Biomedical subjects

Friedrich Scheiflinger

Publications and source records attributed to Friedrich Scheiflinger.

At least 19 recordsLinked to original sources

PubMed2007

Prognostic value of anti-ADAMTS 13 antibody features (Ig isotype, titer, and inhibitory effect) in a cohort of 35 adult French patients undergoing a first episode of thrombotic microangiopathy with undetectable ADAMTS 13 activity.

To study both the pathophysiologic and the prognostic value of ADAMTS13 in thrombotic microangiopathies (TMAs), we enrolled a cohort of 35 adult patients combining a first acute episode of TMA, an undetectable (below 5%) ADAMTS13 activity in plasma, and no clinical background such as sepsis, cancer, HIV, and transplantation. All patients were treated by steroids and plasma exchange, and an 18-month follow-up was scheduled. Remission was obtained in 32 patients (91.4%), and 3 patients died (8.6%) after the first attack. At presentation, ADAMTS13 antigen was decreased in 32 patients (91.4%), an ADAMTS13 inhibitor was detectable in 31 patients (89%), and an anti-ADAMTS13 IgG/IgM/IgA was present in 33 patients (94%). The 3 decedent patients were characterized by the association of several anti-ADAMTS13 Ig isotypes, including very high IgA titers, while mortality was independent of the ADAMTS13 inhibitor titer. In survivors, ADAMTS13 activity in remission increased to levels above 15% in 19 patients (59%) but remained undetectable in 13 patients (41%). Six patients relapsed either once or twice (19%) during the follow-up. High levels of inhibitory anti-ADAMTS13 IgG at presentation were associated with the persistence of an undetectable ADAMTS13 activity in remission, the latter being predictive for relapses within an 18-month delay.

ADAM Proteins↗

PubMed2006

Systemic antithrombotic effects of ADAMTS13.

The metalloprotease ADAMTS13 (a disintegrin-like and metalloprotease with thrombospondin type I repeats 13) cleaves highly adhesive large von Willebrand factor (VWF) multimers after their release from the endothelium. ADAMTS13 deficiency is linked to a life-threatening disorder, thrombotic thrombocytopenic purpura (TTP), characterized by platelet-rich thrombi in the microvasculature. Here, we show spontaneous thrombus formation in activated microvenules of Adamts13-/- mice by intravital microscopy. Strikingly, we found that ADAMTS13 down-regulates both platelet adhesion to exposed subendothelium and thrombus formation in injured arterioles. An inhibitory antibody to ADAMTS13 infused in wild-type mice prolonged adhesion of platelets to endothelium and induced thrombi formation with embolization in the activated microvenules. Absence of ADAMTS13 did not promote thrombi formation in alphaIIbbeta3 integrin-inhibited blood. Recombinant ADAMTS13 reduced platelet adhesion and aggregation in histamine-activated venules and promoted thrombus dissolution in injured arterioles. Our findings reveal that ADAMTS13 has a powerful natural antithrombotic activity and recombinant ADAMTS13 could be used as an antithrombotic agent.

ADAMTS13 Protein↗

PubMed2006

Future aspects of hemophilia research and care.

Key issues in the management of patients with hemophilia include a thorough understanding of the mechanisms of blood coagulation and the complications that follow recurrent joint bleeding. Monoclonal antibodies are powerful tools for dissecting the intrinsic coagulation pathway and deriving reagents that could lead, on the long term, to the identification of molecules that enhance, or perhaps even replace factor (F) VIII concentrates in the management of hemophilia A. In recent in vitro experiments, it was demonstrated that plasmatic thrombin generation and intrinsic FX activation was enhanced by each of two FIXa-specific monoclonal antibodies, one of which had FIXa-agonistic activity only, whereas the other enhanced the activity of the intrinsic FX-activating complex (FVIIIa/FIXa) by at least two distinct mechanisms. Hemophilic synovitis, an inflammatory and proliferative disorder in patients with hemophilia, is the result of bleeding into joints and can lead to debilitating arthritis and chronic arthropathy. A major causative factor in the development of hemophilic synovitis is blood-derived iron deposited in joints. FVIII-deficient knockout mice with trauma-induced hemarthrosis serve as a model system for hemophilic synovitis, reproducing the histological features observed in patients. In addition, this animal model recapitulates the observations made in vitro with synovial cell cultures stimulated by iron. These in vitro experiments suggested a role for iron as an agent capable of inducing proliferation and oncogene expression by human and murine synovial fibroblasts. A better understanding of iron-regulated pathways and oncogene expression may lay the groundwork for targeted molecular interventions in hemophilic synovitis.

