BACKGROUND

Near-infrared (NIR) fluorescent sentinel lymph node (SLN) mapping in breast cancer requires optimized imaging systems and lymphatic tracers.

METHODS

A small, portable version of the FLARE imaging system, termed Mini-FLARE, was developed for capturing color video and two semi-independent channels of NIR fluorescence (700 and 800 nm) in real time. Initial optimization of lymphatic tracer dose was performed using 35-kg Yorkshire pigs and a 6-patient pilot clinical trial. More refined optimization was performed in 24 consecutive breast cancer patients. All patients received the standard of care using (99m)Technetium-nanocolloid and patent blue. In addition, 1.6 ml of indocyanine green adsorbed to human serum albumin (ICG:HSA) was injected directly after patent blue at the same location. Patients were allocated to 1 of 8 escalating ICG:HSA concentration groups from 50 to 1000 μM.

RESULTS

The Mini-FLARE system was positioned easily in the operating room and could be used up to 13 in. from the patient. Mini-FLARE enabled visualization of lymphatic channels and SLNs in all patients. A total of 35 SLNs (mean = 1.45, range 1-3) were detected: 35 radioactive (100%), 30 blue (86%), and 35 NIR fluorescent (100%). Contrast agent quenching at the injection site and dilution within lymphatic channels were major contributors to signal strength of the SLN. Optimal injection dose of ICG:HSA ranged between 400 and 800 μM. No adverse reactions were observed.

CONCLUSIONS

We describe the clinical translation of a new NIR fluorescence imaging system and define the optimal ICG:HSA dose range for SLN mapping in breast cancer.

Although "biocompatible" polymeric elastomers are generally nontoxic, nonimmunogenic, and chemically inert, implants made of these materials may trigger acute and chronic inflammatory responses. Early interactions between implants and inflammatory cells are probably mediated by a layer of host proteins on the material surface. To evaluate the importance of this protein layer, we studied acute inflammatory responses of mice to samples of polyester terephthalate film (PET) that were implanted intraperitoneally for short periods. Material preincubated with albumin is "passivated," accumulating very few adherent neutrophils or macrophages, whereas uncoated or plasma-coated PET attracts large numbers of phagocytes. Neither IgG adsorption nor surface complement activation is necessary for this acute inflammation; phagocyte accumulation on uncoated implants is normal in hypogammaglobulinemic mice and in severely hypocomplementemic mice. Rather, spontaneous adsorption of fibrinogen appears to be critical: (a) PET coated with serum or hypofibrinogenemic plasma attracts as few phagocytes as does albumin-coated material; (b) in contrast, PET preincubated with serum or hypofibrinogenemic plasma containing physiologic amounts of fibrinogen elicits "normal" phagocyte recruitment; (c) most importantly, hypofibrinogenemic mice do not mount an inflammatory response to implanted PET unless the material is coated with fibrinogen or the animals are injected with fibrinogen before implantation. Thus, spontaneous adsorption of fibrinogen appears to initiate the acute inflammatory response to an implanted polymer, suggesting an interesting nexus between two major iatrogenic effects of biomaterials: clotting and inflammation.

OBJECTIVE

To study the relationship between serum albumin level and all-cause mortality in an elderly population and to evaluate the role of albumin level in combination with physical disability status in predicting mortality.

METHODS

Cohort study with a mean follow-up of 3.7 years.

METHODS

Three communities: East Boston, Mass, New Haven, Conn, and Iowa and Washington counties, Iowa.

METHODS

A total of 1486 men and 2630 women aged 71 years and older who consented to have blood drawn.

RESULTS

During follow-up, 447 men and 488 women died. In both sexes, there was a graded increase in mortality rate with decreasing albumin level. After adjusting for age, race, education, chronic conditions, and disability status, hypoalbuminemia (< 35 g/L) was associated with a significantly increased risk of mortality in comparison with the reference group (ie, those with albumin levels greater than 43 g/L) (men: relative risk [RR], 1.9; 95% confidence interval [CI], 1.1 to 3.1; women: RR, 3.7; 95% CI, 2.5 to 5.5). Among those with albumin levels in the range usually considered normal (35 g/L to 50 g/L), there was a graded increase in mortality risk from the highest normal to the lowest normal groups. For women, results were similar in separate analyses that considered deaths occurring during the first year and 1 year or more after baseline, while for men the relationship decreased after 1 year. Lower albumin level was associated with an elevated risk of mortality for all categories of causes of death. A 15-level measure that characterized participants according to albumin level and disability status demonstrated that mortality increased with decreasing albumin level and increasing disability level.

