Leonard T. Rael

Commercial human albumin preparations for clinical use are immunosuppressive in vitro.

Objective:We previously reported significant variations in oxidation status and molecular length among sources and lots of human serum albumin (HSA) commercial preparations intended for clinical use. In this report, we investigated what effect the presence of HSA products have on the immune response in vitro. Design:Laboratory study. Setting:Trauma research basic science laboratory. Subjects:Activated human peripheral blood mononuclear cells. Interventions:Six commercial HSA preparations were tested for their effect on cytokine release from activated human peripheral blood mononuclear cells (PBMCs) and T-lymphocytes. Mass spectrometry analysis of aspartyl-alanyl diketopiperazine (DA-DKP) content of HSA and percentage of HSA having lost its amino terminal dipeptide aspartyl alanyl (HSA-DA) were correlated. Measurements and Main Results:Human PBMCs were cultured in the presence of six commercial HSA preparations and activated via the T-cell receptor complex. A cloned T-lymphocyte cell line, activated with specific antigen, was also cultured with both synthetic DA-DKP and small molecular weight extracts from the commercial HSA tested. Supernatants were quantified by enzyme-linked immunosorbent assay for interferon-γ and tumor necrosis factor-α content. DA-DKP was extracted from HSA by centrifugal filters and quantified by anion exchange liquid chromatography coupled to negative electrospray ionization mass spectrometry. HSA species were determined by reverse phase liquid chromatography coupled to positive electrospray ionization, time of flight mass spectrometry. All HSA preparations significantly inhibited the in vitro production of interferon-γ and tumor necrosis factor-α by activated PBMCs. DA-DKP was detected in all HSA sources at concentrations ranging between 42.0 and 79.6 &mgr;M. A synthetic form of DA-DKP possessed similar immunosuppressive qualities in a dose-dependent manner on T lymphocytes. Conclusions:DA-DKP was present in significant concentrations in all HSA sources tested and was partially responsible for the immunosuppressive effects of HSA on activated PBMCs and T-lymphocytes in vitro. In view of these findings, administering HSA to immunocompromised critically ill patients might be reevaluated.

Copper stimulates the synthesis and release of interleukin-8 in human endothelial cells: A possible early role in systemic inflammatory responses

Endogenous copper can play an important role in postischemic reperfusion injury, a condition associated with endothelial cell activation and increased interleukin 8 (IL-8) production. Excessive endothelial IL-8 secreted during trauma, major surgery, and sepsis may contribute to the development of systemic inflammatory response syndrome (SIRS), adult respiratory distress syndrome (ARDS), and multiple organ failure (MOF). No previous reports have indicated that copper has a direct role in stimulating human endothelial IL-8 secretion. Increased IL-8 in the culture medium of human umbilical vein (HUVEC), lung microvascular, and iliac artery endothelial cells was observed 24 h after the addition of 10 to 50 &mgr;M CuCl2 (cupric ions). HUVEC IL-8 induction by copper was higher than by 50 pg/mL tumor necrosis factor-&agr;, whereas 50 pg/mL IL-1&bgr; and 1 ng/mL platelet-activating factor did not stimulate IL-8 production or release. HUVEC IL-8 mRNA increased 3 h after CuCl2 stimulation and remained elevated after 24 h, implying sustained transcriptional activation. Copper did not stimulate HUVECs to secrete other cytokines. Cu(II) appeared to be the primary copper ion responsible for the observed increase in IL-8 because a specific high-affinity Cu(II)-binding peptide, d-Asp-d-Ala-d-His-d-Lys (d-DAHK), completely abolished this effect in a dose-dependent manner. These results suggest that Cu(II) may induce endothelial IL-8 by a mechanism independent of known Cu(I) generation of reactive oxygen species. Furthermore, in vivo studies are warranted to determine if copper is involved in the pathogenesis of systemic inflammation and if Cu(II) chelation can reduce this IL-8-induced endothelial inflammatory response.

Copper-induced oxidation of epinephrine: protective effect of D-DAHK, a synthetic analogue of the high affinity copper binding site of human albumin.