Animals↗

PubMed2006

Small-volume fluid resuscitation with hypertonic saline prevents inflammation but not mortality in a rat model of hemorrhagic shock.

Hemorrhage remains a primary cause of death in civilian and military trauma. Permissive hypotensive resuscitation is a possible approach to reduce bleeding in patients until they can be stabilized in an appropriate hospital setting. Small-volume resuscitation with hypertonic saline (HS) is of particular interest because it allows one to modulate the inflammatory response to hemorrhage and trauma. Here, we tested the utility of permissive hypotensive resuscitation with hypertonic fluids in a rat model of hemorrhagic shock. Animals were subjected to massive hemorrhage [mean arterial pressure (MAP) = 30 - 35 mmHg for 2 h until decompensation] and partially resuscitated with a bolus dose of 4 mL/kg of 7.5% NaCl (HS), hypertonic hydroxyl ethyl starch (HHES; hydroxyl ethyl starch + 7.5% NaCl), or normal saline (NS) followed by additional infusion of Ringer solution to maintain MAP at 40 to 45 mmHg for 40 min (hypotensive state). Finally, animals were fully resuscitated with Ringer solution and the heparinized shed blood. Hypotensive resuscitation with NS caused a significant increase in plasma interleukin (IL)-1beta, IL-6, IL-2, interferon gamma (IFNgamma), IL-10, and granulocyte-macrophage colony stimulating factor (GM-CSF). This increase was blocked by treatment with HS. HHES treatment significantly reduced the increase of IL-1beta and IL-2 but not that of the other cytokines studied. Despite the strong effects of HS and HHES on cytokine production, both treatments had little effect on plasma lactate, base excess (BE), white blood cell (WBC) count, myeloperoxidase (MPO) content, and the wet/dry weight ratio of the lungs. Moreover, on day 7 after shock, the survival rate in rats treated with HS was markedly, but not significantly, lower than that of NS-treated animals (47% vs. 63%, respectively). In summary, hypotensive resuscitation with hypertonic fluids reduces the inflammatory response but not lung tissue damage or mortality after severe hemorrhagic shock.

Animals↗

PubMed2006

Relation between ADAMTS13 activity and ADAMTS13 antigen levels in healthy donors and patients with thrombotic microangiopathies (TMA).

We have established a new, enzyme-linked immunosorbent assay (ELISA) for the detection of ADAMTS13 antigen using purified polyclonal rabbit anti-human ADAMTS13 IgG. Normal plasma ADAMTS13 antigen levels span a concentration of 740-1420 ng/ml (median 1080 ng/ml) resulting in an ADAMTS13 activity to antigen ratio of 0.48 to 1.68 U/mug. In a cohort of HUS patients, ADAMTS13 antigen was in the normal range, whereas in hereditary TTP patients antigen levels were low to undetectable, in concordance with severe deficient ADAMTS13 activity. Plasma of acquired TTP patients was found to contain free as well as autoantibody-bound ADAMTS13. We also present evidence for circulating anti-ADAMTS13 antibody/ADAMTS13 antigen immune complexes not only in acutely ill or actively treated patients but also in patients who have already achieved clinical remission. This new developed ADAMTS13 antigen ELISA assay allows rapid determination of ADAMTS13 antigen levels in human plasma but is of limited predictive value for the diagnosis or treatment of acquired TTP due to the detection of ADAMTS13 in antibody complexes.

ADAM Proteins↗

PubMed2006

In-vitro and in-vivo consequences of mutations in the von Willebrand factor cleaving protease ADAMTS13 in thrombotic thrombocytopenic purpura.