CONCLUSIONS

Serum albumin level is an independent risk factor for all-cause mortality in older persons. A combined measure of albumin and disability reveals a strong gradient in mortality risk and may serve as a simple but useful index of frailty that can identify a high-risk group of older men and women who could be targeted for preventive and treatment efforts.

Clinical studies of bone marrow (BM) cell therapy for liver cirrhosis are under way but the mechanisms of benefit remain undefined. Cells of the monocyte-macrophage lineage have key roles in the development and resolution of liver fibrosis. Therefore, we tested the therapeutic effects of these cells on murine liver fibrosis. Advanced liver fibrosis was induced in female mice by chronic administration of carbon tetrachloride. Unmanipulated, syngeneic macrophages, their specific BM precursors, or unfractionated BM cells were delivered during liver injury. Mediators of inflammation, fibrosis, and regeneration were measured. Donor cells were tracked by sex-mismatch and green fluorescent protein expression. BM-derived macrophage (BMM) delivery resulted in early chemokine up-regulation with hepatic recruitment of endogenous macrophages and neutrophils. These cells delivered matrix metalloproteinases-13 and -9, respectively, into the hepatic scar. The effector cell infiltrate was accompanied by increased levels of the antiinflammatory cytokine interleukin 10. A reduction in hepatic myofibroblasts was followed by reduced fibrosis detected 4 weeks after macrophage infusion. Serum albumin levels were elevated at this time. Up- regulation of the liver progenitor cell mitogen tumor necrosis factor-like weak inducer of apoptosis (TWEAK) preceded expansion of the progenitor cell compartment. Increased expression of colony stimulating factor-1, insulin-like growth factor-1, and vascular endothelial growth factor also followed BMM delivery. In contrast to the effects of differentiated macrophages, liver fibrosis was not significantly altered by the application of macrophage precursors and was exacerbated by whole BM.

CONCLUSIONS

Macrophage cell therapy improves clinically relevant parameters in experimental chronic liver injury. Paracrine signaling to endogenous cells amplifies the effect. The benefits from this single, defined cell type suggest clinical potential.

The glomerular filtration barrier prevents large serum proteins from being lost into the urine. It is not known, however, why the filter does not routinely clog with large proteins that enter the glomerular basement membrane (GBM). Here, we provide evidence that an active transport mechanism exists to remove immunoglobulins that accumulate at the filtration barrier. We found that FcRn, an IgG and albumin transport receptor, is expressed in podocytes and functions to internalize IgG from the GBM. Mice lacking FcRn accumulated IgG in the GBM as they aged, and tracer studies showed delayed clearance of IgG from the kidneys of FcRn-deficient mice. Supporting a role for this pathway in disease, saturating the clearance mechanism potentiated the pathogenicity of nephrotoxic sera. These studies support the idea that podocytes play an active role in removing proteins from the GBM and suggest that genetic or acquired impairment of the clearance machinery is likely to be a common mechanism promoting glomerular diseases.

BACKGROUND

High C-reactive protein (CRP) and hypoalbuminemia are associated with increased risk of mortality in patients with kidney failure. There are limited data evaluating the relationships between CRP, albumin, and outcomes in chronic kidney disease (CKD) stages 3 and 4.

METHODS

The Modification of Diet in Renal Disease (MDRD) Study was a randomized controlled trial conducted between 1989 and 1993. CRP was measured in frozen samples taken at baseline. Survival status and cause of death, up to December 31, 2000, were obtained from the National Death Index. Multivariable Cox models were used to examine the relationship of CRP [stratified into high CRP>> or =3.0 mg/L (N= 414) versus low CRP<3.0 mg/L (N= 283)], and serum albumin, with all-cause and cardiovascular mortality.

