Decreased levels of ferritin, mild thrombocytosis, and increased erythropoietin are sequential events among frequent plateletpheresis donors: Implication for a ferritin screen.

BACKGROUND: It is generally recognized that repeat apheresis increases the risk for iron deficiency, thus may impact on the blood homeostasis. With regard to donor vigilance, we clarified the mid- to long-term effects of plateletapheresis by comparing the most frequent donors with the first-time ones in hematological and biochemical tests. METHODS: Levels of erythropoietin (EPO), hemoglobin (Hb) and ferritin were analyzed in double-unit (500 mL whole blood or 6 × 1011 apheresis platelets) donations in three male cohorts, with identifiers of first-time whole-blood donors (n = 30), first-time platelet donors transited from maximal whole blood to apheresis (n = 30) and frequent donors subjected to extreme plateletpheresis (n = 90), respectively. According to the number of donations, the last earnest cohort, who donate almost 24 times a year, was further subdivided into three groups- casual (76-120 life-time donations in 5 years), mediocre (121-168 within 7 years) and enthusiastic (≥169 within 7 years and a month). RESULTS: Regardless of the donation experience in whole blood or plateletpheresis, iron deficiency (serum ferritin concentrations <15 µg/L) was identified in all earnest cohorts. The ferritin means were significantly lower in plateletpheresis groups, with the lowest values in the enthusiastic group. EPO levels showed a significant inverse correlation with ferritin (p = 0.015, r = -0.224). Long-term earnest donors had the lowest iron stores accompanied by a later thrombocytosis and a final increase in EPO was revealed. CONCLUSION: Regular ferritin screens are crucial to ensure a high level of donor health protection.

Hyperglycemia exacerbates intracerebral hemorrhage via the downregulation of aquaporin-4: temporal assessment with magnetic resonance imaging.

BACKGROUND AND PURPOSE: Intracerebral hemorrhage (ICH) is associated with high mortality and neurological deficits, and concurrent hyperglycemia usually worsens clinical outcomes. Aquaporin-4 (AQP-4) is important in cerebral water movement. Our aim was to investigate the role of AQP-4 in hyperglycemic ICH. METHODS: Hyperglycemia was induced by intraperitoneal injection of streptozotocin (STZ; 60 mg/kg) in adult Sprague-Dawley male rats. ICH was induced by stereotaxic infusion of collagenase/heparin into the right striatum. One set of rats was repeatedly monitored by MRI at 1, 4, and 7 days after ICH induction so as to acquire information on the formation of hematoma and edema. Another set of rats was killed and brains were examined for differences in the degree of hemorrhage and edema, water content, blood-brain barrier destruction, and AQP-4 expression. RESULTS: Hyperglycemia ICH rats exhibited increased brain water content, more severe blood-brain barrier destruction, and greater vasogenic edema as seen on diffusion-weighted MRI. Significant downregulation of AQP-4 was observed in STZ-treated rats after ICH as compared with non-STZ-treated rats. Apoptosis was greater on day 1 after ICH in STZ-treated rats. CONCLUSIONS: The expression of AQP-4 in the brain is downregulated in hyperglycemic rats as compared with normoglycemic rats after ICH. This change is accompanied by increased vasogenic brain edema and more severe blood-brain barrier destruction.

Investigation of the effect of hyperglycemia on intracerebral hemorrhage by proteomic approaches.

Intracerebral hemorrhage (ICH) is associated with high mortality and disability, and hyperglycemia worsens the clinical and neurological outcomes of patients with ICH. In this study, we utilized proteomic approaches to investigate the role of hyperglycemia in ICH. Hyperglycemia was induced by intraperitoneal injection of streptozotocin (STZ) in adult Sprague-Dawley male rats; ICH was induced by stereotaxic infusion of collagenase/heparin into the right striatum. It was observed that the size of induced hemorrhage was significantly larger in the hyperglycemic group (n=6 in each group). On the first day after ICH, an apparent decrease in the bilateral grasp was also observed for the lesioned hyperglycemic rats compared with normoglycemic ones. When employing 2-DE and MS to examine the proteomes of perihematomal and control regions in individual hyperglycemic and normoglycemic rats, eight differentially expressed protein targets were identified. Most noteworthy, in response to ICH significant increase of albumin was ubiquitously observed in the brains of normoglycemic rats but not in the brains of hyperglycemic rats. Coincidentally, more significant neuronal apoptosis were found in the perihematomal regions of hyperglycemic rats. These observations described suggest the protection role of albumin in acute stage of ICH, which may be dependent on different blood sugar levels.