Thrombotic thrombocytopenic purpura (TTP) is a disease characterized by microvascular thrombosis, often associated with deficiency of the vonWillebrand factor (VWF) cleaving protease ADAMTS13. We investigated the spectrum of ADAMTS13 gene mutations in patients with TTP and congenital ADAMTS13 deficiency to establish the consequences on ADAMTS13 processing and activity. We describe five missense (V88M, G1239V, R1060W, R1123C and R1219W), 1 nonsense (W1016Stop) and 1 insertion (82_83insT) mutations. In two patients no mutation was identified despite undetectable protease activity. Expression in HEK293 mammalian cells (V88M, G1239V, R1123C and R1219W) documented that three missense mutants were not secreted, whereas theV88M was secreted at low levels and with reduced activity. We also provide evidence that impaired secretion of ADAMTS13 mutants observed in vitro translates into severely reduced ADAMTS13 antigen levels in patients in vivo. To evaluate whether the small amounts of mutant protease present in the circulation of patients had VWF cleaving activity, WT and mutant rADAMTS13 were stably expressed in Drosophila S2 cells under the influence of the Drosophila BiP protein signal sequence, which allows protein secretion. Drosophila expression system showed a 40-60% protease activity in the mutants. Several single nucleotide polymorphisms (SNPs) within exons and intron boundaries were found in patients, suggesting that the interplay of SNPs could at least in part account for ADAMTS13 functional abnormalities in patients without mutations. In conclusion, defective secretion and impaired activity of the mutants concur to determine an almost complete deficiency of ADAMTS13 activity in patients with a homozygous or two heterozygous ADAMTS13 mutations.

ADAM Proteins↗

PubMed2005

Modulation of ADAMTS13 secretion and specific activity by a combination of common amino acid polymorphisms and a missense mutation.

Sequence analysis of the ADAMTS13 locus of 2 patients with hereditary thrombotic thrombocytopenic purpura (TTP) revealed the homozygous presence of 4 single nucleotide polymorphisms (SNPs) (R7W, Q448E, P618A, A732V) and a rare missense mutation (R1336W). Analysis of the individual effect of any amino acid exchanges showed that several sequence variations can interact with each other, thereby altering the phenotype of ADAMTS13 deficiency. Introduction of polymorphisms R7W, Q448E, and A732V had no or only minor effects on ADAMTS13 secretion. In contrast, P618A, R1336W, and the A732V-P618A combination strongly reduced ADAMTS13-specific activity and antigen levels. Surprisingly, R7W and Q448E were positive modifiers of ADAMTS13 secretion in the context of P618A and A732V but neither could rescue the severely reduced specific activity conferred by P618A. However, in the context of R1336W, polymorphisms R7W and Q448E enhanced the detrimental effect of the missense mutation and led to undetectable enzyme activity. We show that dependent on the sequence context, the same polymorphisms might be either positive or negative modifiers of gene expression. Our results might therefore be widely relevant to understanding the influence of polymorphisms on the phenotypic expression of complex diseases.

ADAM Proteins↗

PubMed2005

Efficiency of curative and prophylactic treatment with rituximab in ADAMTS13-deficient thrombotic thrombocytopenic purpura: a study of 11 cases.

Thrombotic thrombocytopenic purpura (TTP) is a life-threatening disease that occurs mainly in young adults. Acquired cases are usually a result of antibodies directed against ADAMTS13 (a disintegrin-like and metalloprotease [reprolysin type] with thrombospondin type 1 motif 13), a protease that cleaves the von Willebrand factor multimers. Prognosis has been improved by plasma therapy, but some acute severe forms are refractory to this treatment and achieving a sustained remission is still a challenge in chronic relapsing forms. We therefore conducted a multicentric open-label prospective trial to test the efficacy of rituximab, an anti-B-cell monoclonal antibody, as a curative and prophylactic treatment in patients with TTP as a result of anti-ADAMTS13 antibodies. Six patients were included during an acute refractory TTP episode. Five patients with severe relapsing TTP and persistent anti-ADAMTS13 antibodies were prophylactically treated during remission. All patients received 4 weekly infusions of rituximab. The target of treatment was to restore a significant ADAMTS13 plasma activity (> 10%). Treatment with rituximab led to clinical remission in all cases of acute refractory TTP. In all patients, anti-ADAMTS13 antibodies disappeared, and a significant (18%-75%) plasma ADAMTS13 activity was detected following treatment. Tolerance of rituximab was good. Rituximab is a promising first-line immunosuppressive treatment in patients with acute refractory and severe relapsing TTP related to anti-ADAMTS13 antibodies.

ADAM Proteins↗

PubMed2005

ADAMTS13 autoantibodies in patients with thrombotic microangiopathies and other immunomediated diseases.