RESULTS

Median follow-up time was 125 months, all-cause mortality was 20% (N= 138) and cardiovascular mortality was 10% (N= 71). In multivariable analyses adjusting for demographic, cardiovascular and kidney disease factors, both high CRP (HR, 95% CI = 1.56, 1.07-2.29) and serum albumin (HR = 0.94 per 0.1 g/dL increase, 95% CI = 0.89-0.99) were independent predictors of all-cause mortality. High CRP (HR 1.94, 95% CI 1.13-3.31), but not serum albumin (HR 0.94, 95% CI 0.87-1.02), was an independent predictor of cardiovascular mortality.

CONCLUSIONS

Both high CRP and low albumin, measured in CKD stages 3 and 4, are independent risk factors for all-cause mortality. High CRP, but not serum albumin, is a risk factor for cardiovascular mortality. These results suggest that high CRP and hypoalbuminemia provide prognostic information independent of each other in CKD.

BACKGROUND

The role of surgically placed intra-abdominal drainages after pancreatic resections has not been clearly established. In particular, their effect on morbidity rates and the optimal timing for their removal remains controversial.

METHODS

A total of 114 eligible patients who underwent standard pancreatic resections and at low risk of postoperative pancreatic fistula according to our institutional protocol (amylase value in drains < or =5000 U/L on postoperative day [POD] 1) were randomized on POD 3 to receive either early (POD 3) or standard drain removal (POD 5 or beyond). The primary end point of the study was the incidence of pancreatic fistula. Secondary endpoints included abdominal complications, pulmonary complications, in-hospital stay, and perioperative mortality. Cost-analysis between the 2 groups was also made.

RESULTS

Early drain removal was associated with a decreased rate of pancreatic fistula (P = 0.0001), abdominal complications (P = 0.002), and pulmonary complications (P = 0.007). Median in-hospital stay was shorter (P = 0.018), and hospital costs decreased (P = 0.02). Mortality was nil. A significant association with pancreatic fistula was found for timing of drain removal (P < 0.001), unintentional weight decrease before surgery (P = 0.022), type of pancreas texture (P = 0.015), serum amylase levels on POD 1 (P = 0.001), and albumin levels on POD 1 (P = 0.039). Multivariate analysis showed that timing of drain removal (P = 0.0003) and unintentional weight decrease before surgery (P = 0.02) were independent risk factors of pancreatic fistula.

CONCLUSIONS

In patients at low risk of pancreatic fistula, intra-abdominal drains can be safely removed on POD 3 after standard pancreatic resections. A prolonged period of drain insertion is associated with a higher rate of postoperative complications with increased hospital stay and costs. The manuscript is a randomized trial, registered in the NLM database as NCT00931554.

Proteins are targets of reactive species and detection of oxidatively modified proteins is often used as an index of oxidative stress. Peroxynitrite is a strong oxidant formed by reaction of nitric oxide with superoxide. Using fatty acid-free bovine serum albumin as a model we examined peroxynitrite-mediated protein modifications. The reaction of protein with peroxynitrite resulted in the oxidation of tryptophan and cysteine, in the nitration of tyrosine, in the formation of dityrosine, in the production of 2,4 dinitrophenylhydrazine-reactive carbonyls and in protein fragmentation. The formation of 3-nitrotyrosine represents a specific peroxynitrite-mediated protein modification that is different from modifications mediated by reactive oxygen species.

Diagnosis and monitoring of complex diseases such as cancer require quantitative detection of multiple proteins. Recent work has shown that when specific biomolecular binding occurs on one surface of a microcantilever beam, intermolecular nanomechanics bend the cantilever, which can be optically detected. Although this label-free technique readily lends itself to formation of microcantilever arrays, what has remained unclear is the technologically critical issue of whether it is sufficiently specific and sensitive to detect disease-related proteins at clinically relevant conditions and concentrations. As an example, we report here that microcantilevers of different geometries have been used to detect two forms of prostate-specific antigen (PSA) over a wide range of concentrations from 0.2 ng/ml to 60 microg/ml in a background of human serum albumin (HSA) and human plasminogen (HP) at 1 mg/ml, making this a clinically relevant diagnostic technique for prostate cancer. Because cantilever motion originates from the free-energy change induced by specific biomolecular binding, this technique may offer a common platform for high-throughput label-free analysis of protein-protein binding, DNA hybridization, and DNA-protein interactions, as well as drug discovery.