Evaluating the Effect of Inert Recruiting on Blood Donations Immediately After the Consecutive Earthquakes.

OBJECTIVE: Disasters can have impact on the demand and supply of blood, with such a difficult perspective, planning of an appropriate response to counterbalance the need for blood is of paramount importance. The primary objective of this study was to evaluate how the impact of blood imbalances may be absorbed by inert recruitment of donors during 2 life-threatening earthquakes that shook Taiwan on the same date in 2016 and 2018. METHOD: A retrospective database search from blood bank registries was developed. RESULTS: Despite the public efforts to restrain the flow, a 3- to 4-fold increase in volunteers responded to the earthquakes. This surge alleviated after a day and did not contribute to sub-par collections. Those who donated more than usual immediately after the event were identified as first-time, younger, and female populations. The hospitals providing inpatient care to the injured transfused a slightly decreased amount of packed red cells, whereas the use of whole blood, platelets, and plasma remained stable. The inert recruiting was effective in reducing the duration of donor overabundance. CONCLUSION: Compared with other examples, the inert recruiting approach was effective in reducing the duration of donor overabundance to 1 day and may be useful for disaster preparedness of transfusion supplies.

Cell-based assays and molecular simulation reveal that the anti-cancer harmine is a specific matrix metalloproteinase-3 (MMP-3) inhibitor.

The biological activities of harmine have been a much clearer picture in recent years, which include anti-tumor, anti-inflammation and cytotoxic properties. Numerous in vitro and in vivo animal models have confirmed its activities, but its mode of action remains a relative unsolved issue. We therefore investigated harmine for its effects on MMP-3 and the molecular interaction was also simulated. The human glioma cancer cell line, U-87 MG cells, was subjected to different concentrations (1-10 µM) of harmine for 24 h. Methylthiazol tetrazolium (MTT) test, half maximal inhibitory concentration (IC50), western blot analysis, enzyme-linked immunosorbent assay and molecular docking through BIOVIA DiscoveryStudio™ were performed. These results showed that although harmine stimulation in vitro has very little or no effects on MMP-3 expression by U-87 MG cells, the treatment of harmine decreases MMP-3 activity in a dose dependent manner. It was further calculated that 7.9 µM is the IC50 towards MMP-3. Using a molecular dynamic simulation approach, we identified the N2, methyl of C1 and benzene ring of harmine interact with Zn2+ (2.4 Å), His205 (2.4 Å) and His211 (2.4 Å) as well as Val163 (2.7 Å) at the active site of MMP-3, respectively, and thus conferred a striking specific binding advantage. Taken altogether, the present study evidences that harmine acts as an MMP-3 inhibitor specially targeting the enzymatic active site and possibly efficiently ameliorates MMP-3-driven malignant and inflammatory diseases.

A proteomics-based translational approach reveals an antifolate resistance inherent in human plasma derived from blood donation.

The inhibition of dihydrofolate reductase (DHFR) by antifolates is a common practice both in cell culture and in chemotherapy. Surprisingly, antifolate resistance was also observed in cultured murine myeloma cells (SP2/0) in the presence of human plasma (HP); thus, we used a proteomic approach to identify novel plasma biomarker(s) for this condition. In contrast to the in vitro antifolate response, metabolic enzymes and translation machinery proteins were found to be up-regulated in the presence of HP. The antifolate resistance inherent in HP may be explained by a simultaneous promotion of cell proliferation and the maintenance of DNA integrity. Furthermore, the factor(s) was found to be extrinsic, heat stable and very small in size. Adenine, a supplemented additive in erythrocyte preservation, was subsequently identified as the contributing factor and exogenous addition in cultures reversed the cytotoxicity induced by antifolates. Importantly, adenine-containing blood components, which may provide enhanced survival to otherwise sensitive antifolate-targeted cells, showed a dose-dependent adverse effect in transfusion recipients receiving antifolate (methotrexate) medications. These findings not only highlight a previously unnoticed role of adenine, but also emphasize a novel mechanistic link between transfusion and subsequently reduced survival in patients taking methotrexate.