Autoantibodies neutralizing human ADAMTS13 (a disintegrin-like and metalloproteinase with thrombospondin type 1 motif), the metalloprotease that physiologically cleaves von Willebrand factor, are a major cause of severe deficiency of the protease and of acquired thrombotic thrombocytopenic purpura (TTP). We evaluated prevalence of anti-ADAMTS13 antibodies in 59 patients with thrombotic microangiopathies (TMAs) and in 160 patients with immunologic or thrombocytopenic diseases different from TTP, using an enzyme-linked immunosorbent assay (ELISA). Immunoglobulin G (IgG) antibodies directed against ADAMTS13 were found in 97% of untreated patients with acute acquired TMA who had plasma levels of ADAMTS13 activity below 10%. The corresponding prevalence of IgM antibodies was 11%. In contrast, anti-ADAMTS13 antibodies of G or M isotypes were detected in 20% of patients with TMA with ADAMTS13 activity above 10%. The ELISA was more sensitive than the standard functional inhibitor assay for detecting antibodies against ADAMTS13. Patients with thrombocytopenia from various causes (n = 50), systemic lupus erythematosus (SLE; n = 40), and the antiphospholipid antibody syndrome (APS; n = 55) had prevalences of IgG antibodies of 8%, 13%, and 5% respectively, only slightly higher than the prevalence in 111 healthy donors (4%). A rather high prevalence of anti-ADAMTS13 IgM antibodies was found in patients with SLE and APS (18% each). The clinical significance of IgM antibodies in these groups is unclear. In conclusion, the ELISA method detected anti-ADAMTS13 IgG antibodies in a very large proportion of patients with acquired TMA associated with severe ADAMTS13 deficiency, and was more sensitive than the inhibitor assay.

ADAM Proteins↗

PubMed2005

Rituximab prevents recurrence of thrombotic thrombocytopenic purpura: a case report.

Thrombotic thrombocytopenic purpura (TTP) is a rare disorder of small vessels that is associated with deficiency of the von Willebrand factor-cleaving protease, ADAMTS13. The presence of anti-ADAMTS13 autoantibodies is considered a factor predisposing to relapses. Despite close monitoring and intensive plasma treatment, in these patients acute episodes are still associated with substantial morbidity and mortality rates, and the optimal therapeutic option should be prevention of relapses. This study was conducted in a patient with recurrent TTP due to high titers of ADAMTS13 inhibitors, who used to have 2 relapses of TTP a year. The study compared the standard treatment plasma exchange with rituximab. Results documented that plasma exchange had only a small transient effect on ADAMTS13 activity and inhibitors; on the contrary, prophylaxis with rituximab was associated with disappearance of anti-ADAMTS13 antibodies, a progressive recovery of protease activity, and it allowed the patient to maintain a disease-free state during a more than 2-year follow-up.

ADAM Proteins↗

PubMed2005

Complement factor H mutation in familial thrombotic thrombocytopenic purpura with ADAMTS13 deficiency and renal involvement.

Thrombotic thrombocytopenic purpura is a rare disorder of small vessels that is associated with deficiency of the von Willebrand factor-cleaving protease ADAMTS13, which favors platelet adhesion and aggregation in the microcirculation. The disease manifests mainly with central nervous system symptoms, but cases of renal insufficiency have been reported. Presented are findings of the genetic basis of phenotype heterogeneity in thrombotic thrombocytopenic purpura in two sisters within one family. The patients had ADAMTS13 deficiency as a result of two heterozygous mutations (causing V88M and G1239V changes). In addition, a heterozygous mutation (causing an S890I change) in factor H of complement was found in the patient who developed chronic renal failure but not in her sister, who presented with exclusive neurologic symptoms.

ADAM Proteins↗

PubMed2004

Fatal congenital thrombotic thrombocytopenic purpura with apparent ADAMTS13 inhibitor: in vitro inhibition of ADAMTS13 activity by hemoglobin.

