filaments disperse into the cytoplasmic processes during blebbing and now many appear to point toward the plasmalemma. In isolated epithelia that flatten in response to soluble collagens, laminin, and fibronectin, the actin filaments reform the basal cortical mat typical or epithelial in situ. Thus, extracellular macromolecules influence and organize not only the basal cell surface but also the actin-rich basal cell cortex of epithelial cells.

To identify the structural basis for the observed physiological effects of myosin regulatory light chain phosphorylation in skinned rabbit skeletal muscle fibers (potentiation of force development at low calcium), thick filaments separated from the muscle in the relaxed state, with unphoshorylated light chains, were incubated with specific, intact, myosin light chain kinase at moderate (pCa 5.0) and low (pCa 5.8) calcium and with calcium-independent enzyme in the absence of calcium, then examined as negatively stained preparations, by electron microscopy and optical diffraction. All such experimental filaments became disordered (lost the near-helical array of surface myosin heads typical of the relaxed state). Filaments incubated in control media, including intact enzyme in the absence of calcium, moderate calcium (pCa 5.0) without enzyme, and bovine serum albumin substituting for calcium-independent myosin light chain kinase, all retained their relaxed structure. Finally, filaments disordered by phosphorylation regained their relaxed structure after incubation with a protein phosphatase catalytic subunit. We suggest that the observed disorder is due to phosphorylation-induced increased mobility and/or changed conformation of myosin heads, which places an increased population of them close to thin filaments, thereby potentiating actin-myosin interaction at low calcium levels.

Magnetic resonance imaging (MRI) is a powerful medical diagnostic technique: it can penetrate deep into tissue, provide excellent soft tissue contrast with sub-millimeter resolution, and does not employ ionizing radiation. Targeted contrast agents provide an additional layer of molecular specificity to the wealth of anatomical and functional information already attainable by MRI. However, the major challenge for molecular MR imaging is sensitivity: micromolar concentrations of Gd(III) are required to cause a detectable signal change, which makes detecting proteins by MRI a challenge. Protein-targeted MRI contrast agents are bifunctional molecules comprising a protein-targeting moiety and typically one or more gadolinium chelates for detection by MRI. The ability of the contrast agent to enhance the MR image is termed relaxivity, and it depends upon many molecular factors, including protein binding itself. As in other imaging modalities, protein binding provides the pharmacokinetic effect of concentrating the agent at the region of interest. Unique to MRI, protein binding provides the pharmacodynamic effect of increasing the relaxivity of the contrast agent, thereby increasing the MR signal. In designing new agents, optimization of both the targeting function and the relaxivity is critical. In this Account, we focus on optimization of the relaxivity of targeted agents. Relaxivity depends upon speciation, chemical structure, and dynamic processes, such as water exchange kinetics and rotational tumbling rates. We describe mechanistic studies that relate these factors to the observed relaxivities and use these findings as the basis of rational design of improved agents. In addition to traditional biochemical methods to characterize ligand-protein interactions, the presence of the metal ion enables more obscure biophysical techniques, such as relaxometry and electron nuclear double resonance, to be used to elucidate the mechanism of relaxivity differences. As a case study, we explore the mechanism of action of the serum-albumin-targeted angiography agent MS-325 and closely related compounds and show how small changes in the metal chelate can impact relaxivity. We found that, while protein binding generally improves relaxivity by slowing the tumbling rate of the complex, in some cases, the protein itself can also negatively affect hydration of the metal complex and/or inner-sphere water exchange. Drawing on these findings, we designed next-generation agents targeting albumin, fibrin, or collagen and incorporating up to four gadolinium chelates. Through judicious molecular design, we show that high-relaxivity complexes with high target affinity can be realized.