Recombinant human IL-6 (rhIL-6) is a potent inducer of the synthesis of acute phase proteins in adult human hepatocytes. A wide spectrum of acute phase proteins is regulated by this mediator. After labeling of rhIL-6 stimulated human hepatocytes with [35S]methionine acute phase protein synthesis was measured by immunoprecipitation. Serum amyloid A, C-reactive protein, haptoglobin, alpha 1-antichymotrypsin and fibrinogen were strongly induced (26-, 23-, 8.6-, 4.6- and 3.8-fold increases, respectively). Moderate increases were found for alpha 1-antitrypsin (2.7-fold) and alpha 1-acid glycoprotein (2.7-fold). RhIL-6 had no effect on alpha 2-macroglobulin, whereas fibronectin, albumin and transferrin decreased to 64, 56 and 55% of controls. In the cases of serum amyloid A, haptoglobin, alpha 1-antichymotrypsin, alpha 1-antitrypsin and alpha 1-acid glycoprotein, dexamethasone enhanced the action of rhIL-6. We conclude that rhIL-6 controls the acute phase response in human liver cells.

To interpret or to predict the responses of biopolymer processes in vivo and in vitro to changes in solute concentration and to coupled changes in water activity (osmotic stress), a quantitative understanding of the thermodynamic consequences of interactions of solutes and water with biopolymer surfaces is required. To this end, we report isoosmolal preferential interaction coefficients (Gamma(mu1) determined by vapor pressure osmometry (VPO) over a wide range of concentrations for interactions between native bovine serum albumin (BSA) and six small solutes. These include Escherichia coli cytoplasmic osmolytes [potassium glutamate (K(+)Glu(-)), trehalose], E. coli osmoprotectants (proline, glycine betaine), and also glycerol and trimethylamine N-oxide (TMAO). For all six solutes, Gamma(mu1) and the corresponding dialysis preferential interaction coefficient Gamma(mu1),(mu3) (both calculated from the VPO data) are negative; Gamma(mu1), (mu3) is proportional to bulk solute molality (m(bulk)3) at least up to 1 m (molal). Negative values of Gamma(mu1),(mu3) indicate preferential exclusion of these solutes from a BSA solution at dialysis equilibrium and correspond to local concentrations of these solutes in the vicinity of BSA which are lower than their bulk concentrations. Of the solutes investigated, betaine is the most excluded (Gamma(mu1),(mu3)/m(bulk)3 = -49 +/- 1 m(-1)); glycerol is the least excluded (Gamma(mu1),(mu3)/m(bulk)3 = -10 +/- 1 m(-1)). Between these extremes, the magnitude of Gamma(mu1),(mu3)/m(bulk)3 decreases in the order glycine betaine>>) proline>>TMAO>> trehalose approximately K(+)Glu(-)>> glycerol. The order of exclusion of E. coli osmolytes from BSA surface correlates with their effectiveness as osmoprotectants, which increase the growth rate of E. coli at high external osmolality. For the most excluded solute (betaine), Gamma(mu1),(mu3) provides a minimum estimate of the hydration of native BSA of approximately 2.8 x 10(3) H(2)O/BSA, which corresponds to slightly less than a monolayer (estimated to be approximately 3.2 x 10(3) H(2)O). Consequently, of the solutes investigated here, only betaine might be suitable for use in osmotic stress experiments in vitro as a direct probe to quantify changes in hydration of protein surface in biopolymer processes. More generally, however, our results and analysis lead to the proposal that any of these solutes can be used to quantify changes in water-accessible surface area (ASA) in biopolymer processes once preferential interactions of the solute with biopolymer surface are properly taken into account.