Severe ADAMTS13 deficiency in thrombotic thrombocytopenic purpura (TTP) is either constitutional and caused by ADAMTS13 mutations, or acquired and most often due to ADAMTS13 inhibitory autoantibodies. In strongly hemolytic serum of a pediatric patient, diagnosed with TTP postmortem, ADAMTS13 activity was less than 3%. Both parents had an ADAMTS13 activity of approximately 50%. Sequencing of the ADAMTS13 gene revealed an intronic 687-2A>G substitution affecting exon 7, homozygous in the propositus and heterozygous in both parents, confirming constitutional ADAMTS13 deficiency. ADAMTS13 activity of normal plasma was inhibited by incubation with the propositus' serum, suggesting alloantibody formation to ADAMTS13. However, immunoglobulin purified from serum had no ADAMTS13 inhibitory effect, whereas the immunoglobulin-depleted hemolytic serum inhibited ADAMTS13 activity of normal plasma, suggesting an inhibitory effect of hemolysis products. Incubation of hemoglobin, recombinant and from lysed erythrocytes, with normal plasma revealed an ADAMTS13 inhibitory effect at hemoglobin concentrations of 2 g/L or higher.

ADAM Proteins↗

PubMed2004

An antibody specific for coagulation factor IX enhances the activity of the intrinsic factor X-activating complex.

During hemostasis the zymogen factor X (FX) is converted into its enzymatically active form factor Xa by the intrinsic FX-activating complex. This complex consists of the protease factor IXa (FIXa) that assembles, together with its cofactor, factor VIIIa, on a phospholipid surface. We have studied the functional properties of a FIXa-specific monoclonal antibody, 224AE3, which has the potential to enhance intrinsic FX activation. Binding of the antibody to FIXa improved the catalytic properties of the intrinsic FX-activating complex in two ways: (i) factor VIIIa bound to the FIXa-antibody complex with a more than 18-fold higher affinity than to FIXa, and (ii) the turnover number (kcat) of the enzyme complex increased 2- to 3-fold whereas the Km for FX remained unaffected. The ability of 224AE3 to increase the FXa-generation potential (called the "booster effect") was confirmed in factor VIII (FVIII)-depleted plasma, which was supplemented with different amounts of recombinant FVIII. In the presence of antibody 224AE3 the coagulant activity was increased 2-fold at physiological FVIII concentration and up to 15-fold at low FVIII concentrations. The booster effect that we describe demonstrates the ability of antibodies to function as an additional cofactor in an enzymatic reaction and might open up a new principle for improving the treatment of hemophilia.

Animals↗

PubMed2004

Epitope mapping of ADAMTS13 autoantibodies in acquired thrombotic thrombocytopenic purpura.

Severe deficiency of the von Willebrand factor (VWF)-cleaving protease ADAMTS13 can lead to thrombotic thrombocytopenic purpura (TTP), a disease associated with the widespread formation of platelet-rich thrombi in many organs. Autoantibodies that inactivate ADAMTS13 are the most frequent cause of acquired TTP. Little is known about epitope specificity and reactivity of anti-ADAMTS13 antibodies. In this study, a series of ADAMTS13 domains were expressed in Escherichia coli, and the reactivity of purified recombinant fragments with anti-ADAMTS13 auto-antibodies from 25 patients with severe ADAMTS13 deficiency was evaluated in vitro. All TTP plasmas contained antibodies directed against the cysteine-rich spacer (cys-rich/spacer) domain of ADAMTS13. In the plasma of 3 patients, antibodies were detected that reacted exclusively with the cys-rich/spacer domain, underscoring the importance of this region for functional activity of ADAMTS13. In 64% of the plasmas, antibodies reacted with the 2 CUB domains, and in 56% they reacted with the isolated first thrombospondin type 1 (TSP-1) repeat and with the compound fragment consisting of the catalytic, the disintegrin-like, and the TSP1-1 domain. Less frequently, in 28% of the plasmas, antibodies reacted with the TSP1 repeats 2 to 8. Unexpectedly, antibodies reacted with the propeptide region in 20% of the plasmas. In conclusion, this study shows that even though anti-ADAMTS13 autoantibodies react with multiple domains of the protease, the cys-rich/spacer domain is consistently involved in antibody reactivity.

ADAM Proteins↗

PubMed2004

Native versus recombinant high-mobility group B1 proteins: functional activity in vitro.