Raised urinary albumin excretion (UAE) is associated with an increased risk of cardiovascular disease in non-insulin-dependent diabetes mellitus (NIDDM). We have examined the role of endothelial dysfunction as a possible explanation for this association in 94 NIDDM patients by investigating UAE, new cardiovascular events, and plasma concentration of von Willebrand factor (vWF), an indicator of endothelial dysfunction. At baseline, 66 patients had normal UAE (less than 15 micrograms/min), which remained normal in 33 (group 1) and increased in 33 (to median 31.5 micrograms/min, group 2). In 28 patients, baseline UAE was abnormal (67.1 micrograms/min, group 3). Follow-up ranged between 9 and 53 months. vWF did not change in group 1 (median 128% at baseline and 123% at follow-up), but increased in group 2 (from 116 to 219%, p less than 0.0001) and group 3 (from 157 to 207%, p = 0.0005). Baseline level of and change in vWF were strongly related to the development of microalbuminuria (R2 = 0.60, p less than 0.0001), but cardiovascular risk factors were not (R2 = 0.14). Raised baseline UAE was associated with an increased risk of new cardiovascular events only in patients with vWF concentrations above the median (relative risk 3.66, 95% CI 1.3-11.9) and not in patients with lower vWF (0.19, 0.01-1.33). In addition, the cardiovascular risk associated with increased UAE was modified by low compared with high concentrations of serum high density lipoprotein cholesterol (2.86 [1.03-8.48] vs 0.15 [0.01-1.43]). Dysfunction of vascular endothelium may be a link between albuminuria and atherosclerotic cardiovascular disease in NIDDM.

Leucocin A-UAL 187 is a bacteriocin produced by Leuconostoc gelidum UAL 187, a lactic acid bacterium isolated from vacuum-packaged meat. The bacteriocin was purified by ammonium sulfate or acid (pH 2.5) precipitation, hydrophobic interaction chromatography, gel filtration, and reversed-phase high-performance liquid chromatography with a yield of 58% of the original activity. Leucocin A is stable at low pH and heat resistant, and the activity of the pure form is enhanced by the addition of bovine serum albumin. It is inactivated by a range of proteolytic enzymes. The molecular weight was determined by mass spectrometry to be 3,930.3 +/- 0.4. Leucocin A-UAL 187 contains 37 amino acids with a calculated molecular weight of 3,932.3. A mixed oligonucleotide (24-mer) homologous to the sequence of the already known N terminus of the bacteriocin hybridized to a 2.9-kb HpaII fragment of a 7.6-MDa plasmid from the producer strain. The fragment was cloned into pUC118 and then subcloned into a lactococcal shuttle vector, pNZ19. DNA sequencing revealed an operon consisting of a putative upstream promoter, a downstream terminator, and two open reading frames flanked by a putative upstream promoter and a downstream terminator. The first open reading frame downstream of the promoter contains 61 amino acids and is identified as the leucocin structural gene, consisting of a 37-amino-acid bacteriocin and a 24-residue N-terminal extension. No phenotypic expression of the bacteriocin was evident in several lactic acid bacteria that were electrotransformed with pNZ19 containing the 2.9-kb cloned fragment of the leucocin A plasmid.

Acute-on-chronic liver failure (ACLF) is a severe, life-threatening complication, and new and efficient therapeutic strategies for liver failure are urgently needed. Mesenchymal stem cell (MSC) transfusions have been shown to reverse fulminant hepatic failure in mice and to improve liver function in patients with end-stage liver diseases. We assessed the safety and initial efficacy of umbilical cord-derived MSC (UC-MSC) transfusions for ACLF patients associated with hepatitis B virus (HBV) infection. A total of 43 ACLF patients were enrolled for this open-labeled and controlled study; 24 patients were treated with UC-MSCs, and 19 patients were treated with saline as controls. UC-MSC therapy was given three times at 4-week intervals. The liver function, adverse events, and survival rates were evaluated during the 48-week or 72-week follow-up period. No significant side effects were observed during the trial. The UC-MSC transfusions significantly increased the survival rates in ACLF patients; reduced the model for end-stage liver disease scores; increased serum albumin, cholinesterase, and prothrombin activity; and increased platelet counts. Serum total bilirubin and alanine aminotransferase levels were significantly decreased after the UC-MSC transfusions. UC-MSC transfusions are safe in the clinic and may serve as a novel therapeutic approach for HBV-associated ACLF patients.