Salmonella enterica serotype typhimurium and S. Stanley differ in genomic evolutionary patterns and early immune responses in human THP-1 cell line and CD14+ monocytes.

Salmonella Typhimurium and S. Stanley are the most prevalent serogroup B serovars to infect humans in Taiwan. The aim was to determine possible factors to influence the prevalence between S. Typhimurium and S. Stanley. Genotypes were determined by pulsed field gel electrophoresis (PFGE) analysis and the intracellular survival, phagocytosis, reactive oxygen species (ROS) production of human monocyte THP-1 cell and tumor necrosis factor-α(TNF-α), interleukin-6 (IL-6), and IL-1ßexpression in peripheral blood CD14+ cells after infection were analyzed. 182 S. Stanley was clonal disseminated with main pulsotypes 2 from 2004 to 2007. Overall S. Typhimurium evolved more genotypes, while S. Stanley conserved in genotypes. Human blood CD14+ monocytes expressed TNF-α, IL-6 and IL-1ß differently among serovars and bacterial conditions (live vs. killed). Live S. Stanley and S. Typhimurium suppressed the TNF-α and IL-6 expression compared to killed bacteria. However, live S. Typhimurium stimulated more IL-1ß expression than the killed bacteria, but S. Stanley expressed similar IL-1ß levels in both conditions. Furthermore, S. Stanley and S. Typhimurium differed in intracellular survival in the THP-1 cells, an early decrease for S. Stanley, not for S. Typhimurium. Additionally, higher reactive oxygen species (ROS) production in THP-1 cells was found agsinst S. Stanley infection, not found in S. Typhimurium. However, some isolates of S. Stanley could recover from early loss to become more in the monocytes than S. Typhimurium. Difference in phagocytized number, intracellular survival, ROS production and IL-1ß expression may contribute to prevalence different between two serovars.

Direct visualization of fluorescent signals in protein gels using a backlit blue light plate.

The visualization of fluorescently labeled protein gels is required for the analysis of electrophoretic results or manually picking protein bands or spots from gels. To accomplish this task, an UV light table is generally utilized, but may be hazardous to operators. The blue light transilluminator is another apparatus that may be used for this purpose, but is sometimes insufficient for revealing weak fluorescent signals. In this study, we invented a new setup utilizing a backlit blue light plate for illuminating fluorescently stained protein gels. This method employs Snell's law and allows for the direct visualization of fluorescent signals in protein gels without the use of a filter or filter glasses. This safe, convenient, economic, and effective setup was found to be an ideal alternative for illuminating fluorescent protein gels in proteomic experiments.

Site-directed in vitro immunization leads to a complete human monoclonal IgG4 lambda that binds specifically to the CDR2 region of CTLA-4 (CD152) without interfering the engagement of natural ligands.