This study was initiated to enhance our insight on the health and environmental impact of silver nanoparticles (Ag-np). Using starch and bovine serum albumin (BSA) as capping agents, silver nanoparticles were synthesized to study their deleterious effects and distribution pattern in zebrafish embryos (Danio rerio). Toxicological endpoints like mortality, hatching, pericardial edema and heart rate were recorded. A concentration-dependent increase in mortality and hatching delay was observed in Ag-np treated embryos. Additionally, nanoparticle treatments resulted in concentration-dependent toxicity, typified by phenotypes that had abnormal body axes, twisted notochord, slow blood flow, pericardial edema and cardiac arrhythmia. Ag(+) ions and stabilizing agents showed no significant defects in developing embryos. Transmission electron microscopy (TEM) of the embryos demonstrated that nanoparticles were distributed in the brain, heart, yolk and blood of embryos as evident from the electron-dispersive x-ray analysis (EDS). Furthermore, the acridine orange staining showed an increased apoptosis in Ag-np treated embryos. These results suggest that silver nanoparticles induce a dose-dependent toxicity in embryos, which hinders normal development.

Endothelium is an important target for drug or gene therapy because of its important role in the biological system. In this paper, we have characterized nanoparticle uptake by endothelial cells in cell culture. Nanoparticles were formulated using poly DL-lactide-co-glycolide polymer containing bovine serum albumin as a model protein and 6-coumarin as a fluorescent marker. It was observed that the cellular uptake of nanoparticles depends on the time of incubation and the concentration of nanoparticles in the medium. The uptake of nanoparticles was rapid with confocal microscopy demonstrating their localization mostly in the cytoplasm. The mitogenic study demonstrated biocompatability of nanoparticles with the cells. The study thus demonstrates that nanoparticles could be used for localizing therapeutic agents or gene into endothelial cells. Nanoparticles localized in the endothelium could provide prolonged drug effects because of their sustained release characterics, and also could protect the encapsulated agent from enzymatic degradation.

The aim of this study was to assess the influence of human immunodeficiency virus (HIV) infection on chronic hepatitis B. In a series of 132 (65 anti-HIV positive) homosexual non-drug addicted men with chronic hepatitis B, the liver function was assessed with biochemical tests; the degree of hepatitis B virus (HBV) replication was assessed with serum HBV DNA level and with immunoperoxidase staining of hepatitis B core (HBc) antigen on liver specimens; and the severity of liver lesions was assessed with an histology activity index. Anti-HIV-positive and anti-HIV-negative patients were not different for serum aspartate transaminase activity, bilirubin, prothrombin, and histology activity index. Anti-HIV-positive patients had lower serum alanine transaminase activity levels (P =.0001), lower serum albumin levels (P =.0009), and higher serum HBV DNA levels (P =.01). There was a higher prevalence of cirrhosis in anti-HIV-positive patients (P =.04). In homosexual men with chronic hepatitis B, HIV infection is associated with a higher level of HBV replication and a higher risk for cirrhosis without increased liver necrotico-inflammatory process.

A DNA-activated protein kinase (DNA-PK) was purified from nuclei of HeLa cells. Activity was associated with a single high-molecular-mass (approximately-300,000 Da) polypeptide when analyzed by gel filtration, denaturing polyacrylamide gel electrophoresis, and Western immunoblotting using a monoclonal antibody that also inhibits enzyme activity. Nuclear localization was indicated by subcellular fractionation and confirmed by immunofluorescence on whole cells. Double-stranded DNA stimulated phosphorylation of the 300-kDa polypeptide in purified preparations as well as phosphorylation of the exogenous substrates alpha-casein, simian virus 40 large T antigen, and the human heat shock protein hsp90. Autophosphorylation led to inactivation of the enzyme. The phosphorylation of casein was stimulated over 30-fold by DNA and was specific for serine and threonine residues. Bovine serum albumin and histone H1 were poor substrates for DNA-PK, and no phosphorylation of immunoglobulin G or histones other than H1 was observed. Supercoiled or heat-denatured DNA and synthetic double-stranded RNA or RNA-DNA copolymers did not stimulate casein phosphorylation by DNA-PK. Interaction of the enzyme with DNA in the absence of exogenous substrates was demonstrated by thermal inactivation and gel mobility shifts. These characteristics identify DNA-PK as distinct from other protein kinases described in the literature and suggest that activation by DNA is an important feature of the enzyme's in vivo function.