Oxaliplatin (cis-[(1R,2R)-1,2-cyclohexanediamine-N,N'] oxalato(2-)-O,O'] platinum; Eloxatine) is a novel platinum coordination complex used for the treatment of metastatic colorectal carcinoma in combination with fluoropyrimidines. The objective of this review is to integrate the key data from multiple studies into a single, comprehensive overview of oxaliplatin disposition in cancer patients. The pharmacokinetics (PKs) of unbound platinum in plasma ultrafiltrate after oxaliplatin administration was triphasic, characterized by a short initial distribution phase and a long terminal elimination phase (t1/2, 252-273 h). No accumulation was observed in plasma ultrafiltrate after 130 mg/m2 every 3 weeks or 85 mg/m2 every 2 weeks. Interpatient and intrapatient variability in platinum exposure (area under the curve(0-48)) is moderate to low (33 and 5% respectively). In the blood, platinum binds irreversibly to plasma proteins (predominantly serum albumin) and erythrocytes. Accumulation of platinum in blood cells is not considered to be clinically significant. Platinum is rapidly cleared from plasma by covalent binding to tissues and renal elimination. Urinary excretion (53.8 +/- 9.1%) was the predominant route of platinum elimination, with fecal excretion accounting for only 2.1 +/- 1.9% of the administered dose 5 days postadministration. Tissue binding and renal elimination contribute equally to the clearance of ultrafilterable platinum from plasma. Renal clearance of platinum significantly correlated with glomerular filtration rate, indicating that glomerular filtration is the principal mechanism of platinum elimination by the kidneys. Clearance of ultrafilterable platinum is lower in patients with moderate renal impairment; however, no marked increase in drug toxicity was reported. The effect of severe renal impairment on platinum clearance and toxicity is currently unknown. Covariates such as age, sex, and hepatic impairment had no significant effect on the clearance of ultrafilterable platinum, and dose adjustment due to these variables is not required. Oxaliplatin undergoes rapid and extensive nonenzymatic biotransformation and is not subjected to CYP450-mediated metabolism. Up to 17 platinum-containing products have been observed in plasma ultrafiltrate samples from patients. These include several proximate cytotoxic species, including the monochloro-, dichloro-, and diaquo-diaminocyclohexane platinum complexes, along with several other noncytotoxic products. Oxaliplatin does not inhibit CYP450 isoenzymes in vitro. Platinum was not displaced from plasma proteins by a variety of concomitant medications tested in vitro, and no marked PK interactions between oxaliplatin, 5-fluorouracil, and irinothecan have been observed. These results indicate that the additive/synergistic antitumor activity observed with these agents is not due to major alterations in drug exposure, and the enhanced efficacy is likely to be mechanistically based. Together, these PK, biotransformation, drug-drug interaction analyses and studies in special patient populations provide a firm scientific basis for the safe and effective use of oxaliplatin in the clinic. These analyses also reveal that the pharmacological activity of oxaliplatin may be attributable, at least in part, to the unique pattern of platinum disposition observed in patients.

Objective: To analyze the clinical characteristics of 2019 novel coronavirus (2019-nCoV) pneumonia and to investigate the correlation between serum inflammatory cytokines and severity of the disease. Methods: 29 patients with 2019-ncov admitted to the isolation ward of Tongji hospital affiliated to Tongji medical college of Huazhong University of Science and Technology in January 2020 were selected as the study subjects. Clinical data were collected and the general information, clinical symptoms, blood test and CT imaging characteristics were analyzed. According to the relevant diagnostic criteria, the patients were divided into three groups: mild (15 cases), severe (9 cases) and critical (5 cases). The expression levels of inflammatory cytokines and other markers in the serum of each group were detected, and the changes of these indicators of the three groups were compared and analyzed, as well as their relationship with the clinical classification of the disease. Results: (1) The main symptoms of 2019-nCoV pneumonia was fever (28/29) with or without respiratory and other systemic symptoms. Two patients died with underlying disease and co-bacterial infection, respectively. (2) The blood test of the patients showed normal or decreased white blood cell count (23/29), decreased lymphocyte count (20/29), increased hypersensitive C reactive protein (hs-CRP) (27/29), and normal procalcitonin. In most patients,serum lactate dehydrogenase (LDH) was significantly increased (20/29), while albumin was decreased(15/29). Alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (Tbil), serum creatinine (Scr) and other items showed no significant changes. (3) CT findings of typical cases were single or multiple patchy ground glass shadows accompanied by septal thickening. When the disease progresses, the lesion increases and the scope expands, and the ground glass shadow coexists with the solid shadow or the stripe shadow. (4) There were statistically significant differences in the expression levels of interleukin-2 receptor (IL-2R) and IL-6 in the serum of the three groups (P<0.05), among which the critical group was higher than the severe group and the severe group was higher than the mildgroup. However, there were no statistically significant differences in serum levels of tumor necrosis factor-alpha (TNF-α), IL-1, IL-8, IL-10, hs-CRP, lymphocyte count and LDH among the three groups (P>0.05). Conclusion: The clinical characteristics of 2019-nCoV pneumonia are similar to those of common viral pneumonia. High resolution CT is of great value in the differential diagnosis of this disease. The increased expression of IL-2R and IL-6 in serum is expected to predict the severity of the 2019-nCoV pneumonia and the prognosis of patients.