BACKGROUND: The ability to acquire fully human monoclonal antibodies (mAbs) with pre-defined specificities is critical to the development of molecular tags for the analysis of receptor function in addition to promising immunotherapeutics. Yet most of the arriving affinity maturated and complete human immunoglobulin G (IgG) molecules, which are actually derived from single human B cells, have not widely been used to study the conserved self antigens (Ags) such as CD152 (cytotoxic T lymphocyte antigen-4, CTLA-4) because proper hosts are lacking. RESULTS: Here we developed an optimized protocol for site-directed in vitro immunizing peripheral blood mononuclear cells (PBMC) by using a selected epitope of human CD152, an essential receptor involved in down-regulation of T cell activation. The resultant stable trioma cell lines constantly produce anti-CD152 mAb (gamma4lambdahuCD152), which contains variable (V) regions of the heavy chain and the light chain derived from the VH3 and V lambda human germline genes, respectively, and yet displays an unusual IgG4 isotype. Interestingly, gamma4lambdahuCD152 has a basic pI not commonly found in myeloid monoclonal IgG4 lambdas as revealed by the isoelectric focusing (IEF) analysis. Furthermore, gamma4lambdahuCD152 binds specifically, with nanomolar affinity, to an extracellular constituency encompassing the putative second complementarity determining region (CDR2) of CD152, whereby it can react to activated CD3+ cells. CONCLUSION: In a context of specific cell depletion and conditioned medium,in vitro induction of human Abs against a conserved self Ag was successfully acquired and a relatively basic mAb, gamma4lambdahuCD152, with high affinity to CDR2 of CD152 was thus obtained. Application of such a human IgG4 lambda mAb with designated CDR2 specificity may impact upon and prefer for CD152 labeling both in situ and ex situ, as it does not affect the binding of endogenous B7 ligands and can localize into the confined immunological synapse which may otherwise prevent the access of whole IgG1 molecules.

Development of a Colloidal Gold-based Immunochromatographic Test Strip for Detection of Cetacean Myoglobin.

This protocol describes the development of a colloidal gold immunochromatographic test strip based on the sandwich format that can be used to differentiate the myoglobin (Mb) of cetaceans from that of seals and other animals. The strip provides rapid and on-the-spot screening for cetacean meat, thereby restraining its illegal trade and consumption. Two monoclonal antibodies (mAbs) with reactivity toward the Mb of cetaceans were developed. The amino acid sequences of Mb antigenic reactive regions from various animals were analyzed in order to design two synthetic peptides (a general peptide and a specific peptide) and thereafter raise the mAbs (subclass IgG1). The mAbs were selected from hybridomas screened by indirect ELISA, western blot and dot blot. CGF5H9 was specific to the Mbs of rabbits, dogs, pigs, cows, goats, and cetaceans while it showed weak to no affinity to the Mbs of chickens, tuna and seals. CSF1H13 can bind seals and cetaceans with strong affinity but showed no affinity to other animals. Cetacean samples from four families (Balaenopteridae, Delphinidae, Phocoenidae and Kogiidae) were used in this study, and the results indicated that these two mAbs have broad binding ability to Mbs from different cetaceans. These mAbs were applied on a sandwich-type colloidal gold immunochromatographic test strip. CGF5H9, which recognizes many species, was colloid gold-labeled and used as the detection antibody. CSF1H13, which was coated on the test zone, detected the presence of cetacean and seal Mbs. Muscle samples from tuna, chicken, seal, five species of terrestrial mammals and 15 species of cetaceans were tested in triplicate. All cetacean samples showed positive results and all the other samples showed negative results.

Poor responses to interferon-beta treatment in patients with neuromyelitis optica and multiple sclerosis with long spinal cord lesions.

Interferon-beta (IFN-ß) treatment may not be effective in neuromyelitis optica (NMO). Whether the poor response to IFN-ß is related to long spinal cord lesions (LSCL) or the NMO disease entity itself is unclear. We evaluated the spinal cord involvement of patients with multiple sclerosis (MS) and NMO, as well as the response after receiving IFN-ß. Forty-nine MS and 21 NMO patients treated with IFN-ß for at least 2 years from 2002-2008 were enrolled in this study and the treatment response was analyzed 2 years post-treatment. In the study, spinal cord lesions were present in 57.1% (28/49) of the MS patients, of which 16.3% (8/49) presented spinal cord lesions longer than 3 vertebral segments (LSCL). Responses to IFN-ß treatment were seen in 69.3% (34/49) of all the MS cases, of which the appropriate response rates were 76.1% (16/21) in MS patients without spinal cord lesions and 37.5% (3/8) in patients with LSCL. Only 14.2% (3/21) of NMO patients responded to IFN-ß treatment. In conclusion, spinal cord lesion is common in MS patients in Taiwan. Both NMO and MS patients with LSCL had a poor response to IFN-ß treatment. NMO patients had a worse response to IFN-ß treatment than MS patients with LSCL, which shows that the crucial structural defect is something other than LSCL such as the elevated serum IL17 level in NMO compared to MS.