OBJECTIVE

Autoreactive B cells play a key role in tissue injury in systemic autoimmune disease, and therefore a treatment resulting in B cell depletion could have benefit. This open-label study was undertaken to evaluate the efficacy of the anti-CD20 monoclonal antibody rituximab in the treatment of lupus nephritis.

METHODS

Lupus patients with active proliferative nephritis (4 with focal disease and 6 with diffuse disease) received rituximab (4 weekly infusions of 375 mg/m(2)) combined with oral prednisolone. Clinical, laboratory, and immunologic responses, including peripheral lymphocyte subsets measured by flow cytometry, were prospectively assessed at monthly intervals for 12 months. Complete remission of nephritis was defined as normal serum creatinine and albumin levels, inactive urine sediment, and 24-hour urinary protein <500 mg. Partial remission was defined as >50% improvement in all renal parameters that were abnormal at baseline.

RESULTS

B cell depletion lasted from 1 month to 7 months and was well tolerated. Partial remission was achieved in 8 of 10 patients within a median of 2 months (range 1-4 months); in 5 of them, complete remission was subsequently established (at a median of 3 months from baseline), and it was sustained at 12 months in 4. As early as 1 month from baseline, the expression of the costimulatory molecule CD40 ligand on CD4+ T cells was decreased by 4-fold, and it was almost blocked when partial remission was clinically evident. The expression of T cell activation markers CD69 and HLA-DR was significantly decreased at time points when partial remission was observed, and was further decreased during complete remission. In contrast, in patients who did not exhibit a response or when relapse was detected in patients in whom an initial remission had been achieved, such decreases were not prominent. Serum concentrations of double-stranded DNA autoantibodies were decreased in all patients, regardless of clinical outcome.

CONCLUSIONS

Following B cell depletion, clinical remission of lupus nephritis is associated with a decrease in T helper cell activation, suggesting an additional role for B cells, independent of autoantibody production, in promoting disease. A controlled trial to confirm these promising clinical results is warranted.

OBJECTIVE

To characterize the population pharmacokinetics of bevacizumab and the influence of demographic factors, disease severity, and concomitantly used chemotherapy agents on it's pharmacokinetic behavior.

METHODS

Data from eight clinical trials with bevacizumab administered by intravenous infusion were included. A total of 4,629 bevacizumab concentrations from 491 patients with solid tumors, who received bevacizumab doses ranging from 1 to 20 mg/kg at a dosing frequency ranging from weekly to every 3 weeks, were analyzed using a nonlinear mixed-effects modeling approach (NONMEM).

RESULTS

The best structural model was a two-compartment model with first-order elimination. In the final model, estimated clearance (CL) and central compartment volume of distribution (Vc) were 0.207 L/day and 2.39 L for a typical female. The terminal half-life estimate was approximately 20 days for both men and women. Body weight and gender were the most significant covariates to explain interpatient variability for CL and Vc. Clearance was 26% faster in men than in women. Patients with low serum albumin and high serum alkaline phosphatase had 19 and 23% faster CL, respectively, than a typical patient. Consistent with the long elimination half life, simulations showed that similar steady-state exposures can be maintained when the weekly mg/kg dose rate is maintained, therefore allowing administration of bevacizumab to coincide with the frequency of administration of the cytotoxic agents.

CONCLUSIONS

The PK parameters were consistent with those of other IgG molecules. The results support dosing bevacizumab on a once every 2 weeks or once every 3 weeks dosing schedule on a mg/kg basis.