OBJECTIVE

To study the effect of mesenchymal stem cells (MSC) on experimental liver fibrosis in rats.

METHODS

MSC were derived from bone marrow obtained from femoral and tibial bones of male albino rats. MSC were separated, grown, and propagated in culture for 4 weeks and were characterized morphologically and by detection of CD29 by RT-PCR. They were then infused into the tail vein of female rats that received CCl4 injection to induce liver fibrosis. Rats were divided into 4 groups: control, CCl4, CCl4 plus MSC, and MSC. Liver tissue was examined histopathologically and liver functions (ALT and serum albumin) were estimated for all groups. Y-chromosome gene (sry) was assessed by PCR in liver tissue of the female rats to confirm uptake of the male stem cells. Hydroxyproline content in liver tissue was assessed by chemical methods and expression of the collagen gene (type I) was detected as a marker for liver fibrosis. Results of the present study showed that MSC have a significant antifibrotic effect as evidenced by the significant decrease in liver collagen gene expression as well as the decrease in hydroxyproline content in the CCl4/MSC group (p<0.001) compared to the CCl4 group. The Y-chromosome gene (sry) was detected by RT-PCR in the CCl4/MSC group, but was not detected in control group and other groups. The CD29 gene was expressed in MSC culture, and this confirmed the efficiency of isolation and propagation of MSC in culture. With regard to liver function, there was also a significant improvement and elevation of serum albumin in the CCl4/MSC group compared to the CCl4 group (p<0.05). As regard to the liver enzyme ALT, there was a decrease of its level in the CCl4/MSC group compared to the CCl4 group. However, this was statistically nonsignificant (p>0.05). In conclusion, MSC have a potential therapeutic effect against the fibrotic process through their effect in minimizing collagen deposition in addition to their capacity to differentiate into hepatocytes.

OBJECTIVE

During long-term treatment of various malignant or viral diseases with IFN-alpha up to 20% of patients develop anti-IFN-alpha antibodies for as yet unknown reasons.

METHODS

To address this issue, a mouse model using Balb/C mice was established and the relevance of several potentially anti-IFN-alpha antibodies inducing factors was studied.

RESULTS

The model revealed that both a higher frequency of injections and a higher dosage of IFN-alpha were more immunogenic and that the route of administration affected the antibody response to IFN-alpha. The intrinsic immunostimulatory activity of IFN-alpha itself also enhanced the immune response. IFN-alpha protein aggregates (IFN-alpha-IFN-alpha and human serum albumin (HSA)-IFN-alpha aggregates), which were recently identified in all marketed IFN-alpha products, were significantly more immunogenic than IFN-alpha monomers. These aggregates broke the tolerance against human IFN-alpha monomers in human IFN-alpha transgenic mice.

CONCLUSIONS

Based on these animal studies it is proposed that the immune response to IFN-alpha in humans is most probably elicited by a combination of several factors among which IFN-alpha protein aggregates seem to play a key role.

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.

Decompensated liver cirrhosis (LC), a life-threatening complication of chronic liver disease, is one of the major indications for liver transplantation. Recently, mesenchymal stem cell (MSC) transfusion has been shown to lead to the regression of liver fibrosis in mice and humans. This study examined the safety and efficacy of umbilical cord-derived MSC (UC-MSC) in patients with decompensated LC. A total of 45 chronic hepatitis B patients with decompensated LC, including 30 patients receiving UC-MSC transfusion, and 15 patients receiving saline as the control, were recruited; clinical parameters were detected during a 1-year follow-up period. No significant side-effects and complications were observed in either group. There was a significant reduction in the volume of ascites in patients treated with UC-MSC transfusion compared with controls (P < 0.05). UC-MSC therapy also significantly improved liver function, as indicated by the increase of serum albumin levels, decrease in total serum bilirubin levels, and decrease in the sodium model for end-stage liver disease scores. UC-MSC transfusion is clinically safe and could improve liver function and reduce ascites in patients with decompensated LC. UC-MSC transfusion, therefore, might present a novel therapeutic approach for patients with decompensated LC.