Rapid immune colloidal gold strip for cetacean meat restraining illegal trade and consumption: implications for conservation and public health.

The consumption of cetacean meat is geographically common and often of undetermined sustainability. Besides, it can expose humans to contaminants and zoonotic pathogens. The illegality of possessing cetacean meat was likely under-reported in some countries due to lack of attention paid by the officials although DNA analysis of market products helped to show such practices. We developed two monoclonal antibodies against synthetic peptides of myoglobin (Mb) for constructing a rapid immune colloidal gold strip. Only cetacean Mb is capable of binding to both antibodies and presents positive signal while the Mb from other animals can bind only 1 of the antibodies and presents negative result. The strip for cetacean meat would be an applicable and cost-effective test for field inspectors and even the general public. It contributes to increase the reporting capacity and coverage of illegal cetacean meat possession, which has implications for global cetacean conservation and public health.

Identification of the nanogold particle-induced endoplasmic reticulum stress by omic techniques and systems biology analysis.

Growth inhibition and apoptotic/necrotic phenotype was observed in nanogold particle (AuNP)-treated human chronic myelogenous leukemia cells. To elucidate the underlying cellular mechanisms, proteomic techniques including two-dimensional electrophoresis/mass spectrometry and protein microarrays were utilized to study the differentially expressed proteome and phosphoproteome, respectively. Systems biology analysis of the proteomic data revealed that unfolded protein-associated endoplasmic reticulum (ER) stress response was the predominant event. Concomitant with transcriptomic analysis using mRNA expression, microarrays show ER stress response in the AuNP-treated cells. The ER stress protein markers' expression assay unveiled AuNPs as an efficient cellular ER stress elicitor. Upon ER stress, cellular responses, including reactive oxygen species increase, mitochondrial cytochrome c release, and mitochondria damage, chronologically occurred in the AuNP-treated cells. Conclusively, this study demonstrates that AuNPs cause cell death through induction of unmanageable ER stress.

Increasing hybridoma viability and antibody repertoire after the cell fusion by the use of human plasma as an alternative supplement.

Prion diseases such as Bovine Spongiform Encephalopathy (BSE) and new variant Creutzfeldt-Jakob disease (nvCJD) have caused a major safety concern in cell cultures using fetal calf serum (FCS). In this study, we found that screened and tested human plasma (HP) obtained from blood centers may be an ideal alternate nutrient substitute to FCS for culturing hybridoma. In addition to the inherent safety, a ten-fold increase in the fusion efficiency has been observed if the HP was used as the nutrient supplement instead of FCS. Subsequently, a broader antibody repertoire may be recovered. The HP supplement was found to promote the growth of hybridoma cells but no impact on antibody secretion. Interestingly, this effect of enrichment was only observed for HP, but not plasma from other animals. Unidentified murine hybridoma cloning factors other than IL-6 may specifically reside in human blood.

Immune intervention with monoclonal antibodies targeting CD152 (CTLA-4) for autoimmune and malignant diseases.

CD152 or cytotoxic T lymphocyte antigen-4 (CTLA-4) is an essential receptor involved in the negative regulation of T cell activation. Because of its profound inhibitory role, CD152 has been considered a sound susceptible candidate in autoimmunity and a persuasive target for cancer immunotherapy for over a decade. However, the precise roles played by this molecule continue to emerge. In particular, recent evidence suggests that CD152 is also important in the homeostasis and function of a population of suppressive cells, termed regulatory T cells (Treg). In this review, we discuss the recent progress and main features of monoclonal antibodies (mAbs) targeting CD152 and examine how each mAb prepared to a distinct epitope may impact differently upon CD152 modulation depending on its demonstrated regulatory role acting as an agonist, antagonist, or inverse agonist.