MS-325 is a novel blood pool contrast agent for magnetic resonance imaging currently undergoing clinical trials to assess blockage in arteries. MS-325 functions by binding to human serum albumin (HSA) in plasma. Binding to HSA serves to prolong plasma half-life, retain the agent in the blood pool, and increase the relaxation rate of water protons in plasma. Ultrafiltration studies with a 5 kDa molecular weight cutoff filter show that MS-325 binds to HSA with stepwise stoichiometric affinity constants (mM(-1)) of K(a1) = 11.0 +/- 2.7, K(a2) = 0.84 +/- 0.16, K(a3) = 0.26 +/- 0.14, and K(a4) = 0.43 +/- 0.24. Under the conditions 0.1 mM MS-325, 4.5% HSA, pH 7.4 (phosphate-buffered saline), and 37 degrees C, 88 +/- 2% of MS-325 is bound to albumin. Fluorescent probe displacement studies show that MS-325 can displace dansyl sarcosine and dansyl-L-asparagine from HSA with inhibition constants (K(i)) of 85 +/- 3 microM and 1500 +/- 850 microM, respectively; however, MS-325 is unable to displace warfarin. These results suggest that MS-325 binds primarily to site II on HSA. The relaxivity of MS-325 when bound to HSA is shown to be site dependent. The Eu(III) analogue of MS-325 is shown to contain one inner-sphere water molecule in the presence and in the absence of HSA. The synthesis of an MS-325 analogue, 5, containing no inner-sphere water molecules is described. Compound 5 is used to estimate the contribution to relaxivity from the outer-sphere water molecules surrounding MS-325. The high relaxivity of MS-325 bound to HSA is primarily because of a 60-100-fold increase in the rotational correlation time of the molecule upon binding (tau(R) = 10.1 +/- 2.6 ns bound vs 115 ps free). Analysis of the nuclear magnetic relaxation dispersion (T(1) and T(2)) profiles also suggests a decrease in the electronic relaxation rate (1/T(1e) at 20 MHz = 2.0 x 10(8) s(-1) bound vs 1.1 x 10(9) s(-1) free) and an increase in the inner-sphere water residency time (tau(m) = 170 +/- 40 ns bound vs 69 +/- 20 ns free).

1. Cathepsin B, a tissue (lysosomal) proteinase, and two humoral proteinases, plasmin and kallikrein, activate the latent collagenase ('procollagenase') which is released by mouse bone explants in culture. Other lysosomal proteinases (carboxypeptidase B, cathepsin C and D) and thrombin did not activate the procollagenase. Dialysis of the culture fluids against 3M-NaSCN at 4 degrees C and, for some culture fluids, prolonged preincubation at 25 degrees C also caused the activation of procollagenase. 2. In all these cases, activation of procollagenase involved at least two successive steps: the activation of an endogenous latent activator present in the culture fluids and the activation of procollagenase itself. 3. An assay method was developed for the endogenous activator. Human serum, bovine serum albumin, casein and cysteine inhibited the endogenous activator at concentrations that did not influence the collagenase activity. N-Ethylmaleimide and 4-hydroxy-mercuribenzoate stimulated the endogenous activator, but iodoacetate had no effect. 4. It is proposed that cathepsin B, kallikrein and plasmin may play a role in the physiological activation of latent collagenase and thus initiate degradation of collagen in vivo. This may occur whatever the molecular nature of procollagenase (zymogen or enzyme-inhibitor complex) might be.

Human serum albumin (HSA) is an abundant transport protein found in plasma that binds a wide variety of drugs in two primary binding sites (I and II) and can have a significant impact on their pharmacokinetics. We have determined the crystal structures at 2.5 A-resolution of HSA-myristate complexed with the R-(+) and S-(-) enantiomers of warfarin, a widely used anticoagulant that binds to the protein with high affinity. The structures confirm that warfarin binds to drug site I (in subdomain IIA) in the presence of fatty acids and reveal the molecular details of the protein-drug interaction. The two enantiomers of warfarin adopt very similar conformations when bound to the protein and make many of the same specific contacts with amino acid side chains at the binding site, thus accounting for the relative lack of stereospecificity of the HSA-warfarin interaction. The conformation of the warfarin binding pocket is significantly altered upon binding of fatty acids, and this can explain the observed enhancement of warfarin binding to HSA at low levels of fatty acid.