Epinephrine is known to be rapidly oxidized during sepsis. Ischemia and acidosis, which often accompany sepsis, are associated with the release of weakly bound cupric ions from plasma proteins. We investigated whether copper promotes oxidation of epinephrine at both physiological and acidic pH and whether D-Asp-D-Ala-D-His-D-Lys (D-DAHK), a human albumin (HSA) N-terminus synthetic peptide with a high affinity for cupric ions, attenuates this oxidation. Epinephrine alone [100 microM] or with CuCl(2) [10 microM], and with CuCl(2) [10 microM] and D-DAHK [20 microM] at pH 7.4, 7.0, 6.5, and 6.0 were incubated for 1h at 37 degrees C. Epinephrine oxidation was measured by the spectrophotometric quantification of its oxidation product, adrenochrome. We found that adrenochrome increased, suggesting copper-induced oxidation of epinephrine. At pH 7.4, 7.0, 6.5, and 6.0, adrenochrome increased by 47%, 53%, 24%, and 6% above baseline, respectively. D-DAHK attenuated the copper-induced oxidation of epinephrine to baseline levels. These in vitro results indicate that copper-induced epinephrine oxidation is greatest at the physiological pH 7.4 as well as in severe acidosis, pH 7.0, and that D-DAHK completely inhibits this oxidation.

Inhibitory Effect of Copper on Cystathionine β-Synthase Activity: Protective Effect of an Analog of the Human Albumin N-Terminus

Copper was added to truncated, recombinant cystathionine beta-synthase (CBS), and the enzyme activity was assessed by measuring the production of cystathionine. 10 microM copper significantly decreased CBS activity by 50% while 25 microM copper decreased CBS activity by 70%. This inhibition was negated when an analog of the N-terminus of human albumin, Asp-Ala-His-Lys (DAHK), a strong transition metal binding peptide, was added. The use of copper chelators could significantly reduce in vivo homocysteine levels.

Low Molecular Weight Fraction of Commercial Human Serum Albumin Induces Morphologic and Transcriptional Changes of Bone Marrow-Derived Mesenchymal Stem Cells

Osteoarthritis (OA) is the most common chronic disease of the joint; however, the therapeutic options for severe OA are limited. The low molecular weight fraction of commercial 5% human serum albumin (LMWF5A) has been shown to have anti‐inflammatory properties that are mediated, in part, by a diketopiperazine that is present in the albumin preparation and that was demonstrated to be safe and effective in reducing pain and improving function when administered intra‐articularly in a phase III clinical trial. In the present study, bone marrow‐derived mesenchymal stem cells (BMMSCs) exposed to LMWF5A exhibited an elongated phenotype with diffuse intracellular F‐actin, pronounced migratory leading edges, and filopodia‐like projections. In addition, LMWF5A promoted chondrogenic condensation in “micromass” culture, concurrent with the upregulation of collagen 2α1 mRNA. Furthermore, the transcription of the CXCR4‐CXCL12 axis was significantly regulated in a manner conducive to migration and homing. Several transcription factors involved in stem cell differentiation were also found to bind oligonucleotide response element probes following exposure to LMWF5A. Finally, a rapid increase in PRAS40 phosphorylation was observed following treatment, potentially resulting in the activation mTORC1. Proteomic analysis of synovial fluid taken from a preliminary set of patients indicated that at 12 weeks following administration of LMWF5A, a microenvironment exists in the knee conducive to stem cell infiltration, self‐renewal, and differentiation, in addition to indications of remodeling with a reduction in inflammation. Taken together, these findings imply that LMWF5A treatment may prime stem cells for both mobilization and chondrogenic differentiation, potentially explaining some of the beneficial effects achieved in clinical trials.

