Li-Te Chin

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.

Seroprevalence and demographic characteristics of HTLV-I among blood donors in Taiwan: 1996-1999.

Screening for the human T-cell lymphotropic virus type I (HTLV-I) and-II in blood donors was implemented in Taiwan beginning in February 1996. The purpose of the present study was to investigate the changes in HTLV-I seroprevalence in all unpaid blood donors in Taiwan during the period from February 1996 to December 1999 and to determine the influence of age and sex on the HTLV-I seropositivity of donors. HTLV-I and HTLV-II screening was performed using combined HTLV-I/II immunoassay. Repeated reactive samples were confirmed by Western blot analysis. Of a total of 3,701,087 donors in all 6 blood centers in Taiwan, 2311 (0.058%) were seropositive for HTLV-I. The HTLV-I seropositivity was 0.130%, 0.063%, 0.044%, and 0.032% in the years 1996, 1997, 1998, and 1999, respectively.There was a linear increase of HTLV-I seropositivity with advancing age.The HTLV-I carrier rate for female donors was twice that for the male donors. Ninty-seven percent of HTLV-I seropositive results came from first-time donors. Our findings suggest that Taiwan is a low-prevalence nonendemic area for HTLV-I infection. The large-scale HTLV-I screening program has decreased HTLV-I seropositivity among blood donors and is useful for preventing HTLV-I transmission via blood transfusion.

Molecular characterization of a human anti-HIV 1 monoclonal antibody revealed a CD26-related motif in CDR2

Genes encoding the immunoglobulin variable regions of a human anti-HIV-1 IgG1 kappa monoclonal antibody were rescued from a hybridoma, derived from a sero-negative donor, using PCR cloning and expression in Escherichia coli. The ELISA binding results obtained from the expressed Fab fragment confirmed the anti-V3 loop specificity for HIV-1 (LAI) of the original antibody. In addition, an amino acid sequence derived from the second complementarity determining region (CDRH2) of the heavy chain was found to be very similar to the catalytic motif of CD26, a T-cell activation antigen. Furthermore, synthetic peptides containing both the catalytic domain of CD26 and CDRH2 of the antibody showed specific binding to an HIV peptide representing the V3 region in a dose-dependent manner. This suggests an involvement of CD26 as a possible coreceptor for HIV-1.

Seroprevalence of Cmv Antibodies in a Blood Donor Population and Premature Neonats in The South-Central Taiwan

Infection of cytomegalovirus (CMV) via contaminated blood may endanger immunocompromised patients that require transfusion therapy. The aim of this study is to determine the prevalence of CMV antibodies in the blood donor population in Southern-central Taiwan. A total of 1800 consecutive sera, obtained from Tainan Blood Center of Chinese Blood Services Foundation (CBSF), were tested for CMV antibodies by two commercial enzyme immunoassays (EIAs). Of the sera tested, 150 (8.3%) were found to be CMV seronegative. The frequency of CMV seropositivity revealed no significant difference between male and female donors. The frequency of CMV seronegativity showed a stepwise decrease with the increase of donor age. In addition, the prevalence of HBsAg, antibodies to hepatitis C virus (anti-HCV), antibodies to human immunodeficiency viruses type 1 and 2 (anti-HIV 1 + 2) and antibodies to human T-cell lymphotropic viruses type I and II (anti-HTLV I/II) were compared between CMV seropositive and seronegative groups. Our results showed that there was no significant difference in seroprevalence of these markers between CMV seropositive and seronegative groups. Our findings also showed that six out of twenty (30.0%) premature neonates were CMV-seropositive. These premature specimens and those EIA discrepancy samples were confirmed by specific nucleic acid amplification using polymerase chain reaction (PCR). Our results suggest that a program which aims to supply CMV seronegative blood or blood components to the patients, should not solely depend on current antibody screening methods in an area where CMV infection is highly endemic. Amendments such as PCR testing, leukocyte reduction by filtration before transfusion may be more practical.

Human Th0-type T Helper-Cell Clone Supports Antigen-Specific Immunoglobulin Production in Scid/beige-hu Mice

Tetanus toxoid‐specific T cells have been generated from human splenic lymphocytes by an initial 6‐day stimulation period with antigen, followed by a proliferation period with recombinant IL‐2 and human feeder cells. Proliferating T cells were subsequently cloned by limiting dilution. A human T‐cell clone that was functionally characterized showed: (i) a specific proliferative response to tetanus toxoid in the presence of autotogous Epstein—Barr virus (EBV)‐transformed lymphoblastoid cells; (ii) a phenotype characteristic for the helper/inducer CD4+/CD8−/CD45RO+ T cells, and (iii) a lymphokine profile, as determined by mRNA analysis, representative of Th0‐like human CD4+ T helper cells. This tetanus toxoid‐specific T‐cell clone which showed antigen‐dependent helper activity for antibody production by autologous B cells in vitro could also provide T‐cell help to antigen‐specific human B cells transplanted into severe combined immunodeficiency (scid/beige) mice.

Intrafamilial Transmission and Risk Assessment of HTLV-I among Blood Donors in Southern Taiwan

The human T-lymphotropic virus type I (HTLV-I) is one of the important etiological agents of adult T-cell leukemia/lymphoma and of HTLV-I associated myelopathy or tropical spastic paraparesis. There is still a lack of data concerning HTLV-I transmission by seropositive carriers in Taiwan. We investigated the patterns of HTLV-I intrafamilial transmission in HTLV-I seropositive blood donors and assessed the risk factors of HTLV-I transmission in relatives of HTLV-I carriers in Taiwan. A total of twenty HTLV-I seropositive donors and their 103 relatives were enrolled. Among those 103 relatives, 40 (38.8%) were seropositive for HTLV-I. Their ages ranged from one to 70 years old with a mean age of 31.0 +/- 1.65 year-old. Three of the ten wives of male carriers were HTLV-I seropositive. However, none of the six husbands of female carriers were HTLV-I seropositive. Mother-to-child vertical transmission was found in nine of 48 (18.8%) tested. Significant risk factors of HTLV-I transmission among relatives of HTLV-I carriers were hospital admission, previous transfusion, breast feeding, anti-HCV seropositivity and female relatives of age >/= 30 with odds ratio (OR) of 9.73, 8.64, 4.36, 8.86 and 4.91, respectively (all p < 0.05). Nonsignificant risk factors of HTLV-I transmission were sharing needles, operation history, HBsAg seropositivity and male relatives of age >/= 30. Our findings suggest that mother-to-child and husband-to-wife transmissions are the important forms of intrafamilial transmission of HTLV-I in Taiwan. Screening for HTLV-I in family members of HTLV-I seropositive blood donors may be warranted.

Site-directed in vitro immunization leads to a complete human monoclonal IgG4λ that binds specifically to the CDR2 region of CTLA-4 (CD152) without interfering the engagement of natural ligands

BackgroundThe 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.ResultsHere 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 (γ4λhuCD152), which contains variable (V) regions of the heavy chain and the light chain derived from the VH3 and Vλ human germline genes, respectively, and yet displays an unusual IgG4 isotype. Interestingly, γ4λhuCD152 has a basic pI not commonly found in myeloid monoclonal IgG4λs as revealed by the isoelectric focusing (IEF) analysis. Furthermore, γ4λhuCD152 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.ConclusionIn 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, γ4λhuCD152, with high affinity to CDR2 of CD152 was thus obtained. Application of such a human IgG4λ 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.

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.