The cerebrospinal fluid (CSF)/serum concentration quotients of albumin and the immunoglobulins G, A and M have been determined for 396 patients with a normal blood CSF barrier function or a pure barrier impairment without a humoral immune response within the central nervous system. The ratios between the concentration quotients of the three immunoglobulins and that of albumin increase in a nonlinear fashion with progressive impairments of the blood CSF barrier, i.e. the molecular size dependent discrimination of the protein transfer ('selectivity') decreases. The upper reference values of the concentration quotients for the whole range of passive transfer comply with the hyperbolic function: Q(IgX) = a/b square root Q(Alb)2 + b2 - c with different constants a/b, b2, c for IgG (0.8, 15 X 10(-6), 1.8 X 10(-3), IgA (0.72, 80 X 10(-6), 5.1 X 10(-3) and IgM (0.65, 150 X 10(-6), 7.5 X 10(-3). With these discriminatory functions the locally synthesized fractions IgX(loc) of IgG, IgA and IgM in CSF can be calculated by the general formula: IgX(loc) = [Q(IgX) - a/b square root Q(Alb)2 + b2 + c] X IgX(Ser).

Suppression of experimental autoimmune encephalomyelitis (EAE) in Lewis rats by the oral administration of myelin basic protein (MBP) is mediated by CD8+ T cells that can be isolated from the spleens of MBP-fed animals. These cells adoptively transfer protection to naive animals subsequently immunized with MBP and complete Freund's adjuvant (CFA) and suppress in vitro MBP proliferative responses. Using a transwell system in which the modulator spleen cells from MBP-fed animals are separated by a semipermeable membrane from responder cells, MBP, or OVA-specific T cell lines, we have found that cell contact is not required for in vitro suppression to occur. In vitro suppression is dependent, however, upon antigen-specific triggering of modulator T cells. Once antigen-specific triggering occurs, suppression across the transwell is mediated by an antigen-nonspecific soluble factor that equally suppresses an MBP line or an ovalbumin (OVA) line. This phenomenon of antigen-driven bystander suppression was also demonstrated in vivo. Specifically, Lewis rats fed OVA which were then immunized with MBP/CFA plus OVA given separately subcutaneously were protected from EAE. Animals fed OVA and then immunized with MBP/CFA without OVA given subcutaneously were not protected. The protective effect of feeding OVA could be adoptively transferred by CD8+ T cells from OVA-fed animals into MBP/CFA plus OVA-injected animals. Feeding bovine serum albumin (BSA) or keyhole limpet hemocyanin did not suppress EAE in animals immunized with MBP/CFA plus OVA. EAE was suppressed, however, if BSA was fed and animals then immunized with MBP/CFA plus BSA given subcutaneously. Antigen-driven bystander suppression appears to be an important mechanism by which antigen-driven peripheral tolerance after oral administration of antigen is mediated, and presumably occurs in the microenvironment accounting for the antigen specificity of suppression generated by oral tolerization to antigens.

Castleman's disease, an atypical lymphoproliferative disorder, can be classified into 2 types: hyaline-vascular and plasma cell types according to the histologic features of the affected lymph nodes. The plasma cell type is frequently associated with systemic manifestations and is often refractory to systemic therapy including corticosteroids and chemotherapy, particularly in multicentric form. Dysregulated overproduction of interleukin-6 (IL-6) from affected lymph nodes is thought to be responsible for the systemic manifestations of this disease. Therefore, interference with IL-6 signal transduction may constitute a new therapeutic strategy for this disease. We used humanized anti-IL-6 receptor antibody (rhPM-1) to treat 7 patients with multicentric plasma cell or mixed type Castleman's disease. All patients had systemic manifestations including secondary amyloidosis in 3. With the approval of our institution's ethics committee and the consent of the patients, they were treated with 50 to 100 mg rhPM-1 either once or twice weekly. Immediately after administration of rhPM-1, fever and fatigue disappeared, and anemia as well as serum levels of C-reactive protein (CRP), fibrinogen, and albumin started to improve. After 3 months of treatment, hypergammaglobulinemia and lymphadenopathy were remarkably alleviated, as were renal function abnormalities in patients with amyloidosis. Treatment was well tolerated with only transient leukopenia. Histopathologic examination revealed reduced follicular hyperplasia and vascularity after rhPM-1 treatment. The pathophysiologic significance of IL-6 in Castleman's disease was thus confirmed, and blockade of the IL-6 signal by rhPM-1 is thought to have potential as a new therapy based on the pathophysiologic mechanism of multicentric Castleman's disease. (Blood. 2000;95:56-61)