Use of Saline as a Placebo in Intra-articular Injections in Osteoarthritis: Potential Contributions to Nociceptive Pain Relief

Background: Osteoarthritis of the knee (OAK) is a severe debilitating condition characterized by joint pain, stiffness, and resultant limited mobility. In recent years, intra-articular (IA) injections have been used to relieve symptoms and have succeeded to varying degrees either with sodium hyaluronate preparations or with a biologic. Objective: The objective of this review is to evaluate multiple studies that demonstrate some relief from the symptoms of OAK in the saline arm of various clinical trials. Method: A thorough literature search (PubMed) was performed assessing the pain efficacy of various compounds compared to saline injections in clinical trials. A total of 73 studies were identified in the literature search including a total of 5,816 patients. These clinical trials all involved the IA injection of a viscosupplement (hyaluronate, platelet rich plasma (PRP), etc.) or a biologic (the low molecular weight fraction (< 5kDa) of human serum albumin (LMWF-5A)). For all of these studies, the control arm was injection of sterile physiological saline that approximates the salt concentration and total solute concentration of blood and most tissues. Results: Based on our review of the current literature, the tested compounds performed with mixed results when compared to saline injections. Moreover, OAK is a variable disease, with severity measured on the Kellgren and Lawrence (KL) scale where various hyaluronate preparations have a therapeutic effect mostly on KL 2-3 patients while a biologic works best on KL 3-4 patients. Conclusion: Since the effect of saline injection is always greater than no treatment, the evaluations of these treatments can be confounded in clinical trials. Therefore, the question of whether there are known therapeutic effects of saline injections might explain these results.

Anti-Inflammatory Activity in the Low Molecular Weight Fraction of Commercial Human Serum Albumin (LMWF5A)

The innate immune system is increasingly being recognized as a critical component in osteoarthritis (OA) pathophysiology. An ex vivo immunoassay utilizing human peripheral blood mononuclear cells (PBMC) was developed in order to assess the OA anti-inflammatory properties of the low molecular weight fraction (<5 kDa) of commercial human serum albumin (LMWF5A). PBMC from various donors were pre-incubated with LMWF5A before LPS stimulation. TNFα release was measured by ELISA in supernatants after an overnight incubation. A ≥ 30% decrease in TNFα release was observed. This anti-inflammatory effect is potentially useful in assessing potency of LMWF5A for the treatment of OA.

The low molecular weight fraction of human serum albumin upregulates production of 15d-PGJ2 in Peripheral Blood Mononuclear Cells

Activation of the innate immune system involves a series of events designed to counteract the initial insult followed by the clearance of debris and promotion of healing. Aberrant regulation can lead to systemic inflammatory response syndrome, multiple organ failure, and chronic inflammation. A better understanding of the innate immune response may help manage complications while allowing for proper immune progression. In this study, the ability of several classes of anti-inflammatory drugs to affect LPS-induced cytokine and prostaglandin release from peripheral blood mononuclear cells (PBMC) was evaluated. PBMC were cultured in the presence of dexamethasone (DEX), ibuprofen (IBU), and the low molecular weight fraction of 5% albumin (LMWF5A) followed by stimulation with LPS. After 24xa0h, TNFα, PGE2, and 15d-PGJ2 release was determined by ELISA. Distinct immunomodulation patterns emerged following LPS stimulation of PBMC in the presence of said compounds. DEX, a steroid with strong immunosuppressive properties, reduced TNFα, PGE2, and 15d-PGJ2 release. IBU caused significant reduction in prostaglandin release while TNFα release was unchanged. An emerging biologic with known anti-inflammatory properties, LMWF5A, significantly reduced TNFα release while enhancing PGE2 and 15d-PGJ2 release. Incubating LMWF5A together with IBU negated this observed increased prostaglandin release without affecting the suppression of TNFα release. Additionally, LMWF5A caused an increase in COX-2 transcription and translation. LMWF5A exhibited a unique immune modulation pattern in PBMC, disparate from steroid or NSAID administration. This enhancement of prostaglandin release (specifically 15d-PGJ2), in conjunction with a decrease in TNFα release, suggests a switch that favors resolution and decreased inflammation.

Autoantibody reaction to myelin basic protein by plasma parvovirus B19 IgG in MS patients.

The etiology of multiple sclerosis (MS) remains unclear. To determine if autoantibodies to myelin basic protein (MBP) are produced during parvovirus B19 infection, a competitive ELISA was performed using plasma from MS patients exhibiting high IgG titers for parvovirus. Our results showed the addition of MBP decreased the binding of IgG to B19 antigen in a dose dependent fashion suggesting a possible link between parvovirus B19 and a subset of patients with clinical MS.