Currently, no blood biomarker that specifically indicates injury to the proximal tubule of the kidney has been identified. Kidney injury molecule-1 (KIM-1) is highly upregulated in proximal tubular cells following kidney injury. The ectodomain of KIM-1 is shed into the lumen, and serves as a urinary biomarker of kidney injury. We report that shed KIM-1 also serves as a blood biomarker of kidney injury. Sensitive assays to measure plasma and serum KIM-1 in mice, rats, and humans were developed and validated in the current study. Plasma KIM-1 levels increased with increasing periods of ischemia (10, 20, or 30 minutes) in mice, as early as 3 hours after reperfusion; after unilateral ureteral obstruction (day 7) in mice; and after gentamicin treatment (50 or 200 mg/kg for 10 days) in rats. In humans, plasma KIM-1 levels were higher in patients with AKI than in healthy controls or post-cardiac surgery patients without AKI (area under the curve, 0.96). In patients undergoing cardiopulmonary bypass, plasma KIM-1 levels increased within 2 days after surgery only in patients who developed AKI (P<0.01). Blood KIM-1 levels were also elevated in patients with CKD of varous etiologies. In a cohort of patients with type 1 diabetes and proteinuria, serum KIM-1 level at baseline strongly predicted rate of eGFR loss and risk of ESRD during 5-15 years of follow-up, after adjustment for baseline urinary albumin-to-creatinine ratio, eGFR, and Hb1Ac. These results identify KIM-1 as a blood biomarker that specifically reflects acute and chronic kidney injury.

Numerous previous studies have proposed a role for angiotensin II (AII) in the renal regulation of salt balance. At least one nephron site, the proximal convoluted segment, has been implicated in this role. We used in vitro microperfusion of rabbit proximal convoluted tubules to further examine this question. To insure use of appropriate in vivo concentrations as well as potency of the hormone in vitro, we measured plasma AII levels by radioimmunoassay in normal, sodium-depleted, and adrenalectomized rabbits, and measured AII activity by bioassay after incubation in various microperfusion baths. Plasma levels ranged from approximately 2 X 10(-11) to 5 X 10(-11) M. AII activity was stable in Ringer's solution plus albumin, but not in rabbit serum or Ringer's solution plus fetal calf serum. In Ringer's solution plus albumin, physiologic concentrations of AII stimulated volume reabsorption (Jv). 10(-11) M AII increased Jv by 16% (P less than 0.01). 10(-10) M AII produced a lesser increase, 7.5% (P less than 0.05). At a frequently studied, but probably pharmacologic dose, 10(-7) M AII inhibited Jv by 24% (P less than 0.001). AII at 10(-11) M did not stimulate Jv in the presence of 10(-7) M saralasin. Though previous studies have suggested agonistic effects of saralasin alone in epithelia, we found no significant effect of 10(-7) M saralasin on Jv. None of the AII doses measurably changed transepithelial voltage. We conclude that AII in physiologic doses directly stimulates Jv in proximal convoluted tubules and this effect is probably receptor mediated and, within the limits of detection, electroneutral.

A low molecular weight beta(2)-globulin (beta(2)-microglobulin), albumin, and total protein were measured in concentrated 24-hr urine specimens from 20 healthy subjects and 30 patients with clinical proteinuria of glomerular or tubular type. Classification of proteinuria was made on the basis of clinical diagnosis and size distribution of urinary proteins after gel chromatography. The molecular radii (Stokes' radii) of beta(2)-microglobulin and albumin, estimated by gel chromatography, were 15 A and 35 A. The average 24-hr urinary excretion in healthy subjects was 0.12 mg for beta(2)-microglobulin, 10 mg for albumin, and 80 mg for total protein. The patients with renal glomerular disorders had normal or only somewhat increased excretion of beta(2)-microglobulin, despite considerably increased excretion of albumin and total protein. Most of the patients with tubular dysfunction excreted large amounts of beta(2)-microglobulin, although they excreted normal or only slightly increased amounts of albumin and only moderately increased quantities of total protein. Consequently, the ratio or urinary albumin/urinary beta(2)-microglobulin was high in glomerular proteinuria (1100: 14,200), intermediate in normal proteinuria (33: 163), and low in tubular proteinuria (1.0: 13.3). Determinations of urinary clearances of beta(2)-microglobulin and albumin in four healthy subjects and 11 patients indicated that increased excretions of the two proteins were associated with increased clearances. The results suggest that quantitative determinations of urinary beta(2)-microglobulin and urinary albumin may be useful for detecting disorders of the renal handling of plasma proteins. The findings also seem to suggest a selective tubular reabsorption of the two proteins. Estimates on sera revealed a close correlation between serum levels of beta(2)-microglobulin and creatinine and also a greatly raised serum concentration of beta(2)-microglobulin after bilateral nephrectomy.