To compare the functional activity of native HMGB1 proteins from eukaryotic sources with HMGB1 from prokaryotic sources the cDNAs of human and murine HMGB1 were cloned and the proteins expressed in bacteria. Tissue-derived HMGB1 from calf thymus and HMGB1 secreted from Chinese hamster ovary (CHO) cells were purified. Human whole blood, THP-1 cells, and NIH/3T3 cells were exposed to HMGB1 proteins and the induction of tumor necrosis factor-alpha (TNF-alpha) release in whole blood and monocytic THP-1 cells and a proliferation assay in NIH/3T3 cells were used to study functional activity of HMGB1s in vitro. Native and recombinant HMGB1s induced TNF-alpha release in human blood and in THP-1 cells dose-dependently, but recombinant HMGB1s were more effective. Cell proliferation was induced by native and recombinant HMGB1s. The native HMGB1 proteins from eukaryotic sources exert the same (though less pronounced) biological activity in vitro as recombinant HMGB1 proteins from prokaryotic sources.

Amino Acid Sequence↗

PubMed2004

Expression and characterization of recombinant human ADAMTS-13.

Thrombotic thrombocytopenic purpura (TTP) is a severe disease associated with unusually large, hemostatically hyperactive von Willebrand factor (VWF) and severe deficiency in ADAMTS-13, the protease responsible for the proteolytic degradation of VWF in plasma. ADAMTS-13 prevents inappropriate microvascular platelet aggregation by cleaving VWF between Tyr1605 and Met1606 thereby producing dimers of 176 kd and 140 kd and smaller multimers. Identification of the ADAMTS13 gene and cloning of the corresponding cDNA allowed for the application of recombinant techniques, such as genetic engineering of ADAMTS13 cDNA, cell culture expression, and in vitro activity studies to analyze the functional relationship between ADAMTS-13 and the pathophysiology of ADAMTS-13 deficiency. In vitro expression and characterization of recombinant ADAMTS-13 (rADAMTS-13) clearly established that ADAMTS-13 is deficient in congenital TTP and inhibited in acquired TTP. Recent studies have contributed greatly to our current understanding of the molecular mechanism leading to congenital and acquired TTP. Apart from being a useful tool, availability of rADAMTS-13 raised the prospect of developing a recombinant substitution therapy to improve TTP treatment and allowing present diagnostic assays to be simplified. Here we report on recent advances in cell culture expression and functional characterization of human rADAMTS-13.

ADAM Proteins↗

PubMed2004

Mutation of the surface-exposed amino acid Trp to Ala in the FVIII C2 domain results in defective secretion of the otherwise functional protein.

The C2 domain of factor VIII (FVIII) is important for FVIII-phospholipid (PL) and FVIII-von Willebrand factor (VWF) interactions. A FVIII structural model, derived by electron crystallography, suggests four hydrophobic loops at the FVIII C2 domain-PL interface. Within loop four, the solvent-exposed amino acid, Trp(2313), is believed to contribute to FVIII-PL binding. To analyse this interaction, the amino-acid exchange Trp(2313) to Ala (W2313A) was introduced into the C2 domain of B-domain-deleted FVIII (dBFVIII). Both proteins, dBFVIII and W2313A, were expressed in a mammalian expression system. Labelling experiments showed that the mutation W2313A resulted in reduced secretion but did not affect intracellular synthesis of the protein. Specific activity, kinetic parameters, binding to VWF and haemostatic potential in a murine model of haemophilia A were found to be similar for both proteins. Binding studies to synthetic 4% phosphatidyl-l-serine vesicles showed, however, a 28-fold higher K(D) for W2313A, indicating the important role of Trp(2313) in the FVIII-PL interaction. In conclusion, the C2-domain-surface-exposed residue Trp(2313), is critical for secretion of the protein. The W2313A mutation weakens binding to phosphatidyl-l-serine vesicles but the mutant protein has the same effector function as dBFVIII in vitro and in vivo.

Alanine↗

PubMed2003

Higher expression of Fab antibody fragments in a CHO cell line at reduced temperature.

A chimeric Fab was expressed in Chinese hamster ovary cells under the control of the CMV promoter in a two-stage production process. Cells were first grown to 90% confluence at 37 degrees C in a proliferation phase, followed by a production phase at either 37 degrees C or 28 degrees C. Medium supplemented with serum and medium free from serum was tested in the production phase at both temperatures. Comparison of Fab expression revealed that reducing the temperature to 28 degrees C resulted in a 14-fold increase in product yield when cells were cultivated in serum-containing medium, and in a 38-fold increase in product yield when serum-free medium was applied.

Animals↗