Functional impairment of HDL may contribute to the excess cardiovascular mortality experienced by patients with renal disease, but the effect of advanced renal disease on the composition and function of HDL is not well understood. Here, we used mass spectrometry and biochemical analyses to study alterations in the proteome and lipid composition of HDL isolated from patients on maintenance hemodialysis. We identified a significant increase in the amount of acute phase protein serum amyloid A1, albumin, lipoprotein-associated phospholipase A2, and apoC-III composing uremic HDL. Furthermore, uremic HDL contained reduced phospholipid and increased triglyceride and lysophospholipid. With regard to function, these changes impaired the ability of uremic HDL to promote cholesterol efflux from macrophages. In summary, the altered composition of HDL in renal disease seems to inhibit its cardioprotective properties. Assessing HDL composition and function in renal disease may help identify patients at increased risk for cardiovascular disease.

OBJECTIVE

This multicenter study examined the impact of albumin excretion rate (AER) on the course of estimated glomerular filtration rate (eGFR) and the incidence of sustained eGFR <60 ml/min/1.73 m(2) in type 1 diabetes up to year 14 of the Epidemiology of Diabetes Interventions and Complications (EDIC) study (mean duration of 19 years in the Diabetes Control and Complications Trial [DCCT]/EDIC).

METHODS

Urinary albumin measurements from 4-h urine collections were obtained from participants annually during the DCCT and every other year during the EDIC study, and serum creatinine was measured annually in both the DCCT and EDIC study. GFR was estimated from serum creatinine using the abbreviated Modification of Diet in Renal Disease equation.

RESULTS

A total of 89 of 1,439 subjects developed an eGFR <60 ml/min/1.73 m(2) (stage 3 chronic kidney disease on two or more successive occasions (sustained) during the DCCT/EDIC study (cumulative incidence 11.4%). Of these, 20 (24%) had AER <30 mg/24 h at all prior evaluations, 14 (16%) had developed microalbuminuria (AER 30-300 mg/24 h) before they reached stage 3 chronic kidney disease, and 54 (61%) had macroalbuminuria (AER >300 mg/24 h) before they reached stage 3 chronic kidney disease. Macroalbuminuria is associated with a markedly increased rate of fall in eGFR (5.7%/year vs. 1.2%/year with AER <30 mg/24 h, P < 0.0001) and risk of eGFR <60 ml/min/1.73 m(2) (adjusted hazard ratio 15.3, P < 0.0001), whereas microalbuminuria had weaker and less consistent effects on eGFR.

CONCLUSIONS

Macroalbuminuria was a strong predictor of eGFR loss and risk of developing sustained eGFR <60 ml/min/1.73 m(2). However, screening with AER alone would have missed 24% of cases of sustained impaired eGFR.

Osteopontin (OPN) is a secreted, integrin-binding phosphoprotein that has been implicated in both normal and pathological processes; qualitative increases in OPN blood levels have been reported in a small number of patients with metastatic tumors of various kinds. We measured plasma OPN levels in 70 women with known metastatic breast carcinoma, 44 patient controls who were on follow-up after completion of adjuvant treatment for early breast cancer, and 35 normal volunteers. The median plasma OPN of patients with metastatic disease was 142 microgram/liter (range, 38-1312 microgram/liter) and was significantly different (P < 0.0001, Mann Whitney U test) from both control groups (medians, 60 and 47 microgram/liter; ranges, 15-117 and 22-122 microgram/liter). Furthermore, we found that increasing plasma OPN is associated with shorter survival (P < 0.001) when patients were grouped in terciles for plasma OPN. This was also demonstrated when using a Cox proportional hazards model. Median plasma OPN levels were significantly increased for three or more sites of involvement (median, 232 microgram/liter; n = 13) versus 1 or 2 metastatic sites (medians, 129 and 130 microgram/liter; n = 29 and 28, respectively). Plasma OPN levels were correlated with other biochemical markers related to the extent of disease, such as serum alkaline phosphatase, aspartate succinate aminotransaminase, and albumin (r = 0.81, 0.62, and -0.56, respectively; all P < 0.001). This study demonstrates a statistically significant elevation in plasma OPN in the majority ( approximately 70%) of a large series of patients with metastatic breast cancer when compared (95th percentile) to healthy women or patients who had completed adjuvant treatment for early-stage breast cancer. Furthermore, this is the first study to demonstrate that higher OPN levels in patients with metastatic breast cancer may be associated with an increased number of involved sites and decreased survival.